HK1118573B - Device and method for the production of biologically active compounds by fermentation - Google Patents

Description

Installation and method for the fermentative production of biologically active compounds

The invention relates to a device and a method for the fermentative production of biologically active materials, wherein a fermenter is arranged in an insulator which is arranged in or adjacent to a working chamber and has a pressure drop in relation to the ambient pressure in the insulator and the working chamber.

The work involving biologically active compounds requires increased safety measures in the preparation to keep the personnel away from possible hazards or poisoning. Biologically active compounds are usually prepared or used for pharmaceutical purposes and therefore have to meet high purity requirements, in particular during the preparation process. A conventional construction of a facility for preparing high-purity substances consists of a first internal chamber, which is closed to the outside, and a second chamber containing it. In the second chamber there are personnel handling the biological material, while the processing of the biological material is mainly performed in the first chamber. The two chambers are connected to each other by a closable gate. The first chamber is usually a glove box, whereby the method steps can be carried out from the second chamber by means of gloves. The second chamber is normally closed to the outside, but is connected by a closable gate. In both chambers, sterile or clean room conditions are desired. In order to avoid contamination of the biologically active material by environmental particles, micro-organisms and the like, an overpressure, typically an overpressure of 15Pa, is applied in the first chamber. In the second chamber, there is a low pressure with respect to the first chamber, but an overpressure with respect to the environment. This prevents particles or bacteria from entering the first chamber from the second chamber. The bioactive material is thereby protected against environmental contamination.

In the case of the preparation of biologically active compounds by fermentation processes, there are process steps which are carried out in an apparatus which is switched on at least for a short time. Such process steps are for example inoculation. This step must usually be performed manually. In this process, aerosols (Aerosolbildung) may form when the instrument (container, vial) with the starting material (cell bank) is opened, even briefly. The same is true for the intermediate step from the preculture stage to the start of the fermentation, in which the connection of the fermenter is opened-even briefly-whereby aerosols can form. It is also possible that the biologically active substance forms an aerosol during the finishing of the fermentation product. There is a risk of exposing the environment to fungi, bacteria or toxic substances. Due to the relatively low pressure in the second chamber relative to the first chamber, aerosols in the first chamber may enter the second chamber, which may cause harm to operators in the second chamber. Nevertheless, in order to ensure operational safety, other safety measures must be taken in this case, such as inoculation of the operator, wearing protective clothing, etc.

Thus, there is a need for a method and a facility that can produce biologically active materials by fermentation, wherein the materials are protected from environmental pollution and at the same time exposure of personnel to aerosols is reduced or completely avoided.

This object is achieved according to the invention by a device and a method according to the appended claims.

The invention therefore relates to a facility and a method for producing biologically active substances by means of fermentation, preferably in high purity, wherein contamination of the biological material by environmental particles and fungi is avoided and at the same time the risk potential for personnel handling the biological material is reduced. Preferably the fermentation process comprises at least one inoculation step. In particular, the fermentation process comprises at least one step which is carried out manually and particularly preferably this step is an inoculation step.

The method according to the invention and the installation according to the invention used therefor allow the use of mature production and purification techniques, which are or may be relevant to aerosol formation, without there being associated with substantial risks to the personnel and environment commissioned for preparation and/or finishing by aerosol formation. In turn, the arrangement according to the invention and the method according to the invention reduce the exposure of the relevant preparation personnel and environment to biologically active materials, bacteria and/or fungi, and at the same time allow the application of mature techniques for the efficient production and purification of biological materials despite the associated aerosol formation.

The installation according to the invention comprises at least one insulation silo (isolator) which is enclosed by or borders on the working silo. The working chamber is connected with the environment through a pressure gate. Both in the insulator and in the working chamber there is a low pressure relative to the environment, wherein the pressure in the insulator is lower than the pressure in the working chamber. The pressure sluice has an overpressure relative to the ambient pressure. The insulator here comprises at least one fermenter for producing biologically active material by fermentation.

In the installation according to the invention, the biologically active material is prepared and/or refined in the insulator. An isolator, as used herein, is a system that is hermetically delimited from the environment, which in principle has an energy exchange with its environment, but does not allow an uncontrolled transport of substances with its environment. Controlled substance transport of its environment, e.g. feeding or removing appliances, reagents, starting materials, intermediate products and end products, can take place via one or more sluice, double-valve containers, via a so-called beta-port connection to an alpha-container or alpha-bag. Controlled delivery of substances is possible before, during or after fermentation and/or purification of the fermentation product or bioactive material.

The isolator can be operated at either overpressure or underpressure, regulated by suitable means. It is also possible to create an inert gas atmosphere in the separator. The separator typically employs one or more air inlet and outlet ducts, which are typically equipped with filters. It is therefore preferred to achieve a colony count in the isolator of less than 100, preferably less than 10 and most preferably less than 1 colony/m³. Such filters are known to the skilled person. An example of such an isolator is a HEPA (high efficiency particulate air) filter.

Instruments, devices or items in the isolator can be moved or manipulated via external controls or via an access glove operating from the studio, isolated on the isolator.

The isolator is preferably made in whole or in part of a material that is biologically inert and easy to clean or can be decontaminated or sterilized in situ. Preference is given here to materials which can be sterilized in particular with formalin, ethylene oxide or Vaporous Hydrogen Peroxide (VHP). Such materials are, for example, glass, stainless steel or plastics such as PVC. Preferably, the spacers are made such that their inner faces are entirely or partially made of glass and/or stainless steel or other suitable material such as plastic. Hard and soft plastics (hard-wall and soft-wall spacers) can be used here. Gloves are typically made of neoprene, chlorosulfonated polyethylene (Hypalon), ethylene, or other suitable material.

The isolator is typically provided with means for temperature control and regulation.

The isolators are typically disposed in or bordering a working chamber. There is a pressure drop between the isolator and the working chamber. In this case, the pressure in the isolator is lower than in the working chamber, which in turn has a lower pressure than the environment. The ambient pressure is 101325Pa, but may vary depending on geographical or climatic location, respectively.

Typically, the operating pressure drop in the isolator is from 20 to 200Pa, preferably from 40 to 100Pa and most preferably from 55 to 75Pa, with the pressure drop being relative to ambient pressure (101325 Pa).

In general, the operating pressure in the insulator is 10 to 100Pa, preferably 20 to 80 Pa, particularly preferably 40 to 60Pa, lower than the pressure in the working chamber.

In an embodiment of the invention, the pressure difference between the isolator and the working chamber is typically 35 to 57Pa, preferably about 45Pa, within a range of fluctuations depending on the measurement technique and the instrument, i.e. the pressure in the isolator is about 35 to 57Pa or about 45Pa lower than the pressure in the working chamber.

The air transport into and out of the isolator is preferably carried out by means of a filter, so that the number of colonies in the isolator is at most 100, preferably at most 10 colonies/m³Most preferably at most 1 colony/m³。

The working chamber according to the invention is a gas-tight closed system which is connected in a gas-tight manner for the purpose of performing a material and instrument exchange and isolation, on the one hand, by means of one or more closable switches, and on the other hand is connected in a gas-tight manner to the environment for the purpose of material and instrument exchange and/or access, by means of at least one closable pressure sluice. The working chamber is contained or connected with the isolator. The air supply to and from the working chamber is preferably carried out by means of a filter, so that the colony count in the working chamber is at its maximumAt most 200, preferably at most 100, and most preferably at most 10 colonies/m³. Such filters are commercially available, for example under the trade name HEPA-isolators.

The working chamber has a temperature of preferably about 19 to 26 ℃ and a relative air humidity of 40 to 60%. The first and second insulator, independently of one another, have a temperature and a relative air humidity which are conventional and suitable, for example, for the production implementation and purification of biologically active materials.

There is a higher operating pressure in the working chamber than the isolator, however there is a pressure drop relative to the ambient pressure and the pressure sluice.

Typically, the pressure in the working chamber is 5 to 50Pa, preferably 10 to 30 Pa, particularly preferably 12 to 18Pa, lower than ambient pressure.

In an embodiment of the invention, the typical value of the pressure difference with respect to the ambient pressure is 15Pa, i.e. the pressure in the working chamber is about 15Pa lower than the ambient pressure, within the range of fluctuations depending on the measurement technique and the instrument.

The working chamber is connected to the environment via at least one closable pressure sluice. There is an overpressure in the sluice of 10 to 100Pa, preferably 20 to 80 Pa, most preferably 25 to 35Pa, relative to the ambient pressure.

In general, the pressure difference of the pressure sluice with respect to the working chamber is from 10 to 200Pa, preferably from 20 to 100Pa, particularly preferably from 40 to 60 Pa.

In an embodiment, the pressure difference between the pressure sluice and the working chamber is typically 45Pa over a range of fluctuations depending on the measurement technique and the instrument, i.e. the working pressure in the working chamber is about 45Pa lower than the pressure sluice.

In general, the pressure difference of the pressure sluice with respect to the separator is from 10 to 300, preferably from 50 to 200, particularly preferably from 80 to 100 Pa. Within the range of fluctuations depending on the measurement technique and the instrument, the pressure difference between the pressure sluice and the isolator is typically 90Pa, i.e. the working pressure in the isolator is about 90Pa lower than the pressure sluice.

In a particularly preferred embodiment, the installation of the invention comprises at least one further insulator. The at least one further insulator may be present in or border the same working chamber as the first insulator and it may also be connected to the first insulator in a gas-tight manner, for example via one or more closable doors, double-flap containers or ports. However, the separators may not be connected to each other. The at least one further insulator preferably has the same properties as the first insulator.

Typically, a fermentation step, preferably a fermentation step producing a biologically active material, is carried out in the insulator of the present invention. Preferably, the fermentation step comprises one or more inoculations. The insulator according to the invention therefore comprises at least one fermenter.

Typically, one or more steps of the measures required for purifying the biological material are also carried out in the isolator. The fermentation and purification can be carried out in one and the same isolator or also in different isolators, for example fermentation in a first isolator and purification in a second isolator.

The fermentation step, which is aimed at the synthesis of biologically active material, is carried out in a fermenter. This optionally relates to anaerobic or aerobic fermenters. The fermenter is constructed on the industrial scale of production, corresponding to the respective fermentation conditions, as is known to the person skilled in the art. The fermenter may be a Batch Fermentation (Batch-Fermentation), a Semi-Batch Fermentation (Semi-Batch-Fermentation), a Repeated Batch Fermentation (repeat-Batch-Fermentation) or a continuous Fermentation (kontiinuelization). Preferably to a fermenter for batch fermentation.

The fermentation step may also include a pre-incubation or initial incubation for inoculation of the main fermentor. The different measures and the corresponding equipment of the process claimed here are known to the person skilled in the art and comprise, in particular, one or more fermenters which allow a first and/or second amplification of the inoculum of the production strain producing the biologically active material. Thus, the measures associated therewith also include, for example, the establishment of a working cell bank, the first-stage expansion of the cellular material, the second-stage expansion of the cellular material and the original fermentation step, the understanding of which is in particular the growth of the cells for synthesizing the biologically active compound. This step is preferably carried out at a temperature or temperature gradient defined by the respective production organism. The temperature is here optionally adapted continuously or discontinuously to the production conditions.

In the isolator, one or more of the steps or measures required for purifying the biologically active material may also be implemented. Accordingly, the isolator is changed to become a device required for this.

The purification of the biologically active material can also be carried out in the second insulator of the installation according to the invention. If only a part of the measures for purification is carried out in the second insulator, the other part of the required measures is carried out in the first insulator or in one or more further insulators.

Certain steps required for the preparation of the bioactive material or associated with established measures may also be carried out outside the insulator, particularly if those not associated with aerosol formation or the bioactive material is pre-placed in a form or container that is not hazardous to the personnel or environment involved in the preparation and operation.

Under fermentation to produce bioactive materials, fermentation involving bacteria is preferred, with the strain Clostridium botulinum (Clostridium botulinum) being particularly preferred. The same applies to recombinant or genetically modified other heterologous expression systems (e.g. e.

Essentially all biologically active substances can be synthesized using the installed apparatus, in particular proteins synthesized by the microorganisms by fermentative cultivation.

The biologically active substance produced by the fermentation process is preferably a toxin, in particular a botulinum toxin and most preferably a botulinum neurotoxin.

Botulinum toxin is produced by the bacterium Clostridium botulinum and has been modified by the bacterium Clostridium botulinumThe same serotype exists: botulinum toxin type A, B, C, D, E, F, G. The botulinum toxin produced by Clostridium botulinum is a complex of botulinum neurotoxin (i.e. the protein responsible for the toxic effect) and a complexing protein which is not toxic per se. These proteins are haemagglutinin with different molar masses and at least one protein that does not agglutinate blood cells. The complex consisting of botulinum neurotoxin and bacterial complex protein is generally referred to as botulinum toxin. A, B and form C are commercially available. Type A, for example, as BOTOX. The complex is further processed in complexes of different protein compositions and purities up to the botulinum neurotoxin itself. Thus, in the context of the present invention, the term botulinum neurotoxin is used to describe the protein responsible for the toxic effect, i.e. a botulinum toxin which is free of bacterial complex proteins and has a high purity. Botulinum neurotoxin type A has a molecular weight of about 150kDa and the applicant has the trade name XeominAnd carrying out commercial sale.

Suitable within the scope of the present invention are all forms of "botulinum toxin", in particular different serotypes, different complexes of botulinum neurotoxin and complex proteins, botulinum neurotoxin itself, as well as modified or correspondingly recombinantly prepared botulinum toxins or botulinum neurotoxins including corresponding mutants, deletions and the like. For suitable mutants, we used the relevant mutants described in WO 2006/027207A 1. Furthermore, within the scope of the present invention, mixtures of different serotypes (in complexed or highly pure and/or recombinant form) are prepared, for example mixtures of botulinum toxins of types A and B or mixtures of botulinum neurotoxins of types A and B.

The preparation of botulinum neurotoxins of types a and B is described, for example, in patent application WO 00/74703. Due to the advantageous property of the (isolated) botulinum neurotoxin being substantially devoid of immunity to botulinum neurotoxin complexes and complex proteins, there is thus a particularly high need to provide a facility and a method for the safe and reliable industrial production of botulinum neurotoxin, as is done by the present invention.

Depending on the biological compound produced, the skilled person is free to choose the choice and/or number of separators specifically used. Within the scope of the specific purification of botulinum neurotoxin described in detail herein, it is preferred to use at least two isolators. The separator is distinguished here in particular in view of the prevailing temperature in it or in a partial region thereof. It is therefore preferred within the scope of the present invention that the first isolator be operated at a higher temperature, for example in the range of from about 20 to about 50 c, and the second isolator be operated at a lower temperature, for example in the range of from about-5 to +25 c. Those preparation and purification steps are carried out in the first insulator in this respect, which require a correspondingly higher temperature or can be carried out at this temperature. This includes, in particular, the inoculation, fermentation and/or precipitation of the production strain. In contrast, those steps of the preparation process and in particular the purification of the neurotoxin, which require a correspondingly lower temperature or can be carried out at this temperature in a particularly advantageous manner, are carried out in a second insulator.

It is further recognized that those steps in general in the preparation and those in particular in the fermentation, purification, which can be carried out at two temperature ranges and thus in each of two isolators, are finally carried out in such isolators, which are advantageous from the standpoint of size and the energy consumption, in particular temperature, due to the size, which maintains the respective reaction conditions for carrying out the reaction steps, or from the standpoint of operability or feasibility of the individual measures.

The second insulator is basically of similar design to the first insulator, but has equipment features for carrying out the planned proposed steps or measures required for this in the second insulator, and for equipping the object or the production process. Such corresponding equipment items are known to the person skilled in the art. Preferably, the purification step in the second insulator is a step selected from the group consisting of precipitation, extraction, centrifugation, dialysis and chromatography. Accordingly, the second separator contains one or more sedimentation devices, extraction devices, centrifugation devices, dialysis devices and/or chromatography devices. Preferably, the precipitation device comprises a reaction vessel containing the solution to be extracted for precipitation, equipped with a delivery line for adding the compound required for precipitation. Furthermore, the precipitation device contains in principle a removal agent (Entfernungsmittel) to remove supernatant or precipitate. Suitable devices are known to the skilled worker. The extraction apparatus typically comprises a reaction vessel containing the liquid to be extracted, a transfer line for addition of an extraction medium and means to separate the liquid containing the extract from the extraction liquid. The centrifugation device typically comprises a centrifuge to separate a liquid-liquid mixture or a liquid-solid mixture. Chromatography devices typically comprise a chromatography column, which is packed with chromatography material, as well as an input and an output and a container with a suitable medium for feeding to or eluting from the chromatography column. In the case of chromatography, it may, for example, involve size exclusion chromatography, HPLC or affinity chromatography.

Within the scope of the invention, in the case of the use of two or more isolators, they are connected to one another so that the entirety of the installation comprising the two isolators contains only one inlet and outlet pipe and the two isolators are connected by one or more intermediate pipes. However, it is also within the scope of the invention for the first and second separator and/or each further separator to have an inlet and outlet line, respectively, independently of one another.

The first and second separators preferably have a lower internal pressure relative to the external pressure. Within the scope of the invention, both isolators have the same internal pressure. However, it is also possible within the scope of the invention for the two separators to have different internal pressures. In this case, it is particularly preferred that the method steps carried out in the separator with a lower internal pressure have a higher probability of aerosol formation than in the other separator with a lower internal pressure.

In the case of the use of a second insulator, the connection between the insulators is present in the form of a channel, for example in the form of a sluice, between the two insulators, which is advantageous because it is thereby possible to ensure the transport of the produced or purified material to the different stages of preparation or purification without the need to open the barrier constructed by the insulator. The construction of the installation or each of the individual isolators or at least one isolator of the installation of the invention with a sterilization device is essentially for the same purpose. Corresponding sterilization devices are known to the person skilled in the art and comprise, for example, a motor for generating vaporous hydrogen peroxide. The use of vaporous hydrogen peroxide is particularly advantageous here from the point of view of the low pressure conditions in the separator.

In another aspect, the present invention relates to a process for the fermentative preparation of a biologically active compound, wherein the process comprises a fermentation step for the preparation of the biologically active compound and a purification of the biologically active compound, wherein the fermentative preparation and/or the purification is carried out completely or partially in one or more isolators. Preferably, the fermentation step and one or more portions of the purification are carried out in a first isolator and one or more portions of the purification are carried out in a second isolator. In a preferred embodiment, the fermentation step comprises at least an inoculation step. Preferably this step is performed manually. Other isolators are also within the scope of the invention. The biologically active material is preferably a botulinum toxin, and in particular a botulinum neurotoxin as described herein. Also relevant to the features disclosed in connection with the arrangement according to the invention are features presented in each case and in any combination in the method according to the invention.

The purification-related measures are centrifugation, dialysis, extraction, precipitation, protamine sulfate precipitation, ammonium sulfate precipitation, dissolution of the precipitate, which is typically present as a pellet, dialysis, chromatographic steps or chromatography and filtration. Chromatography preferably involves a single chromatography with a plurality performed sequentially. The same chromatography or different chromatographs can be involved here. Preferably in the case of the pharmaceutically active compound botulinum neurotoxin type A, three chromatography steps under the use of suitable column materials are involved. It is possible and preferred here for each eluate to be subjected to dialysis before being loaded onto the next column.

After substantial fermentation and separation of the fermentation medium from the cells, the fermentation medium is subjected to a first precipitation with the aim of removing large proteins. The precipitation is preferably carried out in a first separator. The centrifugation of the precipitate obtained in this way is preferably already carried out in the second separator. The precipitation is preferably a column precipitation. Reaction conditions for such acidic precipitation are known to those skilled in the art. Typically 3N H is used₂SO₄To acidify the supernatant to a pH of 3.5. The centrifugation is typically carried out at 2400 Xg for 20 minutes at 4 ℃. The pellet obtained by centrifugation is preferably washed here with water.

In the case of the use of Clostridium strains, preference is given to Clostridium botulinum type A, which is stored as a raw culture in a suitable medium at temperatures which ensure stability. For the fermentation, the method described by DasGupta B.R. et al, Toxicon, Vol.22, Nr.3, pp.414-424, 1984 is preferably employed. Here, 2% of N-Z-amine type A medium is mixed with 0.5% of yeast extract and 0.6% of autoclaved yeast paste and adjusted to pH 7.2 with 4N NaOH, and the medium thus prepared is subsequently autoclaved. To this medium was added additionally autoclaved glucose (20 wt% per volume) to obtain a final glucose concentration in the medium of 0.5%. The incubation was carried out without stirring at 37 ℃ where the fermentation was interrupted after 96 hours. Within the scope of the present invention, it is also possible to carry out a semi-batch fermentation, a repeated batch fermentation or a continuous fermentation in addition to the batch fermentation described above. The measures required for this and the preparation process requirements are known to the person skilled in the art.

In a further step, starting from the pellet of the precipitate, this is dissolved again in order to release the toxin from the pellet. Measures for this are known to the skilled worker and are described in particular in DasGupta b.r. et al (aaO). For example, the extraction can be carried out with the aid of 0.1M citric acid-trisodium citrate, pH 5.5, for 1 hour. This extraction is combined with a subsequent further centrifugation step, typically at 9800 Xg for 20 minutes at 4 ℃. The pellet thus obtained can then optionally be extracted again as described before. The supernatant of the extract, and in case of repeated extractions both supernatants, will be subjected to protamine sulfate-precipitation. The precipitation was carried out at 8 ℃ overnight. The precipitate was then centrifuged again at 12000 Xg for 20 min at 4 ℃. In particular, DNA is removed in the protamine sulfate-precipitation step.

The supernatant obtained after centrifugation will be subjected to ammonium sulfate-precipitation in the first separator or in the second separator, wherein other larger proteins are removed. After the ammonium sulfate precipitation, the centrifugation step is again carried out and the pellet obtained in this way is subsequently redissolved and optionally dialyzed. The dialyzed extract from the pellet is preferably subsequently centrifuged again and subjected to successive chromatographic steps with the aim of purifying the botulinum neurotoxin, in particular until homogeneous purification. The individual chromatographic steps are used here in particular for removing protamine sulfate, residual DNA, parts of small and medium-sized proteins and also the haemagglutinin of the botulinum neurotoxin protein complex. For this purpose, a plurality of chromatography steps is carried out in sequence in a preferred embodiment. The eluate was then filtered. The filtration is used here to reduce the colonies in the eluate, and then preferably to obtain pure botulinum neurotoxin free of complexing proteins. The eluate may optionally be diluted prior to filtration or a suitable adjuvant may be added.

In a further step, sterilization-filtration is carried out again after the addition of the auxiliary. The filtration is carried out in a reaction vessel and then, preferably, freeze-dried in a separate process step. The freeze-dried product was stored in this manner.

It is recognized by those skilled in the art that decontamination or sterilization is performed before inoculation and after the different process steps, preferably ammonium sulphate-precipitation and filtration in the first insulator. The sterilization preferably involves the implementation by means of Vaporous Hydrogen Peroxide (VHP).

The features of the invention disclosed in the description and the claims may be essential for the implementation of the invention in different embodiments both individually and in any combination.

Claims (29)

1. Installation for the fermentative production of a biologically active compound, characterized in that the installation comprises at least a first insulator, which comprises a fermenter and which is enclosed by a working chamber, wherein the working chamber is connected to the environment via a pressure sluice, wherein a low pressure prevails in the insulator and in the working chamber, respectively, the pressure prevailing in the insulator relative to the ambient pressure is lower than the pressure prevailing in the working chamber relative to the ambient pressure, and the pressure sluice is an overpressure relative to the ambient pressure, wherein the biologically active compound is botulinum toxin, and wherein the pressure prevailing in the insulator is 20 to 200Pa lower than the ambient pressure and the pressure prevailing in the working chamber is 5 to 50Pa lower than the ambient pressure.

2. The facility according to claim 1, wherein the pressure in the pressure lock is 10 to 100Pa higher than the ambient pressure.

3. The installation according to claim 1, wherein the installation comprises a sterilization device and/or a disinfection device.

4. The facility according to claim 1, wherein the first insulator comprises an anaerobically operated fermenter.

5. The apparatus according to claim 1, wherein the first insulator comprises a precipitation device.

6. The apparatus according to claim 1, wherein the first separator comprises a filtration device.

7. The plant according to any of claims 1 to 6, characterized in that the plant comprises at least a second insulator.

8. The plant according to claim 7, characterized in that the plant and/or the first and/or second insulator comprise at least one input and output pipe, wherein said input and output pipe is provided with a filter.

9. The apparatus according to claim 7, wherein the internal pressures of the first and second isolators are the same.

10. The apparatus according to claim 7, wherein the internal pressure of the first insulator is different from the internal pressure of the second insulator.

11. The apparatus according to claim 7, wherein the first and second separators are connected to each other by a passageway, wherein the passageway allows the transfer of a substance from the first separator to the second separator.

12. The apparatus of claim 11, wherein the passageway is a gate.

13. The apparatus according to claim 7, wherein the second isolator comprises an extraction device.

14. The apparatus according to claim 7, wherein the second isolator comprises a precipitation device.

15. The apparatus according to claim 7, wherein the second insulator comprises at least one chromatographic device.

16. A process for the fermentative preparation of a biologically active material comprising

-a fermentation step for the preparation of a biologically active material; and

-a purification of the biologically active material,

wherein the process is carried out in a plant according to any one of claims 7 to 15.

17. The method of claim 16, wherein the bioactive material is a toxin.

18. The method of claim 16, wherein the biologically active material is botulinum toxin.

19. The method of claim 16, wherein the bioactive material is botulinum neurotoxin.

20. The method of claim 18, wherein the botulinum toxin is A, B, C, D, E, F or Clostridium botulinum toxin G (Clostridium botulinum) or a mixture of two or more of these types.

21. The method of claim 20, wherein the botulinum toxin is botulinum toxin type a.

22. The method of claim 20, wherein the botulinum toxin or mixture of botulinum toxins is a botulinum neurotoxin or a mixture of botulinum neurotoxins.

23. The method of claim 16, wherein the fermentation step is performed in a first isolator and the purification is performed in whole or in part in a second isolator.

24. The method of claim 16, wherein a portion of the fermentation step and the purification is performed in a first isolator and the other portion of the purification is performed in a second isolator.

25. The method of claim 16, wherein the fermentation step and the precipitation and filtration of the fermentation step product as part of the purification are performed in a first isolator.

26. The method of claim 16, wherein the first isolator and the second isolator operate at the same or different temperatures.

27. Method according to claim 16, characterized in that the temperature in the first and/or second isolator is varied depending on the respective method step.

28. The process of claim 27, wherein the temperature in the first insulator is from 20 to 50 ℃ and the temperature in the second insulator is from-5 to +25 ℃.

29. A method according to claim 16, characterized in that the method comprises the steps of:

a) in the first isolator

-inoculating the fermentation medium with a production strain of a biologically active compound;

-fermentation of a production strain;

-separating the supernatant from the cells of the produced strain;

-precipitation of the supernatant;

b) in the second isolator

-centrifuging the precipitated supernatant to form a pellet;

-extracting the pellet, centrifuging and obtaining the supernatant;

-precipitating the supernatant, followed by centrifugation to further form a supernatant;

c) in the first or second isolator:

-precipitating the supernatant;

-centrifuging the precipitate;

-dissolving the pellet obtained by centrifugation of the precipitate;

-dialyzing the solubilized pellet and centrifuging the dialysate;

-subjecting the dialysate to chromatography;

-filtering the eluate obtained.