By Phage Consultants
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Controlling contamination in bioprocessing
Viral contamination is a serious threat for any type of bioprocessing laboratory or manufacturing facility, even biopharmaceutical facilities run under cGMP standards. Phage Consultants are experts in analyzing causes and sources of contamination and prevention measures.
The problem of viral contaminations has gained a lot of attention in recent times. One high profile example was the viral outbreak that closed down the Genzyme Corp. manufacturing plant in Allston Landing, Massachusetts, for several months in 2009, after the virus strain, Vesivirus 2117, was introduced, probably through a cell culture nutrient.
Although this represented a worst-case scenario, viral contaminations occur relatively often in scientific laboratories as well as in production facilities. They can endanger all types of bacterial cultures and eukaryotic cell cultures.
The consequences range from relatively mild workflow disturbances to total paralysis of the productivity within the facility.
Contamination in bioprocesses tends to be poorly reported, leading to underestimation of risk when calculating the costs of running a business based on the use of microbial or eukaryotic cells.
Under-reporting is due to a common view of contamination as a scientific ‘failure’ invalidating experimental results and thus negating publication. More serious, any admission of contamination problems is usually regarded as commercially suicidal. These two aspects contribute to the fact that the problem is not taken seriously enough by both decision makers and production staff.
Causes of contamination
Various types of fermentation show differing susceptibility to contamination. Fungi fermentations, such as yeasts, are mostly endangered by bacteria while animal-derived cell cultures tend to suffer from viral, bacterial and some fungal contaminations.
Bacteriophages form the most dangerous contaminants in bacteria used for production while other bacteria, especially sporulating ones, also pose a threat. A frequent contamination scenario is that a facility runs problem-free for several years until equipment failure or operator error causes an initial contamination. A single fermentation is lost, but due to a lack of proper treatment, contamination spreads within the facility. This in turn usually means that all weak points, masked so far by lack of contaminant in the facility environment, will be an entry point for contamination to recur. In the worst scenario, the facility will become unable to continue production, with all fermentations contaminated and requiring destruction. Breaking this loop may be difficult when the personnel have little experience in dealing with such a situation.
The source of primary infection is usually not easy to detect, and is often impossible to identify unless the contamination originated from serious equipment failure. The spread of the contaminant following infection may mask the original source of the problem. However, one clear conclusion can be made on the basis of contamination occurrence – there are weak points to be fixed. In the case of some contaminations (sporulating bacteria and bacteriophages) there is a seasonal dependence in the frequency of occurrence, with peaks during spring and autumn. This pattern can be linked with agricultural works, which may greatly increase the amount of contaminants released from the ground and spread widely. A moist atmosphere, caused by rain or dew, for example, will accelerate this process and also accelerates passage of contaminants through the air. Relatively large numbers of contaminants circulating in a moist environment increases likelihood of contamination breaching any barriers protecting the bioprocess. Of course, the better the protection, the lower the probability that contamination will occur. However, at some point, it is usually necessary to balance the costs of protection and the increase in security.
Secondary infections originate from the spread of the causative agent from the area of primary infection in the facility. The entry paths of secondary contamination to the process are usually much harder to eliminate, as a high load and frequent occurrence of the contaminant may increase the chances of penetrating the barriers, which otherwise may have successfully stopped infection. The much higher numbers of contaminant also mean that it is more difficult to eliminate: 1ml of bacterial culture may harbor up to 1013 bacteriophage particles. Spillage of that amount of contaminated material may occur relatively easily and may go unnoticed, but it means that the likelihood of another process becoming infected is raised dramatically. A full-scale outbreak may also be triggered, reducing almost to zero the facility’s ability to run even a single successful fermentation without contamination. Worse still, any material transfer, for example a cell bank, may spread contamination to another facility.
The situation can be further complicated by the contaminant constantly evolving to use the available resources. The majority of fungi, bacteria and viruses, especially bacteriophages, are not well adapted to an environment with a high abundance of resource nutrients, host cells, etc., since such conditions rarely occur in nature. During initial contamination, natural selection favors mutations of the contaminant that make the best use of this extremely rich production environment. These mutants will give more progeny and increase the speed of contamination. Usually, this is observed as a reduction in generation time, and in the case of viruses, an increase in progeny formation ratio and the resulting burst size.
After contamination, sporulating bacteria and drying-resistant viruses tend to survive better in a hostile environment, subjected to drying and various cleaning procedures designed to kill contaminants. The most common bacteriophages causing outbreaks in fermentation facilities are T1-like phages, which usually have a very high resistance to drying.
The most important concern in the majority of facilities is that there are no procedures prepared which can be used in case of viral contamination, or that existing procedures are inadequate.
It is important to have emergency procedures that are easy to implement and focused on reducing the spread of the contamination. Use of these procedures should be provisional, with clearly defined conditions for implementation.
Common problems in recovering from viral contaminations are due to use of disinfectants that do not show sufficient virucidal action or to disinfectants not being used correctly. Other problems include wrong choices of equipment, facility architecture; poor work procedures and badly designed workflow habits.
In some cases, the reason lies outside of the facility. The problems can be caused, for example, by the contamination of raw materials or the facilities position near rich sources of viruses.
Proper procedures are necessary; otherwise personnel have to learn by themselves how to fight the contamination, while trying to maintain productivity. Unaided decontamination can be a very long process with little guarantee of ultimate success. It also distracts personnel from their routine work, and thus the process of recovery from contamination is often accompanied with decreased productivity even in areas not affected directly by virus contamination.
However, it can be shortened considerably, and thus costs of outbreak can be reduced, when help of external experts is used.
If contamination has already occurred, the main task is to prevent its spread in the facility. Collection of a sample of contaminated material from failed fermentations is crucial, but often omitted. This action requires proper precautionary measures to be undertaken, but is necessary to identify the problem. If no sample is taken and stored, it becomes impossible to find out if subsequent infections were due to contamination by a previous contaminant, or if they were caused independently. The proper method of storage for contaminated sample is generally the same as for other samples taken from the process.
Bacterial and fungal contaminations of bioprocesses are relatively easy to prevent. Thus the best strategy is usually based on prevention, with eradication of contamination where it does occur.
With viral contaminants, there is an additional factor to consider since these are most common in the dairy industry that depends on acceptance of the ‘normal’ contamination levels in the facility.
The production and use of phage resistant strains, preferentially in cocktails of different strains with different phage resistance, means that it is possible to obtain a product, even in a heavily contaminated environment, but it requires a constant change of production starter cultures, as phages overcoming strain resistance would be constantly selected.
In the case of the dairy industry, the main problem is the contamination of raw material, which always consists of bacteriophages. However, the phages contaminating dairy industry facilities are also the ones to blame for the majority of production failures.
Sometimes contamination is not easy to recognise – it may manifest itself with a change in oxygen consumption, optical density growth, pH, product formation or foaming when compared to uncontaminated processes. Even a viral contamination of the process may not always give obvious signs, so collecting samples from the processes and performing proper tests is highly recommended. The most recommended option is testing samples from all process runs for the presence of contaminants. Tests should be performed by properly trained personnel, especially when checking for viruses, as obtaining a false negative result in these types of tests is relatively easy, when conditions are not properly prepared.
Even in the best-designed facilities, with advanced equipment perfectly adapted to process, there is always a risk that the personnel will form a weak point exploited by infection. Although not generally the case in the majority of facilities, it is still possible that people running the process have a very limited knowledge of contaminants, the way they may be introduced to the process and the way they can be recognized and eliminated. This makes it less likely and much more complicated to avoid contamination. Even perfect procedures may fail if operators do not understand the meaning of key steps.
Lack of understanding also greatly reduces the probability of rapid recovery after contamination, as it can be spread involuntarily by personnel or not efficiently cleaned. It is very important to understand that the personnel are the first and the last line of the defense against contamination, and proper preparation and education as to how to fulfil this function is absolutely necessary.
Phage Consultants Prevention and Decontamination Services
Phage Consultants has extensive knowledge in recognizing and controlling bacteriophage and other viral contaminations.
The Polish-based CRO is headed by Dr. Marcin Los, who is a recognized world expert in bacteriophages and viral infections. The organization shares this experience with clients to help fight contamination in existing facilities and prevent future infections.
Phage Consultants can also plan step-by-step recovery plans, design of robust SOPs and construction of emergency procedures to be implemented in cases of infection.