Chemical inhibitors of hexokinase‐2 enzyme reduce lactate accumulation, alter glycosylation processing, and produce altered glycoforms in CHO cell cultures
Chemical inhibitors of hexokinase‐2 enzyme reduce lactate accumulation, alter glycosylation processing, and produce altered glycoforms in CHO cell cultures
复制标题
DOI：
10.1002/bit.28417
复制
发表时间：
2023-05
影响因子：
3.8
通讯作者：
Harnish Mukesh Naik;Swetha Kumar;J. Reddy;Jacqueline E. Gonzalez;Brian O. McConnell;Venkata Gayatri Dhara;Tiexin Wang;Marcella Yu;M. Antoniewicz;M. Betenbaugh
中科院分区：
文献类型：
--
作者：
Harnish Mukesh Naik;Swetha Kumar;J. Reddy;Jacqueline E. Gonzalez;Brian O. McConnell;Venkata Gayatri Dhara;Tiexin Wang;Marcella Yu;M. Antoniewicz;M. Betenbaugh
中国仓鼠卵巢（CHO）细胞是重组生物治疗药物生产的主要宿主，产生乳酸作为主要的糖酵解副产物。高乳酸水平对细胞生长和生产力产生不利影响。本研究的目的是通过向己糖激酶-2（HK 2）（催化葡萄糖转化为葡萄糖6-磷酸的酶）中添加化学抑制剂来减少CHO细胞培养物中的乳酸盐，并检查其对乳酸盐蓄积、细胞生长、蛋白滴度和N-糖基化的影响。评价了不同浓度的5种HK 2酶抑制剂，其中2-脱氧-d-葡萄糖（2DG）和5-硫代-d-葡萄糖（5 TG）成功降低了乳酸盐蓄积，对CHO细胞生长的影响有限。单独补充2DG和5 TG导致峰值乳酸降低35%-45%，而它们的组合补充导致峰值乳酸降低60%。抑制剂补充导致每摩尔葡萄糖消耗产生的乳酸摩尔数减少至少50%。在补充培养物中，重组EPO-Fc滴度相对于培养持续时间结束时更早达到峰值，导致最终EPO-Fc滴度增加至少11%，高达32%。天冬酰胺，丙酮酸，丝氨酸的消耗率也增加了在指数增长期在2DG和5 TG处理的文化，因此，重新布线中央碳代谢由于低糖酵解通量。EPO-Fc的N-聚糖分析显示，高甘露糖聚糖从对照培养物的5%分别增加至2DG和5 TG补充培养物的25%和37%。抑制剂补充还导致双触角、三触角和四触角结构减少，EPO-Fc唾液酸化降低高达50%。有趣的是，添加2DG导致2-脱氧-己糖（2DH）掺入到EPO-Fc N-聚糖上，添加5 TG导致首次观察到5-硫代-己糖（5TH）的N-聚糖掺入。对于分别用不同浓度的5 TG和2DG处理的培养物，6%至23%的N-聚糖包括5TH部分，最可能是5-硫代-甘露糖和/或5-硫代-半乳糖和/或可能的5-硫代-N-乙酰葡糖胺，并且14%至33%的N-聚糖包括2DH部分，最可能是2-脱氧-甘露糖和/或2-脱氧-半乳糖。我们的研究首次评估了这些葡萄糖类似物对CHO细胞生长、蛋白质产生、细胞代谢、N-糖基化加工和替代糖型形成的影响。
Chinese hamster ovary (CHO) cells, predominant hosts for recombinant biotherapeutics production, generate lactate as a major glycolysis by‐product. High lactate levels adversely impact cell growth and productivity. The goal of this study was to reduce lactate in CHO cell cultures by adding chemical inhibitors to hexokinase‐2 (HK2), the enzyme catalyzing the conversion of glucose to glucose 6‐phosphate, and examine their impact on lactate accumulation, cell growth, protein titers, and N‐glycosylation. Five inhibitors of HK2 enzyme at different concentrations were evaluated, of which 2‐deoxy‐ d‐glucose (2DG) and 5‐thio‐ d‐glucose (5TG) successfully reduced lactate accumulation with only limited impacts on CHO cell growth. Individual 2DG and 5TG supplementation led to a 35%–45% decrease in peak lactate, while their combined supplementation resulted in a 60% decrease in peak lactate. Inhibitor supplementation led to at least 50% decrease in moles of lactate produced per mol of glucose consumed. Recombinant EPO‐Fc titers peaked earlier relative to the end of culture duration in supplemented cultures leading to at least 11% and as high as 32% increase in final EPO‐Fc titers. Asparagine, pyruvate, and serine consumption rates also increased in the exponential growth phase in 2DG and 5TG treated cultures, thus, rewiring central carbon metabolism due to low glycolytic fluxes. N‐glycan analysis of EPO‐Fc revealed an increase in high mannose glycans from 5% in control cultures to 25% and 37% in 2DG and 5TG‐supplemented cultures, respectively. Inhibitor supplementation also led to a decrease in bi‐, tri‐, and tetra‐antennary structures and up to 50% lower EPO‐Fc sialylation. Interestingly, addition of 2DG led to the incorporation of 2‐deoxy‐hexose (2DH) on EPO‐Fc N‐glycans and addition of 5TG resulted in the first‐ever observed N‐glycan incorporation of 5‐thio‐hexose (5TH). Six percent to 23% of N‐glycans included 5TH moieties, most likely 5‐thio‐mannose and/or 5‐thio‐galactose and/or possibly 5‐thio‐N‐acetylglucosamine, and 14%–33% of N‐glycans included 2DH moieties, most likely 2‐deoxy‐mannose and/or 2‐deoxy‐galactose, for cultures treated with different concentrations of 5TG and 2DG, respectively. Our study is the first to evaluate the impact of these glucose analogs on CHO cell growth, protein production, cell metabolism, N‐glycosylation processing, and formation of alternative glycoforms.