Multi-Attribute Monitoring Method for Process Development of Engineered Antibody for Site-Specific Conjugation

Abstract

Antibody drug conjugates, a class of biotherapeutic proteins, have been extensively developed in recent years, resulting in new approvals and improved standard of care for cancer patients. Among the numerous strategies of conjugating cytotoxic payloads to monoclonal antibodies, insertion of a cysteine residue achieves a tightly controlled, site-specific drug to antibody ratio. Tailored analytical tools are required to direct the development of processes capable of manufacturing novel antibody scaffolds with the desired product quality. Here, we describe the development of a 12 min, mass-spectrometry-based method capable of monitoring four distinct quality attributes simultaneously: variations in the thiol state of the inserted cysteines, N-linked glycosylation, reduction of interchain disulfide bonds, and polypeptide fragmentation. This method provides new insight into the properties of the antibody intermediate and associated manufacturing processes. Oxidized thiol states are formed within the bioreactor, of which a variant containing an additional disulfide bond was produced and remained relatively constant throughout the fed-batch process; reduced thiol variants were introduced upon harvest. Nearly 20 percent of N-linked glycans contained sialic acid, substantially higher than anticipated for wildtype IgG1. Lastly, previously unreported polypeptide fragmentation sites were identified in the C239i constant domain, and the relationship between fragmentation and glycoform were explored. This work illustrates the utility of applying a high-throughput liquid chromatography–mass spectrometry multi-attribute monitoring method to support the development of engineered antibody scaffolds.

“Small is beautiful” – the significance of reliable determination of low- abundant therapeutic antibody glycovariants

“…6, <5% relative abundance). Few studies were directly comparing relative glycan quantities obtained from different structural levels, e.g., references [40,60,61]. Hines et al [61] employed a "datasupported glycopeptide detection" with optimized ionization settings.…”

“…Few studies were directly comparing relative glycan quantities obtained from different structural levels, e.g., references [40,60,61]. Hines et al [61] employed a "datasupported glycopeptide detection" with optimized ionization settings. Furthermore, sialylated glycoforms might decompose to smaller glycosylation species falsifying quantification [61].…”

“…Hines et al [61] employed a "datasupported glycopeptide detection" with optimized ionization settings. Furthermore, sialylated glycoforms might decompose to smaller glycosylation species falsifying quantification [61]. Therefore, specific ionization and fragmentation properties of each glycoform to be quantified were considered and data were corrected by conversion factors, with the results showing high correlation of glycopeptide and released glycan quantitative information [61].…”

“…Furthermore, sialylated glycoforms might decompose to smaller glycosylation species falsifying quantification [61]. Therefore, specific ionization and fragmentation properties of each glycoform to be quantified were considered and data were corrected by conversion factors, with the results showing high correlation of glycopeptide and released glycan quantitative information [61]. Furthermore, relative abundances of glycopeptide variants also depend on the charge state of the quantified peptide (i.e., +2 or +3) and whether only full or also miss-cleaved glycopeptides are considered [60].…”

An Online Two-Dimensional Approach to Characterizing the Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies Using a 2D-CEX–AEX–MS Workflow

“…One of the common attributes that can occur is charge heterogeneity, which refers to the presence of different charged forms of the same product. Charge heterogeneity is considered an important product quality attribute for monoclonal antibodies because it can affect their stability, pharmacokinetics, and efficacy. − Charge heterogeneity can arise from a variety of factors, such as changes in the pH or temperature during the production process or enzymatic or chemical modifications of the protein. These modifications can affect the charge of the protein, resulting in different forms of mAbs with varying charge properties.…”