title: Spectroscopic evaluation of the stabilization of humanized monoclonal antibodies in amino acid formulations.
abstract: The protective effects of amino acids on stabilizing protein secondary structure were evaluated using diffuse reflectance FTIR spectroscopy, and interactions between proteins and arginine were detected using solid-state NMR spectroscopy. Upon freeze-drying, excipient-free anti-CD11a and anti-IgE antibodies underwent significant changes in their secondary structures. For both antibodies, the amount of intermolecular beta-sheet substantially increased and the native conformation of intramolecular beta-sheet content decreased considerably. The addition of amino acids to the formulations reduced protein secondary structure alterations in a concentration-dependent manner. Histidine and arginine appeared to be the most protective excipients (of the amino acids studied) in inhibiting protein secondary structural changes. Solid-state NMR illustrated that non-covalent interactions (e.g., hydrogen bonding, ion-dipole interactions) were formed between the arginine side chain and the protein. Glycine is the least effective additive of those studied in preventing secondary structure changes upon freeze-drying. Despite secondary structural changes, freeze-dried protein in the presence and absence of amino acids refolded back into its native conformation upon reconstitution in water.
authors.affiliation: Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, United States.
authors.forename: Fei
authors.initials: F
authors.lastname: Tian
authors.affiliation: Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, United States.
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
authors.affiliation: Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, United States.
authors.forename: Tom
authors.initials: T
authors.lastname: Offerdahl
authors.affiliation: Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, United States.
authors.forename: Eric
authors.initials: E
authors.lastname: Munson
authors.affiliation: Genentech Inc., San Francisco, CA, Unites States.
authors.forename: Samir
authors.initials: S
authors.lastname: Sane
authors.affiliation: Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, United States. Electronic address: rytting@ku.edu.
authors.forename: J Howard
authors.initials: JH
authors.lastname: Rytting
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001120:Arginine / Q000737:chemistry; D040881:CD11a Antigen / Q000276:immunology; D002626:Chemistry, Pharmaceutical; D004355:Drug Stability; D005079:Excipients / Q000737:chemistry*; D005612:Freeze Drying; D005998:Glycine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006860:Hydrogen Bonding; D007073:Immunoglobulin E / Q000276:immunology; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012995:Solubility; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013678:Technology, Pharmaceutical; D014867:Water / Q000737:chemistry
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D040881:CD11a Antigen; D005079:Excipients; D014867:Water; D007073:Immunoglobulin E; D006639:Histidine; D001120:Arginine; D005998:Glycine