WO2007025968A1

WO2007025968A1 - Semi-continuous fermentation process

Info

Publication number: WO2007025968A1
Application number: PCT/EP2006/065779
Authority: WO
Inventors: Ralf Harand
Original assignee: Nycomed Gmbh
Priority date: 2005-08-31
Filing date: 2006-08-29
Publication date: 2007-03-08
Also published as: JP2009505672A; DE102005041526A1; EP1924682A1

Abstract

The invention relates to a method for the biosynthesis of a biomolecule by-microorganisms in a multiplicity of fermentation vessels (1) which have an outer wall that is impermeable to microorganisms, but is gas-permeable at least in a part-region; comprising the steps (a) introducing a nutrient medium into the fermentation vessels; (b) if appropriate closing the fermentation vessels so that mass transfer with the surroundings can only still proceed through the outer wall of the fermentation vessels; (c) if appropriate sterilizing the fermentation vessels; (d) inoculating the nutrient medium in each fermentation vessel by aseptic addition of the microorganisms; (e) introducing the fermentation vessels into a fermentation bath (4) in which a gas or gas mixture is dissolved; and (f) if appropriate removing the fermentation vessels from the fermentation bath. In addition, the invention relates to a fermentation vessel and its use in the inventive method.

Description

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Semi-continuous fermentation process

The invention relates to a method for the biosynthesis of biomolecules by microorganisms and to a fermentation vessel which can be used advantageously in the inventive method.

Numerous pharmaceutical (intermediate) products are currently produced by the biochemical route. In this case frequently microorganisms such as, for example, cell lines, fungi (yeasts) or bacteria, are frequently cultured in suitable media and supplied with the nutrients which are required for biosynthesis of the desired biomolecule. Depending on the microorganism used, customarily it is necessary to maintain accurately well-defined boundary conditions, such as, e.g., pH, temperature, salt concentration and nutrient concentration, type and partial pressure of certain gases etc., so that biosynthesis succeeds and can be maintained over a relatively long period.

Since in this manner not only must optimum growth conditions be created for a certain organism, it is necessary to take care to ensure that no contamination by foreign organisms, or any other contamination, takes place. Customarily, such processes are carried out in fermenters having a number of typical internals, such as, e.g., agitators, feed air and exhaust air filters, heating devices, etc. The apparatus used and also the nutrient media are customarily sterilized before culturing the microorganisms. In addition, aseptic conditions are usually maintained during the entire process.

Biotechnological processes can be carried out in principle continuously, semi-continuously or batchwise. - -

It is known that continuous processes proceed in principle considerably more cheaply, more logically and with smaller quality variations and more reliably than charge processes (batch processes). Nevertheless, in pharmaceutical biotechnology, continuous processes have hitherto scarcely been employed. Two reasons, especially, are responsible for this: firstly on the part of the supervisory authorities there is the wish for a reproducible definition of the individual charges, secondly there is the risk that even the smallest (microbiological) contamination can lead to an unpredictable result, to a buildup or even to a collapse of the process.

In the case of semi-continuous procedures of biochemical processes, customarily use is made of a fermenter for culturing and at defined time points a part of the fermentation medium present is withdrawn and replaced by fresh medium (termed fed-batch culture). The fermentation medium which is withdrawn at regular intervals then contains the biomolecule synthesized by biosynthesis. In this context, for example, reference can be made to US 5,342,765 and US 6,610,516. This type of the semi-continuous process procedure, however, has the disadvantage that the cultured microorganisms, at least in part, remain for a relatively long time in the fermenter, which in the case of microorganisms having comparatively low genetic stability, can lead to problems due to mutations. In addition, the repeated withdrawal and replenishment of medium increases the risk of contaminating the fermentation batch by foreign organisms.

In the case of the discontinuous procedure of biochemical processes (charge or batch processes), per fermenter one process is made up and is then processed completely. Cost minimization in the case of the discontinuous process procedure is virtually only possible if the batch per charge is increased, that is, for example, if the procedure is switched from a reactor having a capacity of 1000 I to a reactor having a capacity of 15 000 I. The batch increase, however, also increases the cost risk, since in the case of contamination the entire large batch may need to be discarded.

In addition to the risk of contamination, with large volumes there is a significant problem of such processes in deficient mass transport. For instance, for example gases which are supplied, such as O₂, must cover comparatively long pathways through the fermentation medium before, from the site of their feed, they reach the microorganisms. This likewise applies to by-products of the process, such as, for example, CO₂, which must be removed. It is known that mass transport problems increase disproportionately with the increase in the process scale. This problem is frequently solved by powerful agitators which introduce high kinetic energy into the fermentation medium. It is disadvantageous, in this case, however, that at the same time high shear forces are generated which can damage the microorganisms.

There is therefore a need for a process for the biosynthesis of a biomolecule which minimises or avoids the abovemetioned disadvantages of the processes of the prior art.

The object underlying the invention is to provide a method for the biosynthesis of a biomolecule by microorganisms which has advantages over the prior art. The method should also be able to be - - carried out on a large scale and reduce the risk that, in the case of contamination, in particular by foreign organisms, the entire (large) batch must be discarded. In addition, the method should be inexpensive, automated and be able to be integrated into clean room designs.

This object is solved by the subject matter of the patent claims. It has surprisingly been found that the cost risk accompanied by the possibility of contamination may be decreased if the fermentation medium is subdivided over a multiplicity of fermentation vessels and these are incubated under suitable conditions during the biosynthesis.

The invention relates to a method for the biosynthesis (fermentation) of a biomolecule by microorganisms in a multiplicity of fermentation vessels which have an outer wall that is impermeable to microorganisms, but is gas-permeable at least in a part-region, comprising the steps

(a) introducing a nutrient medium into the fermentation vessels;

(b) if appropriate closing the fermentation vessels so that mass transfer, preferably gas exchange, with the surroundings can only still proceed through the outer wall of the fermentation vessels;

(c) if appropriate sterilizing the fermentation vessels;

(d) inoculating the nutrient medium in each fermentation vessel by aseptic addition of the microorganisms;

(e) introducing the fermentation vessels into a fermentation bath in which a gas or gas mixture is dissolved, the gas or gas mixture preferably bubbling through the fermentation bath; and

(f) if appropriate removing the fermentation vessels from the fermentation bath.

The inventive method has the advantage that the biosynthesis proceeds in the fermentation vessels as separate compartments, so that any contamination affects only one individual fermentation vessel, but not the entire reaction batch. The individual contaminated fermentation vessel can usually be identified with the naked eye owing to its atypical colouring, and can be eliminated. In this manner the cost risk of the overall method is considerably reduced. Nevertheless, essentially all the advantages which accompany the enlargement of the reaction batch can be utilized. The inventive method may be automated and/or carried out semi-continuously.

Figure 1 shows a diagrammatic representation of a preferred embodiment of the inventive method. Figure 2 shows a preferred embodiment of steps (e) and (f) of the inventive method. Figures 3 to 7 show preferred embodiments of the inventive fermentation vessel. - -

The term "biosynthesis" within the meaning of the description comprises any fermentation, that is the reaction of biological materials by microorganisms, in particular bacterial, fungal or cell cultures.

"Biomolecule" within the meaning of the description is to be taken to mean any organic molecule which can be produced by the route of a biosynthesis (fermentation) by microorganisms which may have been genetically manipulated. Examples of biomolecules are proteins, which can if appropriate be post-translationally modified.

"Nutrient medium" within the meaning of the description is to be taken to mean a medium which is suitable in principle for culturing microorganisms, but does not yet contain them. The term also comprises those media to which, for the later biosynthesis, essential additions (e.g. activators or certain nutrients) are not fed until a later timepoint of the method.

"Fermentation medium" within the meaning of the description is to be taken to mean a medium which already contains microorganisms which have not yet converted, have already in part converted, or have completely converted the nutrient medium.

In step (a) of the inventive method, a nutrient medium is introduced into the fermentation vessels. The type and composition of the nutrient medium depends on the microorganisms which are used for the biosynthesis. Preferably, the nutrient medium is a liquid in which customary nutrients and aids are present in dissolved and/or suspended form. Such nutrients and aids are known to those skilled in the art. In this context, reference can be made, for example, in its entirety to S. Isaac et al., Kultur von Mikroorganismen [Culture of microorganisms], Spektrum Akademischer Verlag, 1996; K. Schϋgerl, Bioreaktionstechnik, Bioprozesse mit Mikroorganismen und Zellen [Bioreaction techniques, bioprocesses with microorganisms and cells], Birkhauser Verlag, 1997; M.A. Harrison et al., General Techniques of Cell Culture (Handbooks in Practical Animal Cell Biology), Cambridge University Press (1997); M. Clynes, Animal Cell Culture Techniques, Springer, Berlin, 1998; T. Lindl, ZeII- und Gewebekultur [Cell and tissue culture], Spektrum Akademischer Verlag, 2002; V. Vinci et al., Handbook of Industrial Cell Culture: Mammalian, Microbial, and Plant Cells, Humana Press, 2002; and CD. Helgason et al., Basic Cell Culture Protocols (Methods in Molecular Biology), Humana Press, 3rd Bk&Cdr ed. (2004).

Customarily such nutrient media contain buffers, electrolytes, monosaccharides, oligosaccharides, polysaccharides, amino acids, peptides, proteins, vitamins and/or coenzymes. However, in addition, for example antibiotics, against which the microorganisms used are resistant, dyes etc may also be present.

Preferably, the nutrient medium at this timepoint contains no further microorganisms with which the biosynthesis is to be later carried out (that is at this timepoint preferably no fermentation medium is present). - -

The nutrient medium can be introduced into the fermentation vessels in various ways. The fermentation vessels can be charged with nutrient medium under aseptic conditions or, if a concluding sterilization of the filled fermentation vessels is to be performed before the inoculation in step (d), under non-aseptic conditions.

Preferably, the fermentation vessels have orifices which are suitable for charging with nutrient medium and are preferably reclosable.

If the nutrient medium is a liquid, this can, for example, be poured, injected, added dropwise, sprayed, or pressed into the fermentation vessels, or introduced in other ways. The filling can proceed on filling machines for liquids available on the market, e.g. high-volume bags or bottles for parenteral compositions.

If the nutrient media comprises a solid which is not dissolved until later, this solid can be charged into the fermentation vessels, for example using funnels, spoons, paddles or spatulas. To dissolve the solids, subsequently, a suitable liquid, customarily water, an aqueous solution or halogenated hydrocarbons, in particular perfluorohydrocarbons, e.g. perflubron, can be charged into the fermentation vessels.

Preferably, at least 70% of the volume of the fermentation vessels is charged with nutrient medium, more preferably at least 80%, and in particular at least 90%, or at least 95%. This ensures that the nutrient or fermentation medium wets the outer wall of the fermentation vessels to a large extent during the biosynthesis, so that the gas exchange which takes place during the biosynthesis through at least a part of the outer wall reaches a high efficiency.

In a preferred embodiment, the inventive method comprises the optional step (b), which is preferably carried out immediately after step (a). In this case the fermentation vessels are closed in such a manner that mass transfer with the surroundings can only still proceed through the outer wall of the fermentation vessels. The outer wall of the fermentation vessels is for this purpose made, at least in part-regions, of a material which makes possible later mass transfer, in particular gas exchange, with the surroundings.

The orifices of the fermentation vessels through which, for example in step (a), the nutrient medium was introduced into the fermentation vessels, are closed in this optional method step. This can be performed irreversibly, for example by welding or gluing. Preferably, however, the closing is performed in a manner which is reversible. For this the fermentation vessels can have, for example screw closures or similarly acting closures, which are suitable for repeated opening and closing. Such closures are known to those skilled in the art. Depending on the connections or orifices situated on the fermentation vessels, a multiplicity of closure techniques and closure machinery are available, for example rubber stoppers, crimped aluminium caps, screw or push-in closures (e.g. Luer-Lock closures). - -

The closure of the fermentation vessels is performed in such a manner that no uncontrolled mass transfer can proceed between the surroundings of the fermentation vessels and their interior. Since the outer wall of the fermentation vessels is impermeable to microorganisms, not only can no microorganisms escape from the interior of the fermentation vessels, but, in particular, no microorganisms can penetrate from the outside into the fermentation vessels either.

Preferably, after step (a) and (b) have been carried out, inside the fermentation vessel there are no microorganisms with which the biosynthesis is to be later carried out (that is at this timepoint no fermentation medium is yet present).

In a preferred embodiment of the inventive method, subsequently to step (c) the fermentation vessels are sterilized. This process step is advisable if the charging in step (a) and if appropriate the closing in step (b) are not performed under aseptic conditions and therefore the filled fermentation vessels are to be finally sterilized, i.e. in the filled state at the end of the charging process. The optional sterilization can be performed in different ways. Those skilled in the art know various possibilities for this, for example the sterilization can be performed by superheated steam, dry heat, ionizing radiation ( - or β- sterilization) or using ethylene oxide.

In a preferred embodiment, the sterilization is performed by superheated steam in counterpressure autoclaves at 121⁰C, for example for 30 minutes. A sufficient counterpressure here is advantageous to prevent damage to the fermentation vessels due to swelling. If appropriate, it can be advantageous to rotate the filled fermentation vessels during the sterilization (rotary autoclave), in order to achieve improved heat transfer and prevent sticking of the nutrient medium within the fermentation vessels owing to local overheating.

The optional sterilization step (c) ensures that any contamination of the nutrient medium by foreign organisms such as, for example, bacteria, cells or fungi, is made harmless.

Subsequently to the optional sterilization of the fermentation vessels in step (c), in step (d) the nutrient medium of each fermentation vessel is inoculated by aseptic addition of the microorganisms. In this manner, the nutrient medium is converted into a fermentation medium. For this, preferably temporarily, access from the outside into the interior of the fermentation vessel is created. This access can be an orifice which, if appropriate, had been closed previously in step (b).

Step (d) of the inventive method can be effected in different ways known to those skilled in the art. For example, the fermentation vessels can be equipped with a device for aseptic addition of a solid or liquid composition which, for the purposes of addition of microorganisms, can be punctured by a needle or a mandrel and closes again spontaneously after removal of the needle or mandrel. Suitable materials for producing such closures are known to those skilled in the art. For example, suitable - - membranes are those such as are also used for producing septa, for example aseptic withdrawal of injection sera using cannulae.

The microorganisms used to inoculate the fermentation vessels can be preferably firstly cultured in a shake culture or preculture according to the standard methods of microbiology and the requirements of the specific microorganism.

Alternatively, for the inoculation, microorganisms can be withdrawn from fermentation vessels which have already passed through the inventive method and contributed to the biosynthesis of the biomolecules to be synthesized. Completely reacted (fermented) fermentation vessels can be withdrawn from the running process for this. If appropriate after testing the fermentation vessels for microbiological contamination and measuring the cell density, fermentation medium is withdrawn from these fermentation vessels under aseptic conditions in order thereby to inoculate fresh fermentation vessels filled with nutrient medium. Which tests are necessary depends on the respective microorganism and the requirements of the biomolecule to be produced by biosynthesis. How often the process can, and may, be reinoculated using completely reacted preceding fermentation vessels from the process itself, or how often new precultures must be cultured, depends on the genetic stability of the organism used or on the occurrence of unwanted genetic changes (mutations, back- mutations, etc.).

The fermentation vessels which are charged with nutrient medium and are sterile are then inoculated with fermentation medium from the shake culture or from a completely reacted (fermented) fermentation vessel. For this, a defined amount of the fermentation medium (typically in the range of a few microlitres to a few millilitres) is aseptical Iy withdrawn from the shake culture or from the completely reacted fermentation vessel and added aseptically to the sterile fermentation vessels which are charged with fresh nutrient medium. In this manner many sterile fermentation vessels which are charged with fresh nutrient medium can be inoculated with the fermentation medium of a shake culture or of a fermented fermentation vessel. This can be performed manually or using corresponding commercially available equipment, e.g. Laborautosampler or pharmaceutical charging machines for small-volume Parenterals.

In a preferred embodiment of the inventive method, the microorganisms are selected from the group consisting of bacteria, cell lines and yeasts. The type of microorganisms selected depends on the biomolecule to be synthesized by biosynthesis.

Preferably, the biomolecule to be synthesized is a peptide or protein which, if appropriate, can be post-translationally modified. For the expression thereof, genetically engineered bacteria, cell lines or yeasts are preferably used.

In a particularly preferred embodiment of the inventive method, the microorganisms are bacteria which produce recombinant SP-C Inclusion Bodies. SP-C is Surfactant-Protein C, a polypeptide which, inter - - alia, is suitable for treating acute respiratory distress syndrome of premature infants and adults. In this context, for example, reference can be made to DE-A 44 18 936 and A. ten Brinke et al., Biochim Biophys Acta. 2002, 1583(3):253-65.

After the inoculation, in the fermentation vessels, the biomolecules are biosynthesized by the microorganisms. For this, in step (e) of the inventive method, the fermentation vessels are introduced into a fermentation bath in which a gas or gas mixture is dissolved. Preferably, a gas or gas mixture is bubbled through the fermentation bath (gas-dispersion trough). Under these conditions, the microorganisms grow, multiply and produce the desired biomolecule by biosynthesis.

A plurality of fermentation vessels can be introduced simultaneously into the fermentation bath. Alternatively, the fermentation vessels can be introduced successively into the fermentation bath. Preferably, at a given timepoint, a plurality of fermentation vessels are situated in the fermentation bath, other fermentation vessels are not yet situated in the fermentation bath, and in turn other fermentation vessels are no longer in the fermentation bath.

The gas-dispersion troughs are preferably charged with a liquid (fermentation bath), in which the gas or gas mixture can dissolve and with which the gas-dispersion troughs are treated with gas. Preferably, this liquid is water, an aqueous solution, or a halogenated hydrocarbon, in particular perfluorohydrocarbon, e.g. perflubron. From the liquid, in which the gas or gas mixture is dissolved and through which, preferably, gas bubbles are passed, although direct passage of gas bubbles into the interior of the fermentation vessel takes place, the majority of the passage takes place from the liquid phase to the fermentation vessel. The majority of the gases available for the gas exchange to the fermentation vessel is dissolved in the liquid.

Treating the fermentation vessels with gas in a liquid has, compared with direct gas-treatment with gas in a gaseous surrounding, the advantage, inter alia, that gas bubbles form in the liquid which, owing to their buoyancy very gently ensure vortexing of the fermentation medium within the fermentation vessels. In addition, a liquid may be better heated/cooled than a gas and can in addition be utilized for transfer of mechanical impulses (vibrations, ultrasound, etc.).

Also, gas passage from the liquid phase to the fermentation vessel can proceed better than passage from the gas phase to the fermentation vessel. Precisely in the case of anaerobic fermentation using inert gas as gas-treatment medium (N₂, He, Ar, etc.), personal safety of a gas-dispersion trough filled with liquid is more easily ensured than that of a chamber filled with gas. In addition, a gas-dispersion trough may be much more readily freed from dissolved oxygen than a gas-filled chamber. The inert gas stream additionally serves for, if appropriate, discharging substances which are toxic to the microorganisms, e.g. oxygen (anaerobic fermentation), hydrogen sulphide, methane and other hydrocarbons, volatile amines etc. Finally, the fermentation current can also be used for introducing and removing the fermentation vessels. - -

The fermentation vessels are preferably completely immersed in the liquid. However, it is also possible to immerse the fermentation vessels only partly.

If the microorganisms are those for which the biosynthesis proceeds under anaerobic conditions, the gas or the gas mixture is preferably an inert gas such as, for example, nitrogen, or a nitrogen- containing gas mixture. If the microorganisms are those for which the biosynthesis proceeds under aerobic conditions, the gas or the gas mixture is preferably oxygen, or an oxygen-containing mixture, such as air.

In a preferred embodiment of the inventive method, in step (e), the gas or gas mixture is at a pressure which is higher than atmospheric pressure. For example, the gas or gas mixture can be introduced into the fermentation vessel at an overpressure of at least 0.1 bar, more preferably at least 0.2 bar, still more preferably at least 0.3 bar, most preferably at least 0.4 bar and in particular at least 0.5 bar, as a result of which the gas exchange can be improved through the outer wall of the fermentation vessel. The overpressure here is based on the ambient pressure of the atmosphere, customarily 1.0 bar (1013 hPa). The said pressures are gas inlet pressures in the feed line, the minimum gas pressure not necessarily having to be at least 0.1 bar overpressure.

The minimum exposure overpressure is calculated from the filling height of the fermentation bath (density of the filling medium • filling height) and, in addition, is determined from the resistances to flow in the gas feed lines and gas distribution devices, and also from the desired gas-introduction rate (in m³/min). Preferably, the pressure when the gas is blown in is greater than the sum of ambient air pressure and hydrostatic pressure in the fermentation bath (density • filling height).

The gas-dispersion trough in this case is preferably constructed as a pressure vessel. The gas exchange between the fermentation bath outside the fermentation vessel and the interior of the fermentation vessel, that is the fermentation medium, preferably proceeds principally via gas which is situated in the fermentation bath in the dissolved state. A direct gas exchange, i.e. transport of gas molecules in the gaseous undissolved state through the outer wall of the fermentation vessel does take place, but plays only a subsidiary role.

Preferably, the gas or gas mixture is blown into the fermentation bath on the underside of the fermentation vessel via suitable inlet nozzles, so that the gas or gas mixture firstly dissolves in the fermentation bath, secondly also ascends in the form of small bubbles in the fermentation bath and thus bubbles through the fermentation vessel situated in the fermentation bath. Since the outside of the fermentation vessels is gas-permeable at least in part-regions, the fermentation vessels take up a part, in particular of the dissolved gas or the gas mixture, from the fermentation bath.

Depending on the gas permeability of the material used to produce the fermentation vessels, the gas exchange with the surroundings, that is with the fermentation bath, is more or less strongly expressed. The predominant part of the gas exchange takes place in dissolved form of the gases. This makes the - - exchange more effective than with additional passage from the liquid phase to the gas phase and vice versa.

In the aerobic process, gas transport of the oxygen proceeds from the gas bubble through the boundary layer into the filling medium of the gas-dispersion trough (fermentation bath), from there to the vicinity of the surface of the fermentation vessel, through the boundary layer of the filling medium of the fermentation bath into the permeable material of the wall of the fermentation vessel, from the outside to the inside of the wall of the fermentation vessel, from the inside of the wall through the boundary layer of the nutrient medium or fermentation medium, through the nutrient medium or fermentation medium to the microorganism, and finally through its cell membrane. Mass transport of the exhaust gases (CO₂, CH₄, H₂S, amines, etc.) takes place in reverse sequence.

In a preferred embodiment, dissolved gases pass through the outer wall of the fermentation vessel and convert to the gaseous state in the interior of the fermentation vessel, so that gas bubbles also form in the interior of the fermentation vessels. The gas bubbles which are situated within the fermentation vessels and ascend therein, owing to their buoyancy, ensure vortexing of the fermentation medium, so that settling of microorganisms situated in the fermentation vessels is prevented and intensive mass transfer is ensured.

Powerful agitators can therefore be avoided, so that also, high sheer forces do not occur. Since the microorganisms are situated within the fermentation vessels and preferably not in the fermentation bath, however, it is also possible, to improve the mass transport within the fermentation bath, to vortex this by the introduction of kinetic energy. Any sheer forces occurring in the course of this are comparatively harmless for the microorganisms, since they are sufficiently protected by the outer wall of the fermentation vessels. High sheer forces also, do not occur, especially because the microorganisms in the fermentation vessels are substantially protected from external forces in the fermentation baths. Powerful agitators in the fermentation baths would not be avoided in all cases, since the agitator energy input decisively affects the gas transfer from the gas bubbles into the filling medium of the fermentation bath.

For the gas treatment, the gas-dispersion troughs have a device by which the gas treatment proceeds. The following can serve for gas introduction: a simple immersion tube having an additional mechanical device for vortexing the gas bubbles, or an immersion tube having many fine boreholes, what is termed a "sparging ring" (that is an annular tube having many fine boreholes), a sparging disc or gas introduction and distribution devices produced from fine-pored materials. Fine-pored materials which come into consideration are, for example, sintered steel, (sintered) plastics, perforated rubber membranes (for example in the case of self-closing sparging discs), filter membrane materials, etc.

In a preferred embodiment of the inventive method, in step (e) the fermentation bath is heated/cooled. The heating/cooling can be performed by suitable heating or cooling elements. Customarily, a temperature is set in the range from 20 to 6O⁰C, preferably 30 to 4O⁰C. - -

In a preferred embodiment, the liquid in the gas-dispersion trough, that is the fermentation bath, is agitated and mixed in order to keep the diffusion path lengths for the gases in the liquid as short as possible. The mixing can proceed via agitators or pumps. Preferably, the fermentation vessels and the fermentation medium contained therein can be kept in agitation by pressure variations, motions of the bath liquid and/or by vibrations, in order to promote mass transport and diffusion in the fermentation bath and in the fermentation medium.

In a preferred embodiment of the inventive method, in step (e), the fermentation bath is sonicated with ultrasound. The frequencies used are preferably in the range from 20 to 120 kHz. The typical energy input should be in the framework of introduction in the case of typical laboratory ultrasound baths. Too little energy input leads to the fact that the energy is absorbed by the fermentation vessels and too little arrives in the nutrient or fermentation medium. Too much energy would destroy the cells. Those skilled in the art can make an accurate setting, depending on the size and geometry of the vessels and the plant.

The sonication with ultrasound has the advantage that settling of the microorganisms and any particles suspended in the fermentation medium are actively inhibited, so that a distribution which is as homogeneous as possible is ensured.

In a preferred embodiment of the inventive method, the fermentation bath, in addition to the gas or the gas mixture, contains at least one substance which is required by the microorganisms for the biosynthesis and to which the outer wall of the fermentation vessels is permeable in at least in a part- region. In this case, the mass transport of the substance proceeds through the outer wall of the fermentation vessel by diffusion (osmosis). Since the microorganisms within the fermentation vessels continuously consume this substance, the gradient of chemical potential of the substance between fermentation medium and fermentation bath is continuously maintained. Preferably, the substance is a molecule of a relatively low molecular weight. Preferably, the molecular weight of the substance is below 5000 g/mol, more preferably below 2500 g/mol and in particular below 1000 g/mol.

In a preferred embodiment of the inventive method, the microorganisms synthesize by biosynthesis a biomolecule to which the outer wall of the fermentation vessel is impermeable. In this manner, the synthesized biomolecule is retained in the fermentation vessels. Isolation does not proceed until after termination of the process for biosynthesis of a biomolecule and emptying of the fermentation vessels. However, in principle, it is also conceivable that the outer wall of the fermentation vessels is permeable to the synthetic biomolecule, so that it can be isolated not only from the fermentation medium in the interior of the fermentation vessels, but also from the liquid of the fermentation bath.

In a preferred embodiment, the fermentation vessels for carrying out step (e) are fixed in a holder device which permits the fermentation vessels to participate equally in mass transport (gas exchange), or which facilitates the defined withdrawal and identification of the individual fermentation vessels in - - the bath, for example during removal. For this purpose, the holder device can be provided with an inscription, e.g. numbering.

In a preferred embodiment, the gas-dispersion trough has a transport system to which the fermentation vessels are attached and by which the fermentation vessels are transported through the gas-dispersion trough. It is possible that a plurality of gas-dispersion troughs are passed through one after the other, so that the transport system transports the individual fermentation vessels from gas- dispersion trough to gas-dispersion trough. Between the individual gas-dispersion troughs, there can proceed various processes for controlling microbial growth, expression and production of the desired biomolecule, for example the addition of starter substances, control substances or messager substances, replenishment of nutrient medium, etc.

Preferably, the fermentation vessels are incubated in the fermentation bath until the biosynthesis has led to a certain minimum conversion rate of the biomolecule and the nutrient substances fed have been substantially consumed. The time which is required to achieve a defined conversion rate under the conditions selected can be determined by simple preliminary experiments. The fermentation vessels can be withdrawn, for instance, under time or progress control, the advance of the biosynthesis also being able to be tested at regular intervals by cell density measurements. Also, if the process requires it, the fermentation vessels can be withdrawn from the fermentation bath for the purpose of reinoculation. This can be necessary with nutrient medium and/or with defined substances for control, for induction or for termination of certain phases of growth and the production of the microorganisms.

Thereafter, the fermentation vessels are removed if appropriate in step (f) from the fermentation bath. Preferably, they are cleaned from the outside and subjected to automatic or visual inspection.

Individual removed fermentation vessels can be used for step (d), that is for inoculating fresh nutrient- medium-filled fermentation vessels.

The remaining fermentation vessels can subsequently be opened, for example by removing closure caps or stoppers. Subsequently, the contents are emptied for further processing, for example homogenization and separation of the cells. The biomolecule obtained by biosynthesis can be isolated thereafter by suitable methods, for example by extraction, crystallization, centrifugation or chromatographic methods, such as, for example, by affinity chromatography. Suitable methods which depend, in particular, on the type of biomolecule synthesized are known to those skilled in the art. In this context, reference can be made, for example, to H. Schott, Affinity Chromatography: Template Chromatography of Nucleic Acids and Proteins (Chromatographic Science), Marcel Dekker (1984); R. K. Scopes, Protein Purification: Principles and Practice, Springer; 3 ed. (1993); S. Roe, Protein Purification Techniques: A Practical Approach (Practical Approach Series), Oxford University Press; 2nd ed. (2001 ); and H. Ahmed, Principles and Reactions of Protein Extraction, Purification, and Characterization, CRC Press (2004). - -

If the biomolecule obtained by biosynthesis is not too sensitive, the fermentation vessels can be sterilized before emptying and further processing.

If the biomolecule, in contrast, is too sensitive, the fermentation vessels are preferably first opened under appropriate aseptic conditions, emptied and subsequently the emptied fermentation vessels are sterilized and thereafter cleaned, or vice versa.

Steps (b), (c) and (f) of the inventive method are optional, and steps (a), (d) and (e) are obligatory. In a preferred embodiment, the inventive method, in addition to steps (a), (d) and (e) comprises at least one, preferably at least two, of the optional steps (b), (c) and (f). In a particularly preferred embodiment, the inventive method comprises all steps (a), (b), (c), (d), (e) and (f), preferably in this sequence.

In a preferred embodiment of the inventive method, the volume of the fermentation vessels is typically in the range from 1 ml to 10 I. Preferably, the volume of the fermentation vessels is in each case at most 10 000 ml, preferably at most 1000 ml, more preferably at most 800 ml, still more preferably at most 600 ml, most preferably at most 500 ml, and in particular at most 400 ml.

This ensures that a batch of a plurality of hundred or thousand litres is subdivided over a sufficiently large number of compartments, as a result of which the risk of contamination of the entire batch is significantly decreased.

Fermentation vessels which have a contamination can generally be recognized by a discoloration of the fermentation medium. In a preferred embodiment, the outer wall of the fermentation vessels is made, at least in a part-region, of a transparent material, so that such a discoloration of the fermentation medium can be established by simple visual control and correspondingly discoloured, that is contaminated, fermentation vessels can be eliminated.

In a preferred embodiment of the inventive method, emptied fermentation vessels are reused in an optional step (g).

For this, if required, after the fermentation medium together with the synthesized biomolecule and the microorganisms have been emptied out, they are washed by current cleaning methods, for example using hot, purified water (in accordance with the European pharmacopoeia) which is sprayed into the interior of the fermentation vessels using a nozzle and can run out via an orifice. After cleaning the fermentation vessels, a drying step can proceed, for example using filtered blown-in air.

Subsequently it is possible to subject the empty, possibly cleaned, and dried fermentation vessels to sterilization. For this, the appropriate standard methods are available, as have already been described above in connection with step (c) of the inventive method. - -

To avoid contamination, leaks and problems in the later process, it is advisable to subject the emptied fermentation vessels to an inspection for damage and material fatigue before reuse. Preferably, visual examination of the empty fermentation vessels for damage and leaks finally proceeds.

In a preferred embodiment of the inventive method, it is carried out semi-continuously, which can be achieved in various ways. A semi-continuous method within the meaning of the description comprises a method in which, of a multiplicity of fermentation vessels filled in step (a), at least two fermentation vessels are subjected one after the other in time to the incubation in step (e).

In a preferred embodiment of the semi-continuously performed inventive method, the totality of all fermentation vessels can, at a given timepoint, be subdivided into at least three groups:

(i) empty fermentation vessels;

(ii) fermentation vessels which have been charged with nutrient medium, but not yet inoculated with microorganisms;

(iii) fermentation vessels which have been charged with fermentation medium, i.e. charged with nutrient medium and also already inoculated with microorganisms.

In a particularly preferred embodiment of the inventive method which is preferably carried out semicontinuously, at a given timepoint, at least N fermentation vessels are used in N different steps of the inventive method, the N different steps being selected from steps (a), (b), (c), (d), (e) and (f), and N being able to be 2, 3, 4, 5 or 6.

Figure 1 shows a diagrammatic presentation of a preferred embodiment of the inventive method. The empty if appropriate cleaned and if appropriate sterilized fermentation vessels are charged in step (a) with nutrient medium, closed in step (b) and if appropriate sterilized in step (c). The fermentation vessels prepared in this manner are subsequently inoculated in step (d) with microorganisms which are indicated by an oval. In step (e) the microorganisms were multiplied and the desired biomolecule (not shown) was biosynthesized in a gas-dispersion trough. In step (f), the completely reacted (fermented) fermentation vessels are opened and the microorganisms are withdrawn together with the synthesized biomolecule. Subsequently, the used fermentation vessels, in step (g), if appropriate after cleaning, drying and/or sterilization, are fed back to the starting point of the method. A part of the microorganisms from fermented fermentation vessels is redrculated to the process and serves for (renewed) inoculation in step (d).

Figure 2 shows a preferred embodiment of steps (e) and (f) of the inventive method. The fermentation vessels (1 ) are attached using suitable external attachment means (2) to a transport device (3) and are moved in the direction of the arrow. In parallel to the direction of transport of the transport device (3), a gas-dispersion trough (4) is arranged, which is charged with a fermentation bath (5) and has a gas-dispersion device (6), via which the air or oxygen is blown into the gas-dispersion trough (4), that - - is into the fermentation bath(s). The air or oxygen bubbles ascend within the gas-dispersion trough (4) in the direction of the arrows (7) in the fermentation bath (5). Instead of oxygen, inert gas (for example He, Ar, N₂, etc.) or CO₂ or other gases or mixtures thereof can also be used, depending on requirements of the respective fermentation process (aerobic vis. anaerobic). In the course of transport of the fermentation vessels (1 ) using the transport device (3), the individual fermentation vessels (1 ) are immersed in the fermentation bath (5) and treated with gas in this manner. After a certain time which depends on the length of the gas-dispersion trough (4) and the velocity of the transport device (3), the fermentation vessels (1 ) are removed again from the fermentation bath (5) in the gas-dispersion trough (4). Those skilled in the art know that subsequently further gas-dispersion troughs can be controlled by the transport device (3) and in the interim the fermentation vessels can be fed to certain process steps, such as replenishment of the nutrient medium, control of growth of the microorganisms, partial withdrawal of the biomolecule to be synthesized, monitoring for contamination, etc.

The inventive, preferably semi-continuous, method can, in addition, be characterized by the following features:

the contents of the fermentation trough (the fermentation bath) can be circulated by pumping;

the fermentation bath can be filtered during the circulation by pumping in order to reduce or control the microbial loading;

the fermentation bath can, during circulation by pumping, be sterilized or reduced in microbial count in continuous flow; suitable methods for this are known to those skilled in the art (heat sterilization, pasteurization, UV sterilization, sterilization using ionizing radiation);

the fermentation bath, during circulation by pumping, can be filtered or ultrafiltered in order to remove solids, precipitates and unwanted substances, for example endotoxins;

the fermentation bath, to remove H₂S (for example in the case of the anaerobic fermentation), can contain a dissolved metal salt whose cation forms slightly soluble sulphides (for example CoCI₂, FeCI₂); the resultant precipitates can be filtered off and removed; in this manner a high concentration gradient of H₂S is continuously maintained between fermentation vessel and fermentation bath, so that continuous diffusion and removal of H₂S from the fermentation vessels can be achieved;

the fermentation bath can contain further substances in dissolved form, which substances form slightly soluble precipitates with unwanted substances which can then be filtered off; and/or

the fermentation bath, during circulation by pumping, can be pumped through a packed column or bed, in order to remove certain unwanted substances; for this, the column packing or the bed should contain an immobilized material which has an affinity to the substance to be removed or - - reacts chemically with it; the packing or bed can be regularly renewed (for example a corresponding packing of sintered pure iron can be used for removing H₂S - iron sulphide forms on the surface of the iron grains).

The inventive method has not only ecological but also economic advantages, in particular when account is taken of the requirements which are made of clean rooms for pharmaceutical production. The construction and operation of clean rooms for pharmaceutical production is cost-intensive. The costs typically increase virtually linearly with the altered clean room volume.

In the case of clean rooms for pharmaceutical production, the protection of the product against external contamination is in the foreground. Therefore, such clean rooms typically have an overpressure compared with the surrounding rooms.

For reasons of protecting the environment and humans from contamination with genetically modified microorganisms, there are efforts to provide the fermentation rooms with reduced pressure compared with the environment.

Both concepts, overpressure on the one hand and reduced pressure on the other, however, can only be combined with one another in a very complex manner, which ultimately leads to mutually interlocking clean room designs having different pressure levels. Such clean room designs further increase the costs for operating production in clean rooms. There is therefore a great requirement for designs which minimize as far as possible the requirement for clean room volumes.

It has surprisingly been found that the concept underlying the invention permits the minimization of clean room volumes and the partial separation of clean rooms having an overpressure and reduced pressure design, so that interlocking can be substantially avoided.

In the operation of a conventional fermentation vessel, virtually all process steps are carried out in one place, customarily in a room. The room requirement, is thus high and therefore requires a comparatively large clean room. Not only product protection but also protection of the environment must then be ensured for this room.

In contrast thereto, the inventive method can be carried out semi-continuously and therefore at different places or rooms having conditions individually matched to the clean room design, which overall, simplifies and reduces the costs of the clean room design.

On charging the fermentation vessels (step (a)), it is only necessary to take into account protection of the product. Since the individual fermentation vessels are relatively small, a small clean room region suffices, for example an isolator. - -

For inoculating the fermentation vessels (step (d)), it is necessary to take into account protection not only of the product but also of the environment. Owing to the small size of the fermentation vessels, here also, a comparatively small clean room region suffices, for example of the size of an isolator.

All transports, intermediate storage and, especially, the fermentation itself (step (e)) take place in closed systems, so that a special clean room design is not necessary. Hazards due to possible leaks from fermentation vessels can be counteracted by adding biocides to the fermentation bath.

To empty the fermentation vessels (step (f)), it is necessary to take into account protection of the product and, if a preceding sterilization is not possible, also the protection of the environment. Owing to the small size of the fermentation vessels, here again a minimum clean room region is sufficient, for example of the size of an isolator.

A further aspect of the invention relates to a fermentation vessel which can advantageously be used in the above-described method. The inventive fermentation vessel comprises

an outer wall which is impermeable to microorganisms, but is gas-permeable at least in a part- region, preferably oxygen-permeable;

a closable charging orifice; and

if appropriate a device for aseptic addition of a solid or liquid composition;

the ratio of the outer surface area of the fermentation vessel to its internal volume being at least 0.5 cm²/ml, preferably at least 1.0 cm²/ml, more preferably at least 2.5 cm²/ml, still more preferably at least 4.0 cm²/ml, most preferably at least 5.0 cm²/ml, and in particular at least 6.0 cm²/ml.

Fermentation vessels of various types are disclosed in the prior art. For example, US 5,686,304 discloses a bag having thin gas-permeable outer walls made of silicone rubber.

The fermentation vessels of the prior art, however, have the disadvantage that the gas exchange between the fermentation medium and the surroundings is not optimal, as a result of which the conditions for biosynthesis are impaired. To achieve a surface area/volume ratio as high as possible, the fermentation vessels of the prior art must be filled to be virtually gas free. To keep the layer thicknesses of the nutrient or fermentation medium within the fermentation vessels as small as possible, the working volume in the case of flexible bags must be only a fraction of the maximum filling capacity which could be achieved with complete bulging of the bag.

The fermentation vessels of the prior art bulge. The bulging proceeds, in particular with increasing bag size, in an undefined manner with increasing extent. For industrial processes, for economic reasons, working capacities greater than 200 ml are preferred. The undefined deformations lead to uneven - - growth conditions. These in turn mean disadvantages in the use of the fermentation vessels for a standardized repeating industrial process. The fermentation vessels of the prior art are therefore suitable for laboratory experiments, but suitable only with restrictions for a semi-continuous industrial process.

It has surprisingly been found that the conditions for biosynthesis within the fermentation vessel can be improved when the outer surface area of the fermentation vessel is in a defined ratio to its internal volume. This ratio can have a significant effect on the course of the biosynthesis.

Firstly, the mass transport is the better, the higher the ratio.

Secondly, however, it is true that the smaller the ratio, the more compact are the individual fermentation vessels, the simpler is the handling, the easier it is to avoid regions having directly contacting vessel walls (collapsing), the smaller the amount of vessel material is required, and the more inexpensive are the fermentation vessels.

For the respective framework conditions, there is therefore an optimum between the requirements of mass transfer, the technical handleability and the vessel costs. This optimum is also affected by the properties of the vessel material and the possible maximum partial pressure differences of dissolved gases between the filling of the fermentation trough and the contents of the fermentation vessel.

At a layer thickness of the vessel material (outer wall) of about 50 to 150 μm, starting from a fermentation vessel having between 50 and 1000 ml working volume, produced from polymer material (polysiloxane film), of medium strength, at an oxygen partial pressure difference (dissolved) of approximately 150 to 200 mbar, the optimum surface area/volume ratio should be between 0.01 cm²/ml and 5.0 cm²/ml, preferably between 0.025 and 2.5 cm²/ml, still more preferably between 0.1 and 1.0 cm²/ml.

Preferably, the inventive fermentation vessel is equipped in such a manner that it is advantageously suitable for use in the inventive method. Preferred embodiments of the inventive fermentation vessel have therefore already been explained above in connection with the description of the inventive method (for example with respect to volume).

The inventive fermentation vessel has an outer wall which is impermeable to microorganisms, but, at least in a part-region, is gas permeable, preferably oxygen-permeable.

In a preferred embodiment of the inventive fermentation vessel, the outer wall, at least in a part-region, has an oxygen permeability of at least 500 cm m^" d^" bar^" , more preferably at least 600 cm m^" d^" bar^" , still more preferably at least 750 cm³m^"2d^"1bar^"1, most preferably at least 1000 cm³m^"2d^"1bar^"1 and in particular at least 1500 cm³m^"2d^"1bar^"1. Suitable methods for determining oxygen permeability are - - known to those skilled in the art. Preferably, the oxygen permeability is determined as specified in DIN ISO 53 380, part 3.

Suitable materials for producing gas-permeable membranes are known to those skilled in the art. In this context, reference can be made, for example, to R.W. Baker, Membrane Technology and Applications, Wiley, 2nd ed., 2004; P. E. Odendaal et al., Water Treatment Membrane Processes, McGraw-Hill Professional, 1996; T. Melin et al., Membranverfahren [Membrane methods], Springer, Berlin, 2003; K. Gorner, Gasreinigung und Luftreinhaltung [Gas purification and clean air maintenance], Springer, 2001 ; R. Rautenbach et al., Membrane Processes, John Wiley & Sons, 1989; R. E. Kesting, Polymeric Gas Separation Membranes, Wiley-lnterscience, 1993; and D. R. Paul et al., Polymeric Gas Separation Membranes, CRC Press, 1993.

In a preferred embodiment of the inventive fermentation vessel, the outer wall, at least in a part-region, is made of a membrane which is based on at least one polysiloxane. Suitable silicone membranes are described, for example, in US 3,489,647, US 3,510,387, US 3,969,240 and US 4,093,515, the contents of which are incorporated by reference into the present description.

Preferably, the gas-permeable part-region of the outer wall of the inventive fermentation vessel extends over at least 50% of the area of the outer wall, preferably at least 60%, still more preferably at least 70%, most preferably at least 80%, and in particular at least 90%, of the area of the outer wall.

Preferably, the outer wall of the inventive fermentation vessel, at least in a part-region, is permeable to CO₂, H₂S, CH₄ and/or volatile amines. Volatile amines within the meaning of the description are preferably amines which, at atmospheric pressure, have a boiling point below 4O⁰C. Examples are CH₃NH₂, (CHa)₂NH, (CH₃)₃N and CH₃CH₂NH₂.

In a preferred embodiment of the inventive fermentation vessel, the outer wall, at least in a part-region, has a thickness of at most 150 μm, more preferably at most 125 μm, still more preferably at most 100 μm, most preferably at most 75 μm, and in particular at most 50 μm. The layer thickness of the membrane can be determined by conventional methods, preferably by microscopy.

In a preferred embodiment, the inventive fermentation vessel has a device for aseptic addition of the solid or liquid composition. Suitable devices are known to those skilled in the art. Preferably, the device, for the purposes, of addition, can be punctured by a needle or mandrel and after, removing the needle or the mandrel, can close again spontaneously.

In a preferred embodiment of the inventive fermentation vessel, the outer wall, at least in a part-region, is permeable to water, preferably to water in the liquid state. In a preferred embodiment, the outer wall of the fermentation vessel, at least in a part-region, is permeable to dissolved substances of a relatively low molecular weight. In this case, the mass transport of the dissolved substance proceeds through the outer wall of the fermentation vessel by diffusion (osmosis). Preferably, the limit molecular - - weight of a substance which is still just let through (also termed "cut-off'), is less than 10 000 g/mol, more preferably less than 5000 g/mol, most preferably less than 2500 g/mol, and in particular less than 1000 g/mol.

In another preferred embodiment of the inventive fermentation vessel, the outer wall, at least in a part- region, is impermeable to water, preferably to water in the liquid state.

In a preferred embodiment of the inventive fermentation vessel, it has an outer attachment means. The outer attachment means can be used for attaching the inventive fermentation vessel to an automated transport device and to use it, for example, in the inventive method.

Preferably, the inventive fermentation vessel, in its interior, has a composition to which the microorganisms can be immobilized, preferably can adhere. Composition and surfaces which are suitable for this purpose depend on the type of microorganism used and are known to those skilled in the art.

Figure 3 shows a preferred embodiment of the inventive fermentation vessel. The fermentation vessel has an outer wall (8) which, at least in part-regions, is gas-permeable. On the side, a device (9) for adding solid compositions is arranged which is closed with a screw closure (10). On the top side there is situated a device (11 ) for aseptic addition of a liquid composition, which, for the purposes of addition, can be punctured by a needle or a mandrel and, after removing the needle or the mandrel, closes again spontaneously. For this, the device (11 ) has a septum (12). In addition, on the top side is situated an attachment device (13) having an eye (14), via which the fermentation vessel can be attached, for example to a transport device.

In a preferred embodiment of the inventive fermentation vessel, it is sterilizable, that is it resists the conditions which are found, depending on the sterilization method. The vessel should preferably be designed in such a manner that it can be sterilized repeatedly. Sterilization methods available are: heat/saturated steam, heat/dry, ethylene oxide gas treatment, ozone gas treatment, UV irradiation, ultrasound, use of ionizing radiation, γ-radiation, β-radiation.

Preferably, the inventive fermentation vessel has a dimensional stability such that it essentially retains, after being filled with a liquid, its original spatial extension. Preferably, the dimensional stability is achieved by inner and/or outer internals (stiffening means).

Preferably, the inventive fermentation vessel is shaped to form a bag. If the fermentation vessel is not a rigid vessel, but a flexible bag, the bag preferably contains inner and/or outer internals (stiffening means), which ensure that the layer thickness of the nutrient medium or fermentation medium within the fermentation vessel is kept relatively constant, bulging or collapsing of the fermentation vessel being prevented. This makes possible the fact that parts as large as possible of the vessel surface prepared and intended for diffusion (mass transport) participate in mass transfer. - -

Preferably according to the invention, the layer thickness of the nutrient medium or fermentation medium is defined as the greatest expansion of the fermentation vessel perpendicular to its main extension plane.

In a preferred embodiment, the inventive fermentation vessel is created in such a manner that the greatest expansion perpendicular to its main extension plane in the emptied state by charging the fermentation vessel with nutrient medium or fermentation medium increases by at most 30%, more preferably at most 25%, still more preferably at most 20%, most preferably at most 15%, and in particular at most 10%. This increase can be considered to be an index for the bulging of the fermentation vessel due to charging, the expansion preferably being measured in the free state, that is not in the state immersed in the fermentation bath.

To improve the mechanical strength, the membranes of the inventive fermentation vessel can be equipped with suitable reinforcing or stiffening means, as are described, for example, in US 4,093,515. In addition, the described fermentation vessel can have the following additional features:

further connections for sterile/aseptic withdrawal and addition of liquids, or suspensions, emulsions, solutions;

connections which permit circulation by pumping and if appropriate filtration of the contents.

To achieve optimum gas diffusion and mass diffusion from the vessel wall to the microorganisms in the fermentation medium, it is advantageous to select the mechanical and geometric properties of the fermentation vessel in such a manner that the layer thickness of the nutrient medium or fermentation medium within the charged fermentation vessel is kept relatively constant in the range of a few mm to a few cm.

The layer thickness to be set varies according to bag working contents, geometric requirements (technical handleability of the fermentation vessels), permitted process time duration, gas and mass requirement for the specific process, microorganism, bag geometry and bag material used, etc. Preferably, the average layer thickness of the nutrient medium or fermentation medium is in the range from 0.1 mm to 200 mm, more preferably 1.0 mm to 50 mm, still more preferably 2.0 mm to 25 mm, most preferably 3.0 mm to 15 mm. The said values preferably apply to a typical aerobic fermentation (for example with E. coli), limited by the oxygen transport at a working capacity of approximately 200 ml and a vessel made of typical medical grade polysiloxane having a material thickness of 75 to 125 μm. These values are dependent on the material used, the layer thickness of the material, the vessel surface participating in mass transfer and the specific diffusion constants of the material for the substance in question. - -

The layer thickness of the nutrient medium or fermentation medium in the interior of the fermentation vessel is of importance, in particular, since preferably in the inventive method no agitator internals are provided in the fermentation vessels, and therefore the mass transport in the nutrient medium or fermentation medium proceeds predominantly via diffusion and only to a lesser extent by flow or cavitation. Flow and cavitation (compared with an agitator) can proceed only to a comparatively low extent via the medium in the fermentation bath by conversion of currents and forces (e.g. ultrasound or mechanical shaking).

For this reason, a simple bag which is only sealed at the rim (welded, glued or clamped) made of a film is at best conditionally suitable for the inventive method. Such a bag, in particular with the use of thin wall thicknesses, bulges greatly, the advantages of the thin layer thickness being lost. In addition, owing to the bulging, only a part of the film of the bag is sufficiently wetted with nutrient medium or fermentation medium, which means that the non-wetted part scarcely participates in the gas exchange. In particular in the case of increasing bag size, as is advantageous, for example, for a method on the industrial scale, the bulging of a conventional bag welded at the rims made of a film cannot always take place uniformly. This has adverse effects on the uniformity and predictability of the overall method.

It has surprisingly been found that this problem can be solved when the fermentation vessel has inner and/or outer internals (stiffening means) which permit the fermentation vessel to retain within certain limits its shape and thus the layer thickness of the nutrient medium or fermentation medium present therein. It is advantageous in this case when the fermentation vessel, despite fixing, still has sufficient mechanical flexibility to permit external force actions (ultrasound, flow, etc.), so that currents and agitations can be induced in the interior of the fermentation vessel in the fermentation medium.

In a preferred embodiment of the inventive fermentation vessel, it is either constructed from a dimensionally stable material, or has inner and/or outer internals (stiffening means) or additional connections which stabilize its shape, so that the layer thickness of the nutrient medium or fermentation medium is kept as constant as possible and/or collapsing of the outer wall is prevented, so that as large a part as possible of the outer wall can participate in the mass transfer.

Such a stabilization of the inventive fermentation vessel can be achieved by:

forming the fermentation vessel from a rigid material having recesses ("windows") from gas- permeable, flexible material;

introducing a fermentation vessel also made of a flexible material into an outer rigid frame (e.g. having transverse ridges or a mesh), which prevents the bulging of the fermentation vessel and is preferably fixed to the fermentation vessel; - - introducing inner internals made of a rigid material into the fermentation vessel which are fixed to the outer wall and thus prevent bulging;

connecting the outer wall of the fermentation vessel (bag film) at one or more points to the opposite outer wall of the fermentation vessel (bag film), in order to limit the maximum layer thickness of the nutrient medium or fermentation medium; and/or

introducing the fermentation vessel into a rigid frame or outer container (which can be used, for example, at the same time as transport mount), the fermentation vessel not being fixed to the frame or outer container, but the shape of the fermentation vessel being fixed and bulging being prevented.

The stabilized inventive fermentation vessel, compared with the fermentation vessels of the prior art, in the mechanical aspect, has in particular the following advantages:

in vertical position: avoidance of the undefined and uncontrolled bulging or deformation after charging and also avoidance of the collapse of the bag film in the upper half of the fermentation vessel; and/or

in horizontal position: avoidance of collapse of the bag film at various points.

This has various advantageous effects:

defined thin layer thicknesses of the nutrient medium or fermentation medium in the interior of the fermentation vessel are achieved;

short and defined average diffusion pathways for mass transport in the nutrient or fermentation medium lead to defined uniform growth conditions;

the great majority of the surface can participate in mass transport, since the collapse of the bag film is minimized - this leads to better and defined growth conditions for all cells in the fermentation medium;

the more defined conditions overall improve the use in a defined, semi-continuous industrial process, the process control and the stability of the process; and/or

the defined layer thicknesses of the nutrient medium or fermentation medium make possible improved standardized visual or physical and optical evaluation of the quality of the fermentation contents (for example colour assessment, turbidity assessment) and the fermentation progress (optical cell density measurement) on the closed fermentation vessel. - -

Figure 4 shows a conventional filled fermentation vessel which contains no stabilization. The fermentation vessel has a rim weld seam (16) and a surface region not participating in mass transfer (17). The side view shows how the material of the fermentation vessel (bag) bulges in the lack of stabilization. The layer thickness (15) of the fermentation medium within the fermentation vessel is therefore much greater than desired.

Figures 5 to 7 show preferred embodiments of the inventive fermentation vessel each in front view and side view. The desired layer thickness (15) of the fermentation medium is effected here via different structural elements.

Figure 5 shows a charged fermentation vessel having stabilizing connections or adhesions (diagrammatic). The connections made between the upper film and the lower film (18).

Figure 6 shows a fermentation vessel having stabilizing internals, here having a fixed integrated frame (19). The frame merely fixes the shape of the fermentation vessel, but does not subdivide it into separate compartments.

Figure 7 shows a fermentation vessel having a stabilizing outer frame (20).

A further aspect of the invention relates to the use of one or more of the above described fermentation vessels for the synthesis of a biomolecule by biosynthesis by microorganisms.

In a preferred embodiment of the inventive use, the biomolecule is synthesized according to the above-described inventive method.

Claims

- -Claims:

1. Method for the biosynthesis of a biomolecule by microorganisms in a multiplicity of fermentation vessels which have an outer wall that is impermeable to microorganisms, but is gas- permeable at least in a part-region comprising the steps

(a) introducing a nutrient medium into the fermentation vessels,

(b) if appropriate closing the fermentation vessels so that mass transfer with the surroundings can only still proceed through the outer wall of the fermentation vessels,

(c) if appropriate sterilizing the fermentation vessels,

(d) inoculating the nutrient medium in each fermentation vessel by aseptic addition of the microorganisms,

(e) introducing the fermentation vessels into a fermentation bath in which a gas or gas mixture is dissolved, and

2. Method according to Claim 1 , characterized in that the volume of each fermentation vessel is at most 1000 ml and/or the ratio of the outer surface area of each fermentation vessel to its internal volume is at least 0.5 cm²/ml.

3. Method according to Claim 1 or 2, characterized in that, in step (e), the gas is oxygen or the gas mixture contains oxygen.

4. Method according to one of the preceding claims, characterized in that, in step (e), the gas or gas mixture is bubbled through the fermentation bath, the gas on being blown in being at a pressure which is equal to or greater than atmospheric pressure.

5. Method according to Claim 4, characterized in that the gas or gas mixture comprises oxygen and serves to introduce the oxygen for an aerobic fermentation and/or to discharge volatile byproducts formed in an aerobic fermentation, or comprises no oxygen and serves to discharge the oxygen formed as by-product in an anaerobic fermentation.

6. Method according to Claim 4 or 5, characterized in that the gas mixture contains CO₂, CH₄ and/or H₂S, together with an inert gas. - -

7. Method according to one of the preceding claims, characterized in that, in step (e), the fermentation bath is heated/cooled.

8. Method according to one of the preceding claims, characterized in that, in step (e), the fermentation bath is sonicated with ultrasound.

9. Method according to one of the preceding claims, characterized in that the microorganisms are selected from the group consisting of bacteria, cell lines and yeasts.

10. Method according to one of the preceding claims, characterized in that the fermentation bath, in addition to the gas or gas mixture, contains at least one substance which is required by the microorganisms for biosynthesis and to which the outer wall of the fermentation vessels is permeable at least in a part-region.

11. Method according to one of the preceding claims, characterized in that the microorganisms synthesize by biosynthesis a biomolecule to which the outer wall of the fermentation vessels is impermeable.

12. Method according to one of the preceding claims, characterized in that it is carried out semi- continuously.

13. Fermentation vessel comprising

an outer wall which is impermeable to microorganisms, but is gas-permeable at least in a part- region,

a closable charging orifice, and

if appropriate a device for aseptic addition of a solid or liquid composition,

the ratio of the outer surface area of the fermentation vessel to its internal volume being at least 0.5 cm²/ml.

14. Fermentation vessel according to Claim 13, characterized in that it has a device for aseptic addition of the solid or liquid composition, which, for the purposes of addition, can be punctured by a needle or a mandrel and, after removing the needle or the mandrel, closes again spontaneously.

15. Fermentation vessel according to Claim 13 or 14, characterized in that the outer wall, at least in a part-region, has an oxygen permeability of at least 500 cm³rrT²d^'1bar^'1. - -

16. Fermentation vessel according to one of claims 13 to 15, characterized in that the outer wall, at least in a part-region, is made of a membrane which is based on at least one polysiloxane.

17. Fermentation vessel according to one of claims 13 to 16, characterized in that the outer wall, at least in a part-region, has a thickness of at most 150 μm.

18. Fermentation vessel according to one of claims 13 to 17, characterized in that the outer wall, at least in a part-region, is impermeable to water.

19. Fermentation vessel according to one of claims 13 to 18, characterized in that the outer wall, at least in a part-region, is permeable to CO₂, H₂S, CH₄ and/or volatile amines.

20. Fermentation vessel according to one of claims 13 to 19, characterized in that it is sterilizable.

21. Fermentation vessel according to one of claims 13 to 20, characterized in that it is shaped to form a bag.

22. Fermentation vessel according to one of claims 13 to 21 , characterized in that it has outer attachment means.

23. Fermentation vessel according to one of claims 13 to 22, characterized in that it has in its interior a composition on which the microorganisms can be immobilized.

24. Fermentation vessel according to one of claims 13 to 23, characterized in that it has a dimensional stability such that, after it is charged with a liquid, it essentially retains its original spatial extension.

25. Fermentation vessel according to Claim 24, characterized in that the dimensional stability is achieved by internal and/or external stiffening means.

26. Use of a fermentation vessel according to one of claims 13 to 25 for synthesis of a biomolecule by biosynthesis by microorganisms.

27. Use according to Claim 26, characterized in that the biomolecule is synthesized by the method according to one of claims 1 to 12.