Enough phage for a farm: How Proteon Pharmaceuticals manufactures phages for livestock farming

Issue 109 | January 15, 2021
19 min read
Capsid and Tail

The Proteon Pharmaceuticals team is using Cellexus CellMakers for optimizing phage production technology. Source: Proteon Pharmaceuticals.

For this week’s feature article, we interviewed Dr. Arkadiusz Wojtasik, a Production Director at Proteon Pharmaceuticals, about how his team uses the Cellexus CellMaker to produce phage for livestock farming.

Name, Arek at Proteon Pharmaceuticals

Dr. Arkadiusz Wojtasik is a Production Director at Proteon Pharmaceuticals. Proteon uses precision biology for microbiome protection to improve animal and human health, increasing environmental sustainability and eliminating the unnecessary use of antibiotics.

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Add your voice to the State of Phage 2020 Survey!

State of Phage 2020 logo

In celebration of our 100th issue of Capsid & Tail, we launched a survey called State of Phage 2020! We created this survey to help us all better understand the phage research community globally, including what kind of phages people are collecting, what methods they’re using, and more. If you work with phages, please fill it out! We’ll compile and share the results in Capsid & Tail in 2021, and repeat the survey annually so we can all follow and share our community’s exciting growth over time.

Thanks so much to the 117 labs who’ve completed the survey, and to those who have shared it!! We’ll be keeping it open a little longer, then sharing results over the course of 2021!

We’d love if you continued sharing it with friends! https://survey.phage.directory/

Take the State of Phage 2020 Survey

What’s New

Paris phage biotech company Eligo Bioscience announced a new agreement worth up to $224M with GlaxoSmithKline, which is aimed at advancing Eligobiotics® for acne treatment/prevention. Eligo employs CRISPR-based phage therapeutics for microbiome modulation.

Biotech newsFunding news

Anushila Chatterjee (University of Colorado School of Medicine) and colleagues published a new paper in PLOS Genetics showing phage infection and sub-lethal antibiotic exposure mediate Enterococcus faecalis type VII secretion system dependent inhibition of bystander bacteria. Read also this perspective on the paper by Felicity Alcock and Tracy Palmer.

PerspectivePhage-host interactionsResearch paper

Fernando Gordillo Altamirano (Monash University, Australia) and colleagues published a new paper in Nature Microbiology showing that phage-resistant Acinetobacter baumannii are resensitized to antimicrobials. They found that phage-resistant strains had diminished fitness in vivo due to loss-of-function mutations in genes related to capsule biosynthesis.

Phage resistancePhage-host interactionsResearch paper

Duhita Sant (Monash University, Australia) and colleagues published a new paper in Nature Ecology & Evolution showing that host diversity slows phage adaptation by selecting generalists over specialists. These findings support the evolutionary mechanisms that favour low-fitness generalists over high-fitness specialists with the addition of more hosts within the community.

Ecology and evolutionPhage-host interactionsResearch paper

Megha Shah (University of Toronto) and colleagues published a new paper in Molecular Cell showing how a phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa.

Quorum sensingResearch paper

Latest Jobs

The Department of Medicine at Baylor College of Medicine is seeking a qualified Researcher at the Professor level. Researchers with research portfolios in genitourinary, respiratory or gastrointestinal pathogens, human immunology and pathogenesis, bacteriophage, or microbiome studies are preferred as they will synergize with Baylor’s outstanding resources, strengths and current funding.

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!

PhageAI 0.7.2 has been released!

What’s new: Life cycle classifier v1.3 to distinguish between virulent and temperate phages (accuracy benchmark); PhageAI as free Python package on GitHub and PyPI; Duplicate samples detection alert in phage display view; “History” tab in phage display view with historical predictions, and more!

PhageAI still offers: a possibility to upload your phage genomes and getting predictions of a lifecycle in less than a few seconds; creating projects and sharing it among your collaborators; building your private repository of bacteriophages; sharing your feedback concerning model predicitions with scientific community; 3D interactive plot tools.

Sign in to PhageAI and feel free to rate our efforts on your social media accounts using #PhageAI tag. You can follow us on Twitter @PhageAI to be up to date with the PhageAI roadmap.

AIBioinformatics Tool

The fifth iVoM event will be Thursday, January 21, 4pm GMT, and the theme will be “Agro-food, veterinary and environmental biotechnology applications”.

It will feature talks by:
Sam R. Nugen - Department of Food Science and Technology, Cornell University
Danish J. Malik - Chemical Engineering Department, Loughborough University
Ana Oliveira - Centre of Biological Engineering, University of Minho

Chairs:
Mathias Middelboe - Department of Biology, Marine Biological Section, University of Copenhagen
Lone Brøndsted - Department of Veterinary and Animal Sciences, University of Copenhagen

Register for this event and/or the whole series at https://ivom.phage.directory.

Virtual EventViruses of microbesiVoM

International Bacteriophage Research Consortium on Twitter: "IBRC springs into action with its lecture series - 2021. We have Prof. Paul Turner onboard with us! An expert in steering phages and INSeq mutagenesis, Prof. Turner is an established authority in bio-therapeutic application of phages. Save the date : Jan 27, 2021. Stay tuned!

Virtual Event

Mayo Clinic is hosting a webinar on machine learning in phage therapy Jan 27 1PM MST. Register here. Featured Speaker: Cedric Lood, Researcher-Arenberg Doctoral Program at KU Leuven University-Belgium. Gina Suh, M.D. and William Faubion, M.D. from Mayo Clinic will moderate.

Machine learningPhage TherapyVirtual Event

We had a great time kicking off PHAVES for 2021 this week — thanks to everyone who attended! We had an awesome casual chat with phage researchers around the world, heard about what people are working on, what they’re proud of from 2020, and what they’re excited about for next year! The new casual format was a big hit, so we’ll likely be hosting more of these this year.

Let us know your thoughts, or if you have suggestions on other ways we can experiment with the virtual medium, or would like to recommend a speaker for this year!

Register for the PHAVES series here.

Virtual EventRecap

Fantastic turnout for the first Africa Phage Forum webinar this week — thanks to the ~70 attendees who were there, and thanks to Dr. Paul Turner who gave a thought-provoking talk and extensive Q&A!

Sign up for future Africa Phage Forum events here. Next speaker: Dr. Evelien Adriaenssens on Feb 15, who will speak about: Basics of phage genome annotation and classification - how to get started.

There will soon be an Africa Phage Forum YouTube channel where talks will be posted — stay tuned! Also, join their What’s App to keep up.

Virtual EventRecap

Enough phage for a farm: How Proteon Pharmaceuticals manufactures phages for livestock farming

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Phage microbiologist and co-founder of Phage Directory
Co-founder
Phage Directory, Atlanta, GA, United States

Jessica Sacher is a co-founder of Phage Directory and has a Ph.D in Microbiology and Biotechnology from the University of Alberta.

For Phage Directory, she takes care of the science, writing, communications, and business aspects.

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Arkadiusz Wojtasik is a Production Director at Proteon Pharmaceuticals, an innovative biotechnological company committed to improving animal and human health. He leads day-to-day production of phage products and veterinary autovaccines, and supervises a qualified team consisted of biotechnologists and production specialists.

Arkadiusz co-developed several products, successfully built up a pilot-scale phage products manufacturing line from the foundation and launched dedicated production site. He manages design and scale up of new bacteriophage cocktail production capability and is responsible for developing new technologies to efficiently and effectively produce bacteriophages.

Phage manufacturing is a known bottleneck for the phage field, so it was great to chat with Dr. Arkadiusz Wojtasik, Production Director at the Polish biotech company Proteon Pharmaceuticals, about this topic. We discussed his experience with phage production, how he’s worked to overcome its associated challenges, and his experience using the Cellexus CellMaker to produce phages for their animal feed additive products.

Jessica: Let’s start with your background — how did you come to work at Proteon? What’s your role there?

Arkadiusz: I graduated with a degree in microbiology and microbial genetics, and then I did my PhD in biotechnology. After that, I worked for several years as a scientist in the Polish Academy of Sciences, where I developed my skills in microbiology, genetics, and bioinformatics. And in 2009, I got the chance to join an early stage biotech company, Proteon Pharmaceuticals, which was aiming to develop phage therapy products. I recognized that phage-based antibacterials have a real future. My role was to do deep genetic and bioinformatics analysis of phages, to support the selection of the best phages for our products. At that time, work on phages at Proteon was concentrated in the laboratory. It was only a few years later that we had the need to develop phage production technologies. Since the goal of industrial scale production was new, we built our approach to phage production from scratch. Based on skills I had acquired during my PhD studies, I took this responsibility and now I work as Production Director, managing the team, process, supplies, and the further development of Proteon’s phage production path.

J: And what does Proteon do with phages? What kinds of phages are you working on, and what kind of applications are you working towards?

A: Proteon uses precision biology for microbiome protection. Our goal is to improve animal and human health, increase environmental sustainability and eliminate the unnecessary use of antibiotics. We use natural and safe solutions developed from our patented phage-platform technology. We aim to cooperate with farmers in the field of animal health, focusing on solutions that improve the economic efficiency of their farms, and also enable ecologically friendly solutions. Since we focus on the livestock industry, our first product, BAFASAL, eliminates Salmonella on the poultry farm. This product is already registered in India and Southeast Asia, and we are at the final steps of the registration process in Europe, North and South America.

J: How was the process for registering your phage products as feed additives?

A: Our products are novel applications everywhere that we register. So it takes a lot of work. For instance, I think we are the first company in Europe that wants to register a phage product as a feed additive. There are questions about how to classify that kind of product, what tests need to be done and things like that. We have had enough success that we are starting to register our other products. We have an aquaculture product, BAFADOR, which prevents and eliminates opportunistic infections in aquaculture, as well as BAFACOL, to combat APEC strains (avian pathogenic E. coli) in poultry. Both are also feed additives and are being registered in selected markets around the world.

J: How much phage are you regularly producing?

A: We can produce a large volume of phages. We are manufacturing phages in lines with the smaller 8-L Cellexus CellMakers and we also have a process line, which is still in the developing stages, where we use 50-L Cellexus machines. I cannot share exact volumes, but when we take into consideration the upstream and downstream processing, we use a significant amount of this capacity.

J: What kind of challenges have been coming up for your phage production?

A: I think the biggest challenge of phage production is to prevent cross-contamination with other phages. Proteon products are all cocktails consisting of several phages. Usually, some phages cover one part of our target production strains and others cover some other part. But sometimes, different phages target the same strains, so more than one phage may be active against a given production strain. This is why cross-contamination can be a serious challenge. The key, in my opinion, is to be extremely careful in all possible fields of operation, and I think one way is to use disposable technology. Cellexus offers that kind of technology, which helps to avoid cross-contamination.

J: That makes sense. So you always produce each phage separately even if they’re going to be combined in a cocktail?

A: Yeah, each phage should be amplified in separate cultures. And, of course, if we want to have a cocktail, we need to make several separate cultures. But the most important way to avoid cross-contamination is to not produce two similar phages one after the other. Of course this may sound difficult, but it’s not. Especially if you are preparing three or four products, as we do, with different phages, you can alternate the phages, and it works.

J: I see. So they all have different host strains that you’re propagating them on. Is that right? Or is there some crossover in the strains you end up using?

A: It’s different every time. Sometimes every phage has separate host ranges, other times several phages have the same host range. It depends on the product.

J: Got it. So you brought in the Cellexus CellMaker specifically to tackle this cross-contamination problem?

A: Yes, we launched the first CellMaker system seven years ago, when I was searching for a solution for phage amplification using disposable technology. At that time, it was very difficult to find a proper device that would ensure effective multiplication of microbes. The market offered mainly bioreactors for cell culture, and most were rather ineffective in aeration, because cell cultures don’t need that. But Cellexus put in an ‘airlift’ technology, and I think this was very important. This is quite a different approach compared to most known bioreactor manufacturers, but this solution actually gives the possibility of multiplication of bacteria and thus phages. Of course the market is changing, but we started with Cellexus, and the CellMaker is very effective, so we continue to use these machines.

J: Are there other ways that the CellMaker has helped you?

A: Yes, the system is quite simple and it allows for a quick transfer of production from laboratory flask to bioreactor. However, it all depends on the phage strain. If it can be easily optimized in a flask, it will be possible to achieve quick success in the bioreactors, I think. And despite the fact that investments related to the purchase of the bags are necessary, the disposable bag technology lets us avoid many problems related to sterilization and cleaning the machines. In this way, we not only reduce the risk of cross-contamination, but also the risk of bacterial contamination of the operators, which is important when working with microbial pathogens.

J: What other potential pitfalls do you face when scaling up phage production? Do you have issues getting titers high enough, for instance?

A: Amplification is one thing, and you can search for solutions, like changing the media, optimizing the parameters, or culturing for longer, but the bioreactor culture is not the only thing that can be optimized. We have also discovered that the process of purification is also very sensitive. In the case of E. coli, you tend to lose phages using typical methods of purification. So every phage is individual, and although you may rely on your previous experience, it’s not the only important thing.

J: So how easy is it to set this up? If you’re ordering a new CellMaker in your lab, would you need a lot of background and experience in production, or is it pretty plug-and-play?

A: If someone had no experience in working with bioreactors, a quick startup of any bioreactor will of course not be easy, but it is worth noting that the CellMaker doesn’t require additional systems like sources of water, aeration, or temperature. You can connect the bioreactors and start to use them in any laboratory. So I think technically speaking, it is easier to start work with the CellMaker.

J: Do you have any tips for someone considering ordering the CellMaker for their phage work? Would you say it’s a good option?

A: Of course, I would recommend trying it. We are also pleased of course that both Cellexus’ devices and bags can be purchased quite economically, though if you have experience in bioreactor cultures, remember that working with the airlift technology can generate some changes in processes that you may have already optimized.

Another very important thing to note, in my opinion, is that Cellexus offers the Regular system, a simple version of the bioreactor without the possibility of pH, or DO control, which is really quite inexpensive. But in fact, in the case of some of our phages, we didn’t need to monitor these parameters at all. In contrast, for other manufacturers, the removal of pH or DO sensor is treated as a custom product, so it’s not cheaper, it’s actually much more expensive than the regular one! That is not very customer-friendly. So that might be another tip for laboratories: you may not need all the bells and whistles of advanced manufacturing — the Cellexus bag has a cheaper option that can be suitable. Of course if you want to work with bacteria, and you need precision control of pH, you need to use the more sophisticated option — the CellMaker Plus.

J: With regards to Proteon’s upcoming directions, what are you excited about?

A: I’m glad that we are still developing new phage products. We are developing products to combat mastitis in dairy cows, as well as to combat infections caused by bacteria like Campylobacter or Vibrio. Of course, I’m very excited about how our production will develop, and what we will need to upgrade for those kinds of cultures. But my biggest challenge is that we are expanding our manufacturing footprint because we need to build our capacity further to meet current and future demand.

J: Are farmers excited about your products? And have they found them to be working?

A: Yes, Proteon has had a lot of interest and our clients have had great results. One challenge is that phage products are really new, and not everybody understands how to use them. Some farmers think they can use them like antibiotics, but it’s not true. So we give them support, show them how to reduce antibiotic usage and take advantage of our products. As we do this, we also get feedback and it helps us better support farmers. So we see that our products are valuable and helpful and also that our dialogue with clients helps everyone learn.

J: Proteon seems to be taking exciting steps forward with phage bioinformatics and AI. How is that going, and how does that integrate with the rest of your activities?

A: Bioinformatics and AI are valuable. We use and develop both as tools for a number of reasons, for instance to help assess whether a given phage is safe, whether it is lysogenic or not, which can be very difficult if the phage’s genes are not similar to known genes. The AI tool we developed, Phage.ai, lets you compare phage genetic sequences with databases and find out if it is likely to be lysogenic or lytic. We see this as an important obstacle for commercial development and have made this platform free for use to the research community because we want other scientists to join us in testing a larger number of phages. With more phages, the machine learns even more and enables the expansion of phage understanding.

J: Awesome. I think it’s so cool what you’re doing, and so encouraging to hear that you’re already at the stage of working with farmers, and that you have this AI angle — I like how it incorporates data from the community too, because that just seems like the smartest way to do it.

A: It’s very nice to hear that. Thank you.

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Thanks to Stephanie Lynch for her work writing summaries for the What’s New section this week!

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