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WO2024258896A1 - Procédés de détermination de la viscosité - Google Patents

Procédés de détermination de la viscosité Download PDF

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Publication number
WO2024258896A1
WO2024258896A1 PCT/US2024/033491 US2024033491W WO2024258896A1 WO 2024258896 A1 WO2024258896 A1 WO 2024258896A1 US 2024033491 W US2024033491 W US 2024033491W WO 2024258896 A1 WO2024258896 A1 WO 2024258896A1
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Prior art keywords
antigen
viscosity
antibody
binding protein
formulation
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PCT/US2024/033491
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English (en)
Inventor
Yangjie WEI
Wei Qin
Michelle LUONG
Christopher Sloey
Erick MAGLALANG
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Amgen Inc
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Amgen Inc
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Priority to AU2024302800A priority Critical patent/AU2024302800A1/en
Publication of WO2024258896A1 publication Critical patent/WO2024258896A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N11/00—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties

Definitions

  • the present disclosure is in the field of determining viscosity of pharmaceutical compositions comprising antigen-binding protein.
  • mAbs Monoclonal antibodies
  • the FDA has approved over 110 therapeutic antibodies and Fc fusion proteins across diverse therapeutic areas covering immunology, oncology, infectious diseases, genetic diseases, etc.
  • Extensive screening and engineering efforts go into the candidate selection process to identify the lead mAb candidate for clinical testing.
  • significant efforts need to be put into the evaluation of their developability profiles, which encompasses expression titer, stability, manufacturability, delivery options, pharmacokinetic properties, and others. Progression of a lead candidate with a desirable developability profile to clinical trial can reduce the risks, timelines, and resources associated with product development and increase the odds of regulatory approval and market competitiveness.
  • SC administration of mAbs with autoinjectors or pre-filled syringes offers several advantages over the common IV route as can potentially allow patients to self-administer at home and reduces hospital visits.
  • the SC route is particularly beneficial for patients with chronic diseases as frequent dosing is often required.
  • SC administration is usually limited to an injection volume less than 2 mL (Mathaes et al.) and this in turn requires a formulation concentration as high as 100-200 mg/mL to deliver enough active ingredients in a single dose.
  • mAbs at high concentrations can show elevated tendency to self-associate and manifest high viscosity, posing challenges during product manufacturing, storage, and SC injection.
  • Direct measurement of viscosity at 100 mg/mL and above is often not feasible due to limited materials at the early stage of candidate screening.
  • Predictive assays with low material consumption would serve as a powerful tool to ensure successful identification of lead candidates with low viscosity.
  • Diffusion interaction parameter (k D ) is such a label-free biophysical measure among many other techniques, and it has been shown to correlate with mAb viscosities at high concentrations (Connolly et al., 2012. 103(1): p. 69-78; Kingsbury et al. 2020. 6(32): p. eabb0372; Yadav et al. 2012. 101 (3): p. 998-1011 ; Zhou et al. U.S. 7,235,641 ).
  • k D Common techniques to measure k D include dynamic light scattering (DLS) and Taylor dispersion (Lavoisier et al., Latunde-Dada et al. 2016. 8(2): p. 386-392), with the former being more commonly used for screening purposes because it can be adapted to high-throughput analyses in microplate-based format.
  • Connolly et al. measured the k D of 29 mAbs in different buffers and found a strong linear dependence between k D and the exponential coefficient of concentration dependent viscosity profiles (Connolly et al., 2012. 103(1 ): p. 69-78).
  • PPIs measured in dilute solutions is not necessarily representative of molecular interactions seen at high concentrations.
  • the strength of various molecular interactions is strongly dependent on intermolecular distances; therefore, their relative contributions can differ remarkably at low versus at high concentrations (Neergaard et al. 2013. 49(3): p. 400-410; Chari et al., 2009. 26(12): p. 2607-2618).
  • the average surface-to-surface intermolecular distance of charged mAb molecules is far larger than their Debye screening length, and mAb molecules can freely rotate and see each other as point charges (Yadav et al., 2011 . 28(7): p.
  • Disclosed herein is a high-throughput assay measuring protein-protein interactions to predict/determine mAb viscosity.
  • the present disclosure provides methods of determining the viscosity of a pharmaceutical formulation comprising an antigen-binding protein, which requires a small amount of antigen-binding product and may be carried out at an early stage in the development process of the antigen-binding product.
  • the diffusion interaction parameter (k D ) of a formulation comprising an antigen binding protein at high concentrations is indicative of the viscosity of that formulation.
  • the disclosed methods comprise the steps of measuring the diffusion coefficient (D) of a plurality of compositions under a condition that reduces the surface charge of the antigen-binding protein in the formulation, and calculating the k D of the formulation based on the measured diffusion coefficient.
  • the monoclonal antibody (mAb) candidates often have pl values well above the formulation pH of 5.2 and therefore carry strong net positive charges in a low ionic buffer.
  • ko reflects primarily long-range electrostatic repulsions because it is measured in dilute solutions where short-range interactions are insignificant. This is further supported by the observation that k D values show a strong correlation with pl values of mAbs. Short-range interactions that play a key role in driving viscosity in concentrated mAb solutions are not being sufficiently captured by the k D measurements in common buffers, such as low ionic buffers.
  • the disclosure provides method of determining the viscosity of a pharmaceutical composition in which salts are added to purposely reduce antigen-binding protein surface charge.
  • the disclosed methods of measuring a k D will capture short-range interactions, if any.
  • the salt type and concentration was optimized to maximize the predictability of k D .
  • the disclosed methods were validated using one engineering panel of 10 mAbs and another mAb panel with 17 mABs with minimal sequence similarity in the complementarity-determining region (CDR). A consolidated data set consisting of these data points suggests a notable improvement in the prediction power of ko for viscosity using the optimized ko method described herein.
  • the disclosure provides for method of determining viscosity of a pharmaceutical formulation comprising an antigen-binding protein, the method comprising a) measuring a plurality of diffusion coefficients (D) of a plurality of compositions comprising different concentrations of the antigen-binding protein under a condition that reduces the surface charge of the antigen-binding protein, and b) calculate k D of the formulation based on the plurality of Ds from step a), wherein ko of the formulation is indicative of the viscosity of the formulation.
  • D diffusion coefficients
  • the plurality of compositions comprises the pharmaceutical formulation but have different concentrations of the antigen-binding protein in order to calculate kD, which is indicative of viscosity of the formulation which comprises a high concentration of an antigen-binding protein.
  • the disclosed method uses lower concentrations of antigen-binding protein to predict the viscosity of the formulation comprising high concentration of said antigen-binding protein.
  • Diffusion interaction parameter measures colloidal self-association in dilute solutions and has been reported to be predictive of mAb viscosity at high concentrations.
  • k D of IgG 1 mAb candidates measured in acetate buffer having a pH of 5.2 shows only weak correlation to their viscosities at 140 mg/mL.
  • Measuring ko in low strength acetate buffers reflects primarily long-range electrostatic repulsions because most internal lgG1 mAb candidates carry strong net positive charges in this low ionic strength formulation with pH (5.2) well below pl values of mAb candidates.
  • the viscosity of high concentration mAbs depends heavily on short-range molecular interactions.
  • the improved methods described herein comprise measuring D under a condition that reduces the surface charge of the antigenbinding protein, such as adding salt to the formulation to reduce the surface charge by suppressing charge repulsions and allowing for detection of key short-range interactions in dilute solutions.
  • the method improved the Pearson R2 between kD and viscosity (6-230 cP) from 0.42 to 0.80 for a data set consisting of 31 mAbs.
  • a low ko is indicative of a high viscosity formulation while a high k D is indicative of a low viscosity formulation.
  • a low viscosity formulation has a k D ranging from 10 cP to 30 cP.
  • a low viscosity formulation has a k D of about 10 cP, or about 1 1 cP, or about 12 cP, or about 13 cP, or about 14 cP, or about 15 cP, or about 16 cP, or about 17 cP, or about 18 cP, or about 19 cP, or about 20 cP, or about 21 cP, or about 22 cP, or about 23 cP, or about 24 cP, or about 25 cP, or about 26 cP, or about 27 cP or about 28 cP or about 29 cP or about 30 cP.
  • a k D ranging from 10 cP to about 30 cP may be used a cut-off for determining if the formulation has a low viscosity or high viscosity.
  • the condition to reduce the surface charge of the antigen-binding protein is the presence of salt in the composition, such as adding salt to the plurality of compositions comprising different concentrations of the antigen-binding protein and measuring the Ds of these plurality of compositions.
  • the salt may be any known salt, such as sodium chloride, potassium chloride, lithium chloride, magnesium chloride, calcium chloride, ammonium chloride, sodium sulfate, ammonium sulfate, potassium sulfate, lithium sulfate, magnesium sulfate, calcium sulfate, sodium phosphate, ammonium phosphate, potassium phosphate, lithium phosphate, magnesium phosphate, calcium phosphate, sodium bromide, ammonium bromide, potassium bromide, lithium bromide, magnesium bromide, calcium bromide, sodium nitrate, ammonium nitrate, potassium nitrate, lithium nitrate, magnesium nitrate, calcium nitrate, sodium chlorate, ammonium chlorate, potassium chlorate, lithium chlorate, magnesium chlorate, calcium chlorate, sodium iodide, ammonium iodide, potassium iodide, lithium iodide, magnesium iodide, calcium iodide, sodium per
  • the salt is sodium chloride (NaCI), for example the NaCI is at a concentration of about 10 mM to about 200 mM, about 10 mM to about 190 mM, about 10 mM to about 180 mM, about 10 mM to about 170 mM, about 10 mM to about 160 mM, about 10 mM to 150 mM, about 20 mM to about 180 mM, about 20 mM to about 160 mM, about 20 mM to about 150 mM, about 50 to about 150 mM, about 50 mM to about 160 mM, about 50 mM to about 150 mM, about 100 mM to about 150 mM, about 100 mM to 160 mM, about 100 mM to about 170 mM, about 100 mM to about 180 mM, about 100 to about 190 mM, about 100 mM to about 200 mM, about 100 mM to about 250 mM,
  • NaCI sodium chloride
  • the NaCI is at a concentration of about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 1 10 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 250 mM, about 300 mM, about 400 mM or about 500 mM.
  • the salt is ammonium sulfate.
  • the ammonium sulfate is at a concentration of about 5 mM to about 100 mM, about 5 mM to about 90 mM, about 5 mM to about 80 mM, about 5 mM to about 70 mM, about 5 mM to about 60 mM, about 5 mM to 50 mM, about 8 mM to about 80 mM, about 8 mM to about 60 mM, about 8 mM to about 50 mM, about 10 to about 50 mM, about 10 mM to about 60 mM.
  • the ammonium sulfate is at a concentration of about 5 mM, 8.33 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.
  • the disclosure also provides for a method of determining viscosity of a pharmaceutical formulation comprising an antigen-binding protein, the method comprising a) measuring a plurality of diffusion coefficients (D) of a plurality of compositions comprising different concentrations of the antigen-binding protein under a condition wherein the pH of the composition is near to the pl of the antigen-binding protein, and b) calculate k D of the formulation based on the plurality of Ds from step a) wherein k D of the formulation is indicative of the viscosity of the formulation.
  • D diffusion coefficients
  • the plurality of compositions comprises the pharmaceutical formulation but have different concentrations of the antigen-binding protein in order to calculate k D , the measurement of the which is indicative of viscosity of the formulation which comprises a high concentration of an antigen-binding protein.
  • the disclosed method uses lower concentrations of antigen-binding protein to predict the viscosity of the formulation comprising high concentration of said antigenbinding protein.
  • the pH of the formulation ranges from about 5.5 to about 9.5.
  • the pH of the formulation ranges from about 5.5 to about 9.0, or from about 5.5 to about 8.0, or from about 5.5 to about 7.7, or from about 5.5 to about 7.5, or from 5.5 to about 7.0, or about 6.0 to about 9.5, or from 6.0 to about 9.0, or from 6.0 to about 8.6, or from 6.0 to about 8.0, or from about 6.0 to about 7.8, or from about 6.0 to about 7.5, or from about 6.5 to about 9.5, or from about 6.5 to about 9.0, or from about 6.5 to about 8.6, or from 6.5 to about 8.0, or from 6.5 to about 7.8, or from about 6.5 to about 7.5, or from 7.5 to about 9.5, or from7.5 to about 9.0, or from 7.5 to about 8.6, or from 7.5 to about 9.5
  • the pH of the formulation is about 5.5, or about 5.8, or about 6.0 or about 6.5 or about 6.8, or about 7.0, or about 7.1 or about 7.2, or about 7.3, or about 7.4, or about 7.5, or about 7.6, or about 7.7, or about 7.8, or about 7.9, or about 8.0, or about 8.1 or about 8.2 or about 8.3 or about 8.4, or about 8.5 or about 8.6, or about 8.7, or about 8.8, or about 8.9, or about 9.0, or about 9.1 or about 9.2 or about 9.3 or about 9.4, or about 9.5.
  • the plurality of compositions comprises different concentrations of the antigen-binding protein.
  • the concentrations of the plurality of antigen-binding protein is less than or equal to ( ⁇ ) 20 mg/ml, such as the concentration of antigen-binding protein ranging from about 2 mg/ml to about 20 mg/ml, about 2 to about 19 mg/ml, about 2 mg/ml to about 18 mg/ml, about 2 mg/ml to about 17 mg/ml, 2 mg/ml to about 16 mg/ml, about 2 mg/ml to about 15 mg/ml, about 5 mg/ml to about 20 mg/ml, about 5 to about 19 mg/ml, about 5 mg/ml to about 18 mg/ml, about 5 mg/ml to about 17 mg/ml, about 5 mg/ml to about 16 mg/ml, about 5 mg/ml to about 15 mg/ml, about 10 mg/ml to about 20 mg/ml, about 10 to about 19 mg/ml, such as the concentration of antigen
  • the disclosure provides for methods of screening an excipient for preparation of a pharmaceutical formulation comprising an antigen-binding protein, wherein the method comprises determining the viscosity of the pharmaceutical formulation comprising the excipient using any of the methods disclosed herein, and comparing the viscosity of the pharmaceutical composition comprising the excipient with the viscosity of the pharmaceutical composition without the excipient.
  • the method further comprises the step of determining the viscosity of the pharmaceutical formulation without the excipient using any of the methods disclosed herein. Any of the disclosed methods may be used to screen a panel of excipients, and the screening may be carried out with high-throughput screening.
  • the disclosure provides a method of screening an excipient for preparation of a pharmaceutical formulation comprising an antigen-binding protein, the method comprising i) determining the viscosity of the pharmaceutical formulation comprising the excipient by: a) measuring a plurality of diffusion coefficients (D) of a plurality of compositions comprising different concentrations of the antigen-binding protein and different concentrations of the excipient under a condition that reduces the surface charge of the antigen-binding protein or under a condition wherein the pH of the formulation is near to the pl of the antigen-binding protein, and b) calculating k D of the formulation based on the plurality of Ds from step a), wherein k D of the formulation is indicative of the viscosity of the formulation, and ii) comparing the k D of the formulation comprising the excipient with the k D of the formulation without the excipient.
  • the term “plurality” refers a number of compositions in which
  • the diffusion coefficient (D) can be measured by any method known in the art.
  • D is measured by dynamic light scattering, including capillary based DLS.
  • the D is measured by Fluorescence Correlation Spectroscopy, nuclear magnetic resonance spectroscopy, macroscopic mass transfer (Taylor dispersion) or by measuring the concentration spatial profile using optical techniques within a diffusion chamber.
  • the pharmaceutical formulation comprises a saccharide and a surfactant.
  • the pharmaceutical formulation further comprises a buffer.
  • the buffer is an isotonic butter, an acetate buffer, a glutamate buffer, a citrate buffer, a lactic buffer, a succinate buffer, a tartrate buffer, a fumarate buffer, a maleate buffer, a histidine buffer, or a phosphate buffe, r2-(N- morpholino)ethanesulfonate buffer or a combination thereof.
  • the buffer is present in the formulation at a concentration ranging from about 5 mM to about 200 mM (or about 10 mM to about 50 mM
  • the pharmaceutical formulation comprises a saccharide.
  • the saccharide is monosaccharide or a disaccharide.
  • the saccharide is a sugar alcohol (e.g., sorbitol).
  • the saccharide is sucrose, galactose, fructose, xylose, xylitol, maltose, trehalose, mannitol, sorbitol or a combination thereof.
  • the saccharide is present in the formulation at a concentration ranging from about 1 to about 15% (w/V) (or about 9 to about 12% (w/V) or about 5% to about 12% (w/V) or about 7% to about 12% (w/V)).
  • the formulation comprises a surfactant.
  • the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, poloxamer 407, triton X-100, polyoxyethylene, PEG 3350, PEG 4000, or a combination thereof.
  • the surfactant is present in the formulation at a concentration ranging from 0.004 Any to about 0.5% (w/V) (or about 0.001 to about 0.01% (w/V), or about 0.001 to about 0.5% (w/V) or about 0.004 to about 0.01 % (w/V)).
  • the method is carried out on a pharmaceutical formulation having a pH of 5.2.
  • the pharmaceutical formulation comprises a 10 mM acetate buffer, sucrose and polysorbate, at pH 5.2.
  • the method is carried out on a pharmaceutical formulation comprising 10 mM glutamate, 9% (w/V) sucrose and 0.01% (w/V) polysorbate 80, and wherein the pH of the pharmaceutical formulation is 4.2.
  • the method is carried out on a pharmaceutical formulation comprising histidine, at pH 6.0, which may also comprise sucrose and a polysorbate.
  • the antigen-binding protein is a protein comprising a domain that binds a specified target antigen.
  • the antigen-binding protein is a polypeptide, large peptide, peptide conjugate, antibody, antibody fragment, antibody fusion peptide or antigen-binding fragment thereof.
  • the antigen-binding protein is a polyclonal antibody, monoclonal antibody or bi-specific antibody construct.
  • the antibody is an IgG monoclonal antibody, such as IgG 1 or lgG2.
  • the pharmaceutical formulation of the disclosure is a liquid formulation.
  • a pH from about pH 4 to about pH 6 could be, but is not limited to, pH 4, 4.2, 4.6, 5.1 , 5.5 etc. and any value in between such values.
  • a pH from about pH 4 to about pH 6 should not be construed to mean that the pH of a formulation in question varies 2 pH units in the range from pH 4 to pH 6 during storage, but rather a value may be picked in that range for the pH of the solution, and the pH remains buffered at about that pH.
  • Figures 1 A-1 H provide DLS D plot of the 10 mAb in Buffer A with different levels of NaCI (A-F: 0-150 mM) or ammonium sulfate (G-H: 8.33 and 50 mM).
  • Figures 2A-2B demonstrate that addition of NaCI (A) or (NH 4 )2SO4 (B) gradually decreased DLS k D values of the 10 mAbs in Buffer A.
  • the Pearson R 2 gradually improves as a function of ionic strength (I). The best linear correlation is observed for ko measured in Buffer A with 150mM NaCI (F).
  • Figures 4A-4D provide DLS k D plots of an engineering panel of 10 IgG 1 (Test set A) measured in Buffer A with and without 150 mM NaCI (A and B).
  • Candidate mAb A5 shows an apparent D o substantially lower than expected values of around 4.0x10 -7 cm 2 /s (B) and is therefore considered as an outlier (D).
  • Figures 5A-5D provides DLS k D plots of a test panel of 11 IgG 1 mAbs (Test set B) measured in Buffer A with and without 150 mM NaCI (A and B).
  • Figures 6A-6B provide correlation between ko (A) or kD.isoNaci (B) and viscosity at 140 mg/mL in Buffer A at 25 e C measured as cP. All samples (31 mAbs) tested in the study are consolidated in this figure. Compared to ko, ko.isoNaci shows improved correlation with viscosity.
  • Figures 7A-7C provide the averaged surface-to-surface intermolecular distance of mAb molecules as a function of protein concentration (A1 , B1 , C1 ).
  • the calculated Debye length is plotted as a function of solution ionic strength (A2, B2, C2).
  • A2, B2, C2 solution ionic strength
  • Figures 8A-8B provides a comparison of mAb k D measured in Buffer A with salts at 25 mM (A) or 150 mM (B) ionic strength.
  • Figures 9 provides a scatter plot of k D , isoNaci versus viscosity (at 140 mg/mL in Buffer A) on logarithmic scale.
  • k D , isoNaci was calculated from the diffusion coefficient at a single concentration of around 12 mg/mL.
  • Figure 10 provides the viscosity profile of several IgG 1 mAbs (denoted as mAb C1 , mAb C2, mAb 03 and mA 04) as a function of concentration at different temperatures 5-37 °C.
  • mAb 01 and mAb 02 show significant increase in the viscosity as the temperature cools down to 5°C.
  • Figures 12A-B provide scatter plots showing correlation between k D and temperature in Buffer without (A) and with 150 NaCI (B).
  • salt was used to suppress long-range electrostatic interactions of proteins so that short-range self-associations can be captured in dilute conditions to predict viscosity at high concentrations.
  • electrostatic interactions both long- and short-range
  • electrostatic interactions are being heavily suppressed.
  • k D isoNaci well predicts viscosity based on the data.
  • measurement of the optimized k D is used to select candidate molecules with high k D , isoNaci values that would translate to low viscosities at high concentrations.
  • k D isoNaci is somewhat biased towards short-range interactions and the electrostatics contribution is suppressed due to charge screening, both false positive (i.e. high ko. isoNaci but high viscosity) and false negative (i.e. low k D , isoNaci but low viscosity) scenarios may occur.
  • k D , isoNaci might fail to flag out candidates that have high viscosity issues primarily driven by electrostatic attractions, since the addition of 150 mM salts would screen out charge attractions and result in a high k D , isoNaci value if other short-range interactions are minimal.
  • This can be an issue in principle, but practically this will be rare or at least can be mitigated for the following two reasons: 1 ) those candidates with charge-driven viscosity issues are often flagged or deselected during an earlier stage of molecular selection because they would most likely have relatively low pl values; 2) ko measurement in common antigen-binding protein buffer will likely be able to flag out those candidates due to their charge driven self-associations.
  • isoNaci Potentially using a combination of k D and k D , isoNaci will be a more effective strategy to identify these viscous mAbs rather than relying on one individual measurement.
  • low k D , isoNaci may not always translate to high viscosity resulting in false negative selection. This can happen if mAbs exhibit both strong electrostatic repulsions and strong short-range attractions with the former being stronger. Under charge screening conditions, these molecules will show low k D . isoNaci driven by short-range interactions. At higher concentrations, although molecules are in closer proximity, strong charge repulsion may still outweigh short-range attractions, resulting a low viscosity.
  • compositions are samples of the pharmaceutical formulation which comprise different concentrations of the antigen-binding protein in order to predict the k D wherein the k D correlates with the predicted viscosity of the pharmaceutical formulation. This method allows for determining the viscosity early in the development process; and allows for determining viscosity when the amount of antigen-binding product is limited, such as early in the development process.
  • the diffusion coefficient (D) of the compositions can be measured using methods known in the art.
  • D is determined using dynamic light scattering (DLS) which measured the intensity fluctuation of light scatted by particles.
  • the D may also be measured using Fluorescence Correlation Spectroscopy (Yu et aL, Front. Phys. 9:110, 2021), which measures the temporal autocorrection of the detected fluorescence signal emitted by the volume of liquid.
  • DLS dynamic light scattering
  • Fluorescence Correlation Spectroscopy Yu et aL, Front. Phys. 9:110, 2021
  • macroscopic mass transfer may be used to measure the D, such as methods based on Taylor dispersion within a Poiseuille flow, or the diaphragm cell may be used to measure the D.
  • Direct detection of D may be carried out by measuring the concentration spatial profile using optical techniques within a diffusion chamber (Hamada & de Anna, T ransport in Porous Media 146: 463-474, 2023). Furthermore, NMR may be used to measure D, e.g. as taught in Patil et aL, AAPS J. 2017 Nov; 19(6): 1760-1766.
  • D is indicative of viscosity of a formulation which comprises a high concentration of an antigen-binding protein.
  • the plurality of compositions used in the disclosed methods comprise the pharmaceutical formulation but have different concentrations of the antigen-binding protein in order to calculate k D .
  • the term “pharmaceutical formulation” relates to a formulation which is suitable for administration to a subject in need thereof.
  • subject or “individual” or “animal” or “patient” are used interchangeably herein to refer to any subject, particularly a mammalian subject, for whom administration of the pharmaceutical formulation of the invention is desired.
  • Mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like, with humans being preferred.
  • the pharmaceutical formulation of the present disclosure is stable and pharmaceutically acceptable, i.e., capable of eliciting the desired therapeutic effect without causing significant undesirable local or systemic effects in the subject to which the pharmaceutical formulation is administered.
  • compositions of the invention may be sterile.
  • pharmaceutically acceptable can mean approved by a regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particularly in humans, but is not limited to those approved by a regulatory agency.
  • Various aspects of the pharmaceutical formulations are described below. The use of section headings is merely for the convenience of reading, and not intended to be limiting per se. The entire document is intended to be viewed as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated.
  • An “antigen-binding protein” is a protein comprising a domain that binds a specified target antigen (such as CD3 and/or CDH19, MSLN, DLL3, FLT3, EGFRvlll, BCMA, PSMA, CD33, CD19, CD70, CLDN18.2 or MUC17).
  • An antigen-binding protein comprises a scaffold or framework portion that allows the antigen binding domain to adopt a conformation that promotes binding of the antigen-binding protein to the antigen.
  • the antigen-binding protein is an antibody or immunoglobulin, or an antigen-binding antibody fragment.
  • an antibody refers to an intact antigen-binding immunoglobulin.
  • An “antibody” is a type of an antigen-binding protein.
  • the antibody can be an IgA, IgD, IgE, IgG, or IgM antibody, including any one of lgG1 , lgG2, lgG3 or lgG4.
  • an intact antibody comprises two full-length heavy chains and two full-length light chains.
  • An antibody has a variable region and a constant region. In IgG formats, a variable region is generally about 100-110 or more amino acids, comprises three complementarity determining regions (CDRs), is primarily responsible for antigen recognition, and substantially varies among other antibodies that bind to different antigens.
  • CDRs complementarity determining regions
  • variable region typically comprises at least three heavy or light chain CDRs (Kabat et al., 1991 , Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901 -917;
  • the antibody of the pharmaceutical formulation is a bispecific antibody, i.e. , an antibody that binds two different targets (e.g., CD3 and a second, different target).
  • antibody constructs that binds to two different target antigens, i.e., it comprises a first binding domain and a second binding domain, wherein the first binding domain binds to one antigen or target (e.g., the target cell surface antigen), and the second binding domain binds to another antigen or target (e.g. CD3).
  • antibody constructs according to the disclosure comprise specificities for two different antigens or targets.
  • target cell surface antigen refers to an antigenic structure expressed by a cell and which is present at the cell surface such that it is accessible for an antibody construct as described herein.
  • the invention may be a protein, preferably the extracellular portion of a protein, or a carbohydrate structure, preferably a carbohydrate structure of a protein, such as a glycoprotein. It is preferably a tumor antigen.
  • the invention also encompasses multispecific antibody constructs such as trispecific antibody constructs, the latter ones including three binding domains, or constructs having more than three (e.g. four, five%) specificities.
  • Bispecific antibodies and/or antibody constructs as understood herein include, but are not limited to, traditional bispecific immunoglobulins (e.g., BsIgG), IgG comprising an appended antigen-binding domain (e.g., the amino or carboxy termini of light or heavy chains are connected to additional antigen-binding domains, such as single domain antibodies or paired antibody variable domains (e.g., Fv or scFv)), BsAb fragments (e.g., bispecific single chain antibodies), bispecific fusion proteins (e.g., antigen binding domains fused to an effector moiety), and BsAb conjugates.
  • BsIgG traditional bispecific immunoglobulins
  • IgG comprising an appended antigen-binding domain
  • additional antigen-binding domains such as single domain antibodies or paired antibody variable domains (e.g., Fv or scFv)
  • BsAb fragments e.g., bispecific single chain antibodies
  • bispecific constructs include, but are not limited to, diabodies, single chain diabodies, tandem scFvs, bispecific T cell engager (BiTE®) format (a fusion protein consisting of two single-chain variable fragments (scFvs) joined by a linker), and Fab2 bispecifics, as well as engineered constructs comprising full length antibodies.
  • BiTE® bispecific T cell engager
  • the pharmaceutical formulations described herein comprise a bispecific antibody construct comprises a first binding domain that binds to a target cell surface antigen, a second binding domain that binds to human CD3 on the surface of a T cell, and optionally a third domain comprising, in an amino to carboxyl order, hinge-CH2 domain-CH3 domain-linker-hinge-CH2 domain-CH3 domain.
  • each of the first and second binding domains comprise a VH region and a VL region.
  • binding domain refers to a domain which (specifically) binds to I interacts with I recognizes a given target epitope or a given target site on the target molecules (antigens), e.g. CDH19, MSLN, DLL3, FLT3, EGFRvlll, BCMA, PSMA, CD33, CD19, CD70, CLDN18.2 or MUC17 and CD3, respectively.
  • the structure and function of the first binding domain (recognizing e.g.
  • the first binding domain is characterized by the presence of three light chain CDRs (i.e.
  • the second binding domain preferably also comprises the minimum structural requirements of an antibody which allow for the target binding. More preferably, the second binding domain comprises at least three light chain CDRs (i.e. CDR1 , CDR2 and CDR3 of the VL region) and/or three heavy chain CDRs (i.e. CDR1 , CDR2 and CDR3 of the VH region). It is envisaged that the first and/or second binding domain is produced by or obtainable by phage-display or library screening methods rather than by grafting CDR sequences from a pre-existing (monoclonal) antibody into a scaffold.
  • the first binding domain which binds to the target cell surface antigen and/or the second binding domain which binds to CD3E is/are human binding domains.
  • Antibodies and antibody constructs comprising at least one human binding domain avoid some of the problems associated with antibodies or antibody constructs that possess non-human such as rodent ⁇ e.g. murine, rat, hamster or rabbit) variable and/or constant regions. The presence of such rodent derived proteins can lead to the rapid clearance of the antibodies or antibody constructs or can lead to the generation of an immune response against the antibody or antibody construct by a patient.
  • rodent derived antibodies or antibody constructs human or fully human antibodies I antibody constructs can be generated through the introduction of human antibody function into a rodent so that the rodent produces fully human antibodies.
  • the antigen binding protein comprises a single chain antibody construct.
  • An scFv comprises a variable heavy chain, an scFv linker, and a variable light domain.
  • the C-terminus of the variable light chain is attached to the N-terminus of the scFv linker, the C-terminus of which is attached to the N-terminus of a variable heavy chain (N-vh-lin ker-vl-C), although the configuration can be switched (N-vl-linker-vh-C).
  • the C-terminus of the variable heavy chain is attached to the N-terminus of the scFv linker, the C-terminus of which is attached to the N-terminus of a variable light chain (N-vl-linker-vh-C), although the configuration can be switched (N-vh-linker-v-C).
  • N-vl-linker-vh-C variable light chain
  • the at least two binding domains and the variable domains (VH/VL) of the antibody construct of the present disclosure may or may not comprise peptide linkers (spacer peptides).
  • the term “peptide linker” comprises in accordance with the present invention an amino acid sequence by which the amino acid sequences of one (variable and/or binding) domain and another (variable and/or binding) domain of the antibody construct of the disclosure are linked with each other.
  • the peptide linkers can also be used to fuse the third domain to the other domains of the antibody construct of the invention.
  • An essential technical feature of such peptide linker is that it does not comprise any polymerization activity.
  • suitable peptide linkers are those described in U.S.
  • the peptide linkers can also be used to attach other domains or modules or regions (such as half-life extending domains) to the bispecific antibody construct described herein.
  • the third domain comprises a “Fc” or “Fc region” or “Fc domain,” which refers to the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain.
  • Fc domain refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains.
  • Fc may include the J chain.
  • the Fc domain comprises immunoglobulin domains Cy2 and Cy3 (Cy2 and Cy3) and the lower hinge region between Cy1 (Cy1 ) and Cy2 (Cy2).
  • the bispecific antibody construct is preferably an IgG antibody (which includes several subclasses, including, but not limited to lgG1 , lgG2, lgG3, and lgG4).
  • the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat.
  • amino acid modifications are made to the Fc region, for example, to alter binding to one or more FcyR receptors or to the FcRn receptor.
  • the pharmaceutical formulations described herein comprise a bispecific antibody construct which binds human CD3 and human CDH19, or human CD3 and human MSLN, or human CD3 and human DLL3, or human CD3 and human FLT3, or human CD3 and human EGFRvI 11 , or human CD3 and human BCMA, or human CD3 and PSMA, or human CD3 and human CD33, or human CD3 and human CD19, human CD3 and human CD70, or human CD3 and human MUC17, or human CD3 and human CLDN18.2.
  • the pharmaceutical formulation comprises an antigen-binding protein in a concentration ranging from about 2 mg/ml to about 20 mg/ml, about 2 to about 19 mg/ml, about 2 mg/ml to about 18 mg/ml, about 2 mg/ml to about 17 mg/ml, 2 mg/ml to about 16 mg/ml, about 2 mg/ml to about 15 mg/ml, about 5 mg/ml to about 20 mg/ml, about 5 to about 19 mg/ml, about 5 mg/ml to about 18 mg/ml, about 5 mg/ml to about 17 mg/ml, about 5 mg/ml to about 16 mg/ml, about 5 mg/ml to about 15 mg/ml, about 10 mg/ml to about 20 mg/ml, about 10 to about 19 mg/ml, about 10 mg/ml to about 18 mg/ml, about 10 mg/ml to about 17 mg/ml, about 10 mg/ml to about 16 mg/ml, about 10 mg/ml to about 16 mg/
  • the pharmaceutical formulation comprises an antigen-binding protein at a concentration of about 2 mg/ml, about 3 mg/ml, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, aboutIO mg/mL, about 1 1 mg/mL, about 12 mg/mL, about 13 mg/mL, about 14 mg/mL, about 15 mg/mL, about 16 mg/mL, about 17 mg/mL, about 18 mg/mL, about 19 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, about 45 mg/mL or about 50 mg/mL.
  • the pharmaceutical formulation comprises an antigen-binding protein in a concentration of about 20 mg/mL.
  • the pharmaceutical formulation of the invention comprises a buffer, which optionally may be acetate, lactate, aspartate, glutamate, citrate, succinate, tartrate, fumarate, maleate, histidine, phosphate, 2-(N-morpholino)ethanesulfonate or combinations thereof.
  • a buffer which optionally may be acetate, lactate, aspartate, glutamate, citrate, succinate, tartrate, fumarate, maleate, histidine, phosphate, 2-(N-morpholino)ethanesulfonate or combinations thereof.
  • Buffering agents are often employed to control pH in the pharmaceutical formulation.
  • the buffer is added in a concentration that maintains pH of the pharmaceutical formulation of about 4.0 to about 6.0, about 4.0 to 5.0, or about 4.2.
  • the effect of pH on pharmaceutical formulations may be characterized using any one or more of several approaches such as accelerated stability studies and calorimetric screening studies (Remmele R.L. Jr., et al., Biochemistry, 38(16): 5241-7 (1999)).
  • the buffer system present in the pharmaceutical formulation is selected to be physiologically compatible and to maintain a desired pH.
  • the buffer may be present at a concentration between about 0.1 mM and about 1000 mM (1 M), or between about 5 mM and about 200 mM, or between about 5 mM to about 100 mM, or between about 10 mM and 50 about mM. Suitable buffer concentrations encompass concentrations of about 200 mM or less.
  • the buffer in the pharmaceutical formulation is present in a concentration of about 190 mM, about 180 mM, about 170 mM, about 160 mM, about 150 mM, about 140 mM, about 130 mM, about 120 mM, about 110 mM, about 100 mM, about 80 mM, about 70 mM, about 60 mM, about 50 mM, about 40 mM, about 30 mM, about 20 mM, about 10 mM or about 5 mM.
  • the concentration of the buffer is at least 0.1 , 0.5, 0.7, 0.8 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, or 900 mM. In some embodiments, the concentration of the buffer is between 1 , 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or 90 mM and 100 mM. In some embodiments, the concentration of the buffer is between 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, or 40 mM and 50 mM. In some embodiments, the concentration of the buffer is about 10 mM.
  • the pharmaceutical formulations described here comprise a surfactant.
  • exemplary surfactants include but are not limited to, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, poloxamer 407, triton X-100, polyoxyethylene, PEG 3350, PEG 4000, or a combination thereof.
  • compositions described herein comprise at least one surfactant, either individually or as a mixture in different ratios.
  • the pharmaceutical formulation comprises a surfactant at a concentration of about 0.001 % to about 5% w/v (or about 0.001% to about 0.5%, or about 0.004 to about 0.5% w/v or about 0.001 to about 0.01 % w/v or about 0.004 to about 0.01 % w/v).
  • the pharmaceutical formulation comprises a surfactant at a concentration of at least 0.001 , at least 0.002, at least 0.003, at least 0.004, at least 0.005, at least 0.007, at least 0.01 , at least 0.05, at least 0.1 , at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1 .0, at least 1 .5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, or at least 4.5% w/v.
  • the pharmaceutical formulation comprises a surfactant at a concentration of about 0.001% to about 0.5% w/v.
  • the pharmaceutical formulation comprises a surfactant at a concentration of about 0.001 to about 0.01% w/v. In some embodiments, the pharmaceutical formulation comprises a surfactant at a concentration of about 0.001 to about 0.01% w/v. In some embodiments, the pharmaceutical formulation comprises a surfactant at a concentration of about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, to about 0.5% w/v.
  • the pharmaceutical formulation comprises a surfactant incorporated in a concentration of about 0.001 % to about 0.01% w/v.
  • the surfactant is polysorbate 80 and the polysorbate 80 is present in a concentration of about 0.01% w/v.
  • the pharmaceutical formulations described herein comprise a saccharide.
  • the saccharide is a monosaccharide or a disaccharide.
  • the saccharide is glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, sorbitol, mannitol or xylitol or a combination thereof.
  • the pharmaceutical formulation comprises a saccharide at a concentration of about 0.01% to about 40% w/v, or about 00.1% to about 20% w/v, or about 1% to about 15%, or about 5% to about 12 %, or about 7% to about 12% w/v.
  • the pharmaceutical formulation comprises at least one saccharide at a concentration of at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 30%, or at least 40% w/v.
  • the pharmaceutical formulation comprises at least one saccharide at a concentration of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% w/v.
  • the pharmaceutical formulation comprises at least one saccharide at a concentration of about 1% to about 15% w/v.
  • the pharmaceutical formulation comprises at least one saccharide at a concentration of about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 1 1%, about 11 .5%, or about 12% w/v.
  • the pharmaceutical formulation comprises at least one saccharide at a concentration of about 7% to about 12% w/v. In some embodiments, the at least one saccharide is in the pharmaceutical formulation at a concentration of about 9% w/v. In some embodiments, the saccharide is sucrose and is present in the pharmaceutical formulation ranging from about 9% to about 12% w/v.
  • the pharmaceutical formulation comprises 10 mM glutamate, 9% (w/V) sucrose and 0.01% (w/V) polysorbate 80, wherein the pH of the pharmaceutical formulation is 4.2.
  • the pharmaceutical formulation is lyophilized.
  • Test set A is an engineering panel of 10 lgG1 molecules with a high level of CDR sequence similarity.
  • Test set B has 17 lgG1 mAbs with a broad viscosity range at high concentrations.
  • 12 molecules are conventional monospecific bivalent lgG1 mAbs
  • the rest 5 are multi-specific IgG 1 molecules (e.g., 3 bispecific molecules with scFv domains; one IgG peptide conjugate; one antibody cytokine conjugate).
  • a dilution series from 2 to 12 mg/mL was prepared in a buffer having a pH of 5.2 with and without salts.
  • “training” mAb set two types of salt were screened: NaCI (10, 20, 100, 150 mM) and ammonium sulfate (8.33, 50 mM).
  • NaCI 10, 20, 100, 150 mM
  • ammonium sulfate 8.33, 50 mM
  • the optimal buffer condition was identified and validated using two additional panel sets. Protein concentrations were measured using a Droplet Quant UV-Vis spectrophotometer (Perkin Elmer, Waltham, MA) prior to DLS k D analysis.
  • D Do (1 + k D * c) where D is diffusion coefficient of samples, D o is diffusion coefficient of mAb at infinite dilution. kD is diffusion interaction parameter, and c is protein concentration. Dividing the slope (D 0 *kD) by the intercept (D o ) returns ko.
  • ko measured in an exemplary pharmaceutical formulations having a pH of 5.2 (Buffer A) by dynamic light scattering shows only weak correlation with viscosity at 140 mg/mL. Therefore, the present experiment was carried out to improve the predictability of k D by screening the buffer conditions in which k D was measured. To achieve this goal, 10 well characterized mAbs with a large viscosity range (5.8-82.5 cP) at 140 mg/mL at 25 e C (Table 1) were used.
  • Table 1 List of the 10 mAbs used to optimize the DLS k D method for viscosity prediction
  • k D in Buffer A was measured in the presence of ammonium sulfate to determine whether a better prediction can be achieved than using NaCI.
  • Two concentrations of ammonium sulfate were tested: 8.33 and 50 mM, which are equivalent to 25 and 150 mM NaCI in terms of ionic strength, respectively.
  • the addition of ammonium sulfate gradually improved the increasing correlation between k D and viscosity ( Figures 3G and 3H).
  • the best prediction for viscosity was still observed for k D measured in Buffer A with 150mM NaCI ( Figure 3I). Therefore, Buffer A with 150 mM was selected as the optimized buffer condition for DLS k D measurement, and the diffusion interaction coefficient measured under this condition was designated as kD,i50Naci-
  • Example 1 The optimized k D method for viscosity prediction described in Example 1 was validated by testing an engineering panel of 10 lgG1 mAbs because screening of an engineering panel is a routine task during the lead molecule optimization stage.
  • the mAbs differ by only a few amino acid residues in the CDR region but show diverse viscosities ranging from 7.5 cP to 36.1 cP at 140 mg/mL in Buffer A. All candidates exhibit relatively large positive k D values in Buffer A, indicative of net self-repulsion (Figure 4A).
  • the modified method further tested our approach to determine whether this method can be applied to screen mAb molecules with even broader viscosity ranges (6-220 cP).
  • the 17 mAbs (12 mABs and 5 multispecifics) tested have diverse CDR sequences and target different proteins.
  • the overall correlation between k D and viscosity was weak in the absence of NaCI, whereas k D ,i 5 oNaci showed improved correlation.
  • Candidates mAb B2 and mAb B11 exhibited lower apparent Do under salt conditions.
  • Candidate mAb B2 also showed a low apparent D o in Buffer A, and this might imply that there are pre-existing aggregates in the sample resulting a low apparent DO in both buffers.
  • candidate mAb B11 showed a normal D o ( ⁇ 4.0x10-7 cm 2 /s) in Buffer A, therefore this candidate may have a high propensity to self-associate in salt solutions, a similar behavior observed for candidate mAb A5 ( Figure 5B).
  • viscosity_140 mg/ml__25 e C 10 A (0.2976 - 0.0759 * k D , 150NaCl)
  • viscosity_140 mg/mL_25 e C in cP
  • kD, 150NaCI in mL/g
  • Ds-s The averaged surface-to-surface intermolecular distance (Ds-s) is a function of protein concentration and can be calculated using the following formula [23]:
  • c is the protein concentration in mg/mL
  • MW is the molecular weight of a mAb
  • NA is the Avogadro constant of 6.022x10 23 mol -1
  • D h is the hydrodynamic diameter of a mAb (a D h of 10 nm for a typical mAb is used).
  • D s-S drastically decreases as protein concentration arises.
  • D s.s ranges from 17.5 to 39.9 nm within the ko concentration regime (2 ⁇ 12 mg/mL) ( Figures 7A1 and 7B1 ), whereas D s.s decreases to 1.84 nm at 140 mg/mL ( Figure 7C1 ).
  • E relative permittivity of water
  • s 0 is the permittivity of free space
  • k B Boltzmann’s constant
  • T temperature
  • z t is charge on electron.
  • I is the ionic strength in mol/L.
  • first-order self-interactions can occur at very low concentrations ( ⁇ 2 mg/mL) and instead capture higher order interactions within the DLS relevant concentration region (2 ⁇ 20 mg/mL). Fitting such a region will result in a low apparent D o .
  • This is a red flag from a broad developability perspective because this behavior often correlates with high viscosity of concentrated mAb solutions at least from the data collected. It may also inform aggregation risks upon subcutaneous injections where highly concentrated mAbs are exposed to a high salt concentration at neutral pH.
  • Example 6 Potential ways to further reduce sample consumption
  • One idea is to use a single-concentration approach to approximate k D . Given that the theoretical D o of an lgG1 in a given buffer is constant (i.e. an averaged value of 3.93x10 -7 cm2/s in Buffer A), a linear plot can be constructed between DO and the diffusion coefficient measured at a relatively high concentration (e.g.
  • k D isoNaci was calculated using the diffusion coefficient at the highest testing concentration only for the monospecific mAbs. As shown in Figure 9, there is some level of differentiation between low and high viscosity molecules. For example, molecules with k D , isoNaci > -10 mL/g are likely to exhibit low viscosity at 140 mg/mL ( ⁇ 10 cP). However, k D , isoNaci calculated based on single concentration data show inferior correlation with viscosity compared ko, isoNaci derived from the linear fit of a concentration gradient ( Figure 6B). In addition, this method may fail to identify some D o outliers.
  • Example 1 To determine if the disclosed optimized k D method for viscosity prediction can be used to predict viscosity at different temperatures, the method described in Example 1 was used to predict the viscosity of mAbs C1 - C4 at different temperatures (5°C, 10°C, 15°C, 25°C and 37°C).
  • the diffusion coefficient (cm 2 /s) of the mAb formulation at each temperature was measured at various concentrations (4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 and 14 mg/mL) in order to calculate the k D of the formulation based on the measured diffusion coefficient.
  • Figure 11 provides the linear plot of the diffusion coefficient vs. the concentration of each mAb composition at each temperature with or without 150 mM NaCI added.
  • Figure 12 demonstrates that the temperature dependency of viscosity correlates with the k D . Therefore, this study demonstrates that the optimized method disclosed herein predicted viscosity of the pharmaceutical formulations comprising antigen-binding proteins at different temperatures.

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Abstract

La présente divulgation concerne le domaine de la détermination de la viscosité de compositions pharmaceutiques comprenant une protéine de liaison à l'antigène, les procédés comprenant la mesure d'une pluralité de coefficients de diffusion (D) d'une pluralité de compositions comprenant différentes concentrations de la protéine de liaison à l'antigène dans une condition qui réduit la charge de surface de la protéine de liaison à l'antigène, et le calcul du kD de la formulation sur la base de la pluralité mesurée de D.
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