Reproducible High Yields of Recombinant Adeno-Associated Virus Produced Using Invertebrate Cells in 0.02- to 200-Liter Cultures

Abstract

The large amounts of recombinant adeno-associated virus (rAAV) vector needed for clinical trials and eventual commercialization require robust, economical, reproducible, and scalable production processes compatible with current good manufacturing practice. rAAV produced using baculovirus and insect cells satisfies these conditions; however, recovering rAAV particles from 200-liter bioreactors is more complicated than bench-scale vector preparations. Using a variety of processing media, we developed a reliable and routine downstream procedure for rAAV production that is scalable from 0.02-to 200-liter cultures. To facilitate the upstream process, we adapted the titerless infected-cell preservation and scale-up process for rAAV production. Single-use aliquots of cryopreserved baculovirus-infected insect cells (BIIC) are thawed and added to the suspension culture to achieve the desired ratio of BIIC to rAAV-producer cells. By using conditions established with small-scale cultures, rAAV was produced in larger volume cultures. Strikingly consistent rAAV yields were attained in cultures ranging from 10 liters to 200 liters. Based on the final yield, each cell produced 18,000 AE 6,800 particles of purified rAAV in 10-, 20-, 100-, and 200-liter cultures. Thus, with an average cell density of 4.32Â10 6 cells/ml, !10 16 purified rAAV particles are produced from 100 to 200 liters. The downstream process resulted in about 20% recovery estimated from comparing the quantities of capsid protein antigen in the crude bioreactor material and in the final, purified product. The ease and reproducibility of rAAV production in 200-liter bioreactors suggest that the limit has not been reached, and 500-liter productions are planned.

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

“…6,22,24,25 The first clinical AAV production using the HSV system in sBHK cells was performed by Applied Genetic Technologies Corporation (AGTC, Alachua, FL) with the final yield approaching 2 × 10 13 VG/liter or 1 × 10 4 VG/cell. 26 Yields from the serum-free sBHK protocol have not been published.…”

Neuropathic and inflammatory pain are modulated by tuberoinfundibular peptide of 39 residues

“…38), and then into pFBGR (gift of Robert Kotin, National Heart, Lung, and Blood Institute, Bethesda, MD). AAV particles (serotype 8; AAV-FLEX-DTR) were produced as described by Cecchini et al (39). AAV encoding Cre-dependent barley lectin (20) expression was produced similarly.…”

Latest developments in the large-scale production of adeno-associated virus vectors in insect cells toward the treatment of neuromuscular diseases

“…The maximum production scale developed is in the range of 50-200 L using WAVE type reactors (50 L: Hermens, 2008) and stirred tank reactors of 50 L (Merten, 2009) or 200 L (Cecchini et al, 2011). In all cases a downstream processing protocol making use of an immuno-affinity chromatography using camelid antibodies directed against the AAV capsid and several other mainly membrane purification steps were employed (e.g., Smith et al, 2009).…”