Molecular engineering of cyclic azobenzene-peptide hybrid ligands for the purification of human blood Factor VIII via photo-affinity chromatography.

The use of benign stimuli to control the binding and release of labile biologics for their isolation from complex feedstocks is a key goal of modern biopharmaceutical technology. This study introduces cyclic azobenzene-peptide (CAP) hybrid ligands for the rapid and discrete photo-responsive capture and release of blood coagulation Factor VIII (FVIII). A predictive method - based on amino acid sequence and molecular architecture of CAPs - was developed to correlate the conformation of cis/trans CAP photo-isomers to FVIII binding and release. The combined in silico and in vitro analysis of FVIII:peptide interactions guided the design of a rational approach to optimize isomerization kinetics and biorecognition of CAPs. A photoaffinity adsorbent, prepared by conjugating selected CAP G-cycloAZOB [Lys-YYKHLYN-Lys]-G on translucent chromatographic beads, featured high binding capacity (> 6 mg of FVIII per mL of resin) and rapid photo-isomerization kinetics ( τ < 30s) when exposed to 420-450 nm light at the intensity of 0.1 W·cm-2 . The adsorbent purified FVIII from a recombinant harvest using a single mobile phase, affording high product yield (>90%), purity (>95%), and blood clotting activity. The CAPs introduced in this report demonstrate a novel route integrating gentle operational conditions in a rapid and efficient bioprocess for the purification of life-saving biotherapeutics.