WO2014001504A2

WO2014001504A2 - Culture of bacteriophage

Info

Publication number: WO2014001504A2
Application number: PCT/EP2013/063633
Authority: WO
Inventors: Michael Mattey
Original assignee: Fixed Phage Limited
Priority date: 2012-06-28
Filing date: 2013-06-28
Publication date: 2014-01-03
Also published as: WO2014001504A3; GB2516581A; GB2516581B; GB201211497D0; GB201420253D0

Abstract

Bacteriophage are produced in a continuous process comprising maintaining a culture of bacteriophage in a first vessel (16); feeding the culture with bacteria maintained in a second vessel (12a, 12b) in the stationary phase and harvesting bacteriophage from the first vessel (16), wherein the first vessel is operated at a dilution rate in excess of 1 / (bacteriophage lysis time).

Description

Culture of Bacteriophage

Field of the Invention

The present invention relates to manufacture of bacteriophages, in particular to manufacturing methods that are suitable for large-scale production purposes, and to apparatus therefor.

Background to the Invention

In recent years, as resistance to conventional antibiotics has continued to grow and the application of chemical biocides becomes increasingly unacceptable on environmental grounds, attention has turned to alternative methods for control of bacterial contamination problems.

One promising approach involves the application of bacteriophages, being naturally occurring ubiquitous viruses that are harmless to humans, animals, plants and fish but lethal for bacteria. Bacteriophages are specific and will infect only particular bacterial types, with several sanitation products now on the market against pathogens such as Salmonella and Listeria. In the laboratory bacteriophage are routinely grown on single plates as and when required. For larger volumes of bacteriophage, it is known to manufacture bacteriophage by a batch process. It is believed that bacteriophage cannot infect bacteria in the stationary phase and, accordingly, in batch manufacture a log-phase growing culture of bacteria is established and then seeded with bacteriophages. After allowing time for infection and lysis throughout the whole culture, the liquor containing bacteriophages is harvested in readiness for purification.

Batch culture is effective but generally requires much equipment for a relatively small output of bacteriophage. Running times are also low as equipment must be cleaned and prepared between each batch. Moreover these costs are multiplied where several bacteriophage strains are required: for some proposed applications of bacteriophage technology, multiple bacteriophage strains are needed, e.g. to provide a mixed coating on a wound dressing. At present the only way to do this is using multiple, separate batch cultures. An alternative culture method is known, though designed less for scale and more to elucidate the developmental relationship between host bacteria and the bacteriophages; using co-cultures of bacteria and bacteriophage (De Haan, Winkler, & Felix, 1955; Mizoguchi et al., 2003; Schwienhorst, Lindemann, & Eigen, 1996). In these examples nutrient media is introduced into a mixture of bacteria and bacteriophages and results in co-growth in culture, essentially as a batch culture.

US 2012/0040329 A (Baldwin et al.) discloses a method of producing bacteriophage by continuous flow-controlled methods, feeding both bacteriophage and bacteria into a proliferation chamber. These require complex control of multiple elements in the process and are not suitable, however, for large scale production of phage.

Lindemann et al. (2002) Journal of Virology 76:5784-5792 describes a system for studying the evolution of bacteriophage and discloses a system wherein log-phase bacteria containing prophage are cultured in a turbidostat and then transferred to a second reactor where the phage are induced to become lytic before these phage are transferred to further reactors containing bacteria to be infected with the phage.

Schwienhorst et al. (1996) Biotechnology and Bioengineering 50:217-221 describes a system for studying the growth kinetics of bacteriophage fed with exponentially growing bacteria in a turbidostat, over the course of four latency periods, and time limited to a little less than four hours in total.

Husimi et al. (1982) Review of Scientific Instruments 53:517-522 describes a system for studying mutation and selection of bacteriophage in a cellstat fed by log-phase bacteria from a turbidostat.

It is desired to achieve higher production volumes, without having to introduce bacteriophage manipulations. A scaleable process is desired, as is production with reduced cost. It is known or feared, however, that long-term or repeated culture can promote isolation of bacteriophage - resistant bacteria. - -

An aim of the present invention is to provide an alternative method of bacteriophage production, and an aim of particular embodiments of the invention is to provide improved bacteriophage production and apparatus therefor. Summary of the Invention

Accordingly, the present invention provides a continuous method for production of bacteriophage, comprising:

maintaining a culture of bacteriophage in a vessel;

feeding the culture with bacteria; and

harvesting bacteriophage from the vessel.

The invention hence provides a method for production of bacteriophages, wherein bacteriophage production is continuous and suitable for long-term, high-yield phage production. In embodiments of the invention, and as described in examples below, the present invention is not a continuous flow process. Further, the method is preferably operated such that the phage in the vessel are in excess compared to the bacterial content of the vessel.

Also provided by the present invention is apparatus suitable for the methods described above, being suitable for continuous production of bacteriophage.

Details of the Invention

Bacteriophage can thus be grown in a continuous process, using a bacterial feedstock. The method may comprise feeding the culture with bacteria from two or more bacterial fermenters. As described below, the individual fermenters may contain the same or different bacteria. The method may also comprise continuously feeding the culture with the bacteria (whether from one or more bacterial fermenters). This facilitates production where, for example, the growth rates respectively of the bacteria in fermenters and of the bacteriophage are such that increased rates of input of bacteria are needed. The feed bacteria are suitably grown in continuous culture, optionally with continuous feeding, agitation and aeration. Hence, in embodiments of the invention there are provided parallel continuous cultures of bacteria and bacteriophage. As described below in more detail in specific embodiments, valves or other devices can be provided so that bacteriophage do not contaminate the bacterial feed, enabling long-term operation of the methods and long-term bacteriophage production.

The methods of the invention enable long-term culture and harvesting of bacteriophage. While continuous culture may in theory be unlimited, in practice the culture can become contaminated, e.g. by undesirable changes in the product or by debris. The methods can be carried out continuously for 5 hours or more, for 10 hours or more, for 2 or more days, for 4 or more days, for a week or more, for 10 days or more, for 2 weeks or more, for 1 month or more and for 2 months or more. The invention hence provides methods that are genuinely continuous and long term, to be contrasted with the prior art in which phage is grown only for a short period, such as over 4 latency periods of growth in Schweinhorst, et al.

It is optional to feed the bacterial feedstock directly into the bacteriophage culture vessel, also referred to as the lysis vessel (as in that vessel there is infection of the bacteria and subsequent lysis and production of bacteriophage). It is further optional to combine bacterial nutrient medium with the bacterial feed prior to transfer to the lysis vessel or at the same time as feeding bacteria into the lysis vessel. The method may comprise feeding the culture with stationary phase bacteria. The method may alternatively comprise feeding the culture with bacteria in the logarithmic phase.

It is preferred that the method of the invention comprises feeding bacteria that are not in log phase growth but are instead in late log-phase growth or preferably substantially in the stationary phase into the culture vessel. To achieve this, the bacterial culture fed into the vessel may be nutrient limited, e.g. in the method a portion of the feed culture containing bacteria is withdrawn and replaced with nutrients that are consumed at a rate higher than the rate at which they are replaced. This contrasts with prior art methods that are not continuous and in which, additionally, bacteria in logarithmic phase are combined with phage. This also contrasts with prior art methods that use a turbidostat for bacterial culture. A turbidostat is a continuous culture device that has feedback between the turbidity of the culture vessel and the dilution rate; in operation, bacteria are removed at their - - growth rate, so the turbidity of the culture remains constant, with bacteria in the log phase. Typically, the dilution rate varies over time as a result of feedback mechanisms so that log-phase growth is achieved constantly. Methods of the invention, however, preferably grow feed bacteria in a chemostat in the stationary phase. Chemostats as used in preferred embodiments have a fixed volume and flow rate, and thus a fixed dilution rate.

The method may be operated continuously and suitably includes one or more process control steps. The method may include monitoring of the bacteriophage harvested and modulating the bacterial input to the vessel. Based on the yield of bacteriophage, the bacterial input may be increased to increase the yield or otherwise modified to obtain improved ratio of product to feedstock.

The continuous cultures of the invention are also suitable for maintaining a culture of a plurality of bacteriophages in a vessel; that is to say bacteriophages of different types, either different bacteriophages that infect the same bacteria or different bacteriophages that infect different bacteria. The invention thus enables processes that produce a mixed bacteriophage product. This is an advantage when multiple bacteriophage types are needed, e.g. to provide protection against multiple pathogenic bacteria.

Following the invention it is possible to produce a mixed population of bacteriophage, infecting different bacteria, wherein the individual types of bacteriophage are present in minimum amounts. An estimate of the respective inputs of the different bacteria can be made based upon the burst size for each bacteriophage / bacteria combination. An embodiment of the invention comprises monitoring the bacteriophages harvested and modulating the feeding of the respective bacterial inputs. This enables a balance between bacteriophages to be maintained. By monitoring the ratio of the bacteriophage that infect different bacterial strains in the harvested material and modulating the feeding of the different bacterial strains a desired product can be obtained. The mixed cultures suitably contain 2 or more bacteriophages, generally up to 10 different bacteriophages, meaning generally up to 10 different bacterial feeds. In practice this can be achieved as many different bacteria can be grown on common amino-acid based growth medium. Typically, a - - mixed culture of bacteriophage of the invention has from 2 to 6 different bacteriophage strains, preferably from 2 to 4.

Methods of the invention can be carried out at dilution rates that enable continuous culture. Generally, dilution rates in the range 0.1 to 10 per hour are believed workable, or more usually from 0.3 to 3 per hour. In examples, we have used dilution rates in the range from about 0.5 to about 2.6 per hour and above. Monitoring of the method can comprise modulating the dilution rate to increase bacteriophage yield. It is expected from the art that dilution rates must be monitored to avoid washout of phage from the lysis vessel, and that based on the known phage lysis time (average time to lysis after initial contact with phage) the dilution rate for phage growth should be no more than, and should preferably be less than, 1/(lysis time). Hence, for a lysis time of 30 minutes (½ hr) the dilution rates should be 2 hr ^"1 or less. In preferred methods of the invention, however, and contrary to this dogma, to achieve increased and more commercially viable yields of phage, higher dilution rates are used than hitherto would have been believed to be operational. Thus it is preferred to carry out the methods at a dilution rate (i) greater than 1/(lysis time), (ii) more preferably (1 .1 )/(lysis time) or greater, or (iii) (1 .25)/(lysis time) or greater. As a worked example, therefore, with a lysis time of 30 minutes, preferred dilution rates are (i) greater than 2 hr ^"1 , (ii) 2.2 hr ^"1 or greater, or (iii) 2.5 hr ¹ or greater. In a specific embodiment of the invention described in an example below, culture of a phage with a lysis time of about 30 minutes was carried out at a dilution rate of 2.6 hr ^~1 and above, stably producing phage in high yield. Further embodiments of the invention can be carried out at higher dilution rates, such as (1 .5)/(lysis time) or greater, or 2/(lysis time) or greater.

In preferred embodiments, the methods comprise growing feed bacteria continuously in a chemostat at a fixed dilution rate. Hence, in specific embodiments, described in examples below, bacteria are cultured in a chemostat at a fixed dilution rate and continuously fed into the lysis vessel which is operated at a fixed dilution rate.

For co-production of two bacteriophage in the same bacterial host, one or more chemostats are suitably operated at constant dilution rates, each generating stationary phage bacteria and are fed into a single lysis vessel operated at a fixed dilution rate. The lysis vessel may be operated at a dilution rate calculated in the ranges described elsewhere for the phage having the longer lysis time, and the dilution rate may also be greater than 1/(lysis time) of the phage with the shorter lysis time, especially if the respective lysis times do not differ substantially.

For co-production of two bacteriophage in separate bacterial hosts, separate chemostats are suitably operated at independent dilution rates, each generating stationary phage bacteria and are fed into a single lysis vessel operated at a fixed dilution rate. The lysis vessel may be operated at a dilution rate calculated in the ranges described elsewhere for the phage having the longer lysis time, and the dilution rate may also be greater than 1 /(lysis time) of the phage with the shorter lysis time, especially if the respective lysis times do not differ substantially. Harvested bacteriophage can be treated according to a variety of conventional means. The methods of the invention typically also include subjecting harvested bacteriophage to downstream processing comprising one or more or all of (i) purification, (ii) filtration, (iii) centrifugation, (iv) combining with a carrier and (v) packaging bacteriophage with or without carrier into a container. Harvesting can be continuous or batch-wise.

In use of the invention, medium for the bacterial culture can be continuously fed into the bacterial culture. Nutrient medium for the bacteria or for the bacteriophage can be recycled, as per known culture techniques. This can result in build up over time of potentially contaminating components, so with any such recycling it may be necessary to carry out continuous culture for a limited period then start afresh - as is known generally in this art.

The invention is suitable for bacteriophage production in general, without limitation to the bacteriophage strain, though preferably with lytic bacteriophage. The methods can operate with lysogenic phage, as there is typically a vast excess of phages in the lysis vessel and the feed bacteria supplied into the lysis vessel have not yet been exposed to the phages before so at least one round of infection and lysis occurs, and that is all that is needed to maintain the culture given the excess phages present. - -

In specific embodiments of the invention, the methods described are distinguished over the prior art by one or more of the features:

1 . The method of continuous culture is genuinely continuous;

2. Dilution rates in the continuous culture are greater than would be expected to be feasible from the art knowledge of phage growth characteristics (thus you can have a greater rate of production as you can run the culture vessels at a higher dilution rate than would be predicted otherwise and the culture is still stable); and

3. The methods feed the phage containing vessel with stationary phase bacteria, when the prior art dogma is that these are not suitable for infection with phage and, instead, bacteria in the log phase of growth must be used.

Also provided by the present invention is apparatus suitable for the methods described above, being suitable for continuous production of bacteriophage. Apparatus of the invention, for production of bacteriophage, may comprise:

a first vessel for culture of bacteriophage; and

a second vessel for culture of bacteria and in fluid connection with the first vessel so that it can feed bacteria into the first vessel.

The apparatus may comprise two or more second vessels, all in fluid connection with the first vessel so that all can feed bacteria into the first vessel. The or each second vessel is preferably a bacterial fermenter in which bacteria can be continuously cultured.

A third vessel intermediate between the first and second vessels is optionally also included, capable of adding nutrient to the bacteria output from the or each second vessel prior to bacteria being fed into the first vessel. This enables addition of nutrient to the output of the bacterial fermenter(s) in case this aids infectivity of bacteria in the lysis vessel.

The apparatus is useful for continuous culture as described. For that purpose, it typically includes comprising one or more pumps, valves and/or pipework so that bacteria from the or each second vessel can be fed continuously into the first vessel. - -

In a typical use, bacteria are continuously fed into the lysis vessel and bacteriophage are continuously harvested therefrom.

A sampler can be provided, for extraction of a sample of bacteriophage from the first vessel for monitoring purposes. Preferably, for automated or semi-automated apparatus, control systems are incorporated into the machinery that modulate the feed of bacteria from the or each second vessel into the first vessel according to the content of the sample. For example, the control systems can monitor the bacteriophages harvested and modulate on that basis the feeding of the respective bacterial inputs. If too much of one bacteriophage is being produced at the expense of another then the ratios of bacterial feedstock can be changed to promote a more even ratio.

Apparatus of the invention can additionally comprise one or more or all of (i) a purification unit, (ii) a filtration unit, (iii) a centrifuge, (iv) a unit to combine bacteriophage with a carrier and (v) a unit to package bacteriophage with or without carrier into a container.

In use of the invention, continuous bacteriophage production can be achieved with improved efficiency. For example, with high dilution rates continuous bacteriophage lysis in excess of 2v/hr is possible. This means the output of a 20000L batch fermenter is achieved by a 200L continuous system. Assuming capital cost is proportional to size, this example represents a potential 100x reduction in cost. Conventionally, nutrient medium is pumped into a bacterial fermenter in a continuous stream and an equal quantity of medium and cells are removed. In essence this ensures a consistent ratio of bacteria and nutrient, relevant to maintaining growth conditions at steady rate. In particular embodiments of the invention, one nutrient in the medium is introduced at a concentration such that it is substantially entirely utilised by the bacteria during the average (fermenter) residence time of the bacteria, with the nutrient substantially completely denuded during bacterial cell growth. Bacteria leaving the fermenter are therefore essentially in the stationary phase with growth tailing off as that nutrient is exhausted. In preferred methods of the invention, - - the culture of bacteriophage is carried out continuously with parallel continuous culture of feed bacteria under nutrient-limiting conditions.

In particular embodiments, for carrying out methods of the invention as described elsewhere herein, the apparatus comprises a chemostat for growth of bacteria to be fed into the lysis vessel. Preferably, hence, the apparatus comprises a chemostat operating at constant dilution rate for growth of bacteria in the stationary phase, for feeding into the lysis vessel operating at constant dilution rate. The present invention advantageously provides a manufacturing system whereby a continuous output of bacteriophage may be obtained. The method can utilise appropriate bacteria from a continuous bacterial culture as a nutrient source, the bacteria being harvested from the continuous system and mixed at appropriate dilution with bacteriophages.

In examples described below, bacteria were fed into a fully developed bacteriophage culture and infection was more or less immediate and at a very high MOI (multiple of infection) making it efficient. In the cultures carried out, initially phages were provided from an external source: phage may be introduced directly into the lysing vessel or into a pre-mixing chamber prior to discharge into the lysing vessel. Once bacteriophage manufacture was underway the lysis vessel was full of bacteriophage; while bacteriophage was withdrawn the bacteriophage supply was replenished by lysis of phage-infected bacteria and this ensured a continuous supply of bacteriophage for the bacteriophage infection step. Bacteria were destroyed in the lysis vessel and withdrawn material contained phage but no live bacteria.

The ratio of bacteriophage to bacteria was initially relatively low but changed rapidly thereafter to a very high ratio; this ensured bacterial lyses occured rapidly and effectively and so maintained a steady state in the lysing vessel.

In examples carried out, bacteria harvested from the continuous culture vessel were in the late logarithmic or stationary growth phase. Their growth may be reactivated in media and soon after harvesting in an activation chamber and prior to introduction into a mixing chamber or in the mixing chamber itself or as the bacteria/phage - - mixture is discharged into the lysing vessel. The precise timing and ratios vary according to bacteriophage.

In general the system provided and enabled by the invention can run continuously, in the sense it can run in a substantially steady state. There can be continuous harvesting and/or feeding or the culture can be run until such time that sufficient bacteriophage has been produced.

The methods of the invention are believed suitable for bacteriophage culture without limitation provided that the bacteriophage is obtainable and its host or target bacteria can be cultured and infected in culture. The bacteriophage can be ssRNA, dsRNA, ssDNA, or dsDNA bacteriophage, with either circular or linear arrangement of the genetic material, and which infect cells of bacteria. The suitable bacteriophage include Myoviridae, Siphoviridae, Podoviridae, Lipothrixviridea, Rudiviridae, Ampullaviridae, Bacilloviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae, Fusseloviridae, Globuloviridae, Guttavirus, Inoviridae, Leviviridae, Microviridae, Plasmaviridae and Tectiviridae.

Examples of potential pathogens the bacteriophage for which may be cultured according to the invention include Streptococci, such as Streptococcus pyogenes; enterococci, such as Enterococcus faecalis; Staphylococci, such as Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus (MRSA); Pseudomonas aeruginosa; Enterobacter species; Escherichia coli; Klebsiella species; Proteus species; Bacteroides; Clostridium; yeasts such as Candida; and Aspergillus.

The invention is now described in more detail in the specific embodiments set out below, which demonstrate that, contrary to expectations, it is possible to attain continuous bacteriophage manufacture. The invention is illustrated with reference to the accompanying diagrams in which:

Fig. 1 shows a schematic diagram of apparatus of the invention for carrying out continuous bacteriophage culture;

Fig. 2 shows the results of bacteriophage production in continuous culture at different dilution rates; - -

Fig. 3 shows a bacteriophage continuous culture at a dilution rate of 2.61 hr , Fig. 4 shows bacteriophage particles at any dilution rate of bacteriophage continuous culture (the concentration of bacteriophages decreases with increasing dilution rate); and

Fig. 5 shows a bacteriophage growth curve of bacteriophage produced in bacteriophage continuous culture at dilution rate 2.61 hr ^"1.

Examples Fermenter Design

Referring to figure 1 , a fermenter system 10 as shown schematically was designed and made for the purpose of the experiments. Two bacterial chemostat fermenters (12a, 12b) were arranged in parallel to provide feedstock of bacteria (Staphylococcus aureus strain 8588, being susceptible to bacteriophage K, was used), with continuous medium feed from the reservoir 14 to provide continuous culture of bacteria. The number and size of such bacterial fermenters can be varied in other system designs. For this example, two bacterial fermenters were used because of the anticipated volume of fermenter output needed for the next stage of the system. The output from these two fermenters was fed into a bacteriophage lysis tank 16. An intermediate nutrient addition tank 18 was positioned for optional input of nutrient medium into the bacterial feed, prior to transfer of bacteria into the lysis fermenter. The output from the bacteriophage lysis tank was directed to a holding tank 20 and into downstream processing. Both continuous and batch harvesting was carried out and then subsequent purification was carried out for both continuous and batch processes.

Bacteriophage continuous culture

Initiation

A batch culture of bacteriophage K with the following operating parameters was used to start the process: temperature 37°C, agitation 200 rpm, aeration 1vvm, initial pH 7.0, working volume 0.6 L.

A 2% inoculum from an overnight bacterial culture was inoculated into 0.75 L of modified M9 medium with 5% yeast extract as carbon source and grown to late log - - phase. Bacteriophages were introduced into the culture, the agitation decreased to 80 rpm and the culture monitored until complete lysis occurred.

Continuous culture

A continuous culture was established as described herein, in which the operating parameters were as follows: temperature 37°C, agitation 80 rpm, aeration 1 vvm, initial pH 7.0, working volume 0.6 L. The dilution rate (D) for the final bacteriophage lysis tank was between 0.50 and 2.61 hr ^"1. Continuous culture was achieved via a fixed volume system in which a feed was continuously pumped in up to a volume of 0.6 L and another pump continuously removed excess liquid via a tube placed at a fixed height. The dilution rate for the bacteriophage lysis stage was determined by the pump rate for the input into the bacterial continuous culture - see Table 1 .

The continuous culture was periodically sampled to determine the concentration of bacteriophages by counting particle per milliliter using a NanoSight (Model LM10, NanoSight Ltd., Salisbury, UK). The results are shown in Figures 2-5. The concentration of bacteriophages with time is shown in Figures 2 and 3; the time to reach steady state depends on the dilution rate.

No wash out was observed in continuous culture even at a dilution rate of 2.61 hr^"1 , at which dilution rate the bacteriophage lysis time is significantly exceeded. The latent period for bacteriophages isolated from the high dilution rate culture (2.61 hr^"1) was measured. The result of one-step growth curve shown in Figure 5 indicates that the lysis time (latent period) was from 25-35 minutes, which was the same as the latent period of the bacteriophages initially so that the accepted explanation is incorrect. With a conventional bacterial culture one bacterium divides into two, so wash out occurs when the average division time is exceeded. However, with bacteriophages a single lysis may result in 30 or more progeny so that even a few lysis events, for example the few at 20 minutes will be sufficient to maintain the steady state. In this particular case, surprisingly and against conventional wisdom in this field, provided 1/30^th of the culture lysed before removal from the culture vessel - - a steady state can be maintained. The methods were thus stably performed, with high yield at dilution rates significantly in excess of 1/(lysis time).

The invention thus provides continuous culture and hence continuous production of bacteriophage.

- -

Table 1 Parameters for bacteriophage production in continuous culture

Residence Time Time Working volume (V) Feed Dilution

(T) Rate Rate

(hr) (min) (L) (L/hr) (hr^"1)

2.00 120 0.6 0.300 0.50

1 .50 90 0.6 0.400 0.67

1 .00 60 0.6 0.600 1 .00

0.67 40 0.6 0.900 1 .50

0.38 23 0.6 1 .565 2.61

Refs

De Haan, P., Winkler, K., & Felix, H. (1955). Phage reproduction in relation to bacterial growth rate. Antonie van Leeuwenhoek, 27(1 ), 103-1 12.

Mizoguchi, K., Morita, M., Fischer, C. R., Yoichi, M., Tanji, Y., & Unno, H. (2003).

The coevolution of PP01 phage and Escherichia coli O157:H7 in continuous culture. Appl. Environ. Microbiol. , 69(1 ), 170-176.

Schwienhorst, A. B., Lindemann, F. , & Eigen, M. (1996). Growth kinetics of a bacteriophage in continuous culture. Biotechnol. Bioeng., 50, 217-221 .

Abedon, S. T. (1989). Selection for bacteriophage latent period length by bacterial density: a theoretical examination. Microb. Ecol., 18, 79-88.

Claims

1. A method for production of bacteriophage, comprising:

maintaining a culture of bacteriophage in a vessel;

feeding the culture with bacteria; and

harvesting bacteriophage from the vessel.

2. A method according to claim 1 , comprising feeding the culture with bacteria in the stationary phase.

3. A method according to claim 2, comprising combining stationary phase feed bacteria with nutrient medium prior to feeding the combination to the bacteriophage culture.

4. A method according to any previous claim, comprising operating the bacteriophage culture at a dilution rate in excess of 1 / (bacteriophage lysis time).

5. A method according to claim 4, comprising operating the bacteriophage culture at a dilution rate of 1.1 / (bacteriophage lysis time) or greater.

6. A method according to claim 4, comprising operating the bacteriophage culture at a dilution rate of 1.25 / (bacteriophage lysis time) or greater.

7. A method according to claim 4, comprising operating the bacteriophage culture at a dilution rate of 1.5 / (bacteriophage lysis time) or greater.

8. A method according to any previous claim, comprising growing bacteria to be fed into the lysis vessel in a chemostat.

9. A method according to any previous claim, comprising monitoring the bacteriophage harvested and modulating the bacterial input to the vessel.

10. A method according to any previous claim, comprising maintaining a culture of a plurality of bacteriophages in the vessel.

1 1 . A method according to claim 10, wherein the plurality of bacteriophages comprise bacteriophage of different strains that infect the same bacteria.

12. A method according to claim 10 or 1 1 , wherein the plurality of bacteriophages comprise bacteriophage that infect bacteria of different species and/or strains.

13. A method according to any of claims 10 to 12, comprising monitoring the bacteriophages harvested and modulating the feeding of the respective bacterial inputs.

14. A method according to claim 12, comprising monitoring the ratio of the bacteriophage that infect different bacterial species and/or strains and modulating the feeding of the different bacterial strains.

15. A method according to any previous claim, comprising subjecting harvested bacteriophage to downstream processing comprising one or more or all of (i) purification, (ii) filtration, (iii) centrifugation, (iv) combining with a carrier substrate and (v) packaging bacteriophage with or without carrier into a container.

16. A method according to any previous claim, wherein bacteriophage production is continuous.

17. A method according to any previous claim, comprising producing bacteriophage continuously for a period of 2 days or more.

18. A method according to any previous claim, comprising producing bacteriophage continuously for a period of 4 days or more.

19. Apparatus, for production of bacteriophage, comprising:

a first vessel for culture of bacteriophage; and

20. Apparatus according to claim 19, wherein the second vessel is a chemostat that can be operated at constant dilution rate.

21. Apparatus according to claim 19 or 20, wherein the first vessel is a chemostat that can be operated at constant dilution rate.

22. Apparatus according to any of claims 19 to 21 , comprising two or more second vessels, all in fluid connection with the first vessel so that all can feed bacteria into the first vessel.

23. Apparatus according to any of claims 19 to 22, wherein the or each second vessel is a bacterial fermenter in which bacteria can be continuously cultured.

24. Apparatus according to any of claims 19 to 23, further comprising a third vessel intermediate between the first and second vessels, capable of adding nutrient to the bacteria output from the or each second vessel prior to bacteria being fed into the first vessel.

25. Apparatus according to any of claims 19 to 24, comprising one or pumps, valves and/or pipework so that bacteria from the or each second vessel can be fed continuously into the first vessel.

26. Apparatus according to any of claims 19 to 25, comprising a sampler for extraction of a sample of bacteriophage in the first vessel for monitoring purposes.

27. Apparatus according to claim 26, comprising control systems that modulate the feed of bacteria from the or each second vessel into the first vessel according to the content of the sample.

28. Apparatus according to any of claims 19 to 27, comprising one or more or all of (i) a purification unit, (ii) a filtration unit, (iii) a centrifuge, (iv) a unit to combine bacteriophage with a carrier and (v) a unit to package bacteriophage with or without carrier into a container.

29. A method for production of bacteriophage substantially as hereinbefore described with reference to the examples.

30. Apparatus for production of bacteriophage substantially as hereinbefore described with reference to figure 1.