Many recombinant therapeutic proteins such as monoclonal antibodies are produced in eukaryotic cells to enable post-translational modifications such as glycosylation. Glycan profiles and charge variants are important quality attributes for the activity, efficacy, and safety of therapeutic proteins.
To meet these parameters highly efficient and targeted media development is crucial. Whereas systematic approaches such as QbD are increasingly applied, a deeper understanding of the interaction between metabolic alterations, protein production, and processing should significantly improve production processes (Figure 1).
Metabolomics is a valuable tool to study underlying mechanisms and involved biochemical pathways—especially for the widely used CHO cell line, for which little is known about its physiology.
In this tutorial we will show how we evaluated the potential of metabolomic tools to support cell-line characterization and subsequent process development, this information was also recently presented at the ESACT meeting in Vienna. As part of the study, we compared a CHO producer and its corresponding mock cell line (transfected with an empty plasmid) on a metabolic level in order to see whether the metabolite profile differences came from the increased protein production capacity. In addition, we were able to observe the complex glycosylation machinery.
Sample-Prep Optimization
First, studies dealing with optimization of sample preparation—more precisely the sampling procedure called quenching—were conducted. This step is responsible for the mandatory immediate stopping of enzymatical reactions in order to preserve the intracellular metabolite environment and can strongly affect subsequent results.
We developed a simple and straightforward protocol, with no need for special equipment, that focuses on the efficiency of stopping metabolic activities and the potential metabolic leakage due to the loss in membrane integrity.
Within this protocol, it is recommended that cells are quenched and washed simultaneously to keep sampling time to a minimum and to prevent further metabolic activity within the cells. Studies also showed that by using this procedure additional washing steps can be omitted. A detailed description of the studies will be published elsewhere. An overview of the key steps of the implemented sampling procedure, which allows simultaneous quenching of a large number of samples, is illustrated in Figure 2.