Dee K U, Shuler M L, Wood H A
School of Chemical Engineering, Cornell University, 340 Olin Hall, Ithaca, New York 14853-5201, USA.
Biotechnol Bioeng. 1997 May 5;54(3):191-205. doi: 10.1002/(SICI)1097-0290(19970505)54:3<191::AID-BIT1>3.0.CO;2-A.
Sulfated polyanions have been successfully used to rapidly obtain and maintain stable single-cell suspension of BTI-TN5B1-4 cells, a cell line which has a high intrinsic capacity for recombinant protein production but clumps severely in suspension reducing its effectiveness as a host for foreign protein production with the baculovirus expression vector system. The efficacy of inducing single-cell suspension correlated positively with the increase in sulfation of the added polyanion. Unsulfated polyanions, neutral polymers, polycations, disaccharides, and monosaccharides were ineffective in inducing single-cell suspension.Elimination of clumping in suspension culture by adding a dispersing agent can lead to enhanced recombinant protein production. Inducing single-cell suspension with dextran sulfate, a highly sulfated polyanion, resulted in a four-fold increase in volumetric yield of the recombinant glycosylated protein, human secreted alkaline phosphatase, and a two-fold increase in volumetric yield of the recombinant cytoplasmic protein, beta-galactosidase. High yields of 82 U/ml (ca. 110 mg/L) for alkaline phosphatase, and 705 U/mL (ca. 2.3 g/L) for beta-galactosidase under elevated oxygen have been obtained. The optimum volumetric yield of alkaline phosphatase in BTI-TN5B1-4 dextran sulfate cells under elevated oxygen but unsupplemented medium is 6 to 11-fold higher than attached cultures, and 3-fold higher than the best yield obtained for SF21 cells in suspension at elevated oxygen and with nutrient supplementation. More importantly, cells can be infected at high density without complications from aggregation, which has important implications for scale-up.
硫酸化聚阴离子已成功用于快速获得并维持BTI-TN5B1-4细胞的稳定单细胞悬液。该细胞系具有较高的重组蛋白生产内在能力，但在悬浮培养时严重聚集，降低了其作为杆状病毒表达载体系统中外源蛋白生产宿主的有效性。诱导单细胞悬液的效果与添加的聚阴离子硫酸化程度的增加呈正相关。未硫酸化的聚阴离子、中性聚合物、聚阳离子、二糖和单糖在诱导单细胞悬液方面无效。通过添加分散剂消除悬浮培养中的聚集可提高重组蛋白产量。用高度硫酸化的聚阴离子硫酸葡聚糖诱导单细胞悬液，使重组糖蛋白人分泌碱性磷酸酶的体积产量提高了四倍，重组细胞质蛋白β-半乳糖苷酶的体积产量提高了两倍。在提高氧气浓度的条件下，碱性磷酸酶的产量高达82 U/ml（约110 mg/L），β-半乳糖苷酶的产量高达705 U/mL（约2.3 g/L）。在提高氧气浓度但未添加培养基的情况下，BTI-TN5B1-4硫酸葡聚糖细胞中碱性磷酸酶的最佳体积产量比贴壁培养高6至11倍，比在提高氧气浓度并添加营养物质的情况下悬浮培养的SF21细胞的最佳产量高3倍。更重要的是，细胞可以高密度感染而不会出现聚集并发症，这对扩大规模具有重要意义。
