In vitro RNA production and purification system and methods of use thereof

Abstract

Systems and methods for the high-throughput production and purification of nucleic acids, and in particular the in vitro transcription of RNA molecules.

Description

[0001] IN VITRO RNA PRODUCTION AND PURIFICATION SYSTEM AND METHODS OF USE THEREOF CROSS REFERENCE TO RELATED APPLICATION This International PCT application claims the benefit of and priority to U.S. Provisional Application No. 63 / 559,436, filed February 29, 2024, and U.S. Provisional Application No. 63 / 701,800, filed October 1, 2024, the specification, claims and drawings of which are incorporated herein by reference in their entirety. TECHNICAL FIELD The present invention relates to the field of in vitro production of polynucleotides, and in particular the in vitro production and purification of mRNA for use in therapeutic applications. BACKGROUND Messenger RNA (mRNA) is the template molecule that is transcribed from cellular DNA and is translated into an amino acid sequence, i.e., a protein, at ribosomes in the cells of an organism. In order to control the expression level of the encoded proteins, mRNAs possess untranslated regions (UTRs) flanking the actual open reading frame (ORF) which contains the genetic information encoding the amino acid sequence. Such UTRs, termed the 5'-UTR and the 3'- UTR, respectively, are sections of the mRNA located before the start codon and after the stop codon. Further, mRNA contains a poly(A) tail region which is a long sequence of adenine nucleotides which promotes export of mRNA from the nucleus, translation and to some extent protects the mRNA from degradation. Scientific and technological advances of the recent years have made mRNA a promising candidate for a variety of uses, including diagnostic applications, and therapeutic products, like vaccines. Due to the increasing demands of the medical community to enable personalized medicine, but also emergency responses in epidemic crisis situations, such as with the recent COVID-19 pandemic, many approaches have been developed for mRNA production at scale. Most current methods utilize fermentation to synthesize mRNA in culture from self-replicating DNA templates, then isolate the total RNA as raw material utilizing volatile organic solvents. These processes are costly, dangerous, produce hazardous waste streams that must be mediated, while the production rate is severely dependent on the performance of the producing strain and the ability to remove impurities from diverse tRNA, rRNA and host mRNA. As can be seen, there exists a long-felt need for an effective in vitro mRNA manufacturing system that does not require volatile organic solvents, produces no hazardous waste stream, and costs significantly less than its fermentation-based counterpart, while generating uniform pure mRNA fit for therapeutic applications. Moreover, this in vitro mRNA manufacturing system needs to be part of a compact assembly that can be easily transported to various location for rapid in situ RNA production for vaccines, and other therapeutic applications. SUMMARY OF THE INVENTION In one aspect, the present invention includes improved systems, methods, and compositions for the production and purification of nucleic acids, and in particular the in vitro transcription of RNA molecules. In another aspect, the present invention includes an integrated IVT assembly and DNase assembly configured to form an automated modular device that operates as a continuous flow RNA production and purification platform, which can be deployed in a GMP, or a development (non- GMP) environment. In another aspect, the present invention includes one or more purification modules positioned in-stream or downstream of the product stream of a continuous flow IVT production and purification platform. In a preferred aspect, the purification module can include single-pass tangential flow filtration (SP-TFF) system comprising one or more hollow-fiber ultra-filtration (HF-UF) cartridges positioned in-stream or downstream of the product stream of a continuous flow IVT production and purification platform. In another preferred aspect, the HF-UF cartridges can include two or more cartridges positioned in series and configured to generate a cross-flow or a counter-flow of the product stream. In another aspect, the present invention includes an in vitro RNA IVT assembly having at least one hollow fiber bioreactor (HFBR) wherein a reaction mixture is introduced continuously through an intra-capillary space (IC) of the HFBR, and a feed mixture is continuously supplied to the extra-capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis, wherein the reaction mixture includes a first quantity of a cap analog, and the feed mixture includes a second quantity of a cap analog, or wherein the feed mixture includes a first quantity of a cap analog. In a preferred embodiment, the reaction mixture includes a first quantity of a cap analog that is introduced into the continuously through an intra-capillary space (IC) of the HFBR, and a feed mixture, that does not contain a quantity of cap analog, is continuously supplied to the extra-capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis. In this embodiment, less cap analog is required to successfully cap the formed RNA molecules in the system. As used herein, the term “quantity” refers to the amount of substrate needed to carry out a specified chemical reaction. In one example, a quantity, whether a first, second, or third quantity means a substrate that is part of a feed or reaction mixture that is used in the synthesis of RNA, preferably using a hollow fiber bioreactor (HFBR) as generally described herein. The “quantity” of a substrate can be generally calculated by one of ordinary skill in the art based on the reaction type, substrate concentrations, and / or desired yield of a synthesis product, which in a preferred embodiment includes RNA synthesized hollow fiber bioreactor (HFBR) as generally described herein. In another aspect, the present invention includes an in vitro RNA IVT assembly having at least one hollow fiber bioreactor (HFBR) wherein a reaction mixture is introduced continuously through an intra-capillary space (IC) of the HFBR, and a feed mixture is continuously supplied to the extra-capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis; and one or more chromatography extraction modules in fluid communication with the HFBR. In another aspect, the present invention includes an in vitro RNA IVT assembly having at least one hollow fiber bioreactor (HFBR) wherein a reaction mixture is introduced continuously through an intra-capillary space (IC) of the HFBR, and a feed mixture is continuously supplied to the extra-capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis, wherein the pressure of the EC is higher than the IC, wherein the pressure differential increases substrate availability during the IVT reaction within the HFBR. In another aspect, the present invention includes an in vitro RNA IVT assembly having at least one hollow fiber bioreactor (HFBR) wherein a reaction mixture is introduced continuously through an intra-capillary space (IC) of the HFBR, and a feed mixture is continuously supplied to the extra-capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis, and wherein a first quantity of ribonucleotide tri-phosphate (rNTP) substrates are introduced to the EC via the feed mixture, and a second quantity of NTP substrates are introduced to the IC via the reaction mixture. Additional aspects of the inventive technology will become apparent from the specification, figures and claims below. BRIEF DESCRIPTION OF THE FIGURES Figure 1A-B. Off-line LC-MS / MS data of in-process samples taken in 5minute intervals from the IC outlet show the synthesis of RNA (red) and steady state flow of NTPs (blue) during the synthesis phase of the continuous flow reaction. B. Off-line LC-MS / MS data of in-process samples taken in 5minute intervals from the IC and EC outlets. Red dots show the synthesis of RNA in the IC, with no RNA in the EC (expected), blue dots show steady state flow of NTPs (blue) in both the EC and IC during the synthesis phase of the continuous flow reaction. NTPs are only added to the EC space, the presence of NTPs in the IC space demonstrate the flow control and TMP generation (Figure 2) are effective as a driving force of NTPs across the porous membrane. Figure 2. Bioanalyzer electropherogram of eGFP mRNA produced using the present IVT production system of the invention. Figure 3. Removal of ionic solutes in co- and counter-current configurations of 2mL and 5mL HF-UF cartridges at increasing diafiltration rates. Figure 4A-B. (A) Co- and Counter-current configuration tests of rNTP removal from IC volume through 2mL and 5mL HF-UF cartridges (B) Cross-flow configuration test of rNTP removal for multiple variations of SP-TFF cassette specifications. Figure 5. Effectiveness of the capping reaction in various ratios of IVT buffer and capping buffer. Figure 6. Two-stage SP-TFF design model of ISP to achieve effective buffer exchange and removal of LMW process impurities. Figure 7A-B. (A) Enzyme removal by single-pass cross-flow through TFF membranes (B) RNA retention in model single-pass TFF system. Figure 8. Reduced enzyme trace (gray) during primary experimental process due to downstream high molecular weight filtration stages relative to before DSP (orange). Figure 9A-B. Show separate continuous flow IVT synthesis and in-line DNase reactions with timepoints T0 and T2. (A) shows first example of reduction of DNA content as a ratio of DNA to RNA content is below the regulatory threshold of .05%. (B) shows first example of reduction of DNA content as a ratio of DNA to RNA content is below the regulatory threshold of .05%. Figure 10. Schematic diagram of an exemplary process and control systems of in vitro RNA transcription and purification assembly including an IVT Module and DNase Module in one embodiment thereof. Figure 11. Schematic diagram of an exemplary 2-Stage Serial Counter-Current Buffer Exchange TFF Unit in one embodiment thereof. Figure 12. Schematic diagram of an exemplary 2-Stage Serial Cross-Flow Single-Pass TFF Unit for RNA Purification in one embodiment thereof. Figure 13. Schematic diagram of an exemplary 4-Stage Serial Cross-Flow Single-Pass TFF Unit for RNA Purification in one embodiment thereof. Figure 14. Schematic of the hollow fiber bioreactor as a reaction vessel to allow for the continuous flow RNA synthesis and continuous feeding of the reaction substrate. Introduction of the cap analog can occur in the Reaction mix or the Feed mix into the reaction space to allow for continuous co-transcriptional capping. Equations shown in red allow for the continuous transport of Feed Mix components into the intercapillary space triggering synthesis. Figure 15A-B. (A.) Comparison of capping efficiency between cap analog introduction into the Feed mix and the Reaction (Rxn) mix showing no difference in capping efficiency. cap analog co-transcriptional capping compared to enzymatic capping in batch with Vaccinia Capping System (VCE) or Fausto Capping System (FCE). (B). Comparison of yield between Cap analog introduction via the Feed mix or the Reaction (Rxn) mix showing similar yield. Figure 16. Schematic of exemplary assembly using ion exchange resin and protein binding resin to remove salts and enzymes prior to solid phase tangential flow filtration as an efficient continuous flow mRNA purification. Figure 17A-B. (A) optimized protein purification IVT incorporating a chromatography stage extraction module; (B) RNA recovery vs, protein removed as percentage of material process through exemplary continuous RNA IVT process incorporating a chromatography stage extraction module. Figure 18. Schematic of the hollow fiber bioreactor as a reaction vessel to allow for the continuous flow RNA synthesis and continuous feeding of the reaction substrate. Introduction of the cap analog can occur in the Reaction mix or the Feed mix into the reaction space to allow for continuous co-transcriptional capping. Equations shown in red allow for the continuous transport of Feed Mix components into the intercapillary space triggering synthesis. Figure 19A-C. In Process sensor data demonstrating flow and pressure differentials. A. Pressure differential between the intercapillary (IC) space and the extracapillary space (EC) generated by flow control on the inputs of both the IC and EC and output on the IC (B.). C. post- processed flow data demonstrating flow control differential to generate the transmembrane pressure (A) as a driving force for substrate transport into the IC space. Figure 20: Bioanalyzer of RNA produced in the continuous flow system. Electropherogram shows a high percentage of full-length intact RNA. DETAILED DESCRIPTION OF THE INVENTION One embodiment of the current invention includes novel in vitro methods and systems for the production of polynucleotides, and in particular mRNA that may be directed to one or more diagnostic or therapeutic applications. In one preferred aspect, the invention includes a fully recombinant stable, reliable and functional in vitro system for continuous flow production of mRNA. The systems and methods for the production of mRNA can include an in vitro bioreactor, and preferably a continuous-flow bioreactor be configured combine a reaction mixture comprising components necessary for in vitro RNA transcription from a DNA template. For example, in one example the reaction mixture of the invention can include a quantity of a buffer, isolated RNA Polymerase (RNAP), a nucleotide template, and preferably a linear, or circular non-self-replicating DNA template, along with a plurality of ribonucleotide triphosphates (NTPs) which may be incorporated into the synthesized mRNA molecules through the action of the RNAP, and an energy source, such as the novel inorganic polyphosphate energy-regeneration system generally described by Koglin and Humbert in PCT Application No. PCT / US2018 / 012121, the description, figures, examples, sequences and claims being incorporated herein by reference in their entirety). In a preferred embodiment, the synthesized mRNA can be purified and used for diagnostic or therapeutic uses, such as vaccines directed to select target pathogens. The IVT system of the invention consists of two-assembly operations, the IVT RNA synthesis reaction performed by an IVT Assembly (1), and the DNase reaction performed by a DNase Assembly (3) for the removal of DNA template in stream. A reaction mixture can be generated in the IVT (1) assembly and transmitted introduced in a continuous manner to an IVT hollow fiber bioreactor (HFBR) (See Figure 18). The IVT reaction in is performed in a HFBR as generally described by Humbert et al., in PCT / US2021 / 027774, (the entirety of which is incorporated here by reference), and preferably with a molecular weight cutoff (MWCO) that has been selected for retention of template, enzymes, and product, while allowing lower molecular weight reactants and by-products to pass through a membrane. In a preferred embodiment, an IVT reaction buffer is introduced continuously through the intra-capillary space (IC) (34) of the HFBR with rNTPs continuously supplied to the extra-capillary space (EC) (36) and exchanged (transported) through the HFBR membrane into the IC space initiating RNA synthesis. As the continuous reaction progresses, the IVT reaction reaches steady state kinetics, as indicated by the constant concentration of RNA product in the effluent for the duration of the IVT reaction As generally shown in Figure 10, the HFBR (3) of the invention is configured to be in fluid communication with an IVT reaction mixture (4) that includes quantity of isolated RNA polymerase (RNAP) enzyme and optionally a quantity of isolated ribonucleotide triphosphates (rNTPs). The HFBR (3) of the invention is further in fluid communication with an IVT feed mixture (5) containing: 1) a quantity of isolated NTPs; optionally the components of an inorganic polyphosphate energy-regeneration system, and optionally one or more co-factors for the production of mRNA polynucleotides. The IVT feed mixture (5) and IVT reaction mixture (4) of the invention can further be in fluid communication with an IVT buffer supply (6) that can supply a controlled a quantity of buffer solution. In this configuration, a feed mixture and a reaction mixture can be combined with a buffer solution and separately introduced to the HFBR (3) which generates a gradient such that the free NTs from the feed solution are be drawn into an internal compartment of the HFBR (3) where they may react with the components of the reaction mixture. In this embodiment, RNAP may associate with a DNA template, and preferably a linear DNA template having a target sequence that encodes a target mRNA, and enzymatically catalyze the incorporation of NTPs into a target mRNA nucleotide. Notably, the introduction of both the feed solution and reaction mixture can be subject to a reaction and feed controller (7, 8) respectively. These controllers (7, 8) can drive a pump to manage the controlled introduction of the reaction and feed solutions to the HFBR (3). In this embodiment, the controllers (7, 8) can be responsive to one or more sensors, or computer devices configured to execute computer executable applications for the automated introduction the reaction and feed solutions to the HFBR (3) through separate input positions which is further positioned within a temperature controlled incubator (9). The HFBR (3) of the invention can further include a plurality of output positions. In one embodiment, an output position can be in fluid communication with an IVT feed waste (10) configured to draw away depleted waste feed solution from the HFBR (3). A separate output position can be in fluid communication with the reaction mixture which contains the newly transcribed RNA, among other components. This can be drawn from the HFBR (3) and transported to a DNase assembly (2) for elimination of impurities, such as DNA templates. As shown in Figure 10 generally, the IVT Assembly operation flows directly into the DNase Assembly operation where DNase from a DNase Mix (11) is combined with a DNase Capping buffer supply (12) can be continuously added to the product stream from the IVT Assembly (1), for example as directed by a DNase controller (13) configured to drive a pump to manage the controlled introduction of the DNase Mix (11) to the DNase Loop (14). In this embodiment, the controller (13) can be responsive to one or more sensors, or computer devices configured to execute computer executable applications for the automated introduction the DNase mixture to the DNase Loop (14) which is further positioned within a temperature controlled incubator (15) to promote the enzymatic action of the DNase enzyme. Additional waste streams from the process can be shunted to a product line wase (16). Additionally, a product stream containing the transcribed RNA can be shunted to a product collection line (17) and collected for further purification or modification. As noted below, the continuous flow design of the in vitro production platform allows for the integration of additional reaction systems in-stream to accomplish the necessary functional modifications, including 5’ capping and 3’ polyadenylation. The present inventors next sought to incorporate into the IVT system of the invention a purification process for the removal of in-process impurities as a replacement for lithium chloride (LiCl) precipitation, affinity chromatography, or off-line traditional batch ultrafiltration / diafiltration (UF / DF) methods. In one embodiment, Single-Pass Tangential Flow Filtration (SP-TFF) through hollow-fiber ultra-filtration (HF-UF) cartridges can be integrated in- flow with the IVT platform of the invention. As generally shown in Figures 11-13, integration of in-flow purification can be most effective as two separate unit operations: an in-stream purification module (between RNA modification operations) and a downstream (end of process) purification module. In this embodiment, the in-stream purification (ISP) module can be configured to remove low molecular weight impurities (digested DNA fragments, residual rNTPs) and act to exchange buffers between RNA modification reactions (capping and tailing). The downstream purification (DSP) module can be configured to remove high molecular weight process enzymes and perform final buffer exchange for the unformulated mRNA drug substance. As described below, the present inventors configured systems for the removal of low molecular weight (LMW) impurities (rNTPs), buffer exchange, as well as larger impurities and enzyme removal allowing for the continuous coupling of the IVT and capping reactions. As shown in Figure 11, the present inventors configure a 2-Stage Serial Counter-Current Buffer Exchange TFF assembly. In this embodiment, raw material containing the transcribed RNA from the process described above is introduced, via a controller (20) driving a pump to a first HF-UF Cartridge (21a). A separate quantity of water / buffer (19) is also introduced, also via a controller (20) driving a pump to the first HF-UF Cartridge (21a). This first HF-UF Cartridge (21a) can further be in fluid communication with a second HF-UF Cartridge (21b), again via a controlled (21) driven pump. In this configuration the second HF-UF Cartridge (21b) is in fluid communication with the water / buffer (19) in a counter-current configuration such that the water / buffer (19) and processed material from the first HF-UF Cartridge (21a) are introduced to the second HF-UF Cartridge (21b) separately. Waste (22) can be drawn away from the second HF-UF Cartridge (21b) while the purified material (23) is extracted via a controller (20) driven pump. As shown in Figure 12, the present inventors configured a 2-Stage Serial Cross-Flow Single-Pass TFF Assembly for the removal of low molecular weight (LMW) impurities (rNTPs), buffer exchange, as well as larger impurities and enzyme removal. In this embodiment, raw material (18) containing the transcribed RNA from the process described above is introduced, via a controller (20) driving a pump to a first HF-UF Cartridge (21a). A separate quantity of water / buffer (19) is also introduced, also via a controller (20) driving a pump to the first HF-UF Cartridge (21a). This first HF-UF Cartridge (21a) can further be in fluid communication with a second HF-UF Cartridge (21b), again via a controlled (21) driven pump. In this configuration the second HF-UF Cartridge (21b) is in fluid communication with the water / buffer (19) in a cross- flow configuration where the processed material from the first HF-UF Cartridge (21a) is combined directly with the second quantify water / buffer (19) prior to introduction to the second HF-UF Cartridge (21b). Again, after processing by the second HF-UF Cartridge (21b), waste (22) can be drawn away while the purified material (23) is extracted via a controller (20) driven pump. As shown in Figure 13, the present inventors configured the present inventors configured a multi-Stage Serial Cross-Flow Single-Pass TFF Unit, having in this example four HF-UF Cartridges (21a,b,c,d) in serial communication with one another in a cross-flow configuration as described above. In another embodiment, a quantity of cap analogs can be added either in the reaction mix, containing template and enzymes, or in the feed mix for improved reaction process control and increased mRNA capping efficiency. Exemplary cap analogs are described in PCT / US2016 / 052670, which is incorporated herein by reference. In a preferred embodiment, the disclosure includes systems, methods, and compositions for a co-transcriptional capping IVT reaction in a continuously flow hollow fiber bioreactor (30) also referred to as a hollow fiber bioreactor (HFBR), to generate capped mRNA products. Most eukaryotic cellular mRNA transcripts and most eukaryotic viral mRNA transcripts are blocked or “capped” at their 5' terminus. In addition to mRNA, some other forms of eukaryotic RNA, such as but not limited to, small nuclear RNA (“snRNA”) and pre-micro RNA (i.e., “pre- miRNA”, the primary transcripts that are processed to miRNA) are also capped. A “cap” is a guanine nucleoside that is joined via its 5 '-carbon to a triphosphate group that is, in turn, joined to the 5 '-carbon of the most 5 '- nucleotide of the primary mRNA transcript, and in most eukaryotes, the nitrogen at the 7 position of guanine in the cap nucleotide is methylated. Such a capped transcript can be represented as m7G(5')ppp(5')Ni(pN)x — OH(3'), or more simply, as m7GpppNi(pN)x, where m7G represents the 7-methylguanosine cap nucleoside, ppp represents the triphosphate bridge between the 5' carbons of the cap nucleoside and the first nucleotide of the primary RNA transcript, and Ni(pN)x — OH(3') represents the primary RNA transcript, of which Ni is the most 5 '-nucleotide. The 5' caps of eukaryotic cellular and viral mRNAs (and some other forms of RNA) play important roles in RNA stability and processing. For example, the cap plays a pivotal role in mRNA metabolism, and is required to varying degrees for processing and maturation of an RNA transcript in the nucleus, transport of mRNA from the nucleus to the cytoplasm, mRNA stability, and efficient translation of the mRNA to protein. As used in the present invention, “capping analog” refers to a structure at the 5' end of mature mRNA that is a chemical analog of a methyl guanosine cap. This structure can prevent the degradation of mRNA at the 5' end, help RNA transcripts pass through the selective pores of the nuclear membrane and enter the cytoplasm, enhance translation, and help complete the entire shearing process. With respect to IVT, synthetic cap analogs are commonly used co-transcriptional chemical capping of mRNA vaccines and therapeutics. Typically, co-transcriptional capping is performed in batch reactions by adding the chemical cap to the reaction mix via the ribonucleotide triphosphates (rNTPs). As shown in Figure 14, in one preferred embodiment, cap analogs can be included in a feed mixture that is introduced to a HFBR (30) which acts as a reaction vessel for the transport and mixing of reaction substrates (Feed Mix) with DNA template and T7 DNA dependent RNA polymerase (Reaction Mix) in a continuous fashion. Peristaltic pump, or other similar devices can be used to control the flow of reaction and feed mixtures through HFBR. The addition of cap analogs to the feed mix allows for their continuous introduction along with rNTPs into the reaction space of the HFBR (30) to achieve in-flow capping in a co-transcriptional fashion. In an alternative embodiment, the cap analogs can be included in a reaction mixture that is introduced to a HFBR (30) which acts as a reaction vessel for the transport and mixing of reaction substrates (Feed Mix) with DNA template and T7 DNA dependent RNA polymerase and cap analogs (Reaction Mix) in a continuous fashion. Peristaltic pump, or other similar devices can be used to control the flow of reaction and feed mixtures through HFBR. The addition of cap analogs to the reaction mix allows for their continuous introduction into the reaction space of the HFBR (30) to achieve in-flow capping in a co-transcriptional fashion. As further shown in Figure 14, a Cap analog can be introduced into the reaction mix to bypass the transport mechanism allowing for transcription to be primed prior to entering the intra-capillary space (IC) (34) of the HFBR (30). In this embodiment, less cap analog is needed to produce similar capped RNA yields compared to if the cap analog is introduced in the feed solution. As further shown in Figure 15A- B, introduction of a Cap analog into the feed mix or the reaction mix results in a similar capping efficiency and yield. When compared to enzymatic capping, Applicant’s achieve comparable capping efficiencies to both vaccinia capping system and Fausto capping system. Notably, As used herein, cap analogs, also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, retain cap function but are natural in their chemical structure. That is, it is different from the endogenous, wild or physiological 5'cap. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized to the polynucleotides of the invention and / or bound to the polynucleotides of the invention. As used herein, “capping” refers to the enzymatic addition of a Nppp-moiety onto the 5' end of an RNA, where N a nucleotide such as is G or a modified G. A modified G may have a methyl group at the N7 position of the guanine ring, or an added label at the 2 or 3 position of the ribose, and, in some embodiments, the label may be an oligonucleotide, a detectable label such as a fluorophore, or a capture moiety such as biotin or desthiobiotin, where the label may be optionally linked to the ribose of the nucleotide by a linker, for example. See, e.g., WO 2015 / 085142. A cap may have a Cap-0 structure, a Cap-1 structure, or a Cap-2 structure (as reviewed in Ramanathan, Nucleic Acids Res.201644: 7511-7526), depending on which enzymes and / or whether SAM is present in the capping reaction. As used herein, a “5’-cap” describes: A 5 ’-cap structure that is typically a modified nucleotide (cap analogue), particularly a guanine nucleotide, added to the 5' -end of an mRNA molecule. Preferably, the 5'-cap is added using a 5' - 5' -triphosphate linkage (also named m7GpppN). Further examples of 5'-cap structures include glyceryl, inverted deoxy abasic residue (moiety), 5' methylene nucleotide, l-(beta-D- erythrofuranosyl) nucleotide, 4' -thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo- pentofuranosyl nucleotide, acyclic 3', 4' -seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3' -3' -inverted nucleotide moiety, 3' -3' -inverted abasic moiety, 3' - 2' -inverted nucleotide moiety, 3' -2' -inverted abasic moiety, 1,4-butanediol phosphate, 3' - phosphoramidate, hexylphosphate, aminohexyl phosphate, 3' -phosphate, 3' phosphorothioate, phosphorodithioate, or bridging or non-bridging methylphosphonate moiety. These modified 5 - cap structures may be used in the context of the present invention to modify the mRNA sequence of the inventive composition. Further modified 5 -cap structures which may be used in the context of the present invention are CAP1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), CAP2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), capz (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA (anti-reverse CAP analogue), modified ARCA (e g. Phosphorothioate modified ARCA), inosine, Nl-methyl- guanosine, 2 '-fluoro-guanosine, 7-deaza-guanosine, 8-oxo- guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. As used herein, “DNA template” refers to a double stranded DNA molecule that is transcribed in an in vitro transcription reaction. DNA templates have a promoter (e.g., a T7, T3 or SP6 promoter) recognized by the RNA polymerase upstream of the region that is transcribed. In another embodiment, the invention includes a modular IVT system incorporating a HFBR (30) positioned upstream, and in fluid communication with one or more a ion exchange resins and protein binding resins. Use of a HFBR (30) allow for continuous in vitro transcription of RNA products, while also allowing the continuous withdrawal of waste products as generally described by Humbert et al., in PCT / US2021 / 027774, (the entirety of which is incorporated here by reference). As shown in Figure 15, in a preferred embodiment a chromatography extraction module (32), such as a chromatography ion exchange resin and / or protein binding resin (32) can be configured to remove salts and enzymes prior to single-pass tangential flow filtration (SP-TFF) system comprising one or more hollow-fiber ultra-filtration (HF-UF) cartridges positioned in- stream or downstream of the product stream of a continuous flow IVT production and purification platform. In a preferred embodiment, the resin include a cation resins configured to remove enzyme from the process stream, while maintaining good RNA recovery. In another embodiment, positive pressure is applied to the feed mix across the HFBR membrane to increase substrate availability during the IVT reaction. As noted above, a HFBR module in vitro transcription relies on effective transport of reaction substrates from the feed mixture across the membrane. In one embodiment, control of the flow rates through of the feed mixture through HFBR creates a convective force which facilitates the efficient transfer of UVT substrate across the membrane of the HFBR via a bulk flow movement. In a preferred embodiment, an IVT reaction buffer is introduced continuously through the intra-capillary space (IC) of the HFBR with rNTPs continuously supplied to the extra-capillary space (EC) and exchanged (transported) through the HFBR membrane into the IC space initiating RNA synthesis. In one preferred embodiment, the introduction of NTP substrates from the feed mix is split between the IC and EC regions of the bioreactor to increase IVT reaction efficiency. In this embodiment, the HFBR module is configured such that NTP substrate introduced partially to the IC inlet allowing substrate to blend with the reaction mix, containing template and enzymes, while the remaining substrate is allowed to enter the EC region for increased IVT reaction efficiency. This embodiment enables IVT reaction to occur sooner as it enters the HFBR column followed by a constant reaction maintained throughout the column. The amount ratio of substrate to the IC and EC can be controlled to optimize reaction efficiency. As can be appreciated, all of the processes and assembly components described above may be accomplished in some embodiments through any appropriate machine and / or device resulting. It should also be noted that in some instance’s software and / or software solution may be utilized to carry out the objectives of the invention and may be defined as software stored on a magnetic or optical disk or other appropriate physical computer readable media including wireless devices and / or smart phones. In alternative embodiments the software and / or data structures can be associated in combination with a computer or processor that operates on the data structure or utilizes the software. Further embodiments may include transmitting and / or loading and / or updating of the software on a computer perhaps remotely over the internet or through any other appropriate transmission machine or device, or even the executing of the software on a computer resulting in the data and / or other physical transformations as herein described. Certain embodiments of the inventive technology may utilize a machine and / or device which may include a general purpose computer, a computer that can perform an algorithm, computer readable medium, software, computer readable medium continuing specific programming, a computer network, a server and receiver network, transmission elements, wireless devices and / or smart phones, internet transmission and receiving element; cloud-based storage and transmission systems, software updateable elements; computer routines and / or subroutines, computer readable memory, data storage elements, random access memory elements, and / or computer interface displays that may represent the data in a physically perceivable transformation such as visually displaying said processed data. In addition, as can be naturally appreciated, any of the steps as herein described may be accomplished in some embodiments through a variety of hardware applications including a keyboard, mouse, computer graphical interface, voice activation or input, server, receiver and any other appropriate hardware device known by those of ordinary skill in the art. A “processor,” “processor system,” or “processing system,” includes any suitable hardware and / or software system, mechanism or component that processes data, signals or other information. A processor can include a system with a general-purpose central processing unit, multiple processing units, dedicated circuitry for achieving functionality, or other systems. Processing need not be limited to a geographic location or have temporal limitations. For example, a processor can perform its functions in “real time,” “offline,” in a “batch mode,” etc. Portions of processing can be performed at different times and at different locations, by different (or the same) processing systems. A computer may be any processor in communication with a memory. The memory may be any suitable processor-readable storage medium, such as random-access memory (RAM), read- only memory (ROM), magnetic or optical disk, or other tangible media suitable for storing instructions for execution by the processor. Particular embodiments may be implemented by using a programmed general purpose digital computer, by using application specific integrated circuits, programmable logic devices, field programmable gate arrays, optical, chemical, biological, quantum or nanoengineered systems, components and mechanisms may be used. In general, the functions of particular embodiments can be achieved by any means as is known in the art. Distributed, networked systems, components, and / or circuits can be used. Communication, or transfer of data may be wired, wireless, or by any other means. As used in the description herein and throughout the claims that follow, “a”, “an”, and “the” includes plural references unless the context clearly dictates otherwise. Also, as used in the description herein and throughout the claims that follow, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise. The foregoing description is merely illustrative in nature and is not intended to limit the embodiments of the subject matter or the application and uses of such embodiments. Furthermore, there is no intention to be bound by any expressed or implied theory presented in the technical field, background, or the detailed description. As used herein, the word “exemplary”, “embodiment” or “preferred embodiment” means “serving as an example, instance, or illustration.” Any implementation described herein as exemplary is not necessarily to be construed as preferred or advantageous over other implementations, and the exemplary embodiments described herein are not intended to limit the scope or applicability of the subject matter in any way. Embodiments of the subject matter may be described herein in terms of functional and / or logical block components and with reference to symbolic representations of operations, processing tasks, and functions that may be performed by various computing components or devices. Such operations, tasks, and functions are sometimes referred to as being computer-executed, computerized, software-implemented, or computer-implemented. In this regard, it should be appreciated that the various block components shown in the figures may be realized by any number of hardware, software, and / or firmware components configured to perform the specified functions. For example, an embodiment of a system or a component may employ various integrated circuit components, e.g., memory elements, digital signal processing elements, logic elements, look-up tables, or the like, which may carry out a variety of functions under the control of one or more microprocessors or other control devices. In this regard, the subject matter described herein can be implemented in the context of any computer-implemented system and / or in connection with two or more separate and distinct computer-implemented systems that cooperate and communicate with one another. While at least one exemplary embodiment has been presented in the foregoing detailed description, it should be appreciated that a vast number of variations exist. It should also be appreciated that the exemplary embodiment or embodiments described herein are not intended to limit the scope, applicability, or configuration of the claimed subject matter in any way. Rather, the foregoing detailed description will provide those skilled in the art with a convenient road map for implementing the described embodiment or embodiments. It should be understood that various changes can be made in the function and arrangement of elements without departing from the scope defined by the claims, which includes known equivalents and foreseeable equivalents at the time of filing this patent application. Accordingly, details of the exemplary embodiments or other limitations described above should not be read into the claims absent a clear intention to the contrary. The invention now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain embodiments of the embodiments of the present invention. The examples are not intended to limit the invention, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed invention. Indeed, while this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. EXAMPLES Example 1: Validation of IVT mRNA production system. The present invention includes novel systems, methods, and compositions for a mRNA manufacturing platform using continuous flow principles to improve the production rate and quality of the mRNA material. in vitro RNA manufacturing systems incorporate a DNA-dependent RNA Polymerase that catalyzes the in vitro transcription (IVT) reaction. As generally shown in Figure 10, the IVT system of the invention consists of two-assembly operations, the IVT RNA synthesis reaction performed by an IVT Assembly, and the DNase reaction performed by a DNase Assembly for the removal of DNA template in stream. The IVT reaction is performed in a hollow fiber bioreactor (HFBR) as generally described by Humbert et al., in PCT / US2021 / 027774, (the entirety of which is incorporated here by reference), and preferably with a molecular weight cutoff (MWCO) that has been selected for retention of template, enzymes, and product, while allowing lower molecular weight reactants and by-products to pass through a membrane. In a preferred embodiment, an IVT reaction buffer is introduced continuously through the intra-capillary space (IC) of the HFBR with rNTPs continuously supplied to the extra-capillary space (EC) and exchanged (transported) through the HFBR membrane into the IC space initiating RNA synthesis. As the continuous reaction progresses, the IVT reaction reaches steady state kinetics, as indicated by the constant concentration of RNA product in the effluent for the duration of the IVT reaction (Figure 1). As shown in Figure 10 generally, the IVT Assembly operation flows directly into the DNase Assembly operation where DNase enzyme is continuously added to the product stream from the IVT unit operation to end the synthesis of RNA in the process. The continuous flow design of the in vitro production platform allows for the integration of additional reaction systems in-stream to accomplish the necessary functional modifications, including 5’ capping and 3’ polyadenylation. Integrating additional reaction systems increases the range of transcripts which can be manufactured on the inventive platform. In this embodiment, the platform can be incorporated into single-use consumables, hardware, and automation to facilitate process control and deployment of the inventive technology. The invention’s continuous flow design, with integrated assembly operations enables integration of in-line or at-line process analytical technology (PAT) for improving in-process monitoring and real-time release. The present inventors performed 3 replicate runs on the IVT system of the invention as generally shown in Figure 10. A total of three Process Performance Runs were completed with an input concentration of 0.05uM template DNA. All Process Performance Runs were completed under the same conditions. The results were similar between the process performance runs for dsRNA and residual template. The present inventors conducted two additional successive test runs in a single day over a period of 6 hours. The two test runs performed produced RNA material that fall within the average + / - one standard deviation for yield, integrity, and residual DNA, as compared to the process performance runs.. Example 2: Validation of continuous instream purification of mRNA product. Initial work by the present inventors on the process of continuous purification focused on the ISP unit operation and aimed to demonstrate removal of LMW impurities (rNTPs) and buffer exchange allowing for the continuous coupling of the IVT and capping reactions. To determine the optimal configuration of the HF-UF modules for effective removal of ionic solutes (buffer) experiments were performed in counter- and co-current configurations. These experiments show that both configurations can effectively transport ionic solutes across the HF-UF membrane at different diafiltration rates, however the counter-current configuration shows higher performance at lower diafiltration rates in both the 2mL and 5mL HF-UF cartridges, achieving >97% removal of ionic solutes as shown in Figure 3. ISP is also responsible for the removal of residual rNTPs. The removal of rNTPs is greater than 70% in both co- and counter-current configurations (Figure 4A). To achieve a greater removal of rNTPs, a cross-flow configuration was employed and showed nearly 90% removal of rNTPs (Figure 4B). Based on these experiments, design options were modeled with the applicable physical design equations. Based on the theoretical calculations, it was determined that a two-stage SP-TFF counter-current configuration was optimal to effectively exchange ionic solutes and remove residual rNTPs (Figure 6). Figure 6 illustrates the two-stage SP-TFF design of the ISP unit operation, where the first dilutant is water to remove IVT buffer followed by the second stage for capping buffer diafiltration. To demonstrate that this configuration would be sufficient, capping reactions were successfully performed in retentate collected from a “mock run” of the two-stage SP-TFF process (Figure 5). Mock runs consist of a full system run with all buffer and enzyme inputs but lack the DNA template for synthesis. This allows for the testing of purification modules with full enzyme loads in the proper buffer matrix. Together these data demonstrate that two-stage SP-TFF is effective at removing low molecular weight impurities and exchanging between IVT and capping buffer allowing for efficient capping in-line. Example 3: Validation of continuous downstream purification of mRNA product In contrast to the removal of LMW solutes and buffer exchange in the ISP module, the challenge with the DSP module is the removal of kilodalton (kDa) from megadalton (MDa) RNA. To address this issue, a similar configuration using HMW (300-750kDA) cutoff HF-UF cartridges in a multi-stage SP-TFF design was employed by the present inventors. The advantage of this design is the ability to add additional stages to achieve greater enzyme removal. Solute tracer experiments were performed to determine the single-species transport properties of typical large biomolecules through the HF-UF cartridges A model enzyme (Bovine Serum Albumin (BSA), MW: 65kDa) was selected based on relative size to typical process enzyme properties (20-130 kDa) for experimental characterization of DSP. Three different HF-UF cutoff ranges demonstrated consistent enzyme removal as a function of diafiltration factor, achieving low-single-log removal of enzyme (Figure 7a). Greater than 90% recovery was achieved, for a 4MDa RNA molecule through the HF-UF process at a diafiltration rate of 6 through the most permeable membrane (750KDa cutoff) (Figure 7b). These experiments demonstrate the potential for multi-stage SP-TFF to be used for in- stream removal of enzyme impurities from RNA product. As a further demonstration of the multi- stage SP-TFF design, the present inventors performed a mock full system operation (IVT, DNase, ISP, Capping, DSP) to characterize the complete enzyme load in the continuous IVT flow system of the invention. Using in-line UV sensors pre- and post-DSP the present inventors observed the low log removal of enzyme levels through the DSP unit relative to the prior unit operation (Figure 10). This suggests substantial reductions of enzyme are achievable using this configuration, and purification targets will be met with additional SP-TFF stages. Further, the in-process enzyme concentration trends were confirmed via in-line sampling during the process. Together, these data demonstrate multi-stage SP-TFF with HMW cutoffs as an effective design for removal of enzyme impurities with little impact to RNA yield. Through the addition of more SP-TFF stages, log removal can be increased to achieve higher purity in a consistent manner. Example 4: Validation of integrated continuous purification system. The integration of a continuous purification system can be most effective as two separate unit operations, specifically an in-stream purification module and a downstream (end of process) purification module. As described above, both the ISP and DSP purification modules are effective for both for buffer exchange and in-process impurity clearance (rNTPs, enzymes). Single-Pass Tangential Flow Filtration integrates well in a continuously flowing process and can be performed with single-use consumables. Moreover, characterization of ISP demonstrates that two-stage SP- TFF is effective at removing low molecular weight impurities and exchanging between IVT and capping buffer allowing for capping in-line. In line DSP with SP-TFF using HMW cutoffs is also an effective design for removal of enzyme impurities with little impact to RNA yield. Together, the data presented herein demonstrates that continuous purification modules to be integrated on with a hollow-fiber-based IVT platform as generally described herein.

[0002] Table 1. Model of relative exchange properties and removal of LMW impurities using SP- TFF Solute First Stage Second stage Relative IVT Buffer 2% <1% Relative Capping Buffer [N / A] >95% Residual NTP <25% <5% RNA step yield 90% 80%

Claims

CLAIMS What is claimed is:

1. An in vitro RNA transcription (IVT) and purification system comprising: − an IVT assembly having a product stream including: − at least one hollow fiber bioreactor (HFBR), wherein a reaction mixture is introduced continuously through an intra-capillary space (IC) of the HFBR, and a feed mixture is continuously supplied to an extra-capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis; − a DNase assembly having a DNAse loop in fluid communication with the HFBR; − a product collection line in fluid communication with the HFBR configured to collect RNA reaction product produced within the IC space of the HFBR; and − a single-pass tangential Flow filtration (SP-TFF) system having one or more hollow-fiber ultra-filtration (HF-UF) cartridges in fluid communication with the HFBR, or product collection line.

2. The system of claim 1, wherein the DNase assembly continuously introduces a quantity of DNase and a buffer to the DNase loop containing the RNA reaction product of the IC space of the HFBR.

3. The system of claim 1, wherein the SP-TFF system is positioned in-stream between the HFBR and the DNase assembly.

4. The system of claim 1, wherein the SP-TFF system is positioned down-stream of the product collection line.

5. The system of claim 1, wherein the one or more HF-UF cartridges comprise two or more cartridges positioned in series and generates a cross-flow of the product stream.

6. The system of claim 1, wherein the one or more HF-UF cartridges comprise two or more cartridges positioned in series and generates a counter-flow of the product stream.

7. The system of claim 1, wherein said reaction mixture comprises one or more components selected from: − a first quantity of a cap analog; − a first quantity of isolated RNA polymerase (RNAP) enzyme;− a quantity of a reaction buffer; and − an first quantity of isolated ribonucleotide triphosphates (rNTPs).

8. The system of claim 1, wherein said feed mixture comprises one or more components selected from: − a second quantity of isolated rNTPs; − a quantity of a reaction buffer; − one or more co-factors for the production of mRNA polynucleotides; and − optionally a second quantity of a cap analog.

9. The system of claim 1, wherein the pressure of the EC is higher than the pressure of the IC, wherein the pressure differential increases substrate availability during the IVT reaction within the HFBR.

10. The system of claim 9, wherein the positive pressure of the EC is generated by a variable pump.

11. The system of any of claims 9-10, wherein: − ECiis greater than ICi. − ECois (ECi +ICi) - ICo− ICois greater than (ECi +ICi).

12. The system of claim 1, further comprising one or more chromatography extraction modules in fluid communication with the HFBR.

13. The system of claim 12, wherein the one or more chromatography extraction modules are selected from: one or more ion exchange resins, one or more protein binding resins, or a combination of the same.

14. The system of claim 13, wherein the resin comprises a cationic resin.

15. The system of claim 12, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR and configured to be in fluid communication with the feed mixture and / or reaction mixture.

16. The system of claim 12, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR and configured to be in fluid communication with a retentate.

17. The system of claim 1, further comprising a nucleotide tri-phosphate (NTP) substrate reservoir in fluid communication with the HFBR, wherein a first quantity of NTP is introduced into the IC where it mixes with the reaction mixture, and a second quantity of NTPs is introduced into the EC where it mixes with the feed mixture.

18. The system of claim 17, wherein the first quantity of NTP substrates from the reservoir is introduced into the reaction mixture prior to introduction into the IC, and wherein the second quantity of NTP substrates from the reservoir is introduced into the feed mixture prior to its introduction into the EC.

19. A method of in vitro RNA transcription (IVT) and purification comprising: − establishing an IVT assembly having a product stream including: − at least one hollow fiber bioreactor (HFBR), wherein a reaction mixture is introduced continuously through an intra-capillary space (IC) of the HFBR, and a feed mixture is continuously supplied to an extra-capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis; − coupling a DNase assembly having a DNAse loop in fluid communication with the HFBR; − collecting RNA reaction product produced within the IC space of the HFBR and transporting it to a product collection line in fluid communication with the HFBR; and − securing a single-pass tangential Flow filtration (SP-TFF) system having one or more hollow-fiber ultra-filtration (HF-UF) cartridges in fluid communication with the HFBR or product collection line.

20. The method of claim 19, further comprising the step of continuously introducing a quantity of DNase and a buffer to the DNase loop via the DNase assembly containing the RNA reaction product of the IC space of the HFBR.

21. The method of claim 19, wherein the step of securing comprises the step of securing the SP-TFF system in-stream between the HFBR and the DNase assembly.

22. The method of claim 19, wherein the step of securing comprises the step of securing the SP-TFF system down-stream of the system of product collection line.

23. The method of claim 19, wherein the step of securing the SP-TFF comprises positioning two or more HF-UF cartridges in series generating a cross-flow of the product stream.

24. The method of claim 19, wherein the step of securing the SP-TFF comprises positioning two or more HF-UF cartridges in series and generating a counter-flow of the product stream.

25. The method of claim 19, wherein said reaction mixture comprises one or more components selected from: − a first quantity of a cap analog; − a first quantity of isolated RNA polymerase (RNAP) enzyme; − a quantity of a reaction buffer; and − an first quantity of isolated ribonucleotide triphosphates (rNTPs).

26. The method of claim 19, wherein said feed mixture comprises one or more components selected from: − a second quantity of isolated rNTPs; − a quantity of a reaction buffer; − one or more co-factors for the production of mRNA polynucleotides; and − optionally a second quantity of a cap analog.

27. The system of claim 19, wherein the pressure of the EC is higher than the pressure of the IC, wherein the pressure differential increases substrate availability during the IVT reaction within the HFBR.

28. The method of any of claims 27, wherein the positive pressure of the EC is generated by a variable pump 29. The method of any of claims 27-28, wherein: − ECiis greater than ICi. − ECois (ECi +ICi) - ICo− ICois greater than (ECi +ICi).

30. The method of claim 19, further comprising placing one or more chromatography extraction modules in fluid communication with the HFBR.

31. The method of claim 30, wherein the one or more chromatography extraction modules are selected from: one or more ion exchange resins, one or more protein binding resins, or a combination of the same.

32. The method of claim 31, wherein the resin comprises a cationic resin.

33. The method of claim 30, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR in fluid communication with the feed mixture and / or reaction mixture.

34. The method of claim 30, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR in fluid communication with a retentate.

35. The system of claim 19, further comprising establishing a rNTP substrate reservoir in fluid communication with the HFBR, wherein a first quantity of rNTPs is introduced into the IC where it mixes with the reaction mixture, and a second quantity of rNTPs is introduced into the EC where it mixes with the feed mixture.

36. The method of claim 35, wherein the first quantity of rNTP substrates from the reservoir are introduced into the reaction mixture prior to introduction into the IC, and wherein the second quantity of NTP substrates from the reservoir are introduced into the feed mixture prior to its introduction into the EC.

37. An in vitro RNA transcription (IVT) and purification system comprising: − an IVT assembly having at least one hollow fiber bioreactor (HFBR) wherein a reaction mixture is introduced continuously through an intra-capillary space (IC) of the HFBR, and a feed mixture is continuously supplied to the extra-capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis; and − wherein the reaction mixture includes a first quantity of a cap analog.

38. The system of claim 37, further comprising a second quantity of cap analog in the feed mixture.

39. The system of claim 37, wherein the pressure of the EC is higher than the pressure of the IC, wherein the pressure differential increases substrate availability during the IVT reaction within the HFBR.

40. The system of any of claims 39, wherein the positive pressure of the EC is generated by a variable pump 41. The system of any of claims 39-40, wherein: − ECiis greater than ICi; − ECois (ECi +ICi) - ICo; and− ICois greater than (ECi +ICi).

42. The system of claim 37, further comprising a DNase assembly having a DNAse loop in fluid communication with the HFBR.

43. The system of claim 37, further comprising a product collection line in fluid communication with the HFBR configured to collect RNA reaction product produced within the IC space of the HFBR.

44. The system of claim 37, further comprising a single-pass tangential Flow filtration (SP-TFF) system having one or more hollow-fiber ultra-filtration (HF-UF) cartridges in fluid communication with the HFBR or product collection line.

45. The system of claim 44, wherein the SP-TFF system is positioned down-stream of the product collection line.

46. The system of claim 44, wherein the one or more HF-UF cartridges comprise two or more cartridges positioned in series and generates a cross-flow of product stream.

47. The system of claim 44, wherein the one or more HF-UF cartridges comprise two or more cartridges positioned in series and generates a counter-flow of product stream.

48. The system of claim 37, further comprising one or more chromatography extraction modules in fluid communication with the HFBR, wherein the one or more chromatography extraction modules are selected from: one or more ion exchange resins, one or more protein binding resins, or a combination of the same.

49. The system of claim 48, wherein the resin comprises a cationic resin.

50. The system of claim 48, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR and configured to be in fluid communication with the feed mixture and / or reaction mixture.

51. The system of claim 48, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR and configured to be in fluid communication with a retentate.

52. The system of claim 37, further comprising a nucleotide tri-phosphate (NTP) substrate reservoir in fluid communication with the HFBR, wherein a first quantity of NTP is introduced into the IC where it mixes with the reaction mixture, and a second quantity of NTPs is introduced into the EC where it mixes with the feed mixture.

53. The system of claim 52, wherein the first quantity of NTP substrates from the reservoir is introduced into the reaction mixture prior to introduction into the IC, and wherein the second quantity of NTP substrates from the reservoir is introduced into the feed mixture prior to its introduction into the EC.

54. A method of in vitro RNA transcription (IVT) and purification comprising: − establishing an IVT assembly having a product stream including at least one hollow fiber bioreactor (HFBR), wherein a reaction mixture is introduced continuously through an intra- capillary space (IC) of the HFBR, and a feed mixture is continuously supplied to an extra- capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis; and − wherein the reaction mixture includes a first quantity of a cap analog.

55. The method of claim 54, further comprising coupling a DNase assembly having a DNAse loop in fluid communication with the HFBR.

56. The method of claim 54, further comprising collecting RNA reaction product produced within the IC space of the HFBR and transporting it to a product collection line in fluid communication with the HFBR.

57. The method of claim 54, further comprising securing a single-pass tangential Flow filtration (SP-TFF) system having one or more hollow-fiber ultra-filtration (HF-UF) cartridges in fluid communication with the HFBR or product collection line.

58. The method of claim 55, further comprising the step of continuously introducing a quantity of DNase and a buffer to the DNase loop via the DNase assembly containing the RNA reaction product of the IC space of the HFBR.

59. The method of claim 57, wherein the step of securing comprises the step of securing the SP-TFF system in-stream between the HFBR and the DNase assembly.

60. The method of claim 57, wherein the step of securing comprises the step of securing the SP-TFF system down-stream of the system of product collection line.

61. The method of claim 57, wherein the step of securing the SP-TFF comprises positioning two or more HF-UF cartridges in series generating a cross-flow of the product stream.

62. The method of claim 57, wherein the step of securing the SP-TFF comprises positioning two or more HF-UF cartridges in series and generating a counter-flow of the product stream.

63. The method of claim 54, wherein the pressure of the EC is higher than the pressure of the IC, wherein the pressure differential increases substrate availability during the IVT reaction within the HFBR.

64. The method of any of claims 63, wherein the positive pressure of the EC is generated by a variable pump.

65. The method of any of claims 63-64, wherein:. − ECiis greater than ICi; − ECois (ECi +ICi) - ICo; and − ICois greater than (ECi +ICi).

66. The method of claim 54, further comprising placing one or more chromatography extraction modules in fluid communication with the HFBR.

67. The method of claim 66, wherein the one or more chromatography extraction modules are selected from: one or more ion exchange resins, one or more protein binding resins, or a combination of the same.

68. The method of claim 67, wherein the resin comprises a cationic resin.

69. The method of claim 66, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR in fluid communication with the feed mixture and / or reaction mixture, or wherein the one or more chromatography extraction modules are positioned downstream from the HFBR in fluid communication with a retentate.

70. The method of claim 71, wherein the fee mixture includes a second quantity of a cap analog.

71. The system of claim 54, further comprising establishing a rNTP substrate reservoir in fluid communication with the HFBR, wherein a first quantity of rNTPs is introduced into the IC where it mixes with the reaction mixture, and a second quantity of rNTPs is introduced into the EC where it mixes with the feed mixture.

72. The method of claim 71, wherein the first quantity of rNTP substrates from the reservoir are introduced into the reaction mixture prior to introduction into the IC, and wherein the second quantity of NTP substrates from the reservoir are introduced into the feed mixture prior to its introduction into the EC.

73. An in vitro RNA transcription (IVT) and purification system comprising: − an IVT assembly having at least one hollow fiber bioreactor (HFBR) wherein a reaction mixture is introduced continuously through an intra-capillary space (IC) of the HFBR, and a feed mixture is continuously supplied to the extra-capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis; and − one or more chromatography extraction modules in fluid communication with the HFBR.

74. The system of claim 73, wherein the one or more chromatography extraction modules are selected from one or more ion exchange resins, and / or one or more protein binding resins, or a combination of the same.

75. The system of claim 74, wherein the resin comprises a cationic resin.

76. The system of claim 73, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR and configured to be in fluid communication with the feed mixture and / or reaction mixture.

77. The system of claim 73, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR and configured to be in fluid communication with the retentate.

78. The system of claim 73, a wherein the reaction mixture includes a first quantity of a cap analog, and second quantity of a cap analog in the feed mixture.

79. The system of claim 73, wherein the pressure of the EC is higher than the pressure of the IC, wherein the pressure differential increases substrate availability during the IVT reaction within the HFBR.

80. The system of any of claims 79, wherein the positive pressure of the EC is generated by a variable pump 81. The system of any of claims 79-80, wherein: − ECiis greater than ICi; − ECois (ECi +ICi) - ICo; and − ICois greater than (ECi +ICi).

82. The system of claim 73, further comprising a DNase assembly having a DNAse loop in fluid communication with the HFBR.

83. The system of claim 73, further comprising a product collection line in fluid communication with the HFBR configured to collect RNA reaction product produced within the IC space of the HFBR.

84. The system of claim 83, further comprising a single-pass tangential Flow filtration (SP-TFF) system having one or more hollow-fiber ultra-filtration (HF-UF) cartridges in fluid communication with the HFBR or the product collection line.

85. The system of claim 84, wherein the SP-TFF system is positioned down-stream of the product collection line.

86. The system of claim 84, wherein the one or more HF-UF cartridges comprise two or more cartridges positioned in series and generates a cross-flow of product stream.

87. The system of claim 84, wherein the one or more HF-UF cartridges comprise two or more cartridges positioned in series and generates a counter-flow of product stream.

88. The system of claim 73, further comprising two or more chromatography extraction modules in fluid communication with the HFBR.

89. The system of claim 88, wherein the two or more chromatography extraction modules is selected from: one or more ion exchange resins, one or more protein binding resins, or a combination of the same.

90. The system of claim 89, wherein the resin comprises a cationic resin.

91. The system of claim 88, wherein two or more chromatography extraction modules are positioned downstream from the HFBR and configured to be in fluid communication with the feed mixture and / or reaction mixture.

92. The system of claim 88, wherein two or more chromatography extraction modules are positioned downstream from the HFBR and configured to be in fluid communication with a retentate.

93. The system of claim 73, further comprising a nucleotide tri-phosphate (NTP) substrate reservoir in fluid communication with the HFBR, wherein a first quantity of NTP is introduced into the IC where it mixes with the reaction mixture, and a second quantity of NTPs is introduced into the EC where it mixes with the feed mixture.

94. The system of claim 93, wherein the first quantity of NTP substrates from the reservoir is introduced into the reaction mixture prior to introduction into the IC, and wherein the second quantity of NTP substrates from the reservoir is introduced into the feed mixture prior to its introduction into the EC.

95. A method of in vitro RNA transcription (IVT) and purification comprising: − establishing an IVT assembly having a product stream including at least one hollow fiber bioreactor (HFBR), wherein a reaction mixture is introduced continuously through an intra-capillary space (IC) of the HFBR, and a feed mixture is continuously supplied to an extra-capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis; and − coupling one or more chromatography extraction modules in fluid communication with the HFBR.

96. The method of claim 95, further comprising coupling a DNase assembly having a DNAse loop in fluid communication with the HFBR.

97. The method of claim 95, further comprising collecting RNA reaction product produced within the IC space of the HFBR and transporting it to a product collection line in fluid communication with the HFBR.

98. The method of claim 95, further comprising securing a single-pass tangential Flow filtration (SP-TFF) system having one or more hollow-fiber ultra-filtration (HF-UF) cartridges in fluid communication with the HFBR or product collection line.

99. The method of claim 96, further comprising the step of continuously introducing a quantity of DNase and a buffer to the DNase loop via the DNase assembly containing the RNA reaction product of the IC space of the HFBR.

100. The method of claim 98, wherein the step of securing comprises the step of securing the SP-TFF system in-stream between the HFBR and the DNase assembly.

101. The method of claim 98, wherein the step of securing comprises the step of securing the SP-TFF system down-stream of the system of product collection line.

102. The method of claim 98, wherein the step of securing the SP-TFF comprises positioning two or more HF-UF cartridges in series generating a cross-flow of the product stream.

103. The method of claim 98, wherein the step of securing the SP-TFF comprises positioning two or more HF-UF cartridges in series and generating a counter-flow of the product stream.

104. The method of claim 95, wherein the pressure of the EC is higher than the pressure of the IC, wherein the pressure differential increases substrate availability during the IVT reaction within the HFBR.

105. The method of any of claims 104, wherein the positive pressure of the EC is generated by a variable pump.

106. The method of any of claims 104-105, wherein: − ECiis greater than ICi. − ECois (ECi +ICi) - ICo− ICois greater than (ECi +ICi).

107. The method of claim 95, further comprising placing one or more chromatography extraction modules in fluid communication with the HFBR.

108. The method of claim 107, wherein the one or more chromatography extraction modules are selected from: one or more ion exchange resins, one or more protein binding resins, or a combination of the same.

109. The method of claim 108, wherein the resin comprises a cationic resin.

110. The method of claim 107, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR in fluid communication with the feed mixture and / or reaction mixture.

111. The method of claim 107, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR in fluid communication with a retentate.

112. The method of claim 95, further comprising establishing a rNTP substrate reservoir in fluid communication with the HFBR, wherein a first quantity of rNTPs is introduced into the IC where it mixes with the reaction mixture, and a second quantity of rNTPs is introduced into the EC where it mixes with the feed mixture.

113. The method of claim 112, wherein the first quantity of rNTP substrates from the reservoir are introduced into the reaction mixture prior to introduction into the IC, and wherein the second quantity of NTP substrates from the reservoir are introduced into the feed mixture prior to its introduction into the EC.

114. The method of claim 95, wherein the reaction mixture includes a first quantity of a cap analog, and optionally the feed mixture includes a second quantity of a cap analog.

115. An in vitro RNA transcription (IVT) and purification system comprising: − an IVT assembly having at least one hollow fiber bioreactor (HFBR) wherein a reaction mixture is introduced continuously through an intra-capillary space (IC) of the HFBR, and a feed mixture is continuously supplied to the extra-capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis; − wherein the pressure of the EC is higher than the pressure of the IC, wherein the pressure differential increases substrate availability during the IVT reaction within the HFBR.

116. The system of claim 115, wherein the pressure in the EC is generated by a positive pressure applied to the feed mix across the HFBR membrane.

117. The system of claim 116, wherein the positive pressure of the EC is generated by a variable pump 118. The system of any of claims 115-117, wherein ECiis greater than ICi.

119. The system of any of claims 115-118, wherein ECois (ECi +ICi) - ICo.

120. The system of any of claims 115-119, wherein ICois greater than (ECi +ICi).

121. The system of claim 115, further comprising one or more chromatography extraction modules in fluid communication with the HFBR.

122. The system of claim 121, wherein the one or more chromatography extraction modules are selected from one or more ion exchange resins, and / or one or more protein binding resins, or a combination of the same.

123. The system of claim 122, wherein the resin comprises a cationic resin.

124. The system of claim 121, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR and configured to be in fluid communication with the feed mixture and / or reaction mixture.

125. The system of claim 121, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR and configured to be in fluid communication with the retentate.

126. The system of claim 115, a wherein the reaction mixture includes a first quantity of a cap analog, and optionally a second quantity of a cap analog in the feed mixture.

127. The system of claim 115, further comprising a DNase assembly having a DNAse loop in fluid communication with the HFBR.

128. The system of claim 115, further comprising a product collection line in fluid communication with the HFBR configured to collect RNA reaction product produced within the IC space of the HFBR.

129. The system of claim 115, further comprising a single-pass tangential Flow filtration (SP-TFF) system having one or more hollow-fiber ultra-filtration (HF-UF) cartridges in fluid communication with the HFBR or product collection line.

130. The system of claim 129, wherein the SP-TFF system is positioned down-stream of the product collection line.

131. The system of claim 129, wherein the one or more HF-UF cartridges comprise two or more cartridges positioned in series and generates a cross-flow of product stream.

132. The system of claim 129, wherein the one or more HF-UF cartridges comprise two or more cartridges positioned in series and generates a counter-flow of product stream.

133. The system of claim 115, further comprising a nucleotide tri-phosphate (NTP) substrate reservoir in fluid communication with the HFBR, wherein a first quantity of NTP is introduced into the IC where it mixes with the reaction mixture, and a second quantity of NTPs is introduced into the EC where it mixes with the feed mixture.

134. The system of claim 133, wherein the first quantity of NTP substrates from the reservoir is introduced into the reaction mixture prior to introduction into the IC, and wherein the second quantity of NTP substrates from the reservoir is introduced into the feed mixture prior to its introduction into the EC.

135. A method of in vitro RNA transcription (IVT) and purification comprising: − establishing an IVT assembly having a product stream including at least one hollow fiber bioreactor (HFBR), wherein a reaction mixture is introduced continuously through an intra- capillary space (IC) of the HFBR, and a feed mixture is continuously supplied to an extra- capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis; and − wherein the pressure of the EC is higher than the pressure of the IC, wherein the pressure differential increases substrate availability during the IVT reaction within the HFBR.

136. The method of claim 135, wherein the pressure of the EC is higher than the pressure of the IC, wherein the pressure differential increases substrate availability during the IVT reaction within the HFBR.

137. The method of any of claims 136, wherein the positive pressure of the EC is generated by a variable pump.

138. The method of any of claims 135-137, wherein:− ECiis greater than ICi; − ECois (ECi +ICi) - ICo; and − ICois greater than (ECi +ICi).

139. The method of claim 138, wherein the feed mixture includes a second quantity of a cap analog.

140. The method of claim 135, further comprising coupling a DNase assembly having a DNAse loop in fluid communication with the HFBR.

141. The method of claim 135, further comprising collecting RNA reaction product produced within the IC space of the HFBR and transporting it to a product collection line in fluid communication with the HFBR.

142. The method of claim 135, further comprising securing a single-pass tangential Flow filtration (SP- TFF) system having one or more hollow-fiber ultra-filtration (HF-UF) cartridges in fluid communication with the HFBR or product collection line.

143. The method of claim 135, further comprising the step of continuously introducing a quantity of DNase and a buffer to the DNase loop via the DNase assembly containing the RNA reaction product of the IC space of the HFBR.

144. The method of claim 142, wherein the step of securing comprises the step of securing the SP-TFF system in-stream between the HFBR and the DNase assembly.

145. The method of claim 142, wherein the step of securing comprises the step of securing the SP-TFF system down-stream of the system of product collection line.

146. The method of claim 142, wherein the step of securing the SP-TFF comprises positioning two or more HF-UF cartridges in series generating a cross-flow of the product stream.

147. The method of claim 142, wherein the step of securing the SP-TFF comprises positioning two or more HF-UF cartridges in series and generating a counter-flow of the product stream.

148. The method of claim 135, further comprising placing one or more chromatography extraction modules in fluid communication with the HFBR.

149. The method of claim 148, wherein the one or more chromatography extraction modules are selected from: one or more ion exchange resins, one or more protein binding resins, or a combination of the same.

150. The method of claim 149, wherein the resin comprises a cationic resin.

151. The method of claim 148, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR in fluid communication with the feed mixture and / or reaction mixture.

152. The method of claim 148, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR in fluid communication with a retentate.

153. The method of claim 135, further comprising establishing a rNTP substrate reservoir in fluid communication with the HFBR, wherein a first quantity of rNTPs is introduced into the IC where it mixes with the reaction mixture, and a second quantity of rNTPs is introduced into the EC where it mixes with the feed mixture.

154. The method of claim 153, wherein the first quantity of rNTP substrates from the reservoir are introduced into the reaction mixture prior to introduction into the IC, and wherein the second quantity of NTP substrates from the reservoir are introduced into the feed mixture prior to its introduction into the EC.

155. The method of claim 135, wherein the reaction mixture includes a first quantity of a cap analog.

156. An in vitro RNA transcription (IVT) and purification system comprising: − an IVT assembly having at least one hollow fiber bioreactor (HFBR) wherein a reaction mixture is introduced continuously through an intra-capillary space (IC) of the HFBR, and a feed mixture is continuously supplied to the extra-capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis when the ICois greater than (ECi +ICi).

157. The system of any of claims 156, wherein ECiis greater than ICi.

158. The system of any of claims 156-157, wherein ECois (ECi +ICi) - ICo.

159. A method of in vitro RNA transcription (IVT) and purification system comprising:− establishing an IVT assembly having at least one hollow fiber bioreactor (HFBR); − introducing a reaction mixture continuously through an intra-capillary space (IC) of the HFBR; and − supplying feed mixture is continuously to the extra-capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis when the ICois greater than (ECi +ICi).

160. The system of any of claims 159, wherein ECiis greater than ICi.

161. The system of any of claims 159-160, wherein ECois (ECi +ICi) - ICo.

162. An in vitro RNA transcription (IVT) and purification system comprising: − an IVT assembly having at least one hollow fiber bioreactor (HFBR) wherein a reaction mixture is introduced continuously through an intra-capillary space (IC) of the HFBR, and a feed mixture is continuously supplied to the extra-capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis; and − a first quantity of nucleotide tri-phosphate (NTP) substrates introduced to the EC via the feed mixture, and a second quantity of NTP substrates are introduced to the IC via the reaction mixture.

163. The system of claim 162, wherein a reservoir of NTP substrates is split and introduced into the feed mixture prior to its introduction into the EC, and the reaction mixture prior to its introduction into the IC.

164. The system of claim 162, wherein the pressure of the EC is higher than the pressure of the IC, wherein the pressure differential increases substrate availability during the IVT reaction within the HFBR.

165. The system of claim 164, wherein the pressure in the EC is generated by a positive pressure applied to the feed mix across the HFBR membrane.

166. The system of claim 165, wherein the positive pressure of the EC is generated by a variable pump.

167. The system of any of claims 164-166, wherein ECiis greater than ICi.

168. The system of any of claims 164-167, wherein ECois (ECi +ICi) - ICo.

169. The system of any of claims 164-168, wherein ICois greater than (ECi +ICi).

170. The system of claim 162, further comprising one or more chromatography extraction modules in fluid communication with the HFBR.

171. The system of claim 170, wherein the one or more chromatography extraction modules are selected from one or more ion exchange resins, and / or one or more protein binding resins, or a combination of the same.

172. The system of claim 171, wherein the resin comprises a cationic resin.

173. The system of claim 170, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR and configured to be in fluid communication with the feed mixture and / or reaction mixture.

174. The system of claim 170, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR and configured to be in fluid communication with the retentate.

175. The system of claim 162, a wherein the reaction mixture includes a first quantity of a cap analog, and optionally a second quantity of a cap analog in the feed mixture.

176. The system of claim 162, further comprising a DNase assembly having a DNAse loop in fluid communication with the HFBR.

177. The system of claim 162, further comprising a product collection line in fluid communication with the HFBR configured to collect RNA reaction product produced within the IC space of the HFBR.

178. The system of claim 162, further comprising a single-pass tangential Flow filtration (SP-TFF) system having one or more hollow-fiber ultra-filtration (HF-UF) cartridges in fluid communication with the HFBR or product collection line.

179. The system of claim 178, wherein the SP-TFF system is positioned down-stream of the product collection line.

180. The system of claim 178, wherein the one or more HF-UF cartridges comprise two or more cartridges positioned in series and generates a cross-flow of product stream.

181. The system of claim 178, wherein the one or more HF-UF cartridges comprise two or more cartridges positioned in series and generates a counter-flow of product stream.

182. The system of claim 163, wherein the nucleotide tri-phosphate (NTP) substrates are positioned in a reservoir in fluid communication with the HFBR.

183. The system of claim 182, wherein the first quantity of NTP substrates from the reservoir is introduced into the reaction mixture prior to introduction into the IC, and wherein the second quantity of NTP substrates from the reservoir is introduced into the feed mixture prior to its introduction into the EC.

184. The system of claim 182, wherein the reservoir of NTP substrates is split and introduced into the feed mixture prior to its introduction into the EC, and the reaction mixture prior to its introduction into the IC.

185. A method of in vitro RNA transcription (IVT) and purification comprising: − establishing an IVT assembly having a product stream including at least one hollow fiber bioreactor (HFBR), wherein a reaction mixture is introduced continuously through an intra- capillary space (IC) of the HFBR, and a feed mixture is continuously supplied to an extra- capillary space (EC) and exchanged through a HFBR membrane into the IC space initiating RNA synthesis; and − introducing a first quantity of nucleotide tri-phosphate (NTP) substrates to the EC via the feed mixture, and introducing a second quantity of NTP substrates to the IC via the reaction mixture.

186. The method of claim 185, wherein the nucleotide tri-phosphate (NTP) substrates are positioned in a reservoir in fluid communication with the HFBR.

187. The method of claim 186, wherein the first quantity of NTP substrates from the reservoir is introduced into the reaction mixture prior to introduction into the IC, and wherein the second quantity of NTP substrates from the reservoir is introduced into the feed mixture prior to its introduction into the EC.

188. The system of claim 186-187, wherein the reservoir of NTP substrates is split and introduced into the feed mixture prior to its introduction into the EC, and the reaction mixture prior to its introduction into the IC.

189. The method of claim 185, wherein the pressure of the EC is higher than the pressure of the IC, wherein the pressure differential increases substrate availability during the IVT reaction within the HFBR.

190. The method of any of claims 189, wherein the positive pressure of the EC is generated by a variable pump.

191. The method of any of claims 189-190, wherein: − ECiis greater than ICi. − ECois (ECi +ICi) - ICo− ICois greater than (ECi +ICi).

192. The method of claim 185, further comprising coupling one or more chromatography extraction modules in fluid communication with the HFBR.

193. The method of claim 185, further comprising coupling a DNase assembly having a DNAse loop in fluid communication with the HFBR.

194. The method of claim 185, further comprising collecting RNA reaction product produced within the IC space of the HFBR and transporting it to a product collection line in fluid communication with the HFBR.

195. The method of claim 185, further comprising securing a single-pass tangential Flow filtration (SP- TFF) system having one or more hollow-fiber ultra-filtration (HF-UF) cartridges in fluid communication with the HFBR or product collection line.

196. The method of claim 185, further comprising the step of continuously introducing a quantity of DNase and a buffer to the DNase loop via the DNase assembly containing the RNA reaction product of the IC space of the HFBR.

197. The method of claim 195, wherein the step of securing comprises the step of securing the SP-TFF system in-stream between the HFBR and the DNase assembly.

198. The method of claim 195, wherein the step of securing comprises the step of securing the SP-TFF system down-stream of the system of product collection line.

199. The method of claim 195, wherein the step of securing the SP-TFF comprises positioning two or more HF-UF cartridges in series generating a cross-flow of the product stream.

200. The method of claim 195, wherein the step of securing the SP-TFF comprises positioning two or more HF-UF cartridges in series and generating a counter-flow of the product stream.

201. The method of claim 185, further comprising placing one or more chromatography extraction modules in fluid communication with the HFBR.

202. The method of claim 201, wherein the one or more chromatography extraction modules are selected from: one or more ion exchange resins, one or more protein binding resins, or a combination of the same.

203. The method of claim 202, wherein the resin comprises a cationic resin.

204. The method of claim 201, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR in fluid communication with the feed mixture and / or reaction mixture.

205. The method of claim 201, wherein the one or more chromatography extraction modules are positioned downstream from the HFBR in fluid communication with a retentate.

206. The method of claim 185, wherein the first quantity of rNTP substrates from the reservoir are introduced into the reaction mixture prior to introduction into the IC, and wherein the second quantity of NTP substrates from the reservoir are introduced into the feed mixture prior to its introduction into the EC.

207. The method of claim 185, wherein the reaction mixture includes a first quantity of a cap analog.

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