Characterization of the Acidic Species of a Monoclonal Antibody Using Weak Cation Exchange Chromatography and LC-MS

Abstract

Charge variants, especially acidic charge variants, of recombinant monoclonal antibodies have been challenging to fully characterize despite the fact that several posttranslational modifications have already been identified. The acidic species of a recombinant monoclonal antibody were collected using weak cation exchange (WCX)-10 chromatography and characterized by LC-MS at multiple levels. In this study, methionine oxidation and asparagine deamidation are the only two modifications identified in the acidic species. Incubation of the collected main chromatographic peak with hydrogen peroxide generated acidic species, which confirmed that acidic species were enriched in oxidized antibody. Differences observed between the original acidic species and the oxidization-induced acidic species indicate that different mechanisms are involved in the formation of acidic species. Additionally, acidic species were generated by thermal stress of the collected main peak from the original sample. Thermal stress of the collected main peak in pH 9 buffer or ammonium bicarbonate generated chromatograms that are highly similar to those from the analysis of the original molecule. LC-MS analysis identified oxidation of the same methionine residue and deamidation of the same asparagine in the corresponding acidic fractions generated by thermal stress; however, relatively lower levels of methionine oxidation and higher levels of asparagine deamdiation were observed. The results support the use of stressed conditions to generate low abundance species for detailed characterization of recombinant monoclonal antibody charge variants, but with caution.

Forced degradation of recombinant monoclonal antibodies: A practical guide

“…The same retention time indicates that variants detected in the drug substance are likely the same as those generated from stressing the samples. 95 However, there are also cases where the degradation products generated during the process are different from those generated from forced degradation studies. A study demonstrated that oxidation of a drug substance resulted in oxidation of methionine residues preferentially on the same heavy chain while incubation with hydrogen peroxide resulted in oxidation of methionine residues randomly on the two heavy chains.…”

Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry

“…7 Deamidated and non-modified species can therefore be distinguished by using adequate charge-sensitive separation techniques. 20,26,[30][31][32] The gold standard for identification of deamidated forms is peak collection after separation, followed by proteolytic digestion and peptide mapping.…”

Impact of cell culture on recombinant monoclonal antibody product heterogeneity

“…The similarity of the charge profiles after Carboxypeptidase B digestion suggests that common modifications irrespective of cell line differences contribute to the charge profiles. Studies have also demonstrated that the charge profiles of recombinant monoclonal antibodies from cell cultures are very similar to the charge profiles obtained from incubation of purified main peak in slightly basic buffers . Apart from enzymatic reactions such as sialylation, removal of C‐terminal lysine, C‐terminal amidation etc., the majority of the charge variants are likely formed due to non‐enzymatic reactions varying with the environmental conditions such as pH, temperature, duration, etc.…”

“…One such common reaction is asparagine deamidation. Non‐enzymatic deamidation of asparagine is one of the most common protein degradation pathways contributing to protein charge variants . There are cases where well‐separated peaks in the acidic region are formed completely due to deamidation .…”