Method validation of Q-PCR detection of host residual DNA in antibody drug based on protein A magnetic beads

Liu, Xiao-zhi; Zhang, Bin; Zhao, Wei; Li, Geng; Zhou, Meiling; Wei, Jingshuang; Zhou, Jinghua; Gao, Jian; Wang, Zhiming

doi:10.1016/j.biologicals.2019.09.004

Cited by 4 publications

(2 citation statements)

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“…In addition, the abundance of CpG and unmethylated sequences in genomic DNA derived from microorganisms increases the risk of immunogenicity of genetically engineered recombinant protein drugs in vivo. In recent years, a large amount of work has been carried out around the problem of rcDNA in research institutions of various countries, and many related articles and technical guidelines have been published 28,29 . WHO and national drug registration regulatory agencies only allow the presence of the amount of rcDNA in biological agents below 100 pg/dose.…”

Section: Introductionmentioning

confidence: 99%

“…In recent years, a large amount of work has been carried out around the problem of rcDNA in research institutions of various countries, and many related articles and technical guidelines have been published. 28,29 WHO and national drug registration regulatory agencies only allow the presence of the amount of rcDNA in biological agents below 100 pg/dose. Under special circumstances, it is allowed to be up to 10 ng/dose according to the source and process of impurity.…”

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confidence: 99%

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

Tian,

Wang,

Shao

et al. 2024

Cell Biochemistry & Function

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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.

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Section: Introductionmentioning

confidence: 99%

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confidence: 99%