Mycoplasmas – the perfect parasite

Mycoplasmas, one of several genera within the class mollicutes, are the smallest and simplest known free-living and self-replicating forms of life. They are ubiquitous in the environment and are present in man, animals and plants.

They have been described as the perfect parasite because they adapt to their host, on which they depend to supply all their nutritional needs, and generally do little harm. However, they can provoke an immune system response causing the host to damage itself.

Mycoplasmas were first isolated in cattle in Africa in the 1890s as a cause of contagious bovine pleuromonia, and are responsible for a wide range of serious livestock diseases. They are also implicated in a range of autoimmune conditions in humans as well as a form of pneumonia.

There are more than 100 known species of mycoplasmas that are further divided into a number of strains. They are very small, measuring between 125 and 500nm, and physically delicate. Through a process of degenerative evolution up to half of all their genetic material has been lost, retaining just those genes that are essential for cell function and a sophisticated defence system.

The first major change in their evolution was the loss of the gene for creating a cell wall. In its place they have a double or triple layer membrane. They are not host-specific and will take on the appearance of the host in which they find themselves, which makes them hard to detect, while their reliance on the host to provide them with nutrients makes them very hard to culture because it requires the growth conditions in the host to be replicated.

The growth in biopharmaceutical manufacturing has made mycoplasmas a particular cause for concern because they:

• Penetrate 0.2µm “sterilising grade” filters
• Thrive in bioreactors
• Can be found in plants, animals and humans
• Can cause occult infections
• Affect host cell metabolism and expression levels.

It has been estimated that between 5% and 35% of cell cultures currently in use are infected with at least one species of mycoplasma.1

Acholeplasma Laidlawii was isolated from filter-sterilised broths in 1993. During a media fill trial a haze in TSB produced typical mollicute colonies on Tryptone Soya Agar (TSA) and was referred to Mycoplasma Experience, a specialist company concerned solely with mycoplasmas and closely related organisms, for identification.

In 2001 during routine sterility testing of blending vessel, WFI (held at 80°C) was used to produce TSB and A. Laidlawii was isolated at Mycoplasma Experience from 90g of the same TSB powder lot in 3L solution plus supplements and antibacterials to hold back rest of bioburden. And in 2006 in Fluid Thioglycolate Medium A. laidlawii was isolated at Mycoplasma Experience from 90g powder in 3L as before, proving that A. laidlawii was present in the powder.

Mycoplasma contamination in pharma-ceutical manufacturing was largely regarded as a myth until in 2002 US biotech company Genentech revealed a case of contamination in a non-animal alternative to Tryptone Soya Broth (TSB). The medium was prepared from powder and filtered using a 0.2µm filter. Genetech found turbid media that was then inoculated into another manufacturer’s TSB medium – after three days turbidity was again noted.

Microscopic analysis revealed small, uniform particles of an unknown nature. No micro-organisms could be recovered from the turbid media using standard microbiological analysis, and no organisms could be detected using Gram stain.

The broth was turbid 48 hours following preparation and after a media fill at the 7-day inspection. It tested positive for mycoplasma in a mycoplasma cultural detection assay, and PCR and sequencing analysis revealed that the contamination was A. laidlawii. From this, Genentech concluded that:

• the non-animal broth was the source of the contamination;
• mycoplasma contamination could be arising through crop fertilisation, i.e. animal-based fertilisers; and
• that this was a new risk associated with vegetable-derived products.

Since Genentech’s revelation a number of myths have grown up around mycoplasma contamination, according to Alison Smith, pharmaceutical marketing manager at Oxoid and member of the PDA Mycoplasma Task Force:

• Mycoplasma can’t pass through a 0.1µm filter – they can be prevented with a validated 0.1µm filtration, but their lack of a cell wall means that they can change their shape and if the wrong process conditions are applied to the filter it may become ineffective.
• Mycoplasma can’t grow without mammalian cells or added sterols – but A. laidlawii has been shown to grow in TSB.
• Physical changes in cell culture media will indicate mycoplasma contamination – but a lack of haze does not always indicate absence of mycoplasma contamination
• Animal-free media equals a mycoplasma risk – customers moved to non-animal to remove regulatory concerns about animal viruses and TSEs but they initially treated non-animal products as risk-free, not realising that they could have mycoplasmas in either plant- or animal-derived material.

“A. laidlawii contamination has been documented from both plant and animal formulations,” said Smith. “None of this was a new revelation.”

However, she believes the main reason the problem arose at Genentech is related to changes in the fill processing, rather than the change to non-animal broth.

In September 2004 guidance for industry, “Sterile Drug Products Produced by Aseptic Processing, Current Good Manufacturing Practice”, was issued by the FDA, the Centre for Drug Evaluation and Research (CDER), Centre for Biologics Evaluation and Research (CBER) and the Office of Regulatory Affairs (ORA).

The guidelines said that aseptic processing should be validated using microbial growth medium in place of the product, i.e. the media should touch everything that the product comes into contact with, including the bulk non-sterile vessels.

As a result TSB medium is prepared in a non-sterile bulk vessel and passed through the production filter-train. Consequently, users are relying on 0.22µm filters for sterilisation, whereas previously autoclaving was used.

Heat is an effective destroyer of myco-plasma contamination. The population starts to decline at around 40°C and goes into serious decline at 60°C. However, although refrigeration may slow growth, A. laidlawii can grow in TSB at refrigeration temperature, and therefore may grow in refrigerated cell culture media. And although it does not grow in water, it may survive at refrigeration temperatures in purified waters for more than seven days.

Manufacturers unaware

Under the previous guidelines traditional media fill trials had been conducted using traditional TSB (bench specification), heat sterilised at 121°C for 15 minutes, connected directly to the filling line and filled into ampoules and vials.

Unfortunately, said Smith, the media manufacturers were not made aware that customers were making this change in use of the media. Traditional DCM (dehydrated culture medium) bioburden makes it unsuitable for filter sterilisation and unsuitable for entry into formulating areas.

It also has low filterability, which means it may have to be heated to pass through the filter, and the filter may become blocked before the full volume is processed. But the product had never had a filter specification before because nobody had ever asked for it.

The media manufacturers then started to look at where the mycoplasmas originated. The most obvious potential source is the raw materials of both plant and animal origin. But more worryingly it is also possible that they could be part of the bioburden in the processing environment, living in the steamy, wet peptone environment and surviving because they are less frail than was originally supposed.

“We still don’t know if we only find A. laidlawii because it survives as it has lower nutritional requirements, or because it is able to become part of the processing plant bioburden,” Smith pointed out. “As manufacturers we have to come up with a new specification.”

The new media specification should be: low bioburden; mycoplasma-free; cold filterable; with pharmaceutical use-based QC testing. Oxoid’s cold filterable product range offers TSB and non-animal TSB suitable for modern media fill requirements

“Although in the past we have been the cause of problems, now that we are talking to each other and we are starting to understand the needs customers have for media fill trials, we have been able to develop products that can be used with peace of mind,” Smith concluded.

According to Steve Spencer, of the Immunologicals Team and the Veterinary Medicines Directorate, the presence of mycoplasmas may indicate that insufficient care has been taken during vaccine manufacture or quality control.

Extensive testing

Therapeutic products for both human and animal use are required to undergo mycoplasma testing throughout various steps in the production process.

Directive 2001/82/EEC forms the basis of all procedures and requirements. This is supplemented by EudraLex: the Rules Governing Medicinal Products in the European Union – Vol 3: guidelines on medicinal products for human use.

The European Pharmacopoea includes a Mycoplasma Monograph (Monograph 2.6.7 Mycoplasmas) that describes three test methods: culture method, indicator cell method and nucleic acid amplification techniques (NAT). Whichever method is chosen, full validation is required even if it is a recommended Eur. Ph. method.

Millipore is at the leading edge of mycoplasma testing and supplies solutions to prevent mycoplasma by filtration and ready-to-use process equipment. The company provides a validated mycoplasma detection service (MicroSafe Services) and validated mycoplasma clearance (Access Services).

MicroSafe Services is a GMP/GLP compliant contract testing facility for the pharmaceutical and biotechnology sectors that offers microbiological, mycoplasma and virus tests. It carries out both routine and custom-made studies. Based in Leiden in the Netherlands, the site has a Class 10,000 area with isolator for sterility testing; and is a bio safety level 2(+) facility with the ability to handle some grade 3 organisms with modifications.

Its key expertise is mycoplasma testing and it carries out around 40 tests a week. It offers testing using agar broth and indicator cell methods, as well as mollicute screening by QPCR (NAT).

Mollicute screening using NAT is quite a robust assay, according to MicroSafe customised studies manager Sandra Jainandunsing. It complies with Eur. Ph. Guidelines, PDA Technical report no 33 and general guidelines for GLP and GMP. Screening takes as little as six days (four days culture, two days testing), compared with 28 days for assay duration compendial.

Filtration technologies are one possible solution to reducing the risk of mycoplasma contamination in biotech processes, according to Joachim Regel, Millipore’s aseptic technology manager for the European market.

Lack of harmonisation

Unlike other bacteria, mycoplasma have the capacity to deform their shape and thereby to be able to penetrate across a standard 0.22µm rated sterilising grade filter. But in spite of the fact that, by default, the use of 0.1µm SG filters is mandatory, no industry guidance is in place to harmonise the validation of such a solution between suppliers and customers.

A new PDA Guideline Nr 26 revision is due to be released in 2008 that will prevent method-dependent results and inequivalent comparisons in general for filter selection, but still no standards exist for qualifying filters for mycoplasma removal.

Log Reduction Values (LRVs) will also be better normalised by the new guidance, said Regel. This will make it easier to compare claims among filter suppliers. But even so, there will be no standardised method for qualification of process filters and each user of a 0.1µm rated filter will have to determine an individual method for applying and qualifying their own process filter.

Validation should be carried out as close as possible to the actual process conditions and should take into account a range of process conditions, including filtration parameters; duration of the filtration; specified filter type; species of bioburden in the feed streams (mycoplasma type); and the way in which media and supplements are transferred into the process.

Millipore’s solution to detection uses NovaSeptum sterile sampling devices, Mycoplasma Tissue Culture Non-isotopic (MTC-NI) rapid detection assay kits and product release testing by MicroSafe Services.

Millipore’s Access Services offer device validation testing for mycoplasma clearance. These services include validated test methods – PDA TR 26; bacterial retention validation; certified analysts; and from the second quarter of this year mycoplasma clearance validation for A. laidlawii.

A problem shared is a problem halved. Now that the issue of mycoplasma contamination has been brought to light, the industry can bring its combined resources to bear to develop comprehensive solutions to this complex but far from intractable problem.