Analysis of extracellular vesicle and particulate production by CHO cells in culture: evaluating the risk to the efficiency of downstream purification operations

In recent years, the production of recombinant proteins and, most importantly, monoclonal antibodies, has driven the rise of productivity in the biopharmaceutical industry. This rise came with an increased demand for producing safe therapeutic bioproducts. To meet the demand, downstream process operations for removing unwanted impurities from the product are employed. The purpose of the research for this thesis is to monitor and analyse the production of extracellular vesicles (EVs), which were found to be produced in large quantities (109 per mL of cell culture) by cells, and to assess the potential risks they impose on the downstream process operations. In the first results chapter of this thesis, the concentration, size distribution and enrichment of different populations of EVs through the course of a CHO cell culture were investigated. A comparison of particle production and sample purity between a mAb-producing and a non-producing CHO cell line was performed. The results show that, although the mAb-producing cell line contained a higher concentration of particles compared to the non-producing one (5.44 ± 0.59·106, 2.26 ± 0.12·106 particles/mL respectively, at day 7 of the culture), the latter produced more particles per cell (1288.65±109, 1794.30±313 particles/ viable cell, respectively). During the analysis, it was also revealed that a high population of particles (7·109 particles/mL) was found in the EV isolation supernatant. Subsequent analysis demonstrated that this population of particles were not of EV nature, however was possibly of protein origin. In the next chapter, the production of EVs in a 3L bioreactor run, provided by AstraZeneca, was analysed. The concentration, size distribution and enrichment of EVs were compared throughout the stages of the bioreactor run and monitored through the stages of each downstream process operation. The analysis revealed that the majority of the large and small vesicles were removed by the Protein A column; however, a small amount remained in the downstream process stream (1.9±0.26·1013 particles per 20 mL sample). In the third chapter, the proteomic content of EVs was analysed using mass spectrometry and compared for any discrepancies between the EVs from the two cell lines. It was found that the non-producing cell line was more enriched in EV biomarkers (1.84, 1.81 log2 fold enrichment, respectively, for TSG101 and CD81). Finally, the extracellular vesicles were subjected to treatment with a protease to investigate the proteome of the EV membrane surface. The subsequent proteomic analysis of treated EVs showed a persistent presence of the mAb in the samples (0.688 log2 fold enrichment in the trypsinated pellet), signifying a possible connection with the EV membrane.