Study of glycosylation of Antibodies produced in serum-free medium

Glycosylation plays a key role in the biological activity of recombinant antibodies (Ab) and their oligosaccharide profile depends on culture conditions. In order to optimize production of monoclonal antibodies in cells grown in serum-free medium and to obtain conform products, characterization of glycosylation of these proteins is needed. Indeed, changes in glycosylation profile can induce a lack of antigen recognition or an inappropriate host response.
In this context, four chemically defined media have been chosen to produce recombinant antibodies. The amount of serum in each culture medium has been decreased progressively to allow adaptation of cells to serum-free conditions. The goal of this study is the analysis of potential changes in the glycosylation profile of antibodies resulting from both the adaptation process of the cells to their serum-free medium and the cell culture age. Data were collected on mouse and human antibodies, respectively.
The first approach was to study the whole protein (here the heavy chains of Ab) by ESI-Q-TOF in order to quickly determine the profiles of glycosylation. Subsequently, we focused the experiments on some samples to determine more precisely the structures of the glycans. In this case, the glycans were extracted, permethylated and then analyzed by MALDI-TOF. Experiences of MS/MS were also allowed to highlight some of these structures. Finally, the location of the glycosylated part of protein was performed using mass spectrometry in tandem CID-ETD.
By combining the currently available results, it appears that, in our cell culture conditions, the glycosylation of antibodies produced in serum-free media is generally close to that obtained for proteins produced in the media containing serum. The main species are indeed identical. In addition, fucosylation is stable in the different samples analyzed (80 to 90% of the glycans contain a fucose). Nevertheless, some differences may appear on the presence and/or the nature of sialic acids found. Regarding the location of the glycosylated parts, the results show that there is only one site on the glycans and that it remains the same in the different conditions tested.