Coagulation factor IX analysis in bioreactor cell culture supernatant predicts quality of the purified product

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, critical for rFIX clinical efficacy. Bioreactor operating conditions can impact production post-translational modifications (PTMs). With the goal optimizing production, we developed suite Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics methods used these to investigate yield, other PTMs, host cell proteins during bioreactor culture after purification. We detail dynamics site-specific PTM occupancy structure on which correlated with efficiency purification purified product. identified new PTMs in near GLA domain could GLA-dependent function. Our workflows are applicable biologics expression systems, should aid optimization control upstream downstream bioprocesses.