It is not always possible to obtain the same production performance simply by making the equipment larger when scaling up from a pilot plant to a production scale bioreactor system. Steve Spreckley, sales and applications manager for Lightnin Mixers in the UK, reviews the complex variables and offers some solutions
Agitators are central to bio-reactor systems, but to fulfil their production role they have to be of a different design from those used in pilot scale production. When scaling up a cell culture system, special attention has to be given to the bioreactor agitator in order to achieve the required results. Although vessel baffles and agitator mounting orientation are mainly down to customer preference, the impeller type does affect process scale-up, for it is not always possible to obtain the desired performance in a bioreactor system using the same type of impeller employed on a pilot scale.
To achieve the desired performance, it is necessary at the outset to define the process parameters. In a cell culture system, the agitator has to fulfil two roles. First it must provide sufficient shear to obtain the desired oxygen and carbon dioxide mass transfer. Second, it has to keep the vessel well blended to minimise variations in temperature, pH and additive concentration.
The first requirement may be characterised by the gas mass transfer effectiveness (kLa) and the second by the blend time. Concurrently, the peak shear rate must be kept low enough to avoid cell culture damage or stress, which is contradictory to the first two parameters. In an ideal situation, all three parameters should be specified by the end-user to assist in the selection of the most appropriate impeller type.
To attain effective gas mass transfer the largest possible impeller should be employed because reducing the fraction of oxygen in the sparge gas or flow of sparge gas can reduce operating costs and reduce cell damage in certain cell lines.
It is possible to measure the kLa experimentally using either sodium sulphite addition or nitrogen stripping to reach 0% dissolved oxygen (DO) concentration. The kLa is proportional to the rate of rise in DO concentration as air sparging continues. The sodium sulphite method is more practical for shop-testing large vessels because it does not release large quantities of nitrogen into the testroom; it also reaches baseline values more quickly.
There is another benefit to this method: it achieves between 30% and 50% higher kLa values than the nitrogen stripping method because of the higher ionic strength, but various agitator types and configurations may still be compared. One point to be noted is that typical media culture is of an even higher ionic strength, so kLa values by either method will be conservative.
kLa variables
For vessels and agitators that are geometrically similar, the kLa depends on:
- the superficial gas velocity rising through the bioreactor;
- power per volume for the agitator;
- size of the bubbles;
- ionic strength.
If the agitator is turning too slowly for the gas rate, then flooding occurs and the kLa will not increase any further if the gas load exceeds this flooding point. With the exception of flooded impeller or zero gas conditions, it is possible to change impeller types as equipment is scaled up and still achieve the desired kLa.
Should the production bioreactors be geometrically similar to the pilot scale plant, the kLa performance may be expected at the same superficial gas velocity and power per unit volume.
While the combination of Rushton impellers and marine impellers work well on smaller scale systems, it does not perform as well in large production scale bioreactors. To obtain blending times in a production scale bioreactor similar to those of the pilot equipment, it is necessary either to increase the rpm of the agitator or change the impeller.
Studying the use of high solidity and high efficiency impellers reveals that these impellers convert a larger fraction of their power input into fluid pumping rather than shear. As a result, they can operate at higher tip speeds without compromising shear. Peak shear rates occur at the impeller tip and vary linearly with tip speed for radial impellers and linearly with rpm for high efficiency and high solidity impellers.
In terms of impeller types, it is well documented that the axial flow impeller is the most effective for flow-controlled applications. In ungassed mixing applications, axial flow impellers are unsurpassed in flow production. However, they are designed specifically for flow and without any consideration for the effect of gas dispersion. Radial impellers, traditionally used for dispersing gas, are inefficient producers of flow.
Lightnin Mixers, part of SPX Process Equipment, has undertaken considerable research into the design of impellers for gas-liquid processing systems resulting in a family of high solidity axial flow impellers designed to balance requirements of gas dispersion shear levels and efficient bulk fluid flow. Typically in today's bioreactor systems, clients are moving from traditional pitched blade or marine propeller technology to A320 high solidity impeller systems.
Larger impeller diameters (for the same kLa) result in shorter blend time and lower peak shear. In cell culture systems, the impeller diameter is usually set at half the vessel internal diameter. Because the outer one-fifth of the vessel is occupied by baffles, there is an upper limit on the physical limit of the diameter. Offsetting these process benefits are the higher cost, increased vibration, larger run-out and the more robust build requirements associated with larger diameter impellers and greater fluid forces.
Where multiple impellers are required, the typical off-bottom clearance is half of the impeller diameter; any lower than that and the increasing proportion of the input power translates into energy dissipation (shear) as the impeller discharge flow decreases with little change in power input.
Developing and engineering mixers for sanitary and hygienic applications is a tough challenge. For a start, there is the matter of size, followed by impeller design and the matter of whether the vessel is open or closed, as this will dictate where to mount the mixer. In addition, there is that all-important factor - the cleaning regime.
Lightnin Mixers has addressed these and many other issues in the development of its SanStar mixer family. This consists of no fewer than 10 separate mixers developed for applications ranging from bulk processing through to bench top laboratory duties with options for stainless steel drives systems. What all the models have in common is their compliance with FDA mandated cGMP guidelines, and the range includes seal and seal-less (magnetic drive) models designed and developed to meet the requirements of bioreactor applications.
The availability of the SanStar mixer family enables companies operating in the pharmaceutical and biotech industry to standardise on a single group of mixers for an extensive range of duties and operating locations. Most significantly, it provides solutions that allow companies to scale up mixing operations from the lab to the large scale processing line employing the same design of impeller.
Recognising that there is a need for geometrically accurate lab-scale impellers, Lightnin has produced a process where exact duplicates of Lightnin-patented high-efficiency axial flow and other commonly used impellers are produced in very small diameters. The employment of these geometrically accurate impellers provides laboratory data that can be used for precise scale-up and process parameters proven in the laboratory and may be used to develop pilot plant and production scale equipment.
The surface finish for the agitator should match that specified for the vessel. Typically, this would be 0.5µm Ra with electro-polish. The required quality of finish for cell culture often exceeds the requirements for bacterial fermentation or those common in the food process industry.
The usual industry practice of applying the finish after completing impeller and shaft assembly does not produce consistently satisfactory results. Corner areas cannot be polished orthogonally and it is difficult to obtain good electrode geometry for electro-polishing.
It is more efficient to apply the mechanical polish to blades, hubs and shaft before welding. In so doing, successive grits at right angles to the previous polish can be used without concern for interference by other parts of the assembly. The components can then be electro-polished and protected during forming and assembly. After the rotating assembly is complete, the welds are ground and then spot electro-polished.
Agitation orientation is generally specified by the customer. On small vessels, such as those used in pilot scale or seed train bioreactors, top-mounted agitators are very common. They are easier to seal, but require longer shafts and larger diameter to control run-out and vibration. If the vessel does not have baffles, then the agitator must be mounted either on an angle or offset from the vessel centre line. Baffled vessels with centre top-mounted agitators are more common.
Bottom-mounted agitators are more prevalent in large production bioreactors. They need much shorter shafts - perhaps as much as 3m shorter on a production scale bioreactor. The shorter and smaller diameter shaft saves money and is easier to handle during servicing. Maintenance on bottom-mounted agitators in production bioreactors is simplified because it is easy to support the shaft while removing the mechanical seal and because vessel entry is not required.
As cell culture systems become larger, the key process parameters (kLa, blend time and peak shear rate) do not remain constant. Large cell culture systems may require a different impeller from existing pilot scale equipment, with high efficiency and high solidity axial flow impellers being recommended.