Zanghi J A, Schmelzer A E, Mendoza T P, Knop R H, Miller W M
Department of Chemical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3120, USA.
Biotechnol Bioeng. 1999 Oct 20;65(2):182-91. doi: 10.1002/(sici)1097-0290(19991020)65:2<182::aid-bit8>3.0.co;2-d.
Accumulation of CO(2) in animal cell cultures can be a significant problem during scale-up and production of recombinant glycoprotein biopharmaceuticals. By examining the cell-surface polysialic acid (PSA) content, we show that elevated CO(2) partial pressure (pCO(2)) can alter protein glycosylation. PSA is a high-molecular-weight polymer attached to several complex N-linked oligosaccharides on the neural cell adhesion molecule (NCAM), so that small changes in either core glycosylation or in polysialylation are amplified and easily measured. Flow-cytometric analysis revealed that PSA levels on Chinese hamster ovary (CHO) cells decrease with increasing pCO(2) in a dose-dependent manner, independent of any change in NCAM content. The results are highly pH-dependent, with a greater decrease in PSA at higher pH. By manipulating medium pH and pCO(2), we showed that decreases in PSA correlate well with bicarbonate concentration ([HCO(3)(-)]). In fact, it was possible to offset a 60% decrease in PSA content at 120 mm Hg pCO(2) by decreasing the pH from 7.3 to 6.9, such that [HCO(3)(-)] was lowered to that of control (38 mm Hg pCO(2)). When the increase in osmolality associated with elevated [HCO(3)(-)] was offset by decreasing the basal medium [NaCl], elevated [HCO(3)(-)] still caused a decrease in PSA, although less extensive than without osmolality control. By increasing [NaCl], we show that hyperosmolality alone decreases PSA content, but to a lesser extent than for the same osmolality increase due to elevated [NaHCO(3)]. In conclusion, we demonstrate the importance of pH and pCO(2) interactions, and show that [HCO(3)(-)] and osmolality can account for the observed changes in PSA content over a wide range of pH and pCO(2) values.
在动物细胞培养中，二氧化碳（CO₂）的积累在重组糖蛋白生物制药的放大培养和生产过程中可能是一个重大问题。通过检测细胞表面多唾液酸（PSA）含量，我们发现升高的二氧化碳分压（pCO₂）会改变蛋白质糖基化。PSA是一种高分子量聚合物，附着在神经细胞黏附分子（NCAM）上的几种复杂的N-连接寡糖上，因此核心糖基化或多唾液酸化的微小变化都会被放大并易于测量。流式细胞术分析显示，中国仓鼠卵巢（CHO）细胞上的PSA水平随着pCO₂的升高呈剂量依赖性降低，与NCAM含量的任何变化无关。结果高度依赖于pH值，在较高pH值下PSA的降低幅度更大。通过调节培养基pH值和pCO₂，我们发现PSA的降低与碳酸氢盐浓度（[HCO₃⁻]）密切相关。事实上，通过将pH值从7.3降至6.9，使得[HCO₃⁻]降低至对照水平（38 mmHg pCO₂），可以抵消在120 mmHg pCO₂时PSA含量60%的降低。当通过降低基础培养基中的[NaCl]来抵消与升高的[HCO₃⁻]相关的渗透压升高时，升高的[HCO₃⁻]仍然会导致PSA降低，尽管程度比不进行渗透压控制时要小。通过增加[NaCl]，我们发现单纯的高渗会降低PSA含量，但程度小于因[NaHCO₃]升高导致相同渗透压增加时的情况。总之，我们证明了pH值和pCO₂相互作用的重要性，并表明[HCO₃⁻]和渗透压可以解释在广泛的pH值和pCO₂值范围内观察到的PSA含量变化。
