Single-Use Bioreactors: Making the Transition

Size: px

Start display at page:

Download "Single-Use Bioreactors: Making the Transition"

Mabel Sybil Hancock
6 years ago
Views:

IPT 20 2006 31/8/06 09:07 Page 54 Biotechnology Single-Use Bioreactors: Making the Transition The biopharmaceutical industry is moving rapidly towards single-use production systems but a number of

1 IPT /8/06 09:07 Page 54 Biotechnology Single-Use Bioreactors: Making the Transition The biopharmaceutical industry is moving rapidly towards single-use production systems but a number of challenges remain; a new disposable bioreactor/mixer offers easier integration, simpler operation and lower investment costs. By Kevin A Auton at Cellexus Biosystems plc Kevin Auton was trained as a Biochemist in protein research and cell culture at Southampton University supervised by Prof Chris Anthony and received a PhD for his research in protein expression analysis and characterisation. Subsequently, his entire career has been in the field of product development, manufacturing, sales and marketing for life science technology companies. Dr Auton has been the inspiration and driving force behind the development of a succession of new products. During his career, he has led the development of eight new products from first concepts through to prototype, into manufacture, marketing launch and closed sales to end users. Dr Auton founded NextGen Sciences i n 2000, which was listed on London s AIM in December 2005 (symbol: NGG). NextGen Sciences was awarded the Frost and Sullivan Technology Leadership Award for one if its products developed under Dr Auton s leadership. Dr Auton founded Cellexus Biosystems PLC in 2005 and listed the company on Ofex in May 2006 (symbol: CBIO). The CellMaker Lite is the company s first product. Throughout 2006, there have been numerous articles describing the acceleration in the use of disposable modules for biopharmaceutical production. Interest in the subject area can be measured indirectly by the increase in the number of conferences dedicated to the subject and their levels of attendance they are usually sold out. The growth of interest in single-use or disposable components for biopharmaceutical production has been discussed elsewhere (1-5), but in summary it has been driven by the need to limit the down time between processes and reduce the costs associated with validation of each component of a production process as well as the ever-growing burden of meeting regulatory approval. INTEGRATION WITH EXISTING SYSTEMS In many areas, it is relatively straightforward to replace stainless steel and glass systems that must be cleaned and steamed in place with single-use systems. A number of innovative suppliers in the industry have responded quickly to the need to integrate these new disposable systems with the incumbent systems already in place. Pall Inc has brought out its Kleenpak TM connectors that remain sterile and can be brought together without exposing the internal surfaces to the environment, enabling integration with legacy systems. The Kleenpak connectors can be sterilised by both gamma irradiation and by autoclaving; this means that one part can be supplied with the disposable system and sterilised by gamma irradiation. However, they can also withstand high temperatures so that the matching part can be sterilised on the steam-in-place (SIP) legacy systems before the two are brought together. The availability of a universal connector of this type makes it much easier to integrate disposable systems with existing systems in a way that was not previously possible. In-Vessel Operations Other transitions to single-use systems are not so easily achieved. Key challenges are often found when a mechanical operation must be performed within a vessel, particularly if the contents of that vessel must remain isolated from the environment. In traditional systems, propellers, valves and probes can all be inserted through highly effective seals and bearings in the vessel s bulk head; the internalised components can then be sterilised along with the rest of the vessel. But when replacing the vessel with a disposable system, it is much harder to make these arrangements without breaching the integrity of the system. To do so would circumvent the benefit of investing in a validated single-use system. A variety of inventive solutions have been developed to overcome some of these problems. In vessels that must be stirred, magnetically-driven impellers can be suspended 54 Innovations in Pharmaceutical Technology

2 IPT /8/06 09:08 Page 55 in solution. Alternative configurations include the provision of entire disposable seals and bearings which can be integrated into the wall of the plastic container, or the ability to invert the vessel. However, all of these attempts to replicate a steel chamber result in an increased cost for the disposable and greater complexity with respect to the instruments that operate their use. Often, considerable effort must be invested to remove the excess heat that is generated by mechanically-driven stirring systems. Rocking Platforms In the field of disposable bioreactors, Wave Biotech Inc (Somerset, NJ) has pioneered the use of rocking platforms, particularly for growing insect and mammalian cell cultures. Here, the use of a gentle agitation process eliminates the need for inclusion of a means for agitation. However, the mass transfer of oxygen into solution that is achieved is related to the energy of the agitation process. For example, oxygen transfer (nmol L -1 h -1 ) in a stirred bioreactor is , a shaker flask is and a Waveagitated system is less than 1 (6). Mammalian cells only require nmols of oxygen per litre per hour, and are therefore less sensitive to oxygen limitation but greatly sensitive to the shear forces created in a stirred system. By contrast, E. coli has a massive requirement for oxygen, 800nmol L -1 h -1, but are less sensitive to the shear forces created within stirred systems. The ideal bioreactor would thus be able to deliver the high rates of oxygen mass transfer achieved in a stirred reactor, but without the shear forces that these generate. Rocked systems seek to reduce shear forces, but this comes at the cost of very low oxygen mass transfer (6). In such systems, aeration rate is a complex function of the frequency of rocking, the acceleration rates between oscillations and degree of pitch. This is a difficult combination, and control of the culture to a specified percentage of air saturation is challenging (9). Nonetheless, these bioreactors have been proven to be highly effective for certain cell cultures that have low requirements for oxygen, and they can be scaled up to 500 litres. The success of the Wave system has encouraged others to adopt a similar approach, and systems from Applikon and CatchMab are available outside of the US. Process Monitoring Further new challenges present themselves when transitioning from CIP/SIP to single-use systems. One such challenge is how to monitor the contents of the vessel. Reusable probes may be inserted into the singleuse vessel in situ or probes may be supplied as a disposable item as part of the assembly. The former violates the integrity of the disposable system, while the latter increases the cost. Fortunately, there are a number of suppliers that have created alternative strategies which replace traditional probes with ones that are non-invasive. Suppliers such as Polestar Technologies (Needham Heights, MA), Oxysense (Dallas, TX) and Fluorometix (Baltimore, MD) have each developed novel systems based on fluorescence-quenching technologies. Here, a small patch is placed on the inner surface of the vessel in which a fluorescent material is immobilised; the chemistry is then manipulated such that fluorescence is either enhanced or quenched in the presence of a particular analyte such as, for example, oxygen or by changes in ph. Each of the above suppliers takes a different approach to how the chemistry of fluorescencequenching works. The Fluorometrix technology (now licensed exclusively to Sartorius AG) uses two coupled fluors. The chemistry invented by Polestar Technologies (US patents and ) uses a single fluorescent molecule and can be readily used with just a single point calibration. Light is delivered to the patch through the bag s wall and fluorescence decay is monitored a simple process requiring no invasive probes. These and other innovations mean that there is considerable scope for systems to be integrated, giving rise to greater flexibility. In most cases, the situation with respect to intellectual property is relatively straightforward and not as complex as has been reported (6). A NEW DISPOSABLE BIOREACTOR At Cellexus, we have developed a new disposable bioreactor and mixing system (the CellMaker Lite TM ) that is simple and inexpensive to operate, and has been designed to take advantage of the recent innovations in disposable integration and non-invasive monitoring technologies. Cells can be grown or solutions mixed in the bioreactor. The active materials (cells or materials being mixed) are retained within a pre-sterilised bag (the CellexusBag TM, patent applied for). The bag and the vessel in which it is housed are arranged into a unique geometry that Innovations in Pharmaceutical Technology 55

IPT 20 2006 31/8/06 09:09 Page 56 Figure 1: CellMaker Lite system (10-50 litre version) The CellMaker Lite Enclosure (left) completely encloses the CellexusBag in which cells are grown.

provides for effective mixing (Figures 1 and 2). In experiments, mixing in a 50-litre vessel can take just 2-5 seconds, with no mechanical agitation.

3 IPT /8/06 09:09 Page 56 Figure 1: CellMaker Lite system (10-50 litre version) The CellMaker Lite Enclosure (left) completely encloses the CellexusBag in which cells are grown. Integrated into the enclosure is a temperature control system. The CellMaker Lite Controller (right) controls the flow of aeration gas, the headspace pressure and the pulsed intervals. provides for effective mixing (Figures 1 and 2). In experiments, mixing in a 50-litre vessel can take just 2-5 seconds, with no mechanical agitation. This property makes the system ideal for mixing materials (aqueous liquids and solids) and for growing cells, both in suspension and on microcarriers, in a fluidised bed. Typically, the bag is filled with sterile media by directly pumping the media through a Pall KleenPak filter into the base. We find it convenient to make a x10 concentrate of media which is then diluted in situ with water passed through the same filter. The aeration or mixing gas is delivered at relatively low flow rates (1-10 litres per minute depending on the volume of the culture) to the base of the bag. This aeration/mixing inlet gas is sterilised again using a Pall Kleenpak filter and enters the vessel through a simple sparging tube, supplied as part of the disposable assembly, that runs along the length of the bag at its base (A in Figure 2). A curtain of small bubbles is created and rises up the narrow lower part of the bag (B in Figure 2); a surface is then presented to this riser which deflects it forward. At the same time, the more dense, air-depleted solution is pulled downwards into the riser current to be mixed with aeration gas (D in Figure 2), and mixing is achieved gently and without mechanical stirring or further agitation. The upper surface of the liquid-air interface (C in Figure 2) is arranged to be as large as possible to further encourage gas exchange at this upper surface; the head space can also be perfused with the same or a different gas. Furthermore, through the provision of a pressure regulation system that is integrated into the instrument, the head space can be pressurised under operator control. Experiments indicate that pressurisation of the bag which is entirely constrained by the enclosure has the effect of increasing mass transfer and reducing the foaming that is often experienced in bioreactors with high oxygen mass transfer rates. Inoculum can be added and culture removed via side ports, using disposable connectors which maintain the integrity of the system. Cell culture samples can be extracted via separate sampling ports. Production-scale airlift fermenters (which can be 20,000 litres or greater (7)) give comparable results to stirred systems (8). Foaming, rather than the presence of bubbles, is the major cause of high mammalian cell mortality in fermenters or bioreactors. Airlift fermenters are typically cylindrical and often suffer from flat spots in which the culture is not effectively mixed. It is not unusual, therefore, to over-perfuse the system to ensure adequate mixing but this gives rise to undesirable foaming. Cell mortality is all but eliminated when antifoaming agents are added (10-11), demonstrating Figure 2: Profile of the asymmetrical geometry of the CellMaker Lite Enclosure and the CellexusBag restrained within C CellMaker Lite Enclosure Pressurised headspace D B A CellexusBag 56 Innovations in Pharmaceutical Technology

IPT 20 2006 31/8/06 09:11 Page 58 that it is the formation of foam that causes cell disruption rather than the presence of bubbles.

4 IPT /8/06 09:11 Page 58 that it is the formation of foam that causes cell disruption rather than the presence of bubbles. The use of an asymmetric profile in the CellMaker Lite gives more effective mixing compared with cylindrical airlift systems, and eliminates these flat spots; at the same time, it can generate the same high rates of oxygen transfer at which airlift fermenters operate. This means that the rate of gas perfusion required for mixing can be minimised to lower operating costs and reduce foaming, while at the same time, providing sufficient aeration for even the most oxygen-hungry cells. The inclusion of a fluorescent patch allows the oxygen concentration to be monitored using the Polestar non-invasive probe, so that the cells are not oxygen-limited but neither is the culture over-aerated. EXPERIMENTAL EVIDENCE The high mass transfer rate of oxygen that can be achieved with the CellMaker Lite was demonstrated by an experiment in which a highly oxygen-dependent E. coli recombinant cell line was used to express a recombinant fluorescent protein. The results were compared with a 500ml baffled shaker flask. Experimental conditions for all growth media and culture parameters were kept as similar as possible throughout. The CellMaker Lite/CellexusBag combination was used to culture E. coli cells to high densities and gave growth profiles comparable to those generated in shaker flasks with vigorous agitation (Figure 3a). Typically, disposable bioreactors are not used to grow E. coli due to the difficulties in achieving sufficient oxygen mass transfer; as such, this was an excellent experimental model with which to demonstrate the system s efficient aeration process. The results indicated that the growth curves and final densities achievable with a 50-litre CellMaker Lite are the same as for a 500ml culture in a flask. In the latter, the challenges of oxygen mass transfer are minimal. Figure 3a also demonstrates that a 2-litre flask culture (4 x 500ml) can be directly scaled-up to a 50-litre culture volume with no interim steps. In a separate experiment, a 500ml flask culture and the 50-litre CellexusBag culture of E. coli were grown and expression of a recombinant fluorescent protein was induced at an OD600 (optical density of liquid medium at 600nm) of 0.6. In this experiment, the lag time was

5 IPT /8/06 09:12 Page 59 longer than seen previously, but both the flask and the 50- litre culture followed the same profile (Figure 3b). In both cultures, the expression of the recombinant fluorescent protein was found to be similar, demonstrating that the cells could be grown and protein expressed to near identical levels in either system (Figure 3c). CONCLUSION The biopharmaceutical industry is moving towards single-use systems in its production facilities; this transition is occurring more rapidly as a result of numerous innovations from suppliers to enable integration. Challenges remain, particularly in how to achieve effective mixing without complex systems or high-cost consumables, and how to build truly scalable systems. The CellMaker Lite has been demonstrated to be suitable for growing suspension cultures of the highly oxygen-dependent E. coli host strain; it has also been shown to be scalable for protein production, delivering the same performance as a highly agitated shaker flask system. The efficiency of aeration and mixing enables culture volumes to be further increased to large-scale bioreactors, whilst retaining the mechanical simplicity and thereby reducing investment costs. The use of suitable connectors and non-invasive probes demonstrate how some of the many challenges involved in creating an end-to-end single-use process can be solved simply, at low cost and within the budgets of most investigators and production environments. The author can be contacted at kevin.auton@cellexusbiosystems.com References OD at 600nm Time after inoculation (minutes) Cells grown were E. coli TUNER(DE3) grown in LB broth transformed with an expression plasmid expressing a recombinant fluorescent protein. An inoculum was prepared so that the inoculating density for each of the two vessels was nearly identical. The culture was sampled at intervals and reported as OD ml flask 50-litre CellMaker Lite The same cell line was grown in both a 500ml flask and in the 50-litre CellMaker Lite. An inoculum was prepared so that the inoculating density for each of the two vessels was nearly identical. The culture was sampled at intervals and reported as OD600. Cells were grown to an OD of 0.6 and protein expression was induced with IPTG (isopropyl thiogalactoside). Cells were harvested, and the relative concentrations of recombinant fluorescent protein were compared by SDS PAGE. OD600 Figure 3a: Growth curves for a 500ml culture in a baffled shaker flask agitated at 225 rpm and growth curve for the 50-litre CellMaker Lite culture Figure 3b: Expression of recombinant fluorescent protein Time after inoculation (minutes) Figure 3c: SDS page gels of cell cultures Flask CellMaker Lite 500ml flask 50-litre CellMaker Lite 1. Cardona M and Allen B (2006). Bioprocess International Supplement Series, p Wilson JS (2006). Bioprocess International Supplement Series, p Forgione P, and Van Trier, Mark (2006). Bioprocess International Supplement Series, p Montgomery SA (2005). Bioprocess International Supplement Series, p Fury J (2005). Bioprocess International Supplement Series, p Reif O-W and Vogt R (2006). Innovations in Pharmaceutical Technology, 19, p Birch J. Lonza presentation to Bioproduction Induction 8. Osman et al. Poster: Stirred Tank and Airlift Bioreactors are Interchangeable Systems for Mammalian Cell Culture, IBC Antibody Conference, San Diego. 9. Pendleton R. Amgen Presentation, IBC conference, Zhang S, et al. (1992). J Biotechnol, 25, van der Pol LA, et al. (1993). Biotechnol Prog, 9, Innovations in Pharmaceutical Technology 59

CHAPTER 2 CULTURE TYPE

CHAPTER 2 CULTURE TYPE All types of micro-organisms are grown in suspension with the exception of cell culture, in which cells can also be anchorage dependent. Suspension the micro-organism can grow successfully