The following is a translation of the bacteriophage preparation methodology for treatment and prophylactic uses. The method below is used in ex-Soviet countries for the preparation of phage medication and phage cocktail production.

OFS.1.7.1.0002.15 Bacteriophages treatment-and-prophylactic

GENERAL PHARMACOPEIA ARTICLE
This pharmacopeia article applies to treatment and prophylactic use of bacteriophages, drugs containing complexes of polyclonal virulent bacterial viruses that cause the death of their respective bacteria due to intracellular reproduction and destruction of the bacterial cell, accompanied by the release of mature phage particles capable of infecting new bacterial cells. Bacteriophages are bacterial viruses. According to the type of interaction with a bacterial cell, phages are subdivided into virulent (causing the death of bacteria) and temperate, which, infecting a bacterial cell, are built into its genetic apparatus and reproduced during cell division, without causing lysis. In medicine, bacteriophages are used for the treatment and prevention of purulent-septic and intestinal infections, as well as for diagnostic purposes for the indication of species and intraspecific differentiation of bacteria.

Treatment and prophylactic bacteriophages contain only virulent phages. These preparations are sterile purified filtrates of phagolysates of the corresponding bacterial species, freed from endo and exotoxins, phagolysis products of bacterial cells, as well as their antigenic complexes and protein components of nutrient media.

Due to the strict specificity of the action, therapeutic and prophylactic bacteriophages, unlike antibiotics, do not inhibit the normal microflora, do not suppress immune defense, do not have a toxic effect, and do not cause allergies. The presence of bacterial resistance to antibiotics does not affect the lytic activity of bacteriophages.

Therapeutic prophylactic bacteriophages are used to treat and prevent purulent-inflammatory diseases and intestinal infections, and to correct dysbiotic conditions. The main condition for clinical efficacy in the appointment of bacteriophages is the phage sensitivity of pathogenic bacteria.

The treatment and prophylactic bacteriophages in their composition are divided into:

• monocomponent bacteriophages – medicines containing virulent phages against one genus or species of bacteria.
• combined bacteriophages – drugs containing several types of monocomponent bacteriophages. The activity of this group of drugs is expressed in terms of the maximum dilution titer, which gives a complete phagolysis of the types of bacterial strains corresponding to the preparation, determined by the Appelman method. Indicators of specific activity for each drug are indicated in the corresponding pharmacopeial articles. Therapeutic prophylactic bacteriophages are used for oral, external, local, rectal administration, intranasal and conjunctival administration, and administration to drained cavities. They are produced in various dosage forms, such as; solutions, in the form of tablets, suppositories, liniments and ointments.

Table – Treatment and prophylactic bacteriophages and their target orientation

Production

The organization of the production of therapeutic and prophylactic bacteriophages is carried out in accordance with the current state sanitary rules, taking into account the requirements of the quality assurance system of the good manufacturing practice. However, there are certain features in the configuration and placement of production facilities.

To work with bacterial strains isolated from different sources (pathological material and environmental objects), and to isolate phages from pathological material and environmental objects (soil, sewage, etc.), boxes isolated from production for microbiological studies are necessary.

Mandatory initial stages of work are:

• isolation of pure cultures of promising bacterial strains (i.e. candidates for industrial bacterial strains)
• isolation of virulent bacteriophages to replenish the collection of phages for production.

Separate microbiological boxes should be provided in the production area for working with industrial bacterial strains and phages found in the current library collection.

In the production of phage preparations, the validation of the technological process, technological equipment, raw materials and control methods are carried out. All raw materials and raw materials used in production must have documents confirming their quality. Excipients that are part of medicines with therapeutic and prophylactic bacteriophages should be approved for medical use and used in doses that do not cause toxic, allergic or other undesirable reactions in humans.

Culture media

The culture media should not contain antibiotics and components that cause allergic or other adverse reactions in humans. Culture media must have good growth properties and be sterile. Raw materials, reactants, and reagents used in the production of culture media should be suitable for the respective purposes and their quality must be documented.

The following nutrient media are recommended for inclusion in the pharmacopeia article: meat-peptone broth (MPB) and meat-peptone agar (1.5 – 2%) or Hottinger broth and agar (1.5 – 2%) with the necessary additives, depending on the specific needs of the target bacteria phage preparations. It is possible to use other nutrient media for special purposes, for example, for pathogens of, especially dangerous infections.

Requirements for Production Strains

Production strains of bacteria producing bacteriophages are isolated from patients with purulent-septic or intestinal infections and are obtained from bacteriological diagnostic laboratories (preferably located in the regions of sale and consumption of therapeutic and prophylactic bacteriophages). The collection of bacterial production strains used in the production of bacteriophages should be updated annually with freshly isolated strains from patients by at least one third.

Production bacterial strains should possess morphological, cultural, biochemical and antigenic properties typical for each species, not producing enterotoxins and not containing temperate bacteriophages in their genome.

Production of bacterial strains should be lysed by phages from the current library according to the Appelman method in titers of at least 1-2 orders of magnitude higher than the specific activity of the final product. Moreover, the stability of the lysis should be maintained after (48 ± 3) hours of incubation at a temperature of 37 ⁰C.

Industrial strains of bacteria stored in specific isolated areas in the lyophilized state in ampoules at 2 – 8 ⁰C for 10 years and in tubes with 0.4 – 0.7% agar nutrient medium (based hydrolyzate Hottinger or BCH) under sterile vaseline oil at a temperature of 2 – 8 ⁰C for not more than 1 year with regular subculturing every 2 – 3 months.

Control bacteria strains. When determining the specific activity of the finished preparations of bacteriophages, strains are selected as control from the collection of industrial bacterial strains. They should not be used in the manufacturing of this series of the drug.

Bacteriophages from current collection. To obtain phage preparations, only virulent bacteriophages are used. They are isolated from natural sources – clinical material, wastewater, soil, passaging on strains of homologous types of bacteria – freshly isolated and industrial. Highly active races (strains) of phages are selected for weakly lysing and phagoresistant bacterial strains. The replenishment of production phage races with various strains from natural sources allows us to overcome the primary phage resistance of pathogens.

Bacteriophages should include only virulent phages with a wide range of action in relation to strains of a homologous bacterial species, have high activity, stability of lysis, a specific direction of antimicrobial action and high “yield”. Characterization of candidate phage races (strains) during selection can be supplemented by electron microscopic study of their morphology and other modern molecular biological methods (for example, by sequencing – determining the sequence of phage DNA). Storage production collection of bacteriophages is carried out on homologous bacterial strains at a temperature from 2 to 8 ⁰C for 5 years, with an annual reseeding.

Working with production strains of bacteria and bacteriophages, namely regular replenishment of production bacterial strains due to clinical isolates and selection of phage races from natural sources for them, makes it possible to adapt therapeutic and prophylactic bacteriophages to circulating pathogens of bacterial infections. Thus, it is possible to produce production batches of bacteriophages for special purposes. For example, during an outbreak or in the focus of infection caused by a phage-resistant pathogen, the transfer of an epidemic significant bacterial strain to production will allow the drug from bacteriophages to adapt to specific epidemiological conditions.

The range of action of therapeutic and prophylactic bacteriophages, their activity against modern pathogens of bacterial infections largely depends on the organization of collection of clinical strains.

Brief Process Description

The production process includes the following main stages: work with pathogens of purulent-septic and intestinal bacterial infections, selection of active phage races to them, work with a collection of bacterial production strains regularly replenished with freshly isolated bacteria.

The cultivation of bacteriophages is carried out in reactors (fermenters) by seeding production strains of bacteria and phages containing several highly active phage races. After the completion of the phagolysis process to purify the phagolysates from fragments of bacterial cells, their metabolites, including enterotoxins and protein components of the nutrient medium, the culture suspension is passed through microfiltration units. The resulting phagolysate is subjected to ultrafiltration and concentration, followed by sterilizing filtration. Then, a liquid preparation is prepared from the obtained sterile concentrate of phagolysates by diluting with 0.9% sodium chloride solution with stabilizers or lyophilized biomass intended for tablets is obtained (lyophilizing the phagolysate concentrate under appropriate conditions).

During the production of bacteriophages, the quality of the initial, intermediate and final products is evaluated at the following main stages and tests.

1. Preparation of bacterial production strains – control of inoculum cultures of bacterial producer strains:

a) cleanliness;

b) typical biological properties;

c) the absence of lysogenesis.

2. Preparation of bacteriophages – control of the bacteriophages:

a) the content of phage particles in 1 ml, determined by the method of agar layers according to Grazia;

b) for specific activity determined by titration in a liquid nutrient medium according to the Appelman method on inoculum bacterial strains;

c) the stability of lysis (preservation of the results obtained by the method of lysis Appelman) for 48 hours of incubation at (37 ± 1) ⁰C;

g) on ​​sterility.

At the final stage of cultivation in reactors, the onset of complete phagolysis is determined visually by the absence of visible bacterial growth.

As the purification of phagolysates by ultrafiltration, concentration and sterilizing filtration is completed, the following indicators are determined: pH, sterility, specific activity by the Appelman method, abnormal toxicity.

In the manufacture of combined preparations, finished monophages are combined in reactors under aseptic conditions.

To obtain a liquid preparation after the final stage, the bacteriophage is poured into bottles and sealed.

To receive drugs in tablets:

a) stabilizers are added to the concentrated bacteriophage and lyophilized. The dry mass of the bacteriophage is monitored in tests for a specific activity and microbiological purity;

b) after adding fillers, tableting is carried out. As auxiliary substances, pectin, calcium gluconate, glucose, talc, calcium stearate are used. Tablets are checked for brittleness, absence of defects (chips, delamination), the average weight of tablets is determined;

c) after the completion of the packing of the tablets, the finished product is monitored according to all indicators: description (appearance of the tablets), authenticity and specific activity, average tablet weight, disintegration, weight loss upon drying, microbiological purity, abnormal toxicity.

Specific research methods used to characterize therapeutic and prophylactic bacteriophages include Appelman and Grazia methods.

Determination of the specific activity of bacteriophages and the stability of lysis by the method of Appelman

The specific activity of bacteriophages and the stability of lysis by the Appelman method is determined using homologous bacterial test strains (control strains). Indicators of specific activity are indicated in the pharmacopeia article. Control strains were selected from a collection of production of bacterial strains. They should not be used in the manufacture of this series of the drug. The composition of the nutrient broth is indicated in a pharmacopeia article on a specific phage preparation.

Analysis technique

Tests are carried out in compliance with aseptic rules. In test tubes containing 4.5 ml of nutrient broth (BCH, Hottinger broth), a series of ten-fold serial dilutions of the bacteriophage from 10 ^-1 to 10 ^-6 – 10 ^-9 is prepared  (depending on the specific activity indicators laid down in the pharmacopoeia article for a specific phage preparation) with a mandatory change of pipettes at each dilution. To prepare the first dilution, add 0.5 ml of a sample of the drug to 4.5 ml of broth. As a control, use a tube with 4.5 ml of broth without phage. After that, 0.03 ml of a suspension of daily bacterial agar culture containing 10 ⁹ is added to all test tubes with the obtained dilutions of the bacteriophage with a pipette. Microbial cells in 1 ml according to the standard turbidity (10 IU). The results are taken into account after (18 ± 1) hour of incubation at a temperature of (37 ± 1) °C. A change in the duration of incubation is possible depending on the species-specific growth of the bacterial target of the phage preparation, as indicated in the pharmacopeia article. The result is determined by the absence of visible bacterial growth in the presence of a bacteriophage. The activity of the bacteriophage is denoted by a negative degree of ten, where the degree indicates the last dilution of the bacteriophage, in which the growth of the control strain is not visually observed. To determine the stability of the lysis, the incubation period is extended to 2 days.

Determination of phage particles in 1 ml by the method of Grazia (on solid nutrient media two-layer method)

Meat peptone 1.5% agar is poured into Petri dishes in (20 ± 2) ml (first layer). Before use, agar plates are dried upside down with a lid closed at a temperature of (37 ± 1) °C for 30-60 minutes. Inoculation strains of bacteria are used for titration. Ten-fold sequential dilutions of the bacteriophage in BCH are prepared according to the Appelman method from 10 ^{– 1} to 10 ^-8 (10 ^-9 or higher, depending on the specific orientation of the bacteriophage). Then mix 1 ml of diluted phage from test tubes with dilutions of 10 ^-6, 10 ^-7, 10 ^-8 dilutions with 5 ml of molten and cooled to a temperature of 45 °C 0.7% meat-peptone agar. Then add (0.2 ± 0.05) ml of an 18-hour broth culture of the inoculum strain prepared from a daily culture grown on a solid nutrient medium, in accordance with the requirements for bacterial production strains. The contents of the tubes are quickly mixed by rotating the tubes between the palms so that the agar does not solidify, and a second layer is poured onto the surface of 1.5% agar in Petri dishes. After solidification of the upper agar layer, the plates are incubated for 18 hours at a temperature of (37 ± 1) °С. To determine the concentration of phage particles, the number of negative colonies (transparent spots on the matte background of the deep growth of bacteria) in each dish is counted. Multiply by the phage dilution factor in vitro with the appropriate dilution. Then calculate the average value of 3 determinations.

This technique is used in the selection of promising phage races in the library collection of bacteriophages. The order of dilutions used in the experiment depends on the type of bacteriophage.

pH

The phagolysate pH should be between 6.6 and 7.8. The test is carried out by the potentiometric method in accordance with the General Pharmacopoeia Monograph “Ionometry”.

Sterility

The purified and concentrated filtrate of phagolysates should be sterile. The test is carried out in accordance with the General Pharmacopoeia Monograph “Sterility”.

Abnormal toxicity

Administration to mice of purified filtrate of phagolysates should not cause the death of animals. The determination is carried out in accordance with the General Pharmacopoeia Monograph “Anomalous toxicity”. A test dose of a bacteriophage preparation in a volume of 1 ml is administered to the animals subcutaneously.

Testing

The necessary tests are carried out for the therapeutic and prophylactic bacteriophages in the appropriate dosage form.

Description

The physical properties of the corresponding dosage form of the drug are described. The test is carried out visually.

Authenticity

The drug is confirmed by its specific activity.

pH

For solutions – from 6.6 to 7.8. The test is carried out by the potentiometric method in accordance with the General Pharmacopoeia Monograph “Ionometry”.

Recoverable volume

For solutions – it must not be less than nominal. The determination is carried out in accordance with the General Pharmacopoeia Monograph “Recoverable volume for parenteral dosage forms”.

Average weight and deviations from average weight

For tablets – the requirements for the average weight and the maximum permissible deviations from the average weight are given in accordance with the General Pharmacopoeia Monograph “Uniformity of mass of dosage forms”.

Disintegration time

For tablets – it should not exceed 30 minutes if there are no other indications in a pharmacopeia article on a specific phage preparation. The test is carried out in accordance with the General Pharmacopoeia Monograph “Disintegration of tablets and capsules”.

Bacteriophages, produced in the form of enteric-soluble tablets, must not disintegrate within 1 h in a 0.1 M hydrochloric acid solution and, after washing with water, must disintegrate in a solution of sodium bicarbonate (pH 7.5 to 8.0) within 30 minutes.

Mass loss on drying

For tablets – it should be no more than 4.0%, unless otherwise indicated in the pharmacopeia article. The determination is carried out in accordance with the General Pharmacopoeia Monograph “Mass loss upon drying” or the General Pharmacopoeia Monograph “Determination of water”.

Sterility (for solutions)

The preparation must be sterile (category 5BII according to the General Pharmacopoeia Monograph “Microbiological purity”). The sterility test is carried out by direct seeding or membrane filtration in accordance with the General Pharmacopoeia Monograph “Sterility”.

Microbiological purity

For tablets – must comply with category 5.2B according to the General Pharmacopoeia Monograph “Microbiological purity”:

• the total number of non-pathogenic aerobic bacteria is not more than 10 ²CFU per unit of the preparation (1 g);
• lack of yeast and mold in a unit of the preparation (1 g);
• the absence of enterobacteria in the unit of the drug (1 g);
• the absence of Pseudomonas aeruginosain the unit of the drug (1 g);
• the absence of Staphylococcus aureusin the unit of the drug (1 g).

The test is carried out in accordance with the requirements of the General Pharmacopoeia Monograph “Microbiological purity”.

Abnormal toxicity

Administration to mice subcutaneously with 1 ml of the bacteriophage preparation should not cause the death of animals. The determination is carried out in accordance with the General Pharmacopoeia Monograph “Anomalous toxicity”. Sample preparation of drugs indicated in the pharmacopeia article on the drug.

Specific activity

The activity of the bacteriophage is denoted by a negative degree of ten, where the degree indicates the last dilution of the bacteriophage, in which the growth of control strains of bacteria is not visually observed. Regulatory requirements, including the number of control strains used (at least 10 strains for single drugs), indicate in the pharmacopeia article on the drug. The determination is carried out by titration in a liquid nutrient medium according to the Appelman method. The methodology and sample preparation indicate in the pharmacopeia article on a specific phage preparation.

Packaging and labeling

In accordance with the General Pharmacopoeia Monograph “Immunobiological drugs”. In the labeling of the dosage form “solution” should be provided with a warning inscription “Do not use in case of turbidity”.

Transportation

At a temperature of 2 to 8 ° C, allowed at a temperature of 9 to 25 °C within 1 month.

Storage

In a dry, dark place and out of the reach of children, at a temperature of 2 to 8 °C.

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