Proteomic Analysis of Chinese Hamster Ovary Cells Producing Glycosylated Monoclonal Antibodies

Abstract

Therapeutic monoclonal antibodies (MAb) are produced as secreted complex glycoproteins from mammalian cell systems and represent one of the most important classes of therapeutic medicines for the treatment of a variety of human diseases. Their benefit in health care and high economic impact provide the driving force for the development of improved production levels with the focus of optimizing clinical efficacy. One important issue is the optimization of monoclonal antibody production. A frequent approach used to address this challenge is the engineering of mammalian cell lines to increase antibody production levels through genetic manipulation. Valuable information can then be obtained by monitoring the effects of genetic changes on the biochemistry of the cell associated with MAb production. Global protein expression profiling of mammalian cells used for the production of biopharmaceuticals may reveal key biochemical characteristics associated with MAb-producing cell lines. A better understanding of these characteristics can in turn lead to more rational strategies for cell line and process development.

The proposed research relates to a larger NSERC Strategic Network (MAbNet) Grant to develop and establish a novel platform for the large-scale manufacture of specific glycoforms of therapeutic monoclonal antibodies. The efficacy of these recombinant MAbs will be enhanced by the control of their glycosylation profiles. The work presented in this thesis will assist MAbNet in meeting their objectives. Specifically, we use 2D-Differential In-Gel Electrophoresis (2D-DIGE) to quantify protein expression differences between EG2-hFc1-producing Chinese Hamster Ovary cells (CHO-1A7) with its parental cell line (CHO-BRI). Here, we identified 34 unique differentially expressed proteins associated with EG2-hFc1 production that relate to various biological processes including protein processing, carbohydrate metabolism, amino acid metabolism, energy metabolism, apoptosis, and cell proliferation pathways.

The majority of identified significant protein expression changes and their associated metabolic processes seem to prioritize energy production in CHO-1A7 cells. Due to the metabolic load of recombinant antibody production, the CHO-1A7 cell line attempts to meet the energy requirements needed for recombinant protein biosynthesis while maintaining cell viability and efficient protein folding mechanisms.

A 2-D proteome reference map was also constructed for the CHO-BRI host cell line containing 131 identified protein spots. The map provides information that will further expand our understanding of this particular cell line. It will be a useful tool for studies investigating physiological responses and protein expression patterns of CHO-BRI to genetic and environmental perturbations.

The set of identified differentially expressed proteins provides data on the downstream changes in protein expression due to genetic manipulation, and furthermore can provide targets for cell-line specific optimization of antibody production. The work described in this thesis furthers our understanding of antibody production in a specific CHO cell line.