Quantification of glycated IgG in CHO supernatants: A practical approach
| Autor: | Karola Vorauer-Uhl, Gerald Striedner, Bernhard Sissolak, Gabriele Lhota, Wolfgang Sommeregger |
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| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
Glycosylation recombinant monoclonal antibody medicine.drug_class post‐translational modification Process Sensing and Control CHO Cells Monoclonal antibody 01 natural sciences Chromatography Affinity RESEARCH ARTICLES Cricetulus Glycation 010608 biotechnology Cricetinae medicine Animals Bioprocess CHO cell culture biology Chemistry Elution 010401 analytical chemistry boronate affinity chromatography Boronic Acids Quantitative determination Recombinant Proteins 0104 chemical sciences Titer Biochemistry Immunoglobulin G biology.protein glycation Antibody Protein glycation Protein Processing Post-Translational Biotechnology Research Article |
| Zdroj: | Biotechnology Progress |
| ISSN: | 1520-6033 |
| Popis: | Post‐translational, nonenzymatic glycation of monoclonal antibodies (mAbs) in the presence of reducing sugars (in bioprocesses) is a widely known phenomenon, which affects protein heterogeneity and potentially has an impact on quality, safety, and efficacy of the end product. Quantification of individual glycation levels is compulsory for each mAb therapeutically applied in humans. We therefore propose an analytical method for monitoring glycation levels of mAb products during the bioprocess. This is a useful tool for process‐design considerations, especially concerning glucose‐feed strategies and temperature as major driving factors of protein glycation. In this study, boronate affinity chromatography (BAC) was optimized for determination of the glycation level of mAbs in supernatants. In fact, the complex matrix found in supernatants is an underlying obstacle to use BAC, but with a simple clean‐up step, we found that the elution profile could be significantly improved so that qualitative and quantitative determination could be reached. Complementary analytical methods confirmed the performance quality, including the correctness and specificity of the results. For quantitative determination of mAb glycation in supernatants, we established a calibration procedure for the retained mAb peak, identified as glycated antibody monomers. For this approach, an available fully characterized mAb standard, Humira®, was successfully applied, and continuous monitoring of mAbs across three repetitive fed‐batch processes was finally performed. With this practical, novel approach, an insight was obtained into glycation levels during bioprocessing, in conjunction with glucose levels and product titer over time, facilitating efficient process development and batch‐consistency monitoring. |
| Databáze: | OpenAIRE |
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