title: Isolation and Characterization of Acetylated Derivative of Recombinant Insulin Lispro Produced in Escherichia coli.
abstract: OBJECTIVE
  Insulin lispro is a rapid-acting insulin analogue produced by recombinant DNA technology. As a biosynthetic drug, the protein undergoes strict monitoring aiming for detection and characterization of impurities. The goal of this study was to isolate and identify a derivative of insulin lispro formed during biosynthesis.
  
  
  METHODS
  For this purpose, ion exchange chromatography in combination with endoproteinase Glu-C digestion, MALDI-TOF/TOF mass spectrometry and Edman sequencing were employed.
  
  
  RESULTS
  Ion exchange chromatography analysis of related proteins in development batches of recombinant insulin lispro revealed the existence of unknown derivative in excess of the assumed limit. Its molecular mass was 42 Da higher than the theoretical mass of Lys(B31) insulin lispro--one of the expected process-related intermediates. Endoproteinase Glu-C cleavage enabled indication of the modified peptide. Tandem mass spectrometry (MS/MS) allowed to explore the location and type of the modification. The 42 amu shift was present in the mass of y-type ions, while b-type ions were in agreement with theoretical values. It suggested that the modification is present on B31 lysine. Further inquiry revealed the presence of two diagnostic ions for lysine acetylation at m/z 143.1 and 126.1. In addition, the peptide was isolated and sequenced by Edman degradation. Standards of phenylthiohydantoin derivatives of N-ε-acetyl-L-lysine and N-ε-trimethyl-L-lysine, not available commercially, were synthesized in the laboratory. The retention time of the modified residue confirmed its identity as N-ε-acetyl-L-lysine.
  
  
  CONCLUSIONS
  The derivative of insulin lispro formed during biosynthesis of the drug was identified to be N-ε-acetyl-L-lysine (B31) insulin lispro.
authors: Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516, Warsaw, Poland.
authors: Joanna
authors: J
authors: Szewczak
authors: Anna
authors: A
authors: Bierczyńska-Krzysik
authors: Marcin
authors: M
authors: Piejko
authors: Paweł
authors: P
authors: Mak
authors: Dorota
authors: D
authors: Stadnik
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D061268:Insulin Lispro / Q000031:analogs & derivatives* / Q000302:isolation & purification* / Q000378:metabolism; D008239:Lysine / Q000031:analogs & derivatives* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D013678:Technology, Pharmaceutical / Q000379:methods*
chemical_list: D061268:Insulin Lispro; D010446:Peptide Fragments; D011994:Recombinant Proteins; C016949:N-epsilon-acetyllysine; D008239:Lysine