title: Investigation of N-terminal glutamate cyclization of recombinant monoclonal antibody in formulation development.
abstract: The N-terminal glutamic acid (Glu) can be cyclized to form pyroglutamate (pGlu). Recent studies have suggested that N-terminal pGlu formation is an important posttranslational or co-translational event and is greatly facilitated by the enzyme glutaminyl cyclase, although the impact of the N-terminal cyclization on the potency and overall stability of mAbs is not been well known. Since most recombinant monoclonal antibodies (mAbs) contain glutamic acid and/or glutamine at their N-terminus, understanding the cyclization mechanisms may shed light on the factors that control the pGlu formation in therapeutic mAb development. Here, two mass spectrometry-based techniques were developed to investigate N-pyroglutamyl formation and the high conversion rate to pGlu at the N-terminus of the mAb was reported in the formulation development. The pGlu formation is favored at pH 4 and 8, but is less common at the neutral pH that is optimum for the enzymatic Glu conversion. These observations suggest that pGlu formation can proceed non-enzymatically at mild conditions and that this cyclization is not driven by glutaminyl cyclase in non-physiological conditions. We also calculate the half-lives of the N-terminal Glu at different pH and temperatures from the kinetics data, which would be very helpful for predicting pGlu formation and for selecting proper formulation and storage conditions.
authors.affiliation: Department of Pharmaceutics, Amgen Inc., One Amgen Center Drive, 2-2-A, Thousand Oaks, CA 91320, USA. leiy@amgen.com
authors.forename: Lei
authors.initials: L
authors.lastname: Yu
authors.forename: Alona
authors.initials: A
authors.lastname: Vizel
authors.forename: Mary Beth
authors.initials: MB
authors.lastname: Huff
authors.forename: Meagan
authors.initials: M
authors.lastname: Young
authors.forename: Richard L
authors.initials: RL
authors.lastname: Remmele
authors.forename: Bing
authors.initials: B
authors.lastname: He
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics; D002626:Chemistry, Pharmaceutical; D002851:Chromatography, High Pressure Liquid; D003500:Cyclization; D004355:Drug Stability; D005079:Excipients / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D011761:Pyrrolidonecarboxylic Acid / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013501:Surface-Active Agents / Q000737:chemistry; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D005079:Excipients; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D018698:Glutamic Acid; D011761:Pyrrolidonecarboxylic Acid