关键词：关节炎;软骨细胞;细胞凋亡;生物物理学;临床医学;系数;一致性;控制;切除;成纤维细胞;摩擦;接头;润滑;单体;预防;生产;蛋白质
摘 要：Recombinant human lubricin (rhPRG4) was expressed by transfected CHO- S cells at a level of secretion that may be scalable and thus enable production of a GMP protein for clinical use. A purification bioprocess was created that achieved a purity level of approximately 95%. A total of 5 bands visualized on SDS-PAGE are all product related following excision and analysis by LC-MS. Full- length and truncated rhPRG4 constructs are being tested for chondroprotective ability. The full-length construct significantly lowers friction between pressurized discs of articular cartilage. The coefficient of friction in the presence of rhPRG4 was 0.03 whereas for a saline control 0.07. Study of the 5 other constructs and confirmation of reduction in levels of chondrocyte apoptosis attributable to lower friction is ongoing. The posttranslational glycosylations on rhPRG4 appear to be (1-3)GalNac-Gal (2,3) NeuAc judging by sequential enzymatic deglycosylation and molecular weight shift on SDS-PAGE. Western blotting of rhPRG4 with mAb 9G3, which reacts with the glycosylations in the lubricin mucin domain, also confirms that the glycosylations are consistent with lubricin secreted by human synovial fibroblasts. Analytical ultracentrifugation shows that there are 5 distinct species in rhPRG4, 3 of which are major forms: monomer, dimer and a higher order tetramer. This biophysical form of analysis may also prove useful in a quality assurance program in GMP protein production as the relative amounts of each major form can be monitored.