Herpes Simplex Virus Clearance During Purification of a Recombinant Adeno-Associated Virus Serotype 1 Vector

Abstract

Gene delivery vectors based on adeno-associated virus (AAV) have potential utility for treatment of many genetic disorders. Current AAV vector manufacturing methods employ helper viruses to deliver functions needed to produce replication-defective recombinant AAV (rAAV) vectors, and clearance of infectious helper virus from the drug substance is essential for ensuring the safety of rAAV-based therapies. We have developed a manufacturing method for the production of rAAV vectors using a recombinant herpes simplex virus type 1 (rHSV) complementation system in suspension baby hamster kidney cells. The manufacturing process includes three primary unit operations, detergent lysis of the cell harvest and two downstream column chromatography steps, which achieve viral clearance. These unit operations inactivate and remove HSV, including replication-competent HSV present at low levels in rHSV helper stocks. Here we report results quantifying the reduction in HSV achieved during rAAV vector purification. Clearance of HSV was at least 6.84 log10 with 1% Triton X-100, 4.34 log10 with CIM Q column chromatography, and 2.86 log10 with AVB affinity chromatography. Combined, these three orthogonal methods achieved clearance of at least 14.04 log10 of HSV. The total input quantity of rHSV in a 100-liter production batch is approximately 1.2×10(12) plaque-forming units (pfu), and after purification, the concentration of residual rHSV in the resulting drug substance of approximately 450 ml would be less than 2.42×10(-5) pfu/ml. A rAAV vector produced using this method was used in a clinical trial in which subjects receive up to 100 intramuscular injections of 1.35 ml each, which would contain a maximum of 3.27×10(-3) pfu of HSV. These results support the safety of rAAV vectors produced using our rHSV complementation method.

Viral contamination in biologic manufacture and implications for emerging therapies

“…If the product itself is a virus or a living cell, how will potential viral contaminants be removed or inactivated? In the case of viral vectors for gene therapy, characteristics of commonly used vectors could be exploited to provide differential clearance from many potential contaminating viruses 53 . Two examples of how common virus removal approaches can be used with different viral vectors are shown in Table 4.…”

Moving from the bench towards a large scale, industrial platform process for adeno‐associated viral vector purification

“…A benefit of this addition is the inactivation of enveloped viruses due to the disruption of the lipid bilayer. As an example, rHSV was shown to be reduced by >6.8 LRV during the detergent step (Triton X-100) used to lyse the cells (Ye et al, 2014).…”

“…Mustang Q Davidoff et al (2004), Okada et al (2009) AAV8 CIMmultus QA Lock et al (2012) 2.9 LRV clearance of HSV (Ye et al, 2014). Comparably to low pH viral inactivation, there is considerable historical knowledge from monoclonal antibody production of the capability of affinity and anion-exchange chromatography as effective viral clearing steps (Mattila et al, 2019).…”

“…When these techniques are operated sequentially in a purification process, considerable viral removal can be attained. For example, a total cumulative HSV clearance of 14 LRV was obtained through a purification process that employed a detergent lysis during harvest (Triton X-100), AEX purification with a CIMmultus™ QA matrix, and affinity capture using AVB Sepharose resin (Ye et al, 2014).…”

Thymosin Beta-4 Recombinant Adeno-associated Virus Enhances Human Nucleus Pulposus Cell Proliferation and Reduces Cell Apoptosis and Senescence

“…[ 27 28 29 ] Recently, these vectors have been evolved and diversified to appeal to a variety of gene delivery needs with excellent safety records and success rates. [ 30 31 ] Compared to other types of viral vectors, recombinant AAV vectors show the capacity for long-term gene expression and can autonomously replicate without a helper virus. In addition, their low pathogenicity and immunogenicity make recombinant AAV vectors a preferred model for researchers in the field of gene therapy.…”