Schmelzer Albert E, Miller William M
Chemical Engineering Department, Northwestern University, Evanston, Illinois 60208-3120, USA.
Biotechnol Prog. 2002 Mar-Apr;18(2):346-53. doi: 10.1021/bp010187d.
Medium osmolality increases with pCO2 at constant pH. Elevated pCO2 and osmolality inhibit hybridoma growth to similar extents in both serum-containing and serum-free media. The combination of osmolality and elevated pCO2 synergizes to negatively impact cell growth. IgG2a glycosylation by hybridoma cells was evaluated under elevated pCO2 (to 250 mmHg pCO2) and/or osmolality (to 476 mOsm/kg). IgG2a site occupancy did not change significantly under any of the conditions studied, which is consistent with the robust glycosylation of other antibodies produced under various environmental stresses. However, changes were observed in the IgG2a charge distribution. Changes in the isoelectric point (pI) were greater under hyperosmotic stress, increasing by 0.32 and 0.41 pH units at 435 mOsm/kg in serum-containing and serum-free medium, respectively. Hyperosmotic stress also resulted in a concomitant increase in the heterogeneity of the charge distribution. The mean pI in serum-containing medium decreased by 0.16 pH units at 250 mmHg pCO2 when osmolality was controlled at 320 mOsm/kg but increased by 0.20 pH units when the osmolality increased with pCO2 (195 mmHg pCO2-435 mOsm/kg). In serum-free medium, elevated pCO2 did not alter pI, regardless of medium osmolality. In contrast to elevated osmolality at control pCO2, elevated pCO2 did not significantly alter the IgG2a charge heterogeneity under any of the conditions studied. The IgG2a was not sialylated, so sialylation changes were not responsible for changes in the charge distribution. IgG2a galactose content decreased with elevated osmolality, as a result of either elevated NaHCO3 or NaCl. However, when osmolality was controlled at elevated pCO2, the galactose content tended to increase. The mannose content decreased with increasing stress, while the fucose content remained relatively unchanged. It is likely that the observed increases in the pI of murine IgG2a were due to increased organellar pH, which is reflected by increased specific beta-galactosidase activity in the supernatant.
在pH值恒定的情况下，中等渗透压随二氧化碳分压（pCO2）升高而增加。在含血清和无血清培养基中，升高的pCO2和渗透压对杂交瘤生长的抑制程度相似。渗透压升高与pCO2升高共同作用，对细胞生长产生负面影响。在升高的pCO2（至250 mmHg pCO2）和/或渗透压（至476 mOsm/kg）条件下，评估杂交瘤细胞对IgG2a的糖基化作用。在所研究的任何条件下，IgG2a位点占有率均无显著变化，这与在各种环境压力下产生的其他抗体的强大糖基化作用一致。然而，观察到IgG2a电荷分布发生了变化。在高渗应激下，等电点（pI）变化更大，在含血清和无血清培养基中，435 mOsm/kg时分别增加0.32和0.41个pH单位。高渗应激还导致电荷分布的异质性同时增加。当渗透压控制在320 mOsm/kg时，在250 mmHg pCO2条件下，含血清培养基中的平均pI降低0.16个pH单位，但当渗透压随pCO2升高（195 mmHg pCO2 - 435 mOsm/kg）时，平均pI升高0.20个pH单位。在无血清培养基中，无论培养基渗透压如何，升高的pCO2均未改变pI。与对照pCO2下渗透压升高相反，在所研究的任何条件下，升高的pCO2均未显著改变IgG2a电荷异质性。IgG2a未发生唾液酸化，因此唾液酸化变化不是电荷分布变化的原因。由于NaHCO3或NaCl升高，IgG2a半乳糖含量随渗透压升高而降低。然而，当在升高的pCO2条件下控制渗透压时，半乳糖含量趋于增加。甘露糖含量随应激增加而降低，而岩藻糖含量相对保持不变。观察到的小鼠IgG2a的pI升高可能是由于细胞器pH值升高，这由上清液中特异性β-半乳糖苷酶活性增加所反映。
