During the production of clinical-grade retroviral vector supernatant, we noted significant differences in the lactate production and glucose consumption of various producer cell lines submitted to the National Gene Vector Laboratory (NGVL). Since differences in growth characteristics could be important in determining the optimal culture conditions for maximizing titer, we studied the growth characteristics of three commonly used packaging cell lines: PA317, PG13 and GP+envAM12. A transformed phenotype, assessed by the ability to form colonies in semisolid media, was evident in all three packaging cell lines tested. In confluent cultures, the rates of glucose consumption and lactate production (per cell per hour) were similar for the three lines tested, but the growth rate and culture density varied. PA317 and PG13 continued to expand after reaching confluence, resulting in higher cell densities and subsequent rapid depletion of glucose within the 24-hr observation period. When the cell lines were evaluated for titer optimization, the slower growing packaging cell line GP+envAM12 generally provided the highest titer after 8 hr of culture in confluent roller bottles, while most vectors introduced into PA317 and PG13 cells yielded optimal titers after 24 hr of culture. We also found that the improved titers obtained by culturing cells at 32 degrees C previously reported for PA317 cells do not apply to other packaging cell lines. In particular, PG13 rapidly lost titer when grown at the lower temperature. Our findings suggest that optimization of titer requires careful consideration of the culture conditions, which should be individualized for the vector producer cell line.

译文

在临床级逆转录病毒载体上清液的生产过程中,我们注意到提交给国家基因载体实验室(NGVL)的各种生产细胞系的乳酸生产和葡萄糖消耗存在显着差异。由于生长特性的差异对于确定最大滴度的最佳培养条件可能很重要,因此我们研究了三种常用包装细胞系的生长特性:PA317,PG13和GP envAM12。通过在半固体培养基中形成菌落的能力评估的转化表型在所有测试的三种包装细胞系中均很明显。在融合培养物中,三个试验品系的葡萄糖消耗速率和乳酸产生速率(每细胞每小时)相似,但是生长速率和培养密度不同。达到汇合后,PA317和PG13继续扩增,导致更高的细胞密度,随后在24小时观察期内葡萄糖迅速耗竭。当评估细胞系的滴度优化时,生长缓慢的包装细胞系GP envAM12在融合滚瓶中培养8小时后通常提供最高滴度,而导入PA317和PG13细胞的大多数载体在培养24小时后产生最佳滴度。我们还发现先前报道的PA317细胞通过在32摄氏度下培养细胞获得的改进的滴度不适用于其他包装细胞系。特别是,PG13在较低温度下生长时会迅速失去效价。我们的发现表明效价的优化需要仔细考虑培养条件,应针对载体生产细胞株进行个性化。

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