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‐cycloAZOB[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 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. Labile therapeutics are unavailable to patients—despite their therapeutic potential—due to a lack of suitable purification tools. Current chromatographic technology controls product capture, washing, and release using aqueous media with different conductivity/pH, which harms delicate biologics. Photo‐affinity chromatography overcomes these challenges by leveraging light as the only process control, thus conducting the purification process under conditions that safeguard product's bioactivity.