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.     

Evaluation of a Dual-Wavelength Size Exclusion HPLC Method With Improved Sensitivity to Detect Protein Aggregates and Its Use to Better Characterize Degradation Pathways of an IgG1 Monoclonal Antibody

The evaluation of a dual wavelength size exclusion high performance liquid chromatography (DW-SE-HPLC) method with improved sensitivity to detect aggregates in a high concentration IgG1 monoclonal antibody formulation is presented. This technique utilizes ultraviolet detection at two different wavelengths to monitor the levels of monomer, aggregate, and fragments and was shown to have improved sensitivity for the detection aggregates and fragments compared to light scattering (LS) detection. After assay optimization including the use of column conditioning, the limit of quantitation for aggregates was determined to be 0.04% with essentially complete recovery of aggregates from the column (> 99.5%). The DW-SE-HPLC method was used to evaluate the level of protein aggregates generated by different environmental conditions such as exposure to elevated temperatures/acidic pH or intense light. The detection and characterization of protein aggregates by DW-SE-HPLC was compared with an orthogonal biophysical technique (sedimentation velocity analytical ultracentrifugation, SV-AUC). A good overall correlation was observed for levels of monomer, aggregates (dimer and multimers), and fragments as measured by the two analytical techniques (e.g., 6.0% vs. 5.3% and 14% vs. 11% for dimeric aggregates generated by elevated temperature/acidic pH and light exposure, respectively). The stability profile of a high concentration IgG1 monoclonal antibody formulation was investigated under stressed storage conditions (40 °C over 3 months) using the DW-SE-HPLC method including the loss of monomeric species with the concomitant accumulation of both aggregates and fragments. The nature and composition of the aggregates (primarily noncovalent dimers) and fragments (primarily loss of Fab from an intact IgG1) formed during storage were further characterized by a combination of LS measurements and mass spectroscopy analysis of deglycosylated IgG1 samples isolated by preparative SE-HPLC. The combination of DW-SE-HPLC, SV-AUC, LS, and mass spectroscopy results provided a detailed overall understanding the monomer, aggregate, fragment degradation pathway(s) for a high concentration IgG1 monoclonal antibody formulation during storage.     

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.     

Application of a High Throughput and Automated Workflow to Therapeutic Protein Formulation Development

Rapid and efficient formulation development is critical to successfully bringing therapeutic protein drug products into a competitive market under increasingly aggressive timelines. Conventional application of high throughput techniques for formulation development have been limited to lower protein concentrations, which are not applicable to late stage development of high concentration therapeutics. In this work, we present a high throughput (HT) formulation workflow that enables screening at representative concentrations via integration of a micro-buffer exchange system with automated analytical instruments. The operational recommendations associated with the use of such HT systems as well as the efficiencies gained (reduction in hands-on time and run time by over 70% and 30%, respectively), which enable practical characterization of an expanded formulation design space, are discussed. To demonstrate that the workflow is fit for purpose, the formulation properties and stability profiles (SEC and CEX) from samples generated by the HT workflow were compared to those processed by ultrafiltration/diafiltration, and the results were shown to be in good agreement. This approach was further applied to two case studies, one focused on a formulation screen that studied the effects of pH and excipient on viscosity and stability, and the other focused on selection of an appropriate viscosity mimic solution for a protein product.     

The Effect of Formulation Excipients on Protein Stability and Aerosol Performance of Spray-Dried Powders of a Recombinant Humanized Anti-IgE Monoclonal Antibody1

Purpose. To study the effect of trehalose, lactose, and mannitol on the biochemical stability and aerosol performance of spray-dried powders of an anti-IgE humanized monoclonal antibody. Methods. Protein aggregation of spray-dried powders stored at various temperature and relative humidity conditions was assayed by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein glycation was determined by isoelectric focusing and affinity chromatography. Crystallization was examined by X-ray powder diffraction. Aerosol performance was assessed as the fine particle fraction (FPF) of the powders blended with coarse carrier lactose, and was determined using a multiple stage liquid impinger. Results. Soluble protein aggregation consisting of non-covalent and disulfide-linked covalent dimers and trimers occurred during storage. Aggregate was minimized by formulation with trehalose at or above a molar ratio in the range of 300:1 to 500:1 (excipient:protein). However, the powders were excessively cohesive and unsuitable for aerosol administration. Lactose had a similar stabilizing effect, and the powders exhibited acceptable aerosol performance, but protein glycation was observed during storage. The addition of mannitol also reduced aggregation, while maintaining the FPF, but only up to a molar ratio of 200:1. Further increased mannitol resulted in crystallization, which had a detrimental effect on protein stability and aerosol performance. Conclusions. Protein stability was improved by formulation with carbohydrate. However, a balance must be achieved between the addition of enough stabilizer to improve protein biochemical stability without compromising blended powder aerosol performance.     

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.     

Characterization of Murine Monoclonal Antibody to Tumor Necrosis Factor (TNF-MAb) Formulation for Freeze-Drying Cycle Development

Purpose. This study was designed to characterize the formulation of protein pharmaceuticals for freeze-drying cycle development. Thermal properties of a protein formulation in a freezing temperature range are important in the development of freezing and primary drying phases. Moisture sorption properties and the relationship between moisture and stability are the bases for the design of the secondary drying phase. Methods. We have characterized the formulation of TNF-MAb for the purpose of freeze-drying cycle development. The methods include: DTA with ER probes, freeze-drying microscopy, isothermal water adsorption, and moisture optimization.Results. The DTA/ER work demonstrated the tendency to noneutectic freezing for the TNF-MAb formulation at cooling rates of –1 to –3°C/min. The probability of glycine crystallization during freezing was quite low. A special treatment, either a high subzero temperature holding or annealing could promote the maximum crystallization of glycine, which could dramatically increase the T_g' of the remaining solution. The freeze-drying microscopy further indicated that, after the product was annealed, the cake structure was fully maintained at a T_p below –25°C during primary drying. The moisture optimization study demonstrated that a drier TNF-MAb product had better stability. Conclusions. An annealing treatment should be implemented in the freezing phase in order for TNF-MAb to be dried at a higher product temperature during primary drying. A secondary drying phase at an elevated temperature was necessary in order to achieve optimum moisture content in the final product.     

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.     

Frozen State Storage Instability of a Monoclonal Antibody: Aggregation as a Consequence of Trehalose Crystallization and Protein Unfolding

Purpose  

To investigate the cause of unexpected and erratic increase in aggregation during long-term storage of an IgG2 monoclonal antibody in a trehalose formulation at −20°C.     

Deciphering the High Viscosity of a Therapeutic Monoclonal Antibody in High Concentration Formulations by Microdialysis-Hydrogen/Deuterium Exchange Mass Spectrometry

High concentration formulations of therapeutic monoclonal antibodies (mAbs) are highly desired for subcutaneous injection. However, high concentration formulations can exhibit unusual molecular behaviors, such as high viscosity or aggregation, that present challenges for manufacturing and administration. To understand the molecular mechanism of the high viscosity exhibited by high concentration protein formulations, we analyzed a human IgG4 (mAb1) at high protein concentrations using sedimentation velocity analytical ultracentrifugation (SV-AUC), X-ray crystallography, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and protein surface patches analysis. Particularly, we developed a microdialysis HDX-MS method to determine intermolecular interactions at different protein concentrations. SV-AUC revealed that mAb1 displayed a propensity for self-association of Fab-Fab, Fab-Fc, and Fc-Fc. mAb1 crystal structure and HDX-MS results demonstrated self-association between complementarity-determining regions (CDRs) and Fc through electrostatic interactions. HDX-MS also indicated Fab-Fab interactions through hydrophobic surface patches constructed by mAb1 CDRs. Our multi-method approach, including fast screening of SV-AUC as well as interface analysis by X-ray crystallography and HDX-MS, helped to elucidate the high viscosity of mAb1 at high concentrations as induced by self-associations of Fab-Fc and Fab-Fab.     

Polar Solvents Decrease the Viscosity of High Concentration IgG1 Solutions Through Hydrophobic Solvation and Interaction: Formulation and Biocompatibility Considerations

Low-volume protein dosage forms for subcutaneous injection pose unique challenges to the pharmaceutical scientist. Indeed, high protein concentrations are often required to achieve acceptable bioavailability and efficacy for many indications. Furthermore, high solution viscosities are often observed with formulations containing protein concentrations well above 150 mg/mL. In this work, we explored the use of polar solvents for reducing solution viscosity of high concentration protein formulations intended for subcutaneous injection. An immunoglobulin, IgG1, was used in this study. The thermodynamic preferential interaction parameter (Г₂₃) measured by differential scanning calorimetry, as well as Fourier transform infrared, Raman, and second-derivative UV spectroscopy, were used to characterize the effects of polar solvents on protein structure and to reveal important mechanistic insight regarding the nature of the protein–solvent interaction. Finally, the hemolytic potential and postdose toxicity in rats were determined to further investigate the feasibility of using these cosolvents for subcutaneous pharmaceutical formulations.     

Correlating Excipient Effects on Conformational and Storage Stability of an IgG1 Monoclonal Antibody with Local Dynamics as Measured by Hydrogen/Deuterium-Exchange Mass Spectrometry

The effects of sucrose and arginine on the conformational and storage stability of an IgG1 monoclonal antibody (mAb) were monitored by differential scanning calorimetry (DSC) and size-exclusion chromatography (SEC), respectively. Excipient effects on protein physical stability were then compared with their effects on the local flexibility of the mAb in solution at pH 6, 25°C using hydrogen/deuterium-exchange mass spectrometry (H/D-MS). Compared with a 0.1 M NaCl control, sucrose (0.5 M) increased conformational stability (T_m values), slowed the rate of monomer loss, reduced the formation of insoluble aggregates, and resulted in a global trend of small decreases in local flexibility across most regions of the mAb. In contrast, the addition of arginine (0.5 M) decreased the mAb's conformational stability, increased the rate of loss of monomer with elevated levels of soluble and insoluble aggregates, and led to significant increases in the local flexibility in specific regions of the mAb, most notably within the constant domain 2 of the heavy chain (C_H2). These results provide new insights into the effect of sucrose and arginine on the local dynamics of IgG1 domains as well as preliminary correlations between local flexibility within specific segments of the C_H2 domain (notably heavy chain 241–251) and the mAb's overall physical stability. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2136–2151, 2013     

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.     

Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay

Programmed death-1 (PD-1) protein is a co-inhibitory receptor which negatively regulates immune cell activation and permits tumors to evade normal immune defense. Anti-PD-1 antibodies have been shown to restore immune cell activation and effector function—an exciting breakthrough in cancer immunotherapy. Recent reports have documented a soluble form of PD-1 (sPD-1) in the circulation of normal and disease state individuals. A clinical assay to quantify sPD-1 would contribute to the understanding of sPD-1-function and facilitate the development of anti-PD-1 drugs. Here, we report the development and validation of a sPD-1 protein assay. The assay validation followed the framework for full validation of a biotherapeutic pharmacokinetic assay. A purified recombinant human PD-1 protein was characterized extensively and was identified as the assay reference material which mimics the endogenous analyte in structure and function. The lower limit of quantitation (LLOQ) was determined to be 100 pg/mL, with a dynamic range spanning three logs to 10,000 pg/mL. The intra- and inter-assay imprecision were ≤15%, and the assay bias (percent deviation) was ≤10%. Potential matrix effects were investigated in sera from both normal healthy volunteers and selected cancer patients. Bulk-prepared frozen standards and pre-coated Streptavidin plates were used in the assay to ensure consistency in assay performance over time. This assay appears to specifically measure total sPD-1 protein since the human anti-PD-1 antibody, nivolumab, and the endogenous ligands of PD-1 protein, PDL-1 and PDL-2, do not interfere with the assay.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-015-9762-4) contains supplementary material, which is available to authorized users.KEY WORDS: biomarker, nivolumab, reagent characterization, reagent characterizationsoluble PD-1, reference standard     

Use of Quantitative Pharmacology in the Development of HAE1, a High-Affinity Anti-IgE Monoclonal Antibody

HAE1, a high-affinity anti-IgE monoclonal antibody, is discussed here as a case study in the use of quantitative pharmacology in the development of a second-generation molecule. In vitro, preclinical, and clinical data from the first-generation molecule, omalizumab, were heavily leveraged in the HAE1 program. A preliminary mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model for HAE1 was developed using an existing model for omalizumab, together with in vitro binding data for HAE1 and omalizumab. When phase I data were available, the model was refined by simultaneously modeling PK/PD data from omalizumab studies with the available HAE1 phase I data. The HAE1 clinical program was based on knowledge of the quantitative relationship between a pharmacodynamic biomarker, suppression of free IgE, and clinical response (e.g., lower exacerbation rates) obtained in pivotal studies with omalizumab. A clinical trial simulation platform was developed to predict free IgE levels and clinical responses following attainment of a target free IgE level (相似文献   

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.     

Development of a Pharmaceutical Composition and Stablity of Liquid Dosage Forms Based on Monoclonal IgG1 Antibodies

Pharmaceutical Chemistry Journal - A scheme for developing an excipient composition for liquid dosage forms based on monoclonal antibodies(mAbs) is proposed and used to develop a stable formulation...     

Effect of pH and Excipients on Structure, Dynamics, and Long-Term Stability of a Model IgG1 Monoclonal Antibody upon Freeze-Drying

Purpose

To investigate the mechanism of IgG1 mAb stabilization after freeze-drying and the interdependence of protein structural preservation in the solid state, glassy state dynamics and long-term storage stability under different formulation conditions.

Methods

IgG1 mAb was formulated with mannitol at pH 3.0, 5.0, and 7.0 in the presence and absence of sucrose and stability was monitored over 1 year at different temperatures. Physical and covalent degradation of lyophilized formulation was monitored using SEC, CEX, and light obscuration technique. Secondary and tertiary structure of the protein in the solid state was characterized using FTIR and fluorescence spectroscopy respectively. Raman spectroscopy was also used to monitor changes in secondary and tertiary structure, while SS-NMR ¹H relaxation was used to monitor glassy state dynamics.

Results

IgG1 mAb underwent significant secondary structural perturbations at pH 3.0 and conditions without sucrose, while pH 5.0 condition with sucrose showed the least structural change over time. The structural changes correlated with long-term stability with respect to protein aggregate formation and SbVP counts. SS-NMR data showed reduced relaxation time at conditions that were more stable.

Conclusions

Native state protein structural preservation and optimal solid-state dynamics correlate with improved long-term stability of the mAb in the different lyophilized formulations.     

Systematic Development of a Size Exclusion Chromatography Method for a Monoclonal Antibody with High Surface Aggregation Propensity (SAP) Index

Size Exclusion Chromatography (SEC) has been widely used to assess aggregate content in bio-pharmaceutical drugs such as monoclonal antibodies (mAbs), and is routinely used during method development and release testing. Electrostatic interactions between protein analytes and SEC column resin are commonly observed besides the primary mode of size separation during SEC method development, which needs to be minimized. An effective method to minimize electrostatic interactions is through increasing mobile phase (MP) salt concentration. However; increasing salt concentration in MP will induce increased hydrophobicity of proteins and increased hydrophobic interactions between protein and stationary phase, as demonstrated for mAb-A in this paper, a protein with high surface aggregation propensity (SAP) score and an isoelectric point near mobile phase pH. In this work, a systematic, Design of Experimental approach was taken to identify optimal SEC method conditions including column type, buffer composition, ionic strength, pH and additives. The optimized method was demonstrated to be robust towards small changes in method operation conditions and was qualified for use in product release and stability studies. Additionally, biophysical and computational studies were performed to elucidate the role of MP additives, which supports the use of arginine as an essential additive to minimize undesirable hydrophobic interactions between proteins and stationary phase.