Volume 7 Supplement 6
Chemical chaperone suppresses the antibody aggregation in CHO cell culture
© Onitsuka et al.; licensee BioMed Central Ltd. 2013
Published: 4 December 2013
Aggregation of therapeutic antibodies could be generated at different steps of the manufacturing process, posing the problem for quality control of produced antibodies. It has been well known that secreted antibodies from recombinant mammalian cells into culture medium can aggregate due to the physicochemical stresses such as media pH and osmolality, cultivation temperature [1, 2]. The antibody aggregation during the cell culture process is difficult to suppress because the cell culture conditions for antibody production are generally optimized for cell culture and growth and not for suppressing the aggregate formation. Here we show the novel strategy to suppress the antibody aggregation; application of chemical chaperone to the cell culture process. It is well established that an addition of some cosolutes serves as chemical chaperone to suppress the protein aggregation. Trehalose, non-reducing sugar formed from two glucose units with α-1,1 linkage, is known as an effective chemical chaperone. In this study, we investigated the anti-aggregation effect of trehalose in the culture process of recombinant Chinese hamster ovary cell (CHO) line producing Ex3-humanized IgG-like bispecific single-chained diabody with Fc (Ex3-scDb-Fc). Ex3-scDb-Fc shows the remarkable anti-tumor activity based on anti-EGFR and anti-CD3 bispecificity . However, our in-house results showed that Ex3-scDb-Fc shows aggregation tendency, demonstrating the necessity of developing a bioprocess for suppressing the aggregation of the bispecific diabody.
Materials and methods
CHO Top-H cell line producing the Ex3-scDb-Fc  was cultivated in 500mL Erlenmeyer flask and 2L-glass bioreactor with serum-free medium containing 150mM trehalose. Viable cell densities and antibody concentrations were determined with Vi-Cell XR™ cell viability analyzer (Beckman Coulter) and by ELISA, respectively. Ex3-scDb-Fc was purified with Hi-Trap protein A column (GE Healthcare). 1M Arg-HCl (pH4.2) was used as eluting solution, which make it possible to prevent the aggregation of the antibody in the affinity purification process. Antibody aggregation was analyzed by sephacryl S-300 column (GE healthcare). Solution structure of Ex3-scDb-Fc was assessed by circular dichroism spectroscopy.
Results and discussion
Cell culture performance in trehalose containing medium
Kinetic parameters of cell culture in Erlenmeyer flasks.
Specific growth rate
(μ; ×10-2 1/h)
antibody production rate
3.07 ± 0.18 a
0.39 ± 0.02 a
1.51 ± 0.04 a
1.55 ± 0.03 a
Anti-aggregation effects by trehalose during the cell culture process
We demonstrated the potential application of chemical chaperon in the culture of antibody-producing mammalian cells. Trehalose can be incorporated in the culture media for CHO cells, and can suppress the antibody aggregation, especially high-order aggregates. In addition, trehalose may be involved in the enhancement of antibody production.
This study was supported by the Advanced research for medical products Mining Programme of the National Institute of Biomedical Innovation (NIBIO). Trehalose was kindly supplied by HAYASHIBARA Biochemical Laboratories, Inc. (Okayama, Japan). This work was collaboration with Assoc. Prof. Ryutaro Asano and Prof. Izumi Kumagai (Tohoku University, Japan).
- Cromwell ME, Hilario E, Jacobson F: Protein aggregation and bioprocessing. AAPS J. 2006, 8: E572-579.PubMed CentralView ArticlePubMedGoogle Scholar
- Vázquez-Rey M, Lang DA: Aggregates in monoclonal antibody manufacturing processes. Biotechnol Bioeng. 2011, 108: 1494-1508.View ArticlePubMedGoogle Scholar
- Asano R, Kawaguchi H, Watanabe Y, Nakanishi T, Umetsu M, Hayashi H, Katayose Y, Unno M, Kudo T, Kumagai I: Diabody-based recombinant formats of humanized IgG-like bispecific antibody with effective retargeting of lymphocytes to tumor cells. J Immunother. 2008, 31: 752-761.View ArticlePubMedGoogle Scholar
- Onitsuka M, Kim WD, Ozaki H, Kawaguchi A, Honda K, Kajiura H, Fujiyama K, Asano R, Kumagai I, Ohtake H, Omasa T: Enhancement of sialylation on humanized IgG-like bispecific antibody by overexpression of α2,6-sialyltransferase derived from Chinese hamster ovary cells. Appl Microbiol Biotechnol. 2012, 94: 69-80.View ArticlePubMedGoogle Scholar
- Rodriguez J, Spearman M, Huzel N, Butler M: Enhanced production of monomeric interferon-beta by CHO cells through the control of culture conditions. Biotechnol Prog. 2005, 21: 22-30.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.