Integrated system for temperature-controlled fast protein liquid chromatography. IV. Continuous ‘one-column’ ‘low-salt’ hydrophobic interaction chromatography

•Green environmentally-friendly & sustainable solutions to bioprocess chromatography.•One-column low-salt continuous hydrophobic interaction chromatography of proteins.•Column mounted travelling cooling device permits elution under isocratic conditions.•High-yield high-purity separation of protein monomers from higher order forms. Systematic development of a temperature-controlled isocratic process for one-column low-salt hydrophobic interaction chromatography (HIC) of proteins employing a travelling cooling zone reactor (TCZR) system, is described. Batch binding and confocal scanning microscopy were employed to define process conditions for temperature-reversible binding of bovine serum albumin (BSA) which were validated in pulse-response temperature switching HIC experiments, before transferring to TCZR-HIC. A thin-walled stainless-steel column mounted with a movable assembly of copper blocks and Peltier elements (travelling cooling zone, TCZ) was used for TCZR-HIC. In pulse-response TCZR-HIC, 12 TCZ movements along the column desorbed 86.3% of the applied BSA monomers in 95.3% purity depleted >6-fold in 2–4 mers and nearly 260-fold in higher molecular weight (HMW) species. For continuous TCZR-HIC, the TCZ was moved 49–58 times during uninterrupted loading of BSA feeds at 0.25, 0.5 or 1 mg·mL-1. Each TCZ movement generated a sharp symmetrical elution peak. In the best case, (condition 1: 0.25 mg·mL-1 BSA; >17 mg BSA applied per mL of bed) the height of TCZ elution peaks approached pseudo-steady midway through the loading phase with no rise in baseline UV280 signal between peaks. Peak composition remained constant averaging 94.4% monomer, 5.6% 2–4 mers and 3.1 fold in 2–4 mers and 92-fold in HMW species cf. the feed (63.6% monomers, 21.8% 2–4 mers, 14.6% HMW). However, increasing the BSA concentration to 1 mg·mL-1 (condition 2) or employing a fouled HIC column with 0.5 mg·mL-1 BSA (condition 3) compromised monomer purification performance.