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

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.

Detection and quantitation of low abundance oligosaccharides in recombinant monoclonal antibodies.

Oligosaccharides are critical for structural integrity, stability, and biological functions of recombinant monoclonal antibodies. It is relatively easy to characterize, quantify, and determine the impact of major glycoforms. While challenging to detect and quantify, certain low abundance oligosaccharides are highly relevant to the stability and functions of recombinant monoclonal antibodies. Methods were established in this study based on enzymatic digestion to consolidate peaks of the same type of oligosaccharides by removing heterogeneity and thus increase detectability of low abundance peaks. Endo H was used to collapse high mannose oligosaccharides to a single peak of GlcNAc for ease of detection and quantitation. ß-Galactosidase and ß-N-acetylhexosaminidase were used to convert complex oligosaccharides into two peaks containing either GlcNAc2Man3Fuc or GlcNAc2Man3, which simplified the chromatograms and data analysis. More importantly, low abundance hybrid oligosaccharides can only be detected and qualified after ß-galactosidase and ß-N-acetylhexosaminidase digestion. Detection and quantitation of low abundance oligosaccharides can also be achieved using a combination of all three enzymes. These methods can be applied to the development of recombinant monoclonal antibody therapeutics.