Advances in bioreactor agitation

By Neil Meadows, Product Manager Lightnin Mixers

Introduction
In the critical part of scaling up a cell culture system it is often found that insufficient attention is given in bid specifications to the bioreactor agitator. Typically, the specifications provide little more than the impeller type, mounting orientation, whether the vessel is baffled, ma¬terials of construction and surface finish. Al¬though vessel baffles and agitator mounting orientation are truly a matter of customer preference, the type of impeller affects process scale-up. It may not be possible to obtain the desired performance in a production bioreactor using the same impeller type successfully employed at pilot scale. Furthermore, process param¬eters needed for scaling up are almost always missing.

In the experience of SPX Process equipment Lightnin Mixers virtually all bid specifications are silent on the design features of the shaft seal. This is unfor¬tunate, especially given the wide variation in price and advances in ease of maintenance, clean ability and sterile operation between seal models.

Process Requirements
The agitator has two jobs in a cell culture system. First, it must produce enough shear to obtain the desired oxygen and carbon dioxide mass transfer. Second, it must keep the vessel well-blended to minimize variations in tem¬perature, pH and additive concentration. The first requirement may be characterized by the gas mass transfer effectiveness (kLa) and the second by the blend time. At the same time, the peak shear rate must be kept low enough to avoid cell damage or stress—contradictory to the first two requirements. Ideally, all three parameters would be specified by the end-user.

Bigger is better for kLa, because reducing the fraction of oxygen in the sparge gas or flow of sparge gas can lower the operating cost and reduce cell damage in certain cell lines. For geometrically similar vessels and agitators, the kLa depends on the superficial gas velocity (F) rising through the bioreactor, power per unit volume (P/V) for the agitator, size of the bubbles and ionic strength. Flooding occurs when the agitator is turning too slowly for the gas rate; kLa will not increase further if gas load exceeds this flooding point. The superficial gas velocity is calculated as though the vessel were empty. Most published correlations of kLa follow the form (where A,  and  are experimentally determined constants):

One very interesting thing about the kLa equation is the fact that all of the variations with impeller type, tip speed and diameter appear in the term for power per unit volume. Except for the two extremes (flooded impeller or zero gas flow), one may change impeller types as equipment is scaled up, yet still be confident of obtaining the required kLa. If the proposed production bioreactors are geometrically similar to existing pilot scale equipment, similar kLa performance may be expected at the same superficial gas velocity and power per unit volume.

The shear produced by an agitator impeller may be mea¬sured by Doppler velocimetry using a laser beam to scan the instantaneous velocity at points throughout the vessel. Lead¬ing agitator vendors have characterized the performance of their impellers using this technique. Peak shear rates occur at the tip of the impeller and vary linearly with tip speed for radial impellers and linearly with rpm for high efficiency and high solidity impellers.

Scale-Up
In production conditions, to obtain similar blending time in the production scale bioreactor, one must either increase agitator rpm or change the impeller. Rather than conduct process development ex¬periments to see if the cell strain can tolerate higher shear, one may study the use of high solidity and high efficiency impellers. These impellers convert a bigger fraction of their power input into fluid pumping rather than shear, and so can operate at much higher tip speeds. Table A shows the results of such a study (all agitators used two impellers).

After considering the kLa requirements of high density mammalian cell cultures together with managing the shear level of such a culture, it is clear that the Rushton/marine combination cannot perform as well in large scale production bioreactors as in the smaller reference system.

As a result, it becomes necessary to consider dual axial flow impellers instead of the Rushton/marine combination that perform effectively at a smaller scale. Though any of the axial flow impellers offer viable performance, a high solidity impeller has good performance at a low tip speed. Peak shear is half that of the Rushton, kLa 130% higher and blend time over seven times faster.

It is worth noting that marine impellers from many manu¬facturers are made from castings rather than fabrications. As such, the impeller surface will almost always have some porosity. For this reason, agitator manufacturers may suggest a high solidity impeller (Fig.1), which is a formed and welded assembly, when specifications call for a marine im¬peller

Agitators for a seed train can be designed as scaled down versions of the one for the largest bioreactor. The same impellers and geometry ratios apply, though the smallest seed train vessels might use a single impeller.

Optimizing Agitator Geometry
Agitation orientation is generally specified by the customer. On small vessels, such as used in pilot scale or seed train bioreactors, top-mounted agitators are very common. They are easier to seal, but do 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.

Bottom-mounted agitators also require much less head¬room to remove the impeller. Rather than remove the shaft assembly through a ceiling hatch (and thereby expose the bioreactor suite to outside air), most users build their bioreactor suite with enough ceiling height (or height in a ceiling bump-out) to keep all the maintenance activity within the classified room area. 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.

Bigger impeller diameters (for the same kLa) result in shorter blend time and lower peak shear. Against these process benefits must be weighed the higher cost, increased vibra¬tion and larger run-out and more robust design require¬ments associated with larger diameter impellers and greater fluid forces.

Mechanical Seals
Mechanical seals are relatively easy to implement on top drive agitators. Bottom drive agitators offer more of a chal¬lenge to comply with biotech industry needs.

Recognizing the unique concerns of the biotech industry, a special double mechanical seal has been developed for bottom mount agitators. This seal is lubricated with con¬densed clean steam. As shown in Figure 2, this seal uses a cartridge-canister design that can be pre-assembled and statically tested before installation. Only the housing, seal sleeve, O-rings and inboard seal faces contact the culture medium. None of the springs are in contact with the culture medium.

Seal face material selection depends on drive size, normal operating conditions, CIP/SIP temperatures, shaft speeds, seal fluid pressure and auxiliary support system used. Be¬cause cell culture agitators run at relatively low speeds, frictional heating at the seal faces is not as much of an issue as in bacterial or yeast fermentors. Some users therefore prefer silicon carbide vs. silicon carbide for the inboard seal faces to eliminate the possibility of carbon wear particles entering the culture medium. If this approach is followed, however, seal fluid flow and temperature must be sufficient to lubricate the seal faces during elevated temperature CIP. Use of dissimilar materials (alpha sintered silicon carbide vs. reaction bonded silicon carbide) will help in this regard.

The outboard seal faces, which do not contact culture medium, are recommended to be carbon vs. silicon carbide. This combination provides the most reliable performance under both normal and adverse lubrication conditions.

Agitator Fabrication
The surface finish for the agitator should match that speci¬fied for the vessel. Typically, this would be 0.5 micron 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 elec¬trode 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 assem¬bly. The components should then be electro-polished and protected by paper during forming and assembly. After the rotating assembly is complete, the welds are ground and then spot electro-polished.

Agitator specialist firms routinely use CFD (computa¬tional fluid dynamics) to study agitator performance in the customer's vessel. The CFD analysis has been validated by laser Doppler velocimetry data collected in test vessels, and so represents a good predictor of actual agitator performance. The CFD study greatly reduces the need to adjust impeller centre lines. If CFD studies are performed, it is recom¬mended that the impellers be welded to the shaft, thus eliminating two hard-to-clean O-ring seals at the hub of each impeller.

Conclusions
As cell culture systems get 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 exist¬ing pilot scale equipment, with high efficiency and high solidity axial flow impellers (Fig.3) being recommended.

Production-scale systems frequently use bottom-drive agitators to reduce ceiling height in the bioreactor suite. Double mechanical seals lubricated by pressurized condensed clean steam are needed for bottom-drive agitators. The car¬tridge seal improves on past practice through fewer parts contacting the culture medium, design features that meet ASME BPE requirements and ability to be removed for service without entering the vessel or removing the gearbox.