title: Purification and identification of high molecular weight products formed during storage of neutral formulation of human insulin.
abstract: OBJECTIVE
  To identify High Molecular Weight Products (HMWP) formed in human insulin formulation during storage.
  
  
  METHODS
  Commercial formulation of human insulin was stored at 37°C for 1 year and HMWP was isolated using preparative size exclusion chromatography (SEC) and reverse phase (RP) chromatography. The primary structure of the isolated species was analysed using liquid chromatography mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS). To test the hypothesis that amino groups of insulin are involved in HMWP formation, the HMWP content of various formulations spiked with amine compounds or formulations of insulin with modified amino groups was measured.
  
  
  RESULTS
  More than 20 species of HMWP were observed and 16 species were identified using LC-MS. All identified species were covalent dimers of human insulin linked via A21Asn and B29Lys, formed via the formation of an anhydride intermediate at A21Asn. Two types of HMWP were identified, with the covalent link in the open or closed (succinimidyl) form. Some species also contained single deamidation at B3 or the desPhe(B1)-N-oxalyl-Val(B2) modification. Reduced rate of HMWP formation was observed after addition of L-lysine, L-arginine or piperazine or when insulin analogues with methylated N-terminals and side chain amines and A21Gly mutation were used. Formulations of human insulin without zinc and m-cresol were found to contain a different pool of HMWP.
  
  
  CONCLUSIONS
  HMWP formed in formulation of human insulin at pH 7.4 with zinc and m-cresol consists primarily of covalent dimers linked via A21Asn and B29Lys. Insulin formulation properties determine the amount and identity of formed HMWP.
authors.affiliation: Diabetes Protein Engineering, Novo Nordisk A/S, Novo Nordisk Park, 2760, Måløv, Denmark, cfth@novonordisk.com.
authors.forename: Christian Fogt
authors.initials: CF
authors.lastname: Hjorth
authors.forename: František
authors.initials: F
authors.lastname: Hubálek
authors.forename: Jonatan
authors.initials: J
authors.lastname: Andersson
authors.forename: Christian
authors.initials: C
authors.lastname: Poulsen
authors.forename: Daniel
authors.initials: D
authors.lastname: Otzen
authors.forename: Helle
authors.initials: H
authors.lastname: Naver
mesh_terms: D000588:Amines / Q000737:chemistry; D000595:Amino Acid Sequence; D002626:Chemistry, Pharmaceutical; D002850:Chromatography, Gel; D056148:Chromatography, Reverse-Phase; D003408:Cresols / Q000737:chemistry; D004340:Drug Contamination; D004355:Drug Stability; D004356:Drug Storage; D006801:Humans; D007004:Hypoglycemic Agents / Q000737:chemistry*; D061386:Insulin, Regular, Human / Q000737:chemistry*; D061389:Insulin, Regular, Pork / Q000737:chemistry*; D008970:Molecular Weight; D055503:Protein Multimerization; D055550:Protein Stability; D053719:Tandem Mass Spectrometry; D013696:Temperature; D013997:Time Factors; D015032:Zinc / Q000737:chemistry
chemical_list: D000588:Amines; D003408:Cresols; D007004:Hypoglycemic Agents; D061386:Insulin, Regular, Human; D061389:Insulin, Regular, Pork; C032538:4-cresol; C000006:insulin, neutral; D015032:Zinc