Sample Preparation of Secreted Mammalian Host Cell Proteins and Their Characterization by Two-Dimensional Electrophoresis and Western Blotting
| Autor: | Anton Posch, Elizabeth Jordan Dreskin, Franziska Kollmann, Thomas R. Berkelman |
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| Rok vydání: | 2021 |
| Předmět: |
Gel electrophoresis
0303 health sciences Two-dimensional gel electrophoresis biology Chemistry education 010401 analytical chemistry Gel electrophoresis of proteins 01 natural sciences 0104 chemical sciences law.invention Blot 03 medical and health sciences Isoelectric point Biochemistry Polyclonal antibodies law Cell culture biology.protein Recombinant DNA 030304 developmental biology |
| Zdroj: | Methods in Molecular Biology ISBN: 9781071611852 |
| Popis: | The manufacturing and purification of therapeutic recombinant proteins expressed by cultivated mammalian cells into the cell culture medium leaves the potential for contamination by host cell proteins (HCPs). Validation and quality control testing of any therapeutic protein needs to include a test to show that HCP contamination is at a minimal level. The presence of residual mammalian HCPs during purification and in the final drug product is typically determined using enzyme linked immunosorbent assay (ELISA), which is regarded as the gold standard. The complexity and heterogeneity of HCPs, which include proteins with significant differences in molecular weight (MW), isoelectric point (pI) and hydrophobicity, poses a challenge to detection and quantitation. Two-dimensional gel electrophoresis (2-DE) is one of the most powerful technologies for studying complex protein profiles and is a valuable analytical method in biologics manufacturing. In the purification process, it is very important to know the nature and composition of HCPs, and this information can be used in a rational process design in order to minimize HCPs from the product. Additionally, 2-DE in combination with western blotting can support ELISA development and quality control for the comprehensive immunochemical detection of HCPs by estimating the recognition capacity of the polyclonal serum used in those assays. Here, we present a standardized 2-DE western blotting protocol which takes into account the latest developments in sample preparation of HCPs, protein electrophoresis, protein transfer, immunostaining, and imaging. |
| Databáze: | OpenAIRE |
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