Optimising Vaccine Process Scale up of Attachment Dependent Cells using Micro Bioreactors and Microcarriers
News Mar 17, 2014
The trend for increased vaccine production1 is being driven by the requirement to produce affordable prophylactic vaccines for use in emerging markets and for newer types of therapeutic vaccines to treat an ever increasing array of diseases including cancer. These drivers coupled with the already present necessity to produce vaccines rapidly for seasonal inﬂuenza prevention and to react to pandemic threats has lead to the investigation of rapid method development for optimising scale-up of cGMP-compliant manufacturing processes.
Cell culture is becoming the route of choice for manufacturing many vaccines as it offers distinct advantages over egg based production which include shorter lead times and greater process flexibility.2
The most commonly used cell lines for vaccine production are Vero, Madin-Darby canine kidney, (MDCK), PER.C6 and insect cells. Several of the commonly used cell lines such as the Vero and MDCK cell lines are attachment dependent and cannot be cultivated well as suspension cultures in bioreactors3 so currently have to be propagated in roller bottle systems over which process control is limited.
This productivity issue can be overcome by attaching the cells to a substrate such as a microcarrier. There is now a great deal of interest in developing vaccines using attachment dependent cells and microcarriers as they allow good propagation in bioreactors and provider better process control, often resulting in high-titre vaccine production in shorter timelines.4,5,6 Additionally, microcarrier processes are seen as a candidate for cost-effective vaccine production at very large scales, to supply the rapidly growing demand for mass vaccination programmes in densely populated countries such as China and India.
One technical problem, which is preventing wider adoption of microcarrier culture in bioreactors, is how to accurately mimic bioreactor conditions to optimise cell growth, adherence and vaccine production with microcarrier cultures. Spinner flasks and benchtop bioreactors are currently used to define optimal media, feed and bioprocessing conditions.7 These types of vessels are both resource- and capital-intensive. Additionally, due to their scale, expense and limited throughput, vaccine producers are restricted in the number of benchtop bioreactors that can be run in parallel. Typically, only a small number of parameters can be evaluated in small (2-10 L) vessels. Hence, the final process scale-up can be sub-optimal adversely affecting outcomes such as cell density and vaccine titre. If a larger number of parameters could be studied under conditions which are representative of the bioreactor environment, then it might be possible to select the optimum process for vaccine manufacturing and shave months off of production timelines, as well as reduce production costs.
This need to conduct many parameter optimisation experiments under benchtop bioreactor conditions has resulted in the development of miniaturised high-throughput culture technologies. The drawback with many of many of these approaches is that they do not mimic stirring or sparging action of a bioreactor so producing evenly distributed microcarriers and performing media exchange because of the fast sedimentation rate of microcarriers can be problematic.
In this article, we discuss how performing microcarrier culture using an automated microscale bioreactor system allows even microcarrier distribution, good cell attachment and propagation therefore offering a method of improved process development for production of vaccine using attachment dependent cells.
Stirred Microbioreactor Technology
With the need to imitate bioreactor mixing, the advanced microbioreactor system (ambr™) was introduced by TAP Biosystems (now part of Sartorius). This stirred; sparged microbioreactor system is now an established technology for improving therapeutic antibody production using Chinese Hamster Ovary (CHO) cultures.8 This is because the ambr system mimics benchtop bioreactors, offering better environmental control and thus more representative culture performance than shake flasks and has been shown to be an excellent tool for high throughput process development.8
An element which is key to the ambr system’s success as a potential mimic for vaccine production using microcarriers is that each disposable bioreactor’s contents are stirred individually by an internal impeller so that scientists can begin their experiments with uniform microcarrier samples, automatically distributed from a well-mixed ‘stock solution’ into the micro bioreactor vessels being used for cell culture. In addition, the ambr’s automated pipetting system enables media exchanges to be carried out without interruption of stirring using an automated ‘pipette tip settling’ method developed by TAP. This involves taking a microcarrier sample from the ambr bioreactor within a pipette and allowing the microcarriers to settle for up to three minutes. The settled microcarriers are then dispensed to the bioreactor and the spent media is discarded. Using this method, a 20% media exchange can be performed at any time of the day or night on 24 ambr bioreactors in approximately four hours, without any manual intervention. A conventional approach can also be used, where the user stops the stirring, allow microcarrier to settle out, then removes and replaces media (in this case using the automated pipetting system).
To demonstrate that the ambr system provides uniform addition of microcarriers to multiple ambr vessels, one of the ambr bioreactors in each workstation station was filled with a Cytodex® 1 microcarrier (GE Healthcare) stock suspension (40g/L) and stirred at 300rpm. The workstation was set to dispense microcarrier amounts (2g/L 3g/L 4g/L 5g/L and 6g/L) from this microcarrier stock bioreactor to the others within the culture station. The uniformity of dispensing was measured across 6 ambr bioreactor replicates. The results showed the weight of microcarriers dispensed is consistent with a CV<1% of the amount programmed to be dispensed. This indicates that the ambr system can reliably and consistently dispense microcarriers from two ‘stock’ vessels to the other 22 micro bioreactor vessels for the cell culture phase.
Cell Attachment and Culture
To demonstrate that the ambr system can support cell attachment and propagation on microcarriers, Cytodex 1 microcarriers and Vero cells (ATCC® CL-160™) were added to ambr bioreactors, to provide 2g/L microcarriers and 1.5x105/mL cells in a 6mL volume for seeding. Vero cells were chosen as these cells are commonly used attachment dependent cells in the production of many prophylactic vaccines. The bioreactors were filled to 15mL for culture and were then set to stir at low speed continuously or intermittently for 16 hours, to allow initial cell attachment and spreading. Microcarriers were sampled every 24 hours and cell growth on the microcarriers was observed by microscopy. The results showed that the Vero cell achieved good attachment using continuous or intermittent stirring and grew well on the microcarriers and had attained confluence by day four of culture.
These studies show that using the ambr system’s unique stirring capability and automated pipetting; microcarriers can automatically be consistently distributed across each bioreactor. Additionally, cells can attach and propagate well. This means that scientists can rapidly assess up to 24 cell-specific culture parameters in parallel, simultaneously including stirring speed, media formulation or feed strategies to determine the optimum conditions for cell attachment, growth rate and vaccine titre, for example.
In summary, stirred, sparged microbioreactor technology can provide a good method of developing optimal process development for propagating cells on microcarriers in vaccine production. Setting up and running benchtop vessels and spinner flasks is manually intensive, while the ambr microbioreactor is more convenient and takes far less time to operate. This reduces reliance on spinner flasks and benchtop bioreactors and means process development for vaccine manufacture can be performed more quickly and efficiently, as well as increases the number of parameters that can be evaluated. Being able to assess so many different parameters and perform process development in weeks rather than months may save valuable time and thus, utilisation of the ambr system could make a significant contribution where more affordable vaccines or a rapid response to unexpected situations such as a pandemic threat is critical.
 Kresse H, Shah M. (2010) Strategic trends in the vaccine market.Nat Rev Drug Discov. 12:913-4.
 Montomoli E, Khadang B, Piccirella S, Trombetta C, Mennitto E, Manini I, Stanzani V, Lapini G. (2012). Cell culture-derived influenza vaccines from Vero cells: a new horizon for vaccine production.Expert Rev Vaccines. 11(5):587-94
 Genzel Y, Dietzsch C, Rapp E, Schwarzer J, Reichl U. (2010). MDCK and Vero cells for influenza virus vaccine production: a one-to-one comparison up to lab-scale bioreactor cultivation. Appl Microbiol Biotechnol. 88(2):461-75.
 Rourou S, Riahi N, Majoul S, Trabelsi K, Kallel H. (2013) Development of an in situ detachment protocol of Vero cells grown on Cytodex1 microcarriers under animal component-free conditions in stirred bioreactor. Appl. Biochem. Biotechnol. 170(7):1724-37.
 Trabelsi K, Majoul S, Rourou S, Kallel H. (2012). Development of a measles vaccine production process in MRC-5 cells grown on Cytodex1 microcarriers and in a stirred bioreactor. Appl Microbiol Biotechnol. 93(3):1031-40.
 Lohr V, Genzel Y, Behrendt I, Scharfenberg K, Reichl U. (2010) A new MDCK suspension line cultivated in a fully defined medium in stirred-tank and wave bioreactor. Vaccine. 31;28(38):6256-64.
 Hundt B, Best C, Schlawin N, Kassner H, Genzel Y, Reichl U. (2007) Establishment of a mink enteritis vaccine production process in stirred-tank reactor and Wave Bioreactor microcarrier culture in 1-10 L scale. Vaccine. 16;25(20):3987-95.
. Hsu WT, Aulakh RP, Traul DL, Yuk IH. (2012) Advanced microscale bioreactor system: a representative scale-down model for bench-top bioreactors. Cytotechnology 64, (6), 667-78.
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