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

Post-translational modifications on recombinant human factor IX from fed-batch and perfusion chinese hamster ovary cell culture (#808)

Dinora Roche Recinos 1 , Lucía F. Zacchi 1 2 , Casandra Pegg 2 , Toan K. Phung 2 , Campbell Aitken 3 , Ellen Otte 3 , Tony Hunt 3 , Mark Napoli 3 , Yih Yean Lee 3 , Ben Schulz 1 2 , Christopher B. Howard 1
  1. Centre for Biopharmaceutical Innovation (CBI), Australian Institute of Bioengineering and Nanotechnology (AIBN) at The University of Queensland (UQ), Brisbane, QLD, Australia
  2. School of Chemistry and Molecular Biology, University of Queensland, Brisbane, QLD, Australia
  3. CSL Limited, Melbourne, Victoria, Australia

Human coagulation factor IX (FIX) is a protein that relies on an extensive spectrum of posttranslational modifications (PTMs) for its correct and efficient function in the coagulation pathway [1, 2]. These PTMs include seven disulfide bridges, two N-glycans, six O-linked glycans, one sulfation site, one phosphorylation site, 12 g-carboxylation (GLA) sites, and one b-hydroxylation site [3-9]. Here, we investigated the differences in the PTMs of human recombinant factor IX (rFIX) produced in CHO fed-batch and perfusion cultures, compared with native plasma-derived factor IX (PD-FIX).  The cell line used was a CHO-K1SV expressing rFIX. Two fed-batch bioreactors were conducted using commercial CD-CHO media and EfficientFeed A and B respectively. Perfusion cultures were conducted in the same base medium using an Applisens Biosep acoustic perfusion unit at a dilution rate of one reactor volume per day. The bioreactors were sampled daily for off-line measurements to track cell growth, metabolism and productivity. These samples were also used for Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) [10] analysis of the culture supernatant proteome during the time-course of fermentation. We also performed detailed LC-MS/MS characterization of purified rFIX from each culture.The fed-batch cultures responded differently to each of the feeds despite achieving similar peak cell densities of ~15x106 cells/mL. Pseudo steady-states were established in the perfusion cultures at 15x106 cells/mL via bleeding of the cultures under the control of an online turbidity probe. Almost all the PTMs present on PD-FIX were also observed in rFIX of fed-batch and perfusion cultures, although they showed partial occupancy and higher heterogeneity in rFIX. The extent of gamma-carboxylation in the rFIX GLA domain, and of diverse other PTMs across the protein, was affected by fermentation conditions, emphasizing the utility of LC-MS/MS techniques in monitoring the quality of recombinant biopharmaceuticals, especially those heavily modified by diverse PTMs.