A protocol to transfer a fed‐batch platform process into semi‐perfusion mode: The benefit of automated small‐scale bioreactors compared to shake flasks as scale‐down model

Abstract

in Wiley Online Library (wileyonlinelibrary.com) Continuous processes such as perfusion processes can offer advantages compared to fed-batch or batch processes in bio-processing: improved product quality (e.g. for labile products), increased product yield, and cost savings. In this work, a semi-perfusion process was established in shake flasks and transferred to an automated small-scale bioreactor by daily media exchange via centrifugation based on an existing fed-batch process platform. At first the development of a suitable medium and feed composition, the glucose concentration required by the cells and the cell-specific perfusion rate were investigated in shake flasks as the conventional scale-down system. This lead to an optimized process with a threefold higher titer of 10 g/L monoclonal antibody compared to the standard fed-batch. To proof the suitability and benefit as a small-scale model, the established semi-perfusion process was transferred to an automated small-scale bioreactor with improved pH and dissolved oxygen control. The average specific productivity improved from 24.16 pg/(c*d) in the fed-batch process and 36.04 pg/c*d in the semi-perfusion shake flask to 38.88 pg/(c*d) in the semi-perfusion process performed in the controlled small-scale bioreactor, thus illustrating the benefits resulting from the applied semi-perfusion approach, especially in combination with controlled DO and pH settings.

A class of low‐cost alternatives to kifunensine for increasing high mannose N‐linked glycosylation for monoclonal antibody production in Chinese hamster ovary cells

“…The fed‐batch shake flasks studies have shown tris as a lever for modulating high mannose glycans similarly to kifunensine and DMJ. The shake flask scale down model has limitations due to lack of pH and DO control, which may impact VCD and viability 51,52 . To confirm the impact of the inhibitors in a process scalable to a 15,000 L bioreactor, the novel inhibitors were evaluated in a 5 L bioreactor scale down model 35,53 …”

“…The shake flask scale down model has limitations due to lack of pH and DO control, which may impact VCD and viability. 51,52 To confirm the impact of the inhibitors in a process scalable to a 15,000 L bioreactor, the novel inhibitors were evaluated in a 5 L bioreactor scale down model. 35,53 3.3 | Confirmation of inhibitor effects in bioreactor scale down model Addition of 10 mM tris to CL1 had no significant impact on VCD, viability, or titer and was comparable to the control process ( Figure 7).…”

Process design and development of a mammalian cell perfusion culture in shake‐tube and benchtop bioreactors

“…The major drawback of the ST system, with respect to the bench‐scale reactors, is the lack of pH and DO control, as well as the different type of agitation. The most recent study of Janoschek et al () uses an automated and well controlled bioreactor system and appears to reproduce the bioreactor conditions in a much better way. Additionally, proper technologies, such as flux balance analysis and metabolic flux analysis, could be used to better understand the metabolic differences observed in the ST and the bench‐scale system.…”

“…The system is operated at 15 ml and shows comparable process performance to large scale reactors (Goletz, Stahn, & Kreye, ). Most recently, Janoschek, Schulze, Zijlstra, Greller, and Matuszczyk () presented a similar semi‐continuous approach in the ambr® 15 system, but instead applied centrifugation to separate cells from the culture medium, and therefore strongly reduced the time without bioreactor control compared with the sedimentation method. The approach allows the control of pH, DO, and agitation during perfusion culture and appears to be a very close representation of a fully automated perfusion reactor with cell retention system (Janoschek et al, ), but still needs further validation with bench‐scale systems.…”

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…The use of polymer-based controlled-release fed-batch microtiter plates during preculture was theoretically studied and tested with an E. coli clone bank containing 32 strains (Keil et al, 2019). The suitability of an automated smallscale bioreactor (ambr 15 with 48 wells) as a small-scale model was confirmed using a perfusion process (Janoschek et al, 2019). In another study, it was reported that an automated microbioreactor system (ambr15) can be utilized to scale down the perfusion process using cell sedimentation (as the cell retention method).…”