Analysis of protein glycosylation of recombinant proteins produced in Chinese Hamster ovary cells on a single cell level

Many proteins produced by the biotechnology industry in recombinant cells are glycoproteins, which require their carbohydrate part for optimal biological function and to avoid an immune response or the premature clearance of the protein from the blood stream. Examples are cytokines, interferon, antibodies, erythropoietin and other therapeutics. Such proteins have to be produced in mammalian cells, as bacteria are not able to add carbohydrate side chains to proteins, while yeast produce only a primitive type of glycosylation. Several parameters were described that influence the correct glycosylation of such proteins, among them culture conditions, the cell line and species used, the overall production rate and others. So far the correct glycosylation of proteins was analysed from the culture supernatant with sophisticated techniques such as HPLC and MALDI-TOF. Typically a pattern of several forms of glycosylation is found, which may change with culture conditions. These different forms of glycosylation, however, can not be traced back to the status of the individual cell that produced them. With a new technique, that allows the association of a product with the cell that secreted it we are now able to establish a connection between the state of the cell and the glycosylation pattern of the proteins produced by this individual cell. We plan to establish a method that will analyse the quality of carbohydrate side chain sequences on secreted proteins on individual, live cells by Flow Cytometry. This method can then be used to analyse the individual cell status in correlation to glycosylation, to analyse different subclones as well as cells in bioreactors during varying culture conditions. The goal of this part of the project is the identification of parameters than need to be kept constant to allow optimal glycosylation. In a second part we want to select cells that have an improved capacity for correct glycosylation from a population of cells by flow cytometric cell sorting, so as to optimise the cell line producing a glycoprotein at high amounts. This holistic approach seems more promising than the transfection of single glycosylation enzymes, which will influence only a single step in this multistep process.