Characterization of charge variants of a monoclonal antibody using weak anion exchange chromatography at subunit levels
| Autor: | Hongcheng Liu, Gomathinayagam Ponniah, Christine Nowak, Alyssa Neill |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
medicine.drug_class Protein subunit Biophysics Monoclonal antibody 01 natural sciences Biochemistry Mass Spectrometry Isoaspartate 03 medical and health sciences chemistry.chemical_compound Isomerism Succinimide medicine Amino Acid Sequence Asparagine Deamidation Molecular Biology Chromatography High Pressure Liquid Chromatography Ion exchange Chemistry 010401 analytical chemistry Antibodies Monoclonal Cell Biology Chromatography Ion Exchange Recombinant Proteins 0104 chemical sciences 030104 developmental biology Immunoglobulin G Peptides Protein Processing Post-Translational Isomerization |
| Zdroj: | Analytical Biochemistry. 520:49-57 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2016.12.017 |
| Popis: | An efficient strategy to characterize recombinant monoclonal antibody charge variants was established using weak anion exchange chromatography, LC-MS and IdeS digestion to allow subunit level characterization. Significantly higher resolution was achieved at subunit levels by weak anion exchange chromatography and LC-MS. In addition, subunit analysis localized potential modifications to either F(ab')2 or Fc fragments to facilitate further characterization. Peptide mapping of fractions from various charge variants after IdeS digestion identified aspartate isomerization, asparagine deamidation and glycation as the modifications. Although, aspartate isomerization does not generate net charge difference directly, it does generate antibody basic species. Antibodies with either isoaspartate or aspartate from deamidation showed different retention times by chromatography. Even more interestingly, the antibody contained succinimide as the isomerization intermediate, which though more basic compared to aspartate, eluted off the weak anion exchange column as an acidic species. The results demonstrated not only the utility of subunit level characterization but also the unpredictable chromatographic behavior of antibody charge variants. |
| Databáze: | OpenAIRE |
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