Direct quantitative detection of host cell residual DNA in recombinant Filgrastim by qPCR

Anal Biochem. 2021 Sep 15:629:114296. doi: 10.1016/j.ab.2021.114296. Epub 2021 Jun 30.

Abstract

Host cell residual DNA is considered as an impurity in recombinant biopharmaceuticals. This study aimed to develop a direct qPCR method to quantify E. Coli residual DNA in recombinant Filgrastim. The specific primers were designed to amplify E. Coli's 16S-rDNA genomic region, which encodes the 16S-rRNA. The developed qPCR method showed that the designed primer has specifically amplified the target genome without any secondary reaction. The designed primer was also able to amplify the target gene as a representative of residual DNA in the drug matrix. Results show that the amount of residual DNA in Filgrastim is undetectable.

Keywords: Direct method; Recombinant protein production; Residual DNA; qPCR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA / metabolism*
  • DNA, Bacterial / metabolism
  • Escherichia coli / genetics*
  • Filgrastim / genetics*
  • Filgrastim / metabolism
  • Limit of Detection
  • RNA, Ribosomal, 16S / metabolism
  • Real-Time Polymerase Chain Reaction
  • Recombinant Proteins / genetics*
  • Recombinant Proteins / metabolism

Substances

  • DNA, Bacterial
  • RNA, Ribosomal, 16S
  • Recombinant Proteins
  • DNA
  • Filgrastim