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首页> 外文期刊>Molecular therapy: the journal of the American Society of Gene Therapy >Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector
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Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector

机译：使用悬浮HEK293细胞生产重组腺相关病毒载体，并从培养基中连续收获GMP FIX和FLT1临床载体

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Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. Using the triple transfection method, the suspension HEK293 cell line generates greater than 1 x 10(5) vector genome containing particles (vg)/cell or greater than 1 x 10(14) vg/l of cell culture when harvested 48 hours post-transfection. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high-purity vector preps of AAV serotypes 1-6, 8, 9 and various chimeric capsids tested. This user-friendly process can be completed within 1 week, results in high full to empty particle ratios (>90% full particles), provides postpurification yields (>1 x 10(13) vg/l) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. To date, this scalable manufacturing technology has been utilized to manufacture GMP phase 1 clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which have been administered into patients. In addition, we report a minimum of a fivefold increase in overall vector production by implementing a perfusion method that entails harvesting rAAV from the culture media at numerous time-points post-transfection.
机译：腺伴随病毒（AAV）作为临床治疗和临床应用多个方面的基因治疗载体已显示出巨大的希望。现在，包括新血清型和自身互补载体在内的许多进展都进入了临床。随着这些载体的不断发展，人们一直在致力于可扩展的生产工艺，这些工艺可以有效地产生高滴度，高纯度和有效量的rAAV载体。以相对简单和高效的HEK293细胞转染系统为起点，我们成功地将来自合格临床主细胞库的贴壁HEK293细胞系改造为可在摇瓶和WAVE生物反应器中在无动物成分的悬浮条件下生长，快速，可扩展的rAAV生产。使用三重转染方法，悬浮液HEK293细胞系在收集48小时后收获时，会产生大于1 x 10（5）vg / l的细胞基因组或大于1 x 10（14）vg / l的细胞培养物。转染。为了获得这些产量，优化了许多变量，例如选择支持生长和转染的兼容无血清悬浮培养基，选择转染试剂，转染条件和细胞密度。还开发了一种基于离子交换色谱方法的通用纯化策略，该策略可得到AAV血清型1-6、8、9和各种嵌合衣壳的高纯度载体制剂。此用户友好型过程可在1周内完成，可实现高的全空颗粒比（> 90％的全颗粒），提纯后的产率（> 1 x 10（13）vg / l）和适用于临床应用的纯度和关于所有血清型和嵌合颗粒是通用的。迄今为止，这种可扩展的制造技术已被用于制造GMP 1期临床AAV载体，用于视网膜新血管形成（AAV2），血友病B（scAAV8），巨大轴突神经病（scAAV9）和色素性视网膜炎（AAV2）。耐心。此外，我们报告了通过实施一种灌注方法将整体载体产量提高至少五倍的方法，该方法需要在转染后的多个时间点从培养基中收获rAAV。

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