Chinese hamster ovary (CHO) cells, a major platform in biopharmaceutical manufacturing, produce and secret recombinant proteins along with host cell proteins (HCPs). While residual HCPs in the final drug formulation can adversely affect patients by causing immune responses, removal of some HCPs can be a challenge. Many studies have reported that a particular set of HCPs is difficult to remove through downstream purification processes as they share similar biophysical properties to biopharmaceuticals. In particular, Cathepsin D (CtsD), an aspartyl protease, was demonstrated to be involved in the enzymatic degradation of some recombinant protein products, suggesting that nearly complete removal of this protein is necessary for product stability. We employed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system to create <i>CtsD</i> knockout cells and isolated dozens of clones without using selective drugs or reporter genes. These clones were screened using immunoblotting and knockout candidates were validated at a DNA and peptide level using Sanger sequencing and multiple reaction monitoring assay, respectively. In conclusion, we demonstrate the possibility of applying the CRISPR/Cas9 system in CHO cells to eliminate difficult-to-remove HCP gene expression in an industry-relevant setting.

Cathepsin D knockout in CHO cells using CRISPR/Cas9

253rd National Meeting (2017)

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