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Applied and Environmental Microbiology, May 2004, p. 2567-2576, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.2567-2576.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of Humanized Antibodies Secreted by Aspergillus niger

Michael Ward,1* Cherry Lin,1 Doreen C. Victoria,1 Bryan P. Fox,1 Judith A. Fox,1 David L. Wong,1 Hendrik J. Meerman,1 Jeff P. Pucci,1 Robin B. Fong,1 Meng H. Heng,1 Naoya Tsurushita,2 Christine Gieswein,2 Minha Park,2 and Huaming Wang1

Genencor International, Inc., Palo Alto, California 94304,1 Protein Design Labs, Inc., Fremont, California 945552

Received 12 September 2003/ Accepted 15 January 2004

Two different humanized immunoglobulin G1({kappa}) antibodies and an Fab' fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex6GlcNAc2 to Hex15GlcNAc2. An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function.

* Corresponding author. Mailing address: Genencor International, Inc., 925 Page Mill Rd., Palo Alto, CA 94304. Phone: (650) 846-5850. Fax: (650) 845-6509. E-mail: mward{at}genencor.com.

Applied and Environmental Microbiology, May 2004, p. 2567-2576, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.2567-2576.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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