Producing phages consistently: then and now

Issue 181 | June 24, 2022
17 min read
Capsid and Tail

Source: Jafral

This week, Kyle Jackson tackles phage manufacturing. He first enlightens us on how phages have historically been produced and preserved. Then, through a chat with Frenk Smrekar and Barbara Hubad, we learn how JAFRAL helps companies and university labs consistently produce safe phages today.

This is an article in Kyle Jackson’s ‘Making your company go viral’ series, where he explores phage biotech companies and interviews the people behind them. Check out his past articles on Adaptive Phage Therapeutics, Cytophage and BiomX.

Sponsor

JAFRAL Sponsorship Logo

Need help with phage manufacturing, and looking for an outsourcing partner? JAFRAL manufactures phages to the highest quality and purity, and develops manufacturing processes and analytical methods for various phage types and phage applications.

JAFRAL is the world’s only CMO/CRO that focuses primarily on production of non-GMP and GMP phages.

More info at https://www.jafral.com or [email protected]

Urgent June 23, 2022

Urgent need for Serratia phages for a patient in the USA

Phage Therapy

We are urgently seeking Serratia phages for a patient in the USA

Ways to help at this stage:

  • By sending your phages for testing on the patient’s strain
  • By receiving the strain and testing your phages
  • By receiving the strain and using it to search for new phages against the organism
  • By offering to prepare phages supplied by others to clinical grade
  • By helping spread the word about this request
  • By providing us with names/email addresses of labs you think we should contact

Please email [email protected] if you can help in any way, or if you would like further details/clarification.

Let’s make a difference,
Phage Directory

What’s New

Paper: Compassionate-use of phages in twenty patients with drug-resistant mycobacterial disease.

Phage TherapyResearch paper

Paper: Reversible bacteriophage resistance by shedding the bacterial cell wall.

Phage resistanceResearch paper

Paper: Assessment of phage-mediated inhibition and removal of multidrug-resistant Pseudomonas aeruginosa biofilm on medical implants.

MDR bacteriaPhages in MedicineResearch paper

Paper: Metagenomic analysis of wastewater phageome from a University Hospital in Turkey.

MetagenomicsPhageomeResearch paper

Paper: Monomodular phage JG004 lysozyme (Pae87) contains a bacterial surface-active antimicrobial peptide-like region and a possible substrate-binding subdomain.

Research paperStructural Biology

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Research Lead: Join BioNTech’s team in Vienna (Austria) as Research Lead to work on the early endolysin pipeline! In this position you can advance your career of leadership in science, push the boundaries of synthetic biology and work on the next frontier of antimicrobial therapy.
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PhD Student at CNRS, France to explore the functionality of Vibrio cholerae chromosome dimer resolution sites.
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Research Fellowship in Braga, Portugal to work on the R&D project, PhageShaper — a high throughput platform to engineer P. aeruginosa phages for infectious disease control.
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Postdoc at UNC Chapel Hill to study the cell wall biogenesis of mycobacteria and to explore the potential of treating mycobacteria with bacteriophages.
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Assistant/Associate/Full Professor at University of Florida Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition. Candidates should have a strong research record of publication in bacteriophage biology and applications in foods and the clinical setting, bacterial resistance, as well as a background in the study of bacterial interaction in the intestine.
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Adjunct Faculty, Biology: The Department of Biology at Gonzaga University in Spokane, Washington, is seeking instructors to teach intro lab and lecture courses (incl. inquiry-based phage discovery laboratory course for first-year students).

Community Board

Anyone can post a message to the phage community — and it could be anything from collaboration requests, post-doc searches, sequencing help — just ask!

Webinar: Embracing the Australian Pathway to Phage Therapy in Canada: Connect Globally, Act Locally.

Hosted by AMMI (Association of Medical Microbiology and Infectious Disease Canada)

Tuesday, June 28th at 6pm - 7pm EDT Ottawa (Wednesday, June 29th at 8am - 9am AEST Sydney).

Join the AMMI Canada Phage Therapy Working Group and invited speakers, Dr. Jon Iredell and team to illustrate how Phage Australia was created and developed research strengths and opportunities.

Joining the discussion are Jessica Sacher and Jan Zheng from Phage Directory, a data partner with Phage Australia, on how anyone, anywhere can connect on phage therapy.

A Q&A period will follow the presentation, tying in Canada’s unique capabilities.

Register here.

Phage TherapyVirtual Event

We warmly invite you to attend you to attend our 12th annual Oxford bacteriophage conference – the longest running phage conferences series in Europe – which will be held on 05-06 September 2022.

We are committed to holding this conference as a ‘virtual-in-person’ hybrid event at our traditional venue of St Hilda’s College, Oxford, UK (COVID-19 situation permitting).

Get involved in shaping the conference agenda, which will include most aspects of bacteriophage research and application in medicine, food and biotechnology. We welcome proposals for speakers and topics.

We invite emerging researchers, including final-year doctoral students and early-/mid-career postdoctoral researchers/junior group leaders to play an active role in putting together ECR symposia on topics of their interest in the field, and to apply to present.

Please email [email protected] with any questions.

ConferencePhage biology

The Ibadan Bacteriophage Research Team (IBRT) is setting up a Twitter space event to discuss the interlink between biomedical research and programming languages via the team’s Twitter handle. Topic: The Confluence: Programming languages and Biomedical Research Date: 01st July, 2022. Time: 6pm Nigerian time (5pm GMT). Venue: Twitter Spaces: @IbadanPhageTeam

Phage TwitterVirtual Event

Producing phages consistently: then and now

Profile Image
PhD Candidate
McMaster University, Hamilton, Canada
Skills

Molecular Biology, Biotechnology, Phage isolation, Phage Therapy, Phage-host interactions

Graduated from McMaster University with a BHSc in Biomedical Discovery and Commercialization. Specialized in strategic business development focusing on novel biologic therapeutics and translational research. I have consulted for several Life Sciences startup companies compiling market intelligence and advising them on their commercialization and regulatory pathways.

My research explores the use of phage therapeutics to combat microbiome-based infections. I investigate phage-based methods of modulating the gut microbiome to prevent opportunistic pathogens from colonizing and inducing inflammation. It is my goal to develop comprehensive preclinical models to assess the efficacy of phage therapy candidates for use in the gut microbiome environment.

Establishing reliable manufacturing and purification procedures to produce modern biological drugs compounds will be crucial if we are to see wider adoption of next generation treatment platforms, including bacteriophage therapy.

For bacteriophage therapy to garner mainstream clinical adoption, like its biologic-based drug brethren like antibodies, it too needs to overcome the barrier of large-scale manufacturing and purification. More specifically, phage therapeutic solutions will need to be pharmaceutical grade bacteriophages and strictly adhere to Good Manufacturing Practices (GMP), which are then approved by regulatory bodies.

Defining Good Manufacturing Practices

The American Food and Drug Administration, like Health Canada and the European Medicine Agency, summarizes the cGMP regulations (“c” standing for “current”) as “[the] minimum requirements for the methods, facilities, and controls used in manufacturing, processing, and packing of a drug product. The regulations make sure that a product is safe for use, and that it has the ingredients and strength it claims to have.”

GMP can be summarized by the following three points:

(i) Defines quality measures for production, process, and product quality control (QC).
(ii) Ensures that processes are clearly defined, validated, reviewed, and documented.
(iii) Guarantees that the personnel, premises, and materials are suitable for the production.

GMP also covers responsibilities for distribution, contract manufacturing, and testing, as well as product defects and complaints. Some requirements are specific to biological medicinal products.

While the exact rules and regulations for pharmaceutical grade manufacturing and purification of biological compounds may differ from region to region based on regulatory guidelines, the prevailing three rules outlined above lay the foundation.

A brief overview of bacteriophage manufacturing in the early 20th century

Felix d’Hérelle first reported the use of phage solutions to treat dysentery and cholera in 1922. These solutions did not adhere to the cGMP as they were considered polyvalent solutions (a solution containing a heterogenous mixture of phages with little-to-no characterization other than host-infectivity profile). A secondary issue was that these solutions presumably contained high levels of contaminating proteins, which was raised by Polish surgeon Jasieński in 1927. The lack of standards to produce pure phage solutions persisted until the early 1950s and led to the production of consistently inefficacious batches, which may have contributed to the lack of wider adoption in the mid 20th century!

The first hypothesized fractional ultrafiltration purification method for phages was demonstrated by Kramer in 1927. He used colloidal membrane to retain the phages but demonstrated that the concentrated solutions were labile and lost their bioactivity after a few days. Despite these setbacks, filtrations procedures through these membranes of various pore sizes led to the first estimates of phage particles being approximately 2 nm in size.

Shortly thereafter, in the 1930s, questions regarding bacteriophage stability began circulating resulting from the observations reported by Kramer. In 1935, Flosdorf and Mudd experimentally tried to improve bacteriophage stability through lyophilization (freeze drying was already an established method to preserve blood plasma, food, and viruses – for vaccines). A few years later in 1939, Northrop described a method of concentrating phage lysates through heated vacuum distillation. In the early 1940s, Schade and Caroline combined the preservations and concentrations methods previously described into a holistic manufacturing process. In doing so, they began assessing how lyophilization efficiency was impacted by pH, salt concentrations, and protective agents. In 1944, they showed that their freeze-dried phage mixture was stable at 37°C for 14 months without any titer loss!

In 1923, the George Eliava Institute of Bacteriophages, Microbiology and Virology was founded and became the centre for large-scale phage manufacturing processes later in the century. As part of the Soviet Ministry of Health, the institute became the largest producer of pharmaceutical grade bacteriophage preparations and, at its peak, was producing two tons of phages annually in the late 20th century. Since 80% of the production from the Eliava institute went to the Soviet Army, little is publicly known about its historical manufacturing procedures.

Spearheading bacteriophage manufacturing in a modern world

Modern standards for GMP have presented many barriers to entry for novel technology platforms, including gene therapy and biological inhibitors like antibodies. Bacteriophages are not immune to these barriers, and present their own challenges resulting from the explosive release of viral progeny. The high standards for modern GMP represent one of several obstacles preventing phage therapy from garnering widespread clinical adoption. To solve this issue, the establishment of a universal GMP to produce phage therapeutics will be crucial if phage therapy is ever to be widely adopted for global consumption.

To this end, Slovenia-based biologic manufacturer JAFRAL has sought to position itself as the key provider of GMP phage. In doing so, they have quickly become known as the global leader in manufacturing pharmaceutical grade bacteriophage solutions.

Founded in 2011, JAFRAL became the first biologic pharmaceutical manufacturer to focus solely on the production of bacteriophages. Led by Dr. Frenk Smrekar (CEO) and Dr. Barbara Hubad (COO), JAFRAL’s mission is simple: to provide purified phage solutions to consumers that are both reliable and economical.

“Even if you have the best drug, you need to figure out how to produce it, otherwise you won’t have a drug”, says Smrekar.

JAFRAL positions itself as a third-party entity that assist companies (large and small) in producing reliable and affordable phage solutions under established GMP. They set themselves apart from other manufacturers in that they are the only contract manufacturing organization (CMO) in the world that focuses on phage manufacturing under GMP protocols.

“The lack of [phage-focused] CMO companies presented us with a really unique opportunity… the focus of which is to help other groups produce [pharmaceutical grade] phage solutions.”

These protocols have enabled JAFRAL to establish robust manufacturing capabilities and have allowed them to work with companies across the world. In doing so, this has allowed JAFRAL to institute stringent internal manufacturing standards that satisfy the requirements of distinct regulatory bodies including Health Canada, the FDA, and the EMA.

“We started off as researchers, but we knew that we wouldn’t be able to do it all. So, we built our technology [leveraging] a wide knowledge net,” explains Smreker. “This will ultimately benefit everyone… as we’re able to decrease the cost of manufacturing for everyone.”

Of significant note, JAFRAL is actively producing phage solutions for pre-clinical and clinical trial development. To date, JAFRAL has created over 30 GMP cell banks and 50 phage banks, as well as more than 30 drug substances (ie. monophages) and 8 drug products (ie. phage cocktails) that have been used in clinical trials. Four of their products are currently being used in ongoing FDA-approved trials.

It is the hope of JAFRAL’s leadership team, that the position they have taken to spearhead the next generation of bacteriophage manufacturing practices will allow the therapeutic practice of applying phages to become mainstream again. A goal that is becoming ever more likely due to rising cases in antimicrobial resistance (AMR) and a shift towards personalized medicine.

As JAFRAL expands its partnerships with key players in the therapeutic space, it will continue to improve the chances of successful clinical outcomes for patients using phage therapy.

“More manufacturing capabilities means more clinical trials, which statistically means [more successful] clinical trial outcomes”.

Developing greater manufacturing capacities at the right time

The resurgence in phage therapy comes at a pivotal crossroads in modern medicine. We are seeing increasing rates of AMR that are already being felt by healthcare systems, particularly those in low and middle income regions including central Africa and Southeast Asia.

The ongoing COVID-19 pandemic has exacerbated the AMR crisis. The World Health Organization has reported increased rates of prescription of antibiotics over the last two years. Specifically, one report out of India reported that physicians were over prescribing antibiotics to treat the pneumonia-like symptoms accompanying a COVID-19 infection, despite the lack of evidence of a bacterial infection.

Notwithstanding the surge in AMR cases, we are also seeing an institutional shift towards personalized medicine approaches that were not seen at the advent of conventional antibiotics treatments. This shift presents a further challenge for manufacturers. Conventional antibiotics are often broad-spectrum compounds that are multipurpose and are not patient-focused. For that reason, they are less expensive to manufacture.

Phage therapy has often been touted as a solution due to its intrinsic personalized medicine capacity. But, with western health systems so dependent on the broad spectrum use of antimicrobial agents, it will be hard to transition away from this decades-long approach. Recently, there has been growing interest to suggest that phages have synergistic tendencies when paired with convention antibiotic compounds. This potential for broader phage therapy applications, apart from a personalized approach, will do well to assist phage clinical adoption and push forward manufacturing capabilities, a sentiment shared by JAFRAL.

“There is a future for both [personalized and broad-spectrum] approaches with phage therapy and its accompanying manufacturing. We should also look at unique pairings [of conventional antibiotics and phage] as both will benefit from the other.”

While phages are still relatively inexpensive to mass produce compared to other biologics, this shift towards personalized medicine will nonetheless be accompanied by increased costs in manufacturing.

“At the moment, we are focusing on the larger scale, but we’re also trying to help individuals create personalized phage solutions. At the end of the day, personalized phage solutions will benefit from conventional GMP because you need that knowledge to produce something efficiently and at a good quality. To get that knowledge, we need to start with the larger scale, which will lead to more cost-effective and efficient personalized manufacturing.”

Enabling academics and small companies to focus on their research

Not only does JAFRAL focus on providing high quality phage solutions to established companies all over the world, but they also take pride in working with academic-based research groups who are looking to enrich newly discovered phages and scale them to ensure reproducibility.

“We started as scientists — even though we have big budget GMP products, we really wanted to also help the smaller start-ups or university groups and try to help them through all the steps… this is something I’m not aware of any other CMO doing.”

The goal of this initiative is to ensure that smaller companies and academic research groups do not need to worry about the intrinsic batch-to-batch variation often seen in traditional laboratory-based phage propagation. Additionally, this style of partnership allows researchers to worry less about the presence of endotoxins, and other contaminants, that are present in these solutions.

The result of this unique philosophy is that JAFRAL positions itself as a bridge for researchers to scale their operations, enabling them to focus on the key scientific variables being studied, and not the confounding variables that may result from inconsistent manufacturing practices. These considerations are even more important when researchers are pursuing pre-clinical animal testing and small-scale compassionate use or clinical trials.

A diversified manufacturing portfolio

Although JAFRAL prides itself on being a world leader in bacteriophage manufacturing, they are equally talented in producing plasmid DNA and recombinant proteins, both of which have demonstrated immense potential in biomedical applications including vaccines and gene therapy.

“We learned quickly that the skillsets that we developed for producing bacteriophages could also be applied to other biologics. This has allowed us to [diversify our ability] to produce other biologics, which has far-reaching applications” explains Hubad.

Recombinant DNA technology has seen a boom in popularity in recent years and has immense potential, including within the bacteriophage research ecosystem. As emerging industries like synthetic biology continue to be explored, having the capacity to manufacture the building blocks of life will become ever more important.

Notably, there has been a push by companies and academic institutions to employ synthetic biology techniques to engineer bacteriophages for specific purposes. It is abundantly clear that JAFRAL is already primed to support these endeavours and help usher in the manufacturing of next-generation biologics at both a personalized and mass scale.

Learn more

Check out JAFRAL’s website


Thanks so much to JAFRAL for being a consistent sponsor of Capsid & Tail since the beginning!

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