Perfusionsodling av CHO celler för produktion av monoklonala antikroppar med biofarmacentisk tillämpning
Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesisAlternative title
Perfusion in Chinese Hamster Ovary cell cultivation producing monoclonal antibody for biopharmaceutical bioprocess application (English)
A recent upstream trend in the biopharmaceutical industry is that the perfusion mode of operation tends to gain interest as relative to batch and fed-batch modes. Major advantages of perfusion are high cell numbers and high total productivity in a relatively small size bioreactor. Moreover, perfusion is optimal when the product of interest is unstable or if the cell line product yield is low. On the other hand, disadvantages are for example technical challenges originating from non-robust cell separation devices as well as sterility concerns from the more complex set-up needed.
In the present work, the use of a WAVE Bioreactor™ system 20/50 in perfusion mode with disposable hollow fibre filters as external cell separating devices was investigated. A comparison between alternating tangential flow (ATF) and tangential flow (TFF) filtration was performed using a recombinant CHO cell line producing an IgG as a model system. Kinetics (growth, consumption of glucose, glutamine and production of lactate, ammonia and monoclonal antibody) were determined during the perfusion cultures and differences between the ATF and TFF filtration modes were studied. Antifoam addition was performed to counter-act excessive bubble formation resulting from the wave motion. Four perfusion cultures using the ATF system and two using the TFF system were performed during the project. Cell densities around 20 MVC/mL and viability above 95% were kept in both systems for prolonged periods of time (more than three weeks) by performing regularly bleeds, daily if required. Stable systems using both filtration devices were achieved and high CHO cell densities were reached, up to 104 million cells per mL (MVC/mL) in the TFF system and 45 MVC/mL in the ATF system. The high cell densities could be maintained at high viability for more than one week. Due to technical problem, the ATF system could not be fully explored and lower cell density was reached compared to the TFF system. High product retention could be observed in both retention devices; the TFF system (up to 90%) withheld the product to a higher extent compared to the ATF system (up to 50%). A hypothesis to explain the high retention was the relatively low flow rate in the filter of both systems. The low shear rate allowed the building up of a secondary layer of cell debris on the membrane. A back flush is automatically created in the ATF system which gives rise to less product retention.
In summary, proof-of-concept experiments for perfusion cultivation of CHO cells in a WAVE Bioreactor™ system with external hollow fibre filters as cell separating devices have been successfully performed. The use of a disposable bioreactor equipped with a disposable separation device offers a solution alleviating technical and sterility challenges occurring in perfusion processes.
Place, publisher, year, edition, pages
Engineering and Technology
IdentifiersURN: urn:nbn:se:kth:diva-49119OAI: oai:DiVA.org:kth-49119DiVA: diva2:459272
Master of Science in Engineering - Biotechnology
Chotteau, Veronique, Dr
Veide, Andres, Dr