title: Mass spectrometrical analysis of recombinant human growth hormone (Genotropin(R)) reveals amino acid substitutions in 2% of the expressed protein.
abstract: BACKGROUND: The structural integrity of recombinant proteins is of critical importance to their application as clinical treatments. Recombinant growth hormone preparations have been examined by several methodologies. In this study recombinant human growth hormone (rhGH; Genotropin(R)), expressed in E. coli K12, was structurally analyzed by two-dimensional gel electrophoresis and MALDI-TOF-TOF, LC-MS and LC-MS/ MS sequencing of the resolved peptides. RESULTS: Electrospray LC-MS analysis revealed one major protein with an average molecular mass of 22126.8 Da and some additional minor components. Electrospray LC-MS/MS evaluation of the enzymatically digested Genotropin(R) sample resulted in the identification of amino acid substitutions at the residues M14, M125, and M170; di-methylation of K70 (or exchange to arginine); deamidation of N149, and N152, and oxidation of M140, M125 and M170. Peak area comparison of the modified and parental peptides indicates that these changes were present in ~2% of the recombinant preparation. CONCLUSION: Modifications of the recombinant human growth hormone may lead to structural or conformational changes, modification of antigenicity and development of antibody formation in treated subjects. Amino acid exchanges may be caused by differences between human and E. coli codon usage and/or unknown copy editing mechanisms. While deamidation and oxidation can be assigned to processing events, the mechanism for possible di-methylation of K70 remains unclear.
authors.affiliation: Department of Pediatrics, Medical University of Vienna, Vienna, Austria. gert.lubec@meduniwien.ac.at.
authors.forename: Felix
authors.initials: F
authors.lastname: Hepner
authors.forename: Edina
authors.initials: E
authors.lastname: Cszasar
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Roitinger
authors.forename: Gert
authors.initials: G
authors.lastname: Lubec