Apparatus for preparing DNA products by capillary polymerase chain reaction

Abstract

The present invention relates to an apparatus for preparing a DNA product by capillary polymerase chain reaction (PCR); an apparatus for manufacturing a pharmaceutical product; a module for manufacturing a pharmaceutical product; a method for preparing a DNA product by capillary polymerase chain reaction; use of an apparatus for preparing a DNA product by polymerase chain reaction; and use of an apparatus for producing a pharmaceutical product. The apparatus for preparing a DNA product by capillary polymerase chain reaction comprises a tube for guiding a PCR liquid, a first compartment and at least a second compartment. The tube is bent at least from the first compartment to the second compartment and from the second compartment to the first compartment, wherein the tube bent from compartment to compartment forms a helical stack of tube windings. The first compartment and the second compartment each comprise means for adjusting a temperature in the compartment to prepare a DNA product based on the PCR liquid, wherein the first compartment is configured to provide a temperature for denaturation and the second compartment is configured to provide a temperature for annealing and / or elongation.

Description

Technical Field

[0001] This invention relates to an apparatus for preparing DNA products via capillary polymerase chain reaction; an apparatus for manufacturing pharmaceutical products; a manufacturing module for pharmaceutical products; a method for preparing DNA products via capillary polymerase chain reaction; the use of the apparatus for preparing DNA products via polymerase chain reaction; and the use of the apparatus for manufacturing pharmaceutical products. Background Technology

[0002] Active pharmaceutical ingredients (APIs) in biomolecular form are now widely used to treat a wide range of diseases. Such biomolecules include, for example, therapeutic antibodies, peptides, and nucleic acids. Therapeutic nucleic acids include DNA and RNA molecules.

[0003] RNA represents an emerging class of drugs. RNA-based therapies involve using mRNA molecules encoding antigens as vaccines. For RNA production, DNA templates are typically used in in vitro RNA transcription reactions. Such DNA templates can be obtained via polymerase chain reaction (PCR). Therefore, producing DNA in the sense of amplifying DNA via PCR is a key step in manufacturing RNA as an API.

[0004] Furthermore, DNA itself can serve as an API; for example, DNA vaccines against coronaviruses are currently in clinical development. To mass-produce DNA, i.e., to amplify DNA, polymerase chain reaction (PCR) can be used.

[0005] Currently established manufacturing processes for APIs in the form of nucleic acids, approved by regulatory agencies, typically involve numerous separate manufacturing steps, all of which must comply with GMP standards. These steps are usually performed in GMP-compliant rooms located within the production facility and are therefore not portable. Furthermore, these rooms are often designed for the production of specific biomolecules, making them inflexible, and requiring significant reconstruction if different nucleic acids need to be produced. In short, the manufacture of API-grade nucleic acids requires extensive manual labor performed by trained technicians in GMP-regulated, rather inflexible, and fixed laboratories. Consequently, the established manufacturing processes are time-consuming, costly, and require substantial laboratory space and equipment. Summary of the Invention

[0006] As mentioned above, there are problems associated with common manufacturing processes for nucleic acids (particularly DNA and RNA) as APIs or intermediates in API production processes, namely that these processes are rather inflexible, non-transferable, and often require a large amount of manual labor by trained technicians. Therefore, there is a need for an improved apparatus for the preparation / amplification of, and especially DNA, and a corresponding manufacturing apparatus that includes such an apparatus, which is more flexible. The manufacturing apparatus should particularly offer the benefits of operating under GMP-compliant conditions, being transferable, and / or automated. Such a manufacturing apparatus would significantly reduce the time required to manufacture APIs (e.g., DNA) or intermediates (e.g., DNA that can serve as a template for RNA production, where RNA is the API), which is particularly important in the case of viral outbreaks (e.g., pandemic outbreaks or localized outbreaks). Such a transferable API / intermediate manufacturing apparatus (including equipment for the preparation / amplification of, and especially DNA, which may be referred to as a "PCT reactor") can be easily transported, installed, and operated in pandemic hotspots globally, thus allowing for rapid vaccine production in these areas. Therefore, most importantly, the manufacturing apparatus of the present invention is configured to produce nucleic acid APIs / intermediates under GMP-compliant conditions, and the corresponding "PCR reactor" is configured to operate within such a manufacturing apparatus.

[0007] The aforementioned problem is addressed by the subject matter of the independent claims, wherein further embodiments thereof are provided in the dependent claims.

[0008] In a first aspect, the present invention relates to an apparatus for preparing DNA products via capillary polymerase chain reaction (PCR). "Preparing DNA products via capillary PCR" may alternatively be referred to as "amplifying DNA via capillary PCR" or "amplifying DNA via PCR." The apparatus includes a tube for guiding PCR liquid, a first compartment, and at least a second compartment. The tube is bent at least from the first compartment to the second compartment and from the second compartment (back) to the first compartment, wherein the bends from compartment to compartment form a helical stack of tube loops. Typically, each tube loop represents one PCR cycle. The first and second compartments each include means for regulating the temperature within the compartments to prepare DNA products based on the PCR liquid, wherein the first compartment is configured to provide a temperature for denaturation and the second compartment is configured to provide a temperature for annealing and / or extension.

[0009] In a preferred embodiment, the apparatus for preparing DNA products via capillary polymerase chain reaction (PCR) is not a microfluidic device, or is not designed to operate as a microfluidic device. In a preferred embodiment, the apparatus for preparing DNA products via capillary polymerase chain reaction (PCR) is not designed or configured to operate as an analytical PCR apparatus. In a preferred embodiment, the apparatus for preparing DNA products via capillary polymerase chain reaction (PCR) is designed or configured to operate as an apparatus for preparative production of DNA (e.g., for producing more than 1 mg of DNA product, or even optionally for producing g-quantities of DNA, e.g., producing 5 g of DNA). If operated in continuous mode, the output is essentially unlimited, making it possible to provide, for example, very large amounts of DNA in g-quantities, particularly when the overall goal is to produce RNA for use as template DNA in subsequent in vitro RNA transcription reactions.

[0010] In one embodiment, the device is not configured to allow the PCR liquid within and / or included in the tubes of the device to be recycled. Instead, as described above, the tubes are bent in a spiral stack of coiled tubes, wherein the tubes are not reattached to the front portion of the tube at any point. Thus, the PCR liquid enters the tube at a first end, remains within the tube during all PCR cycles, and exits the tube at a second end along with the DNA product that has already formed. In one embodiment, the device does not include a closed loop of the PCR liquid (which may, for example, be formed by the tube or by a reaction vessel connected via the tube). In one embodiment, the device does not include a closed loop of the PCR liquid connected to a valve (whereby the valve would allow the PCR liquid and DNA product to flow separately into separate tubes exiting the device).

[0011] The DNA product can be DNA corresponding to an API (e.g., a DNA vaccine) or a DNA template for enzymatic RNA in vitro transcription (and thus an intermediate for the API). RNA in vitro transcription involves the process of synthesizing RNA in a cell-free system. A DNA template is a DNA molecule containing a nucleic acid sequence encoding the RNA sequence to be transcribed in vitro. The DNA template serves as a template for RNA in vitro transcription to produce RNA encoded by the template DNA. The DNA template can be transcribed into mRNA representing the API, which can be formulated into lipid nanoparticles to produce encapsulated mRNA, which can be further formulated into a final pharmaceutical product. Therefore, the DNA product can be a pharmaceutical active ingredient (e.g., in a DNA vaccine), or the DNA product can be a DNA template that can be transcribed into mRNA as a pharmaceutical active ingredient (in which case the DNA is not an API but an intermediate). In the context of this invention, the DNA product is DNA obtained by PCR, i.e., amplified DNA.

[0012] Polymerase chain reaction (PCR) is one of the fastest methods for preparing DNA products. Typically, PCR can be performed in laboratory-scale and mass-produced PCR equipment. It is a technique in molecular biology that synthetically amplifies DNA by several orders of magnitude, resulting in thousands to millions of copies of a specific DNA sequence. PCR relies on thermal cycling, which involves repeated cycles of heating and cooling for DNA denaturation and enzymatic replication using thermostable DNA polymerases. Cycles include denaturation, annealing, and extension temperatures. The individual temperatures in the first and second compartments can be one of the denaturation, annealing, and / or extension temperatures. Specific PCR techniques include, for example, RT-PCR, hot-start PCR, long-fragment PCR, RT-qPCR, etc.

[0013] Capillary polymerase chain reaction (PCR) can be understood as PCR performed in a tube, pipe, or capillary. The tube or capillary can be very thin or narrow. It can have a very small diameter, ranging from about 0.5 to about 50 mm, preferably from about 0.5 mm to about 1.5 mm, more preferably from about 0.75 to about 1.25 mm. When PCR is in progress, i.e., when the PCR liquid flows / travels through the tube during PCR, the diameter of the capillary or tube is appropriately configured to allow for rapid and efficient temperature changes in the PCR liquid within the tube. As described herein, the tube is configured to provide different compartments with different temperatures, exposing the tube and the PCR liquid within it to these different temperatures. The tube or capillary, and particularly its diameter (or external dimensions), can be configured to allow capillary action or capillary effect with the PCR liquid. Capillary action or capillary effect is the ability of a liquid to flow in a narrow space without the aid of external forces (such as gravity) or even against external forces. Capillary action, or capillary effect, can be observed, for example, in the extraction of liquids in thin tubes, porous materials such as paper, and some non-porous materials such as sand. It occurs due to intermolecular forces between the liquid and the surrounding solid surface. If the diameter of the tube or capillary is small enough, the surface tension caused by cohesive forces within the liquid and the adhesive forces between the liquid and the tube or capillary wall work together to propel the liquid.

[0014] The tube is curved at least from the first compartment to the second compartment, and from the second compartment back to the first compartment. "Wound" can be understood as not forming a line (segment) that is the shortest distance between two points. Instead, the curved tube can be longer than the shortest distance between the two points, and it can extend in a circular, curved, or wavy shape. The reason the tube is curved is that the length of the tube in a particular compartment, along with a predefined flow rate, determines the time the PCR liquid is exposed to a specific temperature within that compartment. This, in turn, defines the time the PCR liquid in the tube is exposed to the specific temperature required for PCR (e.g., the denaturation temperature, which may exist, for example, in the first compartment). If the tube travels through the first compartment to the second compartment via the shortest path, the length of the tube exposed to the denaturation temperature provided in the first compartment may be too short, ultimately resulting in insufficient DNA denaturation. However, if the tube travels through the first compartment in a curved manner, the length of the tube exposed to the denaturation temperature provided in the first compartment is long enough that the PCR liquid passing through that tube segment is exposed to that temperature for a sufficiently long time, ultimately resulting in sufficient DNA denaturation.

[0015] It is important to understand that the tubing according to this application does not connect to one (or more) reaction mixture tanks, and in particular, does not connect to a single reaction mixture tank. In other words, the tubing according to this application does not allow for a recirculation path (where the tubing originates from the reaction mixture tank, passes through a different tank (with different temperatures), and then returns to the exact same reaction mixture tank to provide recirculation). Furthermore, it is important to understand that the tubing is not connected to a valve that enables the recirculation path.

[0016] PCR liquid can be understood as a reaction solution containing a DNA template for PCR amplification (wherein the DNA template is initially present in a small amount; preferably, the initial DNA template for PCR amplification is de novo synthesized DNA), one or more DNA primers, dNTPs, a DNA polymerase (e.g., a proofreading DNA polymerase), and a suitable PCR buffer. The PCR liquid is filled or pumped into a tube at one end (wherein the tube generally comprises two ends: an "inlet end" from which the PCR liquid originates and an "outlet end" from which the amplified DNA product is obtained) and guided through the compartments by a tube that bends through these compartments. The PCR liquid is separated from the regions within the compartments by the tube walls, and thus from, for example, a thermal medium (e.g., liquid or gas) or a thermal solid to be filled within the compartments, such that the PCR liquid does not have any direct contact with the compartments and the space within them at any point. The tube walls allow temperature exchange between the PCR liquid and the compartments (and thus the temperature inside the compartments), for example, the thermal medium or thermal solid inside the compartments, which is therefore used to heat or cool the PCR liquid by means of a device for regulating the temperature of the tube (e.g., by means of a thermal medium or thermal solid). As the PCR liquid is guided through the compartment, the template used for PCR amplification is amplified, generating a DNA product. This DNA product then exits the tube at the second end (“outlet end”). Therefore, it is not necessary to remove the DNA product from the reaction mixture container or any corresponding entity in the recycling system.

[0017] The tubes form a spiral stack of tubes wound around each other (basically like...) Figure 3 (As illustrated in the example). A single “tube loop” refers to “one loop” in which the tube returns to the initial first compartment, i.e., the tube bends from the first compartment to the second compartment and from the second compartment (back) to the first compartment, or preferably, through the first compartment and from the first compartment to and through the second compartment, from the second compartment to and through the third compartment, and from the third compartment (back) to the first compartment. It is important to understand that the tube returns to the first compartment, but does not reconnect back to the initial segment of the tube in the first compartment. Instead, after forming the tube loop from the first compartment through the compartments and back to the first compartment, the tube now continues to bend in a helical form, eventually forming a helical stack. In other words, the tube does not form a “two-dimensional” loop, i.e., from the first compartment through different compartments back to the first compartment, where it reconnects to the initial tube to form a loop, but the tube forms a “three-dimensional” helix, i.e., from the first compartment through different compartments back to the first compartment without reconnecting to the initial tube. The helix in the “third dimension” can in particular be substantially perpendicular to each loop in the “second dimension.” This means that "the stacking direction of the helical coils is different from, and preferably substantially perpendicular to, the corrugation direction of the corrugated tube." The helical stacking of the coils allows for multiple PCR cycles, with each coil corresponding to a single PCR cycle.

[0018] The tube has two distinct ends (“PCR liquid inlet” and “DNA product outlet”), with the first end typically located on the tube before it enters the first compartment (where the PCR liquid enters the device) because the denaturation step is the first step of the PCR reaction, and the second end typically located after the tube leaves the second / third compartment (where the DNA product leaves the device) because the extension step is typically the last step of the PCR reaction.

[0019] Apparatus for preparing DNA products comprises at least two (tempering) compartments, a first compartment and a second compartment. A third compartment may also be present, which is generally preferred. Even more compartments are possible. A compartment can be understood as a space, chamber, or container that is at least partially and preferably completely enclosed. The compartments may have a volume of at least 10 mL, preferably at least 100 mL, more preferably at least 1 L, for example, from about 1 L to about 10 L. They may be sealed and leak-proof. The at least two compartments may form at least two separate or different containers to be filled with, for example, a heat medium or heat solid. The at least two compartments may be separated from each other and preferably thermally insulated from each other to allow separate, different, or independent temperature zones in each of the at least two compartments. These temperature zones correspond to the temperature spectrum of PCR. A rapid “gradual change rate,” which indicates the change in temperature over time, is particularly important for the efficiency of PCR. In the context of this invention, the change in temperature over time is determined by the flow rate of the PCR liquid and the time required for heat exchange within the compartment.

[0020] One option is to regulate the temperature in each compartment using a heat medium. The heat medium can be a liquid or a gas. If so, in one embodiment, the heat medium can enter each of at least two compartments through a respective heat medium inlet and exit each of at least two compartments through a respective heat medium outlet. Therefore, in this embodiment, each compartment includes a heat medium inlet and a heat medium outlet. Thus, in at least two compartments at least partially filled with heat medium, there can be a flow of heat medium through their respective containers. The heat medium can be understood as a liquid coolant and heat carrier, such as a high thermal conductivity medium, such as propylene glycol, a silicone resin-based liquid or other synthetic heat medium, and water or any mixture of the above liquids. The heat medium can also be understood as a gaseous coolant or gaseous heat carrier. The heat medium can be, for example... A heat medium can be used to provide, control, and / or regulate the individual temperatures in at least two compartments to prepare DNA products via thermal cycling as part of PCR. The heat media in the different compartments are configured to allow rapid temperature changes in the tubes and thus the PCR liquid within the tubes during PCR. In one embodiment, the compartments do not include a stirring device for mixing the heat media in the compartments. In another embodiment, the compartments include a stirring device for mixing the heat media in the compartments.

[0021] Another option is to regulate the temperature in each compartment using a hot solid. If so, in this embodiment, the temperature of the hot solid is provided and regulated by a heating unit and optionally a cooling unit. In this embodiment, no liquid heat medium is used; therefore, there are no heat medium inlets and outlets in the compartments. The hot solid is a thermally conductive solid, thus providing specific temperatures to the compartments and tubes surrounded by the hot solid (the hot solid is configured to allow rapid temperature changes in the tube and thus the PCR liquid within the tube during PCR). The hot solid can be selected from the group consisting of aluminum, aluminum alloys or other alloys with high thermal conductivity, copper, bronze, and brass, with aluminum or aluminum alloys being preferred. The hot solid can be in the form of pellets, spheres, and / or coarse powder, preferably having a diameter of about 1 to about 3 mm for pellets and spheres. Aluminum pellets are particularly preferred. The hot solid can be contained in a liquid or gel to remove residual air from the compartments. Therefore, in this setup, the compartments surrounding the tubes (or the portion of the compartment formed by the inner compartment wall 30, see...) Figure 8 The compartment is filled with hot solids and liquids or gels as described above, and contains no air. The heating unit is preferably an electric heating unit. Suitablely, the heating unit is integrated into a support (or tube support device) to allow for optimal heating. The electric heating unit within the compartment can be, for example, a heating cylinder, a heating wire, or a heating conductor. The compartment (or the portion of the compartment formed by the inner compartment wall 30, see below) can also be cooled by additional use of a cooling unit, particularly a thermoelectric cooler (i.e., a Peltier element) with a heat pipe connected thereto, which extends into and is therefore also present inside the compartment. Figure 8 This is to provide and / or regulate the required temperature. If operating in a heating mode, the opposite of cooling mode, the thermoelectric cooler can also be used to provide additional heat to the compartment. If operating in cooling mode, a fan may also be present to remove heat from the thermoelectric cooler. However, the presence of a fan is not mandatory, as heat from cooling can also be used as additional heating in another compartment of the equipment. Typically, several independent electronic heating systems exist in each compartment, each connected to its own temperature sensor to implement several independent control circuits.

[0022] The apparatus for preparing DNA products according to the present invention allows for flexible adaptation to different conditions, requirements, and, for example, the amount of DNA. This apparatus allows for the preparation of large quantities of DNA (e.g., more than 2 mg per reaction). Furthermore, the preparation of DNA products can be scalable. In particular, the preparation of DNA products can be continuous, wherein as new PCR liquid is continuously added to the “inlet” while the resulting DNA exits the apparatus through tubes and the “outlet”, g-quantities and substantially unlimited quantities can be produced. This apparatus for preparing DNA products allows for very rapid preparation of DNA products, thereby also allowing for the rapid manufacture of pharmaceutical products or intermediates. The apparatus for preparing DNA products can also be very quickly modified and adapted to specific DNA products. Adjustment or calibration steps may not be required. Cleaning steps may be required little or no. Turnaround time, for example, for vaccine manufacturing, can be less than about one week.

[0023] The apparatus for preparing DNA products according to the present invention is constructed in a very compact manner. Its size can be very small. This apparatus for preparing DNA products via capillary PCR is portable, allowing it to be transported to areas, for example, during pandemic outbreaks. Therefore, it can allow for the local, decentralized production of pharmaceutical products (e.g., DNA vaccines) or intermediates (e.g., DNA templates) for pharmaceutical products (e.g., RNA vaccines).

[0024] The apparatus for preparing DNA products according to the present invention can be used for flexible and rapid DNA vaccine production or for the production of DNA templates for RNA vaccines, particularly for flexible and rapid RNA vaccine production. This apparatus can be specifically used to produce DNA templates for mRNA-based vaccines, for example during infectious disease epidemics and pandemics, and for various cancer diseases, including personalized therapies.

[0025] In one embodiment, partitions are arranged between the compartments to insulate the compartments from each other. This can be understood as a partition being arranged between the first and second compartments. In the case of more (e.g., three) compartments, a partition can be arranged between the first and second compartments and another partition can be arranged between the second and third compartments and / or another partition can be arranged between the third and first compartments. As a result, the (adjacent) compartments can be thermally insulated from each other, thus different temperatures can be established and maintained in the (adjacent) compartments. The partitions can be implemented by only one wall assembly to insulate all compartments by the same wall assembly or more than one wall assembly. The partitions can be made of a material with low thermal conductivity and good mechanical and temperature resistance. Additionally or in part as an alternative, an inner compartment wall can be present that more closely follows the bellows, and thus creates a narrower space around the tube in each compartment. This inner compartment wall also results in insulation of the corresponding compartment, while creating a fairly narrow space around the tube for particularly filling hot solids. If internal compartment walls exist, the walls need not be fully present between the compartments, but may be only partially present, especially in areas where pipes pass from one compartment through the walls into the next, as described below.

[0026] Therefore, in the most preferred embodiment, the partition includes a through-hole for a pipe to extend from one compartment to the next, allowing the pipe to pass through different compartments. The through-hole or channel allows the pipe to enter and / or exit the compartment and its temperature zone. The through-hole can be sealed to improve leak-proofness. The through-hole can also be thermally insulated to improve temperature stability in (adjacent) compartments.

[0027] In one embodiment, and if present, a hot medium inlet and a hot medium outlet are arranged in at least one, and preferably in each compartment, to provide a flow of hot medium through the compartment substantially in the opposite direction to the direction of PCR liquid flow in the tube. This can be understood as follows: if the PCR liquid is flowed clockwise, the hot medium inlet and outlet are arranged to provide a counterclockwise flow of hot medium, or vice versa. Alternatively, if the PCR liquid is flowed from left to right, the hot medium inlet and outlet are arranged to provide a flow of hot medium from right to left, or vice versa. The countercurrent flow of the hot medium to the PCR liquid enhances the temperature exchange between the hot medium and the PCR liquid, thereby improving the temperature control of the PCR liquid. For an efficient PCR reaction, a constant temperature in each compartment of the apparatus is particularly important, and rapid temperature exchange is facilitated as the tube containing the PCR liquid is flowed to the next compartment (which typically has a different temperature).

[0028] In one embodiment, the heat medium inlet (if present) is located at a lower position in the respective compartment, and the heat medium outlet (if present) is located at a higher position in the respective compartment. This configuration has the advantage of making it easier to remove air bubbles.

[0029] Alternatively, the heat medium inlet (if present) may be located at a higher position in the respective compartment, and the heat medium outlet (if present) may be located at a lower position in the respective compartment.

[0030] In a particularly preferred embodiment, the apparatus for preparing DNA products via capillary polymerase chain reaction further includes a third compartment. The tube may bend at least (through and) from the first compartment to (and through) the second compartment, and from the second compartment to (and through) the third compartment. The tube may further bend from the third compartment to the first compartment, again (through and) from the first compartment to (and through) the second compartment, and again from the second compartment to (and through) the third compartment. The third compartment also includes means for regulating the temperature within the compartment. In this regard, it may include a heat medium inlet and a heat medium outlet to provide a heat medium flow through the third compartment, which is separate from the heat medium flow through the first and second compartments. The separate heat medium flow can be used to regulate a separate temperature in the third compartment, separate from the temperatures in the first and second compartments. Instead of a heat medium inlet and outlet, embodiments having a heating unit as described above can be used as means for regulating the temperature within the compartment. The separate temperature in the third compartment is also used for preparing DNA products based on PCR liquids. These three temperature zones typically correspond to the temperature spectrum of PCR denaturation, annealing, and extension.

[0031] The tubes, curved from compartment to compartment, form a helical stack of coils. As defined above, a coil is the portion of the tube that begins in the first compartment and returns to the first compartment, where the coil represents one PCR cycle. Such coils then form helices, ultimately resulting in a helical stack of coils, where each coil represents one PCR cycle. In other words, this can be understood as the tubes having a helical, coiled, or looped shape (when viewed from the side). Furthermore, the tubes can be arranged in loops (however, the loops are not connected to each other), which makes the apparatus for preparing DNA products via capillary polymerase chain reaction very compact and space-saving. Loops can extend from the first compartment to the second compartment and from the second compartment to (optionally) the third compartment. Each loop corresponds to one PCR cycle. The direction of the helical extension or stacking of the tubes can extend away from the so-called x, y plane formed by the bottom of the apparatus used for preparing DNA products or by the floor or ground. The helical extension of the tubes can extend substantially perpendicular to the bottom of the apparatus, the floor, or the ground, or it can extend at an angle to the bottom of the apparatus, the floor, or the ground.

[0032] In one embodiment, the tube extends in a corrugated or zigzag manner in at least one, and preferably in each, compartment, resulting in a specific length of tube in each compartment that reflects the incubation time of the PCR liquid within the tube as it passes through the tube in that compartment, preferably at a predetermined and controlled flow rate. Since this incubation time is typically longer than the time achieved if the tube were linear within the compartment, it is preferable that the tube exists in a corrugated or zigzag manner in each compartment. This can be understood as the tube having a wavy or serpentine shape in one or all compartments (when viewed in a top view). The wavy or serpentine or corrugated shape of the tube may extend in the x and y directions of a plane formed by the bottom of the apparatus for preparing the DNA product or a plane formed by a floor or ground; that is, it should be understood to be associated with a “two-dimensional” form and exclude spiral shapes (i.e., a “three-dimensional” form). The corrugated shape of the tube defines the residence time of the PCR liquid in each compartment, thereby defining the residence time of the PCR liquid within the temperature zone provided by each compartment. Therefore, the corrugated shape of the tube defines the length of the tube in each compartment with a certain temperature, thereby defining the residence time of the PCR liquid in the temperature zone provided by each compartment.

[0033] In one embodiment, the stacking direction of the helical stack of the tube coils is different from, and preferably substantially perpendicular to, the corrugation direction of the corrugated tube. The angle between the stacking direction of the helical stack of the tube coils and the corrugation direction of the corrugated tube may also be different from about 90°, for example, less than about 90°, in the range of 45° to 89°. The stacking direction of the helical stack of the tube coils and the corrugation direction of the corrugated tube may then be inclined or oblique to each other. The stacking direction of the helical stack of the curved tubes may be inclined or oblique to the bottom of the apparatus for preparing DNA products or a plane formed by a floor or ground. Additionally or alternatively, the corrugation direction of the corrugated tube may be inclined relative to the bottom of the apparatus for preparing DNA products or a plane formed by a floor or ground. The tube may then have an inclination relative to the bottom of the apparatus for preparing DNA products or a plane formed by a floor or ground. The inclination may be the same throughout the apparatus for preparing DNA products or throughout one of the compartments, but the inclination may also vary throughout the apparatus for preparing DNA products or throughout one of the compartments.

[0034] In one embodiment, the apparatus for preparing DNA products via capillary polymerase chain reaction further includes at least one support or tube holding device disposed in one of the compartments to provide a fixing point for the tube and / or a deflection point for the corrugations of the tube. The support may be cylindrical. When viewed in top view, it may also be T-shaped or L-shaped. Other shapes are also possible. The support may include at least one mounting structure configured to receive and hold the tube (thereby receiving and holding tube coils and helical stacks of tube coils, respectively). Preferably, the support includes multiple mounting structures to securely hold the tube (thereby holding tube coils and helical stacks of tube coils, respectively) at the support. The mounting structures may be formed as grooves, through holes, hooks, slits, or other means to mount the tube (thereby holding tube coils and helical stacks of tube coils, respectively) to the support. The tube may be secured at the mounting structure by force-fit and / or form-fit. Additionally or alternatively, the support may also include at least one clamping device to securely hold the tube. Preferably, the mounting structure is coated with a soft material, including, for example, rubber or silicone, to prevent damage to the tube such as scratches or cuts. Suitablely, the support may be made of a material that is chemically resistant to the heat medium used.

[0035] In one embodiment, the tube extends partially around the outer dimensions of the support. This can be understood as, in a top view, the tube does not extend completely around the outer dimensions or circumference of the support, but less than about 360°. It may extend around the outer dimensions of the support in the range of about 45° to about 270°. In another embodiment, the tube extends completely around the outer dimensions of the support. This can be understood as, in a top view, the tube extends around the outer dimensions or circumference of the support in about 360°. In another embodiment, the tube extends around the outer dimensions of the support more than once. This can be understood as, in a top view, the tube extends around the outer dimensions or circumference of the support in more than 360°. It may extend around the outer dimensions of the support in the range of about 450° to about 540°, meaning that there may be overlap of the tube around the support.

[0036] In one embodiment, the support is replaceable within the compartment. This can be understood as the support being achievably fixed to the compartment, particularly to the base plate of the compartment. Therefore, the support can be replaced with another support having, for example, different external dimensions (radius and / or height) to accommodate different tubes, different purposes, different PCR durations (e.g., to reflect different PCR protocols), different coiling radii, and / or different coiling patterns, etc.

[0037] In one embodiment, the scaffold can be repositioned within the compartment. This can be understood as the scaffold being positioned at different locations or sites within the compartment, and particularly at different locations relative to the compartment's base plate. Thus, the base plate can be formed as a multi-well plate with (concealed) holes, into which the scaffold can be flexibly inserted. Therefore, the scaffold can be repositioned to adapt the apparatus for preparing DNA products to different tubes, different purposes, different PCR durations (e.g., to reflect different PCR protocols), different coiling radii, and / or different coiling patterns, etc.

[0038] Scaffolds, particularly replaceable and / or repositionable scaffolds, can be used to define the residence time of PCR liquids within the compartment, thereby defining the residence time of the PCR liquids within the temperature range provided by the compartment. Therefore, the size and position of the scaffold can define the temperature spectrum of PCR and can be flexibly adjusted to match the desired PCR spectrum (e.g., for the production of different DNA products, such as DNA products of different lengths).

[0039] The tubes can be continuous throughout the apparatus used for preparing DNA products, which can be understood as being identical throughout the apparatus. The tubes can also be discontinuous throughout the apparatus, which can be understood as including portions having, for example, different sizes or diameters in the tube cross-section. The tubes can be replaceable to adapt the apparatus for preparing DNA products to different PCR or other purposes. For cleanability reasons, the tubes are preferably continuous. Furthermore, the tubes are configured to allow corrugations, for example, through a support, without the risk of breakage or downward bending that would impede PCR efficiency. However, in all cases, the tubes are not a recirculating or closed tube system with an optional outlet, but rather consist of a defined "inlet" end and a defined "outlet" end.

[0040] In one embodiment, the tube includes a PCR liquid inlet connectable to a pump unit for pumping PCR liquid through the tube. The pump unit may be a pump. The pump unit may be an external pump unit. In one embodiment, the pump unit is disposed outside the compartment and preferably outside the housing surrounding the compartment. The housing may be made of a plastic material, such as POM, PEEK, PTFE, but may also be made of metal, alloy, compound, etc., and is resistant to and compatible with heat and the applied heat medium. External placement can save space within the equipment used to prepare DNA products and / or may allow the use of a powerful pump unit. Preferably, the pump unit includes a micro-gear pump configured to produce a constant flow rate.

[0041] In one embodiment, the pump unit is configured to provide a flow rate of PCR liquid in the range of about 0.05 mL / min to about 50 mL / min, preferably about 0.1 mL / min to about 10 mL / min, more preferably about 0.2 mL / min to about 3 mL / min. Such a flow rate defines the residence time of the PCR liquid in the compartment and therefore defines the residence time of the PCR liquid within the temperature range provided by the compartment. Thus, these flow rates define the temperature spectrum of PCR. Preferably, the flow rate is constant, for example, with flow rate fluctuations of less than + / - 10%, preferably less than + / - 5%.

[0042] In one embodiment, the tube includes a DNA product outlet for the continuous preparation of DNA products. This can be understood as the continuous, uninterrupted preparation of DNA products. Therefore, the DNA product outlet can be "always open during the production process." This continuous preparation of DNA products improves the continuous and scalable production of large quantities of DNA products. The DNA product outlet may include a device for shutting off the mechanism, such as a shutter, which can be closed when the continuous preparation of the DNA product is complete.

[0043] In one embodiment, the tube includes a shut-off DNA product outlet for discontinuous preparation of the DNA product. This can be understood as discontinuous, batch-by-batch preparation of the DNA product. Therefore, the DNA product outlet can be shut off by a shut-off mechanism (e.g., a shut-off device).

[0044] In one embodiment, the apparatus for preparing DNA products via capillary polymerase chain reaction further includes at least one sensor disposed in at least one compartment. The sensor may be a temperature sensor, a flow rate sensor, and / or a leakage sensor, configured to detect the temperature, flow rate, and / or leakage of the compartment, respectively. The flow sensor may form a closed-loop control with the pumping unit to monitor and control the flow rate outside the apparatus for preparing the DNA product or the PCR reactor. At least two temperature sensors may be provided in at least one compartment, and preferably in each compartment, for example, one near the liquid inlet and one near the liquid outlet to establish a replication device. Two leakage sensors may be used, one in at least one compartment and preferably in each compartment, and one within the surrounding unit / frame. Other sensors may be disposed outside the compartment, for example, in the pumping unit, or at the PCR liquid inlet, or at the DNA product outlet.

[0045] The device may also include a first part of a proximity sensor, such as a magnetic sensor, which locates its counterpart outside the device according to the first aspect, or a second part of a proximity sensor. Accordingly, the second part of the proximity sensor may be specifically located within the manufacturing equipment according to the second aspect. Once the device has been correctly installed in the manufacturing equipment, the two parts of the proximity sensor are connected and preferably signal successful installation and / or preferably only then can the device be successfully started and operated. Therefore, a proximity sensor including both parts can also be activated during operation and cause an alarm and / or operation to stop once the two parts of the proximity sensor lose their connection.

[0046] In one embodiment, the diameter of the tube (which may roughly correspond to the external dimensions) is in the range of about 0.5 mm to about 50 mm, about 0.5 mm to about 40 mm, preferably about 0.5 mm to about 20 mm, more preferably about 0.5 mm to about 4 mm, even more preferably about 0.5 mm to about 1.5 mm, and even more preferably about 0.75 mm to about 1.25 mm. These dimensions allow for rapid and efficient temperature exchange (“gradual change rate”) between the thermal medium and the PCR liquid.

[0047] The tubes can be made of plastic materials such as PEEK, ETFE, or PTFE, but can also be made of metals, alloys, compounds, etc. Suitablely, the tubes can be made of biocompatible and process-compatible materials (e.g., materials that do not leak into the DNA product, do not oxidize / degrade, and have low surface binding of the PCR compound to minimize the risk of deprivation). PEEK is a preferred material.

[0048] In one embodiment, the length between the tube inlet and outlet is in the range of about 5 m to about 300 m, preferably about 10 m to about 200 m, and more preferably about 25 m to about 50 m. It can, for example, have a length of about 100 m (e.g., about 97 m). The tube portions are comprised of different compartments, and these dimensions define the residence time of the PCR liquid within the compartments, and thus define the residence time of the PCR liquid within the temperature range provided by the compartments. Therefore, these tube length portions can define the temperature spectrum of PCR.

[0049] In one embodiment, the tube is designed to contain PCR liquid in the range of about 10 mL to about 1 L, preferably about 20 mL to about 250 mL, more preferably about 30 mL to about 150 mL. Its size can, for example, be designed to contain PCR liquid in the range of about 80 mL.

[0050] In one embodiment, the helical stack of the tube coils comprises about 10 to about 50 coils or layers, preferably about 15 to about 40, more preferably about 20 to about 30. The number of coils corresponds to the number of PCR cycles.

[0051] As an example only, the tube may have a total length of approximately 15 m, comprising approximately 25 spiral stacks (or loops) around which the tube is wound. In this particular embodiment, within one spiral stack (or loop), the tube may be guided through a first compartment (100 cm), then a second compartment (100 cm), and then a third compartment (400 cm).

[0052] In one embodiment, the device is configured to produce about 1 mg or more of DNA product per reaction, for example up to 5 g, preferably in the range of about 1 mg to about 30 mg, more preferably in the range of about 15 mg to about 25 mg.

[0053] In one embodiment, the device is configured to produce DNA products in the range of about 2 μg / min to 2000 μg / min, preferably about 5 μg / min to 1500 μg / min, and more preferably about 10 μg / min to 1000 μg / min.

[0054] In one embodiment, the device is configured to prepare about 25 μg to about 200 μg of DNA product per mL of PCR liquid, preferably about 50 μg to about 150 μg per mL of PCR liquid, and more preferably about 75 μg to about 125 μg per mL of PCR liquid.

[0055] In one embodiment, the first compartment is configured to have a temperature in the range of about 85°C to about 105°C, preferably about 98°C.

[0056] In one embodiment, the second compartment is configured to have a temperature in the range of about 45°C to about 72°C, preferably about 60°C to about 72°C.

[0057] In one embodiment, an optional third compartment is configured to have a temperature in the range of about 65°C to about 75°C, preferably about 72°C.

[0058] As an example only, the tube may have a total length of approximately 15 m, comprising approximately 25 spiral stacks (or loops) of tube coils. In this particular embodiment, within one spiral stack (or loop), the tube may be guided through a first compartment (100 cm; temperature from approximately 85°C to approximately 105°C), then a second compartment (100 cm; temperature from approximately 45°C to approximately 72°C), and then a third compartment (400 cm; temperature from approximately 65°C to approximately 75°C). The residence time in each compartment can be adjusted by the PCR flow rate and the tube diameter.

[0059] Those skilled in the art will understand that different DNA products (e.g., different sizes and / or sequences) may require different PCR conditions (e.g., due to DNA primer annealing temperature or extension time). Different PCR programs suitable for a wide variety of DNA products can be achieved by adjusting the tube length, the tube length in different compartments, the number of helical stacks (and thus the number of PCR cycles), the flow rate, the tube diameter, and / or the temperature in the compartments. Therefore, equipment for preparing DNA products via capillary polymerase chain reaction can be readily adapted and adjusted to allow for the preparation of DNA products of various sizes and sequences.

[0060] In a second aspect, the present invention relates to a pharmaceutical product manufacturing apparatus comprising the apparatus of the first aspect for preparing DNA products by capillary polymerase chain reaction.

[0061] In one embodiment, the pharmaceutical product manufacturing apparatus includes equipment for preparing DNA products via capillary polymerase chain reaction as described in the first aspect and at least one other functional component (e.g., additional chambers, additional units, housings, etc.). In a preferred embodiment, the pharmaceutical product manufacturing apparatus according to the second aspect includes a processing chamber, a technology chamber, and equipment for preparing DNA products via capillary polymerase chain reaction as described in the first aspect.

[0062] Description of manufacturing equipment and its preferred embodiments

[0063] Pharmaceutical manufacturing equipment can generally be understood as a closed shell with different chambers for different purposes.

[0064] The processing chamber is suitable for and used in the manufacture of pharmaceutical products. In the sense that virtually all wet processes in the manufacturing process occur within the processing chamber, it can also be referred to as the "wet section" of the manufacturing equipment. "Wet process" refers to, for example, the supply of buffer solutions and / or reactants and / or products to and from containers or to and from chromatographic columns, the collection of waste liquids or exhaust gases, and any connections (e.g., connections in the form of hoses) to such buffer solutions and / or reactants and / or products. Ideally, such wet processes should be separated from any technical media supply (particularly electrical supply, such as power cables) to make accidents due to liquid leaks in the processing chamber and their possible consequences (particularly due to voltage) impossible or at least minimized. In the event of a leak in the processing chamber, safety is enhanced due to the aforementioned separation, and only the processing chamber needs to be cleaned. The equipment according to the first aspect is located in the processing chamber.

[0065] A technical room is suitable for providing technical support, such as housing instruments that do not come into direct contact with process media, such as pumps, motors, mixers, processors, etc. A technical room is generally suitable for providing (e.g., housing) the supply of technical media, particularly power, such as power to instruments / units located in the processing room that come into direct contact with process media. In the sense that no wet process as defined above occurs in the technical room, the technical room can also be referred to as the "dry part" of the manufacturing equipment. As mentioned above, this particularly increases safety. One might refer to a technical room as "not as clean as a processing room" because the required level of cleanliness in a technical room is lower than in a processing room. The supply of technical media for the equipment according to the first aspect is located in the technical room.

[0066] The chamber can be separated by a separation element, which is plate-shaped, particularly when viewed from the processing chamber. Therefore, as will be outlined below, in order not to disturb the airflow, the separation element may include at least one accessory having an opening toward the processing chamber and extending in the direction of the technical chamber, in which instruments that come into contact with the process medium can be accommodated, particularly the equipment according to the first aspect.

[0067] Manufacturing apparatus including devices for preparing DNA products via capillary polymerase chain reaction preferably also includes a control unit configured to control airflow through the processing chamber as a gas spray. The airflow may be provided by a flow unit. The flow unit may be at least a pump or HVAC system, and may also be referred to as a fan system. The flow unit may be arranged outside the housing of the manufacturing apparatus (external flow unit) or preferably inside the housing of the manufacturing apparatus (internal flow unit). The flow unit preferably includes a filter unit (which may be referred to as a "fan filter unit" and preferably includes a HEPA filter as described below). The flow unit may be configured to provide a gas, preferably of variable volume. The gas may be clean air, air with additives, an inert gas, or any other gas. The control unit can be understood as any control device for the airflow, such as a valve or processor. The control unit may be configured to regulate different pressures in the airflow. The control unit may be configured to control the airflow through the processing chamber to provide positive pressure in the processing chamber. Positive pressure in the processing chamber can be understood as overpressure relative to the external environment and / or a pressure higher than that in the processing chamber. Positive pressure in the processing chamber can protect open processing steps from particulate contamination.

[0068] The control unit is configured to control the airflow through the processing chamber to provide a gas spray through the chamber. The gas spray can fall from a higher level to a lower level in the processing chamber. The gas spray in the processing chamber can protect open processing steps from particulate contamination. In one embodiment, the gas spray is laminar, which can be understood as substantially similar to laminar flow or substantially unidirectional or substantially non-turbulent. The laminar gas spray or airflow can be downward or horizontal, preferably in a constant flow, toward the bottom of the processing chamber. Laminar gas spray can allow for the reduction and retention of extremely low levels of particulate matter, such as dust, airborne organisms, or evaporated particles, without introducing turbulence and / or contamination, for example, in manufacturing processes within the corresponding housing or chamber. Instruments installed in the gas spray area can be tightly mounted to the separation element or processing chamber wall or covered or encapsulated to reduce or eliminate turbulence or interference from the gas spray. Instruments can also be “hidden” in attachments to the separation element or processing chamber wall to reduce or eliminate turbulence or interference from the gas spray. In one embodiment, the velocity of the gas spray is in the range of about 0.2 to about 0.6 m / s, preferably about 0.36 to about 0.54 m / s. This velocity provides a good balance between reducing and maintaining extremely low particulate levels and not interfering with manufacturing processes, such as those in a processing chamber.

[0069] The pharmaceutical manufacturing equipment according to the second aspect allows for flexible adaptation to different conditions, requirements, and, for example, the quantity of pharmaceutical products, because the process operates in continuous or semi-continuous mode. This manufacturing equipment allows for the production of large quantities of pharmaceutical products. Furthermore, the manufacturing of pharmaceutical products is generally scalable.

[0070] This manufacturing equipment can provide a closed (biosynthetic) process from raw materials (e.g., from dNTPs, DNA primers, DNA templates) to the final product. This manufacturing equipment can be understood as in-box manufacturing. The size of this manufacturing equipment can be designed to be very small. It can have a low footprint and form a low-footprint manufacturing entity. The footprint can be in the range of approximately 100 × approximately 100 × approximately 200 cm, making it easy to transport and store in conventional transport containers of typical sizes. Therefore, this manufacturing equipment is portable, allowing it to be transported to areas such as those experiencing pandemic outbreaks. Thus, it typically allows for the localized, decentralized production of pharmaceutical products (e.g., DNA vaccines, DNA templates).

[0071] This manufacturing equipment typically allows for the very rapid production of pharmaceutical products. Modifications and preparation of the equipment for a specific pharmaceutical product, as well as the production of the specific pharmaceutical product itself, can be very quick. Adjustment or calibration steps may be unnecessary. Cleaning steps may be unnecessary or minimal. Turnaround time, for example, for vaccine manufacturing, can be less than approximately one week.

[0072] This manufacturing equipment is typically very easy to clean, which allows for reduced cleaning efforts. This is due to its clean and hygienic design from the outset. This can include: an aluminum housing, where the aluminum may have been pre-treated with, for example, anodizing and passivation; antimicrobial surface treatments using, for example, silver ion-containing colors; electropolished surfaces; the use of compatible materials throughout the manufacturing equipment; the ability to clean many or every part of the manufacturing equipment; smooth, seamless, and cleanable exposed surfaces; harmonized internal diameters and geometries to enable optimal in-situ automated cleaning (CIP) procedures and / or antimicrobial cleaning; sealing any gaps or hollow areas; reducing or stopping the accumulation of any condensation and deposits, etc. In particular, the housing, chambers, and / or separation elements can be made of aluminum, preferably surface-treated aluminum, to allow for CIP. Additionally or alternatively, the surfaces of the outer and / or inner housing, and especially the surfaces within the chambers, can be treated with antimicrobial surfaces. Furthermore, instruments within the housing, and especially within the processing chambers, can be encapsulated to provide smooth surfaces. Additionally, one or more components of moving parts and guiding technology media can be encapsulated to ensure safety and ease of cleaning. Additionally or alternatively, instruments within the housing, and particularly within the processing chamber, can be embedded in the wall and especially in the separation element to (ideally) be flush with the corresponding wall or surface. These options improve cleanability and prevent airflow from being disturbed or creating undesirable detours by non-flush elements in the processing chamber.

[0073] This manufacturing facility can operate under GMP (Good Manufacturing Practice) conditions. At least the processing room and preferably the manufacturing facility can operate according to the requirements of Annex 1 of the EU Guidelines for Good Manufacturing Practice for Medicinal Products (EU Guidelines for Good Manufacturing Practice for Medicinal Products, Annex 1). In one embodiment, the GMP requirement is the requirement of EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use, Annex 1, Manufacture of Sterile Medicinal Products (corrected version), European Commission, Brussels, 25 November 2008 (revised version). In another embodiment, the GMP requirement is the requirement of FDA (2004) Guidance for Industry, Sterile Drug Products Produced by Aseptic Processing – Current Good Manufacturing Practice, USDapartment of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER), Office of Regulatory Affairs (ORA) Pharmaceutical CGMP. In another embodiment, this manufacturing equipment can operate in accordance with CE, ISO, or GAMP5 standards. This manufacturing equipment allows for GMP-compliant in-box manufacturing. GMP compliance may include compliant machine control and data logging, such as the automatic recording and reporting of key process parameters and / or quality attributes, such as pressure, humidity, temperature, UV absorption, total organic carbon, infrared absorption spectrum, flow rate, power consumption, and events that could lead to errors (door opening, leakage, quick-access methods, pressure loss, etc.).

[0074] In one embodiment, the manufacturing apparatus for preparing DNA products via capillary polymerase chain reaction further includes a separation element that separates the processing chamber from the technical chamber. The processing chamber may be adapted for and used in the manufacture of pharmaceutical products. The technical chamber may be adapted for and used to house instruments such as pumps, motors, mixers, processors, etc. The separation element is plate-shaped. The separation element can be used to mount instruments. The mounted instruments may be covered to reduce airflow turbulence or disturbance, prevent sedimentation, and / or facilitate cleaning. The separation element may extend (only) between the processing chamber and the technical chamber, or it may extend at least partially or completely through the housing. The separation element may also extend outside the housing to separate portions of the surrounding environment, such as the surrounding cleanroom.

[0075] In one embodiment, the separation element includes an attachment having an opening toward the processing chamber. The attachment may protrude in the direction of the processing chamber and may have an extension in the direction of the processing chamber, acting as an extended portion of the processing chamber. The separation element and attachment can be understood as a plate with concealed openings. The attachment can be used to at least partially or completely house an instrument. An apparatus for preparing DNA products (which may alternatively be referred to as a "PCR reactor") can be inserted into the attachment. Thus, an apparatus for preparing DNA products or a PCR reactor can be included and housed in a box for easy insertion into the attachment. The instrument (e.g., the PCR reactor) can then be considered to be located within the processing chamber without or with minimal intrusion. This allows for an increase in available space within the processing chamber without increasing its length. Furthermore, it allows for plug-and-play use of different instruments for, for example, different or new PCR processes (e.g., different PCR reactors reflecting different PCR procedures, e.g., by means of tubes with different lengths).

[0076] In one embodiment, at least one handle for operating the device for preparing DNA products into and / or out of the accessory is positioned on the front of the device for preparing DNA products to facilitate operation of the device for preparing DNA products.

[0077] In one embodiment, the accessory includes an exchange rail system to guide and facilitate the exchange of equipment used for preparing DNA products with another device. The exchange rail system may include exchange rails arranged at the accessory or at the equipment used for preparing DNA products, and particularly at its housing. The equipment used for preparing DNA products may also include operating devices, such as trolleys, lifts, etc., to allow and / or facilitate instrument exchange from, for example, the processing chamber side.

[0078] In one embodiment, when the device for preparing the DNA product is inserted into the accessory, the front of the device for preparing the DNA product or its cartridge end is substantially flush with the opening of the accessory into the processing chamber. This allows airflow, and particularly gas spraying, to be undisturbed or minimally disturbed and / or reduces surface deposits.

[0079] In one embodiment, the inlet of the PCR liquid and / or the outlet of the DNA product are located on the front of the device or its cartridge for preparing the DNA product. In another embodiment, the inlet of the heating medium and / or the outlet of the heating medium are located on the back plate of the device for preparing the DNA product, opposite the front of the device.

[0080] In another, less preferred embodiment, the accessory may also have an opening toward the technical compartment and an extension toward the processing compartment. In all cases, the accessory may be closed with a cover.

[0081] In one embodiment, the manufacturing apparatus including a device for preparing DNA products via the capillary polymerase chain reaction further includes a leakage sensor disposed in an accessory external to the device for preparing the DNA products. This leakage sensor can improve the safety of the device for preparing the DNA products. As described in the first aspect above, the manufacturing apparatus may also include a second portion of a proximity sensor, wherein the first portion is located at the device of the first aspect. Once the device has been correctly installed in the manufacturing apparatus, the two portions of the proximity sensor are connected and preferably signal successful installation and / or preferably only then can the device be successfully started and operated. Therefore, the proximity sensor including these two portions can also be activated during operation and cause an alarm and / or operation stop if the two portions of the proximity sensor lose their connection.

[0082] In one embodiment, a pump unit for pumping PCR liquids through tubes in an apparatus for preparing DNA products is arranged externally to the apparatus of the first aspect. Preferably, the pump unit is arranged in the processing chamber of the pharmaceutical product manufacturing apparatus. Alternatively, the pump unit may be an external pump unit. External arrangement can save space within the manufacturing apparatus and / or may allow the use of a powerful pump unit. The pump unit may be a pump.

[0083] In one embodiment, the manufacturing apparatus including a device for preparing DNA products via capillary polymerase chain reaction further includes a coupling unit disposed within the housing of the pharmaceutical product manufacturing apparatus and configured to couple the pharmaceutical product manufacturing apparatus to another manufacturing apparatus. The coupling unit may allow and facilitate the coupling of two or more manufacturing apparatuses. The resulting manufacturing module is described in a third aspect herein.

[0084] Manufacturing equipment for pharmaceutical products can be understood as an enclosed enclosure having a technical room and a processing room. In one embodiment, the enclosure is sealed relative to the environment. In the same or another embodiment, the processing room is sealed relative to the technical room. Throughout this application, the term "sealed" can be understood as conforming to at least IP64. IP codes are based on the protection level provided by the enclosure (IP code) (IEC 60529:1989+A1:1999+A2:2013); German version EN 60529:1991+A1:2000+A2:2013 and / or ISO 20653:2013-02 Road vehicles - Degrees of protection (IP code) - Protection of electrical equipment against foreign objects, water and access. The term "sealed" can be understood as dustproof and splashproof. "Dustproof" can be understood as no dust ingress and complete prevention of contact. For testing, a vacuum is typically applied, and airflow-based tests last for up to 8 hours; details can be found in the aforementioned standards. "Splash-proof" can be understood as meaning that water splashed onto the enclosure from any direction has no harmful effect after 10 minutes using a vibrating clamp or at least 5 minutes using a nozzle without a protective cover, as detailed in the standards cited above. A sealed enclosure or sealed processing chamber can have the benefit of particularly cleanliness, meaning that it contains and maintains low levels of particulate matter, such as dust, airborne organisms, or evaporated particles.

[0085] In one embodiment, the processing room is a cleanroom and / or configured as at least a Class D room according to Annex 1 of the EU Good Manufacturing Practices (GMP) guidelines for pharmaceutical products described above. Preferably, the processing room is configured as at least a Class C room. More preferably, the processing room is configured as at least a Class B room. Even more preferably, the processing room is configured as a Class A room. Class A to D rooms offer benefits and are designed to be exceptionally clean, meaning they contain and maintain extremely low levels of particulate matter, such as dust, airborne organisms, or evaporated particles. Cleanliness can be quantified by the number of particles per cubic meter at a predetermined molecular measurement. The characteristics of Classes A to D can be found in Table 1 inserted below. Thus, processing rooms can be classified according to the permissible number and size of particles per volume of gas. The number and size of particles can be measured by particle counting based on light scattering, light shielding, or direct imaging. When particles pass through the detection chamber, the particles are illuminated using a high-intensity light source. The particles pass through the light source (typically a laser or halogen lamp), and if light scattering is used, the redirected light is detected by a photodetector. If direct imaging is used, a halogen lamp illuminates the particles from the back of the cell, while a high-resolution, high-magnification camera records the passing particles. The recorded video is then analyzed by computer software to measure particle properties. If light blocking (occlusion) is used, light loss is detected. The amplitude of the scattered or blocked light is measured, the particles are counted, and listed in a normalized counting bin. Direct imaging particle counting uses a high-resolution camera and light to detect particles. A vision-based particle size measurement unit acquires two-dimensional images, which are analyzed by computer software to obtain particle size measurements.

[0086]

[0087] Table 1: Particulate Contamination Limits Recommended by Annex 1 of EU GMP

[0088] A comparison of Class A to D rooms in Annex 1 of the EU Good Manufacturing Practices (GMP) guidelines for pharmaceutical products with the following standards is shown in Table 2 inserted below: FED-STD-209e (1992) Federal Standard 209-e Airborn Particulate Cleanliness Classes in Cleanrooms and Clean Zones; ISO (1999) International Standard 14644-1: Cleanrooms and associated controlled environments—Part 1: Certification of Air Cleanliness. International Organization for Standardisation, Switzerland; and ISO (2003) International Standard ISO 14698-2:2003 Cleanrooms and associated controlled environments—Biocontamination control—Part 2: Evaluation and interpretation of biocontamination data.

[0089] WHO Technical Report Series, No. 902, 2002, Annex 6

[0090] Comparison of different airborne particle classification systems for clean areas (a)

[0091]

[0092] EEC: European Commission; ISO / TC: International Organization for Standardization Technical Committee

[0093] Table 2: Comparison of Classification Systems

[0094] In one embodiment, the control unit is configured to control the airflow to provide airflow from the processing chamber to the technical chamber. This can be understood as gas flowing from the processing chamber to and into the technical chamber. In the same or another embodiment, the two chambers are arranged such that the airflow in the technical chamber is parallel to the airflow in the processing chamber, but in opposite directions. Preferably, the housing contains the following airflow: to the processing chamber, into the processing chamber, through the processing chamber, out of the processing chamber, to the technical chamber, into the technical chamber, through the technical chamber, and out of the technical chamber, although not all steps are necessary.

[0095] Gas can enter the enclosure before entering the processing chamber. Gas can exit the enclosure after entering the processing chamber. Therefore, the manufacturing equipment may also include at least one conduit through the enclosure for airflow between the enclosure and the environment. The conduit can be a gas inlet and / or outlet. Preferably, the conduit is sealed relative to the environment. As mentioned above, the term "sealed" can be understood as dustproof, splashproof, and compliant with at least IP64. The seal may be a silicone seal.

[0096] In one embodiment, the housing includes a passage for airflow from the processing chamber to the technical chamber. The passage can be understood as a channel for gas. The passage or channel can be provided as a duct or intermediate chamber disposed outside the processing chamber and technical chamber and between the processing chamber and technical chamber (downstream). The passage or channel can also serve as a sealed conduit or sealed through-hole traversing the separation element. As mentioned above, the term "sealed" can be understood as dustproof, splashproof, and compliant with at least IP64. Sealed conduits may have the benefit of maintaining low levels of particulate matter. Sealed conduits may also include filters to further reduce particulate levels and / or valve units to control airflow. The valve unit may be a valve, preferably a throttle valve.

[0097] In one embodiment, the control unit is configured to control the airflow to provide a pressure difference between the processing chamber and the environment ranging from about 5 to about 100 Pa, preferably from about 10 to about 20 Pa, or about 15 Pa. The pressure difference is preferably at least about 15 Pa. This can be understood as having a higher pressure in the processing chamber than in the environment (including the technical chamber). At least the pressure in the processing chamber can be higher than atmospheric pressure. The higher pressure in the processing chamber makes it virtually impossible for any particles to enter and contaminate the processing chamber. In one embodiment, the control unit is configured to control the airflow to provide a pressure difference between the technical chamber and the environment ranging from about -5 to about -100 Pa, preferably from about -10 to about -20 Pa. The pressure difference is preferably at least about -15 Pa. In one embodiment, the control unit is configured to control the airflow to provide a pressure difference between the processing chamber and the technical chamber ranging from about 10 to about 200 Pa, preferably from about 20 to about 40 Pa. The pressure difference is preferably at least about 30 Pa.

[0098] In one embodiment, the processing chamber has a higher pressure than the technical chamber. This can be achieved if the pressure in the processing chamber and the technical chamber are both higher than atmospheric pressure, or if the pressure in the processing chamber is higher than atmospheric pressure and the pressure in the technical chamber is the same as atmospheric pressure, or if the pressure in the processing chamber is higher than atmospheric pressure and the pressure in the technical chamber is lower than atmospheric pressure. In other words, in one embodiment, the control unit is configured to control the airflow to provide a negative pressure relative to the environment in the technical chamber.

[0099] In one embodiment, the pharmaceutical product manufacturing apparatus also includes a liquid collection tray to collect liquid leaks. The tray may be located at the bottom of the housing and / or in the passageway from the processing chamber to the technical chamber. This is advantageous because moisture can be collected before damaging the product or instruments disposed within the chamber. Moisture can be collected intentionally or may originate from gas expansion within the technical chamber.

[0100] In one embodiment, the pharmaceutical product manufacturing equipment further includes a sensor unit comprising at least one of a flow sensor, a pressure sensor, a temperature sensor, a humidity sensor, and a leak sensor. The sensor unit may be disposed within a housing (e.g., at a separation element), in a processing chamber, in a technical chamber, in an intermediate chamber between them, or in an intermediate chamber that is part of a technical chamber, in a conduit through the separation element, at the bottom of the housing, etc. The sensor unit can allow conditions to be controlled based on, for example, flow rate, pressure, temperature, humidity, leak, etc. Accordingly, the sensor unit may include at least one of the group consisting of a flow sensor, a pressure sensor, a temperature sensor, a humidity sensor, a microbial sensor, a particulate sensor, an organic matter sensor, and a leak sensor.

[0101] In one embodiment, the pharmaceutical product manufacturing apparatus further includes a coupling unit disposed on the outer wall of the housing and configured to couple the manufacturing apparatus to another manufacturing apparatus. Other manufacturing apparatus may be similar to the pharmaceutical product manufacturing apparatus described above, or it may be a different apparatus. The coupling unit may allow mechanical coupling between at least two manufacturing apparatuses. The coupling unit may allow fluid communication and coupling between at least two manufacturing apparatuses. The coupling unit may allow data communication and coupling between at least two manufacturing apparatuses. Fluid communication specifically includes transferring a pharmaceutical product from one manufacturing apparatus to another, for example, transferring a DNA template purified and / or filtered in a first manufacturing apparatus from that first manufacturing apparatus to a second manufacturing apparatus, wherein the DNA template can then be used by an RNA generation unit included in the second manufacturing apparatus.

[0102] Preferably, the coupling unit is further arranged to include a quality control unit, wherein the quality control unit is adapted to analyze samples acquired and / or provided in communication between the devices, preferably in fluid communication. Thus, as described above, if, for example, a DNA template is transferred from the first device to the second device, the sample can be acquired and analyzed by the quality control unit, which is preferably located within or at the coupling unit.

[0103] A pharmaceutical product can be understood as an active pharmaceutical ingredient (e.g., a DNA vaccine) or any of its precursors or intermediates (e.g., a DNA template for in vitro transcription of RNA). Therefore, a pharmaceutical product can be an active pharmaceutical ingredient or an intermediate thereof used in a pharmaceutical preparation and administered, for example, to a human subject, to treat or prevent a disease.

[0104] Production of DNA-based pharmaceutical products

[0105] It may be desirable to produce DNA-based pharmaceutical products, such as, in particular, DNA vaccines (e.g., DNA vaccines encapsulated in lipid nanoparticles). Essentially, once the target sequence, such as the sequence of a viral protein (e.g., the spike protein in the case of SARS-CoV-2), is known, the production of a DNA vaccine can begin. The process then typically follows these steps: i) de novo synthesis of DNA with the desired sequence; ii) DNA amplification using the apparatus according to the first aspect of this application, optionally followed by DNA modification; iii) encapsulation of the DNA into a formulation; and iv) filling and finishing the product, each step building upon the previous one. Of course, conventional purification and filtration steps, if applicable, are also performed between the above steps.

[0106] In this embodiment, the manufacturing apparatus for the pharmaceutical product according to this application further includes at least one unit from the group consisting of: a process media supply unit, a mixing unit, a de novo DNA synthesis unit, a formulation unit, a purification unit, a filtration unit, a DNA modification unit, a filling and finishing unit, and combinations thereof. The required units may be present in a single manufacturing apparatus or in at least two coupled manufacturing apparatuses (resulting in a manufacturing module as described in the third aspect herein). In a particularly preferred embodiment, each unit includes a technology media supply located in a technology chamber, in a process media supply and / or in a device configured to contact the process media (wherein the process media supply and the device are located in a processing chamber), and in a sealed via (located in a separation element and configured to connect the technology media supply and the device configured to contact the process media). The unit may also include a waste outlet for collecting, for example, washing buffer or byproducts.

[0107] In a preferred embodiment, the manufacturing apparatus for a pharmaceutical product including the device according to the first aspect further includes a process medium supply unit, a mixing unit, a purification unit, an optional DNA modification unit, and a filtration unit, wherein the units are preferably connected in the following order: i) the process medium supply unit, ii) the mixing unit, iii) the device according to the first aspect, optionally followed by the DNA modification unit, iv) the purification unit, and v) the filtration unit, and the apparatus is configured to amplify DNA, wherein the amplified DNA is optionally modified. A DNA template for the device according to the first aspect can be generated as de novo synthesized DNA in another manufacturing apparatus connected to the manufacturing apparatus according to this embodiment. The amplified DNA can be used in a separate manufacturing apparatus connected to the device of this embodiment and including a DNA preparation unit.

[0108] Production of RNA-based drug products

[0109] It may be desirable to produce RNA-based pharmaceutical products, such as, in particular, RNA vaccines (e.g., RNA vaccines encapsulated in lipid nanoparticles). Essentially, once the target sequence, such as the sequence of a viral protein (e.g., the spike protein in the case of SARS-CoV-2), is known, the production of an RNA vaccine can begin. The process then typically follows these steps: i) de novo synthesis of DNA with the desired sequence; ii) DNA template generation using the apparatus according to the first aspect of this application, optionally followed by DNA modification; iii) RNA generation; iv) encapsulation of the RNA into a formulation; and v) filling and finishing the product, each step building upon the previous one. Of course, conventional purification and filtration steps, if applicable, are also performed between the above steps.

[0110] In this embodiment, the manufacturing apparatus for the pharmaceutical product according to this application further includes at least one unit from the group consisting of: a process media supply unit, a mixing unit, a de novo DNA synthesis unit, a DNA modification unit, an RNA generation unit, a formulation unit, a purification unit, a filtration unit, a filling and finishing unit, and combinations thereof. The required units may be present in one manufacturing apparatus or in at least two coupled manufacturing apparatuses (resulting in a manufacturing module as described in the third aspect herein). In a particularly preferred embodiment, each unit includes a technology media supply located in a technology chamber, in a process media supply and / or in a device configured to contact the process media (wherein the process media supply and the device are located in a processing chamber), and in a sealed via (located in a separation element and configured to connect the technology media supply and the device configured to contact the process media). The unit may also include a waste outlet for collecting, for example, wash buffer or byproducts.

[0111] In a preferred embodiment, the manufacturing apparatus for a pharmaceutical product including the device according to the first aspect further includes a process medium supply unit, a mixing unit, a purification unit, an optional DNA modification unit, and a filtration unit, wherein the units are preferably connected in the following order: i) the process medium supply unit, ii) the mixing unit, iii) the device according to the first aspect, optionally followed by the DNA modification unit, iv) the purification unit, and v) the filtration unit, and the apparatus is configured to generate template DNA, wherein the amplified DNA is optionally modified. This template DNA can be used in a separate manufacturing apparatus connected to the device of this embodiment and including an RNA generation unit.

[0112] The units disclosed above are described in more detail in the third aspect below.

[0113] For both the production of DNA-based and RNA-based pharmaceutical products, the apparatus according to the first aspect of the invention (which is included in the manufacturing apparatus according to the second aspect) is naturally suitable for the amplification of a specific DNA to be amplified, particularly by bending the tubes through the compartments and stacking them so that the resulting PCR conditions and cycles are optimal for that specific DNA to be amplified. If a second, different DNA needs to be amplified, the first apparatus or PCR reactor in the manufacturing apparatus can be easily replaced with a different apparatus or PCR reactor according to the first aspect, which is suitable for the amplification of the second DNA to be amplified, particularly by bending the tubes through the compartments and stacking them so that the resulting PCR conditions and cycles are optimal for the second DNA to be amplified. Therefore, the manufacturing apparatus can be easily equipped (or "loaded") with a PCR reactor suitable for amplifying the specific DNA of interest, and this ease of operation and adaptation can be referred to as a "plug-and-play" system.

[0114] In a third aspect, the present invention relates to a manufacturing module for a pharmaceutical product. The manufacturing module comprises at least two manufacturing devices including apparatus according to a second aspect. The at least two manufacturing devices are coupled to each other. Coupling may allow mechanical coupling of the at least two manufacturing devices. Coupling may allow fluid communication and coupling of the at least two manufacturing devices. Coupling may allow data communication and coupling of the at least two manufacturing devices. Fluid communication specifically includes the transfer of a pharmaceutical product from one device to another, for example, the transfer of a purified and / or filtered DNA template from the first device to the second device, wherein the DNA template can then be used by an RNA generation unit included in the second device.

[0115] The manufacturing module can be specifically used for the production of DNA templates and may include a process medium supply unit, a mixing unit, an apparatus according to the first aspect (which may also be referred to as a "DNA template generation unit"), an optional DNA modification unit, a purification unit, and a filtration unit, wherein the units are preferably connected in the listed order, and the module is configured to produce template DNA. An amplification process (or "DNA template generation process") is performed using the apparatus according to the first aspect.

[0116] The manufacturing module includes manufacturing equipment for the pharmaceutical product according to the second aspect, and may further include at least one manufacturing device selected from the group consisting of: manufacturing equipment including a process media supply unit and a mixing unit, manufacturing equipment including a purification unit, and manufacturing equipment including a filtration unit.

[0117] In one embodiment, the manufacturing module includes: (i) a first manufacturing apparatus comprising a process media supply unit and a mixing unit, (ii) a second manufacturing apparatus according to the second aspect, (iii) a third manufacturing apparatus comprising a purification unit, and (iv) a fourth manufacturing apparatus comprising a filtration unit, wherein the apparatuses are preferably connected in the listed order. Such a manufacturing module is configured to produce template DNA, preferably from DNA (optionally de novo synthesized DNA) supplied in the form of process media in PCR liquid. In other words, such a manufacturing module is configured to produce a large quantity of template DNA from a small amount of DNA (which may be de novo synthesized DNA and may be generated by a previous manufacturing module). The template DNA may optionally be modified by a DNA modification unit included in another manufacturing apparatus.

[0118] When RNA, preferably mRNA, is produced via in vitro transcription, the template DNA can be specifically used. Therefore, an RNA generation unit (for transcribing the obtained DNA into RNA) can be included in the combined or separate manufacturing modules.

[0119] In one embodiment, the manufacturing module includes a process medium supply unit, a mixing unit, a de novo DNA synthesis unit, an apparatus according to the first aspect (which may be referred to as a "DNA template generation unit"), an optional DNA modification unit, a purification unit, a filtering unit, a formulation unit, and an RNA generation unit, wherein the units are preferably arranged in the following order: i) process medium supply unit, ii) mixing unit, iii) de novo DNA synthesis unit, iv) purification unit, v) filtering unit, vi) process medium supply unit, vii) mixing unit, viii) apparatus according to the first aspect, optionally combined with DNA modification unit, ix) purification unit, x) filtering unit, xi) process medium supply unit, xii) mixing unit, xiii) RNA generation unit, xiv) purification unit, xv) filtering unit, xvi) process medium supply unit, xvii) formulation unit, xviii) purification unit, and xix) filtering unit, and the module is preferably configured to produce formulated RNA, preferably formulated mRNA, more preferably LNP-formulated mRNA.

[0120] In one embodiment, the manufacturing module for the pharmaceutical product includes i) a manufacturing apparatus comprising a process media supply unit and a mixing unit, ii) a manufacturing apparatus comprising a de novo DNA synthesis unit, iii) a manufacturing apparatus comprising a purification unit, iv) a manufacturing apparatus comprising a filtration unit, v) a manufacturing apparatus comprising a process media supply unit and a mixing unit, vi) the manufacturing apparatus according to the second aspect, optionally combined with a DNA modification unit, vii) a manufacturing apparatus comprising a purification unit, viii) a manufacturing apparatus comprising a filtration unit, ix) a manufacturing apparatus comprising a process media supply unit and a mixing unit, x) a manufacturing apparatus comprising a bioreactor for in vitro transcription of RNA, xi) a manufacturing apparatus comprising a purification unit, xii) a manufacturing apparatus comprising a filtration unit, xiii) a manufacturing apparatus comprising a process media supply unit and a mixing unit, xiv) a manufacturing apparatus comprising a filtration unit, preferably a tangential flow filtration unit, and xv) a manufacturing apparatus comprising a filtration unit, preferably a sterile filter, wherein the apparatuses are coupled in the given order. The module is preferably configured to produce formulated RNA, preferably formulated mRNA, more preferably LNP-formulated mRNA.

[0121] This manufacturing module for pharmaceutical products provides a highly flexible and versatile platform. It can be adapted to various purposes and uses. It allows for the modular manufacturing or production of pharmaceutical products. It can be understood as an entity suitable for modular manufacturing, meaning it can be adapted to various applications. One entity may be sufficient to meet a specific manufacturing method, or several entities can be connected to perform a specific manufacturing method, such as in the form of a production line or chain. Such a modular design facilitates operation, process improvements, and / or maintenance procedures. The technologies and operations used in this manufacturing module, as well as the selection of materials that come into contact with the product, can be pre-evaluated before use to provide high-quality pharmaceutical product manufacturing. Therefore, this manufacturing module allows for very robust and repeatable manufacturing of pharmaceutical products. Furthermore, this manufacturing module typically allows for the (fully or partially) automated manufacturing of pharmaceutical products.

[0122] In one implementation scheme Process media supply unitThis includes mounting elements configured to hold the process media supply container within the processing chamber. Typically, several mounting elements are present, each configured to hold the process media supply container and then connect (e.g., via a sterile tube) to a downstream unit or device, such as, for example, a de novo DNA synthesis unit, a DNA template generation unit, an RNA generation unit, or a formulation unit. The downstream unit or device may also be a purification unit, such as a reversed-phase HPLC column. Process media can be selected from the group consisting of: synthetic DNA used for amplification in PCR reactions, PCR component mixtures, water, in vitro translation buffers, nucleotides (including UTP, GTP, ATP, and CTP), enzymes (including RNA polymerase), buffers (including wash buffers, reagent buffers (e.g., immobilization buffers), and elution buffers), HPLC buffers, formulation solutions (including lipid solutions), and HPLC eluents.

[0123] In one implementation scheme Hybrid unit The device includes a mixer. It may also include a pump unit (e.g., an injection pump, a pulseless flow pump, a peristaltic pump, etc.). The mixing unit can be used, for example, in combination with a DNA template generation unit or an RNA generation unit, wherein the mixing unit is located upstream of the DNA template generation unit or RNA generation unit and provides the respective reaction mixture to the subsequent DNA template generation unit or RNA generation unit. The mixing unit can also be used when producing multivalent drug products, such as multivalent RNA-based drug products containing, for example, at least two different RNA sequences. In the case of such multivalent RNA-based drug products, the mixing unit can be used to mix at least two different RNAs (i.e., at least two RNAs with different sequences) before the RNA is formulated into a formulation unit. Alternatively, at least two already formulated different RNAs can be mixed using the mixing unit to obtain a multivalent RNA-based drug product.

[0124] Mixing units can also be used in the formulation of nucleic acid products, particularly RNA, and in this case can be referred to as formulation units. Therefore, mixing units can be used for formulation when producing RNA encapsulated in lipid nanoparticles (LNPs) or liposomes, or RNA complexed with polycationic peptides or proteins (e.g., protamine or polymeric carriers, such as polyethylene glycol / peptide polymers, e.g., according to WO2012 / 013326). In the case of formulation, the mixing unit may include at least two pumping units (e.g., one for pumping lipids and one for pumping RNA), and optionally a reactor (e.g., a T-connector) for complexation / formulation.

[0125] In one implementation scheme DNA de novo synthesis unitThe de novo DNA synthesis unit includes a solid-phase synthesis unit for DNA, typically using a phosphoramidite method and corresponding phosphoramidite building blocks derived from protected 2'-deoxynucleosides to obtain DNA oligonucleotides of approximately 200 nucleotides in length. Preferably, in a fully automated manner, the phosphoramidite building blocks are sequentially coupled according to the sequence to be produced (also known as a silico-designed sequence), wherein the oligonucleotide product is typically released from the solid phase into solution, then deprotected and collected. Where applicable, the de novo DNA synthesis unit can also be configured to assemble (or couple) at least two oligonucleotides obtained by the phosphoramidite method to obtain longer sequences. As mentioned above, a purification unit is typically located after the de novo DNA synthesis unit. De novo DNA synthesis can also be based on an enzymatic process, such as based on terminal deoxynucleotidyl transferase (TdT), as described in WO2015159023. Additionally, the de novo DNA synthesis unit may include a ligation unit for linking the synthesized DNA to the appropriate DNA backbone.

[0126] In one implementation scheme DNA modification unit The device includes suitable means for enzymatically or chemically modifying amplified DNA. Enzymatic modification can be, for example, a digestion reaction in which DNA is cleaved at a specific location by a restriction endonuclease (e.g., providing DNA with a poly(A) terminus). Chemical modification can be, for example, a chemical modification such as coupling the 5' and / or 3' ends to a tag or the 5' and / or 3' ends. Such a DNA modification unit can be operated in a continuous mode, wherein amplified PCR product, for example, generated by the device according to the first aspect of this application, is supplied to the DNA modification unit. If the DNA modification unit is configured for enzymatic DNA modification by a restriction endonuclease, it is preferable to immobilize the corresponding restriction endonuclease (e.g., as described in WO2016174227). The DNA modification unit as defined above may even be included in the device of the first aspect in some embodiments.

[0127] In one implementation scheme RNA generation unit This includes bioreactors for in vitro transcription of RNA, preferably bioreactors as disclosed in WO 2020 / 002598. In particular, bioreactors as defined in WO 2020 / 002598 or as described in WO 2020 / 002598. Figures 1 to 1 The bioreactor shown in Figure 4 can be used as RNA generation unit Particularly preferred for bioreactors is that the DNA used as a template in the bioreactor is immobilized on magnetic particles, and the bioreactor includes a magnet for mixing a reaction solution containing the DNA immobilized on the magnetic particles.

[0128] In one implementation scheme Purification unitThe system includes an HPLC unit, preferably a unit for performing RP-HPLC. Particularly preferred in this regard is the use of the method disclosed in WO2008 / 077592, preferably using porous, non-alkylated poly(styrene-divinylbenzene) reversed-phase RP-HPLC, wherein the reversed phase is formed by beads or appears as a polymeric block (e.g., monomer). Alternatively, or additionally, the purification unit may include an affinity chromatography unit, preferably an oligo-dT purification unit, for affinity purification of polyadenylated nucleic acids, particularly RNA, via an oligo-dT-functionalized matrix, beads, or column (e.g., as described in WO2014152031A1). Alternatively, or additionally, the purification unit may include an anion exchange chromatography unit. Alternatively, or additionally, the purification unit may include a unit for nucleic acid precipitation and a unit for purifying the precipitated nucleic acids (e.g., using TFF or centrifugation or filtration). Alternatively, or additionally, the purification unit may include a unit for removing dsRNA, such as a unit involving RNase III treatment or a unit involving a cellulose-based dsRNA purification step. The purification unit is particularly useful for purifying nucleic acids, preferably template DNA obtained in the de novo DNA synthesis unit, DNA obtained in the apparatus according to the first aspect, and / or RNA obtained in the RNA generation unit. In a preferred embodiment, the purification unit, particularly the purification unit for RNA purification, includes an RP-HPLC unit and an oligo-dT purification unit, as well as an optional unit for dsRNA removal.

[0129] In one implementation scheme Filter unit This includes a tangential flow filtration unit. Particularly preferred in this regard is tangential flow filtration as described in WO2016 / 193206, where TFF is used for percolation and / or concentration and / or purification of nucleic acids. The filtration unit can be used to specifically filter nucleic acids, preferably template DNA or RNA, after the purification unit. It is also preferable that the filtration unit includes a sterile filter, preferably a sterile filter with a size of 0.22 μm, for particularly sterile filtration of formulations containing RNA, especially mRNA, and more particularly LNP-formulated mRNA.

[0130] In a preferred embodiment, Fill and complete cells This includes a filling and / or dosing device. The filling and finishing unit is configured to fill a formulated pharmaceutical product, particularly a formulated active pharmaceutical ingredient (especially formulated RNA, and more particularly LNP-formulated mRNA), into a suitable vial, such as a glass vial, in a single or multiple dose amount corresponding to the formulated active pharmaceutical ingredient. Typically, the vial (e.g., glass vial) is sealed with a cap (e.g., an aluminum cap) under aseptic conditions. In an embodiment, the filling and finishing unit includes a freezing unit for freezing the obtained pharmaceutical product to at least -20°C, preferably to at least -60°C or -80°C.

[0131] In a fourth aspect, the present invention relates to a method for preparing DNA products via capillary polymerase chain reaction. The method for preparing the DNA products includes the following steps, which do not necessarily follow this order:

[0132] - Provides a first compartment, a second compartment, and a tube, wherein the tube is bent at least from the first compartment to the second compartment and from the second compartment to the first compartment, wherein the tubes bent from compartment to compartment form a helical stack of tube loops.

[0133] - Provides a device for regulating the individual temperature in each compartment, and

[0134] - The PCR liquid is guided through tubes to pass through the individual temperatures in each compartment to prepare DNA products based on the PCR liquid.

[0135] The method for preparing DNA products according to the present invention allows for flexible adaptation to different conditions, requirements, and, for example, the amount of DNA. This method allows for the preparation of large quantities of DNA (e.g., more than 2 mg per reaction, even up to g). Furthermore, the preparation of DNA products can be scalable. This method for preparing DNA products allows for very rapid preparation of DNA products, thereby enabling rapid manufacturing of pharmaceutical products. The equipment used for preparing DNA products can also be very quickly modified and adapted to specific DNA products. Adjustment or calibration steps may not be required. Cleaning steps may be required little or no. Turnaround time, for example, for vaccine manufacturing, can be less than about one week.

[0136] In one embodiment, the temperature in the first compartment is configured for the denaturation step of the polymerase chain reaction, the temperature in the second compartment is configured for the annealing step of the polymerase chain reaction, and optionally also for the extension step of the polymerase chain reaction.

[0137] In one embodiment, the temperature in the first compartment is in the range of about 85°C to about 105°C, preferably about 98°C. In one embodiment, the temperature in the second compartment is in the range of about 45°C to about 72°C, preferably about 60°C to about 72°C.

[0138] In one embodiment, the method includes providing a third compartment. The tube is bent at least from the second compartment to the third compartment and then back to the first compartment. The temperature in the third compartment is configured for an extension step of PCR, preferably in the range of about 65°C to about 75°C, and more preferably about 72°C.

[0139] In one embodiment, the method for preparing the DNA product is carried out under GMP-compliant conditions.

[0140] In one embodiment, the means for regulating the individual temperature in each compartment may be a separate flow of heat medium through each compartment provided by a heat medium inlet and a heat medium outlet in each compartment (to regulate the individual temperature in each compartment).

[0141] In another embodiment, the means for regulating the individual temperature in each compartment may include a heating unit (in particular a heating cylinder) and an optional cooling unit (in particular a thermoelectric cooler) in each compartment (optionally connected to a ventilator).

[0142] In a fifth aspect, the present invention relates to the use of an apparatus for preparing DNA products according to the first aspect for preparing DNA products by capillary polymerase chain reaction, an apparatus for manufacturing pharmaceutical products according to the second aspect for manufacturing pharmaceutical products, and a manufacturing module according to the third aspect for manufacturing pharmaceutical products.

[0143] Pharmaceutical products can be formulated. The pharmaceutical products are preferably active pharmaceutical ingredients in the form of biomolecules or any precursors or intermediates thereof, wherein the biomolecules are preferably nucleic acids, more preferably DNA. Production preferably conforms to GMP standards.

[0144] In one implementation, the above-described uses are automated. This means that the use or operation of the equipment for preparing DNA products via capillary polymerase chain reaction according to the first aspect, the manufacturing equipment for pharmaceutical products according to the second aspect, or the manufacturing module according to the third aspect can be fully or partially automated. This can allow for more reliable, faster, and / or more cost-effective manufacturing of pharmaceutical products.

[0145] It should be noted that the above aspects of the present invention are applicable to apparatus for preparing DNA products by capillary polymerase chain reaction according to the independent claims; pharmaceutical product manufacturing apparatus; pharmaceutical product manufacturing module; method for preparing DNA products by capillary polymerase chain reaction; use of the apparatus for preparing DNA products by polymerase chain reaction; and use of the apparatus for producing pharmaceutical products having similar and / or identical preferred embodiments, particularly as defined in the dependent claims. It should also be understood that preferred embodiments of the present invention may also be any combination of the dependent claims and the corresponding independent claims.

[0146] These and other aspects of the invention will become apparent and elucidated with reference to the embodiments described below. Attached Figure Description

[0147] The accompanying drawings shown below are merely exemplary and should be used to further describe the invention. These drawings should not be construed as limiting the invention thereto.

[0148] Figure 1A top view of an apparatus according to the invention for preparing DNA products by capillary polymerase chain reaction is shown.

[0149] Figure 2 A side view of an apparatus according to the invention for preparing DNA products by capillary polymerase chain reaction is shown.

[0150] Figure 3 A 3D side view of the bent tube is shown.

[0151] Figure 4 A lateral 3D view of a tube bent around multiple supports is shown.

[0152] Figure 5 A 3D view of the manufacturing equipment for the pharmaceutical product is displayed.

[0153] Figure 6a A 3D view shows a box containing equipment for preparing DNA products within a manufacturing facility for inserting a drug product.

[0154] Figure 6b A 3D view shows a box containing equipment for preparing DNA products within a manufacturing facility for inserting a drug product.

[0155] Figure 7 A manufacturing module for a pharmaceutical product according to the present invention is illustrated schematically and exemplary.

[0156] Figure 8 A side 3D view of the device according to the present invention is shown.

[0157] Figure 9 A side 3D view of the top of the device according to the invention is shown.

[0158] Figure 10 A side 3D view of the box that houses the equipment to be inserted into the manufacturing equipment is shown.

[0159] Figure 11 A magnified 3D view of the manufacturing equipment is shown, in which a box accommodates the equipment inserted into the manufacturing equipment.

[0160] Figure 1 A top view of an apparatus 1 according to the invention for preparing DNA products by capillary polymerase chain reaction is shown. Figure 2 A side view of an apparatus 1 according to the invention for preparing DNA products by capillary polymerase chain reaction is shown. Figure 8 A side 3D view of the device according to the present invention is shown. Figure 2 In this process, the apparatus 1 for preparing DNA products is installed in the pharmaceutical product manufacturing apparatus 10 according to the invention, which will refer to... Figure 5To explain in more detail. The DNA product can be DNA to be used as an API or a DNA template for enzymatic RNA in vitro transcription, where the RNA will be the API. Polymerase chain reaction (PCR) is a technique that synthetically amplifies DNA by several orders of magnitude to produce thousands to millions of copies of a specific DNA sequence. Capillary polymerase chain reaction can be understood as PCR performed in a capillary or tube 6. This device 1 for preparing DNA products allows for the flexible and rapid preparation of large quantities of DNA products. This device 1 for preparing DNA products can be used for flexible and rapid vaccine production, particularly DNA vaccine production or the production of DNA templates for enzymatic RNA in vitro transcription. This device 1 can be particularly used for preparing DNA templates for mRNA-based vaccines, for example, during infectious disease epidemics and pandemics.

[0161] like Figure 1 and Figure 8 As shown, the apparatus 1 for preparing DNA products includes a tube 6, a first compartment 7, a second compartment 8, and a third compartment 15. The tube 6, or capillary, has a very small diameter, ranging from about 0.5 mm to about 50 mm, preferably from about 0.5 mm to about 1.5 mm, more preferably from about 0.75 mm to about 1.25 mm. The tube 6 bends from the first compartment 7 to the second compartment 8, and from the second compartment 8 to the third compartment 15. It then returns to the first compartment 7. PCR liquid can be filled into the tube 6, thereby being guided through the three compartments. Figure 2 and 8 As shown, a compartment can be understood as an independent basin, for example, one containing a heat medium (see...). Figure 2 ) or hot solids (see Figure 8 The basin is filled with water. The basin is at least partially, preferably completely, surrounded by insulation, which may be a partition wall 14 (see...). Figure 1 ) or internal compartment wall 30 (see Figure 8 ), which can be optionally combined with the adjacent 14.

[0162] Tube 6 is bent to form a spiral stack of tube loops, which, when viewed in a side view, can be understood as a spiral, coil, or coil. Figure 3 A 3D side view of the curved tube 6 is shown. There can be several layers of coils, for example, 30 layers. The helical extension or stacking direction of the tube 6 extends in the z-direction, which is substantially perpendicular to the x, y plane formed by the bottom of the apparatus 1 for preparing the DNA product or the plane formed by the floor.

[0163] Back Figure 1 and 8Tube 6 not only bends from the first compartment 7 to the second compartment 8, and from the second compartment 8 to the third compartment 15, but also from the third compartment 15 to the first compartment 7, from the first compartment 7 to the second compartment 8, and from the second compartment 8 to the third compartment 15, and so on. Each loop in the tube represents one PCR cycle.

[0164] Tube 6 extends in a corrugated manner within each of the three compartments. This can be understood as appearing wavy or serpentine in all three compartments when viewed from above. The corrugation of tube 6 extends in the x and y directions along a plane formed by the bottom of apparatus 1 used for preparing DNA products or by a floor. The corrugation pattern of tube 6 defines the residence time of the PCR liquid in each compartment. The direction of the corrugation of tube 6 is substantially perpendicular to the stacking direction of tubes 6.

[0165] like Figure 1 As shown, the apparatus 1 for preparing DNA products also includes a PCR liquid inlet 20 for the PCR mixture, which can be connected to an external pump unit for pumping PCR liquid through the tube 6 or a pump unit 37 for pumping PCR liquid. The apparatus 1 for preparing DNA products also includes a DNA product outlet 25 for the PCR liquid product. Alternatively, the PCR apparatus may include an internal pump unit for pumping PCR liquid through the tube 6 or a pump unit 37 for pumping PCR liquid. Figure 1 As shown, the PCR liquid inlet 20 can be located in the first compartment, while the DNA product outlet 25 can be located in the third compartment.

[0166] like Figure 1 and 2 As shown, the apparatus 1 for preparing DNA products in this embodiment further includes a heat medium inlet 12 and a heat medium outlet 13 in each of the three compartments. As described above, other means may be included to regulate the temperature in each compartment. If a heat medium is used, the heat medium can enter each of the three compartments through its respective heat medium inlet 12 and exit each of the three compartments through its respective heat medium outlet 13. This can result in heat medium flow through the respective compartments. The heat medium outlet 13 is preferably positioned at the top to facilitate the removal of air bubbles.

[0167] The heat medium can be understood as a liquid heat carrier and is used to provide three different temperature zones in three compartments to prepare DNA products through thermal cycling. These three temperature zones correspond to the temperature spectrum of denaturation, annealing, and extension in PCR. The PCR liquid is separated from the heat medium in the compartments by the tube walls. The tube walls allow temperature exchange between the PCR liquid and the interior of the compartments (e.g., the heat medium), and can be used to heat or cool the PCR liquid, for example, through the heat medium.

[0168] In this embodiment, a heat medium inlet 12 and a heat medium outlet 13 are arranged in each compartment to provide a heat medium flow through the compartment in a direction substantially opposite to the direction of PCR liquid flow in tube 6. Figure 1 As shown, this can be understood as follows: with the PCR liquid being guided from the upper left to the lower right, the thermal medium inlet 12 and the thermal medium outlet 13 are arranged to provide a thermal medium flow in the opposite direction from the lower right to the upper left (see the arrow in the first compartment 7). The countercurrent flow of the thermal medium and the PCR liquid enhances the temperature exchange between them.

[0169] like Figure 8 As shown, the device 1 in this embodiment includes an isolator 28 and a housing 29, as well as an inner compartment wall 30, which corrugates around the tube in each of the first compartment 7, the second compartment 8, and the third compartment 15. Each compartment includes at least one heating unit 31 (depicted herein as a heating cylinder) and a cooling unit, which includes a heat pipe 32 (part of the cooling unit) for use in cooling if desired. In this embodiment, the space in each compartment surrounded by the inner compartment wall 30 (wherein the space surrounds the tube 6) is filled with a hot solid (e.g., aluminum pellets), optionally in combination with a liquid or gel, wherein the hot solid is heated by at least one heating unit 31 and—if desired—cooled by the cooling unit (including the heat pipe 32) to provide a specific temperature in each compartment. The space between the compartments and / or the surrounding housing 29, i.e., the space not surrounding the tube 6, may be filled with a synthetic material (e.g., synthetic spheres or granules). Figure 9 Showing Figure 8 The diagram shows a side 3D view of the top of device 1, depicting the isolator 28 and housing 29, and also illustrating the top of the heating unit 31 and heat pipe 32. Heat pipe 32 is connected to a thermoelectric cooler 33 (part of the cooling unit, e.g., a copper block with a Peltier element) and a fan 34 for heat removal. Also in this embodiment, three distinct temperature zones are provided in three compartments for preparing DNA products via thermal cycling. The three temperature zones correspond to the temperature spectrum of PCR denaturation, annealing, and extension. The PCR liquid is separated from the hot solids in the compartments by the tube walls.

[0170] like Figure 1 and 8 As shown, the apparatus 1 for preparing DNA products also includes partitions 14 between the compartments to insulate the compartments from each other (in Figure 8In this configuration, partition 14 exists only partially because the inner compartment walls 30 already separate the compartments from each other. One partition 14 is arranged between the first compartment 7 and the second compartment, another partition 14 is arranged between the second compartment and the third compartment, and yet another partition 14 is arranged between the third compartment and the first compartment. Thus, adjacent compartments are thermally insulated from each other, allowing different temperatures to be established and maintained in adjacent compartments. The partition 14 can be a plate-shaped element. The partition 14 between compartments can be implemented by several walls or only one (e.g., T-shaped or star-shaped) wall. The partition 14 includes through-holes for the tube 6 to extend from one compartment to the next.

[0171] like Figure 1 and 9 As shown, the apparatus 1 for preparing DNA products also includes sensors 21 arranged in each compartment, for example, sensor 21. Preferably, several of these temperature sensors are present in each compartment.

[0172] The apparatus 1 for preparing DNA products also includes a support 17 for holding the tube 6. Figure 4 A lateral 3D view of the tube 6, which bends around multiple supports 17, is shown. Supports 17 are also shown. Figure 1 and Figure 8 In this apparatus, a support 17 is arranged in each compartment to hold and guide the tube 6. The support 17 provides anchoring and deflection points for the corrugations of the tube 6. The support 17 may therefore include slits or the like to mount the tube 6 (coiled) to the support 17. The tube 6 extends partially around the outer dimensions of the support 17. When viewed in top view, the support 17 here has a T-shape or L-shape, but other shapes are also possible, such as cylindrical. The support 17 can be replaceable and repositionable within the compartment. Here, the tube 6 is identical throughout the apparatus 1 for preparing DNA products and can be replaceable.

[0173] Figure 5 A 3D view of the manufacturing equipment 10 for the pharmaceutical product is shown. Figure 11 The truncated portion is shown. The pharmaceutical product manufacturing facility 10 includes a housing 5, a processing chamber 2, a technology chamber 3, and equipment 1 as described above for preparing DNA products via capillary polymerase chain reaction. The pharmaceutical product can be understood as an active pharmaceutical ingredient or any of its precursors or intermediates (e.g., a DNA vaccine or a DNA template for RNA synthesis). The pharmaceutical product manufacturing facility 10 can operate under conditions compliant with GMP (Good Manufacturing Practices) guidelines.

[0174] The pharmaceutical manufacturing equipment 10 can be understood as a closed and sealed housing 5 housing different chambers for different purposes. A processing chamber 2 is suitable for and used in the manufacture of pharmaceutical products. The processing chamber 2 is a Class A chamber in this context. The processing chamber 2 is sealed relative to the technical chamber 3. The technical chamber 3 is suitable for and used to house instruments, such as pumps, motors, mixers, processors, etc. The chambers are separated by a separating element 4, which can be understood as a plate. The separating element 4 extends through the housing 5 and can be used to mount instruments onto it.

[0175] Technical chamber 3 and processing chamber 2 are arranged such that the airflow in technical chamber 3 is parallel to the airflow in processing chamber 2, but in the opposite direction. The gas in the airflow is clean air. There is an air inlet duct 9a and an air outlet duct 9b.

[0176] At least one control unit controls the airflow through processing chamber 2 to provide a downward (gravity-driven) gas spray and / or positive pressure within processing chamber 2. The control unit may be a valve or a processor. The gas spray may be laminar. The airflow may be provided by a flow unit, which may be located inside or outside housing 5 and may communicate with the control unit. The flow unit may be a pump, HVAC system, etc. Positive pressure is an overpressure relative to the external environment of housing 5. The flow unit provides a variable volume of gas, while the control unit regulates the different pressures within the airflow.

[0177] like Figure 5 As shown, the separation element 4 includes at least one attachment 16, which has an opening toward the processing chamber 2 and an extension in the direction of the technical chamber 3 (the extended processing chamber). Attachment 16 is used to house instruments, such as the aforementioned apparatus 1 for preparing DNA products (which may also be referred to as a "PCR reactor"). This allows for an increase in the available space in the processing chamber 2. The apparatus 1 for preparing DNA products is housed here in a box 26, which is inserted as an integral element into attachment 16. Box 26 may be replaceable.

[0178] For example Figure 5 As shown, when the apparatus 1 for preparing DNA products is inserted into the attachment 16, the front of the apparatus 1 for preparing DNA products, and particularly the front end of the housing 26, is substantially flush with the opening of the processing chamber 2 of the attachment 16. Thus, the airflow, and particularly the gas spray, is seamless and can be undisturbed or minimally affected by reduced deposition surface. The attachment 16 and its front are sealed by a lid. The lid may be part of the housing 26 of the apparatus 1 for preparing DNA products.

[0179] like Figure 5As shown, the pharmaceutical product manufacturing equipment 10 also includes a liquid collection tray 11 to collect liquid leaks. The liquid collection tray 11 may be arranged at the bottom of the housing 5 and / or in the passage from the processing chamber 2 to the technical chamber 3. The liquid collection tray 11 may be equipped with a sensor 21 (leakage sensor).

[0180] Figure 11 The rear of the manufacturing equipment 10 in which device 1 is inserted is shown. A ventilator 34 is present on the rear facing the technical room 3. Additionally, an air inlet 35 and an electrical plug 36 are present to connect device 1 to the technical medium, particularly a power source, within the technical room. This embodiment particularly relates to equipment in which a heated solid is used to regulate the temperature of the compartment (e.g., such as...). Figure 8 , 9 and as exemplified in 10).

[0181] Figure 6a and 6b as well as Figure 10 A 3D view is shown of a housing 26 of a manufacturing apparatus 10 for inserting a drug product, containing an apparatus 1 for preparing a DNA product. The housing 26 is closed by a lid. The lid includes a handle 22 for operating the apparatus 1 for preparing the DNA product into and / or out of the housing 16. The housing 26 includes a heat medium inlet 12 and a heat medium outlet 13 for each compartment of the apparatus 1 for preparing the DNA product (see [link to documentation]). Figure 6b ) or heating unit 31 (heating cylinder) and heat pipe 32 connected to thermoelectric cooler 33 and fan 34 (see Figure 10 The PCR liquid inlet 23 and / or the DNA product outlet 24 are located on the front of the device 1 for preparing the DNA product and here are located on the lid of the box 26. Annex 16 (see Annex 16) Figure 5 The box 26 includes a corresponding portion of the exchange rail 27 (exchange rail system) to guide and facilitate the exchange of instruments in Annex 16, such as exchanging device 1 for preparing DNA products with another device. Figure 6b and Figure 10 As shown, sensor 21 (e.g., a leak sensor or a temperature sensor) is arranged in housing 26. Housing 26 also includes connections to the processing chamber 2, such as electrical connections. According to... Figure 10 In the implementation scheme, the sensor is preferably a temperature sensor, because Figure 10 Implementation schemes (where hot solids are used as heating devices) typically do not include leak sensors (because...) Figure 10 The embodiment shown does not use a heat transfer medium.

[0182] An external pump unit or pump for pumping PCR liquid through tube 6 of device 1 for preparing DNA products. Figure 7The pump unit 37 for pumping PCR liquid can be arranged outside the device 1 and can be located, for example, in the processing chamber of the pharmaceutical product manufacturing device 10.

[0183] Figure 7 A manufacturing module 100 for a pharmaceutical product according to the present invention is illustrated schematically and exemplary. The manufacturing module 100 includes four manufacturing devices 10. The manufacturing devices are highly flexible, and multiple manufacturing devices can be adapted to multiple manufacturing steps to form, for example, a manufacturing chain.

[0184] Four manufacturing devices 10 are coupled to each other via coupling units 18 disposed on the outer wall of the housing 5. Coupling units 18 allow fluid communication and coupling of the multiple manufacturing devices 10, and preferably also allow mechanical coupling as well as data communication and coupling. The four manufacturing devices 10 are exemplarily shown herein (from left to right): a first device including a process medium supply unit and a mixing unit; a second device including a manufacturing device 10 for preparing DNA products via capillary polymerase chain reaction as described above (device 1 for preparing DNA products via capillary polymerase chain reaction); a third device including a purification unit; and a fourth device including a filtration unit, wherein the filtration unit includes two filtration units, namely a tangential flow filtration unit and a sterile filter. This exemplary module can be used for DNA production and DNA amplification and purification, respectively. The exemplary module for DNA production / amplification can be connected to other manufacturing devices that transcribe the obtained DNA into RNA (e.g., in manufacturing devices including RNA bioreactors, as described in WO2020002598).

[0185] The manufacturing module 100 includes an attachment element 19 disposed on the outer wall of the housing 5 of the manufacturing equipment 10, outside the housing 5. The attachment element 19 is configured to maintain a media supply (not shown), for example in the form of a media storage device.

[0186] definition

[0187] For clarity and readability, the following definitions are provided. Any technical features mentioned in these definitions can be understood in each embodiment of the invention. Additional definitions and explanations may be provided specifically in the context of these embodiments.

[0188] As used in the specification and claims, the singular forms “an” and “a” also include the corresponding plural forms, unless the context clearly specifies otherwise.

[0189] In the context of this invention, the term "about" refers to an accurate range that, as would be understood by those skilled in the art, still ensures the technical effect of the stated feature. This term typically indicates a deviation from the indicated value of ±10%, and preferably ±5%.

[0190] It should be understood that the term "comprising / including" is not limiting. For the purposes of this invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising / including...". If a group is defined below as containing at least a certain number of embodiments, this also means covering a group that preferably consists only of these embodiments.

[0191] As used herein, the term "pharmaceutical product" refers to an active pharmaceutical ingredient or any precursor or intermediate thereof. Thus, a "pharmaceutical product" can be, in particular, an active pharmaceutical ingredient used in pharmaceutical preparations and administered to human or animal subjects to treat or prevent disease, i.e., it has a clinical grade, especially when parameters such as purity and integrity are involved. Such products are typically produced in vitro during synthesis, and the production process includes precursors and intermediates. For the purposes of this invention, it is particularly preferred that the active pharmaceutical ingredient is a biomolecule, particularly a nucleic acid. Nucleic acid can be DNA. DNA can be used, in particular, as a vaccine or as a DNA template for in vitro transcription of RNA. When producing, for example, mRNA as an active pharmaceutical ingredient, the first step may be the production of template DNA (also referred to herein as a "DNA template"), which corresponds to a precursor of mRNA because it acts as a template in the in vitro transcription reaction during RNA production. The intermediate for DNA production may be DNA obtained from a PCR reaction prior to purification, as such DNA does not correspond to the final active pharmaceutical ingredient, but a purification step is particularly necessary to provide the desired clinical grade product.

[0192] The term "DNA" is a common abbreviation for deoxyribonucleic acid. It is a nucleic acid molecule, a polymer composed of nucleotide monomers. These nucleotides are typically monomers of deoxyadenosine monophosphate, deoxythymidine monophosphate, deoxyguanosine monophosphate, and deoxycytidine monophosphate, or their analogues, which themselves consist of a sugar moiety (deoxyribose), a base moiety, and a phosphate moiety, polymerized through a characteristic backbone structure. The backbone structure is typically formed by phosphodiester bonds between the sugar moiety (deoxyribose) of the first monomer and the phosphate moiety of the second adjacent monomer. The specific sequence of the monomers, that is, the sequence of bases attached to the sugar / phosphate backbone, is called the DNA sequence. DNA can be single-stranded or double-stranded. In the double-stranded form, the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, for example, through A / T base pairing and G / C base pairing.

[0193] The term "RNA" is a common abbreviation for ribonucleic acid. It is a nucleic acid molecule, a polymer composed of nucleotide monomers. These nucleotides are typically monomers of adenosine monophosphate (AMP), uridine monophosphate (UMP), guanosine monophosphate (GMP), and cytidine monophosphate (CMP), or their analogues, linked together along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar (ribose) of the first monomer and the phosphate moiety of the second adjacent monomer. The specific sequence of monomers, the sequence of bases attached to the sugar / phosphate backbone, is called the RNA sequence. RNA can be obtained through transcription of a DNA sequence, for example, within a cell. In eukaryotic cells, transcription typically occurs in the nucleus or mitochondria. In vivo, transcription of DNA usually produces so-called immature RNA, which must be processed into so-called messenger RNA, often abbreviated as mRNA. The processing of immature RNA (e.g., in eukaryotes) includes various post-transcriptional modifications such as splicing, 5'-capping, polyadenylation, and export from the nucleus or mitochondria. The sum of these processes is also called RNA maturation. Mature messenger RNA typically provides a nucleotide sequence that can be translated into an amino acid sequence of a specific peptide or protein. Typically, mature mRNA contains a 5' cap, an optional 5' UTR, a coding sequence, an optional 3' UTR, and a poly(A) sequence. If the RNA molecule is of synthetic origin, as in this invention, the RNA molecule does not mean that it is produced in vivo (i.e., within cells) or purified from cells, but rather that it is produced in an in vitro method. One example of a suitable in vitro method is in vitro transcription. Besides messenger RNA, several non-coding types of RNA exist that can participate in the regulation of transcription and / or translation, as well as immune stimulation, and can also be produced by in vitro transcription. The term "RNA" also includes RNA molecules such as viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA), antisense RNA, saRNA (small activating RNA), CRISPR RNA (small guide RNA, sgRNA), ribozymes, aptamers, riboswitch, immune-stimulating RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nucleoRNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA).

[0194] The term "in vitro RNA transcription" refers to the process of synthesizing RNA in a cell-free system. RNA can be obtained through DNA-dependent RNA in vitro transcription using a suitable DNA template, which can be a linearized plasmid DNA template or a PCR-amplified DNA template. The promoter used to control the in vitro RNA transcription can be the promoter of any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases are T7, T3, SP6, or Syn5 RNA polymerases.

[0195] Reagents used for in vitro transcription of RNA typically include: a DNA template (linearized DNA or linear PCR product) having a promoter sequence that has a high binding affinity for its respective RNA polymerase, such as a phage-encoded RNA polymerase (T7, T3, SP6, or Syn5); ribonucleotide triphosphates (NTPs) of four bases (adenine, cytosine, guanine, and uracil); optionally, a cap analog (e.g., m7G(5')ppp(5')G(m7G) or a cap analog derived from the structure disclosed in WO2017 / 053297 or any cap structure derived from the structure defined in WO2018075827); optionally, nucleotides further modified as defined herein; and a DNA-dependent RNA polymerase (e.g., T7, T3, SP6, or Syn5) capable of binding to the promoter sequence within the DNA template. RNA polymerase; optionally, a ribonuclease (RNase) inhibitor to inactivate any potentially contaminating RNase; optionally, a pyrophosphatase to degrade pyrophosphate (an inhibitor of RNA synthesis); MgCl2, which provides Mg2+ ions as a cofactor for the polymerase; a buffer (TRIS or HEPES) to maintain a suitable pH, which may also contain an antioxidant (e.g., DTT) and / or an optimal concentration of a polyamine (e.g., spermidine), such as the buffer system containing citrate and / or betaine disclosed in WO2017 / 109161.

[0196] Nucleotide mixtures used for in vitro transcription of RNA may additionally contain modified nucleotides as defined herein. In this case, preferred modified nucleotides include pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine. Nucleotide mixtures used for in vitro transcription of RNA (i.e., the fraction of each nucleotide in the mixture) can be optimized for a given RNA sequence, preferably as described in WO2015188933.

[0197] The “RNA in vitro transcription (IVT) reaction mixture” may contain the components necessary for carrying out the RNA in vitro transcription reaction as defined above. Therefore, the IVT reaction mixture may contain at least one component selected from nucleotide mixtures, cap analogs, DNA-dependent RNA polymerases, RNase inhibitors, pyrophosphatases, MgCl2, buffers, antioxidants, betaine, and citrate.

[0198] As used herein, the term "template DNA" (or "DNA template") generally refers to a DNA molecule containing a nucleic acid sequence encoding an RNA sequence to be transcribed in vitro. Template DNA serves as a template for in vitro transcription of RNA to produce RNA encoded by the template DNA. Therefore, template DNA contains all the elements necessary for in vitro transcription of RNA, particularly the 5' promoter element of the DNA sequence encoding the target RNA sequence for binding to DNA-dependent RNA polymerases (e.g., T3, T7, and SP6 RNA polymerases). Furthermore, template DNA may contain primer binding sites at the 5' and / or 3' of the DNA sequence encoding the target RNA sequence to determine the presence of the DNA sequence encoding the target RNA sequence, for example, by PCR or DNA sequencing.

[0199] Polymerase chain reaction (PCR) is a technique in molecular biology used to amplify a DNA segment by orders of magnitude, resulting in thousands to millions of copies of a specific DNA sequence. This method relies on thermal cycling, which consists of repeated cycles of heating and cooling for DNA melting and enzymatic replication. Primers (short DNA fragments) containing sequences complementary to the target sequence, along with a thermostable DNA polymerase (such as Taq polymerase), enable selective and repetitive amplification. As PCR proceeds, the generated DNA itself serves as a template for replication, initiating a chain reaction in which the DNA template amplifies exponentially. By using single-stranded DNA as a PCR template and DNA oligonucleotides (also called DNA primers) required to initiate DNA synthesis, the DNA polymerase enzymatically assembles DNA building blocks (nucleotides) into a new DNA strand. Most PCR methods utilize thermal cycling, which involves alternating heating and cooling of the PCR sample through a series of defined temperature steps. In the first step, the two strands of the DNA double helix are physically separated at high temperatures during a process called DNA melting. In the second step, the temperature decreases, and the two DNA strands become templates for the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR stems from the use of primers that are complementary to the target DNA region being amplified under specific thermal cycling conditions.

[0200] A “PCR reaction mixture” or “PCR liquid” may contain the components necessary for PCR as defined above. Therefore, a PCR reaction mixture may contain at least one component selected from a mixture of nucleotides, DNA polymerase, synthetic DNA as a (initial) template, and a buffer.

[0201] The term "lipid nanoparticle" or "LNP" refers to pharmaceutical products, particularly formulations of nucleic acids. In the context of this invention, the term "LNP" is not limited to any particular form and includes any form resulting from the combination of cationic lipids and optionally one or more other lipids, for example, in an aqueous environment and / or in the presence of nucleic acids. For example, liposomes, lipid complexes, lipoplexes, etc., are all within the scope of lipid nanoparticles (LNPs). LNPs typically comprise cationic lipids and one or more excipients selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids (e.g., PEGylated lipids). Nucleic acids may be encapsulated within the lipid portion of the LNP or within an aqueous space surrounded by some or all of the lipid portion of the LNP. In one embodiment, the LNP is essentially composed of: (i) at least one cationic lipid; (ii) a neutral lipid; (iii) a sterol, such as cholesterol; and (iv) a PEG-lipid, such as PEG-DMG or PEG-cDMA, in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol: 0.5-15% PEG-lipid.

[0202] The term "process media" refers to any component that directly and physically participates in any reaction or process step required to produce a pharmaceutical product. Accordingly, since production takes place in a processing chamber, "process media" will be present in the processing chamber when the equipment is used for production. Therefore, process media can specifically be any starting material (such as, for example, nucleotides), any catalytic material (such as, for example, enzymes), and any buffer (such as, for example, reaction buffer or purification buffer). Process media are typically provided in process media supply containers.

[0203] The term "technical medium" refers to a component that does not directly participate in any reaction or process step required to produce the pharmaceutical product. Instead, the technical medium participates indirectly, for example, as an electrical cable providing power to a unit or instrument for handling the process medium, and will be located in the technical room. The supply of the technical medium will be, for example, a power source or power provided by an electrical cable.

[0204] It should be noted that embodiments of the invention have been described with reference to different subject matter. In particular, some embodiments are described with reference to method-type claims, while others are described with reference to device-type claims. However, those skilled in the art will appreciate from the description that, unless otherwise stated, any combination of features relating to different subject matter is also considered to be disclosed with this application, except for any combination of features belonging to one class of subject matter. However, all features can be combined to provide synergies that are not merely a simple addition of features.

[0205] If a group is defined as including at least a certain number of implementations, this also means that it covers a group that preferably consists only of these implementations.

[0206] While the invention has been detailed and described in the accompanying drawings and specification, such descriptions should be considered illustrative or exemplary rather than restrictive. The invention is not limited to the disclosed embodiments. By studying the drawings, disclosure, and dependent claims, those skilled in the art can understand and implement other variations of the disclosed embodiments when practicing the claimed invention.

[0207] In the claims, the word "comprising / including" does not exclude other elements or steps, and the singular forms "a" or "an" do not exclude the plural. The fact that certain measures are referenced in different dependent claims does not indicate that combinations of these measures cannot be used. Any reference numerals in the claims should not be construed as limiting the scope.

[0208] List of reference numerals

[0209] 1. Equipment for preparing DNA products

[0210] 2 processing room

[0211] 3 Technical Room

[0212] 4. Separation element

[0213] 5. Outer shell

[0214] 6 tubes

[0215] 7 First compartment

[0216] 8 Second compartment

[0217] 9 pipes

[0218] 9a Intake pipe

[0219] 9b exhaust pipe

[0220] 10 Manufacturing equipment

[0221] 11 Liquid collection tray

[0222] 12. Heat medium inlet

[0223] 13. Heat medium outlet

[0224] 14 Next door

[0225] 15 Third compartment

[0226] 16. Annex

[0227] 17 stents

[0228] 18 Coupling Units

[0229] 19 Accessory Components

[0230] 20 PCR liquid inlet

[0231] 21 sensors

[0232] 22 handles

[0233] 23 PCR liquid import

[0234] 24 DNA product discharge outlets

[0235] 25 DNA product exports

[0236] 26 boxes

[0237] 27. Exchangeable guide rails

[0238] 28. Isolation components

[0239] 29. Shell

[0240] 30 Inner compartment wall

[0241] 31 Heating Units

[0242] 32 heat pipes

[0243] 33 Thermoelectric cooler

[0244] 34 Ventilation Fan

[0245] 35 Air Inlet

[0246] 36 Electrical plug

[0247] 37 Pump units for pumping PCR liquids

[0248] 100 Manufacturing Modules

[0249] One set of embodiments of this application relates to:

[0250] 1. An apparatus 1 for preparing DNA products via capillary polymerase chain reaction (PCR), comprising:

[0251] - Tube 6, used to guide the PCR liquid.

[0252] -First compartment 7, and

[0253] -At least the second compartment 8,

[0254] The tube 6 is bent at least from the first compartment 7 to the second compartment 8, and

[0255] The first compartment 7 and the second compartment 8 each include a heat medium inlet 12 and a heat medium outlet 13 to provide separate heat medium flows through the first compartment 7 and the second compartment 8 to regulate the individual temperatures in the first compartment 7 and the second compartment 8 for the preparation of DNA products based on PCR liquid.

[0256] 2. The apparatus 1 for preparing DNA products according to embodiment 1, wherein partition walls 14 are arranged between compartments to insulate the compartments from each other.

[0257] 3. The apparatus 1 for preparing DNA products according to the aforementioned embodiment, wherein the partition 14 includes a through hole for the tube 6 to extend from one compartment to the next compartment.

[0258] IV. The apparatus 1 for preparing DNA products according to one of the aforementioned embodiments, wherein a thermal medium inlet 12 and a thermal medium outlet 13 are arranged in at least one compartment, and preferably in each compartment, to provide a thermal medium flow through the compartment in a direction substantially opposite to the direction of PCR liquid in the tube 6.

[0259] V. The apparatus 1 for preparing DNA products according to one of the aforementioned embodiments, wherein the heat medium inlet 12 is arranged at a lower position in each compartment and the heat medium outlet 13 is arranged at a higher position in each compartment.

[0260] VI. The apparatus 1 for preparing DNA products according to one of the aforementioned embodiments further includes a third compartment 15, wherein the tube 6 is bent at least from the first compartment 7 to the second compartment 8 and from the second compartment 8 to the third compartment 15.

[0261] VII. The apparatus 1 for preparing DNA products according to the aforementioned embodiment, wherein the tube 6 is further bent from the third compartment 15 to the first compartment 7, from the first compartment 7 to the second compartment 8, and from the second compartment 8 to the third compartment 15.

[0262] 8. The apparatus 1 for preparing DNA products according to one of the aforementioned embodiments, wherein the tubes 6 that bend from compartment to compartment form a spiral stack of tube loops.

[0263] 9. The apparatus 1 for preparing DNA products according to one of the foregoing embodiments, wherein the tube 6 extends in a corrugated manner in at least one, and preferably in each compartment.

[0264] 10. The apparatus 1 for preparing DNA products according to embodiments 8 and 9, wherein the stacking direction of the spiral stack of tubes is different from and preferably substantially perpendicular to the corrugation direction of the corrugated tube (6).

[0265] XI. The apparatus 1 for preparing DNA products according to one of the aforementioned embodiments, wherein each compartment forms a basin to be filled with a heat medium.

[0266] 12. The apparatus 1 for preparing DNA products according to one of the aforementioned embodiments further includes at least one support 17 arranged in one of the compartments to provide a fixing point for the tube 6 and / or a deflection point for the corrugations of the tube 6.

[0267] 13. The apparatus 1 for preparing DNA products according to the aforementioned implementation scheme, wherein the tube 6 extends partially or completely or more than once around the outer dimensions of the scaffold 17.

[0268] XIV. The apparatus 1 for preparing DNA products according to one of embodiments XII to XIII, wherein the support 17 is replaceable and / or repositionable in the compartment.

[0269] 15. The apparatus 1 for preparing DNA products according to one of the aforementioned embodiments, wherein the tube 6 includes a PCR liquid inlet 20, which can be connected to a pump unit for pumping PCR liquid through the tube 6.

[0270] XVI. The apparatus 1 for preparing DNA products according to the foregoing embodiments, wherein the pump unit is arranged outside the compartment and preferably outside the housing 5 surrounding the compartment.

[0271] 17. The apparatus 1 for preparing DNA products according to embodiment 15 or 16, wherein the pump unit is configured to provide a flow rate of PCR liquid in the range of about 0.1 to about 10 mL / min, preferably about 0.2 to about 3 mL / min.

[0272] 18. The apparatus 1 for preparing DNA products according to one of the aforementioned embodiments, wherein the tube 6 includes a DNA product outlet 25 for continuous preparation of DNA products.

[0273] 19. The apparatus 1 for preparing DNA products according to embodiments 1 to 17, wherein the tube 6 includes a closable DNA product outlet 25 for discontinuous preparation of DNA products.

[0274] 20. The apparatus 1 for preparing DNA products according to one of the foregoing embodiments further includes at least one sensor 21 disposed in at least one compartment, wherein the sensor 21 is configured to detect the temperature, flow rate or leakage of the compartment.

[0275] 21. The apparatus 1 for preparing DNA products according to one of the aforementioned embodiments, wherein the diameter of the tube 6 is in the range of about 0.5 to about 1.5 mm, preferably about 0.75 to about 1.25 mm.

[0276] 22. The apparatus 1 for preparing DNA products according to one of the aforementioned embodiments, wherein the length between the tube inlet and the tube outlet of the tube 6 is in the range of about 10 to about 200 m, preferably about 25 to about 50 m.

[0277] 23. The apparatus 1 for preparing DNA products according to one of the aforementioned embodiments, wherein the tube 6 is designed to contain PCR liquid in the range of about 10 mL to about 1 L, preferably about 20 mL to about 250 mL.

[0278] 24. The apparatus 1 for preparing DNA products according to one of the aforementioned embodiments, wherein the helical stack of the tube coils comprises about 10 to about 50 coils, preferably about 15 to about 40.

[0279] 25. The apparatus 1 for preparing DNA products according to one of the foregoing embodiments, wherein the apparatus 1 for preparing DNA products is configured to prepare about 1 mg and more DNA products per reaction, preferably in the range of about 1 to about 30 mg, more preferably in the range of about 15 to about 25 mg.

[0280] 26. The apparatus 1 for preparing DNA products according to one of the foregoing embodiments, wherein the apparatus 1 for preparing DNA products is configured to prepare about 50 μg to about 150 μg of DNA product per mL of PCR liquid, preferably about 75 μg to about 125 μg per mL of PCR liquid.

[0281] 27. A pharmaceutical product manufacturing apparatus 10, comprising a processing chamber 2, a technical chamber 3, and an apparatus 1 for preparing DNA products by capillary polymerase chain reaction according to one of the foregoing embodiments.

[0282] 28. The pharmaceutical product manufacturing equipment 10 according to the aforementioned embodiment further includes a control unit configured to control the airflow through the processing chamber 2 as a gas spray.

[0283] 29. Manufacturing equipment 10 for pharmaceutical products according to one of the implementation schemes 27 to 28, wherein at least processing room 2, preferably manufacturing equipment 10 can be operated in accordance with the requirements of Annex 1 of the EU Good Manufacturing Practice (GMP) guidelines for pharmaceutical products.

[0284] 30. The pharmaceutical product manufacturing apparatus 10 according to one of embodiments 27 to 29 further includes a separation element 4 separating the processing chamber 2 from the technical chamber 3, wherein the separation element 4 includes an attachment 16 having an opening toward the processing chamber 2 and protruding toward the technical chamber 3, and wherein the apparatus 1 for preparing DNA products can be inserted into the attachment 16.

[0285] 31. The pharmaceutical product manufacturing equipment 10 according to the preceding embodiment, wherein when the equipment 1 for preparing the DNA product is inserted into the attachment 16, the front end of the equipment 1 for preparing the DNA product is substantially flush with the opening of the attachment 16 into the processing chamber 2.

[0286] 32. The manufacturing apparatus 10 for a pharmaceutical product according to one of embodiments 27 to 31, wherein at least one handle 22 for operating the apparatus 1 for preparing the DNA product into and / or out of the attachment 16 is positioned on the front of the apparatus 1 for preparing the DNA product.

[0287] 33. The pharmaceutical product manufacturing equipment 10 according to one of the embodiments 27 to 32, wherein the PCR liquid inlet 23 and / or the DNA product outlet 24 are located on the front of the equipment 1 for preparing the DNA product.

[0288] 34. The pharmaceutical product manufacturing equipment 10 according to one of embodiments 27 to 33, wherein the inlet and / or outlet of the heat medium are located at the back plate of the equipment 1 for preparing DNA products, which is opposite to the front of the equipment 1 for preparing DNA products.

[0289] 35. The manufacturing equipment 10 for the pharmaceutical product according to one of embodiments 27 to 34 further includes a sensor 21 (leakage sensor) disposed in an accessory 16 outside the equipment 1 for preparing the DNA product.

[0290] 36. The pharmaceutical product manufacturing equipment 10 according to one of embodiments 27 to 35, wherein Annex 16 includes an exchange rail 27 to guide the exchange between the device 1 for preparing DNA products and another device 1 for preparing DNA products.

[0291] 37. The pharmaceutical product manufacturing equipment 10 according to one of embodiments 27 to 36, wherein a pump unit for pumping PCR liquid through a tube 6 of the device 1 for preparing DNA products is arranged outside the housing of the pharmaceutical product manufacturing equipment 10.

[0292] 38. The pharmaceutical product manufacturing equipment 10 according to one of embodiments 27 to 37 further includes a coupling unit 18, said coupling unit 18 being disposed at the housing of the pharmaceutical product manufacturing equipment 10 and configured to couple the pharmaceutical product manufacturing equipment 10 to another manufacturing equipment 10.

[0293] 39. A manufacturing module 100 for a pharmaceutical product, comprising a manufacturing apparatus 10 for a pharmaceutical product according to one of embodiments 27 to 38 and at least one of the following: a manufacturing apparatus 10 having a process media supply unit and a mixing unit, a manufacturing apparatus 10 having a purification unit and a filtration unit.

[0294] 40. A method for preparing DNA products by capillary polymerase chain reaction (PCR), comprising:

[0295] - Provides a first compartment 7, a second compartment 8, and a pipe 6, wherein the pipe 6 is curved at least from the first compartment 7 to the second compartment 8.

[0296] - Provides individual hot medium flow through each compartment via hot medium inlet 12 and hot medium outlet 13 to regulate the individual temperature in each compartment, and

[0297] - Guide the PCR liquid through tube 6, thereby passing through the individual temperature in each compartment to prepare DNA products based on the PCR liquid.

[0298] 41. The method according to the aforementioned embodiment, wherein the first temperature in the first compartment 7 is in the range of about 85°C to about 105°C, preferably about 98°C.

[0299] 42. The method according to one of embodiments 40 to 41, wherein the second temperature in the second compartment 8 is in the range of about 45°C to about 72°C, preferably about 60°C to about 72°C.

[0300] 43. The method according to one of embodiments 40 to 42 further includes providing a third compartment 15, wherein the tube 6 is bent at least from the second compartment 8 to the third compartment 15, and wherein the third temperature in the third compartment 15 is in the range of about 65°C to about 75°C, preferably about 72°C.

[0301] 44. The method according to one of embodiments 40 to 43, wherein a first temperature in the first compartment 7 is configured for a denaturation step of the polymerase chain reaction, and a second temperature in the second compartment 8 is configured for an annealing step of the polymerase chain reaction and optionally also for an extension step of the polymerase chain reaction.

[0302] 45. The method according to embodiment 43, wherein a third temperature in the third compartment 15 is configured for the extension step of the polymerase chain reaction.

[0303] 46. ​​The use of the equipment described in one of the embodiments 1 to 26 for the preparation of DNA products by capillary polymerase chain reaction.

[0304] 47. The use of the manufacturing equipment 10 described in one of the implementation schemes 27 to 38 for the production of pharmaceutical products.

[0305] A preferred embodiment of this application relates to:

[0306] 1. An apparatus 1 for amplifying DNA by polymerase chain reaction (PCR), comprising:

[0307] - Tube 6, used to guide the PCR liquid.

[0308] -First compartment 7,

[0309] -Second compartment 8, and

[0310] - Third compartment 15,

[0311] The tube 6 bends through the first compartment 7 and from the first compartment 7 through the second compartment 8, from the second compartment 8 through the third compartment 15, and from the third compartment 15 back to the first compartment 7. The bends of the tube 6 from one compartment to another form a spiral stack of tube loops, and each tube loop represents one PCR cycle. The tube 6 includes a PCR liquid inlet 20 and a DNA product outlet 25.

[0312] The first compartment 7, the second compartment 8, and the third compartment 15 each include means for regulating the temperature in the compartment to prepare DNA products based on PCR liquid, wherein the first compartment is configured to provide a temperature for denaturation, the second compartment 8 is configured to provide a temperature for annealing, and the third compartment 15 is configured to provide a temperature for extension.

[0313] 2. The device 1 for amplifying DNA according to Implementation Scheme 1, wherein the PCR liquid inlet 20 can be connected to a pump unit for pumping PCR liquid through the tube 6.

[0314] 3. The device 1 for amplifying DNA according to embodiment 1 or 2, wherein partition walls 14 are arranged between compartments to insulate the compartments from each other, wherein partition walls 14 include through holes for tubes 6 to extend from one compartment to the next compartment.

[0315] IV. The apparatus 1 for amplifying DNA according to any one of embodiments 1 to 3, wherein each compartment includes a heating unit 31 and an optional cooling unit as a means of regulating the temperature in each compartment.

[0316] V. The apparatus 1 for amplifying DNA according to any one of embodiments 1 to 3, wherein each compartment includes a heat medium inlet 12 and a heat medium outlet 13 as means for regulating the temperature in each compartment, i.e., providing separate heat medium flows through the first compartment 7, the second compartment 8 and the third compartment 15 to regulate the individual temperatures in the first compartment 7, the second compartment 8 and the third compartment 15.

[0317] VI. The apparatus 1 for amplifying DNA according to any one of the foregoing embodiments, wherein the tube 6 extends in a corrugated manner in each compartment.

[0318] 7. The apparatus 1 for amplifying DNA according to any one of the foregoing embodiments, wherein each compartment forms a basin to be filled with a thermal medium or thermal solid.

[0319] 8. The device 1 for amplifying DNA according to any one of the foregoing embodiments further includes at least one support 17 arranged in one of the compartments to provide a fixing point for the tube 6 and / or a deflection point for the corrugations of the tube 6.

[0320] 9. The device 1 for amplifying DNA according to embodiment 8, wherein the support 17 is replaceable and / or repositionable in the compartment.

[0321] 10. The device 1 for amplifying DNA according to any one of the foregoing embodiments, wherein the diameter of the tube 6 is in the range of about 0.5 mm to about 50 mm, preferably about 0.5 mm to about 1.5 mm, more preferably about 0.75 mm to about 1.25 mm.

[0322] XI. The apparatus 1 for amplifying DNA according to any one of the foregoing embodiments, wherein the length of the tube 6 between the tube inlet and the tube outlet is in the range of about 10 m to about 200 m, preferably about 50 m to about 100 m.

[0323] 12. The apparatus 1 for amplifying DNA according to any one of the foregoing embodiments, wherein the tube 6 is designed to contain PCR liquid in the range of about 10 mL to about 500 mL, preferably about 20 mL to about 100 mL.

[0324] 13. The apparatus 1 for amplifying DNA according to any one of the foregoing embodiments, wherein the helical stack of the tube coils comprises about 10 to about 50 coils, preferably about 15 to about 40.

[0325] XIV. A manufacturing apparatus 10, comprising a device 1 for amplifying DNA according to any one of embodiments 1 to 13.

[0326] 15. The manufacturing equipment 10 according to embodiment 14 further includes a processing chamber 2 and a technology chamber 3.

[0327] XVI. The manufacturing apparatus 10 according to embodiment XV further includes a control unit configured to control the airflow through the processing chamber 2 as a gas spray.

[0328] 17. The manufacturing apparatus 10 according to embodiment 15 or 16 further includes a separation element 4 that separates the processing chamber 2 from the technical chamber 3, wherein the separation element 4 includes an attachment 16 having an opening toward the processing chamber 2 and protruding toward the technical chamber 3, and wherein the device 1 for amplifying DNA can be inserted into the attachment 16.

[0329] Eighteen, the manufacturing equipment 10 according to the seventeenth embodiment, wherein when the device 1 for preparing DNA products is inserted into the attachment 16, the front end of the device 1 for preparing DNA products is substantially flush with the opening of the attachment 16 into the processing chamber 2.

[0330] 19. A manufacturing module 100, comprising a manufacturing apparatus 10 according to any one of embodiments 14 to 18 and at least one manufacturing apparatus selected from the group consisting of: a manufacturing apparatus 10 including a process media supply unit and a mixing unit, a manufacturing apparatus 10 including a purification unit, and a manufacturing apparatus 10 including a filtration unit.

[0331] 20. A method for amplifying DNA by polymerase chain reaction (PCR), comprising:

[0332] - Provides a first compartment 7, a second compartment 8, a third compartment 15, and a tube 6, wherein the tube 6 bends through the first compartment 7 and from the first compartment 7 to and through the second compartment 8, and from the second compartment 8 to and through the third compartment 15, and from the third compartment 15 to the first compartment 7, wherein the tube 6 bending from compartment to compartment forms a spiral stack of tube loops.

[0333] - Provides a device for regulating the individual temperature in each compartment, and

[0334] - Guide the PCR liquid through tube 6, thereby passing through the individual temperature in each compartment to prepare DNA products based on the PCR liquid.

[0335] 21. The method according to embodiment 20, wherein the temperature in the first compartment 7 is used for denaturation, preferably in the range of about 85°C to about 105°C, and more preferably about 98°C.

[0336] 22. According to the method of embodiment 20 or 21, the temperature in the second compartment 8 is used for annealing, preferably in the range of about 45°C to about 72°C, more preferably about 60°C to about 72°C.

[0337] 23. The method according to any one of embodiments 20 to 22, wherein the temperature in the third compartment 15 is used for extension, preferably in the range of about 65°C to about 75°C, preferably about 72°C.

[0338] 24. The use of the apparatus 1 according to any one of embodiments 1 to 13 for preparing DNA products by polymerase chain reaction.

[0339] 25. The manufacturing equipment 10 according to any one of embodiments 14 to 18 or the manufacturing module 100 according to embodiment 19 is used for the production of pharmaceutical products.

Claims

1. An apparatus (1) for amplifying DNA by polymerase chain reaction, comprising: - Tube (6) for guiding PCR liquid, - First compartment (7), - Second compartment (8), and - Third compartment (15), The tube (6) bends through the first compartment (7) and from the first compartment (7) to and through the second compartment (8), from the second compartment (8) to and through the third compartment (15), and from the third compartment (15) back to the first compartment (7), wherein the tube (6) bends from one compartment to another to form a spiral stack of tube loops and each tube loop represents one PCR cycle, wherein the tube (6) includes a PCR liquid inlet (20) and a DNA product outlet (25), and the length of the tube (6) between the inlet and outlet is between 10 m and 200 m. The first compartment (7), the second compartment (8), and the third compartment (15) each include means for regulating the temperature within the compartment to prepare the DNA product based on the PCR liquid, wherein the first compartment is configured to provide a denaturation temperature, the second compartment (8) is configured to provide an annealing temperature, and the third compartment (15) is configured to provide an extension temperature. A partition (14) is arranged between the compartments to insulate the compartments from each other, wherein the partition (14) includes a through hole for the tube (6) to extend from one compartment to the next compartment.

2. The device (1) according to claim 1, wherein the PCR liquid inlet (20) is connectable to a pump unit for pumping the PCR liquid through the tube (6).

3. The device (1) according to claim 2, Each compartment includes a heating unit (31) and optional cooling units (32, 33) as means for regulating the temperature in each compartment; or Each compartment includes a heat medium inlet (12) and a heat medium outlet (13) as a means of regulating the temperature in each compartment, i.e., providing separate heat medium flows through the first compartment (7), the second compartment (8), and the third compartment (15) to regulate the individual temperatures in the first compartment (7), the second compartment (8), and the third compartment (15).

4. The device (1) according to claim 1, wherein the tube (6) extends in a corrugated manner in each compartment.

5. The device (1) according to claim 1, wherein each compartment forms a basin to be filled with a heat medium or a heat solid.

6. The device (1) according to claim 1 further includes at least one bracket (17) arranged in one of the compartments to provide a fixing point for the tube (6) and / or a deflection point for the corrugations of the tube (6).

7. The device (1) according to claim 6, wherein the bracket (17) is replaceable and / or repositionable in the compartment.

8. The device (1) according to claim 1, wherein the diameter of the tube (6) is in the range of 0.5 mm to 50 mm; and / or The length of the pipe (6) between the pipe inlet and the pipe outlet is in the range of 50 m to 100 m; and / or The tube (6) is designed to contain PCR liquid in the range of 10 mL to 500 mL.

9. The device (1) according to claim 8, wherein the diameter of the tube (6) is in the range of 0.5 mm to 1.5 mm.

10. The device (1) according to claim 8, wherein the diameter of the tube (6) is in the range of 0.75 mm to 1.25 mm.

11. The device (1) according to claim 8, wherein the tube (6) is sized to contain the PCR liquid in the range of 20 mL to 100 mL.

12. The device (1) according to any one of claims 1 to 11, wherein the spiral stack of the tube windings comprises 10 to 50 windings.

13. The device (1) according to any one of claims 1 to 11, wherein the spiral stack of the tube coils comprises 15 to 40 coils.

14. A manufacturing apparatus (10) comprising an apparatus (1) for amplifying DNA by polymerase chain reaction according to any one of claims 1 to 13, wherein the manufacturing apparatus (10) further comprises a processing chamber (2), a technology chamber (3), and a control unit configured to control an airflow through the processing chamber (2) as a gas spray.

15. The manufacturing apparatus (10) according to claim 14, further comprising a separation element (4) separating the processing chamber (2) from the technical chamber (3), wherein the separation element (4) includes an attachment (16) having an opening toward the processing chamber (2) and protruding in the direction of the technical chamber (3), and wherein the apparatus (1) for amplifying DNA by polymerase chain reaction is insertable into the attachment (16).

16. A method for amplifying DNA by polymerase chain reaction, comprising: - Provides a first compartment (7), a second compartment (8), a third compartment (15), and a tube (6), wherein the tube (6) bends through the first compartment (7) and from the first compartment (7) to and through the second compartment (8), and from the second compartment (8) to and through the third compartment (15), and from the third compartment (15) to the first compartment (7), wherein the tube (6) bends from one compartment to another to form a spiral stack of tube loops, the length of the tube (6) between the tube inlet and the tube outlet is between 10 m and 200 m, and wherein partition walls (14) are arranged between the compartments to insulate the compartments from each other, wherein the partition walls (14) include through holes for the tube (6) to extend from one compartment to the next compartment; - Provides a device for regulating the individual temperature in each compartment; and - The PCR liquid is guided through the tube (6) to pass through the individual temperatures in each compartment to prepare DNA products based on the PCR liquid. The temperature in the first compartment (7) is used for denaturation, the temperature in the second compartment (8) is used for annealing, and the temperature in the third compartment (15) is used for stretching.

17. The method according to claim 16, wherein the temperature in the first compartment (7) is in the range of 85°C to 105°C; and / or The temperature in the second compartment (8) is in the range of 45°C to 72°C; and / or The temperature in the third compartment (15) is in the range of 65°C to 75°C.

18. The method of claim 17, wherein The temperature in the first compartment (7) is 98°C; and / or The temperature in the second compartment (8) is in the range of 60°C to 72°C; and / or The temperature in the third compartment (15) is 72°C.

19. Use of the apparatus (1) according to any one of claims 1 to 13 for preparing DNA products by polymerase chain reaction.

20. Use of the manufacturing equipment (10) according to claim 14 or 15 for the production of pharmaceutical products.

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