Production of human growth hormone antagonist (hGHG120R) in Chinese hamster ovary cells.

摘要

Human growth hormone (hGH) is a polypeptide with 191 amino acids. Previously, it was found that an hGH analog with a single amino acid substitution acted as an hGH antagonist. Previously a genetically engineered anchorage-dependent mouse L cell line was created that produced and secreted the hGH antagonist (hGHG120R) at a level of 10 mg/l in culture media supplemented with 3-5% NuSerum IV. The multistep downstream process that was developed for the purification of hGHG120R consisted of cell clarification, salt precipitation, membrane ultrafiltration, size exclusion chromatography (SEC), reverse phase high performance liquid chromatography (RP-HPLC), phase separation and lyophilization. The total operating time for the purification protocol was approximately 120 hours and the average overall recovery was 51%.; The objective of this work is to produce a system suitable for the large-scale production of hGHG120R, by increasing the specific production rate and versatility of the culture system, and at the same time easing the burden of the purification process, in terms of both, time and efficiency. A proper combination of genetic elements, cell line, and media formulation is employed.; We have used dihydrofolate reductase mutant (DHFR{dollar}sp-){dollar} Chinese hamster ovary (CHO) cells, stably transfected with an expression vector driven by the relatively strong human cytomegalovirus immediate-early gene-regulatory region, to express high levels of hGHG120R. Protein expression levels of 4-5 mg/l were obtained from stably established CHO cells. The hGHG120R tested to be biologically active. These cells were then adapted to grow in suspension in CHO-S-SFM (serum-free media). High cell densities, typically, {dollar}{lcub}sim{rcub}1.0{lcub}-{rcub}2.0times10sp6{dollar} cells/ml were obtained in spinner flask cultures. Partial purification of hGHG120R from CHO cell cultured media using the previously established protocol, revealed that the level of impurities in SFM was significantly lower than the serum-supplemented DMEM. This suggests that the salt precipitation and the SEC step do not need to be employed in the purification of hCHG120R from SFM. This would result in a reduction of the operating time by 50 hours and boost the yield of hGHG120R to 75%.