Ghent University Academic Bibliography

Advanced

Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris

Steve Schoonooghe UGent, Vladimir Kaigorodov, Monika Zawisza UGent, Caroline Dumolyn, Jurgen Haustraete UGent, Johan Grooten UGent and Nico Mertens UGent (2009) BMC BIOTECHNOLOGY. 9. p.AR70-1-AR70-14
abstract
Background: Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic P. pastoris yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs. Results: Based on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers. These recombinant antibody derivatives were efficiently expressed in both mammalian and P. pastoris cells. Stable production in NS0 cells produced 130.5 mg pure bibody and 27 mg pure tribody per litre. This high yield is achieved as a result of the high overall purification efficiency of 77%. Expression and purification of PH1 bibodies and tribodies from Pichia supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers. The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts. Additionally, the yields of 36.8 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in Pichia more efficient than most other previously described trispecific or trivalent molecules produced in E. coli. Conclusion: Bi- and tribody molecules are efficiently produced in P. pastoris. Furthermore, the yeast produced molecules retain the same specific affinity for their antigen. These results establish the value of P. pastoris as an efficient alternative expression system for the production of recombinant multivalent Fab-scFv antibody derivatives.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
IN-VITRO, FRAGMENT, RECOMBINANT ANTIBODY, MONOCLONAL-ANTIBODIES, BISPECIFIC ANTIBODY, HIGH-EXPRESSION, CELL, LARGE-SCALE, CANCER, MUC1
journal title
BMC BIOTECHNOLOGY
BMC Biotechnol.
volume
9
pages
14 pages
publisher
BIOMED CENTRAL LTD
place of publication
LONDON
Web of Science type
Article
Web of Science id
000270293500001
JCR category
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
JCR impact factor
2.723 (2009)
JCR rank
48/150 (2009)
JCR quartile
2 (2009)
ISSN
1472-6750
DOI
10.1186/1472-6750-9-70
language
English
UGent publication?
yes
classification
A1
copyright statement
I don't know the status of the copyright for this publication
id
787978
handle
http://hdl.handle.net/1854/LU-787978
date created
2009-11-23 16:58:48
date last changed
2017-01-02 09:55:56
@article{787978,
  abstract     = {Background: Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic P. pastoris yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs.

Results: Based on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers. These recombinant antibody derivatives were efficiently expressed in both mammalian and P. pastoris cells. Stable production in NS0 cells produced 130.5 mg pure bibody and 27 mg pure tribody per litre. This high yield is achieved as a result of the high overall purification efficiency of 77\%. Expression and purification of PH1 bibodies and tribodies from Pichia supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers. The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts. Additionally, the yields of 36.8 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in Pichia more efficient than most other previously described trispecific or trivalent molecules produced in E. coli.

Conclusion: Bi- and tribody molecules are efficiently produced in P. pastoris. Furthermore, the yeast produced molecules retain the same specific affinity for their antigen. These results establish the value of P. pastoris as an efficient alternative expression system for the production of recombinant multivalent Fab-scFv antibody derivatives.},
  author       = {Schoonooghe, Steve and Kaigorodov, Vladimir and Zawisza, Monika and Dumolyn, Caroline and Haustraete, Jurgen and Grooten, Johan and Mertens, Nico},
  issn         = {1472-6750},
  journal      = {BMC BIOTECHNOLOGY},
  keyword      = {IN-VITRO,FRAGMENT,RECOMBINANT ANTIBODY,MONOCLONAL-ANTIBODIES,BISPECIFIC ANTIBODY,HIGH-EXPRESSION,CELL,LARGE-SCALE,CANCER,MUC1},
  language     = {eng},
  pages        = {AR70-1--AR70-14},
  publisher    = {BIOMED CENTRAL LTD},
  title        = {Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris},
  url          = {http://dx.doi.org/10.1186/1472-6750-9-70},
  volume       = {9},
  year         = {2009},
}

Chicago
Schoonooghe, Steve, Vladimir Kaigorodov, Monika Zawisza, Caroline Dumolyn, Jurgen Haustraete, Johan Grooten, and Nico Mertens. 2009. “Efficient Production of Human Bivalent and Trivalent anti-MUC1 Fab-scFv Antibodies in Pichia Pastoris.” Bmc Biotechnology 9: AR70–1–AR70–14.
APA
Schoonooghe, S., Kaigorodov, V., Zawisza, M., Dumolyn, C., Haustraete, J., Grooten, J., & Mertens, N. (2009). Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris. BMC BIOTECHNOLOGY, 9, AR70–1–AR70–14.
Vancouver
1.
Schoonooghe S, Kaigorodov V, Zawisza M, Dumolyn C, Haustraete J, Grooten J, et al. Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris. BMC BIOTECHNOLOGY. LONDON: BIOMED CENTRAL LTD; 2009;9:AR70–1–AR70–14.
MLA
Schoonooghe, Steve, Vladimir Kaigorodov, Monika Zawisza, et al. “Efficient Production of Human Bivalent and Trivalent anti-MUC1 Fab-scFv Antibodies in Pichia Pastoris.” BMC BIOTECHNOLOGY 9 (2009): AR70–1–AR70–14. Print.