Challenges in Determining Intrinsic Viscosity Under Low Ionic Strength Solution Conditions

Purpose

To determine the intrinsic viscosity of several monoclonal antibodies (mAbs) under varying pH and ionic strength solution conditions.

Methods

An online viscosity detector attached to HPLC (Viscotek®) was used to determine the intrinsic viscosity of mAbs. The Ross and Minton equation was used for viscosity prediction at high protein concentrations. Bulk viscosity was determined by a Cambridge viscometer.

Results

At 15 mM ionic strength, intrinsic viscosity of the mAbs determined by the single-point approach varied from 5.6 to 6.4 mL/g with changes in pH. High ionic strength did not significantly alter intrinsic viscosity, while a significant increase (up to 24.0 mL/g) was observed near zero mM. No difference in bulk viscosity of mAb3 was observed around pH 6 as a function of ionic strength. Data analysis revealed that near zero mM ionic strength limitations of the single-point technique result in erroneously high intrinsic viscosity.

Conclusions

Intrinsic viscosity is a valuable tool that can be used to model baseline viscosity at higher protein concentrations. However, it is not predictive of solution non-ideality at higher protein concentrations. Furthermore, breakdown of numerous assumptions limits the applicability of experimental techniques near zero mM ionic strength conditions. For molecules and conditions studied, the single-point approach produced reliable intrinsic viscosity results at 15 mM. However, this approach must be used with caution near zero mM ionic strength. Data analysis can be used to reveal whether determined intrinsic viscosity is reliable or erroneously high.

     

Conformational Changes and Drivers of Monoclonal Antibody Liquid-Liquid Phase Separation

Liquid-liquid phase separation is a phenomenon within biology whereby proteins can separate into dense and more dilute phases with distinct properties. Three antibodies that undergo liquid-liquid phase separation were characterized in the protein-rich and protein-poor phases. In comparison to the protein-poor phase, the protein-rich phase demonstrates more blue-shift tryptophan emissions and red-shifted amide I absorbances. Large changes involving conformational isomerization around disulfide bonds were observed using Raman spectroscopy. Amide I and protein fluorescence differences between the phases persisted to temperatures above the critical temperature but ceased at the temperature at which aggregation occurred. In addition, large changes occurred in the structural organization of water molecules within the protein-rich phase for all three antibodies. It is hypothesized that as the proteins have the same chemical potential in both phases, the protein viscosity is higher in the protein-rich phase resulting in slowed diffusion dependent protein aggregation in this phase. For all three antibodies we performed accelerated stability studies and found that the protein-rich phase aggregated at the same rate or slower than the protein-poor phase.     

Linking the Solution Viscosity of an IgG2 Monoclonal Antibody to Its Structure as a Function of pH and Temperature

Although the viscosity of concentrated antibody solutions has been the focus of many recent studies, less attention has been concentrated on how changes in protein structure impact viscosity. This study examines viscosity profiles of an immunoglobulin G (IgG) 2 monoclonal antibody at 150 mg/mL as a function of temperature and pH. Although the structure of the antibody at pH 4.0–7.0 was comparable at lower temperatures as measured by second derivative UV absorbance and Fourier transform infrared spectroscopy, differences in 8-anilino-1-naphthalene sulfonate (ANS) fluorescence intensity indicated small structural alterations as a function of pH. Below the structural transition onset temperature, the viscosity profiles were pH dependent and linearly correlated with fluorescence intensity, and followed semilogarithmic behavior as a function of temperature. The transitions of the viscosity profiles correlated well with the major structure transitions at a protein concentration of 150 mg/mL. The viscosity correlated particularly well with ANS fluorescence intensity at 0.2 mg/mL below and above the structural transition temperatures. These results suggest: (1) ANS can be an important measure of the overall structure and (2) hydrophobic interactions and charge-charge interactions are the two major physical factors that contribute collectively to the high viscosity of concentrated IgG solutions.     

Resolving Liquid-Liquid Phase Separation for a Peptide Fused Monoclonal Antibody by Formulation Optimization

Liquid-liquid phase separation (LLPS) of protein solutions has been usually related to strong protein-protein interactions (PPI) under certain conditions. For the first time, we observed the LLPS phenomenon for a novel protein modality, peptide-fused monoclonal antibody (pmAb). LLPS emerged within hours between pH 6.0 to 7.0 and disappeared when solution pH values decreased to pH 5.0 or lower. Negative values of interaction parameter (k_D) and close to zero values of zeta potential (ζ) were correlated to LLPS appearance. However, between pH 6.0 to 7.0, a strong electrostatic repulsion force was expected to potentially avoid LLPS based on the sequence predicted pI value, 8.35. Surprisingly, this is significantly away from experimentally determined pI, 6.25, which readily attributes the LLPS appearances of pmAb to the attenuated electrostatic repulsion force. Such discrepancy between experiment and prediction reminds the necessity of actual measurement for a complicated modality like pmAb. Furthermore, significant protein degradation took place upon thermal stress at pH 5.0 or lower. Therefore, the effects of pH and selected excipients on the thermal stability of pmAb were further assessed. A formulation consisting of arginine at pH 6.5 successfully prevented the appearance of LLPS and enhanced its thermal stability at 40 °C for pmAb. In conclusion, we have reported LLPS for a pmAb and successfully resolved the issue by optimizing formulation with aids from PPI characterization.     

Effects of Temperature and Osmolytes on Competing Degradation Routes for an IgG1 Antibody

Addition of excipients is a common strategy to slow protein aggregation during storage. Excipient effects on the mechanism(s) and temperature (T) dependence of aggregation for a monoclonal antibody solution were tested using size-exclusion chromatography, differential scanning calorimetry (DSC), temperature scanning monomer loss (TSML), and laser light scattering; previous work in buffer-only conditions had shown non-Arrhenius behavior and implicated Fab and/or C_H3 unfolding as a key step in aggregation. Excipients included citrate, amino acid salts (histidine–HCl, arginine–HCl), and polyols (mannitol and glycerol). DSC and TSML showed that Fab, rather than C_H3, unfolding corresponded with the onset of aggregation for each condition. Isothermal incubation at 56.5°C, 40°C, and 2°C–8°C resulted in aggregation, while fragmentation occurred readily at only 40°C. The primary effect of the different excipients appeared to be preferential accumulation/exclusion, affecting the concentrations of partially unfolded monomer key intermediates. In addition, aggregation rates were clearly non-Arrhenius, causing aggregation to dominate over fragmentation at high and low T, and making long-term stability predictions problematic based on commonly employed 40°C conditions. Possible reasons for non-Arrhenius behavior include a strong T-dependence of the Fab unfolding enthalpy and/or a switch from Fab-mediated to Fc-mediated aggregation as one moves from high to low T.     

Effect of Ionic Strength and pH on the Physical and Chemical Stability of a Monoclonal Antibody Antigen-Binding Fragment

Monoclonal antibody (mAb) fragments are emerging as promising alternatives to full-length mAbs as protein therapeutic candidates. Antigen-binding fragments (Fabs) are the most advanced with three Fab-based drug products currently approved. This work presents preformulation characterization data on the effect of pH, NaCl concentration, and various cationic excipients on the physical and chemical stability of a Fab molecule with multiple negatively charged Asp residues in the complementarity-determining region. Conformational stability was evaluated using an empirical phase diagram approach based on circular dichroism, intrinsic Trp and extrinsic 8-anilino-1-naphthalene sulfonate (ANS) fluorescence, and static light scattering measurements. The effect of NaCl concentration, various cationic excipients and pH on the Fab molecule’s conformational stability, aggregation propensity, and chemical stability (Asp isomerization) was determined by differential scanning calorimetry, optical density measurements at 350 nm (OD₃₅₀), and ion-exchange chromatography, respectively. Increasing NaCl concentration increased the overall conformational stability, decreased aggregation rates, and lowered the rates of Asp isomerization. No such trends were noted for pH or cationic excipients. The potential interrelationships between protein conformational and chemical stability are discussed in the context of designing stable protein formulations.     

Opalescent Appearance of an IgG1 Antibody at High Concentrations and Its Relationship to Noncovalent Association

PURPOSE: Therapeutic antibodies are often formulated at a high concentration where they may have an opalescent appearance. The aim of this study is to understand the origin of this opalescence, especially its relationship to noncovalent association and physical stability. METHODS: The turbidity and the association state of an IgG1 antibody were investigated as a function of concentration and temperature using static and dynamic light scattering, nephelometric turbidity, and analytical ultracentrifugation. RESULTS. The antibody had increasingly opalescent appearance in the concentration range 5-50 mg/ml. The opalescence was greater at refrigerated temperature but was readily reversible upon warming to room temperature. Turbidity measured at 25 degrees C was linear with concentration, as expected for Rayleigh scatter in the absence of association. In the concentration range 1-50 mg/ml, the weight average molecular weights were close to that expected for a monomer. Zimm plot analysis of the data yielded a negative second virial coefficient, indicative of attractive solute-solute interactions. The hydrodynamic diameter was independent of concentration and remained unchanged as a function of aging at room temperature. CONCLUSIONS: The results indicate that opalescent appearance is not due to self-association but is a simple consequence of Rayleigh scatter. Opalescent appearance did not result in physical instability.     

High-Throughput Biophysical Analysis and Data Visualization of Conformational Stability of an IgG1 Monoclonal Antibody After Deglycosylation

The structural integrity and conformational stability of an IgG1 monoclonal antibody (mAb), after partial or complete enzymatic removal of the N-linked Fc glycan, were compared with the untreated mAb over a wide range of temperature (10 °C-90 °C) and solution pH (3-8) using circular dichroism, fluorescence spectroscopy, and static light scattering combined with data visualization employing empirical phase diagrams. Subtle-to-larger stability differences between the different glycoforms were observed. Improved detection of physical stability differences was then demonstrated over narrower pH range (4.0-6.0) using smaller temperature increments, especially when combined with an alternative data visualization method (radar plots). Differential scanning calorimetry and differential scanning fluorimetry were then utilized and also showed an improved ability to detect differences in the physical stability of a mAb glycoform. On the basis of these results, a two-step methodology was used in which conformational stability of a mAb glycoform is first screened with a wide variety of instruments and environmental stresses, followed by a second evaluation with optimally sensitive experimental conditions, analytical techniques, and data visualization methods. With this approach, a high-throughput biophysical analysis to assess relatively subtle conformational stability differences in protein glycoforms is demonstrated.     

Arginine and its Derivatives Suppress the Opalescence of an Antibody Solution

Opalescence is a problem concerned with the stability of an antibody solution. It occurs when a high concentration of a protein is present. Arginine (Arg) is a versatile aggregation suppressor of proteins, which is among the candidates that suppress opalescence in antibody solutions. Here, we investigated the effect of various types of small molecular additives on opalescence to reveal the mechanism of Arg in preventing opalescence in antibody solution. As expected, Arg suppressed the opalescence of the immunoglobulin G (IgG) solution. Arg also concentration dependently inhibited the formation of microstructures in IgG molecules. Interestingly, the intrinsic fluorescence spectra of highly concentrated IgG solutions differed from those having low concentrations, even though IgG retained a distinct tertiary structure. Arginine ethylester was more effective in suppressing the opalescence of IgG solutions than Arg, whereas lysine and γ-guanidinobutyric acid were less effective. These results indicated that positively charged groups of both α-amine and guanidinium actively influence Arg as an additive for suppressing opalescence. Diols, which are the suppressors of the liquid–liquid phase separation of proteins were also effective in suppressing the opalescence. These results therefore provide insight into the control of opalescence of antibody solutions at high concentrations using solution additives.     

Effect of Ionic Strength on Solution Stability of PNU-67590A,A Micellar Prodrug of Methylprednisolone

Purpose. PNU-67590A is a water-soluble micellar prodrug of methyl-prednisolone (MP). The major products of degradation of PNU-67590A are MP by hydrolysis and methylprednisolone 17-suleptanate (17-E) by 21 17 acyl migration. The effect of ionic strength on micelle formation and stability of PNU-67590A in aqueous solution was examined. Methods. PNU-67590A solutions at pH 2 and 8 and ionic strength of 0.05, 0.1, 0.2, and 0.4 M were maintained at 25°C in the dark to measure MP and 17-E levels over time. Results. The rate of degradation of micellar PNU-67590A at pH 8 was less than that of monomeric PNU-67590A, and vice versa at pH 2. Increase in ionic strength decreased both the critical micelle concentration of PNU-67590A and the degradation of micelle PNU-67590A at both pHs, resulting in improved overall stability of PNU-67590A. Conclusions. Formulation of PNU-67590A in a concentrated solution with high ionic strength will maximize stability and shelf-life.     

Elucidation of Degradants in Acidic Peak of Cation Exchange Chromatography in an IgG1 Monoclonal Antibody Formed on Long-Term Storage in a Liquid Formulation

Purpose  

An IgG1 therapeutic monoclonal antibody showed an increase in acidic or pre-peak by cation exchange chromatography (CEX) at elevated temperatures, though stable at 2–8°C long-term storage in a liquid formulation. Characterization effort was undertaken to elucidate the degradants in CEX pre-peak and effect on biological activity.     

High Concentration Formulation Studies of an IgG2 Antibody Using Small Angle X-ray Scattering

Purpose

Concentrated protein formulations are strongly influenced by protein-protein interactions. These can be probed at low protein concentration by e.g. virial coefficients. It was recently suggested that interactions are attractive at short distances and repulsive at longer distances. Measurements at low concentrations mainly sample longer distances, hence may not predict high concentration behavior. Here we demonstrate that small angle X-ray scattering (SAXS) measurements simultaneously collect information on interactions at short and long distances.

Methods

IgG2 antibody samples at concentrations up to 122?mg/ml are analyzed using SAXS and compared to Circular Dichroism (CD), Fluorescence, Size Exclusion Chromatography (SEC) and Dynamic Light Scattering (DLS) analysis.

Results

DLS and SEC analyses reveal attraction between antibodies at high concentrations. SAXS data analysis provides an elaborate understanding and shows both attractive and repulsive forces. The protein-protein interactions are strongly affected by excipients. No change in the solution state of IgG2 is observed at pH 4?C8, while samples at pH 3 exhibit heavy oligomerization. The solution conformation of the examined IgG2 derived from SAXS data is a T-shape.

Conclusion

SAXS analysis resolves simultaneous attractive and repulsive interactions, and details the effect of excipients on the interactions, while providing three-dimensional structural information from low-concentration samples.     

过滤对一种IgG2亚型EGFR单抗中不溶性微粒的影响

目的：探讨过滤操作对一种免疫球蛋白G2（immunoglobin G2,IgG2）亚型表皮生长因子受体（epidermal growth factor receptor,EGFR）单克隆抗体（单抗）生物类似药（similar biotherapeutic product,SBP）候选药中不溶性微粒的影响。方法：利用微流数字成像（microflow digital imaging,MDI）技术对某厂家生产的IgG2亚型EGFR单抗SBP候选药与其原研药（reference biotherapeutic product,RBP）中不同粒径的不溶性微粒进行比较;采用0.22 μm的滤器对EGFR单抗SBP候选药进行过滤,并采用MDI法立即对不同粒径和性质的不溶性微粒进行检测和分析;再将过滤后的EGFR单抗SBP候选药在室温静置2 h,并采用MDI法对不同粒径和性质的不溶性微粒进行检测和分析。结果：某IgG2亚型EGFR单抗SBP候选药中不同粒径的不溶性微粒数量明显高于RBP;用0.22 μm的滤器过滤后,EGFR单抗SBP候选药中的不溶性微粒数由8.51×10⁵粒·mL^-1降为1.52×10⁴粒·mL^-1,且主要成分蛋白聚体、气泡和纤维均明显减少;室温静置2 h后,其中的不溶性微粒数由1.52×10⁴粒·mL^-1增加至3.23×10⁴粒·mL^-1,且增加的微粒主要为蛋白聚体。结论：与原研药相比,该EGFR单抗SBP候选药蛋白聚体水平较高,过滤后有明显改善,但随着放置时间延长,蛋白聚体含量又有明显增加。这提示我们需加强研发,以提高SBP候选药的产品质量、安全性和有效性。     

Optimization and Validation of an ELISA to Measure Specific Guinea Pig IgG1 Antibody as an Alternative to the in Vivo Passive Cutaneous Anaphylaxis Assay

Optimization and Validation of an ELISA to Measure Specific Guinea Pig IgG1 Antibody as an Alternative to the in Vivo Passive Cutaneous Anaphylaxis Assay. Kawabata, T. T., Babcock, L. S., Gauggel, D. L., Asquith, T. N., Fletcher, E. R., Horn, P. A., Ratajczak, H. V., and Graziano, F. M. (1995). Fundam. Appl. Toxicol. 24, 238-246.Assessment of the allergenic potency of enzymes involves the use of a guinea pig model in which specific IgG1 antibody titers are used as the endpoint. The in vivo passive cutaneous anaphylaxis (PCA) assay is used to measure specific IgG1 antibody. This report describes the development and validation of an enzyme-linked immunosorbent assay (ELISA) to measure guinea pig specific IgG1 antibody as an in vitro alternative to the PCA assay. Cross reactivity of various rabbit and mouse (monoclonal) anti-guinea pig IgG1 preparations were evaluated using purified IgG1 and IgG2 from serum of guinea pigs immunized with ovalbumin. The two subclasses of guinea pig IgG were purified by first using Protein A affinity chromatography, followed by anion exchange chromatography and fluid phase isoelectric focusing. Affinity-purified rabbit anti-guinea pig IgG1 was shown to have minimal cross reactivity toward IgG2, while providing a strong signal with IgG1. The ELISA was designed as an antigen capture system in which the following are added in sequence: (1) enzyme antigen (passively adsorbed to the plate), (2) diluted serum samples from guinea pigs immunized with enzyme, (3) affinity-purified rabbit anti-guinea pig IgG1, (4) alkaline phosphatase-conjugated donkey anti-rabbit IgG, and (5) p -nitrophenyl phosphate substrate. Three replicate ELISA and PCA analyses were conducted on sera samples of varying titers from guinea pigs immunized with either Alcalase (protease), BPN' (protease), and Termamyl (amylase) enzyme. The correlation coefficients (r²) between the ELISA and PCA assay for Alcalase, BPN', and Termamyl were 0.826, 0.945, and 0.755, respectively. These findings demonstrate that the specific antibody ELISA developed in this study is a valid in vitro alternative to the PCA assay to measure anti-enzyme guinea pig IgG1.     

Development of a Microflow Digital Imaging Assay to Characterize Protein Particulates During Storage of a High Concentration IgG1 Monoclonal Antibody Formulation

The development of a Microflow digital imaging (MDI) method to detect and monitor protein particulates in a high concentration IgG1 monoclonal antibody formulation is presented. The MDI assay was optimized and qualified as a characterization assay in terms of accuracy and precision of particle size and number using polystyrene standards (5–300 μm) and protein particles (2 to > 100 μm). The stability profile of a 90 mg/mL IgG1 formulation stored at 2–8 °C and –70 °C for up to 18 months was then investigated. The MDI assay results showed improved sensitivity to detect subvisible particulates (≥ 5 μm) compared to conventional light obscuration detection, presumably due to the translucent nature of the protein particles. For evaluation of visible protein particles (≥ 70 μm), a good overall correlation was observed for MDI, inverted microscopy and visual assessments. Long-term stability data for a high concentration IgG1 monoclonal antibody formulation demonstrated an accumulation of protein particles in certain size categories with a concomitant increase in the overall particle size distribution over time. The weight amount of protein particulates in the IgG1 formulation was measured experimentally as ~ 0.022% (~ 20 μg/mL) after storage at 2–8 °C for 16 months. Similar results were obtained by calculation from the MDI particle data indicating a low level of protein particulates by weight. The nature and composition of the protein particulates formed during storage were further characterized by a combination of inverted microscopy, FTIR microscopy, and SEM-EDX. Particulates were identified as protein with silicone, although some particles also contained other elements such as aluminum. The combination of MDI results and protein characterization studies have provided an enhanced understanding of protein particulate formation during long-term storage of a high concentration IgG1 monoclonal antibody formulation. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:3343-3361, 2010     

Effects of Drying Process on an IgG1 Monoclonal Antibody Using Solid-State Hydrogen Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS)

Purpose

Lyophilization and spray drying are widely used to manufacture solid forms of therapeutic proteins. Lyophilization is used to stabilize proteins vulnerable to degradation in solution, whereas spray drying is mainly used to prepare inhalation powders or as an alternative to freezing for storing bulk drug substance. Both processes impose stresses that may adversely affect protein structure, stability and bioactivity. Here, we compared lyophilization with and without controlled ice nucleation, and spray drying for their effects on the solid-state conformation and matrix interactions of a model IgG1 monoclonal antibody (mAb).

Methods

Solid-state conformation and matrix interactions of the mAb were probed using solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS), and solid-state Fourier transform infrared (ssFTIR) and solid-state fluorescence spectroscopies.

Results

mAb conformation and/or matrix interactions were most perturbed in mannitol-containing samples and the distribution of states was more heterogeneous in sucrose and trehalose samples that were spray dried.

Conclusions

The findings demonstrate the sensitivity of ssHDX-MS to changes weakly indicated by spectroscopic methods, and support the broader use of ssHDX-MS to probe formulation and process effects on proteins in solid samples.

     

Evaluation of Hydrogen Exchange Mass Spectrometry as a Stability-Indicating Method for Formulation Excipient Screening for an IgG4 Monoclonal Antibody

Antibodies are molecules that exhibit diverse conformational changes on different timescales, and there is ongoing interest to better understand the relationship between antibody conformational dynamics and storage stability. Physical stability data for an IgG4 monoclonal antibody (mAb-D) were gathered through traditional forced degradation (temperature and stirring stresses) and accelerated stability studies, in the presence of different additives and solution conditions, as measured by differential scanning calorimetry, size exclusion chromatography, and microflow imaging. The results were correlated with hydrogen exchange mass spectrometry (HX-MS) data gathered for mAb-D in the same formulations. Certain parameters of the HX-MS data, including hydrogen exchange in specific peptide segments in the C_H2 domain, were found to correlate with stabilization and destabilization of additives on mAb-D during thermal stress. No such correlations between mAb physical stability and HX-MS readouts were observed under agitation stress. These results demonstrate that HX-MS can be set up as a streamlined methodology (using minimal material and focusing on key peptide segments at key time points) to screen excipients for their ability to physically stabilize mAbs. However, useful correlations between HX-MS and either accelerated or real-time stability studies will be dependent on a particular mAb's degradation pathway(s) and the type of stresses used.     

以离子液体为流动相添加剂的HPLC法分离并测定钩藤中钩藤碱和异钩藤碱的含量

目的:建立测定钩藤中钩藤碱和异钩藤碱含量的方法。方法:以离子液体1-丁基-3-甲基咪唑氯化盐(C_4mimCl)为流动相添加剂,与无添加剂的流动相以及加入传统添加剂三乙胺(对色谱柱有损伤)后对高效液相色谱(HPLC)法分离钩藤中钩藤碱和异钩藤碱的分离度进行比较,筛选C_4mimCl的最佳浓度,用新建立的方法测定江西4个产地钩藤中钩藤碱和异钩藤碱的含量。色谱柱为Dikmatech Diamonsil Plus C_(18),流动相为乙腈-缓冲液(0.1%磷酸+3.0 mmol/L C_4mimCl),梯度洗脱,紫外检测波长为245 nm,流速为1 mL/min,进样量为10μL。结果:当流动相中无添加剂时、加入3.0 mmol/L三乙胺或3.0 mmol/L C_4mimCl作添加剂时,钩藤碱与前峰分离度分别为1.02、1.23、1.72,与后峰分离度分别为1.06、6.00、4.25,对称因子分别为0.81、0.86、1.13;异钩藤碱与前峰分离度分别为0.96、3.89、4.05,与后峰分离度分别为1.02、2.34、2.36,对称因子分别为0.88、0.81、0.96。钩藤碱、异钩藤碱检测质量浓度线性范围分别为4.93～157.76(r=0.999 9)、4.98～159.50μg/m L(r=1.000 0),定量限分别为0.486 4、0.793 6μg/mL,精密度、重复性、稳定性和耐用性试验中的RSD均小于5%(n=6),回收率分别为102.9%～107.8%(RSD=1.7%,n=6)、95.4%～106.3%(RSD=3.9%,n=6)。4个产地钩藤中钩藤碱和异钩藤碱的含量范围分别为0.758～1.343、1.511～1.823 mg/g。结论:C_4mimCl加入到流动相中能提高分离度,且以此建立的HPLC法快速、准确、重复性好,可用于钩藤中钩藤碱和异钩藤碱的含量测定。     

A Critical Evaluation of T m(FTIR) Measurements of High-Concentration IgG1 Antibody Formulations as a Formulation Development Tool

Purpose Fourier-transform infrared (FTIR) spectroscopy was applied for the determination of protein melting temperature (T_m(FTIR)) and to assess the stability predictability of a 100-mg/mL liquid IgG₁ antibody formulation. Methods T_m(FTIR) values of various formulations (different pH, buffers, excipients) were compared to the results of a stability study under accelerated conditions (40°C/75% relative humidity), using size-exclusion high-performance liquid chromatography (SE-HPLC) and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for the detection of soluble aggregates and covalent modifications. Results The highest T_m(FTIR) was achieved at pH 5.5, and, similarly, SE-HPLC and SDS-PAGE results suggested a pH optimum between 5.5 and 6.0. Transition temperatures were comparable for all tested buffers. However, the decrease in the monomer fraction upon thermal storage was the lowest for citrate buffers. Whereas sugars and polyols resulted in an increase in T_m(FTIR) and enhanced monomer fraction after storage, amino acids showed a destabilization according to SE-HPLC analysis, albeit no change or even an increase in the melting temperature was observed. Conclusions All examples gave evidence that T_m(FTIR) values did not necessarily correspond to the storage stability at 40°C analyzed by means of SE-HPLC and SDS-PAGE. T_m values, e.g., determined by FTIR, should only be employed as supportive information to the results from both real-time and accelerated stability studies.