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

在生产临床级逆转录病毒载体上清液期间,我们注意到提交给国家基因载体实验室 (NGVL) 的各种生产者细胞系的乳酸产量和葡萄糖消耗量存在显着差异。由于生长特性的差异对于确定使效价最大化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。在所有测试的三个包装细胞系中,通过在半固体培养基中形成菌落的能力评估的转化表型都很明显。在汇合培养物中,测试的三个品系的葡萄糖消耗和乳酸产生速率 (每小时每个细胞) 相似,但生长速率和培养密度各不相同。PA317和PG13在达到汇合后继续膨胀,导致细胞密度更高,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,生长较慢的包装细胞系GP envAM12通常在融合的滚筒瓶中培养8小时后提供最高滴度,而引入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,通过先前报道的PA317细胞在32 ℃ 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迅速失去滴度。我们的发现表明,效价的优化需要仔细考虑培养条件,对于载体生产者细胞系,应将其个性化。

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