Niraj Kumar, Dixat Gopal Gupta, Srikant Kumar, Priyanka Maurya, Ashutosh Tiwari, Babu Mathew, Shubham Banerjee, Sagarika Haldar, Jonathan Pillai, Shinjini Bhatnagar# and Susmita Chaudhuri*
Background: Chinese Hamster Ovary cells (CHO) are the most preferred host cells to meet the increasing demand for high quality ‘human-like’ complex biologics production, but is faced with the challenge of achieving high yield at an affordable price. Secreted proteins critically impact cell growth and product quality and quantity and an integral part of secretome is the packaged microvesicles. In spite of numerous efforts to characterize spent-media proteome, none have identified specific contribution of microvesicles, necessitating further differential analysis of these defined fractions of spent-media proteome, specifically packaged microvesicles. Methods: We have investigated proteome of microvesicles isolated from lag, log, stationary and death phase of CHO batch culture using LC-MS/MS based-proteomic approach to identify proteins that may be involved in regulation of cell growth, viability and productivity in culture. Results: A total of 89 unique proteins were identified in the microvesicles isolated from lag, log, stationary and death-phase of culture; of these only 8.9% were categorized as secretory proteins leaving ~91% proteins of intracellular and non-secretory nature. Microvesicles were observed to contain a number of culture phase-specific proteins which included cell-signaling molecules, transcription and translation regulators and molecular chaperons; many of which are known growth regulators, indicating the potency of microvesicles in regulating culture health. Conclusions: This is the first report of CHO microvesicular proteome and this knowledge is critical in developing rationale design of perfusion process, downstream purification process for rendering improved product stability and also novel cell engineering approaches to maximize growth and improved media formulations to maximize yield and minimize product degradation.
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