Bacteriophage Manufacturing: From Early Twentieth-Century Processes to Current GMP

  • Krzysztof Regulski
  • Patrick Champion-Arnaud
  • Jérôme GabardEmail author
Living reference work entry


Recent clinical progress in the field of phage therapy has led to an increased demand for pharmaceutical grade bacteriophages, manufactured under Good Manufacturing Practices (GMP) conditions and approved by regulatory agencies. However, even if the development of a common standardized process for phage production seems rational, there are no guidelines to pave to way for manufacturing. This chapter reviews phage therapy through the lens of modern medicinal guidelines. It lists manufacturing strategies that can be used for phage production. Finally, it stands quality controls that can be applied to release phage-based drug products in compliance with the latest regulatory requirements.




Agence National de Sécurité des Médicaments et des produits de santé. French drug regulation agency


Code federal regulations in the USA


Colony-forming unit


Contract research organization


Dynamic light scattering


Drug product, i.e., the final product used in clinical trials or for commercialization


A component of a drug product, such as a phage type in a cocktail

EMA (previously EMEA)

European Medicine Agency


Efficiency of plating


The body of European Union legislation in the pharmaceutical sector compiled in Volume 1 and Volume 5 of the publication “The rules governing medicinal products in the European Union”


Food and Drug Administration


Good Laboratory Practices


Genetically modified organism


Good manufacturing practices with “c” standing for current, i.e., the latest updated version


Generally recognized as safe is a FDA product status (sections 201 and 409 of the Federal Food, Drug, and Cosmetic Act) for food substances or additives, which is delivered according to their safety data package


Host cell proteins


International conference on harmonization, leading to international guidelines or procedures, for instance, in the field of technical requirements for registration of pharmaceuticals for human use


The investigational medicinal product dossier is the basis for approval of clinical trials by the competent authorities, such as regulatory agency(ies) end ethics committee(s) in the EU


In the USA, the investigational new drug dossier is the basis for approval of clinical trials, emergency use (in a situation that does not allow time for submission of an IND in accordance with 21CFR) or treatment (for experimental drugs showing promise in clinical testing for serious or immediately life-threatening conditions, while the final clinical work and the data review take place) by FDA


Ion exchange

IU or EU

International unit or enzyme (endotoxin) unit


The Medicines and Healthcare Products Regulatory Agency of the United Kingdom


Master bacteria cell stock


Master phage bank


Next-generation DNA/RNA sequencing


National Institute of Allergy and Infectious Diseases


National Institutes of Health


Polymerase chain reaction


Plaque-forming unit


Quality assurance is a systematic process to ensure that QC tests are properly performed


Quality control is set of tests to ensure that quality (conformity) of a product is respected. When a drug product belongs to a pharmacopeia, this set of tests and procedures is already precisely described


Restriction enzyme


Restriction fragment length polymorphism


Random amplified polymorphic DNA


Size exclusion chromatography


Transmission electronic microscopy


Ultrafiltration is the filtration of a fluid in order to get rid of the particles that it could contain in suspension


World Health Organization


Working bacteria cell stock


Working phage bank


  1. Abedon ST (2016) Phage therapy dosing: the problem(s) with multiplicity of infection (MOI). Bacteriophage 6(3):e1220348CrossRefPubMedPubMedCentralGoogle Scholar
  2. Ackermann HW, Prangishvili D (2012) Prokaryote viruses studied by electron microscopy. Arch Virol 157(10):1843–1849CrossRefPubMedGoogle Scholar
  3. Adriaenssens EM, Lehman SM, Vandersteegen K et al (2012) CIM(®) monolithic anion-exchange chromatography as a useful alternative to CsCl gradient purification of bacteriophage particles. Virology 434(2):265–270CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bachrach U, Friedmann A (1971) Practical procedures for the purification of bacterial viruses. Appl Microbiol 22(4):706–715PubMedPubMedCentralGoogle Scholar
  5. Blom H, Åkerblom A, Kon T et al (2014) Efficient chromatographic reduction of ovalbumin for egg-based influenza virus purification. Vaccine 32(30):3721–3724CrossRefPubMedGoogle Scholar
  6. Bonilla N, Rojas MI, Cruz NF et al (2016) Phage on tap–a quick and efficient protocol for the preparation of bacteriophage laboratory stocks. PeerJ 4:e2261CrossRefPubMedPubMedCentralGoogle Scholar
  7. Boratyński J, Syper D, Weber-Dabrowska B et al (2004) Preparation of endotoxin-free bacteriophages. Cell Mol Biol Lett 9(2):253–259PubMedGoogle Scholar
  8. Bourdin G, Schmitt B, Marvin Guy L et al (2014) Amplification and purification of T4-like Escherichia coli phages for phage therapy: from laboratory to pilot scale. Appl Environ Microbiol 80(4):1469–1476CrossRefPubMedPubMedCentralGoogle Scholar
  9. Brown-Jaque M, Calero-Cáceres W, Muniesa M et al (2015) Transfer of antibiotic-resistance genes via phage-related mobile elements. Plasmid 79:1–7CrossRefPubMedGoogle Scholar
  10. Chaudhari VK, Yadav V, Verma PK et al (2014) A review on good manufacturing practice (GMP) for medicinal products. PharmaTutor 2(9):8–19Google Scholar
  11. Clement CC, Aphkhazava D, Nieves E et al (2013) Protein expression profiles of human lymph and plasma mapped by 2D-DIGE and 1D SDS-PAGE coupled with nano LC-ESI-MS/MS bottom-up proteomics. J Proteome 78:172–187CrossRefGoogle Scholar
  12. Clokie MRJ, Kropinski AW (2009) Bacteriophages – methods and protocols volume 1: isolation, characterization, and interactions, Springer protocols methods in molecular biology, vol 501. Humana Press, Totowa. 307 pagesGoogle Scholar
  13. D’Hérelle F (1922) The bacteriophage: its role in immunity. New edition from the Cornell University Library (August 10, 2009), USA, 300 pGoogle Scholar
  14. Doria F, Napoli C, Costantini A et al (2013) Development of a new method for detection and identification of Oenococcus oeni bacteriophages based on endolysin gene sequence and randomly amplified polymorphic DNA. Appl Environ Microbiol 79(16):4799–4805CrossRefPubMedPubMedCentralGoogle Scholar
  15. Dufour N, Delattre R, Ricard JD et al (2017) The lysis of pathogenic Escherichia coli by bacteriophages releases less endotoxin than by β-lactams. Clin Infect Dis 64(11):1582. CrossRefPubMedPubMedCentralGoogle Scholar
  16. El Haddad L, Ben Abdallah N, Plante PL et al (2014) Improving the safety of Staphylococcus aureus polyvalent phages by their production on a Staphylococcus xylosus strain. PLoS One 9(7):e102600. CrossRefPubMedPubMedCentralGoogle Scholar
  17. European Pharmacopoeia 9.2. (2017a) 5.2.12 Raw materials of biological origin for the production of cell-based and gene therapy medicinal productsGoogle Scholar
  18. European Pharmacopoeia 9.2. (2017b) 5.2.3 Cell substrates for the production of vaccines for human useGoogle Scholar
  19. Fauconnier A (2015) Workshop on the therapeutic use of bacteriophages. EMA June 8, 2015. London.
  20. Flosdorf EW, Mudd S (1935) Procedure and apparatus for preservation in “lyophile” form of serum and other biological substances. J Immunol 29:389–425Google Scholar
  21. Floyd C, McIntire GH et al (1969) Studies on a lipopolysaccharide from Escherichia coli. Heterogeneity and mechanism of reversible inactivation by sodium deoxycholate. Biochemistry 8(10):4063–4067CrossRefGoogle Scholar
  22. Food and Drug Administration (2004) Guidance for industry. Sterile drug products. Produced by aseptic processing. Current good manufacturing practiceGoogle Scholar
  23. Food and Drug Administration (2008) Guidance for industry. CGMP for phase 1. Investigational drugsGoogle Scholar
  24. Fothergill E, Mowat E, Walshaw MJ et al (2011) Effect of antibiotic treatment on bacteriophage production by a cystic fibrosis epidemic strain of Pseudomonas aeruginosa. Antimicrob Agents Chemother 55(1):426–428CrossRefPubMedGoogle Scholar
  25. Galanos C, Luderitz O, Rietschel ET, Westphal O (1977) Newer aspects of the chemistry and biology of bacterial lipopolysaccharides, with special reference to their lipid A component. Int Rev Biochem 14:239–335Google Scholar
  26. Grzenia DL, Carlson JO, Wickramasinghe SR (2008) Tangential flow filtration for virus purification. J Membr Sci 321(2):373–380CrossRefGoogle Scholar
  27. Hatfull GF, Hendrix RW (2011) Bacteriophages and their genomes. Curr Opin Virol 1(4):298–303CrossRefPubMedPubMedCentralGoogle Scholar
  28. Heather KA, Torey L, Darrell OB et al (2011) Antibiotics in feed induce prophages in swine fecal microbiomes. MBio 2(6).
  29. Immel BK (2001) A brief history of the GMPs for pharmaceuticals. Pharmaceutical technology, July 2001Google Scholar
  30. International Pharmacopeia (2016) 5.8 Methods of sterilizationGoogle Scholar
  31. Jacquemart R, Vandersluis M, Zhao M et al (2016) A single-use strategy to enable manufacturing of affordable biologics. Comput Struct Biotechnol J 14:309–318CrossRefPubMedPubMedCentralGoogle Scholar
  32. Jang H, Kim HS, Moon SC et al (2009) Effects of protein concentration and detergent on endotoxin reduction by ultrafiltration. BMB Rep 42(7):462–466CrossRefPubMedGoogle Scholar
  33. Jasieński J (1927) Próby zastosowania bakteriofagii w chirurgii. Polska Gazeta Lekarska 4:67–73Google Scholar
  34. Jin M, Szapiel N, Zhang J et al (2010) Profiling of host cell proteins by two-dimensional difference gel electrophoresis (2D–DIGE): implications for downstream process development. Biotechnol Bioeng 105(2):306–316CrossRefPubMedGoogle Scholar
  35. Jordana V (1959) Study on adsorption of bacteriophage by filters. Appl Microbiol 7:239PubMedCentralGoogle Scholar
  36. Kalmanson G, Bronfenbrenner J (1939) Studies on the purification of bacteriophage. J Gen Physiol 23(2):203–228CrossRefPubMedPubMedCentralGoogle Scholar
  37. Karima R, Matsumoto S, Higashi H, Matsushima K (1999) The molecular pathogenesis of endotoxic shock and organ failure. Mol Med Today 5:123–132CrossRefPubMedGoogle Scholar
  38. Kaźmierczak Z, Górski A, Dąbrowska K (2014) Facing antibiotic resistance: Staphylococcus aureus phages as a medical tool. Virus 6(7):2551–2570CrossRefGoogle Scholar
  39. Klug B, Celis P, Carr M et al (2012) Regulatory structures for gene therapy medicinal products in the European Union. Methods Enzymol 507:337–354. CrossRefPubMedGoogle Scholar
  40. Kramberger P, Honour R, Herman RE et al (2010) Purification of the Staphylococcus aureus bacteriophages VDX-10 on methacrylate monoliths. J Virol Methods 166(1–2):60–64CrossRefPubMedGoogle Scholar
  41. Kramberger P, Urbas L, Štrancar A (2015) Downstream processing and chromatography based analytical methods for production of vaccines, gene therapy vectors, and bacteriophages. Hum Vaccin Immunother 11(4):1010–1021CrossRefPubMedPubMedCentralGoogle Scholar
  42. Kramer SP (1927) Experiments with bacterial filters and filterable viruses. Science 65(1672):45–46CrossRefPubMedGoogle Scholar
  43. Krueger AP, Scribner EJ (1941) The bacteriophage its nature and its therapeutic use. JAMA 116(20):2269–2277CrossRefGoogle Scholar
  44. Krueger AP, Tamada HT (1929) The preparation of relatively pure bacteriophage. J Gen Physiol 13(2):145–151CrossRefPubMedPubMedCentralGoogle Scholar
  45. Kutter E (2009) Phage host range and efficiency of plating. Methods Mol Biol 501:141–149CrossRefPubMedGoogle Scholar
  46. Kutter E, De Vos D, Gvasalia G et al (2010) Phage therapy in clinical practice: treatment of human infections. Curr Pharm Biotechnol 11(1):69–86CrossRefPubMedGoogle Scholar
  47. Lopez MF, Berggren K, Chernokalskaya E et al (2000) A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling. Electrophoresis 21:3673–3683CrossRefPubMedGoogle Scholar
  48. Lu TK, Koeris MS (2011) The next generation of bacteriophage therapy. Curr Opin Microbiol 14(5):524–531. CrossRefPubMedGoogle Scholar
  49. Martin JM (2016) Design and qualification of single-use systems. BioPharm Int 29(7):44Google Scholar
  50. Meessen-Pinard M, Sekulovic O, Fortier LC (2012) Evidence of in vivo prophage induction during Clostridium difficile infection. Appl Environ Microbiol 78(21):7662–7670. CrossRefPubMedPubMedCentralGoogle Scholar
  51. Michen B, Graule T (2010) Isoelectric points of viruses. J Appl Microbiol 109:388–397PubMedGoogle Scholar
  52. Milmo S (2017) EU–US mutual recognition agreement on GMP inspections. Pharm Technol 41:4Google Scholar
  53. Muschel LH, Schmoker K (1966) Activity of mitomycin C, other antibiotics, and serum against lysogenic bacteria. J Bacteriol 92(4):967PubMedPubMedCentralGoogle Scholar
  54. Northrop JH (1938) Concentration and purification of bacteriophage. J Gen Physiol 21(3):335–366CrossRefPubMedPubMedCentralGoogle Scholar
  55. Northrop JH (1939) Increase in bacteriophage and gelatinase concentration in cultures of Bacillus megatherium. J Gen Physiol 23:59–79CrossRefPubMedPubMedCentralGoogle Scholar
  56. Official Journal of the European Union, 2011/C 73/01. European Commission Note for guidance on minimising the risk of transmitting animal spongiform encephalopathy agents via human and veterinary medicinal products (EMA/410/01 rev.3)Google Scholar
  57. Parenteral Drug Association (2010) Technical Report Portal – TR 47Google Scholar
  58. Parracho HM, Burrowes BH, Enright MC et al (2012) The role of regulated clinical trials in the development of bacteriophage therapeutics. J Mol Genet Med 6:279–286CrossRefPubMedPubMedCentralGoogle Scholar
  59. Pirnay JP, Verbeken G, Rose T et al (2012) Introducing yesterday’s phage therapy in today’s medicine. Future Virol 7(4):379–390CrossRefGoogle Scholar
  60. Pirnay JP, Blasdel BG, Bretaudeau L et al (2015) Quality and safety requirements for sustainable phage therapy product. Pharm Res 32:2173–2179CrossRefPubMedPubMedCentralGoogle Scholar
  61. Podgornik A, Lendero Krajnc N (2012) Application of monoliths for bioparticle isolation. J Sep Sci 35(22):3059–3072CrossRefPubMedGoogle Scholar
  62. Prachi P, Donati C, Masciopinto F et al (2013) Deep sequencing in pre- and clinical vaccine research. Public Health Genomics 16(1–2):62–68CrossRefPubMedGoogle Scholar
  63. Reed LJ, Muench H (1938) A simple method of estimating fifty per cent endpoints. Am J Hyg 27:493–497Google Scholar
  64. Ross A, Ward S, Hyman P (2016) More is better: selecting for broad host range bacteriophages. Front Microbiol 7:1352CrossRefPubMedPubMedCentralGoogle Scholar
  65. Sauvageau D, Cooper DG (2010) Two-stage, self-cycling process for the production of bacteriophages. Microb Cell Factories 9:81. CrossRefGoogle Scholar
  66. Schade AL, Caroline L (1943) The preparation of a polyvalent dysentery bacteriophage in a dry and stable form – I. J Bacteriol 46:463–473PubMedPubMedCentralGoogle Scholar
  67. Schade AL, Caroline L (1944a) The preparation of a polyvalent dysentery bacteriophage in a dry and stable form- II. J Bacteriol 48:179–180PubMedPubMedCentralGoogle Scholar
  68. Schade AL, Caroline L (1944b) The preparation of a polyvalent dysentery bacteriophage in a dry and stable form- III. J Bacteriol 48:243–251PubMedPubMedCentralGoogle Scholar
  69. Smith HW, Huggins MB (1982) Successful treatment of experimental Escherichia coli infections in mice using phage: its general superiority over antibiotics. J Gen Microbiol 128(2):307–318PubMedGoogle Scholar
  70. Smrekar F, Ciringer M, Peterka M et al (2008) Purification and concentration of bacteriophage T4 using monolithic chromatographic supports. J Chromatogr B 861(2):177–180CrossRefGoogle Scholar
  71. Sonnedecker G (1970) Contribution of the pharmaceutical profession toward controlling the quality of drugs in the nineteenth century. In: Blake JB (ed) Safeguarding the public: historical aspects of medicinal drug control. Johns Hopkins University Press, Baltimore, pp 97–111Google Scholar
  72. Szermer-Olearnik B, Boratyński J (2015) Removal of endotoxins from bacteriophage preparations by extraction with organic solvents. PLoS One 10(3):e0122672CrossRefPubMedPubMedCentralGoogle Scholar
  73. Touchon M, Bernheim A, Rocha EP (2016) Genetic and life-history traits associated with the distribution of prophages in bacteria. ISME J 10:2744–2754CrossRefPubMedPubMedCentralGoogle Scholar
  74. Urdand G (1951) The development of pharmacopoeias: a review with special reference to the pharmacopoea Internationalis. Bull World Health Organ 4:577–603Google Scholar
  75. Verbeken G, De Vos D, Vaneechoutte M et al (2007) European regulatory conundrum of phage therapy. Future Microbiol 2(5):485–491CrossRefPubMedGoogle Scholar
  76. Verbeken G, Pirnay JP, De Vos D et al (2012) Optimizing the European regulatory framework for sustainable bacteriophage therapy in human medicine. Arch Immunol Ther Exp 60:161–172. CrossRefGoogle Scholar
  77. Verbeken G, Pirnay JP, Lavigne R et al (2014a) Call for a dedicated European legal framework for bacteriophage therapy. Arch Immunol Ther Exp 62:117–129. CrossRefGoogle Scholar
  78. Verbeken G, Huys I, Pirnay JP et al (2014b) Taking bacteriophage therapy seriously: a moral argument. Biomed Res Int 62:13–16. Google Scholar
  79. Weber-Dąbrowska B, Jończyk-Matysiak E, Żaczek M et al (2016) Bacteriophage procurement for therapeutic purposes. Front Microbiol 12:1177. Google Scholar
  80. Withington R (2001) Regulatory issues for phage-based clinical products. J Chem Technol Biotechnol 76:673–676CrossRefGoogle Scholar
  81. Wittebole X, De Roock S, Opal SM (2014) A historical overview of bacteriophage therapy as an alternative to antibiotics for the treatment of bacterial pathogens. Virulence 5(1):226–235. CrossRefPubMedGoogle Scholar
  82. Yohe S, Thyagarajan B (2017) Review of clinical next-generation sequencing. Arch Pathol Lab Med.
  83. Zakharova M, Kozyr AV, Ignatova AN et al (2005) Purification of filamentous bacteriophage for phage display using size- exclusion chromatography. BioTechniques 38(2):194–198CrossRefPubMedGoogle Scholar
  84. Zhilenkov E (2016) “Micromir” phage collection: new developments. Phage Therapy World Congress, Paris, 2–3 June 2016Google Scholar

Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Krzysztof Regulski
    • 1
  • Patrick Champion-Arnaud
    • 1
  • Jérôme Gabard
    • 1
    Email author
  1. 1.Pherecydes Pharma SARomainvilleFrance

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