Peptide ligands targeting GP64 for the purification of Baculovirus from insect cell lysates

Autographa californica multiple nucleopolyhedrovirus, known as Baculovirus, is a widely used platform for producing therapeutic proteins and viral vectors. The purity and infectious activity of Baculovirus stocks determine the quality and productivity of recombinant products produced through this system. Current purification strategies suffer from major limitations: centrifugation lacks productivity and scalability; ion-exchange chromatography affords limited selectivity and purity; and the only commercial affinity resin requires harsh elution conditions that significantly reduce functional product recovery. To overcome these limitations, this study introduces the first peptide affinity ligands targeting the baculoviral envelope glycoprotein GP64 for the purification of active Baculovirus particles. We implemented a combinatorial selection workflow based on dual-fluorescence screening of solid-phase peptide libraries to identify 12-mer sequences that bind GP64 and elute Baculovirus under mild conditions (pH 8.5). As the selected ligands are enriched in histidine and tyrosine residues, product release is effected by the combined modulation of pH and ionic strength. Eight candidate peptides (SB1-SB8) were evaluated on Toyopearl and POROS chromatographic resins, demonstrating that matrix chemistry, pore size, and ligand density govern purification performance. The lead peptide SB4 conjugated to POROS resin at ∼10 µmol/mL achieved 81% recovery of infectious virions (transducing units), robust host cell protein reduction (LRV 1.65), and a dynamic binding capacity (DBC_10%) of 1.9 × 10¹⁰ vg/mL resin. Transmission electron microscopy and multi-angle light scattering confirmed the integrity of purified particles (200 × 50 nm rods with intact nucleocapsids), compared to BacuClear eluates that showed collapsed morphology. The SB4-POROS resin demonstrated storage stability and ∼80% retention of binding capacity over ten purification-regeneration cycles with caustic cleaning. Integration into a three-step downstream process (clarification, affinity capture, and polishing) raised product purity 1,528-fold, from 6.22 × 10⁶ to 9.50 × 10⁹ viral genomes per µg of HCP, while reducing the total HCP burden 1,698-fold, at a cumulative transducing-unit yield of ∼69% relative to the feedstock, establishing SB4-POROS as a promising technology with a favorable projected cost structure for Baculovirus purification.