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)  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.