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Journal of Biochemistry Advance Access published online on July 23, 2007
Journal of Biochemistry, 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

Daisuke Kameoka{ddagger},§, Etsuro Masuzaki{ddagger},§, Tadashi Ueda{ddagger} and Taiji Imoto{ddagger},¶

{ddagger}Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan; §Formulation Technology Research Dept., Chugai Pharmaceutical Co. Ltd., Tokyo 115-8543, Japan;

Correspondence and reprint requests to:Tadashi Ueda, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-3-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, 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


  Abstract

The aggregation propensity of humanized antibody after heat treatment is evaluated in the presence of 6 buffer species. The comparison under equivalent pH showed high aggregation propensity on phosphate and citrate buffer. In contrast, MES, MOPS, acetate and imidazole buffer showed lower aggregation propensity than the above 2 buffers. Meanwhile, unfolding temperature evaluated by DSC 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

¶Present address: Faculty of Biotechnology and Life Science, Sojo University, Kumamoto 860-0082, Japan


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