Understanding the role of C-terminal Lysine in protein secretory pathways

Mammalian cell lines are ubiquitous in their use as hosts for the industrial production of numerous therapeutic proteins. In particular, Chinese Hamster Ovary (CHO) cells are widely used for large-scale production since they are capable of folding, assembling and applying appropriate post-translational modifications to proteins, ensuring their compatibility with humans. Despite the recent publication of the CHO cell genome, there is currently a knowledge gap around the factors which can influence antibody titre, product quality and safety associated Post Translational Modifications (PTM). This limited understanding is predominantly due to the multiple intracellular compartments present in CHO cells and also the diverse enzymatic process involved in product expression. In addition, the majority of recombinant monoclonal antibodies (mAbs) produced in CHO cells, contain heterogeneous variants. These variants are commonly the result of modifications that occur during CHO cell culture production, through enzymatic processes, and can accumulate during production, purification and storage.

Charge variants represent a source of heterogeneity in the majority of mAbs. In particular, the processing and cleavage of C-terminal lysine from antibodies is one of the most commonly detected sources of product charge-heterogeneity. Generally, lysine residues at the heavy-chain C-terminus of recombinant IgGs are removed during cell culture by carboxypeptidases that are endogenous to CHO host cells. However incomplete cleavage of the lysine residues is typically observed and this can lead to the presence of more positively charged mAb variants which cannot be separated from the fully processed product. This not only increases the final product heterogeneity, concomitantly decreasing the batch-to-batch product consistency, but can also potentially affect pharmacokinetics after subcutaneous injection, as an increase in net positive charge generally results in increased tissue retention and increased blood clearance. It is also known now that an incomplete cleavage of lysine residues can also lead to a suboptimal antibody induced complement activation.

Specifically, this proposal will first involve the engineering of a panel of mammalian cell lines to express a variety of proteins with and without C-terminal lysines. Thereafter this project will then leverage expertise in the Centre of Excellence in Biopharmaceuticals (www.coebp.ls.manchester.ac.uk) at the University of Manchester and their newly opened, state-of-the-art Imaging Centre (www.systemsmicroscopycentre. ls.manchester.ac.uk) to further understand how the addition or removal of such C-terminal lysines then impacts spatial, temporal, quantitative and qualitative processing of the proteins produced. In turn it is envisioned this research will result in a better understanding of basic protein cell trafficking and secretion and specifically the role played by C-terminal lysine therein. Additionally, the generic implications of this work extend to (a) embedding technologies that can be applied to the pipeline of optimisation of secretion of novel format biological in CHO cell and (b) illuminate opportunities for bioprocess engineering of enhanced protein expression platforms of the future.