Three Anti-cho Cell Residue Protein Antibody Preparation, Identification, Purification And Preliminary Applications

Host cell proteins (HCPs) are those produced or encoded by the organisms and unrelated to the intended recombinant product. In order to quantify HCPs of recombinant protein drugs that expressed by Chinese hamster ovary (CHO), we prepared soluble host cell proteins from CHO cells by the method of freezing and thawing, and then immunized three kinds of animal of chicken, rabbit and sheep with it to obtain high titers of eggs and anti-sera. Then we detected the antibody titers of the water-diluted egg yolk supernatant by ELISA. Basid on the data of the antibodies level changes, the eggs of high titer were obtained. The chicken IgY anti-CHO HCP were purified by two step ice-ethanol precipitation. The titers of three antibodies were detected by double immune diffusion and ELISA respectively, and their coverage of them to immunogen was identified by western blotting. Follow on, we chose three methods of caprylic acid-ammonium sulfate precipitation, protein A/G affinity purification and antigen coupled affinity purification to purify high titer of rabbit and goat sera. The titers of purified anti-CHO cell protein antibodies were detected by double immune diffusion and antigen-coupled affinity purification, and the purity of the protein was detected by SDS-PAGE. Rabbit and goat F(ab')2 were hydrolyzed by trypsin, then purified by gel filtration. With the method of NaI04 anti-CHO HCP antibodies were labeled with HRP. In the end, we developed ELISA methods to detect CHO HCP with these antibodies and conjugates.The highest titers of chicken IgY, rabbit serum and goat serum were more than 104,107 and 105 by ELISA respectively, and the highest titers of the latter two were 1:128 and 1:32 by performing double immune diffusion respectively. The results of western blotting showed that the coverage of rabbit IgG and goat IgG to CHO HCP was better than that of chicken IgY. The results of SDS-PAGE showed that the purity of purified chichen IgY was 99%. After purified by three methods, the purity of rabbit IgGs were 64.7%,81.3%and 68.3%respectively and goat IgGs were 69.6%,83.5%and 80.6%respectively. But the antibody titers decreased approximately one order of magnitude after purified by protein A/G affinity purification. After rabbit IgG and goat IgG were purified by antigen coupled affinity purification, their specificity increased and their purity were 68.3%and 80.6%respectively. The purity of rabbit and goat F (ab')2 purified by gel filtration were 65.6%and 71.2%respectively. The labeling rate of rabbit IgG and goat IgG were 39.4%and 55.4%respectively and the E/P were 1.58 and 1.716 respectively. The linearity range of indirect ELISA was 19.53 to 1250ng/mL, and the sensitivity of sandwich ELISA was 78.13 ng/mL.We prepared and purfied high titers of rabbit serum and sheep serum anti-CHO HCP successfully. The measuring and quantitative method of CHO HCP is needed to be further explored.