Impact of enzymatic reduction on bivalent bispecific antibody fragmentation and loss of product purity upon reoxidation.

Antibody disulfide bond (DSB) reduction during manufacturing processes is a widely observed phenomenon attributed to host cell reductases present in harvest cell culture fluid. Enzyme-induced antibody reduction leads to product fragments and aggregates that increase the impurity burden on the purification process. The impact of reduction on bivalent bispecific antibodies (BisAbs), which are increasingly entering the clinic, has yet to be investigated. We focused on the reduction and reoxidation properties of a homologous library of bivalent BisAb formats that possess additional single-chain Fv (scFv) fragments with engineered DSBs. Despite all BisAbs having similar susceptibilities to enzymatic reduction, fragmentation pathways were dependent on the scFv-fusion site. Reduced molecules were allowed to reoxidize with and without low pH viral inactivation treatment. Both reoxidation studies demonstrated that multiple, complex BisAb species formed as a result of DSB mispairing. Furthermore, aggregate levels increased for all molecules when no low pH treatment was applied. Combined, our results show that complex DSB mispairing occurs during downstream processes while aggregate formation is dependent on sample treatment. These results are applicable to other novel monoclonal antibody-like formats containing engineered DSBs, thus highlighting the need to prevent reduction of novel protein therapeutics to avoid diminished product quality during manufacturing.

Characterization and quantification of succinimide using peptide mapping under low-pH conditions and hydrophobic interaction chromatography.

Characterization of asparagine deamidation and aspartic acid isomerization is an important aspect of biotherapeutic protein analysis due to the potential negative effect of these modifications on drug efficacy and stability. Succinimide has long been known to be an intermediate product of asparagine deamidation and aspartic acid isomerization, but despite the key role of succinimide in these reactions, its analysis remains challenging due to its instability. We have developed a paradigm in which two interlinked analytical methods are used to develop an optimized approach to analyze succinimide. In the first method, low-pH protein digestion is used for detailed characterization of succinimide with peptide mapping. At low pH, succinimide is stable and can be analyzed with accurate mass measurements and tandem mass spectrometry to confirm its identity and localize its modification site. These results are then used to establish a hydrophobic interaction chromatography (HIC)-based method that can be used for release and stability studies. In this method, unmodified protein, deamidated products, and succinimide are well separated and quantified. Good correlation was obtained between the data from low-pH protein digestion-based peptide mapping and the HIC-based method. Method qualification showed that the HIC-based method is robust, accurate, and precise and has excellent linearity.

Effects of antibody disulfide bond reduction on purification process performance and final drug substance stability.

Antibody disulfide bond reduction during monoclonal antibody (mAb) production is a phenomenon that has been attributed to the reducing enzymes from CHO cells acting on the mAb during the harvest process. However, the impact of antibody reduction on the downstream purification process has not been studied. During the production of an IgG2 mAb, antibody reduction was observed in the harvested cell culture fluid (HCCF), resulting in high fragment levels. In addition, aggregate levels increased during the low pH treatment step in the purification process. A correlation between the level of free thiol in the HCCF (as a result of antibody reduction) and aggregation during the low pH step was established, wherein higher levels of free thiol in the starting sample resulted in increased levels of aggregates during low pH treatment. The elevated levels of free thiol were not reduced over the course of purification, resulting in carry-over of high free thiol content into the formulated drug substance. When the drug substance with high free thiols was monitored for product degradation at room temperature and 2-8°C, faster rates of aggregation were observed compared to the drug substance generated from HCCF that was purified immediately after harvest. Further, when antibody reduction mitigations (e.g., chilling, aeration, and addition of cystine) were applied, HCCF could be held for an extended period of time while providing the same product quality/stability as material that had been purified immediately after harvest. Biotechnol. Bioeng. 2017;114: 1264-1274. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc.

Theoretical predictions and experimental verifications of SERS detection in colorants.

Synthetic colorants added during food processing not only fail to provide nutrients, but also can be harmful to human health when used in excess. To establish a simple, convenient, rapid and low-cost surface-enhanced Raman spectroscopy (SERS) detection method for colorants, an active surface-enhanced substrate of colloidal gold nanoparticles (AuNPs) was prepared in this study. The density functional theory (DFT) method of B3LYP with 6-31G(d) was applied to determine the theoretical Raman spectra of erythrosine, basic orange 2, 21 and 22, and to attribute their characteristic spectral peaks. The SERS spectra of the four colorants were pre-processed using local least squares (LLS) and morphological weighted penalized least squares (MWPLS), and multiple linear regression (MLR) models were established to quantify the four colorants in beverages. The results showed that the prepared AuNPs with a particle size of about 50 nm were reproducible and stable, with a good enhancement of the SERS spectrum of rhodamine 6G at 10-8 mol L-1. The theoretical Raman frequencies were in good agreement with the experimental Raman frequencies, and the peak position differences of the main characteristic peaks of the four colorants were within 20 cm-1. The MLR calibration models for the concentrations of the four colorants showed relative errors of prediction (REP) of 2.97-8.96%, root mean square errors of prediction (RMSEP) of 0.03-0.94, R2 of 0.973-0.999, and limits of detection of 0.06 µg mL-1. The present method could be used to quantify erythrosine, basic orange 2, 21, and 22, revealing its wide range of applications in food safety.

Correction: Theoretical predictions and experimental verifications of SERS detection in colorants.

[This corrects the article DOI: 10.1039/D3RA01584J.].

Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody-Drug Conjugate.

Deamidation, a common post-translational modification, may impact multiple physiochemical properties of a therapeutic protein. MEDI7247, a pyrrolobenzodiazepine (PBD) antibody-drug conjugate (ADC), contains a unique deamidation site, N102, located within the complementarity-determining region (CDR), impacting the affinity of MEDI7247 to its target. Therefore, it was necessary to monitor MEDI7247 deamidation status in vivo. Due to the low dose, a sensitive absolute quantification method using immunocapture coupled with liquid chromatography-tandem mass spectrometry (LBA-LC-MS/MS) was developed and qualified. We characterized the isomerization via Electron-Activated Dissociation (EAD), revealing that deamidation resulted in iso-aspartic acid. The absolute quantification of deamidation requires careful assay optimization in order not to perturb the balance of the deamidated and nondeamidated forms. Moreover, the selection of capture reagents essential for the correct quantitative assessment of deamidation was evaluated. The final assay was qualified with 50 ng/mL LLOQ for ADC for total and nondeamidated antibody quantification, with qualitative monitoring of the deamidated antibody. The impact of deamidation on the pharmacokinetic characteristics of MEDI7247 from clinical trial NCT03106428 was analyzed, revealing a gradual reduction in the nondeamidated form of MEDI7247 in vivo. Careful quantitative biotransformation analyses of complex biotherapeutic conjugates help us understand changes in product PTMs after administration, thus providing a more complete view of in vivo pharmacology.

Atypical Asparagine Deamidation of NW Motif Significantly Attenuates the Biological Activities of an Antibody Drug Conjugate.

Asparagine deamidation is a post-translational modification (PTM) that converts asparagine residues into iso-aspartate and/or aspartate. Non-enzymatic asparagine deamidation is observed frequently during the manufacturing, processing, and/or storage of biotherapeutic proteins. Depending on the site of deamidation, this PTM can significantly impact the therapeutic's potency, stability, and/or immunogenicity. Thus, deamidation is routinely monitored as a potential critical quality attribute. The initial evaluation of an asparagine's potential to deamidate begins with identifying sequence liabilities, in which the n + 1 amino acid is of particular interest. NW is one motif that occurs frequently within the complementarity-determining region (CDR) of therapeutic antibodies, but according to the published literature, has a very low risk of deamidating. Here we report an unusual case of this NW motif readily deamidating within the CDR of an antibody drug conjugate (ADC), which greatly impacts the ADC's biological activities. Furthermore, this NW motif solely deamidates into iso-aspartate, rather than the typical mixture of iso-aspartate and aspartate. Interestingly, biological activities are more severely impacted by the conversion of asparagine into iso-aspartate via deamidation than by conversion into aspartate via mutagenesis. Here, we detail the discovery of this unusual NW deamidation occurrence, characterize its impact on biological activities, and utilize structural data and modeling to explain why conversion to iso-aspartate is favored and impacts biological activities more severely.

Characterization of N-Terminal Glutamate Cyclization in Monoclonal Antibody and Bispecific Antibody Using Charge Heterogeneity Assays and Hydrophobic Interaction Chromatography.

N-terminal glutamate (E) cyclization to form pyroglutamate (pE) generates charge heterogeneities for mAbs and proteins. Thus far, pE formation rate in lyophilized formulation as compared to in liquid formulation has not been reported. Impact of pE on antibody biological activity has only been predicted or assessed using stressed samples that may contain other confounding degradations besides pE. Additionally, application of hydrophobic interaction chromatography (HIC) to separate pE has not been reported. In our study, N-terminal E cyclization was identified as the major degradation pathway in lyophilized formulation at elevated temperature for both monoclonal antibody (mAb-A) and IgG-like bispecific antibody (bsAb-A). pE was enriched in salt-gradient ion exchange chromatography (IEC) as pre-peak and in HIC as post-peak for both mAb-A and bsAb-A. Structure-function studies with pE-enriched IEC and HIC fractions confirmed that pE did not affect binding activities for mAb-A and bsAb-A. In vitro incubation of bsAb-A in serum and PBS revealed that the serum matrix may play a role in pE conversion in human serum, in contrast to the chemical reaction mechanism reported. These techniques can help in characterization of N-terminal E-to-pE cyclization and quality attribute severity assessment during therapeutic protein product development.

Development of a mechanism of action-reflective, dual target cell-based reporter bioassay for a bispecific monoclonal antibody targeting human CTLA-4 and PD-1.

T-cell-mediated immunotherapy has generated much excitement after the success of therapeutic biologics targeting immune checkpoint molecules. Bispecific antibodies (BsAbs) that recognize two antigen targets are a fast-growing class of biologics offering promising clinical benefits for cancer immunotherapy. Due to the complexity of the molecule structure and the potential mechanism of action (MOA) that involves more than one signaling pathway, it is critical to develop appropriate bioassays for measuring potency and characterizing the biological properties of BsAbs. Here, we present a dual target, cell-based reporter bioassay for a BsAb that binds human CTLA-4 and PD-1 and targets two subsequent signaling pathways that negatively regulate T-cell activation. This reporter bioassay is capable of measuring the potency of both antigen target arms in one assay, which would not be achievable using two single target bioassays. This dual target reporter bioassay demonstrates good performance characteristics suitable for lot release, stability testing, critical quality attribute assessment, and biological properties characterization of the CTLA-4/PD-1 BsAb. Furthermore, this assay can capture the synergistic effect of anti-CTLA-4 and anti-PD-1 activity of the BsAb. Compared to single target assays, this dual target bioassay could better reflect the potential MOA of BsAbs and could be used for evaluation of other bispecific biologics, as well as antibody combination therapies.

Characterization and Monitoring of a Novel Light-heavy-light Chain Mispair in a Therapeutic Bispecific Antibody.

Site-specific cysteine engineering, along with other genetic mutations, is broadly implemented in bispecific antibodies (bsAb). Thus far, homodimer, half hole antibody, one-light chain mispaired and light chain swapped variants have been reported as chain-pairing variants for the asymmetric IgG-like bispecific antibodies. Here we report a novel mispair in which the CH3 engineered cysteine on the hole heavy chain (HC) of a knob-into-hole (KiH) bsAb is linked to the engineered cysteine in CL through a disulfide bond, forming a LHL species in a bsAb construct. Due to its impact on bioactivity, it is critical to implement an analytical strategy to monitor this CQA and mitigate risk for the future products. A set of orthogonal physicochemical assays that include hydrophobic interaction chromatography (HIC), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), reverse phase liquid chromatography ultra-performance chromatography mass spectrometry (RP-UPLC MS) and disulfide bond mapping have been utilized to monitor and characterize this chain-pairing impurity for manufacturing process control and product release. Our data shows the LHL mispair in condition medium (CM) is approximately 1.3 - 1.9%. LambdaFabSelect affinity chromatography removes two major chain-pairing variants in CM - i.e. the hole-hole homodimer and hole half-antibody, while retaining the LHL species. Process improvement in Capto Q (anion exchange) and HS50 (cation exchange) chromatography steps removes LHL to as low as 0.2% in the final product. We have demonstrated an orthogonal analytical methodology that is capable of characterizing and monitoring bsAb mispairing, suitable for use in manufacturing process control and product release, and can be potentially implemented for similar bsAb constructs with engineered disulfide bonds.

Identification of a CE-SDS shoulder peak as disulfide-linked fragments from common CH2 cleavages in IgGs and IgG-like bispecific antibodies.

Fragmentation is a well-characterized degradation pathway of therapeutic antibodies and is usually monitored by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). Although fragments due to cleavage in CH2 domains linked by intrachain disulfide bonds are common and can be detected by reduced reversed-phase - liquid chromatography mass spectrometry (RP-LCMS) and reduced CE-SDS methods, their separation in nonreduced CE-SDS (nrCE-SDS) has not been reported but speculated as comigrating with intact IgG. A shoulder peak in nrCE-SDS was observed in the stability samples of an IgG-like bispecific antibody and was determined to be mainly caused by fragments from clipping at the C-terminus of leucine (L)306 or L309 (EU numbering) in the CH2 domain of both heavy chains (HCs) and, to a lesser degree, at the C-terminus of L182 in the CH1 domain of the knob HC. Subunit LCMS analysis verified that the crystallizable fragment contained variants with one or multiple mass additions of ~18 Da due to clipping. Further investigation revealed that CH2 clippings at L306 and L309 were largely due to proteolytic activity, and cleavages were present at various levels in all in-house IgG1 and IgG4 molecules studied. Our study shows that CH2 domain cleavages, with complementary fragments still linked by intrachain disulfide, can be electrophoretically resolved as a front shoulder of the main peak in nrCE-SDS. Given the high occurrence of CH2 cleavages in antibodies, these findings will have broad applicability and could help manufacturers of therapeutic antibodies in process improvement, product characterization, investigations, formulation stability, and stability comparability studies.

Analytical characterization of coformulated antibodies as combination therapy.

When two therapeutic agents are combined in a single formulation, i.e., coformulated, the quality and safety of the individual agents must be preserved. Here we describe an approach to evaluate the quality attributes of two individual monoclonal antibodies (mAbs), designated mAb-A and mAb-B, in coformulation. The mAbs were fractionated from heat-stressed coformulated drug product (DP) by hydrophobic interaction chromatography. Each purified mAb fraction was then compared with mAb-A and mAb-B in their individual formulations from the same drug substance sources used to make the coformulated DP lot, which was subjected to the same stress conditions. Product variants were evaluated and compared by using several analytical tests, including high-performance size exclusion chromatography (HPSEC), reducing and nonreducing gel electrophoresis, ion-exchange chromatography, capillary isoelectric focusing, and peptide mapping with mass spectrometry. Intermolecular interactions in coformulated and photostressed DPs were studied by evaluating aggregates fractionated from coformulated DP by HPSEC. Aggregate fractions of coformulated DP contained dimers, but not coaggregates, of mAb-A or mAb-B. Moreover, extensive assays for higher-order structure and biological interactions confirmed that there was no interaction between the two mAb molecules in the coformulation. These results demonstrate that the two coformulated therapeutic mAbs had the same quality attributes as the individually formulated mAb-A and mAb-B, no new quality attributes were formed, and no physicochemical, intermolecular, or biological interactions occurred between the two components. The approach described here can be used to monitor the product quality of other coformulated antibodies.

Nonspecific cleavages arising from reconstitution of trypsin under mildly acidic conditions.

Tryptic digestion of proteins followed by liquid chromatography with tandem mass spectrometry analysis is an extensively used approach in proteomics research and biopharmaceutical product characterization, owing to the high level of cleavage fidelity produced with this technique. However, nonspecific trypsin cleavages have been frequently reported and shown to be related to a number of digestion conditions and predigestion sample treatments. In this work, we reveal that, for a number of commercial trypsins, reconstitution and storage conditions can have a significant impact on the occurrence of trypsin nonspecific cleavages. We analyzed the tryptic digestion of a variety of biotherapeutics, using trypsins reconstituted under different conditions. The results indicate that, for many commercial trypsins, commonly recommended reconstitution/storage conditions (mildly acidic, e.g., 50 mM acetic acid, 1 mM HCl) can actually promote nonspecific trypsin activities, which are time dependent and can be as high as 20% in total relative abundance. In contrast, using water for reconstitution and storage can effectively limit nonspecific cleavages to 1%. Interestingly, the performances of different commercial trypsins were found to be quite distinct in their levels of nonspecific cleavages and responses to the two reconstitution conditions. Our findings demonstrate the importance of choosing the appropriate trypsin for tryptic digestion and the necessity of assessing the impact of trypsin reconstitution and storage on nonspecific cleavages. We advocate for manufacturers of commercial trypsins to reevaluate manufacturing processes and reconstitution/storage conditions to provide good cleavage specificity.

A method to directly analyze free-drug-related species in antibody-drug conjugates without sample preparation.

Residual free-drug-related species that are present in antibody-drug conjugates (ADC) are a potential safety risk to patients and are therefore categorized as a critical quality attribute that must be strictly monitored and controlled. Among the many analytical methods developed for free-drug analysis, reversed-phase liquid chromatography (RP-LC) is the most common approach. Conventional RP-LC methods for free-drug analysis, however, involve labor-intensive sample preparation. Here we present a new RP-LC method to directly analyze free-drug-related species in an ADC sample without the need for sample preparation. In our work, free-drug-related species were very well separated from ADC peaks in the chromatography gradient. Typical performance issues observed in conventional RP-LC, such as column fouling, detection interference, and carryover, were not observed or were negligible with this new method. Three options were evaluated for free-drug quantitation: Strohl (2017) [1] use of an external free drug calibration curve for determination of absolute concentration; Perez et al. (2014) [2] calculation of relative percentage based on peak area ratio between free drug and ADC at a characteristic wavelength unique for drug payload; and (Beck et al., 2017) [3] calculation of relative percentage based on peak area ratio between free drug and corrected ADC peak area (at any wavelength). The method with calibration curve provides the highest sensitivity, the best accuracy and precision for determination of free drug present in the ADC. However, the second and third options were simpler because they eliminated the need for an external calibration curve, making them worth exploring. Due to its simplicity and compatibility with mass spectrometry, the new method is also a good choice for direct analysis of stability samples.

Site-specific antibody-drug conjugate heterogeneity characterization and heterogeneity root cause analysis.

Site-specific antibody-drug conjugates (ADCs) are designed to overcome the heterogeneity observed with first-generation ADCs that use random conjugation to surface-exposed lysine residues or conjugation to interchain disulfide bonds. Despite significantly enhanced homogeneity, however, the production of site-specific ADCs yields some process-related species heterogeneity, including stereoisomers, unconjugated antibody, underconjugated species, and overconjugated species. An elevated level of size variants, such as heavy chain-light chain species (half ADC), heavy chain-heavy chain-light chain species, and light chain species, is also observed with the final site-specific ADC product. To understand the root cause of heterogeneity generated during the ADC conjugation process, we designed time-course studies for each conjugation step, including reduction, oxidation, conjugation, and quenching. We developed both non-reduced peptide map and LabChip-based capillary electrophoresis sodium dodecyl sulfate methods for time-course sample analysis. On the basis of our time-course data, the half ADC and unconjugated antibody were generated during oxidation as a result of alternative disulfide bond arrangements. During oxidation, two hinge cysteines formed an intra-chain disulfide bond in the half ADC, and three inter-chain hinge disulfide bonds were formed in the unconjugated antibody. Time-course data also showed that the elevated level of size variants, especially heavy chain-heavy chain-light chain species and light chain species, resulted from the quenching step, where the quenching reagent engaged in a disulfide bond exchange reaction with the ADC and broke the disulfide bonds connecting the heavy chain and light chain. Underconjugated and overconjugated species arose from the equilibrium established during the conjugation reaction.

An Automated and Qualified Platform Method for Site-Specific Succinimide and Deamidation Quantitation Using Low-pH Peptide Mapping.

mAbs undergo several post-translational modifications, including the formation of succinimide from the deamidation of asparagine or the isomerization of aspartic acid. Because of the potential impact of succinimide formation on the biological activity of mAbs, detection and quantification of this species is a key area of interest for the pharmaceutical industry. However, studies assessing succinimide stability have been limited, and methods developed to monitor succinimide are either product specific or not robust. Here, we report the development of a platform low-pH peptide-mapping method using a combination of low-pH-resistant Lys-C and modified trypsin to maintain succinimide stability, eliminate deamidation assay artifact, and achieve efficient mAb digestion equivalent to conventional tryptic peptide-mapping method under alkaline condition. Using this method, succinimide stability in serum was accurately assessed in vitro study and the half-life was determined to be 1.5 days. With potential patient exposure to succinimide intermediate, a reliable method was developed to measure site-specific deamidation and succinimide intermediate. Coupled with a single quadrupole mass detector, our method was automated from digestion to data processing and applicable in a good manufacturing practice environment. The method was fully qualified to demonstrate accuracy, precision, linearity, and robustness.

Charge variants characterization and release assay development for co-formulated antibodies as a combination therapy.

Combination therapy is a fast-growing strategy to maximize therapeutic benefits to patients. Co-formulation of two or more therapeutic proteins has advantages over the administration of multiple medications, including reduced medication errors and convenience for patients. Characterization of co-formulated biologics can be challenging due to the high degree of similarity in the physicochemical properties of co-formulated proteins, especially at different concentrations of individual components. We present the results of a deamidation study of one monoclonal antibody component (mAb-B) in co-formulated combination antibodies (referred to as COMBO) that contain various ratios of mAb-A and mAb-B. A single deamidation site in the complementarity-determining region of mAb-B was identified as a critical quality attribute (CQA) due to its impact on biological activity. A conventional charge-based method of monitoring mAb-B deamidation presented specificity and robustness challenges, especially when mAb-B was a minor component in the COMBO, making it unsuitable for lot release and stability testing. We developed and qualified a new, quality-control-friendly, single quadrupole Dalton mass detector (QDa)-based method to monitor site-specific deamidation. Our approach can be also used as a multi-attribute method for monitoring other quality attributes in COMBO. This analytical paradigm is applicable to the identification of CQAs in combination therapeutic molecules, and to the subsequent development of a highly specific, highly sensitive, and sufficiently robust method for routine monitoring CQAs for lot release test and during stability studies.

Cathepsin L Causes Proteolytic Cleavage of Chinese-Hamster-Ovary Cell Expressed Proteins During Processing and Storage: Identification, Characterization, and Mitigation.

A stochastic approach of copurification of the protease Cathepsin L that results in product fragmentation during purification processing and storage is presented. Cathepsin L was identified using mass spectroscopy, characterization of proteolytic activity, and comparison with fragmentation patterns observed using recombinant Cathepsin L. Cathepsin L existed in Chinese hamster ovary cell culture fluids obtained from cell lines expressing different products and cleaved a variety of recombinant proteins including monoclonal antibodies, antibody fragments, bispecific antibodies, and fusion proteins. Therefore, characterization its chromatographic behavior is essential to ensure robust manufacturing and sufficient shelf life. The chromatographic behaviors of Cathepsin L using a variety of techniques including affinity, cation exchange, anion exchange, and mixed mode chromatography were systematically evaluated. Our data demonstrates that copurification of Cathepsin L on nonaffinity modalities is principally because of similar retention on the stationary phase and not through interactions with product. Lastly, Cathespin L exhibits a broad elution profile in cation exchange chromatography (CEX) likely because of its different forms. Affinity purification is free of fragmentation issue, making affinity capture the best mitigation of Cathepsin L. When affinity purification is not feasible, a high pH wash on CEX can effectively remove Cathepsin L but resulted in significant product loss, while anion exchange chromatography operated in flow-through mode does not efficiently remove Cathepsin L. Mixed mode chromatography, using Capto™ adhere in this example, provides robust clearance over wide process parameter range (pH 7.7 ± 0.3 and 100 ± 50 mM NaCl), making it an ideal technique to clear Cathepsin L. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2732, 2019.

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture.

Immunoglobulin G-like bispecific antibodies with asymmetric architecture are among the most widely used bispecific antibody formats for diagnostic and therapeutic applications. The primary technical challenge for this format is how to achieve correctly paired assembly of four unique polypeptide chains. Advances in protein engineering and process development are being used to overcome these challenges and are driving a corresponding demand for sensitive analytical tools to monitor and control mispaired species. Here, we report a systematic approach for analysis and characterization of mispairing in asymmetric bispecific antibodies. This approach consists of three orthogonal components, the first of which is a liquid chromatography (LC)-mass spectrometry (MS)-based method to measure the mass of intact antibodies. This method is used for fast analysis of mispairing and requires minimal method development, which makes it an ideal choice for early-stage development. The second component is a hydrophobic interaction chromatography (HIC)-based mispairing method that is suitable for lot release testing. The HIC method is robust and quality control friendly, and offers great linearity, precision, and accuracy. The third component is a two-dimensional LC-MS method for on-line chromatographic peak identification, which not only expedites this task but also reduces the risk of undesirable modifications during conventional fraction collection. These three methods dovetail to form the foundation of a complementary toolbox for analysis and characterization of mispairing in asymmetric bispecific antibodies and provide guidance and support for process development throughout the drug development life cycle.

Characterization and analysis of scFv-IgG bispecific antibody size variants.

Bispecific antibodies are an emergent class of biologics that is of increasing interest for therapeutic applications. In one bispecific antibody format, single-chain variable fragments (scFv) are linked to or inserted in different locations of an intact immunoglobulin G (IgG) molecule to confer dual epitope binding. To improve biochemical stability, cysteine residues are often engineered on the heavy- and light-chain regions of the scFv to form an intrachain disulfide bond. Although this disulfide bond often improves stability, it can also introduce unexpected challenges to manufacturing or development. We report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, we developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.