Development of perfusion cell culture processes for the manufacturing of therapeutic recombinant proteins
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Author
Date
2019Type
- Doctoral Thesis
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Abstract
Clinical and commercial success of therapeutic proteins has been ever increasing in the past decades. New product classes are emerging while biosimilars, the equivalent of small molecule generics, have entered the market in recent years. Pipelines are growing and the pressure coming from the market is constantly increasing. Manufacturers are therefore challenged to simultaneously increase their production capacity but also to reduce costs. To be more reactive towards the market's rapid fluctuations, technological solution must be implemented to intensify manufacturing capacities and increase their flexibility. Continuous technologies are typically applied for that purpose.
The work presented in this thesis was performed in an industrial environment that is in transition from a well established batch-like production platform, to continuous manufacturing and focuses more specifically on perfusion cell cultures. Chapter 1 describes the context in more detail and is followed by Chapter 2, which is a literature review on the subject. In that chapter, perfusion processes are described in detail. Definitions are given for important process parameters, such as the perfusion rate (P) and the cell specific perfusion rate (CSPR), that are used to describe perfusion operation. Different applications of this technology are then discussed. Process development, process control, medium development and scale-down models are other topics that are reviewed in this chapter. This work was necessary to understand the current state of these technologies and helped to develop the experimental approaches of the following chapters.
This review process highlighted a limitation for perfusion process development that is made difficult by the limited availability of appropriate scale-down models. This is due to the continuous operation that requires complex control and cell retention capacity. For example, the determination of an optimal perfusion and bleed rate for continuous cell culture is often performed in scale-down bioreactors and requires a substantial amount of time and effort. To increase the experimental throughput and decrease the required workload, a semi-continuous procedure, referred to as the VCDmax (viable cell density) approach, has been developed on the basis of shake tubes (ST) and deepwell plates (DWP) and is described in Chapter 3. Its effectiveness has been demonstrated for 12 different CHO-K1-SV cell lines expressing a monoclonal antibody (mAb). Further, its reliability has been investigated through proper comparison with perfusion runs in lab-scale bioreactors. It was found that the volumetric productivity and the CSPRmin (cell specific perfusion rate) determined using the ST and 96-DWP models were successfully (mostly within the experimental error) confirmed in lab-scale bioreactors, which then covered a significant scale-up from the half milliliter to the liter scale. These scale-down models are very useful to design and scale-up optimal bioreactor operating conditions as well as screening for different media and cell lines.
In Chapter 4, a perfusion process for the manufacturing of a conjugated protein was developed and compared to other batch-like processes, both in terms of performance and quality. Perfusion was used as the production stage but also to increase the inoculation density for a so-called high seeding fed-batch process. This chapter reports a unique comparison of low (LS) and high (HS) seeding fed-batch bioreactors, corresponding to traditional and intensified operation using perfusion at the N-1 stage, respectively, with perfusion (PF) bioreactors, using a bispecific conjugated fusion protein as a model. It is found that the gain in daily volumetric productivity compared to the traditional LS fed-batch, increases by a factor 3 with HS and 7 with PF. Critical quality attributes (CQAs) also benefited from the perfusion operation. In particular, levels of clipping, that is the fragmentation of the fusion protein, are significantly reduced compared to both fed-batch operations. In PF the clipping varied between 0.6 and 1.5% while in the LS and HS it reached up to 8.7 and 4.9%, respectively. Aggregate levels were also decreased using PF, while the charge variant distribution was more homogeneous and the glycosylation pattern was also significantly affected. The comparison of LS, HS and PF for the manufacturing of a bispecific conjugated fusion protein reported here highlight some productivity and quality benefits inherent to the nature of continuous processing. Show more
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https://doi.org/10.3929/ethz-b-000368475Publication status
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Contributors
Examiner: Morbidelli, Massimo
Examiner: Guillén Gosálbez, Gonzalo
Examiner: Souquet, Jonathan
Publisher
ETH ZurichSubject
Mammalian cell culture; Perfusion cell culture; Recombinant protein productionOrganisational unit
03451 - Morbidelli, Massimo (emeritus) / Morbidelli, Massimo (emeritus)
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