System and treatment vessel for treating biochemical products by means of magnetic particles

Abstract

The utility model relates to a system and a treatment vessel for treating biochemical products by means of magnetic particles. The system comprises: one or more processing containers; one or more mixing stations, the one or more mixing stations configured to receive the one or more processing containers, and the one or more mixing stations comprising a motor unit configured to drive the one or more processing containers in mechanical motion; one or more magnetic separation stations, the one or more magnetic separation stations comprising one or more magnets, where the one or more treatment containers comprise a wall structure defining a central cavity, where the wall structure comprises an outer wall and an inner wall thereby defining a treatment space between the outer wall and the inner wall, and where the one or more magnetic separation stations comprise one or more magnets, where the one or more magnetic separation stations comprise one or more magnets, where the one or more magnetic separation stations comprise one or more magnets. The wall structure is configured to receive a liquid sample and hold the liquid sample in the interior space.

Description

Technical Field

[0001] The utility model relates to a device and a method for performing a purification step during compound production or purification of molecules such as nucleic acids. More specifically, a device and a method allowing purification by means of magnetic particles are disclosed. Background Art

[0002] The manufacturing methods of biomolecules (e.g. nucleic acids) currently established implement many separate manufacturing steps. In particular, the corresponding manufacturing steps are performed by several different devices. In addition, in the GMP regulated laboratories performed by well-trained technicians, the manufacture of nucleic acids requires a large amount of manual processing. Therefore, the manufacturing process currently established is time-consuming, cost-intensive, and requires a large amount of laboratory space and laboratory equipment.

[0003] US2021 / 0261897 describes a bioreactor for in vitro transcription. However, the system focuses primarily on the biochemical reaction steps for performing RNA production chains and does not provide detailed methods or equipment for purifying the resulting products.

[0004] Magnetic bead separation is a technique used to purify compounds, usually in the field of testing or in the early stages of research, usually used at a laboratory scale. Typically, magnetic particles are linked to a substance that shows affinity for the compound that needs to be purified. By applying a magnetic field, the magnetic particles with the bound compound of interest are attracted to the magnet, allowing any remaining liquid to be removed. Thus, the compound of interest can be separated and / or purified from its liquid environment.

[0005] Magnetic bead purification methods are frequently used to produce biomolecules such as proteins, peptides, antibodies or nucleic acids.

[0006] EP1621890 describes a purification device using magnetic particles. However, it is limited by the use of vessels that can only accommodate small volumes of elution.

[0007] In WO2016123101, magnetic silica nanomembranes are used for nucleic acid extraction. Similar to EP1621890, this method is also constrained to process small volumes.

[0008] Large-scale systems are needed to produce biomolecules such as nucleotides and downstream purification systems are needed. Preferably, such systems are fully automated or semi-automated in order to increase the efficiency of the production process and mitigate human handling errors. Utility Model Content

[0009] One aspect of the utility model relates to a system for processing biochemical products with the aid of magnetic particles, the system comprising: one or more processing containers; one or more mixing stations, the one or more mixing stations being configured to receive the one or more processing containers, and the one or more mixing stations comprising a motor unit, the motor unit being configured to drive the mechanical movement of the one or more processing containers; one or more magnetic separation stations, the one or more magnetic separation stations comprising one or more magnets, wherein the one or more processing containers comprise a wall structure defining a central cavity, wherein the wall structure comprises an outer wall and an inner wall, thereby defining a processing space between the outer wall and the inner wall, and wherein the wall structure is configured to receive a liquid sample and retain the liquid sample in the internal space.

[0010] The distance between the outer wall and the inner wall of the one or more processing containers is less than 80 mm, preferably between 3 mm and 75 mm.

[0011] The one or more processing containers include a bottom wall, wherein the bottom wall has an inclination that causes a level difference of liquid present in the inner space of the one or more processing containers.

[0012] The one or more mixing stations and / or the one or more magnetic separation stations include a raised portion configured to receive the central cavity of the one or more processing vessels.

[0013] The raised portion of the one or more magnetic separation stations includes one or more magnets lining the raised portion.

[0014] The one or more magnetic separation stations include a base including one or more magnets positioned at the base.

[0015] The system further comprises a computer processor, wherein the computer processor comprises instructions for controlling at least one processing device and instructions for calculating and controlling the magnetization time of the magnetic particles binding to the biochemical product and the one or more magnets.

[0016] The system includes one or more robotic arms configured to grip the one or more processing containers and transfer the one or more processing containers between the one or more mixing stations and the one or more magnetic separation stations.

[0017] The system includes a pooling vessel for pooling at least a portion of a plurality of biochemical product samples, preferably generated by a plurality of in vitro transcription reactions, each reaction resulting in a biochemical product sample.

[0018] The system is configured for processing a liquid sample comprising one or more biochemical products, preferably nucleic acid molecules, more preferably RNA or DNA.

[0019] The system is coupled to an upstream production system for producing the biochemical product sample.

[0020] The upstream production system includes a plurality of reaction chambers for producing the biochemical product in a plurality of reactions.

[0021] Another aspect of the present invention relates to a processing container, which includes a central cavity lined with a wall structure, wherein the wall structure includes an outer wall and an inner wall, thereby defining an internal space between the inner wall and the outer wall, and wherein the wall structure is open at the top of the container to allow components and / or liquids to be added to the internal volume. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The following description of the drawings of the specific embodiments of the present invention is only exemplary in nature and is not intended to limit the present teaching, its application or use. In the entire drawings, corresponding reference numerals indicate the same or corresponding parts and features.

[0023] Figure 1 A top view of a processing or purification vessel according to an embodiment of the present invention is shown.

[0024] Figure 2 A bottom view of a processing or purification vessel according to an embodiment of the present invention is shown.

[0025] Figure 3 A cross section of a processing or purification vessel according to an embodiment of the present invention is shown.

[0026] Figure 4A The mixing station is depicted, and Figure 4B Depicted is the fitting of a processing or purification vessel onto a raised portion of a mixing station according to an embodiment of the invention.

[0027] Figure 5A The magnetic separation station is depicted, and Figure 5B Depicted is the raised portion for fitting a processing or purification vessel to a magnetic separation station.

[0028] Figure 6 Depicted are two magnetic separation stations for use with a processing or purification apparatus according to an embodiment of the invention.

[0029] Figure 7 A system for producing and purifying nucleic acids according to an embodiment of the present invention is depicted.

[0030] Fig. 8A A support frame for holding an adapter according to an embodiment of the present invention is depicted, and Figure 8B A representation of a robotic arm grasping an adapter is shown.

[0031] Fig.9A A processing or purification container having a nested arrangement structure according to an embodiment of the present invention is depicted, and Fig. 9B The magnetic separation station is depicted.

[0032] FIG. 10A to FIG. 10C Embodiments of purification vessels according to embodiments of the present invention are depicted.

[0033] Fig.11 The arrangement structure on the magnet on the magnetic separation station according to the embodiment of the present invention is depicted. DETAILED DESCRIPTION

[0034] This article describes systems and methods for producing and / or purifying cell-free synthetic biochemical products (such as nucleic acids). The methods and systems provide large-scale (semi-continuous or continuous) production of these products and are intended to address at least some of the above-mentioned problems and disadvantages.

[0035] definition

[0036] Unless otherwise defined, all terms (including technical and scientific terms) used to disclose the present invention have the meanings commonly understood by ordinary technicians in the field to which the present invention belongs. By means of further guidance, term definitions are included to better understand the teachings of the present invention.

[0037] As used herein, the following terms have the following meanings:

[0038] As used herein, "a", "an", and "the" refer to both the singular and the plural, unless the context clearly indicates otherwise. As an example, "a compartment" refers to one or more than one compartment.

[0039] As used herein, "about" refers to a measurable value, such as a parameter, amount, duration, etc., and is intended to cover variations of + / -20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less, of or from the specified value, until such variations are suitable for implementation in the disclosed utility model. However, it should be understood that the value referred to by the modifier "about" itself is also specifically disclosed.

[0040] As used herein, "comprise," "comprising," and "comprises," and "comprised of" are synonymous with "include," "including," "includes," or "contain," "containing," "contains," and are inclusive or open-ended terms that specify the presence of the following (e.g., components) and do not exclude or preclude the presence of additional, unrecited components, features, elements, members, steps known in the art or disclosed herein.

[0041] In addition, unless otherwise specified, the terms first, second, third, etc. in the specification and claims are used to distinguish between similar elements and are not necessarily used to describe a sequential or chronological order. It should be understood that the terms used in this way are interchangeable under appropriate circumstances, and the embodiments of the utility model described herein can be operated in a different order than described or illustrated herein.

[0042] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0043] Unless defined otherwise, the expressions "weight %", "weight percent", "% wt" or "wt %" herein and throughout the specification refer to the relative weight of the corresponding component based on the total weight of the preparation.

[0044] While the term "one or more" or "at least one" (such as one or more members or at least one member of a group of members) is itself clear, by way of further example, the term particularly covers reference to any one of the members, or reference to any two or more of the members, such as any ≥3, ≥4, ≥5, ≥6 or ≥7 of the members and up to all of the members.

[0045] The terms "purification vessel" and "processing vessel" are used interchangeably herein.

[0046] As used herein, the term "magnetic particles" and variations thereof are intended to represent particles with magnetism, such as paramagnetic cores or superparamagnetic cores, which are coated with at least one material having a surface to which a biochemical product can be reversibly bound. Suitable magnetic particles may include, for example, carboxyl-coated paramagnetic particles, silica-based paramagnetic particles, etc. In embodiments described herein, the magnetic particles are silica-based magnetic particles, wherein, in some embodiments, the silica-based particles may include a paramagnetic core coated with siliceous oxide, thereby providing a hydrous siliceous oxide adsorption surface (e.g., a surface comprising silanol groups) to which nucleic acids can be bound. In other or additional embodiments, the magnetic particles may be coated with a ligand that interacts with a biochemical product, or may be surface-modified to produce a functionalized surface, such as a weak or strong positive charge, a weak or strong negative charge, or a hydrophobic surface (to name a few). In other embodiments, the magnetic particles are coated with a surface that interacts with a protein. Non-limiting examples of such magnetic particles include Dynabeads Protein A / G coated with a mixture of Protein A and Protein G, MagneHis-Ni particles coated with a nickel chelating resin, M-280 Streptavidin Dynabeads, MyOne T1 Streptavidin C1 Dynabeads, or PureProteome TM Protein A magnetic beads, which can bind to the Fc region of immunoglobulins

[0047] The terms "cell-free synthetic biochemical" or "biochemical", used interchangeably herein, refer to any biochemical that is produced, synthesized, expressed, transcribed, or translated outside of a living cell. Non-limiting examples of cell-free synthetic bioproducts include any nucleic acid, such as DNA or RNA, including any known class of RNA, peptides, polypeptides, or proteins, such as enzymes, antibodies, antigens, or vaccines. The process of producing cell-free biochemicals replicates the natural synthetic mechanism without the limitations of living cell membranes.

[0048] Unless otherwise defined, all terms (including technical and scientific terms) used to disclose the present invention have the meanings commonly understood by ordinary technicians in the field to which the present invention belongs. With the help of further guidance, the definitions of the terms used in the description are included to better understand the teachings of the present invention. The terms or definitions used herein are only used to help understand the present invention.

[0049] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present invention. Therefore, the phrases "in an embodiment" or "in an embodiment" appearing in various places throughout this specification do not necessarily all refer to the same embodiment, but may refer to the same embodiment. In addition, in one or more embodiments, particular features, structures, or characteristics may be combined in any suitable manner, which will be apparent to those skilled in the art. In addition, while some embodiments described herein include some but not other features included in other embodiments, the combination of features of different embodiments should be within the scope of the present invention and form different embodiments, as understood by those skilled in the art. For example, in the appended claims, any claimed embodiment may be used in any combination.

[0050] method

[0051] Methods for purifying cell-free synthesized biochemical products

[0052] In the first aspect, a method for processing or purifying a liquid sample is described herein, wherein the liquid sample includes one or more cell-free synthetic biochemical products. The processing or purification includes: combining at least a portion of the liquid sample with a plurality of magnetic particles to form a reactant mixture in a processing or purification container, wherein the plurality of magnetic particles can be bound to at least a portion of the biochemical product in the liquid sample. A magnetic source is applied to a processing container, and the biochemical product bound to the magnetic particles is separated from the unbound portion of the liquid sample. At least a portion of the unbound portion is removed, and a plurality of magnetic particles bound to the biochemical product are maintained. Optionally, the maintained magnetic particles are washed with a washing buffer. The processing container includes at least an inner wall and an outer wall to form a processing space, and wherein the distance between the inner wall and the outer wall is 80 mm or less.

[0053] The method is (semi) automated and allows for continuous or discontinuous purification of cell-free synthetic biochemical products. Typically, when magnetic particles are used for purification purposes, these magnetic particles are present in a tube or vessel carrying a large amount of magnetic particles. Applicants have found that by using a thin layer of magnetic particles in the purification vessel, the process combined with the magnetic particles is more effective and obtains high-quality purified biochemical products in high yield.

[0054] In an embodiment, the magnetic particles are present in the purification vessel as a liquid layer having a layer thickness of less than 100 mm, 95 mm, 90 mm, 85 mm, 80 mm, 75 mm, 70 mm, 65 mm, 60 mm, 55 mm, 50 mm, 45 mm, 40 mm, 35 mm, 30 mm, 25 mm, 20 mm, 15 mm, 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm or 1 mm.

[0055] In another embodiment, the layer thickness is between 3 mm and 75 mm, between 4 mm and 75 mm, between 5 mm and 75 mm, between 6 mm and 75 mm, between 7 mm and 75 mm, between 8 mm and 75 mm, between 9 mm and 75 mm, between 10 mm and 75 mm, between 15 mm and 75 mm, between 20 mm and 75 mm, between 25 mm and 75 mm, between 30 mm and 75 mm, between 35 mm and 75 mm, between 40 mm and 75 mm, between 45 mm and 75 mm, between 50 mm and 75 mm, between 55 mm and 75 mm, between 60 mm and 75 mm or between 65 mm and 75 mm, and all ranges and sub-ranges therebetween.

[0056] Alternatively, the layer thickness is between 3 mm and 65 mm, between 3 mm and 60 mm, between 3 mm and 55 mm, between 3 mm and 50 mm, between 3 mm and 45 mm, between 3 mm and 40 mm, between 3 mm and 35 mm, between 3 mm and 30 mm, between 3 mm and 25 mm, between 3 mm and 20 mm, between 3 mm and 15 mm, between 3 mm and 10 mm, between 3 mm and 9 mm, between 3 mm and 8 mm, between 3 mm and 7 mm, between 3 mm and 6 mm, between 3 mm and 4 mm, and all ranges and sub-ranges therebetween.

[0057] In an embodiment, the liquid layer is formed between the outer wall and the inner wall of the purification container. In an embodiment, such a purification container comprises a hollow central cavity lined with an inner wall. An internal volume is formed between the inner wall and the outer wall, and the internal volume is open at the top of the container to allow the addition of components and / or liquids in the internal volume. The internal volume of the purification container is configured to receive a liquid including a biochemical product and / or magnetic particles. The hollow central cavity is configured to fit on a raised portion of a mixing station and / or a magnetic separation station.

[0058] In some embodiments, a single processing space is formed between the inner wall and the outer wall of the processing or purification container. In other embodiments, multiple processing spaces can be formed between the inner wall and the outer wall of the processing or purification container. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 processing spaces are formed between the inner wall and the outer wall of the processing or purification container. In such an embodiment, multiple processing spaces are formed by subdividing the interior space between the inner wall and the outer wall with additional vertical walls.

[0059] In an embodiment, a certain amount of magnetic particles is added or present between the outer wall and the inner wall of the purification container. In some embodiments, a liquid sample is added or present between the outer wall and the inner wall of the purification container. Alternatively, the purification container includes magnetic particles and the liquid sample is provided to the purification container. In another alternative embodiment, the purification container includes a liquid sample and magnetic particles are provided to the container. In another alternative embodiment, the purification container includes magnetic particles, and additional magnetic particles and / or liquid sample are added to the container.

[0060] The biochemical product in the purification container is allowed to bind to the magnetic particles, after which the unbound liquid portion of the sample is removed and, optionally, the magnetic particles bound to the biochemical product are washed with a washing buffer.

[0061] The step comprising sample-magnetic particle binding and continuous magnetic separation is hereinafter referred to as the "binding step". In some embodiments of the method as disclosed herein, the binding step is repeated multiple times in succession before proceeding to the next step. Therefore, in an embodiment, the binding step of the present method can be repeated once or more times, preferably 1 to 20 times, more preferably 1 to 10 times, before washing. In an embodiment, the binding step is repeated continuously at most 20 times, at most 15 times, at most 10 times, at most 9 times, at most 8 times, at most 7 times, at most 6 times, at most 5 times, at most 4 times, at most 3 times, at most 2 times, preferably between 5 and 15 times.

[0062] Surprisingly observed, before washing and / or eluting, repeating multiple binding steps, thus omitting several rounds of washing and / or eluting in between, increased the yield of purified biochemical products, reducing the amount of buffer required in the purification process, which reflects the use of harmful agents (such as guanidine thiocyanate) and solvents (such as ethanol) commonly used in a large number of practices. Reduced. The method also allows the reuse of magnetic particles and reduces the consumption of magnetic particles overall. The method disclosed herein uses a certain concentration of the molecule of interest on the magnetic particles, making the purification technology safe, sustainable and economical.

[0063] In embodiments, cumulative repeated binding steps can reduce total alcohol consumption per unit nucleic acid by 95%, 90%, 85%, 80%, 75%, 70%, 60%, 40%, 30%, 20%, 10%, 5% compared to methods using a single binding step. In embodiments, the alcohol can be ethanol.

[0064] In embodiments, when continuous binding steps are applied, the reduced use of alcohol can be due to the reduction of alcohol (such as ethanol) used in the wash steps of the disclosed methods. In embodiments, cumulative repeated binding steps can reduce total alcohol consumption per unit nucleic acid by up to 95%, 90%, 85%, 80%, 75%, 70%, 60%, 40%, 30%, 20%, 10%, 5% compared to methods applying a single binding step. In embodiments, the alcohol can be ethanol.

[0065] In an embodiment, these continuous binding steps can be performed by continuously adding magnetic particles and biochemical products at each cycle (hereinafter referred to as the first option) or by placing all magnetic particles in a purification container in one step and continuously placing biochemical products at each cycle (hereinafter referred to as the second option). When the second option is selected, the magnetic particles added in one step should have sufficient concentration to provide enough binding energy for the total amount of biochemical products added in subsequent continuous cycles. It is obvious to those skilled in the art that the ratio of the concentration of magnetic particles to biochemical products in the first cycle of the continuous binding step is higher in the second option than in the first option. In an embodiment, the concentration of magnetic particles added in the first cycle of continuous binding repetition is 20 times higher in the second option than in the first option, for example, for purifying the same amount of biochemical products, in the second option, it is 15 times higher, 10 times higher, 9 times higher, 8 times higher, 7 times higher, 6 times higher, 5 times higher, 4 times higher, 3 times higher, 2 times higher than in the first option.

[0066] In an alternative embodiment, each binding step is followed by successive washing steps.

[0067] In some embodiments of the method, the biochemical product is bound to the magnetic particles, and then the magnetic particles are captured or induced to move toward a magnet present near the purification container, thereby causing the separation of the biochemical product bound to the magnetic particles and any remaining sample liquid. It should be understood that the separation of the magnetic particles from the solution also requires physical separation, such as removing the supernatant from the modified magnetic particles.

[0068] In some embodiments, the binding buffer is used to mediate the reversible binding between the compound of interest and the magnetic particles. In another embodiment, the binding buffer may include a chaotropic agent, an alcohol, PEG, a salt, or a mixture thereof. The chaotropic agent may be selected from: guanidine salts, such as hydrochloride (Gu HCl) and guanidine thiocyanate (Gu SCN); lithium salts, such as lithium acetate and lithium perchlorate; or sodium salts, such as NaCl; and combinations thereof. In an embodiment, the binding buffer does not contain a chaotropic agent.

[0069] The alcohol may be selected from isopropanol, ethanol, methanol, butanol and combinations thereof. In some embodiments, at least one alcohol may be an ethanol more suitable for the purpose of preparing a medicament than the conventional isopropanol taught in the prior art. In an embodiment, the alcohol is present in a concentration of 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 15% to 20% v / v, including all ranges and sub-ranges therebetween.

[0070] In embodiments, the binding buffer may include PEG as an alternative to or in combination with an alcohol. The concentration of PEG in the binding buffer may range from 10% to 40%, 20% to 40%, 20% to 35%, 20% to 30%, or 25% to 35%, including all ranges and subranges therebetween. In embodiments, 30% PEG is used. In embodiments, the PEG used in the binding buffer is selected from PEG 600, PEG 1000, PEG 2000, PEG 3000, PEG 4000, PEG 6000, PEG 8000, PEG 10.000, and PEG 20.000. In embodiments, the PEG used is PEG 8000.

[0071] At least one salt can be present in the binding buffer at a concentration of 0.1M to 5M (e.g., 0.1M to 4M, 0.1M to 3M, 0.1M to 2M, 0.1M to 1M, 0.5M to 1M, 0.5M to 2M, 1M to 2M, 2M to 3M, and 3M to 5M), including all ranges and subranges therebetween. According to various embodiments, the at least one salt can be sodium chloride (NaCl).

[0072] In embodiments, the binding buffer comprises Tris-HCl, NaCl, EDTA, and ethanol. In other embodiments, the binding buffer does not include alcohol, but only comprises Tris-HCl, NaCl, and EDTA. Non-limiting examples of binding buffer compositions include: 40 mM Tris-HCl, 4.8 M NaCl, 4 mM EDTA at a pH of 7.4.

[0073] In embodiments, the binding buffer may have a pH of 5 to 10 (e.g., 5 to 9, 5.5 to 8.5, 6 to 8, or 6.4 to 7.5, and all ranges and subranges therebetween). In embodiments, the binding buffer comprises at least one alcohol and / or PEG, at least one salt, and at least one optional chelating agent (such as EDTA).

[0074] In some embodiments, the binding buffer is free of toxic chaotropic agents such as guanidine salts (guanidine thiocyanate or guanidine thiocyanate), iodide, perchlorate, and trichloroacetate (preferably guanidine salts).

[0075] The magnetic particles can be present in the binding buffer at a concentration of, for example, about 0.1 μg / μl to about 60 μg / μl (e.g., 0.5 μg / μl to about 60 μg / μl, about 0.75 μg / μl to about 55 μg / μl, about 1 μg / μl to about 50 μg / μl, about 2 μg / μl to about 45 μg / μl, about 3 μg / μl to about 40 μg / μl, about 4 μg / μl to about 35 μg / μl, about 5 μg / μl to about 30 μg / μl, about 6 μg / μl to about 25 μg / μl, about 7 μg / μl to about 20 μg / μl, about 8 μg / μl to about 15 μg / μl, or about 9 μg / μl to about 10 μg / μl, including all ranges and sub-ranges therebetween). As a non-limiting embodiment, at least one magnetic particle can be selected from Qbead and can be present in the binding buffer B1 at a concentration of about 0.5 μg / μl to about 5 μg / μl. In an alternative embodiment, at least one magnetic particle can be selected from Grace beads and can be present in the binding buffer B1 at a concentration of about 2 μg / μl to about 60 μg / μl. In another alternative embodiment, the magnetic particles can be selected from GreenMat beads, and the concentration range is 150 mg / ml to 300 mg / ml, preferably 225 mg / ml.

[0076] In specific embodiments, such as in cases where it is preferred to repeat the binding step, additional magnetic particles may be added to the binding buffer. The additional addition of magnetic beads will increase the final concentration of magnetic beads in the binding buffer. In embodiments, as described above, the additional magnetic particles added to the binding buffer may result in a magnetic bead concentration in the binding buffer of up to 20 times (e.g., 15 times, 10 times, 5 times, 2 times) more than 60 μg / μl. For example, the final concentration of magnetic particles present in the binding buffer may be in the range of 0.1 μg / μl to 1200 μg / μl, for example, 1 μg / μl to 1100 μg / μl, 10 μg / μl to 1000 μg / μl, 20 μg / μl to 800 μg / μl, 30 μg / μl to 700 μg / μl, 40 μg / μl to 600 μg / μl, 40 μg / μl to 500 μg / μl. / μl, 50μg / μl to 400μg / μl, 60μg / μl to 300μg / μl, 70μg / μl to 200μg / μl, 80μg / μl to 150μg / μl, 600 μg / μl to 1200μg / μl, 700μg / μl to 1200μg / μl, 800μg / μl to 1200μg / μl, 900μg / μl to 1200μg / μl, 100 0μg / μl to 1200μg / μl, or 0.1μg / μl to 1200μg / μl, 0.1μg / μl to 1100μg / μl, 0.1μg / μl to 1000μg / μl, 0.1μg / μl to 900μg / μl, 0.1μg / μl to 800μg / μl, 0.1μg / μl to 700μg / μl, 0.1μg / μl to 600μg / μl, 0.1μl g / μl to 500 μg / μl, 0.1 μg / μl to 400 μg / μl, 0.1 μg / μl to 300 μg / μl, 0.1 μg / μl to 200 μg / μl, 0.1 μg / μl to 100 μg / μl, 1 μg / μl to 100 μg / μl, 10 μg / μl to 100 μg / μl, or 50 μg / μl to 100 μg / μl, including all ranges and subranges therebetween.

[0077] In embodiments of the methods taught herein, the volume ratio between the sample and the binding buffer can range, for example, from 1:1 to 3:3, such as from 1:1 to 1:1.5, or from 1:1.5 to about 1:2.5, including all ranges and subranges therebetween. The incubation time period for the mixed solution comprising the biochemical product of interest, the binding buffer, and the silica-based magnetic particles can range from 0.1 minutes to 30 minutes, 0.1 minutes to 25 minutes, 0.1 minutes to 20 minutes, 0.1 minutes to 10 minutes, or 0.1 to 5 minutes, 0.1 minutes to 2 minutes, including all ranges and subranges therebetween.

[0078] In a specific embodiment, such as in the case of preferably repeating the binding step, the volume ratio between the additionally added sample and the binding buffer in the repeated step can be, for example, in the range of 1:1 to 3:3, for example 1:1 to 1:1.5, or 1:1.5 to about 1:2.5, including all ranges and subranges therebetween. When repeating the continuous binding step, most of the magnetic beads and most of the samples added in the previous round of binding remain in the purification container to which the additional sample and optional binding buffer are added. It is expected that most of the samples added in the previous round of binding steps are attached to the magnetic particles. It is obvious to those skilled in the art that the ratio of the overall sample (including the samples added in the binding steps of all rounds and the final additionally added samples) to the binding buffer will be higher than the above. The volume ratio between the total sample and the binding buffer can range, for example, from 1:3 to 20:1, such as 1:3 to 1:2, 1:2 to 1:1, 1:3 to 20:1, 1:3 to 19:1, 1:3 to 18:1, 1:3 to 17:1, 1:3 to 16:1, 1:3 to 15:1, 1:3 to 14:1, 1:3 to 13:1, 1:3 to 12:1, 1:3 to 11:1, 1:3 to 10:1, 1:3 to 9:1, 1:3 to 8:1, 1:3 to 7:1, 1:3 to 6:1, 1:3 to 4:1, 1:3 to 3:1, 1:3 to 2:1, including all ranges and subranges therebetween.

[0079] Without being bound by theory, the salts and alcohols introduced by the binding buffer enhance the ability of biochemical products such as nucleic acids (e.g., RNA, DNA or proteins) to reversibly (e.g., non-covalently) bind to the surface of the magnetic particles (e.g., silica surface). The magnetic particles thus modified (e.g., including the reversibly bound biochemical products) can then be separated from unbound contaminants (e.g., salts, proteins, enzymes, oligonucleotides, DNA templates, and nucleotide triphosphates (NTPs)).

[0080] After using a magnet to separate the liquid and the modified magnetic particles, in some embodiments, the modified magnetic particles can be washed with one or more wash buffers. It will be clear to those skilled in the art that washing requires adding wash buffer to the modified beads and removing the wash buffer. In each successive washing step, the modified magnetic particles are separated from the wash buffer by magnetic separation before performing the next wash and / or moving to any other subsequent steps (such as elution steps).

[0081] The washing buffer may include, for example, at least one alcohol and optionally at least one salt. The modified magnetic particles may be rinsed with the washing buffer once or more, and any additional washings may be with the same or different components, concentrations and / or volumes. The washing buffer is preferably added to the purification vessel comprising the modified magnetic particles.

[0082] In a preferred embodiment, the modified magnetic particles are incubated with a wash buffer and then separated at least 2 to 10 times, 2 to 8 times, 2 to 7 times, 2 to 6 times, 2 to 5 times, 2 to 4 times, or 2 to 3 times, preferably at least 2 to 5 times. Alternatively, steps (b) to (d) are repeated at least 3 to 10 times, 4 to 10 times, 5 to 10 times, 6 to 10 times, 7 to 10 times, 8 to 10 times, or 9 to 10 times.

[0083] According to various embodiments, at least one alcohol of the wash buffer may be selected from isopropanol, methanol, ethanol, butanol, and combinations thereof. According to various embodiments, at least one salt of the wash buffer may be selected from ammonium sulfate ((NH4)2SO4), ammonium acetate (NH4Ac), lithium acetate (LiAc), potassium acetate (KAc), sodium acetate (NaAc), sodium chloride (NaCl), and combinations thereof.

[0084] In an embodiment of the method taught herein, the wash buffer includes at least one alcohol in a concentration range of 50% to 100% volume / volume (v / v) (55% to 95%, 60% to 85%, or 60% to 80% v / v, including all ranges and sub-ranges therebetween). The at least one alcohol in the wash buffer may be the same or different from an alcohol in the binding buffer. In some embodiments, the alcohol in the binding buffer may be ethanol. In non-limiting embodiments, the wash buffer may optionally have at least one salt. The optional salt (if present) may have a concentration range of 0.1M to 5M in the binding buffer, such as 0.3M to 4M, 0.1M to 3M, 0.1M to 2M, 0.1M to 1M, and 1M to 2M, including all ranges and sub-ranges therebetween. According to various embodiments, the salt in the wash buffer may be sodium chloride (NaCl). Non-limiting examples of wash buffer compositions include 80% ethanol together with 20% RNAase-free water.

[0085] In some embodiments, the modified magnetic particles can be washed one or more times with at least one of the wash buffers. For example, the modified magnetic particles can be washed once, twice or more times with the wash buffer, wherein the modified magnetic particles are separated by using a magnet between washes.

[0086] After the final binding and / or washing steps, the modified magnetic particles (including the biochemical product of interest reversibly bound to the surface by the multiple purification steps) are substantially free of contaminants, such as salts, proteins, enzymes, etc. According to various embodiments, the modified magnetic particles thus produced can then be incubated with one or more elution buffers to release the bound biochemical product of interest and separate it from the magnetic particles.

[0087] It will be clear to those skilled in the art that in some embodiments, the elution step can be repeated multiple times to increase the yield. For example, the elution is repeated 2 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times and 10 times. However, in some embodiments, a single elution step is performed.

[0088] According to various embodiments, the elution buffer is a low conductivity solution, wherein the conductivity of the buffer ranges from 0.001 mS / cm to 40 mS / cm, more preferably from 0.01 mS / cm to 40 mS / cm, 0.1 mS / cm to 40 mS / cm, 0.5 mS / cm to 40 mS / cm, more preferably from 0.5 mS / cm to 30 mS / cm, 0.5 mS / cm to 20 mS / cm, 0.5 mS / cm to 10 mS / cm, including all ranges and sub-ranges therebetween.

[0089] In another or additional embodiment, the elution buffer comprises a salt concentration between 0.01 mM and 50 mM, more preferably between 0.1 mM and 40 mM, more preferably between 0.1 mM and 30 mM, more preferably between 0.1 mM and 20 mM. Possible salts include sodium citrate, sodium chloride, sodium phosphate, potassium chloride, potassium phosphate, and combinations thereof.

[0090] The pH of the elution buffer may range, for example, from 5 to about 10, such as from 5.5 to about 9, 6 to 8, or 6.4 to about 7.5, including all ranges and subranges therebetween.

[0091] For example, in some cases, the elution buffer may include water, such as but not limited to RNase-free water; in other water and EDTA, or only Tris, or Tris and EDTA, or sodium citrate, or phosphate buffer, or phosphate buffered saline (PBS). If used as elution buffer, the concentration range of sodium citrate can be 0.5mM to 10mM, such as 0.6mM to 5mM, 1mM to 2mM, including all ranges and subranges therebetween. If used as elution buffer, the pH range of sodium citrate can be pH 5.4 to pH 7, pH 6 to pH 6.5, including all ranges and subranges therebetween. According to non-limiting embodiments, the elution buffer may include water or 10mM Tris-HCl, 1mM EDTA, pH 7.4 or 10mM Tris-HCl, pH 7.4 or 1mM citrate Na, pH 6.4.

[0092] In an embodiment, the elution buffer is free of toxic chaotropic agents, such as guanidine salts (guanidine thiocyanate or guanidine thiocyanate), iodide, perchlorate and trichloroacetate (preferably guanidine salts).

[0093] In some embodiments of the methods described herein, the at least one modified particle is incubated with the elution buffer for 30 seconds to 30 minutes, 1 minute to 20 minutes, 1 minute to 10 minutes, including all ranges and subranges therebetween.

[0094] The free magnetic particles (no longer attached to the biochemical particles) can then be removed (e.g., separated using a magnet) from the solution, yielding purified nucleic acids in solution as a final product. The free magnetic particles can further be reused for another round of nucleic acid production.

[0095] The modified magnetic beads can be incubated with the elution buffer for additional rounds (eg, one, two, or more times) for optimal elution of the purified nucleic acids. In another embodiment, the elution buffer including the nucleic acid molecules is removed from the sample container and stored in a collection vessel.

[0096] In embodiments of the methods as disclosed herein, the magnetic particles and the biochemical product in the processing or purification vessel are mixed by means of subjecting the processing or purification vessel to mechanical movement. In some embodiments, the mechanical movement comprises positioning the purification vessel on a mixing station.

[0097] The mixing station allows mixing of magnetic particles with biochemical product and binding buffer and / or mixing of modified magnetic particles with washing or elution buffer.

[0098] In some embodiments, mixing is ensured by rotation of at least one wall of the purification or treatment container. In other embodiments, the inner wall rotates, thereby driving mixing of the contents of the purification or treatment container. In other embodiments, the outer wall rotates, thereby driving mixing of the contents of the purification or treatment container. In yet another embodiment, both walls rotate, thereby driving mixing of the contents of the purification or treatment container. In such an embodiment, the wall in the container that is configured to perform rotation can be a ball bearing, but it is not limited to this.

[0099] In embodiments, the mechanical motion is intermittent rotation, orbital serpentine, or vibration, but it will be apparent to one skilled in the art that any mechanical motion that drives mixing of the contents of a purification or processing vessel may be used with the methods disclosed herein.

[0100] In some embodiments, the entire processing or purification vessel is subjected to mechanical movement. In other embodiments, a portion of the purification or processing vessel is subjected to mechanical movement.

[0101] In another embodiment of the method as disclosed herein, the step of applying a magnetic source occurs at a magnetic separation station, wherein the station comprises one or more magnets and further comprises the processing or purification container comprising the magnetic particles and the biochemical product sample. The purification container comprising the magnetic particles and the biochemical product is positioned at the magnetic separation station, and the magnetic particles are allowed to bind to the magnet during a predefined magnetization time.

[0102] By "binding time" it is understood the time required for the biochemical product to bind to the magnetic particles. By "magnetization time" it is understood the time required for the modified magnetic particles to be attracted and become attached to the magnet.

[0103] The magnetic separation station allows the biochemical product to be incubated with the magnetic particles for a binding time ranging from 5 seconds to 30 minutes, 30 seconds to 20 minutes, and 1 minute to 10 minutes, including all ranges and subranges therebetween. The modified magnetic particles are then combined with the magnet for a predetermined magnetization time ranging from 20 seconds to 15 minutes, 20 seconds to 14 minutes, 20 seconds to 13 minutes, 20 seconds to 12 minutes, 20 seconds to 11 minutes, 20 seconds to 10 minutes, 20 seconds to 9 minutes, 20 seconds to 8 minutes, 20 seconds to 7 minutes, 20 seconds to 6 minutes, 20 seconds to 5 minutes, 20 seconds to 4 minutes, 20 seconds to 3 minutes, 20 seconds to 2 minutes, 20 seconds to 1 minute, 20 seconds to 50 seconds, 20 seconds to 40 seconds, or 20 seconds to 30 seconds, including all ranges and subranges therebetween.

[0104] Alternatively, the modified magnetic particles are then allowed to bind to the magnet for a predetermined magnetization time in the range of 30 seconds to 15 minutes, 40 seconds to 15 minutes, 50 seconds to 15 minutes, 1 minute to 15 minutes, 2 minutes to 15 minutes, 3 minutes to 15 minutes, 4 minutes to 15 minutes, 5 minutes to 15 minutes, 6 minutes to 15 minutes, 7 minutes to 15 minutes, 8 minutes to 15 minutes, 9 minutes to 15 minutes, 10 minutes to 15 minutes, 11 minutes to 15 minutes, 12 minutes to 15 minutes, 13 minutes to 15 minutes, or 14 minutes to 15 minutes, including all ranges and sub-ranges therebetween.

[0105] In embodiments of the methods disclosed herein, the magnetization time of the modified magnetic particles increases with the number of purification steps. In some embodiments, the magnetization time increases with each purification cycle by 10 seconds to 11 minutes, 10 seconds to 10 minutes, 10 seconds to 9 minutes, 10 seconds to 8 minutes, 10 seconds to 7 minutes, 10 seconds to 6 minutes, 10 seconds to 5 minutes, 10 seconds to 4 minutes, 10 seconds to 3 minutes, 10 seconds to 2 minutes, 10 seconds to 1 minute, 10 seconds to 50 seconds, 10 seconds to 40 seconds, 10 seconds to 30 seconds, or 10 seconds to 20 seconds, including all ranges and subranges therebetween.

[0106] Alternatively, the magnetization time increases with each purification cycle by 20 seconds to 11 minutes, 30 seconds to 11 minutes, 40 seconds to 11 minutes, 50 seconds to 11 minutes, 1 minute to 11 minutes, 2 minutes to 11 minutes, 3 minutes to 11 minutes, 4 minutes to 11 minutes, 5 minutes to 11 minutes, 6 minutes to 11 minutes, 7 minutes to 11 minutes, 8 minutes to 11 minutes, 9 minutes to 11 minutes, 10 minutes to 11 minutes, including all ranges and subranges therebetween.

[0107] The magnetization time of the modified magnetic particles increases with each purification step to allow the modified magnetic particles enough time to bind to the magnet. Each purification step increases the saturation of the magnetic particles in the binding molecules. The magnetization characteristics of the magnetic particles decrease and the binding speed with the magnet slows down. In order to avoid that the modified magnetic particles are not captured, the applicant increases the magnetization time of each new purification cycle.

[0108] In an embodiment of the method as disclosed herein, the purification container is moved between the mixing station and the magnetic separation station by means of a robotic arm, which is configured to grip the processing or purification container. In another embodiment, the same purification container is used for the mixing step and the binding step. Alternatively, different containers are used for mixing and binding, and the magnetic particles, biochemical products and buffer are transferred between purification containers by a processing device (e.g., a robotic arm).

[0109] In an embodiment, and in case of multiple purification runs with increasing magnetization time of the magnetic particles, there is asynchrony in the operation of the mixing station and the magnetic separation station, the mixing station operating for a constant time span in each purification cycle, and the operation time at the magnetic separation station increasing with each cycle. This asynchrony allows purification to be performed in multiple purification vessels simultaneously. To further compensate for asynchrony and / or if a mixing / separation station is occupied, a portion of the purification vessels can be kept as a reserve until a free mixing / separation station becomes available.

[0110] In another embodiment, the magnetic particles, liquid sample, binding buffer, wash buffer and / or elution buffer are added to the purification vessel by a pump, an injector or a robotic arm.

[0111] The methods disclosed herein allow for the purification of biochemical products of interest, including biopharmaceutical compounds such as DNA, RNA, modified RNA, proteins, peptides or polypeptides. Advantageously, the methods allow for the automation of separation and / or purification processes, while performing the procedures continuously and with high precision without human intervention. The purification protocols can be implemented directly on the device and / or system without any process amplification, as the device mimics the small-scale manual operations performed in the laboratory.

[0112] In embodiments of the methods disclosed herein, the purified biochemical product is DNA or RNA.

[0113] In embodiments, methods disclosed herein relate to the separation and / or purification of RNA molecules. The term RNA or RNA molecule encompasses long-chain RNA, coding RNA, non-coding RNA, long non-coding RNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA (mRNA), self-amplification mRNA (SAM), Trans amplification mRNA, RNA oligonucleotides, antisense oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (assRNA), CRISPR / Cas9 guide RNA, riboswitch, immunostimulatory RNA (isRNA), ribozyme, aptamer, ribosomal RNA (rRNA), transmission RNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, small nuclear RNA (snRNA), small nucleolar RNA (snRNA), microRNA (miRNA) and Piwi interacting RNA (piRNA). In some embodiments, the RNA includes modified RNA molecules. In some embodiments, the modification of RNA molecule includes chemical modification, which includes backbone modification and sugar modification or base modification. In this article, the modified RNA molecule defined herein includes nucleotide analogs / modification, such as main chain modification, sugar modification or base modification. The backbone modification relevant to the utility model is as follows: the phosphate of the backbone of the nucleotide included in the RNA molecule is chemically modified. The sugar modification relevant to the utility model is the chemical modification of the sugar of the RNA molecule nucleotide. In addition, the base modification relevant to the utility model is the chemical modification of the base part of the nucleotide of the RNA molecule. In this context, nucleotide analogs or modification are selected from nucleotide analogs suitable for transcription and / or translation. In another embodiment, the modified RNA comprises a nucleoside modification selected from the group consisting of 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-isocytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydroacridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxythymidine, 5-methyl -uridine, pyrrole-cytidine, inosine, α-thioguanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-isopropylcytidine, 6-chloro-purine, N6-methyl-adenosine, α-thioadenosine, 8-azido-adenosine, 7-deaza-adenosine.

[0114] In a particularly preferred embodiment, the biochemical product is produced in multiple reactions, preferably multiple in vitro transcription (IVT) reactions, each of which yields a sample of the biochemical product. Without wishing to be bound by theory, the reaction conditions that need to be met in the IVT reaction are to provide: linear DNA to serve as a template for replication into RNA by a polymerase, dNTPs, an RNA polymerase to bind the dNTPs, a magnesium-containing buffer to catalyze the reaction, and a specific reaction temperature, typically 37° C. It will be apparent to one skilled in the art that any reaction conditions that produce mRNA can be used with the methods described herein.

[0115] In some embodiments, the methods disclosed herein relate only to the downstream purification of cell-free obtained biochemical products. In other embodiments, the methods disclosed herein relate to the downstream purification of cell-free obtained biochemical products and their upstream production.

[0116] Methods for producing and purifying cell-free synthetic biochemical products

[0117] In some embodiments, the downstream purification process as described above is preceded by cell-free production of a biochemical product. The biochemical product may be a nucleic acid, such as DNA or RNA, an antibody, a peptide, a polypeptide or a protein, but is not limited thereto.

[0118] In an embodiment, the cell-free reaction produces nucleic acid, such as DNA or RNA. The non-limiting example of DNA production method includes template or amplification of de novo synthesis of DNA. Therefore, a plurality of the nucleic acid production reactions can be PCR reactions and / or DNA synthesis on the solid support that occurs in the reaction chamber. It is obvious to those skilled in the art that in such an embodiment, the reaction chamber is assembled in a device that provides required reaction conditions (such as temperature incubation, mixing, adding and removing reagent buffers, enzymes, nucleotides, etc.). Other examples of cell-free reactions include in vitro transcription (IVT) for producing RNA and any other cell-free method for expressing protein. The non-limiting example of cell-free protein production method includes PURE system, rabbit reticulocyte lysate translation system, wheat germ lysate translation, or any other in vitro translation system that may or may not be ribosomes, tRNA, amino acids and / or energy sources.

[0119] In an embodiment, both purification and downstream processing are independent of each other. In another embodiment, the downstream processing or purification and upstream production are coupled.

[0120] In embodiments, multiple separate cell-free reactions are performed in multiple reaction chambers. In embodiments, reagents for multiple cell-free reactions are added to each reaction chamber. The reagents are added by a handling device such as a robotic arm, a pump, or an injector. In some embodiments, the reaction reagents are premixed; in other embodiments, the reagents are added independently; in other embodiments, some reagents are premixed and other reagents are added independently of the premix.

[0121] In some embodiments, the reagents are thawed, cooled, heated and / or mixed before being added to the reaction chamber. It will be apparent to one skilled in the art that the reactant activities and reaction conditions depend on the type of biochemical product produced, and that the reagents and reaction conditions are adjusted accordingly.

[0122] In another embodiment of the method, as disclosed herein, at least a portion of a plurality of biochemical product samples from a plurality of chambers are collected in a collection vessel. In an embodiment, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of a plurality of biochemical product samples from a plurality of chambers are collected in the collection vessel. Preferably, at least 25% of a plurality of biochemical product samples from the plurality of chambers are collected in the collection vessel. In an embodiment, all biochemical product samples from a plurality of reaction chambers are collected in the collection vessel. The total volume of the collection vessel is preferably equal to or greater than the sum of the volumes of the individual cell-free reactions in the chamber.

[0123] In another embodiment, pooling of multiple biochemical product samples from multiple chambers is performed with the aid of a handling device such as a robotic arm, pump, or ejector.

[0124] Pooling of multiple biochemical product samples from multiple chambers in a pooling vessel provides intermediate storage and allows upstream biochemical product production reactions, such as IVT reactions, to be run continuously. At the same time, the pooling vessel ensures a continuous supply of molecules for downstream purification steps.

[0125] Alternatively, the methods disclosed herein for production and purification can function without a collection vessel. In such an embodiment, the contents of the reaction chamber are collected directly into one or more purification containers.

[0126] In another embodiment, the method disclosed herein relates to the downstream purification of multiple biochemical products, wherein at least a portion of the collected biochemical product samples from the collection vessel is transferred to a processing or purification container and subjected to a processing step such as a purification step. In a preferred embodiment, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the collected nucleic acid samples from the collection vessel are transferred to a purification container. Alternatively, all volumes of the collected biochemical product samples are transferred to a purification container.

[0127] In another embodiment, at least a portion of the volume of the pooled biochemical product sample from the pooling vessel is transferred to a plurality of purification containers. The portion of the volume of the pooled biochemical product sample from the pooling vessel is transferred to at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 purification containers, preferably 4 purification containers.

[0128] Preferably, the transfer of the pooled nucleic acid sample from the pooling vessel to the purification container is performed with the aid of a handling device, such as a robotic arm.

[0129] Therefore, the methods disclosed herein can utilize multiple purification containers. In a preferred embodiment, at least one purification container is used for purification. In another embodiment, at least one purification container is used for purification, and one or more purification containers are kept as a reserve. In another embodiment, multiple purification containers are used for purification, and one or more purification containers are kept as a reserve. Temporary storage of biochemical product samples in reserve purification containers ensures continuous flow of biochemical products between upstream reaction steps and downstream purification steps, regardless of the duration of each step.

[0130] Alternatively, all purification vessels are used for the purification and no purification vessels are in reserve.

[0131] In another embodiment of the method as disclosed herein, another portion of said pooled biochemical product sample is transferred to a processing or purification vessel comprising the biochemical product bound to magnetic particles and is subjected to a binding step.

[0132] After each round of combining and removing the liquid portion of the modified magnetic particles is completed, a new volume portion of the collected biochemical product sample from the collection vessel is transferred to the purification container, above the modified beads. The new volume portion of the collected biochemical product is subjected to a new magnetic purification step, wherein the biochemical product in the volume portion is combined to the modified magnetic particles and separated from the uncombined liquid portion of the sample. Washing with wash buffer is optional. Repeat the steps of transferring and combining the volume portion of the biochemical product until the predetermined volume of the biochemical product sample in the final collection vessel has been used.

[0133] In a preferred embodiment, the steps of transferring and combining volume fractions of the biochemical product are repeated until a majority (preferably all) of the collected biochemical product samples in the collection vessel have been subjected to at least one magnetic purification step. In a preferred embodiment, the steps of transferring and combining volume fractions of the biochemical product are repeated until a range of the collected biochemical product samples in the collection vessel has been subjected to at least one magnetic purification step: at least 25% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100%, including all ranges and sub-ranges therebetween.

[0134] In another preferred embodiment, the steps of transferring and combining volume portions of the biochemical product are repeated until the following range of the pooled biochemical product sample in the pooling vessel has been subjected to at least one magnetic purification step: at least 25% to 100%, 25% to 90%, 25% to 80%, 25% to 70%, 25% to 60%, 25% to 50%, 25% to 40% or 25% to 30%, including all ranges and sub-ranges therebetween.

[0135] In another preferred embodiment, the steps of transferring and combining volumetric portions of the biochemical product are repeated until the majority of the pooled biochemical product sample in the pooling vessel is substantially free of contaminants, such as salts, proteins, enzymes, and the like.

[0136] In an embodiment that has bypassed the need for a pooling vessel, the contents of the purification container (i.e., the sample portion from the reaction chamber) are subjected to a magnetic purification step, after which a new portion of the contents of the reaction chamber is added to the modified magnetic particles. The steps of transferring and binding a portion of the biochemical product are repeated until a predetermined volume of the biochemical product sample in the final reaction chamber has been used.

[0137] In an embodiment of the method, as disclosed herein, when a subsequent purification cycle is initiated, the biochemical product of the earlier purification cycle remains bound to the magnetic particles. Thus, the magnetic particles have an increased accumulation of bound biochemical product as they undergo each purification cycle.

[0138] In a preferred embodiment, the magnetization time of magnetic particles to one or more magnets increases along with the number of processing or purification cycles.The increase of magnetization time is necessary, to compensate for the reduction of the magnetic attraction of the modified magnetic particles saturated in biochemical products.In a preferred embodiment of the method as disclosed herein, the described magnetization time of modified magnetic particles increases along with the number of purification cycles.In some embodiments, the magnetization time increases along with each purification cycle by 110 seconds to 11 minutes, 10 seconds to 10 minutes, 10 seconds to 9 minutes, 10 seconds to 8 minutes, 10 seconds to 7 minutes, 10 seconds to 6 minutes, 10 seconds to 5 minutes, 10 seconds to 4 minutes, 10 seconds to 3 minutes, 10 seconds to 2 minutes, 10 seconds to 1 minute, 10 seconds to 50 seconds, 10 seconds to 40 seconds, 10 seconds to 30 seconds or 10 seconds to 20 seconds, including all ranges and subranges therebetween.

[0139] Alternatively, the binding time increases by 20 seconds to 11 minutes, 30 seconds to 11 minutes, 40 seconds to 11 minutes, 50 seconds to 11 minutes, 1 minute to 11 minutes, 2 minutes to 11 minutes, 3 minutes to 11 minutes, 4 minutes to 11 minutes, 5 minutes to 11 minutes, 6 minutes to 11 minutes, 7 minutes to 11 minutes, 8 minutes to 11 minutes, 9 minutes to 11 minutes or 10 minutes to 11 minutes along with each purification cycle, including all ranges and subranges therebetween. The magnetization time of the magnetic particle of described modification increases along with each purification cycle, to allow the magnetic particle of modification to have enough time to be combined with described magnet. Each purification cycle has increased the saturation degree of magnetic particle in binding molecule. The magnetization characteristic of magnetic particle reduces and slows down with the binding speed of magnet. In order to avoid the magnetic particle of modification not being captured, the applicant has increased the magnetization time of each new purification cycle.

[0140] Surprisingly observed, repeating multiple purification cycles (i.e. combining and optional washing), and omitting elution between the cycles, increased the yield of the biochemical product obtained, reduced the amount of buffer required in the purification process, which reflects the reduction in the use of harmful agents (such as chaotropic agents, such as guanidine thiocyanate) commonly used in practice. The method also allows the reuse of magnetic particles. In short, the method as disclosed herein makes the technology safe and affordable.

[0141] Binding and wash buffers are preferably added to and removed from the purification vessel with the aid of a handling device such as a robotic arm, pump or ejector.

[0142] After the last binding and / or washing step, the modified magnetic particles (including the compounds of interest that are reversibly bound to the surface by multiple purification cycles) are substantially free of contaminants, such as salts, proteins, enzymes, etc. Elution is performed as described in any of the previous embodiments. According to various embodiments, the modified magnetic particles thus produced can then be incubated with one or more elution buffers to release the bound compounds of interest and separate them from the magnetic particles. Elution is performed as described in any of the previous embodiments.

[0143] The method disclosed herein can be used to provide purified biochemical products. In certain embodiments, the method disclosed herein can provide a relative biochemical product yield between 80% and 100%, and provide sufficient purity. In the embodiment of the method taught herein, the integrity of the purified biochemical product is protected, while reducing the use of dangerous or unwanted reagents, and making the technology affordable by reducing costs.

[0144] In an embodiment of the method, as disclosed herein, the biochemical product is a nucleic acid molecule, preferably RNA or DNA.

[0145] In embodiments, the cell-free reaction performed is one or more IVT reactions, thereby producing RNA (such as mRNA). In embodiments, multiple IVT reactions are performed. Each reaction is performed in a chamber.

[0146] In another embodiment, at least a portion of the IVT reagents are combined into an IVT premix. In another embodiment, the IVT premix includes at least dNTPs, template DNA, and one or more RNA polymerase buffer components. In an embodiment, the IVT reagents are stored in a storage unit, wherein the storage unit is fluidly connected to a pump system, and wherein the pump system is configured to provide a desired amount of one or more IVT reagents for preparing an IVT premix to the IVT premix container. As described above, the storage unit can be cooled to a temperature below 10°C, preferably 4°C.

[0147] In embodiments, the total volume of the premix is ​​equal to or greater than the sum of the volumes of the individual IVT reactions in the chamber.

[0148] In an embodiment, prior to providing a portion of the IVT premix to a chamber of the plurality of chambers, the premix is ​​heated to a temperature between 35°C and 55°C. In an embodiment, prior to providing a portion of the IVT premix to a chamber of the plurality of chambers, the premix is ​​heated to a temperature between 35°C and 55°C, preferably between 35°C and 50°C, between 35°C and 45°C, between 35°C and 40°C, or between 35°C and 37°C. Alternatively, prior to providing a portion of the IVT premix to a chamber of the plurality of chambers, the premix is ​​heated to a temperature between 37°C and 55°C, preferably between 40°C and 55°C, between 45°C and 55°C, or between 40°C and 55°C.

[0149] In an embodiment, the IVT premix container is heated at a temperature between 35° C. and 55° C., preferably between 35° C. and 50° C., between 35° C. and 45° C., between 35° C. and 40° C., or between 35° C. and 37° C. Alternatively, the IVT premix container is heated at a temperature between 37° C. and 55° C., preferably between 40° C. and 55° C., between 45° C. and 55° C., or between 40° C. and 55° C. In a preferred embodiment, the premix container is heated at 37° C.

[0150] In an embodiment of the method disclosed herein, an RNA polymerase is added to at least one of the multiple chambers. In other embodiments, multiple RNA polymerases are added to a chamber, preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 RNA polymerases. In another embodiment, the RNA polymerase is added before or after the chamber receives a portion of the IVT premix. Combining the IVT premix with the RNA polymerase triggers the start of the reaction. In some embodiments, it is desirable to heat the premix in the absence of the RNA polymerase, and thus the start of the reaction is delayed until the premix and the RNA polymerase are combined in multiple chambers. In other embodiments, the RNA polymerase is part of the IVT premix. The RNA polymerase used is as described above.

[0151] In some embodiments of the method, the IVT reagent includes a capping agent for co-transcriptional capping. Non-limiting examples of co-transcriptional capping agents include CleanCap (TriLink) or ARCA (CellScript). Alternatively, capping is performed post-transcriptionally using any cap-mimicking structure known in the art.

[0152] In an embodiment of the method, the IVT reaction is terminated by adding at least one DNase. In another embodiment, the IVT reaction is terminated by adding EDTA. In yet another embodiment, the IVT reaction is terminated by adding both DNase and / or EDTA. DNase enzymatically digests the DNA template and thus ends the IVT reaction, while EDTA is a Mg depleting agent. 2+ The chelating agent of the reaction of ions also leads to the termination of the IVT reaction.

[0153] In an embodiment, DNase and / or EDTA are added in a plurality of chambers. In another embodiment, described DNase and / or EDTA are added to an intermediate container for transmitting a sample, for example, a collection vessel. In another embodiment, DNase and / or EDTA are added by means of a pump, an ejector or a robot arm.

[0154] The methods disclosed herein are preferably used to perform multiple IVT reactions in an upstream production step, thereby obtaining multiple RNA molecules that are processed in a downstream purification step. Nevertheless, it is obvious to those skilled in the art that the methods can be used for the upstream production of other molecules (such as but not limited to DNA) that are purified in a downstream step.

[0155] Preferably, the methods as disclosed herein are used for the production and downstream purification of mRNA molecules from an IVT process, or for purification during pre- and / or post-capping. In an embodiment, when the mRNA molecule is co-transcriptionally capped, the mRNA is purified after capping. In another embodiment, when the mRNA is post-transcriptionally capped, the purification step is completed before capping. In another or additional embodiment, the purification is completed at post-capping. In yet another embodiment, the purification step is completed before capping and the second purification step is completed at post-capping. In addition to the desired mRNA product, the resulting product of IVT also contains an array of reaction byproducts, such as salts, nucleotides, enzymes, proteins, DNA templates, or dsRNA. These can interfere with the capping process and reduce the transaction efficiency and overall purity of the final product. Enzymatic capping is performed immediately after IVT without intermediate processing of the reaction products, resulting in a reduced amount or close to 0% of capped mRNA molecules. The method disclosed herein is designed to perform mRNA purification with high accuracy in an automated manner under GMP compliant conditions and is suitable for performing upstream and / or downstream capping of purification. The system allows for continuous production of small or medium volumes of compounds of interest.

[0156] Equipment and systems

[0157] Systems for downstream processing

[0158] In a second aspect, a system for processing biochemical products with the aid of magnetic particles is disclosed, wherein the system comprises: one or more processing or purification containers; one or more mixing stations, wherein the one or more mixing stations are configured to receive the processing or purification containers, and the one or more mixing stations include a motor unit, wherein the motor unit is configured to drive the mechanical movement of the processing or purification containers; and one or more magnetic separation stations, wherein the one or more magnetic separation stations include one or more magnets, and wherein the system also includes at least one robotic arm, wherein the at least one robotic arm is controlled by a computer processor and is configured to grasp the processing or purification containers and transfer the processing or purification containers between the one or more mixing stations and the one or more magnetic separation stations, wherein the processing or purification containers include a wall structure defining a central cavity 1, wherein the wall structure includes an outer wall and an inner wall, thereby defining a processing space between the inner wall and the outer wall, wherein the wall structure is configured to receive a liquid sample and retain the liquid sample in the interior space.

[0159] The system disclosed herein allows for downstream purification of biochemical products with the aid of magnetic particles. Downstream purification platforms are used to purify or separate biochemical products from contaminants, debris, reaction products (such as template moieties, nucleotides, enzymes, buffers, reagents, etc.).

[0160] In an embodiment, a purification system as disclosed herein comprises one or more mixing stations configured to receive a purification vessel and comprising a motor unit capable of driving mechanical movement of the purification vessel. The mixing station enables mixing of the contents found inside the purification vessel. During the binding step, the magnetic particles and nucleic acid molecules are mixed in a binding buffer to ensure uniform distribution of the components suspended in the binding buffer. After magnetic separation, the modified magnetic particles comprising nucleic acids are optionally mixed with a wash buffer. At the end of the purification cycle, in an elution step, the modified magnetic particles are mixed with an elution buffer.

[0161] In an embodiment of the mixing station, the mechanical motion is a rotation, shaking or vibrating motion around an axis. In some embodiments, the rotation around the axis is clockwise, while in other embodiments, it is counterclockwise, or both clockwise and counterclockwise.

[0162] In an embodiment, the mechanical movement of the mixing station is driven by a motor unit or an electromagnetic unit. In a preferred embodiment, an electric motor is used. In another embodiment, a plurality of electromagnets are continuously energized to generate the movement of the mixing station. In another embodiment, the mechanical movement of the mixing station is driven by a shaker unit. The mechanical movement of the mixing station can be activated and deactivated. The purpose of the mechanical movement includes, but is not limited to, homogenization of the mixture formed by the sample and the buffer for separation and / or purification, and / or facilitating contact between the magnetic particles and the sample.

[0163] In some embodiments, mixing is ensured by the rotation of at least one wall of the purification or treatment container. In other embodiments, the inner wall rotates, thereby driving the mixing of the contents of the purification or treatment container. In other embodiments, the outer wall rotates, thereby driving the mixing of the contents of the purification or treatment container. In yet another embodiment, both walls rotate, thereby driving the mixing of the contents of the purification or treatment container. In such an embodiment, the wall of the container configured to perform rotation can be a ball bearing, but it is not limited to this. In some embodiments, mixing can be completed without a mixing station when mixing is ensured by the rotation of at least one wall. Both the mixing step and the combining step can be performed at the same station. In other embodiments, mixing can be performed at a mixing station and magnetic separation can be performed at a magnetic separation station when mixing is ensured by the rotation of at least one wall.

[0164] In another embodiment of the system as disclosed herein, the downstream purification platform comprises one or more magnetic separation stations, comprising one or more magnets. The magnetic separation station is capable of separating magnetic particles in a modified or unmodified state from the liquid of the previous step. After each binding, washing and elution step, the magnetic particles are captured by the magnet, thereby separating the magnetic particles from the liquid in the purification container.

[0165] In an embodiment, the magnet of each magnetic separation station comprises a permanent magnet, a temporary magnet or an electromagnet, preferably a permanent magnet. When an electromagnet is used, the magnetic field can be quickly changed by controlling the amount of current. In another embodiment, the magnetic separation station comprises a magnet array, wherein each magnet can be a permanent magnet, a temporary magnet or an electromagnet. In another embodiment, the magnetic separation station is equipped with at least two, three, four, five, six, seven, eight, nine or ten magnets. In some embodiments, the magnetic separation station comprises a magnetic assembly of a permanent magnet and / or an electromagnetic magnet. Preferably, the mixing station and / or the magnetic separation station comprises a raised portion, which is configured to receive the central cavity of the processing or purification container. The purification container is assembled on the raised portion of the mixing station and / or the magnetic separation station, and is fully fixed to prevent falling, rolling or sliding from the station during mixing and / or separation.

[0166] In other embodiments, the purification or treatment container is assembled at the magnetic separation station and / or mixing station by means of a clamping system. It is obvious that any other method of assembling the container to the station can be used with the system disclosed herein. In a preferred embodiment, the raised portion of the magnetic separation station includes the one or more magnets lined with the raised portion. In another preferred embodiment, the one or more magnets span the entire raised portion of the magnetic separation station. The purification container assembled on the raised portion of the magnetic separation station is in direct contact with the magnet. This ensures that a strong and uniform magnetic field is applied throughout the purification container.

[0167] In some embodiments, one or more magnets are located inside the raised portion and covered by another material. In other embodiments, one or more magnets are located both below and outside the raised portion. Any material can be used in combination with one or more magnets, such as a material that improves the magnetic field generated by the electromagnet.

[0168] The magnets can be arranged in any shape in the raised portion of the magnetic separation station; non-limiting examples include circular, oval, square, rectangular or any polygonal arrangement, cross-shaped arrangement, arrangement in rows or straight lines, X-shaped arrangement, etc. In some embodiments, the magnets can be separated and controlled independently. In other embodiments, the magnets are controlled together. In other embodiments, the magnets can be separated in groups, and the groups can be controlled independently.

[0169] In another embodiment of the system, the magnetic separation station includes a base including one or more magnets positioned at the base. In another embodiment, the base of the magnetic separation station has a slope. The inclined base of the magnetic separation station causes a liquid level difference in the internal space of the purification container to which it is assembled to have multiple heights. The liquid level difference in the container ensures the proper removal of the liquid. In some embodiments, a processing device (such as a robot arm) is therefore configured to remove liquid from the purification container from the area with the highest liquid level of the purification container. Preferably, the magnet is positioned in the base in an area with the minimum thickness of the base, which corresponds to the area with the highest liquid level of the purification container. The applicant observes that the positioning of the magnet in the preferred liquid removal zone prevents the magnetic particles from being removed together with the liquid.

[0170] In a preferred embodiment, the magnetic separation station comprises a magnet lining the raised portion and a magnet positioned on the base. Alternatively, the magnetic separation station comprises only a magnet lining the raised portion.

[0171] The raised portions of the mixing station and the magnetic separation station preferably have the same shape and size to allow assembly of the same purification container. The raised portions preferably have flat side surfaces. However, any shape may be suitable for the raised portions of the mixing station and the magnetic separation station.

[0172] In other embodiments, the raised portion of the magnetic separation station and / or mixing station surrounds the outside of the processing or purification container. In such an embodiment, the magnet is positioned outside the processing or purification container. In such an embodiment, the raised portion is annular in shape. Obviously, the shape of the ring is complementary to the shape of the outer wall of the processing or purification container.

[0173] In some embodiments, the magnetic separation station has a raised portion inside the lumen of the processing or purification vessel, while the mixing station has a raised portion around the outside of the processing or purification vessel. In other embodiments, the magnetic separation station has a raised portion around the outside of the processing or purification vessel, while the mixing station has a raised portion inside the lumen of the processing or purification vessel.

[0174] In another embodiment, the purification system includes at least one purification container. The purification container is configured to keep biochemical products, magnetic particles and buffer during purification cycles and elution. In a preferred embodiment, the purification container includes a hollow central cavity lined with a wall mechanism, wherein the wall structure includes an outer wall and an inner wall, thereby defining an internal volume between the inner wall and the outer wall, wherein the wall structure is open at the top of the container, thereby allowing components and / or liquids to be added to the internal volume. The internal volume of the purification container is configured to receive liquids including biochemical products and / or magnetic particles. The central cavity is configured to be assembled on the raised portion of a mixing station and a magnetic separation station. The space formed between the inner wall and the outer wall is a thin space around the central cavity, and is configured to receive the contents of the purification container, i.e., magnetic particles, liquid samples including biochemical products and buffer. The shape and construction of the purification container allow subsequent binding steps to be performed while omitting elution and / or washing steps therebetween. Elution and / or washing are performed once only at the end when all binding steps have been completed. This significantly increases the yield of the biochemical product obtained, reduces the time required for processing, and reduces the amount of buffer required during purification, reflecting the reduced use of harmful agents commonly used in practice (such as chaotropic agents, like guanidine thiocyanate).

[0175] In a preferred embodiment of the system, as disclosed herein, the distance between the outer wall and the inner wall of the treatment or purification container is less than 80 mm, preferably between 3 mm and 75 mm, between 4 mm and 75 mm, between 5 mm and 75 mm, between 6 mm and 75 mm, between 7 mm and 75 mm, between 8 mm and 75 mm, between 9 mm and 75 mm, between 10 mm and 75 mm, between 15 mm and 75 mm, between 20 mm and 75 mm, between 25 mm and 75 mm, between 30 mm and 75 mm, between 35 mm and 75 mm, between 40 mm and 75 mm, between 45 mm and 75 mm, between 50 mm and 75 mm, between 55 mm and 75 mm, between 60 mm and 75 mm, between 65 mm and 75 mm, or between 70 mm and 75 mm, and all ranges and sub-ranges therebetween.

[0176] Alternatively, the distance between the outer wall and the inner wall of the purification container is between 3 mm and 70 mm, between 3 mm and 65 mm, between 3 mm and 60 mm, between 3 mm and 55 mm, between 3 mm and 50 mm, between 3 mm and 45 mm, between 3 mm and 40 mm, between 3 mm and 35 mm, between 3 mm and 30 mm, between 3 mm and 25 mm, between 3 mm and 20 mm, between 3 mm and 15 mm, between 3 mm and 10 mm, between 3 mm and 9 mm, between 3 mm and 8 mm, between 3 mm and 7 mm, between 3 mm and 6 mm, between 3 mm and 4 mm, and all ranges and sub-ranges therebetween.

[0177] Due to the small distance between the inner and outer walls, the magnetic particles in the purification vessel are close to the magnet and can therefore bind to the magnet in a relatively short time even when saturated with the biochemical product of interest.

[0178] In some embodiments, a single processing space is formed between the inner wall and the outer wall of the processing or purification container. In other embodiments, multiple processing spaces can be formed between the inner wall and the outer wall of the processing or purification container. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 processing spaces are formed between the inner wall and the outer wall of the processing or purification container. In such an embodiment, multiple processing spaces are formed by subdividing the interior space between the inner wall and the outer wall with additional vertical walls.

[0179] In another embodiment, the purification vessel comprises an inner processing space and an outer processing space and a central cavity between the inner processing space and the outer processing space, the central cavity being configured to receive a protrusion of a mixing station and / or a magnetic separation station. In such a nested arrangement, different processing steps can be performed simultaneously in the same vessel. In other embodiments, the purification vessel has more than two nested processing spaces. It is apparent to those skilled in the art that when a processing or purification vessel has more than two nested processing spaces, the mixing station and / or the magnetic separation station has multiple protrusions.

[0180] In one embodiment of the system, as disclosed herein, the container has a bottom wall with a slope, preferably at an angle of 10°. The slope of the bottom wall is configured to cause a liquid added to the internal volume of the purification container to have a liquid level difference throughout the purification container. The slope of the bottom wall of the purification container is complementary to the slope of the base of the magnetic separation station. A higher liquid level difference in the container ensures proper removal of the liquid.

[0181] In an embodiment, the cross-sectional shape of the inner wall and the outer wall of the purification container is the same, which ensures that a thin layer is formed in the internal volume. The cross-section of the inner wall and / or outer wall of the purification container can have any shape, such as but not limited to a pie shape, a regular or irregular polygonal shape, a concave polygonal shape, a convex polygonal shape, a triangular shape, a quadrilateral polygonal shape, a pentagonal shape, a hexagonal shape, an equilateral polygonal shape, an equiangular polygonal shape, a heptagonal shape, an octagonal shape, a non-octagonal shape, a decagonal shape, an eleven-sided shape, a dodecagonal shape, a thirteen-sided shape, a fourteen-sided shape, a pentagonal shape, a hexagonal shape, a hexagonal shape, a heptagonal shape, an eighteen-sided shape, a nineteen-sided shape, an icosagonal shape, an n-sided shape, an elliptical shape or a circular shape, preferably a hexagonal shape. A purification container with a polygonal cross section of the inner wall ensures good contact between the magnet and the purification container, and therefore ensures optimal exposure of the magnetic particles to the magnetic field.

[0182] In some embodiments, the cross-section of the inner wall of the purification container is polygonal, including multiple regular or irregular faces. In a specific embodiment of the container, these faces are square, rectangular, trapezoidal or a mixture thereof. This polygonal configuration enhances the surface area available for interaction between the magnetic particles inside the purification container and the magnetic separation station. In particular, when the inner wall includes multiple trapezoidal faces, the surface of interaction is significantly increased compared to the inner wall with a rectangular or square-shaped face.

[0183] Preferably, the inner wall of the purification container has a cross-sectional shape that is the same as or complementary to the cross-sectional shape of the raised parts of the mixing station and the magnetic separation station. This ensures that the purification container slides onto the raised parts and has optimal contact with the magnets.

[0184] In an alternative embodiment, the cross-sectional shapes of the inner and outer walls of the purification vessel are different.

[0185] It is also preferred that the raised portions of the mixing station and the magnetic separation station comprise a plurality of faces having the same shape or a complementary shape to the faces of the inner wall faces.

[0186] In a preferred embodiment, at least one purification container is used for purification. In another embodiment, at least one purification container is used for purification and one or more purification containers are kept as a reserve. In another embodiment, a plurality of purification containers are used for purification and one or more purification containers are kept as a reserve. Alternatively, all purification containers are used for the purification and no purification container is kept as a reserve.

[0187] In some embodiments, the capacity of the internal volume of the purification container is 10mL to 100mL, 10mL to 500mL, 10mL to 1000mL, 10mL to 1500mL. In a preferred embodiment, the capacity of the internal volume of the purification container is at least 10mL, 12mL, 15mL, 20mL, 25mL, 30mL, 35mL, 40mL, 45mL, 50mL, 55mL, 60mL, 65mL, 70mL, 75mL, 80mL, 85mL, 90mL, 95mL, 100mL, 125mL, 150mL, 200mL or 250mL, preferably at least 12mL.

[0188] As used herein, the magnetic surface of a purification vessel refers to the combined area of ​​all faces of the inner wall that are in contact with the raised portions of the magnetic station. In some embodiments of the vessel, as disclosed herein, the magnetic surface (cm 2 ) and the volume of the liquid inside the container (cm 3 ) is 0.5cm -1 With 1cm -1 In some embodiments, the ratio is between 0.5 cm -1 Up to 0.95cm -1 Between, 0.5cm -1 Up to 0.9cm -1 Between, 0.5cm -1 Up to 0.85cm -1 Between, 0.5cm -1 Up to 0.8cm -1 Between, 0.5cm -1 Up to 0.75cm -1 Between, 0.5cm -1 Up to 0.7cm -1 Between, 0.5cm -1 Up to 0.65cm-1 Between, 0.5cm -1 Up to 0.6cm -1 , or 0.5cm -1 Up to 0.65cm -1 between.

[0189] Alternatively, the ratio is between 0.55 cm -1 Up to 1cm -1 Between, 0.6cm -1 Up to 1cm -1 Between, 0.65cm -1 Up to 1cm -1 Between, 0.7cm -1 Up to 1cm -1 Between, 0.75cm -1 Up to 1cm -1 Between, 0.8cm -1 Up to 1cm -1 Between, 0.85cm -1 Up to 1cm -1 Between, 0.9cm -1 Up to 1cm -1 Between, or 0.95cm -1 Up to 1cm -1 between.

[0190] In yet other alternative embodiments, the ratio is between 0.5 cm -1 Up to 1cm -1 Between, 0.55cm -1 Up to 0.95cm -1 Between, 0.6cm -1 Up to 0.9cm -1 Between, 0.65cm -1 Up to 0.85cm -1 Between or within 0.7cm -1 Up to 8cm -1 Between, such as 0.71cm -1 、0.72cm -1 、0.73cm -1 、0.74cm -1 、0.75cm -1 、0.76cm -1 、0.77cm -1 、0.78cm -1 , or 0.79cm -1 .

[0191] It was observed that a small ratio between the magnetic surface of the purification vessel and the volume of the liquid inside the vessel increases the efficiency of magnetization of the magnetic particles to the magnet. This is evidenced by a reduction in the time required for magnetization and a reduction in the amount of magnetic particles required.

[0192] The purification container according to the present invention advantageously ensures that the liquid in the container is in close proximity to the magnet of the magnetic separation station, preferably at a distance of less than 1 cm, less than 0.95 cm, less than 0.9 cm, less than 0.85 cm, less than 0.8 cm, less than 0.75 cm, less than 0.7 cm, less than 0.65 cm, less than 0.6 cm, less than 0.55 cm, less than 0.5 cm, less than 0.45 cm, less than 0.4 cm, less than 0.35 cm or less than 0.3 cm.

[0193] In some embodiments, the material of the purification container includes, for example, materials that are resistant to cleaning procedures (chemical resistance), extreme temperatures (e.g., nucleic acid denaturation), extreme pH values ​​(sterilization of reactors with bases and acids, such as with NaOH), mechanical forces (e.g., friction caused by magnetic particles), and / or corrosion. In other embodiments, the material of the purification container includes materials that are appropriately light permeable (transparent, translucent, or opaque) for the corresponding purpose. In some embodiments, the material of the purification container includes materials that are appropriately gas permeable for the corresponding purpose. In other embodiments, the material of the purification container should be temperature conductive at an operating temperature of 37° C. to 65° C. (e.g., a W / (mK) value of at least 10, preferably at least 15). In some embodiments, the inner surface of the purification container includes a surface material that does not release undesirable compounds that may contaminate the final product. In another embodiment, the material of the purification container and / or its inner surface is PC (polycarbonate), PP (polypropylene), PAI (polyamide-imide) (e.g., Torlon), PI (polyimide) (e.g., Tecracet), PPS (polyphenylene sulfide) (e.g., Tecatron), PPSU (polyphenylene sulfone) (e.g., Tecatson P), PSU (polysulfone) (e.g., Tecatson S), PEI (polyetherimide) (e.g., Tecapei), glass (e.g., borosilicate glass), technical ceramics (e.g., ), polyaryletherketones (e.g. polyetheretherketone (PEEK)), thermoplastics (e.g. Pa or GF), all of which are chemically resistant, pH resistant and temperature resistant. In other embodiments, the material of the purification container includes materials for single use, including but not limited to polyethylene terephthalate and other polyethylenes, polyvinyl acetate, polyvinyl chloride. In some embodiments, the material of the purification container includes materials that are resistant to sterilization processes (including steam treatment or ethylene oxide (EtO) exposure / gamma radiation) even before adding any reaction-related reagents. In some embodiments, the material of the purification container provides protection for the medium included in the purification container from light (if necessary). In some embodiments, the purification container is made of any non-magnetic or paramagnetic material known in the art.

[0194] In another embodiment, the system includes a processing device configured to dispense, remove and / or transfer a component or liquid.

[0195] The particle component can be a magnetic particle, and the liquid can be deionized water, purified water, a buffer solution, a binding buffer, a washing buffer, an elution buffer, a reagent, a liquid sample including a biochemical product of interest, or a combination thereof, or a waste product. Obviously, the liquid is not limited to these.

[0196] In another embodiment, the handling device is configured to grip a purification vessel and is capable of transferring the purification vessel between the one or more mixing stations and the one or more magnetic separation stations.

[0197] The treatment device can include any device suitable in the art, such as an ejector or a robot arm, for example, provided with one or more nozzles, needles and / or tips. Alternatively, the distribution by the treatment device is completed directly via the pipeline of the treatment device. Any ejector, pump or robot arm known in the art and capable of distributing and / or removing components or liquids can be used together with the system as disclosed herein. The non-limiting example of the treatment device is a syringe pump, a vacuum pump, a peristaltic pump, a centrifugal pump or a combination thereof.

[0198] In a preferred embodiment, the robotic arm grips the purification container by the upper portion and transfers the purification container between the one or more mixing stations and the one or more magnetic separation stations. In another embodiment, the robotic arm is configured to grasp an adapter, the adapter comprising a needle and / or an injector for dispensing, removing and / or transferring components or liquids. The needle and / or injector is connected to one or more reagent storage devices, waste containers and / or collection containers. In another preferred embodiment, the adapter comprising the needle and / or injector is placed on a rack by the robotic arm before and after use.

[0199] In an embodiment, the ejectors and pumps are fixed dispensers placed near said mixing station and / or magnetic separation station.In an embodiment, the robotic arm can move along three separate axes and can access the reaction vessels, purification containers and / or collection vessels.

[0200] In an embodiment, at least one robotic arm is used in combination with a system as disclosed herein. In another embodiment, one or more ejectors are used in combination with the system. In yet another embodiment, one or more pumps are used in combination with the system. In yet another embodiment, a robotic arm and one or more ejectors are used in combination with the system. In yet another embodiment, a robotic arm and one or more pumps are used in combination with the system. In a preferred embodiment, a robotic arm, one or more ejectors, and one or more pumps are used in combination with the system.

[0201] The processing device used in conjunction with the system is connected to one or more reagent storage devices, waste containers and / or collecting vessels. Any reagent storage, waste containers and collecting vessels known in the art can be used in conjunction with the system. Non-limiting examples include bags, vials, pipes, bottles, jars or barrels.

[0202] In an embodiment, a processing device used in conjunction with the system is controlled by a motor unit (preferably an electric motor) and is configured to perform movement. In some embodiments, the movement is vertical, horizontal, centrifugal or 3D movement. In a preferred embodiment, the processing device is operably connected to at least one computer processor for controlling the processing device.

[0203] The system may also include a control system arranged and adapted to control the dispensing and / or removal of components or liquids by the processing device used in conjunction with the system. Based on input information (such as sample ID and sensor data and predefined algorithms), the control system adjusts the performance of the processing device.

[0204] In an embodiment, the system as disclosed herein further comprises at least a computer processor.In another embodiment, the computer processor is operably coupled to the robotic arm and is configured to control a processing device such as a robotic arm.

[0205] In another embodiment, the computer processor is operably connected to a mixing station and / or a magnetic separation station. Preferably, the computer processor includes instructions for calculating and controlling the magnetization time of one or more magnets of the magnetic particles and the magnetic separation station for binding time and modified magnetic particles and the sample comprising biochemical products. The computer processor calculates and controls the magnetization time of the increase of the magnetic particles, and also considers the asynchronism in the operation of the mixing station and the magnetic separation station. In order to further compensate for asynchronism and / or if the mixing station / separation station is occupied, the computer processor controls when the purification container can be kept as a reserve or introduced into an available mixing station / separation station.

[0206] In yet another embodiment, the computer processor is configured to control the transfer of the purification vessel between the mixing station and the magnetic separation station.

[0207] In some embodiments, a single computer processor is coupled to multiple devices and / or apparatuses of the system and is configured to control multiple actions. In other embodiments, each device and / or apparatus of the system is controlled by a separate computer processor. In an embodiment, the system comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 computer processors.

[0208] In some embodiments, the mixing station and the magnetic separation station are operated by the same computer processor, while in other embodiments, the mixing station and the magnetic separation station are operated by different computer processors.

[0209] In a preferred embodiment, the downstream purification system is an independent system that can function independently. In such an embodiment, the system can be arranged in a cabinet or a cartridge. However, the system can be connected to another system (other downstream purification systems or upstream systems, such as a biochemical product production system) to function.

[0210] Systems for upstream production and downstream processing

[0211] The utility model also relates to a system comprising at least one upstream production system connected with at least one downstream purification system.

[0212] The system comprises:

[0213] - a plurality of reaction chambers for producing said biochemical product in a plurality of reactions;

[0214] - a collection vessel for receiving reactants from a plurality of chambers;

[0215] - a downstream purification platform allowing downstream purification of the biochemical product with the aid of magnetic particles, wherein the platform comprises one or more mixing stations configured to receive a purification container and comprising a motor unit capable of driving the mechanical movement of the purification container; and one or more magnetic separation stations, wherein the one or more magnetic separation stations comprise one or more magnets, and wherein the purification platform further comprises at least one robotic arm controlled by a computer processor and configured to grip the purification container and capable of transferring the purification container between the one or more mixing stations and the one or more magnetic separation stations.

[0216] Advantageously, the systems disclosed herein allow for automation of biochemical product production and downstream purification, while performing procedures continuously and with high precision without human intervention. Production protocols can be implemented directly on the systems disclosed herein without any process scale-up, since the device mimics small-scale manual operations performed in the laboratory.

[0217] Reaction Chamber

[0218] The systems disclosed herein include multiple reaction chambers for performing cell-free reactions, such as IVT reactions.

[0219] In some embodiments, the reaction chambers are contained in a cartridge. In another embodiment, the cartridge comprises 2 to 48 chambers.

[0220] In some embodiments, the barrel includes at least 2 chambers, 3 chambers, 4 chambers, 5 chambers, 6 chambers, 7 chambers, 8 chambers, 9 chambers, 10 chambers, 11 chambers, 12 chambers, 13 chambers, 14 chambers, 15 chambers, 16 chambers, 17 chambers, 18 chambers, 19 chambers, 20 chambers, 21 chambers, 22 chambers, 23 chambers, 24 chambers, 25 chambers, 26 chambers, 27 chambers, 28 chambers, 29 chambers, 30 chambers, 31 chambers, 32 chambers, 33 chambers, 34 chambers, 35 chambers, 36 chambers, 37 chambers, 38 chambers, 39 chambers, 40 chambers, 41 chambers, 42 chambers, 43 chambers, 44 chambers, 45 chambers, 46 chambers, 47 chambers, 48 ​​chambers, 49 chambers, or 50 chambers. In some embodiments, the barrel includes at least about 60 chambers, about 70 chambers, about 80 chambers, about 90 chambers, about 100 chambers, about 110 chambers, about 120 chambers, about 130 chambers, about 140 chambers, about 150 chambers, about 160 chambers, about 170 chambers, about 180 chambers, about 190 chambers, or about 200 chambers.

[0221] In an embodiment, the system comprises 1 barrel, 2 barrels, 3 barrels, 4 barrels, 5 barrels, 6 barrels, 7 barrels, 8 barrels, 9 barrels or 10 barrels. In another embodiment, the reaction unit comprises 1 to 10 barrels, 1 to 20 barrels, 1 to 30 barrels, or 1 to 40 barrels, 1 to 50 barrels, 1 to 60 barrels, 1 to 70 barrels, 1 to 80 barrels, 1 to 90 barrels, or 1 to 100 barrels for nucleic acid production.

[0222] In an embodiment, the chamber comprises at least three flat polygonal surfaces. In another embodiment, the chamber comprises one, two, three, four, five, six, seven or eight flat polygonal surfaces, preferably six flat polygonal surfaces. Advantageously, the design of the chamber allows it to be equipped with sensors and / or probes.

[0223] In an embodiment, the cross section of the chamber is a polygon, such as a triangle, quadrilateral, pentagon or hexagon. In a preferred embodiment, the cross section of the chamber is a hexagon. A plurality of hexagonal chambers combined in a barrel can produce a honeycomb shape structure. Without wishing to be bound by theory, the hexagonal shape is recognized in geometry to best fill a plane with equal-sized units without leaving unused space. In addition, the hexagonal packing also minimizes the perimeter of a given area due to its 120-degree angle, and therefore, the hexagonal shape of the cavity ensures less use of raw materials.

[0224] In an embodiment, the open end of the chamber is configured to receive a removable cover for at least partially closing the open end. The cover prevents unwanted components from entering the chamber (e.g., Rnases, microbial contaminants or other degradation compounds or organisms), and shields the content of the chamber from the influence of the external environment. In an embodiment, the cover also prevents the content of the chamber from evaporating. In some embodiments, the chamber includes a cover to limit the exchange with the environment. In some embodiments, the cover of the chamber is removable. In some embodiments, the cover of the chamber is non-removable. Alternatively, the chamber is not covered.

[0225] In some embodiments, the cover can prevent excessive evaporation of water and loss of other critical volatile components. In some embodiments, the cover can prevent oxidation of reagents or any components. In some embodiments, the cover can provide protection from light (if necessary). In some embodiments, the cover prevents contamination from any other potential chemical compounds.

[0226] In some embodiments, the cover comprises at least one opening for filling, draining and sampling. In some embodiments, the at least one opening is positioned on the top of the cover.

[0227] In some embodiments, the bottom of the chamber is rounded. In other embodiments, the bottom of the chamber is flat or pointed.

[0228] In some embodiments, the chamber is removable from the cartridge. In some embodiments, the chamber is not removable from the cartridge.

[0229] In an embodiment, the chamber comprises a volume of about 0.1 mL to 500 mL.

[0230] In some embodiments, the chamber comprises a volume of at least about 0.1 ml, about 0.3 ml, about 0.5 ml, about 1 ml, about 1.5 ml, about 2 ml, about 2.5 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 12 ml, about 15 ml, about 17 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, about 50 ml, about 55 ml, about 60 ml, about 65 ml, about 70 ml, about 75 ml, about 80 ml, about 85 ml, about 90 ml, about 95 ml, or about 100 ml. In some embodiments, the chamber comprises a volume of no more than about 100 ml, no more than about 95 ml, no more than about 90 ml, no more than about 85 ml, no more than about 80 ml, no more than about 75 ml, no more than about 70 ml, no more than about 65 ml, no more than about 60 ml, no more than about 55 ml, no more than about 50 ml, no more than about 45 ml, no more than about 40 ml, no more than about 35 ml, no more than about 30 ml, no more than about 25 ml, no more than about 20 ml, no more than about 15 ml, no more than about 10 ml, no more than about 9 ml, no more than about 8 ml, no more than about 7 ml, no more than about 6 ml, no more than about 5 ml, no more than about 4 ml, no more than about 3 ml, no more than about 2 ml, no more than about 1 ml, no more than about 0.5 ml, no more than about 0.3 ml, or no more than about 0.1 ml. In some embodiments, the chamber comprises a volume between about 1 ml and about 100 ml, between about 10 ml and about 90 ml, between about 15 ml and about 80 ml, between about 20 ml and about 70 ml, between about 25 ml and about 60 ml, or between about 30 ml and about 50 ml.

[0231] In some embodiments, the chamber comprises at least about 150 ml, about 200 ml, about 250 ml, about 300 ml, about 350 ml, about 400 ml, about 450 ml, about 500 ml, about 550 ml, about 600 ml, about 650 ml, about 700 ml, about 750 ml, about 800 ml, about 850 ml, about 900 ml, about 950 ml, about 1000 ml, about 2000 ml, about 3000 ml, about 3500 ml, about 4000 ml, about 4500 ml, about 5000 ml, about 5500 ml, about 6000 ml, about 6500 ml, about 7000 ml, about 7500 ml, about 8000 ml, about 8500 ml, about 9000 ml, about 9500 ml, about 10000 ml, about 20000 ml, about 3 ...0 ml, about 30000000 ml, about 30000000 ml, about 30000000 ml, about 30000000 ml, about 30000000 ml, about 30000000 0ml, about 3000ml, about 4000ml, about 5000ml, about 6000ml, about 7000ml, about 8000ml, about 9000ml, about 10000ml, about 15000ml, about 20000ml, about 25000ml, about 30000ml, about 35000ml, about 40000ml, about 45000ml, or about 50000ml. In some embodiments, the chamber comprises no more than about 50,000 ml, no more than about 45,000 ml, no more than about 40,000 ml, no more than about 35,000 ml, no more than about 30,000 ml, no more than about 25,000 ml, no more than about 20,000 ml, no more than about 15,000 ml, no more than about 10,000 ml, no more than about 9,000 ml, no more than about 8,000 ml, no more than about 7,000 ml, no more than about 6,000 ml, no more than about 5,000 ml, no more than about 4,000 ml, no more than about 3,000 ml. , no more than about 2000ml, no more than about 1000ml, no more than about 950ml, no more than about 900ml, no more than about 850ml, no more than about 800ml, no more than about 750ml, no more than about 700ml, no more than about 650ml, no more than about 600ml, no more than about 550ml, no more than about 500ml, no more than about 450ml, no more than about 400ml, no more than about 350ml, no more than about 300ml, no more than about 250ml, no more than about 200ml, and no more than about 150ml.In some embodiments, the chamber comprises between about 150 ml and about 50,000 ml, between about 200 ml and about 45,000 ml, between about 250 ml and about 40,000 ml, between about 300 ml and about 35,000 ml, between about 350 ml and about 30,000 ml, between about 400 ml and about 25,000 ml, between about 450 ml and about 20,000 ml, between about 500 ml and about 15,000 ml, between about 550 ml and about 10 The present invention relates to a volume of about 1000 ml to about 2000 ml, about 600 ml to about 9000 ml, about 650 ml to about 8000 ml, about 700 ml to about 7000 ml, about 750 ml to about 6000 ml, about 800 ml to about 5000 ml, about 850 ml to about 4000 ml, about 900 ml to about 3000 ml, about 950 ml to about 2000 ml or about 1000 ml to about 1500 ml.

[0232] The chamber is used to produce a biochemical product, such as a nucleic acid molecule, a peptide, a polypeptide or a protein. Preferably, the nucleic acid molecule is RNA, more preferably mRNA. Alternatively, the nucleic acid molecule is DNA.

[0233] In an embodiment of the system, the system comprises an IVT unit for RNA molecule production.

[0234] The IVT unit includes a plurality of chambers, as disclosed in the previous embodiments, wherein each of the plurality of chambers is configured to receive one or more IVT reagents and perform an IVT reaction.

[0235] An IVT unit is to be understood as a part of the system, which regulates an IVT reaction starting from a DNA template with the aid of a DNA-dependent RNA polymerase. In some embodiments, the DNA-dependent RNA polymerase comprises at least one of T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, RNA polymerase V and a single subunit RNA polymerase. The DNA template for RNA in vitro RNA transcription can be obtained by cloning a nucleic acid (in particular a cDNA corresponding to the corresponding RNA to be transcribed in vitro) and introducing it into a suitable vector for RNA in vitro transcription (e.g. in a plasmid circulating plasmid DNA). The cDNA can be obtained by reverse transcription or chemical synthesis of mRNA. In addition, the DNA template for in vitro RNA synthesis can also be obtained by gene synthesis.

[0236] In some embodiments, the DNA template relates to a DNA molecule comprising a nucleic acid sequence encoding an RNA sequence. The template DNA is used as a template for RNA in vitro transcription to produce RNA encoded by the template DNA. Therefore, the template DNA includes all elements necessary for RNA in vitro transcription, particularly promoter elements for binding DNA-dependent RNA polymerases, such as T3, T7 and SP6 RNA polymerases 5' of the DNA sequence encoding the target RNA sequence. The poly (A) tail can be encoded into the DNA template or enzymatically added to the RNA in a separate step after in vitro transcription. In some embodiments, the template DNA includes a primer binding site 5' and / or 3' of the DNA sequence encoding the target RNA sequence, such as to determine the identity of the DNA sequence encoding the target RNA sequence by PCR or DNA sequencing. In some embodiments, the DNA template includes a DNA vector, such as a plasmid DNA, which includes a nucleic acid sequence encoding the RNA sequence. In some embodiments, the DNA template includes a linear or circular DNA molecule.

[0237] In some embodiments, the IVT unit includes at least one processing device configured to distribute and / or remove reagents, reagent mixtures or liquids in the chamber. Without limitation, the liquid distributed and / or removed can be deionized water, purified water, a buffer solution, a cleaning solution, a reagent, an enzyme, a DNA template, a transcription buffer, a solution including nucleotide triphosphates (NTPs), an RNase inhibitor or an RNA polymerase, or a combination thereof, or a waste product. Non-limiting examples of buffers are magnesium acetate, HEPES, Tris or Tris HCl. In a preferred embodiment, the liquid distributed and / or removed is an IVT premix including water, an RNA polymerase buffer, a linearized DNA template and dNTPs. In another preferred embodiment, the IVT premix includes water, a T7 polymerase buffer, a linearized DNA template and dNTPs.

[0238] The processing device of the IVT unit can be an injector or a robot arm provided with one or more nozzles, needles and / or tips. Any injector and robot arm known in the art and capable of dispensing and / or removing components or liquids can be used with the system as disclosed herein. Non-limiting examples of processing devices are syringe pumps, vacuum pumps, peristaltic pumps, centrifugal pumps or combinations thereof. In some embodiments, the pipette, micropipette, needle or tip is removable and / or disposable. In other embodiments, the pipette, micropipette, needle or tip is non-removable.

[0239] Alternatively, the dispensing and / or removal of the components is accomplished by the processing devices directly via the conduits provided by them.

[0240] In an embodiment, an IVT unit as disclosed herein comprises a robotic arm. In another embodiment, the IVT unit comprises one or more ejectors. In yet another embodiment, the IVT unit comprises one or more pumps. In yet another embodiment, the IVT unit comprises a robotic arm and one or more ejectors. In yet another embodiment, the IVT unit comprises a robotic arm and one or more pumps. In a preferred embodiment, the IVT unit comprises a robotic arm, one or more ejectors, and one or more pumps.

[0241] The processing means of the IVT unit is configured to perform a movement. In some embodiments of the IVT unit, the movement is a vertical, horizontal, centrifugal or 3D movement. In an embodiment, the processing means is controlled by a motor unit, preferably an electric motor.

[0242] In some embodiments, the IVT unit includes a control system arranged and adapted to control the dispensing and / or removal of reagents, reagent mixtures or liquids by the processing device. Based on input information (such as sensor data and predefined algorithms), the control system adjusts the performance of the processing device.

[0243] In an embodiment of the IVT unit, the processing device is adapted to dispense an amount of the IVT premix and / or one or more enzymes in said chamber.

[0244] In an embodiment, the system disclosed herein comprises a heating device for heating the IVT premix container. In an embodiment, the IVT premix container is heated at a temperature between 35°C and 55°C (preferably between 35°C and 50°C, between 35°C and 45°C, between 35°C and 40°C, or between 35°C and 37°C). Alternatively, the IVT premix container is heated at a temperature between 37°C and 55°C (preferably between 40°C and 55°C, between 45°C and 55°C, or between 40°C and 55°C). In a preferred embodiment, the premix container is heated at 37°C.

[0245] In an alternative embodiment of the system disclosed herein, the nucleic acid produced is DNA. In such an embodiment, the system includes a device that provides required reaction conditions (e.g., temperature incubation, mixing, adding and removing reagent buffers, enzymes, nucleotides, etc.) for performing PCR reactions and / or DNA synthesis on a solid support.

[0246] In another embodiment, the system is used to produce peptides, polypeptides or proteins. In such an embodiment, the system includes equipment that provides the required reaction conditions (e.g., temperature incubation, mixing, adding and removing reagent buffers, enzymes, tRNAs, aminoacyl-tRNA synthetases, initiation, extension and termination factors, energy sources (ATP, GTP), energy regeneration systems (e.g., creatine phosphate, creatine kinase, phosphoenolpyruvate or pyruvate kinase), cofactors (e.g., Mg2+, K+) and / or starting materials (e.g., DNA or RNA for PCR in vitro protein translation)).

[0247] In an embodiment, the system includes a storage unit for storing one or more reagents, and the storage unit can be cooled to a temperature below 10° C. In an embodiment, one reagent, two reagents, three reagents, four reagents, five reagents, six reagents, seven reagents, eight reagents, nine reagents or ten reagents (preferably four reagents) can be stored in the storage unit. The reagents can be selected from water, T7 reaction buffer, linearized DNA template and NTP, but are not limited to these.

[0248] In some embodiments, the storage unit may be cooled to below 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C or 1°C, preferably cooled storage is at 4°C.

[0249] The storage unit is fluidly connected to a pump system such as a peristaltic pump or a syringe pump. In further embodiments, the pump comprises a vacuum pump, a peristaltic pump, a centrifugal pump, or a combination thereof.

[0250] In an embodiment, the fluid connection is via at least one conduit. In some embodiments, the conduit comprises, and is preferably made of, a material selected from the group consisting of polyethylene (PE), nylon, urethane, copper, stainless steel, aluminum, polyvinyl chloride (PVC), polypropylene (PP), polyurethane (PU), vinyl, polyvinylidene fluoride (PVDF), fiberglass, glass, rubber, and combinations thereof. In some embodiments, the conduit is chemically resistant.

[0251] In an embodiment, the pump system is configured to provide a desired amount of one or more reagents for preparing an IVT premix to an IVT premix container.

[0252] Collection vessel

[0253] In a preferred embodiment, the system includes a collection vessel for receiving reactants from a plurality of chambers. In some embodiments, the collection vessel is in fluid communication with the chamber. In other embodiments, the collection vessel is not in fluid communication with the chamber. In some embodiments, the collection vessel is configured to store biochemical products produced in the reaction chamber. In some embodiments, the collection vessel is configured to detect contamination of the biochemical product. In some embodiments, the collection vessel includes a device for performing certain chemical, physical or mechanical treatments of the biochemical product and / or undesirable residues, such as the combination of products, partial elimination of residues or the adjustment of pH.

[0254] In an embodiment, the volume of the collecting vessel is equal to, preferably higher than, the volume of the reaction chamber. In a preferred embodiment, the volume of the collecting vessel is equal to or higher than the sum of the volumes of the reaction chambers of the cartridges. In an alternative embodiment, the volume of the collecting vessel is equal to or higher than the sum of the volumes of a plurality of cartridges (preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 cartridges).

[0255] In some embodiments, the collection vessel has a volume of 100 mL to 1000 mL, more preferably 250 mL to 500 mL, more preferably 250 mL to 350 mL, and more preferably 300 mL to 350 mL, and all ranges and subranges therebetween. The collection vessel preferably comprises a volume of about 300 ml.

[0256] The collection vessel may be of any shape or size and may be made of any suitable material known in the art, such as any glass or plastic material as disclosed in previous embodiments for purification vessels.

[0257] However, it is obvious to those skilled in the art that the systems disclosed herein for production and purification can function without a collection vessel. In such an embodiment, the contents of the reaction chamber are collected directly into a purification vessel. In such an embodiment, the system can operate in both a continuous and a discontinuous manner.

[0258] Downstream purification platform

[0259] Production and purification systems as disclosed herein can also include a downstream purification platform, for purifying biochemical products produced in multiple reaction chambers and collected in a collection vessel. The downstream purification platform connected to the production platform and the collection vessel is similar to the independent system for biochemical product purification described in the previous embodiment. However, the downstream purification platform is connected to the biochemical product production unit and the collection vessel in such a way that it allows continuous and automated production and purification of biochemical products while simulating a laboratory-based method. The downstream purification platform connected to the production unit and the collection vessel allows a small portion of the biochemical product to be continuously bound to magnetic particles, while washing and / or eluting is performed at the end. The specific construction of the purification container allowing a thin liquid layer together with the connection of the downstream purification platform with the collection vessel and the upstream production platform allows the production and purification of molecules in a continuous manner, which is more effective and provides a high yield.

[0260] The downstream purification platform of the system as disclosed herein may include: one or more mixing stations, wherein the one or more mixing stations are configured to receive the purification container and include a motor unit capable of driving the mechanical movement of the purification container; and one or more magnetic separation stations, wherein the one or more magnetic separation stations include one or more magnets, and wherein the purification platform also includes at least one robotic arm, which is controlled by a computer processor and is configured to grasp the purification container and is capable of transferring the purification container between the one or more mixing stations and the one or more magnetic separation stations.

[0261] In a preferred embodiment, the purification vessel comprises a hollow central cavity lined with a wall structure, wherein the wall structure comprises an outer wall and an inner wall, thereby defining an inner volume between the inner wall and the outer wall, wherein the wall structure is open at the top of the vessel, thereby allowing the addition of components and / or liquids to the inner volume. Thus, the purification vessel has all the characteristics of the purification vessel of the above-described embodiments.

[0262] In an embodiment, the contents of the plurality of reaction chambers are equal to or exceed the contents of the purification vessel. In a preferred embodiment, the contents of the reaction chambers of the cartridge are equal to or exceed the contents of the purification vessel. In an alternative embodiment, the contents of the purification vessel are equal to or greater than the sum of the contents of a plurality of cartridges (preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 cartridges).

[0263] The mixing station and the magnetic separation station of the downstream purification platform coupled to the biochemical product production unit and / or the collection vessel have all the characteristics of the purification container of the above-described embodiment.

[0264] Processing device

[0265] In another embodiment, the system includes a handling device for transferring the contents from the reaction chamber to the collection vessel and the purification container, and transferring the purified biochemical product to the collection vessel. In a preferred embodiment, the handling device is configured to distribute, remove and / or transfer components or liquids. In another embodiment, the handling device is configured to grasp the purification container and is capable of transferring the purification container between one or more mixing stations and one or more magnetic separation stations.

[0266] The particle component may be a magnetic particle, and the liquid may be deionized water, purified water, a buffer solution, a washing buffer, an elution buffer, a reagent or a combination thereof, or a waste product. Obviously, the liquid is not limited to these.

[0267] The treatment device can include any device suitable in the art, such as an injector or a robot arm, such as an injector or a robot arm provided with one or more nozzles, needles and / or tips. Alternatively, the distribution by the treatment device is completed directly via the pipeline of the treatment device. Any injector, pump or robot arm known in the art and capable of distributing and / or removing components or liquids can be used together with the system as disclosed herein. Non-limiting examples of treatment devices are syringe pumps, vacuum pumps, peristaltic pumps, centrifugal pumps or combinations thereof.

[0268] In an embodiment, the robotic arm grasps the purification container by the upper portion and transfers the purification container between the one or more mixing stations and the one or more magnetic separation stations. In another embodiment, the robotic arm is configured to grasp an adapter, the adapter comprising a needle and / or an injector for dispensing, removing and / or transferring components or liquids. The needle and / or injector is connected to one or more reagent storage devices, waste containers and / or collection containers. In another embodiment, the adapter comprising the needle and / or injector is placed on a rack before and after being used by the robotic arm.

[0269] In an embodiment, the ejectors and pumps are fixed dispensers placed near the reaction chamber or mixing station or magnetic separation station.In an embodiment, the robotic arm can move along three separate axes and has access to the reaction chamber, the collection vessel, the purification container and the collection vessel.

[0270] In an embodiment, at least one robotic arm is used in combination with a system as disclosed herein. In another embodiment, one or more ejectors are used in combination with the system. In yet another embodiment, one or more pumps are used in combination with the system. In yet another embodiment, a robotic arm and one or more ejectors are used in combination with the system. In yet another embodiment, a robotic arm and one or more pumps are used in combination with the system. In a preferred embodiment, a robotic arm, one or more ejectors, and one or more pumps are used in combination with the system.

[0271] The processing device used in conjunction with the system is connected to one or more reagent storage devices, waste containers and / or collecting vessels. Any reagent storage, waste containers and collecting vessels known in the art can be used in conjunction with the system. Non-limiting examples include bags, vials, pipes, bottles, jars or barrels.

[0272] In an embodiment, a processing device used in conjunction with the system is controlled by a motor unit (preferably an electric motor) and is configured to perform movement. In some embodiments, the movement is vertical, horizontal, centrifugal or 3D movement. In a preferred embodiment, the processing device is operably connected to at least one computer processor for controlling the processing device.

[0273] The system may also include a control system arranged and adapted to control the dispensing and / or removal of components or liquids by the processing device used in conjunction with the system. Based on input information (such as sample ID and sensor data and predefined algorithms), the control system adjusts the performance of the processing device.

[0274] Computer processor

[0275] In an embodiment, the system as disclosed herein further comprises at least one computer processor.In another embodiment, the computer processor is operably coupled to the robotic arm and is configured to control said robotic arm.

[0276] In another embodiment, the computer processor is operably connected to the downstream purification platform. Preferably, the computer processor includes instructions for calculating and controlling the binding time of the magnetic particles and the volume fraction. The computer processor can calculate and control the increased magnetization time of the magnetic particles, and also consider the asynchrony in the operation of the mixing station and the magnetic separation station. In order to further compensate for asynchrony and / or if the mixing station / separation station is occupied, the computer processor controls when the purification container can be kept as a reserve or introduced into an available mixing station / separation station.

[0277] In an embodiment, the computer processor comprises instructions for calculating and / or controlling the amount of the volume portion of the collection vessel to be transferred to the purification container and the transfer thereof.

[0278] In some embodiments, the computer processor may be configured to control the pooling of samples from multiple chambers in a pooling vessel. In yet another embodiment, the computer processor is configured to control the transfer of the purification container between the mixing station and the magnetic separation station.

[0279] In some embodiments, a single computer processor is coupled to multiple devices and / or apparatuses of the system and is configured to control multiple actions. In other embodiments, each device and / or apparatus of the system is controlled by a separate computer processor. In an embodiment, the system comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 computer processors.

[0280] In an embodiment, the biochemical product production equipment and the downstream processing platform are operably connected to the same computer processor. In another embodiment, the biochemical product production equipment and the downstream processing platform are operably connected to different computer processors. In yet another embodiment, multiple reaction chambers and processing devices of the biochemical product production equipment are operably connected to different computer processors. In yet another embodiment, multiple reaction chambers and processing devices of the biochemical product production equipment are operably connected to the same computer processor. In yet another embodiment, the downstream purification platform and the processing devices of the platform are operably connected to different computer processors. In yet another embodiment, the downstream purification platform and the processing devices of the platform are operably connected to the same computer processor. In yet another embodiment, all processing devices are connected to the same computer processor.

[0281] In some embodiments, the mixing station and the magnetic separation station are operated by the same computer processor, while in other embodiments, the mixing station and the magnetic separation station are operated by different computer processors.

[0282] Cabinet

[0283] Preferably, the system as disclosed herein is arranged in a cabinet, said cabinet preferably having a unit for laminar flow generation. In another embodiment, said system comprises a storage unit for storing ingredients, wherein said storage unit is located outside or inside said cabinet.

[0284] In an embodiment, the cabinet is designed to allow for provision of filtered sterile air that circulates within the unit. The air filtration device may include, for example, an HVAC system with a HEPA filter.

[0285] The housing of the cabinet can be made of any material suitable in the art, such as metal alloy, metal or plastic. In one embodiment, the cabinet is made of a material comprising aluminum or stainless steel. In a particularly preferred embodiment, the cabinet is made of a material comprising stainless steel.

[0286] Preferably, the system and cabinet are designed and operated so that they require only limited handling by the operator. This is to avoid contamination and interference with process conditions. If irregularities are observed, the operator can manipulate the process via one or more control devices present inside or outside the cabinet. These control devices control (a portion of) the process occurring in the cabinet. The cabinet may be coupled to one or more control devices configured to perform multivariate analysis, automatically control the operation of the process, and optionally, remotely communicate with components (using, for example, a network protocol) in order to control operations in the unit.

[0287] Each cabinet is preferably mobile and provided with a transport device. The transport device may include any device suitable for manual and / or electronic control in the art, and includes but is not limited to wheels, tracks or rollers.

[0288] Purification or treatment vessels

[0289] In a third aspect, the utility model relates to a purification container, the purification container comprising a (hollow) central cavity lined with a wall structure, wherein the wall structure comprises an outer wall and an inner wall, thereby defining an internal volume between the inner wall and the outer wall, wherein the wall structure is open at the top of the container, thereby allowing the addition of components and / or liquids to the internal volume. The purification container has a shape, form and volume as described in any of the previous embodiments, and is made of a material as described in any of the previous embodiments.

[0290] The processing or purification vessel is preferably open at the top, or is configured to be open when liquids or components are added or removed from the vessel. In an embodiment, the processing or purification vessel is completely open at the top. In another embodiment, the processing or purification has apertures or holes at the top. In yet another embodiment, the processing vessel is not open at the top, but has a cover that is removable, slidable, openable, etc.

[0291] In a preferred embodiment of the purification vessel as disclosed herein, the distance between the outer wall and the inner wall is between 3 mm and 75 mm, between 4 mm and 75 mm, between 5 mm and 75 mm, between 6 mm and 75 mm, between 7 mm and 75 mm, between 8 mm and 75 mm, between 9 mm and 75 mm, between 10 mm and 75 mm, between 15 mm and 75 mm, between 20 mm and 75 mm, between 25 mm and 75 mm, between 30 mm and 75 mm, between 35 mm and 75 mm, between 40 mm and 75 mm, between 45 mm and 75 mm, between 50 mm and 75 mm, between 55 mm and 75 mm, between 60 mm and 75 mm, between 65 mm and 75 mm, or between 70 mm and 75 mm, and all ranges and sub-ranges therebetween.

[0292] Alternatively, the distance between the outer wall and the inner wall of the purification container is between 3 mm and 70 mm, between 3 mm and 65 mm, between 3 mm and 60 mm, between 3 mm and 55 mm, between 3 mm and 50 mm, between 3 mm and 45 mm, between 3 mm and 40 mm, between 3 mm and 35 mm, between 3 mm and 30 mm, between 3 mm and 25 mm, between 3 mm and 20 mm, between 3 mm and 15 mm, between 3 mm and 10 mm, between 3 mm and 9 mm, between 3 mm and 8 mm, between 3 mm and 7 mm, between 3 mm and 6 mm, between 3 mm and 4 mm, and all ranges and sub-ranges therebetween.

[0293] The shape and configuration of the purification container allow for multiple purification cycles, i.e., binding and liquid removal, while omitting elution and / or washing steps therebetween. Elution is performed once only when at least a portion of the contents of the collection vessel have been subjected to at least one purification cycle. This significantly increases the yield of the biochemical product obtained, reduces the time required for processing, and reduces the amount of buffer required during purification, which reflects the reduction in the use of harmful agents (such as ethanol) commonly used in practice. Due to the small distance between the inner wall and the outer wall, the magnetic particles in the purification container are close to the magnet, so even when saturated with the biochemical product of interest, they can be attached to the magnet in a relatively short time.

[0294] 1. A method for purifying a liquid sample comprising one or more nucleic acid molecules, wherein the purification comprises removing or exchanging at least a portion of the liquid sample, and wherein the purification comprises a magnetic purification step, wherein the nucleic acid molecules are bound to magnetic particles in a purification container during a binding step, after which the unbound liquid portion of the sample is removed and, optionally, the magnetic particles bound to the nucleic acids are washed by a washing buffer, wherein the magnetic particles are present in a liquid layer in the purification container, the liquid layer having a layer thickness of less than 75 mm.

[0295] 2. The method according to embodiment 1, wherein the layer thickness is between 3 mm and 70 mm.

[0296] 3. The method according to embodiment 1 or 2, wherein the liquid layer is formed between the outer wall and the inner wall of the purification container.

[0297] 4. The method according to any one of embodiments 1 to 3, wherein a certain amount of magnetic particles is added to or present between the outer wall and the inner wall of the purification container.

[0298] 5. A method according to any one of the preceding embodiments, wherein the magnetic particles and nucleic acid molecules in the purification container are mixed by subjecting the purification container to mechanical movement, preferably by positioning the purification container on a mixing station so that the purification container is subjected to mechanical movement.

[0299] 6. A method according to any of the preceding embodiments, wherein the magnetic binding step occurs at a magnetic separation station comprising one or more magnets, wherein the purification container comprising the magnetic particles and the nucleic acid sample is positioned at the magnetic separation station, and wherein the magnetic particles are attracted to the magnets during a predefined magnetization time.

[0300] 7. The method according to any of the preceding embodiments, wherein the purification container is moved between the mixing station and the magnetic separation station by means of a robotic arm, the robotic arm being configured to grip the purification container.

[0301] 8. The method according to any one of the preceding embodiments, wherein the nucleic acid molecule is DNA or RNA.

[0302] 9. A method according to embodiment 8, wherein the nucleic acid molecule is produced in multiple nucleic acid reactions, preferably multiple in vitro transcription reactions.

[0303] 10. The method according to embodiment 9, wherein at least a portion of the multiple nucleic acid molecules generated are pooled in a pooling vessel.

[0304] 11. The method according to embodiment 10, wherein at least a volume portion of the pooled nucleic acid sample from the pooling vessel is transferred to the purification container and subjected to a purification step.

[0305] 12. A method according to embodiment 10, wherein an additional volume portion of the pooled nucleic acid sample is transferred to the purification container, which contains nucleic acids bound to magnetic particles, and the portion is subjected to a binding step.

[0306] 13. A method according to embodiment 12, wherein the magnetization time of the magnetic particles to the magnet increases with the number of purification cycles.

[0307] 14. A system for performing purification of nucleic acid molecules with the aid of magnetic particles, wherein the system comprises: one or more purification containers; one or more mixing stations, the one or more mixing stations being configured to receive the purification containers and comprising a motor unit capable of driving mechanical movement of the purification containers; and one or more magnetic separation stations, the one or more magnetic separation stations comprising one or more magnets, and wherein the system further comprises at least one robotic arm, the at least one robotic arm being controlled by a computer processor and being configured to grip the purification containers and to transfer the purification containers between the one or more mixing stations and the one or more magnetic separation stations, wherein the purification container comprises a hollow central cavity defined by a wall structure, wherein the wall structure comprises an outer wall and an inner wall, thereby defining an internal volume between the inner wall and the outer wall, wherein the wall structure is open at the top of the container, thereby allowing the addition of components and / or liquids to the internal volume.

[0308] 15. The system of embodiment 14, wherein the distance between the outer wall and the inner wall of the purification container is between 3 mm and 70 mm.

[0309] 16. The system of any one of embodiments 14 or 15, wherein the purification vessel comprises a sloped bottom configured to allow the liquid added to the interior volume of the purification vessel to have multiple heights throughout the purification vessel.

[0310] 17. The system of any one of embodiments 13 to 15, wherein the one or more mixing stations and / or one or more magnetic stations comprise a raised portion configured to receive the hollow central cavity of the purification vessel.

[0311] 18. The system of embodiment 16, wherein the raised portion of the magnetic separation station comprises one or more magnets lining the raised portion.

[0312] 19. The system of any one of embodiments 13 to 17, wherein the magnetic separation station comprises a base comprising one or more magnets positioned at the base.

[0313] 20. The system of embodiment 13, wherein the computer processor comprises instructions for calculating and controlling magnetization time of magnetic particles and magnets.

[0314] 21. A purification vessel comprising a hollow central cavity defined by a wall structure, wherein the wall structure comprises an outer wall and an inner wall, thereby defining an interior volume between the inner wall and the outer wall, wherein the wall structure is open at the top of the vessel to allow the addition of components and / or liquids to the interior volume.

[0315] Example

[0316] Example 1 RNA production and purification

[0317] IVT reactions were performed in multiple chambers. The contents of 10 reaction chambers (including 20 ml of IVT product) were pooled in a pooling vessel to which EDTA and DNase were added. The resulting mixture of IVT in the pooling vessel included a volume of 250 ml, with an mRNA concentration of 4 mg / ml to 7 mg / ml.

[0318] A volume portion of the 250 ml IVT mixture was added to a processing vessel including 3.5 g to 4 g of silica-coated magnetic beads and subjected to a mixing and binding step. This process was repeated until the entire volume of the 250 ml IVT mixture was used from the pooling vessel. The entire purification cycle of the pooling vessel took 100 minutes and produced 1559 mg of purified mRNA.

[0319] The purification cycle can be continuously repeated as long as the IVT reactor performs the IVT reaction and purifies 935.40 mg RNA / h.

[0320] Example 2 Ratio between the magnetic surface of a purification container and the volume of the liquid inside the container

[0321] The magnetic surface of the purification container according to an embodiment of the present invention was measured, and the ratio between the surface and the volume of the liquid inside the container during both elution and binding operations was calculated (Table 1).

[0322] The same ratio is calculated for the surface area of ​​other containers known in the art to their maximum volume capacity. The container is a standard 1500 μl Tube, 50ml standard Tube and 200ml bottle.

[0323] The container according to the invention has a ratio between the magnetic surface of the purification container and the volume of the liquid inside the container, which is between 0.7 and 0.8 (cm -1 ), which is a ratio smaller than that of other containers used in the art (Table 1), and this increases the magnetization efficiency of the magnetic particles to the magnet.

[0324] Furthermore, the container according to the invention ensures that the maximum distance of the liquid inside the purification container from the magnets of the magnetic station is 0.9 cm, which ensures strong magnetization and low time required for said magnetization. Using the same volume in the prior art container, the distance of the liquid inside the station from the magnetic station is higher.

[0325] Table 1

[0326]

[0327] Description of the drawings

[0328] The present invention will now be further illustrated with reference to the following embodiments. The present invention is in no way limited to the embodiments presented in the given examples or drawings.

[0329] Figure 1 is a top view of a processing or purification container according to an embodiment of the purification system. The container 1 comprises a wall structure comprising an outer wall 3 and an inner wall 2, thereby defining a processing space 4. The wall structure is open at the top of the container, thereby allowing components and / or liquids to be added to the inner space. The liquid can be a liquid sample and / or a buffer, and the component can be a magnetic particle. In some embodiments, the distance 8 between the outer wall and the inner wall is less than 75 mm. The magnetic particles are therefore present in the purification container as a thin layer.

[0330] exist Figure 1 In the embodiment, the container comprises a knob 5 having a shape suitable for being grasped by a robot arm. It will be apparent to those skilled in the art that other adapters may be applied to the purification container so as to make it suitable for transport / movement / grasping by a robot arm.

[0331] like Figure 2 In the embodiment shown in FIG. 1 , the container 1 has a hollow central cavity 6 open at the bottom of the container and lined with an inner wall 2. The processing space 4 between the wall structures is closed at the bottom and has a bottom wall 9. In some embodiments, the inner wall 2 of the container may have a recess 7. This recess has an orientation purpose when the purification container is assembled on the raised part of the mixing station 11 and / or the magnetic separation station 16.

[0332] like Figure 3 As shown, the container 1 has a slope of the bottom wall, which is configured so that the liquid added to the processing space 4 has a liquid level difference of 10', 10" in the entire purification container. Figure 3 In an embodiment, the inner wall of the purification container has uneven heights 2', 2" which result in a slope of the bottom wall. The slope of the bottom wall 9 of the purification container and the slope of the base of the magnetic separation station result in the liquid in the purification container having a difference in liquid level 10', 10". Removal of the liquid is preferably completed where the liquid sample has a maximum liquid level 10', and is preferably avoided where the liquid sample has a minimum liquid level 10".

[0333] like Figure 4A As shown, the mixing station 11 comprises a base 12 having a raised portion 13. The base is positioned on a motor unit that drives the movement of the mixing station. Figure 4B As shown, a processing or purification vessel (eg, vessel 1) fits onto a raised portion 13 of a mixing station. In some embodiments, the raised portion 13 of the mixing station has a notch 15 for properly positioning the purification vessel.

[0334] like Figure 5A As shown, the magnetic separation station 16 includes a base 17 on which a raised portion 20 is placed. In a preferred embodiment, the raised portion is lined with a plurality of magnets 18. The raised portion is provided with a plurality of screw holes 19. After the magnets 18 are inserted into the raised portion, they are held in place by a cover 31 secured in place with screws 32, as shown in FIG. Figure 6 As shown. Figure 5B As shown, a purification vessel (eg, vessel 1) fits onto a raised portion 20 of a magnetic separation station. In some embodiments, the raised portion of the magnetic separation station 16 has a notch 21 for properly positioning the purification vessel.

[0335] like Figure 6 As shown, in some embodiments, the magnetic separation station includes a base 17, which includes one or more magnets 33 positioned at the base. The base 17 of the magnetic separation station has a slope. The inclined base of the magnetic separation station allows the liquid in its purification container to have a liquid level difference. Therefore, the processing device (e.g., a robotic arm) is configured to preferably remove the liquid from the area of ​​the purification container with the highest height of the liquid. The magnet is preferably positioned on the base in the area corresponding to the area of ​​the purification container with the highest height of the liquid.

[0336] Figure 7A system 22 for producing and purifying nucleic acid molecules is shown, the system comprising a plurality of reaction chambers 23. In some embodiments, the reaction chambers are IVT reaction chambers. The system 22 also comprises a collection vessel 24 in which the contents of the plurality of reaction chambers are collected. The system also comprises a mixing station 11, one or more magnetic separation stations 16, and one or more purification containers (e.g., container 1) maintained as a reserve. In addition, the system comprises one or more robotic arms 25, the one or more robotic arms 25 being configured to move the purification containers and to add / remove components or liquids in the purification containers. In other embodiments, the system may comprise other handling devices, such as pumps or injectors and / or reagent storage containers, temperature control devices, etc.

[0337] Fig. 8A A support frame is shown holding an adapter 27, which is equipped with a needle 28 for liquid introduction or aspiration. The adapter 27 is connected to a waste or reactant vessel 30 via a tube 29. The adapter has a shape suitable for being grasped by a robotic arm ( Figure 8B ).

[0338] In some embodiments, a processing or purification vessel (e.g., vessel 1) may include a plurality of processing spaces 4. Fig.9A and Fig. 9B As shown, the processing or purification container with a nested arrangement structure includes an internal processing space 4' and an external processing space 4" and a central cavity between the internal processing space and the external processing space, and the central cavity is configured to receive the protruding portion of the mixing station and / or magnetic separation station 16.

[0339] In some embodiments, the raised portion of the magnetic separation station 16 and / or mixing station surrounds the exterior of the processing or purification vessel (e.g., vessel 1). Fig. 9B In the embodiment of the present invention, magnet 18 is positioned outside the processing or purification container. In such an embodiment, the raised portion is annular. Obviously, the shape of the ring is complementary to the shape of the outer wall of the processing or purification container.

[0340] In some embodiments, the mixing of the liquid sample and the magnetic particles inside the processing or purification container is ensured by the rotation of at least one wall of the purification or treatment container. In other embodiments, the inner wall 2 rotates, thereby driving the mixing of the contents of the purification or treatment container ( Fig. 10C In other embodiments, the outer wall 3 rotates, thereby driving mixing of the contents of the purification or treatment vessel ( Fig. 10B In yet another embodiment, both walls (e.g., inner wall 2 and outer wall 3) rotate, thereby driving mixing of the contents of the purification or treatment vessel ( Fig. 10A ). The wall of the container configured to perform the rotation is a ball bearing 34 and is preferably circular.

[0341] The magnets can be arranged in various shapes in the raised part of the magnetic separation station. Fig.11 In the embodiment, the magnets 18 are arranged in two separate rows 35. The magnets in one row can be operated independently of the magnets in the other row.

[0342] The present invention is by no means limited to the embodiments described in the examples and / or shown in the drawings. On the contrary, the method according to the present invention can be implemented in many different ways without departing from the scope of the present invention.

[0343] 1-Purification or treatment container

[0344] 2-Inner wall

[0345] 2' - inner wall with minimum height

[0346] 2" - Inner wall with maximum height

[0347] 3-Outer wall

[0348] 4- Processing Space

[0349] 4' - Internal processing space

[0350] 4” - External Processing Space

[0351] 5-Knob

[0352] 6-Center cavity

[0353] 7- Notch for purification container

[0354] 8- Distance between outer wall and inner wall

[0355] 9-Bottom wall

[0356] 10′-Maximum liquid level of liquid sample

[0357] 10" - minimum level for liquid samples

[0358] 11-Mixing Station

[0359] 12- Base of the mixing station

[0360] 13- Raised part of the mixing station

[0361] 14-Motor unit

[0362] 15-Notch for mixing station

[0363] 16-Magnetic separation station

[0364] 17- Base of magnetic separation station

[0365] 18-Magnet lining the raised part

[0366] 19-Screw hole

[0367] 20- Raised part of magnetic separation station

[0368] 21-Notch for magnetic separation station

[0369] 22-Systems for nucleic acid production and purification

[0370] 23-Multiple reaction chambers

[0371] 24 Collection Vessels

[0372] 25-Robot Arm

[0373] 26 Support rack

[0374] 27-Adapter

[0375] 28-needle

[0376] 29-Tube

[0377] 30-Waste or reaction vessels

[0378] 31- Cover

[0379] 32-Screw

[0380] 33- Magnet located at the base of the magnetic separation station

[0381] 34-Ball bearing

[0382] Rows of 35 magnets.

Claims

1. A system for processing biochemical products by means of magnetic particles, characterized in that The system comprises: one or more processing vessels; one or more mixing stations configured to receive the one or more processing containers, and the one or more mixing stations comprising a motor unit configured to drive mechanical movement of the one or more processing containers; one or more magnetic separation stations, the one or more magnetic separation stations comprising one or more magnets, wherein the one or more processing containers include a wall structure defining a central cavity, wherein the wall structure includes an outer wall and an inner wall, thereby defining an interior space between the outer wall and the inner wall, and wherein the wall structure is configured to receive a liquid sample and retain the liquid sample in the interior space.

2. The system for processing biochemical products by means of magnetic particles according to claim 1, characterized in that The distance between the outer wall and the inner wall of the one or more processing containers is less than 80 mm.

3. The system for processing biochemical products by means of magnetic particles according to claim 1, characterized in that The distance between the outer wall and the inner wall of the one or more processing containers is between 3 mm and 75 mm.

4. The system for processing biochemical products by means of magnetic particles according to any one of claims 1 to 3, characterized in that The one or more processing containers include a bottom wall, wherein the bottom wall has an inclination that causes a level difference of liquid present in the inner space of the one or more processing containers.

5. The system for processing biochemical products by means of magnetic particles according to any one of claims 1 to 3, characterized in that The one or more mixing stations and / or the one or more magnetic separation stations include a raised portion configured to receive the central cavity of the one or more processing vessels.

6. The system for processing biochemical products by means of magnetic particles according to claim 5, characterized in that The raised portion of the one or more magnetic separation stations includes one or more magnets lining the raised portion.

7. The system for processing biochemical products by means of magnetic particles according to any one of claims 1 to 3, characterized in that The one or more magnetic separation stations include a base including one or more magnets positioned at the base.

8. The system for processing biochemical products by means of magnetic particles according to any one of claims 1 to 3, characterized in that The system includes one or more robotic arms configured to grip the one or more processing containers and transfer the one or more processing containers between the one or more mixing stations and the one or more magnetic separation stations.

9. The system for processing biochemical products by means of magnetic particles according to any one of claims 1 to 3, characterized in that The system includes a pooling vessel for pooling at least a portion of a plurality of biochemical product samples generated by a plurality of in vitro transcription reactions, each reaction resulting in a biochemical product sample.

10. The system for processing biochemical products by means of magnetic particles according to any one of claims 1 to 3, characterized in that The system is configured for processing a liquid sample comprising one or more biochemical products.

11. The system for processing biochemical products by means of magnetic particles according to claim 10, characterized in that The biochemical product is a nucleic acid molecule.

12. The system for processing biochemical products by means of magnetic particles according to claim 10, characterized in that The biochemical product is RNA or DNA.

13. The system for processing biochemical products by means of magnetic particles according to claim 9, characterized in that The system is coupled to an upstream production system for producing the biochemical product sample.

14. System for processing biochemical products by means of magnetic particles according to claim 13, characterized in that The upstream production system includes a plurality of reaction chambers for producing the biochemical product in a plurality of reactions.

15. A processing container, characterized in that: The processing vessel comprises a central cavity lined with a wall structure, wherein the wall structure comprises an outer wall and an inner wall, thereby defining an interior space between the inner wall and the outer wall, wherein the wall structure is open at the top of the processing vessel to allow the addition of components and / or liquids to the interior space.

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