title: Solubility Challenges in High Concentration Monoclonal Antibody Formulations: Relationship with Amino Acid Sequence and Intermolecular Interactions.
abstract: The purpose of this work was to elucidate the molecular interactions leading to monoclonal antibody self-association and precipitation and utilize biophysical measurements to predict solubility behavior at high protein concentration. Two monoclonal antibodies (mAb-G and mAb-R) binding to overlapping epitopes were investigated. Precipitation of mAb-G solutions was most prominent at high ionic strength conditions and demonstrated strong dependence on ionic strength, as well as slight dependence on solution pH. At similar conditions no precipitation was observed for mAb-R solutions. Intermolecular interactions (interaction parameter, kD) related well with high concentration solubility behavior of both antibodies. Upon increasing buffer ionic strength, interactions of mAb-R tended to weaken, while those of mAb-G became more attractive. To investigate the role of amino acid sequence on precipitation behavior, mutants were designed by substituting the CDR of mAb-R into the mAb-G framework (GM-1) or deleting two hydrophobic residues in the CDR of mAb-G (GM-2). No precipitation was observed at high ionic strength for either mutant. The molecular interactions of mutants were similar in magnitude to those of mAb-R. The results suggest that presence of hydrophobic groups in the CDR of mAb-G may be responsible for compromising its solubility at high ionic strength conditions since deleting these residues mitigated the solubility issue.
authors.affiliation: Summer Intern from Department of Pharmaceutical Sciences, University of Connecticut , Storrs, Connecticut 06269, United States.
authors.forename: Mariya
authors.initials: M
authors.lastname: Pindrus
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Shire
authors.forename: Robert F
authors.initials: RF
authors.lastname: Kelley
authors.forename: Barthélemy
authors.initials: B
authors.lastname: Demeule
authors.forename: Rita
authors.initials: R
authors.lastname: Wong
authors.forename: Yiren
authors.initials: Y
authors.lastname: Xu
authors.forename: Sandeep
authors.initials: S
authors.lastname: Yadav
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D022801:Complementarity Determining Regions; D004339:Drug Compounding; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D009994:Osmolar Concentration; D017354:Point Mutation / Q000235:genetics*; D012995:Solubility; D014783:Viscosity
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D007074:Immunoglobulin G
keywords: high concentration proteins; hydrophobic interactions; interaction parameter (kD); molecular interactions; mutant design; solubility/precipitation