title: Good manufacturing practice production of self-complementary serotype 8 adeno-associated viral vector for a hemophilia B clinical trial.
abstract: To generate sufficient clinical-grade vector to support a phase I/II clinical trial of adeno-associated virus serotype 8 (AAV8)-mediated factor IX (FIX) gene transfer for hemophilia B, we have developed a large-scale, good manufacturing practice (GMP)-compatible method for vector production and purification. We used a 293T-based two-plasmid transient transfection system coupled with a three-column chromatography purification process to produce high-quality self-complementary AAV2/8 FIX clinical-grade vector. Two consecutive production campaigns using a total of 432 independent 10-stack culture chambers produced a total of ∼2 × 10(15) vector genomes (VG) by dot-blot hybridization. Benzonase-treated microfluidized lysates generated from pellets of transfected cells were purified by group separation on Sepharose beads followed by anion-exchange chromatography. The virus-containing fractions were further processed by gel filtration and ultrafiltration, using a 100-kDa membrane. The vector was formulated in phosphate-buffered saline plus 0.25% human serum albumin. Spectrophotometric analysis suggested ∼20% full particles, with only low quantities of nonviral proteins were visible on silver-stained sodium dodecyl sulfate-polyacrylamide gels. A sensitive assay for the detection of replication-competent AAV was developed, which did reveal trace quantities of such contaminants in the final product. Additional studies have confirmed the long-term stability of the vector at -80°C for at least 24 months and for at least 24 hr formulated in the clinical diluent and stored at room temperature within intravenous bags. This material has been approved for use in clinical trials in the United States and the United Kingdom.
authors.affiliation: Children's GMP, Memphis, TN 38105, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Allay
authors.forename: Susan
authors.initials: S
authors.lastname: Sleep
authors.forename: Scott
authors.initials: S
authors.lastname: Long
authors.forename: David M
authors.initials: DM
authors.lastname: Tillman
authors.forename: Rob
authors.initials: R
authors.lastname: Clark
authors.forename: Gael
authors.initials: G
authors.lastname: Carney
authors.forename: Paolo
authors.initials: P
authors.lastname: Fagone
authors.forename: Jenny H
authors.initials: JH
authors.lastname: McIntosh
authors.forename: Arthur W
authors.initials: AW
authors.lastname: Nienhuis
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Davidoff
authors.forename: Amit C
authors.initials: AC
authors.lastname: Nathwani
authors.forename: John T
authors.initials: JT
authors.lastname: Gray
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002986:Clinical Trials as Topic; D000229:Dependovirus; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D002836:Hemophilia B / Q000235:genetics / Q000628:therapy*; D006801:Humans; D015151:Immunoblotting; D013053:Spectrophotometry