Determination of CHO Biomass Composition

Chinese hamster ovary (CHO) cells are the primary host organism for the production of protein biopharmaceuticals. Significant improvements in product yield and cell growth were achieved in the past years by bioprocess and media optimization, directed evolution and targeted genetic engineering. However, a deeper understanding of the underlying processes in the cells is still limited. Recently, a CHO-specific genome scale metabolic model was created in a large community effort. This model is a comprehensive resource of CHO metabolism. Using COBRA methods, we are now starting to get valuable insights into the cells’ metabolism, their protein production capabilities and potential limitations. One essential input for the model is biomass composition. It has been shown that using strain and condition specific biomass together with bioprocess data improves the model’s predictions. Currently, however, the model uses estimates and literature values, since comprehensive data about CHO cell composition, specifically of individual cell lines or strains, are lacking. In this work, methods for the determination of CHO biomass composition (proteins and amino acids, lipids, DNA, RNA and cell dry mass) were established. These include chromatography and mass spectrometry determination of amino acids, fluorimetric and spectrophotometric quantification of nucleic acids and gravimetric quantification of cell dry mass. For the first time, biomass compositions of various CHO host and producer cell lines in exponential phase were accurately characterized in media with or without glutamine. The goal was to assess variability of the biomass composition among different strains and conditions to see which components are the most variable and therefore should be measured every time and which could be generalized for all CHO cells.