Spectroscopic evaluation of the stabilization of humanized monoclonal antibodies in amino acid formulations
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- 作者：Fei Tian ; C. Russell Middaugh ; Tom Offerdahl ; Eric Munson ; Samir Sane ; J. Howard Rytting
- 关键词：Antibodies ; Protein ; FTIR ; NMR ; Stabilization ; Amino acids ; Histidine ; Arginine ; Glycine
- 刊名：International Journal of Pharmaceutics
- 出版年：2007
- 出版时间：20 April 2007
- 年：2007
- 卷：335
- 期：1-2
- 页码：20-31
- 全文大小：535 K
文摘
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 β-sheet substantially increased and the native conformation of intramolecular β-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.