Yuetian Yan

Co-formulation of two or more therapeutic monoclonal antibodies (mAbs) into one final drug product has gained significant popularity recently due to its numerous advantages, including enhanced efficacy, reduced overall adverse events, and improved patient convenience and compliance. However, the increased heterogeneity of mAb cocktails introduces additional challenges for both formulation development and analytical characterization. Specifically, the identification and quantitation of various dimer species in mAb cocktails is particularly challenging due to their low abundances and highly similar biophysical properties. Traditional analytical tools used for dimer analysis in mAb monotherapy products often lack the specificity needed to distinguish between heterodimers and homodimers. In this study, we report the development and evaluation of a size-exclusion chromatography method coupled to online native mass spectrometry (nSEC-MS) for analyzing dimers in mAb cocktails. Various sample treatment strategies, such as deglycosylation and immunodepletion, were developed to facilitate the differentiation and quantitation of dimer species in two- and three-mAb cocktails. Additionally, key parameters in SEC separation (e.g., flow rate) and MS data acquisition (e.g., resolution settings) were explored to improve method throughput and sensitivity. Together, this optimized nSEC-MS method and associated analytical strategies were demonstrated to be highly effective in identification and quantitation of various hetero- and homodimer species and can be widely applied to support the development of mAb cocktail products.

Messenger RNAs (mRNAs) have rapidly emerged as a pivotal class of biotherapeutics with great promise in the prevention and treatment of various diseases. As with other biotherapeutics, the sequence accuracy and integrity of mRNAs are critical quality attributes (CQAs), influencing the translation efficiency and protein expression fidelity of mRNAs. Due to the generation of short and repetitive oligonucleotides, traditional sequence mapping methods, which utilize in-solution RNase T1 digestion followed by LC-MS/MS analysis, face challenges in achieving high sequence coverage. In this study, we developed a novel flow through (FT)-based strategy to achieve the limited RNase T1 digestion of therapeutic mRNAs, leading to improved mRNA sequence mapping by LC-MS/MS analysis. Compared with the traditional in-solution digestion methods, the FT-based digestion method could consistently generate an increased number of unique oligonucleotides with miscleavages, which significantly boosted the overall sequence coverage (over 93%) of therapeutic mRNAs of varying sizes. Moreover, the automated digestion workflow using the AssayMAP platform offers significant advantages in method reproducibility and throughput. The high throughput and high sequence coverage features of this method could facilitate its wide application in the development of mRNA-based therapeutics.

Characterizing the cross-links responsible for the covalent high-molecular-weight (HMW) species in therapeutic monoclonal antibodies (mAbs) is of great importance as it not only provides a framework for risk assessment but also offers insights for process improvement. However, owing to the complexity and low abundance, identification of novel and unknown cross-links in mAb products can be very challenging. Here, applying a multipronged MS-based approach, we report the discovery of a novel covalent cross-link formed via an imine bond between lysine and serine residues. In particular, this Ser-Lys cross-link was found to be acid-labile and can be easily overlooked by conventional LC-MS techniques operated at low pH. It is worth noting that although imine-based cross-link has been previously reported in collagen protein cross-linking, this is the first time that a Ser-Lys cross-link has been found in a mAb product that contributes to covalent HMW species formation.

Therapeutic monoclonal antibodies (mAbs) have a propensity to host a large number of chemical and enzymatical modifications that need to be properly assessed for their potential impact on target binding. Traditional strategies of assessing the criticality of these attributes often involve a laborious and low-throughput variant enrichment step prior to binding affinity measurement. Here, we developed a novel competitive binding-based enrichment strategy followed by mass spectrometry analysis (namely, competitive binding-MS) to achieve high-throughput evaluation of potential critical quality attributes in therapeutic mAbs. Leveraging the differences in target binding capability under competitive binding conditions, the criticality of multiple mAb attributes can be simultaneously evaluated by quantitative mass spectrometry analysis. The utility of this new workflow was demonstrated in three mAb case studies, where different post-translational modifications occurring within the complementarity-determining regions were successfully interrogated for their impact on antigen binding. As this workflow does not require prior enrichment (e.g., by forced degradation or liquid chromatography fractionation) of the variants, it is particularly valuable during the mAb candidate developability assessment, where fast turn-around time is highly desired to assist candidate selection. Abbreviations: ACN: acetonitrile; ADCC: antibody-dependent cell-mediated cytotoxicity; AEX: anion exchange chromatography; bsAb: bispecific antibody; CDC: complement-dependent cytotoxicity; CDR: complementarity-determining region; CML: carboxymethylation; CQA: critical 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 interaction 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.

The type 2 l-serine dehydratase from Legionella pneumophila (lpLSD) contains a [4Fe-4S]²⁺ cluster that acts as a Lewis acid to extract the hydroxyl group of l-serine during the dehydration reaction. Surprisingly, the crystal structure shows that all four of the iron atoms in the cluster are coordinated with protein cysteinyl residues and that the cluster is buried and not exposed to solvent. If the crystal structure of lpLSD accurately reflects the structure in solution, then substantial rearrangement at the active site is necessary for the substrate to enter. Furthermore, repair of the oxidized protein when the cluster has degraded would presumably entail exposure of the buried cysteine ligands. Thus, the conformation required for the substrate to enter may be similar to those required for a new cluster to enter the active site. To address this, hydrogen–deuterium exchange combined with mass spectrometry (HDX MS) was used to probe the conformational changes that occur upon oxidative degradation of the Fe–S cluster. The regions that show the most significant differential HDX are adjacent to the cluster location in the holoenzyme or connect regions that are adjacent to the cluster. The observed decrease in flexibility upon cluster binding provides direct evidence that the “tail-in-mouth” conformation observed in the crystal structure also occurs in solution and that the C-terminal peptide is coordinated to the [4Fe-4S] cluster in a precatalytic conformation. This observation is consistent with the requirement of an activation step prior to catalysis and the unusually high level of resistance to oxygen-induced cluster degradation. Furthermore, peptide mapping of the apo form under nonreducing conditions revealed the formation of disulfide bonds between C396 and C485 and possibly between C343 and C385. These observations provide a picture of how the cluster loci are stabilized and poised to receive the cluster in the apo form and the requirement for a reduction step during cluster formation.