Work

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

Year: 2026

Type: article

Abstract: 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). The identified ligands are enriched in histidine and tyrosine residues, which facilitate pH-controlled capture and release. 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 an optimized density (10 µmol/mL) achieved 81% recovery of infectious virions, an ~80-fold reduction of host cell proteins, and a dynamic binding capacity (DBC10%) of 1.9×10^10 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 >90% capacity retention over multiple purification cycles with caustic cleaning. Integration into a three-step downstream process (harvest, affinity capture, and polishing) afforded ~1,530-fold global reduction of HCPs and a process yield of >70%, establishing peptide ligands as a scalable, cost-effective technology for Baculovirus purification.

Source: Journal of Chromatography A

DOI: 10.1016/j.chroma.2026.467433