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J. Biol. Chem., Vol. 280, Issue 50, 41494-41503, December 16, 2005

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Novel Antibody Hinge Regions for Efficient Production of C_H2 Domain-deleted Antibodies^*

Scott M. Glaser1, Ina E. Hughes, Jennifer R. Hopp, Karen Hathaway, Daniel Perret, and Mitchell E. Reff

From the Department of Protein Engineering and the Department of Protein Chemistry, Biogen Idec, Inc., San Diego, California 92122

HuCC49CH2 is a heavy chain constant domain 2 domain-deleted antibody under development as a radioimmunotherapeutic for treating carcinomas overexpressing the TAG-72 tumor antigen. Mammalian cell culture biosynthesis of HuCC49CH2 produces two isoforms (form A and form B) in an approximate 1:1 ratio, and consequently separation and purification of the desired form A isoform adversely impact process and yield. A protein engineering strategy was used to develop a panel of hinge-engineered HuCC49CH2 antibodies to identify hinge sequences to optimize production of the form A isoform. We found that adding a single proline residue at Kabat position 243, immediately adjacent to the carboxyl end of the core middle hinge CPPC domain, resulted in an increase from 39 to 51% form A isoform relative to the parent HuCC49CH2 antibody. Insertion of the amino acids proline-alanine-proline (PAP) at positions 243-245 enhanced production of the form A isoform to 72%. Insertion of a cysteine-rich 15-amino acid IgG3 hinge motif (CPEPKSCDTPPPCPR) in both of these mutant antibodies resulted in secretion of predominantly form A isoform with little or no detectable form B. Yields exceeding 98% of the form A isoform have been realized. Preliminary peptide mapping and mass spectrometry analysis suggest that at least two, and as many as five, inter-heavy chain disulfide linkages may be present.

Received for publication, August 9, 2005 , and in revised form, October 3, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Biogen Idec, Inc., 5200 Research Place San Diego, CA 92122. Tel.: 858-401-8645; Fax: 858-401-8714; E-mail: scott.glaser{at}biogenidec.com.

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