TSB and media fills: Advantages, challenges and considerations

Tryptone soya broth (TSB) is commonly used as a placebo for aseptic process simulations (media fills) as a general-purpose microbiological growth medium (1), in which a broad spectrum of types of microorganisms are expected to survive and multiply.

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Tryptone soy broth is commonly used in aseptic process simulations. But, why use TSB? Is there an alternative? How long should vials be incubated for? These and other questions relating to media fills form the basis of this week’s article.

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What is TSB?

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TSB is equivalent to soyabean casein digest medium and sometimes it is called tryptic soy broth; however described, it is a medium for the isolation and cultivation of non-fastidious and some fastidious microorganisms. Peptone from soybean is frequently used in the preparation of microbiological culture media. Because of its rich nutrient source, the digest is ideal for facilitating growth of a variety of microorganisms. Casein is the major protein in bovine milk.

As well as media fills, this medium is used for sterility testing and as a general growth broth. The medium is referenced in the following pharmacopoeia chapters:

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European Pharmacopeia, 2.6.1 2.6.12 and 2.6.13.        

United States Pharmacopeia, <71> , <61> and <62>        

Japanese Pharmacopeia, 4.06        

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(In other words, the chapters for the sterility test and the microbial limits test).

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The general formula is (2):

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Pancreatic digest of casein (~17g/L)        

Enzymatic digest of soya bean (~3g/L)        

Sodium chloride (~5g/L)        

Dipotassium hydrogen phosphate (~2.5g/L)        

Glucose (~2.5g/L)        

With a pH of 7.3 ± 0.2 @ 25°C.        

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Where there is concern with using animal-derived components, vegetable peptone broth presents a suitable alternative to TSB (3).

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Typical test organisms used to assess media fertility are:

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Bacillus subtilis (ATCC 6633)

Candida albicans (ATCC 10231)

Aspergillus brasilensis (ATCC 16404)

Staphylococcus aureus (ATCC 6538)

Pseudomonas paraeruginosa (ATCC 9027)

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Suitability of TSB

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TSB is a reasonably good all-round, general-purpose microbiological medium, which can support growth of aerobic bacteria when incubated at temperatures in the range 20–35°C (4).

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It is also a reasonably good medium for supporting the growth of yeasts and filamentous fungi, when incubated at 20–25°C.

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Limitations of TSB

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Despite the health laboratory organisms listed above, as used for TSB growth promotion, many microorganisms will not readily grow in TSB and some will not grow at all.

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Generally, TSB is a good recovery medium for Gram-positive and human commensal-type bacteria (5); However, it is not the best recovery medium for all microorganisms (6), including Gram-negative bacteria. The latter grow better with lower nutrient concentrations, and at lower incubation temperatures.

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Neither is TSB the best recovery media for yeasts and filamentous fungi. A mycology specialist would be unlikely to use TSB as the first choice for surveying an environment for fungi.

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Furthermore, it is not the best recovery medium for anaerobic and microaerophilic microorganisms such as the common skin commensal, Cutibacterium acnes.

So why use TSB?

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Why is TSB used so widely if it displays so many limitations? The answer is that it is simply a compromise medium, one commercially available, uncomplicated, easy to manufacture, and robust.

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Due to its longstanding use for the sterility test it is ‘supported’ by the reflected authority of the pharmacopoeias. More importantly, it has become the industry standard.

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Should we be content with TSB?

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In the interests of academic science, it may well be desirable to use a better medium for media fills, or to use distinct types of media for separate media fills. However, in practical terms there is little benefit.

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The media fill is not an exhaustive search for every microorganism that could be contaminating an aseptic process any more than environmental monitoring is. Instead, it is a “snapshot” in time with a recognized and limited “focal range.” The end result is an estimate of process capability and the likelihood of contamination under a given set of (hopefully) representative conditions.

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Alternative media

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Sometimes alternative media are selected (such where there is a risk from anaerobes). Where alternate media are selected such media should possess similar flow characteristics to the product or products that it has been chosen to represent. This is because if the medium does not have these similar characteristics, it might be effectively impossible to simulate the intended process.

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Additionally, the medium must be sterilizable. Radiation is the method of choice for sterilizing media since irradiation is dependable and penetrative through bulk quantities (7). It is also important that the selected medium does not inhibit the growth of microorganisms.

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Reasons to avoid alternative media

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Although other media can be considered, it is generally preferable to use TSB. Here, any inert gas (like carbon dioxide or nitrogen) used to fill or sparge the vial headspace should ideally be disconnected, or, alternatively, compressed air should be substituted for the gas.

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This is because the principle of the use of placebos and culture media is to create conditions where there is the greatest possible likelihood of recovering any contaminants present. Since most contaminants likely to be present in pharmaceutical manufacturing environments metabolize aerobically and the creation of anaerobic conditions in the headspace above the media would decrease the probability of recovering these aerobes.

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Should TSB be prepared in manufacturing areas?

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Some facilities elect to bring in dehydrated culture media into the production facility and prepare it in water and then sterile filter it. The argument for this practice is that by taking dehydrated media through all the stages of dispensing and compounding in production vessels, every potential for contamination of the medium is considered.

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However, dehydrated microbiological media is heavily contaminated with microorganisms reaching levels of around 10^4 colony-forming units (CFU)/g. It is highly unlikely that any product at the final sterilizing stage would have a bioburden anywhere close to this level. Given that the areas of the facility where final formulation and sterile filtration take place need to be tightly restricted and microbiologically controlled (as cleanrooms), what would be the rationale for bringing nonsterile, dehydrated microbiological media into these areas, especially a medium so heavily contaminated? To do so makes a mockery of our enforced controls (8).

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If such a simulation of the filtration process is of value to the media fill, then it is sensible to have the dehydrated media sterilized by radiation (and evaluated to assess the fertility of the medium).

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Another argument against the practice of medium preparation in manufacturing is ‘scope.’ The media fill is intended to detect weaknesses in aseptic processing. Whereas the area of formulation/compounding is intended to be sufficiently clean to prevent increases in contaminants or their byproducts (like endotoxins) resulting from conditions in the manufacturer’s premises, but it is not an aseptic process.

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The media fill should not be seen as an instrument for detection of problems in non-aseptic manufacture; there are other methods based on environmental monitoring and bioburden characterization to achieve this end.

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We can also add the fact that some contend that the media fill is in some way an exact simulation of the process, including the risks associated with sterile filtration. Yet the media fill cannot validate sterile filtration and there are far more challenging and robust assessment criteria for filters.

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Incubation parameters

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Filled units must be incubated as soon as possible after filling. Regulators require that all units are incubated (with the exception of those without caps, obvious cracks, etc.).

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Incubation of media fills is almost universally done for 14 days. This probably originates in the pharmacopoeia sterility tests, although some facilities extend this to 21-days due to concerns over fastidious slow-growing organisms.

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Is this extended time necessary? If the media fill is to be considered as an exhaustive search for potential viable microbial contaminants then the duration of incubation is potentially limitless. For instance, some coryneform bacteria require 28 days or more incubation to produce visible turbidity in TSB.

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Are these organisms likely? Some argue to incubate validation media fills beyond the 14-day period and justify future routine media fill incubation at 14 days or whenever the last contaminant was detected in the extended validation exercise, whichever is the longer.

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Incubation temperature

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There has been some controversy over the temperature of incubation for media fills — 20–25°C or 30–35°C. Any choice will always be open to criticism. Both temperature ranges (and probably some others, too) can be justified.

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Incubation at both temperatures is widely used, but this still leaves the decision over which temperature should be used in the first seven days, and which in the last seven days of incubation (or indeed should there be another pattern?)

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Orientation and volume

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It is usual to incubate the filled units for seven days in their normal orientation, and for seven days upside down. The principle is to ensure that all of the internal surfaces of the container and closure are bathed in media for long enough to allow any adherent contaminants to be resuscitated, recover and grow.

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The amount of media filling each container should be sufficient to reach halfway up the height of the container so that every internal surface is bathed by the medium for at least seven days. This is not always done. This factor should be taken into account when determining how the media fill is conducted.

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Growth promotion

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The first responsibility in the media fill, as a microbiological exercise where the expectation is is to produce “no growth” results, is to ensure that the medium is capable of supporting growth.

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The growth supportiveness of the media should be verified before use. It should also be checked after it has been in contact with the filling equipment and the product containers. This is to ensure that traces of product, antibiotic, detergent, disinfectant, etc. in antimicrobial concentrations have not been passed into the media from any one of these or other production-related sources. Such a test also guards against any inhibitory substance in the vial or container-closure or some other phenomena that might cause the media to become unsuitable (such as excessive aeration).

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Where media fills are incubated at two temperatures, 20–25°C and 30–35°C, it is good practice to replicate the growth promotion test across the two temperature ranges. All test microorganisms should grow profusely in both temperature ranges with seven days’ incubation from an initial inoculum of less than 10 CFU (or, more desirably, 10 to 100 CFU). Separate media samples need to be inoculated with each culture.

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Environmental isolates

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In addition to the sterility test pharmacopoeia media growth support control cultures, many regulatory agencies insist on several isolates from the manufacturing environment being used as a media control.

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The logic applied is that if the TSB is intended to recover microorganisms inhabiting the manufacturing environment, it should be shown to have the ability to support the growth of those environmental microorganisms. Local microorganisms could be frail, injured, disinfectant-damaged, etc. and therefore could be more difficult to recover in TSB compared to the ‘pampered,’ well-nourished subcultures from the laboratory culture collection.

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However, the local environmental isolates used for media controls will have been maintained in a local culture collection for several months (if not for years) and will have recovered from any physiological damage associated with stressful local conditions.

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The discussion around the benefits or otherwise of environmental isolates is reserved for another article.

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The preparation of control cultures should be clearly specified in laboratory documentation, and records of subculturing maintained. It is preferred that working control cultures be separated by no more than five generations from their national or international culture collection origins. This limits the potential for mutation.

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Low inocula must be used in media control, because the intention is to recover microorganisms when they are present only in low numbers (generally between 10 and 100 CFU per inoculum).

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Conclusion

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This article presents the background for the use of TSB in media fills and presents some of the areas for consideration that sterile products pharmaceutical manufacturers need to weigh up in putting together this part of the contamination control strategy.

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References

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1.????? Sandle, T., Leavy, C. and Needham, G. (2012). A Risk Matrix Approach for Media Simulation Trials, Journal of Validation Technology, 18 (4): 70-78

2.????? Polyakov, V.I., Popello, I.A., Grinberg, V. Ya., Tolstoguzov, V.B. (1985) Thermodynamic compatibility of proteins in aqueous media. Part 2. The effect of some physicochemical factors on thermodynamic compatibility of casein and soybean globulin fraction, Nahrung. 29(4):323-33

3.????? Sandle, T. (2018) Microbiological Culture Media: A Complete Guide for Pharmaceutical and Healthcare Manufacturers, DHI/PDA, Bethesda, MD, USA

4.????? Seyfarth, H. (1975) A comparison of the assay regulations for sterility testing between the USP XIX and the European Pharmacopoeia, Zentralbl Bakteriol Orig B., 160 (4-5): 432-42

5.????? Doyle, JE; Mehrhof, WH; Ernst, RR (1968). Limitations of thioglycolate broth as a sterility test medium for materials exposed to gaseous ethylene oxide. Appl Microbiol. 16 (11): 1742–4

6.????? Ishii, M., Matsumoto, Y., Sekimizu, K. Compounds in a particular production lot of tryptic soy broth inhibit Staphylococcus aureus cell growth, Drug Discoveries & Therapeutics, 2015, 9 (3): 178-183

7.????? Tallentire, A., Dwyer, J., Ley, F.J. Microbiological quality control of sterilized products: evaluation of a model relating frequency of contaminated items with increasing radiation treatment. Journal of Applied Bacteriology, 34: 521–534, 1971

8.????? Halls, N. Achieving Sterility in Medical and Pharmaceutical Products. New York: Marcel Dekker, 1994