Methods for purifying bacteriophage and products of manufacture containing purifyied bacteriophage

Abstract

In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for purifying bacteriophage. Provided herein is are practicable methods, or protocols, that in alternative embodiments are ''Good Laboratory Manufacturing Practice" (GLMP), including practicable methods, or protocols for phage isolation, selection, liter-scaled cultivation, and purification. In alternative embodiments, protocols as provided herein employ membrane filtration processes to yield at least about 300 treatment doses at about 109 plaque-forming units with endotoxin levels within human therapeutic regulatory limits. In alternative embodiments, provided are formulations or pharmaceutical preparations of bacteriophage comprising 109 PFU, 1010 PFU, 1011 PFU, or 1012 PFU or more per unit dose and endotoxin levels below about 5.5 EU·mL-1, or below about 5.0 EU·mL-1.

Description

[0001] METHODS FOR PURIFYING BACTERIOPHAGE AND PRODUCTS OF MANUFACTURE CONTAINING PURIFYIED BACTERIOPHAGE

[0002] RELATED APPLICATIONS

[0003] This Patent Convention Treaty (PCT) International Application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Serial No. (USSN) 63 / 600,881, November 20, 2023. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.

[0004] TECHNICAL FIELD

[0005] This invention generally relates to bacteriophage biology and infectious diseases. In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for purifying bacteriophage. In alternative embodiments, provided herein are practicable methods, or protocols, that can be “Good Laboratory Manufacturing Practice” (GLMP), for phage isolation, selection, liter-scaled cultivation, and purification. In alternative embodiments, protocols as provided herein (for example GLMP protocols) employ membrane filtration processes to yield at least about 300 treatment doses at about 109plaque-forming units with endotoxin levels within human therapeutic regulatory limits. In alternative embodiments, provided are formulations or pharmaceutical preparations of bacteriophage comprising 109PFU, 1010PFU, 1011PFU, or 1012PFU or more per unit dose and endotoxin levels below about 5.5 EU mL’1, or below about 5.0 EU mL’1.

[0006] BACKGROUND

[0007] With the usefulness of antibiotics waning, there is an urgent need to develop new treatments against antibiotic resistant infections before they become the leading cause of human death worldwide. Now, researchers and medical doctors have found a way forward - by looking back at how infections were treated before the advent of antibiotics, namely phage therapy. Although bacteriophages (phages) continue to lack drug approval in Western medicine, an increasing number of patients are treated on an emergency investigational new drug (elND) basis.

[0008] Academic and military research institutions are being called to immediate action to produce bacteriophages, i.e., viruses that kill bacteria, for the treatment of antibiotic resistant infections prior to drug approval1-4Sometimes called compassionate use, expanded access provides a patient with an immediately lifethreatening condition or serious disease rapid access to an IND when no satisfactory alternative therapy options are available5. While demand for bacteriophages is increasing in several fields including human and veterinary drugs, biological products, food supply, and cosmetics4’6,7, only a limited number of phage products are produced under good manufacturing practices (GMP)8’9. An effective, consistent, and controllable process for phage production, which also meets safety and efficacy demands for human and animal clinical use, has yet to be achieved10,11. Medicinal phage products have generally been of low purity and titer1’4,12-14.

[0009] Phage producers have largely disregarded unknown gross impurities in phage products that may also risk human health. Indeed, several studies have shown that phage preparations can be safe in animal models22-24 22-25. However, experimental phage therapy studies generally cultivate phages with laboratory-adapted reference bacterial strains. For eIND usage, large quantities of a pathogen, often a clinical multidrug resistant (MDR) isolate, are required to produce a high number of phages. Currently, experimental phage purification is practiced by several ad-hoc approaches 18,26-33 Although these disparate approaches have produced phages, several pitfalls materialized, including low recovery, high gross impurities, inadequate endotoxin removal, and addition of toxic chemicals ms, 31-34 Furthermore, currently practiced phage purification approaches were not developed with human phage therapy in mind. In order to achieve a broader application in humans, phage products must comply with strict quality standards.

[0010] Several ad-hoc laboratory approaches are currently employed for phage cultivation and purification for human use, which have been largely developed for small experimental studies. Large-scale GMP pharmaceutical production of large phage libraries will likely be needed to meet the demands for personalized phage therapy. However, this approach is currently not time and cost-effective, thus creating an unmet need for systematic small batch phage production to excel current efforts for expanded access phage therapy. SUMMARY

[0011] In alternative embodiments, provided are methods for purifying a plurality of bacteriophage (phage) (or generating a purified preparation of phage, wherein optionally the phage are 80%, 90%, 95%, 98%, 99% or completely free of non-phage material or compositions, or are between about 75% to 100% free of non-phage material or compositions), comprising: all or several of the steps as illustrated in FIG. 1 and / or FIG. 2, wherein the method comprises use of a pressure-driven cross-flow ultrafiltration (CFF) comprising a molecular weight cut-off (MWCO) at about 100 kDa, thereby only retaining bacteriophage (phage) particles of greater than about 100 KDa, 150 KDa, 200 KDa, 250 KDa, 300 KDa, 350 KDa or 400 KDa, or between about 50 KDa and 500 KDa.

[0012] In alternative embodiments, provided are methods for purifying a plurality of bacteriophage (phage) (or generating a purified preparation of phage), and generating a substantially endotoxin free preparation of phage, comprising: wherein optionally the phage is from a family Caudoviricetes, Corticoviridae, Tectiviridae, Tubulavirales, Leviviridae, and / or Microviridae, wherein optionally the phage is from a family Inoviridae, Microviridae, Tectiviridae, Corticoviridae, Plasmaviridae, Leviviridae, and / or Cystoviridae, the method comprising:

[0013] (a) providing a sample comprising a bacteriophage (phage), wherein optionally the phage are propagated, and the propagated phage are added during midlog stage of bacterial growth, and optionally phage nucleic acid (optionally the phage nucleic comprise phage genomes) is sequenced, and optionally the phage are filtered, optionally 0.2 pm filtered;

[0014] (b) providing a bacterial strain for propagating a bacteriophage strain, and optionally the bacterial nucleic acid (optionally the bacterial nucleic comprises bacterial genomes) is sequenced, and optionally both the phages and host bacteria genomes are sequenced and annotated to screening for harmful genes, wherein optionally the harmful genes comprise virulence factors, antibiotic resistance and toxin gene from prophages;

[0015] (c) preparing a bacterial host standard curve;

[0016] (d) quantifying phage, optionally by double aliquot spot serial titration;

[0017] (e) amplifying phage, wherein a lysate comprising prophage is generated; (f) detecting prophage in the lysate;

[0018] (g) purifying the amplified phage, optionally filtering and sterilizing, optionally by applying dead-end filtration sterilization to the purified amplified phage, optionally comprising use of crossflow filtration (CFF), ultrafiltration and / or diafiltration, thereby generating a purified amplified phage preparation;

[0019] (h) removing substantially most (optionally removing between about 95% to 99.5%, or 90% to 99.9% of) endotoxin present in the purified amplified phage preparation, optionally using a chromatographic process, thereby generating a purified amplified phage preparation with substantially most endotoxin removed;

[0020] (i) digesting substantially most (optionally removing between about 95% to 99.5%, or 90% to 99.9% of) free nucleic acids, optionally using DNase and / or RNase, to generate an endotoxin-free and a nucleic acid-free phage preparation; and

[0021] (j) sterile fill-finishing the endotoxin-free and a nucleic acid-free phage preparation, optionally processing for use as a drug product, optionally as a sterile injectable, optionally as a sterile liquid filled in vials or syringes, thereby generating a substantially endotoxin free preparation of phage.

[0022] In alternative embodiments of methods as provided herein:

[0023] - the method comprises use of a pressure-driven cross-flow ultrafiltration (CFF) comprising a molecular weight cut-off (MWCO) at about 100 KDa, 150 KDa, 200 KDa, 250 KDa, 300 KDa, 350 KDa or 400 KDa, or between about 50 KDa and 500 KDa, thereby only retaining bacteriophage (phage) particles of greater than about 100 KDa, 150 KDa, 200 KDa, 250 KDa, 300 KDa, 350 KDa or 400 KDa, or between about 50 KDa and 500 KDa;

[0024] - preparation of phage comprises endotoxin levels to below about 5.5 EU mL’ or below about 5.0 EU mL'1;

[0025] - a single production run produces at least about 300 treatment doses, or between about 100 and 1,000 treatment dosages, of phage at about 109PFU, 1010PFU, 1011PFU, or 1012PFU, or more per dose, or between about 105PFU to 1012PFU per dose, or between about 106PFU to 1010PFU per dose, and / or

[0026] - phage lysates or cultures are cooled to below about 37°C, or cooled to between about 4°C to 37°C, at the point where phage-insensitive or phage-resistant bacteria appear in culture. In alternative embodiments, provided are formulations and / or pharmaceutical preparations of bacteriophage (phage), comprising:

[0027] (a) about 109PFU, IO10PFU, 1011PFU, or 1012PFU or more per unit dose, or between about 105PFU to 1012PFU per dose, or between about 106PFU to IO10PFU per dose, and / or endotoxin levels below about 5.5 EU mL’1, or below about 5.0

[0028] EU mL’1, or are between about 1.0 EU mL'1and 5.5 EU mL'1, or are between about 0.1 EU mL'1and 5.0 EU mL'1; or

[0029] (b) a preparation of bacteriophage (phage) prepared by a method of any of the previous claims.

[0030] In alternative embodiments of formulations or pharmaceutical preparations as provided herein:

[0031] - the phage are formulated for enteral or parenteral administration, or are formulated for administration intramuscularly, orally, parenterally, by inhalation spray, nasally, topically, intrathecally, intrathecally, intracerebrally, epidurally, intracranially, vaginally or rectally;

[0032] - the phage are formulated as a lyophilate, a tablet, a pill, a powder, a dragee, a capsule, a liquid, a lozenge, a gel, a syrup, a slurry, an aerosol, or a suspension; and / or

[0033] - the formulations or pharmaceutical preparations comprise or further comprise a pharmaceutically acceptable excipient, or phage dissolved in (optionally sterile) saline, water, polyethylene glycol, propylene glycol, ethanol or oils such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil, tragacanth gum, and / or a buffer.

[0034] In alternative embodiments, provided are methods for treating a bacterial infection in vivo comprising administering to an individual in need thereof a formulation or pharmaceutical preparation as provided herein, wherein optionally the pharmaceutical preparation is administered orally, parenterally, by inhalation spray, nasally, topically, intrathecally, intrathecally, intracerebrally, epidurally, intracranially, vaginally, intravenously, intraoperatively or rectally.

[0035] In alternative embodiments, provided are uses of a formulation or pharmaceutical preparation as provided herein, for treating a bacterial infection in vivo. In alternative embodiments, provided are formulations or pharmaceutical preparations as provided herein, for use in treating a bacterial infection in vivo, thereby generating a substantially endotoxin free preparation of phage.

[0036] In alternative embodiments of the methods, the preparation of phage comprises endotoxin levels to below about 5.5 EU mL’1, or below about 5.0 EU mL’1.

[0037] In alternative embodiments of the methods, a preparation, or a single production run, produces at least about 300, or between about 100 and 10,000, or between about 200 and 1,000, or between about 250 and 500, treatment doses phage at about 109PFU, 1010PFU, 1011PFU, or 1012PFU, or between about 102PFU to 1014PFU, or more, per dose.

[0038] In alternative embodiments of the methods, phage lysates or cultures are cooled to below 37°C, or cooled to between about 4°C to 37°C, at the point where phage-insensitive or phage-resistant bacteria appear in culture.

[0039] In alternative embodiments, provided are pharmaceutical preparations or formulations of bacteriophage (phage), comprising: about 109PFU, 1010PFU, 1011PFU, or 1012PFU or more per unit dose and endotoxin levels below about 5.5 EU mL'1, or below about 5.0 EU mL'1.

[0040] In alternative embodiments of the pharmaceutical preparations or formulations:

[0041] - the phage are formulated for enteral or parenteral administration, or are formulated for administration intramuscularly, orally, parenterally, by inhalation spray, nasally, topically, intrathecally, intrathecally, intracerebrally, epidurally, intracranially, vaginally, intravenously, intraoperatively or rectally;

[0042] - the phage are formulated as a lyophilate, a tablet, a pill, a powder, a dragee, a capsule, a liquid, a lozenge, a gel, a syrup, a slurry, an aerosol, or a suspension; and / or

[0043] - pharmaceutical preparations or formulations comprise or further comprise a pharmaceutically acceptable excipient, or phage dissolved in (optionally sterile) saline, water, polyethylene glycol, propylene glycol, ethanol or oils such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil, tragacanth gum, and / or a buffer.

[0044] In alternative embodiments, provided are methods for treating a bacterial infection in vivo comprising administering to an individual in need thereof a formulations or pharmaceutical preparation as provided herein, wherein optionally the formulations or pharmaceutical preparation is administered orally, parenterally, by inhalation spray, nasally, topically, intrathecally, intrathecally, intracerebrally, epidurally, intracranially, vaginally, intravenously, intraoperatively or rectally.

[0045] In alternative embodiments, provided are uses of a formulation or pharmaceutical preparation as provided herein, for treating a bacterial infection in vivo.

[0046] In alternative embodiments, provided are formulations or pharmaceutical preparations as provided herein, for use in treating a bacterial infection in vivo.

[0047] The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0048] All publications, patents, patent applications cited herein are hereby expressly incorporated by reference in their entireties for all purposes.

[0049] DESCRIPTION OF DRAWINGS

[0050] The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.

[0051] FIG. 1 A-C illustrate a bacterial or Spot+phage titration:

[0052] FIG. 1 A illustrates an exemplary setup using a multichannel pipette and 96- well plate for serial dilutions;

[0053] FIG. IB illustrates exemplary 48-spot plating for bacterial quantification; and

[0054] FIG. 1C illustrates exemplary 48-spot plating for Spot+phage titration, as discussed in detail in Example 2, below.

[0055] FIG. 2A-I illustrate an exemplary phage production and purification process as provided herein:

[0056] FIG. 2A schematically illustrates phage amplification: bacteria should be added to sterile growth medium and grown for one generation with a vented screwcap to allow gas exchange (left), and phages should be added to achieve a MOI of 0.1 (right);

[0057] FIG. 2B schematically illustrates a first centrifugation;

[0058] FIG. 2C schematically illustrate a second centrifugation; FIG. 2D schematically illustrates a dead-end filtration sterilization. Sterilize the lysate by connecting in tandem a 0.8 | 0.45 gm capsule filter to a 0.45 | 0.2 gm filter capsule;

[0059] FIG. 2E schematically illustrates a diafiltration;

[0060] FIG. 2F schematically illustrates an exemplary washing;

[0061] FIG. 2F schematically illustrates exchanging spent lysate for cold ultrapure storage buffer, and stepwise concentrate and dilute phage with storage buffer;

[0062] FIG. 2G schematically illustrates concentrating, when the phage concentrate appears visibly clear, purge the cross-flow ultrafiltration (CFF) and collect phage particles in three or more fractions;

[0063] FIG. 2H schematically illustrates endotoxin removal; and

[0064] FIG. 21 schematically illustrates removal of nucleic acids. Remove free DNA / RNA by 1 h enzyme treatment, as discussed in detail in Example 2, below.

[0065] FIG. 3A-C illustrate an exemplary protocol comprising: phage sterilization, validation, and fill-finish:

[0066] FIG. 3A schematically illustrates syringe sterilization;

[0067] FIG. 3B schematically illustrates endotoxin quantification; and

[0068] FIG. 3C schematically illustrates vial filling. UV sterilize all supplies and vials before use, and filter sterilize the diluted phage preparation before sterile distribution into vials, as discussed in detail in Example 2, below.

[0069] Like reference symbols in the various drawings indicate like elements.

[0070] DETAILED DESCRIPTION

[0071] In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for purifying bacteriophage (phage).

[0072] In alternative embodiments, provided herein are practicable methods, or protocols, for phage isolation, selection, liter-scaled cultivation, and purification. We describe technical issues and solutions to ensure the identity, quality, and purity of phage products for human use. In alternative embodiments, GLMP protocols as provided herein employ modern membrane filtration processes to yield at least 300 treatment doses at 109plaque-forming units with endotoxin levels within human therapeutic regulatory limits.

[0073] In alternative embodiments, protocols or methods as provided herein use cross-flow ultrafiltration (CFF), which is a pressure-driven scalable membrane filtration process that markedly decreases labor and improves purification reproducibility. Use of CFF was able to reliably concentrate phage particles greater than 10-fold, fractionate phages from gross bacterial impurities (for example less than 40 EU rnl'1), and diafilter buffer exchange. In alternative embodiments, exemplary protocols are configured as a cost-effective, programmed, and semi-automated single- step. In alternative embodiments, exemplary protocols using CFF are also scalable with no barrier to running several apparatuses with additional hardware such as for example a multichannel peristaltic pump head or multiple peristaltic pumps. In alternative embodiments, exemplary protocols using CFF implement a molecular weight cut-off (MWCO) at about 100 KDa, 150 KDa, 200 KDa, 250 KDa, 300 KDa, 350 KDa or 400 KDa, or between about 50 KDa and 500 KDa, in combination with several sterile washes; thus, the bulk of the gross impurities (of less than 100 kDa) are removed from phage lysates, including washing away of endotoxin and exotoxins that are smaller than 100 kDa. Thus, exemplary protocols provided herein that comprise use of CFF is a highly cost-efficient methods to concentrate, diafiltrate and purify phages.

[0074] In alternative embodiments, protocols or methods as provided herein do not require use of downstream cesium chloride (CsCl) density gradient ultracentrifugation, dialysis, and / or affinity chromatography purification steps, and because these additional processes are not required to meet the FDA’s drug product endotoxin regulation, exemplary protocols as provided herein can allow phage products to be isolated (for example, de novo isolated), cultivated and purified in less than five (5) days for time-critical emergency phage therapy if needed or desired. While the CsCl density gradient ultracentrifugation, dialysis, and / or affinity chromatography purification steps are optional, their use may result in a homogenous phage band and / or further concentration of phage, so including these steps may be preferred in alternative embodiments.

[0075] Implementation of exemplary protocols as provided herein across research laboratories participating in phage production for expanded access phage therapy can be pivotal to re-introduce phage therapy to Western medicine. In alternative embodiments, protocols or methods as provided herein can produce a phage product that is of high-titer and pure, providing an estimated 3000 treatments at 109plaque forming units (PFU), a commonly prescribed IV phage dose. Thus, implementation of exemplary protocols as provided herein can allow for administration of higher phage titer per treatment dose and maintain a safe patient exposure endotoxin limit of less than (<) 5 EU kg1h1 40.

[0076] In alternative embodiments, protocols or methods as provided herein are exemplary purifications that provide phage for human phage therapy. In alternative embodiments, protocols or methods as provided herein provide high safety standards of phage products.

[0077] In alternative embodiments, protocols or methods as provided herein comprise both bacteriophage cultivation and purification. In alternative embodiments, the procedure starts with sourcing and isolating phages with a target bacterial strain. After multiple rounds of agar plaque isolation, a single plaque is small-scale cultivated overnight. Next, the newly isolated phage genome is sequenced, annotated, and screened for lysogenic and harmful genes. Phages deemed potentially safe for human use are then liter-scale cultivated. After overnight culturing, phage lysate is sterilized by pressure driven double dead-end filtration and cross-flow ultrafiltration. Cross-flow ultrafiltration also diafiltrates to remove growth medium and concentrates phage particles in buffer. As an option, cesium chloride (CsCl) density gradient ultracentrifugation and dialysis can be used to further confirm phage stock homogeneity. LPS-affinity chromatography is used to remove residual endotoxins. Lastly, the final phage preparation purity and safety is tested by quantifying endotoxin level, protein abundance, and cell viability after phage sample exposure.

[0078] In expanded access phage therapy, care must be taken to avoid temperate phages that are able to lysogenize their bacterial host. While there are genetic approaches to inactivate the lysogenic lifecycle, in alternative embodiments it may be preferable to begin with phages that are unable to lysogenize their host. Phage genome sequencing allows rapid confirmation of whether a phage is likely to be temperate or virulent. Whole-genome sequencing may also provide clues to other biological contaminants in the preparation, depending on the abundance of the contaminants relative to the phage. However, in each of these considerations, phage genome sequencing will only identify known features. Computational identification of a toxin, antibiotic resistance gene, or virulence factor should not infer that these elements are not present, rather than known elements are not present.

[0079] In alternative embodiments, protocols as provided herein include additional non-FDA required safeguards, which can include additional checking of preparations by, for example, SDS-PAGE for bacterial proteins, and additional checking of cell culture viability, using for example a 'fast non in vivo test'.

[0080] In alternative embodiments, provided are compositions, a product of manufacture, a food, a drink, a nutraceutical, a formulation, a pharmaceutical or a pharmaceutical preparation comprising, or containing, or mixed with, or formulated with: a preparation of purified or substantially endotoxin-free bacteriophages as provided herein or a formulation or pharmaceutical composition as provided herein; for example, provided are compositions, a product of manufacture, a food, a drink, a nutraceutical, a formulation, a pharmaceutical or a pharmaceutical preparation comprising, or containing, or mixed with, or formulated with: a phage preparation having 109PFU, IO10PFU, 1011PFU, or 1012PFU or more per unit dose and endotoxin levels below about 5.5 EU mL’1, or below about 5.0 EU mL’1.

[0081] In alternative embodiments, a composition, product of manufacture, food, drink, nutraceutical, formulation, pharmaceutical or pharmaceutical preparation as provided herein comprises, contains, or is manufactured as or formulated as or formulated at:

[0082] (a) a capsule, a tablet, a gel, a geltab, a liquid, a solid, an elixir, a spray, a powder, a suppository or an implant, a sachet, a lozenge, a freeze-dried composition, or an infant formula,

[0083] (b) a per dose, or per serving, or per unit dosage at, or a total daily dose of: between about 10(1) (or 101) and 10(20) plaque-forming units (PFUs), or between about 10(3) and 10(17) PFUs, or between about 10(5) and 10(12) PFUs, or between about 10(7) and 10(9) PFUs,

[0084] (c) administration in vivo or for enteral or parenteral administration, or for ophthalmic, topical, oral, intravenous (IV), intramuscular (IM), intrathecal, subcutaneous (SC), intracerebral, epidural, intracranial or rectal administration, or by inhalation, or

[0085] (d) a particle, a nanoparticle, a liposome, a tablet, a pill, a capsule, a gel, a geltab, a liquid, a powder, a suspension, a syrup, an emulsion, a lotion, an ointment, an aerosol, a spray, a lozenge, an ophthalmic preparation, an aqueous or a sterile or an injectable solution, a patch (optionally a transdermal patch or a medicated adhesive patch), an implant, a dietary supplement, an ice cream, an ice, a yogurt, a cheese, an infant formula or infant dietary supplement, a pasteurized milk or milk product or milk-comprising product.

[0086] In alternative embodiments, a composition, product of manufacture, food, drink, nutraceutical, formulation, pharmaceutical or pharmaceutical preparation as provided herein, further comprises, or contains, or is mixed with, or formulated with: a pharmaceutically acceptable excipient; a flavoring or a sweetening agent, an aspartamine, a stevia, monk fruit, a sucralose, a saccharin, a cyclamate, a xylitol, a vanilla, an artificial vanilla or chocolate or strawberry flavor, an artificial chocolate essence, or a mixture or combination thereof; a preservative, a benzoic acid, a potassium sorbate. at least one probiotic or prebiotic, wherein optionally the prebiotic comprises an inulin, lactulose, extracts of artichoke, chicory root, oats, barley, various legumes, garlic, kale, beans or flacks or an herb; at least one congealing agent, wherein optionally the congealing agent comprises an arrowroot or a plant starch, a powdered flour, a powdered potato or potato starch, an absorbant polymer, an Absorbable Modified Polymer, and / or a corn flour or a com starch; at least one an anti-inflammatory agent, wherein optionally the inflammatory agent comprises or is an NSAID, a 4 or a 5-amino-salicylate, an olsalazine, a mesalazine, a sulfasalazine and / or a balsalazide or an equivalent thereof or a combination thereof; an additive selected from one or more of a saline, a media, a defoaming agent, a surfactant agent, a lubricant, an acid neutralizer, a marker, a cell marker, a drug, an antibiotic, a contrast agent, a dispersal agent, a buffer or a buffering agent, a sweetening agent, a debittering agent, a flavoring agent, a pH stabilizer, an acidifying agent, a preservative, a desweetening agent and / or coloring agent, vitamin, mineral and / or dietary supplement, or a prebiotic nutrient; and optionally the buffer or a buffering agent or the pharmaceutically acceptable excipient comprises an inorganic salt, a citric acid, a sodium chloride, a potassium chloride, a sodium sulfate, a potassium nitrate, a sodium phosphate monobasic, a sodium phosphate dibasic or combinations thereof; and optionally the antacid comprises a calcium carbonate, a magnesium hydroxide, a magnesium oxide, a magnesium carbonate, an aluminum hydroxide, a sodium bicarbonate or a dihydroxyaluminum sodium carbonate; or any combination thereof.

[0087] In alternative embodiments, a composition, product of manufacture, food, drink, nutraceutical, formulation, pharmaceutical or pharmaceutical preparation as provided herein, further comprises, or is contained, or mixed with, or formulated with or as: a delayed or gradual enteric release composition or formulation, and optionally the formulation comprises a gastro-resistant coating designed to dissolve at a pH of 7 in the terminal ileum, optionally an active ingredient is coated with an acrylic based resin or equivalent, optionally a poly(meth)acrylate, optionally a methacrylic acid copolymer B or NF.

[0088] In alternative embodiments, bacteriophages or viruses infecting bacteria in a composition, product of manufacture, food, drink, nutraceutical, formulation, pharmaceutical or pharmaceutical preparation as provided herein, or purified using a method as provided herein, are from the Caudoviricetes, a class of phage known as the tailed phages whose hosts are phage and archaea; in alternative embodiments, phase purified in methods as provided herein are from a family Caudoviricetes, Corticoviridae, Tectiviridae, Tubulavirales, Leviviridae, and / or Microviridcte: in alternative embodiments, phase purified in methods as provided herein are from a family Inoviridae, Microviridcte, Tectiviridae, Corticoviridae, Plasmaviridae, Leviviridae, and / or Cystoviridae . In alternative embodiments, provided herein are processes, and tips, on how to safely remove bacterial toxins from phage lysates. Although dependent on the phage strain, exemplary methods as described herein can be used to rapidly generate and purify phages, and exemplary methods also can be extremely useful for a variety of applications.

[0089] With several options at each step of the production process, exemplary processes as provided herein are used for the production and purification of phages, and can be used incorporating different criteria, including cost, equipment, ease, time, yield, and purity of the product. In alternative embodiments, steps intrinsic to exemplary processes as provided herein to rapidly generate highly pure small -batch phage stocks that meet regulatory guidelines for intravenous (IV) administration are timed process execution to match phage-bacteria kinetics and modifications that have been used to optimize ultrafiltration (UF) and diafiltration (DF). In alternative embodiments, exemplary processes as provided herein minimize the quantity of bacterial toxins that accumulate during phage replication by using several techniques to limit bacterial overgrowth and reduce bacterial debris in phage lysates. In alternative embodiments, a semi-permeable membrane with active force applied to drive UF and DF is used to get target phages ready for the next processing step by enabling sample cleanup, purification, concentration, buffer exchange, and desalinization. In alternative embodiments, exemplary processes as provided herein comprise use of affinity chromatography resin to selectively capture the conserved region of the inner core of LPS molecules and thereby remove all kinds of endotoxins from Gram-negative bacteria. In alternative embodiments, exemplary processes as provided herein use aseptic / sterile fill-finish methods, which are ideal for small-batch biomedical applications. Together, exemplary processes as provided herein efficiently produces many phages in as little as 48 hours and economically.

[0090] Exemplary processes:

[0091] Bacteriophase and Bacterial Host Strains

[0092] In alternative embodiments, phage and host bacterial genomes used in phage production have been sequenced and annotated. In alternative embodiments, phage and / or host bacteria genomes are screened for virulence factors, antibiotic resistance and toxin genes, and prophages. Prophages integrated into the host genome may not only harbor additional virulence factors, but also introduce an added risk of horizontal gene transfer (transduction) or may interfere with the infectivity of the target phage. If any undesirable elements are identified in either the phage or host genome, we recommend using alternative strains.

[0093] 1. Bacteriophage stock (recommend sequenced and 0.2 pm filtered)

[0094] 2. Bacterial strain used to propagate bacteriophage strain (recommend sequenced)

[0095] Reagents

[0096] In alternative embodiments, reagents are prepared using sterile water, autoclaved (for example, at 45 min at 121 °C), and optionally stored at room temperature (RT). Pre-Processing: Prepare a Bacterial host standard curve

[0097] 1. Streak bacteria on an agar plate from a glycerol stock using a sterile loop. Incubate overnight at the temperature that is optimal for bacterial growth.

[0098] 2. Pick a colony forming unit (CFU) and culture it in the appropriate nutrient-rich broth, overnight at the optimal temperature for growth.

[0099] 3. In a BSC, air dry 5 agar plates for 1 h.

[0100] 4. Subculture 50 pL in 5 mL of fresh liquid broth in a sterile test tube. Incubate until an ODeoo ~0.8 is reached. Place the test tube on ice.

[0101] 5. Aliquot culture into a sterile cuvette and measure absorbance using a spectrophotometer. Dilute the culture to ODeoo 0.8 with sterile bacterial broth in a sterile cuvette. Continue dilutions in twofold increments to obtain cuvettes with ODeoo 0.8, 0.4, 0.2, 0.1, and 0.05.

[0102] 6. Prepare two identical 8-well columns of PBS diluent in a 96-well microtiter plate. Add 198 pL of PBS to the top 2 wells and 90 pL in the remaining 14 wells.

[0103] 7. Sample 2 pL from the ODeoo 0.8 bacterial culture and add to the first microwell containing 198 pL of PBS and mix by repetitive pipetting (repeatedly dispensing and withdrawing solution from the microwell). Sample 2 pL of bacterial culture again and add to the second microwell containing 198 pL.

[0104] 8. Use a multichannel pipette to serially dilute in tenfold increments by aliquoting 10 pL from the first row of wells to the second and mix by repetitive pipetting. Change pipette tips and repeat for the remaining six wells.

[0105] 9. Withdraw 4 pL from the first column of 8 microwells using a multichannel pipette. Spot 8 * 4 pL aliquots to the leftmost edge of the agar plate. Repeat 8 x 4 pL sampling from the first column and spot on the agar plate next to the first column of spots. Repeat 8 * 4 pL sampling from the first column a third time, spotting on the agar plate next to the second column of spots (Fig. la).

[0106] 10. Repeat 4 pL spotting in triplicate, withdrawing from the second column of 8 microwells. Dry spots completely before closing the Petri plate lid and moving the plate (see Note 3).

[0107] 11. Repeat dilution and titration steps 6-10 for the remaining diluted cuvettes.

[0108] 12. Incubate all agar plates at the appropriate bacterial growth conditions until colonies are countable (Fig. lb).

[0109] 13. Calculate bacterial CFU / mL by averaging the number of CFUs visible in all 6 spots from both dilution series. Selected spots should contain between 10-40 countable CFUs. Divide the average CFUs by 4 (volume spotted), times the dilution factor (column position), and times by 1000 (convert pL to mL).

[0110] 14. Plot the CFU / mL (y-axis) against ODeoo reading (x-axis). Generate a trendline and check that the R2of the line is >0.9. The equation of the trendline will allow you to approximate the host ODeoo value necessary to achieve the desired concentration CFU / mL.

[0111] Traditionally, phages are quantified using the top agar overlay method. We recommend a double aliquot 48-spot serial titration per sample of interest. This quantification method is quick and easily performed with general laboratory equipment. The Spot+method increases sample coverage, depth, and technical repetition to improve accuracy over a typical spot titration method.

[0112] 1. In a BSC, air-dry agar plates for 1 h. Dry one plate for each sample to be titered.

[0113] 2. Pour 2 mL of bacterial culture at ODeoo 0.2 onto a dried agar plate to seed. Gently swirl to spread the liquid evenly over the entire surface. Tilt the Petri plate and immediately collect excess liquid for disposal. Repeat seeding for each dried agar plate needed.

[0114] 3. Air dry seeded plates for 30 min.

[0115] 4. Prepare two identical 8-well columns of PBS diluent in a 96-well microtiter plate. Add 198 pL of PBS to the top 2 wells and 90 pL in the remaining 14 wells.

[0116] 5. Sample 2 pL from the phage preparation and add to the first microwell containing 198 pL of PBS and mix by repetitive pipetting (repeatedly dispensing and withdrawing solution from the microwell). Sample 2 pL of phage again and add to the second microwell containing 198 pL.

[0117] 6. Use a multichannel pipette to serially dilute in tenfold increments by aliquoting 10 pL from the first row of wells to the second and mix by repetitive pipetting. Change tips and repeat for the remaining six wells.

[0118] 7. Withdraw 4 pL from the first column of 8 microwells using a multichannel pipette. Spot the 8 aliquots in the leftmost area of the seeded agar plate. Repeat 8 x 4 pL sampling from the first microwell column and spot onto the agar plate next to the first column of spots. Repeat 8 * 4 pL sampling from the first microwell column a third time, spotting on the agar plate next to the second column of spots (Fig. la).

[0119] 8. Repeat step 7 and perform 4 pL spotting in triplicate, withdrawing from the second column of 8 microwells.

[0120] 9. Dry spots completely before closing Petri plate lid and moving. Incubate at the appropriate bacterial growth conditions until plaques are countable (Fig. 1c).

[0121] 10. Calculate phage PFU / mL by averaging the number of PFUs visible in all 6 spots from both dilution series. Selected rows should contain 10-40 PFUs. Divide the average PFUs by 4 (volume spotted), times the dilution factor (column position), and times by 1000 (convert pL to mL).

[0122] Pilot phage ampli fication

[0123] 1. Pick a fresh CFU from the host strain plate and grow to ~108CFU in 5 mL of broth medium.

[0124] 2. Add phages at approximately (~) 107PFU to achieve a multiplicity of infection (MOI) of 0.1.

[0125] 3. Incubate the culture for 10-12 h or until optically turbid, then harvest (cooling has a negative effect on phage stability) (see Note 4).

[0126] 4. Harvest the phage lysate in a sterile 15 mL conical and centrifuge for 10 min at 8000*g.

[0127] 5. Decant the supernatant into a sterile syringe with an attached filter unit. Filter into a fresh 15 mL conical.

[0128] 6. Aliquot 100 pL of the filtered lysate into a PCR tube.

[0129] 7. Store at 4°C.

[0130] Analyze and image gel using a gel documentation system, and Batch Phage Amplification

[0131] In alternative embodiments phage production begins with long culture incubations (for example, overnight / 18-24 h) followed by lysate centrifugation and 0.2 pm filter sterilization. As mentioned, bacterial toxins accumulate during phage amplification, and a large quantity of free endotoxin results from violent cell lysis. It is also common for target bacterial cells to evolve resistance to phage infection during these long incubations. These evolved mutants thrive in optimal culture conditions and overgrow in the lysate. In alternative embodiments cold growth incubation is used 1-2 hours (h) prior to the visible outgrowth of phage-resistant bacteria. In alternative embodiments time-kill kinetics assays help understand interactions that exist between host bacteria and phages, including time to resistance. For example, we recommend when using Pseudomonas aeruginosa phages to reduce the incubation temperature after ten hours to suppress the outgrowth of evolved phage resistant mutant cells while maintaining phage production. In alternative embodiments, use Staphylococcus aureus phages to reduce the incubation temperature after 22 h (hours) to achieve the same suppression. This temperature drop prevents resistant bacterial growth, which causes a clearer lysate while maintaining long incubation times for higher phage yields. In alternative embodiments two centrifugation steps are used to significantly enhance the removal of intact bacterial cells and bacterial debris, including large endotoxin aggregates. Figure 2 shows a flow diagram briefly describing this process.

[0132] Exemplary steps

[0133] 1. Pick a CFU isolated using the streak plate method on solid growth medium and culture in the appropriate nutrient-rich broth medium overnight to bulk up a pure culture.

[0134] 2. Subculture 50 pL in fresh liquid broth to get the bacterial population growing exponentially (i.e., ODeoo 0.1-0.8).

[0135] 3. Add 1 L of broth medium to each of the 3 sterile autoclaved glass Erlenmeyer flasks with GL45 screw cap and pre-warm to bacterial growth temperature.

[0136] 4. Add exponentially growing bacteria at an optical density that corresponds to a count of ~1O10CFU per flask and exchange the flask’s cap for an autoclaved GL45 vented screw cap with 0.22 pm PTFE hydrophobic membrane. Incubate the flasks for one generation at growth temperature with gentle agitation (Fig. 2a).

[0137] 5. After bacteria have acclimated and grown for one generation, add phages at ~109PFU to achieve a multiplicity of infection (MOI) of 0.1 (Fig. 2a).

[0138] 6. Incubate the flasks for 10-12 h or until culture is visually turbid.

[0139] 7. Harvest amplified phages by decanting lysates into sterile centrifuge bottles (for example, 1 L bottles) and balance the three bottles against one another using a double pan balance scale (Fig. 2b).

[0140] 8. Centrifuge at 8000 x g for 30 min at 4°C. Centrifugation is a more cost-effective option when batch volumes are less than 10 L or greater than 1,000 L.

[0141] 9. Decant supernatant containing phage particles without disturbing the solid pellet into new sterile centrifuge bottles (Fig. 2c). Repeat centrifugation at 8000 x g for 30 min at 4°C 10. Collect phage particles without disturbing the tiny pellet into sterile glass storage bottles.

[0142] Phage Puri fication

[0143] Dead-end filtration sterilization

[0144] 1. In a BSC, connect a 0.8 | 0.45 pm heterogeneous PES double layer filter capsule to a 0.45 | 0.2 pm heterogeneous PES double layer filter capsule with autoclaved YP flexible tubing and clamps as shown in Fig. 2d.

[0145] 2. Prepare a disposable 10 mL serological pipette by removing the cotton or filter from its end. Feed the tubing through the peristaltic pump head, insert the serological pipette into the end of the tubing, and place it into sterile water.

[0146] 3. Precondition the filter membrane by pumping 500 mL of sterile water through the capsule filters. Discard the flow through.

[0147] 4. Switch the serological pipette and tubing to the bottle containing phage lysate and pump it into sterile glass bottles (see Note 11).

[0148] 5. Store all filtered lysates at 4°C. This is a potential pause step; depending on the phage strain, the lysate can be stored at 4°C for up to several months without significant loss in titer.

[0149] Ultrafiltration (UF) and diafiltration (DF)

[0150] In alternative embodiments, processes comprise use of crossflow filtration (CFF) for combined UF and DF of up to 5 L batch volumes. UF and DF are commonly used for the development and manufacturing of biological therapeutics, such as proteins and antibodies, as well as therapies that rely on viral nanoparticle delivery (31, 32). The VIVAFLOW 200™ CFF cassette uses a polyethersulfone membrane (PES), which is popular for phage applications given its hydrophilic properties and low protein binding. The semi-permeable membrane retains larger target phages while allowing smaller molecules to filter out. A 00 kDa molecular weight cut-off (MWCO) is adequate to retain most phages, however large MWCO can be used for larger phages to facilitate greater remove of small particles. DF enables medium to buffer exchange by adding new cold buffer to the phage retentate. By returning the sample to the original volume, UF / DF can be repeated until the sample reaches a targeted level of clarity with the new buffer. The VIVAFLOW™’ s unique switchback channels parallel to the filtration membrane improves phage dissociation from endotoxins and require less pressure to drive filtration. The cassette design is easy to set up, gentle on phage particles, and its transparency allows for monitoring flow.

[0151] 1. In a BSC, connect the CFF cassette with autoclaved 6.4 mm outside diameter (O.D.) tubing as shown in Fig. 2e. Connect the filtrate line to the port on top of the cassette, the retentate line to the upper side port, and the intake line to the lower side port.

[0152] 2. Feed the intake line through the peristaltic pump head, insert a disposable 10 mL serological pipette into the end of the tubing, and place the pipette into a bottle with sterilized chilled (4°C) ultrapure water. Keep the cassette in an open circuit by placing both the retentate and filtrate lines in a waste container.

[0153] 3. Pump 500 mL of autoclaved cold ultrapure water through the cassette to remove storage EtOH and to condition the membrane with pure water.

[0154] 4. Transition the cassette into a continuous circuit. Insert the intake and retentate lines into the glass bottle containing sterilized cold phage lysate, as shown in Fig. 2e. Only the filtrate line should remain in the waste container.

[0155] 5. Recirculate the phage lysate through the cassette until 50% volume remains in the bottle, and add cold ultrapure water to double volume.

[0156] 6. Concentrate to 50% again and repeat the ultrapure water dilution by doubling the volume.

[0157] 7. Concentrate to 50% again and dilute with cold sterile PBS to double the volume (Fig. 2f).

[0158] 8. Concentrate to 30% and dilute with cold sterile PBS to double the volume.

[0159] 9. Concentrate to 20% and dilute with cold sterile PBS to double the volume.

[0160] 10. Concentrate to 10% and dilute with cold sterile PBS to double the volume, repeat until the lysate is visibly clear.

[0161] 11. To collect phage particles, set up and label 3 sterile conical centrifuge tubes as “1st, 2nd, 3rdFractions”. Add 30 mL of sterile cold PBS to tubes 2 and 3.

[0162] 12. Concentrate to ~30 mL and pause the peristaltic pump. Pour the ~30 mL of phage solution into the empty conical tube and carefully insert the intake and retentate lines into the conical. Restart pumping until about 5 mL remains in the conical tube. Pause the pump. See Fig. 2g.

[0163] 13. Collect the 1stfraction by draining the cassette, this will allow you to collect the remaining ~30 mL of dead volume of the cassette and tubing. Remove the intake line from the conical and restart the pump to completely flush the cassette into the 50 mL conical. Pause the pump.

[0164] 14. Immediately insert the intake and retentate lines into the 2ndconical tube containing 30 mL sterile cold PBS and start the pump to recirculate buffer to recover the phage remaining in the cassette. When the volume reaches about 5 mL, pause the pump, remove the intake line, and restart the pump to completely flush the cassette again. Pause the pump after all the liquid is collected.

[0165] 15. Repeat the recirculation and draining from step 14 using the 3rdconical tube containing sterile cold PBS. Pause the pump after all liquid is collected. Immediately refill the cassette with buffer for short-term storage until cleaning using manufacturer’s instructions.

[0166] 16. Store fractions at 4°C.

[0167] Chromatographic removal of endotoxin

[0168] Although all prior processing steps reduce and remove endotoxins from phage samples, cross-flow ultrafiltration (CFF) fractions generally contain endotoxins above regulatory limits for intravenous applications. For example, up to 105-l 06EU / mL. Therefore, chromatographic endotoxin removal may be required depending on phage concentration and intended application. For instance, a 1011PFU / mL phage sample with an endotoxin level of 104EU / mL would not need additional endotoxin removal steps if dilution in pyrogen-free saline was done to achieve a therapeutic dose of 109PFU / mL. The commercially available Pierce High-Capacity Endotoxin Removal Resin and ENDOTRAP HD™ assays have a binding capacity of 2* 106and 5* 106EU / mL, respectively. Below is described the specific use of a 1 mL PIERCE CHROMOGENIC ENDOTOXIN REMOVAL SPIN COLUMN™ with a sample capacity of 10 mL with modifications to the manufacturer’s instructions. Samples should be prepared using endotoxin-free water, and the column should only be uncapped in a BSC. Resin columns can be regenerated for reuse, but each column should be dedicated to only a single phage strain.

[0169] 1. Prepare the 1 mL column according to the manufacturer’s instructions. Each time you spin out a solution, loosen the column cap and remove the column’s bottom plug. When adding a solution to the resin slurry, secure the column cap and replace the bottom plug.

[0170] 2. Prepare buffers for use according to the manufacturer’ s instructions. Kit modification: Spike the endotoxin-free equilibration buffer with sterile-filtered NaCl solution to achieve a final solution of 10 mM PBS 0.4 M NaCl.

[0171] 3. Following the removal of the ethanol storage solution, NaOH regeneration buffer, salting with NaCl buffer, and equilibration with water and sodium phosphate buffer, the column is ready for sample addition. Kit modification: Increase the phage concentrate’s NaCl concentration to 0.4 M NaCl by spiking in sterile NaCl solution (Fig. 2h) (see Note 19).

[0172] 4. Mix end-over-end using a tube rotator at 4°C for 1 h.

[0173] 5. Collect the sample in a sterile conical tube.

[0174] 6. Once endotoxin column removal is completed, filter (0.2 pm syringe filter) the final phage preparation into a sterile conical tube.

[0175] 7. Store at 4°C and maintain sterility for qualification / quantification.

[0176] Free nucleic acids digestion

[0177] In alternative embodiments, phage samples are enzymatically digested to remove free DNA and RNA. Phage products can contain residual DNA from host cell substrates. It is, therefore, possible that such residual DNA could encode or harbor harmful molecules or elicit an innate immune response in the patient. It is not clear what health risk the DNA can pose to the product recipients, but often manufacturing can be designed to minimize the risk by reducing the levels of DNA. The World Health Organization and U.S. Food and Drug Administration guidelines recommend that 10 ng / dose and 200 base pairs be the limits of content and size of residual DNA in the final product dose (33). Enzymatic digestion of phage samples removes free DNA and RNA while intact virions are unharmed, and protected by their protein capsid.

[0178] 1. Pre-heat a heat block to 37°C.

[0179] 2. Dilute DNase I stock to a concentration of 100 U / mL. Place on ice.

[0180] 3. Dilute RNase A stock to a concentration of 100 U / mL. Place on ice.

[0181] 4. Determine how many mLs of phage preparation were produced. If you are not using the entire stock, sterilely aliquot the fraction that will be treated.

[0182] 5. Add 2.5 pL DNase I and 2.5 pL RNase A per 100 pL of phage preparation being treated.

[0183] 6. Treat phage preparation for 1 h at 37°C, with the lid heating off (Fig. 2i).

[0184] 7. Store the phage preparation at 4°C.

[0185] Final Preparation Validation

[0186] Sterilization and titration 1. Prepare a fresh exponentially growing culture of host bacteria.

[0187] 2. Air-dry an agar plate for 1 h in a BSC.

[0188] 3. Pre-wet a 0.22 pm PES syringe filter using sterile phage storage buffer (i.e., PBS).

[0189] 4. Decant the nucleic acid-free phage preparation into the syringe. Filter sterilize into a fresh sterile 15 mL conical tube (Fig. 3a).

[0190] 5. Aliquot 100 pL of the phage preparation into a microcentrifuge tube.

[0191] 6. Store 0.22 pm filtered phage preparation at 4°C and protect from UV for longer- term storage.

[0192] 7. Perform spot titration using the phage aliquot as outlined in subheading 3.1.2. Endotoxin quantification

[0193] We recommend choosing either the ENDOZYME™ recombinant factor C or Pierce chromogenic LAL-based assays. Traditional endotoxin quantification uses the Limulus amebocyte lysate (LAL) test, which contains specialized blue blood cells from the wild Atlantic horseshoe crab, Limulus polyphemus. as a component because they react to the presence of endotoxins in a way that can be measured and quantified. The ENDOZYME™ assay are sustainable tests, using a recombinant version of the first enzyme in the LAL clotting cascade. The ENDOZYME™ assay also has a larger detection range from 0.005 to 50 EU / mL

[0194] 1. Select an assay and prepare the assay reagent mixture and set the microplate reader to the settings as per the manufacturer’s instructions.

[0195] 2. Dilute the phage sample in endotoxin-free water to achieve a dilution series of 10’ 4, 10'6, and 10'8PFU / mL to ensure at least one measurement is within the linear dynamic range of the assay.

[0196] 3. Add reagents and run the microplate reader protocol as per the manufacturer’s instructions (Fig. 3b).

[0197] Sterile fill-finish

[0198] In alternative embodiments, the phage preparation is “sterile fill-finished”, for example, is processed for use as a drug product, optionally as a sterile injectable (for example, liquids filled in vials or syringes). Because there is no process to sterilize the phage product in its final container, it is critical that containers be filled and sealed in an extremely controlled environment. Ensuring sterility is not a trivial task, and failure can have catastrophic, even life-threatening, consequences for a patient. The success of aseptic processing and sterile fill-finish operations relies on mitigating contamination from each of these sources: (i) personnel, (ii) drug product components and containers, (iii) cleanroom facilities, and (iv) equipment and processes.

[0199] 1. Clean a BSC with HEPA filtration using 70% EtOH and UV sterilize for 20 min.

[0200] 2. Place all containers and consumables in the cabinet and UV treat for 20 min (see

[0201] Note 22).

[0202] 3. Dilute the phage stock with the desired sterile and endotoxin-free chilled solution in a sterile 50 mL conical tube. Gently vortex (see Note 23).

[0203] 4. Pre-wet a 0.22 pM PES syringe filter with phage diluent (i.e., PBS), and filter sterilize the diluted phage preparation into a fresh sterile 50 mL conical tube.

[0204] 5. Insert a filtered sterile vented needle into the vial’s rubber stopper (see Note 24).

[0205] 6. Draw 4.5 mL in a 5 mL Luer-Lock tip syringe with a 23G x 1-1 / 2” needle. Pierce the rubber stopper and aliquot 1 mL into the vial (Fig. 3c).

[0206] 7. Carefully remove both the syringe needle and the vented needle (see Note 25).

[0207] 8. Repeat until all vials are filled.

[0208] 9. When finished, protect vials from UV light and store at 4°C. Bacteriophage and Bacterial Host Strains

[0209] In alternative embodiments, methods as provided herein are compatible with most phages between about 20 to 500 nm in length. In alternative embodiments, an exemplary purification process is optimized for the Caudoviricetes, which are most sensitive to degradation during filtration because of their complex tail structures.

[0210] However, in alternative embodiments, methods as provided herein also can be used to purify a phage from a family Caudoviricetes, Corticoviridae, Tectiviridae, Tubulavirales, Leviviridae, and / or Microviridae, or a phage from a family Inoviridae, Microviridae, Tectiviridae, Corticoviridae, Plasmaviridae, Leviviridae, and / or Cystoviridae .

[0211] In alternative embodiments host bacteria genomes are sequenced and genetically screened for prophages. The life cycle of temperate phages includes a lysogenic cycle stage when the phage integrates into the host genome and becomes a prophage. While some of the bacterial DNA content can be accounted for by the presence of fully functional prophages, which are able to undergo a replicative, lytic life cycle, a considerable part of it is made up of prophage-like elements, phage remnants left after incomplete excision events, cryptic prophages, or genetic material acquired by horizontal gene transfer. Cultures of lysogens can exhibit free phages (once a prophage) in the cultivation medium supernatant. Because an induced SOS response (for example, RecA) is responsible for the induction of many lysogens, spontaneous SOS induction in single cells might trigger the induction of prophages. A successful infection by a virulent phage poses a major threat to bacterial survival that may induce SOS responses, as well as environmental stresses during long-term culturing.

[0212] For phages intended for therapeutic human and animal use, in alternative embodiments both the phages and host bacteria genomes are sequenced and annotated to screening for harmful genes, such as virulence factors, antibiotic resistance and toxin genes from prophages [27-29], Prophages integrated into the host genome may not only harbor additional virulence factors, but also introduce an added risk of horizontal gene transfer (transduction) or may interfere with the infectivity of the target phage [27, 30], Phage amplification to determine mid-cell-density growth parameters that favor high phage yields and minimize bacterial biomass

[0213] Phage lysates have classically begun with culturing a low number of host bacteria (for example, approximately 106CFU-mL-1) and inoculating with phages at a common multiplicity of infection (MOI) between about 0.1 to 0.001. There cultures are then incubated for extended periods until the bacteria achieve a high density. However, commonly used high-cell-density cultivation approaches have diminishing returns on phage yields and increase final phage product toxicity. For instance, cultures that allow bacterial cells to reach late-log and stationary phase growth (i.e. high-cell-densities) generally have undesirable characteristics such as; 1) secreting higher levels of exotoxin, 2) secreting greater amounts of extracellular polymeric substances that induce biofilm formation, 3) secreting greater amounts of proteases that degrade viral proteins, 4) having increased amounts of extracellular DNA (eDNA), and 5) allowing cells to communicate more with one another (i.e. quorum sensing) that can promote higher phenotypic phage resistance [31-33], In addition, while entering stationary phase, a bacterium regulates transcription in such a way as to trigger the expression of stress response genes required for survival under stress and starvation conditions and to suppress the transcription of unnecessary genes

[0034] , Importantly, spontaneously induced stress responses also increases spontaneous prophage induction rates [35, 36], To avoid the aforementioned concerns, methods as provided herein comprise a mid-cell-density phage cultivation process. That is, we seed cultures with mid-log phase bacterial densities, at density of about 6.0* 108CFU / mL, assuming that bacteria with doubling time of approximately 30 minutes will stay in relative step with phage burst-fold growth. For slower growing bacteria, we reduce seeding to low-log phase bacterial densities at density of about 2.0* 106CFU / mL. Our approach also assumes that phage infections will result in phage growth that will rapidly outpace bacterial cell growth. That is, the host cell has twofold growth rate (binary fission) and burstfold growth rate implies that phage densities will become at least 1 log higher than host cell within 6-7 replication cycles or 180-360 minutes; markedly dependent on each phage’s latent period. Because phage growth declines significantly thereafter and lysates begin a transition to high-cell-density cultivation, lysates are harvested. This exemplary phage amplification process will aid in confirming the specific growth parameters for a mid-cell-density cultivation. In alternative embodiments, this exemplary phage amplification process comprises:

[0214] 1. Pick a fresh CFU from the host strain plate and grow to a density of about 5* 108CFU / mL for phages with 30 minute latency period, or to about 2* 106CFU / mL for phages with 60 minute latency period, in 5 mL of broth medium.

[0215] 2. Add phages at between about 1 to 10 PFU / mL.

[0216] 3. Incubate the culture, optionally for up to about 8 h, at the temperature that is optimal for bacterial growth.

[0217] 4. Harvest the phage lysate, optionally in a sterile 15 mL conical and centrifuge for 10 min at 5,000-10,000 x g.

[0218] 5. Decant the supernatant, optionally into a sterile syringe with an attached filter unit. Filter into a fresh 15 mL conical.

[0219] 6. Spot+ phage titer the lysate and, optionally store at 4°C. Exemplary phage amplification to determine growth parameters to reduce host cell stress responses.

[0220] As mentioned, the bacterial SOS response triggers a myriad of bacterial cell responses, including DNA repair, elongation of bacterial cells, cell surface alteration, increased mutation rates, induction of latent bacteriophages (prophages), secretion of exotoxins, and inhibition of cell division. Initiation of the SOS pathway promotes activation of RecA, inactivation of LexA repressor, and induction of SOS genes

[0034] , Of course, these are unfavorable during phage production. Above, we describe a process that reduces environmental stresses by limiting cell density and by maintenance of rich nutrients and oxygen. In principle, RecA is inhibited by targeting three functionally important processes: recruitment and polymerization, ATP binding, and DNA binding. As mentioned, SOS responses can be minimized by maintaining lower densities of bacteria. To further prevent a spontaneous SOS response during phage production, we suggest supplementing growth medium with inhibitors (inhibitors listed see Table 1) For example, zinc interferes with the actions of RecA, and protects LexA from RecA-mediated cleavage, an early step in initiation of the SOS response

[0037] , Likewise, phthalocyanine tetrasulfonate are anionic, aromatic molecules that tap into the cationic feature of the DNA-binding site of RecA through cation-7t

[0038] , Because RecA inhibitors may be host species specific, a pilot (or preliminary) study is required to determine which inhibitor is effective at suppressing RecA activity and prevent spontaneous SOS responses during production. In alternative embodiments, a pilot (or preliminary) study comprises:

[0221] 1. Pick a fresh CFU from the host strain plate and grow to a density of about 5* 108CFU / mL for 15-45 minute latency period phages or about 2* 106CFU / mL for >45 min latency period phages, optionally grow in 5 mL of liquid broth medium supplemented with SOS response inhibitor. In alternative embodiments, supplement with 0.2-0.4 mM Zinc acetate or 10 pM zinc pyrithione, or 15-25 pM phthalocyanine tetrasulfonic acid, or 1-2 pM naphthalene polysulfonated compounds.

[0222] 2. Add phages, optionally at a density of 1-10 PFU / mL.

[0223] 3. Incubate the culture, optionally for up to 10 times the phages latency period at the temperature most optimal for rapid bacterial growth.

[0224] 4. Harvest the phage lysate, optionally in a sterile 15 mL conical and centrifuge for 10 min at 5,000-10,000 x g.

[0225] 5. Decant the supernatant, optionally into a sterile syringe with an attached filter unit. Filter into a fresh 15 mL conical.

[0226] 6. Spot+ phage titer lysate and, optionally store at 4°C. Scalable production of phages: Rapid Phage Amplification

[0227] As mentioned, bacterial toxins accumulate during phage amplification, and a large quantity of free endotoxin results from violent cell lysis. In alternative embodiments, two centrifugation steps are used to enhance removal of intact bacterial cells and bacterial debris, including large endotoxin aggregates; and in alternative embodiments, an exemplary protocol comprises:

[0228] 1. Pick a CFU isolated using the streak plate method on solid growth medium and culture in the appropriate nutrient-rich broth medium to bulk up a pure culture.

[0229] 2. Subculture 50 pL in fresh liquid broth to get obtain exponentially growing host cells (i.e., OD600 0.1-0.8).

[0230] 3. Add growth broth medium supplemented with SOS response inhibitor to each of the autoclaved large glass growth vessels (for example, glass Erlenmeyer flasks with GL45 screw cap) and pre-warm to optimal growth temperature.

[0231] 4. Add host bacteria at the optimal culture seeding density, as discussed above. In alternative embodiments, seed at a density of between 3* 108to 6* 108CFU / mL for 15-45 minute latency period phages, or between 2* 106to 3* 106CFU / mL for greater than (>) 45 min latency period phages. If using a screw top flask, cap with an autoclaved GL45 sterile vented screw cap with 0.22 pm PTFE hydrophobic membrane to allow gas exchange.

[0232] 5. Add phages, optionally at a density of 1-10 PFU / mL.

[0233] 6. Incubate the flasks, optionally for up to 8 times the phage latency period at the appropriate host growth temperature (for example, 37°C for most human pathogens).

[0234] 7. Harvest amplified phages, optionally by decanting lysates into sterile centrifuge bottles (for example, 1 L bottles) and balance the three bottles against one another using a double pan balance scale.

[0235] 8. Centrifuge, optionally at 8,000-15,000 x g for 10-40 min at 4°C.

[0236] 9. Decant supernatant containing phage particles without disturbing the solid pellet, optionally into new sterile centrifuge bottles, optionally repeat centrifugation at 8,000-15,000 x g for 10-40 min at 4°C at least one additional time to enhance removal of impurities. Centrifugation is a more cost-effective option when batch volumes are less than 10 L or greater than 1,000 L. 10. Collect phages without disturbing the hard-to-see debris pellet into sterile glass, ideally opaque, storage bottles.

[0237] 11. Spot+ phage titer lysate, optionally and store at 4°C.

[0238] Scalable production of phages: Swift Medium-Free Phage Amplification

[0239] As described below, in one exemplary embodiment, the media component is removed during phage amplification to improve downstream purification: i.e., bacterial growth media is removed before phages are added; this eliminates the need to remove spent medium with CFF diafiltration.

[0240] As described below, in one exemplary embodiment, we switch to a very high concentration of host bacterial cells and only allow 1-3 rounds of phage infection latency (optionally, for about 14 min). Thus, amplification set is done in at most 30-60 minutes.

[0241] As described below, in an alternative exemplary embodiment, free molecules that build up in culture conditions are eliminated, optionally, removed in the pellet step.

[0242] In an alternative exemplary embodiment, spent media is formed by bacterial cell growth and is the 'waste' that remains after phage cultivation step. In alternative embodiments, spent media components and extracellular impurities are washed out by CFF ultrafiltration / diafiltration with a MWCO of great than (>) 30 kda before downstream purification; and in alternative embodiments, an exemplary protocol comprises:

[0243] 1. Pick a CFU isolated using the streak plate method on solid growth medium and culture in the appropriate nutrient-rich broth medium to bulk up a pure culture.

[0244] 2. Subculture 50 pL in fresh liquid broth to get obtain dense exponentially growing host cells (i.e., OD600 0.6-0.8) under optimal growth conditions.

[0245] 3. Transfer culture of centrifuge tube and centrifuge at 8000g for 2-10 min to obtain a bacterial pellet. Remove spent media (supernatant) and resuspended pellet in buffer solution, such as Phosphate buffer solution (PBS), normal saline, Bis-tris buffer, or SM buffer, to obtain a concentration of l * 109to 5* 1010CFU / mL, or higher if possible.

[0246] 4. Add buffer solution to each of the autoclaved large glass growth vessels (for example, glass Erlenmeyer flasks with GL45 sterile vented screw cap) and pre-warm to optimal growth temperature.

[0247] 5. Add host bacteria at the high culture seeding density > 1 x 109CFU / mL as discussed above. In alternative embodiments, seed at a density of between 3* 108to 6* 108CFU / mL for 15-45 min latency period phages, or between 2* 106to 3 * 106CFU / mL for greater than (>) 45 min latency period phages. If using a screw top flask, cap with an autoclaved GL45 vented screw cap with 0.22 pm PTFE hydrophobic membrane to allow gas exchange.

[0248] 6. Add phages, optionally at multiplicity of infection of 1.

[0249] 7. Incubate the flasks, optionally for at least 1 phage latency period at the appropriate host growth temperature (e.g., 37°C for most human pathogens).

[0250] 8. 0.2 M sterile filter solution to harvest amplified phages.

[0251] 9. Optionally to harvest amplified phages, decant lysates into sterile centrifuge bottles and balance bottles against one another and centrifuge, optionally at 8,000-15,000 x g for 10-40 min at 4°C to enhance removal of impurities.

[0252] 10. Spot+ phage titer lysate, optionally and store at 4°C.

[0253] Cross-flow ultrafiltration, diafiltration and concentration

[0254] In alternative embodiments, cross-flow filtration (CFF, also known as tangential flow filtration TFF) is used as a scalable purification method to remove bacterial debris, bacterial proteins (for example, endotoxins, exotoxins, proteases) and free DNA (fDNA) molecules less than the CFF molecular weight cut-off (MWCO) (for example, 100 kDa) in phage solutions as well as can remove spent growth medium and buffer exchange. Thus, improving phage solution safety and biocompatibility. Cross filtration is done by indirectly applying flow across a membrane, for example, using the SARTORIUS VIVAFLOW 200 CFF™ cassette, which uses a polyethersulfone membrane (PES), which we find more suitable for phage applications given its hydrophilic properties and low protein binding. The semi- permeable membrane retains larger target phages while allowing smaller molecules to filter out.

[0255] In alternative embodiments a 100 kDa MWCO is used to retain most tailed phage particle sizes, but 50 kDa MWCO can be used to retain smaller phages. The VIVAFLOW’s unique switchback channels parallel to the filtration membrane improves phage dissociation from endotoxins and requires less pressure to drive filtration. The cassette design is easy to set up, gentle on phage particles, and its transparency allows for monitoring flow.

[0256] In alternative embodiments phages are washed with CFF and a PBS (phosphate-buffered saline) with 0.5 M NaCl washing solution. In alternative embodiments purification and buffer exchange is with phosphate buffered saline with 0.5 M NaCl, this was chosen by considering phage stability, impurity nature, and downstream process compatibility. In alternative embodiments the chemical composition of IX PBS has a final concentration of 10 mM PO43, 500 mM NaCl, and 2.7 mM KC1. A high salt (NaCl, MgCh, or CaCh) washing solution enhances dissociation of DNAfrom viral proteins, making DNA available for filtration and degradation. Phage lysates contain a high amount of free DNA (fDNA), released from their lysed hosts. Upon infection, lytic phages shut down and redirect host cell transcriptional machinery to favor transcription of phage genes. Phages also deploy nucleases that degrade the host chromosome freeing up nucleosides to be incorporated into the rapidly replicating phage genomes. PBS is a buffer solution that is particularly valuable for preconditioning therapeutic phage products because it mimics the ion concentration, osmolarity, and pH of human body fluids. PBS is also not known to have any inhibitory effects on PCR.

[0257] In alternative embodiments an exemplary process comprises:

[0258] 1. In a Biological safety cabinet (BSC), connect the CFF cassette with autoclaved 6.4 mm outside diameter (O.D.) tubing, optionally connect the filtrate line to the port on top of the cassette, the retentate line to the upper side port, and the intake line to the lower side port.

[0259] 2. Feed the intake line through the peristaltic pump head, insert a disposable 10 mL serological pipette into the end of the tubing, and place the pipette into a bottle with sterilized chilled (4°C) ultrapure water, optionally keep the cassette in an open circuit by placing both the retentate and filtrate lines in a waste container.

[0260] 3. Pump (optionally 500 mL) of autoclaved cold ultrapure water through the cassette to remove storage EtOH.

[0261] 4. Transition the cassette into a continuous circuit. Insert the intake and retentate lines into the glass bottle containing sterilized cold phage lysate, optionally only the filtrate line should remain in the waste container.

[0262] 5. Recirculate the phage lysate through the cassette until 50% volume remains in the bottle and add cold sterile ultrapure water to double volume.

[0263] 6. Repeat Step 5.

[0264] 7. Concentrate to 50% again and switch diluent, optionally to cold sterile PBS with 0.5 M NaCl washing solution to double the volume.

[0265] 8. Concentrate to 30% and dilute, optionally with cold sterile PBS with 0.5 M NaCl washing solution to double the volume.

[0266] 9. Concentrate to 20% and dilute, optionally with cold sterile PBS with 0.5 M NaCl washing solution to double the volume.

[0267] 10. Concentrate to 10% and dilute, optionally with room temperature sterile PBS with adjusted salinity 0.3 M NaCl washing solution supplemented with 2mM MgC12 to double the volume, repeat until the lysate is visibly clear.

[0268] 11. optionally Pause peristaltic pump Managing free nucleic acid contamination

[0269] As mentioned, phage lysates can contain a high level of DNA and RNA from lysed host cells. Residual DNA may encode harmful molecules if taken up by commensal bacteria and both microbial DNA and RNA can elicit inflammatory immune response in patients if not removed from phage preparations. The World Health Organization and U.S. Food and Drug Administration guidelines recommend that 10 ng / dose and 200 base pairs (bp) be the content and size limits of residual DNA in the final product dose

[0039] , For instance, we use DENARASE® nuclease (c-LEcta, Germany), derived from Bacillus subtilis, an effective endonuclease that can efficiently degrade all types of DNA and RNA (single stranded, double stranded, linear, and circular) without any proteolytic activity (see Note 18). The endonuclease is effective over a wide range of operating conditions and is an essential tool for phage production. The protein has a molecular weight of 27 kDa (dimer with two identical subunits) and can be filtered out during the CFF process. An exemplary process comprises:

[0270] 1. Add lOU / mL of endonuclease (for example, DENARASE®) to the -100 mL of lysate remaining in the bottle.

[0271] 2. Resume the peristaltic pump and recirculate the lysate for -2 min to allow the nuclease to fully circulate within the tubing, CFF cassette, and the full volume of lysate.

[0272] 3. Pause the peristaltic pump again and incubate the lysate for 4 h at room temperature.

[0273] 4. Add 2x volume of PBS with adjusted salinity 0.3 M NaCl washing solution.

[0274] 5. Resume peristaltic pump and concentrate to 10% and dilute with room temperature sterile PBS with adjusted salinity 0.3 M NaCl washing solution to double the volume, repeat until the lysate is visibly clear.

[0275] Exemplary Concentrate collection process

[0276] An exemplary process comprises:

[0277] 1. To collect phage particles, set up and label 3 sterile conical centrifuge tubes as “1st, 2nd, 3rd Fractions”. Add 30 mL of sterile cold PBS with 0.3 M NaCl washing solution to tubes 2 and 3.

[0278] 2. Concentrate to about 30 mL and pause the peristaltic pump. Pour about 30 mL of phage solution into the empty conical tube and carefully insert the intake and retentate lines into the conical. Restart pumping until about 5 mL remains in the conical tube, optionally pause the pump.

[0279] 3. Collect the 1st fraction by draining the cassette, this will allow you to collect the remaining about 30 mL of dead volume of the cassette and tubing. Remove the intake line from the conical and restart the pump to completely flush the cassette into the 50 mL conical optionally pause the pump.

[0280] 4. Immediately insert the intake and retentate lines into the 2nd conical tube containing 30 mL sterile room temperature PBS with 0.3 M NaCl washing solution and start the pump to recirculate buffer to recover the phage remaining in the cassette, optionally when the volume reaches about 5 mL, pause the pump, remove the intake line, and restart the pump to completely flush the cassette again, optionally pause the pump after all the liquid is collected.

[0281] 5. Repeat the recirculation and draining from step 4 using the 3rd conical tube containing sterile room temperature PBS with 0.3 M NaCl washing solution, optionally pause the pump after all liquid is collected. Immediately refill the cassette with buffer for short-term storage until cleaning using manufacturer’s instructions.

[0282] 6. optionally store fractions at 4°C Chromatographic removal of endotoxin

[0283] Although all prior processing steps reduce and remove endotoxins from phage preparations, CFF fractions generally contain endotoxins above regulatory limits for intravenous applications. For example, up to 105-l 06EU / mL. Therefore, in alternative embodiments, chromatographic endotoxin removal may be required depending on phage concentration and intended application. For instance, a 1011PFU / mL phage sample with an endotoxin level of 104EU / mL would not need additional endotoxin removal steps if dilution in pyrogen-free saline was done to achieve a therapeutic dose of 109PFU / mL. The commercially available PIERCE HIGH-CAPACITY ENDOTOXIN REMOVAL RESIN™ and ENDOTRAP HD™ assays have a binding capacity of 2* 106and 5* 106EU / mL, respectively. Though either kit is applicable, below is described the specific use of a 1 mL PIERCE CHROMOGENIC ENDOTOXIN REMOVAL SPIN COLUMN™ with a sample capacity of 10 mL with modifications to the manufacturer’s instructions. Samples should be prepared using endotoxin-free water, and the column should only be uncapped in a BSC. Resin columns can be regenerated for reuse, but each column should be dedicated to only a single phage strain.

[0284] An exemplary process comprises:

[0285] 1. Prepare the 1 mL column according to the manufacturer’s instructions, optionally each time you spin out a solution, loosen the column cap and remove the column’s bottom plug, optionally when adding a solution to the resin slurry, secure the column cap and replace the bottom plug.

[0286] 2. Prepare buffers for use according to the manufacturer’s instructions, optionally kit modification can be: spike the endotoxin-free equilibration buffer with sterile-filtered NaCl solution to achieve a final solution of 10 mM PBS 0.2-0.4 M NaCl.

[0287] 3. Following the removal of the ethanol storage solution, NaOH regeneration buffer, salting with NaCl buffer, and equilibration with water and sodium phosphate buffer, the column is ready for sample addition.

[0288] 4. Mix end-over-end, optionally using a tube rotator at 4°C for 1 h.

[0289] 5. Collect the sample, optionally in a sterile conical tube. 6. Once endotoxin column removal is completed, optionally filter (0.2 pm syringe filter) the final phage preparation into a sterile conical tube.

[0290] 7. optionally store at 4°C and maintain sterility for qualification / quantification.

[0291] Final Preparation Validation

[0292] Exemplary Sterilization and titration

[0293] An exemplary process comprises:

[0294] 1. Prepare a fresh exponentially growing culture of host bacteria, optionally air-dry an agar plate for 1 h in a BSC.

[0295] 2. Pre-wet a filter, optionally a 0.22 pm PES syringe filter, optionally using sterile phage storage buffer (i.e., PBS).

[0296] 3. Decant the nucleic acid-free phage preparation into the syringe, optionally filter sterilize into a fresh sterile 15 mL conical tube.

[0297] 4. Aliquot 100 pL of the phage preparation into a microcentrifuge tube.

[0298] 5. Store 0.22 pm filtered phage preparation, optionally at 4°C and protect from UV for longer-term storage.

[0299] 6. Perform spot titration using the phage aliquot. Exemplary Endotoxin quantification

[0300] In alternative embodiments use either an ENDOZYME™ recombinant factor C or PIERCE™ chromogenic LAL-based assays. Traditional endotoxin quantification uses the Limulus amebocyte lysate (LAL) test, which contains specialized blue blood cells from the wild Atlantic horseshoe crab, Limulus polyphemus. as a component because they react to the presence of endotoxins in a way that can be measured and quantified. Conversely, the ENDOZYME™ assay is a sustainable test, using a recombinant version of the first enzyme in the LAL clotting cascade. The ENDOZYME™ assay also has a larger detection range from 0.005 to 50 EU / mL. An exemplary process comprises:

[0301] 1. Select an assay and prepare the assay reagent mixture and set the microplate reader to the settings as per the manufacturer’s instructions.

[0302] 2. Dilute the phage sample, optionally in endotoxin-free water to achieve a dilution series, optionally of about 10'4, 10'6, and / or 10'8PFU / mL to ensure at least one measurement is within the linear dynamic range of the assay.

[0303] 3. Add reagents and run the microplate reader protocol as per the manufacturer’s instructions.

[0304] Exemplary Sterile fill-finish process

[0305] The term “sterile fill-finish” is used for drug products that are sterile injectables (for example, liquids filled in vials or syringes). Because there is no process to sterilize the phage product in its final container, it is critical that containers be filled and sealed in an extremely controlled environment

[0040] , Ensuring sterility is not a trivial task, and failure can have catastrophic — even life-threatening — consequences for a patient. The success of aseptic processing and sterile fill-finish operations relies on mitigating contamination from each of these sources: (i) personnel, (ii) drug product components and containers, (iii) cleanroom facilities, and (iv) equipment and processes.

[0306] An exemplary process comprises:

[0307] 1. Clean a BSC with HEPA filtration using 70% EtOH and UV sterilize for 20 min.

[0308] 2. Place all containers and consumables in the cabinet and UV treat for 20 min.

[0309] 3. Dilute the phage stock with the desired sterile and endotoxin-free chilled solution in a sterile 50 mL conical tube, optionally gently vortex.

[0310] 4. Pre-wet a 0.22 pM PES syringe filter with phage diluent (optionally with PBS), and filter sterilize the diluted phage preparation into a fresh sterile 50 mL conical tube.

[0311] 5. Insert a filtered sterile vented needle into the vial’s rubber stopper,

[0312] 6. Draw 4.5 mL in a 5 mL Luer-Lock tip syringe with a 23G x 1-1 / 2” needle. Pierce the rubber stopper and aliquot 1 mL into the vial.

[0313] 7. Carefully remove both the syringe needle and the vented needle,

[0314] 8. Repeat until all vials are filled.

[0315] 9. When finished, protect vials from UV light and store at 4°C.

[0316] Alternative options and optional steps for exemplary processes as provided herein:

[0317] 1. If your incubator is not equipped with refrigeration, phage production will need to be manually paused during pilot production;

[0318] 2. A pump with a convex-roller head design is gentler on phages compared to a flip-type head design; 3. A 115 mm Petri plate will support a 48-spot grid of 6 columns of 8 spots each, optionally use this layout to titer CFU / PFU in two sets of triplicate spots;

[0319] 4. Modern microbiological shaking incubators can be refrigerated and programmed to support temperature cycles; alternatively, cultures can be moved to 4-14°C environment. We find that harvesting phage production after 10 hours achieves titers upwards of 108PFU / mL while minimizing bacterial overgrowth during additional incubation. This timing may change depending on the growth rate of the host bacteria, so perform pilot phage production;

[0320] 5. Once bacterial density reaches ODeoo 0.1-0.2, it should be immediately harvested or cooled to 4°C, additional bacterial growth will result in difficulty during dead-end filtration;

[0321] 6. At the time of this writing, PHASTER is accessible using a web browser and accepts FASTA files or GenBank accession numbers

[0041] , alternate tools include Phi Spy and Prophage Hunter [42, 43];

[0322] 7. A standard PCR protocol (such as

[0044] ) should be used; individual reagent volumes will depend on the polymerase / PCR kit being used, optionally each reaction will require 22.5 pL of the master mix;

[0323] 8. Bacterial strains can also be cured of prophages using mitomycin C induction or UV light treatment

[0045] , However, prophage curing can cause changes in phage susceptibility / bacterial fitness, so optionally recommend repeating pilot phage lysate production if prophage curing is performed.

[0324] 9. Culture in liquid broth medium gives bacteria easy access to the available nutrients compared to static bacterial growth on solid medium, also known as an agar culture, optionally agitation to keep the bacteria dispersed through the medium during incubation can aid this access further. Liquid media will also dilute out waste products as they are formed, distributing them through the culture. Consequently, a greater mass of bacteria may be obtained for an equivalent volume of liquid as opposed to solid media.

[0325] 10. Centrifugation at 10,000 x gwiH remove most cellular debris; however, the centrifugation speed will be limited by the centrifuge bottle’s manufactures suggested capacity; centrifugation is a more cost-effective option when batch volumes are less than 10 L or greater than 1,000 L.

[0326] 11. The lysate supernatant may appear clear; however, it is important to perform at least a second spin to enhance the removal of impurities, while leaving the phages in solution;

[0327] 12. Depending on the bacteria species, in general, up to 4 L can be filtered with the capsule filters without the need for cleaning in between, optionally if the dead-end filter becomes clogged, follow the manufacturer’s instructions to clean and reuse the filter.

[0328] 13. If phage lysates are stored in multiple bottles, continue to add all phage lysate to the CFF before switching to the ultrapure water dilution step.

[0329] 14. The CFF will wash proteins and salts through the membrane while leaving phage particles in the retentate and exchange spent growth media for the desired storage buffer, this buffer exchange will occur simultaneously with concentration, where liters of lysate are concentrated up to 100-fold;

[0330] 15. The purpose of salt is to neutralize DNA molecules making them hydrophilic and helps in detaching protein molecules from the free DNA;

[0331] 16. Reduce to PBS with 0.2-0.4 M NaCl washing solution for compatibility with downstream chromatographic removal of endotoxin; optionally consult manufacturers NaCl recommendations for chromatographic removal and adjust accordingly. An insufficient or excess salt concentration can significantly decrease phage recovery or decrease endotoxin affinity to the resin, respectively;

[0332] 17. Repeat this step until the phage solution has visible clarity, denoting the remaining broth and small particles have been washed away (i.e., diafiltration);

[0333] 18. DENARASE® can be substituted with other endonucleases such as BENZONASE™ (Millipore) or TURBONUCLEASE™ (Accelagen). The pivotal distinction between them emanates from their origin and the organisms they are derived from. For example, BENZONASE™ originates from Serratia marcescens. while DENARASE® is a recombinant endonuclease hailing from Bacillus sublilis. engineered for heightened activity. DENARASE® has potentially better availability owing to non-proprietary nature. 19. This will briefly dry the CFF membrane and proceed through fraction collection quickly to ensure the membrane does not stay dry for long.

[0334] 20. Phage may be collected with further repeats. However, as you collect fractions, phage concentration decreases;

[0335] 21. Typically, CFF concentrates phage lysates by 1-2 orders of magnitude and concentrates endotoxin to about 104-l 05EU / mL of buffer. We recommend proceeding with endotoxin removal using Fraction 2, which has lower endotoxin content than Fraction 1, while the phage concentration typically stays within the same order of magnitude. If time permits, titer the phage concentrate before proceeding;

[0336] 22. For improved endotoxin removal, we recommend column regeneration using 0.2 N NaOH overnight incubation at room temperature. Regenerate the column during phage overnight production if you plan to remove endotoxin immediately after CFF concentration;

[0337] 23. If desired, the column can be immediately regenerated, and the endotoxin removal process can be repeated. A second removal step may further reduce endotoxin concentration by up to twofold. However, we do not recommend going beyond two column passages as the final phage recovery is reduced in each passage;

[0338] 24. Pre-wet the syringe membrane with endotoxin-free buffer to prevent loss of phages during final sterile filtration;

[0339] 25. UV does not penetrate into materials, particularly plastics, very well. Although UV can disinfect an empty BSC, it will only disinfect the outer surface of any material in a BSC;

[0340] 26. Filtration typically causes a loss in phage concentration. Calculate and add at least 10% more phage stock to compensate for diluted phage losses.

[0341] 27. For sterile glass vials, it is necessary to vent the gas from the vial and allow the solution in; and / or

[0342] 28. At minimum, replace the needles each time a new syringe is used.

[0343] Products of manufacture and Kits

[0344] Provided are products of manufacture and kits for practicing methods as provided herein; and optionally, products of manufacture and kits can further comprise instructions for practicing methods as provided herein. In alternative embodiments, provided are kits containing preparations of bacteriophage comprising about 109PFU, IO10PFU, 1011PFU, or 1012PFU or more per unit dose and endotoxin levels below about 5.5 EU mL’1, or below about 5.0 EU mL’1.

[0345] Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary, Figures and / or Detailed Description sections.

[0346] As used in this specification and the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0347] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.

[0348] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12% 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

[0349] Unless specifically stated or obvious from context, as used herein, the terms “substantially all”, “substantially most of’, “substantially all of’ or “majority of’ encompass at least about 90%, 95%, 97%, 98%, 99% or 99.5%, or more of a referenced amount of a composition.

[0350] The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such documents, where appropriate, for providing material deemed essential to the claimed subject matter by an examining authority or court. Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of, and "consisting of' may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.

[0351] The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.

[0352] EXAMPLES

[0353] Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols, for example, as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany. Example 1 : Exemplary protocols: Means and methods for producing high titer bacteriophages

[0354] This example demonstrates that exemplary methods (protocols) for producing high titer bacteriophages as provided herein.

[0355] This exemplary method is an experimentally validated protocol that is modular and adaptable, and ensures the identity, titer, quality, and purity of human and animal phage products and prevent instances of contamination, mix-ups, deviations, and errors.

[0356] 1. Introduction

[0357] Bacteriophage, also known as phage, is a virus that attacks and destroys bacteria. Phages are found anywhere and everywhere bacteria can be found: water, air, soil, and in the body. Their activities control the levels of all bacteria in nature. In 1919, Felix d’Herelle, a French-Canadian microbiologist, who at the time was at the Pasteur Institute in Paris, used phages to treat dysentery bacillus. d’Herelle’ s work set the basis for phage therapy. Currently, only the Eliava Institute in Tbilisi, Georgia, has maintained a mainstream use of phage therapy since its discovery. For the past several years, the practice of using phages for patients has been under redevelopment and modernization [1-3], Antibiotic resistance has become a leading public health threat, and the discovery of new classes of antibiotics has stagnated [4], Phages are not affected by antibiotic resistance mechanisms in general [1, 2], and virions themselves are generally considered safe for human and animal use [5-7],

[0358] Virulent phages exclusively use the lytic cycle for replication in bacterial host cells [8], A phage seeks a host and, when found, binds to the cell surface and injects its DNA genome inside the host. In a matter of seconds, the host cell is hijacked for intensive virion replication that results in the bacterium bursting open, releasing newly produced phages to repeat the process. In this manner, the phage lysate can expand to greater than (>) IO10plaque forming units per milliliter (PFU / mL) in growth medium under optimal host conditions. Many phage strains are easy to produce with standard microbiology equipment.

[0359] However, the production bottleneck arises during phage purification. Phage replication accumulates a high amount of toxins in lysates, such as endotoxins, exotoxins, soluble bacterial proteins, peptidoglycans, nucleic acids, and induced prophages [9, 10], Their presence in the bloodstream may cause septic reactions with a variety of symptoms such as fever, hypotension, nausea, shivering, and in extreme cases, fatal shock. Endotoxins are lipopolysaccharide (LPS) molecules associated with the outer membranes of Gram-negative bacteria and make up the most common toxins that must be removed from pharmaceutical phage products. When these bacterial cells are lysed by phages, their cell membranes rupture and endotoxins are released into the solution. Endotoxins in solution range from molecular weights of 10,000 to 1,000,000 Daltons (Da)

[0011] , However, since phages are in the same molecular weight range as endotoxins, filtration cannot be used to adequately separate the endotoxins from the target phages. A harmonized standard for recommended maximum endotoxin exposure is 5.0 endotoxin units (EU) per kg of body weight (interpreted as within 1 hour) for most drugs based on an average patient weight of 70 kg

[0012] , Troubleshooting endotoxin-contaminated phage suspension may become necessary during phage production. Moreover, cold-stored phage suspensions may see the number of viable phages decrease over time, while the concentration of endotoxins detected may not

[0013] ,

[0360] Double polyethylene glycol (PEG) precipitation is a traditional approach for purifying phages [14, 15], As phage particles are made up mostly of coat proteins around their genetic material, solutions containing PEG and high salt can force phage particles to aggregate into clusters. This low-cost method needs minimal equipment and the precipitation is performed by general centrifugation. PEG precipitation, however faces several disadvantages. The method requires the addition of large amounts of PEG and NaCl, some of which remain as a residue in the phage pellet and are difficult to eliminate. Certain other contaminants, such as LPS and exotoxins, are co-purified with the phage particles [16, 17], For these reasons, depending on the purpose, PEG-precipitated phages usually require further methods of purification, such as density gradient ultracentrifugation (DGU), to remove co-precipitated impurities [14, 15], DGU is a highly versatile method that has been widely used for the concentration and purification of phages [14, 18, 19], This approach allows the separation of phages and contaminants based on buoyant density differences independent of size and shape. In this method, the sample is passed through a steep density gradient that contains a very high concentration of CsCl and ultracentrifugation. Multiple bands may be visible after ultracentrifugation; top band(s) corresponds to debris, LPS, and unassembled phages (for example, genome- free capsids). While the lower band contains the target phage that can be collected by puncturing specialized tubes [18, 20], Drawbacks associated with CsCl DGU are that some phages cannot withstand long periods of ultracentrifugation at more than 100,000xg force, and osmotic shock or interaction with CsCl and / or extensive dialysis may lead to loss of phage infectivity

[0021] , Last, not all laboratories can access the required equipment for this method.

[0361] Diverse chromatography -based methods enable the separation and purification of phages. The target phage can be purified based on its interaction types as well as its characteristics, such as shape, size and total charge, hydrophobicity, and binding capacity. Accordingly, the separation functionality is broadly categorized into 1) size, 2) charge, 3) hydrophobicity, and 4) affinity, etc. Size exclusion chromatography (SEC) separates molecules based on their size by filtration through a gel. Given the large size of phages relative to most bacterial components and other small compounds such as LPS, exotoxins, ions, and salts, SEC can be applied as a complementary step for phage purification

[0016] , The main advantages of SEC are the low costs of resins and simplicity in operation as samples are eluted i Socratically, so there is no need to use different buffers during the separation. However, this technique lacks selectivity, suffers from low productivity, and scaling up is restricted because the column can get saturated with host cell debris, consequently preventing the separation of large phages from endotoxins. Another nonspecific technique is ion exchange chromatography (IEC), which is one of the most efficient methods for separating charged particles. Phages are separated according to the strength of their overall ionic interaction with a solid phase material [14, 17, 22], There are two types of IEC: anion- and cationexchange chromatography (AEC and CEC, respectively). A disadvantage of this method is the risk of eluting contaminant proteins and endotoxins with similar electrostatic interactions as the target phage. Phages have a pH-dependent negative surface charge in most solutions, as do LPS

[0023] , The other drawback is the need to connect the chromatography column to a costly FPLC or HPLC instrument. By contrast, affinity chromatography, also called affinity purification, makes use of specific binding interactions between molecules [14, 17, 18, 22], This method uses synthetic ligands for specific elution and involves the impregnation of poly(s-lysine) into cellulose beads, which provide high endotoxin selectivity

[0024] , A combination of electrostatic, hydrophobic, and hydrogen bond interactions are present in the affinity chromatography. The current drawbacks of affinity chromatography are its low yield and high salt concentration requirement for substance elution.

[0362] More recent methods have been applied to phage purification. Extraction with organic compounds can efficiently remove endotoxins from phage samples, such as 1- octanol [25, 26], The removal of octanol is achieved by multiple centrifugation steps and use of a speed vacuum to remove the residual organic solvent [17, 22, 25], The main drawback is inconsistency in sample phage recovery [17, 25, 26], sometimes being as low as 15%

[0017] ,

[0363] With several options at each step of the production process, this protocol selects appropriate methods for the production and purification of phages using different criteria, including cost, equipment, ease, time, yield, and purity of the product. The steps intrinsic to our process to rapidly generate highly pure small-batch phage stocks that meet regulatory guidelines for intravenous (IV) administration are timed process execution to match phage-bacteria kinetics and modifications that have been used to optimize ultrafiltration and diafiltration by both us

[0018] and others [14, 17, 20], Our process first minimizes the quantity of bacterial toxins that accumulate during phage replication by using several techniques to limit bacterial overgrowth and reduce bacterial debris in phage lysates. Next, we use a semi-permeable membrane with active force applied to drive ultrafiltration and diafiltration to get target phages ready for the next processing step by enabling sample cleanup, purification, concentration, buffer exchange, and desalinization. Last, we use affinity chromatography resin to selectively capture the conserved region of the inner core of LPS molecules and thereby remove all kinds of endotoxins from Gram-negative bacteria. We also include our aseptic / sterile fill-finish methods, which are ideal for small-batch biomedical applications. Together, this streamlined process efficiently produces many phages rapidly, safely, and economically.

[0364] 2. Materials

[0365] 2.1 Equipment

[0366] 1. Biological safety cabinet (BSC)

[0367] 2. Microbiological incubators: static and refrigerated incubator shaker (see Note 1)

[0368] 3. Single and multichannel pipettes: various volumes

[0369] 4. Serological pipette controller 5. Spectrophotometer

[0370] 6. High-speed centrifuge with fixed angle rotors (for example, Beckman Coulter J-Lite JLA-8.1000 fixed angle rotor)

[0371] 7. Thermocycler

[0372] 8. Electrophoresis chamber and gel imager

[0373] 9. Double-pan balance scale

[0374] 10. Peristaltic pump and size 16 pump head (see Note 2)

[0375] 11. Tube rotator

[0376] 12. Multimodal microplate reader

[0377] 2.2 Bacteriophage and Bacterial Host Strains

[0378] This method is compatible with most phages 20-500 nm in length. The purification process has been optimized for the tailed Caudoviricetes, which are most sensitive to virus degradation during filtration because of their complex tail structures.

[0379] In alternative embodiments, host bacteria genomes are sequenced and genetically screening for prophages. The life cycle of temperate phages includes a lysogenic cycle stage when the phage integrates into the host genome and becomes a prophage. While some of the bacterial DNA content can be accounted for by the presence of fully functional prophages, which are able to undergo a replicative, lytic life cycle, a considerable part of it is made up of prophage-like elements, phage remnants left after incomplete excision events, cryptic prophages, or genetic material acquired by horizontal gene transfer. Cultures of lysogens can exhibit free phages (once a prophage) in the cultivation medium supernatant. Because an induced SOS response (see for example Radman M. 1974: SOS repair hypothesis In: Prakash L, et al, editors. Mol Environ Asp Mutagen. Springfield, IL: Charles C Thomas Publisher; pp. 128-142) (for example, RecA) is responsible for the induction of many lysogens, spontaneous SOS induction in single cells might trigger the induction of prophages. A successful infection by a virulent phage poses a major threat to bacterial survival that may induce SOS responses, as well as environmental stresses during long-term culturing.

[0380] Phages intended for therapeutic human and animal use, in alternative embodiments, both the phages and host bacteria genomes are sequenced and annotated to screening for harmful genes, such as virulence factors, antibiotic resistance and toxin genes prophages [27-29], Prophages integrated into the host genome may not only harbor additional virulence factors, but also introduce an added risk of horizontal gene transfer (transduction) or may interfere with the infectivity of the target phage [27, 30],

[0381] 2.3 Reagents

[0382] Reagents should be prepared using sterile ultrapure water, autoclaved (45 min at 121 °C), and stored at room temperature unless otherwise indicated.

[0383] 1. Bacterial broth and solid (agar) media

[0384] 2. IM MgCh solution: weigh 203.3 g of MgCh, and dissolve in 80 mL of sterile water. Add water to lOOmL.

[0385] 3. IM CaCh solution: weigh 147.02 g of CaCh and dissolve in 800 mL of sterile water. Add water to 200mL.

[0386] 4. Phosphate buffered saline (PBS) (10X): weigh 17.8 g of Na2HPO4, 2.4 g of KH2PO4, 80 g of NaCl, 2 g of KC1, and dissolve in 800 mL of sterile water. Add water to IL. Dilute 10: 1 in sterile water to make a IX working solution (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCI)

[0387] 5. PCR primers (10 pM concentration)

[0388] 6. Tris-acetate-EDTA (TAE) buffer (10X): weigh 48.5 g Tris and dissolve in 800 mL of sterile water. Add 11.4 mL glacial acetic acid and 20 mL 0.5 M EDTA (pH 8.0). Add water to 1 L. Dilute 10: 1 in sterile water to make IX working solution (40 mM Tris, 20 mM acetic acid, 1 mM EDTA)

[0389] 7. 2% agarose gel: 0.8 g agarose, 40 mL IX TAE

[0390] 8. 0.5 N NaOH

[0391] 9. Endotoxin-free water

[0392] 10. 95% Ethanol and 0.2 N NaOH solution

[0393] 11. 2 M NaCl

[0394] 12. IX high salt PBS has a final concentration of 10 mM PO43, 0.3- 0.5 M NaCl, and 2.7 mM KCI

[0395] 13. DNase I

[0396] 14. RNase A

[0397] 15. 100% Ethanol

[0398] 16. Endonuclease (for example DENARASE®; c-LEcta, Germany)

[0399] 17. 0.4 mM Zinc acetate

[0400] 2.4 Consumables When appropriate (i.e., if plastics are not sterilely packaged), non-sterile glassware and plastics should be autoclave-sterilized prior to use.

[0401] 1. Disposable inoculating loops: 1 pL

[0402] 2. Disposable plastic cuvettes

[0403] 3. Test tubes and caps

[0404] 4. Petri plates: 100 x 15 mm

[0405] 5. Pipette tips: various volumes

[0406] 6. Serological pipets: various volumes

[0407] 7. Polypropylene conical centrifuge tubes: 15 mL and 50 mL

[0408] 8. DNase / RNase-free strip PCR tubes and caps

[0409] 9. Microcentrifuge tubes: 1.5 and 1.7 mL

[0410] 10. Graduated glass media bottles: various volumes

[0411] 11. Glass Erlenmeyer flasks with GL45™ screw cap: 250 mL and 2 L

[0412] 12. GL45™ screw cap with 0.22 pm polytetrafluoroethylene (PTFE) hydrophobic membrane (Coming)

[0413] 13. Polycarbonate centrifuge bottles with cap assemblies (optionally about 1 liter (L))

[0414] 14. Luer Lock tip syringes: 10 mL and 50 mL

[0415] 15. Syringe filters: 0.2 pm

[0416] 16. Syringe needles: 23G x 1-1 / 2”

[0417] 17. RX-VENT™ Filtered Venting Needles

[0418] 18. 0.8 | 0.45 pm polyethersulfone (PES) double layer dead-end filter capsule

[0419] 19. 0.45 | 0.2 pm PES double layer dead-end filter capsule

[0420] 20. VIVAFLOW® 200 CROSSFLOW CASSETTES™: 100 KDa MWCO PES membrane

[0421] 21. ’A” flexible rubber tubing and clamps

[0422] 22. Depyrogenated glass vials (pre-assembled with stopper and aluminum crimp seal)

[0423] 23. 96-well microplates: clear U-bottom and black flat-bottom

[0424] 2.5 Recommended Kits

[0425] 1. Endotoxin Removal: ENDOTRAP® HD (Lionex) or PIERCE™ HIGH- CAPACITY ENDOTOXIN REMOVAL RESIN ASSAY™ (Thermo Scientific)

[0426] 2. Endotoxin Quantification: ENDONEXT™ ENDOZYME® II - RECOMBINANT FACTOR C (RFC) ENDOTOXIN DETECTION ASSAY™ (BioVendor) or PIERCE™ CHROMOGENIC ENDOTOXIN QUANT ASSAY™ (Thermo Scientific)

[0427] 3. Methods

[0428] All procedures must be performed aseptically / sterilely.

[0429] 3.1 Spot+phage titration

[0430] Traditionally, phages are quantified using the top agar overlay method [8], We recommend a double aliquot 48-spot serial titration per sample of interest. This quantification method is quick and easily performed with general laboratory equipment. The Spot+method increases sample coverage, depth, and technical repetition to improve accuracy over a typical spot titration method.

[0431] 1. In a BSC, air-dry agar plates for 1 h. Dry one plate for each sample to be titered.

[0432] 2. Pour 2 mL of bacterial culture at ODeoo 0.2 onto a dried agar plate to seed. Gently swirl to spread the liquid evenly over the entire surface. Tilt the Petri plate and immediately collect excess liquid for disposal. Repeat seeding for each dried agar plate needed.

[0433] 3. Air dry seeded plates for 30 min.

[0434] 4. Prepare two identical 8-well columns of PBS diluent in a 96-well microtiter plate. Add 198 pL of PBS to the top 2 wells and 90 pL in the remaining 14 wells.

[0435] 5. Sample 2 pL from the phage preparation and add to the first microwell containing 198 pL of PBS and mix by repetitive pipetting (repeatedly dispensing and withdrawing solution from the microwell). Sample 2 pL of phage again and add to the second microwell containing 198 pL.

[0436] 6. Use a multichannel pipette to serially dilute in tenfold increments by aliquoting 10 pL from the first row of wells to the second and mix by repetitive pipetting. Change tips and repeat for the remaining six wells.

[0437] 7. Withdraw 4 pL from the first column of 8 microwells using a multichannel pipette. Spot the 8 aliquots in the leftmost area of the seeded agar plate. Repeat 8 x 4 pL sampling from the first microwell column and spot onto the agar plate next to the first column of spots. Repeat 8 * 4 pL sampling from the first microwell column a third time, spotting on the agar plate next to the second column of spots.

[0438] 8. Repeat step 7 and perform 4 pL spotting in triplicate, withdrawing from the second column of 8 microwells.

[0439] 9. Dry spots completely before closing Petri plate lid and moving. Incubate at the appropriate bacterial growth conditions until plaques are countable.

[0440] 10. Calculate phage PFU / mL by averaging the number of PFUs visible in all 6 spots from both dilution series. Selected rows should contain 10-40 PFUs. Divide the average PFUs by 4 (volume spotted), times the dilution factor (column position), and times by 1000 (convert pL to mL). Pre-production small-batch phage growth parameter determinations 1 Bacterial host standard curve to estimate growth rate in absence of phages Streak bacteria on an agar plate from a glycerol stock using a sterile loop. Incubate overnight at the temperature that is optimal for bacterial growth. . Pick a colony forming unit (CFU) and culture it in the appropriate nutrientrich broth, overnight at the optimal temperature for growth. . In a BSC, air dry 5 agar plates for 1 h. . Subculture 50 pL in 5 mL of fresh liquid broth in a sterile test tube. Incubate until an ODeoo approximately 0.8 is reached. Place the test tube on ice. . Aliquot culture into a sterile cuvette and measure absorbance using a spectrophotometer. Dilute the culture to ODeoo 0.8 with sterile bacterial broth in a sterile cuvette. Continue dilutions in twofold increments to obtain cuvettes with ODeoo 0.8, 0.4, 0.2, 0.1, and 0.05. . Prepare two identical 8-well columns of PBS diluent in a 96-well microtiter plate. Add 198 pL of PBS to the top 2 wells and 90 pL in the remaining 14 wells. . Sample 2 pL from the ODeoo 0.8 bacterial culture and add to the first microwell containing 198 pL of PBS and mix by repetitive pipetting (repeatedly dispensing and withdrawing solution from the microwell). Sample 2 pL of bacterial culture again and add to the second microwell containing 198 pL. . Use a multichannel pipette to serially dilute in tenfold increments by aliquoting 10 pL from the first row of wells to the second and mix by repetitive pipetting. Change pipette tips and repeat for the remaining six wells.

[0441] 9. Withdraw 4 pL from the first column of 8 microwells using a multichannel pipette. Spot 8 * 4 pL aliquots to the leftmost edge of the agar plate. Repeat 8 x 4 pL sampling from the first column and spot on the agar plate next to the first column of spots. Repeat 8 * 4 pL sampling from the first column a third time, spotting on the agar plate next to the second column of spots.

[0442] 10. Repeat 4 pL spotting in triplicate, withdrawing from the second column of 8 microwells. Dry spots completely before closing the Petri plate lid and moving the plate (see Note 3).

[0443] 11. Repeat dilution and titration steps 6-10 for the remaining diluted cuvettes.

[0444] 12. Incubate all agar plates at the appropriate bacterial growth conditions until colonies are countable.

[0445] 13. Calculate bacterial CFU / mL by averaging the number of CFUs visible in all 6 spots from both dilution series. Selected spots should contain between 10-40 countable CFUs. Divide the average CFUs by 4 (volume spotted), times the dilution factor (column position), and times by 1000 (convert pL to mL).

[0446] 14. Plot the CFU / mL (y-axis) against ODeoo reading (x-axis). Generate a trendline and check that the R2of the line is greater than (>) 0.9. The equation of the trendline will allow you to approximate the host ODeoo value necessary to achieve the desired concentration CFU / mL.

[0447] 3.2.2 Exemplary phage amplification to determine mid-cell-density growth parameters that favor high phage yields and minimize bacterial biomass

[0448] Phage lysates have classically begun with culturing a low number of host bacteria (for example, approximately 106CFU-mL1) and inoculating with phages at a common multiplicity of infection (MOI) between 0.1-0.001. There cultures are then incubated for extended periods until the bacteria achieve a high density. However, commonly used high-cell-density cultivation approaches have diminishing returns on phage yields and increase final phage product toxicity. For instance, cultures that allow bacterial cells to reach late-log and stationary phase growth (i.e. high-cell- densities) generally have undesirable characteristics such as; 1) secreting higher levels of exotoxin, 2) secreting greater amounts of extracellular polymeric substances that induce biofilm formation, 3) secreting greater amounts of proteases that degrade viral proteins, 4) having increased amounts of extracellular DNA (eDNA), and 5) allowing cells to communicate more with one another (i.e. quorum sensing) that can promote higher phenotypic phage resistance [31-33], In addition, while entering stationary phase, a bacterium regulates transcription in such a way as to trigger the expression of stress response genes required for survival under stress and starvation conditions and to suppress the transcription of unnecessary genes

[0034] , Importantly, spontaneously induced stress responses also increases spontaneous prophage induction rates [35, 36], To avoid the aforementioned concerns, we have developed a mid-cell-density phage cultivation process. That is, we seed cultures with mid-log phase bacterial densities, at density of approximately 6.0* 108CFU / mL, assuming that bacteria with doubling time of approximately 30 minutes will stay in relative step with phage burstfold growth. For slower growing bacteria, we reduce seeding to low-log phase bacterial densities at density of approximately 2.0* 106CFU / mL. Our approach also assumes that phage infections will result in phage growth that will rapidly outpace bacterial cell growth. That is, the host cell has twofold growth rate (binary fission) and burst-fold growth rate implies that phage densities will become at least 1 log higher than host cell within 6-7 replication cycles or 180-360 minutes; markedly dependent on each phage’s latent period. Because phage growth declines significantly thereafter and lysates begin a transition to high-cell-density cultivation, lysates are harvested or temporally cooled to 4°C until harvest. This exemplary phage amplification process will aid in confirming the specific growth parameters for a mid- cell-density cultivation.

[0449] 1. Pick a fresh CFU from the host strain plate and grow to a density of approximately 5* 108CFU / mL for phages with 30 minute latency period, or to approximately 2* 106CFU / mL for phages with 60 minute latency period, in 5 mL of broth medium.

[0450] 2. Add phages at 1-10 PFU / mL.

[0451] 3. Incubate the culture for up to 8 h at the temperature that is optimal for bacterial growth.

[0452] 4. Harvest the phage lysate in a sterile 15 mL conical and centrifuge for 10 min at 5,000-10,000 x g.

[0453] 5. Decant the supernatant into a sterile syringe with an attached filter unit. Filter into a fresh 15 mL conical. 6. Spot+ phage titer the lysate and store at 4°C.

[0454] 3.2.3 Exemplary phage amplification to determine growth parameters to reduce host cell stress responses.

[0455] As mentioned, the bacterial SOS response triggers a myriad of bacterial cell responses, including DNA repair, elongation of bacterial cells, cell surface alteration, increased mutation rates, induction of latent bacteriophages (prophages), secretion of exotoxins, and inhibition of cell division. Initiation of the SOS pathway promotes activation of RecA, inactivation of LexA repressor, and induction of SOS genes

[0034] , Of course, these are unfavorable during phage production. Above, we describe a process that reduces environmental stresses by limiting cell density and by maintenance of rich nutrients and oxygen. In principle, RecA is inhibited by targeting three functionally important processes: recruitment and polymerization, ATP binding, and DNA binding. As mentioned, SOS responses can be minimized by maintaining lower densities of bacteria. To further prevent a spontaneous SOS response during phage production, we suggest supplementing growth medium with inhibitors (inhibitors listed see Table 1) For example, zinc interferes with the actions of RecA, and protects LexA from RecA-mediated cleavage, an early step in initiation of the SOS response

[0037] , Likewise, phthalocyanine tetrasulfonate are anionic, aromatic molecules that tap into the cationic feature of the DNA-binding site of RecA through cation-7t

[0038] , Because RecA inhibitors may be host species specific, a study may be required to determine which inhibitor is effective at suppressing RecA activity and prevent spontaneous SOS responses during production.

[0456] 1. Pick a fresh CFU from the host strain plate and grow to a density of approximately 5* 108CFU / mL for 15-45 minute latency period phages or approximately 2* 106CFU / mL for greater than (>) 45 min latency period phages in 5 mL of liquid broth medium supplemented with SOS response inhibitor. For example, supplement with 0.2-0.4 mM Zinc acetate or 10 pM zinc pyrithione, or 15-25 pM phthalocyanine tetrasulfonic acid, or 1-2 pM naphthalene polysulfonated compounds.

[0457] 2. Add phages at a density of 1-10 PFU / mL.

[0458] 3. Incubate the culture for up to 10 times the phages latency period at the temperature most optimal for rapid bacterial growth.

[0459] 4. Harvest the phage lysate in a sterile 15 mL conical and centrifuge for 10 min at 5,000-10,000 x g.

[0460] 5. Decant the supernatant into a sterile syringe with an attached filter unit. Filter into a fresh 15 mL conical.

[0461] 6. Spot+ phage titer lysate and store at 4°C.

[0462] 3.3 Lysate prophage detection

[0463] Use the exemplary phage lysates as a PCR template for verifying the absence of excised bacteriophages (prophages) carried by host cells during test phage amplification.

[0464] 1. Identify prophages from a fully assembled host strain genome using PHASTER™ or other software of choice, (see Note 6).

[0465] 2. Obtain the FASTA™ file for the putative prophage(s) from the tool of choice.

[0466] 3. Design primers for the prophage(s) using NCBI Primer-BLAST or other tools of choice.

[0467] 4. Make a master mix for each potential prophage or phage strain tested, (see Note 7)

[0468] 5. Vortex briefly to mix. Centrifuge briefly to settle.

[0469] 6. Distribute 22.5 pL of master mix into freshly labeled PCR tubes.

[0470] 7. Add 2.5 pL of the exemplary phage lysate as template into PCR tubes.

[0471] 8. Set the PCR temperatures according to the polymerase / kit chemistry. Use an annealing temperature specific for the phage primers designed in step 3.

[0472] 9. While the PCR reaction is running, prepare a 2% agarose gel and add DNA gel stain.

[0473] 10. Load the PCR reaction after the gel has set for 20 min. Run gel electrophoresis using an appropriately sized DNA ladder and samples.

[0474] 11. Analyze and image gel using a gel documentation system, (see Note 8).

[0475] 3.4 Scalable production of phages

[0476] 3.4.1 ASAP Phage Amplification

[0477] As mentioned, bacterial toxins accumulate during phage amplification, and a large quantity of free endotoxin results from violent cell lysis. We conduct two centrifugation steps to enhance removal of intact bacterial cells and bacterial debris, including large endotoxin aggregates.

[0478] 1. Pick a CFU isolated using the streak plate method on solid growth medium and culture in the appropriate nutrient-rich broth medium to bulk up a pure culture. (see Note 9).

[0479] 2. Subculture 50 qL in fresh liquid broth to get obtain exponentially growing host cells (i.e., ODeoo 0.1-0.8).

[0480] 3. Add growth broth medium supplemented with SOS response inhibitor determined in section 3.2.3 to each of the autoclaved large glass growth vessels (for example, glass Erlenmeyer flasks with GL45™ screw cap) and pre-warm to optimal growth temperature.

[0481] 4. Add host bacteria at the optimal culture seeding density determined in section 3.2. Expect to seed at a density of between 3* 108to 6* 108CFU / mL for 15-45 minute latency period phages, or between 2* 106to 3 * 106CFU / mL for greater than (>) 45 min latency period phages. If using a screw top flask, cap with an autoclaved GL45™ vented screw cap with 0.22 pm PTFE hydrophobic membrane to allow gas exchange.

[0482] 5. Add phages at a density of 1-10 PFU / mL.

[0483] 6. Incubate the flasks for up to 8 times the phage latency period at the appropriate host growth temperature (for example, 37°C for most human pathogens).

[0484] 7. Harvest amplified phages by decanting lysates into sterile centrifuge bottles (for example, 1 L bottles) and balance the three bottles against one another using a double pan balance scale.

[0485] 8. Centrifuge at 8,000-15,000 x g for 10-40 min at 4°C. (see Note 10)

[0486] 9. Decant supernatant containing phage particles without disturbing the solid pellet into new sterile centrifuge bottles. Repeat centrifugation at 8, GOO- 15, 000 x g for 10-40 min at 4°C at least one additional time to enhance removal of impurities (see Note 11).

[0487] 10. Collect phages without disturbing the hard-to-see debris pellet into sterile glass, ideally opaque, storage bottles.

[0488] 11. Spot+ phage titer lysate and store at 4°C.

[0489] 3.3.2 Variable Temperature Phage Amplification

[0490] Although phages are highly capable killers, it is also common for target bacterial cells to evolve resistance to phage infection during long incubations. Resistant cells will continue to grow in the presence of phages and generate unwanted biomass. For convenience, reducing the production process to low temperatures will preserve the integrity of the phages, reduce activity of enzymes (for example, proteases) and prevent cells from dividing. For example, we recommend when producing Gramnegative human pathogen phages that incubation temperatures are dropped to 4°C no longer than 8 hours after culture start. In contrast, Gram-positive human pathogen phage cultures are dropped not longer than 22 hours after culture start to achieve the appropriate bacterial suppression. However, maintaining long periods of low temperature incubation times may reduce phage yields.

[0491] 1. Pick a CFU isolated using the streak plate method on solid growth medium and culture in the appropriate nutrient-rich broth medium to bulk up a pure culture. (see Note 9).

[0492] 2. Subculture 50 pL in fresh liquid broth to get obtain exponentially growing host cells (i.e., ODeoo 0.1-0.8).

[0493] 3. Add growth broth medium supplemented with SOS response inhibitor determined in section 3.2.3 to each of the autoclaved large glass growth vessels (for example, glass Erlenmeyer flasks with a GL45™ polypropylene screw cap (Coming)) and pre-warm to optimal growth temperature.

[0494] 4. Add host bacteria at the optimal culture seeding density determined in section 3.2. Expect to seed at a density of between 3* 108to 6* 108CFU / mL for 15-45 minute latency period phages, or between 2* 106to 3 * 106CFU / mL for greater than (>) 45 min latency period phages. If using a screw top flask, cap with an autoclaved GL45™ vented screw cap with 0.22 pm PTFE hydrophobic membrane to allow gas exchange.

[0495] 5. Add phages at a density of 1-10 PFU / mL.

[0496] 6. Incubate the flasks for up to 8 times the phage latency period at the appropriate host growth temperature (for example, 37°C for most human pathogens).

[0497] 7. Lower temperature to 4°C if required to stop bacterial growth and prolong time to harvest.

[0498] 8. Harvest amplified phages by decanting lysates into sterile centrifuge bottles (for example, 1 L bottles) and balance the three bottles against one another using a double pan balance scale.

[0499] 9. Centrifuge at 8,000-15,000 x g for 10-40 min at 4°C. (see Note 10)

[0500] 10. Decant supernatant containing phage particles without disturbing the solid pellet into new sterile centrifuge bottles. Repeat centrifugation at 8, GOO- 15, 000 x g for 10-40 min at 4°C at least one additional time to enhance removal of impurities (see Note 11).

[0501] 11. Collect phages without disturbing the hard-to-see debris pellet into sterile glass, ideally opaque, storage bottles.

[0502] 12. Spot+ phage titer lysate and store at 4°C.

[0503] 3.4 Phage Purification

[0504] 3.4.1 Dead-end fdtration sterilization

[0505] 1. In a BSC, connect a 0.8 | 0.45 pm heterogeneous polyethersulfone (PES) double layer filter capsule to a 0.45 0.2 pm heterogeneous PES double layer filter capsule with autoclaved YC flexible tubing and clamps.

[0506] 2. Prepare a disposable 10 mL serological pipette by removing the cotton or filter from its end. Feed the tubing through the peristaltic pump head, insert the serological pipette into the end of the tubing, and place it into sterile water.

[0507] 3. Precondition the filter membrane by pumping 500 mL of sterile water through the capsule filters. Discard the flow through.

[0508] 4. Switch the serological pipette and tubing to the bottle containing phage lysate and pump it into sterile glass bottles (see Note 12).

[0509] 5. Store all filtered lysates at 4°C.

[0510] 3.4.2 Cross-flow ultrafiltration, diafiltration and concentration

[0511] We use cross-flow filtration (CFF, also known as tangential flow filtration TFF) as a scalable purification method to remove bacterial debris, bacterial proteins (for example, endotoxins, exotoxins, proteases) and free DNA (fDNA) molecules less than 100 kDa in size in phage solutions as well as can remove spent growth medium and buffer exchange. Thus, improving phage solution safety and biocompatibility. Cross filtration is done by indirectly applying flow across a membrane. The Sartorius VIVAFLOW 200 CFF™ cassette uses a polyethersulfone membrane (PES), which we find more suitable for phage applications given its hydrophilic properties and low protein binding. The semi-permeable membrane retains larger target phages while allowing smaller molecules to filter out. A 100 kDa MWCO is adequate to retain most tailed phage particle sizes, but 50 kDa MWCO can be used to retain smaller phages. The VIVAFLOW™’ s unique switchback channels parallel to the filtration membrane improves phage dissociation from endotoxins and requires less pressure to drive filtration. The cassette design is easy to set up, gentle on phage particles, and its transparency allows for monitoring flow.

[0512] Moreover, we can easily wash the phages with CFF and a PBS (phosphate-buff ered saline) with 0.5 M NaCl washing solution. Purification and buffer exchange with phosphate buffered saline with 0.5 M NaCl was chosen by considering phage stability, impurity nature, and downstream process compatibility. The chemical composition of IX PBS has a final concentration of 10 mM PO43, 500 mM NaCl, and 2.7 mM KC1. A high salt (NaCl, MgCh, or CaCh) washing solution enhances dissociation of DNA from viral proteins, making DNA available for filtration and degradation. Phage lysates contain a high amount of free DNA (fDNA), released from their lysed hosts. Upon infection, lytic phages shut down and redirect host cell transcriptional machinery to favor transcription of phage genes. Phages also deploy nucleases that degrade the host chromosome freeing up nucleosides to be incorporated into the rapidly replicating phage genomes. PBS is a buffer solution that is particularly valuable for preconditioning therapeutic phage products because it mimics the ion concentration, osmolarity, and pH of human body fluids. PBS is also not known to have any inhibitory effects on PCR.

[0513] 1. In a BSC, connect the CFF cassette with autoclaved 6.4 mm outside diameter (O.D.) tubing. Connect the filtrate line to the port on top of the cassette, the retentate line to the upper side port, and the intake line to the lower side port.

[0514] 2. Feed the intake line through the peristaltic pump head, insert a disposable 10 mL serological pipette into the end of the tubing, and place the pipette into a bottle with sterilized chilled (4°C) ultrapure water. Keep the cassette in an open circuit by placing both the retentate and filtrate lines in a waste container.

[0515] 3. Pump 500 mL of autoclaved cold ultrapure water through the cassette to remove storage EtOH.

[0516] 4. Transition the cassette into a continuous circuit. Insert the intake and retentate lines into the glass bottle containing sterilized cold phage lysate. Only the filtrate line should remain in the waste container.

[0517] 5. Recirculate the phage lysate through the cassette until 50% volume remains in the bottle and add cold sterile ultrapure water to double volume (see Note 13).

[0518] 6. Repeat Step 5 (see Note 14).

[0519] 7. Concentrate to 50% again and switch diluent to cold sterile PBS with 0.5 M NaCl washing solution to double the volume, (see Note 15)

[0520] 8. Concentrate to 30% and dilute with cold sterile PBS with 0.5 M NaCl washing solution to double the volume.

[0521] 9. Concentrate to 20% and dilute with cold sterile PBS with 0.5 M NaCl washing solution to double the volume.

[0522] 10. Concentrate to 10% and dilute with room temperature sterile PBS with adjusted salinity 0.3 M NaCl washing solution supplemented with 2mM MgCh to double the volume, repeat until the lysate is visibly clear (see Notes 16 and 17).

[0523] 11. Pause peristaltic pump for steps in section 3.4.4.

[0524] 3.4.4 Managing nucleic acid contamination

[0525] As mentioned, phage lysates can contain a high level of fDNA and RNA from lysed host cells. Residual DNA may encode harmful molecules if taken up by commensal bacteria and both microbial DNA and RNA can elicit inflammatory immune response in patients if not removed from phage preparations. The World Health Organization and U.S. Food and Drug Administration guidelines recommend that 10 ng / dose and 200 base pairs

[0526] (bp) be the content and size limits of residual DNA in the final product dose

[0039] , For instance, we use DENARASE® nuclease (c-LEcta, Germany), derived from Bacillus subtilis, an effective endonuclease that can efficiently degrade all types of DNA and RNA (single stranded, double stranded, linear, and circular) without any proteolytic activity (see Note 18). The endonuclease is effective over a wide range of operating conditions and is an essential tool for phage production. The protein has a molecular weight of 27 kDa (dimer with two identical subunits) and can be filtered out during the CFF process.

[0527] 1. Add lOU / mL of endonuclease (for example, DENARASE®) to the approximately 100 mL of lysate remaining in the bottle.

[0528] 2. Resume the peristaltic pump and recirculate the lysate for approximately 2 min to allow the nuclease to fully circulate within the tubing, CFF cassette, and the full volume of lysate.

[0529] 3. Pause the peristaltic pump again and incubate the lysate for 4 h at room temperature.

[0530] 4. Add 2x volume of PBS with adjusted salinity 0.3 M NaCl washing solution. 5. Resume peristaltic pump and concentrate to 10% and dilute with room temperature sterile PBS with adjusted salinity 0.3 M NaCl washing solution to double the volume, repeat until the lysate is visibly clear (see Notes 16 and 17).

[0531] 3.4.5 Concentrate collection

[0532] 1. To collect phage particles, set up and label 3 sterile conical centrifuge tubes as “1st, 2nd, 3rdFractions”. Add 30 mL of sterile cold PBS with 0.3 M NaCl washing solution to tubes 2 and 3.

[0533] 2. Concentrate to approximately 30 mL and pause the peristaltic pump. Pour approximately 30 mL of phage solution into the empty conical tube and carefully insert the intake and retentate lines into the conical. Restart pumping until approximately 5 mL remains in the conical tube. Pause the pump.

[0534] 3. Collect the 1stfraction by draining the cassette, this will allow you to collect the remaining approximately 30 mL of dead volume of the cassette and tubing. Remove the intake line from the conical and restart the pump to completely flush the cassette into the 50 mL conical. Pause the pump (see Note 19).

[0535] 4. Immediately insert the intake and retentate lines into the 2ndconical tube containing 30 mL sterile room temperature PBS with 0.3 M NaCl washing solution and start the pump to recirculate buffer to recover the phage remaining in the cassette. When the volume reaches approximately 5 mL, pause the pump, remove the intake line, and restart the pump to completely flush the cassette again. Pause the pump after all the liquid is collected.

[0536] 5. Repeat the recirculation and draining from step 4 using the 3rdconical tube containing sterile room temperature PBS with 0.3 M NaCl washing solution. Pause the pump after all liquid is collected. Immediately refill the cassette with buffer for short-term storage until cleaning using manufacturer’s instructions (see Note 20).

[0537] 6. Store fractions at 4°C (see Note 21).

[0538] 3.3.3 Chromatographic removal of endotoxin

[0539] Although all prior processing steps reduce and remove endotoxins from phage samples, CFF fractions generally contain endotoxins above regulatory limits for intravenous applications. For example, up to 105- 106EU / mL. Therefore, chromatographic endotoxin removal may be required depending on phage concentration and intended application. For instance, a 1011PFU / mL phage sample with an endotoxin level of 104EU / mL would not need additional endotoxin removal steps if dilution in pyrogen-free saline was done to achieve a therapeutic dose of 109PFU / mL. The commercially available PIERCE HIGH-CAPACITY ENDOTOXIN REMOVAL RESIN™ and ENDOTRAP HD™ assays have a binding capacity of 2* 106and 5* 106EU / mL, respectively. Though either kit is applicable, below is described the specific use of a 1 mL PIERCE CHROMOGENIC ENDOTOXIN REMOVAL SPIN COLUMN™ with a sample capacity of 10 mL with modifications to the manufacturer’s instructions. Samples should be prepared using endotoxin-free water, and the column should only be uncapped in a BSC. Resin columns can be regenerated for reuse, but each column should be dedicated to only a single phage strain.

[0540] 1. Prepare the 1 mL column according to the manufacturer’s instructions. Each time you spin out a solution, loosen the column cap and remove the column’s bottom plug. When adding a solution to the resin slurry, secure the column cap and replace the bottom plug.

[0541] 2. Prepare buffers for use according to the manufacturer’ s instructions. Kit modification: Spike the endotoxin-free equilibration buffer with sterile-filtered NaCl solution to achieve a final solution of 10 mM PBS 0.2-0.4 M NaCl.

[0542] 3. Following the removal of the ethanol storage solution, NaOH regeneration buffer, salting with NaCl buffer, and equilibration with water and sodium phosphate buffer, the column is ready for sample addition.

[0543] 4. Mix end-over-end using a tube rotator at 4°C for 1 h.

[0544] 5. Collect the sample in a sterile conical tube (see Note 23).

[0545] 6. Once endotoxin column removal is completed, filter (0.2 pm syringe filter) the final phage preparation into a sterile conical tube (see Note 24).

[0546] 7. Store at 4°C and maintain sterility for qualification / quantification.

[0547] 3.4 Final Preparation Validation

[0548] 3.4.1 Sterilization and titration

[0549] 1. Prepare a fresh exponentially growing culture of host bacteria.

[0550] 2. Air-dry an agar plate for 1 h in a BSC.

[0551] 3. Pre-wet a 0.22 pm poly ethersulfone (PES) syringe filter using sterile phage storage buffer (i.e., PBS). 4. Decant the nucleic acid-free phage preparation into the syringe. Filter sterilize into a fresh sterile 15 mL conical tube.

[0552] 5. Aliquot 100 pL of the phage preparation into a microcentrifuge tube.

[0553] 6. Store 0.22 pm filtered phage preparation at 4°C and protect from UV for longer-term storage.

[0554] 7. Perform spot titration using the phage aliquot as outlined in subheading 3.1.2. 3.4.2 Endotoxin quantification

[0555] We recommend choosing either the ENDOZYME™ recombinant factor C or PIERCE™ chromogenic LAL-based assays. Traditional endotoxin quantification uses the Limulus amebocyte lysate (LAL) test, which contains specialized blue blood cells from the wild Atlantic horseshoe crab, Limulus polyphemus. as a component because they react to the presence of endotoxins in a way that can be measured and quantified. Conversely, the ENDOZYME™ assay is a sustainable test, using a recombinant version of the first enzyme in the LAL clotting cascade. The ENDOZYME™ assay also has a larger detection range from 0.005 to 50 EU / mL.

[0556] 1. Select an assay and prepare the assay reagent mixture and set the microplate reader to the settings as per the manufacturer’s instructions.

[0557] 2. Dilute the phage sample in endotoxin-free water to achieve a dilution series of IO-4, IQ-6,ancj IQ- PFU / mL to ensure at least one measurement is within the linear dynamic range of the assay.

[0558] 3. Add reagents and run the microplate reader protocol as per the manufacturer’s instructions.

[0559] 3.5 Sterile fill-finish

[0560] The term “sterile fill-finish” is used for drug products that are sterile injectables (for example, liquids filled in vials or syringes). Because there is no process to sterilize the phage product in its final container, it is critical that containers be filled and sealed in an extremely controlled environment

[0040] , Ensuring sterility is not a trivial task, and failure can have catastrophic, even life-threatening, consequences for a patient. The success of aseptic processing and sterile fill-finish operations relies on mitigating contamination from each of these sources: (i) personnel, (ii) drug product components and containers, (iii) cleanroom facilities, and (iv) equipment and processes.

[0561] 1. Clean a BSC with HEPA filtration using 70% EtOH and UV sterilize for 20 min.

[0562] 2. Place all containers and consumables in the cabinet and UV treat for 20 min (see Note 25).

[0563] 3. Dilute the phage stock with the desired sterile and endotoxin-free chilled solution in a sterile 50 mL conical tube. Gently vortex (see Note 26).

[0564] 4. Pre-wet a 0.22 pM PES syringe filter with phage diluent (i.e., PBS), and filter sterilize the diluted phage preparation into a fresh sterile 50 mL conical tube.

[0565] 5. Insert a filtered sterile vented needle into the vial’s rubber stopper (see Note 27).

[0566] 6. Draw 4.5 mL in a 5 mL Luer-Lock tip syringe with a 23G x 1-1 / 2” needle. Pierce the rubber stopper and aliquot 1 mL into the vial.

[0567] 7. Carefully remove both the syringe needle and the vented needle (see Note 28).

[0568] 8. Repeat until all vials are filled.

[0569] 9. When finished, protect vials from UV light and store at 4°C.

[0570] Notes

[0571] 1. If your incubator is not equipped with refrigeration, phage production will need to be manually paused during exemplary production.

[0572] 2. A pump with a convex-roller head design is gentler on phages compared to a fliptype head design.

[0573] 3. A 115 mm Petri plate will support a 48-spot grid of 6 columns of 8 spots each. We recommended using this layout to titer CFU / PFU in two sets of triplicate spots.

[0574] 4. Modem microbiological shaking incubators can be refrigerated and programmed to support temperature cycles. Alternatively, cultures can be moved to 4-14°C environment. We find that harvesting phage production after 10 hours achieves titers upwards of 108PFU / mL while minimizing bacterial overgrowth during additional incubation. This timing may change depending on the growth rate of the host bacteria, so perform exemplary phage production.

[0575] 5. Once bacterial density is viably turbid (for example reaches about ODeoo 0.1-0.2) the lysate should be harvested. Additional bacterial growth will result in difficulty during dead-end filtration.

[0576] 6. At the time of this writing, PHASTE™R is accessible using a web browser and accepts FASTA™ files or GenBank accession numbers

[0041] , Alternate tools include PHISPY™ and PROPHAGE HUNTER™ [42, 43],

[0577] 7. A standard PCR protocol (such as

[0044] ) should be used. Individual reagent volumes will depend on the polymerase / PCR kit being used. Each reaction will require 22.5 pL of the master mix. Bacterial strains can also be cured of prophages using mitomycin C induction or UV light treatment

[0045] , However, prophage curing can cause changes in phage susceptibility / bacterial fitness. We recommend repeating exemplary phage lysate production if prophage curing is performed. Culture in liquid broth medium gives bacteria easy access to the available nutrients compared to static bacterial growth on solid medium, also known as an agar culture. Agitation to keep the bacteria dispersed through the medium during incubation can aid this access further. Liquid media will also dilute out waste products as they are formed, distributing them through the culture. Consequently, a greater mass of bacteria may be obtained for an equivalent volume of liquid as opposed to solid media. Centrifugation at 10,000 x gwiH remove most cellular debris; however, the centrifugation speed will be limited by the centrifuge bottle’s manufactures suggested capacity. The lysate supernatant may appear clear; however, it is important to perform at least a second spin to enhance the removal of impurities, while leaving the phages in solution. Depending on the bacteria species, in general, up to 4 L can be filtered with the capsule filters without the need for cleaning in between. If the dead-end filter becomes clogged, follow the manufacturer’s instructions to clean and reuse the filter. If phage lysates are stored in multiple bottles, continue to add all phage lysate to the CFF before switching to the ultrapure water dilution step. The CFF will wash proteins and salts through the membrane while leaving phage particles in the retentate and exchange spent growth media for the desired storage buffer. This buffer exchange will occur simultaneously with concentration, where liters of lysate are concentrated up to 100-fold. The purpose of salt is to neutralize DNA molecules making them hydrophilic and helps in detaching protein molecules from the free DNA. Reduce to PBS with 0.2-0.4 M NaCl washing solution for compatibility with downstream chromatographic removal of endotoxin; consult manufacturers NaCl recommendations for chromatographic removal and adjust accordingly. An insufficient or excess salt concentration can significantly decrease phage recovery or decrease endotoxin affinity to the resin, respectively.

[0578] 17. Repeat this step until the phage solution has visible clarity, denoting the remaining broth and small particles have been washed away (i.e., diafiltration).

[0579] 18. DENARASE® can be substituted with other endonucleases such as BENZONASE™ (Millipore) or TURBONUCLEASE™ (Accelagen). The pivotal distinction between them emanates from their origin and the organisms they are derived from. For example, BENZONASE™ originates from Serratia marcescens. while DENARASE® is a recombinant endonuclease hailing from Bacillus sublilis. engineered for heightened activity. DENARASE® has potentially better availability owing to non-proprietary nature.

[0580] 19. This will briefly dry the CFF membrane and proceed through fraction collection quickly to ensure the membrane does not stay dry for long.

[0581] 20. Phage may be collected with further repeats. However, as you collect fractions, phage concentration decreases.

[0582] 21. Typically, CFF concentrates phage lysates by 1-2 orders of magnitude and concentrates endotoxin to approximately 104-l 05EU / mL of buffer. We recommend proceeding with endotoxin removal using Fraction 2, which has lower endotoxin content than Fraction 1, while the phage concentration typically stays within the same order of magnitude. If time permits, titer the phage concentrate before proceeding.

[0583] 22. For improved endotoxin removal, we recommend column regeneration using 0.2 N NaOH overnight incubation at room temperature. Regenerate the column during phage overnight production if you plan to remove endotoxin immediately after CFF concentration.

[0584] 23. If desired, the column can be immediately regenerated, and the endotoxin removal process can be repeated. A second removal step may further reduce endotoxin concentration by up to twofold. However, we do not recommend going beyond two column passages as the final phage recovery is reduced in each passage.

[0585] 24. Pre-wet the syringe membrane with endotoxin-free buffer to prevent loss of phages during final sterile filtration.

[0586] 25. UV does not penetrate into materials, particularly plastics, very well. Although UV can disinfect an empty BSC, it will only disinfect the outer surface of any material in a BSC.

[0587] 26. Filtration typically causes a loss in phage concentration. Calculate and add at least 10% more phage stock to compensate for diluted phage losses.

[0588] 27. For sterile glass vials, it is necessary to vent the gas from the vial and allow the solution in.

[0589] 28. At minimum, replace the needles each time a new syringe is used.

[0590] Table 1 : Summary of reported RecA inhibitors.

[0591] 1NPS, naphthalene polysulfonated compounds;2M.W., molecular weight;3Da, Dalton;4PTA, phthalocyanine tetrasulfonic acid analogs;

[0592] Example 2: Rapid Bench to Bedside Therapeutic Bacteriophage Production This example demonstrates that exemplary methods (protocols) for producing high titer bacteriophages as provided herein.

[0593] It has been over 100 years since bacteriophages (phages) were used as a human therapeutic. Since then, phage production has dramatically evolved. Current phage preparations have fewer adverse effects due to their low bacterial toxin content. As a result, therapeutic phages have become a predominant class of new antimicrobials and are being widely used for compassionate treatment of multidrugresistant (MDR) infections. We describe herein a protocol for the production and ultrapurification of phages. By this technique, it is possible for a lab experienced with the process to produce greater than (>) 109plaque forming units (PFU) per mL of Gram-negative phages that meet FDA endotoxins limits for intravenous infusions in as little as 48 hours. We provide illustrations of the process and tips on how to safely remove bacterial toxins from phage lysates. Although dependent on the phage strain, the approach described herein to rapidly generate and purify phages is extremely useful for a variety of applications.

[0594] Bacteriophage, also known as phage, is a virus that attacks and destroys bacteria. Phages are found anywhere and everywhere bacteria can be found: water, air, soil, and in the body. Their activities control the levels of all bacteria in nature. In 1919, Felix d’Herelle, a French-Canadian microbiologist, who at the time was at the Pasteur Institute in Paris, used phages to treat dysentery bacillus. d’Herelle’ s work set the basis for phage therapy. Currently, only the Eliava Institute in Tbilisi, Georgia, has maintained a mainstream use of phage therapy since its discovery. For the past several years, the practice of using phages for patients has been under redevelopment and modernization (1-3). Antibiotic resistance has become a leading public health threat, and the discovery of new classes of antibiotics has stagnated (4). Phages are not affected by antibiotic resistance mechanisms in general (1, 2), and virions themselves are generally considered safe for human and animal use (5-7).

[0595] Virulent phages exclusively use the lytic cycle for replication in bacterial host cells (8). A phage seeks a host and, when found, binds to the cell surface and injects its DNA genome inside the host. In a matter of seconds, the host cell is hijacked for intensive virion replication that results in the bacterium bursting open, releasing newly produced phages to repeat the process. In this manner, the phage lysate can expand to greater than (>) 1010plaque forming units per milliliter (PFU / mL) in growth medium under optimal host conditions. Many phage strains are easy to produce with standard microbiology equipment.

[0596] However, the production bottleneck arises during phage purification. Phage replication accumulates a high amount of toxins in lysates, such as endotoxins, exotoxins, soluble bacterial proteins, peptidoglycans, nucleic acids, and induced prophages (9, 10). Their presence in the bloodstream may cause septic reactions with a variety of symptoms such as fever, hypotension, nausea, shivering, and in extreme cases, fatal shock. Endotoxins are lipopolysaccharide (LPS) molecules associated with the outer membranes of Gram-negative bacteria and make up the most common toxins that must be removed from pharmaceutical phage products. When these bacterial cells are lysed by phages, their cell membranes rupture and endotoxins are released into the solution. Endotoxins in solution range from molecular weights of 10,000 to 1,000,000 Daltons (Da) (11). However, since phages are in the same molecular weight range as endotoxins, filtration cannot be used to adequately separate the endotoxins from the target phages. A harmonized standard for recommended maximum endotoxin exposure is 5.0 endotoxin units (EU) per kg of body weight (interpreted as within 1 hour) for most drugs based on an average patient weight of 70 kg (12). Troubleshooting endotoxin-contaminated phage suspension may become necessary during phage production. Moreover, cold-stored phage suspensions may see the number of viable phages decrease over time, while the concentration of endotoxins detected may not (13).

[0597] Double polyethylene glycol (PEG) precipitation is a traditional approach for purifying phages (14, 15). As phage particles are made up mostly of coat proteins around their genetic material, solutions containing PEG and high salt can force phage particles to aggregate into clusters. This low-cost method needs minimal equipment and the precipitation is performed by general centrifugation. PEG precipitation, however faces several disadvantages. The method requires the addition of large amounts of PEG and NaCl, some of which remain as a residue in the phage pellet and are difficult to eliminate. Certain other contaminants, such as LPS and exotoxins, are generally co-precipitated with phages (16, 17). For these reasons, depending on the purpose, PEG-precipitated phages usually require further methods of purification, such as density gradient ultracentrifugation (DGU), to remove co-precipitated impurities (14, 15). DGU is a highly versatile method that has been widely used for the concentration and purification of phages (14, 18, 19). This approach allows the separation of phages and contaminants based on buoyant density differences independent of size and shape. In this method, the sample is passed through a steep density gradient that contains a very high concentration of CsCl and ultracentrifugation. Multiple bands may be visible after ultracentrifugation; top band(s) corresponds to debris, LPS, and unassembled phages (for example, genome- free capsids). While the lower band contains the target phage that can be collected by puncturing specialized tubes (18, 20). Drawbacks associated with CsCl DGU are that some phages cannot withstand long periods of ultracentrifugation at more than 100,000xg force, and osmotic shock or interaction with CsCl and / or extensive dialysis may lead to loss of phage infectivity (21). Last, not all laboratories can access the required equipment for this method.

[0598] Diverse chromatography -based methods enable the separation and purification of phages. The target phage can be purified based on its interaction types as well as its characteristics, such as shape, size and total charge, hydrophobicity, and binding capacity. Accordingly, the separation functionality is broadly categorized into 1) size, 2) charge, 3) hydrophobicity, and 4) affinity, etc. Size exclusion chromatography (SEC) separates molecules based on their size by filtration through a gel. Given the large size of phages relative to most bacterial components and other small compounds such as LPS, exotoxins, ions, and salts, SEC can be applied as a complementary step for phage purification (16). The main advantages of SEC are the low costs of resins and simplicity in operation as samples are eluted i Socratically, so there is no need to use different buffers during the separation. However, this technique lacks selectivity, suffers from low productivity, and scaling up is restricted because the column can get saturated with host cell debris, consequently preventing the separation of large phages from endotoxins. Another nonspecific technique is ion exchange chromatography (IEC), which is one of the most efficient methods for separating charged particles. Phages are separated according to the strength of their overall ionic interaction with a solid phase material (14, 17, 22). There are two types of IEC: anion- and cationexchange chromatography (AEC and CEC, respectively). A disadvantage of this method is the risk of eluting contaminant proteins and endotoxins with similar electrostatic interactions as the target phage. Phages have a pH-dependent negative surface charge in most solutions, as do LPS (23). The other drawback is the need to connect the chromatography column to a costly FPLC or HPLC instrument. By contrast, affinity chromatography, also called affinity purification, makes use of specific binding interactions between molecules (14, 17, 18, 22). This method uses synthetic ligands for specific elution and involves the impregnation of poly(s-lysine) into cellulose beads, which provide high endotoxin selectivity (24). A combination of electrostatic, hydrophobic, and hydrogen bond interactions are present in the affinity chromatography. The current drawbacks of affinity chromatography are its low yield and high salt concentration requirement for substance elution.

[0599] More recent methods have been applied to phage purification. Extraction with organic compounds can efficiently remove endotoxins from phage samples, such as 1- octanol (25, 26). The removal of octanol is achieved by multiple centrifugation steps and use of a speed vacuum to remove the residual organic solvent (17, 22, 25). The main drawback is inconsistency in sample phage recovery (17, 25, 26), sometimes being as low as 15% (17).

[0600] With several options at each step of the production process, this protocol selects appropriate methods for the production and purification of phages using different criteria, including cost, equipment, ease, time, yield, and purity of the product. The steps intrinsic to our process to rapidly generate highly pure small-batch phage stocks that meet regulatory guidelines for intravenous (IV) administration are timed process execution to match phage-bacteria kinetics and modifications that have been used to optimize ultrafiltration (UF) and diafiltration (DF) by both us (18) and others (14, 17, 20).

[0601] Exemplary processes as provided herein minimize the quantity of bacterial toxins that accumulate during phage replication by using several techniques to limit bacterial overgrowth and reduce bacterial debris in phage lysates. Exemplary processes as provided herein use a semi-permeable membrane with active force applied to drive UF and DF to get target phages ready for the next processing step by enabling sample cleanup, purification, concentration, buffer exchange, and desalinization. Exemplary processes as provided herein use affinity chromatography resin to selectively capture the conserved region of the inner core of LPS molecules and thereby remove all kinds of endotoxins from Gram-negative bacteria. Exemplary processes as provided herein also include aseptic / sterile fill-finish methods, which are ideal for small-batch biomedical applications. Together, this exemplary streamlined process efficiently produces many phages in as little as 48 hours and economically. Materials

[0602] 2.1 Equipment

[0603] 1. Biological safety cabinet (BSC)

[0604] 2. Microbiological incubators: static and refrigerated incubator shaker (see Note 1)

[0605] 3. Single and multichannel pipettes: various volumes

[0606] 4. Serological pipette controller

[0607] 5. Spectrophotometer

[0608] 6. High-speed centrifuge with fixed angle rotors (for example, Beckman Coulter J-Lite JLA-8.1000 fixed angle rotor)

[0609] 7. Thermocycler

[0610] 8. Electrophoresis chamber and gel imager

[0611] 9. Double-pan balance scale

[0612] 10. Peristaltic pump and size 16 pump head (see Note 2)

[0613] 11. Tube rotator

[0614] 12. Multimodal microplate reader

[0615] 2.2 Bacteriophage and Bacterial Host Strains

[0616] We recommend before phage production that both phage and host bacterial genomes have been sequenced and annotated. Both phage and host bacteria genomes require screening for virulence factors, antibiotic resistance and toxin genes, and prophages (27-29). Prophages integrated into the host genome may not only harbor additional virulence factors, but also introduce an added risk of horizontal gene transfer (transduction) or may interfere with the infectivity of the target phage (27, 30). If any undesirable elements are identified in either the phage or host genome, we recommend using alternative strains.

[0617] 1. Bacteriophage stock (recommend sequenced and 0.2 pm filtered)

[0618] 2. Bacterial strain used to propagate bacteriophage strain (recommend sequenced)

[0619] 2.3 Reagents

[0620] Reagents should be prepared using sterile MILLI-Q™ water, autoclaved (for example, 45 min at 121°C), and stored at room temperature unless otherwise indicated. 1. Bacterial broth and solid (agar) media

[0621] 2. Phosphate buffered saline (PBS) (10X): weigh 17.8 g of Na2HPO4, 2.4 g of KH2PO4, 80 g of NaCl, 2 g of KC1, and dissolve in 800 mL of sterile water. Add water to IL. Dilute 10: 1 in sterile water to make a IX working solution (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCI)

[0622] 3. PCR primers (10 pM concentration)

[0623] 4. Tris-acetate-EDTA (TAE) buffer (10X): weigh 48.5 g Tris and dissolve in 800 mL of sterile water. Add 11.4 mL glacial acetic acid and 20 mL 0.5 M EDTA (pH 8.0). Add water to 1 L. Dilute 10: 1 in sterile water to make IX working solution (40 mM Tris, 20 mM acetic acid, 1 mM EDTA)

[0624] 5. 2% agarose gel: 0.8 g agarose, 40 mL IX TAE

[0625] 6. 0.5 N NaOH

[0626] 7. Endotoxin-free water

[0627] 8. 95% Ethanol and 0.2 N NaOH solution

[0628] 9. 2 M NaCl

[0629] 10. 10 mM sodium phosphate buffer (PBS): Dilute 10X PBS 10: 1 in endotoxin-free water and filter sterilize

[0630] 11. DNase I

[0631] 12. RNase A

[0632] 13. 100% Ethanol

[0633] 2.4 Consumables

[0634] When appropriate (i.e., if plastics are not sterilely packaged), non-sterile glassware and plastics should be autoclave-sterilized prior to use.

[0635] 1. Disposable inoculating loops: 1 pL

[0636] 2. Disposable plastic cuvettes

[0637] 3. Test tubes and caps

[0638] 4. Petri plates: 100 x 15 mm

[0639] 5. Pipette tips: various volumes

[0640] 6. Serological pipets: various volumes

[0641] 7. Polypropylene conical centrifuge tubes: 15 mL and 50 mL

[0642] 8. DNase / RNase-free strip PCR tubes and caps 9. Microcentrifuge tubes: 1.5 and 1.7 mL

[0643] 10. Graduated glass media bottles: various volumes

[0644] 11. Glass Erlenmeyer flasks with GL45™ screw cap: 250 mL and 2 L

[0645] 12. GL45™ screw cap with 0.22 pm polytetrafluoroethylene (PTFE) hydrophobic membrane (Coming)

[0646] 13. Polycarbonate centrifuge bottles with cap assemblies (recommend 1 L)

[0647] 14. Luer Lock tip syringes: 10 mL and 50 mL

[0648] 15. Syringe filters: 0.2 pm

[0649] 16. Syringe needles: 23G x 1-1 / 2”

[0650] 17. RX-VENT™ Filtered Venting Needles

[0651] 18. 0.8 | 0.45 pm polyethersulfone (PES) double layer dead-end filter capsule

[0652] 19. 0.45 | 0.2 pm PES double layer dead-end filter capsule

[0653] 20. VIVAFLOW® 200 CROSSFLOW CASSETTES™: 100 KDa MWCO PES membrane

[0654] 21. ’A” flexible rubber tubing and clamps

[0655] 22. Depyrogenated glass vials (pre-assembled with stopper and aluminum crimp seal)

[0656] 23. 96-well microplates: clear U-bottom and black flat-bottom

[0657] 2.5 Exemplary Kits

[0658] 1. Endotoxin Removal: ENDOTRAP® HD™ (Lionex) or PIERCE™ HIGH-CAPACITY ENDOTOXIN REMOVAL RESIN ASSAY™ (Thermo Scientific)

[0659] 2. Endotoxin Quantification: ENDONEXT™ ENDOZYME® II™ - RECOMBINANT FACTOR C (RFC) ENDOTOXIN DETECTION ASSAY™ (BioVendor) or PIERCE™ CHROMOGENIC ENDOTOXIN QUANT ASSAY™ (Thermo Scientific)

[0660] 3. Methods

[0661] All procedures must be performed aseptically / sterilely in a biological safety cabinet.

[0662] 3.1 Pre-Processing

[0663] 3.1.1 Bacterial host standard curve

[0664] 1. Streak bacteria on an agar plate from a glycerol stock using a sterile loop. Incubate overnight at the temperature that is optimal for bacterial growth. 2. Pick a colony forming unit (CFU) and culture it in the appropriate nutrient-rich broth, overnight at the optimal temperature for growth.

[0665] 3. In a BSC, air dry 5 agar plates for 1 h.

[0666] 4. Subculture 50 pL in 5 mL of fresh liquid broth in a sterile test tube. Incubate until an ODeoo approximately 0.8 is reached. Place the test tube on ice.

[0667] 5. Aliquot culture into a sterile cuvette and measure absorbance using a spectrophotometer. Dilute the culture to ODeoo 0.8 with sterile bacterial broth in a sterile cuvette. Continue dilutions in twofold increments to obtain cuvettes with ODeoo 0.8, 0.4, 0.2, 0.1, and 0.05.

[0668] 6. Prepare two identical 8-well columns of PBS diluent in a 96-well microtiter plate. Add 198 pL of PBS to the top 2 wells and 90 pL in the remaining 14 wells.

[0669] 7. Sample 2 pL from the ODeoo 0.8 bacterial culture and add to the first microwell containing 198 pL of PBS and mix by repetitive pipetting (repeatedly dispensing and withdrawing solution from the microwell). Sample 2 pL of bacterial culture again and add to the second microwell containing 198 pL.

[0670] 8. Use a multichannel pipette to serially dilute in tenfold increments by aliquoting 10 pL from the first row of wells to the second and mix by repetitive pipetting. Change pipette tips and repeat for the remaining six wells.

[0671] 9. Withdraw 4 pL from the first column of 8 microwells using a multichannel pipette. Spot 8 * 4 pL aliquots to the leftmost edge of the agar plate. Repeat 8 x 4 pL sampling from the first column and spot on the agar plate next to the first column of spots. Repeat 8 * 4 pL sampling from the first column a third time, spotting on the agar plate next to the second column of spots (Fig. la).

[0672] 10. Repeat 4 pL spotting in triplicate, withdrawing from the second column of 8 microwells. Dry spots completely before closing the Petri plate lid and moving the plate (see Note 3).

[0673] 11. Repeat dilution and titration steps 6-10 for the remaining diluted cuvettes.

[0674] 12. Incubate all agar plates at the appropriate bacterial growth conditions until colonies are countable (Fig. lb).

[0675] 13. Calculate bacterial CFU / mL by averaging the number of CFUs visible in all 6 spots from both dilution series. Selected spots should contain between 10-40 countable CFUs. Divide the average CFUs by 4 (volume spotted), times the dilution factor (column position), and times by 1000 (convert pL to mL). 14. Plot the CFU / mL (y-axis) against ODeoo reading (x-axis). Generate a trendline and check that the R2of the line is greater than (>) 0.9. The equation of the trendline will allow you to approximate the host ODeoo value necessary to achieve the desired concentration CFU / mL.

[0676] 3.1.2 Spot+phage titration

[0677] Traditionally, phages are quantified using the top agar overlay method (8). We recommend a double aliquot 48-spot serial titration per sample of interest. This quantification method is quick and easily performed with general laboratory equipment. The Spot+method increases sample coverage, depth, and technical repetition to improve accuracy over a typical spot titration method.

[0678] 1. In a BSC, air-dry agar plates for 1 h. Dry one plate for each sample to be titered.

[0679] 2. Pour 2 mL of bacterial culture at ODeoo 0.2 onto a dried agar plate to seed. Gently swirl to spread the liquid evenly over the entire surface. Tilt the Petri plate and immediately collect excess liquid for disposal. Repeat seeding for each dried agar plate needed.

[0680] 3. Air dry seeded plates for 30 min.

[0681] 4. Prepare two identical 8-well columns of PBS diluent in a 96-well microtiter plate. Add 198 pL of PBS to the top 2 wells and 90 pL in the remaining 14 wells.

[0682] 5. Sample 2 pL from the phage preparation and add to the first microwell containing 198 pL of PBS and mix by repetitive pipetting (repeatedly dispensing and withdrawing solution from the microwell). Sample 2 pL of phage again and add to the second microwell containing 198 pL.

[0683] 6. Use a multichannel pipette to serially dilute in tenfold increments by aliquoting 10 pL from the first row of wells to the second and mix by repetitive pipetting. Change tips and repeat for the remaining six wells.

[0684] 7. Withdraw 4 pL from the first column of 8 microwells using a multichannel pipette. Spot the 8 aliquots in the leftmost area of the seeded agar plate. Repeat 8 x 4 pL sampling from the first microwell column and spot onto the agar plate next to the first column of spots. Repeat 8 * 4 pL sampling from the first microwell column a third time, spotting on the agar plate next to the second column of spots (Fig. la).

[0685] 8. Repeat step 7 and perform 4 pL spotting in triplicate, withdrawing from the second column of 8 microwells. 9. Dry spots completely before closing Petri plate lid and moving. Incubate at the appropriate bacterial growth conditions until plaques are countable (Fig. 1c).

[0686] 10. Calculate phage PFU / mL by averaging the number of PFUs visible in all 6 spots from both dilution series. Selected rows should contain 10-40 PFUs. Divide the average PFUs by 4 (volume spotted), times the dilution factor (column position), and times by 1000 (convert pL to mL).

[0687] 3.1.3 Exemplary phage amplification

[0688] 1. Pick a fresh CFU from the host strain plate and grow to approximately 108CFU in 5 mL of broth medium.

[0689] 2. Add phages at approximately 107PFU to achieve a multiplicity of infection (MOI) of O. l.

[0690] 3. Harvest the phage lysate in a sterile 15 mL conical and centrifuge for 10 min at 8000xg.

[0691] 4. Decant the supernatant into a sterile syringe with an attached filter unit. Filter into a fresh 15 mL conical.

[0692] 5. Aliquot 100 pL of the filtered lysate into a PCR tube.

[0693] 6. Store at 4°C.

[0694] 3.1.4 Lysate prophage detection

[0695] Even if a putative prophage is detected bioinformatically, it may not necessarily be liberated during lysate production. Use the exemplary phage lysate as a PCR template for verification.

[0696] 1. Identify prophages from a fully assembled host strain genome using PHASTER or other software of choice, (see Note 6).

[0697] 2. Obtain the FASTA file for the putative prophage(s) from the tool of choice.

[0698] 3. Design primers for the prophage(s) using NCBI Primer-BLAST or other tools of choice.

[0699] 4. Make a master mix for each potential prophage or phage strain tested, (see Note 7)

[0700] 5. Vortex briefly to mix. Centrifuge briefly to settle.

[0701] 6. Distribute 22.5 pL of master mix into freshly labeled PCR tubes.

[0702] 7. Add 2.5 pL of the exemplary phage lysate as template into PCR tubes.

[0703] 8. Set the PCR temperatures according to the polymerase / kit chemistry. Use an annealing temperature specific for the phage primers designed in step 3. 9. While the PCR reaction is running, prepare a 2% agarose gel and add DNA gel stain.

[0704] 10. Load the PCR reaction after the gel has set for 20 min. Run gel electrophoresis using an appropriately sized DNA ladder and samples.

[0705] 11. Analyze and image gel using a gel documentation system, (see Note 8).

[0706] 3.2 Batch Phage Amplification

[0707] Phage production typically begins with long culture incubations (for example, overnight / 18-24 h) followed by lysate centrifugation and 0.2 pm filter sterilization. As mentioned, bacterial toxins accumulate during phage amplification, and a large quantity of free endotoxin results from violent cell lysis. It is also common for target bacterial cells to evolve resistance to phage infection during these long incubations. These evolved mutants thrive in optimal culture conditions and overgrow in the lysate. We recommend switching to cold growth incubation, 1-2 h prior to the visible outgrowth of phage-resistant bacteria. Time-kill kinetics assays help understand interactions that exist between host bacteria and phages, including time to resistance. For example, we recommend when using Pseudomonas aeruginosa phages to reduce the incubation temperature after ten hours to suppress the outgrowth of evolved phage resistant mutant cells while maintaining phage production. Similarly, we recommend for Staphylococcus aureus phages to reduce the incubation temperature after 22 hours to achieve the same suppression. This temperature drop prevents resistant bacterial growth, which causes a clearer lysate while maintaining long incubation times for higher phage yields. The addition of two centrifugation steps significantly enhances the removal of intact bacterial cells and bacterial debris, including large endotoxin aggregates. Figure 2 shows a flow diagram briefly describing this process.

[0708] 1. Pick a CFU isolated using the streak plate method on solid growth medium and culture in the appropriate nutrient-rich broth medium overnight to bulk up a pure culture, (see Note 9).

[0709] 2. Subculture 50 pL in fresh liquid broth to get the bacterial population growing exponentially (i.e., ODeoo 0.1-0.8).

[0710] 3. Add 1 L of broth medium to each of the 3 sterile autoclaved glass Erlenmeyer flasks with GL45™ screw cap and pre-warm to bacterial growth temperature.

[0711] 4. Add exponentially growing bacteria at an optical density that corresponds to a count of approximately 1010CFU per flask and exchange the flask’s cap for an autoclaved GL45™ vented screw cap with 0.22 pm PTFE hydrophobic membrane. Incubate the flasks for one generation at growth temperature with gentle agitation (Fig. 2a).

[0712] 5. After bacteria have acclimated and grown for one generation, add phages at approximately 109PFU to achieve a multiplicity of infection (MOI) of 0.1 (Fig. 2a).

[0713] 6. Incubate the flasks for 10-12 h, or until visibly turbid.

[0714] 7. Harvest amplified phages by decanting lysates into sterile centrifuge bottles (for example, 1 L bottles) and balance the three bottles against one another using a double pan balance scale (Fig. 2b).

[0715] 8. Centrifuge at 8000 x for 30 min at 4°C.

[0716] 9. Decant supernatant containing phage particles without disturbing the solid pellet into new sterile centrifuge bottles (Fig. 2c). Repeat centrifugation at 8000 x g for 30 min at 4°C (see Note 10).

[0717] 10. Collect phage particles without disturbing the tiny pellet into sterile glass storage bottles.

[0718] 3.3 Phage Purification

[0719] 3.3.1 Dead-end fdtration sterilization

[0720] 1. In a BSC, connect a 0.8 | 0.45 pm heterogeneous PES double layer filter capsule to a 0.45 | 0.2 pm heterogeneous PES double layer filter capsule with autoclaved YD flexible tubing and clamps as shown in Fig. 2d.

[0721] 2. Prepare a disposable 10 mL serological pipette by removing the cotton or filter from its end. Feed the tubing through the peristaltic pump head, insert the serological pipette into the end of the tubing, and place it into sterile water.

[0722] 3. Precondition the filter membrane by pumping 500 mL of sterile water through the capsule filters. Discard the flow through.

[0723] 4. Switch the serological pipette and tubing to the bottle containing phage lysate and pump it into sterile glass bottles (see Note 11).

[0724] 5. Store all filtered lysates at 4°C. This is a potential pause step; depending on the phage strain, the lysate can be stored at 4°C for up to several months without significant loss in titer.

[0725] 3.3.2 Ultrafdtration and diafdtration

[0726] This process uses crossflow filtration (CFF) for combined UF and DF of up to 5 L batch volumes. UF and DF are commonly used for the development and manufacturing of biological therapeutics, such as proteins and antibodies, as well as therapies that rely on viral nanoparticle delivery (31, 32). The VIVAFLOW 200 CFF™ cassette uses a polyethersulfone membrane (PES), which is popular for phage applications given its hydrophilic properties and low protein binding. The semi- permeable membrane retains larger target phages while allowing smaller molecules to filter out. A 100 kDa MWCO is adequate to retain most phages. DF enables medium to buffer exchange by adding new cold buffer to the phage retentate. By returning the sample to the original volume, UF / DF can be repeated until the sample reaches a targeted level of clarity with the new buffer. The VIVAFLOW™’ s unique switchback channels parallel to the filtration membrane improves phage dissociation from endotoxins and require less pressure to drive filtration. The cassette design is easy to set up, gentle on phage particles, and its transparency allows for monitoring flow.

[0727] 1. In a BSC, connect the CFF cassette with autoclaved 6.4 mm outside diameter (O.D.) tubing as shown in Fig. 2e. Connect the filtrate line to the port on top of the cassette, the retentate line to the upper side port, and the intake line to the lower side port.

[0728] 2. Feed the intake line through the peristaltic pump head, insert a disposable 10 mL serological pipette into the end of the tubing, and place the pipette into a bottle with sterilized chilled (4°C) ultrapure water. Keep the cassette in an open circuit by placing both the retentate and filtrate lines in a waste container.

[0729] 3. Pump 500 mL of autoclaved cold ultrapure water through the cassette to remove storage EtOH and to condition the membrane with pure water.

[0730] 4. Transition the cassette into a continuous circuit. Insert the intake and retentate lines into the glass bottle containing sterilized cold phage lysate, as shown in Fig. 2e. Only the filtrate line should remain in the waste container.

[0731] 5. Recirculate the phage lysate through the cassette until 50% volume remains in the bottle, and add cold ultrapure water to double volume (see Note 12).

[0732] 6. Concentrate to 50% again and repeat the ultrapure water dilution by doubling the volume (see Note 13).

[0733] 7. Concentrate to 50% again and dilute with cold sterile PBS to double the volume (Fig. 2f).

[0734] 8. Concentrate to 30% and dilute with cold sterile PBS to double the volume. 9. Concentrate to 20% and dilute with cold sterile PBS to double the volume.

[0735] 10. Concentrate to 10% and dilute with cold sterile PBS to double the volume, repeat until the lysate is visibly clear (see Note 14).

[0736] 11. To collect phage particles, set up and label 3 sterile conical centrifuge tubes as “1st, 2nd, 3rdFractions”. Add 30 mL of sterile cold PBS to tubes 2 and 3.

[0737] 12. Concentrate to approximately 30 mL and pause the peristaltic pump. Pour the approximately 30 mL of phage solution into the empty conical tube and carefully insert the intake and retentate lines into the conical. Restart pumping until approximate^ mL remains in the conical tube. Pause the pump. See Fig. 2g.

[0738] 13. Collect the 1stfraction by draining the cassette, this will allow you to collect the remaining approximately 30 mL of dead volume of the cassette and tubing. Remove the intake line from the conical and restart the pump to completely flush the cassette into the 50 mL conical. Pause the pump (see Note 15).

[0739] 14. Immediately insert the intake and retentate lines into the 2ndconical tube containing 30 mL sterile cold PBS and start the pump to recirculate buffer to recover the phage remaining in the cassette. When the volume reaches approximately 5 mL, pause the pump, remove the intake line, and restart the pump to completely flush the cassette again. Pause the pump after all the liquid is collected.

[0740] 15. Repeat the recirculation and draining from step 14 using the 3rdconical tube containing sterile cold PBS. Pause the pump after all liquid is collected. Immediately refill the cassette with buffer for short-term storage until cleaning using manufacturer’s instructions (see Note 16).

[0741] 16. Store fractions at 4°C (see Note 17).

[0742] 3.3.3 Chromatographic removal of endotoxin

[0743] Although all prior processing steps reduce and remove endotoxins from phage samples, CFF fractions generally contain endotoxins above regulatory limits for intravenous applications. For example, up to 105- 106EU / mL. Therefore, chromatographic endotoxin removal may be required depending on phage concentration and intended application. For instance, a 1011PFU / mL phage sample with an endotoxin level of 104EU / mL would not need additional endotoxin removal steps if dilution in pyrogen-free saline was done to achieve a therapeutic dose of 109PFU / mL. The commercially available Pierce High-Capacity Endotoxin Removal Resin and ENDOTRAP HD™ assays have a binding capacity of 2* 106and 5* 106EU / mL, respectively. Below is described the specific use of a 1 mL Pierce Chromogenic Endotoxin Removal Spin Column with a sample capacity of 10 mL with modifications to the manufacturer’s instructions. Samples should be prepared using endotoxin-free water, and the column should only be uncapped in a BSC. Resin columns can be regenerated for reuse, but each column should be dedicated to only a single phage strain.

[0744] 1. Prepare the 1 mL column according to the manufacturer’s instructions. Each time you spin out a solution, loosen the column cap and remove the column’s bottom plug. When adding a solution to the resin slurry, secure the column cap and replace the bottom plug (see Note 18).

[0745] 2. Prepare buffers for use according to the manufacturer’ s instructions. Kit modification: Spike the endotoxin-free equilibration buffer with sterile-filtered NaCl solution to achieve a final solution of 10 mM PBS 0.4 M NaCl.

[0746] 3. Following the removal of the ethanol storage solution, NaOH regeneration buffer, salting with NaCl buffer, and equilibration with water and sodium phosphate buffer, the column is ready for sample addition. Kit modification: Increase the phage concentrate’s NaCl concentration to 0.4 M NaCl by spiking in sterile NaCl solution (Fig. 2h) (see Note 19).

[0747] 4. Mix end-over-end using a tube rotator at 4°C for 1 h.

[0748] 5. Collect the sample in a sterile conical tube (see Note 20).

[0749] 6. Once endotoxin column removal is completed, filter (0.2 pm syringe filter) the final phage preparation into a sterile conical tube (see Note 21).

[0750] 7. Store at 4°C and maintain sterility for qualification / quantification.

[0751] 3.3.4 Free nucleic acids digestion

[0752] Phage products can contain residual DNA from host cell substrates. It is, therefore, possible that such residual DNA could encode or harbor harmful molecules or elicit an innate immune response in the patient. It is not clear what health risk the DNA can pose to the product recipients, but often manufacturing can be designed to minimize the risk by reducing the levels of DNA. The World Health Organization and U.S. Food and Drug Administration guidelines recommend that 10 ng / dose and 200 base pairs be the limits of content and size of residual DNA in the final product dose (33). Enzymatic digestion of phage samples removes free DNA and RNA while intact virions are unharmed, and protected by their protein capsid.

[0753] 8. Pre-heat a heat block to 37°C.

[0754] 9. Dilute DNase I stock to a concentration of 100 U / mL. Place on ice.

[0755] 10. Dilute RNase A stock to a concentration of 100 U / mL. Place on ice.

[0756] 11. Determine how many mLs of phage preparation were produced. If you are not using the entire stock, sterilely aliquot the fraction that will be treated.

[0757] 12. Add 2.5 pL DNase I and 2.5 pL RNase A per 100 pL of phage preparation being treated.

[0758] 13. Treat phage preparation for 1 h at 37°C, with the lid heating off (Fig. 2i).

[0759] 14. Store the phage preparation at 4°C.

[0760] 3.4 Final Preparation Validation

[0761] 3.4.1 Sterilization and titration

[0762] 8. Prepare a fresh exponentially growing culture of host bacteria.

[0763] 9. Air-dry an agar plate for 1 h in a BSC.

[0764] 10. Pre-wet a 0.22 pm PES syringe filter using sterile phage storage buffer (i.e., PBS).

[0765] 11. Decant the nucleic acid-free phage preparation into the syringe. Filter sterilize into a fresh sterile 15 mL conical tube (Fig. 3a).

[0766] 12. Aliquot 100 pL of the phage preparation into a microcentrifuge tube.

[0767] 13. Store 0.22 pm filtered phage preparation at 4°C and protect from UV for longer- term storage.

[0768] 14. Perform spot titration using the phage aliquot as outlined in subheading 3.1.2.

[0769] 3.4.2 Endotoxin quantification

[0770] We recommend choosing either the ENDOZYME™ recombinant factor C or Pierce chromogenic LAL-based assays. Traditional endotoxin quantification uses the Limulus amebocyte lysate (LAL) test, which contains specialized blue blood cells from the wild Atlantic horseshoe crab, Limulus polyphemus. as a component because they react to the presence of endotoxins in a way that can be measured and quantified. The ENDOZYME™ assay are sustainable tests, using a recombinant version of the first enzyme in the LAL clotting cascade. The ENDOZYME™ assay also has a larger detection range from 0.005 to 50 EU / mL.

[0771] 1. Select an assay and prepare the assay reagent mixture and set the microplate reader to the settings as per the manufacturer’s instructions.

[0772] 2. Dilute the phage sample in endotoxin-free water to achieve a dilution series of 10’4, 10'6, and 10'8PFU / mL to ensure at least one measurement is within the linear dynamic range of the assay.

[0773] 3. Add reagents and run the microplate reader protocol as per the manufacturer’s instructions (Fig. 3b).

[0774] 3.5 Sterile fill-finish

[0775] The term “sterile fill-finish” is used for drug products that are sterile injectables (for example, liquids filled in vials or syringes). Because there is no process to sterilize the phage product in its final container, it is critical that containers be filled and sealed in an extremely controlled environment (34). Ensuring sterility is not a trivial task, and failure can have catastrophic — even life-threatening — consequences for a patient. The success of aseptic processing and sterile fill-finish operations relies on mitigating contamination from each of these sources: (i) personnel, (ii) drug product components and containers, (iii) cleanroom facilities, and (iv) equipment and processes.

[0776] 1. Clean a BSC with HEPA filtration using 70% EtOH and UV sterilize for 20 min.

[0777] 2. Place all containers and consumables in the cabinet and UV treat for 20 min (see Note 22).

[0778] 3. Dilute the phage stock with the desired sterile and endotoxin-free chilled solution in a sterile 50 mL conical tube. Gently vortex (see Note 23).

[0779] 4. Pre-wet a 0.22 pM PES syringe filter with phage diluent (i.e., PBS), and filter sterilize the diluted phage preparation into a fresh sterile 50 mL conical tube.

[0780] 5. Insert a filtered sterile vented needle into the vial’s rubber stopper (see Note 24).

[0781] 6. Draw 4.5 mL in a 5 mL Luer-Lock tip syringe with a 23G x 1-1 / 2” needle. Pierce the rubber stopper and aliquot 1 mL into the vial (Fig. 3c).

[0782] 7. Carefully remove both the syringe needle and the vented needle (see Note 25).

[0783] 8. Repeat until all vials are filled.

[0784] 9. When finished, protect vials from UV light and store at 4°C.

[0785] 4. Notes

[0786] 1. If your incubator is not equipped with refrigeration, phage production will need to be manually paused during exemplary production (see Subheading 3.1.3).

[0787] 2. A pump with a convex-roller head design is gentler on phages compared to a fliptype head design.

[0788] 3. A 115 mm Petri plate will support a 48-spot grid of 6 columns of 8 spots each (see Fig. Ib-lc). We recommended using this layout to titer CFU / PFU in two sets of triplicate spots. Modem microbiological shaking incubators can be refrigerated and programmed to support temperature cycles. Alternatively, cultures can be moved to 4-14°C environment. We find that harvesting phage production after 10 hours achieves titers upwards of 108PFU / mL while minimizing bacterial overgrowth during additional incubation. This timing may change depending on the growth rate of the host bacteria, so perform exemplary phage production (see Subheading 3.1.3). Once bacterial density is optically turbid (for example, ODeoo 0.1-0.2) the lysate should be immediately harvested. Additional bacterial growth will result in difficulty during dead-end filtration. At the time of this writing, PHASTER is accessible using a web browser and accepts FASTA files or GenBank accession numbers (35). Alternate tools include Phi Spy and Prophage Hunter (36, 37). A standard PCR protocol (such as (38)) should be used. Individual reagent volumes will depend on the polymerase / PCR kit being used. Each reaction will require 22.5 pL of the master mix. Bacterial strains can also be cured of prophages using mitomycin C induction or UV light treatment (39). However, prophage curing can cause changes in phage susceptibility / bacterial fitness. We recommend repeating exemplary phage lysate production if prophage curing is performed. Culture in liquid broth medium gives bacteria easy access to the available nutrients compared to static bacterial growth on solid medium, also known as an agar culture. Agitation to keep the bacteria dispersed through the medium during incubation can aid this access further. Liquid media will also dilute out waste products as they are formed, distributing them through the culture. Consequently, a greater mass of bacteria may be obtained for an equivalent volume of liquid as opposed to solid media. When pouring to a new container, the pellet may not be easily observed. Nonetheless, carefully handle the centrifuge bottles to avoid disturbing the pellet. The supernatant may appear clear; however, it is critical to perform a second spin to enhance the removal of gross bacterial debris and prevent clogging of capsule filtration units. By centrifuging lysates twice, 3 L can generally be filtered with the capsule filters without the need for cleaning in between. If the 0.45 | 0.2 gm filter becomes clogged, follow the manufacturer’s instructions to clean the filter (for example, using 0.5 M NaOH), rinse with sterile water, and resume filtration.

[0789] 12. If phage lysates are in multiple storage bottles, continue to add all phage lysate to the CFF before switching to the ultrapure water dilution step.

[0790] 13. The CFF will wash proteins and salts through the membrane while leaving phage particles in the retentate and exchange spent growth media for the desired storage buffer. This buffer exchange will occur simultaneously with concentration, where liters of lysate are concentrated up to 100-fold.

[0791] 14. Repeat this step until the phage solution has visible clarity, denoting the remaining broth and small particles have been washed away (i.e., diafiltration).

[0792] 15. This will briefly dry the CFF membrane and proceed through fraction collection quickly to ensure the membrane does not stay dry for long.

[0793] 16. Phage may be collected with further repeats. However, as you collect fractions, phage concentration decreases.

[0794] 17. Typically, CFF concentrates phage lysates by 1-2 orders of magnitude and concentrates endotoxin to approximately 104- 105EU / mL of buffer. We recommend proceeding with endotoxin removal using Fraction 2, which has lower endotoxin content than Fraction 1, while the phage concentration typically stays within the same order of magnitude. If time permits, titer the phage concentrate before proceeding.

[0795] 18. For improved endotoxin removal, we recommend column regeneration using 0.2N NaOH overnight (16-18 h) incubation at room temperature. Regenerate the column during phage overnight production if you plan to remove endotoxin immediately after CFF concentration.

[0796] 19. When using the Pierce endotoxin removal resin, an insufficient salt concentration significantly decreases final phage sample recovery. Spiking the equilibration buffer and phage concentration to approximately 0.4 M NaCl, as recommended in the manufacturer’s troubleshooting guide, has significantly improved the recovery of P. aeruginosa phage particles. 0. If desired, the column can be immediately regenerated, and the endotoxin removal process can be repeated. A second removal step may further reduce endotoxin concentration by up to twofold. However, we do not recommend going beyond two column passages as the final phage recovery is reduced in each passage.

[0797] 21. Pre-wet the syringe membrane with endotoxin-free buffer to prevent loss of phages during final sterile filtration.

[0798] 22. UV does not penetrate into materials, particularly plastics, very well. Although UV can disinfect an empty BSC, it will only disinfect the outer surface of any material in a BSC.

[0799] 23. Filtration typically causes a loss in phage concentration. Calculate and add at least 10% more phage stock to compensate for diluted phage losses.

[0800] 24. For sterile glass vials, it is necessary to vent the gas from the vial and allow the solution in.

[0801] 25. At minimum, replace the needles each time a new syringe is used.

[0802] Figure legends

[0803] Fig. 1 Illustration of bacterial or Spot+phage titration, (a) Example setup using a multichannel pipette and 96-well plate for serial dilutions, (b) Desired 48-spot plating for bacterial quantification. Counting should proceed from the row containing 10 or more distinct CFUs. (c) Desired 48-spot plating for Spot+phage titration. Counting should proceed from the row containing 10 or more distinct PFUs.

[0804] Fig. 2 Illustrated schematic of phage production and purification process, (a) Phage amplification. Bacteria should be added to sterile growth medium and grown for one generation with a vented screw-cap to allow gas exchange (left). Phages should be added to achieve a MOI of 0.1 (right), (b) First centrifugation. Centrifuge produced phage lysates at 8000 xg for 30 min at 4°C. (c) Second centrifugation. Avoid the bacterial pellet and carefully decant the supernatant into new centrifuge bottles. Re-balance bottles and repeat centrifugation at 8000*g for 30 min at 4°C. (d) Deadend filtration sterilization. Sterilize the lysate by connecting in tandem a 0.8 | 0.45 pm capsule filter to a 0.45 | 0.2 pm filter capsule, (e) Diafiltration. Remove storage buffer from the CFF cassette, flush with water, and set up the CFF cassette as pictured. The intake and retentate lines should recirculate from the bottle containing lysate. Flow all phage lysate through the CFF. Concentrate to 50% volume and dilute with cold ultrapure water, (f) Washing. Exchange spent lysate for cold ultrapure storage buffer. Stepwise concentrate and dilute phage with storage buffer, (g) Concentrating. When the phage concentrate appears visibly clear, purge the CFF and collect phage particles in three or more fractions, (h) Endotoxin removal. Sterilely prepare reagents for endotoxin removal. Perform endotoxin removal per manufacturer’s instructions with suggested modifications to prevent phage loss, (i) Removal of nucleic acids. Remove free DNA / RNA by 1 h enzyme treatment.

[0805] Fig. 3 Illustrated schematic of phage sterilization, validation, and fill-finish, (a) Syringe sterilization. Syringe filter the final phage preparation before obtaining a final phage titer, validation, and / or long-term storage, (b) Endotoxin quantification. Perform endotoxin quantification using a kit with an appropriate detection range, (c) Vial filling. UV sterilize all supplies and vials before use. Filter sterilize the diluted phage preparation before sterile distribution into vials.

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[0859] References Example 2

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[0896] A number of embodiments of the invention have been described.

[0897] Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for purifying bacteriophage (phage) and generating a substantially endotoxin free preparation of phage, wherein optionally the phage is from a family Caudoviricetes, Corticoviridae, Tectiviridae, Tubulavirales, Leviviridae, and / or Microviridae, wherein optionally the phage is from a family Inoviridae, Microviridae, Tectiviridae, Corticoviridae, Plasmaviridae, Leviviridae, and / or Cystoviridae, the method comprising:(a) providing a sample comprising a bacteriophage (phage), wherein optionally the phage are propagated, and the propagated phage are added during midlog stage of bacterial growth, and optionally phage nucleic acids (optionally the phage nucleic comprise phage genomes) are sequenced, and optionally the phage are filtered, optionally 0.2 pm filtered;(b) providing a bacterial strain for propagating a bacteriophage strain, and optionally the bacterial nucleic acid (optionally the bacterial nucleic comprises bacterial genomes) is sequenced, and optionally both the phages and host bacteria genomes are sequenced and annotated to screening for harmful genes, wherein optionally the harmful genes comprise virulence factors, antibiotic resistance and toxin gene from prophages;(c) preparing a bacterial host standard curve;(d) quantifying phage, optionally by double aliquot spot serial titration;(e) amplifying phage and generating a lysate comprising prophage;(f) detecting the prophage in the lysate;(g) purifying the amplified phage, optionally filtering and sterilizing, optionally by applying dead-end filtration sterilization to the purified amplified phage, optionally comprising use of crossflow filtration (CFF), ultrafiltration and / or diafiltration, thereby generating a purified amplified phage preparation;(h) removing substantially most (optionally removing between about 95% to 99.5%, or 90% to 99.9% of) endotoxin present in the purified amplified phagepreparation, optionally using a chromatographic process, thereby generating a purified amplified phage preparation with substantially most endotoxin removed;(i) digesting substantially most (optionally removing between about 95% to 99.5%, or 90% to 99.9% of) free nucleic acids, optionally using DNase and / or RNase, to generate an endotoxin-free and a nucleic acid-free phage preparation; and(j) sterile fill-finishing the endotoxin-free and a nucleic acid-free phage preparation, optionally processing for use as a drug product, optionally as a sterile injectable, optionally as a sterile liquid filled in vials or syringes, thereby generating a substantially endotoxin free preparation of phage.

2. The method of claim 1, wherein the method comprises use of a pressure-driven cross-flow ultrafiltration (CFF) comprising a molecular weight cut-off (MWCO) at about 100 kDa, thereby only retaining bacteriophage (phage) particles of greater than about 100 KDa.

3. The method of claim 1 or claim 2, wherein preparation of phage comprises endotoxin levels to below about 5.5 EU mL'1, or below about 5.0 EU mL’1.

4. The method of any of claims 1 to 3, wherein a single production run produces at least about 300 treatment doses phage at about 109PFU, 1010PFU, 1011PFU, or 1012PFU, or more per dose.

5. The method of any of claims 1 to 4, wherein phage lysates or cultures are cooled to below about 37°C, or cooled to between about 4°C to 37°C, at the point where phage-insensitive or phage-resistant bacteria appear in culture.

6. A formulation or pharmaceutical preparation of bacteriophage (phage), comprising:(a) about 109PFU, 1010PFU, 1011PFU, or 1012PFU or more per unit dose and endotoxin levels below about 5.5 EU mL'1, or below about 5.0 EU mL'1; or(b) a preparation of bacteriophage (phage) prepared by a method of any of the previous claims.

7. The formulation or pharmaceutical preparation of claim 6, wherein the phage are formulated for enteral or parenteral administration, or are formulated for administration intramuscularly, orally, parenterally, by inhalation spray, nasally, topically, intrathecally, intrathecally, intracerebrally, epidurally, intracranially, vaginally or rectally.

8. The formulation or pharmaceutical preparation of claim 6 or claim 7, wherein the phage are formulated as a lyophilate, a tablet, a pill, a powder, a dragee, a capsule, a liquid, a lozenge, a gel, a syrup, a slurry, an aerosol, or a suspension.

9. The formulation or pharmaceutical preparation of any of claims 6 to 8, comprising or further comprising a pharmaceutically acceptable excipient, or phage dissolved in (optionally sterile) saline, water, polyethylene glycol, propylene glycol, ethanol or oils such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil, tragacanth gum, and / or a buffer.

10. A method for treating a bacterial infection in vivo comprising administering to an individual in need thereof a formulation or pharmaceutical preparation of any of claims 6 to 9, wherein optionally the pharmaceutical preparation is administered orally, parenterally, by inhalation spray, nasally, topically, intrathecally, intrathecally, intracerebrally, epidurally, intracranially, vaginally, intravenously, intraoperatively or rectally.

11. Use of a formulation or pharmaceutical preparation of any of claims 6 to 9, for treating a bacterial infection in vivo.

12. A formulation or pharmaceutical preparation of any of claims 6 to 9, for use in treating a bacterial infection in vivo.

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