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) 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—is developed to correlate the conformation of cis/trans-CAP photo-isomers to FVIII binding and release. Combined in silico - in vitro analysis of FVIII:peptide interactions guide the design of a rational approach to optimize isomerization kinetics and biorecognition of CAPs. A photoaffinity adsorbent, prepared by conjugating selected CAP G-cyclo_AZOB[Lys-YYKHLYN-Lys]-G on translucent chromatographic beads, features high binding capacity (>6 mg of FVIII per mL of resin) and rapid photo-isomerization kinetics (τ < 30 s) when exposed to 420–450 nm light at the intensity of 0.1 W cm⁻². The adsorbent purifies 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.