UFDF (ultrafiltration and diafiltration)

Two types of devices were used for UFDF, a centrifugal concentrator, and a flat sheet membrane, depending on the target volume. For process 1, Amicon centrifugal concentrator, with a 10 kDa molecular weight cutoff (MWCO) (MilliporeSigma, Burlington, USA) was used to concentrate the HIC elution pool (2050×g at 4 °C). This allowed concentration to the small volume needed for SEC. For process 2, a flat sheet Pellicon XL Cassette with a Biomax 5 membrane (5 kDa MWCO) and a Labscale TFF System (MilliporeSigma, Burlington, USA) were used to concentrate the HIC elution pool 8-fold, followed by diafiltration with 4 diavolumes of 20 mM Tris-HCl (pH 7.5) and 100 mM NaCl. A crossflow was kept at 25 mL/min over a 0.005-m² membrane area throughout the entire process with an average transmembrane pressure (TMP) of ~15 psi. For process 3, a flat sheet Pellicon 2 Mini Cassette with a Biomax 5 membrane (MilliporeSigma, Burlington, USA) was used for the first UFDF (UFDF-1) to concentrate the FS 4-fold followed by diafiltration with 4 diavolumes of 20 mM sodium citrate (pH 4.2) and 10 mM NaCl. A crossflow was kept constant at 200 mL/min over a 0.1-m² membrane area throughout the entire process with an average TMP of ~8 psi. For the UFDF-2, the CEX elution pool was concentrated 4-fold followed by diafiltration with 4 diavolumes of 25 mM Tris-HCl (pH 7.2) and 5 mM NaCl using the Pellicon XL Cassette as described for process 2.