Quantitative analysis of glycation and its impact on antigen binding
| Autor: | Michael J. Lewis, Ping Hu, Renzhe Jin, Qingrong Yan, Jingjie Mo, Izabela Sokolowska |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Monoclonal antibody Glycosylation medicine.drug_class Immunology Lysine peptide mapping CHO Cells 01 natural sciences 03 medical and health sciences Residue (chemistry) intact LC-MS Structure-Activity Relationship Cricetulus Glycation Report medicine Immunology and Allergy Bioassay Animals Humans antigen binding structural and function biology Chemistry Isoelectric focusing 010401 analytical chemistry Antibodies Monoclonal Deuterium Exchange Measurement HDX MS 0104 chemical sciences cIEF 030104 developmental biology Biochemistry biology.protein glycation Hydrogen–deuterium exchange Antibody Immunoglobulin Heavy Chains |
| Zdroj: | mAbs |
| ISSN: | 1942-0870 1942-0862 |
| Popis: | Glycation has been observed in antibody therapeutics manufactured by the fed-batch fermentation process. It not only increases the heterogeneity of antibodies, but also potentially affects product safety and efficacy. In this study, non-glycated and glycated fractions enriched from a monoclonal antibody (mAb1) as well as glucose-stressed mAb1 were characterized using a variety of biochemical, biophysical and biological assays to determine the effects of glycation on the structure and function of mAb1. Glycation was detected at multiple lysine residues and reduced the antigen binding activity of mAb1. Heavy chain Lys100, which is located in the complementary-determining region of mAb1, had the highest levels of glycation in both stressed and unstressed samples, and glycation of this residue was likely responsible for the loss of antigen binding based on hydrogen/deuterium exchange mass spectrometry analysis. Peptide mapping and intact liquid chromatography-mass spectrometry (LC-MS) can both be used to monitor the glycation levels. Peptide mapping provides site specific glycation results, while intact LC-MS is a quicker and simpler method to quantitate the total glycation levels and is more useful for routine testing. Capillary isoelectric focusing (cIEF) can also be used to monitor glycation because glycation induces an acidic shift in the cIEF profile. As expected, total glycation measured by intact LC-MS correlated very well with the percentage of total acidic peaks or main peak measured by cIEF. In summary, we demonstrated that glycation can affect the function of a representative IgG1 mAb. The analytical characterization, as described here, should be generally applicable for other therapeutic mAbs. |
| Databáze: | OpenAIRE |
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