Characterization of humanized antibodies secreted by Aspergillus niger.
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 Hex(6)GlcNAc(2) to Hex(15)GlcNAc(2). 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.
Ward
Michael
M
Genencor International, Inc, Palo Alto, California 94304, USA. mward@genencor.com
Lin
Cherry
C
Victoria
Doreen C
DC
Fox
Bryan P
BP
Fox
Judith A
JA
Wong
David L
DL
Meerman
Hendrik J
HJ
Pucci
Jeff P
JP
Fong
Robin B
RB
Heng
Meng H
MH
Tsurushita
Naoya
N
Gieswein
Christine
C
Park
Minha
M
Wang
Huaming
H
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D001234:Aspergillus niger / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D005087:Glucan 1,4-alpha-Glucosidase