Mixing four Cell lines to produce an Antibody cocktail as a Next-generation antivenom - a Case study with a “mixed” Outcome

Snakebite envenoming is a neglected tropical disease that is treated with antivenom based on polyclonal antibodies derived from the plasma of hyperimmunized animals, such as horses or sheep. Antivenoms derived through this method can be health-threatening to patients due to their heterologous nature, including the foreign glycan structures that are attached to the antibodies. Additionally, they contain irrelevant antibodies that do not target snake toxins. In this thesis, we generated a mixture of four antibodies of human origin targeting different snake toxins to provide a proof of concept that next-generation antivenoms based on recombinant oligoclonal antibodies are manufacturable. Such recombinant antivenoms are composed of relevant antibodies only and are safe for use in humans when they are manufactured using the right host cells. Here, we use stable CHO cell lines due to their ability to attach more appropriate glycan structures to the antibodies. Instead of producing the antibodies in separate batches, we carefully mixed the cell lines in an oligoclonal fashion, as this approach holds the potential to significantly reduce upstream, downstream, and validation costs in an industrial setting. We show that the cell lines generated by targeted integration of the antibody genes exhibit similar growth, consumption, and byproduct formation patterns. Furthermore, we show that mixtures with two different cell line ratios are reasonably stable, indicating that cell lines can be mixed in several ways to manipulate the product outcome. When changing medium and cell cultivation conditions, cell line ratio divergence increases with increasing stress on the cells. The findings show that next-generation antivenoms can be produced in cell line mixtures and stresses the importance of characterizing cells in different conditions and keeping these as favorable as possible. The divergence of cell lines is consistent and follows a pattern, making it possible to account for by mixing cell lines in different ratios. Testing binding of the antibody cocktail produced in a cell line mixture showed that the antibodies are still functional and, thus, that oligoclonal antibodies could be an attractive solution for creating next-generation antivenom. As antibody cocktails are being used in other fields, such as cancer therapy or in the fight against infectious diseases, the manufacturing approach developed in this study could find even broader utility beyond snakebite envenoming.