F9 and hemophilia B: In the purification process of Rebinyn® (a 40 kDa-PEG coagulation factor IX for hemophilia B), a pH gradient is generated by equilibrating the column with acetate buffer 100 mM pH 5.7, followed by a five column volumes (CV) acidic wash with acetate buffer 250 mM pH 4.3; then elution is accomplished by a 5 CV linear gradient of 10 mM histidine, 50 mM NaCl, 50 mM CaCI2 buffer pH ≈ 6.0 (Stennicke et al., 2008; Wiendahl et al., 2020).