Mechanistic insights into inter-chain disulfide bond reduction of IgG1 and IgG4 antibodies.

Therapeutic monoclonal antibodies (mAbs), primarily immunoglobin G1 (IgG1) and IgG4 with an engineered CPPC motif in its hinge region, are predominant biologics. Inter-chain disulfide bonds of IgG mAbs are crucial to maintaining IgG integrity. Inter-chain disulfide bond-reduced low molecular weight (LMW) is considered as one of quality attributes of IgG drug substance and is observed in drug substance manufacturing. In this study, we demonstrate that IgG1 and IgG4 are susceptible to the reducing agent TCEP differently and they follow different pathways to form LMWs. Our study shows that IgG1 is more sensitive to TCEP than IgG4. Both therapeutic IgG1 and human blood plasma IgG1 follow a heavy-heavy-light chain (HHL) pathway, featured with HHL and HH as intermediate species. Human blood plasma IgG4 with a CPSC motif in its hinge region follows heavy-light chain (HL) pathway, featured with HL as the intermediate species. However, therapeutic IgG4 follows a hybrid pathway with the HL pathway as the primary and the HHL pathway as the secondary. These experimental observations are further explained using solvent accessibility of inter-chain disulfide bonds obtained from computational modeling and molecular dynamics simulations. Findings from this study provide mechanistic insights of LMW formation of IgG1 and IgG4, which suggest selection of IgG1 or IgG4 for bispecific antibodies and cysteine-based antibody-drug conjugates. KEY POINTS: • Experimentally discovered preferable disulfide bond reduction pathways between IgG1 and IgG4 antibodies, driven by the different solvent accessibilities of these disulfide bonds. • Computationally explained the solvent accessibility aided by molecular dynamics simulations. • Provided insights in developing robust biologics process and designing bispecific antibodies and cysteine-based antibody-drug conjugates.

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.

Understanding the pathway and kinetics of aspartic acid isomerization in peptide mapping methods for monoclonal antibodies.

Isomerization of aspartic acid (Asp) in therapeutic proteins could lead to safety and efficacy concerns. Thus, accurate quantitation of various Asp isomerization along with kinetic understanding of the variant formations is needed to ensure optimal process development and sufficient product quality control. In this study, we first observed Asp-succinimide conversion in complementarity-determining regions (CDRs) Asp-Gly motif of a recombinant mAb through ion exchange chromatography, intact protein analysis by mass spectrometry, and LC-MS/MS. Then, we developed a specific peptide mapping method, with optimized sample digestion conditions, to accurately quantitate Asp-succinimide-isoAsp variants at peptide level without method-induced isomerization. Various kinetics of Asp-succinimide-isoAsp isomerization pathways were elucidated using 18O labeling followed by LC-MS analysis. Molecular modeling and molecular dynamic simulation provide additional insight on the kinetics of Asp-succinimide formation and stability of succinimide intermediate. Findings of this work shed light on the molecular construct and the kinetics of the formation of isoAsp and succinimide in peptides and proteins, which facilitates analytical method development, protein engineering, and late phase development for commercialization of therapeutic proteins.

Design of next-generation therapeutic IgG4 with improved manufacturability and bioanalytical characteristics.

Manufacturability of immunoglobulin G4 (IgG4) antibodies from the Chemistry, Manufacture, and Controls (CMC) perspective has received little attention during early drug discovery. Despite the success of protein engineering in improving antibody biophysical properties, a clear gap still exists between rational design of IgG4 candidates and their manufacturing suitability. Here, we illustrate that undesirable two-peak elution profiles in cation-exchange chromatography are attributed to the S228P mutation (in IgG4 core-hinge region) intentionally designed to prevent Fab-arm exchange. A new scaffolding platform for engineering IgG4 antibodies amenable to bioprocessing and bioanalysis is proposed by introducing an "IgG1-like" single-point mutation in the hinge or CH1 region of IgG4S228P. This work offers insight into the design, discovery, and development of innovative therapeutic antibodies that are well suited for robust biomanufacturing and quality control.

Therapeutic protein purity and fragmented species characterization by capillary electrophoresis sodium dodecyl sulfate using systematic hybrid cleavage and forced degradation.

Positive identification of capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) electropherogram peaks provides information to understand protein molecular characteristics at the structural level. It is critical in the design of a robust assay that can accurately resolve, differentiate, and quantify all therapeutic protein components including fragmented species, which are considered as product related impurities. However, direct identification of the impurity peaks observed in CE-SDS is a challenging and oftentimes an ambiguous task. This paper proposed a systematic workflow for characterizing CE-SDS fragmentation peaks. Forced degradation of monoclonal antibody (mAb) by multiple stress methods was utilized to induce fragmentation and species enrichment. The characteristics, such as size and the clipped region of sequence, were then evaluated based on multiple enzymatic treatment and particle reduction. The identified fragments were further confirmed using tryptic digestion and liquid chromatography coupled with mass spectrometry (LC-MS) analysis. Common fragment sizes and clipping locations are identified after evaluating multiple IgG molecules. The methodology and procedure described in this article are readily deployable and will provide necessary information for method, process, and product characterizations. Graphical abstract.

Streamlining the polishing step development process via physicochemical characterization of monoclonal antibody aggregates.

When developing purification processes for monoclonal antibodies (mAbs), ensuring the effective removal of high molecular weight (HMW) species is often challenging and labor intensive. In this work, we present a bottom-up characterization approach to achieve streamlined polishing step development as well as a more fundamental understanding of the protein of interest. Prior to physicochemical characterization, in-process HMW species of two IgG4 mAbs (mAb A and mAb B) were isolated via semi-preparative size exclusion chromatography (SEC). Key differences in approximate molecular weight, net charge, and native surface hydrophobicity were then identified using multi-angle light scattering (SEC-MALS), analytical-scale chromatographic screening, isoelectric focusing, and structural aggregation propensity modeling. SEC-MALS revealed two main HMW isoforms for each mAb: dimers and 1.7-mers for mAb A, and tetramers and dimers for mAb B. Analytical-scale chromatographic screening showed promising trends in charge-based separation for mAb A, and hydrophobic-based separation for mAb B. Isoelectric focusing data detected a 30% increase in acidic variants for mAb A HMW species relative to monomer, and a 20% increase in basic variants for mAb B HMW species. Lastly, analytical-scale characterization data was successfully applied to preparative scale purification conditions, producing results highly similar to those observed during analytical characterization of the isolated species. By using this high-throughput approach as a template for preparative-scale process development, key physicochemical differences between aggregate and monomer species were utilized to determine optimal polishing steps for HMW removal.

Assessment of CE-based baseline disturbances using simulation and targeted experimental evaluation-impact on the purity determination of therapeutic proteins.

The baseline instability for capillary electrophoretic analysis is an intrinsic feature of the technique, which has not been thoroughly examined for its impact on therapeutic protein purity analysis with the capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) applications. For the particular CE-SDS application, this phenomenon was manifested through peak migration time shifts and sliding of the superimposed baseline profile. These dual phenomena are closely associated so that experimental assessment alone may not shed enough light to the underlying drivers. In the current study, both experimental and simulation approaches were employed to assess the systematic drifts. Computer simulation was used to decipher the two underlying factors and test their contributions toward purity and impurity peak determination inaccuracies. The data generated in this study demonstrated that the electrophoretic baseline disturbance had more pronounced impact on the purity data than the migration time shift. In addition, the potential contributing factors to the baseline disturbances were assessed experimentally which indicated that the source is related to thermal disruption during a sample run and the unique baseline patterns came from the background electrolytes. To improve data reproducibility for drug purity testing in the industrial setting and quality control (QC) environment, it is recommended to run shorter injection sequences including fewer samples and closely monitor the baseline drift for accurate integration. Those methods would help reduce the impact of systematic drift and disturbances. Graphical abstract.

Using hydrogen peroxide to prevent antibody disulfide bond reduction during manufacturing process.

During large-scale monoclonal antibody manufacturing, disulfide bond reduction of antibodies, which results in generation of low molecule weight species, is occasionally observed. When this happens, the drug substance does not meet specifications. Many investigations have been conducted across the biopharmaceutical industry to identify the root causes, and multiple strategies have been proposed to mitigate the problem. The reduction is correlated with the release of cellular reducing components and depletion of dissolved oxygen before, during, and after harvest. Consequently, these factors can lead to disulfide reduction over long-duration storage at room temperature prior to Protein A chromatography. Several strategies have been developed to minimize antibody reduction, including chemical inhibition of reducing components, maintaining aeration before and after harvest, and chilling clarified harvest during holding. Here, we explore the use of hydrogen peroxide in clarified harvest bulk or cell culture fluid as a strategy to prevent disulfide reduction. A lab-scale study was performed to demonstrate the effectiveness of hydrogen peroxide in preventing antibody reduction using multiple IgG molecules. Studies were done to define the optimal concentration of hydrogen peroxide needed to avoid unnecessary oxidization of the antibody products. We show that adding a controlled amount of hydrogen peroxide does not change product quality attributes of the protein. Since hydrogen peroxide is soluble in aqueous solutions and decomposes into water and oxygen, there is no additional burden involved in removing it during the downstream purification steps. Due to its ease of use and minimal product impact, we demonstrate that hydrogen peroxide treatment is a powerful, simple tool to quench reducing potential by simply mixing it with harvested cell culture fluid.

Monoclonal antibody higher order structure analysis by high throughput protein conformational array.

The elucidation of antibody higher order structure (HOS) is critical in therapeutic antibody development. Since HOS determines the protein bioactivity and chemo-physical properties, this knowledge can help to ensure that the safety and efficacy attributes are not compromised. Protein conformational array (PCA) is a novel method for determining the HOS of monoclonal antibodies. Previously, we successfully utilized an enzyme-linked immunosorbent assay (ELISA)-based PCA along with other bioanalytical tools to elucidate the structures of antibody aggregates. In this study, applying a new multiplex-based PCA with 48-fold higher throughput than the ELISA-based one we revealed structural differences between different antibody molecules and antibody structure changes affected by various processing conditions. The PCA analysis of antibody molecules clearly demonstrated significant differences between IgG1 and IgG4 subclasses in epitope exposure and folding status. Furthermore, we applied small angle X-ray scattering to decipher mechanistic insights of PCA technology and validate structural information obtained using PCA. These findings enhance our fundamental understanding of mAbs' HOS in general. The PCA analysis of antibody samples from various processing conditions also revealed that antibody aggregation caused significantly higher exposure of antibody epitopes, which potentially led to a "foreign" molecule that could cause immunogenicity. The PCA data correlated well with protein stability results from traditional methods such as size-exclusion chromatography and protein thermal shift assay. Our study demonstrated that high throughput PCA is a suitable method for HOS analysis in the discovery and development of therapeutic antibodies.

Purity Determination by Capillary Electrophoresis Sodium Hexadecyl Sulfate (CE-SHS): A Novel Application For Therapeutic Protein Characterization.

Capillary gel electrophoresis using sodium dodecyl sulfate (CE-SDS) is used commercially to provide quantitative purity data for therapeutic protein characterization and release. In CE-SDS, proteins are denatured under reducing or nonreducing conditions in the presence of SDS and electrophoretically separated by molecular weight and hydrodynamic radius through a sieving polymer matrix. Acceptable performance of this method would yield protein peaks that are baseline resolved and symmetrical. Nominal CE-SDS conditions and parameters are not optimal for all therapeutic proteins, specifically for Recombinant Therapeutic Protein-1 (RTP-1), where acceptable resolution and peak symmetry were not achieved. The application of longer alkyl chain detergents in the running buffer matrix substantially improved assay performance. Matrix running buffer containing sodium hexadecyl sulfate (SHS) increased peak resolution and plate count 3- and 8-fold, respectively, compared to a traditional SDS-based running gel matrix. At Bristol-Myers Squibb (BMS), we developed and qualified a viable method for the characterization and release of RTP-1 using an SHS-containing running buffer matrix. This work underscores the potential of detergents other than SDS to enhance the resolution and separation power of CE-based separation methods.

Imaged capillary isoelectric focusing in native condition: A novel and successful example.

Imaged capillary isoelectric focusing (icIEF) separates ampholytic components of biomolecules in an electric field according to their isoelectric points and has been used for protein charge variants quantification and characterization. Denaturants are ordinarily incorporated into icIEF to stabilize charge species in solution. In certain circumstances, however, denaturants are detrimental to stable isoelectric separation of proteins due to their unique structural and biophysical features, such as an aggregation-prone antibody we encountered recently. Here we report our novel matrix formula non-detergent sulfobetaine and taurine (NDSB-T). It is deprived of denaturants that notably ameliorates the assay robustness and peak resolution for this antibody. NDSB-T is a combination of non-detergent sulfobetaine (NDSB) and taurine possessing the stabilization and separation power while maintaining protein integrity. As a result, assay throughputs are tremendously increased for more than 10 folds along with extraordinarily improved assay accuracy. Furthermore, NDSB-T can separate and quantify protein charge species in native state and therefore avoid partial denaturation derived peaks which are often misleading and hard to characterize. NDSB-T may be a valuable tool for proteins incompatible with conventional icIEF matrices and potentially opens a new window for icIEF assay in native conditions.

Host Cell Protein Profiling by Targeted and Untargeted Analysis of Data Independent Acquisition Mass Spectrometry Data with Parallel Reaction Monitoring Verification.

Host cell proteins (HCPs) are process-related impurities of biopharmaceuticals that remain at trace levels despite multiple stages of downstream purification. Currently, there is interest in implementing LC-MS in biopharmaceutical HCP profiling alongside conventional ELISA, because individual species can be identified and quantitated. Conventional data dependent LC-MS is hampered by the low concentration of HCP-derived peptides, which are 5-6 orders of magnitude less abundant than the biopharmaceutical-derived peptides. In this paper, we present a novel data independent acquisition (DIA)-MS workflow to identify HCP peptides using automatically combined targeted and untargeted data processing, followed by verification and quantitation using parallel reaction monitoring (PRM). Untargeted data processing with DIA-Umpire provided a means of identifying HCPs not represented in the assay library used for targeted, peptide-centric, data analysis. An IgG1 monoclonal antibody (mAb) purified by Protein A column elution, cation exchange chromatography, and ultrafiltration was analyzed using the workflow with 1D-LC. Five protein standards added at 0.5 to 100 ppm concentrations were detected in the background of the purified mAb, demonstrating sensitivity to low ppm levels. A calibration curve was constructed on the basis of the summed peak areas of the three highest intensity fragment ions from the highest intensity peptide of each protein standard. Sixteen HCPs were identified and quantitated on the basis of the calibration curve over the range of low ppm to over 100 ppm in the purified mAb sample. The developed approach achieves rapid HCP profiling using 1D-LC and specific identification exploiting the high mass accuracy and resolution of the mass spectrometer.

A novel reagent significantly improved assay robustness in imaged capillary isoelectric focusing.

Imaged Capillary Isoelectric Focusing (icIEF) has been used as primary method for charge variants analysis of therapeutic antibodies and proteins [1], [9]. Proteins tend to precipitate around their pI values during focusing [14], which directly affects the reproducibility of their charge profiles. Protein concentration, focusing time and various supplementing additives are key parameters to minimize the protein precipitation and aggregation. Urea and sucrose are common additives to reduce protein aggregation, solubilize proteins in sample matrix and therefore improve assay repeatability [15]. However some proteins and antibodies are exceptions, we found urea and sucrose are not sufficient for a typical fusion protein (Fusion protein A) in icIEF assay and high variability is observed. We report a novel reagent, formamide, significantly improved reproducibility of protein charge profiles. Our results show formamide is a good supplementary reagent to reduce aggregation and stabilize proteins in isoelectric focusing. We further confirmed the method robustness, linearity, accuracy and precision after introducing the new reagent; extremely tight pI values, significantly improved method precision and sample on-board stability are achieved by formamide. Formamide is also proven to be equally functional to multiple antibodies as urea, which makes it an extra tool in icIEF method development.

Optimization of microchip-based electrophoresis for monoclonal antibody product quality analysis revealed needs for extra surfactants during denaturation.

Microchip-based electrophoresis has gained increasing popularity in biopharmaceutical development and testing laboratories because of its automation and rapid analysis capabilities. One application of microchip-based electrophoresis is the assessment of size-based variants for product purity analysis. However, monoclonal antibodies analyzed by this technique sometimes exhibited different electrophoretic behaviors. In this study, when three IgG1 and five IgG4 were analyzed using microchip-based electrophoresis under reducing conditions, one of the IgG1s, denoted as mAb1, exhibited an atypical profile attributed to its specific heterogeneity resulting in separation of its heavy chain into two main species. During investigation of the atypical profile, several parameters that were critical to optimal resolution were evaluated, and the data pointed toward incomplete denaturation of mAb1 due to lack of sufficient surfactant in the vendor provided sample buffer (0.7% surfactant). Denaturation studies demonstrated that, although typical antibody profiles could be achieved at 0.7% surfactant for most antibodies analyzed, five out of eight antibodies were not fully denatured until the surfactant concentration reached 0.9% or higher, and mAb1 required a surfactant concentration of 1.3% for complete denaturation. Molecular modeling analysis revealed features in surface charge, hydrophobicity, and structure from mAb1 that led to its unique surfactant concentration-dependent electrophoretic behaviors observed. The optimized method was further evaluated for specificity, linearity, precision, and limit of quantitation for mAb1, and compared with that of conventional CE-SDS.

Process analytics for purification of monoclonal antibodies.

The application of appropriate analytical methods is an essential requirement for the purification of therapeutic antibodies. A range of analytical methods need to be employed to effectively determine the purity, identity, integrity and activity of these important class of pharmaceuticals. These include notably electrophoresis, high performance liquid chromatography and immunoassays. Regulatory and industry demands in recent years have brought the need for improvements and many have been successfully implemented. This article reviews the current analytical methods applied to support the purification of monoclonal antibodies.