Journal of Biochemistry Advance Access originally published online on July 23, 2007
Journal of Biochemistry 2007 142(3):383-391; doi:10.1093/jb/mvm145
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© 2007 The Japanese Biochemical Society.
Effect of Buffer Species on the Unfolding and the Aggregation of Humanized IgG
1Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582; and 2Formulation Technology Research Department, Chugai Pharmaceutical Co. Ltd., Tokyo 115-8543, Japan
To whom correspondence should be addressed. Tel: +81-92-642-6662, Fax: +81-92-642-6667, E-mail: ueda{at}phar.kyushu-u.ac.jp
Received May 23, 2007; Accepted June 27, 2007
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Abstract |
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The aggregation propensity of humanized antibody after heat treatment is evaluated in the presence of six buffer species. The comparison under equivalent pH showed high aggregation propensity on phosphate and citrate buffer. In contrast, 2-(N-Morpholino) ethane sulfonate (MES), 3-(N-Morpholino) propane sulfonate (MOPS), acetate and imidazole buffer showed lower aggregation propensity than the above two buffers. Meanwhile, unfolding temperature evaluated by differential scanning calorimetry measurement was not altered among these buffer species. The light scattering analysis suggested that heat-denatured intermediate was aggregated slightly on MES and acetate buffer. Therefore, it was found that the different aggregation propensity among buffer species was caused from the aggregation propensity of heat-denatured intermediate rather than the unfolding temperature. Furthermore, it was revealed that the aggregation dependency on buffer species is accounted for by the specific molecular interaction between buffer and IgG, rather than the ionic strength. On the contrary, on the analyses of unfolding and aggregation propensity by molecular dissection of IgG into Fab and Fc fragments, aggregation propensity of Fc fragment on MES, acetate and phosphate buffer was almost the same as whole IgG. From the above results, it was suggested that the specific interaction between buffer molecule and Fc domain of IgG was involved in the aggregation propensity of heat-denatured IgG.
Key Words: buffer, DSC, humanized antibody, protein aggregation, protein stabilization
Abbreviations: ANS, 8-Anilino-1-naphthalene sulfonate; DSC, differential scanning calorimetry; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MES, 2-(N-Morpholino) ethane sulfonate; MOPS, 3-(N-Morpholino) propane sulfonate; PAGE, polyacrylamide gel electrophoresis; SDS, Sodium dodecyl sulfate
*Present address: Faculty of Biotechnology and Life Science, Sojo University, Kumamoto 860-0082, Japan