title: The effect of cosolutes on the isomerization of aspartic acid residues and conformational stability in a monoclonal antibody.
abstract: The aspartate residue (Asp 32) located in the complementarity-determining region (CDR) of a recombinant humanized monoclonal antibody (MAb I) is highly susceptible to the isomerization reaction. The modification of Asp 32 residue due to the isomerization reaction results in a significant reduction in the binding affinity of MAb I to IgE. The binding of a MAb I therapeutic to IgE is important for its desired pharmacological effect. In earlier investigations, we demonstrated that the conformational flexibility and residue exposure are factors that are responsible for the observed reactivity of Asp 32 in MAb I. This report explores the role of cosolutes such as glycerol and sucrose in the modulation of Asp 32 reactivity in MAb I. These cosolutes are routinely incorporated in injectable pharmaceutical formulations. The reactivity of the Asp residue in MAb I in these different cosolute-based formulations was compared to its reactivity in a peptide model VDYDG comprising residues 29-33 of MAb I. The formulations of MAb I and VDYDG containing varying concentrations of glycerol and sucrose were incubated at 50 degrees C for a period of 5-7 days. The isomerization of the Asp residue in VDYDG and MAb I was monitored using rp-HPLC and hydrophobic interaction chromatography (HIC), respectively. Structural analysis of MAb I using differential scanning calorimetry (DSC) demonstrated that the structural stability of MAb I was increased in formulations containing glycerol and sucrose. However, the stability of Asp 32 in MAb I was significantly decreased in these formulations. This research suggests that a formulation approach that relies purely on enhancing the structural stability of proteins through addition of these cosolutes could result in problems associated with the chemical stability of these biomolecules.
authors.affiliation: Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, USA. wakankar.aditya@gene.com
authors.forename: Aditya A
authors.initials: AA
authors.lastname: Wakankar
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: David
authors.initials: D
authors.lastname: Vandervelde
authors.forename: Y John
authors.initials: YJ
authors.lastname: Wang
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Shire
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Borchardt
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D000915:Antibody Affinity; D001224:Aspartic Acid / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D002626:Chemistry, Pharmaceutical; D004355:Drug Stability; D005079:Excipients / Q000737:chemistry*; D005990:Glycerol / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007073:Immunoglobulin E / Q000378:metabolism; D007536:Isomerism; D015636:Magnesium Chloride / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009842:Oligopeptides; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013395:Sucrose / Q000737:chemistry*; D013678:Technology, Pharmaceutical / Q000379:methods; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D005079:Excipients; D009842:Oligopeptides; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D001224:Aspartic Acid; D007073:Immunoglobulin E; D012965:Sodium Chloride; D013395:Sucrose; D005990:Glycerol