Non-immunoglobin scaffold binders as efficient affinity ligands for purification of broad-spectrum serum albumins

Although non-immunoglobin scaffold binders with high affinity and broad spectrum for albumin are attractive for lab-scale albumin purification, affinity chromatography based on these binders has not been developed. Here, the albumin-binding capabilities of representative binders, including protein G-derived albumin binding domain (ABD), albumin binding nanofitins (ABNF), and human serum albumin affimer 31 (HSA31) were predicted by interaction structure analysis and verified by experimental assays. Interaction structure prediction suggested that ABD possessed great potential to bind human (HSA), rhesus monkey (RhSA), mouse (MSA), and rat serum albumin (RSA), whereas ABNF might only bind HSA and bovine serum albumin (BSA), and HSA31 might not bind any of the tested albumins. Subsequent experimental assays demonstrated that ABD was similar to ABNF in HSA and RhSA binding but was superior to ABNF in MSA and RSA binding. ABNF exhibited greater BSA-binding capability than that exhibited by ABD. Both ABD and ABNF showed little binding to rabbit serum albumin (RbSA) and HSA31 showed no binding to all tested albumins. ABD and ABNF could be conjugated to prepackaged NHS-activated HP columns at similar ligand densities. For purification of albumin from plasma, the yields of ABD-HP column for HSA, RhSA, MSA, and RSA were approximately 20 mg, which was 3-4 times higher than that of ABNF-HP column using either Gly-HCl or sodium caprylate as elution buffer. Notably, BSA could only be recovered from plasma using ABNF-HP column with a yield of 5-8 mg. However, little albumin could be purified using HSA31-HP column. These results indicate that both ABD- and ABNF-HP columns could be used as novel tools for purification of broad-spectrum albumins.