Proteome Sci. 2005 Feb 11;3(1):1.
Mass spectrometrical analysis of recombinant human growth hormone (Genotropin(R)) reveals amino acid substitutions in 2% of the expressed protein.
Hepner F, Cszasar E, Roitinger E, Lubec G.
Department of Pediatrics, Medical University of Vienna, Vienna, Austria.
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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.

Excerpts from the full text article:

Background. 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 I_{179_}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 R_{183_}H₁₈₃. This single mutation causes autosomal dominant growth hormone deficiency type II by prolonged retention time of R_{183_}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 [12,15-18].

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.

Results

Two dimensional gel electrophoresis

Two dimensional gel electrophoresis (2-DE) of 1 mg Genotropin^® showed a multiple spot pattern with masses between 20 000 and 35 000 Da and pIs from 4.5 to 7.0. Several two dimensional (2D) gels with sample amounts of 0.5, 1, 2, 5, 10, 20, 50, 100, 200 and 500 g of Genotropin^® were performed. Decreasing protein load showed reduction of spot size and number and finally, limitation to two spots of 22000 Da with pI of 5.3 and 5.4.

Neither MALDI-TOF-TOF nor LC-MS analysis of picked gel spots indicated any modifications or isoforms in an amount, that would explain differences between the two spots.

Electrospray LC-MS measurements of the Genotropin^® sample

Electrospray liquid chromatography – mass spectrometry (LC-MS) measurements of the intact Genotropin^® have shown that the main product was a molecule with an average molecular weight (MW) of 22126.8 Da. The manufacturers had determined the average MW of Genotropin^® to be 22124 Da.

The mass difference of approximately 3 Da may originate from the deconvulation of some broader, lower intensity peaks. Several minor components could be also detected. (Figure 1) The mass differences between the main product (Nr. 1) and components Nr. 2–5 respectively indicate the oxidation of several amino acid residues. Components Nr.6 and Nr.7 show a mass discrepancy of approximately +268 Da and -19 Da respectively. According to the ratios of peak areas (Table 1), the sample consists to 84.4% of the unmodified main component; the oxidation products are present to 13.7% of the whole sample and the ratio of other minor components, which may represent additional modifications or amino acid substitutions, is approximately 2%.

Electrospray LC-MS/MS measurements following the tryptic digestion of the Genotropin^® sample

Electrospray tandem liquid chromatography – mass spectrometry (LC-MS/MS) measurements of the samples prepared from one dimensional SDS-PAGE indicated mass differences at several peptides. Doubly or triply charged ions were chosen for all MS/MS experiments due to their better fragmentation pattern. Table 2 shows the sequences of the modified peptides and possible explanations for the mass discrepancies.

A mass difference of +28 Da was detected at the position K₇₀ (Figure 2), could be explained by the di-methylation of this residue, or by the exchange of this lysine to an arginine. These modifications result in a mass difference of 28.03 Da and 28.01 Da respectively. The accuracy of the mass spectrometric detection was not high enough to differentiate between these possibilities. Figure 2 shows the fragment spectrum of the peptide EETQQKSNLELLR. Intensive y ions verify that all residues have unchanged masses except of K₇₀, which makes the localization of the mass discrepancy on that lysine residue unambiguous.

Deamidation of the amino acids N₁₄₉ and N₁₅₂ was also detected. The molecular mass of the peptide RLEDGSPR was decreased with 28 Da. The mass difference could be localized to the N terminus of the peptide and might indicate the substitution of R₁₂₇ with a lysine or glutamine. The mass difference of these residues is only 0.04 Da and the accuracy of the mass spectrometric detection was not high enough to differentiate between these amino acids. Residues M₁₄, M₁₂₅ and M₁₇₀ were observed partly oxidized and in some cases the non oxidized residue showed a mass discrepancy of -18 Da (Table 2). This phenomenon is illustrated by Figure 3, which shows a product ion spectrum of the modified peptide LFDNAMLR. Fragment ions from the y series verify the mass reduction at the M₁₄ residue. This mass difference can be explained by the replacement of these methionines with isoleucines, which can be originate from the substitution of the last base in the genetic codon of methionine (M:ATG; I:ATT/C/A). According to the ratios of the peak areas of the peptides containing the unmodified and possibly substituted methionines, these changes were present at < 2% of the whole protein amount. A mass increase of 57 Da was detected at the peptide LFDNAMLR. It could be localized at the N terminus of the peptide and it is supposed to be an artefact of the alkylation step during sample preparation. All modifications were partial; in each case peptides with both modified and unmodified residues were present. LC-MS/MS spectra for all modified peptides are available as supplementary material.

Genotropin^® was unambiguously identified by MS and MS/MS Data (Figure 4 and 5), with a maximum of 24 matching peptides, representing a sequence-coverage of 86% to human growth hormone sequence present in database (Figure 4, Table 3). All picked and analysed spots showed similar peptide mass fingerprints. Only the oxidation status of M varied, represented by a mass difference (ÄM) of 16 Da. Oxidation at M₁₄ was demonstrated in 59,52% of analysed spots, 80,91% of M₁₂₅ and 54,87% of M₁₇₀ showed oxidation too (Table 3). Neither changes in amino acid sequence, nor post translational modifications like phosphorylation or deamidation could be detected by this method.

Discussion

The dominant protein in the Genotropin^® preparation has an average molecular mass of 22127 Da. Electrospray LC-MS/MS evaluation of the trypsinized recombinant human growth hormone (rhGH) resulted in the identification of amino acid substitutions at residues M₁₄, M₁₂₅ and M₁₇₀. Di-methylation of K₇₀ or exchange to arginine, deamidation of N₁₄₉ and N₁₅₂, and oxidation of M₁₄, M₁₂₅ and M₁₇₀ were also observed. These sequence alteration account for 2% of the recombinant protein.

Amino acid exchanges of a rhGH has been described before: Gellerfors et al. (1990) describe exchanges rhGH Q65_V65 and rhGH Q_{66_}K₆₆[25]. Since the product was not identified we cannot compare our results. Binding of recombinant human growth hormone to the GH receptor may be modified by the five amino acid exchanges observed in the present study. Pal et al. (2003) calculated binding energy differences between modified human growth hormone (hGHv; M_{14_}W₁₄) and wild type human growth hormone (hGHwt; M₁₄) with the result that the hGHv had more binding affinity to its receptor than hGHwt [26,27]. Cunningham et al. showed that M₁₄ influenced binding, even if it is not a “hot spot” for linkage to its receptor. Furthermore, the amino acid exchanges detected may very well lead to antigenic differences and thus form the molecular basis for eliciting immune responses.

The underlying cause of amino acid exchanges may be codon usage and/or absence of copy editing in E. coli: The M_I exchanges may be due to miscast of the third nucleoside of the cognate anticodon at the so-called Wobble-position, i.e. switch cytosine to guanine/adenosine, a phenomenon described by Crick as “Wobble-hypothesis [28]. Crick (1966) postulated a certain amount of wobble at the third base position of the codon allowing more than one possible codon-anticodon-base pairing. Methionine (M)_I exchange of rhGH Genotropin^® may have been generated as the base pair G-G / G-A was replacing G-C. Arginine (R)_K/Q and R_G exchanges of rhGH Genotropin^® may be due to difficulties in translation of the rare codon AGG. Kane et al. (1995) predicted translational problems with an abundant mRNA species containing an excess of rare tRNA codons that may arise after the initiation of transcription of a cloned heterologous gene in the E. coli host [21]. Recent studies suggest clusters of AGG/AGA codons can reduce both quantity and quality of the synthesized protein [22,29]. Translational modification normally does not include amino acid exchanges but rather frameshift mutations/deletions [21,29-31].

In summary, we found two different pathways for amino acid exchanges in Genotropin^®: translation errors due to usage of (1) the rare codon AGG in E. coli and (2) incorrect codon usage consisted with Crick’s “Wobble-hypothesis”.

Oxidative modification of a recombinant human growth hormone has been described by Karlsson et al. (1999) who demonstrated M₁₄ and M₁₂₅ oxidation as detected by LC-MS [32]. No other group have reported oxidation of M₁₇₀ as in our study. Indeed Teh et al. (1987) oxidised natural hGH extracted from pituitary glands and detected M₁₄ and M₁₂₅ oxidation by reversed phase chromatography [33]. Gellerfors et al. (1990) oxidised rhGH with hydrogen peroxide but again failed to show oxidation of M₁₇₀, as detected by reversed phase chromatography [25]. It is not known whether oxidation of methionines in recombinant human growth hormone leads to functional impairment but conformational changes are unlikely as proposed by circular dichroism and 1H-NMR studies [33]. It is worth mentioning that oxidised methionines are not localised at the receptor binding site.

Post translational modification such as N-acetylation, N-glycosylation, deamidation and oxidation have been reported for rhGH, hGH and bovine growth hormone (bGH) [15-17,34]. Dimethylation of K₇₀ in rhGH and hGH have not previously been reported.

Whether transmethylation occurred during processing or is a post translational event during rhGH production in E. coli is unknown. Nevertheless, Martal et al. (1985) demonstrated reduction of biological activity of hGH and bGH by methylation and ethylation of its residues K₄₁, K₇₀, and K₁₁₅[35]. Therefore, dimethylation of K₇₀ in Genotropin^® could have biological relevance, probably reducing its pharmacotherapeutic activity.

Deamidation of N₁₄₉ and N₁₅₂ may be due to technical processing, probably by heat treatment or lyophilisation and has already been reported by Gellerfors et al (1990) and Karlsson et al. (1999) [25,32]. Though these appear to have no function significance [26,27,36].

Modifications of the recombinant human growth hormone, as shown in this study, may effect functionality and safety depending on the prevalence of such forms in the preparation. As already mentioned above, impaired binding to the receptor, conformational changes leading to impaired function, amino acid exchanges as mutations may well lead to immune phenomena or even disease [1-3,37]. In addition, such modifications may act as markers of these proteins in situations like rhGH doping.

Conclusions

Using one- and two-dimensional gel electrophoresis, electrospray LC-MS, LC- MS/MS and MALDI-TOF-TOF mass spectrometry we detected a series of modifications of the recombinant human growth hormone (Genotropin^®) including amino acid exchanges, oxidation, di-methylation and deamidation. This analytical battery is a reliable, specific and sensitive analytical tool for this purpose.

Categories: 2005, Acetylation, Glycosylation, Glycoproteins, Growth hormone physiology, Growth hormone safety, Growth hormone treatment, Methionine, Post-translational modification