Characterization of Recombinant Monoclonal Antibody Charge Variants Using OFFGEL Fractionation, Weak Anion Exchange Chromatography, and Mass Spectrometry

Abstract

Recombinant monoclonal antibody charge heterogeneity has been commonly observed as multiple bands or peaks when analyzed by charge-based analytical methods such as isoelectric focusing electrophoresis and cation or anion exchange chromatography. Those charge variants have been separated by some of the above-mentioned methods and used for detailed characterization. The utility of a combination of OFFGEL fractionation and weak anion exchange chromatography to separate the charge variants of a recombinant monoclonal antibody was demonstrated in the current study. Charge variants were separated into various fractions of high purity and then analyzed thoroughly by liquid chromatography mass spectrometry. Analysis of intact molecular weights identified the presence of heavy chain leader sequence, C-terminal lysine, and C-terminal amidation. The identified modifications were further localized into different regions of the antibody from analysis of antibody fragments obtained from FabRICATOR digestion. Analysis of tryptic peptides from various fractions further confirmed the previously identified modifications in the basic variants. Asparagine deamidation and aspartate isomerization were identified in acidic fractions from analysis of tryptic peptides. Basic variants have been fully accounted for by the identified modifications. However, only a portion of the acidic variants can be explained by deamidation and isomerization, suggesting that additional modifications are yet to be identified or acidic variants are an ensemble of molecules with different structures.

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

“…A recent improvement in the analytical workflow involved the use of an additional fractionating step with the Agilent OFFGEL fractionator before the IEX separation. This led to MS analyses that fully accounted for individual charge variant species in the basic region and partially accounted for those in the acidic region (Neill et al, 2015). Similarly, researchers have used free flow electrophoresis (FFE) to isolate specific acidic species by fractionating protein samples before analysis by IEF samples (Hosken et al, 2016;Timm, Gruber, Wasiliu, Lindhofer, & Chelius, 2010).…”

Qualitative and quantitative characterization of protein biotherapeutics with liquid chromatography mass spectrometry

“…In addition, various impurities with micro‐heterogeneity in size, charge, and structure could be produced by enzymatic or spontaneous degradation and chemical modifications during production, purification, and storage processes (Srebalus Barnes & Lim, ). The most frequently occurring modifications of protein therapeutics are glycosylation, C‐terminal lysine truncation, N‐terminal glutamine cyclization, methionine oxidation, asparagine deamidation, aspartate isomerization, and hydrolysis (Harris et al, ; Lyubarskaya et al, ; Neill et al, ). Because these modifications might alter the stability of the product, half‐life, and activity (Robinson & Jones, ), accurate and reproducible analytical platforms that can reliably evaluate these specifications in a high‐throughput manner are required during development and production.…”

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis

“…1 Many of these modifications lead to changes in surface-exposed charged residues or modify the acid dissociation constant, 2 which changes the overall surface charge distribution of the antibody. 3 Compared with the major constituents, species with a lower apparent isoelectric point (pI) are considered acidic species, 4,5 while basic peaks refer to species with a higher pI value. [6][7][8] Methyl-glyoxal of arginine residues, 9 deamidation of asparagine residues, 6,10 glycation, 11 or the presence of sialic acid and trisulfide bonds typically yield acidic variants.…”