title: Correlating the Effects of Antimicrobial Preservatives on Conformational Stability, Aggregation Propensity, and Backbone Flexibility of an IgG1 mAb.
abstract: Multidose formulations of biotherapeutics, which offer better dosage management and reduced production costs, require the addition of antimicrobial preservatives (APs). APs have been shown, however, to decrease protein stability in solution and cause protein aggregation. In this report, the effect of 4 APs, m-cresol, phenol, phenoxyethanol, and benzyl alcohol on conformational stability, aggregation propensity, and backbone flexibility of an IgG1 mAb, mAb-4, is investigated. Compared with no preservative control, each of the APs decreased the conformational stability of mAb-4 as measured by differential scanning calorimetry and extrinsic fluorescence spectroscopy. The addition of APs resulted in increased monomer loss and aggregate accumulation at 50°C over 28 days, as monitored by size-exclusion chromatography. The extent of conformational destabilization and protein aggregation of mAb-4 induced by APs followed their calculated octanol-water partition coefficients. Increases in backbone flexibility, as measured by hydrogen exchange, of a region located in the CH2 domain of the mAb (heavy chain 237-254) in the presence of APs also correlated with hydrophobicity. Based on these results, the destabilizing effect of APs on mAb-4 correlates with the increased hydrophobicity of the APs and their ability to enhance the local backbone flexibility of an aggregation hot spot within the CH2 domain of the mAb.
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047; Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas 66047.
authors.forename: Jayant
authors.initials: J
authors.lastname: Arora
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047; Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas 66047.
authors.forename: Sangeeta B
authors.initials: SB
authors.lastname: Joshi
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047; Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas 66047.
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047; Department of Chemistry and R.N. Adams Institute of Bioanalytical Chemistry, University of Kansas, Lawrence, Kansas 66045. Electronic address: dweis@ku.edu.
authors.forename: David D
authors.initials: DD
authors.lastname: Weis
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047; Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas 66047. Electronic address: volkin@ku.edu.
authors.forename: David B
authors.initials: DB
authors.lastname: Volkin
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D019905:Benzyl Alcohol / Q000737:chemistry; D003408:Cresols / Q000737:chemistry; D005026:Ethylene Glycols / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D008958:Models, Molecular; D019800:Phenol / Q000737:chemistry; D011310:Preservatives, Pharmaceutical / Q000737:chemistry*; D066329:Protein Aggregates / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects
chemical_list: D000890:Anti-Infective Agents; D000911:Antibodies, Monoclonal; D003408:Cresols; D005026:Ethylene Glycols; D007074:Immunoglobulin G; D011310:Preservatives, Pharmaceutical; D066329:Protein Aggregates; D019800:Phenol; C042041:3-cresol; C005398:phenoxyethanol; D019905:Benzyl Alcohol
keywords: antimicrobial agents; formulation; hydrogen exchange mass spectrometry; local flexibility; mAb; multidose formulations; preservatives; protein aggregation; protein stability