Analysis of coagulation factor IX in bioreactor cell culture medium predicts yield and quality of the purified product

Abstract

Coagulation factor IX (FIX) is a highly complex post-translationally modified human serum glycoprotein and a high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete g-carboxylation, is critical for rFIX clinical efficacy. Changes in bioreactor operating conditions can impact rFIX production and occupancy and structure of rFIX posttranslational modifications (PTMs). We hypothesized that monitoring the bioreactor cell culture supernatant with Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics would allow us to predict product yield and quality after purification. With the goal of optimizing rFIX production, we developed a suite of MS proteomics analytical methods and used these to investigate changes in rFIX yield, g-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. Our methods provided a detailed overview of the dynamics of site-specific PTM occupancy and abundance on rFIX during production, which accurately predicted the efficiency of purification and the quality of the purified product from different culture conditions. In addition, we identified new PTMs in rFIX, some of which were near the GLA domain and could impact rFIX GLA-dependent purification efficiency and protein function. The workflows presented here are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses. Figure 1: Coagulation Factor IX (FIX) structural domains and post-translational modifications (PTMs). Schematic of mature FIX with previously described PTMs in plasma derived FIX and/or recombinant FIX (figure modified from [13]). The GLA domain (red) contains 12 potential sites of g-carboxylation on E 7-40 and one O-glycan at T 38/39 . The EGF-like 1 domain (pink) contains two O-glycans on S 53 and S 61 , b-hydroxylation of D 64 , and phosphorylation of S 68 . The EGF-like 2 domain (violet) has no known PTMs. The short domain (white) linking the EGFlike 2 domain with the activation peptide (AP, green) contains one O-glycan at S 141 . The AP contains two N-glycans at N 157 and N 167 , four O-glycans at T 159 , T 169 , T 172 , and T 179 , sulfation of Y 155 , and phosphorylation of S 158 and T 159 . The serine protease domain (orange) contains one Nlinked glycan at N 258 .FIX is a highly post-translationally modified glycoprotein [7,14] (Fig. 1). Immature FIX is composed of six structural domains: an N-terminal propeptide, the GLA domain, two consecutive EGF-like domains followed by a short linker, the activation peptide (AP), and the C-terminal serine protease domain (Fig. 1). Most described PTMs are on or near the GLA, the EGF-like 1, and the AP domains. The propeptide and the AP are removed through proteolysis events, and both proteolysis are required for function [1,9,15]. FIX's propeptide is cleaved by the trans-Golgi protease PACE/Furin during transit through the secretory pathway [16,17]. FIX's AP is removed in the blood by Factor XIa as part of the c...

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“…The top 25 mL of supernatant were transferred to 3 kDa Amicon columns (Amicon Ultra-15 Centrifugal Filter Unit, UFC 900324) and concentrated following the manufacturer’s recommendations. 100 μL of the concentrated sample were precipitated overnight in 4 volumes of methanol:acetone in a LoBind Eppendorf tube, as previously described ( Zacchi et al, 2020a , 2020b ). The supernatant was eliminated after high speed centrifugation, and air-dried proteins were resuspended in 50 mM ammonium bicarbonate supplemented with 10 mM DTT and 1 μg of trypsin.…”