or better:which one is active? genotropin not.

.Mass spectrometrical analysis of recombinant human growth hormone (Genotropin®) reveals amino acid substitutions in 2% of the expressed protein
Felix Hepner1, Edina Cszasar2, Elisabeth Roitinger2 and Gert Lubec1
1Department of Pediatrics, Medical University of Vienna, Vienna, Austria
2Mass Spectrometry Unit, University of Vienna, Austria

Proteome Science 2005, 3:1 doi:10.1186/1477-5956-3-1


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®), 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.


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® 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.


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.

The structural integrity of recombinant products generated by prokaryotic and eukaryotic organisms is a major concern. Modifications such as amino acid sequence substitution/mutations of recombinant
proteins may lead to pharmacological inactivation, autoimmune phenomena [1-3] and adverse effects [4,5]. Human growth hormone (hGH) replacement is a frequent therapeutic intervention [6,7]. Genetic changes in human growth hormone have been linked to biological inactivity and disease: Lewis et al (2004) reported that a growth hormone variant I179_M179 showed decreased ability to activate the extracellular signal-regulated kinase pathway and Binder et al. (2002) described hGH deficiency due to mutations of the coding regions of the growth hormone-1 gene [8,9]. Zhu et al. (2002) reported a case of hGH R183_H183. This single mutation causes autosomal dominant growth hormone deficiency type II by prolonged retention time of R183_H183 aggregates into secretory granules [10].

However, although such changes can be detrimental, non functional sequence alteration induced by poor editing of recombinant
proteins may act as a marker of growth hormone abuse in situations such as athlete doping. We therefore were highly interested in the homogeneity and structure of rhGH preparations.

Genotropin® is expressed by E. coli, strain K12. It consists of a single polypeptide chain containing 191 amino acids and two disulfide bonds (C53-C165; C182-C189) [
11] with a molecular mass of 22 124 Da – representing the most abundant growth hormone form in humans [12].

In humans two major hGH splicing variants have been described, a 22 kDa
protein and a 20 kDa protein, that bind different sites at the growth hormone receptor and serve different biological activities [13,14].

The genetic origin of hGH is the hGH-N gene, located on the long arm of chromosome 17, in a 66-kbp cluster region closely related to four other genes: hGH-V, hCS-A, hCS-B and hCS-L. The hGH-N gene is expressed in both, pituitary and several nonpituitary sites [
12], all other gene products are produced by placental syncytio-trophoblasts.

A series of posttranslational modifications of hGH have been described and range from N-glycosylation, acetylation, deamidation, oxidation at M14 and M125 to polymerisation [

As mentioned above, Genotropin® is expressed by E. coli. Since the fidelity of hGH translation in E. coli cannot rely on copy editing [
19,20], nor on correct codon usage [21-23], there is a large potential for sequence errors. That's why investigations of structural/sequential integrity, including amino acid exchanges/mutations, and post translational modifications of rhGH Genotropin® is of particular interest to for modern medicine and pharmacotherapy.

The aim of the present study was to investigate the homogeneity of a commercial available rhGH, Genotropin®. This was achieved using two dimensional gel electrophoresis (2-DE), matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) followed by tandem mass spectrometry (MALDI-MS/MS) and liquid chromatography mass spectrometry (LC-MS) followed by tandem mass spectrometry (LC-MS/MS). These modern analytical tools provide definitive structural analysis independent of antibody availability and specificity.