FIELD OF THE INVENTION

The present invention relates to a method of producing bacteriophage preparations, with improved yield. The invention also relates to apparatus for use for producing bacteriophage preparations, and a cassette that can be used vertically in bacteriophage production.

BACKGROUND OF THE INVENTION

Since their discovery almost 100 years ago, bacteriophages have been investigated extensively and a plethora of literature reviewing these studies exists (Ellis and Delbruck, 1938; Carlton, 1999; Sulakvelidze et al., 2001 ; Skurnik and Strauch, 2006; Loc-Carrillo and Abedon, 201 1 ; Abedon, 201 1 ; Ormala and Jalasvuori, 2013). Bacteriophages (commonly called phage) are a group of naturally occurring antibacterial agents (viral in nature) that infect only bacterial cells. It is estimated that in excess of 10 ³⁰ phage exist, making them the most abundant entities on earth. In all known environments, phages exist as part of a complex microbial ecosystem. They can be part of a free-living environment such as soils, vegetation and oceans, or as part of a microbial environment within a macroorganism such as an animal system. Phages play a crucial role in the regulation of nutrient cycling, as sources of diagnostic and genetic tools and as novel therapeutic agents. To date, phages have been used in a number of areas of biotechnology and medical science including rapid bacterial detection and diagnosis of disease (phage typing), prevention of bacterial disease (phage vaccine), treatment (phage therapy) and biocontrol (Haq et al., 2012).

Bacteriophages are highly specific and can only infect bacterial cells that present cell surface receptors matching those of the phage (similar to a lock and key mechanism) (Lindberg, 1973; Kutter and Sulakvelidze, 2005). Without the matching receptors, phages are unable to multiply and can quickly be degraded in the environment. Phages can either multiply via the lytic cycle ("virulent phage") or lysogenic cycle ("temperate phage"). While virulent phages kill the cells they infect (lytic cycle), temperate phages can establish a persistent infection of the cell without killing it (lysogenic cycle).

Virulent phages are effective at controlling bacterial populations with no known side effects to human, animal or plant. The method by which virulent phages kill their specific host bacterium is called 'lysis' (Kutter and Sulakvelidze, 2005; Sulakvelidze, 201 1 ). Virulent phages attach to receptors on the surface of bacteria, insert their genetic material through the bacterial membrane and take over the bacterium's transcription and translation machinery to synthesize many new phages. Finally, the bacterial cell wall is destroyed (lysed), releasing large numbers of newly assembled phage to the environment, where they can attack new bacteria. This entire process can take as little as 25 minutes (Gill and Hyman, 2010). Virulent phages have been intensely investigated for their bactericidal properties and are particularly suitable for applications that require destruction of the host bacterium such as biological control and phage therapy, making them an attractive treatment alternative to antibiotics.

Conversely, phages that replicate without immediately killing their host bacteria are termed temperate phage. These phages can either multiply via the lytic cycle (cell death) or enter a dormant state in the cell (lysogeny). In most cases the phage DNA integrates into the host chromosome ("prophage") and is replicated along with the host bacterium, being passed on to the daughter cells (Kutter and Sulakvelidze, 2005). The host bacterium continues to replicate without adverse effects to the host until host conditions become unfavourable. At this point the lytic cycle is initiated and the host cell is destroyed, releasing progeny phage. A bacterial cell that harbors a prophage may occasionally carry genes that are expressed in the cell and present new properties to the bacteria such as host virulence (toxin production), increased pathogenicity or antimicrobial resistance (Skurnik and Strauch, 2006; Kutter et al., 2010; Gill and Hyman, 2010). Temperate phages have various applications and are particularly suited to purposes that require the transport or expression of genes such as phage display, phage typing and phage vaccines (Haq et al., 2012). Due to the largely non-lytic nature of temperate phage and their ability to exchange genes, these phages are not good candidates for therapeutic applications that require immediate destruction of the host cell, such as in the treatment or control of disease (Haq et al., 2012).

Phage therapy can be largely described as the use of bacteriophages to control specific pathogenic or problematic bacteria. In human and animal health sectors, phage therapy has been practiced in regions of Eastern Europe with proven success for more than 60 years (Kutter et al., 2010). Early phage trials often produced unreliable and inconsistent results due to a poor understanding of phage biology and quality control during the preparation of phage therapeutic formulations. As phage therapy was gaining momentum in 1930-1940, antibiotics were discovered and western countries all but forgot about phage. Due to the isolation of many Eastern European countries from the advancements in antibiotic production during this time, a number of countries continued to develop and perfect phage treatments (Chanishvili, 2012). Today phage therapy is a widespread form of treatment in a number of Eastern European countries such as Russia, Poland and Georgia (Chanishvili, 2012).

Due to the specificity of phages, virulent phage can be considered a natural and effective way to target difficult or problem bacteria, without affecting normal beneficial bacteria and without negatively affecting the environment. Importantly, phages are able to infect bacteria regardless of their susceptibility to antibiotics and are capable of penetrating biofilms (Sulakvelidze et al., 2001 ; Kutter et al., 2010). As described above, virulent phages kill their bacterial hosts and liberate large numbers of progeny, which are able to infect neighbouring susceptible bacteria and start the cycle again. This replication continues until the phage can no longer find the specific targeted bacterial cells, significantly reducing bacterial biomass. It is for this reason that virulent only phages are used in phage therapies. The use of bacteriophage preparations has advantages and challenges, the critical points being high bacterial specificity, transference of virulence or toxin genes, appropriate administration of phage preparations and the development of phage resistant bacteria.

With advances in technologies and better understanding of bacteriophage biology, these challenges can be addressed. For example, virulent phages are very good candidates for phage therapy, whereas temperate phages are not. With rigorous testing, favourable virulent phages can be selected and safe phage preparations for therapeutic use can be developed. It is crucial that the perceived challenges associated with phage therapy do not impede the advancement of this technology in the Western world.

The use of phage products in the food industry, human medicine, agriculture and aquaculture has gathered momentum recently. A range of products have been approved by the FDA (Food and Drug Administration), US Environmental Protection Agency (EPA) and FSANZ (Food Standards Australia and New Zealand) for the control of Listeria monocytogenes, Salmonella, pathogenic E. coli and Pseudomonas putida. However, commercial scale production of bacteriophage preparations for therapeutic use still has its limitations, current methods using large scale biofermenters such as those used for the production of bacterial probiotics, to ensure adequate bacteriophage titres (concentrations). Biofermenters require large volumes of liquid, are difficult to operate, optimise and clean, have a large footprint and are very expensive to purchase, setup and maintain. In addition, the preparation of bacteriophage 'cocktails' requires several biofermenters, one for each individual bacteriophage, further adding to the cost of production. The downstream treating of the crude bacterial/bacteriophage preparations from the biofermenting process is also difficult and expensive due to the high level of bacterial debris, toxins and other undesirable metabolic products that must be removed or neutralised before being used as a treatment. Setup costs are a major consideration and drawback for those in the field and downstream processing adds to the difficulties and cost. There is a definite and increasing need for a method that enables the commercial production of bacteriophage preparations that reduces manufacturing volumes, maximises bacteriophage titres and reduces downstream processing of the final product thus providing a cost effective method for large commercial scale production of bacteriophage preparations.

The inventor has developed a new method and apparatus for production of bacteriophage preparations in commercially useful quantities, without the need for a biofermenter. The new method and apparatus, subject of the invention, will reduce the production volume and increase the bacteriophage production yield, providing significant cost savings and benefits, over the prior art.

For clarity, any prior art referred to herein, does not constitute an admission that the prior art forms part of the common general knowledge, in Australia or elsewhere.

It is an object of the present invention to provide a method of bacteriophage production that at least ameliorates one or more of the aforementioned problems of the prior art. It is a further object of the invention to provide apparatus or a cassette that at least ameliorates one or more of the aforementioned problems of the prior art.

DISCLOSURE OF THE INVENTION

Accordingly, the present invention provides a method of bacteriophage

production, the method including the following steps: a) Loading "cassettes" with a substrate, suitable for growth of a bacteria strain; b) Inoculating loaded "cassettes" with the bacteria and bacteriophage culture; c) Incubating inoculated "cassettes" for a suitable time for bacteria lysis to occur;

d) Extraction of bacteriophage lysate into extraction media; and e) Purification of bacteriophage lysate,

wherein the yield of bacteriophage is substantially a commercially useful quantity.

The method may also be used for other production, other than bacteria and bacteriophage, where use of the method is also suitable.

The purification step may be omitted, when using the invention, to be performed later. It is intended that the claimed invention include each of the stages but it is not intended that delay or omission of the purification step, for some reason, would avoid potential infringement.

The loading of the cassettes may take any suitable form. The loading may be the introduction of substrate into the cassette in any suitable manner. The loading may be by a method chosen from the following group: immersion; pouring; or spraying. The loading may be known as "casting" of the substrate into the cassettes for use. Preferably, the loading of the cassettes is casting of substrate onto the cassettes. Other loading may be used instead, suitable to the medium to be used. The loading may be the polymerisation of a gel substrate. The loading or casting of the substrate may include filling a casting module with cassettes and introduction of the substrate such that on removal of the cassettes some of the substrate is retained about the cassette, suitable for growth of bacteria and bacteriophage culture. The casting module may take any suitable form. Preferably, the casting module is formed of a plurality of parts assembled to form a tank into which one or more cassette can be placed and substrate filled to load the cassettes. More preferably, the cassettes are placed and loaded vertically. The casting module may be adapted to receive a plurality of the cassettes so they can be loaded with substrate simultaneously. A casting module may be used to load a plurality of the cassettes with substrate at the same time. The number of cassettes used can be varied to suit the needs of the user and the dimensions of the apparatus. Suitable numbers of cassettes may be 16 or 32. The cassette may be a gel cassette. The gel cassette may be adapted for receipt of a gel substrate suitable for growing bacteria and bacteriophage culture. Preferably, at least 16 gel cassettes can be loaded with gel at the same time. Preferably, 32 gel cassettes can be loaded with gel substrate simultaneously. Any suitable number of gel cassettes may be loaded at the same time. Preferably, the gel cassettes are loaded with gel while in the substantially vertical orientation. Preferably, a plurality of gel cassettes are loaded while in a gel casting module in the substantially vertical orientation. Preferably, the configuration of at least part of the cassette is adapted to help maintain the gel substrate in the cassette, including while in the vertical orientation.

Preferably, the cassettes are loaded with substrate while in the vertical orientation. Most preferably, the particular configuration of the cassette is particularly adapted to casting with gel substrate in the vertical orientation. Preferably, a plurality of cassettes are loaded into a casting module and substrate introduced to cover at least part of the cassettes whereby a substrate surface is created suitable for growth of bacteria and bacteriophage culture. Most preferably, the substrate is a semi-solid gel and the gel "sets" around the cassettes during loading.

Preferably, a matrix is included in the cassette and the substrate is loaded about the matrix structure. Preferably, the substrate may be loaded, in, on, around, under and below parts of the cassette. Preferably, the substrate loads to the matrix of the cassette only, during loading. However, any part of the cassette may be loaded with substrate, not limited to the matrix.

The substrate may be any suitable substrate. Preferably, the substrate is a gel substrate. The gel substrate may be agar. Other suitable gels may be used. Preferably, the gel substrate is a semi-solid substrate. Preferably, a 1-1.5% agar concentration is used. Preferably, the gel is very suitable for the growth of bacteria and bacteriophage. Most preferably, the matrix is a geometric matrix which encourages the adhering of a gel substrate during loading. Most preferably, the gel substrate is substantially maintained in the matrix when the cassette is vertical. Preferably, the gel substrate is loaded into the matrix of the gel cassette in the substantially vertical orientation. Preferably, the configuration of the matrix resists loss of gel from the cassette in the vertical orientation. Most preferably, the configuration of the matrix of the cassette strongly resists loss of substrate in the vertical orientation. In another form of the invention the cassettes could be configured for use in the horizontal orientation. Preferably, the configuration of the matrix resists loss of gel from the cassette in any orientation.

The matrix may take any suitable form. The matrix may include cells of a shape chosen from the following group: rectangular, including square; hexagonal; triangular; circular; or irregular shaped. Preferably, the matrix is formed of tessellating shapes. Preferably, the matrix is formed of tessellated square cells. The cells may, in another form of the invention, not tessellate and be formed with another configuration of matrix. Most preferably, the configuration of the matrix encourages efficient polymerisation of a gel suitable for the growth of bacteria and bacteriophage around the matrix. Preferably, the adhesion of the gel to the matrix is suitable to provide a surface on which bacteria and bacteriophage can be grown. Most preferably, the configuration of the matrix provides two surfaces of gel on which bacteria and bacteriophage can be grown, one on either side of the matrix of the cassette. More than one matrix may be included. For example, one on either side of the cassette.

Most preferably, the configuration of the matrix is such that gel adheres readily, to create surfaces for bacteria and bacteriophage culture and in use resists loss of gel, when in the vertical orientation.

The cassette may include sheets on either side of the matrix, used with spacers to separate the loaded matrix layers. Clips may be included to maintain the layers of sheets, spacers and matrix together. The cassette may be formed of a matrix, surrounded by a sheet on either side spaced from the matrix by spacers and held together by suitable clips. The cassette may be simply the matrix or may be the combination of the matrix, sheets, spacers and clips. Each of the components may be used in individual combinations as is suitable to the particular application. Different numbers of each of the components of the cassette may be used. Preferably, the spacers maintain a space between the sheet and a side of the matrix. Preferably, a pair of sheets surround the matrix and a space is maintained on either side so that a growing surface of substrate is created and protected. Preferably, the cassette is formed of a matrix which has a growing surface on either side surrounded by a space defined by a pair of sheets, maintained in place by one or more clip. Preferably, a space is maintained around the culture growing surface of the substrate of the matrix of the cassette.

Preferably, the bacteria strain is chosen from the following group: Coccus; Bacillus; Vibrio; Spirillum; or Spirochete. The bacteria strain and bacteriophage used can be varied to suit the particular needs for production as would be understood by a person skilled in the art.

Preferably, inoculation is by manually spreading a fine layer of the bacteria and bacteriophage culture onto the cassette. Most preferably, both sides of the cassette can be inoculated, substantially improving yield. Preferably, a matrix is included and it is the substrate on the surface of the matrix that is inoculated. Preferably, in this form of the invention, the matrix provides two surfaces for inoculation with bacteria and bacteriophage culture. Inoculation may take any suitable form. Any suitable form of inoculation may be used, suitable to the bacteria culture. Inoculation of cassettes could also be automated, processing each cassette in turn.

Incubation of the cassettes can be in any suitable manner, suitable for the bacteria strain. The incubation may be for several hours until the culture has grown sufficiently for bacteria lysis to occur. The incubation may be for 16 hours, for example. Any suitable incubation period may be used. Preferably, very high incidence of bacteria lysis is observable at the end of the suitable incubation period.

Extraction may take any suitable form. Preferably, an extraction module is used and the cassettes are loaded into the extraction module filled with extraction media for extraction to occur. Preferably, the extraction module is adapted to receive a plurality of cassettes. Preferably, the cassettes can be loaded into the extraction module in a substantially vertical orientation. Preferably, at least 16 cassettes can be extracted at the same time. There may be 32 or more cassettes included and extracted at the same time. Any suitable number of cassettes may be extracted at the same time. Most preferably, the cassettes have the bacteriophage product extracted while in the substantially vertical orientation. Preferably, the cassette is used with a holder to facilitate handling of the cassette. Preferably, the holder is slid into a slot in the extraction module. Preferably, the cassette is substantially covered in extraction media during extraction. Preferably, use of the holder substantially prevents the cassette from touching the sides of the extraction module. Preferably, the holder includes means to suspend the cassette within the extraction module, so that the cassette is covered by the extraction media but does not touch the extraction module. Preferably, a plurality of cassettes in holders are used in the extraction module, suspended within the extraction media. The extraction step may take place separate from loading step, and vice versa. Preferably, loading and extraction occur. Most preferably, the method is used in full including loading and extraction substantially in the vertical orientation.

Purification may take any suitable form to produce the usable bacteriophage product. Purification may include a series of physical or chemical filters.

Preferably, the yield of bacteriophage product is suitable for commercial use. The bacteriophage product can also be used by the producer themselves. Preferably, the yield of bacteriophage product is significantly greater than produced using laboratory techniques. Preferably, the disclosed method is of improved efficiency over known laboratory methods. Preferably, the method produces a significantly improved yield of bacteriophage product over known smaller scale methods.

Preferably, bacteriophage product can be produced in high yield quantities by use of lower volumes of materials. Preferably, the method has improved efficiency and lower costs than known methods, and can be performed on a small or larger scale to suit the needs of the user.

Most preferably, the yield of bacteriophage product is significantly greater than known laboratory methods with lower volumes of materials required. In this way an economic and efficient means of bacteriophage product, without the need for large pieces of equipment is disclosed. The methods disclosed can be used without the need to sterilise large and expensive pieces of equipment, the cassettes can be readily reused through standard sterilisation. Disposable forms of the invention are also anticipated, with use of paper or other matrix.

Preferably, the invention also relates to an apparatus for use with the method. The apparatus may be as described with reference to the Figures. The invention also relates to a cassette. The cassette may be as described with reference to the figures.

Accordingly, the present invention provides an apparatus for production of a bacteriophage preparation, the apparatus including:

a cassette including a matrix which can be loaded with a substrate suitable for growth of bacteria;

a casting module, into which a plurality of cassettes can be loaded with substrate;

an extraction module, into which a plurality of cassettes can be loaded for extraction of the bacteriophage lysate; wherein, in use, the cassette is loaded with substrate, inoculated with bacteria and bacteriophage culture, incubated and the cassette loaded into the extraction module for extraction of the lysate. Preferably, purification of the resultant solution occurs to produce bacteriophage product. Preferably, the cassettes are configured to be used substantially vertically. Preferably, a plurality of cassettes can be used vertically at the same time. Preferably, the use may be to load with substrate. The use may be to extract the lysate product. The substrate is preferably, a suitable gel. A holder may be used with the cassettes for the extraction stage. Most preferably, the apparatus is used to produce a commercially useful quantity of bacteriophage product.

Accordingly, the invention also provides a cassette for culture of bacteria, the cassette including:

a matrix for loading with a substrate for growing bacteria culture; wherein, cultures can be grown on the substrate and the cassette used vertically and the substrate be substantially maintained in the matrix, such as during extraction of a bacteriophage product.

Accordingly the invention also provides a cassette for culture of bacteria, the cassette including

a matrix for loading with a substrate for growing bacteria culture;

a pair of sheets for surrounding the matrix, on either side of the growing surfaces, whereby a space is maintained, wherein, cultures can be grown on the substrate and the cassette used vertically and the substrate be substantially maintained in the matrix.

Preferably, the cassettes can be used for loading substrate into the matrix. Preferably, the cassettes can be used for bacteria culture and ultimate extraction. Preferably, the matrix includes cells and it is the cells of the matrix in which substrate is retained to create a growing surface. Most preferably, the matrix has two growing surfaces once loaded with substrate, one on either side. One or more matrix may be included in the cassette.

Accordingly, the invention also provides a casting module for use with the cassettes of the invention, including a body into which cassettes can be placed substantially vertically before filling with substrate, which "sets" about the matrix ready for bacteria culture.

Accordingly, the invention also provides an extraction module for use with the cassettes of the invention including a body and one or more slots whereby cassettes can be positioned vertically through the slots and be immersed in extraction fluid within the body. Preferably, extraction can occur for a plurality of cassettes simultaneously. Preferably, the slots are formed in a top or cover of the extraction module. Preferably, the cassettes are suspended in extraction media within the extraction module.

Accordingly, the invention also provides a further method of bacteriophage production using a cassette including a matrix and an extraction module, the method including the following steps:

a) preparing a semi-solid medium, gel;

b) loading the prepared gel into the matrix of gel cassette;

c) inoculating the gel with bacteria and bacteriophage culture;

d) incubating the bacteria and bacteriophage culture until sufficient bacteria lysis is observable; and

e) placing vertically each cassette in extraction medium in the extraction module to extract the bacteriophage product.

Accordingly, the invention also provides a further method of the invention provides a method of preparing gel cassettes including the followings steps:

1. Loosening spring loaded screws on a sealing plate and remove from the gel casting module;

2. Putting the open body of gel casting module up on a bench top, or other suitable surface;

3. Placing one gel separation sheet into body of gel casting module so that it sits flush with the module bottom and back; 4. Positioning a pair of spacers, one on either side, with all edges flush with the sides and bottom of the gel casting module;

5. Placing one geometric matrix on top of half of the pair spacers ensuring that all edges are flush with the sides and bottom of the casting module;

6. Positioning the other halves of spacers on top of geometric matrix one on either side with all edges flush with the sides and bottom of gel casting module;

7. Placing one gel separation sheet on top of the spacers ensuring that all edges are flush with the sides and bottom of the casting module;

8. Repeating steps 3 to 7 until the desired number of gel cassettes have been added or gel casting module is full;

9. Guiding sealing plate into place and tightening spring loaded screws to achieve a complete seal with the gasket on gel casting module;

10. Standing gel casting module up, on a flat surface ready for introduction of gel.

Accordingly, the invention also provides a method of casting the cassettes including the followings steps:

1. Preparing suitable gel (agar or other) medium under sterile conditions (if this step has not already occurred);

2. Attaching one end of sterile silicon tubing of pipe arrangement to an inlet located at the bottom of sealing plat and the other end to container containing the gel;

3. Using a peristaltic pump, the gel is slowly introduce into gel casting module until the gel has reached the top of all gel cassettes; and

4. Allow full polymerisation of the gels at room temperature- at least 1 hour.

5. After the polymerisation of the gels has occurred the cassettes can be removed for use or storage.

Accordingly, a method of extraction of the bacteriophage product, using gel cassettes, holder, and extraction module with slotted lid is provided, the method including the following steps:

a) placing gel cassettes, in the holder; b) suspending the gel cassettes in holder vertically in the extraction module by sliding the holder into a slot in the lid;

c) Covering the gel cassettes in the holder with extraction media and allowing sufficient time for extraction to occur.

A purification, filtration step may follow for any of the disclosed methods.

INDUSTRIAL APPLICABILITY

The apparatus, cassettes and parts useful for the method of the invention may be manufactured industrially, and supplied for use to produce bacteriophage. The bacteriophage product itself is made in commercially useful quantities, such as for supply to the research and health industries.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in connection with a non-limiting preferred embodiment with reference to the accompanying drawings, in which:

Figure 1 is a view of a gel cassette casting module, according to a preferred embodiment of the invention:

Figure 1 a is a schematic perspective view of the gel cassette casting module of Figure 1 ;

Figure 1 b is a schematic view from above of the gel cassette casting module of Figure 1 a;

Figure 2 is a front view of an example geometric matrix of the gel cassette of a preferred embodiment of the invention for use in the casting module of Figures 1 , 1 a and 1 b, with gel cast onto the matrix represented by black circles;

Figure 3 is a schematic perspective view of the components of the assembled gel cassette, according to the preferred embodiment of the invention including two sheets, geometric matrix and spacers, and less the gel for ease of illustration, as used in the casting module of Figures 1 , 1 a and 1 b; Figure 4 is a perspective front view of the peristaltic pump of gel for filling the gel cassette of Figures 2 and 3 in the casting module of Figure 1 to produce the gel cassette ready for use;

Figure 5 is a front view of the assembled gel cassette of Figures 2 and 3, after filling with gel in the gel cassette casting module of Figures 1 , 1 a and 1 b, with the peristaltic pump of Figure 4, ready to be inoculated with bacteria and bacteriophage culture;

Figure 6 is the cross-sectional view through line A-A of the assembled gel cassette of Figure 5, illustrating the clip;

Figure 7 is a front view of an open cassette holder, of the preferred embodiment of the invention, with the assembled, inoculated and incubated gel filled cassette of Figure 6, in place ready for immersion and extraction;

Figure 8 is a schematic perspective view of the extraction chamber with gel cassettes hanging vertically into the extraction medium; and

Figure 9 is a flow diagram of the steps of the method of bacteriophage production according to a preferred method of the invention.

DETAILED DESCRIPTION OF THE INVENTION INCLUDING A BEST MODE

Referring to Figures 1 , 1 a, and 1 b to 9, a preferred embodiment of the invention will be described, and corresponding method of use, where gel cassette casting module 10 has body 12 and sealing plate 14. Gel cassette casting module 10 is used to fill or cast gel cassettes 16 with a suitable gel medium for the particular bacteria and bacteriophage culture of interest. The gel used may be an agar or similar gel. Clearly, there would be different suitable mediums used for different preparations, as would be understood by a person skilled in the art. The gel itself is not shown for ease of illustration, with the exception of Figure 2.

As illustrated the dimensions of gel cassette casting module 10 are length 320 millimetres, height 230 millimetres with a width of 120 millimetres. These dimensions are useful but clearly these dimensions may be varied to suit the particular needs of the user. The illustrated dimensions of gel cassette casting module 10 are suitable to allow up to sixteen, 2 millimetre thick gel cassettes 16, to be cast simultaneously. Alternatively, thirty-two 1 millimetre thick gel cassettes may be cast instead. In either case, it is beneficial that many gel cassettes can be cast in a single action, improving efficiency. Use of the gel cassettes in a vertical orientation enables numerous cassettes to be cast, at the same time, a great benefit.

Base 18 of gel cassette casting module 10 includes V-shaped groove 20 to assist in filling with gel. Inclusion of V-shaped groove 20 enables filling of module 10 from the bottom to ensure even filling and avoid bubble formation. More than one V- shaped groove 20 may be included instead, for example a pair of V-shaped grooves may be included one towards either side of base 18, in another form of the invention. Peristaltic pump 24, refer Figure 4), of a known form is used to fill module 10, including container 26, for containing the gel medium, and pipe arrangement 28. Module 10 is filled from the bottom via peristaltic pump 24, in the preferred embodiment of the invention, for best loading of the cassette with gel. Filling of module 10 could also occur via the top, by means of pouring the gel medium into module 10. Module 10 as illustrated is made of polypropylene, as is a particular suitable material, and readily cleaned. In this way, module 10 is readily reusable after sterilisation which can be achieved easily, as there is no large pieces of equipment to clean and sterilise. Module 10 can be made from any material able to be sterilised by autoclave or chemical sterilisation, including other plastics and metal.

Referring to Figure 3 in particular, module 10 is closed by means of captive spring loaded screws 30, received in corresponding holes 32; only some of screws 30 and holes 32 are labelled for ease of illustration. Use of spring loaded screws 30, in holes 32, enables module 10 to be sealed so sealing plate 14 is tightly held against body 12. Inlet 34 is used with peristaltic pump 24 to fill module 10 with gel medium by attachment to pipe arrangement 28, from the bottom, again refer Figure 4. In this manner a number of gel cassettes 16 can be cast at the same time as is illustrated in Figures 1 a and 1 b. Figures 1 a and 1 b show casting module 10, illustrated schematically, showing how the many gel cassettes are loaded vertically, for very space efficient casting. Each of the gel cassettes as illustrated includes geometric matrix 36 of cassette 16, as is illustrated in Figure 2. The cast gel is not shown in most of the drawings, except for Figure 2 for the ease of illustration. The geometric form of matrix 36 is very useful to ensure stability of the gel, in particular when held vertically. Square cells are shown, but other geometric shapes such as circular or hexagonal cells could be used instead. As shown in Figure 2 gel forms around the matrix, on both sides providing a medium for bacteria and bacteriophage growth. The matrix provides an efficient and stable base to which the gel adheres, useful for the invention. In alternative forms of the invention another form of matrix may be used instead to which the gel is cast and adheres. Paper may be used instead of the plastic geometric matrix. In these forms of the invention less water is used and the cost of overall production is reduced.

As disclosed gel cassette 16 is made of a disposable plastics material, removing the need for sterilisation after use. Other materials may be used such as other paper, plastics or metals, for example, suitable for sterilisation by autoclave or chemically, in a reusable form of the invention, or being disposable. Each cassette 16 has dimensions as follows 270 millimetres long, 200 millimetres high with a width of 1 millimetres thick, as is suitable for the described embodiment. Other dimensions can be used, and the other parts of the invention adapted accordingly, to suit a particular use or user, for example.

Clips 37, only one of which is labelled, are used to clip and hold geometric matrix

36 between gel separation sheets 38 and 39, with spaces between the layers. Clip

37 also assists to keep the loaded gel off the bench top during the inoculation process. Clip 37 is of a known form that has two sides bias towards one another to firmly hold together several sheets of material. As shown, clip 37 is made of a plastic that may be readily sterilised in the usual manner. Gel separation sheets

38 and 39 separate individual gels, so they do not contact one another and enable gels to be removed singularly as described further below with the steps of the method. Gel separation sheets 38 and 39 can be made from any disposable plastic, able to be sterilised by autoclave or chemically. As illustrated gel separation sheets 38 and 39 have dimensions of length 270 millimetres, a height of 200 millimetres and a width of 200 micrometre. The particular size and shape can be varied with the gel cassette to which the sheet is to be applied. The thickness of 200 micrometres is useful, but may also be varied to suit a particular application.

Pairs of gel cassette spacers 40 and 41 , one pair on either side of the cassette, are also included to ensure even distribution of gel around gel cassette 16. Gel separation sheets 38 and 39 and spacers act to create a space around geometric matrix 36 to enable gel filling during casting and to prevent contact with other parts of the cassette. As illustrated, gel cassette spacers 40 and 41 are made from disposable plastic, able to be sterilised by autoclave or chemical, for example polypropylene. Gel cassette spacers 40 and 41 as shown have dimensions 1 10 millimetres, height 210 millimetres width 15 millimetres and are 1 millimetre thick; other suitable dimensions may be used and the other parts of the apparatus adapted accordingly. In an alternative form of the invention gel cassette spacers 40 and 41 can be made integrally with the other parts of the cassette.

Referring to Figures 7 and 8 in particular, gel cassette holder 42 is shown, in an open state with a filled gel cassette 16 in place. Gel cassette holder 42 is used to encase the gel loaded, inoculated cassette 16 for immersion in extraction fluid. Cassette holder 42 has two mirror image sides, 44 and 46, with pairs of hangers 48 and 50 and numerous openings 52. Parts 44 and 46 of cassette holder 42 are held together by hinge 54 at the bottom enabling the gel loaded inoculated cassette 16 to be held firmly in place during extraction. Extraction takes place when gel cassette holders 42 are slid vertically into extraction module 56 through slots 58. Extraction module 56 is filed with suitable extraction medium 60 to a level marked 62 to cover gel cassette 16 within holder 42, as discussed in further detail below. Again cassette holder 42 is made from any disposable plastic, able to be sterilised by autoclave or chemical. Other materials including other plastics or metals can be used instead. Each cassette holder has the following dimensions, length 400 millimetres and height 250 millimetres. Again these dimensions can be varied to suit the rest of the apparatus.

Extraction module 56 is made of a food grade plastic suitable for sterilisation by autoclave or chemical sterilisation. Up to 23 gel cassettes 16 can be placed in the extraction module, such that hangers 48 and 50 suspend cassettes 16 away from the floor of the module. The size of the extraction module can be varied to vary the number of cassettes. The free hanging arrangement is efficient to enable extraction of all the cassettes at the same time with minimal extraction medium, therefore leading to high yield and high concentrations of bacteriophage product. If 23 cassettes are used, approximately 60 litres of extraction medium 60 is used to fill the module to fill level 62.

A key advantage of the invention is to enable production in a laboratory, or other smaller scale operation, of the large scale commercial culture of bacterium and bacteriophage on a semi-solid medium (agar, gel or other). Specific semi-solid medium developed to provide optimum growth conditions will be used for each bacterial species and associated bacteriophage leading to a high titre bacteriophage preparation, significantly reducing the production volume and eliminating the need to use large biofermenters.

The propagation of small quantities of bacteriophage on solid and in semi-solid media, at laboratory level, is known in the field. International patent application PCT/IL2003/001041 in the name of BUJANOVER, published as WO/2004/052274, discloses the combined use of semi-solid and solid hydrocolloid medium for the increased production of bacteriophage. The BUJUNOVER patent application is, however, fully directed to use of specific hydrocolloid concentrations, to improve production. For example, the primary claim for monopoly requires a hydrocolloid concentration of below 0.5%. The subject invention, is distinctly different, in use of a cassette, used vertically, on which the gel medium is loaded, before inoculation, incubation and extraction of the bacteriophage product. The product produced by the method of the invention is significantly improved in terms of reduced volume but increased bacteriophage concentrations. The invention, as described, in its new method and apparatus in its various forms produces improved bacteriophage yield, a significant advance over the prior art.

The method of the subject invention, as is listed in the steps in Figure 9, is a straightforward method, which produces a high yield of bacteriophage product. As illustrated, production of bacteriophage preparations with titres in excess of 10 ¹¹ pfu/ millilitres (plaque forming units) on semi-solid medium can be produced. The use of such an easy to use, efficient method is one of the many strong advantages of the subject invention over the prior art.

Taking each step in turn, first a preparation of a semi-solid medium, a gel such as agar, is made, suitable for the particular bacteria strain used. The preparation of gel for incubation of bacteria is well known in the art. A gel concentration of 1-1.5% is used for all bacteria strains.

Second, the prepared gel is loaded into geometric matrix 36 of gel cassette 16. Use of gel cassette 16 provides significant and surprising advantages over the prior art, for example as the medium will not fall out when placed vertically. Using gel casting module 10, the medium is prepared, and pumped into gel casting module 10 which has been loaded with one or more gel cassettes 16. The gel is allowed to solidify around the individual gel cassettes, so that gel forms in the geometric matrix 36, refer Figure 2, ready for use

The method of preparing gel cassettes 16 in casting module 10 can summarised as follows: 1. Loosening spring loaded screws 30 on sealing plate 14 and remove from the gel casting module 10;

2. Putting the open body 12 of gel casting module 10 up on a bench top, or other suitable surface;

3. Placing one gel separation sheet 38 into body 12 of gel casting module 10 so that it sits flush with the module bottom and back;

4. Positioning spacers 40 and 41 , one on either side, with all edges flush with the sides and bottom of the gel casting module 10.

5. Placing one geometric matrix 36 on top of half of the pair spacers 40 and 41 ensuring that all edges are flush with the sides and bottom of the casting module;

6. Positioning the other halves of spacers 40 and 41 on top of geometric matrix 36, one on either side with all edges flush with the sides and bottom of gel casting module 10;

7. Placing one gel separation sheet 39 on top of spacers 40 and 41 ensuring that all edges are flush with the sides and bottom of the casting module 10;

8. Repeating steps 3 to 7 until the desired number of gel cassettes 16 have been added or gel casting module 10 is full;

9. Guiding sealing plate 14 into place and tightening spring loaded screws 30 to achieve a complete seal with the gasket on gel casting module 10;

10. Standing gel casting module 10 up, on a flat surface ready for introduction of gel.

The method of casting gel cassettes 16, from the bottom of gel casting module 10 can be summarised in the following steps:

1. Preparing suitable gel (agar or other) medium under sterile conditions (if this step has not already occurred);

2. Attaching one end of sterile silicon tubing of pipe arrangement 28 to inlet 34 located at the bottom of sealing plate 14 and the other end to container 26 containing the gel; 3. Using peristaltic pump 24, the gel is slowly introduce into gel casting module 10 until the gel has reached the top of all gel cassettes 16; and

4. Allow full polymerisation of the gels at room temperature- at least 1 hour.

After the polymerisation of the gels has occurred the cassettes 16 can be removed for use or storage. To do so sealing plate 14 is removed after loosening screws 30, so gel cassettes 16 can be lifted out. Individual cassettes 16 may be lifted out or the whole stack lifted as one. Once lifted from gel casting module 10, sheets 38 and 39 can be removed from the individual gel cast cassettes. If necessary a long flat blade may be used to separate sheets 38 and 39 and cassettes 16. If the gel loaded cassettes are not be used immediate they are stored in sterile tightly sealed containers or plastic bags and maintained at 4 degrees Centigrade for up to one month.

During casting the gel medium solidifies around geometric matrix 36, in and around the matrix structure. The geometric matrix 36 loaded with gel provides two culture surfaces, one on each side of gel cassette 16, effectively doubling the culture area and thus reducing the extraction media volume whilst maintain a high bacteriophage titre. Clearly, use of cassette 16, loaded with gel and the increase surface area is one of many advantages of the subject invention over the prior art. The vertical stability of the gel once loaded into the geometric matrix is another clear and significant advantage.

Third, the gel surface on both sides of geometric cell matrix 36 is inoculated with a bacteria and bacteriophage culture. The culture and mix can be varied to suit the desired end product. The bacteria and bacteriophage combination is spread on both surfaces of the solid medium creating a lawn and incubated until bacterial lysis occurs. As illustrated the incubation step is 16 hours, as is suitable, but this can be varied to obtain maximum bacterial lysis and maximise product yield. The method of inoculation of the gel surfaces with bacteria and bacteriophage culture can be summarised in the following steps, all being performed under sterile conditions:

1. Allowing the required number of gel loaded cassettes 16 to come to room temperature (if taken from the refrigerator);

2. Combining the appropriate concentration of bacteria and bacteriophage in minimal media and allow to bind for an appropriate time, for example for Vibrio species and Vibrio bacteriophage a binding time of 10 to 15 minutes. The concentration of bacteria and bacteriophage and binding time will differ with bacterial species and previous laboratory based trials are undertaken to provide this information;

3. Removing an individual gel loaded cassette and attaching clips 37 to the four corners. Clips 37 are used to elevate and keep the gel surface clear of the bench top;

4. Inoculating the top surface of geometric cell matrix 36 with bacteria and bacteriophage culture to produce a very thin even coat ("lawn").

5. Allowing the lawn to dry completely;

6. Turning gel loaded cassette 16 over and repeat points 4 and 5, to inoculate the other surface;

7. Repeating steps 2 to 6 until the desired number of inoculated gel loaded cassettes 16 have been prepared; and

8. Incubating cassettes 16 at optimum temperature for example 37 Centigrade for clinical samples, for bacterial growth for 16 hours or until maximum lysis is obtained.

Fourth, is extraction of the free bacteriophage particles, by placing gel cassettes 16, in holder 42 and suspending vertically in extraction module 56. The positioning of geometric matrix 36 within holder 42 is such that matrix 36 is fully covered with extraction media 60, to maximise extraction. Extraction module 56 can support 23 gel cassettes 16 with a surface area of 888cm ² per cassette, having the dimensions as described, and 60 litres of extraction media 60. The cultures are incubated at room temperature for 3 or 4 hours.

The extraction is followed by filtration and purification, the fifth step, where the crude bacteriophage filtrate is pumped from extraction module 56 through a series of commercial filters to ensure total eradication of all bacterial debris. Purification of toxins and other undesirable components is undertaken. Due to the nature of the culture conditions, and compared to other bacteriophage production methods, only very low quantities of bacterial debris are produced reducing the downstream processing costs.

The method of extraction of the bacteriophage product is summarised in the following steps:

1. Placing individual inoculated gel loaded cassettes 16 in gel cassette holder 42 and closing both sides 44 and 46 together so gel cassette 16 is contained therein;

2. Loading gel cassette holders 42 into extraction module 56 by inserting holders 42 through slots 58 in the extraction module lid;

3. Adding 60 litres of extraction media 60 to extraction module 56 to completely cover all inoculated gel loaded cassettes 16 in holders 42.

4. Allowing to stand at room temperature for 4 hours;

5. Removing through use of a pump extraction media containing a high titre of bacteriophage (bacteriophage lysate) and pass through a series of commercial filters to extract all bacterial debris, toxins and other undesirable components; and

6. Purification of bacteriophage product, using methods widely known in the art. The resultant bacteriophage product is a high yield product with little by-product, very useful to researchers and for bacteriophage therapy. The uses of the resultant product are too numerous to list but are generally discussed above in the background, giving some of the very many beneficial advantages. Clearly, being able to readily produce high yield bacteriophage product, without the need for a biofermenter or other large pieces of equipment, will enable further research and rapid application and availability of bacteriophage therapies in the future. It is hoped that the method and apparatus of the invention will be the industry standard once the information is made publicly available.

It will be apparent to a person skilled in the art that changes may be made to the embodiments disclosed herein, and the method and apparatus specifically described without departing from the spirit and scope of the invention in its various aspects.

REFERENCE SIGNS LIST:

10 Gel cassette casting module 44 First part of 42

12 Body of 10 46 Second part of 42

14 Sealing plate of 10 47 Hanger of 42

16 Gel cassette 48 Hanger of 42

18 Base of 10 49 Hanger of 42

20 V-shaped groove 50 Hanger of 42

24 Peristaltic pump 52 Openings of 42

26 Container of 24 54 Hinges of 42

28 Pipe arrangement of 24 56 Extraction module

30 Spring loaded screws of 10 58 Slots in extraction module

32 Holes of 10 60 Extraction medium

34 Inlet of 10 62 Level of top of extraction medium

36 Geometric matrix of 16

37 Clip of 16

38 Gel separation sheet of 16

39 Gel separation sheet of 16

40 Spacer of 16

41 Spacer of 16