Establishment and evaluation of detection methods for process‐specific residual host cell protein and residual host cell DNA in biological preparation

To establish accurate detection methods of process‐specific Escherichia coli residual host cell protein (HCP) and residual host cell DNA (rcDNA) in recombinant biological preparations. Taking the purification process of GLP expressed by E. coli as a specific‐process model, the HCP of empty E. coli was intercepted to immunize mice and rabbits. Using IgG from immunized rabbits as the coating antibody and mouse immune serum as the second sandwich antibody, a process‐specific enzyme‐linked immunosorbent assay (ELISA) for E. coli HCP was established. Targeting the 16S gene of E. coli, ddPCR was used to obtain the absolute copies of rcDNA in samples. Non‐process‐specific commercial ELISA kit and the process‐specific ELISA established in this study were used to detect the HCP in GLP preparation. About 62% of HCPs, which should be process‐specific HCPs, could not be detected by the non‐process‐specific commercial ELISA kit. The sensitivity of established ELISA can reach 338 pg/mL. The rcDNA could be absolutely quantitated by ddPCR, for the copies of rcDNA in three multiple diluted samples showed a reduced gradient. While the copies of rcDNA in three multiple diluted samples could not be distinguished by the qPCR. Process‐specific ELISA has high sensitivity in detecting process‐specific E. coli HCP. The absolutely quantitative ddPCR has much higher accuracy than the relatively quantitative qPCR, it is a nucleic acid quantitative method that is expected to replace qPCR in the future. Significance statement Process‐specific immunological detection methods like a process‐specific double antibody sandwich enzyme‐linked immunosorbent assay method for HCP and a ddPCR absolute quantification method for rcDNA were established in this study, which provided a reference to improve the HCP and rcDNA detection system. It could be conducive to improving the process, improving the product quality, and achieving convergence with the standards of biopharmaceuticals in developed countries in Europe and America.