(3c) bar graph comparison showing the relative abundances of different variants in bsAb-1 between the control and the unbound portion samples. AEX: anion exchange chromatography; CB30865 bsAb: bispecific antibody; CDC: complement-dependent cytotoxicity; CDR: complementarity-determining region; CML: carboxymethylation; CQA: crucial quality attribute; DDA: data-dependent acquisition; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; FA: formic acid; Fab: Fragment antigen-binding; FcRn: neonatal Fc receptor; HC: heavy chain; HIC: hydrophobic conversation chromatography; IAA: iodoacetamide; IEX: ion exchange chromatography; LC: light chain; mAb monoclonal antibody; msAb: monospecific antibody; MS: mass spectrometry; PBS: phosphate-buffered saline; pI: isoelectric point; PTM: post-translational modification; SCX: strong cation exchange chromatography; SEC: size exclusion chromatography; SPR: surface plasmon resonance; XIC: extracted ion chromatography. KEYWORDS: Therapeutic antibody, antigen-antibody binding, crucial quality attribute, competitive binding, native LC-MS, bottom-up MS, high-throughput Introduction Development of therapeutic monoclonal antibodies (mAbs) remains a challenging process despite its huge success in the past two decades.1 Due to their large size and complexity, mAb molecules often host a large number of modifications (e.g., characteristics) that need to be thoroughly characterized to support their development.2,3 These attributes can be introduced from both post-translational modifications (e.g., deamidation, oxidation, glycation)4C7 and physicochemical degradations (e.g., aggregation, fragmentation) during mAb production, purification, and storage.8 Of these attributes, those that can impact the efficacy or safety of the drug products are defined as critical quality attributes (CQAs).9C11 Frequently, CQAs that impair the target binding affinity are found within the mAb complementarity-determining regions (CDRs), although they could also occur on residues outside the CDRs through allosteric effects. Conversely, some CDR modifications might not be considered as CQAs if they do not directly or indirectly impact the epitope-paratope interactions.12 Therefore, in addition to empirical knowledge or computational modeling methods, it is also critical to experimentally assess each CDR modification for its effect on mAb target binding. Identification of potential CQAs (pCQAs) that impact mAb target binding is particularly important during drug candidate developability assessment, which is a vital step to select drug candidates with favorable drug-like properties, and therefore, reduce failure rates.13,14 Currently, this task is performed in a low-throughput fashion that requires enrichment of the attribute-bearing variants followed by either in vitro target binding measurement or cell-based potency testing.15 For example, enrichment of mAb variants can often be achieved through liquid chromatography fractionation under different separation modes, such as ion exchange chromatography (IEX),16,17 hydrophobic conversation chromatography (HIC),18,19 and size exclusion chromatography (SEC).20,21 This approach is highly laborious and may not CB30865 isolate low-abundance variants. Therefore, specific stress conditions to artificially generate variants at higher levels are commonly applied prior to fractionation and subsequent assessment.22 Furthermore, to isolate the variants with CB30865 sufficient purity, substantial efforts are often needed to optimize a liquid chromatography method. Finally, as each attribute needs to be evaluated one at a time, this approach is usually low-throughput, and CB30865 therefore, not ideally suited during candidate developability assessment, where multiple candidates might need to be evaluated simultaneously with fast turn-around occasions. To address these limitations, SEC has recently been used as an alternative means to enrich mAb variants with compromised target binding affinity. Following mAb-antigen incubation, the unbound mAb species were separated from your mAb-antigen complexes by SEC. Subsequent bottom-up MS analysis of these SEC fractions could identify attributes that were enriched in the unbound portion due to impaired antigen binding.23 Shi et al.24 further incorporated a competitive binding step to this workflow and showed improved CB30865 method sensitivity in identifying attributes with less significant effects on target binding. This SEC fractionation and bottom-up MS-based approach significantly Ywhaz improved the method throughput in pCQA evaluation. However, due to the need for SEC fractionation,.
(3c) bar graph comparison showing the relative abundances of different variants in bsAb-1 between the control and the unbound portion samples