Analysis of recombinant human growth hormone directly in osmotic shock fluids

• Published in: Journal of Chromatography A Vol. 782; no. 2; pp. 199 - 210
• Main Authors: Dalmora, Sergio, de Oliveira, João Ezequiel, Affonso, Regina, Gimbo, Elizabeth, Ribela, Maria Teresa C.P., Bartolini, Paolo
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 10-10-1997; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, High Pressure Liquid - methods; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Growth hormones; Hormones; Human Growth Hormone - analysis; Human Growth Hormone - genetics; Humans; Osmotic Pressure; Osmotic shock fluids; Protein hormones. Growth factors. Cytokines; Proteins; Recombinant Proteins - analysis; Recombinant Proteins - genetics; Proteins; Growth hormones; Osmotic shock fluids; Hormones; Human; Chemical analysis; STH; HPLC chromatography; Isocratic condition; Protein hormone; Reversed phase chromatography; Ultraviolet detector; Analysis method; Adenohypophyseal hormone; Qualitative analysis; Recombinant protein; Quantitative analysis
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(97)00493-7

An isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of human growth hormone (hGH) directly in osmotic shock fluids is described. This methodology allows an initial rapid evaluation of the quality and quantity of hGH being secreted in the bacterial periplasmic space right after, or even during fermentation. Considering that RP-HPLC does not identify size isomers, these were determined via a parallel run of the same osmotic shock fluid on high-performance size-exclusion chromatography, coupled with radioimmunoassay, of the eluted fractions. The methodology provides a complete picture, within 24 h from the beginning of the fermentation process, of the recombinant protein being produced with respect to its activity, identity, yield, and hGH-related contaminants. These latter include sulfoxide and desamido derivatives, dimer and high-molecular-mass forms.