Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/34—Internal compartments or partitions
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
  • C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/38—Caps; Covers; Plugs; Pouring means
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
  • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
  • C12M29/20—Degassing; Venting; Bubble traps

Definitions

• the invention relates to a bioreaction module for biochemical reactions, in particular for the cell-free biosynthesis of proteins or other polypeptides.
• biochemical reactions are often carried out in small, easy-to-use reaction vessels, which are referred to here as the bioreaction module. They are primarily used for the biosynthesis of polypeptides, such as peptide hormones, antibiotics and - more recently - human proteins.
• the biochemical systems used here include pure translation systems in which the biosynthesis is based on the genetic code of a messenger RNA (mRNA) and transcriptions / translation systems which also include the preceding step of forming messenger RNA from DNA.
• mRNA messenger RNA
• transcriptions / translation systems which also include the preceding step of forming messenger RNA from DNA.
• bioreaction modules are also used for other biochemical reactions, in particular enzymatic reactions.
• Cell-free biosynthesis is of particular importance, as it has significant advantages over in-vivo systems. For example, it enables the expression of toxic and unstable gene products. Cell-free biosynthesis is often used for analytical purposes, in particular to quickly and conveniently verify that the desired gene products are actually synthesized from a cloned gene. There are also many others important applications, such as the study of protein: protein interactions and protein: DNA interactions.
• a method for cell-free biosynthesis of polypeptides is described, for example, in US Pat. No. 5,478,730.
• the translation system used there contains a source of mRNA that encodes the polypeptide.
• the cell-free synthesis system also contains ribosomes, tRNA, amino acids, ATP and GTP.
• the translation of the mRNA with the aid of the tRNA leads to the production of the polypeptide, whereby at the same time by-products and waste materials with a lower molecular weight are produced.
• These can pass into the supply space through a semipermeable membrane which separates the space in which the synthesis system is contained from a supply space.
• the supply room contains a liquid which acts as a supply medium and contains ATP, GTP and amino acids.
• the semipermeable membrane is an ultrafiltration membrane in the form of hollow membrane fibers.
• the components that are biochemically active in biochemical reactions can generally be referred to as a "producing system".
• a production system adapted to the respective application is used.
• a manufacturing system can basically be anything that is capable of enabling or accelerating the implementation or synthesis of desired compounds by biochemical means.
• enzymes and enzyme complexes in particular are suitable in conjunction with the auxiliary substances customary in such processes.
• the invention is particularly directed to micro-bio-reaction modules in which the chamber which contains the producing system (“system chamber”) preferably has a volume of at most 2 ml, in any case less than 10 ml. Since these are disposable products intended for single use, it is particularly important that such micro-bioreaction modules can be produced at low cost.
• system chamber the chamber which contains the producing system
• 5,478,730 describes an experiment with a synthesis chamber volume of 1 ml, in which the feed rate was varied between 2 ml / h and 3 ml / h. The results show that the higher feed rate resulted in high production output, while the lower feed rate hardly increased the amount of product.
• the dispodialyzer is placed in a conical 15 ml laboratory tube that contains between 3.5 and 7 ml Contains supply liquid.
• the laboratory tube with the DispoDialyzer is shaken during the reaction time (20 hours) using a laboratory shaker.
• the object of the invention is therefore to provide a bioreaction module with which bioreactions can be carried out simply, inexpensively and with the highest possible yield.
• a bioreaction module for biochemical reactions in particular for cell-free polypeptide biosynthesis, with a housing which encloses a system chamber and a supply chamber, the system chamber containing a producing system during the biochemical reaction, the supply chamber during the biochemical reaction Contains supply liquid and the system chamber and the supply chamber are separated by a semipermeable membrane, which is characterized in that the housing has a system chamber part and at least one supply chamber part and the semipermeable membrane is clamped between the chamber elements such that the system chamber from the system chamber part and the semipermeable Membrane and the supply chamber is delimited by the supply chamber part and the semipermeable membrane.
• the invention also relates to a bioreaction process using such a module.
• a bioreaction module according to the invention consists of a few parts that can be easily manufactured and assembled. Despite the resulting low manufacturing costs, it enables simple reactions to be carried out with high yields.
• the bioreactor according to the invention is suitable for different bioreaction processes, including for continuous flow processes. However, it is preferably used in processes in which no pumping process takes place, i.e. the supply liquid in the supply chamber is not subjected to external pressure during the biochemical reaction. As a result, the exchange of substances between the chambers is based exclusively on diffusion through the membrane, i.e. on dialysis. To the extent that the concentration of substances consumed in the biosynthesis decreases in the middle chamber, they flow in from the at least one supply chamber due to the concentration gradient. The reverse happens with waste from biosynthesis.
• the semipermeable membrane is preferably a dialysis membrane.
• Dialysis membranes are mechanically less stable (with the same exclusion limit) than the ultrafiltration membranes used in flow-through methods. Nevertheless, mechanical damage to the membrane occurs only to a very small extent in the bioreaction module according to the invention. Compared to an ultrafiltration membrane with the same transmission limit, a much more effective biosynthesis (better yield) is achieved.
• the pass limit is preferably (molecular cutoff) at least about 10 kD and at most about 20 kD.
• a construction is advantageous in which the housing includes two supply chambers which adjoin a system chamber and are separated from it by a first and a second semipermeable membrane.
• the housing of such a 3-chamber module has two supply chamber parts, the first semipermeable membrane being clamped between the system chamber part and the first supply chamber part and the second semipermeable membrane between the system chamber part and the second supply chamber part.
• At least one, preferably each, chamber of the module contains a magnetic stirring element which can be set into a rotary movement by means of an external magnetic field.
• the bioreaction module is manufactured as a single-use, disposable part, ready for use, including a magnetic stirring element if necessary.
• FIG. 1 is an external perspective view of a bioreaction module according to the invention
• FIG. 2 shows an exploded view of the module according to FIG. 1,
• FIG. 3 shows a cross section through a slightly modified embodiment of a bioreaction module
• 4 shows a detailed illustration from FIG. 3 to illustrate a preferred construction for clamping the edge region of a membrane
• FIG. 5 shows a cross section through a further preferred embodiment of a bioreaction module according to the invention.
• the housing 1 of the bioreaction module 2 shown in FIGS. 1 to 4 essentially consists of three chamber parts, namely a first supply chamber part 3, a second supply chamber part 4 and a system chamber part 5 arranged between the two supply chamber parts and connected to them.
• a first semipermeable membrane 7 is clamped between the first supply chamber part 3 and the system chamber part 5 in such a way that a peripheral edge strip 14 surrounding the exchange surface of the membrane is clamped between the adjacent chamber parts 3, 5 and the membrane is thereby tensioned.
• a second semipermeable membrane 8 is clamped between the second supply chamber part and the system chamber part.
• a first supply chamber 10 is defined by the walls of a recess in the first supply chamber part 3 which is only open towards the system chamber part 5 and the first which delimits the semipermeable membrane 7 spanning the opening of the recess.
• a second supply chamber 11 is enclosed by the walls of a recess of the second supply chamber part 4, which is also open on one side, and the second semipermeable membrane.
• a system chamber 12 which is formed by the two membranes and the walls of a recess of the system chamber part 5 which is open on both sides.
• the walls of the recesses in the chamber parts 3, 4 and 5 aligned, ie their edges adjoining the membranes 7, 8 frame the membranes along a corresponding boundary line.
• the membranes 7, 8 are clamped between the chamber parts 3, 5 and 4, respectively, in such a sealing manner that a molecular exchange between the system chamber 12 and the adjacent supply chamber 10 or 11 is only possible through the membrane 7 or 8. This is achieved by means of annular sealing means 9 provided on the chamber parts 3, 5 and 4, respectively, which surround the exchange surfaces 7a, 8a of the membranes 7, 8, between which the peripheral edge region 14 of the membranes 7, 8 is tightly clamped.
• FIG. 4 A preferred embodiment of suitable sealing means 9 is shown in FIG. 4.
• One of the chamber parts clamping the membrane, in the case shown the system chamber part 5, a circumferential groove 13 and the other chamber part, in the case shown the supply chamber part 3 has a projecting rib 16 which is aligned with the groove and penetrates into it, through which the between the Chamber parts 3.5 clamped peripheral edge region 14 of the membrane is pressed into the groove 13.
• the annular region of the chamber parts 3, 5 in which the peripheral edge region 14 of the membrane 7 is clamped is referred to as the clamping region 18.
• the groove 13 has a width of approximately 1 mm and a depth of approximately 0.6 mm.
• the height of the rib 16 is about 0.3 mm, its foot width is slightly smaller than the width of the groove and its edges penetrating into the groove are rounded, as shown in FIG. 4.
• the chamber parts 3, 4, 5 are preferably made of plastic, such as polyether sulfone, PVC or PMMA, and are manufactured, for example, as cast parts.
• the membrane consists of cellulose acetate, possibly cellulose nitrate.
• the volume of the system chamber was 1 ml and the volume of the two adjacent supply chambers was 5 ml each, so that the relationship between the volume of the system chamber and the total volume of the supply chambers adjacent to the system chamber and separated by a semipermeable membrane 1: 10 was. This relation is preferably at least 1: 5 and at most 1:20, values of at least 1: 7 and at most 1:15 being preferred.
• the connection of the chamber parts 3, 4, 5 can take place using means customary in plastics technology, for example by screwing, gluing or as a plastic plug connection. It is essential that the chamber parts 3 to 5 have sufficient rigidity to ensure a uniform pressure on the edge area of the membranes. In order to save weight and improve the rigidity, the chamber parts can have additional cavities that are not filled with liquid when the bioreaction module is used, such as the cavities 15 shown in FIG. 3.
• At least one of the chambers preferably contains a magnetic stirring element 17 which, for the sake of clarity, is only shown in one of the supply chambers in FIG. 3. If there are several supply chambers, it is preferably provided in both supply chambers. It can also be advantageous to provide a magnetic stirring element 17 in each of the chambers, ie also in the system chamber 12. As a rule, it is preferred to produce the bioreaction module 2 as largely as possible ready for use, possibly including the magnetic stirring elements, and to deliver it to the user.
• a magnetic stirring element has proven to be particularly advantageous in supply chambers with a volume of more than 2 ml, in particular more than 3 ml. With even smaller volumes of the supply chamber (and thus very small bioreaction modules), it is advantageous to shake the entire bioreaction module during the biochemical reaction.
• a stirring ball can be enclosed in at least one of the chambers in order to promote mixing.
• the chambers can be filled using an ordinary laboratory pipette. For this purpose, they each have connection openings, from which lines lead into the respective chambers.
• the supply chambers 10, 11 each have two connection openings 20, 21 and 22, 23 and the system chamber 12 also has two connection openings 23, 24.
• Seals for example cylindrical silicone rubber seals 26, are provided in the mouth area of the connection openings, which enable a tight connection of the pipette tip.
• the connection openings 20 to 25 can be closed by means of a cover part 28 which has locking pins 29 which penetrate into the connection openings when the cover part 28 is put on and bring about a tight seal by means of the seals 26.
• the supply chambers 10 and 11 are filled with a supply liquid and the system chamber 12 with a producing system.
• the biochemical reaction sets the at least one magnetic stirring element 17 in rotation by means of an external magnetic field, so that the supply liquid or the producing system is constantly stirred during the reaction.
• the housing can be set up in a position in which the connection openings 20 to 25 are directed obliquely upwards.
• This can be achieved in that the housing has a foot plane extending transversely to the membrane surface, which is oriented in such a way that when the module is in a filling position in which it stands on a horizontal surface with support points lying in the foot plane, the membrane runs essentially vertically, the connection openings 20 to 25 being located in the upper part of the module, that is to say at least above its center.
• This position is called the fill position.
• the inclined surfaces 30 and 31 of the overall rombically designed housing each form a base level that fulfills these conditions.
• the base level does not have to be formed by a closed, flat housing surface. Since only the orientation of the housing 1 is important, three support points are sufficient, which form a foot plane that fulfills the condition mentioned.
• the supply chambers each have a line 19 which, when the module is in the filling position, lead from the respective connection opening into the lower region of the respective chamber 10 or 11 in such a way that the mouth located in the lower area of the chamber, at least below its center.
• the invention has been described with the aid of a 3-chamber module, embodiments with a different number of chambers, in particular a 2-chamber module, are also possible, as explained.
• the construction of the chamber parts and the membrane clamping can be designed similarly to the figures above, but of course the system chamber part is closed on one side (on the side facing away from the supply chamber).
• the bioreaction module 2 contains a system chamber 12 and a supply chamber 10. Both chambers are separated from one another by a semipermeable dialysis membrane 7.
• the system chamber 12 is delimited by a system chamber part 5 and the semipermeable membrane 7.
• the supply chamber 10 is delimited by a supply chamber part 3 and the semipermeable membrane 7.
• the membrane 7 is clamped in a sealing manner at its peripheral edge region 14 by means of annular sealing means 9, the construction described with reference to FIG. 4 being preferably used.
• the membrane 7 For centering and fixing the membrane 7, several (for example six) pins 36 cast over the circumference and cast onto one of the chamber parts (here the supply chamber part 3) are provided in the embodiment shown.
• the membrane 7 has holes 35 aligned with the pins 36.
• connection of the chamber parts 3 and 5 takes place in the preferred embodiment shown that the system chamber part 5 is pressed into a corresponding recessed receptacle 34 of the supply chamber part 3.
• the outer diameter of the system chamber part 5 is made with a slight oversize compared to the inner diameter of the receptacle 34.
• a filling opening 40 or 41 is provided in a wall 38 or 39 opposite the membrane 7 for filling the chamber enclosed by the respective chamber part (ie the supply chamber 10 or the system chamber 12).
• the filling openings 40, 41 preferably each have a round cross section and merge into a filling channel 42, 43, which is delimited by a tubular wall 44 which extends into the respective chamber 10 or 12.
• the filling channel primarily serves the purpose of reducing the foaming of the biochemical liquid to be filled into the chamber.
• the bioreaction module shown is intended for cases in which only the liquid to be filled into the system chamber 12 tends to foam. For this reason, the filling channel 42 leads there close to the membrane 7.
• the filling channel 43 provided in the supply chamber 10, which is very short in relation to the length of the supply chamber, is suitable for filling in a non-foaming liquid. For cases in which the supply liquid also tends to foam, the filling channel 43 would also have to be brought close to the membrane 7.
• a vent 45 and 46 is also provided in each of the walls 38, 40 opposite the membrane 7. It preferably has a shape (for example in the form of a very narrow elongated hole) which makes it impossible to attach a pipette so that it cannot be accidentally filled through the ventilation opening.
• the filling openings 40, 41 and the ventilation openings 45, 46 can each be closed by means of a sealing plug 47, which projects from a folding part 49 to 52 perpendicular to its surface area.
• the folding parts 49 to 52 are pivoted in such a way - inexpensively by means of film hinges 53 - on the wall 38 or 39 opposite the semipermeable membrane 7 that the plug 47 penetrates into the respective opening 40, 41, 45, 46 in a sealing manner when the folding - Part 49 to 52 is brought into the position shown in Figure 5.
• the folding part is pivoted upward, as is shown in dashed lines for the folding part 50, for example.
• FIG. 5 there are preferably magnetic stirring elements (not shown for the sake of clarity) in the chambers 10, 12, which were introduced into the chambers during the manufacture of the module 2, so that the module delivered to the user ready for use contains the magnetic stirrer 17 prepacked.
• the bioreaction modules according to the invention enable a very effective biochemical reaction with a comparatively small exchange area 7a of the membrane 7 in relation to the volume of the respective system chamber 12.
• the relation between the total exchange area of one or more membranes delimiting the system chamber 12 with respect to one or more supply chambers and the total volume of the system chamber 12 is at most 4.0 unit -1 , whereby a lower limit of 0.2 unit -1 is not undershot should be.
• the bioreaction module shown in FIG. 5 enables particularly simple handling without restrictions in effectiveness. It is very small, as the dimensions (in mm) entered in FIG. 4 make clear.
• the walls 38, 39 which contain the filling openings 40, 41 and preferably also the ventilation openings 45, 46, each form a standing surface 54 or 55, which is designed and oriented in such a way that the other chambers 12 and 10 are filled can if the module 2 stands on a standing surface 54 or 55 delimiting one of the chambers 10 or 12.

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Wood Science & Technology (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Organic Chemistry (AREA)
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• Zoology (AREA)
• Genetics & Genomics (AREA)
• Biotechnology (AREA)
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• General Engineering & Computer Science (AREA)
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• Biomedical Technology (AREA)
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• Apparatus Associated With Microorganisms And Enzymes (AREA)
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Citations (4)

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• 2000
  • 2000-01-21 US US09/890,090 patent/US6670173B1/en not_active Expired - Fee Related
  • 2000-01-21 DE DE50002990T patent/DE50002990D1/de not_active Expired - Fee Related
  • 2000-01-21 WO PCT/DE2000/000190 patent/WO2000044877A1/de not_active Ceased
  • 2000-01-21 JP JP2000596122A patent/JP2002538778A/ja active Pending
  • 2000-01-21 DE DE10080164T patent/DE10080164D2/de not_active Ceased
  • 2000-01-21 CA CA002360077A patent/CA2360077A1/en not_active Abandoned
  • 2000-01-21 EP EP00906160A patent/EP1144587B1/de not_active Expired - Lifetime
  • 2000-01-21 AT AT00906160T patent/ATE245693T1/de not_active IP Right Cessation

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