Preparation of antibodies and preparation of standards: There are two options for the preparation of antibodies. One is to ferment and purify the host cells according to the established production process, and then immunize the animals with the obtained HCP protein to prepare antibodies; the other option is to remove the antibodies from the host cell stock solution obtained by the process and then immunize the animals. The preparation of standards can be transformed into host cells using empty plasmids, and fermented and purified according to the established production process.
Downstream purification: Generally, the recovery of recombinant products expressed in cultured cell systems begins with the harvesting of cell culture fluid (HCCF), followed by a series of downstream processing (DSP) steps. These DSP steps not only allow product recovery, but also emphasize the removal of process-related impurities (including HCP) and product-related impurities (such as aggregates). The final result is a recombinant protein with an HCP level of less than 100 ppm and a high molecular weight immunogenic aggregate of less than 5%.
HCP characterization and quantification: The most widely used method for HCP characterization and quantification is enzyme-linked immunosorbent assay (ELISA); with the development of proteomics technology, orthogonal methods relative to ELISA, such as two-dimensional fluorescence difference electrophoresis (2D-DIGE), liquid chromatography-mass spectrometry (LC-MS) and other mass spectrometry technologies can be used for HCP characterization and analysis, and can more objectively and comprehensively verify the specificity of HCP ELISA detection and the actual HCP coverage. Now, taking 2D-DIGE technology as an example, the HCP detection steps are summarized: take two identical gels to perform two-dimensional electrophoresis on the same purified HCP sample, one of which is used for immunoblotting experiment, transfer HCP protein in the gel to the membrane, and then incubate and bind with HCP antibody, wash and scan by ECL or fluorescence, etc.; the other is scanned by silver staining; the protein spots in the two gels are matched and analyzed in a dedicated analysis software, and the coverage results are calculated.