A scalable method for the production of high-titer and high-quality adeno-associated type 9 vectors using the HSV platform

Abstract

Recombinant adeno-associated vectors based on serotype 9 (rAAV9) have demonstrated highly effective gene transfer in multiple animal models of muscular dystrophies and other neurological indications. Current limitations in vector production and purification have hampered widespread implementation of clinical candidate vectors, particularly when systemic administration is considered. In this study, we describe a complete herpes simplex virus (HSV)-based production and purification process capable of generating greater than 1 × 1014 rAAV9 vector genomes per 10-layer CellSTACK of HEK 293 producer cells, or greater than 1 × 105 vector genome per cell, in a final, fully purified product. This represents a 5- to 10-fold increase over transfection-based methods. In addition, rAAV vectors produced by this method demonstrated improved biological characteristics when compared to transfection-based production, including increased infectivity as shown by higher transducing unit-to-vector genome ratios and decreased total capsid protein amounts, shown by lower empty-to-full ratios. Together, this data establishes a significant improvement in both rAAV9 yields and vector quality. Further, the method can be readily adapted to large-scale good laboratory practice (GLP) and good manufacturing practice (GMP) production of rAAV9 vectors to enable preclinical and clinical studies and provide a platform to build on toward late-phases and commercial production.

A novel scalable, robust downstream process for oncolytic rat parvovirus: isoelectric point-based elimination of empty particles

“…The virus titer of 1E10 PFU/ml is optimal, because the virus particles start to aggregate at 1E11 PFU/ml (as attested by high variance of the titer) and because preparations with a virus titer lower 1E8 PFU/ml are unstable over the years (data not shown). The overall recovery of 40% with our new purification strategy is comparable with or superior to those obtained in other virus/viral vector productions (Adamson-Small et al 2016; Merten et al 2016). …”

Sodium Chloride Enhances Recombinant Adeno-Associated Virus Production in a Serum-Free Suspension Manufacturing Platform Using the Herpes Simplex Virus System

“…12 When DMEM was supplemented with 60 m M sodium chloride, there was a significant, 1.6- and 2.2-fold increase in rAAV vector genomes and transducing units, respectively (Fig. 4A and B, solid columns; p  < 0.05).…”

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

“…Similar to monoclonal antibodies, AAV is stable at acidic conditions, with no loss in infectivity of AAV9 when held for 2 hr at pH values between 2.5 and 8 (Potter et al, 2014). While infectivity remained, it was found that during an acid flocculation step of the harvest of AAV9 produced using HSV, HSV particles were undetectable (<100 pfu/ml) after the flocculation step (Adamson-Small et al, 2016).…”