June 1, 2012 (Vol. 32, No. 11)

Phage Consultants Aims to Prevent Microbial Attacks on Fermentation Systems

Bioprocessing operations face the threat that bacteriophages (viruses that infect bacteria) will infect the cell cultures used to manufacture biotherapeutic proteins in bioreactors. Contamination with bacteriophages, also known as phages, may completely paralyze an operation and even cause a shortage of a biopharmaceutical. Marcin Los, Ph.D., started Phage Consultants to help companies fight and prevent phages from attacking their microbial fermentation systems.

While completing his doctoral studies, Dr. Los published several scientific papers about phages in industrial microbial systems. Researchers at biotechnology companies who read his papers contacted him for help with phage contamination problems. This inspired him to start Phage Consultants in 2007.

The experts at Phage Consultants assist bioprocessing facilities worldwide. About 70% of their clients manufacture biopharmaceuticals or secondary metabolites, such as antibiotics, and 30% are industrial biotechnology firms, such as food, enzyme, and chemical manufacturers. Sometimes a client sends samples from fermentation or a production strain to Poland for analysis, and the problem can be solved remotely. More often, the company’s scientists travel to a customer’s facility to track down the culprit and find solutions.

Phages infect bacterial cells, but not human cells, so contaminated products generally are not a threat to people. However, a phage infection can devastate a biomanufacturing production schedule. Phage infections release proteins that cause foaming and may even clog filters, resulting in the buildup of pressure in bioreactors.

This can lead to leakage and equipment damage, which spreads phages. Just one milliliter of a bacterial fermentation culture can be infected with up to 1013 phages, and spillage of a few milliliters can severely contaminate an entire facility. In the worst cases, production may be forced to shut down, and a biotherapeutic may become unavailable to sell for several months.

Both upstream and downstream bioprocesses can be infected with phages. Phages are nanosized and readily pass through filters used to sterilize process solutions. Media, buffers, induction agents, vitamin mixes, and trace minerals that are fed into bioreactors are all capable of introducing a phage if not properly treated. Particularly worrisome are raw materials generated by bacterial fermentation, such as amino acids and antibiotics.

Drs. Marcin Los and Joanna Los during the phage identification process.

Each Case Unique

The experts at Phage Consultants track down the origin of a phage contamination, eliminate the phage, and develop ways to reduce the risk of a recurrence. Solutions to phage problems are not based on well-defined protocols, off-the-shelf products, or trade secrets. Each case is unique, and Dr. Los’ team uses their experience and expertise to identify the root cause of contamination and the weak points that allow it to spread. The solution depends on the properties of the phage and the site of contamination.

Sources of contamination in a fermentation facility include raw materials, equipment malfunction, improper maintenance of equipment, and operator handling errors. Various methods for inactivation of phages include exposing them to gamma-irradiation or ultraviolet-C irradiation, filtration, or treatment with high-temperatures.

Phage Consultants specialists analyze biomanufacturing sites and select the best cleaning procedures for a contaminant, which vary in their resistance to chemical solvents. “We advise clients on how to clean equipment properly or suggest how to modify procedures and processes,” says Dr. Los. Other solutions may involve engineering bacterial cell lines or manipulating growth conditions to prevent the growth of phages.

Overall, phage contamination is relatively uncommon among companies that manufacture biologics in bioreactors. However, when a phage strikes, these companies “do not have the knowledge and training to troubleshoot phage contamination. We deal with it on a daily basis, so it’s easier for us to fix the problem. No one else offers the scope of services that we do,” says Dr. Los.

E. coli cells lysed by phage under transmission electron microscopy

Expanding Services

Current customers are located in Europe, North America, and Asia, and Dr. Los plans to expand to Australia and South America. The company’s customers range from huge pharmaceutical companies to small startups. When a fermentation facility asks Phage Consultants for help, they are assured that all interactions will be performed with a high level of confidentially. “We sign strict nondisclosure agreements with customers,” Dr. Los reports.

Most companies currently contact Phage Consultants to solve phage problems, but a few request help with preventing phage contamination. Dr. Los believes that preventive services could save money in the long term. Phage Consultants also helps companies that are building new bioprocessing facilities. “If we have the blueprints for a new facility, we can advise them about how to prevent contamination,” he says.

Dr. Los sees a growing need for preventive phage advice as biomanufacturing services, such as contract manufacturing organizations, increase and evolve. In emerging markets like India and China, new facilities are being built with the goal of operating at or near 100% capacity. This likely will increase contamination risk. “People who do microbial fermentation need to pay more attention to the risk of phage contamination,” says Dr. Los.

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