WO2014048407A2 - Medicinal preparation in the form of antistaphylococcal phage lysate, process of its preparation and use - Google Patents

Medicinal preparation in the form of antistaphylococcal phage lysate, process of its preparation and use Download PDF

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Publication number
WO2014048407A2
WO2014048407A2 PCT/CZ2013/000117 CZ2013000117W WO2014048407A2 WO 2014048407 A2 WO2014048407 A2 WO 2014048407A2 CZ 2013000117 W CZ2013000117 W CZ 2013000117W WO 2014048407 A2 WO2014048407 A2 WO 2014048407A2
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phage
lysate
preparation
medicinal product
production
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PCT/CZ2013/000117
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French (fr)
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WO2014048407A3 (en
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Marek MOŠA
Josef BOŠTIK
Roman PANTŬČEK
Jiri DOŠKAŘ
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Imuna S.R.O.
Masarykova Univerzita
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Publication of WO2014048407A2 publication Critical patent/WO2014048407A2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/02—Recovery or purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00051—Methods of production or purification of viral material
    • C12N2795/00052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10111—Myoviridae
    • C12N2795/10133—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory

Definitions

  • the invention relates to a medicinal product with the active ingredient in a form of antistaphylococcal phage lysate containing high-virulent and specifically effective phage particles which have a very fast, strong and polyvalent lytic effect on the agents of staphylococcal infections.
  • Cell components occurring after destruction of bacterial cells during lysis after release of phage virions can also have an immune-modulating effect and stimulate of non-specific immunity.
  • the invention also includes the production method of the medicinal product and its use.
  • Infectious diseases caused by pathogenic bacteria are worldwide one of the main causes of human death.
  • the most often occurring pathogenic bacteria include staphylococci triggering a number of diseases, from common skin infections through serious organ disorders like osteomyelitis, endocarditis and pneumonia to severe sepses ending with patient's death (individuals with weakened immunity, after tumour treatment, with implants, elderly persons, cystic fibrosis) .
  • Staphylococcus aureus which belongs to the main agents of community and hospital (nosocomial) infections, has a special significance.
  • a serious problem which complicates treatment of patients is occurrence and spreading of species resistant to antimicrobial products (particularly antibiotics) as a result of irrational use of these substances for treatment of often banal diseases.
  • phage K The best-studied phages of the group include phage K, the complete genome sequence of which is known (0' Flaherty et al. 2004, J. Bacteriol. 186: 2862-2871, Kwan et al. 2005, Proc. Natl. Acad. Sci. USA 102:5174-5179) .
  • phage 812 which is distinguished by a wide range of hosts within the Staphylococcus genus.
  • the principle of the invention is antistaphylococcal phage lysate for local use; it is exclusively intended for local application in infections caused by staphylococcal strains. It can be used both in human and veterinary medicine for all forms of staphylococcal infections. It serves for destruction of staphylococcal cells in the area of current infection.
  • Bacteriophage DSM 26144 (a mixture of several subtypes of a polyvalent bacteriophage, isolated via a genetic selection in clinical isolates of Staphylococcus aureus)
  • Taxonomic classification Order Caudovirales, family Myoviridae, genus SPOl-like viruses
  • Type of nucleic acid linear double stranded DNA without cohesive ends; size 138.7 kb; molar content of G+C 30.42 %; genetic diversity of subtypes 0.2 % of the DNA sequence includes 27 sequence variations (short insertions, deletions and substitutions) and 287 point mutations described in the sequence in a form of degenerated bases according to the IUPAC nomenclature.
  • Host range strains of various species of the Staphylococcus genus, in particular Staphylococcus aureus
  • Life-cycle entirely virulent bacterial virus which does not undergo a lysogenic cycle; in the DNA sequence no genes for intergrases or antirepressors which are typical of temperate bacteriophages.
  • the virion structure Head and tail with contractile sheath, basal plate with spikes, head size 80 - 100 nm, tail length 200 - 250 nm (an electron-microscope image of the bacteriophage enclosed) .
  • Staphylococcus aureus strain for bacteriophage propagation Identification Staphylococcus aureus CCM 8428
  • Taxonomic classification Bacteria; Firmicutes; Bacilli; Bacillales; Staphylococcaceae
  • Phenotype characteristics G+ cocci, diameter 0.8-1 um, single, pairs, clusters, clumps. On blood agar or tryptone agar they produce colonies of 1-2 mm, smooth edges, smooth surface, convex, beige colour, opaque. On blood agar - production of haemolysin alpha and beta. Clumping- factor positive, hyaluronidase positive. The RPLA method has not demonstrated production of enterotoxins SEA - SEE, TSST-1, ETA and ETB. Sensitivity to bacteriophages: high-sensitive to temperate bacteriophages 29, 52, 52A, 79, 80, 6, 42E, 47, 53, 54, 75, 77, 83A, 84, 85, 81 and 95.
  • Genotype characteristics The strain is without prophages, without plasmids, the PCR method has not demonstrated genes coding for enterotoxins ⁇ sea - see, seh - sen, sep - seu) and genes for PVL, TSST-1, ETA, ETB, ETD; in addition, antibiotic resistance genes mecA, blaZ, tetK, tetM, ermA, ermB and ermC has not been proven.
  • the starting material for production of lysate are subtypes of a polyvalent bacteriophage which are kept in a lyophilized state while keeping the system of uniform inoculation (the seed lot system) .
  • the bacteriophages are propagated on a non-pathogenic propagation strain of Staphylococcus aureus till a high titre (10 8 - 10 9 PFU/ml) is achieved, and processed to the final product whereas a PFU unit (Plague forming units) is a measure of the amount of infectious viral particles.
  • staphylococcal cells from infection foci (suppurative processes of skin, subcutis and skin adnexa) and potential reservoirs (in particular rhinopharynx, intestinal and urinary tracts) . It is a significant means in a complex treatment of chronic forms of staphylococcal infections (suppurative affections, abscesses, fistulas, infections of deep soft tissues) which prevents them from a septic state. It is also an important part of preventive measures in pre-operative preparation in order to prevent occurrence of superposed pyogenic complications after surgical interventions.
  • An alternative use of the product is an application in preserving and storing of food, in production of feed for farm animals and use in decontamination of surfaces (hospital environment, medical aids) .
  • the bacterial host culture is incubated in a liquid tryptone medium.
  • MI multiplicity of infection
  • the active component is prepared as a mixture of high potent virulent phage particles with a polyvalent effect prepared by incubation . of bacteriophage subtypes and standardized in its efficiency according to concentrations of specified phage particles resulting in 1 ml.
  • lyophilized strain is re-inoculated to 1.5 % tryptone agar and incubated 24 hours at +37 °C + 1 °C.
  • the grown-up bacterial strain is re-inoculated to the required number of plates with 1.5 % tryptone agar and incubated for 24 hours at + 37
  • the grown-up culture is used for inoculation of liquid tryptone medium.
  • In-process control Cultivation and microscopic checking of the strain purity.
  • Bacterial culture is re-inoculated from the solid medium to an Erlenmayer flask with 100 ml of tryptone broth and incubated for 24 hours at + 37 °C ⁇ 1 °C.
  • the number of inoculated Erlenmayer flasks depends on the required number of subcultures for the production (1 - 3 pc) .
  • In-process control Microscopic checking of subculture purity.
  • the resuspended phage strain is revived at first and propagated via a method of double-layer agar and incubated in a liquid medium.
  • Petri dishes with 1.5 % tryptone agar which act as a base are overlayed with 3 ml of 0.36 % tryptone agar which contains bacterial suspension of the appropriate host bacterium Staphylococcus aureus and suspension of bacteriophage obtained by re-suspending of the lyophilized phage
  • In-process control Determination of the number of phage particles. . Preparation in liquid medium
  • the subculture of the host bacteria Staphylococcus aureus is used for inoculation of a tryptone broth. Incubation proceeds in a thermostat at +37 °C ⁇ 1 °C until optical density of biomass is 0.3 - 0.4 (measured in a spectrophotometer at wavelength 500 nm) .
  • Into the staphylococcal suspension 15 - 20 ml of the phage lysate is added (prepared according to the previous step, with the min. titre 1 x 10 10 PFU/ml) .
  • the bacteriophage is propagated at a laboratory temperature for 18 - 20 hours while being agitated continuously. Centrifugation for 1 hour at 5000 RPM follows. The bacteriophage is contained in the supernatant .
  • In-process control Checking of the bacterial suspension density and determination of the number of phage particles of the phage starter during bacteriolysis.
  • Bacterial suspension is inoculated from Erlenmayer flasks into 3000 ml of tryptone broth in a 5L bottle.
  • Incubation at + 37 °C ⁇ 1°C proceeds till the required density is achieved (optical density 0.25 - 0.35) .
  • Duration of incubation usually ranges from 4 to 5 hours. The first sample for the check of density is taken after 3 hours of incubation; other samples are taken depending on bacterial growth.
  • In-process control Operation checking is performed during incubation by measuring of density, and via a microscopic check upon the end of incubatio .
  • phage starter of min. titre 1 x 10 10 PFU/ml is added in a volume of 50 - 60 ml.
  • the starter is added aseptically at the moment when the value of optical density of the sample is within the interval (0.25 - 0.35 ) .
  • Bacteriolysis proceeds at a room temperature for 20 - 30 hours. In the course of bacteriolysis the liquid culture clarifies. Optical density of lysed bacterial suspension must fall under the value of 0.1.
  • In-process control The operation check performs measuring of density.
  • the prepared tryptone agar and tryptone broth are sterilised in a steam sterilizer at 121 °C for 30 minutes.
  • Most of sensitive strains of Staphylococcus aureus are classified in clonal lineages STl, ST5, ST8, ST20, ST22, ST30, ST36, ST111, ST154, ST225, ST228, ST247, ST624.
  • genotypes and characteristics of staphylococcal strains undergo relatively fast evolution, and in the future occurrence of insensitive strains can be expected.
  • Subtypes of bacteriophage with a wider spectrum of the host are isolated from plaques grown up after plating of the starting phage on insensitive bacterial strains of Staphylococcus aureus. Isolation of new efficient subtypes is performed by plating of phage lysates of a high titre onto growth of resistant strains. Single plaques that grow on these strains with a very low frequency (1CT 9 - 1Q ⁇ 6 ) are then used for initiation of lysates containing mutant phages. In order to achieve a wide lytic spectrum, the selection can be made in several stages during which the phage mutants obtained in one resistant strain are subsequently plated on other (different) resistant strains.
  • the final product is lyophilized powder for preparation of solution (lyophilizate) produced by sterile filtration and lyophilisation of a mixture of antistaphylococcal phage lysate containing active phage particles of a polyvalent staphylococcal bacteriophage.
  • a medicinal product for local use contains high-potent virulent phage particles with a polyvalent effect. It is standardized in its efficiency according to concentration of specific phage particles after re-constitution of the powder for preparation of solution in a solvent (water for injection) for 1 ml.
  • ⁇ therapeutic dose contains minimum 10 6 particles.
  • Production of a batch of the final product follows continuously the production of phage lysates of particular subtypes of the phage strain.
  • a batch of the final product is made by mixing of the equal amount of phage lysates of the particular subtypes of the bacteriophage; the prepared mixture is sterile-filtered, filled in vials and lyophilized.
  • vials with the finished product are closed with a stopper secured by crimp.
  • a mixture of lysates after sampling for microbial burden (100 ml) is filtered through a filtration layer of porosity 0.22 um.
  • Filtered lysate is filled out by 10 ml in PNC 20 ml vials.
  • In-process control checking of the filled volume using a calibrated graduated cylinder.
  • Filled vials covered with a lyophilisation stopper are lyophilized at vacuum from deep freezing (-45 °C) by heating on plates to the specified temperature (+30 °C) .
  • Phase I Freezing of plates down to -50 °C, putting on material, freezing of material till temperature of the plates and material is stabilized at the same value (duration of the phase is 1 hour)
  • Phase II The freezing temperature of plates is maintained at -50 °C and the vacuum pump is heated (duration of the phase is 30 minutes)
  • Phase III Main drying - evacuation of the drying space to the set up vacuum (30-80 Pa) .
  • pressure value When the pressure value is achieved, heating of plates adjusted at the temperature of +30 °C according to the time regime starts. This phase of the lyophilisation process takes app 90 % of the total time and lasts for approx. 24 hours.
  • Phase IV Final drying at the maximum vacuum (2 - 0.5 Pa) and the maximum temperature +30 °C. If the product temperature is consistent with the temperature of heating plates and values of vacuum do not change in the test of the end of drying, lyophilization is finished by stopping vacuum.
  • stoppers are secured by crimps.
  • Fig. 1 illustrates a flow-chart of preparation of phage lysate
  • Fig. 2 illustrates a flow-chart of production of the final product
  • the method of production of a medicinal product in a form of staphylococcal phage lysate includes the following successive steps:
  • the step of revival of a bacterial strain includes inoculation of thawed lyophilized bacterial strain onto 1.5 % agar and following incubation for 24 hours at +37 °C ⁇ 1 °C .
  • the step of passaging of the bacterial strain includes re-inoculation of the grown-up bacterial strain onto the required number of plates with 1.5 % tryptone agar and its incubation for 24 hours at + 37 °C ⁇ 1 °C.
  • the step of preparation of cell sub-cultures includes re-inoculation of the bacterial culture from the solid medium to an Erlenmayer flask with 100 ml of tryptone medium whereas the amount of inoculated Erlenmayer flasks depends on the required number of sub-cultures for production and incubation lasts for 24 hours at + 37 °C ⁇ 1 °C.
  • the step of preparation of the phage starter via a double-layer agar method includes revival of the resuspended phage strain and its propagation on Petri dishes with 1.5 % tryptone agar which serve as the base which is overlayed with 3 ml of 0.36 % tryptone agar which contains bacterial suspension of the appropriate host bacterium Staphylococcus aureus and suspension of bacteriophage obtained by re- suspending of the lyophilized phage strain in an isotonic solution. Incubation for 24 h at +37 °C ⁇ 1 °C follows. Then surface of each plate is overlayed with tryptone broth liquid medium. After 30 minutes the soft surface with 0.36 % tryptone soft agar is washed off and centrifuged 3 x 1 hour at 5000 RPM. Bacteriophage is contained in the supernatant .
  • the step of preparation of the phage starter in a liquid medium includes inoculation of tryptone medium with a subculture of the host bacterium Staphylococcus aureus; incubation proceeds in a thermostat at +37 °C ⁇ 1 °C until optical density of biomass is 0.3 - 0.4, measured in a spectrophotometer at wavelength 500 ran; into the staphylococcal suspension 15 - 20 ml of the phage lysate is added (prepared according to the previous step) , with the min. titre 1 x 10 10 PFU/ml) ; bacteriophage is propagated at a laboratory temperature for 18 - 20 hours while being agitated continuously; centrifugation for 1 hour at 5000 RPM follows. The bacteriophage is contained in the supernatant .
  • the step of preparation of bacterial suspension includes inoculation of bacterial suspension from Erlenmayer flasks into 3000 ml of tryptone broth in a 51 bottle, while incubation at + 37 °C ⁇ 1°C proceeds till the required density 0.25 - 0.35 is achieved; duration of incubation usually ranges from 4 to 5 hours; the first sample for the check of density is taken after 3 hours of incubation and other samples are taken depending on bacterial growth.
  • the step of preparation of phage lysate or a mixture of phage lysates includes addition of the phage starter of min. titre 1 x 10 10 PFU/ml in an amount of the volume 50 - 60 ml to the bacterial suspension, while the starter is added aseptically at the moment when the value of optical density of the sample is within the interval 0.25 - 0.35; bacteriolysis proceeds at a room temperature for 20 - 30 hours; in the course of bacteriolysis the liquid culture clarifies and optical density of lysed bacterial suspension must fall under the value of 0.1.
  • the phage lysate or lysate mixture is filtered through a filtration layer of porosity 0.22 um whereas microbial burden of the product corresponds to max. 10 PFU/100 ml.
  • Filtered lysate is filled by 10 ml in PNC 20ml vials.
  • Vials with lysate of a mixture of lysates covered with a lyophilisation stopper are lyophilized at vacuum from deep freezing (- 45 °C) by heating on plates to the specified temperature (+30 °C) , while the lyophilisation step consists of the following phases, and when lyophilisation is finished, stoppers are secured by crimps:
  • Phase I Freezing of plates down to -50 °C, putting on material, freezing of material till temperature of the plates and material is stabilized at the same value (duration of the phase is 1 hour)
  • Phase II The freezing temperature of plates is maintained at -50 °C and the vacuum pump is heated (duration of the phase is 30 minutes)
  • Phase III Main drying - evacuation of the drying space to the set up vacuum (30-80 Pa) .
  • Heating of plates adjusted at the temperature of +30 °C according to the time regime starts.
  • This phase of the lyophilisation process takes approx. 90 % of the total time and lasts for approx. 24 hours.
  • Phase IV Final drying at the maximum vacuum (2 - 0.5 Pa) and the maximum temperature +30 °C. If the product temperature is consistent with the temperature of heating plates and values of vacuum do not change in the test of the end of drying, lyophilisation is finished by stopping vacuum.
  • tryptone broth consisting of a set of substances containing Tryptone Oxoid, Yeast extract Oxoid, sodium chloride and purified water was used; and tryptone agar consisting of a combination of tryptone broth and Agar Oxoid W 1 was used.
  • the prepared tryptone agar and tryptone broth were sterilized in a steam sterilizer at the temperature 121 °C for 30 minutes.
  • a medicinal product in a form of antistaphylococcal phage lysate containing active phage particles of polyvalent staphylococcal bacteriophage prepared via the method according to Example 1.
  • One therapeutic dose contains minimum 10 6 particles of bacteriophage.
  • the invention is utilisable in the field of both human and veterinary medicine in all forms of staphylococcal infections. It serves for destruction of staphylococcal cells in the spot of a developing infection.

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Abstract

A process of production of a medicinal product in the form of antistaphylococcal phage lysate, including the following successive steps: revival of a bacterial strain, passaging of the bacterial strain, preparation of cell sub-cultures, preparation of the phage starter via a double-layer agar method, preparation of the phage starter in a liquid medium, preparation of bacterial suspension, preparation of phage lysate, filtration of phage lysate or a lysate mixture, filling of filtered lysate or a lysate mixture in vials, lyophilisation of lysate or a lysate mixture in vials; whereas the final medicinal product is lyophilized powder for preparation of solution (lyophilisate) produced via sterile filtration and lyophilisation of a mixture of antistaphylococcal phage lysate containing active phage particles of a polyvalent staphylococcal bacteriophage. The medicinal product for local use contains high-potent virulent phage particles with a polyvalent effect.

Description

Medicinal preparation in the form of antistaphylococcal phage lysate, process of its preparation and use
Technical Field
The invention relates to a medicinal product with the active ingredient in a form of antistaphylococcal phage lysate containing high-virulent and specifically effective phage particles which have a very fast, strong and polyvalent lytic effect on the agents of staphylococcal infections. Cell components occurring after destruction of bacterial cells during lysis after release of phage virions can also have an immune-modulating effect and stimulate of non-specific immunity. The invention also includes the production method of the medicinal product and its use.
Background Art
Infectious diseases caused by pathogenic bacteria are worldwide one of the main causes of human death. The most often occurring pathogenic bacteria include staphylococci triggering a number of diseases, from common skin infections through serious organ disorders like osteomyelitis, endocarditis and pneumonia to severe sepses ending with patient's death (individuals with weakened immunity, after tumour treatment, with implants, elderly persons, cystic fibrosis) . Staphylococcus aureus which belongs to the main agents of community and hospital (nosocomial) infections, has a special significance. A serious problem which complicates treatment of patients is occurrence and spreading of species resistant to antimicrobial products (particularly antibiotics) as a result of irrational use of these substances for treatment of often banal diseases. The problem of the increasing number of bacterial strains resistant to antibiotics can be resolved by introducing a rational phage therapy, i.e. use of bacteriophages for treatment of bacterial infections as an alternative to or replacement of the antibiotic treatment, particularly as it is time-consuming and financially difficult to develop new efficient antibiotics (Gill and Hyman 2010, Curr. Pharm. Biotechnol. 11:2-14). Polyvalent staphylococcal bacteriophages of the Myoviridae family have become the point of interest with regard to their potential practical use in phage therapy which represents a suitable alternative to the antibiotic treatment of staphylococcal infections (Mann 2008, Res. Microbiol. 59:400-405) . The best-studied phages of the group include phage K, the complete genome sequence of which is known (0' Flaherty et al. 2004, J. Bacteriol. 186: 2862-2871, Kwan et al. 2005, Proc. Natl. Acad. Sci. USA 102:5174-5179) . In our earlier work (Pantucek et al. 1998, Virology 246:241-252) we characterized in detail phage 812 which is distinguished by a wide range of hosts within the Staphylococcus genus. By testing the lytic effect of phage 812 and its mutants on a wide number of S. aureus strain it was found out that 95 % of strains are sensitive to at least one host range mutant of polyvalent phage 812. Studying the restriction-deficient mutants of S. aureus implied that resistance of the S. aureus strains to polyvalent phage 812 is conditioned by specific restriction-modification systems (Doskar 1989, Scripta Fac. Sci. Nat. Univ. Purk. Brun. 19:391-401) .
Disclosure of the Invention
The principle of the invention is antistaphylococcal phage lysate for local use; it is exclusively intended for local application in infections caused by staphylococcal strains. It can be used both in human and veterinary medicine for all forms of staphylococcal infections. It serves for destruction of staphylococcal cells in the area of current infection.
A description of the biological material is as follows. Polyvalent staphylococcal bacteriophage
Identification: Bacteriophage DSM 26144 (a mixture of several subtypes of a polyvalent bacteriophage, isolated via a genetic selection in clinical isolates of Staphylococcus aureus)
Taxonomic classification: Order Caudovirales, family Myoviridae, genus SPOl-like viruses Type of nucleic acid: linear double stranded DNA without cohesive ends; size 138.7 kb; molar content of G+C 30.42 %; genetic diversity of subtypes 0.2 % of the DNA sequence includes 27 sequence variations (short insertions, deletions and substitutions) and 287 point mutations described in the sequence in a form of degenerated bases according to the IUPAC nomenclature.
Genome sequence: enclosed on CD
Host range: strains of various species of the Staphylococcus genus, in particular Staphylococcus aureus
Life-cycle: entirely virulent bacterial virus which does not undergo a lysogenic cycle; in the DNA sequence no genes for intergrases or antirepressors which are typical of temperate bacteriophages.
The virion structure: Head and tail with contractile sheath, basal plate with spikes, head size 80 - 100 nm, tail length 200 - 250 nm (an electron-microscope image of the bacteriophage enclosed) .
Deposition in collection: The biological material has been deposited in compliance with the Budapest Agreement in DSMZ-Deutsc e Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrafle 7B, 38124
Braunschweig, Germany with the priority of 03/07/2012 (the number in the database: DSM 26144)
Staphylococcus aureus strain for bacteriophage propagation Identification: Staphylococcus aureus CCM 8428
Taxonomic classification: Bacteria; Firmicutes; Bacilli; Bacillales; Staphylococcaceae
Concept of genome sequence: a genome sequence 2446 kb long, representing 87 % of the total length of genome DNA and it is enclosed on CD in the form of contigs
Clonal classification: sequence type ST30, spa-type t021
Phenotype characteristics: G+ cocci, diameter 0.8-1 um, single, pairs, clusters, clumps. On blood agar or tryptone agar they produce colonies of 1-2 mm, smooth edges, smooth surface, convex, beige colour, opaque. On blood agar - production of haemolysin alpha and beta. Clumping- factor positive, hyaluronidase positive. The RPLA method has not demonstrated production of enterotoxins SEA - SEE, TSST-1, ETA and ETB. Sensitivity to bacteriophages: high-sensitive to temperate bacteriophages 29, 52, 52A, 79, 80, 6, 42E, 47, 53, 54, 75, 77, 83A, 84, 85, 81 and 95.
Genotype characteristics: The strain is without prophages, without plasmids, the PCR method has not demonstrated genes coding for enterotoxins {sea - see, seh - sen, sep - seu) and genes for PVL, TSST-1, ETA, ETB, ETD; in addition, antibiotic resistance genes mecA, blaZ, tetK, tetM, ermA, ermB and ermC has not been proven.
Depositing in a Collection: The biological material has been deposited in compliance with the Budapest Agreement in CC -Czech Collection of Microorganisms with the priority of 21/06/2012 (the number in the database CCM 8428) .
The starting material for production of lysate are subtypes of a polyvalent bacteriophage which are kept in a lyophilized state while keeping the system of uniform inoculation (the seed lot system) . In the production of phage lysate, the bacteriophages are propagated on a non-pathogenic propagation strain of Staphylococcus aureus till a high titre (108 - 109 PFU/ml) is achieved, and processed to the final product whereas a PFU unit (Plague forming units) is a measure of the amount of infectious viral particles.
It is particularly used for elimination of staphylococcal cells from infection foci (suppurative processes of skin, subcutis and skin adnexa) and potential reservoirs (in particular rhinopharynx, intestinal and urinary tracts) . It is a significant means in a complex treatment of chronic forms of staphylococcal infections (suppurative affections, abscesses, fistulas, infections of deep soft tissues) which prevents them from a septic state. It is also an important part of preventive measures in pre-operative preparation in order to prevent occurrence of superposed pyogenic complications after surgical interventions.
An alternative use of the product is an application in preserving and storing of food, in production of feed for farm animals and use in decontamination of surfaces (hospital environment, medical aids) .
Principle of manufacturing process
The bacterial host culture is incubated in a liquid tryptone medium. When growth approaching the value of lxlO9 PFU/ml is achieved, the active phage strain, is added in an approximate MI ratio 1 (MI = multiplicity of infection) , which means that a phage particle falls upon 1 bacterial cell. When lysis of the bacterial culture is finished, the phage lysate is put in a refrigerator where it is stored till further processing. The active component is prepared as a mixture of high potent virulent phage particles with a polyvalent effect prepared by incubation . of bacteriophage subtypes and standardized in its efficiency according to concentrations of specified phage particles resulting in 1 ml.
1. Revival of bacterial strain
Immediately after thawing, lyophilized strain is re-inoculated to 1.5 % tryptone agar and incubated 24 hours at +37 °C + 1 °C.
Passaging of the strain
The grown-up bacterial strain is re-inoculated to the required number of plates with 1.5 % tryptone agar and incubated for 24 hours at + 37
°C ± 1 °C. The grown-up culture is used for inoculation of liquid tryptone medium. In-process control: Cultivation and microscopic checking of the strain purity.
2. Preparation of subcultures
Bacterial culture is re-inoculated from the solid medium to an Erlenmayer flask with 100 ml of tryptone broth and incubated for 24 hours at + 37 °C ± 1 °C. The number of inoculated Erlenmayer flasks depends on the required number of subcultures for the production (1 - 3 pc) .
In-process control: Microscopic checking of subculture purity.
3. Preparation of phage starter
The resuspended phage strain is revived at first and propagated via a method of double-layer agar and incubated in a liquid medium.
The double-layer agar
Petri dishes with 1.5 % tryptone agar which act as a base are overlayed with 3 ml of 0.36 % tryptone agar which contains bacterial suspension of the appropriate host bacterium Staphylococcus aureus and suspension of bacteriophage obtained by re-suspending of the lyophilized phage
S strain in an isotonic solution. Incubation for 24 h at +37 °C ± 1 °C follows. Then surface of each plate is overlayed with tryptone broth liquid medium. After 30 minutes the soft surface with 0.36 % tryptone agar is washed off and centrifuged 3 1 hour at 5000 RPM. Bacteriophage is contained in the supernatant.
In-process control: Determination of the number of phage particles. . Preparation in liquid medium
The subculture of the host bacteria Staphylococcus aureus is used for inoculation of a tryptone broth. Incubation proceeds in a thermostat at +37 °C ± 1 °C until optical density of biomass is 0.3 - 0.4 (measured in a spectrophotometer at wavelength 500 nm) . Into the staphylococcal suspension 15 - 20 ml of the phage lysate is added (prepared according to the previous step, with the min. titre 1 x 1010 PFU/ml) .
The bacteriophage is propagated at a laboratory temperature for 18 - 20 hours while being agitated continuously. Centrifugation for 1 hour at 5000 RPM follows. The bacteriophage is contained in the supernatant .
In-process control: Checking of the bacterial suspension density and determination of the number of phage particles of the phage starter during bacteriolysis.
5. Preparation of bacterial suspension
Bacterial suspension is inoculated from Erlenmayer flasks into 3000 ml of tryptone broth in a 5L bottle.
Incubation at + 37 °C ± 1°C proceeds till the required density is achieved (optical density 0.25 - 0.35) . Duration of incubation usually ranges from 4 to 5 hours. The first sample for the check of density is taken after 3 hours of incubation; other samples are taken depending on bacterial growth.
In-process control: Operation checking is performed during incubation by measuring of density, and via a microscopic check upon the end of incubatio .
6. Preparation of phage lysate To the bacterial suspension the phage starter of min. titre 1 x 1010 PFU/ml is added in a volume of 50 - 60 ml. The starter is added aseptically at the moment when the value of optical density of the sample is within the interval (0.25 - 0.35 ) .
Bacteriolysis proceeds at a room temperature for 20 - 30 hours. In the course of bacteriolysis the liquid culture clarifies. Optical density of lysed bacterial suspension must fall under the value of 0.1.
In-process control: The operation check performs measuring of density.
Culture media for production of phage lysate:
Tryptone broth
- raw materials Tryptone Oxoid, Yeast extract Oxoid, sodium chloride, purified water
Tryptone agar
tryptone broth, Agar Oxoid N! 1
The prepared tryptone agar and tryptone broth are sterilised in a steam sterilizer at 121 °C for 30 minutes.
Checking of culture media is based on verification of growth characteristics using the strain of Staphylococcus aureus.
Checking of critical steps and intermediate products
Figure imgf000009_0001
subcultures clumps. No extraneous
microorganisms present
3. Preparation of Determination of
phage starter the number of min. 1 . 1010 PFU/ml
double-layer agar phage particles
4. Preparation of Spectrophotometric Growth of the host bacteria on phage starter - measuring of OD 0.3 - 0.4
liquid medium density
Determination of
the number of min. 1 . 1010 PFU/ml
phage particles
5.preparation of Microscopic purity G+ cocci, diameter 0.8 - 1 um, bacterial single, pairs, clusters, suspension clumps . No extraneous
microorganisms present
Spectrophotometric Growth of the host bacteria on measuring of OD 0.25 -0.35
density
6. Preparation of Spectrophotometric Checking of growth of
phage lysate measuring of bacteriophage + decrease of OD density under 0, 1
Use for:
Extension of the efficacy spectrum of the product on the currently- occurring strains
Product efficiency
The product is currently effective on 85 % of examined strains (n=100) including hospital and community strains of serious infections and MRSA collected in hospital facilities and clinical laboratories of several regions. Most of sensitive strains of Staphylococcus aureus are classified in clonal lineages STl, ST5, ST8, ST20, ST22, ST30, ST36, ST111, ST154, ST225, ST228, ST247, ST624. However, genotypes and characteristics of staphylococcal strains undergo relatively fast evolution, and in the future occurrence of insensitive strains can be expected. In order to extend the efficacy spectra based on testing of sensitivity of currently occurring strains of Staphylococcus aureus, use of a mixture of more phage lysates containing several subtypes of bacteriophages is proposed (phage mutants with the extended range of the host) . Isolation of new subtypes of bacteriophage and innovation of the product
Subtypes of bacteriophage with a wider spectrum of the host are isolated from plaques grown up after plating of the starting phage on insensitive bacterial strains of Staphylococcus aureus. Isolation of new efficient subtypes is performed by plating of phage lysates of a high titre onto growth of resistant strains. Single plaques that grow on these strains with a very low frequency (1CT9 - 1Q~6) are then used for initiation of lysates containing mutant phages. In order to achieve a wide lytic spectrum, the selection can be made in several stages during which the phage mutants obtained in one resistant strain are subsequently plated on other (different) resistant strains. During this process, there are mutations developing in phage genomes which affect the lytic capacities of the phage and lead to extending of its host range. The possibility that the selected phages may be a result of modification induced by the host strain used for the selection is verified by determination of the value of efficiency of plating (EOP) on the tested strains and their capability to maintain a wide host range after repeated propagation on a propagation strain (reversion testing) . However, based on long-term experience most of thus obtained bacteriophage subtypes have a character of mutants which maintain permanently the extended host range.
Manufacturing of the final product
Description and composition of the final product
The final product is lyophilized powder for preparation of solution (lyophilizate) produced by sterile filtration and lyophilisation of a mixture of antistaphylococcal phage lysate containing active phage particles of a polyvalent staphylococcal bacteriophage.
A medicinal product for local use contains high-potent virulent phage particles with a polyvalent effect. It is standardized in its efficiency according to concentration of specific phage particles after re-constitution of the powder for preparation of solution in a solvent (water for injection) for 1 ml.
Ά therapeutic dose contains minimum 106 particles. Manu acturing process of the final medicinal product
Production of a batch of the final product follows continuously the production of phage lysates of particular subtypes of the phage strain. A batch of the final product is made by mixing of the equal amount of phage lysates of the particular subtypes of the bacteriophage; the prepared mixture is sterile-filtered, filled in vials and lyophilized. When lyophilisation is finished, vials with the finished product are closed with a stopper secured by crimp.
Filtration
A mixture of lysates after sampling for microbial burden (100 ml) is filtered through a filtration layer of porosity 0.22 um.
In-process control:
Determination of microbial burden of the product - max. 10 PFU/10Q ml. Filling
Filtered lysate is filled out by 10 ml in PNC 20 ml vials.
In-process control: checking of the filled volume using a calibrated graduated cylinder.
Lyophilisation
Filled vials covered with a lyophilisation stopper are lyophilized at vacuum from deep freezing (-45 °C) by heating on plates to the specified temperature (+30 °C) .
Description of a lyophilisation cycle:
Phase I : Freezing of plates down to -50 °C, putting on material, freezing of material till temperature of the plates and material is stabilized at the same value (duration of the phase is 1 hour)
Phase II: The freezing temperature of plates is maintained at -50 °C and the vacuum pump is heated (duration of the phase is 30 minutes)
Phase III: Main drying - evacuation of the drying space to the set up vacuum (30-80 Pa) . When the pressure value is achieved, heating of plates adjusted at the temperature of +30 °C according to the time regime starts. This phase of the lyophilisation process takes app 90 % of the total time and lasts for approx. 24 hours.
Phase IV: Final drying at the maximum vacuum (2 - 0.5 Pa) and the maximum temperature +30 °C. If the product temperature is consistent with the temperature of heating plates and values of vacuum do not change in the test of the end of drying, lyophilization is finished by stopping vacuum.
When lyophilisation is finished, stoppers are secured by crimps.
Example of Embodiment of the Invention
Fig. 1 illustrates a flow-chart of preparation of phage lysate
Fig. 2 illustrates a flow-chart of production of the final product
Example of Embodiment of the Invention
Example 1
The method of production of a medicinal product in a form of staphylococcal phage lysate includes the following successive steps:
- revival of a bacterial strain;
- passaging of the bacterial strain;
- preparation of cell sub-cultures;
- preparation of the phage starter via a double-layer agar method;
- preparation of the phage starter in a liquid medium;
- preparation of bacterial suspension;
- preparation of phage lysate;
- filtration of phage lysate or a lysate mixture;
- filling of filtered lysate or a lysate mixture in vials;
- lyophilisation of vials with lysate or a lysate mixture.
The step of revival of a bacterial strain includes inoculation of thawed lyophilized bacterial strain onto 1.5 % agar and following incubation for 24 hours at +37 °C ± 1 °C . The step of passaging of the bacterial strain includes re-inoculation of the grown-up bacterial strain onto the required number of plates with 1.5 % tryptone agar and its incubation for 24 hours at + 37 °C ± 1 °C.
The step of preparation of cell sub-cultures includes re-inoculation of the bacterial culture from the solid medium to an Erlenmayer flask with 100 ml of tryptone medium whereas the amount of inoculated Erlenmayer flasks depends on the required number of sub-cultures for production and incubation lasts for 24 hours at + 37 °C ± 1 °C.
The step of preparation of the phage starter via a double-layer agar method includes revival of the resuspended phage strain and its propagation on Petri dishes with 1.5 % tryptone agar which serve as the base which is overlayed with 3 ml of 0.36 % tryptone agar which contains bacterial suspension of the appropriate host bacterium Staphylococcus aureus and suspension of bacteriophage obtained by re- suspending of the lyophilized phage strain in an isotonic solution. Incubation for 24 h at +37 °C ± 1 °C follows. Then surface of each plate is overlayed with tryptone broth liquid medium. After 30 minutes the soft surface with 0.36 % tryptone soft agar is washed off and centrifuged 3 x 1 hour at 5000 RPM. Bacteriophage is contained in the supernatant .
The step of preparation of the phage starter in a liquid medium includes inoculation of tryptone medium with a subculture of the host bacterium Staphylococcus aureus; incubation proceeds in a thermostat at +37 °C ± 1 °C until optical density of biomass is 0.3 - 0.4, measured in a spectrophotometer at wavelength 500 ran; into the staphylococcal suspension 15 - 20 ml of the phage lysate is added (prepared according to the previous step) , with the min. titre 1 x 1010 PFU/ml) ; bacteriophage is propagated at a laboratory temperature for 18 - 20 hours while being agitated continuously; centrifugation for 1 hour at 5000 RPM follows. The bacteriophage is contained in the supernatant .
The step of preparation of bacterial suspension includes inoculation of bacterial suspension from Erlenmayer flasks into 3000 ml of tryptone broth in a 51 bottle, while incubation at + 37 °C ± 1°C proceeds till the required density 0.25 - 0.35 is achieved; duration of incubation usually ranges from 4 to 5 hours; the first sample for the check of density is taken after 3 hours of incubation and other samples are taken depending on bacterial growth.
The step of preparation of phage lysate or a mixture of phage lysates includes addition of the phage starter of min. titre 1 x 1010 PFU/ml in an amount of the volume 50 - 60 ml to the bacterial suspension, while the starter is added aseptically at the moment when the value of optical density of the sample is within the interval 0.25 - 0.35; bacteriolysis proceeds at a room temperature for 20 - 30 hours; in the course of bacteriolysis the liquid culture clarifies and optical density of lysed bacterial suspension must fall under the value of 0.1.
When a sample for microbial burden of the sample content of 100 ml is taken, the phage lysate or lysate mixture is filtered through a filtration layer of porosity 0.22 um whereas microbial burden of the product corresponds to max. 10 PFU/100 ml.
Filtered lysate is filled by 10 ml in PNC 20ml vials.
Vials with lysate of a mixture of lysates covered with a lyophilisation stopper are lyophilized at vacuum from deep freezing (- 45 °C) by heating on plates to the specified temperature (+30 °C) , while the lyophilisation step consists of the following phases, and when lyophilisation is finished, stoppers are secured by crimps:
Phase I : Freezing of plates down to -50 °C, putting on material, freezing of material till temperature of the plates and material is stabilized at the same value (duration of the phase is 1 hour)
Phase II: The freezing temperature of plates is maintained at -50 °C and the vacuum pump is heated (duration of the phase is 30 minutes)
Phase III: Main drying - evacuation of the drying space to the set up vacuum (30-80 Pa) . When the pressure value is achieved, heating of plates adjusted at the temperature of +30 °C according to the time regime starts. This phase of the lyophilisation process takes approx. 90 % of the total time and lasts for approx. 24 hours. Phase IV: Final drying at the maximum vacuum (2 - 0.5 Pa) and the maximum temperature +30 °C. If the product temperature is consistent with the temperature of heating plates and values of vacuum do not change in the test of the end of drying, lyophilisation is finished by stopping vacuum.
In the production, a tryptone broth consisting of a set of substances containing Tryptone Oxoid, Yeast extract Oxoid, sodium chloride and purified water was used; and tryptone agar consisting of a combination of tryptone broth and Agar Oxoid W 1 was used. The prepared tryptone agar and tryptone broth were sterilized in a steam sterilizer at the temperature 121 °C for 30 minutes.
Example 2
A medicinal product in a form of antistaphylococcal phage lysate containing active phage particles of polyvalent staphylococcal bacteriophage prepared via the method according to Example 1. One therapeutic dose contains minimum 106 particles of bacteriophage.
Industrial utilization
The invention is utilisable in the field of both human and veterinary medicine in all forms of staphylococcal infections. It serves for destruction of staphylococcal cells in the spot of a developing infection.

Claims

PATENT CLAIMS
1. The process of production of a medicinal product in the form of antistaphylococcal phage lysate, characterized by including the following successive steps:
- revival of the bacterial strain;
- passaging of the bacterial strain;
- preparation of cell sub-cultures;
- preparation of the phage starter via a double-layer agar method;
- preparation of the phage starter in a liquid medium;
- preparation of bacterial suspension;
- preparation of phage lysate;
- filtration of phage lysate or a lysate mixture;
- filling of filtered lysate or a lysate mixture in vials;
- lyophilisation of vials with lysate or a lysate mixture.
Or including the following successive steps:
- revival of the bacterial strain;
- passaging of the bacterial strain;
- preparation of cell sub-cultures;
- preparation of bacterial suspension;
- preparation of the phage starter via a double-layer agar method;
- preparation of the phage starter in a liquid medium;
- preparation of phage lysate;
- filtration of phage lysate or a lysate mixture;
- filling of filtered lysate or a lysate mixture in vials;
- lyophilisation of vials with lysate or a lysate mixture.
2. The process of production of a medicinal product according to claim 1 characterized by the step of revival of a bacterial strain including inoculation of lyophilized bacterial strain after its thawing onto 1.5 % agar and following incubation for 24 hours at +37 °C + 1 °C.
3. The process of production of a medicinal product according to claim 1 characterized by the step of passaging of the bacterial strain including re-inoculation of the grown-up bacterial strain onto the required number of plates with 1.5 % tryptone agar and its incubation for 24 hours at + 37 °C ± 1 °C.
4. The process of production of a medicinal product according to claim 1 characterized by the step of preparation of cell subcultures including re-inoculation of the bacterial culture from the solid medium to an Erlenmayer flask with 100 ml of tryptone broth whereas the amount of inoculated Erlenmayer flasks depends on the required number of sub-cultures for production and incubation lasts for 24 hours at + 37 °C ± 1 °C.
5. The process of production of a medicinal product according to claim 1 characterized by the step of preparation of the phage starter via a double-layer agar method including revival of the resuspended phage strain and its propagation whereas Petri dishes with 1.5 % tryptone agar which serve as the base are overlayed with 3 ml of 0.36% tryptone agar which contains bacterial suspension of the appropriate host bacterium Staphylococcus aureus and suspension of bacteriophage obtained by re-suspending of the lyophilized phage strain in an isotonic solution. Incubation for 24 h at +37 °C ± 1 °C follows. Then surface of each plate is overlayed with tryptone broth liquid medium; after 30 minutes the soft surface with 0.36% tryptone agar is washed off and centrifuged 3 times 1 hour at 5000 RPM while bacteriophage is contained in the supernatant.
6. The process of production of a medicinal product according to claim 1 characterized by the step of preparation of the phage starter in a liquid medium including inoculation of tryptone medium with a subculture of the host bacterium Staphylococcus aureus; incubation proceeds in a thermostat at +37 °C ± 1 °C until optical density of biomass is 0.3 - 0.4, measured in a spectrophotometer at wavelength 500 run; into the staphylococcal suspension 15 - 20 ml of the phage lysate is added (prepared according to the previous step) , with the min. titre 1 x 1010 PFU/ml; bacteriophage is propagated at a laboratory temperature for 18 - 20 hours while being agitated continuously; centrifugation for 1 hour at 5000 RPM follows. The bacteriophage is contained in the supernatant.
7. The process of production of a medicinal product according to claim 1 characterized by the step of preparation of bacterial suspension including inoculation of bacterial suspension from Erlenmayer flasks into 3000 ml of tryptone broth in a 51 bottle, while incubation at + 37 °C ± 1°C proceeds till the required density 0.25 - 0.35 is achieved; duration of incubation usually ranges from 4 to 5 hours; the first sample for the check of density is taken after 3 hours of incubation and other samples are taken depending on bacterial growth.
8. The process of production of a medicinal product according to claim 1 characterized by the step of preparation of phage lysate or a mixture of phage lysates including addition of the phage starter of min. titre 1 x 1010 PFU/ml in an amount of the volume 50 - 60 ml to the bacterial suspension, while the starter is added aseptically at the moment when the value of optical density of the sample is within the interval 0.25 - 0.35; bacteriolysis proceeds at a room temperature for 20 - 30 hours; in the course of bacteriolysis the liquid culture clarifies and optical density of lysed bacterial suspension must fall under the value of 0.1.
9. The process of production of a medicinal product according to claim 1 characterized by that after taking of a sample for microbial burden with the sample content of 100 ml, the phage lysate or lysate mixture is filtered through a filtration layer of porosity 0.22 um whereas microbial burden of the product corresponds to max. 10 PFU/100 ml.
10. The process of production of a medicinal product according to claim 1 characterized by the filtered lysate being filled by 10 ml in PNC 20 ml vials.
11. The process of production of a medicinal product according to claim 1 characterized by vials with lysate of a mixture of lysates covered with a lyophilisation stopper being lyophilized at vacuum from deep freezing (-45 °C) by heating on plates to the specified temperature (+30 °C) , while the lyophilisation step consists of the following phases, and when lyophilisation is finished, stoppers are secured by crimps: Phase I: Freezing of plates down to -50 °C, putting on material, freezing of material till temperature of the plates and material is stabilized at the same value (duration of the phase is 1 hour)
Phase II: The freezing temperature of plates is maintained at -50 °C and the vacuum pump is heated (duration of the phase is 30 minutes )
Phase III: Main drying - evacuation of the drying space to the set up vacuum (30-80 Pa) . When the pressure value is achieved, heating of plates adjusted at the temperature of +30 °C according to the time regime starts. This phase of the lyophilisation process takes approx. 90 % of the total time and lasts for approx. 24 hours.
Phase IV: Final drying at the maximum vacuum (2 - 0.5 Pa) and the maximum temperature +30 °C. If the product temperature is consistent with the temperature of heating plates and values of vacuum do not change in the test of the end of drying, lyophilisation is finished by stopping vacuum.
12. The process of production of a medicinal product according to any of claims 1 to 7 characterized by including a tryptone broth consisting of a set of substances containing Tryptone Oxoid, Yeast extract Oxoid, sodium chloride and purified water.
13. The process of production of a medicinal product according to any of claims 1 to 7 and 10 characterized by including tryptone agar consisting of a combination of tryptone broth and Agar Oxoid N' 1.
14. The process of production of a medicinal product according to claim 1 characterized by including sterilization of the prepared tryptone agar and/or tryptone broth in a steam sterilizer at the temperature 121 °C for 30 minutes.
15. A medicinal product in the form of antistaphylococcal phage lysate preparable via a process according to claims 1 to 14 containing active phage particles of more than one subtype polyvalent staphylococcal bacteriophage.
16. A medicinal product in the form of antistaphylococcal phage lysate preparable via a process according to claims 1 to 14 containing active phage particles of more than one subtype of a polyvalent staphylococcal bacteriophage and phage mutants with the extended range of the host.
17. The process of production of a medicinal product according to claim 16 characterized by the subtypes of bacteriophage with a wider spectrum of the host being isolated from plaques grown up after plating of the starting phage on insensitive bacterial strains of Staphylococcus aureus, while new efficient subtypes are isolated by plating of phage lysates of a high titre onto growth of resistant strains; single plaques that grow on these strains with a very low frequency (1CT9 - 1CT6) are then used for initiation of lysates containing mutant phages; selection is made in several stages during which the phage mutants obtained in one resistant strain subsequently plated on other resistant strains in order to achieve a wide lytic spectrum.
18. A medicinal product according to claim 15 or 16 characterized by- containing minimum 106 particles of bacteriophage in one therapeutic dose.
19. Use of the medicinal product in the form of antistaphylococcal phage lysate according to claims 15 or 16 produced by the process according to claims 1 to 14 and/or 17 for local application in the treatment of infections caused by staphylococcal strains.
20. Use of the medicinal product in the form of antistaphylococcal phage lysate according to claims 15 or 16 produced by the process according to claims 1 to 14 and/or 17 as a part of preventive measures in pre-operative preparation.
-Use of the medicinal product in the form of antistaphylococcal phage lysate according to claims 15 or 16 produced by the process according to claims 1 to 14 and/or 17 to prevent occurrence superposed pyogenic complications after surgical interventions.
22. Use of the medicinal product in the form of antistaphylococcal phage lysate according to claims 15 or 16 produced by the process according to claims 1 to 14 and/or 17 for preserving and storing of food, or in production of feed for farm animals.
23. Use of the medicinal product in the form of antistaphylococcal phage lysate according to claims 15 or 16 produced by the process according to claims 1 to 14 and/or 17 for use in decontamination of surfaces, particularly in hospital environment or in decontamination of medical instruments and aids .
PCT/CZ2013/000117 2012-09-27 2013-09-27 Medicinal preparation in the form of antistaphylococcal phage lysate, process of its preparation and use WO2014048407A2 (en)

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EP3839031A1 (en) * 2019-12-16 2021-06-23 Fachhochschule Südwestfalen Phage culturing device, phage preparation method and filter device therefor
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Publication number Priority date Publication date Assignee Title
WO2016030871A1 (en) * 2014-08-31 2016-03-03 Instytut Biochemii I Biofizyki Polskiej Akademii Nauk Staphylococcus aureus strains for production of monoclonal phage preparations deprived of contamination with plasmid dna
EP3460048A1 (en) * 2014-08-31 2019-03-27 Instytut Biochemii I Biofizyki Polskiej Akademii Nauk Staphylococcus aureus strains for production of monoclonal phage preparations deprived of contamination with plasmid dna
RU2677290C1 (en) * 2017-08-17 2019-01-16 Наталья Анатольевна Ковязина Biodegradable medicinal plates and method for production thereof
EP3839031A1 (en) * 2019-12-16 2021-06-23 Fachhochschule Südwestfalen Phage culturing device, phage preparation method and filter device therefor
WO2021122776A1 (en) * 2019-12-16 2021-06-24 Fachhochschule Südwestfalen Phage culturing device, method for preparing phages, and filtration device for same
WO2021197517A1 (en) * 2020-03-31 2021-10-07 MB PHARMA s.r.o. A lyophilized test kit for determination of antimicrobial biological activity of phage preparations against the bacteria staphylococcus aureus

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