470. Efficient Clearance of Herpes Simplex Virus Using a GMP-Compliant Method for Production of Recombinant Adeno-Associated Virus Vectors
Authors: Guo-jie Ye, Chantelle Gaskin, Charlotte Butts, Royce Threadgill, David R. Knop, Jeffrey D. Chulay
Journal: Molecular Therapy (2015)

AGTC's proprietary rAAV manufacturing system employs two mutant, replication incompetent rHSV helper viruses. To ensure the safety of rAAV gene therapy products, infectious rHSV in the unprocessed bulk must be inactivated and removed during the harvest and purification processes that generate the rAAV drug substance. Here we report the viral clearance of a new HSV-based manufacturing process developed for rAAV serotype 2 vectors with 3 surface tyrosine residues substituted by phenylalanine (rAAV2tYF) which is being used to manufacture drug products for treatment of patients with XLRS and ACHM. The wtHSV-1 strain KOS 1.1 was used as a spiking reagent for the viral clearance studies. Matrices generated for use in this study: (1) unprocessed bulk (pre-harvest) material for detergent studies, (2) a mock clarified lysate pool for AVB column studies, and (3) AVB pool for CIM SO3- column studies. The unprocessed bulk, clarified lysate pool and AVB pool were obtained as aliquots from a 10 L production batch of rAAV2tYF-CB-hRS1. Viral clearance of each operation unit was determined based on the HSV titers in samples before and after each of inactivation or purification steps. The three orthogonal methods employed during harvest and purification of the drug substance achieved clearance of a total of at least 7.34 + 4.54 + 1.41 = 13.29 log10 of wtHSV. From this total clearance a simple deterministic risk assessment of the theoretical residual level of rHSV in a single dose of rAAV2tYF-CB-hRS1 can be made. The total input quantity of rHSV infectious particles in a 25 L production batch is 3 × 10^11 pfu and a 13.29 log10 reduction of this value is 1.54 × 10^-2 pfu remaining in the final formulated bulk product which has a volume of 100 mL. The volume of the highest proposed single dose for a patient is 0.1 mL which therefore contains 1.54 × 10^-5 pfu of rHSV. However, this is the worst case estimation which does not account for >100-fold loss of input rHSV after infection and during culture of rHSV-infected cells. Measurement of infectious rHSV in development batches of unprocessed bulk has shown the titer of residual rHSV is <10^5 pfu/mL or <2.5 × 10^9 pfu of HSV in 25 L. A 13.29 log10 reduction of this value imply that the highest proposed single dose of 0.1 mL would contain 1.28 × 10^-7 pfu of rHSV, which is more representative of the actual situation in manufacture. The manufacturing process for rAAV vectors for use in patients with XLRS and ACHM effectively inactivates and removes HSV particles from rAAV vectors produced using an rHSV complementation system in sBHK cells, resulting in greater than a 13.29 log10 reduction in HSV, corresponding to a maximum of amount of rcHSV or replication-incompetent HSV in a single patient dose of 1.54 × 10^-5 under the worst-case estimation, and a maximum of 1.28 × 10^-7 pfu of replication-incompetent HSV when loss of input rHSV during culture of rHSV-infected cells is taken into consideration. Therefore, the risk of HSV-related adverse events in patients receiving rAAV vectors purified in this manner is very low.