Stable Production of a Human Growth Hormone Antagonist from CHO Cells Adapted to Serum-Free Suspension Culture

• Published in: Biotechnology progress Vol. 15; no. 3; pp. 336 - 346
• Main Authors: Haldankar, Raj, Kopchick, John J., Ridgway, Darin
• Format: Journal Article
• Language: English
• Published: USA American Chemical Society 01.05.1999; American Institute of Chemical Engineers
• Subjects: Adaptation, Physiological; Amino acids; Animals; Biological and medical sciences; Biotechnology; CHO Cells; Cricetinae; Culture Media, Serum-Free; Fundamental and applied biological sciences. Psychology; Genetic engineering; Growth kinetics; Health. Pharmaceutical industry; High performance liquid chromatography; Hormones; Human Growth Hormone - analogs & derivatives; Human Growth Hormone - antagonists & inhibitors; Human Growth Hormone - genetics; Humans; Industrial applications and implications. Economical aspects; L Cells (Cell Line); Mice; Other active biomolecules; Phase separation; Polypeptides; Production of active biomolecules; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Size exclusion chromatography; Suspensions (fluids); Transfection; Ultrafiltration; Human; Cell culture; Serum free medium; Purification; Industrial application; Rodentia; Somatotropin hormone; Biological activity; CHO cell line; Vertebrata; Cell line; Mammalia; Animal; Production; Antagonist; Recombinant protein; Pharmaceutical industry; Hamster
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1021/bp9900460

Human growth hormone (hGH) is a polypeptide with 191 amino acids and a molecular mass of 22 kDa. An hGH analogue was created with a single amino acid substitution (glycine[G] 120 to arginine[R]) in the third α‐helix of the hGH molecule. This hGH analogue, named hGHG120R, was found to be an hGH antagonist. It is a parenteral drug candidate for treating conditions in which hGH levels are abnormally high, as found in type I diabetics. Previously, a genetically engineered anchorage‐dependent mouse L cell line was created that produced and secreted hGHG120R in culture media (Dulbecco's modified Eagle's medium, DMEM) supplemented with 5% NuSerum IV. A multistep downstream process was developed to purify hGHG120R. The process consisted of cell clarification, salt precipitation, membrane ultrafiltration, size exclusion chromatography, reversed phase high‐performance liquid chromatography, phase separation, and lyophilization. Here, we present the development of a superior eukaryotic system using a proper combination of genetic elements, cell line, and media formulation. This system is suitable for the large‐scale production of the recombinant protein and is superior to the previously developed system in that it increases the specific production rate and at the same time eases the burden of the purification process, in both time and efficiency. Dihydrofolate reductase mutant (DHFR−) Chinese hamster ovary (CHO) cells were used that were stably transfected with an expression vector in which the hGHG120R gene is driven by the relatively strong human cytomegalovirus−early gene regulatory region. 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 2.0 × 106 cells/mL were obtained from spinner flask cultures. Partial purification of hGHG120R from CHO cell cultured media 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 need not be employed in the purification of hGHG120R from SFM. This would result in a reduction of the operating time by 50 h and boost the recovery yield of hGHG120R to 75%.