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

Abstract

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

“…Although HIC has been broadly implemented for the monitoring of various quality attributes for mAbs, bsAbs, and antibody-drug conjugates, [19][20][21][22][23][24][25][26][27] the use of this technique to separate N-terminal pE from main species and other variants has not been reported. In our study, pE was enriched exclusively in HIC post-peak 1 by ninefold, indicating that the retention of pE in HIC is not affected by other modifications.…”

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

“…Various post-translational modifications (PTMs) such as deamidation, isomerization, and succinimide formation, have been reported extensively in therapeutic proteins, especially in mAbs. [1][2][3][4][5][6][7][8][9][10] Succinimide is the common product intermediate of 2 important protein degradation pathways, asparagine deamidation and aspartic acid isomerization, both in vitro 1,2,[11][12][13][14][15] and in vivo. 2,3,16,17 In asparagine deamidation, the amide backbone nitrogen engages in a nucleophilic attack on the side chain carbonyl carbon and subsequently loses an ammonia, resulting in the formation of a succinimide intermediate.…”

“…24 Because of the wide occurrence of succinimide formation and its potential impact on the biological activity of therapeutic proteins, the detection and quantitation of this variant is a key area of interest in the pharmaceutical industry. 13,25 Methods to separate succinimide-containing variants from native protein include hydrophobic interaction chromatography, 1,2,7,12,15,26 ion exchange chromatography (IEC), 4,9,10,26,27 sizeexclusion chromatography, 2,27 and capillary isoelectric focusing (cIEF). 2,27 With hydrophobic interaction chromatography method, succinimide variants elute later than the main peak because of their higher hydrophobicity compared with the native protein.…”

“…2,27 With hydrophobic interaction chromatography method, succinimide variants elute later than the main peak because of their higher hydrophobicity compared with the native protein. 7,12,15 With IEC and cIEF, succinimide formed by aspartic acid isomerization is detected as a basic peak due to the loss of a negative charge when forming a cyclic imide. Interestingly, succinimide formed by asparagine deamidation was also reported to appear as an IEC basic peak, although there is no net charge difference between asparagine and succinimide.…”

Systematic workflow for studying domain contributions of bispecific antibodies to selectivity in multimodal chromatography

“…The use of different light chain isotypes (Kappa and Lambda light chains) within the same molecule enabling the use of affinity ligands such as KappaSelect and LambdaFabSelect, has also been investigated (Wang et al, 2018). Regardless of the strategies implemented, productrelated variants such as parental mAbs, mispaired species, half IgG molecules, and fragments are inevitably generated and can pose a significant challenge for downstream processing.…”