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WO2025054112A2 - Procédés de régulation de la dégradation du polysorbate dans des formulations biothérapeutiques - Google Patents

Procédés de régulation de la dégradation du polysorbate dans des formulations biothérapeutiques Download PDF

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Publication number
WO2025054112A2
WO2025054112A2 PCT/US2024/044968 US2024044968W WO2025054112A2 WO 2025054112 A2 WO2025054112 A2 WO 2025054112A2 US 2024044968 W US2024044968 W US 2024044968W WO 2025054112 A2 WO2025054112 A2 WO 2025054112A2
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pharmaceutical composition
incubating
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WO2025054112A3 (fr
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Ralf J. CARRILLO
Xuanwen Li
Taku TSUKIDATE
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Merck Sharp and Dohme LLC
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Merck Sharp and Dohme LLC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01013—Sterol esterase (3.1.1.13)

Definitions

  • This disclosure relates generally to methods for removing enzymatic activity from a pharmaceutical composition.
  • PS Polysorbates
  • PS degradation causes for PS degradation include chemical stress and residual host cell-derived enzymes (Ref. 3).
  • PSDE PS- degradative enzymes
  • lipases a chemical proteomics approach was used to identify phospholipase A2, group VII (PLA2G7) in an ion-exchange column pool that exhibited considerable PS degradation, and the enzymatic activity of PLA2G7 on PS was confirmed using a recombinant protein (Ref. 8).
  • HCP-derived polysorbate degradation is an ongoing and growing challenge in the pharmaceutical industry as more commercial formulations move towards subcutaneous dosing.
  • Subcutaneous dosing requires monoclonal antibody (mAb) formulations to be dosed at higher concentrations due to loss of absorption of drug product (DP) through subcutaneous layers.
  • mAb monoclonal antibody
  • DP drug product
  • the required increase in mAb concentration subsequently is accompanied with increased HCP levels needed to be removed in the DP.
  • the competitive market advantage of offering subcutaneous dosing versus l.V. dosing is enormous but as mentioned comes with additional challenges in titer, purification, and stability of DP.
  • the present disclosure provides a method for reducing serine hydrolase activity in a pharmaceutical composition
  • a method for reducing serine hydrolase activity in a pharmaceutical composition comprising: a) incubating the pharmaceutical composition at a temperature of from about 40°C to about 70°C; wherein the pharmaceutical composition comprises i) an active biological ingredient, ii) a serine hydrolase that cleaves polysorbate while in its native conformation, and iii) polysorbate; wherein the serine hydrolase is irreversibly misfolded after the incubating step; and wherein the active biological ingredient in the pharmaceutical composition is not irreversibly misfolded after the incubating step.
  • the serine hydrolase is Phospholipase A2 Group VII (PLA2G7) or Sialic Acid Acetylesterase (SIAE).
  • the active biological ingredient has a melting temperature that is about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, or about 15°C above the melting temperature of PLA2G7, SIAE, or both PLA2G7 and SIAE, wherein the melting temperature is measured by differential scanning fluorimetry (DSF).
  • DFS differential scanning fluorimetry
  • the active biological ingredient has a melting temperature that is 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, or 15°C above the melting temperature of PLA2G7, SIAE, or both PLA2G7 and SIAE, wherein the melting temperature is measured by differential scanning fluorimetry’ (DSF).
  • DFS differential scanning fluorimetry
  • the active biological ingredient has a melting temperature that is about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, or about 15°C above the melting temperature of PLA2G7, SIAE, or both PLA2G7 and SIAE, wherein the melting temperature is measured by differential scanning calorimetry 7 (DSC).
  • DSC differential scanning calorimetry 7
  • the active biological ingredient has a melting temperature that is 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, or 15°C above the melting temperature of PLA2G7, S1AE. or both PLA2G7 and SIAE, wherein the melting temperature is measured by differential scanning calorimetry (DSC).
  • DSC differential scanning calorimetry
  • the step of incubating the pharmaceutical composition is performed at a temperature from about 45°C to about 55°C. from about 47°C to about 53°C, or at about 50°C. In some embodiments, the step of incubating the pharmaceutical composition is performed at a temperature from 45°C to 55°C, from 47°C to 53°C, or at 50°C. In some embodiments, the step of incubating the pharmaceutical composition is performed at a temperature from about 45°C to about 65°C, from about 50°C to about 60°C, from about 53°C to about 57°C, or at about 55°C.
  • the step of incubating the pharmaceutical composition is performed at a temperature from 45°C to 65°C, from 50°C to 60°C, from 53°C to about 57°C, or at 55°C. In some embodiments, incubating the pharmaceutical composition is performed at a temperature from about 50°C to about 67°C, from about 55°C to about 65°C, from to 57°C to about 63°C, or at about 60°C. In some embodiments, incubating the pharmaceutical composition is performed at a temperature from 50°C to 67°C. from 55°C to 65°C, from 57°C to 63°C, or at 60°C.
  • incubating the pharmaceutical composition is performed from about 1 hour to about 20 hours, from about 2 hours to about 20 hours, from about 3 hours to about 20 hours, from about 4 hours to about 20 hours, from about 5 hours to about 20 hours, from about 6 hours to about 20 hours, from about 7 hours to about 20 hours, from about 8 hours to about 20 hours, from about 9 hours to about 20 hours, from about 10 hours to about 20 hours, from about 12 hours to about 20 hours, from about 15 hours to about 20 hours, or from about 17 hours to about 20 hours.
  • incubating the pharmaceutical composition is performed from 1 to 20 hours, from 2 hours to 20 hours, from 3 hours to 20 hours, from 4 hours to 20 hours, from 5 hours to 20 hours, from 6 hours to 20 hours, from 7 hours to 20 hours, from 8 hours to 20 hours, from 9 hours to 20 hours, from 10 hours to 20 hours, from 12 hours to 20 hours, from 15 hours to 20 hours, or from 17 hours to 20 hours. In some embodiments.
  • incubating the pharmaceutical composition is performed for at least about 5 minutes, at least about 7 minutes, at least about 10 minutes, at least about 12 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 7 hours, at least about 10 hours, at least about 15 hours, or at least about 20 hours.
  • incubating the pharmaceutical composition is performed for at least 5 minutes, at least 7 minutes, at least 10 minutes, at least 12 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 7 hours, at least 10 hours, at least 15 hours, or at least 20 hours.
  • incubating the pharmaceutical composition is performed for about 5 minutes, about 7 minutes, about 10 minutes, about 12 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 7 hours, about 10 hours, about 15 hours, or about 20 hours. In some embodiments, incubating the pharmaceutical composition is performed for 5 minutes, 7 minutes, 10 minutes, 12 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 7 hours, 10 hours, 15 hours, or 20 hours.
  • the pharmaceutical composition has a volume of from about 1 ml to about 5 ml, from about 2.5 ml to about 7.5 ml, from about 5 ml to about 10 ml, from about 7.5 ml to about 12.5 ml, from about 10 ml to about 15 ml, from about 12.5 ml to about 17.5 ml, or from about 15 ml to about 20 ml.
  • the pharmaceutical composition has a volume of from 1 ml to 5 ml, from 2.5 ml to 7.5 ml, from 5 ml to 10 ml, from 7.5 ml to 12.5 ml, from 10 ml to 15 ml.
  • the pharmaceutical composition has a volume of about 20 ml or less, about 17 ml or less, about 15 ml or less, about 12 ml or less, about 10 ml or less, about 9 ml or less, about 8 ml or less, about 7 ml or less, about 6 ml or less, about 5 ml or less, about 4 ml or less, about 3 ml or less, about 2 ml or less, or about 1 ml or less. In some embodiments, the pharmaceutical composition has a volume of 20 ml or less. 17 ml or less.
  • the step of incubating is performed during upstream or downstream processing prior to any filling of the drug product into containers for storage or administration. In some embodiments, the step of incubating is performed post filling of the drug product into containers for storage or administration.
  • the active biological ingredient is selected from the group consisting of: an enzyme; a hormone; a fusion protein, an Fc-containing protein; an immunoconjugate; a cytokine; and an antibody or antigen binding fragment thereof.
  • the antibody is selected from the group consisting of: a monoclonal antibody; a chimeric antibody; a humanized antibody; a human antibody; and a multispecific antibody .
  • the multispecific antibody is a bispecific antibody or a trispecific antibody.
  • the antigen binding fragment thereof is selected from the group consisting of: a Fab fragment; a Fab' fragment; a F(ab')2 fragment; an scFv; a di-scFv; a bi-scFv; a tandem (di, tri) scFv; an Fv; an sdAb; a tri-functional antibody; a BiTE; a diabody; and a triabody.
  • the serine hydrolase is selected from the group consisting of: RB Binding Protein 9 (RBBP9), Palmitoyl-Protein Thioesterase 2 (PPT2), Phospholipase A2 Group VII (PLA2G7; known to cleave polysorbate), Cathepsin A (CTSA), and Acylaminoacyl-Peptide Hydrolase.
  • RBBP9 RB Binding Protein 9
  • PPT2 Palmitoyl-Protein Thioesterase 2
  • Phospholipase A2G7 Phospholipase A2 Group VII
  • CTSA Cathepsin A
  • Acylaminoacyl-Peptide Hydrolase is selected from the group consisting of: PLA2G7, Sialic Acid Acetylesterase (SIAE), and Phospholipase B Domain Containing 2 (PLBL2).
  • the polysorbate is selected from the group consisting of: polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the disclosure provides a pharmaceutical composition produced by any one of the methods embodied above. In some embodiments, the disclosure provides a pharmaceutical composition obtainable by any one of the methods embodied above.
  • FIG. IB is a line graph of heat inactivation of PLA2G7 in PAP. The intensity values corresponding to PLA2G7 are extracted from FIG. 1A and plotted against incubation temperature.
  • FIG. 2A is a line graph that plots the fluorescence of unfolding for mAbl utilizing Spyro Orange extrinsic dye, a melting temperature comparison between extrinsic fluorescence and differential scanning calorimetry (DSC) reported Tms are given.
  • FIG. 2B is a line graph plotting the 2nd derivative of unfolding for PL2GA7, SIAE and PLBL2, superimposed onto the mAbl unfolding profile.
  • FIG. 2C is a line graph plotting the aggregation (diameter in nm) of PL2AG7, S1AE. PLBL2 and mAbl measured by Dynamic Light Scatering (DLS). The 95% confidence band is shaded around the line for each molecule.
  • FIG. 2D is a line graph that fits the normalized DLS unfolding and aggregation profiles of PL2AG7, SIAE, PLBL2 and mAbl. The 95% confidence band is shaded around the line for each.
  • FIGs. 3A-3C show PS degradation in PAP over four weeks of incubation at 25. 37, 40 and 50°C.
  • FIG. 3 A is a line graph ploting the relative change of PS concentration in mAbl over four weeks.
  • FIG. 3B is a line graph plotting the relative change of PS concentration of mAb2 over four weeks.
  • FIG. 3C is a line graph ploting the relative change of PS concentration of formulation buffer with no protein over four weeks.
  • N 1.
  • FIG. 4A is a line graph of the decrease in PS-80 between heat inactivated mAbl drug product and control (non-heat inactivated) drug product.
  • FIG. 4B is a bar graph showing sub- visible particle counts for heat inactivated mAbl drug product.
  • FIG. 4C is a line graph of the percentage of high molecular weight species over time for heat inactivated mAbl drug product and control.
  • the present disclosure describes methods for heat inactivating serine hydrolases to reduce PS degradation and particle formation in drug product.
  • the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.
  • the term “about” refers to plus or minus up to 10% of the value it modifies (rounded up to the nearest whole number if the value is not sub-dividable, such as a number of molecules or nucleotides).
  • such variation can occur through typical measuring, handling and sampling procedures involved in the preparation, characterization and/or use of the substance or composition, through inadvertent error in these procedures, through differences in the manufacture, source, or purity of the ingredients employed to make or use the compositions or carry out the procedures.
  • the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of.”
  • the terms “comprise(s).” “include(s).” ‘‘having,” “has.” “may.” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients/steps and permit the presence of other ingredients/steps.
  • such description should be construed as also describing compositions or processes as “consisting of’ and “consisting essentially of’ the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.
  • the term “pharmaceutical composition” refers to a liquid composition containing an active biological ingredient (e.g., an antibody or antigen binding fragment thereof)), along with one or more additional excipients.
  • exemplary excipients may include additives, diluents, buffers, sugars, amino acids, chelating agents, surfactants, polyols, bulking agents, stabilizers, lyo-protectants, solubilizers, emulsifiers, salts, adjuvants, tonicity enhancing agents, delivery vehicles, and anti-microbial preservatives.
  • Active biological ingredients may include, but are not limited to: antibodies; antigen binding fragments of antibodies; multi-specific antibodies (e.g., bispecific antibodies); chimeric proteins that are a fusion of a ligand-binding protein and an effector protein (e.g., etanercept); or any other protein or polypeptide used to treat a disease or disorder in a human or animal.
  • the term “antibody” or “immunoglobulin” as used herein refers to a glycoprotein comprising at least two heavy chains (HCs) and two light chains (LCs) interconnected by disulfide bonds.
  • Each HC is comprised of a heavy chain variable region or domain (VH) and a heavy chain constant region or domain.
  • Each light chain is comprised of an LC variable region or domain (VL) and a LC constant domain.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • the basic antibody structural unit for antibodies is a Y-shaped tetramer comprising two HC/LC pairs (2H).
  • Each tetramer includes two identical pairs of polypeptide chains, each pair having one LC (about 25 kDa) and HC chain (about 50-70 kDa) (H+L).
  • Each HC:LC pair comprises one VH: one VL pair.
  • the one VH:one VL pair may be referred to by the term “Fab’'.
  • each antibody tetramer comprises two Fabs, one per each arm of the Y-shaped antibody.
  • the antibody may include post-translational modifications thereof (e.g., C-terminal Lysine clipping in the heavy chain, conversion of glutamine or glutamic acid to pyroglutamate or pyroglutamic acid) which may occur when recombinantly expressed in host cells (e.g., CHO cells), or during purification/storage.
  • post-translational modifications thereof e.g., C-terminal Lysine clipping in the heavy chain, conversion of glutamine or glutamic acid to pyroglutamate or pyroglutamic acid
  • host cells e.g., CHO cells
  • antigen binding fragment refers to a polypeptide or polypeptides comprising a fragment of a full-length antibody, which retains the ability to specifically bind to the antigen bound by the full length antibody, and/or to compete with the full length antibody for specifically binding to the antigen.
  • antigen binding fragments include, but are not limited to: Fab fragments; Fab’ fragments; F(ab’)2 fragments; Fv regions; scFv proteins; and diabodies.
  • Fab fragment refers to an antigen binder comprising one antibody light chain and the CHI and Vj-[ of one antibody heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • a Fab fragment can be the product of papain cleavage of an antibody.
  • Fab 1 fragment refers to an antigen binder comprising one antibody light chain and a portion or fragment of one antibody heavy chain that contains the Vp
  • F(ab')2 fragment refers to an antigen binder comprising two antibody light chains and two heavy chains containing the Vj-[ and the CHI domain up to a region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains.
  • An F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.
  • An F(ab')2 fragment can be the product of pepsin cleavage of an antibody.
  • v region refers to an antigen binder comprising the variable regions from both the heavy and light chains of an antibody but lacks the constant regions.
  • the term “ScFv” or “single-chain variable fragment” refers to a fusion protein comprising a V
  • the linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the V [ with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.
  • the term “diabody” refers to an antigen binder comprising a small antibody fragment with two antigen-binding regions, which fragments comprise a heavy chain variable domain (V [) connected to a light chain variable domain (VL) in the same polypeptide chain (Vpj-Vp or VL-VJL).
  • V [ heavy chain variable domain
  • VL light chain variable domain
  • Vpj-Vp or VL-VJL linker that is too short to allow pairing between the two domains on the same chain
  • the domains are forced to pair with the complementarity domains of another chain and create two antigen-binding regions.
  • Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.
  • Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136 For a review of engineered antibody
  • serine hydrolase refers to a class of enzymes that contains a serine at the active site which is important for hydrolyzing a particular protein.
  • the serine hydrolase is capable of cleaving polysorbate.
  • the serine hydrolase is capable of cleaving polysorbate 80 (PS-80).
  • the serine hydrolase is from a CHO cell or from E. Coli
  • the serine hydrolase is PLA2G7 or SIAE.
  • polysorbate refers to a heterogenous mixture of fatty acylated sorbitan or isosorbide.
  • exemplary polysorbates include, but are not limited to, polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).
  • the term “irreversibly misfolded” or “irreversibly denatured” refers to a protein which has been forced out of its native conformation due to heating beyond its melting temperature and is unable to return to its native conformation due to the kinetic forces preventing the protein from properly refolding to its native conformation. In its misfolded or denatured state, the protein is unable to catalyze enzymatic reactions or bind to other proteins as it would in its native conformation.
  • Example 1 Profiling Heat Sensitivity of serine hydrolase activity
  • the inventors profiled the heat sensitivity of serine hydrolase activity in a monoclonal antibody (mAbl) Protein A pool (PAP) with the active site-directed chemical reporter FP-biotin, which enables the sensitive detection of active serine hydrolases.
  • the PAP was incubated at various temperatures between 40-75°C for 5 minutes. The samples were subsequently cooled on ice. buffer-exchanged with Tris-HCl pH 8.0, and treated with FP-biotin to label serine hydrolases having residual activity. Labelled proteins were then enriched with streptavidin magnetic beads, digested with trypsin, and identified with liquid chromatography-tandem mass spectrometry (LC- MS/MS).
  • LC- MS/MS liquid chromatography-tandem mass spectrometry
  • the inventors identified 48 proteins with at least two unique peptides, including five serine hydrolases: RB Binding Protein 9 (RBBP9), Palmitoyl-Protein Thioesterase 2 (PPT2), Phospholipase A2 Group VII (PLA2G7; known to cleave polysorbate), Cathepsin A (CTSA), and Acylaminoacyl-Peptide Hydrolase (APEH) (FIG. 1 A).
  • Five proteins, including the mAbl light and heavy chains, were consistently identified across the entire temperature range. These proteins were highly abundant and likely bound the streptavidin magnetic beads non-specifically. In contrast, many other proteins showed a decreased intensity as the incubation temperature increased.
  • FIG. IB is a line graph of heat inactivation of PLA2G7 in PAP. The intensity values corresponding to PLA2G7 are extracted from FIG. 1A and plotted against incubation temperature.
  • the inventors profiled the heat sensitivity' of serine hydrolase activity in harvested cell culture fluid (HCCF) for mAbl (FIG. 1C). While serine hydrolase activity decreased upon heating, the activity curves for several enzymes including PLA2G7 shifted to higher temperatures in HCCF. This observation points to a potential matrix effect on enzyme activity such as pH and the presence of stabilizing proteins in the sample. These findings suggest that brief heat treatment could serve as a control method for PS degradation and beyond either through the dissociation of heat-sensitive enzymes from mAbl to facilitate their removal or through inactivation of such enzymes in formulation.
  • Example 2 Profiling Heat Sensitivity of serine hydrolase activity for a single mAb
  • the inventors further characterized the thermal stability of mAbl and several PS- degradative enzymes.
  • Each of mAbl, PLA2G7, Sialic Acid Acetylesterase (SIAE), and Phospholipase B Domain Containing 2 (PLBL2) was formulated in 10 mM L-histidine, 7% sucrose, pH 6.0.
  • DSF Differential scanning fluorimetry
  • DSC differential scanning calorimetry
  • PLA2G7 and SIAE unfolded at lower temperatures of 53.2°C and 57.8°C, respectively, while PLBL2 turned out to be as thermally stable as mAbl with its Tm at 65.4°C (FIG. 2B).
  • label-free dynamic light scattering (DLS) measurements revealed a dramatic increase in hydrodynamic diameters of PLA2G7, SIAE, and PLBL2 at temperatures between 50°C and 60°C, which is indicative of protein aggregation (FIG. 2C).
  • DLS dynamic light scattering
  • PS degradation slowed down at 50°C to a level that is comparable to 5°C samples; this is consistent with the observation that PLA2G7 lost ⁇ 90% of activity at 50°C relative to 40°C (see FIG. IB).
  • the inventors executed a similar experiment with mAb2 PAP and observed the same trend (FIG. 3B).
  • PS-80 concentration remained unchanged in buffer controls at all temperatures (FIG. 3C).
  • Example 4 Impact of heat inactivation on PS degradation and particle formation
  • the inventors evaluated the impact of brief heat inactivation on PS degradation and particle formation.
  • the inventors incubated mAbl drug product at 5°C or 50°C for 20 h and measured changes in PS-80 concentration, sub-visible particle count, and high-molecular weight species fraction over 28 days.
  • PS-80 concentration was measured with a high performance liquid chromatography (HPLC)charged aerosol detector (CAD) assay and its decrease was slower for the heat-inactivated sample, with relative decreases of 31% versus 58% after 28 days (FIG. 4A).
  • Sub-visible particle counts as measured with a HIACTM light obstruction assay was lower for the heat-inactivated sample (FIG. 4B).
  • Heat inactivation of the drug product (DP) formulation at smaller volumes such as 2 mL glass crimped vials or pre-filled syringes may be more advantageous than performing the heat inactivation at larger scale such as 5L drug substance (DS) during processing. It may be easier to implement the inactivation step at the final stages of manufacturing post filling in comparison to engineering an additional step during upstream or downstream processing because determining the correct column or vessel size, hold-time and filter would require resources and development in comparison to determining the hold-time alone for heat inactivation of post-filled DP.
  • Heat inactivation may also be more effective in small volumes, as assurance of heat distribution is greater at smaller volumes. Heat inactivation of serine hydrolase in larger volumes would require longer hold times than inactivating in smaller glass vials. The observed similarity in percentage of high molecular weight particles between heat inactivated and control samples throughout the disclosed analysis shows that heat inactivation is viable post-DP fill.
  • the heat inactivation step could be part of the original processing and DP filling at release (e.g., filling glass vials, plastic or glass syringes, or other containers) or could potentially be implemented as an emergency contingency alleviating DP already in circulation.
  • DP filling at release e.g., filling glass vials, plastic or glass syringes, or other containers
  • the heat inactivation of HCP during both the process and post DP filling stages offers the assurance that proteolytic activity will be decreased or diminished completely in manufactured DP, this potentially translates to purer DP with anticipated extended shelf-life.
  • FP-biotin was synthesized by WuXi AppTec.
  • the recombinant proteins PLBD2, PLA2G7, and SIAE were expressed in CHO-Express cells and purified by Genscript Biotech.
  • mAbl and mAb2 materials were internally prepared.
  • the mAbl HCCF (0. 1 mL) or PAP (24.56 g/L, 0.2 mL) was heated at 40, 45, 50, 55, 60, 65. 70, or 75 °C for 5 min on a thermomixer with 500 rpm agitation and cooled on ice.
  • the PAP sample was diluted with 50 mM Tris-HCl pH 8.0 (0.8 mL). To this was added FP-biotin (final concentration: 1 pM) at 25 °C, and the reaction mixture was incubated for 1 h on a thermomixer with 500 rpm agitation.
  • the reaction mixture was filtered through a Zeba spin column that had been equilibrated with 0.2 % SDS in PBS pH 7. 1.
  • the HCCF sample was diluted with 0.2 % SDS.
  • magnetic streptavidin beads (20 pL), and the suspension was incubated at room temperature for 1 h on an end-over-end rotator.
  • the beads were collected and washed three times with 0.2% SDS (1 mL) and once with PBS (1 mL).
  • sequencing- grade modified trypsin (1 pg) in Tns-HCl pH 8.0 (50 pL). and the reaction mixture was incubated at 37°C for 18 h on a thermomixer with 500 rpm agitation.
  • mAbl drug product 100 mg/mL mAbl, lOmM L-histidine, 7% sucrose, 0.02% (w/v) polysorbate 80, pH 6.0 (15 ml) was incubated at 5°C or 50°C for 20 h and filtered through a 0.22 pm filter. Aliquots were made for HPLC-CAD, UP-SEC, and sub- visible particle analyses (day 0). Remaining samples were incubated at 40°C and aliquoted for analyses on days 13 and 28.
  • Sypro Orange dye (Molecular Probes; Cat # S6650, Lot # 1001304) was added to MilliQ water (1:250) dilution. Protein samples of PLA2G7, SIAE, PLBL2, or mAbl formulated in lOmM L-histidine, 7% (w/v) sucrose, pH 6.0 were aliquoted (19pl each) into individual wells in a 96-well plate. Additionally, 1 pL of diluted Sypro Orange dye mix was added to each sample well and mixed. Plates were then covered, sealed and centrifuged at 1200 rpm for 3 min. Finally, plates were loaded into a thermal cycler for extrinsic fluorescence measurements.
  • DSC Differential scanning calorimetry
  • DLS measurements were performed in a Malvern Nano Zetasizer utilizing Non-Invasive Back Scatter (NIBS). lOOpL of each sample formulated at Img/mL in lOmM L-histidine, 7% (w/v) sucrose, pH 6.0 was added to a lOOpL quartz cuvette. DLS measurements were made between the 30°C to 90°C temperature range at 5°C increments with additional 5 minute incubation time between measurements.
  • NIBS Non-Invasive Back Scatter

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  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract

L'invention concerne des procédés de traitement thermique de compositions pharmaceutiques contenant au moins un ingrédient biologique actif (par exemple, une protéine), une sérine hydrolase qui clive le polysorbate lorsqu'elle est dans sa conformation native, et du polysorbate, ce qui permet de réduire l'activité de la sérine hydrolase dans la composition pharmaceutique.
PCT/US2024/044968 2023-09-08 2024-09-03 Procédés de régulation de la dégradation du polysorbate dans des formulations biothérapeutiques Pending WO2025054112A2 (fr)

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US202363581328P 2023-09-08 2023-09-08
US63/581,328 2023-09-08

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WO2025054112A3 WO2025054112A3 (fr) 2025-05-01

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2600488T3 (es) * 2014-05-23 2017-02-09 Ares Trading S.A. Composición farmacéutica líquida
AU2021228763A1 (en) * 2020-02-27 2022-09-15 Regeneron Pharmaceuticals, Inc. Activity based host cell protein profiling

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