Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Understanding complex post-translational modifications in CHO cells  (#429)

Aiden Beauglehole 1 , Lucia Zacchi 1 2 , Axayacatl Gonzalez 1 , Esteban Marcellin 1 , Ben Schulz 1 2
  1. Australian Institute for Bioengineering and Nanotechnology, AIBN, Brisbane, Queensland, Australia
  2. School of Chemistry and Molecular Biosciences, SCMB, Brisbane City, Queensland

Biologics production in CHO cells is a rapidly expanding field that makes up over 1/3 of the pharmaceutical industry. The expansion of biologics in yield and quality has been focused around physical processes such as bioreactors, media quality, and cell density rather than focusing on cellular machinery. The lack of focus on cellular machinery has left complex proteins with large amounts of post-translational modifications (PTMs) to have comparatively small yields and qualitative issues to other biologics. Compounding issues, biologics production is centered on the stable integration of DNA into the genome. Stable integration into the genome is random meaning that the gene can be inserted anywhere with multiple gene copy numbers, collectively called ‘position effects’. In order to understand biologic production variation, position effects need to be minimised. Factor IX is a biologic that has numerous PTMs with 10 fold lower yields in contrast to other biologics. Utilising CRISPR/Cas9, we have developed CHO cell lines that are able to study protein production independent of position effects. By removing position effects, comparative SWATH based proteomics and metabolomics can occur to understand why Factor IX has poor yields compared to high producing biologics. Additionally, SWATH based proteomics allowed us to observe recombinant protein expressions effect on Factor IX’s post-translational modifications and other biologics as a whole. Our findings can be used to engineer CHO cells to cope with the metabolic and biological demands of making Factor IX and other biologics with large amounts of PTMs.