Ribonucleic acid (RNA)

Abstract

The invention relates to ribonucleic acid (RNA), and to methods for synthesizing RNA. More specifically, the invention relates to methods for preparing RNA that is formulated in a carrier, such as a lipid nanoparticle (LNP). The invention is especially concerned with an apparatus and a system, and consumable components therefore, for the production of RNA and / or formulated RNA, such as RNA encapsulated in an LNP. The invention further relates to methods to utilize the apparatus and consumable for clinical use, and ideally in compliance with current GMP regulations. The invention further relates to methods for manufacturing the apparatus, and to methods for testing the integrity of the apparatus. Finally, the invention also relates to methods of quality control and a method to use self-optimizing feedback loops.

Description

[0001] Ribonucleic acid (RNA)

[0002] The invention relates to ribonucleic acid (RIMA), and particularly, although not exclusively, to methods for synthesizing RNA. More specifically, the invention relates to methods for preparing RNA that is formulated in a carrier, such as a lipid nanoparticle (LNP). The invention is especially concerned with an apparatus and a system, and consumable components therefor, for the production of RNA and / or formulated RNA, such as RNA encapsulated in an LNP. The invention further relates to methods to utilize the apparatus and consumable for clinical use, and ideally in compliance with current GMP regulations. The invention further relates to methods for manufacturing the apparatus, and to methods for testing the integrity of the apparatus, and to methods for ratiometrically-defined mixing of two or more fluid streams. The invention also relates to methods of quality control and a method to use self-optimizing feedback loops.

[0003] All living organisms make use of DNA to store their genetic information. Most of these genes code for specific proteins which control all cellular functions, including metabolic processes, cellular signalling to allow cells to respond to their environment, the structure of cells and ultimately the organism, and also intercellular communication through cellcell contacts and hormones. Key to translating the genetic information from the level of DNA into proteins is a molecule called messenger RNA (mRNA). Each gene is first transcribed into an mRNA which is then translated through ribosomes into a protein. The key elements of this process are very similar across all kingdoms of life from bacteria to fungi to plants to animals. Following transcription of the mRNA, so-called posttranscriptional modifications occur that further modify the primary mRNA structure, however, these posttranscriptional processes and / or their functional implications differ between kingdoms of life.

[0004] Messenger RNA is a polymer of ribonucleic acids with a backbone formed from the pentose sugar ribose and phosphate. The nucleobases adenine, guanine, cytosine and uridine are attached to these sugars via the l'C-atom. The resulting nucleosides are coupled to each other via phosphate molecules that form esters with the hydroxy group of the 3'C-atom of one ribose and of the hydroxy group of the 5'C-atom of another ribose. The 5'- and 3'C-atoms give the mRNA molecule a directionality. During transcription, the mRNA is synthesized from 5' to 3' along the DNA template. Also, translation through the ribosome occurs from 5' to 3'. One unit of the mRNA polymer consisting of nucleoside and phosphate is called a nucleotide.

[0005] As one of the structural modifications, for all eukaryotic mRNAs, a stretch of adeninenucleotides is attached to the 3'-end of the mRNA molecule. This poly-A tail plays a key role for controlling the lifetime and expression of the mRNA as the poly-A tail is bound through proteins that protect the mRNA from degradation by RNA exonucleases. Poly-A tails also occur in prokaryotes, however, in this case rather destabilize than stabilize mRNA.

[0006] A further important modification of the mRNA is the addition of a so-called 5' cap structure, which is a chemical modification of the 5'-end of the mRNA. The 5' cap is required to initiate assembly of ribosomes at the mRNA and thus protein expression from the mRNA. Bacteria and eukaryotes differ in the structure of the cap and these structural differences also form the basis for the recognition of foreign mRNA through the innate immune system of higher organisms as explained, below. Importantly, in eukaryotes, the 5' cap is added in the nucleus which means that all cytosolic mRNA that does not carry a proper 5' cap is most likely of foreign (pathogen) origin and so potentially poses a threat.

[0007] Next to mediating initiation of translation through ribosome binding, the 5' cap also protects the mRNA from 5'-to-3' exonucleases, and prevents the activation of 5' phosphate triggering of RIG-I, which mediates a potent intracellular anti-viral response. Considering that (i) mRNA can code for virtually any protein and that (ii) the presence of mRNA in the cytosol is transient, thereby not posing the risk of permanent changes of the genome, and also that (iii) mRNA, unlike DNA, does not have to enter the nucleus to show expression, thus can also be applied to non-dividing, differentiated cells, synthetic mRNA introduced into cells may be considered a highly interesting modality to yield cellular protein expression. In 1990, it was first demonstrated that injection of naked mRNA yielded an immune response against the protein that the mRNA encoded for, thus demonstrating that mRNAs could be applied in vivo as a message for protein production (Wolff JA, et al. Science. 1990;247: 1465-1468). Two years later, it was also demonstrated that in vivo delivery of mRNA could yield expression of a bioactive protein (Jirikowski GF, et al. Science. 1992;255 :996-998).

[0008] However, it took more than twenty years until the full potential of mRNA as a highly valuable molecular modality in medicine and biotechnology was more broadly recognized. This reservation was a result of the fact that mRNA was broadly recognized as a biologically very unstable molecule that is rapidly degraded by RNases which are omnipresent in the body and also on the skin and thus complicate handling of mRNA. Furthermore, because of its large molecular weight and negative charge, delivery vehicles had to be developed that complex with the mRNA and thereby protect the mRNA from degradation and mediate cellular entry and cytosolic delivery. Finally, at about the same time that exploration of mRNA as a therapeutic modality started, also mechanisms of innate immunity were discovered that showed that exogenously added mRNA can act as a strong immunostimulatory molecule that causes inflammatory reactions and shuts down cellular protein biosynthesis as explained above.

[0009] It, therefore, became clear that in vitro transcription of mRNA was more challenging than initially thought, as only mRNA that mimics as closely as possible endogenous mRNA can avoid recognition through innate immunity receptors and thus yield high protein expression without side effects. Therefore, innovations that improve the in vitro synthesis of mRNA, including synthesis yields, and also structural changes that reduce innate immunity are of enormous economic value, as exemplified by the patent applicants for inclusion of chemically modified bases (PCT / US06 / 32372 - RNA containing modified nucleosides and methods of use thereof) and of modified 5' cap structures (US7074596B2 - Synthesis and use of anti-reverse mRNA cap analogues).

[0010] Following the break-through success of the mRNA-based SARS-CoV2 vaccines, Comirnaty (marketed by Pfizer / BioNTech) and SpikeVax (marketed by Moderna), which were made possible through the above-mentioned insights into mRNA biology, RNA (in particular, mRNA) is considered to be one of the fastest growing molecular modalities in therapy development and biotechnology with a multi-billion dollar market, the ultimate scope of which is still difficult to predict but which will, to a major extent, depend on costs-of-goods and safety. Costs-of-goods directly relate to methods of manufacturing, and safety to the avoidance of side products which also includes incompletely 5'-capped mRNAs. In particular, for applications in medicine that require more frequent applications of larger amounts of mRNA as for enzyme replacement therapy where the mRNA codes for proteins that a patient is missing due to a genetic defect, both factors will be crucial for the long-term success of therapeutic mRNA.

[0011] In addition to playing a role during regular commercial manufacturing, cost-effectiveness and quality of manufacturing also play a role during pandemic response. Current systems capable of pandemic response are unique suited for the manufacturing of large amounts of RNA drug substance or drug product. In non-pandemic situations and a developing RNA market, such large-scale systems are typically severely under used and carry a heavy cost to the operator, in part of the requirement of frequent updates, cleanings and other activities to remain pandemic prepared. Therefore, there is an urgent need for systems that can scale from manufacturing smaller amounts of RNA, relevant to research and clinical use, to manufacturing for pandemic response, so-called use. Novel continuous flow and fed-batch systems are first attempts at making RNA manufacturing systems more scalable, but cannot thus far claim true scalability, wherein all relevant process parameters, including flow rate remain constant.

[0012] One of the main issues of current RIMA manufacturing systems is that the multi-step process is disconnected over a variety of containers, equipment and locations (within a manufacturing environment). Each transfer is sensitive to contamination and crosscontamination, which can ruin a batch. These issues become increasingly evident in multi-product facilities, wherein the separation of the manufacturing of each individual product in separate cleanrooms becomes cost-inefficient. Especially when entering the era of personalized medicine, there is a great need for equipment that can manufacture one or more products in a completely closed manner, preventing (Cross)contamination. Such system may also negate the need for cleaning of the cleanroom in between batches of the same of different RNA products, and thus improves turn-around time.

[0013] Furthermore, progress in the clinic of a new modality like RNA depends on the correlation between pre-clinical and clinical outcomes, which is often complicated by process-related impurities in non-GMP manufacturing lots. Therefore, there is an urgent need for a system that can produce both small and large quantities of RNA at equal quality, preferably with automatically generated GMP-ready documentation. The availability of constant quality, always accompanied with future-proof documentation will aid in rapidly developing new therapeutics with RNA.

[0014] There is, therefore, a need to provide improved methods and apparatus for in vitro transcription, optional modification and formulation of RNA that enable to seamlessly scale from amounts relevant for discovery and early research, to amounts relevant for commercial operation and even pandemic preparedness. Further, there is an urgent need for methods and apparatus for such methods that enable performing the manufacturing of RNA and formulations thereof (e.g. encapsulation in an LNP) in a manner sufficiently closed as to avoid contamination with microbial elements and RNAses, and cross-contaminations with other products produced in the same multiproduct manufacturing site. Also, there is an urgent need for methods, and apparatus for such methods, that shorten the turn-around time and / or accelerate the optimization of the protocol for the manufacturing of RNA and formulations thereof. Finally, there is an urgent need for apparatus and associated consumables that facilitate the manufacturing of RNA in remote, challenging environments, and by less experienced personnel.

[0015] The inventors have devised a novel method for the manufacturing and optionally formulating RNA utilizing a consumable microfluidic cartridge allowing precise and autonomous mixing and separation of the reaction mixtures. However, as a consequence, the volume of such microfluidic reactions are only sufficient for small scale manufacturing needs. They have, therefore, provided a protocol for continuous RIMA synthesis in seamlessly scalable reservoirs included on the microfluidic device. The microfluidic device with integrated reservoirs enables the prevention of (cross-) contamination and multi-product manufacturing sites by means of its closed nature.

[0016] Accordingly, in a first aspect of the invention, there is provided a method of preparing an RNA molecule, optionally capped, or a formulation thereof, wherein the method comprises:

[0017] (i) contacting a template DNA molecule with raw materials of a DNA amplification reaction and incubating the resultant reaction mixture to amplify the DNA molecule, and optionally modifying the amplified DNA molecule;

[0018] (ii) extracting the amplified, and optionally post-amplification modified, DNA molecule from the reaction mixture;

[0019] (iii) contacting the extracted amplified DNA molecule from (ii) with raw materials of an in vitro transcription (IVT) reaction and incubating the resultant reaction mixture to produce a transcribed RNA molecule;

[0020] (iv) extracting the transcribed RNA molecule, and optionally removing residual DNA;

[0021] (v) optionally post-modifying the extracted RNA molecule;

[0022] (vi) optionally contacting the transcribed RNA molecule from (iv) or (v) with a buffer solution and contacting the buffered RNA with one or more delivery component to produce formulated RNA, and

[0023] (vii) subjecting the naked RNA from (iv or v) or the formulated RNA from (vi) to one or more purification step to prepare an RNA molecule, optionally capped, or a formulation thereof.

[0024] The formulated RNA may comprise a delivery vehicle encapsulating the RNA.

[0025] In one embodiment, the method comprises continuously preparing an RNA molecule. As such, step (ii) may comprise continuously extracting the amplified, and optionally postamplification modified, DNA molecule from the reaction mixture. Step (ii) may comprise continuously extracting the transcribed RNA molecule, and optionally removing residual DNA. The method may continue for at least one hour, two hours, three hours or more. The

[0026] In another embodiment, however, the method comprises intermittently preparing an RNA molecule. As such, step (ii) may comprise intermittently extracting the amplified, and optionally post-amplification modified, DNA molecule from the reaction mixture. Step (ii) may comprise intermittently extracting the transcribed RIMA molecule, and optionally removing residual DNA.

[0027] The method may continue for at least one hour, two hours, three hours or more. The method may continue for at least five hours, ten hours, fifteen hours or more. The method may continue for at least twenty hours, twenty five hours, fifteen hours or more.

[0028] As shown in Figure 1, the inventors have also developed a sophisticated apparatus for performing the method of the first aspect.

[0029] Therefore, in a second aspect, there is provided a microfluidic RNA manufacturing apparatus for preparing an RNA molecule, or a formulation thereof, the apparatus comprising :

[0030] (i) one or more DNA amplification chamber in which, in use, a template DNA molecule is amplified to produce amplified DNA, and optionally in which the amplified DNA is modified;

[0031] (ii) one or more IVT chamber in which, in use, in vitro transcription (IVT) is performed to produce transcribed RNA, and optionally in which the transcribed RNA is modified;

[0032] (iii) optionally one or more purification chamber or channel in which, in use, the RNA is purified;

[0033] (iv) one or more DNA substrate reservoir for containing DNA substrates, or one or more DNA substrate connector connectable to a DNA substrate reservoir, wherein the DNA substrates, in use, are required for amplifying DNA;

[0034] (v) one or more RNA substrate reservoirs for containing RNA substrates, or one or more RNA substrate connector connectable to an RNA substrate reservoir, wherein the RNA substrates, in use, are required for performing IVT;

[0035] (vi) one or more RNA product storage reservoir or one or more RNA product connector connectable to an RNA product storage reservoir;

[0036] (vii) one or more DNA microfluidic channel along which, in use, DNA substrates are fed from the one or more DNA substrate reservoir to the one or more DNA amplification chamber and / or the one or more IVT chamber;

[0037] (viii) one or more RNA microfluidic channel along which, in use, RNA substrates are fed from the one or more RNA substrate reservoir to the one or more IVT chamber;

[0038] (ix) one or more amplified DNA microfluidic channel along which, in use, amplified DNA is fed from the one or more DNA amplification chamber, to the one or more IVT chamber; and (x) one or more transcribed RIMA microfluidic channel along which, in use, transcribed RNA is fed from the one or more IVT chamber to an optional purification chamber and / or the RNA product storage reservoir.

[0039] As shown in Figure 2, the apparatus may comprise two or more substrates which are attached, joined or fused together defining a layer therebetween. Each substrate may comprise or consist of a substantially planar plate. Two or more substrates, when attached, joined or fused together, define the one or more channel, reservoir and / or chamber therebetween. Additionally, or alternatively, the two or more substrates may independently comprise one or more channel therethrough, the one or more channel being configured to fluidly connect two or more layers and / or a layer to an external fluid source.

[0040] In some embodiments, the apparatus comprises at least three, four or five substrates, which are attached, joined or fused together defining layers therebetween. The substrates may or may not comprise the same area. As can be seen in the embodiment shown in Figure 2, for example, a first and a second substrate comprise substantially the same area, whereas the area of other substrates is less. In an embodiment, the apparatus comprises at least two, three or four layers of attached, joined or fused substrates.

[0041] In one embodiment, the apparatus is for continuously preparing an RNA molecule. As such, the apparatus is configured to continuously extract the amplified, and optionally post-amplification modified, DNA molecule from the reaction mixture. The apparatus is configured to continuously extract the transcribed RNA molecule, and optionally remove residual DNA.

[0042] In another embodiment, the apparatus is for intermittently preparing an RNA molecule. As such, the apparatus is configured to intermittently extract the amplified, and optionally post-amplification modified, DNA molecule from the reaction mixture. The apparatus is configured to intermittently extract the transcribed RNA molecule, and optionally remove residual DNA.

[0043] The apparatus may further comprise:

[0044] (xi) one or more RNA formulation reservoirs for containing one or more delivery component, wherein the one or more delivery component, in use, is required for formulating the RNA; (xii) one or more mixer configured, in use, to mix transcribed RIMA and one or more delivery component to provide formulated RNA;

[0045] (xiii) one or more further transcribed RNA microfluidic channel along which, in use, RNA substrates are fed from the RNA product storage reservoir to the one or more mixer; and

[0046] (xiv) one or more delivery component microfluidic channel along which, in use, the one or more delivery component is fed from the one or more RNA formulation reservoir, to the one or more mixer.

[0047] The mixer may be a microfluidic mixer. Accordingly, the mixer may comprise one or more microfluidic channels configured to mix one or more fluids travelling therethrough. In certain embodiments, a series of mixers may be used to sequentially mix multiple fluids. Advantageously, this prevents sensitive components from being damaged, precipitated or otherwise affected by the temporary improper fluid composition.

[0048] The apparatus may further comprise:

[0049] (xv) one or more buffer reservoirs for containing a buffer therein; and

[0050] (xvi) one or more buffer microfluidic channel along which, in use, the buffer is fed from the one or more buffer reservoir, to the one or more mixer or to the one or more further transcribed RNA microfluidic channel.

[0051] Typically, the apparatus is configured to carry out the method of the first aspect of the invention.

[0052] Advantageously, the methods and apparatus of the invention achieve surprisingly uniform product characteristics over a wide-range of manufacturing volumes, without significant changes to the principal parameters of the manufacturing steps, such as flow rate and incubation time, providing a major advantage of the current state-of-the-art. Furthermore, the continuous use of the enzymes, input DNA, and cap-analogues, as well as the integrated DNA amplification step (thus limiting the volume of input DNA required), significantly reduce the cost-of-good of manufacturing RNA, achieving 10-100- fold higher yields than the current state-of-the-art batch and fed-batch methods. Importantly, by performing continuous DNA amplification, any losses of DNA template during the repeated IVT cycles may be compensated, enabling the system to continue longer. In addition, the integrated path from DNA amplification, or IVT, to formulation and downstream processing enables shorter and simpler manufacturing protocols, with virtually no losses or risk of (cross-) contamination. Integrated quality control enables in-process monitoring and fine-tuning of the reaction and the reaction products, and even allows for automated optimization of the reaction conditions for a particular RIMA construct or formulation.

[0053] Advantageously, the apparatus comprises a fully integrated microfluidic cartridge in which all of the various reservoirs, microfluidic channels, mixers, filters etc, are combined, with a connected flow path that does not need to interact with the outside world before downstream processing is finished. The apparatus may be referred to as a "consumable", because it may be discarded after use, and then replaced with a new, replacement apparatus. The method and apparatus are configured to optionally run in a continuously cycling reaction or so-called "infinite loop", in which a first incubation chamber is emptied into a secondary incubation chamber, while, meanwhile, the reaction keeps ongoing, and the DNA or RNA extraction beads donated in the initial incubation chamber or in a dedicated reservoir can then be washed, optionally incubated with a post-modification solution or eluted. The eluted extraction beads can then be recombined with the ongoing reaction mix, capturing additional DNA or RNA. By feeding intermittently or continuously, the ingredients that are consumed and compensating for losses (in activity) of the ingredients that are in principle not consumed, the reaction can proceed indefinitely. This infinite loop means that the apparatus can run continually with no down-time, meaning the product yields can be enormous. For example, it is possible to produce 20kg of RNA product within only 24 hours. The high yield that can be obtained over time from the infinite loop reactions avoid the use of large reaction reservoirs, which introduce complications with convection, thermal management, etc.

[0054] It will be appreciated that step (vi) of the method of the first aspect is optional if unformulated RNA drug substance is required. However, it will also be appreciated that step (i) and / or (ii) of the method of the first aspect may be optional if sufficient IVT- ready DNA is available. Therefore, in an embodiment, the method comprises:

[0055] (i) contacting an IVT-ready DNA molecule with raw materials of an in vitro transcription (IVT) reaction and incubating the resultant reaction mixture to produce a transcribed (optionally capped) RNA molecule;

[0056] (ii) continuously or intermittently extracting the transcribed RNA molecule, and optionally removing residual DNA;

[0057] (iii) optionally post-modifying the extracted RNA molecule;

[0058] (iv) optionally contacting the transcribed RNA molecule from (ii) or (iii) with a buffer solution and contacting the buffered RNA with one or more delivery component to produce formulated RNA, and

[0059] (v) subjecting the naked RNA from (ii or iii) or the formulated RNA from (iv) to one or more purification step to prepare an RIMA molecule, optionally capped, or a formulation thereof.

[0060] Similarly, component (i) and (ix) of the apparatus as defined above may also be optional. Accordingly, in an embodiment, the apparatus comprises:

[0061] (i) one or more IVT chamber in which, in use, in vitro transcription (IVT) is performed to produce transcribed RNA, and optionally in which the transcribed RNA is modified;

[0062] (ii) optionally one or more purification chamber or channel in which, in use, the RNA is purified;

[0063] (iii) one or more DNA substrate reservoir for containing DNA substrates, or one or more DNA substrate connector connectable to a DNA substrate reservoir, wherein the DNA substrates, in use, are required for performing IVT;

[0064] (iv) one or more RNA substrate reservoirs for containing RNA substrates, or one or more RNA substrate connector connectable to an RNA substrate reservoir, wherein the RNA substrates, in use, are required for performing IVT;

[0065] (v) one or more RNA product storage reservoir or one or more RNA product connector connectable to an RNA product storage reservoir;

[0066] (vi) one or more DNA microfluidic channel along which, in use, DNA substrates are fed from the one or more DNA substrate reservoir to the IVT chamber;

[0067] (vii) one or more RNA microfluidic channel along which, in use, RNA substrates are fed from the one or more RNA substrate reservoir to the one or more IVT chamber; and

[0068] (viii) one or more transcribed RNA microfluidic channel along which, in use, transcribed RNA is fed from the one or more IVT chamber to an optional purification chamber and / or the RNA product storage reservoir.

[0069] In some embodiments, however, the formulation step is essential. Therefore, in an embodiment, the method comprises:

[0070] (i) contacting an IVT-ready DNA molecule with raw materials of an in vitro transcription (IVT) reaction and incubating the resultant reaction mixture to produce a transcribed RNA molecule;

[0071] (ii) continuously or intermittently extracting the transcribed RNA molecule, and optionally removing residual DNA;

[0072] (iii) optionally post-modifying the extracted RNA molecule;

[0073] (iv) contacting the transcribed RNA molecule from (ii) or (iii) with a buffer solution and contacting the buffered RNA with one or more delivery component to produce formulated RNA, and

[0074] (v) subjecting the formulated RNA from (iv) to one or more purification step to prepare an RNA molecule, optionally capped, or a formulation thereof. Similarly, in an embodiment, the apparatus comprises:

[0075] (i) one or more IVT chamber in which, in use, in vitro transcription (IVT) is performed to produce transcribed RIMA, and optionally in which the transcribed RNA is modified;

[0076] (ii) optionally one or more purification chamber or channel in which, in use, the RNA is purified;

[0077] (iii) one or more DNA substrate reservoir for containing DNA substrates, or one or more DNA substrate connector connectable to a DNA substrate reservoir, wherein the DNA substrates, in use, are required for performing IVT;

[0078] (iv) one or more RNA substrate reservoirs for containing RNA substrates, or one or more RNA substrate connector connectable to an RNA substrate reservoir, wherein the RNA substrates, in use, are required for performing IVT;

[0079] (v) one or more RNA product storage reservoir or one or more RNA product connector connectable to an RNA product storage reservoir;

[0080] (vi) one or more DNA microfluidic channel along which, in use, DNA substrates are fed from the one or more DNA substrate reservoir to the IVT chamber;

[0081] (vii) one or more RNA microfluidic channel along which, in use, RNA substrates are fed from the one or more RNA substrate reservoir to the one or more IVT chamber;

[0082] (viii) one or more transcribed RNA microfluidic channel along which, in use, transcribed RNA is fed from the one or more IVT chamber to an optional purification chamber and / or the RNA product storage reservoir;

[0083] (ix) one or more RNA formulation reservoirs for containing one or more delivery component, wherein the one or more delivery component, in use, is required for formulating the RNA;

[0084] (xi) one or more mixer configured, in use, to mix transcribed RNA and one or more delivery component to provide formulated RNA;

[0085] (xii) one or more further transcribed RNA microfluidic channel along which, in use, RNA substrates are fed from the RNA product storage reservoir to the one or more mixer; and

[0086] (xiii) one or more delivery component microfluidic channel along which, in use, the one or more delivery component is fed from the one or more RNA formulation reservoir, to the one or more mixer.

[0087] The apparatus may further comprise:

[0088] (xv) one or more buffer reservoirs for containing a buffer therein; and

[0089] (xvi) one or more buffer microfluidic channel along which, in use, the buffer is fed from the one or more buffer reservoir, to the one or more mixer or to the one or more further transcribed RNA microfluidic channel. In a preferred embodiment, the apparatus is positioned and operated substantially vertically, wherein the direction of gravity is parallel to the face of the apparatus, such that reservoirs and channels are gravity-assisted. Advantageously, this means that the reservoirs and channels may be emptied (e.g. partially or even completely) due to their shape and orientation in relationship to gravity.

[0090] In a preferred embodiment, the filling and emptying of the reservoirs of the apparatus is performed in a manner that substantially follows the first-in, first out principle, and causes unidirectional fluid movement through the reservoir. A reservoir may, during initial loading or during operation, be filled from top to bottom, or from bottom to top.

[0091] The initial loading of the apparatus may be performed by any of the following methods, or combinations thereof:

[0092] (i) Reactants are loaded by hand, by injecting the solutions in the appropriate reservoir, by connecting a syringe via the corresponding connector;

[0093] (ii) Reactants are loaded by hand, by injecting the solutions in the appropriate reservoir, by piercing a membrane (e.g. rubber) with a needle on top of a syringe;

[0094] (iii) Reactants may be loaded by a dedicated external machine, connecting to the appropriate connectors and filling, either sequentially or in parallel, the reservoirs of the apparatus;

[0095] (iv) Reactants may be loaded by placing an external container (which is preferably hermetically sealed) with one or more pierceable face and / or side, such that attachment of the external container to a corresponding face of the apparatus results in one or more faces being pierced and a leak-free seal is formed between the inside of the external container and one or more chambers or channels in the apparatus. The contents of the sealed container may be either fluidic, and can thus be pushed into the reservoir(s) of the apparatus by applying air pressure to the back or side of the sealed container, or are dried, and can thus be pushed into the reservoir(s) of the apparatus by applying a liquid via the back or side of the sealed container. A brief incubation period may be applied to allow the dried contents to be dissolved;

[0096] (v) The reservoirs may be preloaded with liquid (preferably, frozen) or dried contents and no initial loading is required by the end-user, except for the specific DNA template, which may be applied as under (i), (ii), or (iii) above.

[0097] In an embodiment in which the container may be a single reservoir, or a plurality of reservoirs, the reservoir(s) may have simple geometric shapes mutually convenient to the apparatus (i.e. cartridge), or shapes that are convenient for storage and / or usage of the container after disconnecting from the single substrate cartridge / consumable.

[0098] In an embodiment, the containers may be a continuous part of the apparatus (i.e. single substrate cartridge / consumable) (e.g., be a continuous piece of plastic), or may be temporary connected, but not forming a continuous substrate. When temporarily connected, the containers may be connected via a connector on the surface of the apparatus of the invention and be disconnected by twisting, pulling, snapping or any other motion.

[0099] If the containers are a continuous part of the apparatus (i.e. consumable cartridge) during operation and are functionally connected to the remainder of the consumable via one or more microfluidic channels, there is provided a method to close the microfluidic channels temporarily or preferably permanently, wherein a method to close such channels is selected from: a one-way valve, more preferably by sealing the channel, more preferably by heat-sealing the channel (across or longitudinally over the channel), whereafter closing of the microfluidic channel(s), the container may be disconnected from the consumable via heat-cutting, laser-cutting, breaking / snapping along a perforated line, mechanical punch-out, milling, or any other suitable operation. Heatcutting is preferred.

[0100] In some embodiments, the template DNA may comprise (e.g. linearized) plasmid DNA, doggybone DNA, linear double-stranded DNA, linear single stranded DNA, minicircle DNA, de novo synthesized DNA, or pre-amplified DNA .

[0101] In some embodiments, the raw materials of the DNA amplification reaction may comprise template DNA, dNTPs (such as dATP, dCTP, dTTP, dGTP), a DNA polymerase (e.g., phi29 DNA polymerase, Bst DNA polymerase, Taq polymerase, Sequenase 2.0, T7 DNA polymerase, Pfu polymerase, any other suitable DNA polymerase, or mutants thereof), a buffer (comprising salts, including a magnesium or manganese salt, a buffering agent such as Tris-HCI, (RNAse-free) water, optional chelating agents) at suitable pH, optional DNAse-inhibitors, optional endonucleases (e.g., nickases), optional DNA primer(s) complementary to at least one element of the template DNA, and / or any other ingredient known to those skilled in the art to be part of a DNA amplification reaction.

[0102] In some embodiments, the incubation conditions of the DNA amplification reaction may comprise incubating at: room temperature, 30°C, preferably 37°C, 42°C, 50°C, 72°C, and incubating for more than 5 minutes, more than 15 minutes, more than 30 minutes, more than 1 hour, more than 2 hours, more than 3 hours, more than 6 hours, more than 16 hours, more than 24 hours, and incubating; and / or stationary, intermittently mixing, or continuously mixing. Advantageously, amplifying DNA in this way means that significantly less input DNA is required.

[0103] In some embodiments, the amplified DNA may be modified, for example by an endonuclease, an exonuclease, a kinase, a phosphatase, a ligase, a methylase, and / or any other DNA modifying enzyme known to those skilled in the art.

[0104] In some embodiments, extraction of the amplified DNA or modified amplified DNA may be achieved by hybridization of the optional poly(T)tail on the DNA to oligo(d)A nucleic acids coupled to a solid support, hybridization of a sequence-specific oligo nucleic acid coupled to a solid support, wherein the sequence-specific oligo is reverse complementary to at least one element of the DNA of interest, precipitation or electrostatic binding to a solid support, and / or capture of a secondary structure in the DNA of interest by a complementing protein, aptamer, or other polymer coupled to a solid support. In some preferred embodiments, the solid support is a bead, such as a superparamagnetic bead. In other embodiments, the solid support is a surface provided by a channel or reservoir, a column material, a membrane, sponge, or hollow fibre, each either enclosed by a channel or reservoir, or attached to the outside of the apparatus and functionally connected via a connector.

[0105] In some embodiments, the raw materials of the IVT reaction may comprise template DNA containing a suitable promoter for a RNA polymerase and preferably being linearized at the desired 3' end of the run-off transcript, NTPs (such as ATP, CTP, UTP, GTP, or modified variants thereof, including pseudollTP, Nl-methyl-UTP, m5CTP, m6ATP, mlATP, Inosine triphosphate, hm5CTP, mlGTP, m7GTP, or m6AmTP), an RNA polymerase (e.g., T7; T3; SP6; KP34; Syn5; Vsw-3; or other DNA-dependent RNA polymerase, or hybrids or mutants thereof, including temperature stabilized versions thereof), a buffer (comprised of salts, including a magnesium or manganese salt, a buffering agent such as Tris-HCI, RNAse-free water, optional chelating agents) at suitable pH, optional RNAse-inhibitors, optional endonucleases (e.g., nickases), optional DNA primer(s) to complement the RNA polymerase promoter by hybridizing to the template DNA, and / or any other ingredient known to those skilled in the art to be part of an IVT reaction. In some embodiments, the incubation conditions of the IVT reaction may comprise incubating at: room temperature, 30°C, preferably 30-37°C, more preferably 37°C, 42°C, 50°C, 72°C, and incubating for; more than 5 minutes, more than 15 minutes, more than 30 minutes, more than 1 hour, more than 2 hours, more than 3 hours, more than 6 hours, more than 16 hours, more than 24 hours, and incubating; and / or stationary, intermittently mixing, or continuously mixing.

[0106] In some embodiments, extraction of the transcribed RIMA may be achieved by hybridization of the poly(A)ta II on the RNA to oligo(d)T nucleic acids coupled to a solid support, hybridization of a sequence-specific oligo nucleic acid coupled to a solid support wherein the sequence-specific oligo is reverse complementary to at least one element of the RNA of interest, precipitation or electrostatic binding to a solid support, and / or capture of a secondary structure in the RNA of interest by a complementing protein, aptamer, or other polymer coupled to a solid support. In some preferred embodiments, the solid support is a bead, such as a superparamagnetic bead. In other embodiments, the solid support is a surface provided by a channel or reservoir, a column material, a membrane, sponge, or hollow fibre, each either enclosed by a channel or reservoir, or attached to the outside of the apparatus and functionally connected via a connector.

[0107] In some embodiments, any residual DNA contaminating the transcribed RNA may be removed by a DNA-specific exonuclease, a DNA-specific endonuclease such as DNAsel, or a combination thereof, specifically capturing the DNA by hybridization of an element unique or selective to the DNA over the RNA, such as hybridization of the poly(T)ta II on the DNA to oligo(d)A nucleic acids coupled to a solid support, or hybridization of a sequence-specific oligo nucleic acid coupled to a solid support wherein the sequencespecific oligo is reverse complementary to at least one element of the DNA.

[0108] In some embodiments, the transcribed RNA may be modified, for example, 5' cap addition and / or 5' cap methylation, site-selectively (base)modified (e.g., methylated or treated with a deaminase), 5' modified with a non-canonical or non-natural cap, 3' modified with a stabilizing or binding element, site-selectively digested, folded by chaperone proteins, folded by heating and subsequent controlled cooling, or any other method of modifying the RNA known to those skilled in the art.

[0109] In some embodiments, the buffer solution to be combined with the RNA prior to combination with one or more component of a delivery vehicle may comprise a citrate buffer at a pH of 4-6, an acetate buffer at a pH of 4-6, a tris-buffer at a pH 6-8, a phosphate buffer at a pH of 6-8, or a multi-component buffer (e.g., citrate-phosphate) at an appropriate pH.

[0110] In some embodiments, the one or more component of the delivery vehicle may comprise a lipid, such as: ionizable lipid (such as l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[ 1,3] -dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-15 dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), 1,2- dioleoyl-3-trimethylammonium-propane (DOTAP), l,2-Dioleoyl-3-trimethylammonium propane (DODAP), di((9Z,12Z)-octadeca-9,12-dien-l-yl) 2-((2- (dimethylamino)ethyl)thio)succinate maleate, di((9Z,12Z)-octadeca-9,12-dien-l-yl) 2- ((2-(dimethylamino)methyl)thio)succinate maleate, di((9Z,12Z)-octadeca-9,12-dien-l- 20 yl) 2-((2-(dimethylamino)propyl)thio)succinate maleate, di((9Z,12Z)-octadeca-9,12- dien-l-yl) 2-((2-(methylethylamino)ethyl)thio)succinate maleate, di((9Z,12Z)-octadeca- 9,12-dien-l-yl) 2-((2-(diethylamino)ethyl)thio)succinate maleate or dioleyl 2-((2- (diethylamino)ethyl)thio)succinate maleate), core or structural lipids (such as cholesterol, cholesterol-derivatives (such as Vitamin D2, Vitamin D3, Calcipotriol, Stigmasterol, Campesterol, Fucosterol, Brassicasterol, Ergosterol, 9,11- dehydroergosterol, Daucosterol, beta-Sitosterol-Acetate, Betutin, Lupeol, Ursotic acid, or Oleanotic acid), resveratrol, resveratrol derivatives (such as dihydroresveratrol, deoxyrhapontigenin, isorhapontigenin, piceatannol, pterostilbene, polydatin), or other small hydrophobic compounds), phospholipids (such as di-oleoyl- phosphatidylethanolamine (DOPE), di-oleoyl-phosphatidylcholine (DOPC), Di-oleoyl- phosphatidylserine (DOPS), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or any naturally occurring phospholipid), and / or a shielding lipid (such as a lipid with an attached shielding polymer (such as a polyethylene glycol (PEG) group, a poly-sarcosine group, an oligopeptide or a polypeptide, a hydroxyl-containing non-ionic water-soluble polymer, polyvinylpyrrolidone (PVP), a poly(2-alkyl-2-oxazoline) or a zwitterionic polymer or any other suitable shielding polymer. The oligopeptide or polypeptide may be PAS. PAS may be understood to be an oligopeptide or polypeptide consisting or comprising proline, alanine and serine residues. The hydroxyl-containing non-ionic water-soluble polymer may be poly(glycerol) (PG), poly(N (2 hydroxypropyl)methacrylamide) (pHPMA), polysarcosine, or poly(vinyl alcohol). The zwitterionic polymer may be a polybetaine)).

[0111] In some embodiments, the buffered RIMA is contacted with the one or more delivery vehicle component under conditions comprising a rapid mixing of the buffered RNA with the one or more delivery component, resulting in the formation of defined nanoparticles. If the delivery vehicle is a lipid nanoparticle (LNP), the lipids are dissolved in an organic solvent, such as ethanol, which is mixed at a suitable ratio (preferably, 1 :3) with the buffered RIMA at low pH, to ensure a rapid increase in polarity, causing the lipids to form nanoparticles, and associate with the RNA by the ionization of the ionizable lipids in the lipid mix due to the low pH. Optionally, one or more components may be heated prior, during and / or after the mixing. Heating may occur from room temperature to 30°C, to 37°C, to 42°C, to 50°C or higher. A (e.g. microfluidic) mixer may be mixing under chaotic or controlled mixing regimes. For specific microfluidic mixers, the flow rate is a crucial determinant of the mixing rate and thus the size (and / or uniformity) of the nanoparticles formed, and the total flow rate may be at least 1 ml / min, 3 ml / min, 5ml / min, 7 ml / min, 9 ml / min, 10 ml / min, preferably more than 12ml / min, more than 15 ml / min, or more than 20 ml / min.

[0112] In some embodiments, the mixer may comprise a T-junction mixer, a -shaped mixer, serpentine mixers, a spiral mixer, a chaotic advection mixer, a toroidal mixer, a (staggered) herringbone mixer, a hydrodynamic focusing mixer, a multi-lamination mixer, a 3D mixer, or a variant or combination thereof. In some embodiments, the receiving reservoir may have additional mixing elements, such as strategically placed obstacles to the incoming flow. In some embodiments, multiple mixers and / or mixertypes may be combined in series to achieve the desired mixing regime.

[0113] In some embodiments, the purification step may comprise an extraction by binding of the RNA or delivery vehicle to a solid support, a (ultra)filtration, preferably a tangential- flow filtration (TFF), more typically a single-pass tangential-flow filtration (Sp-TFF), a particle selection method based on diverting fluid streams, a centrifugation, a precipitation, or other method known to those skilled in the art. One or more purification step may be performed on the naked RNA or the formulated RNA.

[0114] In some embodiments, naked RNA from (iv or v) or the formulated RNA from (vi) may be stored. In some embodiments, the naked RNA from (iv or v) or the formulated RNA from (vi) is stored in bulk. In other embodiments, the naked RNA from (iv or v) or the formulated RNA from (vi) is stored in one or more single- or multi-use vials.

[0115] In a most typical embodiment, therefore, the method comprises:

[0116] (i) contacting a template DNA with raw materials of a DNA amplification reaction and incubating the resultant reaction mixture in one or more reservoirs of a microfluidic device,

[0117] (ii) continuously or intermittently extracting the amplified, and optionally post- amplification modified, DNA from the reaction mixture,

[0118] (iii) contacting the amplified DNA from (ii) with raw materials of an in vitro transcription (IVT) reaction and incubating the resultant reaction mixture in one or more reservoirs of the microfluidic device to produce transcribed RNA,

[0119] (iv) continuously or intermittently extracting the transcribed RNA and optionally removing residual DNA,

[0120] (v) optionally post-modifying the RNA, such as enzymatic 5' cap addition and / or 5' cap methylation,

[0121] (vi) contacting the transcribed RNA from (iv) or (v) with a buffer solution and contacting the buffered RNA with the one or more delivery component via a microfluidic mixer,

[0122] (vii) subjecting the naked RNA from (iv or v) or the formulated RNA from (vi) to a final (series of) purification step(s), and

[0123] (viii) storing the RNA drug substance or drug product in a bulk container, wherein step (i-ii) and step (vi) are optional if sufficient IVT-ready DNA is available and unformulated RNA drug substance is desired, respectively.

[0124] In further embodiments, the method may comprise preheating and maintaining the DNA amplification, IVT reaction and / or RNA formulation reaction (i.e. buffer solution and the one or more delivery component) at the desired incubation temperature. Therefore, this means it is possible to get large volumes of reagents to temperature in very short periods of time. Thus, the apparatus may comprise a pre-heater.

[0125] The pre-heater may comprise a bifurcating channel, typically a repeatedly bifurcating channel, configured to slow the local flow rate of fluid passing therethrough, while maintaining a very high total flow rate over the cartridge. Advantageously, this allows the heater cartridge to (pre)heat the fluid at relatively low temperatures of the heater cartridge.

[0126] The apparatus of the second aspect may comprise one or more filter disposed in the one or more microfluidic DNA channel and / or one or more microfluidic RNA channel. The one or more filter may be a size-selective filter. Such filters are preferably composed of a material suitable for contact with pharmaceutical products, and has a pore size suitable to prevent extraction beads and / or other particulate material from entering the downstream process. The pore size is preferably smaller than 1pm, more preferably smaller than 0.45pm, preferably smaller than 0.2pm. Advantageously, filters with a pore size smaller than 0.2pm have the additional benefit of being sterilizing- or bioburdenreducing filters. In some embodiments, the apparatus comprises one or more entry-guarding filter, which is configured to prevent contamination with bacterial elements. Such filters are preferably composed of a material suitable for contact with pharmaceutical products, and has a pore size smaller than 0.45pm, preferably smaller than 0.2pm. Advantageously, filters with a pore size smaller than 0.2pm are required under GMP to qualify as bioburden-reducing or sterilizing filters.

[0127] In some embodiments, the apparatus may comprise one or more internal filter configured to prevent DNA extraction means (e.g., beads) from reaching the IVT process, and / or RIMA extraction means (e.g., beads) from mixing with RNA formulated with the one or more delivery component and / or contaminating the final product. Such filters are preferably composed of a material suitable for contact with pharmaceutical products, and has a pore size smaller than 1pm, more preferably smaller than 0.45pm, preferably smaller than 0.2pm. Advantageously, filters with a pore size smaller than 0.2pm have the additional benefit of being sterilizing- or bioburden-reducing filters.

[0128] In some embodiments, the apparatus may comprise one or more exit-guarding filter, which is configured to prevent any particulate material from the apparatus entering in the fill and finish process (e.g., filling of individual vials with the drug product for individual use) or reverse flow introducing foreign material in the exit channels of the apparatus.

[0129] In addition to the filters of any of the embodiments above, the geometry and / or orientation of the channel(s) (including openings) may be constructed such that particulates may be trapped, removed from the fluid stream or otherwise prevented from entering or continuing into the process / apparatus. Such elements may be combined with additional features, such as bubble traps and / or seals.

[0130] Accordingly, it will be appreciated that the apparatus is hermetically sealed with a plurality of filters thereby reducing the risk of contamination from the external environment. Alternatively, or additionally, one or more entry or exit channels may be temporarily sealed from the outside world by a mechanically opening and closing (e.g. one-way) valve, a puncturable seal, a twist connector, a connector suitable for welding to tubing, or any other means known to a person skilled in the art to provide a sterile connection.

[0131] The apparatus may comprise one or more valve controlling the access and / or direction of fluids through the one or more (e.g. microfluidic) DNA channel and / or one or more (e.g. microfluidic) RIMA channel. Such valves may be selected from a group of; check valves, capillary valves, hydrogel valves, solenoid valves, thermally actuated valves, pneumatic valves, electrokinetic valves, magnetically actuated valves, elastomeric valves, phase change valves, membrane valves, quake valves, optically controlled valves, and most preferably rotational valves. Such valves may connect channels parallel or preferably perpendicular to the surface of the substrate of the apparatus. The valves may be normally open, preferably normally closed, or a combination thereof. In a preferred embodiment, a single valve can connect multiple combinations of channels, either 1-by- 1, simultaneously, or combinations thereof depending on the valve state. In one embodiment, the apparatus comprises a single type of valve and optionally multiple designs of such valve type. In another embodiment, the apparatus comprises multiple valve types.

[0132] In a preferred embodiment, a rotational valve is used, wherein the surface of the valve contains one or more channels, connecting channels perpendicular to the face whereon the valve is pressed. The valve may be held in place by a protrusion of the first substrate (the valve dome), pressing on the top of the valve, thereby pressing the opposite face of the valve (containing the channel(s)) on the face of the second substrate. The first substrate has a hole, through which the valve can be reached by (the shaft of) an actuator. In a preferred embodiment, the valve dome, or a female insert therein, has an inward facing threading, allowing a male insert to ride along the threading perpendicular to the face of the second surface. The male insert contains a tight-fitting, smooth cavity, wherein the valve resides. By screwing the male insert, the valve is moved towards or away from the face of the second substrate, and is therefore pressed more or less tightly against the face of the second surface, allowing to adjust the valve tension. The collection of threaded dome / threaded female insert and threaded male insert is coined the valve tensioning system (VTS). In one embodiment, the threading or another part of the VTS is prevented from slipping or modified to be unidirectional, preventing the tensioned valve to become untensioned by the rotational movement of the valve. The VTS may therefore be modified with an anti-slip surface, a ratcheting system, a pressure activated glue, or any other method known to those skilled in the art related to making rotational movement unidirectional.

[0133] In another embodiment, the rotational position of the valve is held in place by the shaft of the actuator of said valve, while a second actuator rotates the male insert of the VTS, preventing the valve from losing rotational alignment while the VTS is rotated.

[0134] In another embodiment, the rotational position of the male insert of the VTS is held in place by the secondary actuator, while the primary actuator of said valve rotates the valve, preventing the (repeated) rotation of the valve from loosening or further fastening the VTS and thus loosening or seizing the valve.

[0135] In some embodiments, the system is capable of detecting the tension of the VTS by a method comprising :

[0136] (i) a cam mechanism allowing the actuator to click when the preset torque is reached;

[0137] (ii) a digital torque sensor, that provides feedback when the preset torque is reached;

[0138] (iii) measuring the power / current of the electric motor (e.g., servo motor), and calculating the torque applied by the motor;

[0139] (iv) measuring the torque applied by the actuator of the valve; when the valve has less tension, the torque required to reach a new rotational position is lowered, providing feedback to the need of (re)tensioning the valve by the VTS; and / or

[0140] (v) measuring the leak-tightness of the valve by applying a known, constant pressure on the valve while in a closed position via a connected channel; loss of pressure and / or volume is an indication of the need to (re)tensioning the valve by the VTS.

[0141] In some embodiments, the valve has a female connector for the (shaft of the) actuator. In other embodiments, the valve has a male connector for the actuator. For reasons of robustness during transport and use, the female connector is preferred. In addition, in some embodiments, the male or female connector has an irregular shape, ensuring only 1 mating orientation wherein connection between the shaft and the shaft mating element of the valve is allowed. Such irregular shape may be on one side of a regular shape, such as a protrusion.

[0142] In some embodiments, the shaft of the actuator and corresponding shaft mating element in the valve may have a complementary shape with at least 1 conical feature, to facilitate entry of the shaft into the shaft mating element. Advantageously, including a non-conical part of the of the mating surface (i.e. the complementary shape) prevents the shaft from being dislodged (by generating a perpendicular force - in the direction of the shaft) from the shaft mating element during high friction rotation. In a preferred embodiment, the non-conical part is larger than the conical part, preferably at least 2 times larger, more preferably at least 4 times larger, even more preferably more than 10 times larger.

[0143] In some embodiments, the one or more DNA amplification chamber may comprise a reservoir formed by a void between a second face of a first substrate and a first face of a second substrate, or an equivalent between two other substrates; an external container connected to a connector on a first face of the first substrate, or an equivalent on the outside facing face of any other substrate, wherein such container may be a tube, a bottle, a vat, barrel or tank, a tubing, a (collection of) hollow fibre(s), or any other suitable closed container with a suitable connector; or a channel between the second face of the first substrate and the first face of the second substrate, or an equivalent between two other substrates. It will be appreciated that, in use, a template DNA molecule may be amplified in the one or more DNA amplification chamber to produce amplified DNA, as described herein with reference to the methods of the invention. Similarly, the amplified DNA may be modified in the one or more DNA amplification chamber. The DNA amplification chamber may have any suitable form and may have a (repeating) irregular form, such form is preferably designed to empty completely. In one embodiment, the shape of the DNA amplification chamber is such that homogenous magnetic fields may be applied to the contents, and rapid (e.g. meaning short range) magnetic extraction of beads may be performed.

[0144] In some embodiments, the one or more IVT chamber may comprise a reservoir formed by a void between a second face of a first substrate and a first face of a second substrate, or an equivalent between two other substrates; an external container connected to a connector on a first face of the first substrate, or an equivalent on the outside facing face of any other substrate, wherein such container may be a tube, a bottle, a vat, barrel or tank, a tubing, a (collection of) hollow fibre(s), or any other suitable closed container with a suitable connector; or a channel between the second face of the first substrate and the first face of the second substrate, or an equivalent between two other substrates. The skilled person will understand that, in use, IVT may be performed in the one or more IVT chamber to produce transcribed RNA. Also, in an embodiment, the transcribed RNA may be modified in the one or more IVT chamber. The IVT chamber may have any suitable form and may have a (repeating) irregular form, such form is preferably designed to empty completely. In one embodiment, the shape of the IVT chamber is such that homogenous magnetic fields may be applied to the contents, and rapid (e.g. meaning short range) magnetic extraction of beads may be performed.

[0145] In an embodiment, the apparatus comprises one or more purification chamber or channel in which, in use, the RNA is purified. The purification chamber or channel may comprise a reservoir formed by a void between a second face of a first substrate and a first face of a second substrate, or an equivalent between two other substrates; an external container connected to a connector on a first face of the first substrate, or an equivalent on the outside facing face of any other substrate, wherein such container may be a tube, a bottle, a vat, barrel or tank, a tubing, a (collection of) hollow fibre(s), or any other suitable closed container with a suitable connector; or a channel between the second face of the first substrate and the first face of the second substrate, or an equivalent between two other substrates. The purification chamber may have any suitable form and may have a (repeating) irregular form, such form is preferably designed to empty completely. In one embodiment, the shape of the IVT chamber is such that homogenous magnetic fields may be applied to the contents, and rapid (meaning short range) magnetic extraction of beads may be performed.

[0146] In a particular embodiment, the purification chamber comprises two adjacent / overlapping chambers or channels with a filter functionally separating the two adjacent / overlapping chambers or channels, enabling fluid to be pumped and / or drawn through the filter, retaining molecules and / or particulates (such as a solid support for nucleic acid extraction) from the fluid that passes through the filter.

[0147] In an embodiment, the one or more DNA substrate reservoir for containing DNA substrates may comprise a reservoir formed by a void between a second face of a first substrate and a first face of a second substrate, or an equivalent between two other substrates; an external container connected to a connector on a first face of the first substrate, or an equivalent on the outside facing face of any other substrate, wherein such container may be a tube, a bottle, a vat, barrel or tank, a tubing, a (collection of) hollow fibre(s), or any other suitable closed container with a suitable connector; or a channel between the second face of the first substrate and the first face of the second substrate, or an equivalent between two other substrates.

[0148] However, in some embodiments, instead of the apparatus comprising one or more DNA substrate reservoir, the apparatus comprises one or more DNA substrate connector which is connectable to an external DNA substrate reservoir. In these embodiments, the DNA substrate connector may comprise a Luer-lock connector, a Luer-slip connector, a barbed connector (straight, reducing, T-junction), quick-disconnect coupling (single-use or reusable), compression fittings (plastic or metal), threaded connectors, flare fittings, push-to-connect fittings, CPC couplings, Swagelok fittings, tri-clamp fittings, ferrule fittings, or any other connector known to those skilled in the art.

[0149] In one particular embodiment, the one or more DNA substrate reservoir for containing DNA substrates comprise an external container (e.g. hermetically sealed) with one or more pierceable face and / or side, such that attachment of the external container to a corresponding face of the apparatus results in one or more faces being pierced and a leak-free seal is formed between the inside of the external container and one or more chambers or channels in the apparatus.

[0150] In an embodiment, the one or more RIMA substrate reservoir for containing RNA substrates may comprise a reservoir formed by a void between a second face of afirst substrate and a first face of a second substrate, or an equivalent between two other substrates; an external container connected to a connector on afirst face of the first substrate, or an equivalent on the outside facing face of any other substrate, wherein such container may be a tube, a bottle, a vat, barrel or tank, a tubing, a (collection of) hollow fibre(s), or any other suitable closed container with a suitable connector; or a channel between the second face of the first substrate and the first face of the second substrate, or an equivalent between two other substrates.

[0151] However, in some embodiments, instead of the apparatus comprising one or more RNA substrate reservoir, the apparatus may comprise one or more RNA substrate connector which is connectable to an external RNA substrate reservoir. In such embodiments, the RNA substrate connector may comprise a Luer-lock connector, a Luer-slip connector, a barbed connector (straight, reducing, T-junction), quick-disconnect coupling (single-use or reusable), compression fittings (plastic or metal), threaded connectors, flare fittings, push-to-connect fittings, CPC couplings, Swagelok fittings, tri-clamp fittings, ferrule fittings, or any other connector known to those skilled in the art. In one particular embodiment, the one or more RNA substrate reservoir for containing RNA substrates comprise an external container (e.g. hermetically sealed) with one or more pierceable face and / or side, such that attachment of the external container to a corresponding face of the apparatus results in one or more faces being pierced and a leak-free seal is formed between the inside of the external container and one or more chambers or channels in the apparatus.

[0152] In some embodiments, the one or more RNA product storage reservoir may comprise a reservoir formed by a void between a second face of a first substrate and a first face of a second substrate, or an equivalent between two other substrates; an external container connected to a connector on a first face of a first substrate, or an equivalent on the outside facing face of any other substrate, wherein such container may be a tube, a bottle, a vat, barrel or tank, a tubing, a (collection of) hollow fibre(s), or any other suitable closed container with a suitable connector; or a channel between the second face of the first substrate and the first face of the second substrate, or an equivalent between two other substrates. In other embodiments, the one or more RIMA product connector which is connectable to an RNA product storage reservoir may comprise a Luer-lock connector, a Luer-slip connector, a barbed connector (straight, reducing, T-junction), quick-disconnect coupling (single-use or reusable), compression fittings (plastic or metal), threaded connectors, flare fittings, push-to-connect fittings, CPC couplings, Swagelok fittings, tri-clamp fittings, ferrule fittings, or any other connector known to those skilled in the art.

[0153] In a particular embodiment, the one or more RNA product connector connects a tubing attached in parallel to more than one container and each container may be filled with a defined volume.

[0154] In another embodiment, the one or more RNA product storage reservoir may comprise one or more single- or multi-use vials. In embodiments in which the vial may be a single reservoir, or a plurality of reservoirs, the reservoir(s) may have simple geometric shapes mutually convenient to the cartridge, or shapes that are convenient for storage and / or usage of the container after disconnecting from the single substrate cartridge / consumable.

[0155] The containers may be a continuous part of the single substrate cartridge / consumable (e.g., be a continuous piece of plastic), or may be temporarily connected, but not forming a continuous substrate. When temporarily connected, the containers may be connected via a connector on the surface of the apparatus of the invention and be disconnected by twisting, pulling, snapping or any other motion.

[0156] If the containers are a continuous part of the consumable during operation and are functionally connected to the remainder of the consumable via one or more microfluidic channels, there is provided a method to close the microfluidic channels temporarily or preferably permanently, selected from: a one-way valve, more preferably by sealing the channel, more preferably by heat-sealing the channel (across or longitudinally over the channel), whereafter closing of the microfluidic channel(s), the container may be disconnected from the consumable via heat-cutting, laser-cutting, breaking / snapping along a perforated line, mechanical punch-out, milling, or any other suitable operation. Heat-cutting is preferred.

[0157] In an embodiment, the one or more DNA microfluidic channel may comprise a microfluidic channel, a channel with at least 1 dimension belonging to the microfluidic domain, or a channel, wherein such channel may have a square, rectangular, rectangular with 2 rounded corners, a circular, a semi-circular or irregular cross-section, optional with radii consequential to the manufacturing method, and wherein such channel may follow a straight, curved, or irregular path, and wherein such channel may be perpendicular, angled or parallel, or a combination thereof, to the face of the substrate wherein such channel is contained. It will be understood that, in use, DNA substrates may be fed along these channels from the one or more DNA substrate reservoir to the one or more DNA amplification chamber and / or the one or more IVT chamber.

[0158] In an embodiment, the one or more RNA microfluidic channel may comprise a microfluidic channel, a channel with at least 1 dimension belonging to the microfluidic domain, or a channel, wherein such channel may have a square, rectangular, rectangular with 2 rounded corners, a circular, a semi-circular or irregular cross-section, optional with radii consequential to the manufacturing method, and wherein such channel may follow a straight, curved, or irregular path, and wherein such channel may be perpendicular, angled or parallel, or a combination thereof, to the face of the substrate wherein such channel is contained. It will be appreciated that, in use, RNA substrates may be fed along these channels from the one or more RNA substrate reservoir to the one or more IVT chamber.

[0159] In an embodiment, the one or more amplified DNA microfluidic channel may comprise a microfluidic channel, a channel with at least 1 dimension belonging to the microfluidic domain, or a channel, wherein such channel may have a square, rectangular, rectangular with 2 rounded corners, a circular, a semi-circular or irregular cross-section, optional with radii consequential to the manufacturing method, and wherein such channel may follow a straight, curved, or irregular path, and wherein such channel may be perpendicular, angled or parallel, or a combination thereof, to the face of the substrate wherein such channel is contained. It will be understood that, in use, amplified DNA is fed from the one or more DNA amplification chamber, to the one or more IVT chamber.

[0160] In some embodiments, the one or more transcribed RNA microfluidic channel may comprise a microfluidic channel, a channel with at least 1 dimension belonging to the microfluidic domain, or a channel, wherein such channel may have a square, rectangular, rectangular with 2 rounded corners, a circular, a semi-circular or irregular cross-section, optional with radii consequential to the manufacturing method, and wherein such channel may follow a straight, curved, or irregular path, and wherein such channel may be perpendicular, angled or parallel, or a combination thereof, to the face of the substrate wherein such channel is contained. The skilled person will appreciate that, in use, transcribed RNA is fed from the one or more IVT chamber to an optional purification chamber and / or the RNA product storage reservoir. In one embodiment of the invention, the apparatus of the second aspect is manufactured from plastic, more preferably from transparent plastic, even more preferably from plastic with substantial transparency in the UV-spectrum, even more preferably with substantial transparency at 230-280nm. The plastic used has, after processing, a low leachable and extractable profile with any of the fluids and subject to the process parameters of the method of the first aspect of the invention. The plastic may be selected from a group consisting of: polyethylene (PE, both HDPE and LDPE), polypropylene (PP), polyethylene terephthalate (PET) and glycol-modified PET (PETG), polystyrene (PS), polycarbonate (PC), polyvinyl chloride (PVC, medical-grade), Cyclic Olefin Copolymer (COC), Cyclic Olefin Polymer (COP), Polymethyl Methacrylate (PMMA), Fluoropolymers (PTFE, FEP, PFA), PolyEther ether ketone (PEEK), Ethylene vinyl acetate (EVA), Thermoplastic polyurethane (TPU), or polyamide (Nylon), or combinations thereof. The plastic is typically a suitable (medical / GMP) grade and type of COC plastic, with high UV-spectrum transparency and a suitable melting temperature / profile matching the manufacturing methods used.

[0161] The type, thickness and processing of the plastic of the apparatus is selected such that each reservoir in its final size has a wall thickness that is capable to withstand the positive and / or negative pressure applied to the reservoir during all steps of the process. Furthermore, in one embodiment, the thickness of the reservoirs is matched to the size and pressures experienced by that reservoir. In another embodiment, the thickness of the walls of a reservoir is non-uniform, i.e., it may be thicker or thinner in specific areas, such as corners or long sides / and faces. In addition, the walls of one or more reservoirs may have a shape or additional supporting structures to prevent buckling, such as a structure known as crinkum crankum, wherein waves in the wall distribute the load such that no single point is overloaded by the sum of the force on the wall. In some embodiments, the shape of the reservoir is such that the optimal distribution of force is achieved upon positive pressure on the reservoir, and / or that a uniform distribution of the available plastic is achieved, such as during stretching of the plastic during vacuum forming.

[0162] In a third aspect, there is provided a method of manufacturing the apparatus of the second aspect, the method comprising :

[0163] (a) attaching, joining or fusing two or more substrates together to define a microfluidic layer therebetween; or (b) vacuum forming or blow moulding millilitre to litre scale reservoirs in one substrate and subsequently attaching, joining or fusing the substrate to another substrate to form a reservoir to hold fluid.

[0164] In one embodiment, the apparatus is manufactured substrate by substrate, followed by assembly of all the substrates into the finished apparatus, which is consumable. In another embodiment, the apparatus is manufactured by manufacturing the first substrate, then adding the second substrate directly in contact with the first or by assembling both substrates and then assembling the substrates, followed by the manufacturing of the third substrate, and the process is repeated until all substrates are assembled into a finished apparatus of the second aspect.

[0165] Bonding of the substrates of the cartridge may be performed by;

[0166] (i) exposing one or more substrates to a solvent;

[0167] (ii) optionally soaking the substrate (surface) in the solvent;

[0168] (iii) optionally allowing the solvent to evaporate until the substrate is tacky;

[0169] (iv) assembling the substrates in a stack, either one-by one, or all together;

[0170] (v) applying pressure and optionally heating;

[0171] (vi) optionally holding the pressure, allowing the solvent to penetrate deeper and the bond to establish; and

[0172] (vii) releasing the pressure.

[0173] In one embodiment, the solvent is exclusively applied to only those areas that require a bond to be formed. More importantly, the solvent is not applied to those areas that will form part of the internal faces of a channel, reservoir, or connector, or any other surface in contact with any fluid from the method of the first aspect of the invention. The sitespecific application of the solvent may be achieved by spraying (similar to inkjet printing), brushing, screen-printing, or any other method known to those skilled in the art. The use of very fine nozzles, similar to inkjet technology allows precise and rapid deposition of the solvent site-selectively on the surface of the substrate.

[0174] During bonding a significant pressure may be applied, which is directly proportional to the mating surfaces and which may cause significant internal stress, potentially deforming the microfluidic structures. Therefore, in one embodiment, the first and second substrate are bonded, as well as the third and fourth substrate, next, the two assemblies are combined with a thin film, matching the layout of the channels from the first assembly to those of the second assembly, sandwiched in between. The thin film has those parts resected that are devoid of any channels running from the first to the second assembly, and thereby significantly reduces the pressure required for bonding. In another embodiment, the faces of the substrates to be bonded together have matching recessed and protruded parts restricting the bonding to those elements.

[0175] In one embodiment of the invention, the substrates of the apparatus are manufactured by hot embossing the channels and other microfluidic elements into extruded plates of the plastic, followed by placement and optional fixation of the filters, valves and other integrated elements, subsequently followed by one or more bonding step(s) of the substrates. The substrate containing the macrofluidic reservoirs is optionally manufactured with vacuum forming.

[0176] In another embodiment, the substrates of the apparatus are manufactured by injection moulding, creating the substrate, its channels, its reservoirs, its optional connectors, and other structural elements in a single manufacturing step. Following the injection moulding of the substrate, filters, valves and other integrated elements may be placed, and optionally fixed with a second component injection. Finally, the substrates are assembled via one or more bonding steps. Alternatively, the filters, valves and other integrated elements are placed immediately prior to the bonding step.

[0177] In another embodiment, a combination of hot-embossing, injection moulding, and one or more optional alternative manufacturing methods is used to create the apparatus. Such alternative manufacturing methods comprise; (injection) blow moulding, extrusion, thermoforming / vacuum forming, compression moulding, rotomolding (rotational moulding), 3D printing (fusion deposition modeling (FDM), multi-jet fusion (MJF), stereolithography (SLA)), casting, calendering, transfer moulding, foam moulding, CNC- milling, any combination thereof, or any other suitable manufacturing method known to those skilled in the art.

[0178] The filters, valves and optional other integral elements may be placed by hand or automatically with a pick and place machine, or a combination thereof. The accurate placement of filters, valves and other integral elements is aided by any (combination of) the following methods and features:

[0179] (i) receptacles are slightly concave, optionally matching the valve or other element, providing a guiding / centring function;

[0180] (ii) filters and optionally other elements contain where possible rounded corners,

[0181] (iii) jigs, fiducials, mating elements, and combinations thereof; (iv) irregular shapes, allowing assembly only in the correct orientation; and / or

[0182] (v) indentations matching the size and shape of a filter.

[0183] Besides aiding in the placement, positioning and orientation of the filters, the recessed / indented receptacle in the substrate of the apparatus reduces the pressure on the filter during bonding, preventing lateral compression of the filter, thereby reducing the effective surface by closing pores or reducing the pore size, as was surprisingly found by the inventors. In a preferred embodiment, the depth of the recessed receptacle is around 50% of the thickness of the filter, ensuring sufficient holding power during bonding. For example, the depth of the receptacle is 60pm for a filter of 120pm thickness.

[0184] Filters and optionally other integrated elements may be fixed by compression only (applied by the bonding of the plates), by the application of a solvent to the mating area (preferably only to the substrate and not to the filter), by application of a glue or hot plastic to the mating area, by local heating, or a combination thereof, wherein the glue is non-leaching, and preferably has a high viscosity, and the solvent in the solvent bonding method is allowed to evaporate to the point of leaving the substrate tacky. Both prevent the porous structure of the filter from soaking up the glue or solvent, respectively.

[0185] The valves may be orientation dependent in their function, therefore, in one embodiment, the valves contain a visual or physical reference aiding in the correct orientation during or subsequent to placement. The valves may further contain an irregular mating slot for a shaft or tool that operates / rotates the valve, thereby communicating automatically the orientation of the valve to the actuator.

[0186] In one embodiment, the shaft of the actuator operating the rotational valve is spring- loaded and compression of the spring (by a mismatch between the slot and the shaft prevents the shaft from entering the slot) is sensed by for example a proximity sensor, an end-stop sensor, or another suitable sensor. Upon activation of the sensor, the actuator is slowly rotated until the shaft matches with the slot and the shaft is by action of the spring pushed into the slot, which resets the sensor. Next, the actuator is returned to the intended zero-position. A smart search sequence may be used, wherein the actuator rotates in both directions and / or uses optical feedback to find the correct position.

[0187] As shown in the Figures, the apparatus of the invention can be fitted into, or attached to, a main housing unit, and then controlled by a computer. Thus, in a fourth aspect, there is provided a system comprising a housing configured to operably receive one or more apparatus according to the second aspect or manufactured by the method of the third aspect.

[0188] The system may comprise at least two, three, four or five apparatuses of the second aspect. Typically, the system may comprise at least six, seven, eight or nine apparatuses of the second aspect. Typically, the system may comprise at least 11, 12, 13, 14 or 15 apparatuses of the second aspect. Most typically, the system may comprise at least 16, 17, 18, 19 or 20 apparatuses of the second aspect. In another embodiments, the system may comprise a plurality of independent apparatuses, wherein each apparatus comprises an independent housing. The, or each, apparatus of the second aspect may be referred to as a cartridge, which comprises a substrate comprising a network of microfluidic channels and reservoirs, as described herein.

[0189] The apparatus may be described as a consumable, and so may be replaced as and when required with a new or alternative embodiment with different scales (e.g. reservoirs or channels etc.) for producing different RIMA molecules, and yet can still fit into the housing and so still be used and controlled by the same system. Furthermore, it is a huge advantage that the system comprises a plurality of apparatuses, each with fixed microfluidic channels, mixers, filters, and that only the reservoir size is modified to scale, depending on the amount of product that is required.

[0190] The system may comprise a processor configured to control the one or more apparatus. It will be appreciated that each apparatus may be controlled independently.

[0191] The system may comprise one or more gas compressor for providing pressure to the apparatus.

[0192] The system may comprise one or more independent manufacturing module, each configured to receive or be operably connected to an apparatus of the second aspect. In typical embodiments, the one or each independent manufacturing module is configured to independently operate an apparatus of the second aspect, preferably when it is attached to the housing.

[0193] In a fifth aspect, therefore, there is provided an independent manufacturing module configured to receive or be operably connected to an apparatus of the second aspect. The or each independent manufacturing module may comprise an independent set of flow and / or pressure controllers (such as proportional valves, one-way valves, solenoid valves, etc.), heating elements, optical detectors and light sources, electromotors (such as servos, stepper motors, etc.) for rotating rotational valves or linearly moving sliding valves, movable permanent magnets, neutralizable permanent magnets, and / or electronically controllable electromagnets, each positioned and capable of interacting with the relevant apparatus of the second aspect, ideally when in situ in the housing.

[0194] The propulsion of the fluids in the apparatus of the second aspect, according to the method of the first aspect of the invention, is typically by the application of positive and / or negative air pressure in a channel or connector in a specific manner. Typically, the positive or negative pressure is centrally generated by the system in a volume sufficient that typical consumption does not significantly deplete the pressure reservoir and a minimum of pressure can be guaranteed. Subsequently, the centrally generated pressure may be periodically applied and reduced to the desired application pressure by the proportional valves of each independent manufacturing module of the system. Thus, the or each independent manufacturing module may comprise a central pressure system for applying positive and / or negative pressure to the apparatus.

[0195] The central pressure system may have a redundancy setup, wherein two reservoirs are pressurized, and while one is used, the other is repressurized by a compressor. By alternating between reservoirs, the system can be used continuously. The volume of each of the reservoirs may be more than 10L, preferably more than 20L, more preferably more than 30L, more preferably more than 40L, more preferably more than 50L, but no more than could fit in the system, optionally an upper compartment thereof. Optionally, the system may comprise more than two pressure reservoirs, or a multiple of two reservoirs, each with a smaller volume, enabling quicker re-pressurization.

[0196] Alternatively, two sets of two smaller and larger reservoirs may be provided, wherein the smaller reservoirs are used to perform an initiation test and initial testing runs, while the larger reservoirs are pressurized.

[0197] The or each independent manufacturing module may comprise one or more pressure controllers and / or mass flow controllers, for applying the final application pressure to the channels and reservoirs of the apparatus of the second aspect, so as to move fluid therein. In typical embodiments, the pressure controllers and / or mass flow controllers may comprise a feedback loop which is configured to vary the final application pressure suitable for ratiometrically-defined mixing of fluids. As such, in another aspect, there is provided a method for ratiometrically-defined mixing of two or more fluid streams, the method comprising applying gas pressure to two or more separate fluid-containing reservoirs, each reservoir containing a fluid, via feedback-controlled mass flow controllers, so as to create two or more defined fluid volumes, and then feeding the two or more defined fluid volumes to a microfluidic mixer where they are mixed to thereby produce a ratiometrically-defined fluid stream comprising the two or more fluid streams.

[0198] The pressure controllers and / or mass flow controllers are preferably selected to have a high precision (preferably 0.1-10 mbar), a low latency (preferably l-10ms), and / or a high repeatability (preferably <5% deviation).

[0199] The or each independent manufacturing module may comprise one or more magnet configured to separate RIMA capture means, typically RNA capture beads, from the IVT reaction. The or each independent manufacturing module may comprise one or more movable permanent magnets, which may be moved perpendicular to the face of the apparatus of the second aspect, changing its effect by the increase or decrease of the distance between the magnet and the contents of the apparatus. Alternatively, or additionally, the one or more movable permanent magnets may be moved parallel to the face of the apparatus, changing its effect by the increase or decrease of the overlapping with one or more reservoirs.

[0200] The or each independent manufacturing module may comprise one or more neutralizable magnets which may contain multiple magnets in an orientation, wherein in the 'off' position, the magnetic poles are aligned to neutralize each other's magnetic field at the device's surface, and wherein in the 'on' position, the magnets align their poles to create a concentrated magnetic field at the device's surface.

[0201] The or each independent manufacturing module may comprise one or more electromagnets which may be static and may be switched 'on' and 'off' by permitting a current of sufficient strength through the coils of the electromagnet. The amount of current may be used to adjust the magnetic strength. Electro and permanent magnets may be combined to achieve stronger or better controllable effects. In some embodiments, the magnets may be gradually disengaged to gradually, preferably proportionally to the fluid volume, release the superparamagnetic beads to the volume.

[0202] In some embodiments, the or each independent manufacturing module may comprise one or more optical or magnetic decoders to read the partial, full or multiple of rotations of the motor, and / or the connected elements, such as the rotational valves in the apparatus of the second aspect.

[0203] In some embodiments, or each independent manufacturing module may comprise one or more (e.g. spring-loaded) magnetic switches or hall sensors to determine the magnetic strength of the permanent or electromagnets in a particular location of the apparatus of the second aspect.

[0204] In some embodiments, the or each independent manufacturing module may comprise one or more optical sensors to detect absorption, fluorescence, colour changes, optical diffraction, time-of-flight / reflections, phase-contrast changes, transparency, or other optical parameters and / or changes thereto to the contents of the consumable. Such optical sensors may measure any of the following aspects:

[0205] (i) Changes in fluid levels in a reservoir (confirming filling or emptying);

[0206] (ii) Changes in optical transparency / density to quantify the amount of solid support added to, or extracted from, a solution;

[0207] (iii) Changes in, or absolute values of, absorption in the UV-range, particularly 230-280nm, to measure the concentration and purity of nucleic acids;

[0208] (iv) Changes in, or absolute values of, absorption in the UV-Vis range, to measure the concentration of specific reactants;

[0209] (v) Changes in, or absolute values of, fluorescence after mixing of sample with DNA or RIMA intercalating dye, optionally in the presence of a delivery vehicle disrupting substance;

[0210] (vi) Changes in, or absolute values of, the colour of the sample, to measure and quantify an ELISA-type of assay;

[0211] (vii) Changes in phase-contrast optical signal to measure the fluid front or end in a channel, can be used to measure the presence and flowrate of the fluid;

[0212] (viii) Changes in, or absolute values of, optical diffraction or laser diffraction as part of the DLS-method to determine (compute) nanoparticle size;

[0213] (ix) Changes in, or absolute values of, transparency as indicator of precipitates and properly dissolved solutions; and / or

[0214] (x) Changes in optical transparency or diffraction to detect undesired particulates in the fluids.

[0215] The optical sensors may be positioned parallel, under an angle, or preferably perpendicular to the face of one of the substrates of the apparatus. Said optical sensors are preferably measuring in a location, wherein a minimal number and thickness of substrates are sandwiched together. In a typical embodiment, the optical measurements are of a nature and / or intensity that does not damage the biomolecules in the consumable, enabling subsequent use. In particular, UV-measurement of the nucleic acid (DNA or RIMA) must be limited in exposure duration and / or intensity to avoid damage to the nucleic acid.

[0216] In a typical embodiment, the destructive quality control, including UV-measurements of nucleic acids and / or mixing of the (formulated) nucleic acids with a fluorescent dye or other QC substrate may be performed in an aliquot of the reaction product, preventing the final products of having undesired contamination or mutation. Such aliquots may be continuously (via a (smaller) channel branching off the main channel, continuously receiving a fraction of the fluid flowing through the channel) or intermittently sampled from the main flow or reservoir.

[0217] In some embodiments, or each independent manufacturing module may comprise one or more dedicated reservoirs, optimized for optical measurements, including a minimal substrate (plastic) thickness, defined depth of the reservoir or channel, shaped to be cleaned completely (preventing transfer of remnants of previous samples) and optional valve control over the flow through such channels or reservoirs (providing a temporary stationary fluid during measurement).

[0218] In a sixth aspect, there is provided a method for testing the integrity and / or selective filtering of the entry and / or exit filters on the apparatus of the second aspect, the method comprising analysing the entry and / or exit filters for any damage.

[0219] The method may be performed during or directly after manufacture of the apparatus of the second aspect, directly prior to use, during and / or after the completion of RNA production. In one embodiment, beads, preferably polystyrene or COC-plastic beads of a defined size, similar to the particulates the filter is designed to deny passage, are added to the retentate side of the filters under an appropriate amount of pressure. Any damage to the filter membrane would allow the beads to pass through the membrane and is detected by optical sensors that monitor the downstream channels and / or reservoirs for the bead-specific optical signature.

[0220] Upon completion of the test, the beads may optionally (preferably) be removed from the filter surface by reversing the flow, or rinsing the filter by a tangential flow, or a combination thereof. Ideally, the beads are supplied in a fluid that is easily removed (optionally by evaporation) from the reservoirs, channels and filter medium, and / or is innocuous if not completely removed. The fluid may be an organic solvent, such as ethanol, which can be evaporated quickly and completely. The reservoirs, channels and / or filter may be subjected to (optionally heated) airflow to dry. Alternative, or supplementary, the integrity of the filter is determined by the amount of resistance provided by the filter to the flow of the fluid.

[0221] The or each independent manufacturing module may comprise a preheater which is configured to preheat the reactants and / or the reaction mix or a fraction thereof to the incubation temperature before entering the incubation reservoirs, such that the incubation reservoirs do not need to heat the solution to the incubation temperature, but may instead maintain the incubation temperature. The pre-heater means that excessive heating on the incubation reservoirs can be avoided, thereby avoiding denaturing the proteins (e.g. enzymes) therein. Thus, the preheater, in use, is configured to pre-heat the DNA amplification, IVT reaction and / or RIMA formulation reactions (i.e. buffer solution and the one or more delivery component) at the desired incubation temperatures. The preheater may comprise a bifurcating channel, typically a repeatedly bifurcating channel, configured to slow the local flow rate of fluid passing therethrough, while maintaining a very high total flow rate over the cartridge.

[0222] Advantageously, with the preheater, it is possible to get large volumes of reagents to temperature in very short periods of time without physics getting in the way, that is to say, both the time it takes for fluid to heat and convection induction by temperature differences of large bodies of fluid. Importantly, the (walls of the) reaction reservoirs need to be re-heated to the incubation temperature to avoid the pre-heated fluid cooling down on contact with the walls of the reaction reservoirs.

[0223] To enable reaching the incubation temperature with a certain flow rate, the following measures may be typically allowed :

[0224] (i) the pre-heater may heat the plastic around the flow channel to a temperature higher than the incubation temperature to enable sufficient heat being transferred into the fluid at a given flow rate. Importantly, the applied temperature may not damage, deform or release (a higher degree of) leachables (from) the plastic, and may also not damage or permanently alter the reactants flowing through the channel. Therefore, the temperature is limited to 100°C, more preferably to 90°C, more preferably to 80°C, and possibly cooler;

[0225] (ii) the pre-heater may be a heater-cooler element, such as a Peltier element, to rapidly fine-tune the temperature of the plastic to maintain a very precise temperature / heat transfer, preventing overshoot; and / or (iii) the pre-heater may receive feedback from thermometers (contact, infrared, or any other suitable means) measuring the surface temperature of the plastic, the temperature (difference) of the incoming and outgoing fluid from the preheater channel, and / or the temperature of the fluid in a downstream element, such as the reaction reservoir.

[0226] The pre-heater may be any suitable heating element compatible with the desired heat transfer. In one embodiment of the invention, the pre-heater is not physically connected with the plastic it heats, but rather comprises an infrared heating element, or a heating element that transfer heat via air (flow) or heatpipe.

[0227] In some embodiments, one or more reservoirs in the apparatus is heated, preferably to maintain the fluid temperature at the desired incubation temperature. In one embodiment, the reservoir(s) are heated indirectly, preferably with an infrared heater or by heating the air around the reservoir. When heating the air around the cartridge of the apparatus, it is preferred that the system comprises a container surrounding the heated reservoir, wherein such container contains one or more heating elements, such as heating mats, and optionally a fan to circulate the air through the container, homogenizing the temperature.

[0228] The benefit of indirect heating may be that the reservoir may be scaled seamlessly without influencing the system's ability to heat the walls of the reservoir.

[0229] The heater may receive feedback from thermometers (contact, infrared, or any other suitable means) measuring the surface temperature of the plastic, the temperature (difference) of the incoming and outgoing fluid from the preheater channel, and / or the temperature of the fluid in a downstream element, such as the reaction reservoir.

[0230] In one embodiment of the invention, the apparatus or the or each independent manufacturing module comprises thermochromic dyes contained in or around the reservoirs of interest, wherein the thermochromic dye changes colour or transparency at an exact temperature set-point. Optical sensors may determine the colour or transparency to determine the local temperature.

[0231] In some embodiments, the apparatus or the or each independent manufacturing module comprises channels, reservoirs, membranes, or complete chips to perform on-board capillary gel electrophoresis (CGE), wherein a microfluidic channel may be used as capillary. The capillary channel is filled with the gel, which is subsequently allowed to polymerize, and a buffer is applied. Reservoirs up- and downstream of the capillary channel comprise an electrode to perform electrophoresis. One or more nucleic acid samples may be loaded onto the gel by injection or pulling the fluid through the channel, or injecting the sample via a T-junction. Prior to loading onto the gel, the sample may be mixed, heated or otherwise prepared for CGE in an upstream reservoir or channel.

[0232] In some embodiments, the apparatus or the or each independent manufacturing module comprises channels, reservoirs, membranes, or complete chips to perform on-board Sanger, next generation or preferably nanopore sequencing. In a particular embodiment, a nanopore sequencing chip is connected to the surface of the apparatus, such that a continuous, leak-free connection is made, wherethrough one or more samples of the nucleic acid may be added to the sequencing chip. In one embodiment, the chip is bolted on the surface of the apparatus, optionally sealed with a thin rubber sheet with a matching hole. In a preferred embodiment, the nanopore sequencing chip is the minion sequencing chip by Oxford nanopore.

[0233] In some embodiments, the method of the first aspect, the apparatus of the second aspect, system of the fourth aspect, or independent manufacturing module of the fifth aspect, are capable of manufacturing RIMA, within typical desired specifications known to those skilled in the art, for a wide range of sequences and RNA types, from a single generic protocol / set of parameters.

[0234] However, in rare instances, complicated sequences may require adjustment of the process parameters. Therefore, the method may comprise the use of one or more small volume reactions, each comprising a fraction of the available reactants, which may be processed sequentially or in parallel in a small volume independent circuit comprising a mixer, reaction reservoir, optionally an extraction or purification chamber, and one or more QC elements.

[0235] Alternatively, the reaction may be performed at full scale using the volume of reactants as intended for the manufacturing run. From the first extraction of the nucleic acid, or the first volume of formulation of the RNA with a delivery vehicle, automated QC may be performed, and process parameters are automatically adjusted based on the readout of the QC modules.

[0236] The products of the small volume reactions or the initial products of the full-scale reaction may be analysed by the one or more QC elements and the most optimal set of process parameters may be selected to perform the actual, larger volume manufacturing run. The selection of the most optimal set of process parameters may be automated based on preset definitions, or may be manual, optionally with an automated recommendation to the user. As an example, channel dynamic light scattering (DLS, wherein a laser is used to determine the nanoparticle size and size distribution) may provide information on the size of the nanoparticles, which may be used to adjust the flowrate of the buffered RIMA and / or delivery vehicle components, affecting the size of the nanoparticles subsequently produced, which is measured again via DLS to adjust further if needed.

[0237] For each portion of the reaction products that is out of-specification, automated or manual decisions may be used to divert the material to the waste channel, rather than continue into downstream process steps or the finished product reservoir.

[0238] The reservoirs in the apparatus may be proportionally or disproportionally scaled, depending on the need. For example, reactant reservoirs may be scaled disproportionally (larger) than reaction reservoirs, to provide sufficient substrate (fed-batch) for a manufacturing run with an increased number of manufacturing cycles.

[0239] During manufacturing or post-processing of the apparatus, the reservoirs may be scaled by:

[0240] (i) using a mould or stamp for one or more of the substrates of the apparatus with a larger positive and / or negative mould element corresponding to the desired reservoir size, wherein a dedicated mould is used for each (combination of) reservoir size(s);

[0241] (ii) using the same mould for all, or a collection of, reservoir sizes, wherein insets are used to enlarge positive or reduce negative mould elements to adjust the size of the reservoirs of the apparatus made with the mould; and / or

[0242] (iii) using the same mould for all, or a collection of, reservoir sizes, wherein the (substrate of the) apparatus produced with the mould has a single reservoir size matching the mould, whereafter the apparatus is post-processed (optionally after assembly of the substrates) by heating the plastic walls of the reservoir and applying a positive or negative mould to stretch the plastic into a larger reservoir size.

[0243] In a particular embodiment of the method of (iii) above, a standard sized reservoir may be manufactured and the size adjusted via post-processing, and is performed in situ by the end-user with a dedicated machine, directly prior to use of the apparatus.

[0244] In principle, scaling of the reaction only requires changes to the size of the reservoirs. However, should scaling of the channels be required to maintain similar or identical flow parameters, the following methods may be used to scale the throughput of the channels: (i) Multiple channels may be positioned parallel to each other, wherein each is identical to or larger (e.g., wider and optionally deeper) than the previous, each leading from and to the same reservoir, and through the positioning of the valve one or more of the channels are connected;

[0245] (ii) An existing channel may be embossed deeper, increasing its flow throughput proportional to the extra depth of the channel. This may be particularly suitable for two-dimensional mixers, wherein the depth of the mixer channel does not affect the mixing; and / or

[0246] (iii) A channel or mixer in a substrate may be overlaid with an identical channel or mixer in another substrate and connected through channels perpendicular to the surface, through the prior substrate.

[0247] Finally, the inventors have developed a method for calibrating and / or testing the system of the invention in which a so-called calibration cartridge containing (sealed-in) standards (such a pure RIMA sample and / or a concentration gradient of superparamagnetic beads) is inserted into the housing, and an absolute or relative correct response is expected from the system.

[0248] Thus, in a seventh aspect, there is provided a method for calibrating and / or testing the system of the fourth aspect, wherein the method comprises inserting, into the system, a calibration cartridge comprising known substrates and / or products, and detecting a response from the system.

[0249] Based on the response, the system may automatically adjust the settings and / or position of an actuator, sensor, or provide an error message to the user. Optionally, the system may provide a diagnostic report and may provide that diagnostic report via wireless connection to a receiver.

[0250] The calibration cartridge may have a similar or substantially the same dimensions or layout and size as the apparatus of the second aspect. The calibration cartridge may contain elements normally not included in the apparatus, such as in channel thermometers, thermogenic dyes, integrated hall sensors, and / or integrated cameras. The calibration cartridge may have an electrical connection to power, control and receive data from the integrated electronic components. Typically, the calibration cartridge is connected or inserted into an independent manufacturing module of the fifth aspect, which is inserted into the housing. The inventors believe that the valves used in their apparatus is a novel and important feature thereof.

[0251] In an eighth aspect, there is provided a valve system, the valve system comprising : first and second substrates defining a valve chamber therebetween, the first substrate comprising an aperture therethrough providing access to the valve chamber, and the second substrate comprising a planar portion comprising first and second fluid inlets; and a valve component disposed in the valve chamber, the valve component comprising opposite first and second surfaces wherein the first surface defines a valve slot configured to reversibly receive a valve actuator therein and second surface defines a cavity, such that a cross section of the cavity has one or more lobes, wherein the valve component is positioned in the valve chamber such that the valve slot is accessible via the aperture in the first substrate and the second surface is disposed adjacent to the planar portion of the second substrate, the valve component being capable of rotational movement between a first position, wherein the first and second fluid inlets are not fluidly connected, and a second position, wherein the first and second fluid inlets are fluidly connected due to the position of the one or more lobes.

[0252] It may be appreciated that the apparatus of the first aspect may define and / or comprise a valve system as defined in the eighth aspect.

[0253] It may be appreciated that the valve actuator can be used to rotate the valve component between the first and second positions.

[0254] It may be appreciated that the valve component may rotate about an axis substantially perpendicular to the planar portion.

[0255] In one embodiment, the first substrate is configured to act on the valve component, and thereby maintain the second surface of the valve component against the planar portion of the second substrate.

[0256] In an alternative embodiment, the valve system may further comprise a valve tensioning component configured to act on the valve component, and thereby maintain the second surface of the valve component against the planar portion of the second substrate. The valve tensioning component may be disposed in the valve chamber. The valve tensioning component may comprise or consist of a first tensioning component, the first tensioning component may comprise a first surface defining a tensioning slot configured to reversibly receive a tensioning actuator therein and a second surface configured to act on the valve component and thereby maintain the second surface of the valve component against the planar portion of the second substrate. The first tensioning component may be positioned such that the tensioning slot is accessible via the aperture in the first substrate and the valve slot is accessible via the aperture in the first substrate and the tensioning slot. The first tensioning component may be capable of rotational movement relative to the first and second substrate. The first tensioning component may comprise a threaded outer surface. The valve system may comprise a corresponding threaded surface. The first tensioning component may be configured such that rotational movement of the first tensioning component causes the first tensioning component to move closer to or further from the second substrate. Rotational movement of the first tensioning component may cause the first tensioning component to move along an axis substantially perpendicular to the planar portion.

[0257] The male component may be capable of rotating independently of the valve component. The tensioning slot may be larger than the valve slot. Accordingly, the valve system may be configured to enable the valve actuator to rotatably manipulate the valve component while the male component may remain unmoved.

[0258] In one embodiment, the valve tensioning component is configured to prevent slipping or modified to be unidirectional. Advantageously, this will prevent the tensioned valve to become untensioned by the rotational movement of the valve. The valve tensioning component may therefore comprise an anti-slip surface, a ratcheting system, a pressure activated glue, or any other method known to those skilled in the art related to making rotational movement unidirectional.

[0259] Alternatively, the valve actuator or the tensioning actuator may act on the tensioning slot to prevent movement of the first tensioning component while the valve actuator rotates the valve component.

[0260] The corresponding threaded surface may be defined by the first substrate. Alternatively, the valve tensioning component may comprise a second tensioning component defining the corresponding threaded surface, the second tensioning component may be disposed in the valve chamber. The first substrate and the second tensioning component may be shaped to prevent rotation movement of the second tensioning component relative to the first substrate. The male component may be capable of rotating about an axis substantially perpendicular to the planar portion.

[0261] In use, it may be possible to determine that a desired tension is reached by using a cam mechanism allowing the actuator to click when a preset torque is reached. Alternatively, or additionally, in use, it may be possible to determine that a desired tension is reached by using a digital torque sensor, that provides feedback when a preset torque is reached, use, it may be possible to determine that a desired tension is reached by measuring the power / current of an electric motor driving the tensioning actuator, and calculating the torque applied by the motor. Alternatively, or additionally, in use, it may be possible to determine that a desired tension is reached by measuring torque applied by the valve actuator. It may be appreciated that when the valve has less tension, the torque required to reach a new rotational position is lowered. Accordingly, measuring the torque applied by the valve actuator provides feedback regarding the need of (re)tensioning the valve.

[0262] Alternatively, or additionally, in use, the leak-tightness of the valve may be measured by applying a known, constant pressure on the valve while in closed position via a connected channel. It may be appreciated that loss of pressure and / or volume is an indication of the need to (re)tensioning the valve.

[0263] All features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0264] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which:-

[0265] Figure 1 is an apparatus containing twenty cartridges, each cartridge being an independent manufacturing module for producing RIMA, or a formulation comprising RNA; Figure 2 is an exploded diagram of a cartridge used in the apparatus of Figure 1;

[0266] Figure 3 shows examples of different cartridges which can be used in the apparatus of Figure 1, specifically showing the variants of the integrated storage of the finished RNA DS or DP (formulated RNA);

[0267] Figure 4 is a flow diagram describing a method of producing RNA or a formulation comprising RNA;

[0268] Figure 5 is a schematic diagram illustrating the method shown in Figure 4; Figure 6 is a schematic diagram showing how a continuous in vitro transcription (IVT) method may be conducted with a static bead or static solid support;

[0269] Figure 7 is a schematic diagram showing how a continuous, one-pot IVT method may be conducted with a dynamic solid support, such as a bead;

[0270] Figure 8 is a schematic diagram showing how a continuous IVT and enzymatic post modification method may be conducted with a static bead or static solid support;

[0271] Figure 9 is a plan view of a cartridge;

[0272] Figure 10 is a plan view of an alternative cartridge;

[0273] Figure 11 is a plan view of a further alternative cartridge;

[0274] Figure 12 is a plan view of a DNA amplification module portion of the cartridge of Figure

[0275] 11;

[0276] Figure 13 is a plan view of an RIMA mixing module portion of the cartridge of Figure 11;

[0277] Figure 14 is a plan view of an RNA purification module portion of the cartridge of Figure 11;

[0278] Figure 15 is a plan view of a first LNP encapsulation module portion of the cartridge of

[0279] Figure 11;

[0280] Figure 16 is a plan view of a second LNP encapsulation module portion of the cartridge of Figure 11;

[0281] Figure 17 is a plan view of an end product module portion of the cartridge of Figure 11;

[0282] Figure 18 is a plan view of a reservoir;

[0283] Figure 19 is a perspective view of a valve system;

[0284] Figure 20 is section view of the valve system of Figure 19;

[0285] Figure 21 is a plan view of the valve system of Figures 19 and 20;

[0286] Figure 22 is a plan view of a valve which is a component part of the valve system shown in Figures 19 to 21;

[0287] Figure 23 is a schematic diagram showing how a continuous IVT method may be conducted, and is a variant of the IVT method shown in Figure 6;

[0288] Figure 24 is a schematic diagram showing how an alternative continuous IVT method may be conducted, and is a variant of the IVT method shown in Figure 7;

[0289] Figure 25 is a schematic diagram showing the mixing of nucleic acid synthesis reactions from individual fluid components. Examples using such fluid mixing can include the assembly of the DNA amplification reaction, the In vitro transcription reaction, and the formulation reaction.

[0290] Figure 26 (A) shows an embodiment of a first generation prototype of a one manufacturing module of the system from Figure 1, (B) shows one first-generation microfluidic cartridge containing the end-to-end RNA DS / DP manufacturing process, and (C) shows an embodiment of a second generation prototype of one manufacturing module of the system of the invention; Figure 27 shows three different embodiments of the arrangement of magnets used to separate beads from the IVT reaction in the microfluidic cartridge: (A) shows a schematic view of magnets moving perpendicular to the cartridge surface, (B) shows a schematic view of magnets moving parallel to the cartridge surface, and (C) a schematic view of magnets of increasing strength forming a magnetic gradient over one axis of a reservoir in the cartridge;

[0291] Figure 28 shows a plan view of a valve which is a component part of the valve system embedded between layers of the cartridge;

[0292] Figure 29 shows a plan view of an apparatus with five modules, wherein each module is attached on a lateral face to the previous module and contains its own structural frame; Figure 30 shows a schematic view of the channel pattern of a bifurcating pre-heating section of the cartridge of the invention, wherein the channel thickness is displayed dimensionless and may be adjusted based on the desired flowthrough; and

[0293] Figure 31 shows a table summarizing independently verified results from three runs on a second-generation prototype of the apparatus of the invention, demonstrating adherence to quality standards for RIMA DS / DP manufacturing.

[0294] Example 1

[0295] The inventors have developed an apparatus 2 which can be used to produce mRNA or a lipid nanoparticle (LNP) formulation comprising mRNA. An embodiment of the apparatus 2 is shown in Figure 1. In the embodiment shown, the apparatus contains twenty identical or similar manufacturing modules 33, each of which can be used independently to produce mRNA or a lipid nanoparticle (LNP) formulation comprising mRNA.

[0296] The apparatus 2 shown in Figure 1 comprises a housing 4 comprising six sides, namely a top 6, a base 8, a front 10, a back 12, a first side 14 and a second side 16. In the embodiment shown in Figure 1, the housing defines a height of 1.8-2 m, a width of 1-2 m and a depth of 1-1.5 m. Castor wheels 18 are disposed on the base 8 of the housing 4, and are configured to enable movement of the apparatus. One or more of the castor wheels 18 may have a mechanical break to prevent the apparatus 2 from moving when in operation.

[0297] The housing 4 defines three layers; a top layer 20 disposed adjacent to the top 6 of the housing 4, a bottom layer 22 disposed adjacent to the base 8 of the housing 4 and a middle layer 14 disposed between the top and bottom layers 20, 22.

[0298] The top layer 20 comprises a compartment 26. The compartment 26 is accessible for maintenance via a door 28 disposed in the front 10 of the housing 4. A controller (not shown) and one or preferably two, or more compressors 30 are disposed in the compartment 26. The controller is configured to control the apparatus 2, and the compressor 30 is configured to provide a base pressure (e.g., 6-10 bar), which is further controlled (adjusted) by the controller in each of the cartridges 34 to pressurise and thus move fluids during use of the apparatus 2, as described in more detail below.

[0299] In the embodiment shown, the bottom and middle layers 22, 24 each define ten slots 32 therein. However, it will be appreciated that in alternative embodiments, the number of slots 32 may vary. For instance, the bottom and middle layers 22, 24 may define between five and twenty slots 32 therein. Each slot 32 defines an opening 35 in the front 10 of the housing 4. Each slot 32 may contain an independent manufacturing module 33. The independent manufacturing module 33 contains a handle 37 enabling the user to at least partially pull the independent manufacturing module 33 out of the slot 32. The independent manufacturing module 33 is configured to receive a removable cartridge 34 therein which can be inserted or removed when the independent manufacturing module 33 is pulled out of the slot 32. In the embodiment shown in Figure 1, one independent manufacturing module 33, containing one cartridge 34 is shown extending from a slot 32. The other slots 32 may each independently be empty or contain an independent manufacturing module 33.

[0300] A screen 36 and control panel 38 disposed on the first side 14 of the housing 4 provide users with information and enable them to operate the apparatus 2.

[0301] The cartridges 34 are shown in more detail in Figures 2 and 3. In the embodiment shown in Figures 2 and 3, the cartridges are 700 mm x 700 mm, and have a thickness of between 10mm and 100mm.

[0302] As shown in Figure 2, each cartridge 34 comprises a first substrate 102 comprising a planar first face 104 and an opposing planar second face (not shown) and a second substrate 108 also comprising a planar first face 110 and an opposing planar second face (not shown). In the assembled cartridge 34, the first and second substrates 104 and 108 are sandwiched together. A pattern may be defined in the second face of the first substrate 102 and / or the first face 110 of the second substrate 108, such that the pattern defines reservoirs and / or chambers and optionally also channels, between the first and second substrates 102 and 108. The first substrate 102 comprises channels 106 extending between the first 104 and second faces thereof. The channels are configured to fluidly connect reservoirs or connectors which disposed on the first face 104 of the first substrate 102 with the reservoirs, chambers and / or channels disposed between the first and second substrates 104 and 108.

[0303] Each cartridge 34 further comprises one or more third substrates 112. In the embodiment shown in Figure 2, there are multiple third substrates. Each third substrate comprises a planar first face 114 and an opposing planar second face (not shown). In the assembled cartridge 34, the second and third substrates 108 and 112 are sandwiched together. A pattern may be defined in the second face of the second substrate 108 and / or the first faces 114 of the third substrates 112, such that the pattern defines microfluidic channels and optionally chambers, between the second and third substrates 102 and 108. Each third substrate 112 preferably defines a separate, preferably complete, reaction module, as described in more details in example 9. The second substrate 108 further comprises channels (not shown) extending between the first and second faces thereof, fluidly connecting the channels 106 in the first substrate 102 or the reservoirs or channels contained between the first and second substrates 102 and 108, with channels or chambers contained between the second and third substrates 108 and 112. The third substrates 112 may further comprise channels (not shown) extending between the first and second faces thereof.

[0304] The reaction chambers disposed either between the first and second substrates 102 and 108 or between the second and third substrates 108 and 112 may include one or more DNA amplification chamber for amplifying a template DNA molecule and one or more IVT chamber for producing transcribed RIMA. In some embodiments, a portion of the microfluidic channels downstream of the one or more IVT chamber are configured to cause turbulence in a solution flowing therethrough. These channels may be defined as one or more mixer channel.

[0305] Each cartridge further optionally comprises one or multiple fourth substrates 116, each fourth substrate comprises a planar first face 118 and an opposing planar second face (not shown). The fourth substrates 116 may further comprise channels (not shown) extending between the first and second faces thereof. In the assembled cartridge 34, the third and fourth substrates 114 and 116 are sandwiched together with a filter 120 disposed therebetween.

[0306] Reservoirs 122 which may be configured to contain consumables (e.g. reactants and / or solvents), products and / or waste may be disposed on the first face 104 of the first substrate 102. The reservoirs 122 may include one or more DNA reactant reservoirs containing reactants to be used in DNA amplification reaction. The reservoirs 122 may also include one or more RIMA reactant reservoirs containing reactants to be used in an in vitro transcription (IVT) reaction. The reservoirs 122 may also include one or more solvent reservoirs and / or one or more lipid reservoirs.

[0307] Depending upon the application, and the amount of RNA required, the reservoirs 122 may vary. The size of the reservoirs may be scaled proportionally or non-proportionally. In particular, for small-scale applications (e.g., for a cartridge producing between 1 mg and lOOmg of RNA) the reactant and / or final product holding reservoirs 122 may comprise vials. For instance, as shown in Figure 3 (A), the vials could be integrated vials 122A extruded from the first face 104 of the first substrate 102. While not shown in Figure 3, the bottom of these reservoirs 122A is defined by the first face 110 of the second substrate 108. Alternatively, as shown in Figure 3 (B) the first substrate 102 may comprise one or more connectors 124 disposed on the first face 104 thereof configured to connect the first substrate 102 to one or more removable vials 122B. For larger-scale applications (e.g., for a cartridge producing between 5 gr and 50 gr of RNA), (vacuum formed) reservoirs 122C and / or 122D may be protruded from the first substrate 102, as shown in Figure 3 (C) and (D). Again, the bottom of the reservoirs 122C and 122D is defined by the first face 110 of the second substrate 108. For even larger scale applications (e.g., a cartridge producing between 50 and 1.5 kg of RNA a day), instead of the cartridge comprising (optionally removable) reservoirs 122 formed directly on the first substrate 102, the cartridge 34 may instead comprise one or more reservoir connectors (not shown) disposed on the first face 104 of the first substrate 102. The reservoir connectors being configured to connect to an external reservoir (not shown). The external reservoir may be disposed externally to the apparatus 2 and piping may extend between the external reservoir and the connector.

[0308] The channels through the substrates and / or the microfluidic channels defined by the patterns in the faces of the substrates are configured such that reactants may be caused to flow from the one or more DNA reactant reservoir to the one or more DNA amplification chamber, optionally via one or more mixer channel.

[0309] Similarly, the channels through the substrates and / or the microfluidic channels defined by the patterns in the faces of the substrates are configured such that amplified DNA may be caused to flow from the one or more DNA amplification chamber to the one or more IVT chamber, optionally via one or more other chambers. Additionally, the channels through the substrates and / or the microfluidic channels defined by the patterns in the faces of the substrates are configured such that reactants may be caused to flow from the one or more RNA reactant reservoirs to the one or more IVT chamber, optionally via one or more mixer channel.

[0310] In embodiments where the apparatus comprises one or more lipid reservoirs, the channels through the substrates and / or the microfluidic channels defined by the patterns in the faces of the substrates are configured such that lipids may be caused to flow from the one or more lipid reservoirs to the one or more mixer channel. Similarly, the channels through the substrates and / or the microfluidic channels defined by the patterns in the faces of the substrates may be configured such that RNA may be caused to flow from the one or more IVT chamber to the one or more mixer channel.

[0311] The channels through the substrates and / or the microfluidic channels defined by the patterns in the faces of the substrates are configured such that a product may be caused to flow from the one or more IVT chamber and / or the one or more mixer channel to a product reservoir. More preferably, the channels through the substrates and / or the microfluidic channels defined by the patterns in the faces of the substrates are configured such that a product may be caused to flow from the one or more IVT chamber and / or the one or more mixer channel to a TFF module.

[0312] The channels through the substrates and / or the microfluidic channels defined by the patterns in the faces of the substrates are configured such that a waste product may be caused to flow from the one or more DNA amplification chamber and / or the one or more IVT chamber to the one or more waste reservoirs. In addition, the channels through the substrates and / or the microfluidic channels defined by the patterns in the faces of the substrates are configured such that a waste product may be caused to flow from the one or more extraction chambers associated with a DNA amplification module and / or one or more IVT modules to the one or more waste reservoirs.

[0313] Referring to Figure 26(A), there is shown an embodiment of a first generation prototype of a one manufacturing module 33 of the apparatus 2 shown in Figure 1. Figure 26(B) shows a first-generation microfluidic cartridge 34 containing the end-to-end RNA DS / DP manufacturing process, and Figure 26(C) shows a second generation prototype of the manufacturing module 33 of the apparatus 2.

[0314] Example 2

[0315] The apparatus described in example 1 may be used in a method of producing RNA. A flow diagram showing the steps of the method is shown in Figure 4 and a schematic of the method is shown in Figure 5. In brief, in use, reactants to flow from the one or more DNA reactant reservoirs through the channels through the substrates and / or the microfluidic channels defined by the patterns in the faces of the substrates to the one or more DNA amplification chamber. Preferably, the reactants combine via a (microfluidic) mixer channel before entering the DNA amplification chamber. Once in the one or more DNA amplification chamber, a DNA amplification reaction would be conducted to amplify the DNA, as shown in the first box in Figure 4. The DNA may be ssDNA.

[0316] The amplified DNA would then be extracted from the DNA amplification chamber, as shown in the second box of Figure 4. The extracted DNA would flow through the channels through the substrates and / or the microfluidic channels defined by the patterns in the faces of the substrates to the one or more IVT chamber. Additionally, reactants would flow from the one or more RNA reactant reservoirs through the channels through the substrates and / or the microfluidic channels defined by the patterns in the faces of the substrates to the one or more IVT chamber. Preferably, the extracted DNA and the reactants combine via a (microfluidic) mixer channel before entering the IVT chamber. A continuous IVT reaction would then be conducted to produce transcribed RNA, as described in the third box of Figure 4. The RNA which is produced may be mRNA.

[0317] The continuous IVT reaction could be conducted in a number of ways. Three alternative embodiments of how the cartridge 34 may be configured to conduct the DNA amplification and / or IVT reaction are discussed in detail in examples 3 to 5.

[0318] As the RNA is produced it may be continuously or intermittently removed from the one or more IVT chambers and residual DNA may be removed therefrom, as described in the fourth box of Figure 4.

[0319] The RNA could optionally be post-modified, for instance it could be capped, as described in the fifth box of Figure 4.

[0320] The RNA could then be combined with a buffer solution and then fed into the one or more mixer channel. Lipids from the one or more lipid reservoirs could be caused to flow through the channels through the substrates and / or the microfluidic channels defined by the patterns in the faces of the substrates to the one or more mixer channel. The turbulence caused by the buffered solution comprising the RNA and the lipids flowing through the one or more mixer channel would cause the components to become mixed and form LNPs comprising the RNA, as described in the sixth box of Figure 4. The transcribed RIMA and / or the LNP formulation could optionally be purified, as described in the seventh box of Figure 4.

[0321] Finally, the transcribed RNA and / or the LNP formulation could be fed into a product store.

[0322] Example 3

[0323] In one embodiment of how the cartridge 34 may be configured to conduct the IVT reaction is illustrated in Figure 6. In this embodiment, the cartridge 34 comprises a first IVT chamber 202a and a second IVT chamber 202b. Each IVT chamber 202a and 202b comprises a reactant inlet 204 and a product and waste outlet 206. The apparatus also comprises an IVT microfluidic channel 208 which extends between the first and second IVT chambers 202a and 202b. Static beads / columns 210 are disposed in each IVT chamber 202a and 202b.

[0324] When enough amplified DNA is produced, it is fed through the inlet 204 into the first IVT chamber 202a. Additionally, reactants from the one or more RNA reactant reservoirs are also fed through the inlet 204 into the first IVT chamber 202a. Preferably, the reactants combine via a (microfluidic) mixer channel before entering the IVT chamber. The mixture is incubated and will form RNA, which will be captured by the static beads / columns 210 as it is formed.

[0325] As shown in image 4 of Figure 6, when the static beads / columns 210 in the first IVT chamber 202a are saturated (or partially saturated), the reaction mixture will be pumped from the first IVT chamber 202a, along the IVT microfluidic channel 208 and into the second IVT chamber 202b. The static beads / columns 210 which were present in the first IVT chamber 202a are retained therein. For instance, this could be done by engaging magnets if the solid support comprises superparamagnetic beads. The static beads / columns 210 in the second IVT chamber are non-saturated.

[0326] As shown in image 5 of Figure 6, a washing solution is pumped through the inlet 204 into the first IVT chamber 202a, and waste is pumped out of the outlet 206. Meanwhile, optionally further reactants from the one or more RNA reactant reservoirs are fed through the inlet 204 into the second IVT chamber 202b, which already contains the IVT mix originating from the first IVT chamber 202a (image 4 of figure 6). The reaction mixture in the second IVT chamber 202b is incubated causing further RNA to form. The RNA will bind to the static beads / columns 210 in the second IVT chamber 202b as it is formed.

[0327] As shown in image 6 of Figure 6, an elution solution (preferably RNAse-free water) is then pumped into the first IVT chamber 202a, to elute the RNA from the static beads / columns 210. The solution comprising the RNA is pumped out of the outlet of the first IVT chamber. Meanwhile, the IVT reaction mixture in second IVT chamber 202b is further incubated causing the static beads / columns 210 in the second IVT chamber 202b to become saturated (or partially saturated).

[0328] Once the static beads / columns 210 in the second IVT chamber 202b are saturated (or partially saturated), the reaction mixture will be pumped from the second IVT chamber 202b, along the IVT microfluidic channel 208 and into the first IVT chamber 202a, as shown in image 1 of Figure 6. The static beads / columns 210 in the second IVT chamber 202b may then be washed and the RNA subsequently eluted while the IVT reaction continues to occur in the first IVT chamber, as shown in images 2 and 3 of Figure 6.

[0329] This process may be repeated as many times as required to produce a desired amount of RNA.

[0330] Example 4

[0331] In a further embodiment of how the cartridge may be configured to conduct the IVT reaction is illustrated in Figure 7. In this embodiment, the cartridge 34 again comprises a first IVT chamber 202a and a second IVT chamber 202b. Each IVT chamber 202a and 202b comprises a reactant inlet 204. Additionally, the cartridge also comprises a first elution chamber 212a and a second elution chamber 212b. Each elution chamber 212a and 212b comprises an elution inlet 214 and a product and waste outlet 216. The elution chambers 212a and 212b are preferably much smaller than the IVT chambers 202a and 202b, so as to obtain a more concentrated elution.

[0332] The apparatus also comprises a first IVT microfluidic channel 214a which extends between the first IVT chamber 202a and the first elution chamber 212a, a second IVT microfluidic channel 214b which extends between the second IVT chamber 202b and the second elution chamber 212b and an elution microfluidic channel 215 which extends between the first and second elution chambers 212a and 212b.

[0333] First dynamic beads / columns 218a are disposed in the first IVT chamber 202a and / or the first elution chamber 212a. The cartridge also comprises a first (set of) movable permanent magnets or electromagnet(s) 220a disposed adjacent the first elution chamber 212a and a second (set of) permanent or electromagnet 220b disposed adjacent the second elution chamber 212b.

[0334] When enough amplified DNA is produced, it is fed through the inlet 204 into the first IVT chamber 202a. Additionally, reactants from the one or more RIMA reactant reservoirs are also fed through the inlet 204 into the first IVT chamber 202a. Preferably, the reactants combine via a (microfluidic) mixer channel before entering the IVT chamber. The mixture is incubated and will form RNA and will be captured by the static beads / columns 210 as it is formed, this is shown in images SI and S2 of Figure 7.

[0335] As shown in image 1 of Figure 7, when the first dynamic beads / columns 218a in the first IVT chamber 202a are saturated (or partially saturated), the reaction mixture will be pumped from the first IVT chamber 202a, along the first IVT microfluidic channel 214a and into the first elution chamber 212a. The first dynamic beads / columns 218a will be carried into the first elution chamber 212a along with the reaction mixture. The electromagnet 220a is then activated, ensuring that the first dynamic beads / columns 218a are held in the first elution chamber 212a. The reaction mixture will then be pumped from the first elution chamber 212a, along the elution microfluidic channel 215 and into the second elution chamber 212b, as shown in image 2 of Figure 7.

[0336] The second electromagnet 220b will be disactivated on at this stage, and the reaction mixture will be pumped from the second elution chamber 212b along the second IVT microfluidic channel 214b and into the second IVT chamber 202b, this is shown in image 3 of Figure 7. Since the second electromagnet 220b is (over the flow duration proportionally) disactivated, the second dynamic beads / columns 218b will flow into the second IVT chamber 202b with the reaction mixture. Reactants from the one or more RNA reactant reservoirs may also be fed through the inlet 204 into the second IVT chamber 202b. At the same time, a washing solution is pumped through the inlet 214 into the first elution chamber 212a, and waste is pumped out of the outlet 216 of the first elution chamber 212a.

[0337] As shown in image 4 of Figure 7, an elution solution (preferably RNAse-free water) is then pumped into the first elution chamber 212a, to elute the RNA from the first dynamic beads / columns 218a. The solution comprising the RNA is pumped out of the outlet 216 of the first elution chamber 212a. At the same time, the reaction mixture is incubated in the second IVT chamber 202b causing further RNA to form. The RNA will bind to the second dynamic beads / columns 218b in the second IVT chamber 202b as it is formed.

[0338] As shown in image 5 of Figure 7, when the second dynamic beads / columns 218b in the second IVT chamber 202b are saturated (or partially saturated), the reaction mixture will be pumped from the second IVT chamber 202b, along the second IVT microfluidic channel 214b and into the second elution chamber 212b. The second dynamic beads / columns 218b will be carried into the second elution chamber 212b along with the reaction mixture. The second electromagnet 220b is activated, ensuring that the second dynamic beads / columns 218b are held in the second elution chamber 212b. The reaction mixture will then be pumped from the second elution chamber 212b, along the elution microfluidic channel 215 and into the first elution chamber 212a, as shown in image 5 of Figure 7.

[0339] The first electromagnet 220a will then be disactivated, and the reaction mixture will be pumped from the first elution chamber 212a along the first IVT microfluidic channel 214a and into the first IVT chamber 202a, this is shown in image 7 of Figure 7. Since the first electromagnet 220a is disactivated, the first dynamic beads / columns 218a will flow into the first IVT chamber 202a with the reaction mixture. Reactants from the one or more RNA reactant reservoirs may also be fed through the inlet 204 into the first IVT chamber 202a. At the same time, a washing solution is pumped through the inlet 214 into the second elution chamber 212b, and waste is pumped out of the outlet 216 of the second elution chamber 212b.

[0340] As shown in image 8 of Figure 7, the elution solution is then pumped into the second elution chamber 212b, to elute the RNA from the second dynamic beads / columns 218b. The solution comprising the RNA is pumped out of the outlet 216 of the second elution chamber 212b. At the same time, the reaction mixture is incubated in the first IVT chamber 202a causing further RNA to form. The RNA will bind to the first dynamic beads / columns 218a in the first IVT chamber 202a as it is formed.

[0341] The process as shown in images 1 to 8 may be repeated as many times as required to produce a desired amount of RNA.

[0342] Example 5

[0343] In a further embodiment of how the cartridge may be configured to conduct the IVT reaction is illustrated in Figure 8. In this embodiment, the cartridge 34 comprises a first IVT chamber 202a, a second IVT chamber 202b, a third IVT chamber 202c and a fourth IVT chamber 202d. Each IVT chamber 202a-d comprises a reactant inlet 204 and a product and waste outlet 206. The apparatus also comprises a first IVT microfluidic channel 208a which extends between the first and second IVT chambers 202a and 202b, a second IVT microfluidic channel 208b which extends between the second and third IVT chambers 202b and 202c, a third IVT microfluidic channel 208c which extends between the third and fourth IVT chambers 202c and 202d and a fourth IVT microfluidic channel 208c which extends between the first and fourth IVT chambers 202a and 202d. Static beads / columns 210 are disposed in each IVT chamber 202a-d.

[0344] The first cycle of the IVT reaction occurs, as described below.

[0345] When enough amplified DNA is produced it is fed through the inlet 204 into the first IVT chamber 202a. Additionally, reactants from the one or more RIMA reactant reservoirs are also fed through the inlet 204 into the first IVT chamber 202a. Preferably, the reactants combine via a (microfluidic) mixer channel before entering the chamber. The mixture is incubated and will form RNA, which will be captured by the static beads / columns 210 as it is formed.

[0346] When the static beads / columns 210 in the first IVT chamber 202a are saturated (or partially saturated), the reaction mixture will be pumped from the first IVT chamber 202a, along the first IVT microfluidic channel 208a and into the second IVT chamber 202b. The static beads / columns 210 which were present in the first IVT chamber 202a are retained therein.

[0347] A washing solution is pumped through the inlet 204 into the first IVT chamber 202a, and waste is pumped out of the outlet 206. Meanwhile, further reactants from the one or more RNA reactant reservoirs may be fed through the inlet 204 into the second IVT chamber 202b. The reaction mixture in the second IVT chamber 202b is incubated causing further RNA to form and bind to the static beads / columns 210 in the second IVT chamber 202b as it is formed.

[0348] Once the static beads / columns 210 in the second IVT chamber 202b are saturated (or partially saturated), the reaction mixture will be pumped from the second IVT chamber 202b, along the second IVT microfluidic channel 208b and into the third IVT chamber 202c. The static beads / columns 210 which were present in the second IVT chamber 202b are retained therein. Reactants from the one or more RNA reactant reservoirs may be fed through the inlet 204 into the third IVT chamber 202c. Additionally, enzymes and reagents configured to modify the transcribed RNA may be fed through the inlet 204 into the first IVT chamber 202a. The mixture in the third IVT chamber 202c is incubated and will form RIMA, which will be captured by the static beads / columns 210 as it is formed. The mixture in the first IVT chamber 202a is also incubated and will modify that RNA captured on the solid support 210 in the first IVT chamber 202a.

[0349] While the mixtures in the first and third IVT chambers 202a and 202c are incubated, a washing solution is pumped through the inlet 204 into the second IVT chamber 202b, and waste is pumped out of the outlet 206.

[0350] The reaction may then be conducted in a continuous manner, as described below.

[0351] As shown in image 7 of Figure 8, once the static beads / columns 210 in the third IVT chamber 202c are saturated (or partially saturated) and the modification reaction in the first IVT chamber 202a is complete, the reaction mixture from the first IVT chamber 202a will be pumped along the first IVT microfluidic channel 208a and into the second IVT chamber 202b, and the reaction mixture from the third IVT chamber 202c will be pumped along the third IVT microfluidic channel 208c and into the fourth IVT chamber 202d. The static beads / columns 210 which were present in the first and third IVT chambers 202a and 202c are retained therein. Reactants from the one or more RNA reactant reservoirs may be fed through the inlet 204 into the fourth IVT chamber 202d.

[0352] As shown in image 8 of Figure 8, the mixture in the fourth IVT chamber 202d is incubated and will form RNA, which will be captured by the static beads / columns 210 as it is formed. The mixture in the second IVT chamber 202b is also incubated and will modify the RNA captured on the solid support 210 in the second IVT chamber 202b. While the mixtures in the second and fourth IVT chambers 202b and 202d are incubated, a washing solution is pumped through the inlets 204 into the first and third IVT chambers 202a and 202c, and waste is pumped out of the outlets 206.

[0353] As shown in image 9 of Figure 8, an elution solution (preferably RNAse-free water) is then pumped into the first elution chamber 212a, to elute the RNA from the first static beads / columns 210. The solution comprising the RNA is pumped out of the outlet 206 of the first IVT chamber 202a. At the same time, a washing solution is pumped through the inlet 204 into the third IVT chamber 202c, and waste is pumped out of the outlet 206. Furthermore, the reaction mixtures are still being incubated in the second and fourth IVT chambers 202b and 202d. As shown in image 10 of Figure 8, once the static beads / columns 210 in the fourth IVT chamber 202d are saturated (or partially saturated) and the modification reaction in the second IVT chamber 202b is complete, the reaction mixture from the second IVT chamber 202b will be pumped along the second IVT microfluidic channel 208b and into the third IVT chamber 202c, and the reaction mixture from the fourth IVT chamber 202d will be pumped along the fourth IVT microfluidic channel 208d and into the first IVT chamber 202a. The static beads / columns 210 which were present in the second and fourth IVT chambers 202b and 202d are retained therein. Reactants from the one or more RNA reactant reservoirs may be fed through the inlet 204 into the first IVT chamber 202a.

[0354] The process may continue, and be repeated as many times as desired, as shown in Figure 8.

[0355] In particular, in a specific chamber an IVT reaction mixture is pumped in and incubated to produce RNA which is captured by the static beads / columns 210 therein. The IVT reaction mixture is then pumped out, to the next chamber, and the beads / columns 210 are retained in the present chamber and washed. An enzymatic reaction mixture is then pumped into the specific chamber and the RNA retained on the beads / columns 210 is modified. The enzymatic reaction mixture is then pumped out, to the next chamber, and the beads / columns 210 are retained in the present chamber and washed. Finally, the modified RNA is eluted and carried out of the chamber. The process may then be repeated.

[0356] Example 6

[0357] In a further embodiment of how the cartridge may be configured to conduct the IVT reaction is illustrated in Figure 23, which is a variant of the embodiment of example 3 (Figure 6), wherein 2 more reservoirs for RNA modification is provided.

[0358] In this embodiment, the cartridge 34 comprises a first IVT chamber R1 and a second IVT chamber R2, and a first RNA modification chamber R3 and a second RNA modification chamber R4. Each IVT chamber R1 and R2, and RNA modification chamber R3 and R4 comprises a reactant inlet 204 and a product and waste outlet 206. The apparatus also comprises an IVT microfluidic channel 208 which extends between the first and second IVT chambers R1 and R2, an RNA modification microfluidic channel which extends between the first and second RNA modification chamber R3 and R4, a microfluidic channel between the first IVT chamber R1 and the first RNA modification chamber R3, and a microfluidic channel between the second IVT chamber R2 and the second RNA modification chamber R4. Static beads / columns 210 are disposed in each IVT chamber Rl and R2, and in RIMA modification chamber R3 and R4.

[0359] When enough amplified DNA is produced, it is fed through the inlet 204 into the first IVT chamber Rl. Additionally, reactants from the one or more RNA reactant reservoirs are also fed through the inlet 204 into the first IVT chamber Rl. Preferably, the reactants combine via a (microfluidic) mixer channel before entering the IVT chamber. The mixture is incubated and will form RNA, which will be captured by the static beads / columns 210 as it is formed.

[0360] As shown in image 4 of Figure 23, when the static beads / columns 210 in the first IVT chamber Rl are saturated (or partially saturated), the reaction mixture will be pumped from the first IVT chamber Rl, along the IVT microfluidic channel 208 and into the second IVT chamber R2. The static beads / columns 210 which were present in the first IVT chamber Rl are retained therein. For instance, this could be done by engaging magnets if the solid support comprises superparamagnetic beads. The static beads / columns 210 in the second IVT chamber are at that moment non-saturated.

[0361] As shown in image 6 of Figure 23, a washing solution is pumped through the inlet 204 into the first IVT chamber Rl, and waste is pumped out of the outlet 206. Meanwhile, optionally further reactants from the one or more RNA reactant reservoirs are fed through the inlet 204 into the second IVT chamber R2, which already contains the IVT mix originating from the first IVT chamber Rl (image 5 of Figure 23). The reaction mixture in the second IVT chamber R2 is incubated causing further RNA to form. The RNA will bind to the static beads / columns 210 in the second IVT chamber R2 as it is formed.

[0362] As shown in image 8 of Figure 23, an elution solution (preferably RNAse-free water) is then pumped into the first IVT chamber Rl, to elute the RNA from the static beads / columns 210. The solution comprising the RNA is pumped to the first RNA modification chamber R3, where it is combined with concentrated buffer solution pumped in via inlet 204 of the first RNA modification chamber to enable binding of the RNA to the beads. Meanwhile, the IVT reaction mixture in second IVT chamber R2 is further incubated causing the static beads / columns 210 in the second IVT chamber 202b to become saturated (or partially saturated).

[0363] Once the static beads / columns 210 in the second IVT chamber R2 are saturated (or partially saturated), the reaction mixture will be pumped from the second IVT chamber R2, along the IVT microfluidic channel 208 and into the first IVT chamber Rl, as shown in image 1 of Figure 23. The static beads / columns 210 in the second IVT chamber R.2 may then be washed and the RNA subsequently eluted and pumped to the second RNA modification chamber R4, while the IVT reaction continues to occur in the first IVT chamber, as shown in images 2, 3 and 4 of Figure 23.

[0364] After the elution of the RNA from the static beads in one of the IVT chambers, and rebinding of the RNA to the static beads in one of the RNA modification chambers subsequent to mixing with concentrated buffer solution (images 4 and 8 of Figure 23), the RNA modification solution is pumped in from the other RNA modification chamber and allowed to incubate with the bead bound RNA to modify such RNA (images 5 and 1 of Figure 23). Subsequent incubation results in modified RNA, which is washed by pumping in wash buffer via said RNA modification chamber to a waste outlet channel. Following washing, the modified RNA is eluted from the static beads in said RNA modification chamber by pumping in an elution buffer or water via the inlet 204 and he solution comprising the RNA is pumped out of the outlet of said RNA modification chamber.

[0365] This process may be repeated as many times as required to produce a desired amount of RNA.

[0366] Example 7

[0367] In one embodiment of how the cartridge may be configured to conduct the IVT reaction is illustrated in Figure 24, which is a variant of the embodiment of example 4 (Figure 7), wherein 2 more reservoirs for RNA modification is provided.

[0368] In this embodiment, the cartridge 34 again comprises a first IVT chamber Rl and a second IVT chamber R2. Each IVT chamber Rl and R2 comprises a reactant inlet 204. Further, the cartridge 34 comprises a first RNA modification chamber R3 and a second RNA modification chamber R4. Additionally, the cartridge also comprises a first elution chamber El, a second elution chamber E2, a third elution chamber E3 and a fourth elution chamber E4. Each elution chamber El, E2, E3 and E4 comprises an elution inlet 214 and a product and waste outlet 216.

[0369] The apparatus also comprises a first IVT microfluidic channel 214a which extends between the first IVT chamber 202a and the first elution chamber 212a, a second IVT microfluidic channel 214b which extends between the second IVT chamber 202b and the second elution chamber 212b and an elution microfluidic channel 215 which extends between the first and second elution chambers 212a and 212b.

[0370] Similarly, the apparatus also comprises a first microfluidic channel which extends between the first RIMA modification chamber R3 and the third elution chamber E3, a second IVT microfluidic channel, which extends between the second RNA modification chamber R4 and the fourth elution chamber E4, a microfluidic channel 215 which extends between the first and second elution chambers El and E2, a microfluidic channel, which extends between the third and fourth elution chambers E3 and E4, a microfluidic channel, which extends between the first and third elution chambers El and E3, a microfluidic channel, which extends between the second and fourth elution chambers E2 and E4, and a microfluidic channel, which extends between the third and first elution chambers E3 and El.

[0371] First dynamic beads / columns are disposed in the first IVT chamber R1 and / or the first elution chamber El. Meanwhile, an RNA modification solution is disposed in the second RNA modification chamber R4. Upon incubation of the IVT solution in the first IVT chamber, the beads in said chamber are saturated, and the IVT solution is pumped via elution chambers El and E2 to the second IVT chamber R2, while a magnet activated on elution chamber El removes the saturated beads originated from the first IVT chamber Rl, and a gradual deactivation of a magnet on elution chamber E2 gradually adds unsaturated beads to the IVT solution entering the second IVT chamber R2.

[0372] The saturated beads in the first elution chamber El are washed by pumping of washing solution via inlet 204 of first elution chamber El and deactivation of the magnet of the first elution chamber El during the final wash step allows transfer of the saturated beads to elution chamber E3, where the beads are retained upon activation of the magnet of ethe third elution chamber E3. Subsequently, RNA modification solution is pumped from the second RNA modification chamber R4 via the fourth elution chamber E4 and third elution chamber E3 to the first RNA modification chamber R3, where the saturated beads are incubated with the RNA modification solution. Meanwhile, unsaturated beads are added via inlet 204 of the first elution chamber El and retained there by activation of the magnet of said first elution chamber El.

[0373] Upon saturation of the beads in the second IVT chamber R2, the IVT solution is pumped via the second elution chamber E2 (where the saturated beads are deposited upon magnet activation) and the first elution chamber El (where unsaturated beads are released in the IVT solution upon gradual deactivation of the magnet) to the first IVT chamber Rl.

[0374] Next, the saturated beads in the second elution chamber E2 are washed by pumping of washing solution via inlet 204 of second elution chamber E2 and deactivation of the magnet of the second elution chamber E2 during the final wash step allows transfer of the saturated beads to the fourth elution chamber E4, where the beads are retained upon activation of the magnet of the fourth elution chamber E4. Subsequently, RNA modification solution is pumped from the first RNA modification chamber R3 via the third elution chamber E3 and fourth elution chamber E4 to the second RNA modification chamber R4, where the saturated beads are incubated with the RNA modification solution. Next, the beads saturated with modified RNA are washed and in the final wash steps transferred to the second elution chamber E2, where they are retained by a magnet, and subsequently eluted by pumping of elution solution via inlet 204 of the second elution chamber E2 and removed via outlet 206 of the second elution chamber. The process may be repeated as many times as needed to obtain the desired amount of material.

[0375] Example 8

[0376] Examples 3 to 7 discuss how the IVT reaction may be conducted. While not discussed in detail, it may be appreciated that these methods could also be used mutatis mutandis to conduct the DNA amplification reaction. Accordingly, these examples also demonstrate how a DNA amplification module may be operated.

[0377] In particular, the IVT chambers in an IVT module would be replaced by DNA amplification chambers in a DNA amplification module.

[0378] Reactants from the one or more DNA amplification reactant reservoirs would feed through an inlet into a DNA amplification chamber. As described above, the reactants may combine via a (microfluidic) mixer channel before entering the IVT chamber. The mixture may be incubated and form amplified DNA, which will be captured by the static beads / columns as it is formed.

[0379] In an initial start-up period, the DNA amplification module may produce sufficient DNA to commence the IVT reaction. Once the start-up period is complete, the IVT reaction may commence (as described in examples 3 to 7). After this start-up period, the DNA amplification module may continue to run, optionally continuously, either at a similar capacity to during the start-up period or at a reduced capacity, to produce further DNA template. This further DNA template may be used to replenish the DNA template in the IVT module, and thereby compensate for losses in the IVT reaction.

[0380] Example 9

[0381] Figure 9 shows an embodiment of the cartridge 34. In the embodiment shown in Figure 9, the cartridge comprises four substrates. Each substrate is 700 mm by 700 mm. Reservoirs 304 are disposed between the first and second substrates 102 and 108. These are described in more detail in examples 12 and 13. Additionally, microfluidic channels 326 are disposed between the second and third substrates 108 and 112. Sterility filters 307 are disposed in between the third substrate and fourth substrates 112 and 116. To load the cartridge, liquids are side injected from external reactant reservoirs through the sterility filters and are directed through microfluidic channels to the reservoirs between the first and second substrates. They are then directed, as required, through microfluidic channels to further reservoirs and / or chambers.

[0382] Example 10

[0383] Figure 10 shows a further embodiment of the cartridge 34. Similarly to the embodiment shown in Figure 9, the cartridge comprises four substrates. Each substrate is 700 mm by 700 mm. Reservoirs, chambers and valve enclosures are disposed between the first and second substrates 102 and 108. These are described in more detail in examples 12 and 13. Additionally, microfluidic channels 326 are disposed between the second and third substrates 108 and 112. Sterility filters 307 are disposed in between the third substrate and fourth substrates 112 and 116. To load the cartridge, liquids are side injected from external reactant reservoirs through the sterility filters and are directed through microfluidic channels to the reservoirs. They are then directed, as required, through microfluidic channels to further reservoirs and / or chambers.

[0384] Example 11

[0385] Figure 11 shows a further embodiment of the cartridge 34. The cartridge 34 comprises first and second substrates 102 and 108 which are each 700 mm by 700 mm, and 750 by 750 mm respectively. The cartridge 34 further comprises six third substrates 112a-f and six corresponding fourth substrates 116a-f. Sterility filters 307 are disposed in between the third substrates 112a-f and fourth substrates 116a-f, and liquids pumped into the cartridge 34 from external reactant reservoirs pass therethrough.

[0386] Conduits 302 and internal reservoirs 304 are defined between the first second substrates 102 and 108. Valves 306 between the first and second substrates 102 and 108, with a functional face against the second substrate 108, enable fluids to be directed and stopped. Additionally, microfluidic channels are defined between the second and third substrates 108 and 112a-f.

[0387] Accurate positioning of the substrates 102, 108, 112a-f and 116a-f is achieved due to positioning holes 308 being provided therein.

[0388] As discussed in more detail below, the cartridge 34 may be viewed as comprising six different modules. The different third and corresponding fourth substrates 112a-f and 116a-f, each relate to different modules in the cartridge 34. These modules are illustrated in Figures 12 to 17.

[0389] In particular, Figure 12 shows a DNA amplification module 310. The DNA amplification module 310 comprises a wash buffer reservoir 312, an enzymes reservoir 314, a magnetic beads reservoir 316, a DNA reservoir 318, an NTP buffers reservoir 320, first and second incubation chambers 322a and 322b and first and second extraction chambers 324a and 324b. The reservoirs and chambers are interconnected by microfluidic channels 326 and the flow of fluids along these is controlled by valves 306.

[0390] Figure 13 shows the RNA mixing module 330. The RNA mixing module 330 comprises a magnetic beads reservoir 332, an NTP buffers reservoir 334, an amplified DNA reservoir 336, an enzymes reservoir 338 and a wash buffer reservoir 340. The reservoirs and chambers are interconnected by microfluidic channels 326 and the flow of fluids along these is controlled by valves 306. Microfluidic mixing channels 342 ensure that the components are well mixed. The conduit 302a which connects the DNA amplification module 310 to the RNA mixing module 330 has been omitted from Figures 12 and 13 for simplicity, but can be seen in Figure 11.

[0391] Figure 14 shows the RNA purification module 350. The RNA purification module 350 comprises first and second incubation chambers 352a and 352b and six extraction chambers 354a-f. The chambers are interconnected by microfluidic channels 326 and the flow of fluids along these is controlled by valves 306. The conduits 302b which connect the RNA mixing module 330 to the RNA purification module 350 have been omitted from Figures 13 and 14 for simplicity, but can be seen in Figure 11.

[0392] Figure 15 shows the first LNP encapsulation module 360. The first LNP encapsulation module 360 comprises a purified RNA reservoir 362, a citrate reservoir 364, a lipids reservoir 366 and an RFW reservoir 368. The reservoirs are interconnected by microfluidic channels 326 and the flow of fluids along these is controlled by valves 306. Microfluidic mixing channels 342 ensure that the components are well mixed. The conduit 302c which connects the RIMA purification module 350 to the first LNP encapsulation module 360 has been omitted from Figures 14 and 15 for simplicity, but can be seen in Figure 11.

[0393] Figure 16 shows the second LNP encapsulation module 370. The second LNP encapsulation module 370 comprises first and second waste collection reservoirs 372a and 372b, first and second encapsulated RNA reservoirs 374a and 374b and first and second RFW distribution reservoirs 376a and 376b. The reservoirs are interconnected by microfluidic channels 326 and the flow of fluids along these is controlled by valves 306. The conduit 302d which connects the first and second LNP encapsulation modules 360 and 370 has been omitted from Figures 15 and 16 for simplicity, but can be seen in Figure 11.

[0394] Figure 17 shows the end product module 380. The end product module 380 comprises an end product reservoir 382. The conduit 302e which connects the second LNP encapsulation module 370 and the end product module 380 is shown in Figures 11 and 17, but has been omitted from Figure 16 for simplicity. A microfluidic channel 326 extends between the conduit 302e and the end product reservoir 382.

[0395] Example 12

[0396] A reservoir 400 is shown in more detail in Figure 18. The reservoir 400 is shaped to define a lowermost tip 402 and an uppermost tip 404. Input liquid is fed through a sterility filter 406 along a microfluidic channel 326 and into an inlet 408. The inlet is substantially adjacent to the lowermost tip 402 of the reservoir 400. Accordingly, in use, liquid will fill the reservoir 400 from the bottom. As liquid fills the reservoir, the air therein will be displaced and will be forced out of an outlet 410, which is substantially adjacent to the uppermost tip 404 of the reservoir 400. The pushed out air is driven through an optional filter 412 to ensure that the exit is closed off from the outside world. The presence of the lowermost tip 402 and uppermost tip 404 and the positioning of the inlets 408 and outlet 410 ensure that air is forced out without leaving excessive residue / bubbles. The bottom of the reservoir, close to a lower tip, has a slight (at least 1°) angle towards the lower tip, ensuring complete emptying. The upper and lower tip may be located anywhere along the top or the bottom of the reservoir respectively, and do not need to be aligned in the vertical orientation. Example 13

[0397] A valve system 500 is shown in more detail in Figures 19 to 22. The valve system 500 comprises a valve housing 502, a valve tensioning system 504 and a valve 506.

[0398] As shown in Figure 19, the valve housing 502 is a (vacuum formed) dome 508, which a protruded portion of the first substrate 102. An aperture 510 through the first substrate 102, in the centre of the dome 508, allows external access to both the valve tensioning system 504 and the valve 500, as explained in more detail below.

[0399] The valve tensioning system 504 comprises female and male parts, 512 and 514, respectively. Please note, for simplicity, the male part 514 is only shown in the valve system 500 to the top right of Figure 19 and is not shown in the other two illustrated valve systems in Figure 19. The female part 512 has an external profile / shape matching the internal profile / shape of the dome 508. The dome 508 and female part 512 are shaped to prevent rotational movement of the female part 512 relative to the dome 508, when the female part is disposed therein. The female part 512 has an internal surface 516, which is threaded. Similarly, the male part 514 has an external surface 518, which is also threaded, such that the female part 512 is configured to receive the male part 514 therein.

[0400] The valve 506 is sized and shaped to fit within the male part 514 of the valve tensioning system 504. The valve 506 is capable of rotating relative to the male part 514. When the cartridge 34 is assembled, the valve 505 will be disposed against the first face 110 of the second substrate 108.

[0401] The male part 514 defines a first slot 520, such that when the valve tensioning system 504 is disposed in the dome 508, the first slot 520 is disposed adjacent to and is accessible via the aperture 510. The first slot 520 allows a tool to mate and rotate the male part 504. In the embodiment shown in the figures, the first slot 520 has a hexagonal cross section. Rotation of the male part 514 relative to the female part 512 will cause it reduce or increase the distance between the male part 514 and the second substrate 108. Accordingly, the male part 514 may be manipulated to ensure that it acts on the valve 506, thereby causing the valve 506 to be held against the second substrate 108.

[0402] It may be appreciated that in an alternative embodiment (not shown) the valve system 500 may not comprise a female part 512. In this embodiment, an internal surface of the dome 506 may be threaded and sized to receive the male part 514. The valve 506 defines a second slot 522, which is smaller than the first slot 520. When the valve 506 is disposed in the valve tensioning system 504 and the valve tensioning system 504 is disposed in the dome 508, the first slot 522 is disposed adjacent to and is accessible via the first slot 520. The second slot 522 is sized such that rotation of the valve by a suitable tool mating with the first slot 522 does not cause rotation of the male part 514. In the embodiment shown in the figures, the second slot 522 has a hexagonal cross section.

[0403] In any embodiment, valve 506 defines a cavity 524 therein, such that when the valve 506 is disposed adjacent to the second substrate 108, the cavity 524 will define a chamber 526. As shown in Figure 22, the cavity 524 has an essentially circular cross section with a lobe 528 extending one portion of the circle outward. As shown in Figure 22, the cavity may be disposed directly over an inlet 530 from a first channel 532. When the valve 506 is in a first configuration (shown in Figure 21) an inlet 534 from a second channel 536 may be disposed outside of the cavity 524. However, rotation movement of the valve 506 90° clockwise would cause the lobe 528 to be positioned over the inlet 534, thereby fluidly connecting the channels 532 and 536.

[0404] Example 14

[0405] Referring now to Figure 25, the various raw materials required to make the RNA molecule or formulation thereof (e.g. nucleic acid and nanoparticles) are each stored in independent reservoirs (Rl, R2, R3 and Rn) disposed in the microfluidic cartridge 34. The cartridge 34 is supplied with gas from a connected gas cylinder, as shown.

[0406] By means of separate mass flow controllers (MFC1, MFC2, MFC3, MFCn), each one being configured inline with its respective reservoir (Rl, R2, R3 and Rn), a defined pressure is applied to the raw material fluid by the gas, such that a defined separate fluid flow rate of each raw material is achieved, which is illustrated as flow rate 1, 2, 3, n. This flow rate can be separately adjusted, such that the raw materials from the multiple reservoirs can be combined at pre-specified ratios, and thus a very specific ratiometrically defined mixture (Rl :R2:R3:Rn) can be obtained upon transfer through the microfluidic mixer.

[0407] This exquisite ability of the microfluidic cartridge 34 to very precisely mix the separate components in the raw materials from separate feeds via the mass flow controllers is hugely advantageous for the performance of the subsequent molecular biological or physical (e.g., formulation of LNPs) processes. Example 15

[0408] As described herein, the RIMA that is produced in the IVT reaction is removed from the reaction mix using capture beads, which firstly attach to the RNA molecules, and which are then magnetically separated from the reaction mix, to thereby remove the RNA.

[0409] Various embodiments of how the apparatus 2 can be configured to capture and magnetically attract, and thereby separate, the RNA-capture beads from the reaction mix is illustrated in Figure 27.

[0410] As illustrated in the embodiment shown in Figure 27A, a magnet is moved perpendicularly to the surface of the cartridge 34, such that the magnetic force applied to the superparamagnetic beads in the reservoir El is increased by shortening the distance and the beads are attracted thereto (step 2), or decreased by increasing the distance and the beads are released therefrom (step 3). One ordinarily skilled in the art will know that the magnetic force that is applied to the beads is quadratically related to the distance between the magnet and the beads. As such, it is possible to attract the beads (which have captured the RNA molecules) and move them within the reservoir El to desired locations.

[0411] In the alternative embodiment shown in Figure 27B, however, a magnet is positioned at a constant distance from the cartridge and is moved parallel to the surface of the cartridge, preferably parallel to the surface of reservoir El, enabling the movement of attracted beads along the reservoir. Step 1 shows the magnet and beads (which have captured the RNA molecules) at one end of the reservoir or channel, step 2 shows the magnet and beads moving along the reservoir or channel, and step 3 shows the magnet and beads at the opposite end of the reservoir or channel.

[0412] The embodiments and methods shown in Figures (A) and (B) can be combined in a way such that the magnet is first moved perpendicularly towards the surface to attract the beads, and then laterally to move the beads through the reservoir or channel from one end to the other, and then perpendicularly again away from the reservoir surface to release the beads.

[0413] In the embodiment shown in Figure 27C, permanent magnets or electromagnets of increasing strength (represented by the larger curved lines) are positioned along a reservoir or channel in such a manner that a magnetic gradient is created, and applied over the reservoir or channel. Magnet 2 is stronger than magnet 1, and magnet 3 is stronger than magnet 2. The magnetic gradient from magnet 1 to magnet 2 to magnet 3 effectively moves the beads through the reservoir of channel.

[0414] Example 16

[0415] In one embodiment of the cartridge 34, the valves 306 are located in between substrate layers 102, 112, and have a thickness which is equal to 1, 2 or more intermediate substrate layers. In Figure 28, the valve (306) is located in between the surfaces of layer 1 and layer 3.

[0416] The valve 306 rotates on an indentation 610 in layer 3 enabling precise location to the through holes in layer 3 to make precise fluidic connections. The valve 306 extends through a circular hole in layer 1 to be actuated upon from the top. The valve 306 is shown in a closed position, meaning its cavity is connected to only 1 of the through holes in layer 3.

[0417] Example 17

[0418] In one embodiment of the apparatus 2 of the invention, each independent manufacturing module 33 has an independent structural frame 700 and can be moved and positioned independently. Installation of the independent module 33 occurs by electronically and physically connecting it to either the main module (containing shared facilities) or a previously installed independent manufacturing module. Each independent manufacturing module can, upon installation, be independently accessed, operated and cleaned. In the embodiment shown in Figure 29, each module 33 can be accessed by sliding the module 33 forward, extending it beyond its independent structural frame.

[0419] Example 18

[0420] In some embodiments, the apparatus comprises a preheater which is configured to preheat the reactants and / or the reaction mix or a fraction thereof to the incubation temperature before entering the incubation reservoirs. This means that the incubation reservoirs do not need to heat the solution to the incubation temperature, but may instead maintain the incubation temperature. As such, the preheater, in use, is configured to pre-heat the DNA amplification, IVT reaction and / or RIMA formulation reactions (i.e. buffer solution and the one or more delivery component) at the desired incubation temperatures.

[0421] As shown in Figure 30, in one embodiment of the cartridge 34, the cartridge 34 contains a repeatedly bifurcating channel 800 which drastically slows the local flow rate of the fluid passing therethrough, but maintains a very high total flow rate over the element. This allows for a heater cartridge to (pre)heat the fluid at relative low temperature of the heater cartridge.

[0422] Example 19

[0423] To demonstrate the performance of a prototype of the apparatus 2, a RNA / LNP manufacturing run was performed under simulated GMP conditions. The resultant material was tested according to FDA and USP standards by third parties and the results are summarized in Figure 31, demonstrating the capability of the apparatus 2 to achieve acceptable levels for each critical quality attribute.

[0424] 60 minutes before the start of the manufacturing run, the apparatus 2 was powered on and, from the GUI 36, the INIT command was selected to initiate all components. Afterwards the CALIBRATE command was selected to put all components in the correct starting position. The cartridge 34 was then installed, the LOT number was registered (RPO-FS-038), and afterwards, the HEATING command was selected (37°C) to ensure pre-heating of the reservoir.

[0425] A pre-mix of starting reagents was made for the starting reservoirs. For the enzymestorage reservoir, 250pL RiboPerfect™ RNA Polymerase (RIBOPRO), 31 pL Inorganic pyrophosphatase (Thermo Fisher Scientific) and 100 pL RNase inhibitor (Thermo Fisher Scientific) was thoroughly mixed. For the aqueous-storage reservoir, 150 pL ATP, 100 pL GTP, CTP, and UTP, together with 100 pL of lOx RiboPerfect™ IVT buffer, 80 pL CleanCap AG (3' OMe) (TriLink BioTechnologies), and 444 pL nuclease-free Water was thoroughly mixed. For the DNA-storage reservoir, 100 pL of DNA construct (2.6 kb) was thoroughly mixed with 444 pL of nuclease-free water. For the beads-storage reservoir, OdT(25) PS-beads (NEB, catalog#: S1419S) were resuspended to ensure homogeneity. 6500 pL of beads were first equilibrated by washing in 13000 pL nuclease-free water and equilibration in IVT buffer to remove storage buffer remnants. For the washing buffer compartment, 50 mL wash-buffer was prepared (10 mM Tris-HCL pH 7.5, 150 mM LiCI). For the lipid-storage compartment, a premix of 2658 pL a proprietary lipid mixture (LNP47N, Genevant Sciences) was thawed at 37°C. For the LNP formulation buffer storage compartment, 2278 pL of Acetate buffer was premixed. Afterwards, all premixed reagents were transferred to separate syringes. Each syringe was connected to the corresponding input port on the cartridge 34 and the contents were injected to the cartridge 34.

[0426] For cycle 1, the command RPO-FS-036 was selected, and the machine 2 performed all steps required under supervision of the machine controller to ensure that the machine functioned as expected. For every one of 10 cycles, after completion of the mRNA purification, samples were taken of each elute for internal process development and QC testing. Subsequently, the DS was formulated into LNPs. In the cartridge 34, the RIMA was premixed with acetate and nuclease-free water to the desired starting concentration and directly afterwards mixed with a 4: 1 aqueous:ethanol(lipids) ratio. The LNPs were buffer exchanged in multiple rounds into a final buffer content of 5 mM TRIS, 10% Sucrose (w / v), pH8. The material was then transferred to a Grade A / B cleanroom for aseptic fill and finish. Samples were taken for internal process development and QC testing, and the results are shown in Figure 31.

Claims

Claims1. A microfluidic RIMA manufacturing apparatus for preparing an RNA molecule, or a formulation thereof, the apparatus comprising :(i) one or more DNA amplification chamber in which, in use, a template DNA molecule is amplified to produce amplified DNA, and optionally in which the amplified DNA is modified;(ii) one or more IVT chamber in which, in use, in vitro transcription (IVT) is performed to produce transcribed RNA, and optionally in which the transcribed RNA is modified;(iii) optionally one or more purification chamber or channel in which, in use, the RNA is purified;(iv) one or more DNA substrate reservoir for containing DNA substrates, or one or more DNA substrate connector connectable to a DNA substrate reservoir, wherein the DNA substrates, in use, are required for amplifying DNA;(v) one or more RNA substrate reservoirs for containing RNA substrates, or one or more RNA substrate connector connectable to an RNA substrate reservoir, wherein the RNA substrates, in use, are required for performing IVT;(vi) one or more RNA product storage reservoir or one or more RNA product connector connectable to an RNA product storage reservoir;(vii) one or more DNA microfluidic channel along which, in use, DNA substrates are fed from the one or more DNA substrate reservoir to the one or more DNA amplification chamber and / or the one or more IVT chamber;(viii) one or more RNA microfluidic channel along which, in use, RNA substrates are fed from the one or more RNA substrate reservoir to the one or more IVT chamber;(ix) one or more amplified DNA microfluidic channel along which, in use, amplified DNA is fed from the one or more DNA amplification chamber, to the one or more IVT chamber; and(x) one or more transcribed RNA microfluidic channel along which, in use, transcribed RNA is fed from the one or more IVT chamber to an optional purification chamber and / or the RNA product storage reservoir.

2. The apparatus according to claim 1, wherein the apparatus comprises two or more substrates which are attached, joined or fused together defining a layer therebetween, optionally wherein two or more substrates, when attached, joined or fused together, define the one or more channel, reservoir and / or chamber therebetween, and / or the two or more substrates independently comprise one ormore channel therethrough, the one or more channel being configured to fluidly connect two or more layers and / or a layer to an external fluid source.

3. The apparatus according to either claim 1 or claim 2, wherein the apparatus is for continuously preparing an RIMA molecule, optionally wherein the apparatus is configured to continuously extract the amplified, and optionally post-amplification modified, DNA molecule from the reaction mixture; and / or wherein the apparatus is configured to continuously extract the transcribed RNA molecule, and optionally remove residual DNA.

4. The apparatus according to either claim 1 or claim 2, wherein the apparatus is for intermittently preparing an RNA molecule, optionally wherein the apparatus is configured to intermittently extract the amplified, and optionally postamplification modified, DNA molecule from the reaction mixture; and / or wherein the apparatus is configured to intermittently extract the transcribed RNA molecule, and optionally remove residual DNA.

5. The apparatus according to any preceding claim, wherein the apparatus further comprises:(xi) one or more RNA formulation reservoirs for containing one or more delivery component, wherein the one or more delivery component, in use, is required for formulating the RNA;(xii) one or more mixer configured, in use, to mix transcribed RNA and one or more delivery component to provide formulated RNA;(xiii) one or more further transcribed RNA microfluidic channel along which, in use, RNA substrates are fed from the RNA product storage reservoir to the one or more mixer; and(xiv) one or more delivery component microfluidic channel along which, in use, the one or more delivery component is fed from the one or more RNA formulation reservoir, to the one or more mixer.

6. The apparatus according to claim 5, wherein the mixer comprises one or more microfluidic channels configured to mix one or more fluids travelling therethrough, optionally wherein a series of mixers is used to sequentially mix multiple fluids.

7. The apparatus according to any preceding claim, wherein the apparatus further comprises:(xv) one or more buffer reservoirs for containing a buffer therein; and(xvi) one or more buffer microfluidic channel along which, in use, the buffer is fed from the one or more buffer reservoir, to the one or more mixer or to the one or more further transcribed RIMA microfluidic channel.

8. The apparatus according to any preceding claim, wherein the apparatus comprises:(i) one or more IVT chamber in which, in use, in vitro transcription (IVT) is performed to produce transcribed RNA, and optionally in which the transcribed RNA is modified;(ii) optionally one or more purification chamber or channel in which, in use, the RNA is purified;(iii) one or more DNA substrate reservoir for containing DNA substrates, or one or more DNA substrate connector connectable to a DNA substrate reservoir, wherein the DNA substrates, in use, are required for performing IVT;(iv) one or more RNA substrate reservoirs for containing RNA substrates, or one or more RNA substrate connector connectable to an RNA substrate reservoir, wherein the RNA substrates, in use, are required for performing IVT;(v) one or more RNA product storage reservoir or one or more RNA product connector connectable to an RNA product storage reservoir;(vi) one or more DNA microfluidic channel along which, in use, DNA substrates are fed from the one or more DNA substrate reservoir to the IVT chamber;(vii) one or more RNA microfluidic channel along which, in use, RNA substrates are fed from the one or more RNA substrate reservoir to the one or more IVT chamber;(viii) one or more transcribed RNA microfluidic channel along which, in use, transcribed RNA is fed from the one or more IVT chamber to an optional purification chamber and / or the RNA product storage reservoir;(ix) one or more RNA formulation reservoirs for containing one or more delivery component, wherein the one or more delivery component, in use, is required for formulating the RNA;(xi) one or more mixer configured, in use, to mix transcribed RNA and one or more delivery component to provide formulated RNA;(xii) one or more further transcribed RNA microfluidic channel along which, in use, RNA substrates are fed from the RNA product storage reservoir to the one or more mixer; and(xiii) one or more delivery component microfluidic channel along which, in use, the one or more delivery component is fed from the one or more RIMA formulation reservoir, to the one or more mixer.

9. The apparatus according to any preceding claim, wherein the apparatus further comprises:(xv) one or more buffer reservoirs for containing a buffer therein; and(xvi) one or more buffer microfluidic channel along which, in use, the buffer is fed from the one or more buffer reservoir, to the one or more mixer or to the one or more further transcribed RNA microfluidic channel.

10. The apparatus according to any preceding claim, wherein the apparatus is positioned and operated substantially vertically, wherein the direction of gravity is parallel to the face of the apparatus, such that reservoirs and channels are gravity-assisted.

11. The apparatus according to any preceding claim, wherein the apparatus comprises one or more filter disposed in the one or more microfluidic DNA channel and / or one or more microfluidic RNA channel, optionally wherein the one or more filter is a size-selective filter, optionally wherein the apparatus comprises (i) one or more entry-guarding filter, which is configured to prevent contamination with bacterial elements, (ii) one or more internal filter configured to prevent DNA extraction means from reaching the IVT process, and / or RNA extraction means from mixing with RNA formulated with the one or more delivery component and / or contaminating the final product; and / or (iii) one or more exitguarding filter, which is configured to prevent any particulate material from the apparatus entering in the fill and finish process or reverse flow introducing foreign material in the exit channels of the apparatus.

12. The apparatus according to any preceding claim, wherein the apparatus comprises one or more valve controlling the access and / or direction of fluids through the one or more DNA channel and / or one or more RNA channel.

13. The apparatus according to any preceding claim, wherein the one or more DNA amplification chamber comprises a reservoir formed by a void between a second face of a first substrate and a first face of a second substrate, or an equivalent between two other substrates; an external container connected to a connector on a first face of the first substrate, or an equivalent on the outside facing face ofany other substrate, wherein such container may be a tube, a bottle, a vat, barrel or tank, a tubing, a (collection of) hollow fibre(s), or any other suitable closed container with a suitable connector; or a channel between the second face of the first substrate and the first face of the second substrate, or an equivalent between two other substrates.

14. The apparatus according to any preceding claim, wherein the one or more IVT chamber comprises a reservoir formed by a void between a second face of a first substrate and a first face of a second substrate, or an equivalent between two other substrates; an external container connected to a connector on a first face of the first substrate, or an equivalent on the outside facing face of any other substrate, wherein such container may be a tube, a bottle, a vat, barrel or tank, a tubing, a (collection of) hollow fibre(s), or any other suitable closed container with a suitable connector; or a channel between the second face of the first substrate and the first face of the second substrate, or an equivalent between two other substrates.

15. The apparatus according to any preceding claim, wherein the apparatus comprises one or more purification chamber or channel in which, in use, the RIMA is purified, optionally wherein the purification chamber or channel comprises a reservoir formed by a void between a second face of a first substrate and a first face of a second substrate, or an equivalent between two other substrates; an external container connected to a connector on a first face of the first substrate, or an equivalent on the outside facing face of any other substrate, wherein such container may be a tube, a bottle, a vat, barrel or tank, a tubing, a (collection of) hollow fibre(s), or any other suitable closed container with a suitable connector; or a channel between the second face of the first substrate and the first face of the second substrate, or an equivalent between two other substrates.

16. The apparatus according to any preceding claim, wherein the one or more DNA substrate reservoir for containing DNA substrates comprises a reservoir formed by a void between a second face of a first substrate and a first face of a second substrate, or an equivalent between two other substrates; an external container connected to a connector on a first face of the first substrate, or an equivalent on the outside facing face of any other substrate, wherein such container may be a tube, a bottle, a vat, barrel or tank, a tubing, a (collection of) hollow fibre(s), or any other suitable closed container with a suitable connector; or a channel between the second face of the first substrate and the first face of the second substrate, or an equivalent between two other substrates.

17. The apparatus according to any preceding claim, wherein the apparatus comprises one or more DNA substrate connector which is connectable to an external DNA substrate reservoir.

18. The apparatus according to any preceding claim, wherein the one or more DNA substrate reservoir for containing DNA substrates comprise an external container with one or more pierceable face and / or side, such that attachment of the external container to a corresponding face of the apparatus results in one or more faces being pierced and a leak-free seal is formed between the inside of the external container and one or more chambers or channels in the apparatus.

19. The apparatus according to any preceding claim, wherein the one or more RNA substrate reservoir for containing RNA substrates comprises a reservoir formed by a void between a second face of a first substrate and a first face of a second substrate, or an equivalent between two other substrates; an external container connected to a connector on a first face of the first substrate, or an equivalent on the outside facing face of any other substrate, wherein such container may be a tube, a bottle, a vat, barrel or tank, a tubing, a (collection of) hollow fibre(s), or any other suitable closed container with a suitable connector; or a channel between the second face of the first substrate and the first face of the second substrate, or an equivalent between two other substrates.

20. The apparatus according to any preceding claim, wherein the apparatus comprises one or more RNA substrate connector which is connectable to an external RNA substrate reservoir.

21. The apparatus according to any preceding claim, wherein the one or more RNA substrate reservoir for containing RNA substrates comprises an external container with one or more pierceable face and / or side, such that attachment of the external container to a corresponding face of the apparatus results in one or more faces being pierced and a leak-free seal is formed between the inside of the external container and one or more chambers or channels in the apparatus.

22. The apparatus according to any preceding claim, wherein the one or more RNA product storage reservoir comprises a reservoir formed by a void between a second face of a first substrate and a first face of a second substrate, or an equivalent between two other substrates; an external container connected to a connector on a first face of a first substrate, or an equivalent on the outsidefacing face of any other substrate, wherein such container may be a tube, a bottle, a vat, barrel or tank, a tubing, a (collection of) hollow fibre(s), or any other suitable closed container with a suitable connector; or a channel between the second face of the first substrate and the first face of the second substrate, or an equivalent between two other substrates.

23. A method of manufacturing the apparatus according to any one of claims 1-22, the method comprising :(a) attaching, joining or fusing two or more substrates together to define a microfluidic layer therebetween; or(b) vacuum forming or blow moulding millilitre to litre scale reservoirs in one substrate and subsequently attaching, joining or fusing the substrate to another substrate to form a reservoir to hold fluid.

24. An independent manufacturing module configured to receive or be operably connected to an apparatus according to any one of claims 1-22 or manufactured by the method according to claim 23.

25. The independent manufacturing module according to claim 24, wherein the or each independent manufacturing module comprises an independent set of flow and / or pressure controllers (such as proportional valves, one-way valves, solenoid valves, etc.), heating elements, optical detectors and light sources, electromotors (such as servos, stepper motors, etc.) for rotating rotational valves or linearly moving sliding valves, movable permanent magnets, neutralizable permanent magnets, and / or electronically controllable electromagnets, each positioned and capable of interacting with the relevant apparatus, ideally when in situ in the housing.

26. The independent manufacturing module according to either claim 24 or claim 25, wherein the independent manufacturing module comprises a central pressure system for applying positive and / or negative pressure to the apparatus.

27. The independent manufacturing module according to any one of claims 24-26, wherein the independent manufacturing module comprises one or more pressure controllers, preferably mass flow controllers, for applying the final application pressure to the channels and reservoirs of the apparatus, optionally wherein the pressure controllers and / or mass flow controllers comprise a feedback loop which is configured to vary the final application pressure suitable for ratiometrically-defined mixing of fluids.

28. The independent manufacturing module according to any one of claims 24-27, wherein the independent manufacturing module comprises:(i) one or more magnet configured to separate RIMA capture means, typically RNA capture beads, from the IVT reaction;(ii) one or more movable permanent magnets, which may be moved perpendicular to the face of the apparatus, changing its effect by the increase or decrease of the distance between the magnet and the contents of the apparatus;(iii) one or more neutralizable magnets which may contain multiple magnets in an orientation, wherein in the 'off' position, the magnetic poles are aligned to neutralize each other's magnetic field at the device's surface, and wherein in the 'on' position, the magnets align their poles to create a concentrated magnetic field at the device's surface;(iv) one or more electromagnets which may be static and may be switched 'on' and 'off' by permitting a current of sufficient strength through the coils of the electromagnet;(v) one or more movable permanent magnets, which may be moved parallel to the face of the apparatus, changing the location of its effect relative to the contents of the apparatus; and / or(vi) a combination of magnets of differing strength, disposed along the surface of the apparatus, creating a magnetic gradient over the contents of the apparatus.

29. The independent manufacturing module according to any one of claims 24-28, wherein the independent manufacturing module comprises one or more optical or magnetic decoders to read the partial, full or multiple of rotations of the motor, and / or the connected elements, such as the rotational valves in the apparatus.

30. The independent manufacturing module according to any one of claims 24-29, wherein the independent manufacturing module comprises one or more magnetic switches or hall sensors to determine the magnetic strength of the permanent or electromagnets in a particular location of the apparatus.

31. The independent manufacturing module according to any one of claims 24-30, wherein the independent manufacturing module comprises one or more optical sensors to detect absorption, fluorescence, colour changes, optical diffraction,time-of-flig ht / reflections, phase-contrast changes, transparency, or other optical parameters and / or changes thereto to the contents of the consumable.

32. The independent manufacturing module according to any one of claims 24-31, wherein the independent manufacturing module comprises a preheater which is configured to preheat the reactants and / or the reaction mix or a fraction thereof to the incubation temperature before entering the incubation reservoirs, such that the incubation reservoirs do not need to heat the solution to the incubation temperature, but may instead maintain the incubation temperature.

33. A system comprising a housing configured to operably receive one or more apparatus according to any one of claims 1-22 or manufactured by the method according to claim 23.

34. The system according to claim 33, wherein the system comprises:(i) at least two, three, four or five apparatuses;(ii) at least six, seven, eight or nine apparatuses;(iii) at least 11, 12, 13, 14 or 15 apparatuses;(iv) at least 16, 17, 18, 19 or 20 apparatuses; or(v) a plurality of independent apparatuses, wherein each apparatus comprises an independent housing.

35. The system according to either claim 33 or claim 34, the system comprises a processor configured to control the one or more apparatus, optionally wherein each apparatus is controlled independently.

36. The system according to any one of claims 33-35, wherein the system comprises one or more gas compressor for providing pressure to the apparatus.

37. The system according to any one of claims 33-36, wherein the system comprises one or more independent manufacturing module, each configured to receive or be operably connected to the apparatus, and wherein the one or each independent manufacturing module is configured to independently operate the apparatus, preferably when it is attached to the housing.

38. The system according to claim 37, wherein the one or more independent manufacturing module is as defined in any one of claims 24-32.

39. A method for testing the integrity and / or selective filtering of the entry and / or exit filters on the apparatus according to any one of claims 1-22, the method comprising analysing the entry and / or exit filters for any damage.

40. A method for calibrating and / or testing the system according to any one of claims 33-28, wherein the method comprises inserting, into the system, a calibration cartridge comprising known substrates and / or products, and detecting a response from the system.

41. A method of preparing an RIMA molecule, optionally capped, or a formulation thereof, wherein the method comprises:(i) contacting a template DNA molecule with raw materials of a DNA amplification reaction and incubating the resultant reaction mixture to amplify the DNA molecule, and optionally modifying the amplified DNA molecule;(ii) extracting the amplified, and optionally post-amplification modified, DNA molecule from the reaction mixture;(iii) contacting the extracted amplified DNA molecule from (ii) with raw materials of an in vitro transcription (IVT) reaction and incubating the resultant reaction mixture to produce a transcribed RNA molecule;(iv) extracting the transcribed RNA molecule, and optionally removing residual DNA;(v) optionally post-modifying the extracted RNA molecule;(vi) optionally contacting the transcribed RNA molecule from (iv) or (v) with a buffer solution and contacting the buffered RNA with one or more delivery component to produce formulated RNA, and(vii) subjecting the naked RNA from (iv or v) or the formulated RNA from (vi) to one or more purification step to prepare an RNA molecule, optionally capped, or a formulation thereof.

42. The method according to claim 41, wherein the method comprises continuously preparing an RNA molecule, optionally wherein step (ii) comprises continuously extracting the amplified, and optionally post-amplification modified, DNA molecule from the reaction mixture; and / or step (ii) comprises continuously extracting the transcribed RNA molecule, and optionally removing residual DNA.

43. The method according to claim 41, wherein the method comprises intermittently preparing an RNA molecule, optionally wherein step (ii) comprises intermittently extracting the amplified, and optionally post-amplification modified, DNAmolecule from the reaction mixture; and / or step (ii) comprises intermittently extracting the transcribed RIMA molecule, and optionally removing residual DNA.

44. The method according to any one of claims 41-43, wherein the method comprises:(i) contacting an IVT-ready DNA molecule with raw materials of an in vitro transcription (IVT) reaction and incubating the resultant reaction mixture to produce a transcribed (optionally capped) RNA molecule;(ii) continuously or intermittently extracting the transcribed RNA molecule, and optionally removing residual DNA;(iii) optionally post-modifying the extracted RNA molecule;(iv) optionally contacting the transcribed RNA molecule from (ii) or (iii) with a buffer solution and contacting the buffered RNA with one or more delivery component to produce formulated RNA, and(v) subjecting the naked RNA from (ii or iii) or the formulated RNA from (iv) to one or more purification step to prepare an RNA molecule, optionally capped, or a formulation thereof.

45. The method according to any one of claims 41-44, wherein the method comprises:(i) contacting an IVT-ready DNA molecule with raw materials of an in vitro transcription (IVT) reaction and incubating the resultant reaction mixture to produce a transcribed RNA molecule;(ii) continuously or intermittently extracting the transcribed RNA molecule, and optionally removing residual DNA;(iii) optionally post-modifying the extracted RNA molecule;(iv) contacting the transcribed RNA molecule from (ii) or (iii) with a buffer solution and contacting the buffered RNA with one or more delivery component to produce formulated RNA, and(v) subjecting the formulated RNA from (iv) to one or more purification step to prepare an RNA molecule, optionally capped, or a formulation thereof.

46. The method according to any one of claims 41-45, wherein the extraction of the amplified DNA or modified amplified DNA is achieved by hybridization of the optional poly(T)tail on the DNA to oligo(d)A nucleic acids coupled to a solid support, hybridization of a sequence-specific oligo nucleic acid coupled to a solid support, wherein the sequence-specific oligo is reverse complementary to at least one element of the DNA of interest, precipitation or electrostatic binding to a solidsupport, and / or capture of a secondary structure in the DNA of interest by a complementing protein, aptamer, or other polymer coupled to a solid support.

47. The method according to any one of claims 41-46, wherein the extraction of the transcribed RIMA is achieved by hybridization of the poly(A)tail on the RNA to oligo(d)T nucleic acids coupled to a solid support, hybridization of a sequencespecific oligo nucleic acid coupled to a solid support wherein the sequence-specific oligo is reverse complementary to at least one element of the RNA of interest, precipitation or electrostatic binding to a solid support, and / or capture of a secondary structure in the RNA of interest by a complementing protein, aptamer, or other polymer coupled to a solid support.

48. The method according to any one of claims 41-47, wherein any residual DNA contaminating the transcribed RNA is removed by a DNA-specific exonuclease, a DNA-specific endonuclease such as DNAsel, or a combination thereof, specifically capturing the DNA by hybridization of an element unique or selective to the DNA over the RNA, such as hybridization of the poly(T)ta II on the DNA to oligo(d)A nucleic acids coupled to a solid support, or hybridization of a sequence-specific oligo nucleic acid coupled to a solid support wherein the sequence-specific oligo is reverse complementary to at least one element of the DNA.

49. The method according to any one of claims 41-48, wherein the one or more component of the delivery vehicle comprises a lipid, such as: ionizable lipid (such as l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31- tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2- 15 dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), l,2-Dioleoyl-3-trimethylammonium propane (DODAP), di((9Z,12Z)-octadeca-9,12-dien-l-yl) 2-((2- (dimethylamino)ethyl)thio)succinate maleate, di((9Z,12Z)-octadeca-9,12-dien-l- yl) 2-((2-(dimethylamino)methyl)thio)succinate maleate, di((9Z,12Z)-octadeca- 9,12-dien-l-20 yl) 2-((2-(dimethylamino)propyl)thio)succinate maleate, di((9Z,12Z)-octadeca-9,12-dien-l-yl) 2-((2- (methylethylamino)ethyl)thio)succinate maleate, di((9Z,12Z)-octadeca-9,12- dien-l-yl) 2-((2-(diethylamino)ethyl)thio)succinate maleate or dioleyl 2-((2- (diethylamino)ethyl)thio)succinate maleate), core or structural lipids (such as cholesterol, cholesterol-derivatives (such as Vitamin D2, Vitamin D3, Calcipotriol, Stigmasterol, Campesterol, Fucosterol, Brassicasterol, Ergosterol, 9,11- dehydroergosterol, Daucosterol, beta-Sitosterol-Acetate, Betutin, Lupeol, Ursoticacid, or Oleanotic acid), resveratrol, resveratrol derivatives (such as dihydroresveratrol, deoxyrhapontigenin, isorhapontigenin, piceatannol, pterostilbene, polydatin), or other small hydrophobic compounds), phospholipids (such as di-oleoyl-phosphatidylethanolamine (DOPE), di-oleoyl- phosphatidylcholine (DOPC), Di-oleoyl-phosphatidylserine (DOPS), 1,2-distearoyl- sn-glycero-3-phosphocholine (DSPC), l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE) or any naturally occurring phospholipid), and / or a shielding lipid (such as a lipid with an attached shielding polymer (such as a polyethylene glycol (PEG) group, a poly-sarcosine group, an oligopeptide or a polypeptide, a hydroxyl-containing non-ionic water-soluble polymer, polyvinylpyrrolidone (PVP), a poly(2-alkyl-2-oxazoline) or a zwitterionic polymer or any other suitable shielding polymer.

50. The method according to any one of claims 41-49, wherein the buffered RIMA is contacted with the one or more delivery vehicle component under conditions comprising a rapid mixing of the buffered RNA with the one or more delivery component, resulting in the formation of defined nanoparticles.

51. The method according to any one of claims 41-50, wherein the purification step comprises an extraction by binding of the RNA or delivery vehicle to a solid support, a (ultra)filtration, optionally a tangential-flow filtration (TFF), preferably a single-pass tangential-flow filtration (Sp-TFF), a particle selection method based on diverting fluid streams, a centrifugation, a precipitation, or other method known to those skilled in the art.

52. The method according to any one of claims 41-51, wherein the method comprises:(i) contacting a template DNA with raw materials of a DNA amplification reaction and incubating the resultant reaction mixture in one or more reservoirs of a microfluidic device,(ii) continuously or intermittently extracting the amplified, and optionally postamplification modified, DNA from the reaction mixture,(iii) contacting the amplified DNA from (ii) with raw materials of an in vitro transcription (IVT) reaction and incubating the resultant reaction mixture in one or more reservoirs of the microfluidic device to produce transcribed RNA,(iv) continuously or intermittently extracting the transcribed RNA and optionally removing residual DNA,(v) optionally post-modifying the RNA, such as enzymatic 5' cap addition and / or 5' cap methylation,(vi) contacting the transcribed RIMA from (iv) or (v) with a buffer solution and contacting the buffered RNA with the one or more delivery component via a microfluidic mixer,(vii) subjecting the naked RNA from (iv or v) or the formulated RNA from (vi) to a final (series of) purification step(s), and(viii) storing the RNA drug substance or drug product in a bulk container, wherein step (i-ii) and step (vi) are optional if sufficient IVT-ready DNA is available and unformulated RNA drug substance is desired, respectively.

53. A valve system, the valve system comprising : first and second substrates defining a valve chamber therebetween, the first substrate comprising an aperture therethrough providing access to the valve chamber, and the second substrate comprising a planar portion comprising first and second fluid inlets; and a valve component disposed in the valve chamber, the valve component comprising opposite first and second surfaces wherein the first surface defines a valve slot configured to reversibly receive a valve actuator therein and second surface defines a cavity, such that a cross section of the cavity has one or more lobes, wherein the valve component is positioned in the valve chamber such that the valve slot is accessible via the aperture in the first substrate and the second surface is disposed adjacent to the planar portion of the second substrate, the valve component being capable of rotational movement between a first position, wherein the first and second fluid inlets are not fluidly connected, and a second position, wherein the first and second fluid inlets are fluidly connected due to the position of the one or more lobes.

54. A method for ratiometrically-defined mixing of two or more fluid streams, the method comprising applying gas pressure to two or more separate fluidcontaining reservoirs, each reservoir containing a fluid, via feedback-controlled mass flow controllers, so as to create two or more defined fluid volumes, and then feeding the two or more defined fluid volumes to a microfluidic mixer where they are mixed to thereby produce a ratiometrically-defined fluid stream comprising the two or more fluid streams.

55. The method of claim 54, wherein the method comprises using the apparatus according to any one of claims 1-22.

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