Cole E S, Lee K, Lauziere K, Kelton C, Chappel S, Weintraub B, Ferrara D, Peterson P, Bernasconi R, Edmunds T
Therapeutic Protein Development Department, Genzyme Corporation, Framingham, MA 01701-9322.
Biotechnology (N Y). 1993 Sep;11(9):1014-24. doi: 10.1038/nbt0993-1014.
We have genetically engineered a cell line, and developed a reproducible process, for the expression and purification of biologically active recombinant human thyroid stimulating hormone (rhTSH).rhTSH was expressed by co-transfecting a human alpha-subunit cDNA with a human beta-subunit partial genomic clone into Chinese Hamster Ovary (CHO) cells. Stable transfectants which expressed high levels of rhTSH were selected, and subsequently cultured on microcarrier beads. The rhTSH-containing media, produced under serum-free conditions, was clarified and purified by a combination of ion exchange, dye and gel filtration chromatographies. Individual step recoveries were greater than 90% with the exception of a very conservative pooling of the final gel filtration step (78% recovery) that resulted in a cumulative yield of 54% for the purification process. Purity of the final bulk material was judged to be > 99% by SDS polyacrylamide gel electrophoresis (SDS-PAGE), reverse phase HPLC, and size exclusion chromatography. Initial characterization of the oligosaccharide composition indicated the presence of partially sialylated bi- and triantenary complex oligosaccharides. Purified rhTSH was active in a thyroid membrane bioactivity assay with a specific activity of 8.2 IU/mg. The in vivo activity of rhTSH in cynomolgus monkeys appeared to be equal to or greater than that reported for bovine TSH (bTSH) in human subjects. The rapid clearance phase half-life of rhTSH was approximately 35 minutes while the post-distribution phase half life was approximately 9.8 hours. Furthermore, the monkeys showed cumulative increases in minimum plasma rhTSH levels when given three daily intramuscular (IM) rhTSH injections; a phenomenon not observed when bTSH had been administered to humans. The rhTSH showed no evidence of toxic or adverse effects when administered at doses up to 7.2 IU/kg and 0.52 IU/kg in rat and monkey, respectively. These are 50X and 4X multiples of the bTSH doses of 0.143 IU/kg (10 IU/70kg) previously administered to humans.
我们通过基因工程构建了一种细胞系，并开发了一种可重复的方法，用于表达和纯化具有生物活性的重组人促甲状腺激素（rhTSH）。通过将人α亚基cDNA与人β亚基部分基因组克隆共转染到中国仓鼠卵巢（CHO）细胞中来表达rhTSH。筛选出表达高水平rhTSH的稳定转染子，随后在微载体珠上进行培养。在无血清条件下产生的含rhTSH的培养基，通过离子交换、染料和凝胶过滤色谱法相结合的方法进行澄清和纯化。除了最终凝胶过滤步骤非常保守的合并（回收率78%）导致纯化过程的累积产率为54%外，各个步骤的回收率均大于90%。通过SDS聚丙烯酰胺凝胶电泳（SDS-PAGE）、反相HPLC和尺寸排阻色谱法判断最终原料药的纯度>99%。对寡糖组成的初步表征表明存在部分唾液酸化的双天线和三天线复合寡糖。纯化的rhTSH在甲状腺膜生物活性测定中具有活性，比活性为8.2 IU/mg。rhTSH在食蟹猴体内的活性似乎等于或大于人类受试者中报道的牛促甲状腺激素（bTSH）的活性。rhTSH的快速清除相半衰期约为35分钟，而分布后相半衰期约为9.8小时。此外，当每天给猴子进行三次肌肉注射（IM）rhTSH时，猴子的最低血浆rhTSH水平出现累积增加；在给人类施用bTSH时未观察到这种现象。当分别以高达7.2 IU/kg和0.52 IU/kg的剂量在大鼠和猴子中施用时，rhTSH没有显示出毒性或不良反应的迹象。这些剂量分别是先前施用于人类的0.143 IU/kg（10 IU/70kg）bTSH剂量的50倍和4倍。
