The Art of Bioreactor/Fermenter Scale-Up (or Scale-Down)

by Dr. Deb Quick

Effective bioreactor or fermenter scale-up/down is essential for successful bioprocessing. During development, small scale systems are employed to quickly evaluate and optimize the process, but larger scale systems are necessary for producing commercial quantities at a reasonable cost. But how does one effectively transfer the process between scales so that the process performs the same?

In an ideal world, the physiological microenvironment within the cells/microorganisms will be conserved at the different scales, but with no direct measure of that microenvironment the scientist identifies relevant macroproperties to measure and control to ensure comparability. There are many macroproperties and operating parameters that define the process at each scale, and while the goal is to keep as many of those parameters constant between the scales, it simply isn’t possible to keep them all the same.

When using the same operating parameters at small and large scale is impractical, there are several correlations that are commonly used: mass transfer coefficient (kLa [the volumetric transfer coefficient, 1/hr] or OTR [oxygen transfer rate, mmol/hr]; volumetric power consumption (P/V, agitation power per unit volume); agitator tip speed; and mixing time.

Matching the kLa at different scales is generally considered the most important factor in scaling cell culture and microbial processes. The second most common approach is to match the power consumption. For both of these correlations, there are often multiple combinations of operating parameters that provide the same kLa or the same power consumption at the different scale. And herein lies the art of bioreactor and fermenter scale-up/down. Selecting the best combination of parameters to match process performance at different scales is an art. There is no magic combination that works best for all cell types and products.

To establish comparability at different scales, you’ll make your life significantly easier if you start with the same vessel design at the different scales, but this luxury is rarely reality. More often, the development lab has significantly different equipment than the manufacturing facility. But even with different reactor designs, comparable performance can be obtained at different scales through appropriate experimentation.

• First, you’ll need to understand your equipment at all scales: measure the kLa and P/V of the different scales over a wide range of air flows, agitation rates, working volumes, and backpressures. It’s best to perform the testing in your process media, if possible. If you can find the time, it’s useful to evaluate different mixing schemes at small scale - different impeller styles and positions, baffles, and sparger styles and positions (particularly valuable if you already know the differences in these features between small and large scale systems available to you).
• Second, you’ll need to understand how your product responds to the different operating parameters. Those dreaded statistically designed experiments (DoE) are particularly useful for understanding the effects and interactions of the many parameters that can be changed. Performing DoE experiments at small scale with your product to evaluate the effects of aeration, agitation, and volume will not only help you with scale-up, but will also provide useful information for setting acceptable ranges for the operating parameters at large scale. As with the kLa studies, it’s useful to study different mixing schemes at small scale if time allows. One set of experiments that is highly useful but rarely performed is the evaluation of the process performance at the same kLa (or P/V) obtained using different operating parameters.

Understanding your equipment and how your product responds to various operating conditions is the key to effective process scale-up and scale-down. Despite the historical and ongoing need for scaling bioprocesses up and down, there is no strategy that works in all situations. The art of successful scale-up lies in thoughtful experimental design and thorough data analysis in order to obtain the information that allows equivalent performance at all scales.

TFF Under Pressure

By Dr. Scott Rudge

Are there scale up issues for cross flow filtration?  In general, this step is overlooked as a scale up concern, and usually, given the primarily clean feed streams encountered in simple buffer exchange, this is warranted.  However, forewarned is forearmed when scale up is concerned.

Primarily, there is just one scale up issue with cross flow filtration, and that is the path length on the retentate side of the filter.  The flow on the retentate side of the filter is meant to continuously clean the filter surface, and prevent fouling, or at least limit it to a thin boundary layer.  The shear rate created by the fluid at the filter surface increases as the square of the linear velocity of the fluid.  The pressure drop through the filter module, from inlet to outlet, depends linearly on the length of the module, and also on the square of the linear velocity.  In many cases, a manufacturing scale module is about a meter in length.  However, on the lab scale, a module is likely to be closer to 10 cm.  Therefore, the pressure drop from the inlet to outlet on the retentate side will be 10 times higher at constant linear velocity on scale up from lab to manufacturing.  Since decreasing the flow rate will dramatically decrease the shear rate, the increased pressure will drive higher flux towards the membrane surface, increasing the thickness of the boundary layer and resulting in more surface polarization (fouling or gel formation, potentially). 

One approach taken to this predicament is to keep the pathlength constant on scale up.  This is analogous to maintaining constant bed height on chromatography scale up, an approach I disfavor.  The result of this approach is a “horizontal” scale up, where more and more units of lab proportion are lined up side by side.  This approach works, but is cumbersome and requires more and more manifolding for flow distribution, and other inconveniences.  It also assumes that the length of filter the manufacturer provides is the best and only length for every application, which is absurd.  However, this is an approach commonly pursued, and recommended by the filter manufacturers for its speed and certainty.

Another approach that is taken to this phenomenon is to increase the back pressure on the permeate.  This slows down the permeate independent of changes on the retentate side of the filter.  However, if the back pressure on the retentate side is greater than the pressure at any point along the filter on the retentate side, permeate will flow back to the retentate side.  This is clearly inefficient, it means a particular fluid element will be filtered at least three times, crossing from retentate to permeate, then back to retentate, and then eventually back to permeate on a subsequent pass.  This also means that the effective filtration area is decreased, as some portion of the filter is working in reverse, and another portion is working to correct the back flow.  The negative flow counts against filter area that is filtering in the positive direction.

Finally, employing a constant pressure gradient along the retentate side is worth trying.  Presuming the membrane geometry is essentially maintained on scale up (including spacers in the flow channel) maintaining constant pressure gradient along the retentate channel length means shear will be constant on scale up.  Pressure drop from retentate to permeate will be higher at the retentate inlet, but if the shear is appropriate and the boundary layer controlled, this will only lead to higher flux, which may be preferred.  This can be tested on the small scale by applying back pressure on the retentate and looking for leveling off of the flux vs. pressure curve.  As long as flux vs. back pressure is increasing linearly, you can get improved performance at higher pressure.  Then upon scale up, the pressure at the retentate inlet is held constant.  It is certainly worth exploring longer path lengths on scale up, performance may improve!

In the end, either horizontal scale up will be used, or some reduction in retentate flow rate will probably be required.  The result of the latter will be less shear at the membrane surface, but the payback will be in increased filtration efficiency.  Some back pressure should be applied to the retentate side on the lab scale, as more pressure due to path length will almost surely need to be applied in manufacturing.  Maintaining pressure drop on the retentate side with increased module length, along with back pressure on the permeate side usually results in successful scale up of a lab cross flow filtration procedure.

What's That in My Protein? Degraded Polysorbate Again?

By Dr. Sheri Glaub

Mahler, et. al. have recently published a paper in Pharmaceutical Research entitled, “The Degradation of Polysorbates 20 and 80 and its Potential Impact on the Stability of Biotherapeutics.” (Subscription required.) As discussed in the paper, polysorbates are the most widely used non-ionic surfactants for stabilizing protein pharmaceuticals against interface-induced aggregation and surface adsorption.

Unknown Blogger uses a beater to induce aggregation in a protein solution

Concerns with polysorbate lot-to-lot variability, as well as potential degradation products prompted the authors to investigate the impact on four different monoclonal antibodies (mAbs).  They performed an extensive characterization of polysorbate degradation products, both volatile and insoluble, which included a number of ketones, aldehydes, furanones, fatty acids, and fatty acid esters. They then examined the effect of degraded PS on these proteins.

They concluded that as long as threshold levels of PS20 and PS80 were present (in this case >0.01%), the stability of the four mAbs in pharmaceutically relevant storage conditions (2-8 °C) was maintained despite observed polysorbate degradation.

The authors also suggest during formulation development one evaluate carefully the amount of PS to be used, considering the shelf life and potential behavior during storage.

Getting a grip on prophage

by Dr. Ray Nims

In a previous post, we discussed bacteriophage as a risk for the manufacture of biopharmaceuticals by bacterial fermentation. We mentioned briefly that bacteriophage may integrate within the genome of bacterial cells and that this may also represent a problem. Now we will explain why.

Bacteriophage are viruses that infect bacteria, and they have evolved two mutually exclusive strategies for survival. One involves a lytic growth cycle leading to death (lysis) of the host cell and release of progeny phage that may then infect additional host cells (so-called horizontal transmission). The other strategy is called lysogeny and involves integration of phage coding sequences into the host (bacterial) cell genome. The integrated phage is termed a prophage. This strategy for phage survival is referred to as vertical transmission since the phage genomic material is reproduced along with that of the host cell as the latter proliferates. Under certain circumstances, however, the integrated prophage can excise itself from the host cell chromosome in a process referred to as induction. The excised phage then may initiate a lytic infection of the host cell, causing all of the problems discussed in the previous post.

Illustration of a T4 phage infecting E. coli by Jonathan Heras

The relative success (i.e., from the perspective of the phage!) of the lytic vs. lysogenic survival strategies changes with the probability of host cell survival. Lysogeny appears to be a strategy that allows phage to persist during periods of low host cell availability or poor environmental (e.g., nutrient) conditions. Induction of prophage is an adaptation of the phage to host cell damage. This damage usually takes the form of a major stress to the host cell.

If stess can lead to prophage induction, the worry then becomes that some manipulation of a bacterial production cell during biopharmaceutical manufacture could lead to induction and initiation of a lytic phage infection. How can we assess and mitigate the potential for this to occur? There are two approaches: first, we can perform chemical or physical induction studies to determine the likelihood of encountering a prophage in a given production cell; and second, we can engineer the conditions of bacterial growth such that induction of a prophage is discouraged.

Phage induction studies may be performed on the bacterial production cell following initial engineering of the cell or during characterization of the cell bank. The inducing agent most often employed is mitomycin C. Other types of inducing agents (conditions) include carcinogens (such as the N-nitrosamines), hydrogen peroxide, high temperature, starvation, and UV radiation. The cells are treated with the inducing agent or condition, then one of various endpoints is used to detect the initiation of a lytic phage infection. These could include culture assays as well as molecular techniques such as PCR, microarray, or DNA chips.

Suppose you have an E. coli production cell harboring a problematic prophage. What can be done to discourage phage induction? Certain growth procedures have been shown to reduce spontaneous phage induction in E. coli cultures. These include using lower bacterial growth rates, replacement of glucose in growth medium with glycerol, and engineering the production cell through introduction of a plasmid conferring over-expression of the phage cI gene.

In summary, there are approaches that can identify the likelihood of encountering prophage induction from a bacterial production cell. The time to perform this type of testing is during development of the fermentation process (following the engineering of the production cell), or following banking of the production cell. If prophage induction appears to be a problem, bacterial growth procedures can help to reduce the potential. If this is not sufficient, the production cell may need to be re-engineered to produce a phage-resistant mutant.

Cold Facts About Dissolved Oxygen

By Dr. Scott Rudge

What’s the solubility of oxygen in water? Everyone knows that the answer to this question is “low”, and that’s enough to know for many practical applications. But it’s high enough to rust unprotected metal surfaces, and high enough to grow cells, provided that it’s replenished at some rate. It’s easy to find a number on the internet, at a temperature and pressure that the author of the internet resource thinks is interesting. But my interesting condition always varies from the internet’s, and finding the constants that I need to calculate the actual value is always difficult.


Most references that you can find say that the solubility of oxygen in water follows Henry’s Law. Henry’s Law is a very simple expression that says the concentration of a substance in a liquid phase is related to the partial pressure of that substance in the gas phase by a “constant”. Constant is a relative term in thermodynamics, because the constant in this and most cases varies with temperature and other components in both phases. But we’ll go with it.

In this equation, k_H is Henry’s constant, p is the partial pressure of the substance in the gas phase and c is the corresponding concentration of that substance in the liquid phase. Partial pressure is simply the amount of the total pressure due to that substance. In a room full of air at sea level, the partial pressure of oxygen is approximately 21% of the atmospheric pressure, or 0.21 atmospheres (159.6 torr or mm Hg, or 3.1 psi). At 25°C, Henry’s constant for oxygen in pure water is 769.2 L*atm/mol.

To correct for temperature, an approximation using a reference temperature can be used, although the results will not be exact. The correlation is:

Where T_r is the reference temperature. Again, the use of these equations will give approximate values.

Adding salt to the water further decreases the solubility of oxygen, such that in sea water the solubility of oxygen is about 80% of that in pure water. In fermentation medium, the saturation concentration is probably even less.

For those who don’t like math, here’s the table for the dissolved oxygen concentration under air atmospheres containing 21% oxygen (dissolved oxygen concentrations in moles/L):