Good Manufacturing Practice Production of Self-Complementary Serotype 8 Adeno-Associated Viral Vector for a Hemophilia B Clinical Trial
Good Manufacturing Practice Production of Self-Complementary Serotype 8 Adeno-Associated Viral Vector for a Hemophilia B Clinical Trial
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DOI：
10.1089/hum.2010.202
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发表时间：
2011-05-01
影响因子：
4.2
通讯作者：
Gray, John T.
中科院分区：
文献类型：
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作者：
Allay, James A.;Sleep, Susan;Gray, John T.
为了产生足够的临床级载体以支持用于血友病B的腺相关病毒血清型8（AAV 8）介导的因子IX（FIX）基因转移的I/II期临床试验，我们开发了用于载体生产和纯化的大规模、良好生产规范（GMP）兼容的方法。我们使用基于293 T的双质粒瞬时转染系统结合三柱层析纯化工艺来生产高质量的自身互补的AAV 2/8 FIX临床级载体。使用总共432个独立的10叠层培养室的两个连续生产活动通过斑点印迹杂交产生总共类似于2 X 10（15）个载体基因组（VG）。从转染细胞的沉淀物产生的Benzonase处理的微流化裂解物通过在Sepharose珠上进行组分离，然后通过阴离子交换色谱法进行纯化。使用100-kDa膜通过凝胶过滤和超滤进一步处理含病毒的级分。将载体配制在磷酸盐缓冲盐水加0.25%人血清白蛋白中。分光光度分析表明，与20%的完整颗粒相似，在银染十二烷基硫酸钠-聚丙烯酰胺凝胶上仅可见少量的非病毒蛋白。开发了用于检测可复制AAV的灵敏测定法，其确实揭示了最终产物中痕量的此类污染物。另外的研究已经证实了在临床稀释剂中配制并在室温下储存在静脉内袋中的载体在-80 ℃下至少24个月和至少24小时的长期稳定性。该材料已被批准用于美国和英国的临床试验。
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 similar to 2 X 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 similar to 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 degrees 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.