Phage Production

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Bacteriophages, also known as phages,are viruses that infect bacteria but areunable to infect higher organisms (seeFigure 1). They are normal componentsof every environment containingbacteria, including soil, fresh and sea water, and sediments. They are also present in animals, includinghumans, where they are an importantcomponent of different microbialcommunities including gut microflora.Although they are subject tophagocytosis and some of them possesmotifs, which allow them to bind tointegrins, they are safe for humans andanimals (1,2). In biopharma, they areusually considered a dangerouscontaminant in both laboratories and inbacteria-based production facilities, asthey can paralyse the productivity ofany facility and, once spread, are usuallyhard to eradicate (3). However, they alsooffer many potential benefits due to afew features that are the result of their make up, which have been re-discovered recently. After decades of being neglected by the vast majorityof the scientific community, in the lastdecade they were the basis for settingup several companies.

Why are BacteriophagesAttractive to Biopharma?

Bacteriophages kill bacteria and arepotentially helpful in replacing oraugmenting antibiotics actions. In orderto kill bacteria, they must first attach tothem, which requires the detection of

A new surge of interest in bacteriophages has led to revivedinvestment in this niche research area despite a history offailed commercialisation. But how can today’s techniquesovercome yesterday’s mistakes?

specific structures on their surface. Thisproperty can be used to construct testsfor diagnostic purposes. Bacteriophagesalso possess highly effective enzymes,which have had to adapt to their rapiddevelopment cycles. One of the mainadvantages is that, due to their compactgenomes, it is very easy to modify them.

Figure 1: Bacteriophage T4 under electron microscopy All images: Phage Consultants

Marcin Los of Phage Consultants

The Age of Phage

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Especially attractive is their ability tomodify proteins that are exposed on thesurface of capsid, which allows for use ofthe very robust technique of phagedisplay. On the top of that, theirproduction is relatively cheap.

Phage Display

This technique allows for easyexpression and selection of peptidesthat bind specifically to the selectedtarget. Expressed peptides may berandomised or they may come from acDNA library. This allows for easy accessto cDNA libraries of antibodiesexpressed in animals such as camels orsharks. Following this, clones are selectedwhich will bind to a given antigen andthe selected antibody can then be easilysequenced and amplified in form of a phage, which expresses variabledomains of antibody on the surface in the form of single chain fragmentvariable (scFv). When selecting scFv fromrandomised phage libraries – contrary to the use of libraries generated fromlymphocytes of animals – it is alsopossible to obtain scFv, which binds totargets that are not immunogenic, suchas metal oxides (4).

Another use of the phage involves theexact opposite – the ability to expressalmost any peptide on the surface of aphage makes it a very good carrier forthe construction of vaccines. Moreover,their physical appearance reflects somemammalian viruses. Thus, when properlyexpressed, the peptides on the surface ofa virus may look exactly the same aswith the real virus.

DNA Vaccines

DNA vaccines are another example of the use of phage in biopharma.The concept relies on the ability ofbacteriophage to deliver DNA to antigenpresenting cells, which have a capabilityto express genes from cassettes insertedinto phage genome. Such an approachleads to a slower build-up of immunity,but the final antibody titers may exceedthose obtained by use of standardvaccines. Moreover, there are noobstacles when using this combinedapproach of a DNA phage vaccine withantigens presented on the capsid

surface. This could potentially lead torapid immunity build up with muchhigher antibodies titers present aftercomplete immunisation (5).

Phage Therapy

Bacteriophages are a major concern in biopharma in cases where theyconstitute a major risk in bacterialfermentation (see Figure 2). However,this feature is beneficial if bacteria are tobe killed. Phages as bactericide havesome drawbacks, for example bacteriamay become resistant to the phage.However, contrary to when usingantibiotics, such resistance is seldomencoded on plasmids and thus is much harder for horizontal transfer.Also, phages can easily evolvecountermeasures in order to bypassbacterial resistance to it (6). In this case,as both sides are biological entitieswhich evolve and can mutate, itbecomes very hard for bacteria to evolveresistance to the phage in a way whichthe phage cannot overcome. Even if sucha situation did arise, it is easy to selectalternate phages to which the bacteriawould have not yet become resistant to.Moreover, phages have a better capacityfor rapid evolution, as they multiply atmuch higher rates, offer a larger amountof progeny (up to several thousandprogeny virions per phage with only two

daughter cells per bacterium), and thusproduction of a mutation overcominghost defence is much more probable.However, if the naturally occurring highrates of evolution among phages are notenough, it is also possible to producephages, which have pre-made variants of receptors and can bind to mutatedversions of the host receptor (7). Suchpre-adapted phages can quicklyovercome mutations in host receptors,which otherwise would block phageadsorption and make bacteria immune.

The use of phage in therapy has been in use longer than sulfonamides. Theirdiscovery was a great, but unfulfilled,promise to be a universal medicineagainst bacterial infections. After initialsuccessful treatments, they later failedwhen used and prepared by unskilledpeople, who did not understand theirnature, and thus their possibilities andlimitations. Rapid commercialisation ofthe phage therapy was unreliable, andthus, when chemotherapy of bacterialinfection became available, the phagetherapy was discontinued. There was anexception from this rule – in Georgiaphage therapy was administered and improved by professionals. Itscommercialisation was not possiblethere, as it was a part of the Soviet Unionat the time. This caused a particular

Figure 2: Electron microghraph of bacterium killed by phage T4

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situation, whereby phage therapy wasinvestigated using scientific methods,without the rush to create a profit out of it.For about 60 years, Georgia became thelast bastion of phage therapy. Moreover, itappeared to be very successful there.Theresults obtained in Georgia, although veryencouraging, lacked a double-blind testregime, and thus they could not be usedfor registration of bacteriophage cocktailsas a therapeutic agent in most countries.This drawback was soon noticed, and newphage therapeutics were put into standardclinical trials.Their effectiveness wasproven under this regime; for example,phage mix appeared to be effective in thetreatment of chronic otitis caused byPseudomonas aeruginosa in humans (8).

What is interesting is that phages do nothave to be considered as a replacementfor antibiotics. They act synergisticallywith some antibiotic classes. The result isthe increase of phage burst size from thecell treated with some antibiotics. Thisallows phages to kill bacteria faster, aswell as increases the effectiveness of antibiotics (9).

Phage Enzymes

Most bacteriophages in their life cycleuse various enzymes in order to enterthe bacterial cell, and then destroy it torelease phage progeny. These enzymesare extremely active, and thus they maybe used for rapid bacteria inactivation.Moreover, these enzymes utilisestructures in bacterial cells, which aredifficult, or even impossible, for bacterialcells to modify. The reason for this is thevery long co-evolution of phages andbacteria, where selected phages targetsuch structures. This field of researchseems to be very promising, as enzymeaction is almost instant (10).

Bacterial Detection and Phage Antibodies

Other bacterial proteins may soon beused in bacteria detection assays. Phage

receptors have proven to be very stable proteins, showing a much higherresistance against unfavourableenvironmental conditions thanantibodies. They have good potential for replacing antibodies in standardbacteria-detection assays and for thispurpose it is possible to use wholephage particles. This approach mimicsphage typing to some extent, whichrelies on the ability of various phages tomultiply on given bacterial strains. Thedifference is that phages are just used to capture bacteria and generate adetectable signal (11). Although theability of native phages to bindspecifically is limited to bacteria, it ispossible to generate and use it in thedetection assay phage antibodies. This is created using the aforementionedphage display technique.

Except for phage typing – a wellestablished method for strainidentification – there are other types ofphage-based detections which utilisethe ability to multiply in host cells. Phagemultiplication can be an indicator of the presence of pathogenic bacterialcells in samples (12). There are a fewapproaches; for example the detectionof phage multiplication by plaque assayor by the incorporation of the luciferasegene into bacteriophage genome, whichmakes bacteria glow upon phageinfection (13,14).

Phage Production

To use phages in any of the previouslymentioned methods, it is necessary tofirst of all produce it. Phage production isrelatively cheap, as it is based primarilyon high density bacterial fermentation. Ittends to be very effective, as icosahedralphages may obtain titers of 1013/ml (15).This offers excellent possibilities, as 108

phages is enough for the construction ofsensitive ELISA-like assays for thedetection of bacterial cells (10). However,phage production is challenging for anumber of reasons. Originating

from the process itself, there is the needfor optimisation in order to obtain highproduction yields. A more problematicaspect can be the behaviour of infectedculture, especially when cultures areinfected by lytic phages that tend tofoam extensively and may causeproblems in the suppression of foaming,causing damage or clogging up theexhaust filter. Some phages areextensively resistant to harsh conditions,and there is the risk of problems inequipment cleaning and sterilisation.Another problem is contamination, whicheasily occurs in such facilities duringmaterial handling, potentially affectingfacility productivity. Damage can rangefrom premature infection in bacterialculture, which leads to sub-optimalproductivity and lower phage yields,through the mixed phage infections,where a contaminating phage developsin culture in parallel with the producedphage. Released phage may also causegeneralised facility contamination,leading to total paralysis of the facility.

The problems with phage productionincreases dramatically during scale-upprocesses. It is much harder to controlfoaming in large fermentation volumes,in the same way that it is harder to

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properly clean and sterilise equipment.Also, the contamination of the facility ismore probable when high volumefermentation is used. However, there aremethods that allow for the productionof relatively high volumes of phagelysate when employing relatively lowvolumes of fermentation. A methodpublished by Sauvageau and Cooperemploys a two-stage fermentationprocess, where the first stage deliversuninfected high density culture to thesecond fermentor where infection andphage propagation occurs (16). This kindof process can be run automatically,greatly improving the effectiveness ofphage production in relatively smallvolumes of fermentation.

Conclusion

Phages are gaining more attention inbiopharma, as well as in related fields, asthey emerge to be a versatile and effectivetool.There is the hope that increasedsuccessful use of phages for our benefitwill arise from our greater understandingof their mechanisms, structure and role in the environment.They seem to be anattractive option for the construction of a broad range of products relevant forhuman and animal health, and thus, theyare more frequently present in R&Dprogrammes. However, due to a relativelack of interest in phage biology in thepast – as well as too few scientists skilledin various phage-related microbiologicalprocedures – there has been, to someextent, a reluctance to employ phages inbiopharma. Despite that, the greatpotential of phage-based technologieshas already started the new age of phage.

References1. Nelstrop AE, Taylor G and Collard P,

Studies on phagocytosis I Antigenclearance studies in rabbits,Immunology 14: pp325-337, 1968

2. Dabrowska K, Zembala M, BoratynskiJ, Switala-Jelen K, Wietrzyk J, OpolskiA, Szczaurska K, Kujawa M,Godlewska J and Gorski A, Hoc proteinregulates the biological effects of T4phage in mammals, Arch Microbiol187: pp489-498, 2007

3. Los M, Contamination concerns,European Biopharmaceutical Review51: pp78-80, 2010

4. Okochi M, Sugita T, Furusawa S,Umetsu M, Adschiri T and HondaH, Peptide array-basedcharacterization and design ofZnO-high affinity peptides,Biotechnology and Bioengineering106: pp845-851, 2010

5. Clark JR, Bartley K, Jepson CD, Craik Vand March JB, Comparison of abacteriophage-delivered DNA vaccineand a commercially availablerecombinant protein vaccine againsthepatitis B, FEMS Immunology &Medical Microbiology 61: pp197-204, 2011

6. Labrie SJ, Samson JE and Moineau S,Bacteriophage resistancemechanisms, Nature ReviewsMicrobiology 8: pp317-327, 2010

7. Pouillot F, Blois H and Iris F, Geneticallyengineered virulent phage banks in thedetection and control of emergentpathogenic bacteria, Biosecurity andBioterrorism: Biodefense Strategy,Practice, and Science 8: pp155-169, 2010

8. Wright A, Hawkins CH, Änggård EE and Harper DR, A controlled clinical trial of a therapeuticbacteriophage preparation in chronicotitis due to antibiotic-resistantPseudomonas aeruginosa; apreliminary report of efficacy, Clinical Otolaryngology 34: pp349-357, 2009

9. Comeau AM, Tétart F, TrojetSN, Prère MF and KrischHM, Phage-antibioticsynergy (PAS): beta-lactamand quinolone antibioticsstimulate virulent phagegrowth, PLoS One, 2: e799, 2007

10. Fischetti VA, Using phagelytic enzymes to controlpathogenic bacteria, BMCOral Health 6 (Suppl 1): S16, 2006

11. Galikowska E, Kunikowska D,Tokarska-Pietrzak E,Dziadziuszko H, Los JM,Golec P, Wegrzyn G and LosM, Specific detection ofSalmonella enterica andEscherichia coli strains by using ELISA withbacteriophages asrecognition

agents, Eur J Clin Microbiol Infect Dis, DOI: 10.1007/s10096-011-1193-2, 2011

12. Kalantri S, Pai M, Pascopella L, Riley L and Reingold A, Bacteriophage-based tests for the detection ofmycobacterium tuberculosis in clinicalspecimens: a systematic review andmeta-analysis, BMC InfectiousDiseases 5: p59, 2005

13. Muzaffar R, Batool S, Aziz F, Naqvi A and Rizvi A, Evaluation of theFASTPlaqueTB assay for directdetection of mycobacteriumtuberculosis in sputum specimens,Int J Tuberc Lung Dis 6: pp635-640, 2002

14. Carrière C, Riska PF, Zimhony O,Kriakov J, Bardarov S, Burns J, ChanJ and Jacobs WR Jr, Conditionallyreplicating luciferase reporterphages: improved sensitivity for rapiddetection and assessment of drugsusceptibility of Mycobacteriumtuberculosis, J Clin Microbiol 35:pp3,232-3,239, 1997

15. Seregant K and Yeo RG, Theproduction of bacteriophage µ2,Biotechnology and Bioengineering 8:pp195-215, 1966

16. Sauvageau D and Cooper DG, Two-stage, self-cycling process for theproduction of bacteriophages,Microbial Cell Factories 9: p81, 2010

About the author

Marcin Los studied at theIntercollegiate Faculty ofBiotechnology of the University ofGdansk, Poland, and the MedicalUniversity of Gdansk in 1995, andearned his MSc at the University ofBradford, UK. In September 1999,

he started his PhD studies in Molecular Biology atthe University of Gdansk and obtained his DScdegree in 2011 on the basis of study on phagedevelopment and detection. He spent one year at theFraunhofer Institute for Silicon Technology in Itzehoe,Germany, as a researcher involved in the constructionof novel virus detection methods, before obtaining aPhD in 2004. Marcin was a Secretary of the MainBoard of Polish Genetic Society and is currentlyemployed as an adjunct professor at the University ofGdansk and adjunct professor at Institute of PhysicalChemistry in Warsaw. He is currently CEO of PhageConsultants. Email: mlos@phageconsultants.com

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