Peptide ligands for the affinity purification of adeno-associated viruses from HEK 293 cell lysates

Abstract

Adeno-associated viruses (AAVs) are the vector of choice for delivering gene therapies that can cure inherited and acquired diseases. Clinical research on various AAV serotypes significantly increased in recent years alongside regulatory approvals of AAV-based therapies. The current AAV purification platform hinges on the capture step, for which several affinity resins are commercially available. These adsorbents rely on protein ligands - typically camelid antibodies - that provide high binding capacity and selectivity, but suffer from low biochemical stability and high cost, and impose harsh elution conditions (pH < 3) that can harm the transduction activity of recovered AAVs. Addressing these challenges, this study introduces peptide ligands that selectively capture AAVs and release them under mild conditions (pH 6.0). The peptide sequences were identified by screening a focused library and modeled in silico against AAV serotypes 2 and 9 (AAV2 and AAV9) to select candidate ligands that target homologous sites at the interface of the VP1-VP2 and VP2-VP3 virion proteins with mild binding strength (KD ~ 10-5-10-6 M). Selected peptides were conjugated to Toyopearl resin and evaluated via binding studies against AAV2 and AAV9, demonstrating the ability to target both serotypes with values of dynamic binding capacity (DBC10% > 1013 vp per mL of resin) and product yields (~50-80%) on par with commercial adsorbents. The peptide-based adsorbents were finally utilized to purify AAV2 from a HEK 293 cell lysate, affording high recovery (50-80%), 80-to-400-fold reduction of host cell proteins (HCPs), and high transduction activity (up to 80%) of the purified viruses.

Peptide ligands targeting the vesicular stomatitis virus G (VSV‐G) protein for the affinity purification of lentivirus particles

“…The ability of peptide ligands to target multiple binding sites on the target surface with true affinity suggests the formation of a multi‐site interaction network between the virus and the peptide‐functionalized surface. This mechanism has been observed in prior studies on the de novo discovery of peptide ligands for AAV purification (Chu et al, 2023) to be conducive to high capacity and selective binding as well as efficient product recovery under mild elution conditions. The LV coat displays approximately 216 VSV‐G proteins (Croyle et al, 2004), suggesting that VSV‐G are placed at 12.5–31.3 Å from each other (assuming the LV radius to be 40 – 50 nm); additionally, based on the peptide density on the resin (~30 μmol per gram) and the resin's specific surface (~30 m 2 /g), the peptides are displayed at ~18 Å from each other.…”

“…The ability of peptide ligands to target multiple binding sites on the target surface with true affinity suggests the formation of a multi-site interaction network between the virus and the peptide-functionalized surface. This mechanism has been observed in prior studies on the de novo discovery of peptide ligands for AAV purification (Chu et al, 2023) to be conducive to high capacity and selective binding as well as efficient product recovery under mild elution conditions. The LV coat displays approximately 216 VSV-G proteins (Croyle et al, 2004),…”

Rational design and experimental evaluation of peptide ligands for the purification of adeno‐associated viruses via affinity chromatography

“…Furthermore, a milder binding strength does not necessarily translate into weaker binding. As noted in prior work, [ 9 ] the peptide density on the surface of the resin is sufficient to form multiple interactions with a single capsid, wherein multiple affinity interactions with modest binding energy are synergized into a strong avidity‐like binding that efficient AAV capture. The values of peptide density on the resin (≈0.12–0.15 mmol g −1 ), the resin's specific surface (≈30 m 2 g −1 ), and the projection area of the triangular unit formed by 3 VPs on the icosahedral capsid (≈81 nm 2 ) indeed suggest that up to 30 peptides are displayed on pore surface that is impacted by a single capsid, enabling the formation of 3–5 VP:peptide interactions per bound capsid).…”

“…[ 59,60 ] Combining the cost of synthesis with the average values of number of residues (≈15–17) and molecular weight (≈1.5–1.7 kg mol −1 ) of the peptide ligands, their density on the resin surface (≈0.03 mol per liter), and the cost of the base resin (≈$2500 per liter) indicates that direct material cost of the peptide‐functionalized adsorbent ranges between $7900 and $9500 per liter, when produced at the ≈100 L scale ( note : direct labor and manufacturing overhead are not factored). These considerations, combined with the purification performance of peptide‐functionalized adsorbents presented by our team and by several others in the literature, [ 9,11,55–58,61–65 ] show the promise of this technology to transform the biomanufacturing of modern medicines and reduce their cost ( note : the latter is of particular concern, given the price tag of gene therapies well above US$1M per patient). Under the light of these considerations, our team plans to demonstrate further the technology introduced in this study by purifying AAVs of different serotypes from a variety of HEK293 and Sf9 fluids.…”

“…This is well exemplified by the purification pipeline of adeno‐associated viruses (AAVs, the vector of choice in gene therapy) where protein ligands that resemble the Protein A used for mAb purification are employed at the product capture step. [ 8,9 ]…”