Background and novelty
Human coagulation factor IX (FIX) is a protein that relies on an extensive spectrum of posttranslational modifications that enable it to function correctly and efficiently in the coagulation pathway [1, 2]. These consist of seven disulfide bridges, two N-glycans and six O-linked glycans, one sulfation site, one phosphorylation site, 12 -carboxylation (GLA) sites as well as one -hydroxylation sites [3-9]. This study aims to investigate the differences in the posttranslational modifications of human recombinant factor IX (rFIX) produced in CHO fed-batch and perfusion cultures, compared with plasma-derived factor IX (PD-FIX).
Experimental approach
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 a 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) analysis [10]. In parallel, purified rFIX from these cultures was analyzed after an in-gel digestion using Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC/ESI-MS/MS) to characterize the PTMs.
Results and discussion
The fed-batch cultures responded differently to each of the feeds despite achieving similar peak cell densities of ~15x106 cells/mL. Almost all the PTMs of PD-FIX were observed in rFIX of both fed-batch cultures, although they showed partial occupancy and higher heterogeneity. Preliminary qualitative analysis also suggested that gamma-carboxylation in the rFIX GLA domain is more complete in one fed-batch compared to the other. As a comparison, 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. Samples were collected from these steady-states and purified for PTM analysis to establish comparison across the different modes of cultures and the native PD-FIX.
Acknowledgements & Funding
CSL Limited, Melbourne, Australia, supported this research. We would also like to thank our colleagues from both CSL and the Australian Research Council, Centre for Biopharmaceutical Innovation (CBI) who provided insight and expertise that greatly assisted the research.
References
1.Zogg, T. and H. Brandstetter, Activation mechanisms of coagulation factor IX. Biol Chem, 2009. 390(5-6): p. 391-400.
2.Zögg, T. and H. Brandstetter, Structural Basis of the Cofactor- and Substrate-Assisted Activation of Human Coagulation Factor IXa. Structure, 2009. 17(12): p. 1669-1678.
3.Kumar, S.R., L.L.C. Adimab, and N.H. Lebanon, Industrial production of clotting factors: Challenges of expression, and choice of host cells. Biotechnology Journal, 2015. 10: p. 995 - 1004.
4.Monroe, D.M., et al., Characterization of IXINITY(R) (Trenonacog Alfa), a Recombinant Factor IX with Primary Sequence Corresponding to the Threonine-148 Polymorph. Adv Hematol, 2016. 2016: p. 7678901.
5.Kovnir, S.V., et al., A Highly Productive CHO Cell Line Secreting Human Blood Clotting Factor IX. Acta Naturae, 2018. 10(1): p. 51-65.
6.Monahan, P.E., W.H. Velander, and S.P. Bajaj, Coagulation factor IXa, in Handbook of proteolytic enzymes, N.D. Rawlings and G. Salvasen, Editors. 2013, Elseviert, Ltd.
7.Kurachi, K. and E.W. Davie, Isolation and characterization of a cDNA coding for human factor IX. Proc Natl Acad Sci USA, 1982. 79: p. 6461 - 6464.
8.Lee, M.H., et al., Recombinant human factor IX produced from transgenic porcine milk. Biomed Res Int, 2014. 2014: p. 315375.