Abstract
Coagulation factor IX (FIX) is a complex post-translationally modified human serum glycoprotein and high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete γ-carboxylation, is critical for rFIX clinical efficacy. Bioreactor operating conditions can impact rFIX production and post-translational modifications (PTMs). With the goal of optimizing rFIX production, we developed a suite of Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics methods and used these to investigate rFIX yield, γ-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. We detail the dynamics of site-specific PTM occupancy and structure on rFIX during production, which correlated with the efficiency of purification and the quality of the purified product. We identified new PTMs in rFIX near the GLA domain which could impact rFIX GLA-dependent purification and function. Our workflows are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses.
Highlights
Coagulation factor IX (FIX) is a complex post-translationally modified human serum glycoprotein and high-value biopharmaceutical
The GLA domain is a 46 amino acid long region that contains 12 γ-carboxylated Glu residues in plasma-derived FIX18–20. γ-Carboxylation is the enzymatic addition of a CO2 moiety to the γ-carbon of Glu residues by the γ-glutamyl carboxylase, a vitamin K-dependent enzyme localized in the endoplasmic reticulum (ER)4,19,21,22. γ-Carboxylation of the GLA domain is required for calcium binding and for the formation of the tenase complex, and is absolutely essential for FIX function[2,3,19]
We developed a suite of liquid chromatography (LC)-Mass spectrometry proteomics (MS)/MS Data independent acquisition (DIA) workflows to measure the relative quantity of recombinant FIX (rFIX) and its post-translational modifications (PTMs) produced in fed-batch bioreactor cultures during bioreactor operation and after purification (Supplementary Fig. S1)
Summary
Coagulation factor IX (FIX) is a complex post-translationally modified human serum glycoprotein and high-value biopharmaceutical. Several rFIX products are available on the market, including BeneFIX (Pfizer, 1997), Rixubis (Baxter, 2013), Alprolix (Biogen Idec, 2014), Ixinity (Emergent Biosolutions, 2015), Idelvion (CSL, 2016), and Refixia/Rebinyn (Novo-Nordisk, 2017)[8] These rFIX are produced in mammalian expression systems to ensure native posttranslational modifications (PTMs), which are required for rFIX activity, stability, and serum half-life. Γ-Carboxylation of the GLA domain is required for calcium binding and for the formation of the tenase complex, and is absolutely essential for FIX function[2,3,19]. For this reason, ensuring optimal γ-carboxylation of the GLA domain is a priority for the production of rFIX. The PTMs in FIX are diverse and heterogeneous, and the precise functions of most
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