Spectroscopic evaluation of the stabilization of humanized monoclonal antibodies in amino acid formulations.
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.
Tian
Fei
F
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, United States.
Middaugh
C Russell
CR
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, United States.
Offerdahl
Tom
T
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, United States.
Munson
Eric
E
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, United States.
Sane
Samir
S
Genentech Inc., San Francisco, CA, Unites States.
Rytting
J Howard
JH
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, United States. Electronic address: rytting@ku.edu.
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
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D040881:CD11a Antigen; D005079:Excipients; D014867:Water; D007073:Immunoglobulin E; D006639:Histidine; D001120:Arginine; D005998:Glycine