HK1259772A1 - Use of tryptophan derivatives for protein formulations - Google Patents

Description

Use of tryptophan derivatives for protein formulations

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application No.62/273,273 filed on 30.12.2015 and U.S. provisional application No.62/321,636 filed on 12.4.2016, each of which is hereby incorporated by reference in its entirety.

Technical Field

The present invention relates to liquid formulations comprising a protein and further comprising N-acetyl-tryptophan, and methods for making and using the same.

Background

Oxidative degradation of amino acid residues is a phenomenon often observed in protein pharmaceuticals. Some amino acid residues are susceptible to oxidation, particularly methionine (Met), cysteine (Cys), histidine (His), tryptophan (Trp), and tyrosine (Tyr) (Li et al, Biotechnology and Bioengineering48:490-500 (1995)). Oxidation is typically observed when proteins are exposed to hydrogen peroxide, light, metal ions or combinations of these during various processing steps (Li et al, Biotechnology and Bioengineering48:490-500 (1995)). In particular, proteins exposed to light (Wei, et al, Analytical Chemistry 79(7): 2797-. Light exposure can lead to protein oxidation via the formation of Reactive Oxygen Species (ROS), including singlet oxygen, hydrogen peroxide and superoxide (Li et al, Biotechnology and Bioengineering48:490-500 (1995); Wei, et al, Analytical Chemistry 79(7):2797-2805 (2007); Ji et al, J Pharm Sci 98(12):4485-500 (2009); Frokjaer et al, Nat Rev Drug Discov 4(4):298-306(2005)), while protein oxidation typically occurs via hydroxyl radicals in a fragrance-mediated reaction (Prouseket et al, Pure and Applied Chemistry 79(12):2325-2338(2007)) and via alkoxy peroxides in an AAPH-mediated reaction (Wermer et al, J werer et 20117): 2011 7) (33015). Oxidation of tryptophan produces a myriad of oxidation products, including hydroxytryptophan, kynurenine (Kyn), and N-formyl kynurenine, and has the potential to affect formulation safety and efficacy (Li et al, Biotechnology and Bioengineering48:490-500 (1995); Ji et al, J Pharm Sci 98(12):4485-500 (2009); Frokjaer et al, Nat Rev Drug Discov 4(4):298-306 (2005)). Oxidation of specific tryptophan residues in the heavy chain Complementarity Determining Regions (CDRs) of monoclonal antibodies associated with loss of biological function has been reported (Wei, et al, analytical chemistry 79(7):2797-2805 (2007)). More recently, Trp oxidation mediated by histidine-coordinated metal ions has been reported for Fab molecules (Lam et al, Pharm Res 28(10):2543-55 (2011)). Autoxidation of polysorbate 20 in Fab formulations in the same study was also reported, which resulted in the formation of various peroxides. The formation of these peroxides induced by autoxidation may also lead to the oxidation of methionine in proteins during long-term storage, as the Met residue in proteins has been proposed to act as an internal antioxidant (Levine et al, Proceedings of the National Academy of sciences of the United States of America 93(26):15036-15040(1996)) and is susceptible to peroxide oxidation. Oxidation of amino acid residues of a protein has the potential to affect its biological activity. This may be particularly true for monoclonal antibodies (mabs). Methionine oxidation at Met254 and Met430 in IgG1 monoclonal antibodies potentially affected serum half-life in transgenic mice (Wang et al, Molecular Immunology 48(6-7):860-866(2011)) and also affected binding of human IgG1 to FcRn and Fc γ receptors (Bertolotti-Ciarlet et al, Molecular Immunology 46(8-9)1878-82 (2009)).

The stability of proteins, especially in the liquid state, needs to be assessed during manufacture and storage of the drug product. The development of pharmaceutical formulations sometimes includes the addition of antioxidants to prevent oxidation of the active ingredient. The addition of L-methionine to the formulations results in a reduction in the oxidation of methionine residues in proteins and peptides (Ji et al, J Pharm Sci 98(12):4485-500 (2009); Lam et al, Journal of Pharmaceutical Sciences 86(11):1250-1255 (1997)). Similarly, the addition of L-tryptophan has been shown to reduce the oxidation of tryptophan residues (Ji et al, J Pharm Sci 98(12):4485-500 (2009); Lam et al, Pharm Res 28(10):2543-55 (2011)). However, L-Trp possesses strong absorption in the UV region (260-290nm), making it the primary target during photooxidation (Creed, D., Photochemistry and Photobiology 39(4):537-562 (1984)). Trp has been hypothesized to be an endogenous photosensitizer that enhances oxygen-dependent photooxidation of tyrosine (Babuet al, Indian J Biochem Biophys 29(3):296-8(1992)) and other amino acids (Bent et al, Journal of the American Chemical Society 97(10):2612-2619 (1975)). It has been demonstrated that L-Trp is capable of generating hydrogen peroxide when exposed to light and that L-Trp generates hydrogen peroxide via superoxide anion under UV light (McCormick et al, Science 191(4226):468-9 (1976); Wentworth et al, Science 293(5536):1806-11 (2001); McCormick et al, Journal of the American chemical Society 100: 312-. In addition, tryptophan is known to generate singlet oxygen upon exposure to light (Davies, M.J., Biochem Biophys Res Commun 305(3):761-70 (2003)). Similar to protein oxidation induced by autoxidation of polysorbate 20, it is likely that protein oxidation may occur when ROS are generated from other excipients (e.g., L-Trp) in the protein formulation under normal operating conditions.

The susceptibility of a particular protein residue in a liquid formulation to oxidation may depend on the accessibility of the residue to oxidizing agents (e.g., ROS) in the formulation. Solvent Accessibility Surface Area (SASA) is a measure of the surface area accessible to a solvent by a biomolecule (e.g., amino acid residues). The SASA of amino acid residues in a protein may indicate the availability of the residue for oxidation. SASA can be calculated using a variety of methods, including the Shrake-Rupley algorithm, the pairwise overlapping Linear Combination (LCPO) method, and the Power map method (Shrake, A & Rupley, JA., J.mol.biol.79(2): 351-371, 1973; Weiser et al, J.Comp.Chem.20(2): 217-230, 1999; Klenin et al, J.Comp.Chem.32(12): 2653, 2011). More recently, full-atom Molecular Dynamics (MD) simulations have been used to calculate the SASA of amino acid residues and exhibit a binary dependence on the oxidative susceptibility of% SASA and Trp (Sharma, V.et al, PNAS.111(52):18601-18606, 2014). SASA may therefore be a useful parameter for determining the suitability of including an antioxidant in a given protein formulation.

It is evident from recent studies that the addition of standard excipients, such as L-Trp and polysorbates, to protein compositions where it is desired to stabilize proteins can lead to unexpected and unwanted consequences, such as ROS-induced protein oxidation. This is of particular interest for protein compositions having oxidation-prone residues. Thus, there remains a need to identify alternative excipients for use in protein compositions and to develop such compositions. U.S. patent publication nos. 2014/0322203 and 2014/0314778 provide examples of the use of tryptophan derivatives in protein formulations.

The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are hereby incorporated by reference in their entirety.

Summary of The Invention

The present invention provides a method of reducing oxidation of a polypeptide in an aqueous formulation comprising adding to the formulation an amount of N-acetyl tryptophan that prevents oxidation of the polypeptide, wherein the polypeptide comprisesAt least one Solvent Accessibility Surface Area (SASA) greater than aboutA tryptophan residue of (a). The present invention also provides a method of reducing oxidation of a polypeptide in an aqueous formulation comprising adding to the formulation an amount of N-acetyl tryptophan that prevents oxidation of the polypeptide, wherein the polypeptide comprises at least one tryptophan residue having a Solvent Accessible Surface Area (SASA) greater than about 30%. The invention also provides a method of reducing oxidation of a polypeptide in an aqueous formulation comprising determining the SASA value of a tryptophan residue in the polypeptide and if at least one tryptophan residue has a value greater than aboutIs added to the formulation in an amount that prevents oxidation of the polypeptide. In some embodiments, the SASA value for the tryptophan residue is calculated by molecular dynamics simulation.

In some embodiments, the N-acetyl tryptophan is added to the formulation to a concentration of about 0.1mM to about 5 mM. In some embodiments, the N-acetyl tryptophan is added to the formulation to a concentration of about 0.1mM to about 1 mM. In some embodiments, the N-acetyl tryptophan is added to the formulation to a concentration of about 0.3 mM.

In some embodiments, the oxidation of the polypeptide is reduced by about 50%, 75%, 80%, 85%, 90%, 95% or 99%. In some embodiments, the formulation is stable at about 2 ℃ to about 8 ℃ for about 1095 days.

In some embodiments, the protein concentration in the formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents.

In some embodiments, the formulation is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment.

In some aspects, the invention provides a liquid formulation comprising a polypeptide and an amount of N-acetyl tryptophan to prevent oxidation of the polypeptide, wherein the polypeptide has at least one SASA greater than aboutA tryptophan residue of (a). In some embodiments, the N-acetyl tryptophan is added to the formulation to a concentration of about 0.1mM to about 5 mM. In some embodiments, the N-acetyl tryptophan is added to the formulation to a concentration of about 0.1mM to about 1 mM. In some embodiments, the N-acetyl tryptophan is added to the formulation to a concentration of about 0.3 mM. In some embodiments, the oxidation of the polypeptide is reduced by about 50%, 75%, 80%, 85%, 90%, 95% or 99%. In some embodiments, the formulation is stable at about 2 ℃ to about 8 ℃ for about 1065 days.

In some embodiments, the protein concentration in the formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents.

In some embodiments, the formulation is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment.

In some aspects, the invention provides a method for screening a formulation for reduced polypeptide oxidation, whereinThe polypeptide comprises at least one SASA greater than aboutThe method comprising adding an amount of N-acetyl tryptophan to an aqueous composition comprising the polypeptide, adding 2, 2' -azobis (2-aminopropane) dihydrochloride (AAPH) to the composition, incubating the composition comprising the polypeptide, N-acetyl tryptophan, and AAPH at about 40 ℃ for about 14 hours, measuring oxidation of tryptophan residues in the polypeptide for the polypeptide, wherein a formulation comprising an amount of N-acetyl tryptophan that results in no more than about 20% oxidation of tryptophan residues of the polypeptide is a suitable formulation for reducing oxidation of the polypeptide. In some embodiments, the N-acetyl tryptophan and AAPH are incubated for less than any of about 10 hours, 11 hours, 12 hours, 14 hours, 16 hours, 20 hours, or 24 hours. In some embodiments, no more than about 15%, 20%, 25%, 30%, or 35% of any oxidation of tryptophan residues of the polypeptide is a suitable formulation for reducing oxidation of the polypeptide.

In some aspects, the invention provides a method for screening a formulation for reduced polypeptide oxidation comprising determining the SASA value of a tryptophan residue in the polypeptide, wherein SASA is greater than aboutIs subjected to oxidation, an amount of N-acetyl tryptophan is added to an aqueous composition comprising the polypeptide, 2' -azobis (2-aminopropane) dihydrochloride (AAPH) is added to the composition, the composition comprising the polypeptide, N-acetyl tryptophan and AAPH is incubated at about 40 ℃ for about 14 hours, oxidation of tryptophan residues in the polypeptide is measured for the polypeptide, wherein an amount of N-acetyl tryptophan is included, and formulations that result in no more than about 20% oxidation of tryptophan residues of the polypeptide are suitable formulations for reducing oxidation of the polypeptide.

In some embodiments of the above aspects, the SASA value of the tryptophan residue is calculated by molecular dynamics simulation.

In some aspects, the present invention provides a kit comprising the liquid formulation of any embodiment described herein. In some aspects, the present invention provides an article of manufacture comprising the liquid formulation of any embodiment described herein.

Provided herein are formulations comprising a protein and N-acetyl-tryptophan (NAT), and methods of making and using the same.

In some embodiments, the liquid formulation is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the formulation is aqueous.

In some embodiments, the NAT prevents oxidation of tryptophan in the protein.

In some embodiments, the protein in the formulation is susceptible to oxidation. In some embodiments, the tryptophan in the protein is susceptible to oxidation. In some embodiments, the protein is an antibody (e.g., a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, or an antibody fragment). In some embodiments, the protein concentration in the formulation is about 1mg/mL to about 250 mg/mL.

In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation has a pH of about 4.5 to about 7.0.

The invention also provides a method for determining whether a polypeptide in a liquid formulation comprises tryptophan residues susceptible to oxidation, the method comprising calculating one or more molecular descriptors for each tryptophan residue in the polypeptide based on the amino acid sequence of the polypeptide and applying the one or more molecular descriptors to a machine learning algorithm practiced on the one or more molecular descriptors to predict tryptophan oxidation, wherein the molecular descriptors comprise one or more of: a) colour of pitchAmino acid having a carbon number ofNumber of internal aspartic acid side chain oxygens, b) side chain Solvent Accessibility Surface Area (SASA), c) delta carbon SASA, d) delta carbon from tryptophanInternal overall positive charge, e) backbone SASA, f) Tryptophan side chain Angle, g) delta carbon from TryptophanInternal packing density, h) tryptophan backbone angle, i) SASA of pseudo-pi orbitals, j) backbone flexibility, or k) delta carbon to tryptophanThe total negative charge in. In some embodiments, 2,3, 4,5, 6,7, 8, 9, 10, or 11 molecular descriptors are used. In some embodiments, the molecular descriptor comprises the following: a) at a position delta to tryptophanNumber of internal aspartic acid side chain oxygens, b) side chain Solvent Accessibility Surface Area (SASA), c) delta carbon SASA, d) delta carbon from tryptophanInternal overall positive charge, e) backbone SASA, f) tryptophan side chain angle, and g) delta carbon from tryptophanPacking density of the inner part. In some embodiments, greater than 35% oxidation of tryptophan residues at a particular site is indicative of susceptibility to oxidation. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment.

In some embodiments, the machine learning algorithm is practiced by matching molecular descriptors from a molecular dynamics simulation of a polypeptide based on the amino acid sequence of the polypeptide with experimental data for each tryptophan residue in the polypeptide. In some embodiments, the one or more molecular descriptors are computed using a computer.

The present invention also provides a method for reducing oxidation of a polypeptide, comprising identifying tryptophan residues susceptible to oxidation according to any of the embodiments described above comprising a machine learning algorithm, and introducing an amino acid substitution in the polypeptide to replace one or more tryptophan residues susceptible to oxidation with an amino acid residue that is not subject to oxidation. In some embodiments, provided is a method for reducing oxidation of a polypeptide, comprising introducing an amino acid substitution in the polypeptide to replace one or more oxidation-susceptible tryptophan residues, wherein the one or more oxidation-susceptible tryptophan residues are identified by a method according to any of the embodiments described above comprising a machine learning algorithm. In some embodiments, the tryptophan residue is replaced with an amino acid residue selected from the group consisting of tyrosine, phenylalanine, leucine, isoleucine, alanine, and valine.

The present invention also provides a method for reducing oxidation of a polypeptide in an aqueous formulation comprising determining the presence of one or more tryptophan residues susceptible to oxidation in the polypeptide according to the method of any of the embodiments described above comprising a computer learning algorithm and adding an effective amount of an antioxidant to an aqueous formulation comprising a polypeptide having one or more tryptophan residues susceptible to oxidation. In some embodiments, provided is a method for reducing oxidation of a polypeptide in an aqueous formulation comprising adding an amount of an antioxidant to the aqueous formulation to prevent oxidation, wherein the polypeptide comprises one or more tryptophan residues susceptible to oxidation identified by the method of any of the above-described embodiments comprising a machine learning algorithm. In some embodiments, the antioxidant is N-acetyl tryptophan. In some embodiments, the N-acetyl tryptophan is added to the formulation to a concentration of about 0.1mM to about 5 mM. In some embodiments, the N-acetyl tryptophan is added to the formulation to a concentration of about 0.1mM to about 1 mM. In some embodiments, the N-acetyl tryptophan is added to the formulation to a concentration of about 0.3 mM. In some embodiments, the oxidation of the polypeptide is reduced by about 50%, 75%, 80%, 85%, 90%, 95% or 99%. In some embodiments, the formulation is stable at about 2 ℃ to about 8 ℃ for about 1095 days. In some embodiments, the protein concentration in the formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment.

The present invention also provides a liquid formulation comprising a polypeptide and an amount of N-acetyl tryptophan to prevent oxidation of the polypeptide, wherein the polypeptide has at least one tryptophan residue susceptible to oxidation as measured by the method of any of the embodiments described above comprising a machine learning algorithm. In some embodiments, the N-acetyl tryptophan is added to the formulation to a concentration of about 0.1mM to about 5 mM. In some embodiments, the N-acetyl tryptophan is added to the formulation to a concentration of about 0.1mM to about 1 mM. In some embodiments, the N-acetyl tryptophan is added to the formulation to a concentration of about 0.3 mM. In some embodiments, the oxidation of the polypeptide is reduced by about 50%, 75%, 80%, 85%, 90%, 95% or 99%. In some embodiments, the formulation is stable at about 2 ℃ to about 8 ℃ for about 1065 days. In some embodiments, the protein concentration in the formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In some embodiments, provided is a kit comprising the liquid formulation. In some embodiments, provided is an article of manufacture comprising the liquid formulation.

The invention also provides a method for screening a formulation for reduced oxidation of a polypeptide, wherein the polypeptide comprises at least one tryptophan susceptible to oxidation identified by the method of any of the embodiments described above comprising a machine learning algorithm, the method comprises adding an amount of N-acetyl tryptophan to an aqueous composition comprising the polypeptide, adding 2, 2' -azobis (2-aminopropane) dihydrochloride (AAPH) to the composition, incubating the composition comprising the polypeptide, N-acetyl tryptophan, and AAPH at about 40 ℃ for about 14 hours, measuring oxidation of tryptophan residues in the polypeptide for the polypeptide, formulations which contain an amount of N-acetyl tryptophan that results in no more than about 20% oxidation of the tryptophan residues of the polypeptide are suitable formulations for reducing oxidation of the polypeptide. In some embodiments, provided is a method for screening a formulation for reduced polypeptide oxidation, comprising a) identifying a polypeptide comprising one or more tryptophan residues susceptible to oxidation by a method comprising any of the embodiments described above for a machine learning algorithm, b) adding an amount of N-acetyl tryptophan to an aqueous composition comprising the polypeptide identified in step a), c) adding 2, 2' -azobis (2-aminopropane) dihydrochloride (AAPH) to the composition, d) incubating the composition comprising the polypeptide, N-acetyl tryptophan and AAPH at about 40 ℃ for about 14 hours, e) measuring oxidation of tryptophan residues in the polypeptide for the polypeptide, formulations which contain an amount of N-acetyl tryptophan that results in no more than about 20% oxidation of the tryptophan residues of the polypeptide are suitable formulations for reducing oxidation of the polypeptide.

In some aspects, the invention provides a method for measuring degradation of N-acetyl tryptophan (NAT) in a composition comprising N-acetyl tryptophan, the method comprising a) applying the composition to a reverse phase chromatography material, wherein the composition is loaded onto a chromatography material that has been equilibrated in a solution comprising mobile phase a and mobile phase B, wherein mobile phase a comprises an acid in water and mobile phase B comprises an acid in acetonitrile, B) eluting the composition from the reverse phase chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is elevated compared to step a), wherein NAT degradant elutes from the chromatography separately from intact NAT, c) quantifying the NAT degradant and the intact NAT. In some embodiments, the ratio of mobile phase B to mobile phase a in step a) is about 2: 98. In some embodiments, the ratio of mobile phase B to mobile phase a in step B) increases linearly. In some embodiments, the ratio of mobile phase B to mobile phase a in step B) is increased stepwise. In some embodiments, the flow rate for the chromatography is about 1.0 mL/min. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about 30: 70. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about 30:70 in about 16 minutes. In some embodiments, the ratio of mobile phase B to mobile phase a is further increased to about 90: 70. In some embodiments, the ratio of mobile phase B to mobile phase a is further increased to about 90:70 in about 18.1 minutes. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about 26: 74. In some embodiments, the ratio of mobile phase B to mobile phase a increases to about 26:74 in about 14 minutes. In some embodiments, the ratio of mobile phase B to mobile phase a is further increased to about 90: 70. In some embodiments, the ratio of mobile phase B to mobile phase a is further increased to about 90:70 in about 16.5 minutes. In some embodiments, mobile phase a comprises about 0.1% acid in water. In some embodiments, mobile phase B comprises about 0.1% acid in acetonitrile. In some embodiments, the acid is formic acid. In some embodiments, the reverse phase chromatography material comprises a C18 module. In some embodiments, the reverse phase chromatography material comprises a solid support. In some embodiments, the solid support comprises silica. In some embodiments, the reverse phase chromatography material is contained in a column. In some embodiments, the reverse phase chromatography material is a High Performance Liquid Chromatography (HPLC) material or an ultra high performance liquid chromatography (UPLC) material. In some embodiments, NAT and NAT degradation products are detected by absorbance at 240 nm. In some embodiments, NAT degradation products are identified by mass spectrometry. In some embodiments, the concentration of NAT in the composition is from about 10nM to about 1 mM. In some embodiments, the NAT degradation product comprises one or more of N-Ac- (H,1,2,3,3a,8,8 a-hexahydro-3 a-hydroxypyrrolo [2,3-b ] -indole 2-carboxylic acid) (N-Ac-PIC), N-Ac-oxindolyl alanine (N-Ac-Oia), N-Ac-N-formyl-kynurenine (N-Ac-NFK), N-Ac-kynurenine (N-Ac-Kyn), and N-Ac-2a,8 a-dihydroxy-PIC.

In some aspects, the invention provides a method for measuring degradation of N-acetyl tryptophan (NAT) in a composition comprising N-acetyl tryptophan and a polypeptide, the method comprises a) diluting the composition with about 8M guanidine, b) removing the polypeptide from the composition, c) applying the composition to a reverse phase chromatography material, wherein the composition is loaded onto a chromatography material that has been equilibrated in a solution comprising mobile phase A and mobile phase B, wherein mobile phase A comprises an acid in water and mobile phase B comprises an acid in acetonitrile, d) eluting the composition from the reverse phase chromatography material with a solution comprising mobile phase A and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is increased compared to step a), wherein NAT degradant elutes from the chromatography separately from intact NAT, e) quantifying the NAT degradant and the intact NAT. In some embodiments, the composition is diluted in about 8M guanidine such that the final concentration of NAT in the composition ranges from about 0.05mM to about 0.2 mM. In some embodiments, the composition is diluted in about 8M guanidine such that the final concentration of the polypeptide in the composition is less than or equal to about 25 mg/mL. In some embodiments, the polypeptide is removed from the composition by filtration. In some embodiments, the filtration uses a filtration membrane with a molecular weight cut-off of about 30 kDal. In some embodiments, the ratio of mobile phase B to mobile phase a in step a) is about 2: 98. In some embodiments, the ratio of mobile phase B to mobile phase a in step B) increases linearly. In some embodiments, the ratio of mobile phase B to mobile phase a in step B) is increased stepwise. In some embodiments, the flow rate for the chromatography is about 1.0 mL/min. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about 30: 70. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about 30:70 in about 16 minutes. In some embodiments, the ratio of mobile phase B to mobile phase a is further increased to about 90: 70. In some embodiments, the ratio of mobile phase B to mobile phase a is further increased to about 90:70 in about 18.1 minutes. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about 26: 74. In some embodiments, the ratio of mobile phase B to mobile phase a increases to about 26:74 in about 14 minutes. In some embodiments, the ratio of mobile phase B to mobile phase a is further increased to about 90: 70. In some embodiments, the ratio of mobile phase B to mobile phase a is further increased to about 90:70 in about 16.5 minutes. In some embodiments, mobile phase a comprises about 0.1% acid in water. In some embodiments, mobile phase B comprises about 0.1% acid in acetonitrile. In some embodiments, the acid is formic acid. In some embodiments, the reverse phase chromatography material comprises a C18 module. In some embodiments, the reverse phase chromatography material comprises a solid support. In some embodiments, the solid support comprises silica. In some embodiments, the reverse phase chromatography material is contained in a column. In some embodiments, the reverse phase chromatography material is a High Performance Liquid Chromatography (HPLC) material or an ultra high performance liquid chromatography (UPLC) material. In some embodiments, NAT and NAT degradation products are detected by absorbance at 240 nm. In some embodiments, NAT degradation products are identified by mass spectrometry. In some embodiments, the concentration of NAT in the composition is from about 0.1mM to about 5 mM. In some embodiments, the concentration of NAT in the composition is about 0.3 mM. In some embodiments, the NAT degradation product comprises one or more of N-Ac-PIC, N-Ac-Oia, N-Ac-NFK, N-Ac-Kyn, and N-Ac-2a,8 a-dihydroxy-PIC.

In some embodiments of the above aspect, the protein concentration in the formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody or antibody fragment.

In some aspects, the invention provides a method for monitoring degradation of NAT in a composition, comprising measuring degradation of NAT in a sample of the composition according to the method of any one of claims 74-134, wherein the method is repeated one or more times. In some embodiments, the method is repeated every month, every 2 months, every 4 months, or every 6 months.

In some aspects, the invention provides a quality assay for a pharmaceutical composition, the quality assay comprising measuring degradation of NAT in a sample of the pharmaceutical composition according to the method of any one of claims 74-134, wherein the amount of NAT degradant measured in the composition determines whether the pharmaceutical composition is suitable for administration to an animal. In some embodiments, an amount of NAT degrader in the pharmaceutical composition of less than about 10ppm indicates that the pharmaceutical composition is suitable for administration to the animal.

It is to be understood that one, some, or all of the features of the various embodiments described herein may be combined to form further embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following detailed description.

Brief Description of Drawings

Figure 1 shows protection of individual tryptophan residues from AAPH pressure-induced oxidation by NAT in protein mab 2, mab 4, mab 1, and mab 6. Both figures present the same data, except on the x-axis scale. The legend includes the computer calculated solvent accessibility surface area for each residue tested.

FIGS. 2A and 2B show the relationship between AAPH-induced tryptophan oxidation and% side chain SASA. Fig. 2A shows results from a data set including 38 IgG1 mabs. Figure 2B shows results from a dataset comprising 121 mabs, spanning a diverse framework, including IgG1, IgG2, IgG4, and murine.

FIG. 3 shows the random decision forest accuracy as a function of the number of estimates used during the exercise.

Figure 4 shows the random decision forest accuracy as a function of the number of features considered during the exercise.

FIG. 5 shows the random decision forest accuracy as a function of tree depth used during the exercise.

Figure 6 shows the characteristic importance (gini) of the 14 most relevant simulation-based molecular descriptors for an optimized random decision forest. The exercise parameters include: 5000 estimates, each section considering 3 features, and a tree depth of 10.

Figure 7 shows potential degradants of NAT (series b), along with the corresponding Trp degradants (series a).

FIG. 8A shows a reverse phase chromatogram of 0.2mM NAT after being subjected to different pressure conditions. The peaks of the stars represent the peaks observed only under ICH light pressure. Fig. 8B shows a comparison of fluorescence and absorbance between wavelengths for NAT and NAT degradants in AAPH pressure samples. The curves have been normalized such that the NAT peak is set to 1 AU. Note that only NAT degradants with measurable fluorescence (excitation wavelength 240nm, emission wavelength 342nm) are peak 4 (assigned as N-Ac-PIC, based on this data and MS fragmentation data in fig. 15E).

Figure 9 shows the retention time alignment of synthetic NAT standards and AAPH-induced NAT degradants.

Figure 10 shows the effect of co-formulation of 5mM Met on total NAT oxidation (in both histidine and non-histidine containing formulations). Standard deviation of duplicate injections is shown.

Figure 11 shows the effect of proteins on AAPH-induced NAT degradation. A histidine-based buffer containing 0.3mM NAT with or without 1mg/ml (0.0067mM) of protein was subjected to AAPH pressure. The distribution and level of NAT degradants is largely independent of the presence of proteins. Standard deviation of duplicate injections is shown.

Figure 12 shows a comparison of NAT degradation in protein 1 stability samples and AAPH pressure model. The inset shows an enlarged view of the labeled area.

FIG. 13 shows the linearity of NAT UV-HPLC response (at 240 nm).

Figure 14 depicts that NAT degradants show linear responses in a 1-20 fold dilution series of AAPH stressed NAT in His buffer samples. All peaks were detected at 240 nm.

FIGS. 15A-15F show Mass spectrometry fragmentation analyses (fragmentation literature is reported in Todorovski, T., M.Fedorova, and R.Hoffmann, Mass spectra characterization of peptidic ligation modulated tryptophan reactions. J Mass spectra, 2011.46(10): p.1030-8 and references therein). Figure 15A shows that peak 2 and 3MS data support the identification as the N-Ac-Oia diastereomer. The fragments of the stars are Oia characteristic. Figure 15B shows that peak 6MS data supports identification as N-Ac-Kyn. The fragments of the stars are characteristic of kynurenine-containing molecules. Figure 15C shows that peak 5MS data supports identification as N-Ac-NFK. The fragments of the stars are characteristic of kynurenine-containing molecules. Fig. 15D shows that 263.1 ion and peak 4 in peak set 1 have similar MS fragmentation patterns and are not both N-Ac-HTPs. Fragments of the stars are 5-HTP characteristic. FIG. 15E shows that the fragmentation pattern in Peak 4 is consistent with the potential N-Ac-PIC fragmentation and is reported in the literature (Fang, L., R.Parti, and P.Hu, Characterization of N-acetyltryptophan degradation products in centralized human serum solutions and Characterization of an automated high performance liquid chromatography method for the amplification. J chromatography A,2011.1218(41): p.7316-24). The heuristic ratio was enhanced based on the fluorescence data shown in fig. 8B. FIG. 15F shows that the doubly oxidized species in peak set 1 is likely N-Ac-DiOia or N-Ac-3a,8 a-dihydroxy-PIC.

Detailed Description

In some aspects, the present invention provides methods of reducing oxidation of a polypeptide in an aqueous formulation comprising adding to the formulation an amount of N-acetyl tryptophan that prevents oxidation of the polypeptide, wherein the polypeptide comprises at least one Solvent Accessible Surface Area (SASA) greater than aboutA tryptophan residue of (a). In some aspects, the present invention provides methods of reducing oxidation of a polypeptide in an aqueous formulation, comprising adding to the formulation an amount of N-acetyl tryptophan that prevents oxidation of the polypeptide, wherein the polypeptide comprises at least one tryptophan residue having a Solvent Accessible Surface Area (SASA) greater than about 30%. In some aspects, the invention provides methods of reducing oxidation of a polypeptide in an aqueous formulation comprising determining the SASA value of a tryptophan residue in the polypeptide and if at least one tryptophan residue has a value greater than aboutIs added to the formulation in an amount that prevents oxidation of the polypeptide.

In some aspects, the invention provides methods for screening for formulations for reduced polypeptide oxidation, wherein the polypeptide comprises at least one SASA greater than aboutThe tryptophan residue of (1), which comprisesFormulations in which the amount of N-acetyl tryptophan is such that no more than about 20% of the tryptophan residues of the polypeptide are oxidized are suitable formulations for reducing oxidation of the polypeptide. In some aspects, the invention provides methods for screening a formulation for reduced polypeptide oxidation comprising determining the SASA value of a tryptophan residue in the polypeptide, wherein the SASA is greater than aboutAre subjected to oxidation, wherein a formulation comprising an amount of N-acetyl tryptophan that results in no more than about 20% oxidation of the tryptophan residues of the polypeptide is a suitable formulation for reducing oxidation of the polypeptide.

I. Definition of

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The term "pharmaceutical formulation" refers to a preparation that is in a form effective to allow the biological activity of the active ingredient to be effective and that is free of additional ingredients that would be unacceptably toxic to a subject to which the formulation will be administered. Such formulations are sterile.

"sterile" formulations are free of viable bacteria or are free or substantially free of all viable microorganisms and their spores.

A "stable" formulation is one in which the protein substantially retains its physical and/or chemical stability and/or biological activity upon storage. Preferably, the formulation substantially retains its physical and chemical stability, as well as its biological activity, upon storage. The shelf life is generally selected based on the intended shelf life of the formulation. A variety of analytical techniques for measuring Protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247. sup. -, 301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A.Adv.drug Delivery Rev.10:29-90 (1993). Stability can be measured after a selected amount of light exposure and/or temperature for a selected period of time. Stability can be assessed qualitatively and/or quantitatively in a number of different ways, including assessing the aggregation form (e.g., using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); assessing ROS formation (e.g., by using a photopressure assay or a 2, 2' -azobis (2-amidinopropane) dihydrochloride (AAPH) pressure assay); oxidation of specific amino acid residues of the protein (e.g., Trp residues and/or Met residues of monoclonal antibodies); charge heterogeneity was assessed using cation exchange chromatography, image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometry analysis; SDS-PAGE analysis to compare reduced (reduced) and intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; assessing the biological activity or target binding function of the protein (e.g., antigen binding function of an antibody); and the like. Instability may involve any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation and/or Trp oxidation), isomerization (e.g., Asp isomerization), clipping/hydrolysis/fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extension, C-terminal processing, glycosylation differences, and the like.

A protein "retains its physical stability" in a pharmaceutical formulation if it exhibits little or no aggregation, precipitation, fragmentation, and/or denaturation as measured by visual inspection of color and/or clarity, or by UV light scattering or size exclusion chromatography.

A protein "retains its chemical stability" in a pharmaceutical formulation if the chemical stability at a given time is such that the protein is deemed to still retain its biological activity as defined below. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Chemical changes may involve protein oxidation, which may be assessed using, for example, tryptic peptide mapping, reverse phase High Performance Liquid Chromatography (HPLC), and liquid chromatography-mass spectrometry (LC/MS). Other types of chemical changes include changes in the charge of the protein, which can be assessed by, for example, ion exchange chromatography or icIEF.

A protein "retains its biological activity" in a pharmaceutical formulation if the biological activity of the protein at a given time is within about 20% (such as within about 10%) of the biological activity exhibited when the pharmaceutical formulation was prepared (within the error of the assay), as determined in, for example, an antigen binding assay for a monoclonal antibody.

As used herein, "biological activity" of a protein refers to the ability of the protein to bind to its target, e.g., the ability of a monoclonal antibody to bind to an antigen. It may further comprise a biological response that can be measured in vitro or in vivo. Such activity may be antagonistic or agonistic.

An "oxidation-susceptible" protein is a protein that contains one or more residues that have been found to be prone to oxidation, such as, but not limited to, methionine (Met), cysteine (Cys), histidine (His), tryptophan (Trp), and tyrosine (Tyr). For example, a tryptophan amino acid in the Fab portion of a monoclonal antibody or a methionine amino acid in the Fc portion of a monoclonal antibody may be susceptible to oxidation.

"Oxidato mutated" residues of a protein are residues that have greater than 35% oxidation in an oxidation assay (e.g., AAPH-induced or heat-induced oxidation). The percent oxidation of residues in a protein can be determined by any method known in the art, such as trypsin digestion for site-specific Trp oxidation, followed by LC-MS/MS.

The "solvent accessible surface area" or "SASA" of a biomolecule in a solvent is the surface area of the biomolecule accessible to the solvent. The SASA can be expressed in units of measurement (e.g., square angstroms) or as a percentage of the surface area accessible to the solvent. For example, the SASA of an amino acid residue in a polypeptide may beOr 30%. The SASA may be determined by any method known in the art, including the Shrake-Rupley algorithm, the LCPO method, the Power map method, or molecular dynamics simulations.

By "isotonic" is meant that the formulation of interest has substantially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmolality of about 250 to 350 mOsm. Isotonicity can be measured using, for example, a vapor pressure or freezing type osmometer.

As used herein, "buffer" refers to a buffered solution that resists changes in pH by the action of its acid-base complexing components. The buffer of the present invention preferably has a pH in the range of about 4.5 to about 8.0. For example, one example of a buffer that would control the pH within this range is histidine acetate.

A "preservative" is a compound that may optionally be included in a formulation to substantially reduce the bacterial action therein, thus facilitating production, e.g., multiple use, of the formulation. Examples of potential preservatives include octadecyl dimethyl benzyl ammonium chloride, chlorohexidine, benzalkonium chloride (a mixture of alkyl benzyl dimethyl ammonium chlorides, where the alkyl group is a long chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butanol and benzyl alcohol, hydrocarbyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. In one embodiment, the preservative herein is benzyl alcohol.

As used herein, "surfactant" refers to a surface active substance, preferably a nonionic surfactant. Examples of surfactants herein include polysorbates (e.g., polysorbate 20 and polysorbate 80); poloxamers (e.g., poloxamer 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl, myristyl, linoleyl (linoleyl), or stearyl sulfobetaine; lauryl, myristyl, linoleyl or stearylsarcosine; linoleic acid radical, myristic acid radical, or whaleWax-based betaines; lauramidopropyl, cocamido (cocamido) propyl, linoleamidopropyl, myristoamidopropyl, palmitamido (palmido) propyl, or isostearamidopropyl betaine (e.g. lauramidopropyl); myristamidopropyl, palmitamido (palmido) propyl, or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate, or disodium methyl oleoyl taurate; and MONAQUAT^TMSeries (mona industries, inc., Paterson, n.j.); polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics, PF68, etc.); and the like. In one embodiment, the surfactant herein is polysorbate 20. In yet another embodiment, the surfactant herein is poloxamer 188.

As used herein, a "pharmaceutically acceptable" excipient or carrier includes a pharmaceutically acceptable carrier, stabilizer, buffer, acid, base, sugar, preservative, surfactant, tonicity agent, and the like, which are well known in the art (Remington: the science and Practice of Pharmacy, 22)^ndEd, Pharmaceutical Press, 2012). Examples of pharmaceutically acceptable excipients include buffers such as phosphates, citrates, acetates, and other organic acids; antioxidants including ascorbic acid, L-tryptophan, and methionine; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; metal complexes, such as Zn-protein complexes; chelating agents, such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as polysorbates, poloxamers, polyethylene glycols (PEG), and PLURONICS^TM. "pharmaceutically acceptable" excipients or carriers are those which can be reasonably administered to a subject to provide an effective dose of the active ingredient employed and at the dose and concentration employed to the subject exposed theretoIs non-toxic.

The formulated protein is preferably substantially pure and desirably substantially homogeneous (e.g., free of contaminating proteins, etc.). By "substantially pure" protein is meant that the composition comprises at least about 90% by weight, preferably at least about 95% by weight, of the protein (e.g., monoclonal antibody), based on the total weight of the composition. By "substantially homogeneous" protein is meant that the composition comprises at least about 99% by weight of the protein (e.g., monoclonal antibody), based on the total weight of the composition.

The terms "protein", "polypeptide" and "peptide" are used interchangeably herein to refer to amino acid polymers of any length, which may be linear or branched and which may comprise modified amino acids and which may be interrupted by non-amino acids, the term also encompasses amino acid polymers which have been modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a labeling member, and also includes within this definition, e.g., proteins which contain one or more amino acid analogs (including, e.g., non-natural amino acids, etc.) and which are known in the art, such as the human growth factor receptor, such as the TGF-receptor, such as the VEGF-receptor, such as the thrombopoietin receptor, such as the VEGF-receptor, such as the VEGF-receptor-related protein, such as the VEGF-receptor factor-receptor, such as the VEGF-receptor-stimulating factor-VEGF-or the endothelial growth factor-receptor-factor, such as the VEGF-receptor factor-VEGF-receptor stimulating factor-receptor, such as the VEGF-factor-VEGF-receptor-factor-VEGF-receptor stimulating factor-VEGF-stimulating factor-VEGF-receptor, such as the TNF-VEGF-factor-VEGF-receptor stimulating factor-VEGF-factor-receptor stimulating factor-VEGF-receptor, such as the VEGF-factor-stimulating factor-VEGF-factor-VEGF-factor-VEGF-factor, such as the VEGF-factor-VEGF-factor-VEGF-factor-VEGF-factor inducing or VEGF-factor inducing or VEGF-factor inducing or VEGF-factor inducing or VEGF-factor inducing or VEGF-receptor stimulating factor inducing or VEGF-stimulating factor inducing growth-receptor stimulating factor inducing or VEGF-stimulating factor inducing or VEGF-stimulating factor inducing growth-stimulating factor inducing growth-stimulating factor inducing growth-stimulating factor inducing growth-stimulating factor-factor receptor stimulating factor-receptor stimulating factor-factor inducing growth-factor inducing factor-stimulating factor inducing growth-stimulating factor inducing growth-factor-stimulating factor inducing factor receptor stimulating factor-factor inducing factor-VEGF-factor-stimulating factor-factor inducing factor, such as VEGF-factor inducing factor, such as VEGF-factor (e.as VEGF-factor inducing growth factor inducing factor-factor inducing factor-factor inducing factor receptor stimulating factor (e, such as VEGF-factor receptor stimulating factor inducing factor-VEGF-factor inducing factor-factor inducing factor-VEGF-factor-VEGF-factor receptor-factor-stimulating factor inducing factor, such as VEGF-factor inducing factor-stimulating factor-factor inducing factor-factor receptor, such as VEGF-factor inducing growth factor inducing factor, such as VEGF-factor inducing factor.

The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

An "isolated" protein (e.g., an isolated antibody) refers to a protein (e.g., an antibody) that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment refer to substances that would interfere with the research, diagnostic or therapeutic uses of the protein and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. An isolated protein includes a protein in situ within a recombinant cell, since at least one component of the protein's natural environment will not be present. However, the isolated protein will generally be prepared by at least one purification step.

"Natural antibody" refers to about 150,000 channels, typically composed of two identical light (L) chains and two identical heavy (H) chainsHeterotetrameric glycoprotein from erton. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V) at one end_H) Followed by a plurality of constant domains. Each light chain has a variable domain (V) at one end_L) And the other end is a constant domain. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that particular amino acid residues form the interface between the light and heavy chain variable domains.

The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to the other portions of the immunoglobulin, i.e., the variable domains that contain the antigen binding site. Constant domain heavy chain-containing C_H1，C_H2 and C_H3 domains (collectively CH) and the CHL (or CL) domain of the light chain.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "V_H". The variable domain of the light chain may be referred to as "V_L". These domains are generally the most variable parts of an antibody and contain an antigen binding site.

The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequence among antibodies and are used for the binding and specificity of each particular antibody for its particular antigen, however, the variability is not evenly distributed throughout the variable domains of the antibodies it focuses on three segments of the light and heavy chain variable domains called hypervariable regions (HVRs). the more highly conserved portions of the variable domains are called Framework Regions (FRs). the variable domains of the native heavy and light chains each comprise four FR regions, mostly adopting an β -fold conformation, linked by three HVRs forming loops and in some cases forming part of the β -fold structure.

The "light chain" of an antibody (immunoglobulin) from any mammalian species can be classified into one of two distinct types, called kappa ("κ") and lambda ("λ"), depending on the amino acid sequence of its constant domains.

As used herein, the term IgG "isotype" or "subclass" means any immunoglobulin subclass defined by the chemical and antigenic characteristics of its constant regions. Antibodies (immunoglobulins) can be assigned to different classes depending on the amino acid sequence of their heavy chain constant domains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which may be further divided into subclasses (isotypes), e.g. IgG₁，IgG₂，IgG₃，IgG₄，IgA₁And IgA₂The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al, Cellular and mol.

The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in substantially intact form, rather than an antibody fragment as defined below. The term specifically refers to antibodies in which the heavy chain comprises an Fc region.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having an antigen-binding site, and a remaining "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment produced an F (ab')₂A fragment which has two antigen binding sites and is still capable of cross-linking antigens. The Fab fragment comprises the heavy and light chain variable domains, and further comprises the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residues of the constant domain carry a free thiol group. F (ab')₂Antibody fragments were originally generated as pairs of Fab 'fragments with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

"Fv" is the smallest antibody fragment that contains the entire antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy and one light variable domain in tight, non-covalent association. In the single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain may be covalently linked by a flexible peptide linker, such that the light and heavy chains may associate in a "dimeric" structure analogous to that of a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Together, the six HVRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, with only a lower affinity than the entire binding site.

For reviews on scFv see for example Pl ü ckthun, in The Pharmacology of Monoclonal Antibodies, Vol 113, eds Rosenburg and Moore, Springer-Verlag, NewYork, pp.269-315, 1994.

The term "diabodies" refers to antibody fragments having two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) linked in the same polypeptide chain (VH-VL). By using linkers that are too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of the other chain, thereby creating two antigen binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP404,097; WO 1993/01161; hudson et al, nat. Med.9: 129-; and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-. Tri-antibodies (Triabody) and tetra-antibodies (tetrabody) are also described in Hudson et al, nat. Med.9:129-134 (2003).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal" indicates that the antibody is not characteristic of a mixture of discrete antibodies. In certain embodiments, such monoclonal antibodies typically comprise an antibody comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select unique clones from a collection of multiple clones such as hybridoma clones, phage clones or recombinant DNA clones. It will be appreciated that the target binding sequence selected may be further altered, for example to improve affinity for the target, humanize the target binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of the invention. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are generally uncontaminated by other immunoglobulins.

The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, Monoclonal Antibodies used In accordance with the present invention may be generated by a variety of techniques, including, for example, the Hybridoma method (e.g., Kohler and Milstein, Nature,256: 495-97 (1975); Hongo et al, Hybridoma,14(3):253-260(1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd edition 1988); Hammerling et al, In: Monoclonal Antibodies and T-Cell hybrids: 681(Elsevier, N.Y.,1981)), the recombinant DNA method (see, for example, U.S. Pat. No.4,816,567), the phage display technique (see, for example, Clackson et al., 352: 624: 628 (1991); J.Acad.1247, Biokl et al, WO 32: 96; 20. J.31, Biokl et al., 2000: 96; (Lellson et al., USA) 340. 99.72. J.55, Biokl et al: 96; (Legend et al., 3. 31, USA) and No. 32, methods 284(1-2):119-132(2004)), and techniques for producing human or human-like antibodies in animals having part or all of a human immunoglobulin locus or a gene encoding a human immunoglobulin sequence (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; jakobovits et al, Proc.Natl.Acad.Sci.USA 90:2551 (1993); jakobovits et al, Nature 362:255-258 (1993); bruggemann et al, Yeast in Immuno.7:33 (1993); U.S. patent nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; and 5,661,016; marks et al, Bio/Technology10:779-783 (1992); lonberg et al, Nature 368:856-859 (1994); morrison, Nature 368: 812-; fishwild et al, Nature Biotechnol.14: 845-; neuberger, Nature Biotechnol.14:826 (1996); lonberg and Huszar, Intern.Rev.Immunol.13:65-93 (1995)).

Monoclonal antibodies specifically include "chimeric" antibodies wherein a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No.4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include "primatized" antibodies in which the antigen binding region of the antibody is derived from an antibody produced, for example, by immunizing macaques with an antigen of interest.

"humanized" forms of non-human (e.g., murine) antibodies refer to chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. In one embodiment, a humanized antibody is one in which residues from HVRs in a human immunoglobulin (recipient antibody) are replaced with residues from HVRs of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and/or capacity. In some instances, FR residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details see, e.g., Jones et al, Nature 321:522-525 (1986); riechmann et al, Nature 332: 323-; and Presta, curr, Op, Structure, biol.2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol.1:105-115 (1998); harris, biochem. Soc. Transactions23: 1035-; hurle and Gross, curr. Op. Biotech.5: 428-; and U.S. patent nos. 6,982,321 and 7,087,409.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or produced using any of the techniques disclosed herein for producing human antibodies. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J.mol.biol.227:381 (1991); Marks et al, J.mol.biol.222:581 (1991)). Also useful for the preparation of human monoclonal antibodies are the methods described in the following references: cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985); boerner et al, J.Immunol.147(1):86-95 (1991). See also van Dijk and van deWinkel, curr, opin, pharmacol, 5:368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal, such as an immunized XENOMOUSE (xenomic), that has been modified to produce human antibodies in response to antigenic stimuli but whose endogenous loci have been disabled (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for Xenomose^TMA technique). See also, e.g., Li et al, Proc. Natl.Acad.Sci.USA,103:3557-3562(2006), for human antibodies generated via human B-cell hybridoma technology.

The terms "hypervariable region", "HVR" or "HV", when used herein, refer to regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Typically, an antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Among natural antibodies, H3 and L3 show the greatest diversity of these six HVRs, and H3 in particular is thought to play a unique role in conferring precise specificity to antibodies. See, e.g., Xu et al, Immunity 13:37-45 (2000); johnson and Wu, In: Methods In Molecular Biology 248:1-25(Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies, consisting of only heavy chains, are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al Nature 363:446 + 448 (1993); sheffet al Nature Structure biol.3:733-736, (1996). In some embodiments, the HVRs are Complementarity Determining Regions (CDRs).

A description of many HVRs is used and is contemplated herein. Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al, Sequences of Proteins of immunological interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia instead refers to the position of the structural loops (Chothia and Lesk J.mol.biol.196:901-917 (1987)). The AbM HVR represents a compromise between the Kabat HVR and Chothia structural loops, and results in the use of oxford molecular's AbM antibody modeling software. The "contact" HVRs are based on analysis of the available complex crystal structure. The residues for each of these HVRs are recorded below.

The HVRs can include the following "extended HVRs": 24-36 or 24-34(L1), 46-56 or 50-56(L2) and 89-97 or 89-96(L3) in VL and 26-35(H1), 50-65 or 49-65(H2) and 93-102, 94-102 or 95-102(H3) in VH. For each of these definitions, the variable domain residues are numbered according to Kabat et al, supra.

"framework" or "FR" residues refer to those residues in the variable domain other than the HVR residues as defined herein.

The term "variable domain residue numbering according to Kabat" or "amino acid position numbering according to Kabat" and variations thereof refers to Kabat et al, supra, for the numbering system used for antibody heavy chain variable domain or light chain variable domain editing. Using this numbering system, the actual linear amino acid sequence may comprise fewer or additional amino acids, corresponding to a shortening or insertion of the variable domain FR or HVR. For example, the heavy chain variable domain may comprise a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c according to Kabat, etc.) after heavy chain FR residue 82. The Kabat residue numbering for a given antibody can be determined by aligning the antibody sequence to the region of homology with a "standard" Kabat numbered sequence.

The Kabat numbering system is generally used when referring to residues in the variable domain (approximately light chain residues 1-107 and heavy chain residues 1-113) (e.g., Kabat et al, Sequences of Immunological interest,5th ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in an immunoglobulin heavy chain constant region (e.g., Kabatet al, see EU index reported above). "EU index as in Kabat" refers to the residue numbering of the human IgG1EU antibody.

The term "multispecific antibody" is used in the broadest sense and specifically covers antibodies comprising antigen binding domains with polyepitopic specificity (i.e., capable of specifically binding to two or more different epitopes on one biological molecule or capable of specifically binding to epitopes on two or more different biological molecules). In some embodiments, the antigen binding domain of a multispecific antibody (such as a bispecific antibody) comprises two VH/VL units, wherein a first VH/VL unit specifically binds a first epitope and a second VH/VL unit specifically binds a second epitope, wherein each VH/VL unit comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). Such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies having two or more VL and VH domains, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, and triabodies, antibody fragments that have been covalently or non-covalently linked. A VH/VL unit further comprising at least part of the heavy chain constant region and/or at least part of the light chain constant region may also be referred to as a "half body" or "half antibody". In some embodiments, the half-antibody comprises at least a portion of a single heavy chain variable region and at least a portion of a single light chain variable region. In some such embodiments, a bispecific antibody comprising two half-antibodies and binding to two antigens comprises a first half-antibody that binds to a first antigen or first epitope but not to a second antigen or second epitope and a second half-antibody that binds to a second antigen or second epitope and not to the first antigen or first epitope. In accordance with some embodiments, the multispecific antibody is an IgG antibody that binds each antigen or epitope with an affinity of 5M to 0.001pM, 3M to 0.001pM, 1M to 0.001pM, 0.5M to 0.001pM, or 0.1M to 0.001 pM. In some embodiments, a moiety comprises a portion of the heavy chain variable region sufficient to allow formation of an intramolecular disulfide bond with the second moiety. In some embodiments, a moiety comprises a hole mutation or knob mutation, e.g., to allow heterodimerization with a second moiety or half-antibody comprising a complementary hole mutation or knob mutation. Section mutations and hole mutations are discussed further below.

"bispecific antibody" refers to a multispecific antibody comprising an antigen-binding domain capable of specifically binding to two different epitopes on one biological molecule or capable of specifically binding to an epitope on two different biological molecules. Bispecific antibodies may also be referred to herein as having "dual specificity" or being "dual specific". Unless otherwise indicated, the order in which the antigens bound by the bispecific antibody are listed in the bispecific antibody name is arbitrary. In some embodiments, the bispecific antibody comprises two half antibodies, wherein each half antibody comprises a single heavy chain variable region and optionally at least a portion of a heavy chain constant region, and a single light chain variable region and optionally at least a portion of a light chain constant region. In certain embodiments, the bispecific antibody comprises two half antibodies, wherein each half antibody comprises a single heavy chain variable region and a single light chain variable region and does not comprise more than one single heavy chain variable region and does not comprise more than one single light chain variable region. In some embodiments, the bispecific antibody comprises two half antibodies, wherein each half antibody comprises a single heavy chain variable region and a single light chain variable region, and wherein the first half antibody binds to a first antigen and does not bind to a second antigen and the second half antibody binds to the second antigen and does not bind to the first antigen.

As used herein, the term "knob-to-hole" or "KnH" technology refers to a technology for pairing two polypeptides together in vitro or in vivo by introducing a protuberance (knob) into one polypeptide and a cavity (hole) into the other polypeptide at the interface where the two polypeptides interact. For example, KnH has been introduced into the Fc: Fc binding interface, CL: CH1 interface or VH/VL interface of an antibody (see, e.g., US2011/0287009, US2007/0178552, WO 96/027011, WO 98/050431, and Zhu et al (1997) Protein Science 6: 781-. In some embodiments, KnH drives pairing together of two different heavy chains during the manufacture of multispecific antibodies. For example, multispecific antibodies having KnH in their Fc regions may further comprise a single variable domain linked to each Fc region, or further comprise different heavy chain variable domains paired with similar or different light chain variable domains. The KnH technique can also be used to pair together two different receptor ectodomains or any other polypeptide sequences comprising different target recognition sequences, including for example affinity antibodies (affibodies), peptibodies (peptibodies) and other Fc fusions.

As used herein, the term "knob mutation" refers to a mutation that introduces a protuberance into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a hole mutation (see, e.g., US 5,731,168, US 5,807,706, US 5,821,333, US 7,695,936, US 8,216,805, each of which is incorporated herein by reference in its entirety).

As used herein, the term "hole mutation" refers to a mutation that introduces a cavity (hole) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a node mutation (see, e.g., US 5,731,168, US 5,807,706, US 5,821,333, US 7,695,936, US 8,216,805, each of which is incorporated herein by reference in its entirety).

The expression "linear antibody" refers to the antibodies described in Zapata et al (1995) Protein Eng,8(10): 1057-1062. Briefly, these antibodies comprise a pair of Fd segments (VH-CH1-VH-CH1) in tandem that form, with a complementary light chain polypeptide, a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

As used herein, the term "about" refers to an acceptable error range for a corresponding numerical value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, following practice in the art. References herein to "about" a value or parameter include and describe embodiments that relate to that value or parameter per se. For example, a description referring to "about X" includes a description of "X".

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a compound" optionally includes a combination of two or more such compounds, and the like.

It is understood that the various aspects and embodiments of the invention described herein include aspects and embodiments that "comprise," consist of … …, "and" consist essentially of … ….

Protein formulations and preparations

The invention herein relates to formulations (e.g., liquid formulations) comprising a protein and N-acetyl-tryptophan (NAT), wherein the NAT prevents oxidation of the protein. In some embodiments, the protein is susceptible to oxidation. In some embodiments, methionine, cysteine, histidine, tryptophan, and/or tyrosine in the protein is susceptible to oxidation. In some embodiments, the tryptophan in the protein is susceptible to oxidation. In some embodiments, the protein comprises at least one Solvent Accessibility Surface Area (SASA) greater than aboutTo about(such as greater than about 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, orAny, including any range between these values) of tryptophan residues. In some embodiments, the SASA is greater than aboutIn some embodiments, the protein comprises greater than about 15% to about 45% (such as greater than about any of 15, 20, 25, 30, 35, 40, or 45%) tryptophan residues for at least one SASA. In some embodiments, the SASA is greater than about 30%. The SASA may be calculated using any method known in the art, such as the computer all-atom Molecular Dynamics (MD) simulation method described in Sharma, V.et al, PNAS.111(52):18601-18606, 2014. In some embodiments, the SASA of tryptophan residues is measured at a pH range from about 4.0 to about 8.5. In some embodiments, the SASA of the tryptophan residue is measured at a temperature ranging from about 5 ℃ to about 40 ℃. In some embodiments, the SASA of tryptophan residues is measured at a salt concentration ranging from about 0mM to about 500 mM. In some embodiments, the SASA of tryptophan residues is measured at a pH of about 5.0 to about 7.5, a temperature of about 5 ℃ to about 25 ℃, and a salt concentration of about 0mM to about 200 mM. In some embodiments, the protein comprises at least one tryptophan residue predicted to be susceptible to oxidation by a machine learning algorithm practiced in association with the tryptophan residue oxidation susceptibility and a plurality of molecular descriptors of the tryptophan residue based on MD simulation. In some embodiments, the formulation further comprises at least one additional protein according to any of the proteins described herein. In some embodiments, the formulation is a liquid formulation. In some embodiments, the formulation is an aqueous formulation.

In some embodiments, the NAT in the formulation is from about 0.1mM to about 10mM (such as about any of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0mM, including thisAny range between these values), or up to the highest concentration at which the NAT is soluble in the formulation. In some embodiments, the NAT in the formulation is about 1 mM. In some embodiments, the NAT prevents oxidation of one or more tryptophan amino acids in the protein. In some embodiments, the NAT prevents oxidation of the protein by Reactive Oxygen Species (ROS). In yet another embodiment, the reactive oxygen species is selected from the group consisting of singlet oxygen, superoxide (O)₂-, alkoxy radical, peroxy radical, hydrogen peroxide (H2O)₂) Dihydrogen trioxide (H)₂O₃) Radical of hydrogen trioxide (HO)₃H), ozone (O)₃) Hydroxyl radicals, and alkyl peroxides. For example, a tryptophan amino acid in the Fab portion of a monoclonal antibody and/or a methionine amino acid in the Fc portion of a monoclonal antibody may be susceptible to oxidation.

In some embodiments, the protein (e.g., antibody) concentration in the formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein is a therapeutic protein. Exemplary protein concentrations in the formulation include from about 1mg/mL to more than about 250mg/mL, from about 1mg/mL to about 250mg/mL, from about 10mg/mL to about 250mg/mL, from about 15mg/mL to about 225mg/mL, from about 20mg/mL to about 200mg/mL, from about 25mg/mL to about 175mg/mL, from about 25mg/mL to about 150mg/mL, from about 25mg/mL to about 100mg/mL, from about 30mg/mL to about 100mg/mL, or from about 45mg/mL to about 55 mg/mL.

In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In some embodiments, the antibody is derived from an IgG1 antibody sequence. In yet another embodiment, the NAT prevents oxidation of one or more amino acids in the Fab portion of the antibody. In another further embodiment, the NAT prevents oxidation of one or more amino acids in the Fc portion of the antibody.

In some embodiments, the formulation is aqueous. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. For example, a formulation of the invention may comprise a monoclonal antibody, a NAT provided herein that prevents oxidation of the protein, and a buffer to maintain the pH of the formulation at a desired level. In some embodiments, the formulations provided herein have a pH of about 4.5 to about 9.0. In some embodiments, the formulations provided herein have a pH of about 4.5 to about 7.0. In certain embodiments, the pH is in the range from pH4.0 to 8.5, in the range from pH4.0 to 8.0, in the range from pH4.0 to 7.5, in the range from pH4.0 to 7.0, in the range from pH4.0 to 6.5, in the range from pH4.0 to 6.0, in the range from pH4.0 to 5.5, in the range from pH4.0 to 5.0, in the range from pH4.0 to 4.5, in the range from pH 4.5 to 9.0, in the range from pH 5.0 to 9.0, in the range from pH 5.5 to 9.0, in the range from pH 6.0 to 9.0, in the range from pH6.5 to 9.0, in the range from pH 7.0 to 9.5, in the range from pH 5.0 to 8.5, in the range from pH5 to 8.0, in the range from pH5 to 8.5, in the range from pH5 to 8.0, in the range from pH 6.0 to 6.5, or in the range from pH 6.2 to 6.5. In certain embodiments of the invention, the formulation has a pH of 6.2 or about 6.2. In certain embodiments of the invention, the formulation has a pH of 6.0 or about 6.0. In some embodiments, the formulation further comprises at least one additional protein according to any of the proteins described herein.

In some embodiments, the formulations provided herein are pharmaceutical formulations suitable for administration to a subject. As used herein, "subject" or "individual" for the purposes of treatment or administration refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cattle, etc. In some embodiments, the mammal is a human.

The proteins and antibodies in the formulation can be prepared using methods known in the art. Antibodies (e.g., full length antibodies, antibody fragments, and multispecific antibodies) in this formulation can be prepared using techniques available in the art, non-limiting exemplary methods of which are described in more detail in the following sections. One skilled in the art can adapt the methods herein to prepare formulations comprising other proteins, such as peptide-based inhibitors. For well understood and commonly employed techniques and protocols for the production of therapeutic proteins in general, see Molecular Cloning: A Laboratory Manual (Sambrook et al, 4)^thed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,2012)；Current Protocols in Molecular Biology(F.M.Ausubel,et al.eds.,2003)；Short Protocols in Molecular Biology(Ausubel et al.,eds.,J.Wiley and Sons,2002)；Current Protocols in Protein Science,(Horswill et al.,2006)；Antibodies，A Laboratory Manual(Harlow and Lane,eds.,1988)；Culture of Animal Cells:AManual of Basic Technique and Specialized Applications(R.I.Freshney,6^thed., j.wiley and Sons,2010), which are all herein incorporated by reference in their entirety.

In some embodiments, the formulation comprises two or more proteins in accordance with any of the formulations described above (e.g., a liquid formulation) (e.g., the formulation is a co-formulation of two or more proteins). For example, in some embodiments, the formulation is a co-formulation comprising two or more proteins and N-acetyl-tryptophan (NAT), wherein the NAT prevents oxidation of at least one of the two or more proteins. In some embodiments, the NAT prevents oxidation of multiple of the two or more proteins. In some embodiments, the NAT prevents oxidation of each of the two or more proteins. In some embodiments, at least one of the two or more proteins comprises at least one tryptophan residue as follows: a) a Solvent Accessibility Surface Area (SASA) of greater than aboutTo about(such as greater than about 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, orAny, including any range between these values); b) SASA is greater than about 15% to about 45% (such as greater than any of about 15, 20, 25, 30, 35, 40, or 45%); or c) predicting susceptibility to oxidation by a machine learning algorithm practiced in association with the tryptophan residue oxidation susceptibility and a plurality of molecular descriptors of the tryptophan residue based on MD simulation. In some embodiments, a plurality of the two or more proteins comprises at least one tryptophan residue as follows: a) a Solvent Accessibility Surface Area (SASA) of greater than aboutTo about(such as greater than about 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, orAny, including any range between these values); b) SASA is greater than about 15% to about 45% (such as greater than any of about 15, 20, 25, 30, 35, 40, or 45%); or c) predicting susceptibility to oxidation by a machine learning algorithm practiced in association with the tryptophan residue oxidation susceptibility and a plurality of molecular descriptors of the tryptophan residue based on MD simulation. In some embodiments, each of the two or more proteins comprises at least one tryptophan residue as follows: a) a Solvent Accessibility Surface Area (SASA) of greater than aboutTo about(such as greater than about 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, orAny, including any range between these values); b) SASA is greater than about 15% to about 45% (such as greater than any of about 15, 20, 25, 30, 35, 40, or 45%); or c) predicting susceptibility to oxidation by a machine learning algorithm practiced in association with the tryptophan residue oxidation susceptibility and a plurality of molecular descriptors of the tryptophan residue based on MD simulation. In some embodiments, at least one of the two or more proteins is an antibody, such as a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In some embodiments, a plurality of the two or more proteins are antibodies, such as antibodies independently selected from polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, chimeric antibodies, multispecific antibodies, or antibody fragments. In some embodiments, each of the two or more proteins is an antibody, such as an antibody independently selected from a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In some embodiments, one or more antibodies in the formulation are derived from an IgG1 antibody sequence. In some embodiments, the formulation is a liquid formulation. In some embodiments, the formulation is an aqueous formulation.

A. Antibody preparation

The antibodies in the liquid formulations provided herein are directed against an antigen of interest. Preferably, the antigen is a biologically important polypeptide and administration of the antibody to a mammal suffering from the disorder results in a therapeutic benefit in that mammal. However, antibodies directed against non-polypeptide antigens are also contemplated.

In the case where the antigen is a polypeptide, it may be a transmembrane molecule (e.g. receptor) or ligand such as growth factor exemplary antigens include molecules such as Vascular Endothelial Growth Factor (VEGF), CD 20; ox-LDL; ox-ApoB 100; renin; growth hormones including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; α -1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX, tissue factor, and (von) von Willebrand (von Willebrand) factor; anti-clotting factors such as protein C; atrial natriuretic factor; lung surfactant; plasminogen activator, such as urokinase or human urokinase or tissue type or factor (TNF), such as TNF receptor TGF-TNF-receptor agonist (TGF-5) or TGF-derived), such as TGF-TNF-receptor agonist, such as TGF-TNF-receptor agonist, TGF-5, TGF-TNF-receptor agonist, such as TGF-receptor agonist, such as TGF-receptor agonist, TGF-7, or TGF-TNF-rat-7, or TNF-rat-derived factor receptor, such as TNF-5, or mouse growth factor-receptor cytokine, or mouse growth hormone receptor cytokine, such as TNF-2, or mouse growth hormone receptor cytokine, or mouse growth hormone receptor, or mouse growth factor, or mouse growth hormone receptor cytokine, or mouse growth hormone receptor cytokine, or mouse growth hormone receptor, or human growth hormone receptor cytokine, or human growth hormone receptor cytokine, such as TNF-or human factor III (e.e.g, or human factor III, or human, e.g, or human, e.g, or human factor III, or human growth hormone receptor cytokine-growth hormone receptor cytokine-TNF-receptor cytokine-TNF-receptor, e.g, or mouse, or human, e.g, or rat, e.g-TNF-growth hormone receptor, e.g-TNF-receptor, or mouse, or rat, or mouse, e.g-TNF-17, or mouse, or rat, or human, or mouse.

(i) Antigen preparation

Soluble antigens or fragments thereof (optionally conjugated with other molecules) can be used as immunogens for generating antibodies. For transmembrane molecules, such as receptors, fragments of these (e.g., the extracellular domain of the receptor) can be used as immunogens. Alternatively, cells expressing transmembrane molecules can be used as immunogens. Such cells may be derived from natural sources (e.g., cancer cell lines), or may be cells transformed by recombinant techniques to express the transmembrane molecule. Other antigens and forms thereof useful for making antibodies will be apparent to those skilled in the art.

(ii) Certain antibody-based methods

Polyclonal antibodies are preferably generated by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant in the animal. Using bifunctional or derivatizing reagents, e.g. maleimidobenzoyl sulphosuccinimide ester (conjugated via a cysteine residue), N-hydroxysuccinimide (conjugated via a lysine residue), glutaraldehyde, succinic anhydride, SOCl₂Or R¹N ═ C ═ NR, where R and R¹Being different hydrocarbon groups, it may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor.

Animals are immunized against an antigen, immunogenic conjugate or derivative by mixing, for example, 100 μ g or 5 μ g of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, animals were boosted with an initial amount of 1/5-1/10 of the peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7-14 days, blood was collected from the animals, and the antibody titer of the serum was determined. Animals were boosted until the titer reached a plateau (pateau). Preferably, the animal is boosted with the same antigen but conjugated to a different protein and/or by a different cross-linking agent. Conjugates can also be prepared as protein fusions in recombinant cell culture. Also, a coagulant such as alum is suitably used to enhance the immune response.

Monoclonal Antibodies of interest can be generated using Hybridoma methods, first described in Kohler et al, Nature,256:495(1975), and further described in, for example, Hongo et al, Hybridoma,14(3):253-260(1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring harbor Laboratory Press,2nd ed.1988); hammerling et al, in: Monoclonal Antibodies and Cell hybrids 563-. Other methods include those described, for example, in U.S. Pat. No.7,189,826 for the production of monoclonal human native IgM antibodies from hybridoma cell lines. The human hybridoma technique (Trioma technology) is described in Vollmers and Brandlein, Histology and Histopathology,20(3):927-937(2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology,27(3):185-91 (2005).

For various other hybridoma techniques, see, e.g., US 2006/258841; US 2006/183887 (fully human antibody); US 2006/059575; US 2005/287149; US 2005/100546; US 2005/026229; and U.S. Pat. nos.7,078,492 and 7,153,507. An exemplary protocol for generating monoclonal antibodies using the hybridoma method is described below. In one embodiment, a mouse or other suitable host animal (such as a hamster) is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Antibodies are produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of a polypeptide of interest or a fragment thereof and an adjuvant such as monophosphoryl lipid a (mpl)/trehalose two-stick mycolate (TDM) (Ribi immunochem. research, inc., Hamilton, Mont.). The polypeptide of interest (e.g., antigen) or fragment thereof can be prepared using methods well known in the art, such as recombinant methods, some of which are further described herein. Sera from immunized animals are assayed for anti-antigen antibodies and optionally administered for booster immunizations. Lymphocytes are isolated from an animal that produces anti-antigen antibodies. Alternatively, lymphocytes are immunized in vitro.

The lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells. See, for example, Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103(Academic Press, 1986). Myeloma cells that support stable high-level antibody production by selected antibody-producing cells and are sensitive to a medium such as HAT medium can be used with efficient fusion. Exemplary myeloma cells include, but are not limited to, murine myeloma lines such as those derived from MOPC-21 and MPC-11 mouse tumors (available from the cell distribution center of the institute of Soleck (Salk), San Diego, Calif. USA), and SP-2 or X63-Ag8-653 cells (available from the American type culture Collection, Rockville, Md. USA). Human myeloma and mouse-human heteromyeloma cell lines are also described for the production of human Monoclonal antibodies (Kozbor, J.Immunol.,133:3001 (1984); Brodeur et al, Monoclonal antibody production Techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York, 1987)).

The hybridoma cells so prepared are seeded and cultured in a suitable medium, such as one containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent HGPRT-deficient cells from growing. Preferably, serum-free hybridoma cell culture methods are used to reduce the use of animal-derived serum, such as fetal bovine serum, as described, for example, in Evenet al, Trends in Biotechnology, 24(3),105-108 (2006).

Oligopeptides that are tools for increasing productivity of hybridoma cell cultures are described in Franek, Trends in monoclonal Antibody Research, 111-122 (2005). In particular, standard media are rich in certain amino acids (alanine, serine, asparagine, proline) or protein hydrolysate fractions and apoptosis can be significantly suppressed by synthetic oligopeptides consisting of 3-6 amino acid residues. The peptide is present in millimolar or higher concentrations.

The culture broth in which the hybridoma cells are growing can be assayed for production of monoclonal antibodies that bind to the antigens described herein. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of monoclonal antibodies can be determined by, for example, Scatchard analysis. See, e.g., Munson et al, anal. biochem.107:220 (1980).

After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and/or activity, the clones can be subcloned by limiting dilution procedures and cultured by standard methods (see, e.g., Goding, supra). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be cultured in vivo in animals as ascites tumors. Monoclonal antibodies secreted by the subclones can be suitably separated from the culture fluid, ascites fluid, or serum by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. One procedure for isolating proteins from hybridoma cells is described in US 2005/176122 and U.S. Pat. No.6,919,436. The method involves the use of minimally salts, such as lyotropic salts, during the binding process, and preferably also small amounts of organic solvents during the elution process.

(iii) Certain library screening methods

The antibodies in the formulations and compositions described herein can be generated by screening for antibodies with the desired activity using combinatorial libraries. For example, various methods are known in the art for constructing phage display libraries and screening such libraries for antibodies with desired binding properties. Such Methods are generally described in Hoogenboom et al in Methods in molecular Biology 178:1-37 (O' Brien et al, ed., Human Press, Totowa, N.J., 2001). For example, one method of producing an antibody of interest is via the use of a phage antibody library as described in Lee et al, j.mol.biol. (2004),340(5): 1073-93.

In principle, synthetic antibody clones are selected by screening phage libraries containing phage displaying various antibody variable region (Fv) fragments fused to phage coat proteins. Such phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and are thus separated from those not bound in the library. The bound clones are then eluted from the antigen and can be further enriched by additional antigen adsorption/elution cycles. Any antibody can be obtained by designing an appropriate antigen screening protocol to select phage clones of Interest, followed by construction of full-length antibody clones using Fv sequences from the phage clones of Interest and appropriate constant region (Fc) sequences as described in Kabat et al, sequences of Proteins, 5th edition, NIH Publication 91-3242, Bethesdamd, (1991), volumes 1-3.

In certain embodiments, the antigen-binding domain of an antibody is formed by two variable (V) regions of about 110 amino acids, each from a light chain (VL) and a heavy chain (VH), that both exhibit three hypervariable loops (HVRs) or Complementarity Determining Regions (CDRs). The variable domains can be functionally displayed on phage, either as single chain fv (scFv) fragments, in which VH and VL are covalently linked by a short, flexible peptide, or as Fab fragments, in which each is fused to a constant domain and interacts non-covalently, as described in Winter et al, Ann.Rev.Immunol.,12:433-455 (1994). As used herein, scFv-encoding phage clones and Fab-encoding phage clones are collectively referred to as "Fv phage clones" or "Fv clones".

The repertoire of VH and VL genes can be separately cloned by Polymerase Chain Reaction (PCR) and recombined randomly in a phage library, and then can be searched for antigen binding clones as described in Winter et al, Ann. Rev. Immunol.,12:433-455 (1994). Libraries from immunized sources provide antibodies with high affinity for the immunogen without the need to construct hybridomas. Alternatively, the non-immunized repertoire can be cloned to provide a single human antibody source for a broad range of non-self and self antigens without any immunization as described in Griffiths et al, EMBO J,12: 725-. Finally, unimmunized libraries can also be constructed synthetically, i.e., by cloning unrearranged V gene segments from stem cells and using PCR primers comprising random sequences to encode the highly variable CDR3 regions and to effect rearrangement in vitro, as described in Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992).

In certain embodiments, filamentous phage is used to display antibody fragments by fusion to the minor coat protein pIII. Antibody fragments may be displayed as single chain Fv fragments in which the VH and VL domains are linked by a flexible polypeptide spacer on the same polypeptide chain, for example as described in Marks et al, J.mol.biol.,222: 581-.

Generally, nucleic acids encoding antibody gene fragments are obtained from immune cells harvested from humans or animals. If it is desired to bias the library towards anti-antigen clones, the subject may be immunized with antigen to generate an antibody response and the spleen cells and/or circulating B cells or other Peripheral Blood Lymphocytes (PBLs) recovered for library construction. In one embodiment, a library of human antibody gene fragments biased for anti-antigen cloning is obtained by generating an anti-antigen antibody response in transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system) such that antigen immunization generates B cells that produce human antibodies against the antigen. The generation of human antibody-producing transgenic mice is described below.

Further enrichment of the population of anti-antigen reactive cells can be obtained by isolating B cells expressing antigen-specific membrane-bound antibodies using a suitable screening protocol, e.g.by cell separation using antigen affinity chromatography or adsorption of fluorescent dye-labelled antigen by the cells followed by fluorescence-activated cell sorting (FACS).

Alternatively, the use of splenocytes and/or B cells or other PBLs from non-immunized donors provides a better representation of the possible repertoire of antibodies, and also allows the construction of antibody libraries using any animal (human or non-human) species in which the antigen is not antigenic. For the construction of libraries incorporating antibody genes in vitro, stem cells are harvested from a subject to provide nucleic acids encoding unrearranged antibody gene segments. Immune cells of interest can be obtained from a variety of animal species (such as human, mouse, rat, lagomorph, luprine, canine, feline, porcine, bovine, equine, and avian species, among others).

Nucleic acids encoding antibody variable gene segments (including VH and VL segments) are recovered from the cells of interest and amplified. For the rearranged VH and VL gene libraries, the desired DNA may be obtained by isolating genomic DNA or mRNA from lymphocytes followed by Polymerase Chain Reaction (PCR) using primers matched to the 5 'and 3' ends of the rearranged VH and VL genes, as described in Orlandi et al, Proc. Natl. Acad. Sci. (USA),86: 3833-. The V gene can be amplified from cDNA and genomic DNA, with a reverse primer located 5' to the exon encoding the mature V domain, and a forward primer based within the J segment, as described by Orlandi et al (1989) and Ward et al, Nature,341: 544-. However, for amplification from cDNA, the reverse primer may also be based within the leader exon, as described by Jones et al, Biotechnol., 9:88-89(1991), and the forward primer is based within the constant region, as described by Sastry et al, Proc. Natl. Acad. Sci. (USA),86: 5728-. To maximize complementarity, degeneracy may be incorporated into the primers, as described by Orlandi et al (1989) or by Sasty et al (1989). In certain embodiments, library diversity is maximized by amplifying all available VH and VL rearrangements present in an immune cell Nucleic acid sample using PCR primers targeting each V gene family, for example as described in the methods of Marks et al, J.mol.biol.,222:581-597(1991) or the methods of Orum et al, Nucleic Acids Res.,21:4491-4498 (1993). For cloning the amplified DNA into an expression vector, rare restriction sites may be introduced as tags at one end of the PCR primers, as described by Orlandi et al (1989), or further PCR amplification may be performed using tagged primers, as described by Clackson et al, Nature,352: 624-.

The synthetic rearranged complete set of V genes can be derived in vitro from V gene segments. Most human VH gene segments have been cloned and sequenced (Tomlinson et al, J.mol.biol.,227:776-798(1992)), and located (Matsuda et al, Nature Genet.,3:88-94 (1993)); segments of these clones (including all major constructs of the H1 and H2 loops) can be used to generate a diverse VH gene repertoire using PCR primers for the H3 loop with diverse coding sequences and lengths, as described in Hoogenbomom and Winter, J.mol.biol.,227:381-388 (1992). VH repertoires can also be generated by concentrating all sequence diversity in a single length long H3 loop, as described by Barbas et al, Proc. Natl. Acad. Sci. USA,89:4457-4461 (1992). The human V.kappa.and V.lambda.segments have been cloned and sequenced (Williams and Winter, Eur.J.Immunol.,23: 1456-. A repertoire of synthetic V genes based on a range of VH and VL fold structures and lengths of L3 and H3 will encode antibodies with considerable structural diversity. After amplification of the DNA encoding the V gene, the germline V gene segments can be rearranged in vitro according to the method of Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992).

The repertoire of antibody fragments can be constructed by combining VH and VL gene repertoires together in several ways. Each repertoire can be created in different vectors and recombined in vitro, e.g., as described in Hogrefe et al, Gene,128:119-126(1993), or in vivo by combinatorial infectionGroup vectors, such as the loxP system described in Waterhouse et al, Nucl. acids Res.,21: 2265-. The in vivo recombination method exploits the double-stranded nature of the Fab fragment to overcome the library volume limitation imposed by e. The non-immunized VH and VL repertoires were cloned separately, one into the phagemid and the other into the phage vector. The two libraries were then combined by infecting phage-containing bacteria with phage so that each cell contained a different combination, the library capacity being limited only by the number of cells present (about 10)¹²Individual clones). Both vectors contain in vivo recombination signals, such that the VH and VL genes are recombined onto a single replicon and co-packaged into phage virions. These giant libraries provide large numbers of libraries with excellent affinity (K)_d ^-1Is about 10^-8M) of a diverse antibody.

Alternatively, the complete set may be cloned sequentially into the same vector, e.g., as described in Barbas et al, Proc. Natl. Acad. Sci. USA,88: 7978-. PCR assembly can also be used to link VH and VLDNA to DNA encoding flexible peptide spacers to form single chain fv (scfv) repertoires. In another technique, "intracellular PCR assembly" is used to combine VH and VL genes in lymphocytes by PCR, and then to clone a repertoire of linked genes, as described in Embleton et al, Nucl. acids Res.,20: 3831-.

Antibodies generated by the unimmunized library (natural or synthetic) may have intermediate affinity (K)_d ^-1Is about 10⁶-10⁷M^-1) However, affinity maturation can also be simulated in vitro by constructing a secondary library and selecting again, as described by Winter et al (1994), supra. For example, in the method of Hawkins et al, J.mol.biol.,226:889-896(1992) or the method of Gram et al, Proc.Natl.Acad.Sci USA,89:3576-3580(1992), mutations were randomly introduced in vitro using an error-prone polymerase (Leung et al, Technique,1:11-15 (1989)). In addition, affinity maturation can be performed by randomly mutating one or more CDRs, e.g., using carryover spanning in selected individual Fv clonesPrimers for random sequences of the CDRs of interest were PCR and screened for higher affinity clones. WO 9607754 (published on 3/14 1996) describes a method for inducing mutagenesis in the complementarity determining regions of immunoglobulin light chains to create a light chain gene library. Another highly efficient method is to recombine VH or VL domains selected by phage display with a repertoire of naturally occurring V domain variants from an unimmunized donor and screen for higher affinity in rounds of chain shuffling, as described in Marks et al, Biotechnol.,10:779-783 (1992). This technique allows the generation of an affinity of about 10^-9M or less than 10^-9Antibodies and antibody fragments to M.

Screening of the library can be accomplished by a variety of techniques known in the art. For example, the antigen may be used to coat the wells of an adsorption plate, expressed on host cells attached to an adsorption plate, or used for cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or any other method for panning phage display libraries.

Contacting the phage library sample with the immobilized antigen under conditions suitable for binding of at least a portion of the phage particles to the adsorbent. Normally, conditions including pH, ionic strength, temperature, and the like are selected to mimic physiological conditions. The phage bound to the solid phase is washed and then eluted with an acid, for example as described in Barbas et al, Proc.Natl.Acad.Sci USA,88: 7978-. Phages can be enriched 20-1,000-fold in a single round of selection. In addition, the enriched phage can be cultured in bacterial culture and subjected to more rounds of selection.

The efficiency of selection depends on many factors, including the kinetics of dissociation during washing, and whether multiple antibody fragments on a single phage are capable of binding antigen simultaneously. Antibodies with faster dissociation kinetics (and weak binding affinity) can be retained by using short washes, multivalent phage display, and high antigen coating density in the solid phase. The high density not only stabilizes the phage through multivalent interactions, but also facilitates the recombination of dissociated phage. Selection of antibodies with slower dissociation kinetics (and strong binding affinity) can be facilitated by the use of prolonged washing and monovalent phage display (as described in Bass et al, Proteins,8: 309-.

It is possible to select between phage antibodies with different affinities for the antigen, even if the affinities differ slightly. However, random mutagenesis of selected antibodies (e.g., as performed in some affinity maturation techniques) has the potential to produce many mutants, most of which bind antigen, and a few of which have higher affinity. By limiting the antigen, rare high affinity phages can compete out. To retain all higher affinity mutants, the phage can be incubated with an excess of biotinylated antigen, but the concentration of biotinylated antigen is lower on a molar basis than the target molar affinity constant of the antigen. High affinity binding phage can then be captured with streptavidin-coated paramagnetic beads. Such "equilibrium capture" allows selection of antibodies according to binding affinity, with sensitivity that allows isolation of mutant clones with only 2-fold the original affinity from a large excess of low affinity phage. Conditions for washing phage bound to the solid phase can also be manipulated to perform dissociation kinetic-based differentiation.

Anti-antigen clones may be selected based on activity. In certain embodiments, the invention provides anti-antigen antibodies that bind to live cells that naturally express the antigen or to antigens that are free floating or attached to other cellular structures. Fv clones corresponding to such anti-antigen antibodies can be selected as follows: (1) isolating anti-antigen clones from the phage library as described above, and optionally amplifying the population of isolated phage clones by culturing the population in a suitable bacterial host; (2) selecting a second protein and antigen whose activity is to be blocked and not blocked, respectively; (3) adsorbing anti-antigen phage clones to the immobilized antigen; (4) using an excess of the second protein to elute any unwanted clones that recognize antigen binding determinants (which overlap or share with the binding determinants of the second protein); and (5) eluting the clones still adsorbed after step (4). Optionally, clones with the desired blocking/non-blocking properties can be further enriched by repeating the selection procedure described herein one or more times.

DNA encoding the hybridoma-derived monoclonal antibodies or phage display Fv clones is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from a hybridoma or phage DNA template). Once isolated, the DNA may be placed into an expression vector, which is then transfected into a host cell that does not otherwise produce immunoglobulin protein, such as an escherichia coli cell, simian COS cell, Chinese Hamster Ovary (CHO) cell, or myeloma cell, to obtain synthesis of the desired monoclonal antibody in the recombinant host cell. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al, curr. opinion in immunol, 5:256(1993) and Pluckthun, immunol. revs,130:151 (1992).

DNA encoding Fv clones can be combined with known DNA sequences encoding the constant regions of the heavy and/or light chains (e.g., suitable DNA sequences are available from Kabat et al, supra) to form clones encoding full-length or partial-length heavy and/or light chains. It will be appreciated that constant regions of any isotype may be used for this purpose, including IgG, IgM, IgA, IgD and IgE constant regions, and that such constant regions may be derived from any human or animal species. Fv clones derived from variable domain DNA of one animal species (such as human) and then fused with constant region DNA of another animal species to form coding sequences for "hybrid" full-length heavy and/or light chains are included in the definition of "chimeric" and "hybrid" antibodies as used herein. In certain embodiments, Fv clones derived from human variable DNA are fused to human constant region DNA to form coding sequences for full or partial length human heavy and/or light chains.

The DNA encoding anti-antigen antibodies derived from hybridomas may also be modified, for example, by replacing, i.e., replacing, the homologous murine sequences derived from hybridoma clones with the coding sequences for the human heavy and light chain constant domains (e.g., as in Morrison et al, Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The DNA encoding the antibody or fragment derived from the hybridoma or Fv clone can be further modified by covalently linking the immunoglobulin coding sequence to all or part of the coding sequence for a non-immunoglobulin polypeptide. "chimeric" or "hybrid" antibodies having the binding specificity of Fv clone or hybridoma clone-derived antibodies can be prepared in this manner.

(iv) Humanized antibody and human antibody

Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al, Nature,321:522-525 (1986); Riechmann et al, Nature,332:323-327 (1988); Verhoeyen et al, Science,239:1534-1536(1988)), using rodent CDRs or CDR sequences in place of the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No.4,816,567) in which significantly less than the entire human variable domain is replaced with the corresponding sequence of a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of human light and heavy chain variable domains for making humanized antibodies is very important for reducing antigenicity. The entire library of known human variable domain sequences is screened with the variable domain sequences of rodent antibodies according to the so-called "best-fit" method. The closest human sequence to rodents is then selected as the human Framework (FR) for the humanized antibody (Sims et al, J.Immunol., 151:2296 (1993); Chothia et al, J.mol.biol., 196:901 (1987)). Another approach uses a specific framework derived from the consensus sequence of all human antibodies of a specific subclass of light or heavy chains (subgroups). The same framework can be used for several different humanized antibodies (Carter et al, Proc. Natl. Acadsi. USA,89:4285 (1992); Presta et al, J. Immunol., 151:2623 (1993)).

It is even more important that the antibody retains high affinity for the antigen and other favorable biological properties after humanization. To achieve this, according to one embodiment of the method, a humanized antibody is prepared by a process of analyzing a parent sequence and various conceptual humanized products using three-dimensional models of the parent sequence and the humanized sequence. Three-dimensional models of immunoglobulins are generally available, as will be familiar to those skilled in the art. Computer programs are also available that illustrate and display the likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. By examining these display images, one can analyze the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the acceptor and import sequences and combined to obtain a desired antibody characteristic, such as increased affinity for the target antigen. In general, hypervariable region residues are directly and most substantially involved in the effect on antigen binding.

The human antibodies in the formulations and compositions described herein can be constructed by combining Fv clone variable domain sequences selected from human-derived phage display libraries with known human constant domain sequences as described above. Alternatively, human monoclonal antibodies can be generated by hybridoma methods. Human myeloma and mouse-human heteromyeloma cell lines used to produce human monoclonal antibodies have been described, for example, Kozbor j.immunol.,133:3001 (1984); brodeur et al, monoclonal antibody Production Techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York, 1987); and Boerner et al, J.Immunol.147: 86(1991).

For example, it is possible to generate transgenic animals (e.g., mice) that are capable of generating a complete repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, antibody heavy chain ligation has been described in chimeric and germline mutant miceRegion (J)_H) Homozygous deletion of the gene results in complete suppression of endogenous antibody production. Transfer of large numbers of human germline immunoglobulin genes in such germline mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc.Natl.Acad.Sci.USA,90:2551 (1993); jakobovits et al, Nature,362: 255-; bruggermann et al, Yeast in Immuno, 7:33 (1993); and Duchosal et al Nature355:258 (1992).

Gene shuffling can also be used to derive human antibodies from non-human (e.g., rodent) antibodies, where the human antibodies have similar affinity and specificity as the starting non-human antibody. According to this method, which is also known as "epitope imprinting", the variable regions of the heavy or light chains of non-human antibody fragments obtained by phage display techniques as described herein are replaced with a repertoire of human V domain genes, resulting in a population of non-human chain scFv or Fab chimeras. Selection with antigen results in the isolation of a non-human chain/human chain chimeric scFv or Fab, wherein the human chain restores the antigen binding site that was destroyed upon elimination of the corresponding non-human chain in the primary phage display clone, i.e. the epitope determines (imprints) the selection of the human chain partner. When this procedure is repeated to replace the remaining non-human chains, human antibodies are obtained (see PCT WO 93/06213, published at 1/4/1993). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides fully human antibodies that do not contain FR or CDR residues of non-human origin.

(v) Antibody fragments

Antibody fragments may be generated by conventional means, such as enzymatic digestion, or by recombinant techniques. In some cases, it may be advantageous to use antibody fragments rather than whole antibodies. The smaller size of the fragments allows for rapid clearance and may result in easier access to solid tumors. For a review of certain antibody fragments see Hudson et al (2003) nat. Med.9: 129-.

Various techniques have been developed for generating antibody fragments. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, Jourbal of Biochemical and biophysical methods 24:107-117 (1992); and Brennan et al, Science 229:81 (1985)). However, these fragments can now be produced directly from recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted by E.coli, thus allowing easy production of large quantities of these fragments. Antibody fragments can be isolated from the phage antibody libraries discussed above. Alternatively, Fab '-SH fragments can be recovered directly from E.coli and chemically coupled to form F (ab')₂Fragments (Carteret et al, Bio/Technology10: 163-. According to another method, F (ab') can be isolated directly from recombinant host cell cultures₂And (3) fragment. Fab and F (ab') with extended in vivo half-life comprising salvage receptor binding epitope residues₂Fragments are described in U.S. Pat. No.5,869,046. Other techniques for generating antibody fragments will be apparent to the skilled practitioner. In certain embodiments, the antibody is a single chain Fv fragment (scFv). See WO 93/16185; U.S. patent nos. 5,571,894; and 5,587,458. Fv and scFv are the only types with intact binding sites, lacking constant regions; as such, they may be suitable for reducing non-specific binding when used in vivo. scFv fusion proteins can be constructed to generate fusion of the effector protein at the amino or carboxy terminus of the scFv. See, for example, Antibody Engineering, eds. Borebaeck, supra. Antibody fragments may also be "linear antibodies," for example as described in U.S. Pat. No.5,641,870. Such linear antibodies may be monospecific or bispecific.

(vi) Multispecific antibodies

Multispecific antibodies have binding specificities for at least two different epitopes, wherein the epitopes are typically derived from different antigens. Although such molecules typically bind only two different epitopes (i.e., bispecific antibodies, BsAb), this expression, as used herein, encompasses antibodies with additional specificity, such as trispecific antibodies. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F (ab')₂Bispecific antibodies).

Methods for constructing bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al, Nature, 305:537-539 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, which is usually performed by an affinity chromatography step, is rather cumbersome and the product yield is low. Similar procedures are disclosed in WO 93/08829 and Travecker et al, EMBO J.,10:3655-3659 (1991).

According to a different approach, antibody variable domains with the desired binding specificity (antibody-antigen binding site) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is to an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. Typically, a first heavy chain constant region (CH1) is present in at least one of the fusions that includes the site necessary for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields, this provides great flexibility in adjusting the mutual ratios of the three polypeptide fragments. However, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when expression of at least two polypeptide chains in the same ratio leads to high yields or when the ratio is of no particular significance.

In one embodiment of the method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) on the other arm. Since the presence of immunoglobulin light chains in only half of the bispecific molecule provides a convenient separation route, it was found that this asymmetric structure facilitates the separation of the desired bispecific compound from the unwanted immunoglobulin chain combinations. The method is disclosed in WO 94/04690. For further details on the generation of bispecific antibodies see, e.g., Suresh et al, Methods in Enzymology,121:210 (1986).

According to another approach described in WO96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. An interface comprising at least part of C of an antibody constant domain_H3 domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with a larger side chain (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size to the large side chains are created at the interface of the second antibody molecule by replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, one antibody in the heterologous conjugate may be coupled to avidin, while the other antibody is coupled to biotin. Such antibodies have been suggested for use, for example, in targeting immune system cells to unwanted cells (U.S. Pat. No.4,676,980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugated antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents are well known in the art, as well as a number of crosslinking techniques, and are disclosed in U.S. Pat. No.4,676,980.

Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al, Science 229:81(1985) describes proteolytic cleavage of intact antibodies to F (ab')₂Protocol for fragmentation. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. One of the Fab '-TNB derivatives is then restored to the Fab' -thiol by reduction with mercaptoethylamine,and mixed with an equimolar amount of another Fab' -TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as selective immobilization reagents for enzymes.

Recent advances have facilitated the direct recovery of Fab' -SH fragments from E.coli, which can be chemically coupled to form bispecific antibodies. A fully humanized bispecific antibody F (ab') is described in Shalaby et al, J.Exp.Med.,175:217-225(1992)₂And (4) generation of molecules. Each Fab' fragment was secreted separately from E.coli and subjected to directed chemical coupling in vitro to form bispecific antibodies.

Various techniques for the direct production and isolation of bispecific antibody fragments from recombinant cell cultures are also described. For example, bispecific antibodies have been generated using leucine zippers. Kostelny et al, J.Immunol.,148(5):1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. The "diabody" technique described by Hollinger et al, Proc. Natl. Acad. Sci. USA,90: 6444-. The fragment comprises heavy chain variable domains (V) connected by a linker_H) And a light chain variable domain (V)_L) The linker is too short to allow pairing between the two domains on the same strand. Thus, V on a segment is forced_HAnd V_LDomain and complementary V on another fragment_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another strategy for constructing bispecific antibody fragments by using single chain fv (sfv) dimers has also been reported. See Gruber et al, J.Immunol.,152:5368 (1994).

Antibodies with more than two titers are contemplated. For example, trispecific antibodies can be prepared. Tuft et al, j.immunol.,147:60 (1991).

(vii) Single domain antibodies

In some embodiments, the antibodies described herein are single-domain antibodies. Single domain antibodies are single polypeptide chains that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No.6,248,516B 1). In one embodiment, the single domain antibody consists of all or part of the heavy chain variable domain of the antibody.

(viii) Antibody variants

In some embodiments, amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes can be introduced into the amino acid sequence of a subject antibody at the time of sequence preparation.

(ix) Antibody derivatives

The antibodies in the formulations and compositions of the invention can be further modified to include additional non-proteinaceous moieties known in the art and readily available. In certain embodiments, the moiety suitable for derivatization of the antibody is a water-soluble polymer. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the specific properties or function of the antibody to be improved, whether the antibody derivative is to be used in a treatment under specified conditions, and the like.

(x) Vectors, host cells, and recombinant methods

Antibodies can also be generated using recombinant methods. For recombinant production of anti-antigen antibodies, nucleic acids encoding the antibodies are isolated and inserted into replicable vectors for further cloning (DNA amplification) or expression. DNA encoding the antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody). Many vectors are available. Carrier members typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

(a) Signal sequence component

The heterologous signal sequence selected is preferably recognized and processed (e.g., cleaved by a signal peptidase) by a host cell for prokaryotic host cells that do not recognize and process native antibody signal sequences, the signal sequence may be replaced with, for example, a prokaryotic signal sequence selected from the group consisting of the alkaline phosphatase, penicillinase, lpp, or heat stable enterotoxin II leaders.

(b) Origin of replication

Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Generally, in cloning vectors, such sequences are those which enable the vector to replicate independently of the host chromosomal DNA, including origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeasts and viruses. The origin of replication from the plasmid pBR322 is suitable for most gram-negative bacteria, the origin of replication from the 2. mu. plasmid is suitable for yeast, and various viral origins of replication (SV40, polyoma, adenovirus, VSV or BPV) can be used for cloning vectors in mammalian cells. In general, mammalian expression vectors do not require an origin of replication component (the SV40 origin may generally be used simply because it contains an early promoter).

(c) Selection gene components

Expression and cloning vectors may comprise a selection gene, also referred to as a selectable marker. Typical selection genes encode the following proteins: (a) conferring resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) supplementing the nutritional deficiency; or (c) provide key nutrients not available from complex media, such as a gene encoding a bacillus D-alanine racemase.

One example of a selection scheme utilizes drugs to retard the growth of host cells. Those cells successfully transformed with the heterologous gene produce proteins that confer drug resistance and thus survive the selection protocol. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

Another example of a suitable selectable marker for mammalian cells is one that can identify cells competent to take up antibody-encoding nucleic acids, such as DHFR, Glutamine Synthase (GS), thymidine kinase, metallothionein-I and-II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like.

For example, cells transformed with the DHFR gene are identified by culturing the transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Under these conditions, the DHFR gene is amplified along with any other co-transformed nucleic acids. A Chinese Hamster Ovary (CHO) cell line deficient in endogenous DHFR activity (e.g., ATCC CRL-9096) can be used.

Alternatively, GS gene-transformed cells were identified by culturing the transformants in a medium containing L-methionine sulfoximine (Msx), an inhibitor of GS. Under these conditions, the GS gene is amplified along with any other co-transformed nucleic acids. The GS selection/amplification system may be used in combination with the DHFR selection/amplification system described above.

Alternatively, host cells (particularly wild-type hosts comprising endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody of interest, a wild-type DHFR gene, and another selectable marker such as aminoglycoside 3' -phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No.4,965,199.

A suitable selection gene for yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al, Nature 282:39 (1979)). the trp1 gene provides a selectable marker for yeast mutants lacking the ability to grow in tryptophan, such as ATCC No.44076 or PEP 4-1. The presence of a trp1 lesion in the genome of a yeast host cell then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2 deficient yeast strains (ATCC 20,622 or 38,626) were complemented with known plasmids carrying the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 can be used to transform Kluyveromyces yeast. Alternatively, expression systems for large-scale production of recombinant calf chymosin in Kluyveromyces lactis have been reported. Van den berg, Bio/Technology 8:135 (1990). Also disclosed are stable multi-copy expression vectors suitable for secretion of mature recombinant human serum albumin by industrial strains of the genus Kluyveromyces. Fleer et al, Bio/Technology 9: 968-.

(d) Promoter component

Promoters suitable for use in prokaryotic hosts include the phoA promoter, the β -lactamase and lactose promoter systems, the alkaline phosphatase promoter, the tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter.

Promoter sequences for eukaryotic cells are known. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is the CNCAAT region, where N can be any nucleotide. At the 3 'end of most eukaryotic genes is the AATAAA sequence, which may be the signal to add a poly A tail to the 3' end of the coding sequence. All these sequences are suitably inserted into eukaryotic expression vectors.

Examples of promoter sequences suitable for use in a yeast host include the promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters which are inducible promoters having the additional advantage of controlling transcription by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Vectors and promoters suitable for yeast expression are further described in EP 73,657. Yeast enhancers may also be advantageously used with yeast promoters.

Transcription of antibodies from vectors in mammalian host cells can be controlled by, for example, promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis b virus, simian virus 40(SV40), or from heterologous mammalian promoters such as the actin promoter or an immunoglobulin promoter, and from heat shock promoters, provided such promoters are compatible with the host cell system.

The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment, which also contains the SV40 viral origin of replication, the immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment, U.S. Pat. No.4,419,446 discloses a system for expressing DNA in a mammalian host using the bovine papilloma virus as a vector, an improvement of this system is described in U.S. Pat. No.4,601,978, see also Reyes et al, Nature 297: 598-.

(e) Enhancer element component

Transcription of antibody-encoding DNA by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein and insulin). However, typically an enhancer from a eukaryotic cell virus is used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. For enhanced elements for activation of eukaryotic promoters see also Yaniv, Nature 297:17-18 (1982). Enhancers may be spliced into the vector at positions 5' or 3' to the antibody coding sequence, but are preferably located at sites 5' to the promoter.

(f) Transcription termination component

Expression vectors for eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are typically available from the 5 'and occasionally 3' ends of untranslated regions of eukaryotic or viral DNA or cDNA. These regions comprise nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vectors disclosed therein.

(g) Selection and transformation of host cells

Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotes, yeast or higher eukaryotes as described above. Prokaryotes suitable for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example enterobacteriaceae, such as Escherichia (Escherichia) e.g. Escherichia coli or Escherichia coli (e.coli), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella) e.g. Salmonella typhimurium, Serratia (Serratia) e.g. Serratia marcescens (Serratia marcans), Shigella (Shigella), and bacillus (bacillus) e.g. bacillus subtilis (b.licheniformis) and bacillus (b.heliformis) (e.g. bacillus licheniformis 41P disclosed in DD 266,710 published 4.12.1989), Pseudomonas (Pseudomonas) such as Pseudomonas aeruginosa, Pseudomonas aeruginosa. A preferred E.coli cloning host is E.coli 294(ATCC 31,446), although other strains such as E.coli B, E.coli X1776(ATCC 31,537) and E.coli W3110(ATCC 27,325) are also suitable. These examples are illustrative and not restrictive.

Full-length antibodies, antibody fusion proteins, and antibody fragments can be produced in bacteria, particularly when glycosylation and Fc effector function are not required, such as when a therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin) that itself exhibits efficacy in tumor cell destruction. Full-length antibodies have a longer half-life in circulation. Production in E.coli is faster and more cost effective. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. No.5,648,237(Carter et al.), U.S. Pat. No.5,789,199 (Joly et al.), U.S. Pat. No.5,840,523(Simmons et al.), which describes a Translation Initiation Region (TIR) and signal sequences that optimize expression and secretion. See also Charlton, Methods in Molecular Biology, volume 248(B.K.C.Lo, eds., Humana Press, Totowa, NJ,2003), pp.245-254, which describes the expression of antibody fragments in E.coli. After expression, the antibody can be isolated from the E.coli cell slurry in a soluble fraction and purified by, for example, a protein A or G column (depending on the isotype). A final purification can be performed similar to the process used for purifying antibodies expressed in e.g. CHO cells.

In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae or commonly used baker's yeast are among the most commonly used lower eukaryotic host microorganisms. However, many other genera, species and strains are generally available and useful in the present invention, such as Schizosaccharomyces pombe (Schizosaccharomyces pombe); kluyveromyces hosts such as, for example, Kluyveromyces lactis (k.lactis), Kluyveromyces fragilis (k.fragilis) (ATCC 12,424), Kluyveromyces bulgaricus (k.bulgaricus) (ATCC 16,045), Kluyveromyces williamsii (k.wickraimi) (ATCC 24,178), k.wallidi (ATCC 56,500), Kluyveromyces drosophilus (k.drosophilarium) (ATCC 36,906), Kluyveromyces thermotolerans (k.thermotolerans), and Kluyveromyces marxianus (k.marxianus); yarrowia (EP 402,226); pichia pastoris (Pichia pastoris) (EP 183,070); candida genus (Candida); trichoderma reesei (Trichoderma reesei) (EP244,234); neurospora crassa (Neurospora crassa); schwanniomyces (Schwanniomyces), such as Schwanniomyces occidentalis; and filamentous fungi such as, for example, Neurospora (Neurospora), Penicillium (Penicillium), torticollis (Tolypocladium), and Aspergillus (Aspergillus) hosts such as Aspergillus nidulans (a. nidulans) and Aspergillus niger (a. niger). For a review discussing the use of yeasts and filamentous fungi for the production of therapeutic proteins see, e.g., Gerngross, nat. Biotech.22:1409-1414 (2004).

Certain fungal and yeast strains may be selected in which the glycosylation pathway has been "humanized" resulting in the production of antibodies having a partially or fully human glycosylation pattern. See, e.g., Li et al, nat. Biotech.24:210-215(2006) (which describes humanization of the glycosylation pathway in Pichia pastoris); and Gerngross et al, supra.

Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains and variants and corresponding permissive insect host cells have been identified which are derived from hosts such as Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori (Bombyx mori). A variety of viral strains are publicly available for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used in accordance with the present invention as viruses herein, particularly for transfecting Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, lemna (Leninaceae), alfalfa (m.truncatula), and tobacco may also be utilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177; 6,040,498; 6,420,548, respectively; 7,125,978, respectively; and 6,417,429 (PLANTIBODIIES described for antibody production in transgenic plants^TMA technique).

Vertebrate cells can be used as hosts, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7, ATCCRL 1651); human embryonic kidney lines (293 or 293 cells subcloned for growth in suspension culture, Graham et al, J.Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli (sertoli) cells (TM4, Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV1, ATCC CCL 70); vero cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); bovine murine (buffalo rat) hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCCCCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982); MRC5 cells; FS4 cells; and human hepatoma line (Hep G2)^-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NS0 and Sp 2/0. For reviews of certain mammalian host cell lines suitable for antibody production see, for example, Yazaki and wu, Methods in Molecular Biology, volume 248(b.k.c.lo, eds., Humana Press, Totowa, NJ,2003), pp.255-268.

Host cells are transformed with the expression or cloning vectors described above for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants or amplifying the genes encoding the desired sequences.

(h) Culturing host cells

Host cells for the production of antibodies can be cultured in a variety of media. Commercial culture media such as Ham's F10(Sigma), minimal essential medium (MEM, Sigma), RPMI-1640(Sigma), and Dulbecco's modified Eagle's medium (DMEM, Sigma) are suitable for culturing the host cells. In addition, any of the media described in the following documents can be used as the medium for the host cells: ham et al, meth.enz.58:44 (1979); barnes et al, anal. biochem.102:255 (1980); U.S. patent nos. 4,767,704; 4,657,866, respectively; 4,927,762, respectively; 4,560,655, respectively; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re.30,985. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN)^TMDrugs), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art may also be included in suitable concentrations. It will be apparent to the skilled person that the culture conditions (such as temperature, pH, etc.) are those previously selected for the host cell for expression.

(xi) Purification of antibodies

When recombinant techniques are used, the antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, particulate debris of the host cells or lysed fragments is removed, for example, by centrifugation or ultrafiltration. Carter et al, Bio/Technology10: 163-. Briefly, the cell paste was thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant from such an expression system is typically first concentrated using a commercial protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. In any of the above steps, a protease inhibitor such as PMSF may be included to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being one of the generally preferred purification steps. The suitability of protein a as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein a can be used to purify antibodies based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al, j. immunol. meth.62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human gamma 3(Guss et al, EMBO J.5:1567-1575 (1986)). Protein L can be used to purify kappa light chain-based antibodies (Nilson et al, J.Immunol.meth.164(1):33-40 (1993)). The matrix to which the affinity ligand is attached is most commonly agarose, but other matrices may be used. Physically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene enable faster flow rates and shorter processing times than agarose. If the antibody comprises C_H3 Domain, then Bakerbond ABX can be used^TMPurification was performed on resin (j.t. baker, phillips burg, NJ). Depending on the antibody to be recovered, other protein purification techniques may also be used, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, heparin Sepharose^TMChromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

In general, various methodologies for preparing antibodies for research, testing, and clinical use are well established in the art, are consistent with the methodologies described above, and/or are deemed appropriate for a particular antibody of interest by one of skill in the art.

B. Selection of biologically active antibodies

One or more "biological activity" assays may be performed on the antibodies generated as described above to select antibodies having beneficial properties from a therapeutic standpoint. Antibodies can be screened for their ability to bind to the antigen against which they were generated. For example, for an anti-DR 5 antibody (e.g., hydrozitumab), the antigen binding properties of the antibody can be assessed in an assay that detects the ability to bind to death receptor 5(DR 5).

In another embodiment, binding may be by, for example, saturation; ELISA (enzyme-Linked immuno sorbent assay); and/or competition assays (e.g., RIA) to determine the affinity of the antibody.

Also, the antibody may be subjected to other biological activity assays, for example, to assess its effectiveness as a therapeutic agent. Such assays are known in the art and depend on the target antigen and intended use of the antibody.

To screen for Antibodies that bind to a particular epitope on an antigen of interest, a conventional cross-blocking assay can be performed, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Alternatively, epitope mapping can be performed, for example, as described by Champe et al, j.biol.chem.270: 1388 1394(1995) to determine whether antibodies bind to the epitope of interest.

C. Preparation of the formulations

Provided herein are methods of making a liquid formulation comprising a protein and NAT that prevents oxidation of the protein in the liquid formulation. The liquid formulation may be prepared by mixing the protein with the desired degree of purity with NAT that prevents oxidation of the protein in the liquid formulation. In certain embodiments, the protein to be formulated has not been subjected to prior lyophilization and the formulation of interest herein is an aqueous formulation. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antibody. In still other embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In certain embodiments, the antibody is a full-length antibody. In one embodiment, the anti-cancer agent in the formulationThe body is an antibody fragment, such as F (ab')₂In this case, it may be desirable to address issues that may not occur with full-length antibodies (such as the trimming of antibodies to Fab). The therapeutically effective amount of protein present in the formulation is determined by taking into account, for example, the desired dose volume and mode of administration. Exemplary protein concentrations in the formulation include from about 1mg/mL to more than about 250mg/mL, from about 1mg/mL to about 250mg/mL, from about 10mg/mL to about 250mg/mL, from about 15mg/mL to about 225mg/mL, from about 20mg/mL to about 200mg/mL, from about 25mg/mL to about 175mg/mL, from about 25mg/mL to about 150mg/mL, from about 25mg/mL to about 100mg/mL, from about 30mg/mL to about 100mg/mL, or from about 45mg/mL to about 55 mg/mL. In some embodiments, the proteins described herein are susceptible to oxidation. In some embodiments, one or more of the amino acids in the protein selected from the group consisting of methionine, cysteine, histidine, tryptophan, and tyrosine are susceptible to oxidation. In some embodiments, the tryptophan in the protein is susceptible to oxidation. In some embodiments, the protein comprises a Solvent Accessibility Surface Area (SASA) of greater than aboutTo about(such as greater than about 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, orAny, including any range between these values) of tryptophan residues. In some embodiments, the SASA is greater than aboutIn some embodiments, the protein comprises greater than about 15% to about 45% (such as greater than any of about 15, 20, 25, 30, 35, 40, or 45%) tryptophan residues of the SASA. In some embodiments, the SASA is greater than about 30%. In some embodiments, the SA of a tryptophan residue is measured at a pH range from about 4.0 to about 8.5And (SA). In some embodiments, the SASA of tryptophan residues is measured at a temperature ranging from about 5 ℃ to about 40 ℃. In some embodiments, the SASA of tryptophan residues is measured at a salt concentration ranging from about 0mM to about 500 mM. In some embodiments, the SASA of tryptophan residues is measured at a pH of about 5.0 to about 7.5, a temperature of about 5 ℃ to about 25 ℃, and a salt concentration of about 0mM to about 500 mM. In some embodiments, the protein comprises at least one tryptophan residue predicted to be susceptible to oxidation by a machine learning algorithm practiced in association with the tryptophan residue oxidation susceptibility and a plurality of molecular descriptors of the tryptophan residue based on MD simulation. In some embodiments, an antibody provided herein is susceptible to oxidation in the Fab portion and/or the Fc portion of the antibody. In some embodiments, an antibody provided herein is susceptible to oxidation at a tryptophan amino acid in the Fab portion of the antibody. In yet another embodiment, tryptophan amino acids susceptible to oxidation are in the CDRs of the antibody. In some embodiments, an antibody provided herein is susceptible to oxidation at a methionine amino acid in the Fc portion of the antibody. In some embodiments, the liquid formulation further comprises at least one additional protein in accordance with any of the proteins described herein.

The liquid formulation provided by the invention herein comprises a protein and NAT that prevents oxidation of the protein in the liquid formulation. In some embodiments, the NAT in the formulation is at a concentration from about 0.1mM to more than about 10mM, or up to the highest concentration at which the NAT is soluble in the formulation. In certain embodiments, the NAT in the formulation is at a concentration from about 0.1mM to about 10mM, about 0.1mM to about 9mM, from about 0.1mM to about 8mM, from about 0.1mM to about 7mM, from about 0.1mM to about 6mM, from about 0.1mM to about 5mM, from about 0.1mM to about 4mM, from about 0.1mM to about 3mM, from about 0.1mM to about 2mM, from about 0.3mM to about 2mM, from about 0.5mM to about 2mM, from about 0.6mM to about 1.5mM, or from about 0.8mM to about 1.25 mM. In some embodiments, the NAT in the formulation is about 1 mM. In some embodiments, the NAT prevents oxidation of one or more amino acids in the protein. In some embodiments, the NAT preventionOne or more amino acids selected from the group consisting of tryptophan, methionine, tyrosine, histidine, and/or cysteine are oxidized in the protein. In some embodiments, the NAT prevents oxidation of tryptophan in the protein. In some embodiments, the NAT prevents oxidation of the protein by Reactive Oxygen Species (ROS). In yet another embodiment, the reactive oxygen species is selected from the group consisting of singlet oxygen, superoxide (O)₂-, alkoxy radical, peroxy radical, hydrogen peroxide (H)₂O₂) Dihydrogen trioxide (H)₂O₃) Radical of hydrogen trioxide (HO)₃H), ozone (O)₃) Hydroxyl radicals, and alkyl peroxides. In yet another embodiment, the NAT prevents oxidation of one or more amino acids in the Fab portion of the antibody. In another further embodiment, the NAT prevents oxidation of one or more amino acids in the Fc portion of an antibody.

In some embodiments, the liquid formulation provided by the invention herein comprises a protein and NAT that prevents oxidation of the protein in the liquid formulation, wherein oxidation of the protein is reduced by about 40% to about 100%. In some embodiments, the oxidation of the protein is reduced by about any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, including any range between these values.

The amount of oxidation in the protein can be determined, for example, using one or more of RP-HPLC, LC/MS, or tryptic peptide mapping. In some embodiments, oxidation in a protein is determined as a percentage using one or more of RP-HPLC, LC/MS, or tryptic peptide mapping and the following formula:

in some embodiments, the liquid formulation provided by the invention herein comprises a protein and NAT that prevents oxidation of the protein in the liquid formulation, wherein no more than about 40% to about 0% of the protein is oxidized. In some embodiments, no more than about any of 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0%, including any range between these values, of the protein is oxidized.

In some embodiments, the liquid formulation provided by the invention herein comprises a protein and a NAT that prevents oxidation of the protein in the liquid formulation, wherein oxidation of at least one oxidized isotretinoin in the protein is reduced by about 40% to about 100%. In some embodiments, the reduction in oxidation of the oxoisotretinoin is about any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, including any range between these values. In some embodiments, the oxidation of each of the oxoisotretinoin residues in the protein is reduced by about 40% to about 100% (such as about any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, including any range between these values).

In some embodiments, the liquid formulations provided by the invention herein comprise a protein and NAT that prevents oxidation of the protein in the liquid formulation, wherein no more than about 40% to about 0% of at least one oxoisotretinoin in the protein is oxidized. In some embodiments, no more than about any of 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0%, inclusive of any range between these values, of oxoisotretinoin oxidation. In some embodiments, no more than about 40% to about 0% (such as no more than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0% of any one, including any range between these values) of each of the oxoisotretinoin residues in the protein is oxidized.

In some embodiments, the liquid formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. The liquid formulations of the present invention are prepared in a pH buffered solution. The buffers of the present invention have a pH in the range of from about 4.0 to about 9.0. In certain embodiments, the pH is in the range from pH4.0 to 8.5, in the range from pH4.0 to 8.0, in the range from pH4.0 to 7.5, in the range from pH4.0 to 7.0, in the range from pH4.0 to 6.5, in the range from pH4.0 to 6.0, in the range from pH4.0 to 5.5, in the range from pH4.0 to 5.0, in the range from pH4.0 to 4.5, in the range from pH 4.5 to 9.0, in the range from pH 5.0 to 9.0, in the range from pH 5.5 to 9.0, in the range from pH 6.0 to 9.0, in the range from pH6.5 to 9.0, in the range from pH 7.0 to 9.0, in the range from pH 5.0 to 8.5, in the range from pH5 to 8.0, in the range from pH5 to 8.5, in the range from pH5 to 6.0, in the range from pH5 to 8.5, in the range from pH5 to 8.0, in the range from pH 6.0 to 6.5, or in the range from pH 6.2 to 6.5. In certain embodiments of the invention, the liquid formulation has a pH of 6.2 or about 6.2. In certain embodiments of the invention, the liquid formulation has a pH of 6.0 or about 6.0. Examples of buffering agents that will control the pH within this range include organic and inorganic acids and salts thereof. For example, acetates (e.g., histidine acetate, arginine acetate, sodium acetate), succinates (e.g., histidine succinate, arginine succinate, sodium succinate), gluconates, phosphates, fumarates, oxalates, lactates, citrates, and combinations thereof. The buffer concentration may be from about 1mM to about 600mM, depending, for example, on the buffer and the desired isotonicity of the formulation. In certain embodiments, the formulation comprises histidine buffer (e.g., at a concentration from about 5mM to 100 mM). Examples of histidine buffers include histidine chloride, histidine acetate, histidine phosphate, histidine sulfate, histidine succinate, and the like. In certain embodiments, the formulation comprises histidine and arginine (e.g., histidine chloride-arginine chloride, histidine acetate-arginine acetate, histidine phosphate-arginine phosphate, histidine sulfate-arginine sulfate, histidine succinate-arginine succinate, etc.). In certain embodiments, the formulation comprises histidine at a concentration of from about 5mM to 100mM and arginine at a concentration of 50mM to 500 mM. In one embodiment, the formulation comprises histidine acetate (e.g., about 20mM) -arginine acetate (e.g., about 150 mM). In certain embodiments, the formulation comprises histidine succinate (e.g., about 20mM) -arginine succinate (e.g., about 150 mM). In certain embodiments, histidine in the formulation is from about 10mM to about 35mM, about 10mM to about 30mM, about 10mM to about 25mM, about 10mM to about 20mM, about 10mM to about 15mM, about 15mM to about 35mM, about 20mM to about 30mM or about 20mM to about 25 mM. In yet other embodiments, the arginine in the formulation is from about 50mM to about 500mM (e.g., about 100mM, about 150mM, or about 200 mM).

The liquid formulation of the present invention may further comprise a sugar, such as a disaccharide (e.g. trehalose or sucrose). As used herein, "sugar" includes the general Composition (CH)₂O) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. Examples of sugars herein include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol (glycerol), dextran, erythritol, glycerol (glycerol), arabitol, xylitol (sylitol), sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, and the like.

A surfactant may optionally be added to the antibody formulation. Exemplary surfactants include nonionic surfactants such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188, etc.). The amount of surfactant added is such that it reduces aggregation of the formulated antibody and/or minimizes particle formation in the formulation and/or reduces adsorption. For example, the surfactant may be present in the formulation in an amount from about 0.001% to more than about 1.0%, weight/volume. In some embodiments, the surfactant is present in the formulation in an amount from about 0.001% to about 1.0%, from about 0.001% to about 0.5%, from about 0.005% to about 0.2%, from about 0.01% to about 0.1%, from about 0.02% to about 0.06%, or from about 0.03% to about 0.05%, weight/volume. In certain embodiments, the surfactant is present in the formulation in an amount of 0.04% or about 0.04%, weight/volume. In certain embodiments, the surfactant is present in the formulation in an amount of 0.02% or about 0.02%, weight/volume. In one embodiment, the formulation does not comprise a surfactant.

In one embodiment, the formulation contains an agent identified above (e.g., an antibody, a buffer, a sugar, and/or a surfactant) and is essentially free of one or more preservatives, such as benzyl alcohol, phenol, m-cresol, chlorobutanol, and benzethonium chloride. In another embodiment, a preservative may be included in the formulation, particularly where the formulation is a multi-dose formulation. The concentration of the preservative may range from about 0.1% to about 2%, preferably from about 0.5% to about 1%. One or more other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Remington's pharmaceutical Sciences 16th edition, Osol, a.ed. (1980), may be included in the formulation, provided they do not adversely affect the desired characteristics of the formulation. Exemplary pharmaceutically acceptable excipients herein further include interstitial drug dispersing agents such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (r: (r) ())Baxter International, Inc.). Certain exemplary shasegps and methods of use (including rHuPH20) are described in U.S. patent publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycoaminoglycanases, such as chondroitinase.

The metal ion chelating agents are well known to those skilled in the art and include, but are not necessarily limited to, aminopolycarboxylates, EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol-bis (β -aminoethyl ether) -N, N, N ', N' -tetraacetic acid), NTA (nitrilotriacetic acid), EDDS (ethylenediamine disuccinate), PDTA (1, 3-propylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), ADA (β -alanine diacetic acid), MGCA (methylglycine diacetic acid), and the like.

Tonicity agents are present in the composition to adjust or maintain the tonicity of the liquid. They can also act as "stabilizers" when used with large charged biomolecules such as proteins and antibodies, because they can interact with the charged groups of the amino acid side chains, thereby reducing the potential for intermolecular and internal interactions. Tonicity agents may be present in any amount between 0.1% and 25% by weight, or more preferably between 1% and 5% by weight, taking into account the relative amounts of the other components. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol.

The formulations herein may also contain more than one protein or small molecule drug necessary for the particular indication being treated, preferably those with complementary activity, without adversely affecting other proteins. For example, where the antibody is anti-DR 5 (e.g., drozitumab), it can be combined with another agent (e.g., a chemotherapeutic agent and an anti-tumor agent).

In some embodiments, the formulation is for in vivo administration. In some embodiments, the formulation is sterile. The formulation may be rendered sterile by filtration through sterile filtration membranes. The therapeutic formulations herein are generally placed into a container having a sterile access port, such as a vial or intravenous solution bag having a stopper pierceable by a hypodermic injection needle. The route of administration is according to known and recognized methods, such as injection or infusion in a suitable manner, for example by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional, intraarticular, or intravitreal routes, topical administration, inhalation or by sustained or extended release means, by single or multiple bolus injections or infusion over a longer period of time.

The liquid formulations of the present invention may be stable after storage. In some embodiments, the protein in the liquid formulation is stable after storage for at least about 12 months (such as at least about any of 15, 18, 21, 24, 27, 30, 33, 36 months, or more) from about 0 to about 5 ℃ (such as about any of 1,2,3, or 4 ℃). In some embodiments, the physical stability, chemical stability, or biological activity of the protein in the liquid formulation is assessed or measured. Stability and biological activity can be assessed using any method known in the art. In some embodiments, stability is measured by oxidation of the protein of the liquid formulation after storage. Stability can be measured by assessing the physical stability, chemical stability, and/or biological activity of the antibody in the formulation around the time of formulation and after storage. Physical and/or stability can be assessed qualitatively and/or quantitatively in a number of different ways, including assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); (ii) assessing charge heterogeneity by using cation exchange chromatography or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometry analysis; SDS-PAGE analysis to compare reduced and intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; assessing the biological activity or antigen binding function of the antibody; and the like. Instability can lead to aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Trp oxidation), isomerization (e.g., Asp isomerization), clipping/hydrolysis/fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extension, C-terminal processing, glycosylation differences, and the like. In some embodiments, oxidation in the protein is determined using one or more of RP-HPLC, LC/MS, or tryptic peptide mapping. In some embodiments, oxidation in the antibody is determined as a percentage using one or more of RP-HPLC, LC/MS, or tryptic peptide mapping and the following formula:

the formulation to be used for in vivo administration should be sterile. This is readily achieved by filtration through sterile filtration membranes either before or after preparation of the formulation.

Also provided herein are methods of generating a liquid formulation or preventing oxidation of a protein in a liquid formulation, comprising adding an amount of NAT that prevents oxidation of the protein to the liquid formulation. In certain embodiments, the liquid formulation comprises an antibody. The amount of NAT provided herein to prevent protein oxidation is from about 0.1mM to about 10mM or any amount disclosed herein. In some embodiments, the liquid formulation further comprises at least one additional protein in accordance with any of the proteins described herein.

Method for predicting tryptophan oxidation

The invention herein also provides a method of predicting the susceptibility of a residue of a protein (such as tryptophan) in a liquid formulation to oxidation. The protein in the liquid formulation can be classified as having a residue susceptible to oxidation (such as a tryptophan residue) using a molecular descriptor determined by Molecular Dynamics (MD) simulation (such as an all-atom MD simulation) using protein sequence information in a computer. It is desirable to have a model, such as a computer learning algorithm, that can accurately predict or classify proteins in a liquid formulation as having oxidation-susceptible residues across a diverse array of molecular descriptors.

Methods are provided for generating computer learning algorithms to predict the susceptibility of residues of proteins (such as tryptophan) in liquid formulations to oxidation. In some embodiments, the method involves a) providing an exercise set comprising oxidation hotspot residues associated with i) values for a plurality of molecular descriptors (e.g., nearby aspartate side chain oxygens, side chain SASA, delta carbon SASA, nearby positive charges, backbone SASA, etc.) of the oxidation hotspot residues and ii) whether the oxidation hotspot residues are susceptible to oxidation; and b) applying the set of exercises to a machine learning algorithm (e.g., a random decision forest), whereby the machine learning algorithm is exercised to predict susceptibility to oxidation. In some embodiments, the method further comprises providing the machine learning algorithm (e.g., a random decision forest) to predict susceptibility to oxidation of one or more test residues having values of the plurality of molecular descriptors, including applying the plurality of molecular descriptors of each of the one or more test residues to the machine learning algorithm (e.g., a random decision forest) and classifying the one or more test residues as being susceptible to oxidation using a majority vote of the machine learning algorithm. In some embodiments, the molecular descriptor is determined in silico by MD simulation. In some embodiments, oxidation of at least about 30% (such as at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of any) of the residues in the oxidation assay is indicative of susceptibility to oxidation. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In some embodiments, the liquid formulation is an aqueous formulation.

In some embodiments, the machine learning algorithm is a random decision forest algorithm in which a guided technique in combination with random variable selection grows multiple decision trees (Ho, t.k. proceedings of the 3rd international conference on Document Analysis and Recognition, Montreal, QC,14-16 August 1995)Pp.278-282; ho, T.K.IEEE Transactions on Pattern Analysis and machinery analysis.20 (8)832-844, 1998). These multiple decision trees are sometimes referred to herein as collective or random decision forests of trees. In some embodiments, the random decision forest comprises at least about 20 (such at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000, or more of any) decision trees (also referred to herein as "evaluation quantities"). In some embodiments, the number of variables (also referred to herein as "features") that are randomly selected for consideration at each branch of each tree in the random decision forest is at least about 1 (such as at least about any of 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more). In some embodiments, the number of variables (also referred to herein as "tree depth") that are randomly selected for consideration at each branch of each tree in the random decision forest is between about 1 to about 20 (such as between about 1 to 15, 1 to 10, 1 to 5, 5 to 20, 5, 15, or 5 to 10, including any range between these values). The variables include molecular descriptors of amino acid residues in the polypeptide chain. In some embodiments, the molecular descriptor is determined in silico by MD simulation. In some embodiments, the molecular descriptor includes a delta carbon from the test residueNumber of aspartic acid side chain oxygens in, side chain SASA (stdev), delta carbon from test residueTotal positive charge (stdev) in, main chain SASA (stdev), test residue side chain angle, delta carbon from test residueInternal packing density, test residue backbone angle (stdev), SASA of pseudo-pi orbitals, backbone flexibility, and delta carbon from test residueThe total negative charge in. In some embodiments, the maximum number of times the observation set is divided into sub-branches of each tree is at least about 2 (such as at least about any of 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more). In some embodiments, the maximum number of times the observation set is divided into sub-branches of each tree is between about 2 to about 30 (such as between about 2 to 20,2 to 15, 2 to 10, 2 to 5, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10, including any range between these values). Information about the molecular descriptors of the test residues can be applied to the collective of the trees to obtain a prediction as to whether the test residue is susceptible to oxidation. The prediction is made by taking a majority vote of the predictions for all trees in the collection.

In some embodiments, to determine the distance test residue δ carbon at using MD simulationThe number of aspartic acid side chain oxygens in, for each frame of each molecular simulation, the delta carbon from the test residue was tracedAll atoms in (b), and among these atoms, those oxygen atoms on the side chain that are any aspartic acid residues are counted and the final value is calculated as the time average of this count over the duration of the simulation.

In some embodiments, to determine side chain SASA using MD simulation, for each frame of each molecular simulation, the points of the sphere concentrated on each atom in the simulation are generated by adding together each atomic radius and the radius of a water molecule, eliminating all points within the radius of adjacent spheres, and summing the areas between all remaining points to generate the value of SASA, and calculating the final value of this descriptor as the average SASA or the standard deviation of this calculated for the test residue side chain atom over the duration of the simulation.

In some embodiments, to determine the δ -carbon SASA using MD simulation, for each frame of each simulation, the SASA for the test residue δ -carbon is calculated as described above, and the final value of this descriptor is calculated as the average SASA or the standard deviation of this calculation for the test residue δ -carbon over the duration of the simulation.

In some embodiments, to determine the distance test residue δ carbon at using MD simulationTotal positive charge in, for each frame of each simulation, the delta carbon from the test residue was tracedAll the atoms associated with the charged amino acid side chains in the series are added together with the total positive charge of these atoms and the final value is calculated as the average of this number over the duration of the simulation or the standard deviation of this calculation.

In some embodiments, to determine the backbone SASA using MD simulation, for each frame of each molecular simulation, the SASA of the backbone nitrogen atom of the test residue is calculated as described above, and the final value of this descriptor is calculated as the average SASA or the standard deviation of this calculation over the duration of the simulation.

In some embodiments, to determine the test residue side chain angle using MD simulations, the χ 1 and χ 2 angles of the test residue side chain are tracked through the simulation, and this descriptor is calculated as the percentage of time the test residue spends in the angular region most predictive of oxidation, determined by running many different simulations for many different residues of the same amino acid as the test residue, plotting all χ 1 and χ 2 angles over the simulations, and clustering combinations of co-occurring angles.

In some embodiments, with a radius centered on the δ carbon of the test residueUsing MD simulation to calculate the delta carbon from the test residuePacking density of the inner part.

In some embodiments, the test residue backbone angle is calculated using MD simulations at the average ψ angle or the standard deviation of this calculation relative to the backbone of the test residue over the duration of the simulation.

In some embodiments, to determine the occupied volume of a false pi orbital using MD simulations, the side chain of a test residue is treated with the bottom of a cylinder that highly closely approximates the space occupied by the pi orbital of the test residue, all atoms that fall within the volume of the cylinder during the simulation are tracked, the total volume of all protein atoms that fall within the volume of the cylinder is calculated for each frame of the simulation, and the final value is calculated as the time-averaged volume of the pi orbital of the test residue occupied by other protein atoms.

In some embodiments, to determine backbone flexibility using MD simulations, the root mean square fluctuation of the backbone nitrogen of the test residue was calculated on each simulation. Comparing each frame in the simulation, calculating the distance traveled by each nitrogen atom for each frame, squaring the distance for each frame, determining the average of the squared distance across all frames, and calculating the final value of the descriptor as the square root of the average of the squared distances.

In some embodiments, to determine the distance test residue δ carbon at using MD simulationTotal negative charge in, for each frame of each simulation, the delta carbon from the test residue was tracedThe total negative charge of all the atoms involved in the charged amino acid side chain is added together and the final value is calculated as the time average of this number over the duration of the simulation.

For example, in some embodiments, provided is a method of generating a random decision forest for predicting whether a test residue of a protein in a liquid formulation is susceptible to oxidation, comprising a) providing a training set comprising oxidation hotspot residues, wherein each residue is associated with i) values of a plurality of molecular descriptors of the residue and ii) whether the residue is susceptible to oxidation; and b) applying the exercise set to a random decision forest, thereby exercising the random decision forest to predict susceptibility to oxidation, wherein the number of individual decision trees in the random decision forest is at least about 20 (such as at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000 or more of any), the maximum number of variables to consider random selection at each branch of each decision tree in the random decision forest is at least about 1 (such as at least about 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more of any), and the maximum number of times that the observation set is divided into sub-branches of each tree is at least about 2 (such as at least about 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more). In some embodiments, the plurality of molecular descriptors includes a delta carbon from the test residueNumber of aspartic acid side chain oxygens in, side chain SASA (stdev), delta carbon from test residueTotal positive charge (stdev) in, main chain SASA (stdev), test residue side chain angle, delta carbon from test residueInner packing density, measurementTest residue backbone Angle (stdev), SASA of pseudo- π orbital, backbone flexibility, and δ carbon from test residueThe total negative charge in. In some embodiments, the plurality of molecular descriptors includes a delta carbon from the test residueNumber of aspartic acid side chain oxygens in, side chain SASA (stdev), delta carbon from test residueTotal positive charge (stdev) in (c), main chain sasa (stdev), and test residue side chain angle. In some embodiments, the plurality of molecular descriptors comprises between 2 and 11 molecular descriptors, such as any of 2,3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments, the molecular descriptor is determined based on the amino acid sequence of the protein comprising the test residue, such as the Fv region, when the protein is an antibody. In some embodiments, the molecular descriptor is determined by MD simulation using parameters of the protein in the liquid formulation in a computer. In some embodiments, the test residue and the oxidation hotspot residue are residues of the same amino acid (e.g., they are both tryptophan residues). In some embodiments, the test residue and the oxidation hotspot residue are tryptophan residues. In some embodiments, oxidation of at least about 30% (such as at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of any) of the residues in the oxidation assay is indicative of susceptibility to oxidation. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In some embodiments, the liquid formulation is an aqueous formulation.

In some embodiments, provided is a predictive liquid formulationA method of determining whether a test residue of a protein in a formulation is susceptible to oxidation, comprising a) determining the values of a plurality of molecular descriptors for the test residue; and b) applying the plurality of molecular descriptors of the test residue to a random decision forest trained on the plurality of molecular descriptors to predict susceptibility to oxidation, wherein majority voting of the random decision forest classifies the test residue as susceptible to oxidation. In some embodiments, the random decision forest is trained by providing a training set comprising oxidative hotspot residues, wherein each residue is associated with i) values of a plurality of molecular descriptors of the residue; and ii) whether the residue is susceptible to oxidation; and applying the exercise set to a random decision forest, thereby exercising the random decision forest to predict susceptibility to oxidation, wherein the number of decision tree individuals in the random decision forest is at least about 20 (such as at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000 or more of any), the maximum number of variables selected at random for consideration at each branch of each decision tree in the random decision forest is at least about 1 (such as at least about 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more of any), and the maximum number of times that the observation set is divided into sub-branches of each tree is at least about 2 (such as at least about 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more). In some embodiments, the plurality of molecular descriptors includes a delta carbon from the test residueNumber of aspartic acid side chain oxygens in, side chain SASA (stdev), delta carbon from test residueTotal positive charge (stdev) in, main chain SASA (stdev), test residue side chain angle, delta carbon from test residueInner packageDensity, test residue backbone Angle (stdev), SASA of pseudo-pi orbital, backbone flexibility, and delta carbon from test residueThe total negative charge in. In some embodiments, the plurality of molecular descriptors includes a delta carbon from the test residueNumber of aspartic acid side chain oxygens in, side chain SASA (stdev), delta carbon from test residueTotal positive charge (stdev) in (c), main chain sasa (stdev), and test residue side chain angle. In some embodiments, the plurality of molecular descriptors comprises between 2 and 11 molecular descriptors, such as any of 2,3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments, the molecular descriptor is determined based on the amino acid sequence of the protein comprising the test residue, such as the Fv region, when the protein is an antibody. In some embodiments, the value of the molecular descriptor is determined by MD simulation using parameters of the protein in the liquid formulation in a computer. In some embodiments, the test residue and the oxidation hotspot residue are residues of the same amino acid (e.g., they are both tryptophan residues). In some embodiments, the test residue and the oxidation hotspot residue are tryptophan residues. In some embodiments, oxidation of at least about 30% (such as at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of any) of the residues in the oxidation assay is indicative of susceptibility to oxidation. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In some embodiments, the liquid formulation is an aqueous formulation.

In some embodiments, provided is aA method of predicting whether a test tryptophan residue of a protein in a liquid formulation is susceptible to oxidation, comprising a) determining values for a plurality of molecular descriptors for the test tryptophan residue; and b) applying the plurality of molecular descriptors of the test tryptophan residue to a random decision forest trained on the plurality of molecular descriptors to predict susceptibility to oxidation, wherein majority voting of the random decision forest classifies the test tryptophan residue as susceptible to oxidation. In some embodiments, the random decision forest is trained by providing a training set comprising tryptophan oxidation hotspot residues, wherein each residue is associated with i) values of a plurality of molecular descriptors of the residue; and ii) whether the residue is susceptible to oxidation; and applying the exercise set to a random decision forest, thereby exercising the random decision forest to predict tryptophan oxidation susceptibility, wherein the number of decision tree individuals in the random decision forest is at least about 20 (such as at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000 or more of any), the maximum number of variables considered for random selection at each branch of each decision tree in the random decision forest is at least about 1 (such as at least about 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15 or more of any), and the maximum number of times that the observation set is divided into sub-branches of each tree is at least about 2 (such as at least about 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more). In some embodiments, the plurality of molecular descriptors comprises a delta carbon from tryptophanNumber of aspartic acid side chain oxygens in, side chain SASA (stdev), delta carbon from tryptophanTotal positive charge (stdev) in, main chain SASA (stdev), tryptophan side chain angle, delta carbon from tryptophanInternal packing density, tryptophan main chain angle (stdev), SASA for pseudo-pi orbitals, main chain flexibility, and delta carbon to tryptophanThe total negative charge in. In some embodiments, the plurality of molecular descriptors comprises a delta carbon from tryptophanNumber of aspartic acid side chain oxygens in, side chain SASA (stdev), delta carbon from tryptophanTotal positive charge (stdev) in (c), main chain sasa (stdev), and tryptophan side chain angle. In some embodiments, the plurality of molecular descriptors comprises between 2 and 11 molecular descriptors, such as any of 2,3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments, the molecular descriptor is determined based on the amino acid sequence of the protein comprising the test tryptophan, such as the Fv region, when the protein is an antibody. In some embodiments, the value of the molecular descriptor is determined by MD simulation using parameters of the protein in the liquid formulation in a computer. In some embodiments, oxidation of at least about 30% (such as at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of any) of the residues in the oxidation assay is indicative of susceptibility to oxidation. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In some embodiments, the liquid formulation is an aqueous formulation.

In some embodiments, provided is a method of determining whether a protein comprising tryptophan residues in a liquid formulation is susceptible to oxidation, comprising a) determining a plurality of molecular profiles for each tryptophan residue in the proteinThe value of the character; and b) applying the plurality of molecular descriptors to a random decision forest trained on the plurality of molecular descriptors to predict susceptibility to oxidation, wherein a majority vote of the random decision forest for each tryptophan residue classifies that residue as susceptible to oxidation, and wherein the protein is determined to comprise tryptophan residues susceptible to oxidation if the random decision forest classifies at least one tryptophan residue as susceptible to oxidation. In some embodiments, the random decision forest is trained by providing a training set comprising tryptophan oxidation hotspot residues, wherein each residue is associated with i) values of a plurality of molecular descriptors of the residue; and ii) whether the residue is susceptible to oxidation; and applying the exercise set to a random decision forest, thereby exercising the random decision forest to predict tryptophan oxidation susceptibility, wherein the number of decision tree individuals in the random decision forest is at least about 20 (such as at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000 or more of any), the maximum number of variables considered for random selection at each branch of each decision tree in the random decision forest is at least about 1 (such as at least about 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15 or more of any), and the maximum number of times that the observation set is divided into sub-branches of each tree is at least about 2 (such as at least about 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more). In some embodiments, the plurality of molecular descriptors comprises a delta carbon from tryptophanNumber of aspartic acid side chain oxygens in, side chain SASA (stdev), delta carbon from tryptophanTotal positive charge (stdev) in, main chain SASA (stdev), tryptophan side chain angle, delta carbon from tryptophanInternal packing density, tryptophan main chain angle (stdev), SASA for pseudo-pi orbitals, main chain flexibility, and delta carbon to tryptophanThe total negative charge in. In some embodiments, the plurality of molecular descriptors comprises a delta carbon from tryptophanNumber of aspartic acid side chain oxygens in, side chain SASA (stdev), delta carbon from tryptophanTotal positive charge (stdev) in (c), main chain sasa (stdev), and tryptophan side chain angle. In some embodiments, the plurality of molecular descriptors comprises between 2 and 11 molecular descriptors, such as any of 2,3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments, the molecular descriptor for each tryptophan residue is determined based on the amino acid sequence of the protein comprising the tryptophan residue, such as the Fv region, when the protein is an antibody. In some embodiments, the value of the molecular descriptor is determined by MD simulation using parameters of the protein in the liquid formulation in a computer. In some embodiments, oxidation of at least about 30% (such as at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of any) of the residues in the oxidation assay is indicative of susceptibility to oxidation. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In some embodiments, the liquid formulation is an aqueous formulation.

Method for reducing oxidation

The invention herein also provides a method of reducing oxidation of a protein in a liquid formulation comprising addingAn amount of NAT that prevents oxidation of proteins in the liquid formulation. In some embodiments, the protein is susceptible to oxidation. In some embodiments, methionine, cysteine, histidine, tryptophan, and/or tyrosine in the protein is susceptible to oxidation. In some embodiments, the tryptophan in the protein is susceptible to oxidation. In some embodiments, the protein comprises at least one Solvent Accessibility Surface Area (SASA) greater than aboutTo about(such as greater than about 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, orAny, including any range between these values) of tryptophan residues. In some embodiments, the SASA is greater than aboutIn some embodiments, the protein comprises greater than about 15% to about 45% (such as greater than about any of 15, 20, 25, 30, 35, 40, or 45%) tryptophan residues for at least one SASA. In some embodiments, the SASA is greater than about 30%. In some embodiments, the SASA of tryptophan residues is measured at a pH ranging from about 4.0 to about 8.5. In some embodiments, the SASA of tryptophan residues is measured at a temperature ranging from about 5 ℃ to about 40 ℃. In some embodiments, the SASA of tryptophan residues is measured at a salt concentration ranging from about 0mM to about 500 mM. In some embodiments, the SASA of tryptophan residues is measured at a pH of about 5.0 to about 7.5, a temperature of about 5 ℃ to about 25 ℃, and a salt concentration of about 0mM to about 200 mM. In some embodiments, the SASA is computer-determined by full atomic molecular dynamics simulation. In some embodiments, the protein comprises at least one molecular descriptor mimicking the tryptophan residue by susceptibility to oxidation at the tryptophan residue based on MDIn association with the exercise of machine learning algorithms to predict tryptophan residues susceptible to oxidation. In some embodiments, the amount of NAT added to the formulation is from about 0.1mM to about 10mM (such as any of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0mM, including any range between these values), or up to the highest concentration at which the NAT is soluble in the formulation. In some embodiments, the amount of NAT added to the formulation is about 1 mM. In some embodiments, the NAT prevents oxidation of one or more tryptophan amino acids in the protein. In some embodiments, the oxidation of the protein is reduced by about 40% to about 100% (such as by about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of either, including any range between these values). In some embodiments, no more than about 40% to about 0% (such as no more than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0%, including any range between these values) of the protein is oxidized. In some embodiments, the NAT prevents oxidation of the protein by Reactive Oxygen Species (ROS). In yet another embodiment, the reactive oxygen species is selected from the group consisting of singlet oxygen, superoxide (O)₂-, alkoxy radical, peroxy radical, hydrogen peroxide (H)₂O₂) Dihydrogen trioxide (H)₂O₃) Radical of hydrogen trioxide (HO)₃H), ozone (O)₃) Hydroxyl radicals, and alkyl peroxides. In some embodiments, the protein (e.g., antibody) concentration in the formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody or antibody fragment. In some embodiments, the formulation further comprises one or more agents selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agentsGroup of excipients. For example, a formulation of the invention may comprise a monoclonal antibody, NAT provided herein to prevent protein oxidation, and a buffer to maintain the pH of the formulation at a desired level. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the formulation is aqueous. In some embodiments, the formulation further comprises at least one additional protein in accordance with any of the proteins described herein (e.g., the formulation is a co-formulation comprising two or more proteins).

In some embodiments, provided is a method of reducing protein oxidation in a liquid formulation comprising adding an amount of NAT to the formulation that prevents protein oxidation, wherein the protein comprises at least one SASA greater than aboutTo about(such as greater than about 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, orAny, including any range between these values) of tryptophan residues. In some embodiments, the protein comprises at least one SASA greater than aboutA tryptophan residue of (a). In some embodiments, the SASA is computer-determined by full atomic molecular dynamics simulation. In some embodiments, the amount of NAT added to the formulation is from about 0.1mM to about 10mM (such as any of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0mM, including any range between these values), or up to the highest concentration of NAT that is soluble in the formulation. In some embodiments, the amount of NAT added to the formulation is about 1 mM. In some casesIn embodiments, the NAT prevents oxidation of one or more tryptophan amino acids in the protein. In some embodiments, the oxidation of the protein is reduced by about 40% to about 100% (such as about any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, including any range between these values). In some embodiments, no more than about 40% to about 0% (such as no more than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0%, including any range between these values) of the protein is oxidized. In some embodiments, the NAT prevents oxidation of the protein by Reactive Oxygen Species (ROS). In some embodiments, the protein (e.g., antibody) concentration in the formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody or antibody fragment. In some embodiments, the antibody is derived from an IgG1 antibody sequence. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the liquid formulation is an aqueous formulation. In some embodiments, the formulation further comprises at least one additional protein according to any of the proteins described herein.

In some embodiments, provided is a method of reducing protein oxidation in a liquid formulation comprising determining the SASA value of tryptophan residues in the protein and if at least one tryptophan residue has a SASA value greater than aboutTo about(such as greater than about 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, orEither, including any range between these values), an amount of NAT that prevents protein oxidation is added to the formulation. In some embodiments, the protein comprises at least one SASA greater than aboutA tryptophan residue of (a). In some embodiments, the SASA is computer-determined by full atomic molecular dynamics simulation. In some embodiments, the amount of NAT added to the formulation is from about 0.1mM to about 10mM (such as any of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0mM, including any range between these values), or up to the highest concentration at which the NAT is soluble in the formulation. In some embodiments, the amount of NAT added to the formulation is about 1 mM. In some embodiments, the NAT prevents oxidation of one or more tryptophan amino acids in the protein. In some embodiments, the oxidation of the protein is reduced by about 40% to about 100% (such as about any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, including any range between these values). In some embodiments, no more than about 40% to about 0% (such as no more than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0%, including any range between these values) of the protein is oxidized. In some embodiments, the NAT prevents oxidation of the protein by Reactive Oxygen Species (ROS). In some embodiments, the protein (e.g., antibody) concentration in the formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antibody. In some embodiments of the present invention, the substrate is,the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody or an antibody fragment. In some embodiments, the antibody is derived from an IgG1 antibody sequence. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the liquid formulation is an aqueous formulation. In some embodiments, the formulation further comprises at least one additional protein according to any of the proteins described herein.

In some embodiments, provided is a method of reducing protein oxidation in a liquid formulation comprising determining the SASA value of a tryptophan residue in the protein and based on having a color of greater than aboutTo about(such as greater than about 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, orEither, including any range between these values) of tryptophan residues of the SASA, adding an amount of NAT to the formulation, wherein the amount of NAT added to the formulation prevents oxidation of the protein. In some embodiments, the SASA is computer-determined by full atomic molecular dynamics simulation. In some embodiments, the amount of NAT added to the formulation is from about 0.1mM to about 10mM (such as about any of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0mM, including any range between these values). In some embodiments, to theThe amount of NAT in the formulation is about 1 mM. In some embodiments, the NAT prevents oxidation of one or more tryptophan amino acids in the protein. In some embodiments, the protein has more than one SASA greater than(or greater than 30%) tryptophan residues and a sufficient amount of NAT added to prevent oxidation of each tryptophan residue. In some embodiments, the oxidation of the protein is reduced by about 40% to about 100% (such as about any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, including any range between these values). In some embodiments, no more than about 40% to about 0% (such as no more than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0%, including any range between these values) of the protein is oxidized. In some embodiments, the NAT prevents oxidation of the protein by Reactive Oxygen Species (ROS). In some embodiments, the protein (e.g., antibody) concentration in the formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody or antibody fragment. In some embodiments, the antibody is derived from an IgG1 antibody sequence. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the liquid formulation is an aqueous formulation. In some embodiments, the formulation further comprises at least one additional protein according to any of the proteins described herein.

In some embodiments, provided is a method of reducing protein oxidation in a liquid formulation, comprising adding an amount of NAT that prevents protein oxidation to the formulation, wherein the protein comprises greater than about 15% to about 45% (such as greater than about any of 15, 20, 25, 30, 35, 40, or 45%) tryptophan residues for at least one SASA. In some embodiments, the protein comprises greater than about 30% tryptophan residues for at least one SASA. In some embodiments, the SASA is computer-determined by full atomic molecular dynamics simulation. In some embodiments, the amount of NAT added to the formulation is from about 0.1mM to about 10mM (such as any of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0mM, including any range between these values), or up to the highest concentration of NAT that is soluble in the formulation. In some embodiments, the amount of NAT added to the formulation is about 1 mM. In some embodiments, the NAT prevents oxidation of one or more tryptophan amino acids in the protein. In some embodiments, the oxidation of the protein is reduced by about 40% to about 100% (such as about any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, including any range between these values). In some embodiments, no more than about 40% to about 0% (such as no more than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0%, including any range between these values) of the protein is oxidized. In some embodiments, the NAT prevents oxidation of the protein by Reactive Oxygen Species (ROS). In some embodiments, the protein (e.g., antibody) concentration in the formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody or antibody fragment. In some embodiments, the antibody is derived from an IgG1 antibody sequence. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the liquid formulation is an aqueous formulation. In some embodiments, the formulation further comprises at least one additional protein according to any of the proteins described herein.

In some embodiments, provided is a method of reducing protein oxidation in a liquid formulation comprising determining the SASA value of tryptophan residues in the protein and adding an amount of NAT that prevents protein oxidation to the formulation if at least one tryptophan residue has a SASA of greater than about 15% to about 45% (such as greater than any of about 15, 20, 25, 30, 35, 40, or 45%). In some embodiments, the protein comprises greater than about 30% tryptophan residues for at least one SASA. In some embodiments, the SASA is computer-determined by full atomic molecular dynamics simulation. In some embodiments, the amount of NAT added to the formulation is from about 0.1mM to about 10mM (such as any of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0mM, including any range between these values), or up to the highest concentration of NAT that is soluble in the formulation. In some embodiments, the amount of NAT added to the formulation is about 1 mM. In some embodiments, the NAT prevents oxidation of one or more tryptophan amino acids in the protein. In some embodiments, the oxidation of the protein is reduced by about 40% to about 100% (such as about any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, including any range between these values). In some embodiments, no more than about 40% to about 0% (such as no more than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0%, including any range between these values) of the protein is oxidized. In some embodiments, the NAT prevents oxidation of the protein by Reactive Oxygen Species (ROS). In some embodiments, the protein (e.g., antibody) concentration in the formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody or antibody fragment. In some embodiments, the antibody is derived from an IgG1 antibody sequence. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the liquid formulation is an aqueous formulation. In some embodiments, the formulation further comprises at least one additional protein according to any of the proteins described herein.

In some embodiments, provided is a method of reducing protein oxidation in a liquid formulation, comprising determining an SASA value of tryptophan residues in the protein and adding an amount of NAT to the formulation based on the number of tryptophan residues having an SASA of greater than about 15% to about 45% (such as greater than about any of 15, 20, 25, 30, 35, 40, or 45%), wherein the amount of NAT added to the formulation prevents oxidation of the protein. In some embodiments, the SASA is computer-determined by full atomic molecular dynamics simulation. In some embodiments, the amount of NAT added to the formulation is from about 0.1mM to about 10mM (such as about any of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0mM, including any range between these values). In some embodiments, the amount of NAT added to the formulation is about 1 mM. In some embodiments, the NAT prevents oxidation of one or more tryptophan amino acids in the protein. In some embodiments, the oxidation of the protein is reduced by about 40% to about 100% (such as about any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, including any range between these values). In some embodiments, no more than about 40% to about 0% (such as no more than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0%, including any range between these values) of the protein is oxidized. In some embodiments, the NAT prevents oxidation of the protein by Reactive Oxygen Species (ROS). In some embodiments, the protein (e.g., antibody) concentration in the formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody or antibody fragment. In some embodiments, the antibody is derived from an IgG1 antibody sequence. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the liquid formulation is an aqueous formulation. In some embodiments, the formulation further comprises at least one additional protein according to any of the proteins described herein.

SASA can be calculated using a computer full atom molecular dynamics simulation method, which is described in shara, v.

In some embodiments, provided is a method of reducing oxidation of a protein in a liquid formulation, comprising adding to the formulation an amount of an antioxidant that prevents oxidation of the protein, wherein the protein comprises at least one tryptophan residue that is predicted to be susceptible to oxidation by a machine learning algorithm practiced in connection with a tryptophan residue oxidation susceptibility and a plurality of molecular descriptors for the tryptophan residue based on MD simulation. In some embodiments, the antioxidant is N-acetyl tryptophan (NAT). In some embodiments, the amount of NAT added to the formulation is from about 0From 1mM to about 10mM (such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0mM, including any range therebetween), or up to the highest concentration at which the NAT is soluble in the formulation. In some embodiments, the amount of NAT added to the formulation is about 1 mM. In some embodiments, the NAT prevents oxidation of one or more tryptophan amino acids in the protein. In some embodiments, the oxidation of the protein is reduced by about 40% to about 100% (such as about any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, including any range between these values). In some embodiments, no more than about 40% to about 0% (such as no more than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0%, including any range between these values) of the protein is oxidized. In some embodiments, the NAT prevents oxidation of the protein by Reactive Oxygen Species (ROS). In some embodiments, the protein (e.g., antibody) concentration in the formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody or antibody fragment. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the liquid formulation is an aqueous formulation. In some embodiments, the formulation further comprises at least one additional protein according to any of the proteins described herein. In some embodiments, the machine learning algorithm is a random decision forest according to any of the random decision forests described above. In some embodiments, the random decision forest is a forest of at least about 20 (such at least about 20, 30, 40, 50, 60,70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000, or more any) estimates, at least about 1 (such as at least about 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more any) features, and at least about 2 (such as at least about 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more any) tree depth exercises. In some embodiments, the plurality of molecular descriptors comprises a delta carbon from tryptophanNumber of aspartic acid side chain oxygens in, side chain SASA (stdev), delta carbon from tryptophanTotal positive charge (stdev) in, main chain SASA (stdev), tryptophan side chain angle, delta carbon from tryptophanInternal packing density, tryptophan main chain angle (stdev), SASA for pseudo-pi orbitals, main chain flexibility, and delta carbon to tryptophanThe total negative charge in. In some embodiments, the plurality of molecular descriptors comprises between 2 and 11 molecular descriptors, such as any of 2,3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments, the value of the tryptophan molecule descriptor is determined by MD simulation using parameters of the protein in the liquid formulation in a computer. In some embodiments, oxidation of at least about 30% (such as at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of any) of the residues in the oxidation assay is indicative of susceptibility to oxidation.

In some embodiments, provided is a method of reducing protein oxidation in a liquid formulation comprising adding an amount of a protein inhibitor to the liquid formulationOxidized NAT is added to the formulation, wherein the protein comprises at least one tryptophan residue predicted to be susceptible to oxidation by a machine learning algorithm practiced in association with the tryptophan residue oxidation susceptibility and molecular descriptors of the tryptophan residue based on MD simulation. In some embodiments, the amount of NAT added to the formulation is from about 0.1mM to about 10mM (such as any of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0mM, including any range between these values), or up to the highest concentration of NAT that is soluble in the formulation. In some embodiments, the amount of NAT added to the formulation is about 1 mM. In some embodiments, the NAT prevents oxidation of one or more tryptophan amino acids in the protein. In some embodiments, the oxidation of the protein is reduced by about 40% to about 100% (such as about any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, including any range between these values). In some embodiments, no more than about 40% to about 0% (such as no more than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0%, including any range between these values) of the protein is oxidized. In some embodiments, the NAT prevents oxidation of the protein by Reactive Oxygen Species (ROS). In some embodiments, the protein (e.g., antibody) concentration in the formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody or antibody fragment. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the liquid formulation is an aqueous formulation. In some embodiments, the formulationThe agent further comprises at least one additional protein according to any of the proteins described herein. In some embodiments, the machine learning algorithm is a random decision forest according to any of the random decision forests described above. In some embodiments, the random decision forest is practiced with at least about 20 (such at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000, or more) estimates, at least about 1 (such as at least about 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more) features, and at least about 2 (such as at least about 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more) tree depths. In some embodiments, the plurality of molecular descriptors comprises a delta carbon from tryptophanNumber of aspartic acid side chain oxygens in, side chain SASA (stdev), delta carbon from tryptophanTotal positive charge (stdev) in, main chain SASA (stdev), tryptophan side chain angle, delta carbon from tryptophanInternal packing density, tryptophan main chain angle (stdev), SASA for pseudo-pi orbitals, main chain flexibility, and delta carbon to tryptophanThe total negative charge in. In some embodiments, the plurality of molecular descriptors comprises between 2 and 11 molecular descriptors, such as any of 2,3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments, the value of the tryptophan molecule descriptor is determined by MD simulation using parameters of the protein in the liquid formulation in a computer. In some embodiments, at least about 30% (such as at least about 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) of the residue oxidation indicates susceptibility to oxidation.

In some embodiments, provided is a method of reducing protein oxidation in a liquid formulation comprising adding an amount of NAT to the formulation based on predicting the number of tryptophan residues susceptible to oxidation by a machine learning algorithm practiced in association with a tryptophan residue oxidation susceptibility and a plurality of molecular descriptors of the tryptophan residue based on MD simulation. In some embodiments, the amount of NAT added to the formulation is from about 0.1mM to about 10mM (such as any of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0mM, including any range between these values), or up to the highest concentration of NAT that is soluble in the formulation. In some embodiments, the amount of NAT added to the formulation is about 1 mM. In some embodiments, the NAT prevents oxidation of one or more tryptophan amino acids in the protein. In some embodiments, the oxidation of the protein is reduced by about 40% to about 100% (such as about any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, including any range between these values). In some embodiments, no more than about 40% to about 0% (such as no more than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0%, including any range between these values) of the protein is oxidized. In some embodiments, the NAT prevents oxidation of the protein by Reactive Oxygen Species (ROS). In some embodiments, the protein (e.g., antibody) concentration in the formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody or antibody fragment. In some embodiments, the formulation further comprises aOne or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the liquid formulation is an aqueous formulation. In some embodiments, the formulation further comprises at least one additional protein according to any of the proteins described herein. In some embodiments, the machine learning algorithm is a random decision forest according to any of the random decision forests described above. In some embodiments, the random decision forest is practiced with at least about 20 (such at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000, or more) estimates, at least about 1 (such as at least about any one of 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more) features, and at least about 2 (such as at least about any one of 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more) tree depths. In some embodiments, the plurality of molecular descriptors comprises a delta carbon from tryptophanNumber of aspartic acid side chain oxygens in, side chain SASA (stdev), delta carbon from tryptophanTotal positive charge (stdev) in, main chain SASA (stdev), tryptophan side chain angle, delta carbon from tryptophanInternal packing density, tryptophan main chain angle (stdev), SASA for pseudo-pi orbitals, main chain flexibility, and delta carbon to tryptophanThe total negative charge in. In some embodiments, the plurality of molecular descriptors comprises between 2 and 11 (such as any of 2,3, 4, 5, 6, 7, 8, 9, 10, or 11)A molecular descriptor. In some embodiments, the value of the tryptophan molecule descriptor is determined by MD simulation using parameters of the protein in the liquid formulation in a computer. In some embodiments, oxidation of at least about 30% (such as at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of any) of the residues in the oxidation assay is indicative of susceptibility to oxidation.

In some embodiments, provided is a method of reducing protein oxidation in a liquid formulation comprising introducing an amino acid substitution in the protein to replace one or more tryptophan residues predicted to be susceptible to oxidation with an amino acid residue that is not subject to oxidation, wherein the prediction is by a machine learning algorithm practiced in association with the tryptophan residue oxidation susceptibility and the tryptophan residue based on MD simulated multiple molecular descriptors. In some embodiments, each of the one or more tryptophan residues is replaced with a residue independently selected from the group consisting of tyrosine, phenylalanine, leucine, isoleucine, alanine, and valine. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody or antibody fragment. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the liquid formulation is an aqueous formulation. In some embodiments, the formulation further comprises at least one additional protein according to any of the proteins described herein. In some embodiments, the machine learning algorithm is a random decision forest according to any of the random decision forests described above. In some embodiments, the random decision forest is a forest of at least about 20 (such at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400,500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000, or more any) estimates, at least about 1 (such as at least about 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more any) features, and at least about 2 (such as at least about 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more any) tree depth exercises. In some embodiments, the plurality of molecular descriptors comprises a delta carbon from tryptophanNumber of aspartic acid side chain oxygens in, side chain SASA (stdev), delta carbon from tryptophanTotal positive charge (stdev) in, main chain SASA (stdev), tryptophan side chain angle, delta carbon from tryptophanInternal packing density, tryptophan main chain angle (stdev), SASA for pseudo-pi orbitals, main chain flexibility, and delta carbon to tryptophanThe total negative charge in. In some embodiments, the plurality of molecular descriptors comprises between 2 and 11 molecular descriptors, such as any of 2,3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments, the value of the tryptophan molecule descriptor is determined by MD simulation using parameters of the protein in the liquid formulation in a computer. In some embodiments, oxidation of at least about 30% (such as at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of any) of the residues in the oxidation assay is indicative of susceptibility to oxidation.

V. screening method

The invention herein also provides a method of screening a liquid formulation for reduced protein oxidation. In some embodiments, the proteinIs susceptible to oxidation. In some embodiments, methionine, cysteine, histidine, tryptophan, and/or tyrosine in the protein is susceptible to oxidation. In some embodiments, the tryptophan in the protein is susceptible to oxidation. In some embodiments, the protein comprises at least one Solvent Accessibility Surface Area (SASA) greater than aboutTo about(such as greater than about 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, orAny, including any range between these values) of tryptophan residues. In some embodiments, the SASA is greater than aboutIn some embodiments, the protein comprises greater than about 15% to about 45% (such as greater than about any of 15, 20, 25, 30, 35, 40, or 45%) tryptophan residues for at least one SASA. In some embodiments, the SASA is greater than about 30%. In some embodiments, the tryptophan in the protein predicts susceptibility to oxidation by a machine learning algorithm practiced in association with a tryptophan residue oxidation susceptibility to a plurality of molecular descriptors of the tryptophan residue based on MD simulation. In some embodiments, the oxidation of the protein is reduced by about 40% to about 100% (such as about any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, including any range between these values). In some embodiments, no more than about 40% to about 0% (such as no more than about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0%, including any range between these values) of the protein is oxidized. In some embodiments, the protein(s) in the formulationE.g., antibody) concentration is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody or antibody fragment. In some embodiments, the antibody is derived from an IgG1 antibody sequence. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the liquid formulation is an aqueous formulation.

In some embodiments, provided is a method of screening a liquid formulation for reduced protein oxidation, wherein the protein comprises at least one i) SASA of greater than aboutTo about(such as greater than about 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, orAny, including any range between these values); or ii) tryptophan residues having a SASA of greater than about 15% to about 45% (such as greater than about 15, 20, 25, 30, 35, 40, or 45% of any), the method comprising a) adding from about 0.1mM to about 10mM (such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0mM of any, including any range therebetween) of N-acetyl-tryptophan (NAT) to a liquid formulation comprising the protein, b) adding from about 0.1mM to about 10mM (such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.0.0.0, 0.0.5, 0.0.0.0, 0.5, 0.6, 0.0.0.8, 0.9, 0.0.0, or any range between these values or 2mM of azo-2, including any range therebetween of any of two(2-aminopropane) dihydrochloride (AAPH) is added to the liquid formulation, c) the liquid formulation comprising the protein, NAT and AAPH is added to the liquid formulation at a temperature of from about 35 ℃ to about 45 ℃ (such as about 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 ℃, including any range therebetween) for about 10 hours to about 20 hours (such as about 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 hours, including any range between these values), and d) measuring the oxidation of tryptophan residues in the protein for the protein, in which NAT is included in an amount that results in no more than about 20% (such as no more than about 20, 15, 10, 5, 4, 3, 2, or 1% of either, including any range between these values) oxidized liquid formulations are suitable formulations for reducing the oxidation of the protein. In some embodiments, the SASA is computer-determined by full atomic molecular dynamics simulation. In some embodiments, the protein (e.g., antibody) concentration in the liquid formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In some embodiments, the antibody is derived from an IgG1 antibody sequence. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the liquid formulation is an aqueous formulation.

In some embodiments, provided is a method of screening a liquid formulation for reduced oxidation of a protein comprising a) determining the SASA value of a tryptophan residue in the protein, b) based on having i) greater than aboutTo about(such as greater than about 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, orAny, including any range between these values); or ii) a number of tryptophan residues of the SASA greater than about 15% to about 45% (such as greater than about 15, 20, 25, 30, 35, 40, or 45% of any) adding an amount of NAT to the liquid formulation, c) adding 2,2' -azobis (2-aminopropane) dihydrochloride (AAPH) from about 0.1mM to about 10mM (such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0mM of any of these values, including any range therebetween) to the liquid formulation, d) incubating the liquid formulation comprising the protein, N-acetyl-tryptophan and AAPH at about 35 ℃ to about 45 ℃ such as about 35, 36, 37, 38, 39, 40, or 42 ℃, including any range between these values for about 10 hours (such as about 10 hours), 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 hours, including any range between these values), and e) measuring oxidation of tryptophan residues in the protein for the protein, wherein NAT is included in an amount that results in oxidation of no more than about 20% (such as no more than about any of 20, 15, 10, 5, 4, 3, 2, or 1%) of tryptophan residues in the protein, is a suitable formulation for reducing oxidation of the protein. In some embodiments, the SASA is computer-determined by full atomic molecular dynamics simulation. In some embodiments, the protein (e.g., antibody) concentration in the liquid formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In some embodiments, the antibody is derived from an IgG1 antibody sequence. In some embodiments, theThe formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the liquid formulation is an aqueous formulation.

In some embodiments, provided is a method of screening a liquid formulation for reduced oxidation of a protein, wherein the protein comprises at least one tryptophan residue that is predicted to be susceptible to oxidation by a machine learning algorithm practiced in association with a tryptophan residue oxidation susceptibility and a plurality of molecular descriptors based on MD simulation, the method comprising a) adding from about 0.1mM to about 10mM (such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0mM, including any range between these values) of N-acetyl-tryptophan (NAT) to the liquid formulation comprising the protein, b) adding from about 0.1mM to about 10mM (such as about 0.1, 0.2, 0.0, 0.5.0, 0, 0.0, 9.0, or 10.0mM, including any range between these values), 6.0, 7.0, 8.0, 9.0, or 10.0mM, including any range between these values) of 2,2' -azobis (2-aminopropane) dihydrochloride (AAPH) is added to the liquid formulation, c) the liquid formulation comprising the protein, NAT and AAPH is incubated at about 35 ℃ to about 45 ℃ (such as about 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 ℃ any, including any range between these values) for about 10 hours to about 20 hours (such as about 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 hours any, including any range between these values), and d) the oxidation of tryptophan residues in the protein is measured for the protein, wherein NAT is included in an amount that results in about no more than 20% tryptophan residues in the protein (such as about no more than 20, 15, 10, 5, 4, 3, 2, or 1%, including any range between these values) oxidized liquid formulations are suitable formulations for reducing the oxidation of the protein. In some embodiments, the protein (e.g., antibody) concentration in the liquid formulation is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein is a polypeptideA therapeutic protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the liquid formulation is an aqueous formulation. In some embodiments, the machine learning algorithm is a random decision forest according to any of the random decision forests described above. In some embodiments, the random decision forest is practiced with at least about 20 (such at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000, or more) estimates, at least about 1 (such as at least about 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more) features, and at least about 2 (such as at least about 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more) tree depths. In some embodiments, the plurality of molecular descriptors comprises a delta carbon from tryptophanNumber of aspartic acid side chain oxygens in, side chain SASA (stdev), delta carbon from tryptophanTotal positive charge (stdev) in, main chain SASA (stdev), tryptophan side chain angle, delta carbon from tryptophanInternal packing density, tryptophan main chain angle (stdev), SASA for pseudo-pi orbitals, main chain flexibility, and delta carbon to tryptophanThe total negative charge in. In some embodiments, the plurality of molecular descriptors comprises between 2 and 11 molecular descriptors, such as any of 2,3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments, the value of the tryptophan molecule descriptor is determined by MD simulation using parameters of the protein in the liquid formulation in a computer. In some embodiments, oxidation of at least about 30% (such as at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of any) of the residues in the oxidation assay is indicative of susceptibility to oxidation.

Administration of protein formulations

The liquid formulation is administered to a mammal, preferably a human, in need of treatment with a protein (e.g., an antibody) according to known methods, such as intravenous administration, as a bolus injection or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, inhalation, or intravitreal route. In one embodiment, the liquid formulation is administered to the mammal by intravenous administration. For such purposes, the formulation may be injected, for example, using a syringe or via an IV line. In one embodiment, the liquid formulation is administered to the mammal by subcutaneous administration. In yet another embodiment, the liquid formulation is administered by intravitreal administration.

An appropriate dose of the protein ("therapeutically effective amount") will depend upon, for example, the condition being treated, the severity and course of the condition, whether the protein is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the protein, the type of protein used, and the discretion of the attending physician. The protein is suitably administered to the patient at one time or over a series of treatments, and may be administered to the patient at any time after diagnosis. The protein may be administered as the sole therapy or in combination with other drugs or therapies useful in treating the condition in question. As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. As used herein, "disorder" refers to any condition that would benefit from treatment, including but not limited to chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the disorder in question.

In a pharmacological sense, in the context of the present invention, a "therapeutically effective amount" of a protein (e.g., an antibody) refers to an amount effective to prevent or treat the disorder for which the antibody is effective. In some embodiments, a therapeutically effective amount of protein administered will be in the range of about 0.1 to about 50mg/kg (such as about 0.3 to about 20mg/kg, or about 0.3 to about 15mg/kg) of the patient's body weight, whether by one or more administrations. In some embodiments, a therapeutically effective amount of the protein is administered as a daily dose, or as multiple daily doses. In some embodiments, the therapeutically effective amount of the protein is administered less frequently than daily, such as weekly or monthly. For example, the protein may be administered at a dose of about 100 to about 400mg (such as about 100, 150, 200, 250, 300, 350, or 400mg, including any range between these values) per one or more weeks (such as every 1,2,3, or 4 weeks or more, or every 1,2,3, 4, 5, or 6 months or more), or about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 15.0, or 20.0mg/kg per one or more weeks (such as every 1,2,3, or 4 weeks or more, or every 1,2,3, 4, 5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 15.0, or 20.0 mg/kg). The dose may be administered as a single dose or as multiple doses (e.g., 2,3, 4, or more doses), such as infusion. The progress of this therapy is readily monitored by conventional techniques.

Method for measuring NAT degradation

The invention herein also provides a method of screening a liquid formulation for reduced protein oxidation. In order to effectively protect the proteins in the formulation, NAT in the formulation must outcompete the susceptible Trp residues for sacrificial oxidation; thus, NAT degradation can be expected to form during handling and storage of NAT-containing drug products. It is important to know the rate (rate) and degradation pathways of NAT because degraded NAT species present in the drug product will be administered to the patient along with the therapeutic protein. One report in the literature on NAT degradation uses a two-dimensional size exclusion chromatography trapping approach, together with a multiplex reaction monitoring LC-MS approach, to identify and quantify the two NAT degradants (N-Ac-PIC, 2b, and N-Ac-3a,8 a-dihydroxy-PIC, 3b) observed in concentrated HSA solutions after prolonged storage at elevated temperatures (Fang, l., et al, J Chromatogr a,2011,1218(41): 7316-24). The degradation of Trp itself has been more fully studied (Ji, J.A., et al, J Pharm Sci,2009, 98(12): 4485-.

In some aspects, the invention provides a method for measuring degradation of N-acetyl tryptophan (NAT) in a composition comprising N-acetyl tryptophan, the method comprising a) applying the composition to a reverse phase chromatography material, wherein the composition is loaded onto the chromatography material equilibrated in a solution comprising mobile phase a and mobile phase B, wherein mobile phase a comprises an acid in water and mobile phase B comprises an acid in an organic solvent, B) eluting the composition from the reverse phase chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is elevated compared to step a), wherein NAT degradant elutes from the chromatography separately from intact NAT, c) quantifying the NAT degradant and the intact NAT. In various embodiments, the ratio of mobile phase B to mobile phase a in step a) is about any of 1:99, 2:98, 3:97, 4:96, 5:95, 6:94, 7:93, 8:92, 9:91, or 10: 90. In various embodiments, the ratio of mobile phase B to mobile phase a in step a) is about 2: 98. In some embodiments, the ratio of mobile phase B to mobile phase a in step B) increases linearly. In other embodiments, the ratio of mobile phase B to mobile phase a in step B) is increased stepwise. In some embodiments, the organic solvent is acetonitrile.

In some aspects, the invention provides a method for measuring degradation of N-acetyl tryptophan (NAT) in a composition comprising N-acetyl tryptophan, the method comprising a) applying the composition to a reverse phase chromatography material, wherein the composition is loaded onto the chromatography material equilibrated in a solution comprising mobile phase a and mobile phase B, wherein mobile phase a comprises an acid in water and mobile phase B comprises an acid in acetonitrile, B) eluting the composition from the reverse phase chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is elevated compared to step a), wherein NAT degradant elutes from the chromatography separately from intact NAT, c) quantifying the NAT degradant and the intact NAT. In various embodiments, the ratio of mobile phase B to mobile phase a in step a) is about any of 1:99, 2:98, 3:97, 4:96, 5:95, 6:94, 7:93, 8:92, 9:91, or 10: 90. In various embodiments, the ratio of mobile phase B to mobile phase a in step a) is about 2: 98. In some embodiments, the ratio of mobile phase B to mobile phase a in step B) increases linearly. In other embodiments, the ratio of mobile phase B to mobile phase a in step B) is increased stepwise.

In some embodiments, the flow rate for the chromatography is any of about 0.25 mL/min, 0.5 mL/min, 0.75 mL/min, 1.0mL/min, 1.25 mL/min, 1.5 mL/min, 1.75 mL/min, 2.0 mL/min, or 2.5 mL/min. In some embodiments, the flow rate for the chromatography is about 1.0 mL/min.

In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about any of 25:75, 28:72, 30:70, 32:68, or 35: 65. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about 30: 70. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to any of about 25:75, 28:72, 30:70, 32:68, or 35:65 in any of about 14, 15, 16, 17, or 18 minutes from the start of chromatography. In some embodiments, the ratio of mobile phase B to mobile phase a increases to any one of about 25:75, 28:72, 30:70, 32:68, or 35:65 in about 16 minutes from the start of chromatography. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about 30: 70. In some embodiments, the ratio of mobile phase B to mobile phase a increases to about 30:70 in about 16 minutes from the start of chromatography. In some embodiments, the flow rate is about 1 mL/min.

In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about any of 85:15, 90:10, or 95: 5. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about 30: 70. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to any of about 85:15, 90:10, or 95:5 in any of about 16, 17, 18, 19, or 20 minutes from the start of chromatography. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to any of about 85:15, 90:10, or 95:5 in about 18.1 minutes from the start of chromatography. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about 90: 10. In some embodiments, the ratio of mobile phase B to mobile phase a increases to about 90:10 in about 18.1 minutes from the start of chromatography. In some embodiments, the flow rate is about 1 mL/min.

In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about any of 24:76, 25:75, 26:70, 27:73, or 28: 71. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about 26: 74. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to any of about 24:76, 25:75, 26:70, 27:73, or 28:71 in any of about 12, 13, 14, 15, or 16 minutes from the start of chromatography. In some embodiments, the ratio of mobile phase B to mobile phase a increases to any one of about 24:76, 25:75, 26:70, 27:73, or 28:71 in about 14 minutes from the start of chromatography. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about 26: 74. In some embodiments, the ratio of mobile phase B to mobile phase a increases to about 26:74 in about 14 minutes from the start of chromatography. In some embodiments, the flow rate is about 1 mL/min.

In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about any of 85:15, 90:10, or 95: 5. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about 30: 70. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to any of about 85:15, 90:10, or 95:5 in any of about 14.5, 15.5, 16.5, 17.5, or 18.5 minutes from the start of chromatography. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to any of about 85:15, 90:10, or 95:5 in about 16.5 minutes from the start of chromatography. In some embodiments, the ratio of mobile phase B to mobile phase a is increased to about 90: 10. In some embodiments, the ratio of mobile phase B to mobile phase a increases to about 90:10 in about 16.5 minutes from the start of chromatography. In some embodiments, the flow rate is about 1 mL/min.

In some embodiments, mobile phase a comprises about any one of 0.01%, 0.05%, 0.1%, 0.5%, or 1.0% (v/v) acid in water. In some embodiments, mobile phase a comprises about 0.1% acid in water. In some embodiments, the acid is formic acid. In some embodiments, mobile phase a comprises about 0.1% formic acid in water. In some embodiments, mobile phase B comprises about any one of 0.01%, 0.05%, 0.1%, 0.5%, or 1.0% (v/v) of an acid in acetonitrile. In some embodiments, mobile phase B comprises about 0.1% acid in acetonitrile. In some embodiments, the acid is formic acid. In some embodiments, mobile phase B comprises about 0.1% formic acid in acetonitrile. In some embodiments, mobile phase a comprises about 0.1% formic acid in water and mobile phase B comprises about 0.1% formic acid in acetonitrile.

In some aspects, the invention provides a method for measuring degradation of N-acetyl tryptophan (NAT) in a composition comprising N-acetyl tryptophan, the method comprising a) applying the composition to a reverse phase chromatography material, wherein the composition is loaded onto the chromatography material equilibrated in a solution comprising mobile phase a and mobile phase B, wherein mobile phase a comprises 0.1% (v/v) formic acid in water and mobile phase B comprises 0.1% (v/v) formic acid in an organic solvent, the ratio of mobile phase B to mobile phase a is 2: 98; b) eluting the composition from the reverse phase chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is raised to about 70:30 and then to about 90:10, wherein NAT degradants elute from the chromatography separately from intact NAT, c) quantifying the NAT degradants and the intact NAT. In some embodiments, the flow rate is about 1.0mL/min and the ratio of mobile phase B to mobile phase a is increased to about 70:30 in about 16 minutes after the start of chromatography and then to about 90:10 after about 18 minutes from the start of chromatography. In some embodiments, the organic solvent is acetonitrile.

In some aspects, the invention provides a method for measuring degradation of N-acetyl tryptophan (NAT) in a composition comprising N-acetyl tryptophan, the method comprising a) applying the composition to a reverse phase chromatography material, wherein the composition is loaded onto the chromatography material equilibrated in a solution comprising mobile phase a and mobile phase B, wherein mobile phase a comprises 0.1% (v/v) formic acid in water and mobile phase B comprises 0.1% (v/v) formic acid in acetonitrile, the ratio of mobile phase B to mobile phase a is 2: 98; b) eluting the composition from the reverse phase chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is raised to about 70:30 and then to about 90:10, wherein NAT degradants elute from the chromatography separately from intact NAT, c) quantifying the NAT degradants and the intact NAT. In some embodiments, the flow rate is about 1.0mL/min and the ratio of mobile phase B to mobile phase a is increased to about 70:30 in about 16 minutes after the start of chromatography and then to about 90:10 after about 18 minutes from the start of chromatography.

In some aspects, the invention provides a method for measuring degradation of N-acetyl tryptophan (NAT) in a composition comprising N-acetyl tryptophan, the method comprising a) applying the composition to a reverse phase chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising mobile phase a and mobile phase B, wherein mobile phase a comprises 0.1% (v/v) formic acid in water and mobile phase B comprises 0.1% (v/v) formic acid in acetonitrile, the ratio of mobile phase B to mobile phase a is 2: 98; b) eluting the composition from the reverse phase chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a rises to about 74:26 and then to about 90:10, wherein NAT degradation elutes from the chromatography separately from intact NAT, c) quantifying the NAT degradation and the intact NAT. In some embodiments, the flow rate is about 1.0mL/min and the ratio of mobile phase B to mobile phase a is increased to about 74:26 about 14 minutes after the start of chromatography and then to about 90:10 about 16.5 minutes from the start of chromatography.

In some embodiments, the reverse phase chromatography material comprises a C8 module or a C18 module. In some embodiments, the reverse phase chromatography material comprises a C18 module. In some embodiments, the reverse phase chromatography material comprises a solid support. In some embodiments, the solid support comprises silica. In some embodiments, the reverse phase chromatography material is contained in a column. In some embodiments, the reverse phase chromatography material is a High Performance Liquid Chromatography (HPLC) material or an ultra high performance liquid chromatography (UPLC) material. In some embodiments, the reverse phase chromatography column is AgilentSB-C18 chromatography column. In some embodiments, the reverse phase chromatography column is AgilentSB-C183.5 μm, 4.6X 75mm column.

In some embodiments, the chromatography is performed at a temperature ranging from about 0 ℃ to about 30 ℃. In some embodiments, the chromatography is performed at any of about 0 ℃,5 ℃,20 ℃, or 30 ℃. In some embodiments, the chromatography is performed at room temperature. In some embodiments, the chromatography is performed at about 5 ℃. In some embodiments, the chromatography is performed at 5 ℃ ± 3 ℃.

In some embodiments, NAT and NAT degradation products are detected by absorbance at 240 nm. In some embodiments, NAT degradation products are identified by mass spectrometry. In some embodiments, the concentration of NAT in the composition is from about 10nM to about 1 mM. In some embodiments, the concentration of NAT in the composition is less than any of about 10nM, 25nM, 50nM, 75nM, 100nM, 250nM, 500nM, 750nM, 1 μ M, 2.5 μ M, 5 μ M, 7.5 μ M, 10 μ M, 25 μ M, 50 μ M, 75 μ M, 100 μ M, 250 μ M, 500 μ M, 750 μ M, or 1 mM. In some embodiments, the concentration of NAT in the composition ranges between about 10nM and about 100nM, about 100nM and about 500nM, about 500nM and about 1 μ M, about 1 μ M and about 100 μ M, about 100 μ M and about 500 μ M, or about 500 μ M and about 1 mM.

In some embodiments of the above method, the NAT degradation product comprises one or more of N-Ac- (H,1,2,3,3a,8,8 a-hexahydro-3 a-hydroxypyrrolo [2,3-b ] -indole 2-carboxylic acid) (N-Ac-PIC), N-Ac-oxindolyl alanine (N-Ac-Oia), N-Ac-N-formyl-kynurenine (N-Ac-NFK), N-Ac-kynurenine (N-Ac-Kyn), and N-Ac-2a,8 a-dihydroxy-PIC.

In some embodiments, the invention provides a method for measuring N-acetyl tryptophan (NAT) degradation in a composition comprising N-acetyl tryptophan and a polypeptide, the method comprising a) denaturing the polypeptide, B) removing the polypeptide from the composition, c) applying the composition to a reverse phase chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising mobile phase a and mobile phase B, wherein mobile phase a comprises an acid in water and mobile phase B comprises an acid in acetonitrile, d) eluting the composition from the reverse phase chromatography material with a solution comprising mobile phase a and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is elevated compared to step a), wherein a NAT degradation elutes from the chromatography separate from intact NAT, e) quantifying the NAT degradation and the intact NAT. In some embodiments, the polypeptide is denatured by guanidine treatment. In some embodiments, the polypeptide is denatured with guanidine, wherein guanidine is added to the composition to a final concentration of about 7M to about 9M. In some embodiments, the polypeptide is denatured with guanidine, wherein guanidine is added to the composition to a final concentration of about 8M.

In some embodiments, the invention provides methods for measuring degradation of N-acetyl tryptophan (NAT) in a composition comprising N-acetyl tryptophan and a polypeptide, the method comprises a) diluting the composition with about 8M guanidine, b) removing the polypeptide from the composition, c) applying the composition to a reverse phase chromatography material, wherein the composition is loaded onto the chromatography material in a solution comprising mobile phase A and mobile phase B, wherein mobile phase A comprises an acid in water and mobile phase B comprises an acid in acetonitrile, d) eluting the composition from the reverse phase chromatography material with a solution comprising mobile phase A and mobile phase B, wherein the ratio of mobile phase B to mobile phase a is increased compared to step a), wherein NAT degradant elutes from the chromatography separately from intact NAT, e) quantifying the NAT degradant and the intact NAT.

In some of the above embodiments, the composition is diluted in about 8M guanidine such that the final concentration of NAT in the composition ranges from about 0.01mM to about 0.5 mM. In some of the above embodiments, the composition is diluted in about 8M guanidine such that the final concentration of NAT in the composition ranges from about 0.05mM to about 0.2 mM. In some of the above embodiments, the composition is diluted in about 8M guanidine such that the final concentration of NAT in the composition is less than any of about 0.05mM, 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.10mM, 0.12mM, 0.14mM, 0.16mM, 0.18mM, or about 0.2 mM. In some embodiments, the composition is diluted in about 8M guanidine such that the final concentration of polypeptide in the composition is less than or equal to any of about 5mg/mL, about 10mg/mL, about 15mg/mL, about 20mg/mL, about 25mg/mL, about 30mg/mL, about 35mg/mL, about 40mg/mL, about 45mg/mL, about 50mg/mL, or about 100 mg/mL. In some embodiments, the composition is diluted in about 8M guanidine such that the final concentration of polypeptide in the composition is about 5mg/mL to about 10mg/mL, about 10mg/mL to about 15mg/mL, about 15mg/mL to about 20mg/mL, about 20mg/mL to about 25mg/mL, about 25mg/mL to about 30mg/mL, about 30mg/mL to about 35mg/mL, about 35mg/mL to about 40mg/mL, about 40mg/mL to about 45mg/mL, about 145mg/mL to about 50mg/mL, or about 50mg/mL to about 100 mg/mL.

In some embodiments, the polypeptide is removed from the composition by filtration. In some embodiments, the filtration uses a filtration membrane with a molecular weight cut-off of about 30 kDal.

In some of the above embodiments, the formulation has a pH of about 3.5 to about 7.0. In some of the above embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the formulation has a pH of about any one of 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 7.5, or 8.0.

In some embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents.

In some embodiments, the formulation is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody or antibody fragment.

In some embodiments, the present invention provides a method for monitoring degradation of NAT in a composition comprising measuring degradation of NAT in a sample of the composition according to the method described above, wherein the method is repeated one or more times. In some embodiments, the method is repeated at least about any of 2,3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the method is repeated daily, weekly, or monthly, or any combination thereof. In some embodiments, the method is repeated at least about once per month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 9 months, or at least about once a year.

In some embodiments, the present invention provides a quality assay for a pharmaceutical composition, the quality assay comprising measuring degradation of NAT in a sample of the pharmaceutical composition according to the methods described above, wherein the amount of NAT degradant measured in the composition determines whether the pharmaceutical composition is suitable for administration to an animal. In some embodiments, an amount of NAT degradant in the pharmaceutical composition that is less than any of about 1ppm, 2ppm, 3ppm, 4ppm, 5ppm, 6ppm, 7ppm, 8ppm, 9ppm, 10ppm, 20ppm, 30ppm, 40ppm, 50ppm, 60ppm, 70ppm, 80ppm, 90ppm, or 100ppm indicates that the pharmaceutical composition is suitable for administration to the animal. In some embodiments, an amount of NAT degrader in the pharmaceutical composition of less than about 10ppm indicates that the pharmaceutical composition is suitable for administration to an animal.

VIII. preparation of

In another embodiment of the present invention, an article of manufacture is provided comprising a container containing a liquid formulation of the present invention, and optionally instructions for its use. In some embodiments, the liquid formulation comprises a protein (e.g., an antibody) and N-acetyl-tryptophan (NAT), wherein the protein comprises at least one of a) SASA is greater than aboutTo about(such as greater than about 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, orAny, including any range between these values); b) SASA is greater than about 15% to about 45% (such as greater than any of about 15, 20, 25, 30, 35, 40, or 45%); or c) predicting tryptophan residues susceptible to oxidation by a machine learning algorithm practiced in association with the tryptophan residue oxidation susceptibility and a plurality of molecular descriptors of the tryptophan residue based on MD simulation. In some embodiments, the amount of NAT in the liquid formulation is from about 0.1mM to about 10mM (such as about any of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0mM, including any range between these values). In some embodiments, the oxidation of the protein is reduced by about 40% to about 100% (such as about any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, including any range between these values). In some embodiments, the liquid formulation is stable for at least about 12 months (such as at least about any of 12, 15, 18, 21, 24, 27, 30, 33, or 36 months, including any range between these values) at about 0 ℃ to about 5 ℃ (such as about any of 0, 1,2,3, 4, or 5 ℃, including any range between these values). In some casesIn embodiments, the concentration of the protein in the liquid formulation is from about 1mg/mL to about 250 mg/mL. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody or antibody fragment. In some embodiments, the antibody is derived from an IgG1 antibody sequence. In some embodiments, the liquid formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the liquid formulation has a pH of about 4.5 to about 7.0. In some embodiments, the liquid formulation is an aqueous formulation.

Suitable containers include, for example, bottles, vials, and syringes. The container may be formed from a variety of materials, such as glass or plastic. An exemplary container is a 2-20cc single use glass vial. Alternatively, for multi-dose formulations, the container may be a 2-100cc glass vial. The container contains the formulation and a label on or associated with the container may indicate instructions for use. The article of manufacture may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with printed instructions for use.

The package insert refers to instructions for use typically included in commercial packages of therapeutic products, containing information regarding the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

Kits useful for a variety of purposes are also provided, such as for reducing oxidation of proteins in liquid formulations or for screening liquid formulations for reduced protein oxidation, optionally in combination with an article of manufacture. The kits of the invention comprise one or more containers comprising a protein (e.g., an antibody) comprising at least one of a) a SASA of greater than aboutTo about(such as greater than about 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, orAny, including any range between these values); b) SASA is greater than about 15% to about 45% (such as greater than any of about 15, 20, 25, 30, 35, 40, or 45%); or c) predicting tryptophan residues susceptible to oxidation by a machine learning algorithm practiced in association with the tryptophan residue oxidation susceptibility and a plurality of molecular descriptors of the tryptophan residue based on MD simulation. The instructions provided in the kits of the invention are typically written instructions on a label or package insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

This description is deemed to be sufficient to enable those skilled in the art to practice the invention. Various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Examples

The present invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications and changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Example 1. Evaluation of NAT protection from oxidation.

SASA calculation

The SASA for a given protein residue was calculated using the computer full atom molecular dynamics simulation method described in Sharma, v.et al (supra). Briefly, the structure of the protein is obtained from either the 3D crystal structure or homology model, with ions and explicit (explicit) solvent molecules added as needed. SASA is calculated using g _ sas of GROMACS, and mutual information calculation is performed locally (Eisenhaber F. et al., J. Compout. chem.16(3):273-284, 1995; Lange O. F. et. proteins 70(4):1294-1312, 2008). Root mean square fluctuations, hydrogen bonds, and secondary structures were calculated using status g _ rsmf, g _ hbond, and dssp of GROMACS, respectively. Shannon entropy and mutual information were calculated using previously published methods (Kortkhonjia E, et al, MAbs 5(2): 306-. MD simulations were performed using Amber 11 (FF99SB fixed charge force field; SASA was calculated using areaimol (Bailey S, acta. Crystallogr.D. biol. Crystallogr.50(Pt 5):760- > 763, 1994); 100-ns traces were used as they provided sufficient data within the available computational power.

AAPH pressure

The proteins mab 1, mab 2, and mab 3/mab 4 were dialyzed into sodium acetate buffer (20mM sodium acetate pH5.5) and mab 5/mab 6 was dialyzed into histidine-based buffer (20mM histidine hydrochloride pH 5.5). The protein solution was diluted to a final concentration of 1mg/mL protein in the corresponding buffer and 1mM 2,2' -azobis (2-amidinopropane) dihydrochloride (AAPH) was added. N-acetyl-trp (nat) was added to each protein solution from the concentrated storage solution at a concentration of 0 to 5 mM. The samples were incubated at 40 ℃ for 16 hours, then quenched with methionine and buffer exchanged to the initial dialysis buffer plus 100mM sucrose.

LC-MS tryptic peptide mapping

Digestion with micro-scale tryptic peptide followed by liquid chromatography-mass spectrometry (LC-MS)) Site-specific modification of AAPH stressed samples of mab 2, mab 1, and mab 3/mab 4 were monitored (Anderson, n.et al, nov.20,2014, american pharmaceutical Review). 30 μ L (250 μ g) of each stressed sample was diluted with 190 μ L of reduced carboxymethylation buffer (6M guanidine hydrochloride, 360mM Tris, 2mM EDTA, pH 8.6) to denature the protein. After denaturation, 4 μ L of 1M DTT was added to each mixture and the reduction reaction was incubated at 37 ℃ for 1 hour. The samples were then carboxymethylated by adding 10.4 μ L iodoacetic acid and stored in the dark at room temperature for 15 minutes. The alkylation reaction was quenched by the addition of 2 μ L of 1M DTT. The reduced and alkylated sample buffer was exchanged into tryptic digestion buffer (25mM Tris, 2mM CaCl) on a PD-10 column (GE Healthcare)₂pH 8.2). The sample was then digested by adding sequencing grade trypsin (Promega) in an enzyme to protein ratio of 1:50 (by weight). The digestion reaction was incubated at 37 ℃ for 4 hours and then quenched by adding 100% Formic Acid (FA) to the sample to a final FA concentration of 3.0% (v/v).

Peptide mapping of each digested sample was performed on a Waters acquisition H grade UHPLC coupled to a Thermo Q active Plus high resolution mass spectrometry system (HRMS). Splitting of a 10 μ g injection of digested sample was performed in a CSH C18 column (Waters, 1.7 μm particle size, 2.1mm x 150mm) running at a flow rate of 0.3mL/min and with column temperature controlled at 77 ℃. Solvent a consisted of 0.1% FA in water and solvent B consisted of 0.1% FA in acetonitrile. The gradients are shown in table 1. The column effluent was monitored at 214 nm. Full MS-SIM data was collected over a scan range of 200-2000m/z at a resolution of 17,500. The electron jet ionization in positive ion mode was achieved by using a needle jet voltage of 3.50 kV. Sites of interest for oxidative propensity were previously characterized for each mab using the same micro-scale trypsin digestion followed by LC/MS-MS (MS/MS fragmentation for residue-specific localization of PTMs). The oxidation level at each site was determined by Extractive Ion Chromatography (EIC) using Thermo XCalibur biopharmaceutical characterization software. The relative percentage of oxidation was calculated by dividing the peak area of the oxidized peptide species by the sum of the peak areas of the native and oxidized peptides. Total oxidation number reported for tryptophan sites is only W_ox1(+16) and NFK/W_ox2The sum of (+ 32). For more details on Peptide Mapping see Andersen, n.et al, Rapid UHPLC-HRMS Peptide Mapping for monoclonal antibodies.

TABLE 1

Time (min)	% solvent A	% solvent B
			0.0	99	1
2.0	87	13
			9.5	62	38
12.5	25	75
			12.6	10	90
13.0	10	90
			13.1	99	1
22.0	99	1

NAT protection from oxidation

NAT protection against AAPH-induced oxidation was evaluated for the following oxidation-prone tryptophan residues: mab 2W53 and W106; monoclonal antibody 4W 52; monoclonal antibody 1W 103; and 6W103/104 monoclonal antibody. Each protein was subjected to AAPH pressure using 1mM AAPH as described above. NAT was added at 0, 0.05, 0.1, and 0.3mM for mab 2, mab 4, and mab 1, and at 0, 0.1, and 1mM for mab 6. As shown in figure 1 and tables 2 and 3, NAT is able to protect each of the tested residues from oxidation by AAPH pressure.

Table 2. Oxidation of tryptophan residues

Table 3. Protection of proteins

Protein	Residue of	At 0.1mM NProtection of AT (%)1
			Monoclonal antibody 2	W53	79
Monoclonal antibody 2	W106	56
			Monoclonal antibody 4	W52	43
Monoclonal antibody 1	W103	17
			Monoclonal antibody 6	W103/104	44

1: calculated as (delta oxidation at 0mM NAT-delta oxidation at 0.1mM NAT)/delta oxidation at 0mM NAT

Prediction of tryptophan oxidation susceptibility

As shown in fig. 2A, AAPH induced% oxidation was plotted as a function of tryptophan residue SASA using data from 38 IgG1 mabs. With a 30% SASA cut-off, 87% of the examined residues had an oxidation level of greater than 35%. One molecular descriptor of tryptophan residues,% SASA, is highly accurate in predicting susceptibility to oxidation in this antibody population. However, as shown in fig. 2B, expanding the data set to include tryptophan residues from 121 mabs with a diverse framework (e.g., IgG1, IgG2, IgG4, murine) resulted in less accurate prediction of oxidative susceptibility based on% SASA alone.

Example 2. Machine learning for prediction of tryptophan oxidation susceptibility.

The use of a mimetic-based molecular descriptor, such as SASA, can yield highly accurate predictions about tryptophan oxidation susceptibility in certain conditions, such as for a particular IgG subclass. However, in predicting tryptophan oxidation susceptibility of a wide variety of residues between frameworks, accuracy can be improved by using a variety of molecular descriptors. We used machine learning to correlate a set of MD simulation-based molecular descriptors with tryptophan oxidation susceptibility, resulting in a model that can be used to accurately predict the stability of test tryptophan residues where experimental data on oxidation susceptibility is unavailable, allowing for a faster pipeline to select candidate molecules. Moreover, the relative importance of molecular descriptors in the model was determined, potentially pointing to the underlying mechanisms that drive stability.

Molecular descriptors

The following molecular descriptors were calculated using the computer full atomic molecular dynamics simulation method described above. Six MD simulations were run for each tryptophan residue using the following parameters: fv zone only, 100ns snapshot per simulation, explicit water, constant pressure, 3 simulations with protonated HIS, 3 simulations with deprotonated HIS ("pH"), and 3fs step size. The MD-derived molecular descriptors include cyclic fingerprints of the local chemical environment: charge, hydrophobicity; forming hydrogen bonds; and partial structures of the main chain and the amino acid side chains.

At a position delta to tryptophanNumber of internal aspartic acid side chain oxygens

For each frame of each molecular simulation, the delta carbon from each tryptophan was tracedAll atoms in the column. In thatOf these, the number of oxygen atoms on the side chain of any aspartic acid residue is counted. The final value represents the time average of this count over the duration of the simulation.

Side chain SASA (stdev)

For each frame of each molecular simulation, the Solvent Accessible Surface Area (SASA) of each tryptophan side chain was calculated. In short, the points of the sphere concentrated on each atom in the simulation are generated by adding together the radius of each atom and the radius of a water molecule. All points within the radius of the neighboring sphere are eliminated and the area is summed among all remaining points to generate the SASA value. The final value of this descriptor represents the standard deviation of the SASA for the tryptophan side chain atom over the simulated duration.

Delta carbon SASA (stdev)

For each frame of each simulation, the Solvent Accessibility Surface Area (SASA) was calculated as previously described for each tryptophan delta carbon. The value of this descriptor represents the standard deviation of the SASA for the tryptophan delta carbon over the simulated duration.

At a position delta to tryptophanTotal positive charge in (stdev)

For each frame of each simulation, the delta carbon from each tryptophan was trackedAll the atoms in the inner group associated with the charged amino acid side chain. The total positive charges of these atoms are added together. The final value represents the standard deviation of this quantity over the duration of the simulation.

Main chain SASA (stdev)

For each frame of each molecular simulation, the Solvent Accessible Surface Area (SASA) of the backbone nitrogen atom of each tryptophan was calculated. This descriptor is the standard deviation of the SASA of the backbone nitrogen atoms over the duration of the simulation.

Tryptophan side chain angle

The χ 1 and χ 2 angles of the tryptophan side chains were traced through the simulation. When plotting a number of different tryptophan residues and all simulated χ 1 and χ 2 angles, the cluster of co-occurring angle combinations becomes apparent. K-means clustering was used to define the center of each of the 12 regions.

The angular region most predictive of tryptophan oxidation is "cluster 5" concentrated at 76 degrees χ 1 and 98 degrees χ 2. For each individual tryptophan residue, the percentage of time spent in cluster 5 was mock tracked and added as a descriptor to the random decision forest.

At a position delta to tryptophanPacking density of inner

As a radius concentrated at the tryptophan delta carbonThe time-averaged number of protein atoms within the sphere of (a) calculates the packing density.

Tryptophan main chain angle (stdev)

This descriptor was calculated by measuring the standard deviation of the ψ angle relating to the backbone of each tryptophan residue over the duration of the simulation.

Occupied volume of pseudo-pi orbit

As a height ofThe side chain of each tryptophan residue is treated with the base of a cylinder approximating the space occupied by the tryptophan pi orbital. All atoms falling within the volume of the cylinder during the simulation are tracked. For each frame of the simulation, all proproteins falling within the volume of the cylinder were calculatedTotal volume of seeds. The final value represents the time-averaged volume of tryptophan pi orbitals occupied by other protein atoms.

Flexibility of main chain

The root mean square fluctuation of the backbone nitrogen for each tryptophan residue was calculated on each simulation. Briefly, each frame in the simulation was aligned. The distance traveled by each nitrogen atom is calculated for each frame. The distance for each frame is squared and the average of the squared distances is taken across all frames. Finally, the square root of the mean of the squared distances is taken to produce the root mean square fluctuation of the tryptophan backbone nitrogen.

At a position delta to tryptophanTotal negative charge in

For each frame of each simulation, the delta carbon from each tryptophan was trackedAll the atoms in the group associated with the charged amino acid side chain. The total negative charge of these atoms is added together. The final value represents the time average of this number over the duration of the simulation.

Generation of random decision forest

The values for a set of molecular descriptors of 121 tryptophan residues in 68 molecules with experimentally determined oxidation levels were calculated as described above. Tryptophan residues with greater than 35% oxidation are classified as "unstable", while those with less than 35% oxidation are classified as "stable". The universal molecular descriptors also relate to each tryptophan residue, including IgG class, IgG framework information, CDR positions of tryptophan residues, CDR lengths, preceding and succeeding residues in the sequence, and the number of other oxidative hotspots.

A combination of experimental data (stable or unstable tryptophan residues) and tryptophan molecule descriptors (mock data) was used to exercise a random decision forest to learn which mock-based outputs correspond to tryptophan stability. The accuracy of the random decision forest was evaluated over a series of parameters to identify the best conditions for exercise, and the descriptors most important for predicting tryptophan oxidation were ranked using random decision forests generated using optimized parameters.

Two methods were used to evaluate the accuracy of random decision forests. In one approach, an "out of bag" error is calculated. Off-bag errors have been demonstrated in the machine learning literature to be a reliable estimate of predictive model accuracy (James, g., et al., An introduction to Statistical learning. spring. pp. 316-charge 321, 2013). Briefly, guided clustering is applied to a training set X ═ X₁，…，x_nAnd calculating the mean prediction error x for each of the training samples_iUsing only those samples having no x in their pilot samples_iThe tree of (2). In another approach, the data set is split into an exercise set (80% of the data) for practicing random decision forests and a test set (the remaining 20% of the data) for applying to the resulting random decision forests. The prediction error of the test set is used to calculate the model accuracy.

To determine the optimal training conditions for random decision forests, the following parameters were varied and the model accuracy was evaluated: the number of decision tree individuals (or estimates) included in the random decision forest, the number of variables (or features) at each branch of each tree in the forest for random decision taking into account random selection, and the maximum number of times the observation set is divided into sub-branches (or tree depth). The optimal number of estimates of the accuracy of the tryptophan oxidation model is greater than or equal to 200 (see FIG. 3). Accuracy of over 85% was still achieved with as few as 30 estimates. The optimum number of features ranges between 1 and 4 (see fig. 4). The optimal tree depth is greater than or equal to 5 (see fig. 5). An accurate model is still achieved with tree depths as low as 2. Based on these results, a random decision forest is generated using the following optimized parameters: 5000 estimates, each section considering 3 features, and a tree depth of 10. The resulting out-of-bag error calculation for the optimized random decision forest resulted in an accuracy of 89.2%. The data was split into training and test sets and the accuracy of the optimized random decision forest was found to be 88%, sensitivity 80% and specificity 89% (see table 4).

Table 4: random decision forest accuracy

	The prediction is stable	Predicting that the solution is unstable
			Stabilized	17	2
Unstable	1	4

The relative importance of the simulation-based molecular descriptors in the optimized random decision forest was evaluated using the kini importance and the top 14 molecular descriptors are shown in fig. 6. The most important descriptors are nearby aspartate side chain oxygen, followed in rank order by side chain SASA (stdev), δ carbon SASA (stdev), nearby positive charge (stdev), backbone SASA (stdev), tryptophan side chain angle at pH7, packing density at pH7, backbone angle (stdev), backbone undulation, SASA with pseudo-pi orbitals, packing density at pH5, tryptophan side chain angle at pH5, nearby negative charge at pH5, and nearby negative charge at pH 7.

Example 3. Characterization of NAT degradation under different stress conditions and formulations.

To systematically evaluate NAT stability, we developed a combination of Reverse Phase (RP) chromatography with UV detection to quantify NAT degradation. NAT is added to the typical buffer system of a protein formulation and subjected to pressures designed to mimic those that recombinant proteins may be subjected to during typical manufacturing and storage conditions: alkyl peroxides, fenton chemistry, UV light, and thermal pressure (Grewal, p., et al., Mol Pharm,2014.11(4): 1259-72; Ji, j.a., et al., J Pharm sci,2009.98(12): 4485-500; Torosantucci, r., et al., Pharm Res,2014.31(3): 541-53). In our study, over 10 different NAT degradants were observed and the main species identified.

Chemical medicine

Chemicals were purchased from Sigma unless noted. All chemicals used were of analytical grade. Protein therapeutic samples are produced in chinese hamster ovary cells or e.coli and purified by a series of chromatographic steps, including affinity chromatography and/or ion exchange chromatography. The synthesis of the main NAT degradants (NAT degradant structures see figure 7) was achieved by adapting the literature methods as described below.

General Synthesis protocol

Anhydrous solvents are used where possible. Phenomenex Gemini-NX 10 uC 18 was used100mm X30 mm preparative HPLC columns preparative reverse phase chromatography was performed on a Waters 2525 HPLC system. Mobile phase a ═ Milli-QH₂O, 0.1% formic acid. Mobile phase B-acetonitrile, gradient 0-12 min 0-20% B, passing UV signal threshold at 254nm (10)^-1Au) to trigger fraction collection. Fractions were analyzed by LC/MS for the presence of the desired product. For all preparative separations, the pooled fractions did not include fractions containing the beginning and ending portions of the desired peak in order to improve purity.

LC/MS sample analysis of the final product was performed in tandem with a Thermo Scientific Orbitrap Mass spectrometer using Waters H-Class UPLC and the chromatographic conditions described in the text. Full scan accurate mass data was collected in positive ion mode at 15,000 resolution over a scan range of 50-800 m/z. MS2 was performed for the first three ions, with dynamic exclusion disabled.

NMR analysis was performed in fully deuterated DMF.

General procedure for the acetylation of tryptophan derivatives

Tryptophan derivatives were added to acetonitrile (anhydrous, j.t. baker) (final concentration 200 mM). Diisopropylethylamine (DIPEA, 5eq) was added followed by 1.1eq acetic anhydride (Ac)₂O). The reaction was stirred at room temperature for 16 hours. The mixture was filtered to remove unreacted starting material and the solvent was removed in vacuo. The material was dissolved in Dimethylformamide (DMF) and the desired product was purified by prep-RPLC.

N-Ac-Kyn (N-Ac-DL-kynurenine) 6b

DL-kynurenine (Sigma Aldrich) was acetylated by the general procedure above. DL-kynurenine (800mg, 3.84mmol) was added to 40ml acetonitrile to form a light yellow suspension. DIPEA (5eq) and Ac were added₂O (1.1 eq). After 16 hours of agitation at room temperature, most of the suspended solids dissolved and the solution was dark yellow/orange in color. Separation by preparative chromatography and lyophilization of the partially separated fraction material yielded 428mg of a fluffy pale yellow solid. The obtained product was characterized by LC-MS (m/z-251.103) and NMR (RP-UPLC purity 99%, 44% yield).

The absence of starting material was confirmed by UV and ninhydrin stained TLC analysis.

N-Ac-NFK (N α -acetyl-N' -formyl-kynurenine) 7b

N-Ac-NFK was synthesized by adapting the literature protocol reported in C.E.Dalgliesh in J.chem.Soc.1952, 137-141. A mixture of formic acid (Sigma, 98-100%, 360. mu.l) and acetic anhydride (J.T. Baker, 99% anhydrous, 105. mu.l) was stirred for 30 minutes, after which 100mg of N-acetyl-DL-kynurenine was added. After 2 hours of reaction, LC/MS analysis still showed the presence of starting material, at which point a second addition of formic acid (120. mu.l) and acetic anhydride (35. mu.l) was made to force the reaction to completion. LC/MS analysis performed 1 hour after the second addition showed a loss of starting material and formation of the desired product. The reaction mixture was added to 15ml of water at room temperature and frozen. Light brown crystals formed overnight. These were filtered, washed with ice cold water and the wet residue was freeze dried to yield 10mg of the desired product as a fluffy beige solid. The obtained product was characterized by LC-MS (m/z-279.098) and NMR (RP-UPLC purity 90%, 9.0% yield).

Oia (Hydroxyindoyl-DL-alanine) diastereomer 4a

By adaptation Itakura, k.; uchida, k.; oia was synthesized according to the literature protocol reported by Kawakishi, S.in chem.Res.Toxicol.1994,7, 185-190. DMSO (900. mu.l) and phenol (100mg, 1.06mmol) were premixed with 5mL of 37% HCl at ambient temperature. DL-Trp (1.0g, 4.9mmol) was suspended in 30ml of glacial acetic acid and added to the mixture. The reaction was stirred at ambient temperature. Progress was checked periodically by LC-MS. After 4 hours LC/MS confirmed the loss of starting material and the formation of two close eluting peaks with the desired product mass (m/z 221). The solvent was removed under vacuum to yield a dark brown paste. The material was dissolved in 4ml DMF. No attempt was made to separate the diastereomers; purification of the desired diastereoisomeric product was performed using preparative RPLC. The desired fractions were combined and lyophilized to yield 305mg of a fluffy white solid. The obtained product was characterized by LC-MS (m/z 221) (28% yield).

N-Ac-Oia (N-acetyl-indolylalanine) diastereomer 4b

The isoindolyl-DL-alanine diastereomer 4a (100mg) was acetylated according to the general procedure above. After 16 hours, LC/MS confirmed the reaction was complete. The solvent was removed in vacuo. Preparative chromatography and lyophilization yielded 56mg of a fluffy white powder. No attempt was made to separate the diastereomers. The obtained diastereomeric product was characterized by LC-MS (m/z-263.102) and NMR (RP-UPLC purity 89%, 47% yield). The absence of starting material was confirmed by UV and ninhydrin stained TLC analysis.

N-Ac-5-HTP (N-acetyl-5-hydroxy-tryptophan) 8b

5-HTP 8a (150mg) was acetylated according to the general procedure above. After 16 hours, LC/MS confirmed the reaction was complete. The solvent was removed in vacuo. Preparative chromatography and lyophilization yielded 58mg of a fluffy white powder. The obtained product was characterized by LC-MS (m/z-263.1) and NMR (32% yield). The absence of starting material was confirmed by UV and ninhydrin stained TLC analysis.

2,2' -azobis (2-amidinopropane) dihydrochloride (AAPH) oxidation pressure

AAPH (Calbio Chem, 99.8%) was used to simulate oxidative degradation by alkyl peroxides. Histidine at pH5.5 and a non-histidine buffer containing 0.3mM NAT + -5 mM L-Met pH5.5 were treated with an aqueous AAPH solution to a final concentration of 1.0mM AAPH. Will equal the volume of Milli-Q H₂O was added to the control sample. The samples were incubated at 40 ℃ for 16 hours. The oxidation was quenched by addition of L-Met to a final concentration of 20 mM. After addition of the quench solution, the final NAT concentration was 0.2 mM.

Fenton pressure

FeCl is added₂(Sigma Aldrich, 98% purity) and H₂O₂(Sigma Aldrich, 30% w/w in H₂O) to a histidine containing buffer at pH5.5 containing 0.3mM NAT ± 5mM L-Met, pH5.5 to final concentrations of 0.2mM and 10ppm, respectively. Addition of H₂O₂After this time, the vial was briefly vortexed and incubated at 40 ℃ for 3 hours. The oxidation was quenched by addition of L-Met to a final concentration of 100 mM. After addition of the quench solution, the final NAT concentration was 0.2 mM.

Light pressure

Optical cassettes designed for photostability testing of drugs/products were utilized as recommended by the International conference on harmonization (ICH) expert working group [ Atlas SunTEST CPS + Xenon optical cassette (Chicago, IL)]The samples containing NAT were provided with light pressure. ICH photostability test is defined as 1.2x10⁶Lux-smallWhite light and 200W-hr/m²UV light; the light box was programmed to provide pressure over a 24 hour period. Histidine and non-histidine buffers containing 0.3mM NAT + -5 mM L-Met were dispensed into sterile glass vials (1 ml/vial). The vial cap was placed side-by-side in a light box to maximize exposure to the light source. One control sample of each buffer condition was covered in foil and placed in a light box for the duration of exposure. For consistency with other pressure models, prior to HPLC analysis, the buffer solution was analyzed with Milli-Q H₂O is diluted to a final NAT concentration of 0.2 mM.

Thermal pressure

Histidine and non-histidine buffers containing 1.0mM NAT were dispensed into sterile glass vials (5 ml/vial, 6 vials each buffer). Vials were stored in dark boxes at the indicated temperature during the pressure and transferred to-70 ℃ for storage until analysis (time point taken every month for 5 months). The initial time point for each sample of buffer solution was immediately transferred to-70 ℃. Prior to analysis, the samples were thawed and subjected to Milli-Q H₂O is diluted to a final NAT concentration of 0.2 mM.

HPLC analysis

NAT and NAT degradants were separated on an Agilent 1200 series HPLC or Waters grade H UPLC using an Agilent ZORBAX SB-C183.5 μm, 4.6X 75mm reverse phase column. The column temperature was maintained at 30. + -. 0.8 ℃ by means of a thermostat control. The gradients for HPLC and UPLC are listed in tables 5 and 6, respectively (note: shorter gradients on UPLC are designed to accommodate earlier residence times and extend column re-equilibration periods due to the wider range of system pressures at 1.0ml/min flow rate). NAT degradation products were detected at 240 nm. Analysis was performed on each instrument using standard bandwidth settings (8nm for Agilent 1200 HPLC, 4.8nm for Waters class H). The autosampler was maintained at 5. + -. 3 ℃.10 nmol of NAT and/or NAT degradation products (most samples 50. mu.l) were injected onto the column for analysis. The chromatogram was processed using Dionex Chromeleon software.

Table 5. Gradient for Agilent 1200 HPLC.

Table 6. Gradient for Waters class H UPLC.

Analysis of samples by LC/MS

LC/MS sample analysis was performed in tandem with a Thermo Scientific Orbitrap Mass spectrometer using Waters grade H UPLC and the chromatographic conditions described above. Full scan accurate mass data was collected in positive ion mode at 15,000 resolution over a scan range of 50-800 m/z. MS2 was performed for the first three ions, with dynamic exclusion disabled.

Results

Compression sample panel design

NAT stability was evaluated after exposure to four different representative stresses: 1) fenton chemistry (H)₂O₂+Fe²⁺) Simulating potential oxidation by iron leachates resulting from contact with stainless steel during drug production, 2) AAPH pressure, simulating alkyl peroxides produced by degradation of polysorbate detergents, 3) international conference on harmonization (ICH) light pressure (1.2x 10)⁶Lux hour, 200w hr/m²) The pharmaceutical industry used to evaluate the harsh light pressure of light stability, and 4) accelerated thermal pressure to simulate long-term degradation of biopharmaceuticals. The strength of each pressure is chosen to be harsh compared to typical storage and manufacture and the degradation strengths are similar to each other, enabling a comparison between the changes induced by the different pressure models. These studies were conducted with formulations consistent with those commonly used for mabs. Since histidine is known to be oxidatively active, both histidine-containing and non-histidine-containing buffers were employed when relevant.

Method development

Reverse phase chromatography using a C18 column was used to monitor NAT degradation. Gradient conditions were chosen to ensure proper resolution of NAT and NAT degradants (fig. 8A). The eluate is monitored at 240nm, the wavelength being chosen to ensure sufficient sensitivity for low level species [ some species are not detectable at the larger wavelength (e.g. 280nm) and the signal: noise is reduced at the lower wavelength (e.g. 214nm) [ see FIG. 8B ]. The final chromatographic conditions provide a linear response throughout the relevant range: 0.01-1.0mM NAT (FIG. 13) and 1-20 fold dilution of degradants in AAPH stressed NAT samples (FIG. 14). Sample injector stability for NAT and major degradants was established at 5 ℃ for up to 12 hours (data not shown).

The protein containing sample was diluted in guanidine and the protein was removed via ultrafiltration (Amicon spin filter, 30kDa molecular weight cut-off). Incomplete recovery was observed for some mabs in the absence of chaotropes, suggesting that non-covalent interactions between NAT and proteins may occur. NAT HAS previously been demonstrated to bind to human serum albumin (HAS) (Anraku, M., et al., Biochim Biophys Acta,2004.1702(1): p.9-17), but binding to monoclonal antibodies HAS not been reported to date. Using final sample preparation conditions, NAT recovery from the three tested antibodies/antibody derivatives was 94-99% and NAT degradants 98-100% (data not shown).

Analysis of stressed sample groups

Multiple NAT degradants were observed for all pressure conditions, represented by six major new peaks and multiple minor peaks (fig. 8A, NAT degradant structure see fig. 7). The total NAT degradation for each sample was calculated by comparing the NAT peak area to a control sample for each pressure model (table 7). NAT degradation ranged from 3% (thermal stress, non-His buffer) to 83% (ICH optical stress, His buffer).

Table 7. Model pressure conditions and corresponding NAT degradation

The peaks of the stars represent the peaks observed only at ICH light pressure.

Approximately 30-40% NAT degradation was observed at fenton and AAPH pressures under all conditions tested, and levels and distribution of degradants were generally independent of the presence of buffer histidine (fig. 8A, NAT degradant structure see fig. 7). NAT experiences greater buffer sensitivity (i.e., the difference between histidine and non-histidine formulations) under ICH light pressure (fig. 8A). Although stability of NAT in non-histidine buffers under light pressure caused NAT degradation similar to AAPH and fenton pressure quantification (28% loss compared to 33-41%), the distribution of degradants changed and new peaks were observed (see the peaks marked with an "×", fig. 8A). Significantly higher levels of degradation (> 80%) were observed in histidine buffer under light pressure, leading to elevated levels of previously observed NAT degradants, along with new peaks (fig. 8A).

In summary, a striking consistency between the curves in the degraded sample panel was observed, with the exception of the minor peaks observed under ICH light pressure conditions (peaks marked with an "+" in fig. 8A). This suggests that hydrogen peroxide/hydroxyl radicals (fenton pressure) and alkyl peroxides (AAPH) can degrade NAT via a common pathway, while Reactive Oxygen Species (ROS) (H) induced by UV light₂O₂Singlet oxygen, superoxide) may present an additional degradation pathway. The observation that the presence of histidine enhances NAT degradation under ICH light conditions is consistent with the report that histidine itself is photoreactive and thus increases ROS levels and types (Stroop, s.d. et al, J Pharm Sci,2011.100(12): p.5142-55). Given the common NAT degradation profiles observed under these diverse testing stress conditions, it is likely that any NAT degradation in the drug product will cause these same species to be generated.

Identification of degradants

Next, the identity of the degraded NAT degradant species was explored using LC/MS. The molecular ions of the major peaks are listed in table 8 (a complete list of all peaks exhibiting sufficient signal intensity according to LC/MS is included in table 9). The major peaks 2,3, and 4 have an m/z of 263.1(NAT +16), consistent with the primary oxidation event of NAT. Since the major peaks 2 and 3 had similar MS1 and MS2 spectra (fig. 15A) and consistent ratios between all pressure and absorbance wavelengths monitored (fig. 8B), these peaks were tentatively assigned as the tautomeric diastereomer of N-Ac-Oia 4B (see structure fig. 7). Similar Trp species have been reported after treatment of tryptophan with hydrogen peroxide (Simat, T.J. and H.Steinhart, J Agric Food Chem,1998.46(2): p.490-498). This assignment is further supported by the observation of the MS2 ion at 130.1, which previous reports indicated an oxyindolylalanine (Oia) -containing peptide (Todorovski, t.et al, J Mass spectra, 2011.46(10): p.1030-8) (fig. 15A).

Table 8. NAT degradation product identification

Table 9. Identity of NAT degradant species

The major peaks 5 and 6 had m/z of 279.10(NAT +32) and 251.10(NAT +4), respectively, with no or weak absorbance at 280nm (FIG. 8B). These properties suggesting loss of the indole ring are consistent with two of the major known physiological degradants of Trp, NFK (7a, Trp +32) and Kyn (6a, Trp +4) (Dreaden k., et al, PLoS One,2012.7(7): p.e42220) (see structure figure 7). To assess whether these species represent the corresponding N-acetylated versions, N-Ac-NFK 7b and N-Ac-Kyn 6b (see structure in fig. 7), collision-induced dissociation was used to generate MS2 spectra for both species. Each showed a strong signal at m/z 174.1 (fig. 15B and 15C), which was previously reported to be characteristic of kynurenine (Todorovski, t.et., J Mass spectra, 2011.46(10): p.1030-8). Based on this information, these classes were tentatively assigned as N-Ac-NFK 7b and N-Ac-Kyn 6b, respectively (see FIG. 7 for structure).

To confirm the identity of these species, authentic standards of N-Ac-Oia 4b, N-Ac-NFK 7b, and N-Ac-Kyn 6b were synthesized using the synthesis protocol described above. The chromatography of peaks in stressed NAT samples and MS2 profiles were both aligned with those of authentic samples (fig. 9 and fig. 15A-15C), giving additional support for the identification of these peaks.

Given that 5-OH-Trp 8a is the major physiological catabolite of Trp, synthetic standards of N-Ac-5-OH-Trp 8b (see structure figure 7) were also prepared to assess whether this species follows the major degradation pathway of NAT. Analysis of authentic N-Ac-5-OH-Trp 8b standards by LC-MS/MS indicated that the compound was not present in any significant amount in any stressed NAT sample, as neither retention time nor mass spectroscopy data were consistent with observed NAT degradation (fig. 9 and 15D). MS2 fragment ion 146.1(Todorovski, t.et al, JMass spectra, 2011.46(10): p.1030-8) derived from a Trp derivative that has been oxidized on the benzene portion of the indole ring was not observed in any of the primary oxidative NAT degradation species, suggesting that minimal levels of hydroxylation occurred at positions 4, 5, 6, or 7 of the indole ring during NAT oxidation (fig. 15A and 15D).

The single-oxidation NAT species and peak 4 in peak set 1 were tentatively identified as stereoisomers of N-Ac-PIC 2b (see structure figure 7), respectively, and the double-oxidation NAT species in peak set 1 were similarly tentatively assigned as stereoisomers of N-Ac-3a,8 a-dihydroxy PIC 3 b. These molecules are the only NAT degradants reported by NAT-containing HSA formulations after prolonged hot pressing (up to 3 years at 25 ℃) (Fang, l.et al, Chromatogr a,2011.1218(41): p.7316-24), and the MS2 fragmentation pattern observed in our study is consistent with this report (fig. 15E and 15F). Furthermore, peak 4 is the only NAT degradant observed in these studies using fluorescence detection (FIG. 8B), consistent with the report that H,1,2,3,3a,8,8 a-hexahydro-3 a-hydroxypyrrolo [2,3-B ] -indole 2-carboxylic acid (PIC)2a is one of the only common TRp degradants that fluoresce (Simat, T.J.et al., J.Agric Food Chem,1998.46(2): 490-498). Since no synthetic standards of these species were made, these identifications could not be finalized and it is still possible that doubly oxidized N-Ac-dioxoindolylalanine (N-Ac-DiOia) was still present in the incompletely resolved peak set 1. Peak assignments are summarized in table 8.

The NAT degradants observed in these studies (N-Ac-PIC, N-Ac-Oia, N-Ac-NFK, N-Ac-Kyn, and N-Ac-2a,8 a-dihydroxy-PIC) were largely identical to those reported by Simat et al for free tryptophan oxidized by hydrogen peroxide treatment (PIC, Oia, NFK, Kyn, DiOia, and 5-OH-Trp) (Simat, T.J.J.Agric FoodChem,1998.46(2): p.490-498). The definitive identification of tryptophan degradants in peptides and proteins is limited (because the isobaric nature of many tryptophan derivatives complicates the identification of degradants at the peptide and protein levels and the isolation of individual residues may lead to decomposition), but similarly the peptide/protein literature is consistent with NAT studies (Simat, T.J.et., J Agric Food Chem,1998.46(2): p.490-498; Fedorova, M., et al., Proteomics,2010.10(14): p.2692-700; Li, Y., et al., Anal Chem,2014.86(14): p.6850-7; Ronsein, G.E., et al., J.am Soc Mass Spectrum, 2009.20(2): p.188-97). One notable inconsistency is 5-OH-Trp-which is the major degradant of Trp in vivo (via the tryptophan hydroxylase pathway) and observed in Simat et al studies on free Trp-however, it is only observed at trace levels after oxidation of the tripeptide Ala-Trp-Ala under the same hydrogen peroxide conditions, is not definitively identified in the peptide and protein literature investigated, and is not observed in our studies on NAT.

Altogether, this suggests that Trp derivatives containing an amidated N-terminus relative to free Trp (as in NAT and in peptides/proteins) may be less susceptible to oxidation at the 5-position under non-enzymatic conditions.

Effect of NAT degradation of other excipients and proteins

Next, the effect of other excipients on NAT degradation was evaluated. Of particular interest is the presence of Met (another antioxidant often added as an antioxidant to pharmaceutical product formulations). In general, the inclusion of 5mM Met in the buffer formulation resulted in an overall reduction in total NAT oxidation (table 7, fig. 10), consistent with the assumption that the thioether moiety in Met can act as an oxidation pool. The effect of Met on NAT stability varied between oxidation models: met made modest improvements in NAT stability in the AAPH model (4-8% total NAT loss, depending on buffer), slightly better improvement under ICH light pressure conditions (10-16%), and significant improvement in the fenton model (25%) (table 8). The significant reduction in NAT oxidation when formulated with Met in fenton conditions may be due to the quenching of hydrogen peroxide by Met (Ji, j.a., et al, J Pharm Sci,2009.98(12): p.4485-500). The addition of Met did not appear to alter the oxidation mechanism in each model system, as the distribution of the major species observed in the pressure samples formulated for Met was unchanged from those formulated without Met (data not shown).

The effect of low concentrations of protein on AAPH-induced NAT degradation was also analyzed. Both antibodies (protein 1 and protein 2) were diluted to 1.0mg/ml in buffer and formulated with 0.3mM NAT (approximately 45:1mol NAT: mol protein). The level of NAT degradation after AAPH pressure was largely consistent between protein-containing and protein-free solutions (approximately 40% loss of NAT peak area), as was the distribution of oxidant species (fig. 11). These results suggest that the presence of low levels of protein does not intrinsically affect NAT degradation levels/pathways under the oxidative (alkylperoxide-induced) stress conditions tested.

Real-time stability of NAT in pharmaceutical product formulations

With this model experience in hand, the amount of NAT oxidation expected to occur during manufacture and storage of mabs was next explored. FIG. 12 is a graphical representation of a comparison of the AAPH model pressure system containing 150mg/ml (1.0mM) antibody co-formulated with NAT, Met, and other excipients typical of monoclonal antibody formulations. Results are shown for the initial time points, -20 ℃ and 5 ℃ for 6 months (representing typical storage conditions), and 25 ℃ for 6 months (representing accelerated stability conditions). The oxidation level after manufacture and under typical storage conditions tested is directly negligible. After up to 6 months at 25 ℃, some degradation was observed (total NAT loss 16.8%). Of interest, the corresponding vehicle showed significantly lower NAT degradation-whereas the protein containing sample had 7.5% NAT loss after 3 months at 25 ℃, the corresponding vehicle showed only 1% loss of NAT. Even at higher temperatures (fig. 12), the vehicle showed minimal NAT degradation, suggesting that in some cases the presence of high concentrations of protein under accelerated thermal conditions may enhance NAT degradation. The 5 major species present in the accelerated stability samples corresponded to the major species identified in the stress model (fig. 12), suggesting that the model faithfully reproduces the NAT degradation pathway in the drug product samples.

In summary, using a chromatographic method for assessing the stability of N-Ac-tryptophan, an antioxidant known to protect Trp residues in protein therapeutics against oxidative stress, NAT shows degradation into a common set of degradants-including N-Ac-Oia, N-Ac-PIC, N-Ac-Kyn, and N-Ac-NFK (see structure figure 7) -largely independent of stress type under a wide range of stress conditions and in different model formulations, a discovery that has not been previously reported. These degradants are generally in agreement with the literature on Trp oxidation, with the exception that NAT degradation in the studied stress model does not lead to the formation of the N-acetylated form of 5-hydroxytryptophan (the most common physiological Trp degradant). Without being bound by theory, this suggests that under non-enzymatic conditions NAT (and possibly, by extension, Trp residues in proteins) is not degraded via the same intermediates as Trp catabolism. Indeed, without being bound by theory, the data suggest that oxidation of NAT occurs predominantly at the 2 and 3 positions of the indole ring.

Claims (137)

1. A method of reducing oxidation of a polypeptide in an aqueous formulation comprising adding to the formulation an amount of N-acetyl tryptophan that prevents oxidation of the polypeptide, wherein the polypeptide comprises at least one Solvent Accessible Surface Area (SASA) greater than aboutA tryptophan residue of (a).

2. A method of reducing oxidation of a polypeptide in an aqueous formulation comprising adding to the formulation an amount of N-acetyl tryptophan that prevents oxidation of the polypeptide, wherein the polypeptide comprises at least one tryptophan residue having a Solvent Accessible Surface Area (SASA) greater than about 30%.

3. A method of reducing oxidation of a polypeptide in an aqueous formulation comprising determining the SASA value of tryptophan residues in the polypeptide and if at least one tryptophan residue has an SASA value greater than aboutIs added to the formulation in an amount that prevents oxidation of the polypeptide.

4. The method of claim 3, wherein the SASA value of the tryptophan residue is calculated by molecular dynamics simulation.

5. The method of any one of claims 1-4, wherein the N-acetyl tryptophan is added to the formulation to a concentration of about 0.1mM to about 5 mM.

6. The method of any one of claims 1-5, wherein the N-acetyl tryptophan is added to the formulation to a concentration of about 0.1mM to about 1 mM.

7. The method of any one of claims 1-6, wherein the N-acetyl tryptophan is added to the formulation to a concentration of about 0.3 mM.

8. The method of any one of claims 1-7, wherein the oxidation of the polypeptide is reduced by about 50%, 75%, 80%, 85%, 90%, 95%, or 99%.

9. The method of any one of claims 1-8, wherein the formulation is stable at about 2 ℃ to about 8 ℃ for about 1095 days.

10. The method of any one of claims 1-9, wherein the concentration of protein in the formulation is about 1mg/mL to about 250 mg/mL.

11. The method of any one of claims 1-10, wherein the formulation has a pH of about 4.5 to about 7.0.

12. The method of any one of claims 1-11, wherein the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents.

13. The method of any one of claims 1-12, wherein the formulation is a pharmaceutical formulation suitable for administration to a subject.

14. The method of any one of claims 1-13, wherein the protein is an antibody.

15. The method of claim 14, wherein the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment.

16. A liquid formulation comprising a polypeptide and an amount of N-acetyl tryptophan to prevent oxidation of the polypeptide, wherein the polypeptide has at least one SASA greater than aboutA tryptophan residue of (a).

17. The liquid formulation of claim 16 wherein the N-acetyl tryptophan is added to the formulation to a concentration of about 0.1mM to about 5 mM.

18. The liquid formulation of claim 16 or 17, wherein the N-acetyl tryptophan is added to the formulation to a concentration of about 0.1mM to about 1 mM.

19. The liquid formulation of any one of claims 16-18, wherein the N-acetyl tryptophan is added to the formulation to a concentration of about 0.3 mM.

20. The liquid formulation of any one of claims 16-19 wherein the oxidation of the polypeptide is reduced by about 50%, 75%, 80%, 85%, 90%, 95% or 99%.

21. The liquid formulation of any one of claims 16-20, wherein the formulation is stable for about 1065 days at about 2 ℃ to about 8 ℃.

22. The liquid formulation of any one of claims 16-21, wherein the concentration of protein in the formulation is about 1mg/mL to about 250 mg/mL.

23. The liquid formulation of any one of claims 16-22 wherein the formulation has a pH of about 4.5 to about 7.0.

24. The liquid formulation of any one of claims 16-23, wherein the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents.

25. The liquid formulation of any one of claims 16-24, wherein the formulation is a pharmaceutical formulation suitable for administration to a subject.

26. The liquid formulation of any one of claims 16-25 wherein the protein is an antibody.

27. The liquid formulation of claim 26, wherein the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment.

28. A method for screening a formulation for reduced polypeptide oxidation, wherein the polypeptide comprises at least one SASA greater than aboutThe method comprising

Adding an amount of N-acetyl tryptophan to an aqueous composition comprising the polypeptide,

adding 2, 2' -azobis (2-aminopropane) dihydrochloride (AAPH) to the composition,

incubating a composition comprising the polypeptide, N-acetyl tryptophan and AAPH at about 40 ℃ for about 14 hours,

measuring the oxidation of a tryptophan residue in the polypeptide,

formulations which contain an amount of N-acetyl tryptophan that results in no more than about 20% oxidation of the tryptophan residues of the polypeptide are suitable formulations for reducing oxidation of the polypeptide.

29. A method for screening a formulation for reduced polypeptide oxidation comprising

Determining the SASA value of the tryptophan residue in the polypeptide, wherein the SASA is greater than aboutThe tryptophan residue of (a) is subjected to oxidation,

adding an amount of N-acetyl tryptophan to an aqueous composition comprising the polypeptide,

adding 2, 2' -azobis (2-aminopropane) dihydrochloride (AAPH) to the composition,

incubating a composition comprising the polypeptide, N-acetyl tryptophan and AAPH at about 40 ℃ for about 14 hours,

measuring the oxidation of a tryptophan residue in the polypeptide,

formulations which contain an amount of N-acetyl tryptophan that results in no more than about 20% oxidation of the tryptophan residues of the polypeptide are suitable formulations for reducing oxidation of the polypeptide.

30. The method of claim 29, wherein the SASA value for the tryptophan residue is calculated by molecular dynamics simulation.

31. A kit comprising the liquid formulation of any one of claims 16-26.

32. An article of manufacture comprising the liquid formulation of any one of claims 16-26.

33. A method for determining whether a polypeptide in a liquid formulation comprises tryptophan residues susceptible to oxidation, the method comprising calculating one or more molecular descriptors for each tryptophan residue in the polypeptide based on the amino acid sequence of the polypeptide and applying the one or more molecular descriptors to a machine learning algorithm practiced on the one or more molecular descriptors to predict tryptophan oxidation, wherein the molecular descriptors comprise one or more of:

a) at a position delta to tryptophanThe number of aspartic acid side chain oxygens in,

b) side chain Solvent Accessibility Surface Area (SASA),

c) the delta carbon of the SASA is added,

d) at a position delta to tryptophanThe total positive charge in the positive electrode stack,

e) the main chain of the SASA is provided,

f) the side chain angle of the tryptophan is shown,

g) at a position delta to tryptophanThe packing density of the inner part of the bag,

h) the angle of the tryptophan main chain is shown,

i) the SASA of the pseudo-pi track,

j) main chain flexibility, or

k) At a position delta to tryptophanThe total negative charge in.

34. The method of claim 33, wherein 2,3, 4,5, 6,7, 8, 9, 10, or 11 of the molecular descriptors are used in the molecular simulation.

35. The method of claim 33, wherein the molecular descriptor comprises the following:

a) at a position delta to tryptophanThe number of aspartic acid side chain oxygens in,

b) side chain Solvent Accessibility Surface Area (SASA),

c) the delta carbon of the SASA is added,

d) at a position delta to tryptophanThe total positive charge in the positive electrode stack,

e) the main chain of the SASA is provided,

f) tryptophan side chain angle, and

g) at a position delta to tryptophanPacking density of the inner part.

36. The method of any one of claims 33-35, wherein the machine learning algorithm is practiced by matching molecular descriptors from a molecular dynamics simulation of a polypeptide based on the amino acid sequence of the polypeptide to experimental data for each tryptophan residue in the polypeptide.

37. The method of any one of claims 33-36, wherein oxidation of greater than 35% of the tryptophan residues at a particular site is indicative of susceptibility to oxidation.

38. The method of any one of claims 33-37, wherein the one or more molecular descriptors are computed using a computer.

39. The method of any one of claims 33-38, wherein the protein is an antibody.

40. The method of claim 39, wherein the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment.

41. A method for reducing oxidation of a polypeptide, comprising identifying tryptophan residues susceptible to oxidation according to the method of any one of claims 33-40 and introducing an amino acid substitution in the polypeptide to replace one or more tryptophan residues susceptible to oxidation with an amino acid residue that is not subject to oxidation.

42. A method for reducing oxidation of a polypeptide, comprising introducing an amino acid substitution in the polypeptide to replace one or more tryptophan residues susceptible to oxidation, wherein the one or more tryptophan residues susceptible to oxidation are identified by the method of any one of claims 33-40.

43. The method of claim 41 or 42, wherein the tryptophan residue is replaced with an amino acid residue selected from the group consisting of tyrosine, phenylalanine, leucine, isoleucine, alanine, and valine.

44. A method for reducing oxidation of a polypeptide in an aqueous formulation comprising determining the presence of one or more oxidation-susceptible tryptophan residues in the polypeptide according to the method of any one of claims 33-38, and adding an effective amount of an antioxidant to an aqueous formulation comprising a polypeptide having one or more oxidation-susceptible tryptophan residues.

45. A method for reducing oxidation of a polypeptide in an aqueous formulation comprising adding an amount of an antioxidant to the aqueous formulation to prevent oxidation, wherein the polypeptide comprises one or more tryptophan residues susceptible to oxidation identified by the method of any one of claims 33-38.

46. The method of claim 45, wherein the antioxidant is N-acetyl tryptophan.

47. The method of claim 46, wherein the N-acetyl tryptophan is added to the formulation to a concentration of about 0.1mM to about 5 mM.

48. The method of claim 46 or 47, wherein the N-acetyl tryptophan is added to the formulation to a concentration of about 0.1mM to about 1 mM.

49. The method of any one of claims 46-48, wherein the N-acetyl tryptophan is added to the formulation to a concentration of about 0.3 mM.

50. The method of any one of claims 44-49, wherein the oxidation of the polypeptide is reduced by about 50%, 75%, 80%, 85%, 90%, 95%, or 99%.

51. The method of any one of claims 44-50, wherein the formulation is stable at about 2 ℃ to about 8 ℃ for about 1095 days.

52. The method of any one of claims 44-51, wherein the concentration of protein in the formulation is about 1mg/mL to about 250 mg/mL.

53. The method of any one of claims 44-52, wherein the formulation has a pH of about 4.5 to about 7.0.

54. The method of any one of claims 44-53, wherein the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents.

55. The method of any one of claims 44-54, wherein the formulation is a pharmaceutical formulation suitable for administration to a subject.

56. The method of any one of claims 44-55, wherein the protein is an antibody.

57. The method of claim 56, wherein the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment.

58. A liquid formulation comprising a polypeptide and an amount of N-acetyl tryptophan to prevent oxidation of the polypeptide, wherein the polypeptide has at least one tryptophan residue susceptible to oxidation as measured by the method of any one of claims 33-38.

59. The liquid formulation of claim 58, wherein the N-acetyl tryptophan is added to the formulation to a concentration of about 0.1mM to about 5 mM.

60. The liquid formulation of claim 58 or 60, wherein the N-acetyl tryptophan is added to the formulation to a concentration of about 0.1mM to about 1 mM.

61. The liquid formulation of any one of claims 58-60, wherein the N-acetyl tryptophan is added to the formulation to a concentration of about 0.3 mM.

62. The liquid formulation of any one of claims 58-61, wherein the oxidation of the polypeptide is reduced by about 50%, 75%, 80%, 85%, 90%, 95%, or 99%.

63. The liquid formulation of any one of claims 58-62, wherein the formulation is stable for about 1065 days at about 2 ℃ to about 8 ℃.

64. The liquid formulation of any one of claims 58-63, wherein the concentration of protein in the formulation is about 1mg/mL to about 250 mg/mL.

65. The liquid formulation of any one of claims 58-64, wherein the formulation has a pH of about 4.5 to about 7.0.

66. The liquid formulation of any one of claims 58-65, wherein the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents.

67. The liquid formulation of any one of claims 58-66, wherein the formulation is a pharmaceutical formulation suitable for administration to a subject.

68. The liquid formulation of any one of claims 58-67, wherein the protein is an antibody.

69. The liquid formulation of claim 68, wherein the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment.

70. A method for screening a formulation for reduced oxidation of a polypeptide, wherein the polypeptide comprises at least one tryptophan susceptible to oxidation identified by the method of any one of claims 33-40, comprising

Adding an amount of N-acetyl tryptophan to an aqueous composition comprising the polypeptide,

adding 2, 2' -azobis (2-aminopropane) dihydrochloride (AAPH) to the composition,

incubating a composition comprising the polypeptide, N-acetyl tryptophan and AAPH at about 40 ℃ for about 14 hours,

measuring the oxidation of a tryptophan residue in the polypeptide,

formulations which contain an amount of N-acetyl tryptophan that results in no more than about 20% oxidation of the tryptophan residues of the polypeptide are suitable formulations for reducing oxidation of the polypeptide.

71. A method for screening a formulation for reduced polypeptide oxidation comprising

a) Identifying a polypeptide comprising one or more tryptophan residues susceptible to oxidation by the method of any one of claims 33-40,

b) adding an amount of N-acetyl tryptophan to an aqueous composition comprising the polypeptide identified in step a),

c) adding 2, 2' -azobis (2-aminopropane) dihydrochloride (AAPH) to the composition,

d) incubating a composition comprising the polypeptide, N-acetyl tryptophan and AAPH at about 40 ℃ for about 14 hours,

e) measuring the oxidation of a tryptophan residue in the polypeptide,

formulations which contain an amount of N-acetyl tryptophan that results in no more than about 20% oxidation of the tryptophan residues of the polypeptide are suitable formulations for reducing oxidation of the polypeptide.

72. A kit comprising the liquid formulation of any one of claims 58-69.

73. An article of manufacture comprising the liquid formulation of any one of claims 58-72.

74. A method for measuring degradation of N-acetyl tryptophan (NAT) in a composition comprising N-acetyl tryptophan, the method comprising

a) Applying the composition to a reverse phase chromatography material, wherein the composition is loaded onto a chromatography material equilibrated in a solution comprising mobile phase A and mobile phase B, wherein mobile phase A comprises an acid in water and mobile phase B comprises an acid in acetonitrile,

b) eluting the composition from the reverse phase chromatography material with a solution comprising mobile phase A and mobile phase B, wherein the ratio of mobile phase B to mobile phase A is increased compared to step a), wherein NAT degradation products are eluted from the chromatography separately from intact NAT,

c) quantifying the NAT degradant and the intact NAT.

75. The method of claim 74, wherein the ratio of mobile phase B to mobile phase A in step a) is about 2: 98.

76. The method of claim 74 or 75, wherein the ratio of mobile phase B to mobile phase A in step B) increases linearly.

77. The method of claim 74 or 75, wherein the ratio of mobile phase B to mobile phase A in step B) is increased stepwise.

78. The method of any one of claims 74-77, wherein the flow rate for chromatography is about 1.0 mL/min.

79. The method of claim 78, wherein the ratio of mobile phase B to mobile phase A is increased to about 30: 70.

80. The method of claim 79, wherein the ratio of mobile phase B to mobile phase A is increased to about 30:70 in about 16 minutes.

81. The method of claim 79 or 80, wherein the ratio of mobile phase B to mobile phase A is further increased to about 90: 70.

82. The method of claim 81, wherein the ratio of mobile phase B to mobile phase A is further increased to about 90:70 in about 18.1 minutes.

83. The method of claim 78, wherein the ratio of mobile phase B to mobile phase A is increased to about 26: 74.

84. The method of claim 83, wherein the ratio of mobile phase B to mobile phase A is increased to about 26:74 in about 14 minutes.

85. The method of claim 83 or 84, wherein the ratio of mobile phase B to mobile phase A is further increased to about 90: 70.

86. The method of claim 85, wherein the ratio of mobile phase B to mobile phase A is further increased to about 90:70 in about 16.5 minutes.

87. The method of any one of claims 74-86, wherein mobile phase A comprises about 0.1% acid in water.

88. The process of any one of claims 74-87, wherein mobile phase B comprises about 0.1% acid in acetonitrile.

89. The method of any one of claims 74-88, wherein the acid is formic acid.

90. The method of any one of claims 74-89, wherein the reverse phase chromatography material comprises a C18 module.

91. The method of any one of claims 74-90, wherein the reverse phase chromatography material comprises a solid support.

92. The method of claim 91, wherein the solid support comprises silica.

93. The method of any one of claims 74-92, wherein the reverse phase chromatography material is contained in a column.

94. The method of any one of claims 74-93, wherein the reverse phase chromatography material is a High Performance Liquid Chromatography (HPLC) material or an ultra high performance liquid chromatography (UPLC) material.

95. The method of any one of claims 74-94, wherein NAT and NAT degradation products are detected by absorbance at 240 nm.

96. The method of any one of claims 74-95, wherein NAT degradation products are identified by mass spectrometry.

97. The method of any one of claims 74-96, wherein the concentration of NAT in the composition is about 10nM to about 1 mM.

98. The method of any one of claims 74-97 wherein the NAT degradation products comprise one or more of N-Ac- (H,1,2,3,3a,8,8 a-hexahydro-3 a-hydroxypyrrolo [2,3-b ] -indole 2-carboxylic acid) (N-Ac-PIC), N-Ac-oxindolyl alanine (N-Ac-Oia), N-Ac-N-formyl-kynurenine (N-Ac-NFK), N-Ac-kynurenine (N-Ac-Kyn), and N-Ac-2a,8 a-dihydroxy-PIC.

99. A method for measuring degradation of N-acetyl tryptophan (NAT) in a composition comprising N-acetyl tryptophan and a polypeptide, the method comprising

a) The composition was diluted with about 8M guanidine,

b) removing the polypeptide from the composition to form a composition,

c) applying the composition to a reverse phase chromatography material, wherein the composition is loaded onto a chromatography material equilibrated in a solution comprising mobile phase A and mobile phase B, wherein mobile phase A comprises an acid in water and mobile phase B comprises an acid in acetonitrile,

d) eluting the composition from the reverse phase chromatography material with a solution comprising mobile phase A and mobile phase B, wherein the ratio of mobile phase B to mobile phase A is increased compared to step a), wherein NAT degradation products are eluted from the chromatography separately from intact NAT,

e) quantifying the NAT degradant and the intact NAT.

100. The method of claim 99, wherein the composition is diluted in about 8M guanidine such that the final concentration of NAT in the composition ranges from about 0.05mM to about 0.2 mM.

101. The method of claim 99 or 100, wherein the composition is diluted in about 8M guanidine such that the final concentration of polypeptide in the composition is less than or equal to about 25 mg/mL.

102. The method of any one of claims 99-101, wherein the polypeptide is removed from the composition by filtration.

103. The process of claim 102, wherein the filtration uses a filtration membrane having a molecular weight cut-off of about 30 kDal.

104. The method of any one of claims 99-103, wherein the ratio of mobile phase B to mobile phase a in step a) is about 2: 98.

105. The method of any one of claims 99-104, wherein the ratio of mobile phase B to mobile phase a in step B) increases linearly.

106. The method of any one of claims 99-105, wherein the ratio of mobile phase B to mobile phase a in step B) is increased stepwise.

107. The method of any one of claims 99-106, wherein the flow rate for chromatography is about 1.0 mL/min.

108. The method of claim 107, wherein the ratio of mobile phase B to mobile phase a is increased to about 30: 70.

109. The method of claim 108, wherein the ratio of mobile phase B to mobile phase a is increased to about 30:70 in about 16 minutes.

110. The method of claim 108 or 109, wherein the ratio of mobile phase B to mobile phase a is further increased to about 90: 70.

111. The method of claim 110, wherein the ratio of mobile phase B to mobile phase a is further increased to about 90:70 in about 18.1 minutes.

112. The method of claim 111, wherein the ratio of mobile phase B to mobile phase a is increased to about 26: 74.

113. The method of claim 112, wherein the ratio of mobile phase B to mobile phase a is increased to about 26:74 in about 14 minutes.

114. The method of claim 112 or 113, wherein the ratio of mobile phase B to mobile phase a is further increased to about 90: 70.

115. The method of claim 114, wherein the ratio of mobile phase B to mobile phase a is further increased to about 90:70 in about 16.5 minutes.

116. The method of any one of claims 99-115, wherein mobile phase a comprises about 0.1% acid in water.

117. The process of any one of claims 99-116, wherein mobile phase B comprises about 0.1% acid in acetonitrile.

118. The method of any one of claims 99-117, wherein the acid is formic acid.

119. The method of any one of claims 99-118, wherein the reverse phase chromatography material comprises a C18 module.

120. The method of any one of claims 99-119, wherein the reverse phase chromatography material comprises a solid support.

121. The method of claim 120, wherein the solid support comprises silica.

122. The method of any one of claims 99-121, wherein the reverse phase chromatography material is contained in a column.

123. The method of any one of claims 99-122, wherein the reverse phase chromatography material is a High Performance Liquid Chromatography (HPLC) material or an ultra high performance liquid chromatography (UPLC) material.

124. The method of any one of claims 99-123, wherein NAT and NAT degradation products are detected by absorbance at 240 nm.

125. The method of any one of claims 99-124, wherein NAT degradation products are identified by mass spectrometry.

126. The method of any one of claims 99-125, wherein the concentration of NAT in the composition is between about 0.1mM and about 5 mM.

127. The method of any one of claims 99-126, wherein the concentration of NAT in the composition is about 0.3 mM.

128. The method of any of claims 99-127, wherein the NAT degradation product comprises one or more of N-Ac-PIC, N-Ac-Oia, N-Ac-NFK, N-Ac-Kyn, and N-Ac-2a,8 a-dihydroxy-PIC.

129. The method of any one of claims 99-128, wherein the concentration of protein in the formulation is about 1mg/mL to about 250 mg/mL.

130. The method of any one of claims 99-129, wherein the formulation has a pH of about 4.5 to about 7.0.

131. The method of any one of claims 99-130, wherein the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents.

132. The method of any one of claims 99-131, wherein the formulation is a pharmaceutical formulation suitable for administration to a subject.

133. The method of any one of claims 99-132, wherein the polypeptide is an antibody.

134. The method of claim 133, wherein the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody or antibody fragment.

135. A method for monitoring degradation of NAT in a composition comprising measuring degradation of NAT in a sample of the composition according to the method of any one of claims 74-134, wherein the method is repeated one or more times.

136. The method of claim 135, wherein the method is repeated every month, every 2 months, every 4 months, or every 6 months.

137. A mass assay for a pharmaceutical composition, the mass assay comprising measuring degradation of NAT in a sample of the pharmaceutical composition according to the method of any one of claims 74-134, wherein the amount of NAT degradant measured in the composition determines whether the pharmaceutical composition is suitable for administration to an animal.