Biotechnology Advances 49 (2021) 107758 Available online 22 April 2021 0734-9750/© 2021 Published by Elsevier Inc. Research review paper Manufacturing of bacteriophages for therapeutic applications Jorge Jo˜ ao 1 , Jo˜ ao Lampreia 1, 2 , Duarte Miguel F. Prazeres, Ana M. Azevedo * iBB – Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior T´ ecnico, Universidade de Lisboa, Av. Rovisco Pais, 1, 1049-001 Lisbon, Portugal A R T I C L E INFO Keywords: Bacteriophages Bacteriophage therapy Bacteriophage biomanufacturing Upstream bioprocessing Downstream bioprocessing Purifcation ABSTRACT Bacteriophages, or simply phages, are the most abundant biological entities on Earth. One of the most interesting characteristics of these viruses, which infect and use bacteria as their host organisms, is their high level of specifcity. Since their discovery, phages became a tool for the comprehension of basic molecular biology and originated applications in a variety of areas such as agriculture, biotechnology, food safety, veterinary, pollution remediation and wastewater treatment. In particular, phages offer a solution to one of the major problems in public health nowadays, i.e. the emergence of multidrug-resistant bacteria. In these situations, the use of virulent phages as therapeutic agents offers an alternative to the classic, antibiotic–based strategies. The development of phage therapies should be accompanied by the improvement of phage biomanufacturing processes, both at laboratory and industrial scales. In this review, we frst present some historical and general aspects related with the discovery, usage and biology of phages and provide a brief overview of the most relevant phage therapy applications. Then, we showcase current processes used for the production and purifcation of phages and future alternatives in development. On the production side, key factors such as the bacterial physiological state, the conditions of phage infection and the operation parameters are described alongside with the different operation modes, from batch to semi-continuous and continuous. Traditional purifcation methods used in the initial phage isolation steps are then described followed by the presentation of current state-of-the-art purifcation approaches. Continuous purifcation of phages is fnally presented as a future biomanufacturing trend. 1. Introduction The treatment of infectious diseases caused by pathogenic bacteria has been largely based on the use of antibiotics. This antibiotherapy is however becoming progressively less effective due to the development of antibiotic-resistant bacteria, which now stand as one of the greatest threats to human health (Harada et al., 2018; Kutter and Sulakvelidze, 2005; Parisien et al., 2008; Viertel et al., 2014). Antibiotic-resistant opportunistic bacteria are a very serious and worrying issue, particu- larly in hospital settings, strongly affecting immunocompromised pa- tients, for example. These highly resistant pathogenic species are commonly known by the acronym ESKAPE and include the Gram- Abbreviations: AEC, Anion-exchange chromatography; ATMP, Advanced therapy medicinal product; ATPS, Aqueous two-phase systems; CER, Carbon evolution rate; CF, Continuous fermentation; CI, Continuous infection; CIM, Convective interaction media; DBC, Dynamic binding capacity; DEAE, Diethylaminoethyl; DF, Diafltration; DLS, Dynamic light scattering; DO, Dissolved oxygen; dsDNA, Double-strand DNA; dsRNA, Double-strand RNA; EBA, Expanded-bed adsorption; eIND, Emergency investigational new drug; EMA, European Medicines Agency; ESKAPE, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species pathogenic bacteria; EU, Endotoxin Unit; FDA, Food and Drug Administration; gDNA, Genomic DNA; cGMP, Current good manufacturing practice; GST, Glutathione-S-transferase; IFN-α, Interferon alpha; IL-6, Interleukin-6; LPS, Lipopolysaccharides; MALS, Multi- angle light scattering; MIR, Mid-infrared; MOI, Multiplicity of infection; NIR, Near-infrared; OD, Optical density; OUR, Oxygen uptake rate; PAT, Process analytical technologies; PEG, Polyethylene glycol; PFU, Plaque forming units; PILs, Polymeric ionic liquids; QA, Quaternary amine; RI, Refractive index; SCF, Self-cycling fermentation; SCI, Self-cycling infection; SEC, Size-exclusion chromatography; SLS, Static light scattering; SMB, Simulated moving beds; SP-TFF, Single-pass tangential fow fltration; ssDNA, Single-strand DNA; ssRNA, Single-strand RNA; TFF, Tangential fow fltration; TMB, True moving bed; TNF-α, Tumor necrosis factor alpha; tRNA, Transfer RNA; UF, Ultrafltration; UV-vis, UV-visible.. * Corresponding author. E-mail addresses: jorgejoao@tecnico.ulisboa.pt (J. Jo˜ ao), jlampreia@oxgene.com (J. Lampreia), miguelprazeres@tecnico.ulisboa.pt (D.M.F. Prazeres), a. azevedo@tecnico.ulisboa.pt (A.M. Azevedo). 1 These authors have contributed equally to this work. 2 Present Address: Oxgene, Medawar Centre, Robert Robinson Avenue, Littlemore, Oxford OX4 4HG, United Kingdom Contents lists available at ScienceDirect Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv https://doi.org/10.1016/j.biotechadv.2021.107758 Received 17 November 2020; Received in revised form 14 March 2021; Accepted 20 April 2021