Viral clearance studies …. are they needed for proteins produced using bacterial or yeast fermentation processes?

Dr. Ray Nims

In E. coli or Pichia pastoris-based bioproduction of recombinant proteins, there are no suitable host cells for amplification of viruses that are infectious for humans. Bacteria such as E. coli and yeast such as P. pastoris can only support the growth of certain bacteriophage or yeast viruses, respectively. These types of viruses are not infectious for humans or animals.

The potential for viral infection of a bacterial cell process or a yeast cell process used for production of a recombinant protein is therefore a business risk, not a patient safety risk. This is the reason why case studies of bacteriophage infection of bacterial fermentation processes or of viral contamination of yeast cell processes have not appeared in the literature (or in the news). Unlike virus contamination of animal cell-based production processes, the events involving bacterial or yeast do not have patient safety implications. When a bacteriophage or yeast virus contamination occurs, manufacturers quietly go about the business of restarting the fermentation. Remediation and prevention of future occurrences is driven primarily by business concerns, as opposed to regulatory concerns.

Manufacturers of recombinant proteins produced using bacterial or yeast cell substrates are not required to conduct cell line viral testing (though phage induction studies may be performed as part of bacterial cell line characterization – to mitigate business risk!), nor are they required to conduct lot-by-lot viral testing of bulk harvest samples. Such requirements are mandated for production processes using animal or human cells. As the title of the document “Quality of Biotechnological Products: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin” indicates, the ICH Q5A (R1) guidance mandating such testing is clearly directed toward manufacturing processes utilizing the types of cells which may support growth of viruses infectious for human or animal cells. This ICH document also provides guidance on the conduct of viral clearance studies.

Viral clearance studies are intended to provide evidence of the ability of downstream purification steps of a manufacturing process to remove or inactivate viruses that potentially may contaminate the process, make their way into final product, and represent a health risk to patients. As such, the evaluation typically involves both relevant viruses (i.e., viruses that are known to contaminate these kinds of processes) and model viruses (i.e., viruses representative of the types of viruses that could contaminate these processes). In the case of bacteria- and yeast-based manufacturing processes, the relevant viruses (bacteriophage and yeast viruses, respectively) are not infectious for humans or animals. In addition, there are no model viruses that are capable of infecting these kinds of cells that are of concern for humans or animals. Therefore, viral clearance studies are typically not required or conducted.

Are there any circumstances where a bacterial or yeast fermentation process could be expected to harbor a virus capable of infecting humans? The only that I can imagine is a process utilizing primary or secondary animal-derived raw materials in rather large quantities. The worry would not be that viral amplification might occur, but that some carryover of surviving animal viruses to the final product might be possible. The use of such animal-derived materials by a manufacturer of a therapeutic protein must be justified based on risk analysis. If substantial risk is introduced by the use of such raw materials, then perhaps a raw material treatment approach would need to be validated.

In the absence of the use of animal derived materials or plant materials not subject to processing steps that would inactivate contaminating animal viruses, incorporation and validation of viral clearance steps into protein production processes using bacteria or yeast cell substrates is not expected, nor would this be of practical value in assuring patient safety.

Small, non-enveloped viruses: number 1 threat to biologics manufacture

by Dr. Ray Nims

Perhaps surprisingly, few types of viruses have infected biologics manufacture since the 1980s when the first recombinant proteins began to be produced in mammalian cells. While the list of contaminating viruses has included some relatively large enveloped and non-enveloped viruses (Reovirus type 2, epizootic hemorrhagic disease virus, Cache Valley virus, human adenovirus), by far the most problematic contaminants have been the small non-enveloped viruses. Why? For the most part, the contaminations involving the larger viruses have been attributed to the use of non-gamma irradiated bovine serum or to operators conducting open vessel manipulations. Remediating the manufacturing processes to include gamma irradiation of the serum (or elimination of the use of serum altogether), and eliminating wherever possible open vessel operations should mitigate the risk of experiencing these viruses.

Now we come to the small non-enveloped viruses, the real problem. Foremost among these has been murine minute virus (MMV). This 20-25 nm non-enveloped parvovirus has infected biologics manufacturing processes using Chinese hamster cell substrates on at least four occasions, affecting at least three different manufacturers (Genentech, Amgen, and Merrimack). In each case, the source of the contamination has been unclear, making remediation of the processes difficult. Due to the ability of these viruses to survive on surfaces and their resistance to inactivation by detergents and solvents, eliminating the agent from contaminated facilities may require drastic measures such as fumigation with vaporous hydrogen peroxide .

A second problem virus is the 27-40 nm non-enveloped calicivirus, vesivirus 2117. This is the virus that was found to have infected the Genzyme Allston manufacturing facility in 2009. The same virus had appeared already once in the past, at a manufacturing facility in Germany. Both of the infected processes involved Chinese hamster production cells and both involved the use of bovine serum at some point in the manufacturing process. Whether or not the animal-derived material was the actual source of the infection was not proven in either case. Unfortunately, if the source was the bovine serum, gamma irradiation probably would not mitigate the risk, as gamma irradiation is less effective for inactivating the smaller non-enveloped viruses. This is another virus that may be able to survive on facility surfaces. As in the case with MMV, ridding a manufacturing facility of vesivirus may require entire facility fumigation with vaporous hydrogen peroxide, as was done at Genzyme.

Another problem virus is the 17-20 nm porcine circovirus that was found to contaminate a rotavirus vaccine in 2010. This virus was thought to have originated in contaminated porcine trypsin used in the manufacturing process. Wouldn’t this contaminant have shown up in the raw material testing done for the trypsin, or in the extensive cell bank testing required for vaccine production substrates? The answer is no. The circovirus would not have been detected using the 9CFR-based detection methods used for trypsin at this time (and at present). And the required testing for cell banks used to produce vaccines would not have detected this particular virus. To make matters worse, gamma irradiation of the trypsin would not be expected to inactivate this virus. How can we mitigate the risk of this virus going forward? As described in a previous posting, manufacturers may need to apply specific nucleic acid tests for the circovirus as part of the raw material release process for trypsin.

These and other small non-enveloped viruses represent the greatest risk for biologics manufacturing because they are more difficult to inactivate in raw materials, and more difficult to eradicate from the facility once infected, and because the source of the infection is not always clear. There must be analogous small-non-enveloped bacteriophage lurking out there that represent, for the same reasons, special threats to the fermentation industry.

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.

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):

Remember....bacteriophage are viruses too

by Dr. Ray Nims

Are you using bacterial cells to produce a biologic? Do not make the mistake of thinking that your upstream process is safe from infection by adventitious viruses. True, you are not required to test for the usual viruses of concern using a lot release adventitious virus assay. But bacterial production systems are susceptible to introduction of viruses just as mammalian cell processes are. In this case, the viruses just happen to be referred to as bacteriophage. Other than this, the putative contaminants have the same nasty property exhibited by viruses that can contaminate mammalian cell processes, that is … their small size (24-200 nm) allows them to readily pass through the filters used to “sterilize” process solutions. So media, buffers, induction agents, vitamin mixes, trace metal mixes, etc. that are fed into the fermenter without proper treatment can introduce a bacteriophage. Especially worrisome in this regard are raw materials that are generated through bacterial fermentation (such as amino acids, antibiotics). A fermenter infected with a lytic phage exhibits a clear signal that the bacterial substrate is unhappy. The trick then is to discover where the phage originated and to mitigate the risk of experiencing it again.

How can you mitigate the risk of experiencing a bacteriophage infection? Many of the same strategies used to protect mammalian cell processes may be applicable to the bacterial fermentation world. Raw materials and/or process solutions may be subjected to gamma-irradiation, to ultraviolet light in the C range, to prolonged heating or to high temperature short time treatment, to viral filtration, etc. In addition, mitigation of risk of bacteriophage contamination may require filtration of incoming gasses using appropriate filters. 

A sampling of the data available on inactivation of bacteriophage by various methods is shown in the table below. The literature is extensive, and as with viral inactivation, the inactivation of phage by certain of the methods (e.g., UVC, gamma-irradiation) may be dependent both upon the matrix in which the phage is suspended as well as the physical properties of the phage (e.g., genome or particle size, strandness, etc.). For fairly dilute aqueous solutions, gamma-irradiation, UVC treatment, or parvovirus filtration should represent effective inactivation/removal methods. HTST at temperatures effective for parvoviruses (102°C, 10 seconds) should be effective for most bacteriophage, although this is an area that needs further exploration.

Mitigating the risk of experiencing a bacteriophage contamination of a bacterial fermentation process is possible if one remembers that bacteriophage are similar to mammalian viruses. Strategies that are effective for small-non-enveloped mammalian viruses (i.e., the worst case for mammalian viruses) should also be effective for most bacteriophage.

A possible exception to this is prophage. In analogy with the presence of endogenous retroviruses in certain mammalian cells (i.e., rodent, human, monkey), there is a possibility of encountering integrated bacteriophage (prophage) in certain bacterial cell lines. Like endogenous retroviruses, prophage may result in the production of infectious particles under certain conditions. This phenomenon deserves some discussion, but this will have to be deferred to a future blog.

References: Purtel et al., 2006; Ward, 1979; Sommer et al., 2001.