PURE FILAMENTOUS BACTERIOPHAGE AND METHODS OF

PRODUCING SAME

[001] This application ciaims the benefit of priority of U.S. Provisional Patent Appiication No. 61/515,728, filed August 5, 2011 , which is incorporated by reference in its entirety herein.

[002] The invention relates to compositions of filamentous

bacteriophage having sufficiently low levels of host cell contaminants, such as bacterial endotoxin, for use in the preparation of therapeutically effective pharmaceutical compositions, as well as drug product and pharmaceutical compositions prepared therefrom. The invention also relates to methods for producing such compositions.

[003] Filamentous bacteriophage are emerging as therapeutic agents for treatment of neurodegenerative diseases and disorders, including Parkinson's disease or susceptibility to Parkinson's disease (see PCT Patent Publication WO20100080073), and diseases and disorders characterized by amyloid plaque formation in the brain and elsewhere in the body (see, e.g., U.S. Patent Publication 201 10142803, U.S. Patent Pubiication 20090180991 , and PCT patent pubiication WO2008011503). Filamentous bacteriophage are also emerging as therapeutic agents for treatment of neurodegenerative tauopathies (see PCT Patent Application No. PCT/US2012/028762, filed March 12, 2012). These references also indicate that filamentous

bacteriophage can reduce susceptibility to neurodegenerative tauopathies and/or plaque forming diseases. In addition, filamentous bacteriophage engineered to express a therapeutic agent, antigen, or antibody have also been suggested as useful therapeutic agents. See, for example, PCT patent publications WO2002074243, WO2004030694, WO2007094003, and

WO2007001302; and U.S. Patent Publication US20020044922.

[004] Filamentous bacteriophage are produced by fermentation, using gram-negative bacteria! cell hosts for their growth. Gram-negative bacteria are cultured with a complex growth medium, containing sugars, amino acids, and growth factors, usually supplied from preparations of animal serum. Bacterial DNA and proteins are undesirable contaminants that are typically found in the fermentatiqn media along with the phage. Moreover, gram-negative bacteria produce endotoxin, a toxic and highly undesirable contaminant in any therapeutic agent, which is difficult to separate from the filamentous bacteriophage. The United States Food and Drug Administration has set forth guidelines for the maximum amount of endotoxin allowed in drug products at 5.0 endotoxin units ("EU")/kg body weight/dose and at 0.2

EU/kg/dose for intrathecaily injected drug products. See Food and Drug Administration Inspection Technical Guide No. 40, March 20, 1985, available as file ucm07298.htm in the

ICECI/lnspections/lnspectionGuides/SnspectionTechnicalGuides subdirectory of the FDA website (URL:

http://www.fda.gov/ICECI/lnspections/lnspectionGuides/lns pectionTechnicalG uides/ucm07291 S.htm). Accordingly, the difficulties associated with large- scale, economic purification of filamentous bacteriophage are an increasingly important problem for the biotechnology Industry.

[005] Advances in fermentation techniques have greatly increased the concentration of filamentous bacteriophage capable of being produced in any given composition. This increase in upstream efficiency has led, however, to difficulties in downstream processing. Producing higher concentrations of bacte iophage requires higher concentrations of bacterial hosts and concomitantly higher concentrations of bacteria! DNA, proteins and endotoxin. Bacteriophage must be separated from the bacteria! hosts in which they grow and these bacteria! by-products present in the fermentation media in order to be used as therapeutic compositions,

[008] Procedures for purification of filamentous bacteriophage have typically relied on PEG precipitation and CsC! gradients formed by

ultracentrifugation. See, for example, Sambrook J. and Russe!! D. W.

"Molecular Cloning. A Laboratory Manual"; Third Edition (2001 ) at Chapter 3. The bacteriophage produced by these procedures are not adequate for therapeutic use because the procedures do not remove sufficient quantities of bacteria! cell by-products to allow for administration to humans. Thus, improved methods for purifying compositions of filamentous bacteriophage are greatly needed.

[007] The purification techniques must be sca!eable, efficient, cost- effective, reliable, and meet the rigorous purity requirements of the final product.

[008] The present invention is based in part on the discovery of novel purification techniques resulting in filamentous bacteriophage

compositions comprising acceptably low levels of bacterial cell contaminants, such as, for example, endotoxin. These novel purification techniques are sca!eable, efficient, cost-effective and reliable. Most importantly, however, the purification techniques of this invention are useful to produce filamentous bacteriophage compositions that are suitable for administration to humans. The levels of endotoxin are low enough to allow for any type of administration, including, for example, direct injection into the brain, which may be the preferred delivery method in many diseases characterized by plaque formation in the brain.

[009] Methods for purifying high concentrations of filamentous bacteriophage on a large scale are vital for the commercial preparation of therapeutic filamentous bacteriophage to be used in the treatment and prevention of neuronal diseases and disorders.

[010] Embodiments of the invention include compositions comprising filamentous bacteriophage having an endotoxin to phage ratio of less than 5 x 10 ^"14 endotoxin units ("EU") per phage. The compositions may also comprise filamentous bacteriophage having an endotoxin to phage ratio of less than

1 x 10 ^~13 EU per phage, less than 1 x 10 ^"12 EU per phage, less than 1 x 10 ^~11 EU per phage, and less than 1 x 1 G ^~10 EU per phage.

[01 1] Further embodiments of the invention include compositions comprising wild-type filamentous bacteriophage or fiiamentous phage which does not display an antibody or a non-filamentous bacteriophage antigen on its surface, said composition comprising less than 1 x 10 ^{" 10} endotoxin units per filamentous bacteriophage, less than 1 x 10 ^"1 EU per phage, less than 1 x 10 ^" EU per phage, less than 1 x 0 ^"13 EU per phage, or less than 5 x 10 ^"14 EU per phage.

[012] Additional embodiments of the invention include compositions comprising filamentous bacteriophage for use in the diagnosis, treatment or prevention of a brain disease or a disease characterized by the presence of amyloid plaque, said composition comprising less than 1 x 10 ^"10 endotoxin units per filamentous bacteriophage, less than 1 x 1 Q ^~11 EU per phage, less than 1 x 10 ^"12 EU per phage, less than 1 x 10 ^"13 EU per phage, or less than 5 x 10 ^*14 EU per phage. In still further embodiments, the invention provides methods for the diagnosis, treatment or prevention of a brain disease or a disease characterized by the presence of amyloid plaque, comprising administering to a subject in need thereof a composition comprising less than 1 x 10 ^"10 endotoxin units per filamentous bacteriophage, less than 1 x 10 ^{"1 '} EU per phage, less than 1 x 1 G ^~12 EU per phage, less than 1 x 10 ^~13 EU per phage, or less than 5 x 10 ^"14 EU per phage.

BRIEF DESCRIPTION OF THE DRAWUMGS

[013] Figure 1 is a chromatogram from the Phenyl HiC step.

Fluorescence emission at 334 nrn is measured after excitation at 242 nm. The 13 peak is labeled. In this example, 420 mLs of the M13 containing retentate from the first ultrafiltration step was diluted with equal volume of 25 mM Tris pH 7.4/4M NaCL and loaded onto the Phenyl HIC column with a peristaltic pump at 100 mL/min. M13 was eiuted with a step gradient of 25 mM Tris, pH 7.4, 250 mM NaCI after a wash step with 25 mM Tris, pH 7.4, 2M NaCL

[014] Figure 2 is a chromatogram from the Phenyl HIC step.

Fluorescence is shown (Ex. 242 nm; Em 334 nm). The M13 peak is labeled. In this example, 320 mLs of the M13 containing retentate from the first ultrafiltration step was diluted with an equal volume of 25 mM Tris pH 7.4/4M NaCI and loaded onto a Phenyl HIC column. M13 was eiuted with a step gradient of 25 mM Tris, pH 7.4, 250 m NaC!, after a wash step with 25 mM Tris, pH 7.4, 2M NaCi.

[015] Figure 3 is a chromatogram from the Phenyl HIC step.

Absorbance at A254 nm is shown. The 13 peak is labeled. In this example, 320 mLs of the M13 containing refentate from the first ultrafiltration step was diluted with equal volume of 25 mM Tris pH 7.4/4M NaCi and loaded onto a Phenyl HIC column. M13 was eluted with a step gradient of 25 mM Tris, pH 7.4, 250 mM NaCi, after a wash step with 25 mM Tris, pH 7.4, 2M NaCi

[016] Figure 4 is a chromatogram from the DEAE AEX step.

Fluorescence is shown (Ex. 242 nm; Em 334 nm). The M 3 peak is labeled. In this example, eluate from the HIC Phenyl step, which contains M13, was diluted six times with 25 mM Phosphate, pH 6.5 and loaded onto the DEAE column with a peristalitic pump at 100 m!/min, M13 was eluted with a step gradient in 25 mM Phosphate, pH 6.5, 300 mM NaCi after successive washes with 25 mM Phosphate, pH 7.4, 150 mM NaCi and 25 mM Phosphate, pH 7.4, 250 mM NaCi at a flow rate of 100 ml/min.

[017] Figure 5 is a chromatogram from the DEAE AEX step.

Fluorescence at excitation at 242 nm and Emission at 334 nm is shown. The M13 peak is labeled. In this example, approximately 2L of the eluate from the HIC Phenyl step, which contains M13, was diluted with 10L of 25 mM

Phosphate pH 8.5 and loaded onto the DEAE column. M13 was eluted with a step gradient of 25 mM Phosphate, pH 6.5, 300 mM IMaCI after successive washes with 25 mM Phosphate, pH 7.4, 150 mM NaCi and 25 mM

Phosphate, pH 7.4, 250 mM NaCi. [018] Figure 8 is a chromatogram from the AEX Q step.

Fluorescence at excitation 242 nm and Emission at 334 nm is shown (the corresponding adsorbance trace for this run is provided in Figure 7). The M13 peak is labeled, in this example, approximately 750 mL of the eluate from the DEAE AEX step, which contains M13, was diiuted with 750 mL of 25 mM Tris pH 7.4 and loaded onto the AEX Q column. M13 was eluted with a step gradient of 25 mM Tris, pH 7.4, 280 mM NaCI after a wash step with 25 mM Tris, pH 7.4, 200 mM NaCI.

[019] Figure 7 is a chromatogram from the AEX Q step. Absorbance at A254 nm is shown (the corresponding fluorescence trace for this run is provided in Figure 6). The M13 peak is labeled In this example,

approximately 750 mL of the eluate from the DEAE AEX step, which contains M13, was diluted with 750 mL of 25 mM Tris pH 7.4 and loaded onto the AEX Q column, M13 was eluted with a step gradient of 25 mM Tris, pH 7.4, 280 mM NaCI after a wash step with 25 mM Tris, pH 7.4, 200 mM NaCI.

[020] Figure 8 is a chromatogram from the Phenyl HIC step.

Fluorescence at excitation 242 nm and Emission at 334 nm is shown. The M13 peak is labeled. In this example, 400 mLs of the M13 containing retentate from the first ultrafiltration step was diluted with equal volume of 25 mM Tris pH 7.4/4M NaCI, and loaded onto the Phenyl HIC column with a peristaltic pump at 100 mL/min. M13 was eluted with a step gradient of 25 mM Tris, pH 7.4, 250 mM NaCI after a wash step with 25 mM Tris, pH 7.4, 2M NaCI.

[021] Figure 9 is a chromatogram from the DEAE step.

Fluorescence at excitation 242 nm and Emission at 334 nm is shown. The M13 peak is labeled. In this example, eluate from the HIC Phenyl step, which contains M13, was diluted six times with 25 mM Phosphate, pH 6.5 and loaded onto the DEAE column with a perista!itic pump at 100 ml/min. M13 was e!uted with a step gradientof 25 mM Phosphate, pH 6.5, 300 mM NaCI after successive washes with 25 mM Phosphate, pH 7.4, 150 mM NaCI and 25 mM Phosphate, pH 7.4, 250 mM NaCI.

[022] Figure 10 is a chromatogram from the AEX Q step.

Fluorescence at excitation 242 nm and Emission at 334 nm is shown. The M13 peak is labeled. In this example, the eluate from the DEAE AEX step, which contains M13, was diluted with an equal volume of 25 mM Tris pH 7,4 and loaded onto the AEX Q column. M13 was eluted with a step gradient of 25 mM Tris, pH 7.4, 280 mM NaCI after a wash step with 25 mM Tris, pH 7.4, 200 mM NaCI.

[023] Figure 1 1 is a chromatogram from the Phenyl H!C step.

Fluorescence at excitation 242 nm and Emission at 334 nm is shown. The M13 peak is labeled. In this example, the supernatant from the depth filtration step, which contains M13, was diluted with equal volume of 25 mM Tris pH 7.5/4M NaCI, and loaded onto the Phenyl H!C column with a peristaltic pump at 100 mL/min. M13 was eluted with a step gradient of 25 mM Tris, pH 7.4, 250 mM NaCI, after a wash step with 25 mM Tris, pH 7.4, 2M NaCI.

[024] Figure 12 is a chromatogram from the DEAE step.

Fluorescence at excitation 242 nm and Emission at 334 nm is shown. The M13 peak is labeled. In this example, 3L of the eluate from the HIC Phenyl step, which contains 13, was diluted with 0L 25 mM Phosphate, pH 6.5 and loaded onto the DEAE column with a peristalitic pump at 100 mi/min. M13 was eluted with a step gradient of 25 rnM Tris, pH 7.4, 300 mM NaCI after a wash step with 25 mM Tris, pH 7.4, 200 mM NaC!.

[025] Figure 13 is a chromatogram from the AEX Q step.

Fluorescence at excitation 242 nm and Emission at 334 nrn is shown. The M13 peak is labeled, in this example, approximately 3L of the eluate from the DEAE AEX step, which contains M13, was diluted with 2L of 25 mM Tris pH

7.4 and loaded onto the AEX Q column. M13 was eluted with a step gradient of 25 mM Tris, pH 7.4, 280 mM NaCI after a wash step with 25 mM Tris, pH 7.4, 200 mM NaC!.

[026] Figure 14 shows the eiution profile of M13 purified with the process described in Example 5 from an analytical AEX column (ProSwift WAX-1 S). 5μ! of neat M13 was diluted with 75 μ! of Buffer A (50 mM

Phosphate, pH 7.5). M13 was eluted in a linear gradient from 100% Buffer A to 100% Buffer B (50 mM Phosphate, pH 2.2/2M NaCI).

[027] Figure 15 shows an image of an SDS PAGE Gel stained with Coomassie, where column 1 is loaded with the filamentous bacteriophage produced by the purification procedure outlined in Example 5. Column 2 is loaded with 10 μΙ of a molecular weight marker (Marker 12; Invitrogen), and column 3 with a positive control (reference M13; Batch 5). M13 is loaded at

1.5 x 10 ¹¹ in all lanes (except marker). This gel shows the presence of the major coat protein g8p and the lack of other major protein contaminant bands.

[028] Figure 16 shows the eiution profile of M13 purified with the process described in Example 4 (Batch 2) from an analytical AEX column (ProSwift WAX-1 S). 5μΙ of neat M13 was diluted with 75 μΙ of Buffer A (50 mM Phosphate, pH 7.5). M13 was eluted in a linear gradient from 100% Buffer A to 100% Buffer B (50 mM Phosphate, pH 2.2/2M NaCI).

[029] Figure 17 shows an SDS PAGE Gel, where column 1 is loaded with a positive control (Batch 5), column 2 is loaded with the filamentous bacteriophage produced by the purification procedure outlined in Example 4 (Batch 2), and column 3 with 10 μΙ of a molecular weight marker (Marker 12; Invitrogen). M13 is loaded at 1.5 x 10 ¹¹ in all lanes (except marker). This gel shows the presence of the major coat protein g8p and the lack of other major protein contaminant bands.

[030] Figure 18 shows the eiution profile of M13 produced with the PEG precipitation and 2 x CsC! density gradient (ultracentrifugation method) from an analytical AEX column (ProSwift WAX-1 S). See, Example 7. 5μΙ of neat M13 was diluted with 75 μΙ of Buffer A (50 mM Phosphate, pH 7.5). M13 was eluted in a linear gradient from 100% Buffer A to 100% Buffer B (50 mM Phosphate, pH 2.2/2M NaCI).

[031] Figure 19 shows an SDS PAGE Gel, where column 1 is loaded with an M13 batch generated using the PEG precipitation and 2 x CsCI density gradient method. See, Example 7. Column 2 is loaded with 10 μΙ of a molecular weight marker (Marker 12; Invitrogen), and column 3 is loaded with a positive control (Batch 2; Example 4) sample of purified filamentous bacteriophage (Batch 2: Example 4), and column 3 with a marker. M13 is loaded at 1.5 x 10 ¹¹ in ail lanes (except marker). This gel shows the presence of the major coat protein g8p and the lack of other major protein contaminant bands. DESCRIPTION OF EMBODIMENTS

Definitions

[032] Filamentous bacteriophage are a group of related viruses that infect gram negative bacteria, such as ,e.g., E. cols ^' . See, e.g., Rasched and Oberer, Microbiology Reviews (1986) Dec:401-427. In the present

application, filamentous bacteriophage may also be referred to as

"bacteriophage," or "phage." Unless otherwise specified, the term

"filamentous bacteriophage" includes both wild type filamentous

bacteriophage and recombinant filamentous bacteriophage.

[033] "Wild type filamentous bacteriophage" refers to filamentous bacteriophage thai express only filamentous phage proteins and do not contain any heterologous nucleic acid sequences, e.g. non-phage sequences that have been added to the bacteriophage through genetic engineering or manipulation. One such wild-type filamentous bacteriophage useful in the invention is M13. The term "Ml 3" is used herein to denote a form of M13 phage that only expresses M13 proteins and does not contain any

heterologous nucleic acid sequences. M13 proteins include those encoded by M13 genes I, II, ill, lllp, !V, V, VI, VII, VIII, Vlllp, IX and X. van Wezenbeek et al. Gene (1980) 1 1 :129-148.

[034] Suitable wild type filamentous bacteriophage for use in the compositions and methods of the invention include at least M13, f 1 , or fd, or mixtures thereof. Although M13 was used in the Examples presented below, any closely related wild type filamentous bacteriophage is expected to behave and function similarly to M13. Closely related wild type filamentous

bacteriophage refer to bacteriophage that share at least 85%, at least 88%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity, to the sequence of M13, f1 , or fd at the nucleotide or amino acid level. In some embodiments, closely related filamentous bacteriophage refers to bacteriophage that share at least 95% identity to the DNA sequence of M13 (See, e.g., GenBank:V00604; Refseq: NC 003287).

[035] "Recombinant filamentous bacteriophage" refers to filamentous bacteriophage that have been geneticaliy engineered to express at least one non-filamentous phage protein and/or comprise at least one heterologous nucleic acid sequence. For example, recombinant filamentous bacteriophage may be engineered to express a therapeutic protein, including, e.g., an antibody, an antigen, a detectable marker (for diagnostic use), a peptide that modulates a receptor, a peptide composed of beta-breaker amino acids like proline, cyclic peptides made of alternating D and L residues that form nanotubes, and a metal binding protein.

[036] The filamentous bacteriophage compositions of the invention may be purified in any desired volume by adjusting the processes set forth below as necessary and as would be readily understood by those of skill in the art. In each embodiment, the compositions comprise filamentous bacteriophage or recombinant filamentous bacteriophage that have been purified to reduce the levels of bacterial ceil contaminants, such as, for example, endotoxin. The levels of endotoxin are sufficiently low to administer to humans via any route of administration, including, for example, direct injection into the brain. In one embodiment, the purified filamentous bacteriophage have a concentration of at least 4 x 10 ¹² phage/m!, at least 1 x

10 ¹³ phage/mi, at ieast 5 x 10 ¹³ phage/mi, at least 9 x 10 ¹³ phage/mi, or at least 1 x 10 ¹⁴ phage/ml. Importantly, the EU/phage ratio is less than 1 x 10 ^"10

EU/phage, less than 1 x 1 Q ^~11 EU/phage, less than

1 x 10 ^"12 EU/phage, !ess than 1 x 10 ^{' 13} EU/ phage, or less than 5 x 10 ^{" 14}

EU/phage,

[037] "Endotoxin" is found in the outer cell membrane of all gram- negative bacteria. "Endotoxin" may also be referred to as

"lipopolysaccharide" or "LPS" throughout.

[038] As used herein a "pharmaceutical composition" refers to a preparation of filamentous bacteriophage described herein with other chemical components such as a physiologically suitable carrier and/or excipient.

[039] The phrases "physiologically acceptable carrier" and

"pharmaceutically acceptable carrier" which may be used interchangeably refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the bioiogica! activity and properties of the administered filamentous bacteriophage compound. An adjuvant is included under these phrases.

[040] The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, include, for example, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, poiysorbate 20. [041 J The term "dose" refers to an amount administered to a patient, particularly a human, over not more than one hour. "Dose" includes single bolus or solid dosage forms, as well as infusions and amounts delivered by implanted pumps.

[042] The term "unit dosage form" or "single dosage form" generally refers to the drug product of the invention that is intended to provide delivery of a single dose of a drug to the patient at the time of administration for use, e.g., in homes, hospitals, facilities, etc. The drug product is dispensed in a unit dose container-a non-reusable container, tablet, pill, etc. designed to hold a quantity of drug intended for administration (other than the parenteral route) as a single dose, directly from the container, tablet, pill, etc., employed generally in a unit dose system. The advantages of unit dose dispensing are that the drug is fully identifiable and the integrity of the dosage form is protected until the actual moment of administration. If the drug is not used and the container, tablet, pill, etc. is intact, the drug may be retrieved and redispensed without compromising its integrity.

[043] The term "retenfate" refers to the part of a solution that does not cross a filtration membrane. This is in contrast to the "permeate" part of the solution that passes across the membrane.

[044] As used herein, the term "eluate" generally refers to an entity that is released from another entity by a changing solvent condition (e.g. the release of bound Ml 3 from a charged chromatography matrix by increasing the salt concentration).

[045] The term "treating" is intended to mean substantially inhibiting, slowing or reversing the progression of a disease, substantially ameliorating clinical symptoms of a disease or substantially preventing the appearance of clinical symptoms of a disease. Also as used herein, the term "plaque forming disease" refers to d characterized by formation of plaques by an aggregating protein (plaque forming peptide), such as, but not limited to, alpha-synuc!ein, beta-amyloid, serum amyloid A, cystatin C, IgG kappa light chain, tau protein, or prion protein. Such diseases include, but are not limited to, early onset Alzheimer's disease, late onset Alzheimer's disease, presymptomatic Alzheimer's disease, SAA amyloidosis, hereditary Icelandic syndrome, senility, multiple myeloma, to prion diseases that are known to affect humans (such as for example, kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal familial insomnia (FFI)) or animals (such as, for example, scrapie and bovine spongiform encephalitis (BSE)), Parkinson's Disease, Argyrophilic grain dementia, Corticobasai degeneration, Dementia pugilistica. diffuse neurofibrillary tangles with calcification, Down's syndrome, Frontotemporal dementia with

parkinsonism linked to chromosome 17, Hallervorden-Spatz disease,

Myotonic dystrophy, Niemann-Pick disease type C, Non-Guamanian motor neuron disease with neurofibrillary tangles, Pick's disease, Postencephalitic parkinsonism, Progressive subcortical gliosis, Progressive supranuclear palsy, Subacute sclerosing panencephalitis, and Tangle only dementia.

Compositions

[046] In some embodiments, the invention provides large-scale compositions of filamentous bacteriophage. The term "large-scale

composition" refers to a composition that comprises a sufficient number of filamentous bactenophage for at least 10, 100, 1 ,000, 10,000, 100,000, or more therapeutically effective doses. In some aspects of this embodiment, the compositions comprise at least 2 x 10 ¹⁶ to 4.5 x 10 ²¹ total filamentous bactenophage. The filamentous bacteriophage in these compositions have a concentration of at least 4 x 10 ¹² phage/ml, or at least 1 x 10 ¹⁴ phage/ml. The EU/phage ratio of the composition is less than 1 x 10 ^"10 EU/phage, less than

1 x 10 ^"11 EU/phage, less than 1 x 1 G ^~12 EU/phage, less than 1 x 10 ^~13

EU/phage, or less than 5 x 10 ^'14 EU/phage.

[047] In some aspects of the invention, the compositions comprise less than 20 ng/mL bacterial cell DNA, and less than 10 ng/ml bacteria! cell protein (also referred to as host cell protein or HCP).

[048] In some embodiments, the large-scale compositions of this invention may be concentrated or converted to a solid form for subsequent reconstitution by methods well known in the art, such as ultrafiltration, evaporation, spray-drying, lyophi!ization, etc. When such methods are applied and the resulting form is still liquid, the concentrations of

bacteriophage and endotoxin (and in some cases, bacterial ceil DNA and bacterial cell protein) will increase, but the ratio of endotoxin to bacteriophage will remain approximately the same as in the large scale composition. When such methods are applied and the resulting form is solid, the ratio of bacteriophage to endotoxin will remain approximately the same as in the Iarge scale composition. Such solid form or concentrated compositions are also part of the present invention. [049] In certain embodiments, the invention provides pharmaceutically acceptable compositions comprising filamentous

bacteriophage having an EU/phage ratio of less than 5 x 10 ^"14 EU/phage. Pharmaceutically acceptable compositions may, for example, be in the form of a saline solution.

[050] !n some embodiments, the invention provides pharmaceutically acceptable compositions in single dosage forms. In some aspects, single dosage forms comprise a portion of the large-scale pharmaceutical

composition of the invention. The ratio of endotoxin to bacteriophage will remain approximately the same in the single dosage form as in the large- scale composition. Single dosage forms may be in a liquid or a solid form. Single dosage forms may be administered directly to a patient without modification or may be diluted or reconstituted prior to administration. In certain embodiments, the single dosage forms contain less than 200 endotoxin units, less than 100 endotoxin units, less than 50 endotoxin units, less than 20 endotoxin units, less than 10 endotoxin units, less than 8 endotoxin units, less than 5 endotoxin units, less than 3 endotoxin units, less than 2 endotoxin units, less than 1 endotoxin units, less than 0.5 endotoxin units, or less than 0.2 endotoxin units.

[051] In certain embodiments, a single dosage form may be administered in bolus form, e.g., single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc. In alternate embodiments, a single dosage form may be administered over a period of time, such as by infusion, or via an implanted pump, such as an ICV pump. In the latter embodiment, the single dosage form may be an infusion bag or pump reservoir pre~fil!ed with the indicated number of filamentous

bacteriophage. Alternatively, the infusion bag or pump reservoir may be prepared just prior to administration to a patient by mixing a single dose of the filamentous bacteriophage with the infusion bag or pump reservoir solution.

[052] in some embodiments, when administered to a human patient, the pharmaceutically acceptable composition or single dosage form thereof provides less than 5.0 endotoxin units per kilogram body weight per dose, in a more specific aspect of this embodiment, when administered to a human patient, the pharmaceutically acceptable composition or single dosage form thereof provides less than 0.2 endotoxin units per kilogram body weight per dose.

[053] In one embodiment, the pharmaceutical compositions described above are prepared by admixing all or a portion of the large-scale composition with at least one pharmaceutically acceptable excipient.

Accordingly, methods for preparing a pharmaceutical composition of filamentous bacteriophage comprising admixing a portion of the iarge-scaie composition comprising filamentous bacteriophage with at least one pharmaceutically acceptable excipient are also encompassed.

[054] In certain embodiments, the pharmaceutical compositions are further subjected to dilution or concentration; or to tabletting, iyophilization, direct compression, melt methods, or spray drying to form tablets, granulates, nano-particles, nano-capsules, micro-capsules, micro-tablets, pellets, or powders.

[055] Single dosage forms of the pharmaceutical composition of the invention may be prepared by portioning the large-scale composition or the pharmaceutical composition into smaller a!iquots or into single dose containers or formulating the large-scale composition or the pharmaceutical composition into single dose solid forms, such as tablets, granulates, nano- particles, nano-capsules, micro-capsules, micro-tablets, pellets, or powders. Containers for the smaller aSiquots or the single dose containers include vials, infusion bags and pump reservoirs. Vials contemplated for single dose include 1 ml vials, 2 ml vials, 3 ml vials, 5 ml vials, 10 ml vials, 20 ml vials, 30 ml vials, 40 ml vials, 50 ml vials, 60 ml vials, 70 ml vials, 80 ml vials, 90 ml vials, and 100 ml vials. Vials may contain a single dose in a liquid form or a solid form. Vials containing a single dose in a solid form may be reconstituted by adding liquid, typically sterile water or saline solution, prior to

administration to a patient. Vials containing a single dose in a liquid form are typically filled with the filamentous bacteriophage composition or

pharmaceutical composition at 50% to 90% of the vial volume or from 60% to 80% of the vial volume.

[056] In some embodiments, compositions according to the invention comprise an amount of endotoxin that when administered to a human provides less than 5.0 endotoxin units per kilogram body weight per dose, or less than 0.2 endotoxin units per kilogram body weight per dose. For purposes of this calculation, the human may be assumed to have a weight of at least 40 kg or 50 kg, and the dose may be assumed to have a maximum volume of 10 mL for liquid dosage forms. The dose may be for administration as a bolus (e.g. , an injection) or over an amount of time of up to 1 hour (e.g., an infusion). Accordingly, single dosage forms according to the invention can comprise less than 250 endotoxin units; less than 200 endotoxin units; less than 10 endotoxin units; less than 8 endotoxin units; less than 25 endotoxin units per mL; less than 20 endotoxin units per mL; less than 1 endotoxin unit per mL; or less than 0.8 endotoxin units per mL. Multiple dosage forms according to the invention can comprise less than 250 endotoxin units per dose; less than 200 endotoxin units per dose; less than 10 endotoxin units per dose; less than 8 endotoxin units per dose; less than 25 endotoxin units per mL per dose; less than 20 endotoxin units per mL per dose; less than 1 endotoxin unit per mL per dose; or less than 0.8 endotoxin units per mL per dose.

[057] Further embodiments of the invention include:

- a composition comprising filamentous bacteriophage according to the invention and an endotoxin that when administered to a human provides less than 5.0 endotoxin units per kilogram body weight per dose, wherein the human has a body weight of at least 40 kg and the dose has a maximum volume of 10 mL;

- a composition comprising filamentous bacteriophage according to the invention and an endotoxin that when administered to a human provides less than 0.2 endotoxin units per kilogram body weight per dose, wherein the human has a body weight of at least 40 kg and the dose has a maximum volume of 10 mL;

- a composition comprising filamentous bacteriophage according to the invention and an endotoxin that when administered to a human provides less than 5.0 endotoxin units per kilogram body weight per dose, wherein the human has a body weight of at least 50 kg and the dose has a maximum volume of 10 mL; and - a composition comprising filamentous bacteriophage according to the invention and an endotoxin that when administered to a human provides less than 0.2 endotoxin units per kilogram body weight per dose, wherein the human has a body weight of at least 50 kg and the dose has a maximum volume of 10 mL.

[058] Another aspect of the invention includes methods for preparing a pharmaceutical composition of the invention wherein the method comprises subjecting the large scale composition or the pharmaceutical composition to tabietting, lyophilization, direct compression, melt methods, or spray drying to form tablets, granulates, nano-particles, nano-capsules, micro-capsules, micro-tablets, pellets, or powders.

[059] Formulating the large-scale composition or the pharmaceutical composition into nano-particles, nano-capsu!es, micro-capsules, micro- tablets, pellets, or powders that are subsequently put into capsules is likewise encompassed,

[060] In some embodiments, compositions according to the invention are wild-type filamentous bacteriophage or filamentous bacteriophage which do not display an antibody or a non-filamentous bacteriophage antigen on its surface, The filamentous bacteriophage can be any filamentous

bacteriophage such as M13, f1 , or fd. Any filamentous bacteriophage is expected to behave and function in a similar manner as they have similar structure and as their genomes have greater than 95% genome identity, In some embodiments, the compositions according to the invention do not comprise a filamentous bacteriophage which displays an antibody on its surface. In some embodiments, the compositions according to the invention do not comprise a filamentous bacteriophage which displays a non- filamentous bacteriophage antigen on its surface.

Purification Methods

[061] Purification methods for obtaining the compositions of the invention are also encompassed and are described in detail below. Utilizing these methods allows for a percent recovery of bacteriophage of at least 10%, preferably 30, 40, 50, 60, or 70%.

Exemplary Purification Procedures

[062] Filamentous bacteriophage to be purified according purification methods according to the invention are obtained in solution, for example, in culture media, after growth in gram-negative bacteria. In some aspects of the invention, the filamentous bacteriophage are obtained according to the exemplary processes described in U.S. Application No. 61/512,169, filed July 27, 2011 , incorporated herein in its entirety.

[063] As a general matter, the purification methods according to the invention can comprise a series of chromatography steps. Exemplary steps and combinations of steps are provided below.

[064] In some embodiments, the methods comprise providing bacteriophage material that has been subjected to one or more steps such as centrifugation, nuclease treatment, an/or filtration.

[065] In some embodiments, nuclease treatment was or can be performed before or during the filtration step, for example as described in Examples 10 and 1 1 below, respectively.

[066] In some embodiments, the methods comprise at least one hydrophobic interaction chromatography step. [067] In some embodiments, the methods comprise at least one anion exchange chromatography step, which may be a reductive or binding- type step. (In reductive steps, the bacteriophage material is not retained on the column for a wash step but rather progresses through the column; this type of step is commonly run isocraticaily until the product has been collected. In binding type-steps, the bacteriophage material is loaded onto the column and is eluted by a buffer that tends to reduce the interaction of the

bacteriophage material with the column matrix relative to the strength of interaction in loading buffer.) In some embodiments, the methods comprise at least two anion exchange chromatography steps. When at least two anion exchange chromatography steps are used, it is possible for one step to be a binding anion exchange step and the other to be a reductive anion exchange step.

[088] In some embodiments, the material loaded onto a column for one or more of the chromatography steps comprises detergent. For an exemplary list of detergents compatible with bacteriophage, see Example 13. In some embodiments, the column loaded with material comprising detergent is an anion exchange column. The bacteriophage can be incubated with the detergent for a period before column loading, for example, 1 hour. The chromatography step following loading with material comprising detergent can be a binding-type step or reductive-type step.

[089] In some embodiments, the methods comprise at least one chromatography step using a cationicaliy charged polyamine-based resin that binds endotoxin. The resin for this step can be Etoxiclear resin (available from ProMetic Biosciences Ltd., Rockvil!e, Maryland, USA). [070] Etoxic!ear columns are characterized by the manufacturer as follows:

Mean partic!e size of 100 ± 10 μηι

Cross-linked 8% near-monodisperse agarose (PuraBead 6XL)

Dynamic binding capacity >500,000 EU/mL of adsorbent (loading at 120 cm/hr, 5 minute residence time)

Maximum operational flow rate of up to 400 cm/hr (5 mL Pre-Packed EtoxiClear Column)

Recommended operational flow rate of up to 200 cm/hr

Operational pH range of pH 4.0 to pH 8.0.

Centrifygatiosi

[071] A starting volume of filamentous bacteriophage in solution are centrifuged for a time and speed sufficient to separate the filamentous bacteriophage from bacterial cells and bacterial cell by-products in the starting solution, such as, for example, cellular material from the E. co// cells in which the bacteriophage are grown. In one exemplary embodiment, a starting solution of filamentous bacteriophage is centrifuged at about 4000 rpm for 40 minutes at between 2 and 8°C in a Sorvall RC-3 centrifuge, or the like, using a Sorvall HG 4L rotor, or the like. After centrifugation, the supernatant is collected and the pellet is discarded.

DNase Treatment

[072] The supernatant may next be treated with a DNase enzyme for a time and at a concentration sufficient to degrade any E. coll cellular DNA that may be present. In one exemplary embodiment, 0.5 - 1 L of supernatant from the centrifuge step above is incubated with the DNase enzyme Benzonase at a concentration of 10 units/mL in the presence of 5 mM MgCI ₂ . The supernatant and DNase enzyme are incubated in a shake flask at room temperature for about 80 minutes and agitated at a speed of 95 rpm. The benzonase step can be performed before or directly after the centrifugation step, or in some embodiments after the depth filtration step.

Depth Filtration

[073] The DNase-treated supernatant is next subjected to depth filtration, which involves passing the supernatant across at least three filters containing various filter media in series and collecting the flow through, which comprises the filamentous bacteriophage. Depth filtration (in contrast to surface filtration) generally refers to a "thick" filter that captures particulate matter and contaminating organisms based on size, hydrodynamic diameter and structure that are greater than the nominal cut-off of the membrane or membranes (for multiple filters operated in series). Depth filtration materials and methods are well known to one of skill in the art. For example, the filter material is typically composed of a thick and fibrous structure made of, for example, Poly Ether Sulfone (PES) or Cellulose Acetate (CA) with inorganic filter aids such as dsatomaceous earth particles embedded in the openings of the fibers. This filter material has a large internal surface area, which is key to particle capture and filter capacity. Such depth filtration modules contains pores of from 1 .0 μηι to 4.5 pm, including filter sizes of at least 1 .0, 1 .5, 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 pm, and fractional filter sizes between. Exemplary depth filtration modules include, but are not limited to, Whatman Polycap HD modules {Whatman Inc.; F!orham Park, N.J.), Sartorius Sartoclear P modules (Sartorius Corp. ; Edgewood, N.Y.) and iiiipore Millistak HC modules (Miliipore; Billerica, Mass.). In one particular embodiment, the cell culture fluid is clarified via depth filtration (performed at room temperature) and the filamentous bacteriophage are recovered in the filtrate.

[074] In some embodiments, depth filtration is carried out before DNAse treatment.

[075] In one exemplary embodiment, depth filtration of 0.5 - 1 L occurs across three filters in series. The soiution from the centrifugation step or DNase treatment step is passed over each filter with a peristaltic pump. In each case the flow through is collected. The filters may be as follows:

Table 1 - Exem lary Depth Filtration Filters

[076] This series of filtration sub-steps serves to clarify and reduce bioburden. An increase in scale can be achieved by increasing the

membrane surface area (e.g., larger filters) or a greater number of smaller filters.

Ultrafiltration and Diafi!tration

[077] After the final depth filtration step, the flow through is applied to an uitrafiltration/diaftltration step, where the filamentous bacteriophage are retained by the membrane (500 or 750 KD NMWCO). The goal of diafiltration is to complete buffer exchange, and the goal of the ultrafiltration is purification, or removal of components having a molecular weight lower than 500 or 750 KDa. in one exemplary embodiment, 500 mL of clarified supernatant ÷/- benzonase treatment is diafiltered using a Poly Ether Sulfone ("PES") 500 or 750 KD Net Molecular Weight Cut Off ("NMWCO") against 5-10 volumes of 25 mM Tris, 100 mM NaCI, pH 8.0. Alternatively, the clarified supernatant is diafiltered against 5-10 volumes of 25 mM Tris, 100 mM NaCI, pH 7.4. The cross flow, or transmembrane pressure (dP) is about 5 psi. The permeate rate is set at about 100 mL/min. Filamentous bacteriophage, such as, for example, M13, are retained by the membrane ("the retentate fraction"), and the permeate passes across the membrane.

[078] The ultrafiltration/diafiltration step may also be referred to as "ultrafiltration (UF)", or "tangential flow filtration (TFF)".

[079] In some embodiments, the material coming off of the TFF step (i.e., the ultrafiltration/diafiltration step) is depth filtered using, for example, a Sartoguard PES Capsule 0.2pm (Sartorius), 0.021 m ² at a manufacturers recommended flowrate of 150 mL/min.

HIC Phenyl

[080] Material derived from the TFF step is loaded in a high salt buffer (e.g., 2 - 2.1 M NaCS) onto a 3 L column containing Toyopeari Phenyl 650M (Tosoh Bioscience) with a bed height about 21 cm. This is achieved by diluting 2 fold (1 :1 dilution) with 25 mM Tris-HCI 4M NaCI pH 7.4 or the like. The column is pre-equilibrated with about 3 column volumes ("CV") of 25mM Tris-HCI pH 7.4, 2M NaCI or the like at a linear flowrate of 97.5 cm/h.

Typically, 300-500 mL of filamentous bacteriophage in solution at a

concentration of at least 4 x 10 ¹² phage/mL are loaded onto the column at a linear flowrate of 48.7 cm/h. This is foilowed by a wash step of about 3 CV of 25mM Tris-HCI pH 7.4, 2 M NaCI at a linear flowrate of 97.5 cm/h. The phage fraction is eluted in 3 CV of 25mM Tris-HCI pH 7.4 250mM NaCI or the like at a linear fiowrate of 97.5 cm/h. The filamentous bacteriophage peak is collected (typically 2 - 2.5 L) based on inline detection. Filamentous bacteriophage are eluted in a step or linear gradient. When using a step gradient, there is a sharp decrease to 250 mM NaCI rather than a gradual linear gradient to change the NaCI concentration. The column step yield is typically 90% or greater for M13. Similar yields are expected with other filamentous bacteriophage. The purpose of this step is to increase product purity by decreasing host cell contaminants through hydrophobic interaction chromatography (Functional group Phenyl) run in bind and elute mode. In other embodiments a linear gradient may be used.

[081] In order to ensure consistent collection of the peak and to provide a starting point and end point for peak collection, peak collection criteria is based on fluorescence or absorbance (this is also useful when transferring the process step between sites to ensure that the same peak collection parameters are applied). The absorbance is typically detected in real time after flowing through the column. Further analysis on peak fractions can provide further (more specific and supplemental) information regarding where and how much of the product has eluted from the column (e.g. off line ELISA). The product enriched fraction can also be tested off line for contaminants such as endotoxin.

[082] Fluorescence detection (excitation wavelength - ^■ 242 nm, emission wavelength - 334 nm) provides a sensitive method to detect filamentous bacteriophage such as 13. Alternatively, filamentous bacteriophage can also be detected by absorbance using a wavelength of 254 nm or 280 nm (A269nm). For fluorescence detection, the peak is usually collected starting at 0,1 U (fluorescence units) or 0.05 AU (absorbance units) at A254nm or 0,01 AU (absorbance units) on the leading edge (upward slope) and collection is stopped on the trailing edge (downside slope) of the peak. In one embodiment, collection is started upon observing a peak, an increase that can be less or greater than 1 % of the peak height at the expected retention time or volume and collection is stopped when the signal drops to about 5% of the maximum peak height. In a further embodiment, peak collection is started at a defined process time (based on the expected eiution time or elution volume). In one exemplary embodiment, collection may begin and end at an absorbance unit of .05 to .05 U (254 nm) and .01 to .01 U (280 nm). Other absorbance wavelengths and emission wavelengths may also used.

[083] The column is stripped with 3 CV of 25mM Tris-HCI pH 7.4 2M NaCI or the like followed by a NaOH wash of the matrix (CIP).

Weak anion exchange resin (e.g., DEAE AEX)

[084] Next, the eluate fraction from the preceding Phenyl HIC step is diluted with about 5 voiumes of 25mM Phosphate pH 6.5 buffer or the like and filtered through a weak anion exchange resin, such as, for example, a

Sartopore 2, 150, 0.45 pm/0.2 m filter or the like, The pH is typically pH 6.0- 7.0, including 6.5, and the conductivity 16,8 mS/cm. In one exemplary embodiment, the 3 L column (bed height circa 22 cm) is equilibrated with 3 CV of 25mM Phosphate 100m NaCi pH 6.5 at a linear flo vrate of 97.5 crn/h. The filamentous bacteriophage fraction from the previous step (diluted and filtered as described above) is loaded at a flowrate of 97.5 cm/h. The column is washed with 2 CV of 25mM Phosphate 150m NaCI pH 8.5 followed by 4 CV with 25mM Phosphate 250mM NaC! pH 8.5, the wash steps are run at a f!owrate of 97.5 cm/h. Filamentous bacteriophage are eluted with 3 CV of 25mM Phosphate 300m!VI NaCI pH 6.5 at a flowrate of 97.5 cm/h or the like. The phage peak is collected (typically 3 - 3.5 L) based on in-line defection of fluorescence and/or absorbance. In-line detection is detection in real time after flowing through the column. Further analysis on peak fractions can provide further (more specific and supplemental) information regarding where and how much of the product has eluted from the column (e.g. off line ELSSA). The product enriched fraction can also be tested off line for contaminants such as endotoxin.

[085] Fluorescence detection (excitation wavelength - 242 nm, emission wavelength - 334 nm) provides a sensitive method to detect filamentous bacteriophage such as M13. Alternatively, filamentous

bacteriophage can also be detected by absorbance using a wavelength of 254 nm or 280 nm (A269nm). For fluorescence detection, the peak is usually collected starting at 0.1 U (fluorescence units) or 0.05 AU (absorbance units) at A254nm or 0.01 AU (absorbance units) on the leading edge (upward slope) and collection is stopped on the trailing edge (downside slope) of the peak. In one embodiment, collection is started upon observing a peak, an increase that can be less or greater than 1 % of the peak height at the expected retention time or volume, and collection is stopped when the signal drops to about 5% of the maximum peak height, in a further embodiment, peak collection is started at a defined process time (based on the expected elution time or elution volume). In one exemplary embodiment, collection may begin and end at an absorbance unit of .05 to .05 U (254 nm) and ,01 to .01 U (280 nm). Other absorbance wavelengths and emission wavelengths may also used.

[086] The column is stripped with 3 CV of 25mM Phosphate 1 M IMaCi pH 6.5 or the like followed by a NaOH wash of the matrix (CIP).

[087] Filamentous bacteriophage are eluted in a step or linear gradient. When using a step gradient, the column step yield is typically 55% or greater for M13. Other filamentous bacteriophage are expected to have similar yields. The purpose of this step is to increase product purity by decreasing host cell contaminants through weak anion exchange (functional group diethylaminoethyl (DEAE)) chromatography run in bind and elute mode.

Strong anion exchange resin (e.g., AEX Q)

[088] The M13 eluate from the weak anion exchange resin (e.g., DEAE) is diluted with an equal volume (1 :1 ) of 25m Tris pH 7.4 or the like, and filtered across a suitable filter, such as, for example, a Sartopore 300 0.45 + 0.2 pm filter (Sartorius). The pH is typically 7.3 and the conductivity 15.8 mS/cm, In one embodiment, a Source 15Q (GE Healthcare) column is equilibrated with 3 CV of 20mM Tris-HCi pH 7.4 or the like at a linear flowrate of 189.5 cm/h. Filamentous bacteriophage, such as, for example, M13, is loaded at 169.5 cm/h. The column is washed with 3 CV of 25mM Tris 200 mM NaCi pH 7.4 or the like. Filamentous bacteriophage, such as, for example, M13, are eluted with 5 CV of 25mlV1 Tris-HCI pH 7.4, 280 mM or 300m NaCI (or the like) at a flowrate of 169.5 cm/hr. The phage peak is collected (typically 0.5 L) based on in-line detection. The absorbance or fluorescence is typically detected in real time after flowing through the column. Further analysis on peak fractions can provide further (more specific and supplementai) information regarding where and how much of the product has eluted from the column (e.g. off line EL!SA). The product enriched fraction can also be tested off line for contaminants such as endotoxin.

[089] Fluorescence detection (excitation wavelength - 242 nm, emission wavelength - 334 nm) provides a sensitive method to detect filamentous bacteriophage such as Ml 3. Alternatively, filamentous

bacteriophage can also be detected by absorbance using a wavelength of 254 nm or 280 nm. For fluorescence defection, the peak is usually collected starting at 0.1 U (fluorescence units) or 0.05 AU (absorbance units) at

A254nm or 0.01 AU (absorbance units) on the leading edge (upward slope), and collection is stopped on the trailing edge (downside slope) of the peak. In one embodiment, collection is started upon observing a peak, an increase that can be less or greater than 1 % of the peak height at the expected retention time or volume and coiiection is stopped when the signal drops to about 5% of the maximum peak height. In a further embodiment, peak collection is started at a defined process time (based on the expected elution time or elution volume), in one exemplary embodiment, collection may begin and end at an absorbance unit of .05 to .05 U (254 nm) and .01 to .01 U (280 nm). Other absorbance wavelengths (e.g., A269nm) and emission wavelengths may also used.

[090] The column is stripped with 3 CV of 25m Phosphate 1 M NaCi pH 7.4 followed by a NaOH wash of the matrix (CIP).

[091] Filamentous bacteriophage are eluted in a step or linear gradient. When using a step gradient, the column step yield is typically 80% or greater for M13. Other filamentous bacteriophage are expected to have similar yields. The purpose of this step is to increase product purity by decreasing host cell contaminants through strong anion exchange (Functional group Quaternary Ammonium (Q)) chromatography run in bind and elute mode.

Mustang Q/Clearance Filter

[092] The eluate from the previous step (strong anion exchange resin; AEX Q) is loaded directly onto one or more 10 mL Mustang Q (Pall) membrane at a flo vrate of about 150 mL/min. "Mustang Q" may also be referred to herein as "clearance filter," or "final clearance filter." A Sartobind filter (Sartorious) may be used in place of a Mustang Q filter. The charged filter (functional group Q) is operated in "flow through" mode. The filamentous bacteriophage product (e.g., M13) containing flow through fraction is collected. This step serves to remove remaining negatively charged contaminants, which are primarily endotoxin, but may also remove host eel! DNA and negatively charged host ce!S proteins.

Ultrafi Strati on

[093] Filamentous bacteriophage, such as, for example, M13, are concentrated and diafiltered into PBS (155 mM NaCI, 106 mM KH2P04, 2.97 m Na2HP04.7H20 - pH7.4) using a 500kD NMVVCO PES hollow fiber filter.

[094] The system is washed with approximately 5 system volumes (25mL) of water followed by 5 system volumes (25mL) of 0.5 M NaOH (50°C). 0.5 NaOH is re-circulated over the filter for about 20 to 40 minutes. The NaOH is removed by a 5 system volume wash with Water for Injection (WFI) water or the like followed by a five system volume wash with 25mM Tris 280mM NaCI pH 7.4 or the like. The product (M13 flow through from the previous Mustang Q process step) is added to the system and concentrated to target concentration of about 1 ,0 - 1.5 x 10 ¹⁴ phage/mL, circulated and diafsltered by the addition of 5-10 volumes of Phosphate Buffered Saline (PBS) pH 7.4.

[095] Typically, the yield for M13 after this step is 70% or greater. Other filamentous bacteriophage are expected to have similar yields. Steri!e FHtratson

[098] The supernatant recovered from the ultrafiltration step is filtered across one or more Whatman PURADISC 25 filters or Sartoscale Sartopore 2, 0.2 pm (or the like) at an approximate rate of 2 mL/min, or any other suitable flow rate. The concentration post filtration is adjusted to the target concentration of, for example, 4 x 1Q ⁱ² phage/mL, or in some embodiments 1 ,0 x 10 ¹⁴ phage/mL, or 1.0 x 10 ¹³ phage/mL with Phosphate Buffered Saline pH 7.4.

Tafo!e 2 - Exemplary Process Steps

Centrifugation j Centrifuging culture media comprising

I filamentous bacteriophage for a time I and speed sufficient to separate cellui material from the supernatant.

Example: 4000 rpm 40 minutes, 2-8 °C in a Sorvali RC-3 with a Sorvaii HG 4L

I supernatant is collected and cell pellet Step Short Description is discarded,

DNase Treatment ^* treating the supernatant with a

enzyme thereby facilitating DNA

*steps 2 and 3 may be

reversed. removal by generating smaller

fragments and nucleotides, in the event that DNase treatment precedes depth filtration, facilitates passage across depth filters.

Example: 0.5 - 1 L of culture

supernatant or TFF centrate (where steps 2 and 3 are reversed) is incubated in a 2 L flask with Benzonase at a concentration of 10 units/mL in the presence of 5 rnM MgC . This is incubated at a shaker speed of 95 rpm at room temperature for 60 minutes.

3 Depth Filtration* Applying the DNase-treated supernatant to depth filtration, and collecting the flow

^* steps 2 and 3 may be

through comprising the filamentous reversed.

bacteriophage. Further purposes of clarification/particuiate reduction.

Example: Depth filtration of 0.5 - 1 L (as an example) occurs across three filters in series, material is passed over each filter with a peristaltic pump. In each case the flow through is collected. The filters may be as follows: Short Description

(1) Sartopure GF+ 1.2 ϊη (Sartorius) 500 cm ²

Filter is operated according to the manufacturers recommendations (50 150 mL/min)

(2) Sartopure GF+ 0.85 μηι, 1000 cm

Filter is operated according to the manufacturers recommendations 150 mL/min)

(3) Sartopore 2 XLG, 2000 cm ²

Filter is operated according to the manufacturers recommendations (100 150 mL/min)

Ulirafiltraiion and Ultrafi lie ring to reduce any low

Diafiitration molecular weight contaminants such as host cell proteins, spent fermentation media contaminants, digested host cell DNA, and D ase enzyme using 500 KDa or 750 KDa net molecular weight cut off membrane ("IMMWCO"); and diafiiterlng to exchange the buffer. (<500KD/<750 KD) and buffer exchange (diafiitration).

Example: Ultrafiltration - 500 mL of clarified supernatant +/- benzonase treatment is diafiltered using a 500 or

Step Short D scri tion Det ils

[lililll

j preceding Phenyl 850 HIC Step is i diluted with 5 volumes of 25mM

1 Phosphate pH 6.5 buffer and filtered through a Sartopore 2 150 .45 /.2 μηι filter. The pH of the sample fraction is typically pH 8.3 - 6.4 and the conductivity 16.8 mS/cm. The 3 L column (bed height circa 22 cm) is equilibrated with 3 CV of 25m

Phosphate 100mM NaCI pH 6,5 at a linear flowrate of 97.5 cm/h. The phage fraction from the previous step (diluted and filtered as described above) is loaded at a flowrate of 97.5 cm/h. The coiumn is washed with 2 CV of 25m

Phosphate 150mM NaCI pH 6.5 followed by 4 CV with 25m Phosphate

250mM NaCI pH 8,5, the wash steps are run at a flowrate of 97.5 cm/h. M13 is eluted with 3 CV of 25mlv1 Phosphate

300mM NaCI pH 8.5 at a flowrate of

97.5 cm/h. The phage peak is collected

(typically 3 -- 3.5 L) based on in-line detection. The column is stripped with 3

CV of 25mM Phosphate 1 M NaCI pH

6.5 followed by a NaOH wash of the matrix (CIP).

7 AEX Q applying the collected material from

step 6 to strong anion exchange Short Description Details on anion exchange chemistry

Example: The eluate from the previous step (circa 0.5 L) is loaded directly onto one or more 10 mL Mustang Q (Pali) membrane at a f!owrate of 150 mL/min. The charged filter (functional group Q) is operated in "flow through" mode. The product (M13) containing flow through fraction is collected. This step serves to remove remaining negatively charged contaminants, this is primarily endotoxin (but also has the potential to take out host cell DNA and negatively charged host cell proteins)

Step I Short Details

I with Hi-cione water followed by a five system volume wash with 25m Tris ! 280mlV1 NaCl pH 7.4. the product is added to the system and circulated, and followed by concentration to 1.0 - 1.5 x 10 ¹⁴ phage/mL and then diafiltered by the addition of 5-10 volumes of

Phosphate Buffered Saline (PBS) pH 7.4. The concentration is checked by measuring the absorbance at 269nm, further concentration as needed can be applied at this stage to reach the required final target concentration of 1.0 - 1.5 x 10 ¹⁴ phage/rnL .

The filamentous bacteriophage from the Mustang Q step may be split into batches for this phage, i.e., the bacteriophage product may be equally divided in three sub-Sots and run through the ultrafiltration process in parallel.

Sterile Filtration sterile filter the supernatant

Example: The supernatant is filtered across one or more Whatman

PURADSSC 25, 0.22 prn or Sartoscale Sartopore 2, 0.22 μπι (Sartorius) filter at an approximate rate of 2 mL/min. The concentration post filtration is adjusted Step j Short Description to the target concentration of 1.0 x 10 phage/mL or between 1 .0 x 10 ¹⁴ and 1.5 x 10 ¹⁴ phage/mL with Phosphate Buffered Saline (PBS) pH 7.4.

[097] The following is a list of exemplary embodiments of phage purification methods according to the invention. 1. A method for preparing a composition comprising filamentous bacteriophage and less than 1 x 1 Q ^~10 endotoxin units per filamentous bacteriophage comprising: a) providing a first loading buffer comprising filamentous bacteriophage, wherein the filamentous bacteriophage were centrifuged, treated with a nuclease, and filtered after the filamentous bacteriophage were grown; b) performing a first chromatography step comprising contacting a first chromatography resin with the first loading buffer comprising the filamentous bacteriophage, contacting the resin with fresh buffer, and coilecting a first elution fraction comprising the filamentous bacteriophage; c) performing a second chromatography step comprising contacting a second chromatography resin with a second loading buffer comprising the previously collected filamentous bacteriophage, contacting the resin with fresh buffer, and coilecting a second elution fraction comprising the filamentous bacteriophage; d) performing a final chromatography step comprising contacting a final chromatography resin with a final loading buffer comprising the filamentous bacteriophage, contacting the resin with fresh buffer, and collecting a final elution fraction comprising the filamentous

bacteriophage and less than 1 x 1 G ^"10 endotoxin units per filamentous bacteriophage,

wherein at least one of the chromatography steps is an anion exchange step.

. The method of embodiment 1 above, wherein the nuclease treatment of the preparation occurred prior to a filtration step.

. The method of embodiment 1 above, wherein the nuclease treatment of the preparation occurred during a filtration step.

The method of any one of embodiments 1 to 3 above, wherein the final elution fraction comprises less than 1 x 1 G ^~11 endotoxin units per filamentous bacteriophage,

, The method of any one of embodiments 1 to 4 above, wherein the final elution fraction comprises less than 1 x 1CT ¹² endotoxin units per filamentous bacteriophage.

, The method of any one of embodiments 1 to 5 above, wherein the final elution fraction comprises less than 1 x 1 CT ³ endotoxin units per filamentous bacteriophage.

. The method of any one of embodiments 1 to 6 above, wherein the final elution fraction comprises less than 5 x 1 Q ^{" !4} endotoxin units per filamentous bacteriophage. , The method of any one of embodiments 1 to 7 above, wherein the filamentous bacteriophage comprise phage that do not display an antibody or a non-fi!amentous bacteriophage surface antigen,

, The method of any one of embodiments 1 to 8 above, wherein the filamentous bacteriophage comprise wi!d-type phage.

0, The method of any one of embodiments 1 to 9 above, wherein the filamentous bacteriophage comprise 13 phage.

1 . The method of any one of embodiments 1 to 10 above, wherein the first chromatography resin comprises a hydrophobic interaction chromatography resin or an anion exchange resin,

2, The method of any one of embodiments 1 to 1 1 above, wherein the second chromatography resin comprises an anion exchange resin. 3, The method of any one of embodiments 1 to 12 above, wherein the first or second chromatography resin comprises a weak anion exchange resin.

4. The method of embodiment 13 above, wherein, before contacting the weak anion exchange resin with a loading buffer, a detergent is added to the loading buffer.

5. The method of embodiment 14 above, wherein the detergent is chosen from Triton X-100 and Zwitfergent Z3-12.

6. The method of any one of embodiments 13 to 14 above, wherein the detergent is present at a concentration ranging from 0.05% to 2%.7. The method of any one of embodiments 1 to 16 above, wherein the final chromatography resin comprises an anion exchange resin. The method of any one of embodiments 1 to 17 above, wherein the final chromatography resin comprises a cationicai!y charged

polyamine-based resin that binds endotoxin.

The method of embodiment 18 above, wherein the affinity resin is Etoxic!ear resin.

The method of any one of embodiments 1 to 19 above, wherein the first chromatography resin comprises a weak anion exchange resin and the first chromatography step is performed as a reductive

chromatography step in the presence of a detergent, the second chromatography resin comprises a weak anion exchange resin and the second chromatography step is performed as a binding

chromatography step, and the final chromatography resin comprises a cationica!Sy charged polyamine-based resin that binds endotoxin.

The method of any one of embodiments 1 to 19 above, wherein the first chromatography resin comprises a hydrophobic interaction chromatography resin, the second chromatography resin comprises a weak anion exchange resin and the second chromatography step is performed as a binding chromatography step, and the final

chromatography resin comprises a cationicaiiy charged polyamine- based resin that binds endotoxin.

The method of any one of embodiments 1 to 19 above, further comprising, between the second and final chromatography steps, performing an additional chromatography step comprising contacting an additional chromatography resin with an additional loading buffer comprising the previously collected filamentous bacteriophage, contacting the additional resin with fresh buffer, and collecting a second elution fraction comprising the filamentous bacteriophage., The method of embodiment 22 above, wherein the first

chromatography resin comprises a hydrophobic interaction

chromatography resin, the second chromatography resin comprises a weak anion exchange resin and the second chromatography step is performed as a reductive chromatography step in the presence of a detergent, the additiona! chromatography resin comprises a weak anion exchange resin and the additional chromatography step is performed as a binding chromatography step, and the final chromatography resin comprises a cationically charged polyamine- based resin that binds endotoxin.

, The method of embodiment 22 above, wherein the first

chromatography resin comprises a hydrophobic interaction

chromatography resin, the second chromatography resin comprises a weak anion exchange resin and the second chromatography step is performed as a binding chromatography step, the additional chromatography resin comprises a strong anion exchange resin and the additiona! chromatography step is performed as a binding chromatography step, and the final chromatography resin comprises a strong anion exchange resin and the final chromatography step is performed as a reductive chromatography step.

, The method of any one of embodiments 1 to 19 above, wherein the method yields phage particles in an amount of at least 10% relative to input as measured by OD or ELISA. , The method of any one of embodiments 1 to 25 above, wherein the final elution fraction comprises at least 1 Q ^{1 J} phage particles.

, The method of any one of embodiments 1 to 26 above, wherein the final elution fraction comprises phage particies at a concentration of at least 10 ¹² per mL as measured by OD or ELISA.

, The method of any one of embodiments 1 to 27 above, further

comprising formulating the bacteriophage obtained from the final chromatography step and at least one pharmaceutical excipient into a pharmaceutical composition.

A method for purifying a culture of filamentous bacteriophage comprising:

a) centrifuging culture media comprising filamentous bacteriophage for a time and speed sufficient to separate cellular material from the supernatant;

b) treating the supernatant of the centrifuged media with a DNase enzyme;

c) applying the supernatant from step b) to depth filtration;

d) ultrafi!tering the depth filtered supernatant with a 500 or 700 KDa molecular weight cut off membrane and diafiltering the retentate to exchange the buffer;

e) applying the diafiltered retentate to a chromatography column comprising HSC Phenyl;

f) applying the M13 elution fraction from the HIC Phenyl column to a chromatography column comprising a weak anion exchange resin; g) applying the M13 elution fraction from the weak anion exchange resin to a strong anion exchange resin;

h) applying the Ml 3 elut on fraction from the strong anion exchange resin to a filter clearance step;

i) ultrafiltering the flow through, concentrating and diafiltering against the final formulation buffer; and

j) sterile filtering the retentate.

Formulations

[098] Techniques for formulation of drugs may be found, for example, in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference in its entirety.

[099] Suitable routes of administration for the pharmaceutical compositions of the invention may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0100] Alternatively, one may administer a pharmaceutical

composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into the brain of a patient.

[0101] Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophi!izing processes. [0102] Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into

compositions which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0103] For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosai administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0104] For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG) , If desired, disintegrating agents may be added, such as cross-!inked polyvinyl pyrrolidone, agar, or aiginic acid or a sa!t thereof such as sodium alginate.

[0105] in one embodiment, tablets, granulates, nano-particles, nano- capsules, micro-capsules, micro-tablets, pellets, or powders are

encompassed, either uncoated or enterically coated. The nano-particles, nano-capsules, micro-capsules, micro-tablets, pellets, or powders may be put into capsules.

[0108] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arable, talc, polyvinyl pyrrolidone, carbopoi gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0107] Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols, !n addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration. [0108] For bucca! administration, the compositions may take the form of tablets or lozenges formuiaied in conventional manner.

[0109] For administration by nasal inhalation, the filamentous bacteriophage of the present invention are conveniently delivered in the form of an aerosol spray from a pressurized pack or a nebulizer with the use of a suitable propeilant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide, in the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[01 10] The compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.

Formulations for injection may be presented in unit dosage form, e.g., in vials, ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formu!atory agents such as suspending, stabilizing and/or dispersing agents,

[01 1 1 ] Pharmaceutical compositions for parenteral administration include aqueous solutions of the filamentous bacteriophage in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as oi!y or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection

suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents (e.g., surfactants such as polysorbate (Tween 20)) which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions. A protein based agent such as, for example, albumin may be used to prevent adsorption of M13 to the delivery surface (i.e., IV bag, catheter, needle, etc.).

[01 12] Alternatively, the filamentous bacteriophage may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

[01 13] The pharmaceutical compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

FiHarnentous Bacteriophage for Use in Treatment and !Wllethods of

Treatment Comprising Administering Filamentous Bacteriophage

[01 14] in some embodiments, the invention provides a filamentous bacteriophage composition according to the invention for use in treating a plaque-forming disease, for reducing the amount of amyloid plaque in a patient suffering from a plaque-forming disease, for inhibiting the formation of amyloid deposits or for disaggregating pre-formed amyloid deposits, or for reducing susceptibility to a plaque-forming disease.

[01 15] In some embodiments, the invention provides methods for treating a plaque-forming disease, for inhibiting the formation of amyloid deposits or for disaggregating pre-formed amyloid deposits in a patient, for reducing the amount of amyloid plaque in a patient suffering from a plaque- forming disease, or for reducing susceptibility to a plaque-forming disease, each of which comprise administering a filamentous bacteriophage

composition according to the invention to a patient in need thereof,

[01 16] In certain aspects of these embodiments, the filamentous bacteriophage provided in the uses and methods according to the invention does not display any non-filamentous bacteriophage antigen on its surface. In certain aspects of these embodiments, the filamentous bacteriophage provided in the uses and methods according to the invention is a wild-type bacteriophage. In a more specific aspect, the bacteriophage is a wild-type bacteriophage. In an even more specific aspect, the filamentous

bacteriophage is selected from M13, f1 , or fd. Each of these filamentous bacteriophage is expected to behave and function in a similar manner as they have similar structure and their genomes have greater than 95% genome identity, in an even more specific embodiment, the filamentous bacteriophage used in the methods and compositions for the uses described above according to the present invention is wild-type M13.

[01 17] In certain aspects of these embodiments, the plaque-forming disease is selected from early onset Alzheimer's disease, Sate onset

Alzheimer's disease, presymptomatic Alzheimer's disease, SAA amyloidosis, hereditary Icelandic syndrome, senility, multiple myeloma, to prion diseases that are known to affect humans (such as for example, kuru, Creutzfeldt- Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal familial insomnia (FFI)) or animals (such as, for example, scrapie and bovine spongiform encephalitis (BSE)), Parkinson's Disease, Argyrophilic grain dementia, Corticobasai degeneration, Dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down's syndrome, Frontotemporal dementia with parkinsonism linked to chromosome 17, Hallervorden-Spatz disease, Myotonic dystrophy, Niemann-Pick disease type C, Non-Guamanian motor neuron disease with neurofibrillary tangles, Pick's disease,

Postencephalitic parkinsonism, Progressive subcortical gliosis, Progressive supranuclear palsy, Subacute sclerosing panencephalitis, and Tangle only dementia. In more specific aspects of these embodiments, the plaque- forming disease is selected from early onset Alzheimer's disease, late onset Alzheimer's disease or pre-symptomatic Alzheimer's disease.

[01 18] Methods involving disaggregating pre-formed amyloid deposits may comprise directly contacting any of the filamentous bacteriophage compositions of the invention with the pre-formed amyloid deposits.

[01 19] in one aspect of methods according to the invention, the bacteriophage is administered to the patient as part of a pharmaceutically acceptable composition additionally comprising a pharmaceutically acceptable carrier. For example, the pharmaceutically acceptable carrier can be saline.

[0120] In one embodiment of methods according to the invention, the filamentous bacteriophage composition is administered irUranasa!!y. !n one embodiment of compositions for the uses described above, the filamentous bacteriophage composition is formulated for intranasal administration.

[0121] In another embodiment of methods according to the invention, the filamentous bacteriophage are administered directly to the brain of the subject. Administration "directly to the brain" includes injection or infusion into the brain itself, e.g., intracranial administration, as well as injection or infusion into the cerebrospinal fluid. In one aspect of this embodiment, administration is by intrathecal injection or infusion, intraventricular injection injection or infusion, intraparenchymal injection or infusion, or intracerebroventricular injection or infusion, In more specific aspects, administration is by intraparenchymal injection; intracerebroventricular injection; or

intracerebroventricular infusion. In one embodiment of compositions for the uses described above, the filamentous bacteriophage composition is formulated for administration directly to the brain of a subject, such as by intracranial administration, as well as injection or infusion into the

cerebrospinal fluid, intrathecal injection or infusion, intraventricular injection injection or infusion, intraparenchymal injection or infusion, or

intracerebroventricular injection or infusion.

[0122] Methods delineated herein also include those wherein the patient is identified as in need of a particular stated treatment. Identifying a patient in need of such treatment can be in the judgment of a patient or a health care professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method).

[0123] it is to be understood that both the foregoing and following description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

Example 1 - Exemplary Production Process Steps of the Invention

[0124] Tables 3 through 13 show in table format exemplary specifications for purification processes according to the invention. Those of skill in the art will know where modifications may be made without compromising the novel methods described herein. Table 3 - Upstream Process

Table 4 - Exemplary Filtration Specifications

centrifuqation. Table 5 - Exem lary DNase Treatment

emperature m en room tempera ure

[0125] The benzonase step can be performed before or directly after the centrifug ation step, or after the three stage } depth filtration. - Exemplary Diafiftration Steps

Membrane Area (cm ² ) i 280

Table 7 - Exemplary Fiitration Steps

Filtration: Sartogt sard PES Capsule ϋ,2μιτ

Function Parameter _ I Requirements

Maximum scale Membrane area (m ^z ) ^" L021 — — ^■■ - performed

previously

Filter Filter type 1 Polyethersu!fone Ϊ

Capacity LJnT ^"

Flowrate mL/min I 150 I

Pressure

Table 8 - Exemplary Chromatography Step

possible^

- Exemplary Fractogel DEAE Co!

chromatogram

Table 10 - Exempfary 15Q Column Specifications

I Chromatography Ste 3 - Source 15Q Column

Function iiii

Maximum scale Column size 2GGmL

performed

previously

Column Media type Source 15Q

Binding capacity (g/L) ϊ .86 χ Ϊ 0 ¹³

Bed height (cm)

Net pore size (microns)

Linear flowrate (cm/hr) or 169.5 cm/hr residence time (min)

Packing | Pressure or Flow

technique I flow pack

i Flowrate 15mL/min

j Pressures Less than 50 psi. j Packing buffer 25m M Tris-HCi pH

7.4

Pack test details

Asymmetry specification

Plate number specification

Pre run column Sanitisation solution [ Q.5M aOH

C!P CIP method (including number 3 column volumes of CV's / hold times) with a 30 minute hold time after 1 and ½ column volumes. Wash -with MilliQ

possible)

Table 11 - Exemplary Wfustang Q Filtration Steps

I Mustang Q Filtra

j Function I Parameter Requirements

I Maximum scale j Membrane volume (mL)

performed |

previously [

Filter [ Filter Material Hydrophiliic polyethersulfone j j Capacity g/m ²

I Fiowrate mL/rn n 50mL/min

I Pressure Less than 50 psi

Other j The Source 15Q elution

material is over the mustang Q filter in the exclusion mode.

Tabie 12 - Exem lary Ultrafiltration Steps

- Exemplary Sterile Filtration Step

Whatman PURAD1SC 25:

unction Parameter:

Other After the sample is filtered

absorbance concentration calculations are determined and the sample is diluted with 1 PBS buffer pH 7.4 to reach a concentration of 1.0 x 10 ¹⁴ phage/mL or other desired tarqet concentration.

Example 2 - Exemplary buffers and solutions

Table 14 - Exemplary buffers ivie ia / Chemical W fitraiit uide pH & Specific Buffer/Feed Cons it ents C nducti ity Requirements Name Range

Benzonase 203 3 ^" ΐ.θΎβδ ^'

gCI ₂

Solution

Fermentation Tris Base 121 ,14 3.0285 HCI 8.0

Diafiitration

Buffer Sodium 58,44 5.844

Chloride

HIC Column Tris Base 121.14 3.0285 HCi 7.4

Sample

Preparation Sodium 233.76

Buffer Chloride

HIC Column Tris Base 121.14 3.0285 HC! 7.4

Equilibration

Buffer

Sodium 58.44 116.88

Chloride

HIC Column Tris Base 121.14 ^{' '} 3.0285 ^{'' '} HCi 7.4 I I Wash Buffer

89

Example 3 - Representative Purification Process for H H13 - Batch 1

[0128] A purification process according to the invention was followed according to the steps provided in Table 2 and Example 1 for 0,32 Liters of M13 at a starting concentration of 2.45 x 10 ¹³ phage/ml. For Batch 1 , the hollow fiber was equilibrated with 1 x PBS. Subsequent batches were equilibrated with 25mM Tris 280mM NaCI pH 7.4.

[0127] Table 15 shows the phage recovery results from this experimental purification, including, for example, the totai number of phage recovered after each step of the purification process, as well as the % recoveries, Table 15 - Phage Recovery for Batch 1

[0128] Table 16 shows the removal of endotoxin after each step of the purification process for Batch 1. Purified (post second UF step) materials from Batch 1 contain 4.8 x 10 ^"13 EU/phage. Table 16 - Endotoxin Removal for Batch 1

Example 4 - Representative Purification Process for - Batch 2

[0129] A purification process according to the invention was followed according to the steps provided in Table 2 and Example 1 for 0.35 Liters of Ml 3 at a starting concentration of 2.4 x 10 ¹³ phage/ml. Table 17 shows the phage recovery results from this experimental purification, including, for example, the total number of phage recovered after each step of the purification process, as well as the % recoveries.

Table 17 - Pha e Recovery for Batch 2

[0130] Tab le 18 shows the removal o ^: endotoxin after each ste purification process for Batch 2. Purified (post UF step) M13 material from Batch 2 contains 9.2 x 1G ^~13 EU/phage. The purity after the HIC Phenyl step is 5.8 x 1 G ^~8 EU/phage. A 8.3 x 10 ⁴ increase in purity (EU/phage) is observed from the DEAE step to the final purified material

Table 18 - Endotoxin Removal for Batch 2

[0131] Table 19 shows an exemplary certificate of analysis for Batch Table 19 - Exemplary Certificate of Analysis - Batch 2

Example 5 - Representative Purification Process for H H3 - Batch 3

[0132] A purification process according to the invention was followed according to the steps provided in Tabie 2 and Exampie 1 for 0.4 Liters of

M13 at a starting concentration of 7.2 x 10 ¹³ phage/ml.

[0133] Table 20 shows the phage recovery results from this experimental purification, including, for example, the total number of phage recovered after each step of the purification process, as well as the % recoveries. Table 20 - Phage Recovery for Batch 3

[0134] Table 21 shows the removal of endotoxin after each step of the purification process for Batch 3.

Table 21 - Endotoxin Removal for Batch 3

[0135] Table 22 shows an exemplary certificate of analysis for Batch 3. Purified (post UF step) M13 material from batch 3 contains 8.5 x 10 ^'14 EU/phage. The purity after the HIC Phenyl step is 7.3 x 1 G ^"8 EU/phage. An 8.6 x 10 ⁵ increase in purity (EU/phage) is observed from the DEAE step to the final purified material.

Table 22 - Exemplary Certificate of ArtaHysiis - Batch 3 ssay Result

M13 Concentration (by I ELISA) 1.1 x 10 ¹⁴ pbage/ml

M13 Concentratic (by / ¾>bsorbance) 1 .0 x 10 ¹⁴ phage/ml

Endotoxin (by Ch romog enic LAL) 8.5 EU/mi

88.4 % Main Peak

A EX 8.1 % Pre Peak

3.5 % Post Peak

I AEX Peak Area 31741 100 UV*sec

I AEX Peak Height 1015.968 mV

I Conforms to reference material. SDS PAGE

Major band at 5 kDa.

Bioburden Pass (No growth after 5 days)

Example 6 - Rep resenl ative Purificat! on Process for M13 - Batch 4

[0138] A purification process according to the invention was followed according to the steps provided in Table 2 and Example 1 for 0.4 Liters of M13 at a starting concentration of 2.2 x 10 ¹³ phage/ml,

[0137] Table 23 shows the phage recovery results from this experimental purification, including, for example, the total number of phage recovered after each step of the purification process, as well as the % recoveries.

Table 23 - Pha e Recovery for Batch 4

[0138] Tabie 24 shows the removai of endotoxin after each step of the purification process for Batch 4. Purified (post UF step) M13 material from Batch 4 contains 2.2 x 10 ^"12 EU/phage. The purity after the HIC Phenyl step is 8.7 x 10 ^"8 EU/phage. A 4.0 x 10 ⁴ increase in purity (EU/phage) is observed from the DEAE step to the final purified material.

- Endotoxin Removal for Batch 4

Example 7 - Comparison Purification Process for HI S - Utilizing CsCl purification methods and not the methods of the invention

[0139] Tafaie 25 shows the results for a purification process CsCl purification techniques, and not the inventive techniques described in Table 2 or Example 1 . M13 material corresponding to the "CsCl" batch was produced by infection of E.cols JM109 grown in batch culture. M13 containing supernatants were harvested by centrifugation and PEG precipitated. Further purification was achieved by two successive rounds of Cesium Chloride ("CsCl") density gradient purification (generated by ultracentrifugation).

[0140] In contrast to the purities observed for the batches described in Examples 1 - 6, a CsCl purified batch yielded a purity of only 2.8 x 10 ^"10 EU/phage. Tabie 25 - Exemplary Certificate of A nalysis - CsCI purification methods

Example 8 - Set points for Endotoxin levels

[0141] Table 26 below outlines calculations that were made in order to set the draft target endotoxin release specifications for Ml 3.

Table 26 - Target endotoxin specifications

! weight (40 Kg)

Draft target specification set at 5 EU/rnL based on the current estimation of potential routes of administration and estimated maximum amounts dosed (worst case)

Target specification may be modified at a later date subject to potential changes to route of administration and the maximum projected amount dosed

Example 9 - Exemplary Drug Substance Specification

[0142] Table 27 shows the attributes and specifications for an exemplary drug substance comprising M13 filamentous bacteriophage. This specification covers the purified bu!k drug substance.

Table 27 - Exemplary Drug Substance Specification

Attribute 1 resting site/Metho d Specification

Physical Characteristics

Coior, Appearance and Clear to opalescent, Clarity colorless to straw yellow liquid, no visible particles pH 7.0 -7.8

Osmolality Report result

Concentration

Virus concentration by 10 x 10 ¹⁴ ± 0.1 x 10 ¹⁴ Absorbance (A269nm) ^a phage/mL

Identity and Purity

Attribute T esting site/Metho d Specification

Infectivity assay tbd Report result as

infectious units/mL

Identity by Western Blot Comparable to reference

Identity by ELISA Report result as

phage/mL

Identity by qPCR

Report result as copy number

Identity by SDS-PAGE

(reduced, Coomassie

stain) Report Major bands,

Comparable to reference

Purity by A EX

>90% monomer by peak area, <10% aggregates and fragments

Purity by Size Exclusion

Chromatography (SEC) Report result as % peak area of main peak, pre- peaks and post peaks

Potency (activity/binding)

Subject to the development

of a suitable, robust and

reproducible assay

Impurities

Host Cell DNA b qPCR <20 ng/mL

Host cell protein Report result as ng/mL (ELISA)

Safety

Endotoxin (LAL) ^b <5 EU/mL

Extended bioburden by No growth detected after direct transfer method 14 days ^a UVabs (A269nm) is currently the method of choice for determining concentration, other alternative include the product specific ELISA and qPCR, there is a possibility that one of these method replaces the ELISA prior to IND filing.

Specification subject to change dependent on amount dosed, route of

administration and further regulatory input. [0143] Table 28 shows the attributes and specifications for an exemplary drug product comprising IV113 filamentous bacteriophage. This specification covers the filled drug product, derived from drug substance by passing over two sterile filters in series followed by filling into glass vials, for example.

Table 28 - Exemplary Drug Product Specification

Attribute T&simg Site/Method Specification

Physical Characteristics

Color, Appearance and Clear to opalescent, Clarity colorless to straw yellow liquid, no visible particles

7.0-7.8

Volume in Container Per cUSP <1> NLT 0.6mL/vial

Concentration

Virus concentration by 1.0 x 10 ⁴ + 0.1 x 10 ¹ Ahsorbance (A288nrn phage/mL

Identity and Purity

! nfectivity, assay tbd Report result as infectious particles/mL

Report

Identity by Western Blot result/comparabi e to reference standard

Identity by ELISA

Report result as phage/mL

Identity by qPCR Report result as copy number

Identity by SDS-PAGE Report Major (reduced, Coomassie bands, stain) Comparable to reference

Purity by SEC >90% monomer by peak area. <10%

aggregates and Purity by AEX fragments

Report % peak area of main peak, pre-peaks and post peaks

Potency (activity/binding)

Subject to the

development of a

suitable, robust and

reproducible assay impurities

Host Cell DNA by <20 ng/mL qPCR

Report result as

Host Cell Protein ng/mL

(ELISA) Safety

Particulates Per cUSP 28 <788>

Sterility ⁸³ <71 > No growth

detected after 14 day incubation, passes USP sterility test

Endotoxin by LAL <5 EU/mL

a UVabs (A269nm) is currently the method of choice for determining

concentration, other alternative include the product specific ELISA and qPCR, there is a possibility that one of these method replaces the ELISA prior to SND filing.

bBacteriostasis and fungistasis will be performed on the first cG P lot released c Specification subject to change dependent on amount dosed

Example 10 - Alternative Production Process 70065

[0144] This example sets forth an exemplary process according to the invention for purification of filamentous bacteriophage having low endotoxin contamination.

[0145] Supernatant containing M13 phage from a 5 L fermentation was provided.

[0146] Benzonase treatment: Benzonase was added to the supernatant to achieve a final concentration of 10 units per mL and 1 M MgC , added to give a final concentration of 5m ; the material was incubated for 60 minutes at room temperature. The material was then clarified by depth fiitration using 0.6pm, 0.6/0.2pm and 0.2μηι ULTA Prime capsules (GE). Only 1993.8g of material was carried forward at this point due to blockage of the filters. [0147] TFF1 step: The clarified material was diafiltered for 10 turnover volumes (TOV), using a 500kDa MWCO hollow fibre cartridge (0.48m ² ) until the pH and conductivity of the permeate was comparable to the diafiltration buffer (25mM Tris, l OOmM NaCI, pH 8.0), The inlet pressure

(~5psi) was maintained throughout the diafiltration. The recovered retentate (1864.6g) was 0.45/0.2 m filtered (1795.6g) and sampled for analysis with the remaining bulk stored at 2~8°C.

[0148] HIC step: The post TFF1 material (1792.3g) was diluted 1 :1 with 25mlv1 Tris, 4M NaCI, pH 7.4, 0.2μηι filtered (3739.5g) and sampled for analysis. The material was at pH 8.0 and had a conductivity of 152.9mS following dilution.

[0149] A Toyopearl Phenyl 850M Vantage 90 column (1 144.5mL column volume (CV)) was sanitised prior to use and equilibrated with 25m Tris-HCl, 2 NaCI, pH 7.4. The diluted sample (3739.5g) was loaded onto the Toyopearl Phenyl 850IV1 column at a flow rate of 48.7cm/hr (50.5mL/min). The flow through (F T) unbound material was washed out with 3CV of 25m Tris- HCl, 2 NaC!, pH 7.4, before the NPT002 material was eluted with 250mM NaCI and the column striped with 2M NaCI. All steps were performed at a flow rate of 97.5cm/hr (101 mL/min). The phage peak was collected as a single pool (776.1 g) starting from when the A254 increased from baseline and stopped when the peak decreased to baseline (Figure 2). The product peak was sampled for analysis and stored at 2-8°C overnight before performing the DEAE step.

[0150] DEAE Anion Exchange Step: The post HIC materia! (772.1 g) was removed from 2-8°C storage diluted with 5 volumes of 25mM phosphate pH6.5 and filtered through a 0.45/0.2pm filter (4614.5g). The material was at pH 6.05 and had a conductivity of 21.1 mS following dilution. A Fractogel EMD DEAE (M) Vantage 90 column (864.6mL column volume (CV) was sanitised prior to use and equilibrated with 25mfv1 phosphate, 100mM ad pH 6.5. The diluted post HIC material (4607.8g) was loaded onto the column at a now rate of 97.5cm/hr (101 mL min). The column was then washed with buffer containing 50m NaCI followed a wash at 250mM NaCi. it was noted that the 150mM NaCI wash buffer had a conductivity of 17.7mS which was lower than the conductivity of the sample (2 .1mS). The phage were eluted with SOOmfvl NaCI and collected as a single pool (1 107.8g) starting from when the absorbance at 254 nm (A254) increased from baseline and stopped when the peak decreased to 5% of baseline. The product peak was sampled for analysis and stored at 2-8°C overnight before performing the Source 15Q step.

[0151] Source 15Q step: Post DEAE material (1 102.5g) was removed from 2-8°C storage, diluted 1 :1 with 25mM Tris-HCI pH7.4, and filtered through a 0.45/0.2pm filter (2189.9g). The material was at pH 6.79 and had a conductivity of 15.29mS following dilution, A Source 15Q Fineline 35 column (182.4mL column volume (CV)) was sanitised prior to use and equilibrated with 25mM Tris-HCI, pH 7.4. The diluted post DEAE sample (2183.5g) and 1 CV of wash buffer containing 200 mM NaCI was loaded onto the column at a flow rate of ~60cm/hr (9.5mL/min). This reduced flow rate was used due to the small bead size of the media and the upper limit of pressure provided by the chromatography system. [0152] The remaining wash step and elution of the phage (in buffer containing 280 mM NaCI) was performed at 169.5cm/hr (27.1 mL/min) using the AKTA Pilot system pump with the manual system outlet flow path. The eiuted material was collected as a single pool (295.9g) starting from when the A254 increased from baseline and stopped when the peak decreased to 5% of baseline. The product peak was sampled for analysis and stored at 2-8°C overnight before performing the Mustang Q step.

[0153] Mustang Q step: A 10mL Mustang Q capsule was prepared as per the manufacturers instructions and equilibrated in 25mM Tris, 280mM NaCI, pH7.4. The post Source 15Q pool (291 .3g) was removed from 2-8°C storage and loaded onto the Mustang Q capsule followed by a flush with ~50ml_ buffer at a flow rate of 150mL/min. The material was collected as a single pool from start of loading until end of flush (333.5g). The material was sampled for analysis with the samples stored.

[0154] TFF2 step: The initial filter cartridge was found to give a low flow rate, so after the post Mustang Q pool (330.5g) was initially concentrated approximately 2.8-fold using the 500kDa MWCO hollow fibre cartridge (0.004 m ² ) was initially concentrated approximately 2.8-fold using a SOOkDa MWCO hollow fibre cartridge (0.0041 m ² ), the retentate (1 16.8g) was recovered and the TFF system was rinsed with ~47mL of formulation buffer. The TFF retentate was filtered using a Sartopore 2 150 0.45/0.2pm filter (108.01 g). The material was sampled and the TFF 2 intermediate bulk stored at 2-8X for 7 days. A replacement hollow fiber was obtained, flushed, and wetted out so that its permeability was 474 LMH/barg; then it was sanitised and ready for use. The TFF 2 intermediate bulk material was concentrated to -1.1 x 0 ¹⁴ particles/mL (~30mL) as determined by UV anaiysis. The materia! was then buffer exchanged for 6 turn over volumes (TOV) into formulation buffer. The material was then further concentrated to ~1.5 x 10 ¹⁴ particies/mL before being recovered from the system (20.21 g).

[0155] The materia! was sampled and then 0,2μηι filtered using 5 x Whatman Puradisc 0.2μηη PES 25mm syringe filters (Cat no 6780-2502). The materia! was then diluted with formulation buffer based on UV analysis to give 25mL at a concentration of 9.93 x 10 ¹³ virions/mL by UV.

[0156] Data collected during the process are shown in the following table.

Table 29. Phage and endotoxin concentrations measured at various stages of process 70065.

Example 11 - Alternative Production Process 70078

[0157J The steps in this process were similar to those of process 70065 (Example 10), but had the following changes. The process began with supernatant from two 5L fermentations; as a general matter, in light of the amount of material, column chromatography was generally performed by splitting the material into two aiiquots and performing two column runs.

[0158] TFF1 and Benzonase steps: Treatment with Benzonase occurred during the TFF1 filtration step. Specifically, after depth filtration using 4 x 1.2pm and 2 x O.GSpm Sartopure GF+ filters (Sartorius), 5011.5g of clarified material was diafiitered for 5 turn-over volumes (TOV), using a 500kDa MWCO hollow fibre cartridge (0.48m ² , 60cm path length, cat No RTPUFP-500-C-6S) into 25mM Tris, 100mM sodium chloride, pH 8.0. The inlet pressure (~5psi) was maintained throughout the diafiltration. The

Benzonase treatment occurred in the TFF system used for the TFF1 step rather than before the TFF1 step. Specifically, the appropriate volume of benzonase solution to achieve a final concentration of 10 units per niL and 1 M MgCI ₂ solution to give a final concentration of 5m in the diafiitered materia! were mixed together and injected into the TFF system reservoir bag through the syringe port. The material was then mixed by agitation before being recirculated in the TFF system at approximately 20% of the running flow rate with the permeate lines closed for 60 minutes at room temperature.

[0159] The material was further diafiitered for 5 turn-over volumes (TOV), until the pH and conductivity of the permeate was comparable to the diafiltration buffer (25mM Tris, 1 GGmM NaCI, pH 8.0). The inlet pressure (~5psi) was maintained throughout the diafiltration. [0160] The recovered retentate (5036, 7g) was 0.8/0.2μηΊ filtered using Sartopore 2 XLG icliCap filters (3 filters used to give 4600. Sg (cat no

5445307G9— OO)) and sampled for analysis with the remaining bulk stored at 2-8°C.

[0161 ] DEAE, Source 15Q, and Mustang Q steps were performed.

[0162] TFF 2 step: The post Mustang Q pool (646.7g) was

concentrated to -1 .5 x 10 ¹⁴ particles/mL based on UV analysis (~70mL) using a 500kDa MWCO hollow fibre cartridge (0.014m ² , 30cm path length (UFP- 500-C-3MA)).

[0163] The material was then buffer exchanged for 5 turn over volumes (TOV) into formulation buffer (1 .06mM potassium phosphate, 2.97m sodium phosphate, 155.17m sodium chloride, pH 7.4). The shear rate was maintained between 6500 and SOOOsec ^"1 throughout processing. The retentate was recovered from the system to give 75.6g.

[0164] The material was sampled and then 0.45/0.2pm filtered using a sterile Sartopore 2 150 filter (Cat no 5441307H4— 00-B). The materia! was then diluted with formulation buffer to target a titre of 1 .05x10 ¹⁴ particles/mL based on UV analysis. Following dilution 74.79g of final material was generated at a concentration of 9.24 x 10 ¹³ virions/mL by UV analysis. The final material had 58.2 EU per 10 ¹⁴ phage particles (i.e. , less than 0 ^~ EU per phage particle).

[0165] Data coiiected during the process are shown in the following table. Table 30: Endotoxin data from the purification using process 70078,

- Processes 70101

166] The steps in these process were similar to those of process 70078 (Example 1 1 ), including depth filtration, Benzonase treatment during the TFF1 step, a sequence of chromatography (H!C, DEAE, 15 Q), then Mustang Q filtration and a TFF2 step.

[0167] Data collected during the processes are shown in the foliowinc tables.

Table 31 : Endotoxin data from the purification using process 70101.

Tabfe 32: Endotoxin data from the purification using process

70107.

[0168] Process 70107 was run later in time than the other processes in Examples 1 1 and 12, with much of the same equipment. The overall lower endotoxin reduction across process 70107 in comparison to the previous processes suggested that reuse of the columns may have impacted the contaminant removal efficiency. Example 13 - Screening of Detergents for Use in Purification Processes

[0169] The following detergents were added to TFF1 buffer (25mM Tris, 100mM NaCI, pH 8.0) at 1 % (w/v) concentration and analysed for interference in the endotoxin assay described above (QCSOP296) by preparing mock samples mimicking dilutions of a TFF1 sample containing 1 x10 ⁵ EU/mL endotoxin:

1 . Zwittergent 3-12

2. Zwittergent 3-14

3. Triton X-100

4. Triton X-1 14

5. Tween 20

[0170] The detergents were initially prepared as 5% (w/v) in TFF1 buffer and then diluted to 1 % (w/v) in TFF1 buffer to mirror actual process steps. Interference in the Endotoxin assay was measured by the Positive Product Control (PPC) recovery of spiked-in Endotoxin added to each sample. No interference effect was observed, in that %PPC values were within an acceptable range (between 50% and 200% was considered acceptable;

values were in the range of 83-1 17%; data not shown).

[0171 ] A partially processed phage preparation was provided which had been taken through the TFF 1 step in the order of Example 10 ("post TFF

1 material"). The detergents listed in the previous paragraph were added to the post TFF 1 material at two different final concentrations as detailed in

Table 33. A run was also performed in the absence of detergent as a control

(Run 1 ). The material was incubated at room temperature for 1 hr with continuous gentle mixing on a roller mixer. Eleven columns of approximately

30mL Sepharose 6 Fast Flow (XK16 columns) with a 15-17cm bed height were packed as per the manufacturer's instructions. Each column was sanitised, equilibrated and loaded to -20% of the column volume to run as a group separation. The detergents were observed to interfere with

chromatographic profiles to varying degrees due to their absorbance at 280nm.

Table 33, Columns Run with Various Detergents

[0172] The endotoxin levels and titre as determined by ELiSA for the post SEC materia! from runs 1 -1 1 are shown in Table 34, The 0.1 % Triton X- 100 (Run 2) and 1 % Zwittergent Z3-12 (Run 9) were shown to give the most significant reduction in endotoxin levels. No significant endotoxin removal was observed for the control and other detergents.

Table 34. Results for SEC Runs 1 -11

using the post TFF 1 resuit and dividing by the 1.25 dilution factor performed during the detergent or buffer (control) addition.

Example 14 - Assessment of Use of Detergents In Column Chromatography Steps

[0173] A partiaily processed phage preparation was provided which had been taken through the TFF1 step in the order of Example 10, and HIC (Toyopearl Phenyl 650M) and DEAE (Fractogel E D DEAE (M)) steps were performed in the presence of detergent. [0174] Sn detail, a 21 .3mL HIC column (10.6cm bed height) and a 21 .1 mL DEAE column (10.5cm bed height) were packed as per the

manufacturer's instructions. The columns were re-used for the 4 runs with a cleaning-in-place (CIP) method performed between each run.

[0175] Post TFF 1 material was adjusted to the required detergent concentration, or diluted with the corresponding buffer without detergent for the control runs. The material was mixed gently for one hour at room temperature using a magnetic stirrer bar and platform (HIC runs 1 -4) or using a roller mixer (DEAE Runs 1-4), The material was then adjusted to the required level of sodium chloride (Table 8) and 0.8/0.2μηι filtered before being immediately loaded onto the respective column. The HIC column was loaded at 5.5x10 ¹² particles/mL resin based on the theoretical titre as calculated using the Post TFF 1 titre (ELISA) and taking into account the total 2.5 x dilution factors applied through adjustment of the material. The reductive DEAE column was loaded at 0.81 mL/mL resin which equates to G.5mL Post TFF 1 material /mL resin when taking into account the total 1.61 x dilution factor applied through adjustment of the material. The phage material was collected as a single peak for the HIC runs and as 2mL fractions for the DEAE runs. A small proportion of the reductive DEAE fractions were combined to generate a pool sample for subsequent endotoxin analysis. Endotoxin levels were measured in the samples and the results from the HIC and DEAE runs are shown in Table 35. Table 35. Endotoxin reduction in HfC steps using detergent.

loading. Table 36. Endotoxin reduction in DEAE steps using detergent

loading.

[0176] The reductive DEAE step containing 0.1 % Triton was shown to be most effective for endotoxin removal with a 4.4 fold reduction compared to the control where only a 0.5 log was observed. Run 4 containing 1 %

Zwittergent 3-12 demonstrated a 1 .6 log reduction in endotoxin levels when a proportion of the flow though material was pooled (fractions A1 -A8). However a later fraction (A1 1 ) of the flow through material was shown to contain higher levels of endotoxin.

[0177] Based on the above results, the use of Fractogel EMD DEAE (M) with buffer containing 0.1 % Triton X-100 was investigated further (see below). Example 15 - Characterization of DEAE capacity to bind endotoxin

[0178] A new 10.9mL DEAE column (13,9cm bed height) was packed as per the manufacturer's instructions, A partially processed phage preparation was provided which had been taken through the TFF1 step in the order of Example 1 1 .

[0179] This material was adjusted to a final concentration of 0.1 % Triton X-100 and incubated for one hour at room temperature, with gentle mixing using a magnetic stirrer bar and platform. The sodium chloride concentration was adjusted to 300mM and the material 0.8/0.2 m filtered before being immediately loaded onto the column. The reductive DEAE column was loaded at 0.81 mL/mL resin which equates to 5mL Post TFF 1 material /mL resin when taking into account the total 1 ,61x dilution factor applied through adjustment of the material. The chromatography run was performed as per stage 4a DEAE runs 1 -4. Fractions were collected throughout the run at 3mL intervals. Selected fractions were submitted for endotoxin analysis; the results are shown in Table 37.

Table 37. Endotoxin content of Fractions From Reductive DEAE Column

[0180] The levels of endotoxin were observed to significantly increase above those observed at the start of column loading after fraction A5, corresponding to a loading of 0.77mL post TFF 1 material /mL media. Based on this result, it was expected that a loading capacity of 80% (i.e., 0.6 mL of Post TFF 1 material /mL media) or less would provide consistent and optimal reduction in endotoxin ieveis across this step for the reductive DEAE column.

Example 18 - Studies on Alternative Chromatographic Resins [0181 ] A partially processed phage preparation was provided which had been taken through the TFF1 step in the order of Example 10. This material was diluted with an equal volume of 4M NaC! buffer followed by 0.8/G.2pm filtration. An HIC Toyopear! Phenyl 650M Column (446mL CV, 22.8cm bed height in an XK50 column, new resin) was sanitised and equilibrated prior to use. The column was loaded at 5.5x10 ^{i 2} phage/mL resin based on a theoretical titre caicuiated using the post TFF1 titre (by EL!SA) and taking into account the 1 in 2 dilution performed, assuming no loss on the filtration step. The column was run as follows:

Flow rate: 97.5cm/hr (steps other than sample load)

Sample load flow rate: 48.7cm/hr

Column Equilibration -- 25mM Tris, 2M NaCI, pH 7.4

3CV Wash - 25mM Tris, 2 M NaCI, pH 7.4

3CV Eiution - 25mM Tris, 250mM NaCI, pH 7.4

[0 82] Post HIC material was diluted with 5 volumes of DEAE dilution buffer followed by 0.45/0.2pm filtration. A binding DEAE (Fractoge! EMD DEAE) Column (421.4mL CV, 21.5cm bed height in an XK 50 column, new resin) was sanitised and equilibrated prior to use. The column was loaded at 5x10 ¹² phage/mL resin based on a theoretical DEAE load titre calculated using the post HIC titre (by OD) and taking into account the 1 in 6 dilution performed, assuming no loss on the filtration step. The column was run as follows:

Flow rate: 97.46cm/hr (all steps)

Column Equilibration - 25mM phosphate, 1 G0mM NaCI, pH 8.5 2CV Wash - 25mM phosphate, 150mM NaCI, pH 6.5

4CV Wash - 25mM phosphate, 250mM NaCI, pH 6.5

3CV Eiution - 25mM phosphate, 300mM NaCI, pH 6.5

[0183] The post binding DEAE material thus produced was used to assess the efficacy of possible subsequent steps for further reduction of endotoxin levels. Unless otherwise indicated, for the columns described in the following paragraphs, the phage flow through peak was collected as 5mL fractions when A256 started at and dropped down to 5% of the peak maximum. [0184] Post binding DEAE material was loaded onto a reductive DEAE column based on a loading of 4mL/mL resin. The reductive DEAE column (17.08mL column volume, Fractogel EMD DEAE, 8.5cm bed in an XK 16 column, new resin) was sanitised and equilibrated prior to use.

[0185] Post binding DEAE material was loaded onto a reductive Q Sepharose XL column based on a loading of 4mL/mL resin. The reductive QXL column (14,67mL column volume, 7.3cm bed height in an XK 16 column, new resin) was sanitised and equilibrated prior to use.

[0186] 5 mL pre-packed EtoxiClear columns (Pro etic Biosciences Ltd., Rockvilie, Maryland) were sanitised and equilibrated in the appropriate buffer prior to use. A new EtoxiClear column was used for runs 1 , 2 and 3 and the column used for run 1 was re-used for both runs 4 and 5 with a

sanitisation step performed between runs. Post binding DEAE material was loaded without dilution for runs 1 and 4, diluted to give a final NaCI

concentration of 200 mM NaCI for runs 2 and 5, and diluted to give a final NaCI concentration of 100 mM for run 3. Runs 4-5, performed at pH 5.0, utilised post binding DEAE materia! that had been buffer exchanged via dialysis to reduce pH using snakeskin tubing (10kDa MWCO) at 2-8°C.

[0187] The columns were loaded at ~40mL post binding DEAE material / mL resin (see Table 38). The endotoxin loading was subsequently determined as 32,800EU/mL resin and 27,200EU/mL resin for runs 1 and 2 respectively and ~15 _s 000EU/mL resin for runs 4 and 5. The flow through unbound material was washed out with the appropriate equilibration buffer. The phage peak was collected as multipie fractions when A ₂ SB started at and dropped down to 20mAU. The fraction size was adjusted to account for the dilution in the load material (Table 38). The column load and run in 1 Q0mM NaCI (Run 3) showed partial binding of the phage material, which was eiuted from the column using 25mM phosphate, 300mM NaCI, pH 6.5.

Table 38. EtoxiC!ear Run Conditions.

[0188] The Endotoxin results for the reductive anion exchange (AEX) and EtoxiCiear runs are shown in Table 39. As the capacity for both the reductive AEX and EtoxiCiear for endotoxin was initially unknown, the flow through fractions were not pooled but selected fractions analysed separately for endotoxin and titre by OD to evaluate the performance of the column steps.

[0189] The reductive AEX (DEAE and QXL) showed less than 1 log reduction in endotoxin when comparing the endotoxin levels (EU/mL) in the load materia! to the flow through fractions analysed. [0190] The EtoxiClear chromatography performed in the presence of 200-300mM NaCI (Runs 1 , 2, 3 and 4) demonstrated an approximate 3.4-4.9 log reduction in endotoxin comparing endotoxin levels (EU/mL) in the load material to the flow through fractions analysed. The titre measurements indicated that there was no significant loss in yield over the EtoxICiear step for runs 1 , 2, 4 and 5.

[0191] Thus, the screen of the EtoxiClear resin demonstrated promising results for significant reductions in endotoxin levels and was selected for further investigation. It was noted that performing the EtoxiClear chromatography in 25mM phosphate, 300mM NaCI, pH 8.5 could be carried out following the DEAE chromatography step without an additional buffer exchange step.

Table 39. Endotoxin Results for Reductive AEX and EtoxICiear Steps

E luted

Example 17 - Studies on Alternative Combinations of Purification Steps

[0192] A partiaiiy processed phage preparation ("post-TFF1 material") was provided which had been taken through the TFF 1 step in the order of Example 1 1. Three combinations of purification steps were performed and the level of endotoxin removal was evaluated.

[0193] in the first combination of steps (Run 1 in Table 40 below), Triton X-100 was added to the post-TFF1 material to a final concentration of 0.1 % and NaC! was added to a final concentration of 300 mM. After addition of Triton X-100 and NaCl, the material was incubated for 1 hour followed by 0.45/0.2pm filtration (2 x Sartopore 2 150 filters). Reductive DEAE chromatography was performed using 25mM Tris, 300 mM NaC!, pH7.4 as the buffer conditions on a Fractoge! EMD DEAE column (415mL column volume (CV), 21 .17cm bed height in a XK50 column (new resin)) which was sanitized and equilibrated prior to use. The post filtered, NaCI and Triton X- 100 adjusted material was loaded onto the DEAE column based on a loading of 0.5mL Post TFF 1 material/mL resin (taking into account the total dilution factor of x1.31 following adjustment to generate the load material). The phage peak was collected as a single pool when A ₂₅₄ started at and dropped down to 20mAU. Samples that required storage at -85°C were snap frozen with liquid nitrogen and stored at <-65°C at the end of the processing day. All other samples and bulk material were held at 2-8°C.

[0194] The flowthrough containing phage was diluted with 5 volumes of 25 mM phosphate, pH 6.5 followed by 0.45/0.2 m filtration (1 x Sartopore 2 300 filter). This material was then loaded on a binding DEAE chromatography column (Fractoge! EMD DEAE, 229mL CV, 1 1.68cm bed height in a XK50 column (new resin)) at 5x10 ¹² phage/mL resin based on a theoretical tifre calculated using the post TFF1 titre as determined by ELISA, taking into account the dilution of materia! through adjustment and also the increase in volume over the reductive DEAE step and assuming a 90% step yield for the reductive DEAE step. A sample of the DEAE load was taken and analysed retrospectively for titre as determined by ELISA. The binding DEAE column loading was retrospectively determined as 1.8 x 1 Q ' ² particles/mL resin by ELISA. The binding DEAE column was washed with buffer containing 250 mM NaCI and eiuted with 25 mM phosphate, 300 mM NaCI, pH 8.5. The post binding DEAE material was analysed on-line (the same day) for endotoxin and determined to be at 1.17 x 10 ³ EU/mL and then passed through a 5 mL new, pre-packed, santised, equilibrated EtoxiClear column (without

adjustment of buffer) at 10000 EU/mL resin based on the on-line

measurement. A second sample of DEAE Pool material sampled the following day and termed EtoxiClear load was analysed retrospectively for endotoxin as 931 EU/mL giving a column loading of 7960EU/mi_ resin. The difference in column loading determination between the two results is likely to be due to the variation of the assay. The phage product was loaded onto the column and collected as the flow through fraction when the A254 increased to 20mAU and dropped back down to 20mAU following a wash step with equilibration buffer. The fractions collected were pooled at the end of the run. Samples requiring snap freezing were performed at the end of the processing day with liquid nitrogen and stored at <-65 . Remaining samples were held at 2-8°C.

[0195] In the second combination of steps (Run 2 in Table 40 below), the post-TFF1 materia! was diluted 1 :1 with 25mM Tris, 4 NaCI, pH 7.4 followed by 0.45/0.2 m filtration (1 x Sartopore 2 150), and then loaded on a sanitised, equilibrated HIC column (Toyopearl Phenyl 650M, 440mL CV, 22.4cm bed height in an XK50 column, resin used for one cycle previously). The material was e!uted with 2SmM Tris, 250mM NaCI, pH 7.4. The phage peak was collected as a single pool when A254 started at and dropped down to 20rnAU. It was observed that the phage peak began to elute shortly after the conductivity of the eluafe began to drop, resulting in an NaCI concentration greater than 250 mM. Samples requiring snap freezing were performed at the end of the processing day with liquid nitrogen and stored at <-65°C,

Remaining samples were held at 2-8°C. [0196] Triton X-100 was added to a final concentration of 0,1 % and NaCI was added to a calculated final concentration of 300 mM based on the assumption that the eluate from the previous step contained NaCI at 250 mM. The material was then back-diluted 2.5 fold with 25 mM Tris pH 7.4, giving a conductivity matching the column equilibration buffer for the next column (29.1 mS). Additional Triton X-100 was added as well to maintain a 0.1 %

concentration. This material was incubated for 1 hour followed by 0.45/0.2 m filtration (1 x Sartopore 2 150).

[0197] Reductive DEAE chromatography was performed using 25mlv! Tris, 300 mM NaCI, pH7.4 as the buffer conditions; a reductive DEAE column (Fractogei EMD DEAE, 80mL column volume (CV), 15cm bed height in a XK28 column (new resin)) was loaded at 3.09 mL post-HIC material per mL resin (calculated taking into account the total dilution factor of x2.9 for the dilution and adjustment steps). The flow through phage material was collected as a single pool when A25 started at and washed down to 20mAU with equilibration buffer. Samples requiring snap freezing were performed at the end of the processing day with liquid nitrogen and stored at <-85°C.

Remaining samples were held at 2-8°C.

[0 98] The post-reductive DEAE material was diluted with 5 volumes of 25 mM phosphate, pH 6.5, followed by 0.45/0.2pm filtration (1 x Sartopore 2 300). This diluted material was then loaded on a sanitised, equilibrated binding DEAE chromatography column (Fractogei EMD DEAE, 372mL CV, 19cm bed height in an XK50 column (new resin)), washed with buffer containing 250 mM NaCi, and eluted with 25 mM phosphate, 300 mM NaCi, pH 6.5. The loading of the binding DEAE step could not be determined by OD due to the presence of Triton-XI OO in the load sample and the low

concentration at this point. Therefore the column loading was based on a theoretical titre calculated using the titre of the post HIC material as

determined by OD and an assumption of a 90% reductive DEAE step yield whilst taking into account the material adjustment / dilution steps and the volume increase over the reductive DEAE step. Using this theoretical titre the column was loaded at 5x10 ¹² phage/mL resin. The actual binding DEAE column loading was retrospectively determined as 4.4 x 10 ^{i 2} particles/mL resin by ELISA. The phage peak was collected as a single pool when A254 started at and dropped down to 2QmAU. Samples requiring snap freezing were performed at the end of the processing day with liquid nitrogen and stored at <-65°C. Remaining samples were held at 2-8°C.

[0199] The post-reductive DEAE material was analysed on-line (the same day) for endotoxin and determined to be at 1.57EU/m!_. As the on-line endotoxin level was determined to be significantly lower than runs 1 and 3, the column could not be loaded at 10000EU/mL resin. Therefore, all of the available post binding DEAE materia! was loaded onto the column to give a column loading of 63EU/mL resin, then passed through a 5 mL pre-packed, sanitised, equilibrated EtoxiClear column (used previously for 1 cycle). Phage product was collected as the flow through fraction when the A254 increased to 20mAU and dropped back down to 20mAU following a wash step with equilibration buffer (25mM phosphate, 3Q0mM NaCL pH 6.5).

[0200] In the third combination of steps (Run 3 in Table 40 below), post-HIC material generated from Run 2 was used. It was diluted with 5 volumes of dilution buffer followed by 0.45/0.2μηι filtration (1 x Sartopore 2 150). Post filtration material was loaded onto the binding DEAE column at 5x10 ' ² pbage/mL resin based on a theoretical binding DEAE load litre calculated using the post HIC pool titre as determined by OD and taking into account the 1 in 6 dilution of the binding DEAE load material, assuming no loss on filtration. The binding DEAE column loading was retrospectively determined as 5.3 x 10 ¹² particles / mL resin by ELISA.

[0201] The binding DEAE column (Fractogel EMD DEAE, 34mL CV, 16.9cm bed height in an XK16 column (new resin)) was sanitised and equilibrated prior to use. The material was loaded onto the column and a wash step performed using wash buffer containing 250 m NaCI, before the phage was eluted with 300mM NaCI. The phage peak was collected as a single pool when A254 started at and dropped down to 20mAU. Samples requiring snap freezing were performed at the end of the processing day with liquid nitrogen and stored at <-85°C. Remaining samples were held at 2-S°C.

[0202] The post binding DEAE material was analysed on -line (the same day) for endotoxin and determined to be at 1.34E+4EU/mL The column was loaded at 0720EU/mL resin based on the on-line endotoxin data. A second sample of DEAE Pool material sampled the following day and termed EtoxiCiear load was analysed retrospectively for endotoxin as 7.03E+3EU/mL giving a column loading of 5624EU/mL resin.

[0203] An EtoxiCiear column (5mL pre-packed new column) was sanitised and equilibrated prior to use. The phage material was loaded onto the column and collected as the flow through fraction when the A ₂ s ₄ increased to 20mAU and dropped back down to 2GmAU following a wash step with equilibration buffer (25mM phosphate, 300mfv1 NaCI, pH 6.5). Samples requiring snap freezing were performed at the end of the processing day with liquid nitrogen and stored at <-65 ^a C. Remaining samples were held at 2~8°C.

[0204] General Notes Regarding Runs 1 -3 of this Example: Analysis of post EtoxiClear material for the three process runs showed that there was no residua! Benzonase detected and the infectivity as determined by plaque assay was comparable between runs (5.1 -8,3 x 10 pftj/mL), within the error of the assay. There is a known inherent variation for the EL!SA assay as the assay is non-specific. The ELiSA assay uses a commercial G3 protein capture antibody which actually binds the G8 protein.

[0205] Results from Runs 1 , 2, and 3 are shown in the following tables.

Table 40. Endotoxin Analysis for Runs 1 , 2, and 3.

Table 41. Host C&U Protein Analysis for Runs 1 , 2, and 3. Tabte 42, Titre and Ste Yield Data for Runs 1 2 and 3 ELISA and OD .

[0206] Based on these results, the binding DEAE step was shown to give the greatest reduction in HCP levels, which appeared to be more effective when Triton X-100 was present in the load materia! for this column (runs 1 and 2 compared to run 3 without detergent). Process runs 2 and 3 with the inclusion of the H!C step were shown to generate post EtoxiClear material with the iowest levels of HCP at 1 .5-1.8ng/mL (Tabie 41 ) which standardised to 1 x10 ¹⁴ partic!es gives 95 and 70ng/1x10 ¹⁴ particles for runs 2 and 3 respectively (Table 42). The best performing steps for endotoxin reduction were indicated to be the binding DEAE when performed following a HIC step and with Triton X-100 present in the load material (run 2, 5.78 log reduction (Table 40)) and the EtoxiClear step with 2.7 to 5.7 log reduction (runs 1 -3). The post EtoxiClear material from runs 2 and 3 achieved endotoxin levels of <0.01 EU/mL which standardised to 1x10 ¹⁴ particles gives <0.5EU/1x10 ¹⁴ particles.

Example 18. Additional Purification Protocols

[0207] The following protocol for purifying filamentous bacteriophage are also within the methods according to the invention. It is understood that one skilled in the art would carry out filtration of material, and sanitization and equilibration of columns at appropriate times.

[0208] According to Run 3b, post-TFF1 material is provided and diluted 1 :1 with 25miV1 Tris, 4M NaCI, pH 7.4. HIC chromatography is then performed with elution using 25mM Tris, 250m NaCI, pH 7.4. The post-HIC material is then diluted with 5 volumes of 25mM phosphate, pH 8.5. Next, the diluted material is subjected to binding DEAE chromatography with a wash step followed by elution at 25mM phosphate, 300mM NaCI, pH 6.5. The post- binding DEAE material is then passed through an EtoxiClear column, also using 25mM phosphate, 300mM NaCI, pH 6.5.

[0209] According to Run 4, post-TFF1 material is provided and diluted 1 : 1 with 25mM Tris, 4M NaCI, pH 7.4. HIC chromatography is then performed with elution using 25mM Tris, 250mM NaCI, pH 7.4. The post-HIC material is then diluted with 5 volumes of 0.12% Triton-X100, 25mM phosphate, pH 6.5 (such that the diluted material contains 0.1 % Triton X-100). Next, the diluted material is subjected to binding DEAE chromatography with a wash step followed by elution at 25mM phosphate, SOOmlvl NaCI, pH 8.5. The post-binding DEAE materia! is then passed through an EtoxiClear column, also using 25mM phosphate, 300m NaCI, pH 8.5. - *

[0210] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.