Inclusion of PF68 Surfactant Improves Stability of rAAV Titer when Passed through a Surgical Device Used in Retinal Gene Therapy

Abstract

Recent advances in recombinant adeno-associated virus (rAAV) gene therapy for choroideremia show gene replacement to be a promising approach. It is, however, well known that contact of vector solution with plastic materials in the surgical device may result in non-specific adsorption with resulting loss of physical titer and/or level of protein expression and activity. Here we assessed the biocompatibility and stability of rAAV2-REP1 (Rab Escort Protein-1) before and following passage through the injection device over a period of time to mimic the clinical scenario. Three identical devices were screened using two concentrations of vector: high (1E+12 DNase-resistant particles [DRP]/mL) and low (1E+11 DRP/mL), to mimic high-and low-dose administrations of vector product. The low dose was prepared using either formulation buffer that contained 0.001% of a non-ionic surfactant (PF68) or balanced salt solution (BSS). We observed significant losses in the genomic titer of samples diluted with BSS for all time points. The addition of 0.001% PF68 did not, however, affect rAAV physical titer, or REP1 protein expression and biological activity. Hence we observed that neither the genomic titer nor the biological activity of a rAAV2-REP1-containing solution was affected following passage through the surgical device when PF68 was present as a surfactant and this was maintained over a period up to 10 h.

Manufacturing Challenges and Rational Formulation Development for AAV Viral Vectors

“…9). 98 Significant losses in the genomic titer of samples diluted with BSS occurred for all time points. The addition of 0.001% PF-68 prevented loss at both dose levels.…”

“…At least there are two reports demonstrating the inhibition of AAV2 viral product loss when formulated with nonionic surfactants. 79,98 Stability of the genomic materials encapsulated in viral capsids is also a concern while manufacturing AAV drug products. Nucleic acids are not stable in an aqueous solution under non-physiological temperature; pH and ionic strength disrupt the DNA helix and cause denaturation.…”

“…29 Non-specific absorption of rAAV2-REP1 (Rab Escort Protein-1) on administration devices was observed during dose preparation and administration. 98…”

“…173 58,174 In another example, the biocompatibility and stability of rAAV2-REP1 (Rab Escort Protein-1) was assessed before and after passage through the injection device over a period of time to mimic the clinical scenario. 98 The dose was prepared using either formulation buffer that contained 0.001% of a non-ionic surfactant (PF-68) or balanced salt solution (BSS). The PF-68 formulation dose was made at two different dose levels; however, BSS buffer was tested only at one dose.…”

“…al. 98 are susceptible to oxidation. Capsid proteins can oxidize upon exposure to light and due to metal ion impurities in the raw materials and excipients, 139 leading to a loss in functionality.…”