Modulation of Antibody Galactosylation Through Feeding of Uridine, Manganese Chloride, and Galactose
Modulation of Antibody Galactosylation Through Feeding of Uridine, Manganese Chloride, and Galactose
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DOI：
10.1002/bit.23075
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发表时间：
2011-07-01
影响因子：
3.8
通讯作者：
van Berkel, Patrick H. C.
中科院分区：
文献类型：
--
作者：
Gramer, Michael J.;Eckblad, Jackie J.;van Berkel, Patrick H. C.
通过将GS-CHO细胞的抗体产量提高到3g/L的工艺转移和优化，观察到抗体Fc半乳糖化水平出现了不希望的下降。尿苷(U)、氯化锰(M)和半乳糖(G)是参与细胞内半乳糖化过程的成分，它们在2-L生物反应器中被评估为它们特异性地增加抗体半乳糖化的潜力。将这些组分以从0到20xUMG的比例增加的浓度放置在进料介质中，其中U的1倍浓度为1 mm，M的1倍浓度为0.002 mm，G的1倍浓度为5 mm。抗体半乳糖化从0×UMG的3%迅速增加到8×UMG的21%，然后缓慢增加到20×UMG的23%。增加的主要原因是从G0F转移到G1F，对其他糖形式或产品质量属性的影响很小。添加高达20倍的UMG对细胞培养性能基本没有影响，除了在16和20倍UMG时抑制葡萄糖消耗和乳酸的产生，以及在20倍UMG时抗体浓度略有下降。在8×UMG及以上时，培养液中的游离半乳糖积累量较高，与达到最大半乳糖基化的平台相一致。4倍UMG的浓度导致了2-L级别的18%半乳糖基化的目标，这一结果在1,000-L的实验中重现。对单独添加每种成分直到12倍浓度的后续研究表明，这种作用是协同的；所有三种成分的组合比单独添加每种作用产生更高的半乳糖化水平。这种方法被发现通常是有用的，因为第二个细胞系的反应类似，半乳糖化从5%增加到29%，从0增加到8×UMG，而对培养性能没有进一步的增加或影响，最高可达12×UMG。这些结果表明，通过控制细胞培养液中尿苷、氯化锰和半乳糖的浓度，可以提供准确和特异的抗体半乳糖化控制。生物技术。比昂斯。2011；108：1591-1602。(C)2011年威利期刊公司。
Through process transfer and optimization for increased antibody production to 3 g/L for a GS-CHO cell line, an undesirable drop in antibody Fc galactosylation was observed. Uridine (U), manganese chloride (M), and galactose (G), constituents involved in the intracellular galactosylation process, were evaluated in 2-L bioreactors for their potential to specifically increase antibody galactosylation. These components were placed in the feed medium at proportionally increasing concentrations from 0 to 20 x UMG, where a 1 x concentration of U was 1 mM, a 1 x concentration of M was 0.002 mM, and a 1 x concentration of G was 5 mM. Antibody galactosylation increased rapidly from 3% at 0 x UMG up to 21% at 8 x UMG and then more slowly to 23% at 20 x UMG. The increase was primarily due to a shift from G0F to G1F, with minimal impact on other glycoforms or product quality attributes. Cell culture performance was largely not impacted by addition of up to 20 x UMG except for suppression of glucose consumption and lactate production at 16 and 20 x UMG and a slight drop in antibody concentration at 20 x UMG. Higher accumulation of free galactose in the medium was observed at 8 x UMG and above, coincident with achieving the plateau of maximal galactosylation. A concentration of 4 x UMG resulted in achieving the target of 18% galactosylation at 2-L scale, a result that was reproduced in a 1,000-L run. Follow-up studies to evaluate the addition of each component individually up to 12 x concentration revealed that the effect was synergistic; the combination of all three components gave a higher level of galactosylation than addition of the each effect independently. The approach was found generally useful since a second cell line responded similarly, with an increase in galactosylation from 5% to 29% from 0 to 8 x UMG and no further increase or impact on culture performance up to 12 x UMG. These results demonstrate a useful approach to provide exact and specific control of antibody galactosylation through manipulation of the concentrations of uridine, manganese chloride, and galactose in the cell culture medium. Biotechnol. Bioeng. 2011; 108: 1591-1602. (C) 2011 Wiley Periodicals, Inc.