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.

Figure 1

Reconstructed electrospray LC/MS spectrum of the Genotropin^® sample. The spectrum was recorded in positive ionization modus; 1 pM of the protein was injected. Detected average molecular masses: Nr.1.:22126.8 Da; Nr.2: 22143.5 Da; Nr.3: 22158.7 Da; Nr.4: 22174.4 Da; Nr.5: 22240.7 Da; Nr.6: 22395.9 Da; Nr7.: 22107.6 Da

Figure 2

Electrospray LC-MS/MS spectrum of the modified peptide EETQQK*SNLELLR. Positive ionization product ion spectrum of the tryptic peptide m/z 808.4 generated by a linear ion trap mass spectrometer. Intensive y fragment ions verify that the K₁₇₀ residue shows a mass increment of 28 Da compared to its theoretical mass.

Figure 3

Electrospray LC-MS/MS spectrum of the peptide LFDNAM*LR. Positive ionization product ion spectrum of the tryptic peptide m/z 482.3 generated by a linear ion trap mass spectrometer. Ions of the y series verify the mass discrepancy of -18 Da at the M₁₄ residue compared to its theoretical value.

Figure 4

MS spectrum of Genotropin showing oxidation of M₁₄, M₁₂₅ and M₁₇₀.(a) MS spectrum of Genotropin^® generated by an Ultraflex™ TOF/TOF (Bruker Daltonics) operated in the reflector mode for MALDI-TOF peptide mass fingerprint (PMF). Enlarged sections (b-d) from PMF of Genotropin^® showing oxidised/non-oxidised status (ÄM+16Da) of Methionine. (b) first peak: LFDNAMLR (979.529 Da)/ second peak: LFDNAMLR + oxidation of Methionine (995.519 Da); (c) first peak: DMDKVETFLR (1253.587 Da) / second peak: DMDKVETFLR + oxidation of Methionine (1269.572); (d) first peak: SVFANSLVYGASDSNVYDLLKDLEE-GIQTLMGR (3605.040) / second peak: SVFANSLVYGASDSNVYDLLKDLEE-GIQTLMGR + oxidation of Methionine (3621.064)

Figure 5

MS/MS spectra of Genotropin^® .LIFT-TOF/TOF (MS/MS) (a, b) spectra of Genotropin^® generated by an Ultraflex™ TOF/TOF (Bruker Daltonics) operated in LIFT mode for MALDI-TOF/TOF fully automated using the FlexControl™ software. The parent ions (m/z 1205.56 and m/z 2342.12) were selected for further analysis by MS/MS and the amino acid sequences NYGLLYCFR (5a) and LHQLAFDTYQEFEEAYIPK (5b) were unambiguously assigned to human growth hormone.