Disposable Bioreactors for Viral Vaccine Production: Challenges and Opportunities

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BioPharm International, BioPharm International-11-02-2010, Volume 2010 Supplement, Issue 9

Switching to single-use bioreactors can have financial and performance benefits.


Disposables historically have been used in biotechnology processes for the past three decades, initiated with the use of single-use plastic support for cell culture (e.g., vials, shakers, T-flasks, and roller bottles). Another step was made more recently by the implementation of plastic bags into these processes, either used in the process itself or in supportive steps, such as media and buffer preparation and storage.

One of the key trends for biotech manufacturing is the development of disposable bioreactors. This trend was initiated by the introduction of the Wave system. The implementation of this technology was in line with the use of disposable plastic bags. The Wave bag technology quickly captured the interest of the biotech community. These systems mainly are used for cell expansion to replace shake flasks or intermediate small- and pilot-scale bioreactors, simplifying the process. Despite many advantages, this technology presents some limitations for its use as a final production-scale bioreactor, such as scalability, specificity of the agitation, and the bioreactor geometry.


Because of these limitations, an industrial need drove the development of more conventional and scalable disposable bioreactors. One of the first systems introduced on the market was the Single-Use Bioreactor (SUB) from Hyclone. The SUB opened a new range of applications because of its potential use either as seed vessels or as production vessels for cell-based processes.

Today, single-use bioreactors are used extensively for the production of monoclonal antibodies (MAbs) and recombinant proteins. Based on this market trend, vaccine manufacturers such as GSK Biologicals decided to investigate the potential use of this emerging technology for vaccine manufacturing. The focus of this article will be on the use of disposable bioreactors in the context of viral vaccine production.

Viral Vaccines Specificity

Viral vaccine manufacturing processes present some specific constraints as compared to other biotech products linked to the cell substrate used and to the viral production. These specificities are:

  • Multiple cell lines are used for these productions such as VERO, MDCK, MRC5, BHK, and CHO cells, making it more challenging to develop a platform process.

  • Cell substrates for viral production often are cell-anchored cell lines, such as VERO cells, requiring the use of micro-carriers for bioreactor process steps.

  • Viral production must be handled in the right biosafety containment, i.e., biosafety level 2 or 3 environments.

  • Production scales generally are smaller compared to MAb processes (ranging from 500 to 2,000 L).

Main Drivers for Implementing Disposable Bioreactors in Vaccine Production

There are many benefits of using disposable systems in biotech processes. Two of these benefits justify the evaluation of disposable bioreactors for viral production processes.

  • Maximizing facility output because of the fast turnover of disposable systems (no clean-in-place and steam-in-place operations in production vessels).

  • The reduction of capital investments linked to the reduction and simplification of the facility design and to the reduction of equipment investment.

In addition to these points, other drivers specific to vaccine manufacturing were considered.

  • Simplified biosafety level 2 and 3 production areas (because of the smaller footprint, lower ceiling height, and removal of water-for-injection and steam utilities).

  • Minimizing the harvest size to reduce the size of purification equipment and suites.


As previously mentioned, generic processes are difficult to define in the context of viral vaccine production. Therefore, a worst-case process that would cover all other company viral vaccine processes was defined. The disposable bioreactor technology selection based on this process will then be recommended as a standard single-use bioreactor platform for viral vaccine applications.

The following set of parameters was used to define the worst-case process:

  • animal-free media to decrease shear protection and nutritive support from serum containing formulations

  • cell cultures using micro-carriers (at a high concentration) because cells grown in this condition are more sensitive to shear stress compared to suspension cultures

  • VERO-adherent cell lines

  • medium renewal by sedimentation

  • lytic virus.

The combination of these process criteria made this process challenging enough to cover a large range of current and future in-house processes.

Figure 1. Disposable bioreactors from four companies were evaluated

Single-Use Bioreactor Selection

Based on market availability, system maturity, available scale, and mixing systems, four disposable bioreactors were selected (Figure 1).

  • Cultibag STR from Sartorius Stedim Biotech

  • Nucleo from ATMI—Pierre Guerin

  • Hyclone SUB from ThermoFisher

  • XDR from Xcellerex.

Criteria for Selection

To evaluate these four disposable bioreactor technologies, several criteria were selected. These criteria can be divided in two sections:

  • Process criteria such as cell culture and viral production performances, mixing and aeration characteristics, and scale-up predictability.

  • General criteria such as film type, biosafety, procurement, assurance of supply, and price.

Characterization Results

Mixing and aeration performance evaluation remains mandatory to ensure robust scale-up of cell culture processes, especially for adherent cell line applications (microcarrier use). In this study, these performances were evaluated for the four bioreactor technologies that were identified.


Three tools for mixing characterization were used: mixing time experiments, correlation software, and the particle image velocimetry (PIV). Typical results of the PIV technique are shown in Figure 2.

Figure 2. Results generated by the particle image velocimetry technique

Mixing characterization was performed on the four selected technologies; their mixing configurations are shown in Figure 3. Mixing performances were evaluated based on several criteria such as mixing time, maximum shear levels, etc. These performances were then compared to the application's specific requirements: minimizing shear stress while assuring good homogeneity levels and maintaining all the microcarriers in complete suspension. Some results of this study are depicted in Figure 4.

Figure 3. Mixing configuration of the four single-use bioreactors (SUB) evaluated

This figure shows the combination of maximum shear stress (represented by the tip speed) and mixing time in the minimum operating conditions necessary to maintain microcarriers in complete suspension. In this graph, two technologies seem to show better results in terms of acceptable mixing time combined with a low maximum shear stress (tip speed).

Figure 4. Mixing performances comparison of the four disposable bioreactors


Aeration performances also were compared based on gas transfer capacity measurements (kLa) in our end-of-cell-growth conditions for each bioreactor.

Gas transfer capacities in our operating conditions (minimum agitation speed required to maintain microcarriers in complete suspension) were evaluated for the four bioreactor technologies. A high gas transfer capacity is useful to decrease the amount of oxygen needed to maintain the dissolved-oxygen concentration to its set point. With low sparging flowrates, shear induced by bubble break-up at the liquid surface will be decreased. These low flow rates also have a positive effect on foaming.

Cell Growth and Viral Production Results

Figure 5 shows a typical example of four cell growths obtained in one of the selected disposable bioreactors. These data demonstrate that the cell growths obtained in this system are consistent and also equivalent to our control bioreactor (the control bioreactor is a small-scale 10 L bioreactor that was validated as a representative scale-down model of the larger stainless steel vessels).

Figure 5. Cell growth profiles in one disposable bioreactor

A critical feature of microcarrier-based cell culture is the homogeneity of cell adhesion to the beads. This point was monitored in all experiments performed in the different disposable systems selected and compared to the control bioreactor. Microcarrier pictures by microscopy at different time points (days 0, 2, and 5) were analyzed for each culture. These pictures show that a homogenous cell adhesion to the bead can be achieved using the right disposable bioreactor system, and the level of homogeneity is similar to that obtained in a stainless steel bioreactor.

The most important process criterion for evaluating the performance of these systems was their ability to support the same level of viral production as in a conventional bioreactor. To evaluate this point, several serotypes were produced in the different disposable bioreactors selected. Figure 6 shows an example of the results obtained for one viral serotype with the three disposable bioreactor systems. Viral production obtained with two systems are equivalent to the one obtained with the control bioreactor.

Figure 6. Viral production in the three disposable bioreactors evaluated

By the end of this evaluation, we demonstrated that with the right disposable systems, it is possible to achieve process performances equivalent to stainless steel bioreactors.

Risk Assessments

Disposable bioreactors are a new technology in the biotechnology field. To evaluate potential risks associated with implementing this new technology in future manufacturing processes, risk assessments based on the FMEA methodology were performed. The main risk assessments performed were related to the two critical risks: assurance of supply, and biosafety, a risk specific to viral vaccine applications. The biosafety risk assessment will be used as an example.

Biosafety is one of the main concerns of viral vaccine production, especially when biosafety level 2 and 3 viruses must be produced at large scale. One concept of biosafety is that the equipment itself is considered the first barrier to isolate the pathogenic micro-organism from the environment. The second barrier is the room where the equipment is located. The move from stainless steel to disposable equipment has weakened the first barrier. The main problem in terms of biosafety is the loss of integrity of the disposable bag leading to a leak of the viral contaminant, and potentially operator contamination. To identify all potential root causes for the loss of integrity of the disposable bioreactor, a risk assessment was conducted. Risks were scored according to four criteria, each scaled from 1 to 3: impact, occurrence, detection, and action response time. A risk priority number (RPN) was calculated as the multiplication of these four criteria. Based on the associated RPN, risks were classified as follows:

  • 1 to 3 RPN: low risk

  • 4 to 6 RPN from: medium risk

  • >7 RPN: high risk.

Figure 7 gives a summary of the risks identified associated with their respective RPN. Based on this risk assessment, a set of corrective actions were defined. The following gives examples of improvements made to the systems to mitigate potential biosafety issues:

  • automation, aeration, and pump stops when an overpressure is detected

  • external protection was developed to avoid liquid projections in case of leak and to avoid contact with cutting objects

  • a retention vessel will be part of the system skid to keep the liquid contained in case of spill

  • integrity testing of the disposable bag is under development using pressure to detect bag defaults.

Implementing all of the corrective actions identified in the risk assessment will help secure the disposable system for manufacturing operations.

Figure 7. Summary of biosafety risk assessment and associated risk priority numbers

Cost of Goods

Most of the studies performed today are in favor of disposable implementation from a cost perspective. Despite this, two scenarios should be considered in which the effect of disposable use on cost can be significantly different.

  • Introducing disposables to an existing process in an existing facility.

  • Introducing disposables to a new facility.

In the first scenario (existing facility), the effect of disposables may be marginal and can sometimes even result in increased cost of goods. The reason is that savings linked to disposable implementation are minimized because operating costs are fixed. Costs linked to full time employees will not be affected because production teams are in place. Building depreciation and maintenance also will be equivalent.

In the second scenario (new facility), the effect of implementing disposables can be more significant if the new facility is designed for using disposables. In this case, the facility footprint can be significantly reduced, utility sizing and distribution can be minimized, and production headcount can be adjusted.

At GSK Biologicals, we decided to compare two greenfield manufacturing plants for viral bulk production, one using the old process as a reference, the other one using a similar process but implementing disposable bioreactors along with other disposable systems for media and buffer preparations and purification intermediates. It is important to mention that the manufacturing scheduling was changed along with disposable bioreactor implementation. This point has a major effect on costs.

The model used to make this cost calculation was developed in-house and was validated on existing marketed vaccines. The first component of the cost analysis was establishing a bill of material analysis. Regarding the new process, 50% of the raw material cost was linked to the medium, ~25% was linked to micro-carriers, and the disposable bioreactor represents 6% of our raw material cost.

The output of our model shows that 35% can be saved on facility investment, and the manufacturing headcount (production and maintenance) can be reduced by 30%. If we consider the effect on total direct cost, 25% can be saved on the cost per dose driven by saving on building depreciation and labor. As mentioned previously, a significant amount (~50%) of the savings are linked to the optimization of manufacturing scheduling.


We demonstrated the feasibility to achieve equivalent process performance using the right disposable bioreactor systems compared to stainless steel bioreactors, even in the case of challenging processes, such as the one described in this article.

Additionally, cost of goods analyses show a significant savings when disposable technologies are implemented in new facilities, along with redesigning the manufacturing schedule.

Disposable bioreactors are an attractive technology for viral vaccine production if biosafety risk can be mitigated. One major point is that supply assurance is still a major problem because back-up supply is difficult to establish because of the specificity of these disposable bioreactors.

Jean-François Chaubard is a director, Sandrine Dessoy and Yves Ghislain are expert scientists, Benoit Barbier and Raphael Battisti are technicians, and Pascal Gerkens, PhD, and Ludovic Peeters are associate scientists, all at Viral Industrial Bulk, GSK Biologicals, Rixensart, Belgium, jean-francois.x.chaubard@gskbio.com