Negrete Alejandro, Esteban Geoffrey, Kotin Robert M
Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, US National Institutes of Health, 10 Center Drive, NIH, Building 10, Room 7D05, Bethesda, MD 20892, USA.
Appl Microbiol Biotechnol. 2007 Sep;76(4):761-72. doi: 10.1007/s00253-007-1030-9. Epub 2007 Aug 7.
A well-characterized manufacturing process for the large-scale production of recombinant adeno-associated vectors (rAAV) for gene therapy applications is required to meet current and future demands for pre-clinical and clinical studies and potential commercialization. Economic considerations argue in favor of suspension culture-based production. Currently, the only feasible method for large-scale rAAV production utilizes baculovirus expression vectors and insect cells in suspension cultures. To maximize yields and achieve reproducibility between batches, online monitoring of various metabolic and physical parameters is useful for characterizing early stages of baculovirus-infected insect cells. In this study, rAAVs were produced at 40-l scale yielding ~1 x 10(15) particles. During the process, dielectric spectroscopy was performed by real time scanning in radio frequencies between 300 kHz and 10 MHz. The corresponding permittivity values were correlated with the rAAV production. Both infected and uninfected reached a maximum value; however, only infected cell cultures permittivity profile reached a second maximum value. This effect was correlated with the optimal harvest time for rAAV production. Analysis of rAAV indicated the harvesting time around 48 h post-infection (hpi), and 72 hpi produced similar quantities of biologically active rAAV. Thus, if operated continuously, the 24-h reduction in the production process of rAAV gives sufficient time for additional 18 runs a year corresponding to an extra production of ~2 x 10(16) particles. As part of large-scale optimization studies, this new finding will facilitate the bioprocessing scale-up of rAAV and other bioproducts.
为满足临床前和临床研究以及潜在商业化的当前和未来需求，需要一种特征明确的大规模生产用于基因治疗的重组腺相关病毒（rAAV）的制造工艺。经济因素支持基于悬浮培养的生产方式。目前，大规模生产rAAV的唯一可行方法是在悬浮培养中利用杆状病毒表达载体和昆虫细胞。为了使产量最大化并实现批次间的可重复性，对各种代谢和物理参数进行在线监测有助于表征杆状病毒感染昆虫细胞的早期阶段。在本研究中，以40升规模生产rAAV，产量约为1×10¹⁵个颗粒。在此过程中，通过在300 kHz至10 MHz的射频范围内进行实时扫描来进行介电谱分析。相应的介电常数与rAAV的生产相关。受感染和未受感染的细胞都达到了最大值；然而，只有受感染细胞培养物的介电常数曲线达到了第二个最大值。这种效应与rAAV生产的最佳收获时间相关。对rAAV的分析表明，感染后约48小时（hpi）收获，72 hpi产生的生物活性rAAV数量相似。因此，如果连续运行，rAAV生产过程中减少24小时可提供足够的时间，每年额外增加18批次，相当于额外生产约2×10¹⁶个颗粒。作为大规模优化研究的一部分，这一新发现将促进rAAV和其他生物制品的生物加工放大。
