Statistics and reproducibility

One bioreactor per condition was run. Technical replicate samples of bioreactor culture supernatant for each bioreactor were obtained daily for proteomic analysis of supernatant (15 mL), and at day 13 for proteomic analysis of purified product (2 L). Viability and metabolic measurements were performed once/day/bioreactor. For proteomic analysis, all samples were prepared as independent replicates: N = 2 for FIX characterization (DDA analyses) and N = 3 for FIX and FIX PTMs quantification (DIA analyses), and each sample was analyzed and measured once. Statistical analysis of rFIX/trypsin values during bioreactor operation was performed using multiple t tests, with the two-stage linear setup procedure of Benjamini, Krieger, and Yekutieli to assign P values, and Q = 1%, in GraphPad Prism. Statistical analyses of the HCPs and rFIX abundance in purified rFIX samples (N = 3) and at days 3, 6, 9, and 13 in the supernatant were performed using MSstats in R (refs. ^55,133,138). Statistical analyses for the posttranslationally modified peptides were performed using one-tailed t test in Excel (Microsoft). A P < 0.05 was considered significant for t test, and P < 10⁻⁵ for MSstats.