Spectral monitoring of in vitro transcription

Abstract

The present invention provides a method for monitoring an in vitro transcription (IVT) reaction for the production of RNA in a reaction vessel. The method includes acquiring a spectrum of a reactant or product during the IVT reaction and comparing the acquired spectrum to a predetermined reference spectrum of a reactant or product of the IVT reaction. The method can be used to determine changes in the amount of a reactant or product during the IVT reaction.

Description

[Technical Field]

[0001] The present invention relates to a method for monitoring in vitro transcription (IVT) for the production of RNA (e.g., mRNA) by acquiring spectra of reactants or products during the IVT reaction. The spectra can then be compared to predetermined reference spectra of the reactants or products of the IVT reaction. Differences between the acquired spectra and the predetermined reference spectra indicate changes in the amount of reactant or product. [Background technology]

[0002] RNA-based therapeutics, including messenger RNA (mRNA)-based vaccines, have emerged as novel therapeutic and preventative modalities that can be rapidly developed in a short period of time. However, the development chain for such products currently lacks a quality-by-design (QbD) framework for their manufacture. The first key step in the manufacturing process is an in vitro transcription (IVT) reaction, in which RNA is transcribed from a DNA template. The template contains the sequences required for transcription of a specific RNA and is operably linked to an RNA polymerase promoter. In the presence of ribonucleotides (NTPs) and RNA polymerase, this DNA sequence is transcribed into RNA.

[0003] The lack of data during the manufacturing process necessitates the implementation of process analytical engineering (PAT) to define critical quality attributes (CQAs). The CQA most relevant for optimization during the manufacturing process is RNA yield. Many parameters can affect the final yield of the IVT reaction. These parameters include NTPs, RNA polymerase, cofactors (e.g., Mg 2+ These include the concentrations of reactants such as the DNA template and the DNA template. RNA yield can be improved by monitoring these critical process parameters (CPPs). Typically, this involves performing multiple experiments to optimize the concentrations of individual reactants for a particular template sequence. Therefore, there is a need to reduce the number of experiments to optimize CPPs for RNA yield during IVT.

[0004] Furthermore, current methods for monitoring IVT reactions typically require removing and processing an aliquot of the reaction mixture from the reaction vessel beforehand so that the RNA yield can be determined. Generally, such methods are limited to determining the amount of a single product or reactant (e.g., the amount of RNA) of the IVT reaction. In addition, the presence of enzyme components can interfere with accurately determining the amount of RNA in the reaction vessel. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is a need for a method that can monitor the amount of reactants or products of an IVT reaction in a reaction vessel without having to take aliquots or remove enzyme components. Ideally, such a method would be capable of simultaneously monitoring multiple reactants and products of an IVT reaction and could be used for both process optimization and process control during large-scale production of RNA. [Means for solving the problem]

[0006] In particular, the present invention relates to a method for monitoring in vitro transcription (IVT) reactions for the production of RNA, particularly messenger RNA (mRNA), using spectroscopic probes within the reaction vessel.

[0007] When incident light (e.g., a laser) irradiates a molecule, it is scattered inelastically, resulting in a change in initial wavelength. The inventors have discovered that the shift that occurs between the frequency of the incident light and the frequency of the scattered light as the IVT reaction occurs in the reaction vessel can provide a molecular fingerprint of one or more reactants and / or products. Spectra acquired during the reaction can reveal the concentrations of major products and reactants (i.e., RNA, NTP, and H2PO4 -The results can be automatically converted into molar concentrations (molar concentrations). This in-line method can increase experimental throughput during process development and can provide useful process and quality data during large-scale manufacturing of RNA-based therapeutics, particularly mRNA-based therapeutics, including mRNA-based vaccines. The data can be used to optimize reaction conditions or to determine whether a batch of RNA can be advanced to downstream manufacturing steps, thereby reducing the time and costs associated with large-scale manufacturing of RNA, particularly mRNA.

[0008] In particular, the present invention relates to a method for monitoring an in vitro transcription (IVT) reaction for the production of RNA, e.g., mRNA, in a reaction vessel, comprising: (i) acquiring a spectrum of a reactant or product during the IVT reaction; and (ii) comparing the spectrum acquired in step (i) with a predetermined reference spectrum of a reactant or product of the IVT reaction, wherein a difference between the spectrum acquired in step (i) and the predetermined reference spectrum indicates a change in the amount of the reactant or product.

[0009] In some embodiments, the reactants or products include RNA, pyrophosphate (PPi), H + , inorganic phosphate (Pi), and ribonucleotides (NTPs). In some embodiments, the product is RNA or Pi. In some embodiments, the reactant is one or more ribonucleotides (NTPs).

[0010] In some embodiments, the method determines changes in the amounts of two or more reactants or two or more products of an IVT reaction. In some embodiments, the method determines changes in the amounts of two or more reactants and two or more products of an IVT reaction. In some embodiments, the two or more reactants are adenosine triphosphate (ATP) and guanosine triphosphate (GTP). In some embodiments, the two or more reactants are cytidine triphosphate (CTP) and uridine triphosphate (UTP). In some embodiments, the two or more products are RNA and Pi.

[0011] In some embodiments, the reaction vessel is a bioreactor. In some embodiments, the reaction vessel has an access port or bypass for inserting a spectroscopic probe. In some embodiments, the spectroscopic probe is immersed in the solution in which the IVT reaction occurs. In some embodiments, the spectroscopic probe is not immersed in the solution in which the IVT reaction occurs. In some embodiments, the spectroscopic probe is separated from the solution by a barrier that does not interfere with detection.

[0012] In some embodiments, step (i) comprises acquiring a series of spectra over the course of the IVT reaction. In some embodiments, each spectrum in the series is acquired over a period of 10 to 60 seconds. In some embodiments, the series of spectra comprises a set of at least 3, at least 5, or at least 9 spectra. In some embodiments, a plurality of series of spectra are acquired over the course of the IVT reaction.

[0013] In some embodiments, each of the spectra in the series is pre-processed prior to step (ii), hi some embodiments, the spectra in the series are acquired sequentially, and the pre-processing comprises smoothing the spectra by applying a digital filter that fits a low-order polynomial to the sequentially acquired spectra by linear least squares.

[0014] In some embodiments, the spectrum or series of spectra obtained in step (i) is normalized to a wavelength region in which background noise has been reduced or is not present prior to step (ii). In some embodiments, the series of spectra is normalized to a wavelength region in which background noise has been reduced or is not present prior to any pre-processing steps.

[0015] In some embodiments, step (ii) comprises a qualitative spectral comparison between the series of spectra acquired in step (i) and a predetermined reference spectrum. In some embodiments, the qualitative spectral comparison comprises calculating a weighted spectral difference (WSD) value. In some embodiments, a WSD value of one standard deviation or less indicates no significant difference between the spectrum acquired in step (i) and the predetermined reference spectrum. In some embodiments, a WSD value of more than one standard deviation indicates a significant difference between the spectrum acquired in step (i) and the predetermined reference spectrum.

[0016] In some embodiments, the predetermined reference spectrum correlates with a specified concentration of the reactant or product. In some embodiments, step (ii) further comprises determining the concentration of the reactant or product. In some embodiments, determining the concentration of the reactant or product comprises linear regression analysis. In some embodiments, a partial least squares (PLS) model is used to determine the concentration of the reactant or product from a series of predetermined reference spectra, where each spectrum in the series correlates with a different concentration of the reactant or product.

[0017] In some embodiments, the spectrum, series of spectra, and predetermined reference spectrum / series of predetermined reference spectra, if applicable, are obtained using a spectrometer for vibrational spectroscopy. In some embodiments, the spectrometer is selected from a Raman spectrometer, an infrared (IR) spectrometer, and a turbidimeter. In specific embodiments, the spectrometer is a Raman spectrometer.

[0018] In some embodiments, the spectrum acquired in step (i) spans a wavelength range suitable for monitoring the overall evolution of multiple reactants and products during an IVT reaction. In some embodiments, the multiple reactants and products include RNA, inorganic phosphate (Pi), and one or more ribonucleotides (NTPs). In some embodiments, the wavelength range is 300 cm -1 ~3000cm -1 and the spectrum is obtained using a Raman spectrometer.

[0019] In some embodiments, the spectrum acquired in step (i) spans a wavelength region specific to one product or reactant of the IVT reaction.

[0020] In some embodiments, the product is RNA. In some embodiments, the wavelength region is 801 cm -1 ~831cm -1 and the spectrum is obtained using a Raman spectrometer.

[0021] In some embodiments, the product is Pi. In some embodiments, the wavelength region is 875 cm -1 ~900cm -1 and the spectrum is obtained using a Raman spectrometer.

[0022] In some embodiments, the reactant is one or more ribonucleotides (NTPs). In some embodiments, the wavelength region is 600 cm -1 ~1300cm -1 and the spectrum is obtained using a Raman spectrometer. In some embodiments, the wavelength region is 1107 cm to 1107 cm to determine the amount of NTP. -1 ~1146cm -1 or 1113cm -1 ~1115cm -1 In some embodiments, the wavelength range comprises or consists of 633 cm to determine the amount of ATP. -1 In some embodiments, the wavelength range comprises or consists of 1300 cm to determine the amount of ATP and GTP. -1 ~1600cm -1 In some embodiments, the wavelength range comprises or consists of 780 cm -1 In some embodiments, the wavelength range comprises or consists of 786 cm -1 ~789cm -1 In some embodiments, the wavelength range comprises or consists of 1230 cm to determine the amount of CTP and UTP. -1 ~1245cm -1It comprises or consists of:

[0023] In some embodiments, the spectrum acquired in step (i) detects the turbidity of the solution in which the IVT reaction occurs. In some embodiments, the turbidity indicates the accumulation of an insoluble precipitate. In some embodiments, the insoluble precipitate is Mg2PPi. In some embodiments, the turbidity is measured using a turbidimeter, an ultraviolet spectrometer, or a nephelometer. In some embodiments, the turbidity is measured using an ultraviolet spectrometer at wavelengths in the range of 290 nm to 410 nm (e.g., 300 nm to 350 nm), inclusive, such as 310 nm, 320 nm, 330 nm, 340 nm, or 350 nm.

[0024] In some aspects, the present invention also relates to methods for producing RNA, e.g., mRNA, using an in vitro transcription (IVT) reaction, comprising: (a) providing a DNA template comprising the nucleotide sequence of an RNA operably linked to an RNA polymerase promoter; (b) adding the DNA template to a reaction vessel containing an RNA polymerase and reactants necessary to initiate the IVT reaction; and (c) monitoring one or more of the reactants or products of the IVT reaction using the methods of the invention as set forth in the preceding paragraph.

[0025] In some embodiments, step (c) comprises acquiring a series of spectra, the spectra spanning a wavelength range suitable for monitoring one or more enzymatic components, such as a DNA template and / or an RNA polymerase, during (or after completion of) the IVT reaction.

[0026] In some embodiments, the spectrum acquired in step (c) is used to monitor the amount of DNA template. In some embodiments, the wavelength region acquired in step (c) is 500 cm to determine the amount of plasmid DNA. -1 ~710cm -1 , 1325cm -1 ~1365cm -1 and / or 1585cm -1 ~1725cm -1 It comprises or consists of:

[0027] In some embodiments, the spectrum acquired in step (c) is used to monitor the amount of RNA polymerase. In some embodiments, the RNA polymerase is SP6 RNA polymerase. In some embodiments, the wavelength region acquired in step (c) is 780 cm to determine the amount or addition of SP6 RNA polymerase. -1 ~1200cm -1 and / or 1430cm -1 ~1510cm -1 It comprises or consists of:

[0028] In some embodiments, the method includes adding a nuclease (e.g., DNase I) to terminate the IVT reaction. In some embodiments, the spectrum acquired in step (c) is used to monitor the amount or addition of nuclease. In some embodiments, the wavelength region acquired in step (c) is between 450 cm and 450 cm to determine the amount or addition of nuclease. -1 ~520cm -1 , 1000cm -1 ~1090cm -1 and / or 2915 cm -1 ~3000cm -1 It comprises or consists of:

[0029] In some embodiments, the method includes adding a protease (e.g., proteinase K) to terminate the IVT reaction (e.g., by digesting RNA polymerase) or to inactivate nucleases. In some embodiments, the spectrum acquired in step (c) is used to monitor the amount or addition of protease. In some embodiments, the wavelength region acquired in step (c) is between 505 cm and 510 cm to determine the amount or addition of protease. -1 ~610cm -1 (For example, 550cm -1 ~600cm -1 ), 715cm -1 ~775cm -1 and / or 1385cm-1 ~1395cm -1 It comprises or consists of:

[0030] In some embodiments, one or more reactants or products is RNA. In some embodiments, the monitoring in step (i) of the methods of the invention comprises obtaining a series of spectra at one or more designated time points during the IVT reaction, and step (ii) comprises comparing the series of spectra to a predetermined reference spectrum to determine the amount or change in amount of RNA between the one or more designated time points.

[0031] In some embodiments, the IVT reaction is terminated if the RNA does not reach a target amount at one or more specified time points, hi some embodiments, the batch of RNA resulting from this IVT reaction is discarded.

[0032] In some embodiments, the IVT reaction is terminated if the change in the amount of RNA is less than a predetermined value at two or more specified time points. In some embodiments, the batch of RNA resulting from this IVT reaction is discarded.

[0033] In some embodiments, the two or more designated time points are equally spaced throughout the IVT reaction, hi some embodiments, each interval is 10 minutes or less, 5 minutes or less, 1 minute or less, or 30 seconds or less.

[0034] In some embodiments, the IVT reaction is terminated when the change in the amount of RNA is about zero between at least two or more time points, in some embodiments, the at least two or more time points are at least 5 minutes apart.

[0035] In some embodiments, the IVT reaction is terminated if the spectrum of the IVT reaction obtained in step (i) deviates by more than one standard deviation from either (A) a predetermined reference spectrum of the reactants or products, or (B) a predetermined kinetic model for an IVT reaction using similar or identical conditions and reactants.

[0036] In some embodiments, a batch of RNA resulting from an IVT reaction is discarded if the spectrum of the IVT reaction obtained in step (i) deviates by more than one standard deviation from either (A) a predetermined reference spectrum of the reactants or products, or (B) a predetermined kinetic model for an IVT reaction using similar or identical conditions and reactants.

[0037] In some aspects, the invention also relates to methods of producing RNA, e.g., mRNA, using an in vitro transcription (IVT) reaction, comprising: (a) monitoring production of RNA in a reaction vessel by (i) acquiring a spectrum of the RNA during the IVT reaction to determine a first value; and (ii) comparing the first value acquired in step (i) to a second value derived from a predetermined reference spectrum of the RNA; and (b) purifying the RNA if the first value is greater than or equal to the second value.

[0038] In some embodiments, the second value corresponds to a target concentration, hi some embodiments, the target concentration is at least 3 g / L.

[0039] In some embodiments, steps (i) and (ii) are repeated at equal intervals, and the RNA is purified if a first value obtained at each interval is greater than or equal to a second value at the corresponding interval, derived from a predetermined reference spectrum.

[0040] In some embodiments, the first and second values ​​are considered equal if they are within one standard deviation of each other.

[0041] In some embodiments, the reaction vessel is a bioreactor. In some embodiments, the reaction vessel has an access port or bypass for inserting a spectroscopic probe. In some embodiments, the spectroscopic probe is immersed in the solution in which the IVT reaction occurs. In some embodiments, the spectroscopic probe is not immersed in the solution in which the IVT reaction occurs. In some embodiments, the spectroscopic probe is separated from the solution by a barrier that does not interfere with detection.

[0042] In some embodiments, the reaction vessel has an access port for adding reactants during the IVT reaction. In some embodiments, a reactant in the IVT reaction is magnesium (Mg 2+ ) and NTPs. In some embodiments, the IVT reaction is replenished with NTPs at least once during the process of producing the RNA. In some embodiments, the IVT reaction is replenished with NTPs periodically during the process of producing the RNA.

[0043] In some embodiments, the IVT reaction is supplemented with NTP when the concentration of NTP approaches depletion. In some embodiments, depletion is approached when the NTP concentration is 5% or less of the NTP concentration present when the IVT reaction is initiated. In some embodiments, depletion is approached when the NTP concentration is 5 mM or less. In some embodiments, depletion is approached when the NTP concentration is 3 mM or less.

[0044] In some embodiments, the IVT reaction is continuously supplemented with NTPs during the process of producing RNA.

[0045] In some embodiments, each NTP is present at a concentration of 1-10 mM, 1-6 mM, 2-6 mM, or 3-6 mM when the IVT reaction is initiated. In some embodiments, the IVT reaction is supplemented with NTPs to maintain or return the concentration to the concentration of NTP present when the IVT reaction was initiated. In some embodiments, the IVT reaction is supplemented with NTPs to maintain the concentration of each NTP within 20%-100%, 20%-75%, or 25%-50% of the concentration present when the IVT reaction was initiated.

[0046] In some embodiments, the total NTP concentration in the IVT reaction is maintained above a lower limit of 2 mM, hi some embodiments, the total NTP concentration in the IVT reaction is maintained between 10 mM and 20 mM.

[0047] In some aspects, the present invention also provides methods for producing RNA using an in vitro transcription (IVT) reaction, the methods including: (a) providing a DNA template comprising a nucleotide sequence of RNA operably linked to an RNA polymerase promoter; (b) adding the DNA template to a reaction vessel containing an RNA polymerase and reactants necessary to initiate the IVT reaction; and (c) monitoring the IVT reaction by acquiring a series of spectra, the spectra spanning a wavelength range suitable for monitoring the DNA template and / or the RNA polymerase during the IVT reaction.

[0048] In some embodiments, the spectrum acquired in step (c) is used to monitor the amount of DNA template. In some embodiments, the wavelength region acquired in step (c) is 500 cm to determine the amount of plasmid DNA. -1 ~710cm -1 , 1325cm -1 ~1365cm -1 and / or 1585cm -1 ~1725cm -1 It comprises or consists of:

[0049] In some embodiments, the spectrum acquired in step (c) is used to monitor the amount of RNA polymerase. In some embodiments, the RNA polymerase is SP6 RNA polymerase. In some embodiments, the wavelength region acquired in step (c) is 780 cm to determine the amount or addition of SP6 RNA polymerase. -1 ~1200cm -1 and / or 1430cm -1 ~1510cm -1 It comprises or consists of:

[0050] In some embodiments, the method includes adding a nuclease (e.g., DNase I) to terminate the IVT reaction. In some embodiments, the spectrum acquired in step (c) is used to monitor the amount or addition of nuclease. In some embodiments, the wavelength region acquired in step (c) is between 450 cm and 450 cm to determine the amount or addition of nuclease. -1 ~520cm -1 , 1000cm -1 ~1090cm -1 and / or 2915 cm -1 ~3000cm -1 It comprises or consists of:

[0051] In some embodiments, the method includes adding a protease (e.g., proteinase K) to terminate the IVT reaction or nuclease activity. In some embodiments, the spectrum acquired in step (c) is used to monitor the amount or addition of protease. In some embodiments, the wavelength region acquired in step (c) is between 505 cm and 510 cm to determine the amount or addition of protease. -1 ~610cm -1 , (e.g., 550 cm -1 ~600cm -1 ), 715cm -1 ~775cm -1 and / or 1385cm -1 ~1395cm -1 It comprises or consists of:

[0052] Embodiments of the invention will now be described, by way of example only, with reference to the following drawings, in which: [Brief explanation of the drawings]

[0053] [Figure 1]Figure 1 shows an example of spectral assignments for Raman spectroscopy profiles during an IVT reaction over a selected wavelength range from 800 to 1300 cm. The dotted, dotted, shaded area below the spectrum corresponds to the wavelength region in which changes in the amount of RNA, NTPs (including CTP and UTP), or reaction byproducts H2PO4- or Pi can be detected, as shown in this figure. Peaks are labeled to indicate representative wavelengths for detection of these reactants and products by Raman spectroscopy. The dashed line corresponds to the first spectrum recorded 8 minutes after the start of the IVT reaction. The dark gray solid line is the spectrum recorded at the end of the IVT reaction after 90 minutes. The light gray lines correspond to intermediate spectra recorded during the IVT reaction. [Figure 2] The graph shows the change in weighted spectral difference (WSD) values ​​calculated for (a) the 801-831 cm wavelength region (RNA) and (b) the 1107-1146 cm wavelength region (NTP) from a series of spectra acquired on a Raman spectrometer. Spectra were acquired during IVT reactions carried out at 37°C. Results acquired on a 2 mL scale are shown as solid lines (mean values ​​of n=3) ± 1 standard deviation (shown as dashed lines). Results from an exemplary experiment (Ambr® 250, n=1) carried out on a 250 mL scale are shown as solid lines with open circles. [Figure 3] RNA concentrations in IVT reactions are shown, either determined inline by a univariate PLS model based on Raman spectroscopy data acquired from within the reaction vessel (dotted line) or determined offline by RiboGreen assay from aliquots taken from the reaction vessel (dashed line). [Figure 4] Figure 1 shows the real-time concentrations of purine-based NTPs (ATP-GTP), pyrimidine-based NTPs (CTP-UTP), and inorganic phosphate (Pi) determined by univariate PLS models based on a series of Raman spectra acquired during a representative IVT reaction. [Figure 5]The concentration profiles (filled circles) of reactants (ATP-GTP, CTP-UTP) and products (RNA, Pi, and PPi) of the IVT reaction are overlaid with kinetic model predictions (solid lines) based on a series of Raman spectra acquired during a representative IVT reaction. RNA concentration was determined by both Raman spectroscopy (open circles) and the RiboGreen assay (open triangles). [Figure 6] The predicted progression of turbidity during IVT reactions performed at 31°C, 37°C, and 42°C is illustrated. In panel (a), the predicted turbidity is displayed as a line up to 5 hours at 31°C, 37°C, and 42°C, with the predicted bands corresponding to the 95% prediction interval shown as dashed lines. The turbidity data (absorbance at 320 nm) used for kinetic modeling are displayed as solid circles. Additional experimental data obtained after 1.5 hours are displayed as open circles and were not used for kinetic modeling. Panel (b) shows a time-temperature transformation (TTT) diagram encompassing the 1% to 99% isoconversion range. The gray-filled range indicates the time-temperature window that prevents significant turbidity during the IVT reaction. [Figure 7] This figure illustrates the RNA concentrations obtained during IVT reactions for three different batch sizes. Specifically, the IVT reactions produced 150 mg, 1 g, or 20 g of mRNA, as represented by open circles, filled squares, and open triangles, respectively. Concentrations were determined by a univariate PLS model based on Raman spectroscopy, as described in Example 3. The IVT reactions were monitored over a 2-hour period, as indicated in the figure. [Figure 8]The concentrations of the products RNA and PPi and two of the nucleotide reactants (GTP and ATP) during IVT reactions performed with different batch sizes are shown. Panels (a)-(c) of Figure 8 show the concentrations of RNA (mg / mL), PPi (mM), and GTP-ATP (mM), respectively, as determined using a univariate PLS model based on Raman spectroscopy data (see Example 3). The reactions were monitored over a period of approximately 4 hours, as indicated in the figure. IVT reactions were performed with mRNA batch sizes of 1 g or 20 g, as represented by open circles or filled squares, respectively. Numbered lines indicate reaction stages: 1 is the IVT reaction stage, 2 is the termination stage (initiated by adding DNase I followed by proteinase K), and 3 is the quench stage (initiated by adding DTT). [Figure 9] This figure illustrates the RNA concentration obtained during an IVT reaction producing 1 g of mRNA, measured inline by Raman spectroscopy or offline by RiboGreen assay (depicted by filled circles or open squares, respectively). Numbered lines indicate reaction stages: 1 is the IVT reaction stage, 2 is the termination stage (initiated by adding DNase I followed by proteinase K), and 3 is the quench stage (initiated by adding DTT). The reaction was monitored over a period of approximately 4 hours, as indicated in the figure. [Figure 10]Figure 10 shows an example of the spectral assignments for ATP and GTP obtained from a Kaiser Raman spectrometer. In panels (a) and (b) of Figure 10, four different concentrations of ATP and GTP were tested in a spike experiment to identify the wavelength regions in which ATP and GTP could be identified. Panels (a) and (b) represent the spectra obtained for the wavelength regions from 1550 cm to 1600 cm and from 1550 cm to approximately 1605 cm, respectively. In panels (c) and (d) of Figure 10, the regions in which ATP and GTP were identified are overlaid. Panel (c) represents the spectrum obtained for the wavelength region from 1550 cm to 1590 cm, and panel (d) represents the spectrum obtained for the wavelength region from approximately 640 cm to approximately 775 cm, respectively. [Figure 11] 11 illustrates the spectral assignments of plasmid DNA as determined by Raman spectroscopy. As shown in panel (a) of Figure 11, IVT reaction mixtures containing spiked plasmid DNA at concentrations of 0 mg / mL, 0.075 mg / mL, 0.15 mg / mL, 0.3 mg / mL, and 0.6 mg / mL were monitored over the wavelength range of 400 cm-1 to 3000 cm-1. Regions A to C are boxed and illustrated separately in panels (b) to (d) of Figure 11. Panel (b) represents the spectrum acquired over the wavelength range of 505 cm-1 to 705 cm-1. Panel (c) represents the spectrum acquired over the wavelength range of 1325 cm-1 to 1370 cm-1. Panel (d) represents the spectrum acquired over the wavelength range of 1585 cm-1 to 1720 cm-1. [Figure 11-1] Same as above. [Figure 12]12. Illustrative spectral assignments of DNase I as determined by Raman spectroscopy. As shown in panel (a) of Figure 12, IVT reaction mixtures containing spiked DNase I at concentrations of 0 ku / mL, 0.063 ku / mL, 0.125 ku / mL, 0.25 ku / mL, and 0.5 ku / mL were monitored over the wavelength range of 400 cm-1 to 3000 cm-1. Regions A to C are boxed and illustrated separately in panels (b) to (d) of Figure 12. Panel (b) represents the spectrum acquired over the wavelength range of 440 cm-1 to 515 cm-1. Panel (c) represents the spectrum acquired over the wavelength range of approximately 1005 cm-1 to approximately 1085 cm-1. Panel (d) represents the spectrum acquired over the wavelength range of 2915 cm-1 to 3000 cm-1. [Figure 12-1] Same as above. [Figure 13] 11A and 11B are illustrations of the spectral assignments of SP6 RNA polymerase as determined by Raman spectroscopy. As shown in panel (a) of Figure 11, IVT reaction mixtures containing spiked SP6 RNA polymerase at concentrations of 0 mg / mL, 0.045 mg / mL, 0.09 mg / mL, 0.18 mg / mL, and 0.36 mg / mL, respectively, were monitored over the wavelength region from 350 cm to 1700 cm. Regions A and B are boxed and illustrated separately in panels (b) and (c) of Figure 13. Panel (b) represents the spectrum acquired over the wavelength region from 780 cm to 1140 cm. Panel (c) represents the spectrum acquired over the wavelength region from 1430 cm to 1500 cm. DETAILED DESCRIPTION OF THE INVENTION

[0054] definition To facilitate understanding of the present invention, certain terms are first defined below. Further definitions for these and other terms are set forth throughout the specification.

[0055] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents and plural terms include the singular, unless the context clearly dictates otherwise. For example, a "spectrum" is understood to refer to one or more spectra. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0056] Unless specifically stated or clear from the context, as used herein, the term "or" is understood to be inclusive, including both "or" and "and." Furthermore, "and / or," when used herein, should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. Thus, when the term "and / or" is used herein in phrases such as "A and / or B," it is intended to include "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, when the term "and / or" is used in phrases such as "A, B and / or C," it is intended to encompass each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0057] Whenever an embodiment is described herein with the phrase "comprising," it is understood that otherwise similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0058] As used herein, the term "about" refers to an interval of accuracy that a person skilled in the art would understand to still ensure the technical effect of the feature. This term indicates a deviation of ±10% from the indicated numerical value. In some embodiments, the deviation is ±5% of the indicated numerical value. In certain embodiments, the deviation is ±1% of the indicated numerical value.

[0059] The term "reaction vessel" refers to any vessel suitable for carrying out an IVT reaction. The reaction vessel contains the reactants, enzyme components, and any additional components (such as buffering reagents) necessary to carry out the IVT reaction. The volume and / or configuration of the reaction vessel may depend on the scale of the IVT reaction and / or the application for which the RNA is being prepared. Suitable reaction vessels may be made of glass, plastic, or stainless steel. In some embodiments, the reaction vessel may be sterilized and sealed to prevent contamination (e.g., a disposable, sterilizable, and sealable plastic bag).

[0060] As used herein, the term "bioreactor" refers to a reaction vessel that may include, or is adapted to be operably linked to, heating means and / or means for providing agitation to the reaction mixture. Often, bioreactors also include ports for adding reactants and / or inserting probes. In some embodiments, a bioreactor may include a bypass for inserting a spectroscopic probe.

[0061] As used herein, the term "RNA" refers to any polyribonucleotide. More typically, in the context of the present invention, the term refers to a polyribonucleotide of at least 100 ribonucleotides in length, such as at least 200 ribonucleotides or at least 400 ribonucleotides. In addition to messenger RNA (mRNA), this may include ribosomal RNA, ribozymes, riboswitches, and / or other long non-coding RNAs (lncRNAs), such as Kcnq1ot1, Xlsirt, Xist, and HOTAIR.

[0062] As used herein, the term "mRNA" refers to a polyribonucleotide that encodes at least one polypeptide. mRNA may contain one or more coding and non-coding regions (e.g., a 5' untranslated region and a 3' untranslated region). As used herein, mRNA encompasses both modified and unmodified RNA. For example, mRNA may contain one or more nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise specified. A typical mRNA includes a 5' cap, a 5' untranslated region (5' UTR), a protein-coding region, a 3' untranslated region (3' UTR), and a 3' tail. In some embodiments, the tail structure is a poly(C) tail. More typically, the tail structure is a poly(A) tail.

[0063] As used herein, the term "sequence optimized" refers to a nucleotide sequence that has been modified relative to a naturally occurring or wild-type nucleic acid. Such modifications can include, for example, codon optimization and / or the use of 5' and 3' UTRs not normally associated with that naturally occurring or wild-type nucleic acid. As used herein, the terms "codon optimization" and "codon-optimized" refer to modifications of the codon composition of a naturally occurring or wild-type nucleic acid encoding a peptide, polypeptide, or protein that do not change its amino acid sequence, thereby improving protein expression of the nucleic acid. In the context of the present invention, "codon optimization" can also refer to the process of filtering out suboptimal nucleotide sequences from a list of nucleotide sequences, such as by filtering for guanine-cytosine content, codon adaptability index, the presence of destabilizing nucleic acid sequences or motifs, and / or the presence of pause sites and / or termination signals, thereby arriving at one or more optimized nucleotide sequences.

[0064] As used herein, the term "spectrum" refers to multiple signals detected as light emission or absorption from a sample, recorded by a spectrometer at two or more time points at one wavelength or at one or more time points at two or more wavelengths. For example, a spectrum can be acquired at a single wavelength over a period of time, such as the duration of an IVT reaction. In some embodiments, two or more spectra are acquired at two or more wavelengths. In a typical embodiment, two or more spectra are acquired at two or more wavelengths at two or more time points during an IVT reaction.

[0065] As used herein, the term "template DNA" (or "DNA template") refers to a DNA molecule containing a nucleotide sequence encoding an RNA transcript to be synthesized by in vitro transcription (IVT). The template DNA is used as a template for IVT to produce the RNA transcript encoded by the template DNA. The template DNA contains all the elements necessary for IVT, in particular a promoter element operably linked to the DNA sequence encoding the desired RNA transcript for binding of a DNA-dependent RNA polymerase, such as T3, T7, or SP6 RNA polymerase. Furthermore, the template DNA may contain primer binding sites 5' and / or 3' to the DNA sequence encoding the RNA transcript so that the identity of the DNA sequence encoding the RNA transcript can be determined, for example, by PCR or DNA sequencing. In the context of the present invention, "template DNA" may be a linear or circular DNA molecule. As used herein, the term "template DNA" may refer to a DNA vector, such as a plasmid DNA, containing a nucleotide sequence encoding the desired RNA transcript.

[0066] Unless otherwise defined herein, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs and as commonly used in the technical field to which this application belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this disclosure. In case of conflict, the present specification, including definitions, will control.

[0067] Generally, the nomenclatures used in connection with and techniques of cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein.

[0068] Throughout this specification and the embodiments, the words "have" and "comprise" or variations such as "has," "having," "comprises," or "including" will be understood to imply the inclusion of the specified integer or group of integers, but not the exclusion of any other integer or group of integers.

[0069] All publications and other reference materials referred to herein are hereby incorporated by reference in their entirety. Although certain documents are cited in this specification, the citation does not constitute an admission that any of those documents form part of the common general knowledge in the art.

[0070] The present invention relates to a method for monitoring an in vitro transcription (IVT) reaction for the production of RNA, particularly messenger RNA (mRNA), in a reaction vessel, comprising (i) acquiring spectra of reactants or products during the IVT reaction, and (ii) comparing the spectra acquired in step (i) with predetermined reference spectra of reactants or products of the IVT reaction, wherein a difference between the spectra acquired in step (i) and the predetermined reference spectra indicates a change in the amount of reactant or product.

[0071] The methods of the present invention are particularly advantageous because they can monitor the amount of reactant or product in a reaction vessel. Unlike some prior art methods, the methods of the present invention do not require taking an aliquot or removing enzyme components from the IVT reaction before acquiring a spectrum. Furthermore, they can provide information regarding the amount of reactant or product in an IVT reaction very quickly. In some embodiments, the amount of RNA in a reaction vessel can be monitored essentially in real time. Another advantage of the present invention is that it allows for multiple reactants and / or products to be monitored simultaneously. In some embodiments, the spectrum acquired in step (i) can be used to monitor one or more reactants (e.g., NTP) and the main product (RNA) and by-products (e.g., PPi, Pi, and / or H) of the IVT reaction. + ) provides information about the amount of

[0072] In vitro transcription "In vitro transcription" or "IVT" refers to a process in which transcription to produce a synthetic RNA product occurs in vitro (i.e., not within an organism, but rather in an artificial environment such as a reaction vessel). An IVT reaction is typically carried out in the presence of an RNA polymerase and a template. The template is typically a DNA template, such as a linearized or circular plasmid. In some embodiments, the template (e.g., DNA template) is monitored during the IVT reaction. In some embodiments, the amount of template (e.g., DNA template) is monitored in the reaction vessel by collecting spectra that can provide information about the amount of template.

[0073] The DNA template comprises a nucleotide sequence operably linked to an RNA polymerase promoter. In some embodiments, the promoter is an SP6 RNA polymerase promoter. In other embodiments, the promoter is a T7 RNA polymerase promoter. In other embodiments, the promoter is a T3 RNA polymerase promoter. In some embodiments, the RNA polymerase, e.g., SP6, T7, or T3 RNA polymerase, is monitored during the IVT reaction. In some embodiments, the amount of RNA polymerase is monitored in the reaction vessel by collecting spectra that can provide information about the amount of template.

[0074] In some embodiments, DNA templates can be optimized to facilitate more efficient transcription and / or downstream translation. For example, DNA templates can be optimized for cis-regulatory elements (e.g., TATA boxes, termination signals, and protein binding sites), artificial recombination sites, χ sites, CpG dinucleotide content, negative CpG islands, GC content, polymerase slippage sites, and / or other elements relevant to transcription; DNA templates can be optimized for cryptic splice sites, RNA transcript secondary structure, RNA transcript stable free energy, repetitive sequences, instability motifs, and / or other elements relevant to RNA processing and stability; DNA templates can be optimized for codon usage bias, codon adaptability, internal χ sites, ribosome binding sites (e.g., IRES), premature poly(A) sites, Shine-Dalgarno (SD) sequences, and / or other elements relevant to translation; and / or DNA templates can be optimized for codon context, codon-anticodon interactions, translational pause sites, and / or other elements relevant to protein folding. The present invention may use optimization methods known in the art, such as those described in WO 2021 / 226461 or GeneOptimizer by ThermoFisher and OptimumGene™ described in U.S. Patent Application Publication No. 2011 / 0081708, the contents of which are incorporated herein by reference in their entireties.

[0075] During the reaction, the RNA polymerase synthesizes RNA in a template-dependent manner. In some embodiments, the RNA polymerase is SP6 RNA polymerase. In other embodiments, the RNA polymerase is T7 RNA polymerase. In some embodiments, the RNA polymerase is T3 RNA polymerase.

[0076] As RNA polymerase transcribes the template nucleotide sequence into RNA, it incorporates ribonucleotides (NTPs) into the nascent RNA transcript, releasing pyrophosphate (PPi) upon incorporation of NTPs.

[0077] In some embodiments, the IVT reaction further comprises pyrophosphatase, which hydrolyzes PPi to inorganic phosphate (Pi).

[0078] RNA polymerase requires divalent cations as cofactors to function efficiently. Suitable divalent cations include magnesium (Mg 2+ ) or manganese (Mn 2+ In some embodiments, the IVT reaction includes Mg 2+ or Mn 2+ Includes.

[0079] reactants In some embodiments, the methods of the invention are used to monitor changes in the amount of one reactant, hi some embodiments, changes in the amount of two or more reactants are monitored.

[0080] The primary reactant in the IVT reaction is NTP. NTPs are used to form RNA, the primary product of the IVT reaction. The progress of the IVT reaction can be monitored by determining changes in the amount of NTP. Indeed, NTP levels will decrease as they are incorporated into RNA. Thus, in some embodiments, the methods of the present invention monitor changes in the amount of one or more NTPs. In some embodiments, changes in the amount of one, two, or three NTPs are monitored simultaneously. For example, depending on the spectroscopic method selected for acquiring the spectrum in step (i) of the methods of the present invention, it may be possible to monitor changes in the amount of individual NTPs of interest (e.g., ATP, CTP, GTP, or UTP). In some embodiments, the methods of the present invention are used to monitor changes in the amount of ATP. In some embodiments, the methods of the present invention are used to monitor changes in the amount of CTP. In some embodiments, the methods of the present invention are used to monitor changes in the amount of GTP. In some embodiments, the methods of the present invention are used to monitor changes in the amount of UTP. In some embodiments, the methods of the invention are used to monitor changes in the amount of ATP and UTP, the amount of ATP and CTP, the amount of ATP and GTP, the amount of GTP and CTP, the amount of GTP and UTP, the amount of UTP and CTP, the amount of ATP, CTP and GTP, the amount of ATP, CTP and UTP, or the amount of GTP, UTP and CTP.

[0081] RNA can be synthesized from NTPs, including naturally occurring nucleosides (also referred to herein as "unmodified nucleosides," i.e., adenosine, guanosine, cytidine, and uridine). Thus, the NTPs in an IVT reaction can be adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytosine triphosphate (CTP), and uridine triphosphate (UTP).

[0082] In some embodiments, RNA can be synthesized by including one or more modified nucleosides in the IVT reaction. Thus, one or more NTPs in the IVT reaction can be modified NTPs. The modified NTPs can include nucleoside analogs (e.g., adenosine analogs, guanosine analogs, cytidine analogs, and / or uridine analogs). In some embodiments, the modified NTP is modified UTP (also referred to herein as UTPm), e.g., a uridine analog such as N1-methylpseudouridine.

[0083] In some embodiments, the one or more modified NTPs comprise a nucleoside analog selected from the group consisting of 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N1-methylpseudouridine), 2-thiouridine, and 2-thiocytidine.

[0084] In some embodiments, the modified NTP comprises a nucleoside analog selected from pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the modified NTP comprises the nucleoside analog N1-methylpseudouridine.

[0085] In some embodiments, the modified NTP is 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N 4 -acetylcytidine, 5-formyl-cytidine, N 4 -methylcytidine, 5-methylcytidine, 5-halocytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1 -methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thiozebularine, 2-thio-zebularine, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxypseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine, α-thio-cytidine, 2'-O-methylcytidine, 5,2'-O-dimethylcytidine, N 4 -acetyl-2'-O-methylcytidine, N 4 ,2'-O-dimethylcytidine, 5-formyl-2'-O-methylcytidine, N 4 ,N 4 In some embodiments, the modified NTP comprises a nucleoside analog selected from 2'-O-trimethylcytidine, 1-thio-cytidine, 2'-F-aracytidine, 2'-F-cytidine, and 2'-OH-aracytidine. In some embodiments, the modified NTP comprises the nucleoside analog 5-methylcytidine.

[0086] In some embodiments, the modified NTP comprises a nucleoside analog selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and any combination thereof.

[0087] In some embodiments, the IVT reaction includes both unmodified and modified NTPs. For example, the NTPs in the IVT reaction can be adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytosine triphosphate (CTP), and N1-methylpseudouridine triphosphate.

[0088] In some embodiments, the amount of one or more unmodified NTPs is monitored during IVT reaction.Modified NTPs can be detected in substantially the same manner as unmodified NTPs.Typically, the monitoring method of the present invention may not require any or minimal adjustments to be adapted for use with one or more modified NTPs.Therefore, in some embodiments, the amount of one or more modified NTPs is monitored during IVT reaction.In a specific embodiment, the method of the present invention is used to monitor the amount of one or more unmodified NTPs and the amount of one or more modified NTPs.

[0089] Depending on the method used to acquire spectra during an IVT reaction, it can be difficult to distinguish between purine nucleotides (e.g., adenine and guanine) on the one hand and pyrimidine nucleotides (e.g., cytosine and uracil) on the other hand, given their similar structures and, therefore, the resulting spectral characteristics. Thus, in some embodiments, the methods of the present invention are used to monitor changes in purine nucleotides and / or changes in pyrimidine nucleotides. For example, in some embodiments, changes in purine nucleotides (e.g., ATP and GTP) are monitored separately from changes in pyrimidine nucleotides (e.g., CTP and UTP). In some embodiments, spectra are acquired to monitor changes in all NTPs in the IVT reaction. Specifically, in some embodiments, changes in ATP, UTP, GTP, and CTP are all monitored separately.

[0090] product The major products of the IVT reaction are RNA, inorganic pyrophosphate (PPi), and H +and inorganic phosphate (Pi). During the course of the IVT reaction, the amount of RNA increases. As reactants are exhausted during the IVT reaction, the amount of RNA may plateau. According to the methods of the present invention, it is possible to monitor the progress of the IVT reaction by determining changes in the amount of RNA. Monitoring the amount of RNA is useful because it allows an operator to terminate the reaction early, for example, because the amount of RNA does not increase as expected early in the IVT reaction. Furthermore, monitoring the amount of RNA as the IVT reaction progresses may allow an operator to stop the IVT reaction when the amount of RNA begins to plateau, thereby shortening the time required for the IVT reaction. Either intervention may be useful for increasing the yield of RNA during manufacturing. Thus, in some embodiments, the methods of the present invention monitor changes in the amount of RNA throughout the IVT reaction.

[0091] While NTPs are incorporated, PPi and H are produced as by-products of the IVT reaction. + The progress of the IVT reaction can be monitored by determining changes in the amount of such by-products. In some embodiments, PPi and / or H + The change in the amount of

[0092] As a by-product of the IVT reaction, PPi can form an insoluble precipitate with the magnesium typically included as a reagent. Incorporation of magnesium into the Mg2PPi insoluble precipitate reduces the amount of Mg available to act as a cofactor for RNA polymerase. 2+ This means that the amount of PPi is decreasing. Therefore, the presence of PPi during an IVT reaction is associated with a decrease in transfer efficiency. Therefore, in some embodiments, changes in the amount of PPi are monitored during the IVT reaction.

[0093] In some embodiments, the IVT reaction includes pyrophosphatase. Pyrophosphatase can hydrolyze PPi to Pi. As a result, the amount of Mg2PPi precipitate formed during the IVT reaction can be reduced. Changes in the amount of Pi during the course of the IVT reaction can provide an indirect measure of the progress of the IVT reaction. Therefore, in some embodiments, changes in the amount of Pi are monitored during the IVT reaction.

[0094] During the IVT reaction, H + The pH typically increases due to the production of H. An increase in pH can adversely affect the efficiency of the IVT reaction. Therefore, in some embodiments, H is added during the IVT reaction. + The change in the amount of

[0095] In some embodiments, changes in the amount of one product (e.g., RNA) are monitored. In some embodiments, changes in the amount of two or more products are monitored. For example, changes in the amount of RNA can be monitored at the same time as changes in the amount of other products. In some embodiments, the amount of RNA and the amount of PPi and / or Pi in the reaction vessel are monitored by collecting spectra that can provide information about the amounts of these products.

[0096] In some embodiments, changes in the amount of one or more reactants (e.g., NTP) and one product (e.g., RNA) are monitored. For example, in addition to the amount of RNA, the same spectrum may also provide information about the amount of NTP (e.g., NTP) in the IVT reaction. In some embodiments, changes in the amount of one or more reactants and one or more products (e.g., RNA and PPi and / or Pi) are monitored.

[0097] In some embodiments, the amounts of RNA and NTPs (e.g., purine and / or pyrimidine nucleotides) are monitored throughout the IVT reaction. In some embodiments, the amounts of RNA and PPi and / or Pi are monitored throughout the IVT reaction. In some embodiments, the amounts of (i) RNA, (ii) NTPs (e.g., purine and / or pyrimidine nucleotides), and (iii) PPi and / or Pi are monitored throughout the IVT reaction. In some embodiments, the amounts of (i) RNA, (ii) NTPs (e.g., purine and / or pyrimidine nucleotides), (iii) PPi and / or Pi, and optionally (iv) H are monitored throughout the IVT reaction. + The amount of is monitored.

[0098] Additional ingredients IVT reactions typically contain a buffering agent, such as Tris, HEPES, citrate, acetate, or phosphate. In some embodiments, the buffering agent is selected based on whether it does not interfere with the spectral readout of the reactants and / or products of interest in the IVT reaction. A suitable buffering agent is, for example, Tris. In some embodiments, the Tris in the IVT reaction has a concentration of 10 to 100 mM. In some embodiments, the Tris in the IVT reaction has a concentration of 15 to 35 mM. In one embodiment, the Tris in the IVT reaction has a concentration of 25 mM.

[0099] In some embodiments, the IVT reaction also includes one or more salts, such as sodium chloride and magnesium chloride. Magnesium chloride can be used to provide divalent cations that can act as cofactors to increase the efficiency of RNA polymerase. In some embodiments, the IVT reaction further includes an RNase inhibitor. In some embodiments, the IVT reaction further includes DTT.

[0100] end The IVT reaction can be terminated, for example, by adding a nuclease (e.g., DNase I) to digest the template (e.g., DNA template). Template concentration can be monitored throughout the IVT reaction or only during the termination phase. In some embodiments, the amount of template (e.g., DNA template) is monitored in the reaction vessel by collecting spectra that can provide information about the amount of template. This information can be used to monitor the digestion of the template (e.g., DNA template) after adding a nuclease (e.g., DNase I). Being able to monitor the completion of the IVT reaction is useful, for example, to ensure that a batch of mRNA is ready to proceed to the next manufacturing step (e.g., purification).

[0101] Alternatively, the template may be able to be removed from the solution without being decomposed. For example, the template may be immobilized on magnetic beads. Monitoring the amount of template (e.g., DNA plasmid) may demonstrate whether the template has been removed, for example, by removing the beads with a magnet.

[0102] A protease (e.g., proteinase K) may be added to inactivate any enzymatic components (e.g., RNA polymerase and nuclease, if present). DTT may also be added to quench the reaction as needed. The concentration of proteases (e.g., proteinase K) and / or other enzymatic components may be monitored throughout the IVT reaction or only during the termination phase.

[0103] In some embodiments, the presence and / or amount of one or more of (i) template, (ii) nuclease (e.g., DNase I), (iii) protease (e.g., proteinase K), and (iv) RNA polymerase is monitored in the reaction vessel by collecting spectra that can obtain information about the amount of one or more of the template, nuclease, protease, and RNA polymerase. For example, information about the presence of nuclease or protease can be useful in an automated process to confirm that the enzyme components were added to the reaction vessel at the appropriate time (i.e., during the termination phase after completion of the IVT reaction) and / or to confirm that the protease has degraded the enzyme components.

[0104] In some embodiments, digestion of the template and the presence of a nuclease (e.g., DNase I) are monitored. In some embodiments, digestion of the template (e.g., by DNase I) and digestion of the RNA polymerase (e.g., by proteinase K) are monitored. In some embodiments, digestion of the template is monitored, followed by digestion of the RNA polymerase and nuclease (e.g., DNase I) by a protease (e.g., proteinase K) monitored. In some embodiments, autodigestion of the protease (e.g., proteinase K) is monitored.

[0105] spectrometer A variety of spectrometers can be used to obtain the spectrum in step (i) and the predetermined reference spectrum in step (ii) of the method of the present invention. For example, light scattering (LS)-based techniques can be used to practice the claimed method, as opposed to absorbance-based methods. As demonstrated in the examples of this application, Raman spectrometers or turbidimeters can be used in the operation of the present invention. Thus, in some embodiments, the spectrum in step (i) and the reference spectrum in step (ii) of the method of the present invention are obtained using LS-based techniques.

[0106] Suitable LS-based techniques include Raman spectroscopy, and thus in some embodiments the spectrometer used in the methods of the present invention is a Raman spectrometer.

[0107] LS-based methods can be complex in terms of equipment and required data analysis. A relatively simple way to implement the methods of the present invention involves the use of a turbidity meter. During the IVT reaction, an insoluble Mg2PPi precipitate can be formed as a by-product. As the IVT reaction progresses and the amount of this by-product increases, turbidity increases. Because the formation of the Mg2PPi precipitate depends on the by-products from the IVT reaction, the rate of appearance of turbidity is an indirect indicator of RNA production. Thus, in some embodiments, the spectrometer used in the methods of the present invention is a turbidity meter. In some embodiments, determining turbidity can provide a complementary method of monitoring the IVT reaction.

[0108] In some embodiments, vibrational methods can be used to perform the claimed methods. Vibrational spectroscopy is based on the periodic changes in polarizability (Raman) or dipole moment (infrared) caused by molecular vibrations and discrete energy transitions of molecules or atomic groups within molecules, and the change in vibrational frequency during scattering or adsorption of electromagnetic radiation. An advantage of using vibrational methods such as Raman or infrared (IR) spectroscopy is that multiple components, such as reactants and / or products, can be monitored simultaneously during an IVT reaction.

[0109] As demonstrated in one exemplary embodiment of the present invention, wavelength regions corresponding to key reactants (e.g., NTPs) and products (e.g., mRNA, PPi, and / or Pi) of an IVT reaction can be identified in the vibrational spectrum. Indeed, software-based comparison with predetermined reference spectra allows not only quantitative analysis of reactants or products, but also quantification of specific reactants or products as the IVT reaction progresses in a reaction vessel over a specified time frame.

[0110] Quantitative analysis can include determining changes in the amount of one or more reactants or products relative to a baseline - for example, a baseline spectrum immediately before the IVT reaction is initiated (e.g., by adding RNA polymerase). Quantitation can include monitoring the concentration of one or more reactants or products during the IVT reaction.

[0111] Advantageously, vibrational methods such as Raman or IR spectroscopy allow the reactants and / or products of an IVT reaction to be monitored without the need to label the reactants or products or to take aliquots from the reaction vessel.

[0112] Thus, in some embodiments, the spectrometer used in the methods of the present invention is a Raman spectrometer. In some embodiments, the spectrometer used in the methods of the present invention is an IR spectrometer. In some embodiments, the spectrometer used in the methods of the present invention is a near-infrared (NIR) spectrometer. In some embodiments, the spectrometer used in the methods of the present invention is a mid-infrared (MIR) spectrometer.

[0113] In some embodiments, it may be advantageous to use two or more spectroscopic methods to monitor the IVT response. In particular, a Raman spectrometer, an IR spectrometer (e.g., a NIR or MIR spectrometer), and a turbidimeter, or any combination of two or more thereof, may be used to monitor the IVT response. In some embodiments, a Raman spectrometer and a turbidimeter are used to monitor the IVT response. In other embodiments, an IR spectrometer and a turbidimeter are used to monitor the IVT response. In some embodiments, a Raman spectrometer and an IR spectrometer are used to monitor the IVT response.

[0114] Spectral acquisition The present invention involves acquiring at least one spectrum during the course of an IVT reaction to monitor the amount of reactant and / or product in a reaction vessel. In some embodiments, the present invention also involves acquiring at least one spectrum during the course of an IVT reaction to monitor the amount of RNA polymerase and / or template (e.g., DNA template) in a reaction vessel. In some embodiments, the present invention also involves acquiring at least one spectrum during the course of an IVT reaction to monitor quenching or termination of the IVT reaction by the addition of a nuclease (e.g., DNase I) or a protease (proteinase K) into the reaction vessel.

[0115] In some embodiments, spectra containing information about one or more reactants (e.g., one or more NTPs) are acquired during the course of an IVT reaction. In some embodiments, spectra of one or more products (e.g., RNA) are acquired during the course of an IVT reaction.

[0116] The collection time of a spectrum is related to the width of the wavelength range, i.e., acquiring a spectrum of a wide wavelength range takes longer than acquiring a spectrum of a narrow wavelength range. To reduce the collection time of a spectrum, it may be preferable to limit the step of acquiring a spectrum to a wavelength range specific to one particular reactant or product. For example, in some embodiments, the spectrum acquired in step (i) of the method of the present invention is limited to a wavelength range that provides information about the amount of RNA present in the reaction vessel. Reducing the collection time by reducing the wavelength range allows for essentially real-time monitoring of the reactants or products. Thus, in some embodiments, a 600 cm -1 ~1300cm -1 In some embodiments, a Raman spectrum is acquired in a wavelength range including 800 cm -1 ~1250cm -1 A Raman spectrum is acquired in a wavelength range including

[0117] In some embodiments, it may be desirable to acquire a spectrum that encompasses multiple reactants and products of an IVT reaction. Thus, in some embodiments, the spectrum acquired in step (i) spans a wavelength range suitable for monitoring the overall progress of multiple reactants and products during the IVT reaction. In some embodiments, a wavelength range is selected that provides information about the amount of (i) RNA, (ii) NTPs (e.g., purine and / or pyrimidine nucleotides), (iii) PPi and / or Pi, and optionally (iv) H+ in the IVT reaction. For example, the inventors have used a 150 cm -1 ~4000cm -1 It was found that collecting a Raman spectrum in the wavelength region including ##STR1## required 50 seconds and provided information about all of the aforementioned products and reactants.

[0118] In some embodiments, each spectrum in the sequence is acquired over a period of less than 1 minute. In some embodiments, each spectrum in the sequence is acquired over a period of less than 60 seconds, e.g., less than 50 seconds, less than 30 seconds, less than 20 seconds, less than 10 seconds, or less than 5 seconds. In some embodiments, each spectrum in the sequence is acquired over a period of 1 to 60 seconds, e.g., 10 to 50 seconds, or 5 to 20 seconds.

[0119] Collecting one or more spectra during the course of an IVT reaction can provide qualitative and / or quantitative data, thereby informing a user of the progress of the reaction. The term "information" can be used to refer to such collected qualitative and / or quantitative data. In some embodiments, spectra containing information about one or more reactants and one or more products are acquired during the course of an IVT reaction. For example, when optimizing reaction conditions, particularly for the production of a specific RNA, such as to take into account nucleotide composition and / or RNA length, it can be advantageous to acquire spectra over a broader wavelength range that includes information about multiple reactants and products of the IVT reaction.

[0120] In some embodiments, a series of spectra is acquired. Acquiring multiple spectra allows data merging to reduce background noise. Optimizing the signal-to-noise ratio helps better distinguish the signal from background noise associated with any detection method. In some embodiments, the series of spectra includes a set of at least 3, at least 5, or at least 9 spectra. In some embodiments, a series of 3 to 20 spectra is acquired. In some embodiments, a series of 5 to 15 spectra is acquired. In some embodiments, a series of 8 to 12 spectra is acquired.

[0121] An important consideration when acquiring spectra is the impact of collection on spectral resolution. Acquiring fewer spectra can reduce collection time, but this can result in a loss of quality. For example, spectral noise may be less easily distinguishable from signal, thus affecting spectral interpretation and analysis. The number of spectra that can be collected to obtain a useful data set may also depend on the specific spectroscopic method and / or spectrometer used to acquire the spectra in step (i) of the methods of the present invention. For example, the inventors have found that acquiring a series of 10 Raman spectra reduced background noise. Thus, in specific embodiments, a series of 10 spectra is acquired. In some embodiments, each spectrum in the series is acquired over a period of 1 to 60 seconds, e.g., 10 to 50 seconds.

[0122] Once the IVT reaction is initiated, the amounts of one or more reactants and one or more products may change throughout the course of the reaction until the reaction is terminated, for example, by adding a nuclease (e.g., DNase I) to digest the template. Thus, in some embodiments, a series of spectra is acquired over the course of the IVT reaction. It may be desirable to acquire spectra at intervals during the course of the IVT reaction to determine, for example, changes in the amounts of one or more reactants and / or one or more products. In some embodiments, a series of spectra is acquired over the course of the IVT reaction, typically at evenly spaced intervals. In some embodiments, multiple series of spectra are acquired over the course of the IVT reaction to reduce the signal-to-noise ratio at individual data points.

[0123] In some embodiments, a spectrum or series of spectra is acquired at 1-30 minute intervals over the course of the IVT reaction. In some embodiments, a spectrum or series of spectra is acquired at 30 minute intervals. In some embodiments, a spectrum or series of spectra is acquired at 15 minute intervals. In some embodiments, a spectrum or series of spectra is acquired at 10 minute intervals. In some embodiments, a spectrum or series of spectra is acquired at 5 minute intervals. In some embodiments, a spectrum or series of spectra is acquired at 4 minute intervals. In some embodiments, a spectrum or series of spectra is acquired at 3 minute intervals. In some embodiments, a spectrum or series of spectra is acquired at 2 minute intervals. In some embodiments, a spectrum or series of spectra is acquired at 1 minute intervals.

[0124] In some embodiments, the spectrum or series of spectra are acquired at intervals of less than 1 minute, for example, at intervals of 50 seconds or less, 40 seconds or less, 30 seconds or less, 20 seconds or less, or 10 seconds or less.

[0125] In some embodiments, spectra are acquired continuously over the course of an IVT reaction. The acquired spectra may be combined into a set of spectra, as described above, to improve the signal-to-noise ratio of individual data points. For example, 3, 4, 5, 6, 7, 8, 9, or 10 spectra may be acquired and combined into a set. A set of spectra may be represented as a single data point, but it corresponds to a series of spectra collected over a period of time (e.g., 1, 2, 3, 5, 6, 7, 8, 9, or 10 minutes).

[0126] Monitoring reactants and / or products during an IVT reaction In some embodiments, spectra over a wavelength range are acquired to monitor the overall change in the amount of at least one reactant (e.g., one or more NTPs) and at least one product (e.g., RNA) of the IVT reaction.

[0127] In some embodiments, the wavelength range provides information about all of the major products and reactants of the IVT reaction, including RNA, PPi / Pi, and NTP. A suitable wavelength range for monitoring these products and reactants using a Raman spectrometer is 300 cm. -1 ~3000cm -1 In some embodiments, the wavelength range includes 600 cm -1 ~1300cm -1 In other embodiments, the wavelength range includes 800 cm -1 ~1250cm -1 Includes.

[0128] In some embodiments, the spectrum acquired in step (i) of the methods of the invention spans a wavelength region specific to a single product or reactant of the IVT reaction.

[0129] In some embodiments, the product is RNA (e.g., mRNA). In some embodiments, the amount of RNA is monitored using a Raman spectrometer. A suitable wavelength range for monitoring the amount of RNA in an IVT reaction using a Raman spectrometer is 801 cm -1 ~831cm-1 In some embodiments, the amount of RNA in the IVT reaction is about 810 cm -1 The wavelength is monitored.

[0130] In some embodiments, the product is Pi. In some embodiments, the amount of Pi is monitored using a Raman spectrometer. A suitable wavelength range for monitoring the amount of Pi in an IVT reaction using a Raman spectrometer is 875 cm -1 ~900cm -1 Includes.

[0131] In some embodiments, the reactants are one or more NTPs (e.g., one, two, or three individual NTPs). In some embodiments, the reactants include two or more NTPs (e.g., purine nucleotides or pyrimidine nucleotides). In some embodiments, the amount of one or more NTPs is monitored using a Raman spectrometer. A suitable wavelength range for monitoring the amount of NTPs in an IVT reaction using a Raman spectrometer is, for example, 700 cm -1 ~800cm -1 Including, 600cm -1 ~1300cm -1 In some embodiments, a narrower wavelength range is used to monitor the amount of NTP in an IVT reaction.

[0132] In some embodiments, a suitable wavelength region for monitoring the amount of NTP in an IVT reaction using a Raman spectrometer is 1100 cm -1 ~1120cm -1 or 1107cm -1 ~1146cm -1 Includes: 1113cm -1 ~1115cm -1 The wavelength range is specifically related to the PO2 - Therefore, in some embodiments, a suitable wavelength region for monitoring the amount of NTP in an IVT reaction using a Raman spectrometer is 1113 cm -1 ~1115cm -1 (For example, 1115cm -1 ) is included.

[0133] NTPs can be classified by the presence of a purine or pyrimidine base. In some embodiments, wavelength ranges are selected that allow for separate detection of purine nucleotides (e.g., ATP and GTP) and pyrimidine nucleotides (e.g., CTP and UTP).

[0134] In some embodiments, the reactants are purine nucleotides. In some embodiments, the amount of purine nucleotides is monitored using a Raman spectrometer. For example, using a Raman spectrometer, GTP and ATP are detected at 1300 cm -1 ~1600cm -1 Thus, in some embodiments, a suitable wavelength range for monitoring the amount of purine nucleotides in an IVT reaction using a Raman spectrometer is 1300 cm -1 ~1600cm -1 In some embodiments, a suitable wavelength range for monitoring the amount of GTP and ATP in an IVT reaction using a Raman spectrometer includes 1580 cm -1 Including 1580cm -1 The wavelength at 1580 cm corresponds to the stretching of the C=N bond. Therefore, in some embodiments, a suitable wavelength region for monitoring the amount of GTP and ATP in an IVT reaction using a Raman spectrometer is 1580 cm. -1 Includes.

[0135] 1560cm -1 ~1580cm -1 The first order Raman shift in the wavelength region including 1560 cm is attributed to the amount of GTP in the IVT reaction. Thus, in some embodiments, a suitable wavelength region for monitoring the amount of GTP in an IVT reaction using a Raman spectrometer is 1560 cm -1 ~1600cm -1 , for example 1560cm -1 ~1590cm -1 or 1560cm -1 ~1580cm -1 Includes.

[0136] Furthermore, the characteristic peaks of GTP and ATP in the Raman spectrum are at 650 cm -1 ~750cm -1 The wavelength range suitable for monitoring the amount of GTP in the IVT reaction using a Raman spectrometer is 650 cm -1 ~700cm -1 The preferred wavelength range for monitoring the amount of ATP in the IVT reaction using Raman spectroscopy is 700 cm -1 ~750cm -1 In some embodiments, 650 cm -1 ~750cm -1 and the wavelength range including 1560 cm -1 ~1580cm -1 The amounts of ATP and GTP are determined by monitoring both wavelength regions including ATP and GTP with a Raman spectrometer.

[0137] In some embodiments, the reactant is a pyrimidine nucleotide. In some embodiments, the amount of pyrimidine nucleotide is monitored using a Raman spectrometer. Using a Raman spectrometer, CTP is detected at 770 cm -1 ~785cm -1 , for example, 780 cm corresponding to the vibration of the cytosine ring -1 UTP (including modified UTP) can be detected at 785 cm due to the stretching of the C=C bond corresponding to the 5th and 6th carbons of uracil. -1 ~810cm -1 wavelength region, e.g., 786 cm -1 ~789cm -1 In one embodiment, a suitable wavelength range for monitoring the amount of pyrimidine nucleotides in an IVT reaction using a Raman spectrometer is 780 cm -1 ~789cm -1 Includes.

[0138] Alternatively or additionally, CTP-UTP exhibits a stretching of the C=N bond at 1220 cm -1 ~1250cm -1 , for example 1230cm -1 ~1245cm-1 Thus, in some embodiments, a suitable wavelength range for monitoring the amount of pyrimidine nucleotides in an IVT reaction using a Raman spectrometer is 1230 cm -1 ~1245cm -1 Includes.

[0139] Using a Raman spectrometer, CTP was detected at 1240 cm -1 and 1290 cm -1 It can be detected by monitoring at 1240 cm -1 The wavelength corresponds to the C-N stretching of unsaturated amines. Thus, in some embodiments, a suitable wavelength region for monitoring the amount of CTP in an IVT reaction using a Raman spectrometer is 1240 cm -1 and 1290 cm -1 Includes.

[0140] In some embodiments, the spectrum acquired in step (i) of the method of the invention is measured at a wavelength specific to a single product or reactant of the IVT reaction. For example, using a Raman spectrometer, ATP is measured at a wavelength specific to a single product or reactant of the IVT reaction at 633 cm, which corresponds to a vibration of the purine ring. -1 Using a Raman spectrometer, the preferred wavelength region for monitoring the amount of UTP is the 1670 cm peak associated with the ketone functional group at carbon 4. -1 Thus, in some embodiments, suitable wavelengths for monitoring the amount of ATP and UTP in an IVT reaction using a Raman spectrometer include 633 cm and 633 cm, respectively. -1 and 1670 cm -1 is.

[0141] Mg2PPi is a by-product of the IVT reaction and accumulates as an insoluble precipitate. The amount of Mg2PPi can be detected by monitoring the turbidity of the IVT reaction solution. Turbidity can be monitored using a turbidimeter, ultraviolet spectrometer, or nephelometer. Turbidity can be measured in wavelength ranges ranging from 290 nm to 410 nm, including 300 nm to 350 nm. Exemplary wavelengths for measuring turbidity using an ultraviolet spectrometer are 310 nm, 320 nm, 330 nm, 340 nm, or 350 nm. For example, using an ultraviolet spectrometer, turbidity can be measured by absorbance at 320 nm. The presence of insoluble components can also be measured using a turbidimeter (measurements are typically given in nephelometric turbidity units [NTU]).

[0142] End of IVT response monitoring In some embodiments, spectra of certain wavelength ranges are acquired to monitor the template and RNA polymerase during the IVT reaction, for example, changes in the amount of template or RNA polymerase can indicate contamination of the IVT reaction.

[0143] In some embodiments, the template (e.g., DNA template) is monitored throughout the entire IVT reaction. In some embodiments, the template is monitored only during the termination phase, e.g., to confirm destruction or removal of the template. In some embodiments, the template is monitored using a Raman spectrometer. A suitable wavelength range for monitoring the template using a Raman spectrometer is 505 cm -1 ~705cm -1 In some embodiments, a suitable wavelength region for monitoring the template using a Raman spectrometer is 1325 cm -1 ~1365cm -1 or 1585cm -1 ~1720cm -1 is.

[0144] In some embodiments, the RNA polymerase (e.g., SP6 RNA polymerase) is monitored throughout the entire IVT reaction. In some embodiments, the RNA polymerase is monitored only during the termination phase to confirm destruction of the enzyme (e.g., by protease digestion). In some embodiments, the RNA polymerase is monitored using a Raman spectrometer. A suitable wavelength range for monitoring RNA polymerases, such as SP6 RNA polymerase, using a Raman spectrometer is 780 cm -1 ~1200cm -1 In some embodiments, a suitable wavelength region for monitoring RNA polymerases, such as SP6 RNA polymerase, using a Raman spectrometer is 1200 cm -1 or 1430cm -1 ~1510cm -1 is.

[0145] In some embodiments, spectra of a wavelength range are acquired to monitor the termination of the IVT reaction upon addition of a component (e.g., an enzyme component). In automated systems, it may be useful to confirm the addition of an enzyme component at the appropriate time in the IVT reaction.

[0146] In some embodiments, the addition or amount of nuclease (e.g., DNase I) is monitored. In some embodiments, the amount of nuclease (e.g., DNase I) is monitored only during the termination phase to confirm destruction of the enzyme (e.g., by proteinase K digestion). In some embodiments, the addition or amount of nuclease (e.g., DNase I) is monitored using a Raman spectrometer. A suitable wavelength range for monitoring nucleases such as DNase I using a Raman spectrometer is 450 cm -1 ~520cm -1 In some embodiments, a suitable wavelength range for monitoring DNase I using a Raman spectrometer is 1000 cm -1 ~1090cm -1 or 2915 cm -1 ~3000cm -1 is.

[0147] In some embodiments, the addition of a protease (e.g., proteinase K) is monitored. In some embodiments, the addition of a protease is monitored using a Raman spectrometer. A suitable wavelength range for monitoring a protease, such as proteinase K, using a Raman spectrometer is 505 cm -1 ~610cm -1 , for example 550cm -1 ~600cm -1 In some embodiments, a suitable wavelength region for monitoring proteases, such as proteinase K, using a Raman spectrometer is 715 cm -1 ~775cm -1 or 1385cm -1 ~1395cm -1 is.

[0148] Spectral preprocessing The raw spectral data acquired in step (i) of the method of the present invention may be processed to improve the signal-to-noise ratio. Thus, in some embodiments, the spectrum acquired in step (i) is preprocessed before step (ii). In some embodiments, each acquired spectrum in the series is preprocessed before step (ii). Preprocessing may facilitate comparative analysis of the spectra. For example, the wavelength region of the spectrum acquired in step (i) may be normalized to a wavelength region with low or no background noise. Using a Raman spectrometer, a suitable wavelength region with low background noise is 3100 cm -1 and 3600 cm -1 Normalization is typically performed by using a suitable algorithm, such as the Standard Normal Variate (SNV) algorithm. In some embodiments, the spectrum or series of spectra obtained in step (i) is normalized in a suitable wavelength region. In one embodiment, the spectrum or series of spectra is collected with a Raman spectrometer and is measured at 3100 cm -1 ~3600cm -1 The wavelength range is used for normalization.

[0149] Alternatively or additionally, preprocessing may involve smoothing the data using a series of spectra by applying a digital filter. Applying such a filter increases the accuracy of the data without distorting the signal trends. In some embodiments, the digital filter fits a low-order polynomial using linear least-squares to successively acquired spectra containing adjacent data points. In one exemplary embodiment, the series of spectra is acquired at equal intervals. Thus, because the data points are evenly spaced, an analytical solution to the least-squares equation can be found in the form of a single set of "convolution coefficients," which can be applied to all spectra in the series to provide a smoothed data set. For example, a suitable digital filter may use the Savitzky-Golay (SG) algorithm.

[0150] Comparing a spectrum with a reference spectrum According to the method of the present invention, the spectrum of the reactant or product obtained in step (i) is compared with a predetermined reference spectrum of the reactant or product in step (ii).

[0151] In some embodiments, the predetermined reference spectrum is the first spectrum or series of first spectra acquired during the IVT reaction. In these embodiments, the first spectrum or series of spectra serves as a baseline for the IVT reaction. Any changes in the amount of the reactant or product of interest reflect the progress of the IVT reaction.

[0152] In some embodiments, the predetermined reference spectrum is a spectrum or series of spectra acquired during an IVT reaction using known conditions and reactants. In some embodiments, the predetermined reference spectrum is a spectrum or series of spectra of reactants or products of an IVT reaction, typically acquired with the reactants or products dissolved or suspended in the reaction buffer used for the IVT reaction. In either scenario, the concentrations of the reactants and / or products with respect to the predetermined reference spectrum are known and can therefore be used to infer the concentrations of the reactants and / or products of the spectrum or series of spectra acquired in step (i) of the methods of the invention.

[0153] Thus, in some embodiments, a predetermined reference spectrum correlates with a specified concentration of a reactant or product. In some embodiments, the predetermined reference spectrum is used to determine the concentration of a reactant or product. In some embodiments, a series of predetermined reference spectra is used to calculate the concentration of a reactant or product, with each spectrum in the series correlating with a different concentration.

[0154] In some embodiments, determining the concentration of the reactant or product comprises linear regression analysis, hi some embodiments, a partial least squares (PLS) model is used to determine the concentration of the reactant or product from a series of predetermined reference spectra, where each spectrum in the series correlates with a different concentration of the reactant or product.

[0155] In some embodiments, the concentrations of reactants or products are determined using linear regression analysis. In one embodiment, the concentrations of reactants or products are determined using a PLS model. PLS finds a linear regression model by projecting predictor and observed variables into a new space. In this new space, the underlying relationships between the variables are established, which are then projected back into the original space. PLS regression is widely used in chemometrics and related fields.

[0156] In some embodiments, step (ii) comprises a qualitative spectral comparison between the spectrum or series of spectra obtained in step (i) and a predetermined reference spectrum, hi some embodiments, the qualitative spectral comparison comprises calculating a weighted spectral difference (WSD) value between the spectrum or series of spectra obtained in step (i) and the predetermined reference spectrum.

[0157] In some embodiments, a WSD value within one standard deviation or less indicates a high degree of similarity, e.g., no significant difference, between the spectrum acquired in step (i) and the predetermined reference spectrum. Thus, in these embodiments, the amount of reactant or product in the spectrum acquired in step (i) is considered to be the same as the amount of reactant or product in the predetermined reference spectrum, or, if applicable, the amount of reactant or product in the spectrum acquired in step (i) is considered to remain unchanged compared to the amount of reactant or product in the predetermined reference spectrum.

[0158] In some embodiments, a WSD value greater than one standard deviation (e.g., two standard deviations) indicates a low similarity, e.g., a significant difference, between the spectrum acquired in step (i) and a given reference spectrum. Thus, in these embodiments, the amount of reactant or product in the spectrum acquired in step (i) is considered to be greater or less, as applicable, than the amount of reactant or product in the reference spectrum.

[0159] Process Optimization In some embodiments, the methods of the present invention can be used to optimize reaction conditions prior to large-scale production of RNA (e.g., mRNA). For example, the acquisition of a series of spectra by monitoring an IVT reaction can be used to generate a kinetic model for one or more reactants and / or products. This kinetic model can be used to predict or simulate changes in the amount of one or more products under various conditions, for example, with various starting concentrations of one or more reactants. Thus, the use of a kinetic model can reduce the number of experiments that need to be performed to identify an optimal set of conditions for producing RNA using an IVT reaction.

[0160] In some embodiments, kinetic model is used to predict the yield of RNA produced from IVT reaction.In some embodiments, kinetic model is used to simulate IVT reaction and predict the consumption of reactants.In specific embodiments, kinetic model is used to predict the consumption of NTP during IVT reaction.

[0161] The kinetic model can also be used as a reference to compare spectra acquired during an IVT reaction to determine whether the IVT reaction is proceeding at an expected efficiency level. Typically, the kinetic model is generated from a series of spectra acquired from an IVT reaction performed under similar or identical conditions to the IVT reaction being monitored. In some embodiments, the kinetic model is used to predict the yield of a product (e.g., RNA) of the IVT reaction.

[0162] RNA production The monitoring methods of the invention also find use as process or quality control tools during RNA manufacturing, particularly during large-scale production of RNA. For example, the monitoring methods of the invention may be particularly useful in the large-scale production of RNA, such as mRNA, for use in therapeutic applications.

[0163] Accordingly, the present invention also relates to methods for producing RNA (e.g., mRNA) using an in vitro transcription (IVT) reaction, comprising: (a) providing a DNA template comprising the nucleotide sequence of an RNA operably linked to an RNA polymerase promoter; (b) adding the DNA template to a reaction vessel containing an RNA polymerase and reactants necessary to initiate the IVT reaction; and (c) monitoring one or more of the reactants or products of the IVT reaction using the methods for monitoring an IVT reaction described herein. In some embodiments, one or more of the reactants or products is RNA.

[0164] The present invention further relates to a method for producing RNA (e.g., mRNA) using an in vitro transcription (IVT) reaction, the method comprising: (a) monitoring the production of RNA in a reaction vessel by (i) determining a first value by acquiring a spectrum of the RNA during the IVT reaction; and (ii) comparing the first value acquired in step (i) to a second value derived from a predetermined reference spectrum of the RNA; and (b) purifying the RNA if the first value is greater than or equal to the second value.

[0165] quality control Monitoring the IVT reaction through spectroscopic methods, such as Raman spectroscopy, as described herein can be used to determine whether the IVT reaction during the production of a batch of RNA (e.g., mRNA) meets one or more predetermined parameters. The decision to continue or terminate the IVT reaction can depend on one or more predetermined parameters.

[0166] In some embodiments, one or more of the reactants or products are monitored throughout the course of the IVT reaction during production of RNA (e.g., mRNA). In some embodiments, one or more of the reactants is NTP. In some embodiments, one or more of the products is RNA, PPi, and / or H + In some embodiments, one or more reactants and one or more products are monitored during the RNA production process.

[0167] For example, the spectrum of one or more reactants or products acquired in step (i) of the monitoring method of the present invention can be compared to one or more predetermined reference spectra for that reactant or product. If the acquired spectrum sufficiently corresponds to the predetermined reference spectrum, the IVT reaction can proceed. If not, it can be terminated.

[0168] Comparison of one or more spectra acquired during the production of a batch of RNA (e.g., mRNA) to a predetermined reference spectrum can provide confirmation that the reaction is proceeding efficiently and within a set of predetermined parameters corresponding to one or more reactants (e.g., NTPs) and / or one or more products (e.g., RNA). In some embodiments, a series of spectra is acquired at one or more designated time points during the IVT reaction, where the series of spectra is compared to a predetermined reference spectrum. This comparison between the series of spectra between one or more designated time points can be used to determine, for example, the amount or change in amount of RNA.

[0169] By monitoring multiple time points throughout the reaction, it is possible to understand the performance of the IVT reaction.For example, it can be used to check whether the amount of RNA increases as expected at various intervals during the IVT reaction.In some embodiments, two or more designated time points are equally spaced throughout the IVT reaction.In some embodiments, each interval is 30 minutes or less, 20 minutes or less, 5 minutes or less, 2 minutes or less, 1 minute or less, or 30 seconds or less.

[0170] In some embodiments, the RNA production rate during the IVT reaction can be measured at various time points. For example, in some embodiments, the RNA production rate is measured in the early phase of the reaction, i.e., during the first 20 minutes. In some embodiments, the RNA production rate is measured in the later phase of the reaction, i.e., after 20 minutes. In some embodiments, the RNA production rate is measured after 30 minutes. In some embodiments, the RNA production rate is measured after 45 minutes. In some embodiments, the amount of RNA is measured after 60 minutes. In some embodiments, the amount of RNA is measured after 75 minutes. In some embodiments, it may be useful to continuously monitor the RNA production rate throughout the course of the IVT reaction and terminate the reaction when the RNA production rate reaches approximately 0 (meaning that the amount of RNA in the IVT reaction has plateaued).

[0171] In some embodiments, spectral comparison may be sufficient to determine whether the IVT reaction performed as expected while producing a batch of RNA (e.g., mRNA). For example, if the spectrum acquired during the IVT reaction deviates significantly from a predetermined reference spectrum, the performance of the IVT reaction may be inefficient, and therefore the operator may decide to terminate it. If the spectrum from the IVT reaction is sufficiently similar to the predetermined reference spectrum, the operator may proceed with the production run.

[0172] In some embodiments, the spectrum acquired from the IVT reaction deviates significantly from a predetermined reference spectrum, e.g., the acquired spectrum deviates by more than one standard deviation from the predetermined reference spectrum. In such embodiments, an operator may decide that the batch of RNA (e.g., mRNA) resulting from the IVT reaction should be discarded.

[0173] In some embodiments, if the spectrum from an IVT reaction deviates significantly from a predetermined reference spectrum, e.g., the acquired spectrum deviates by more than one standard deviation from the predetermined reference spectrum, an operator may decide that the reaction should be terminated.

[0174] In some embodiments, the spectra acquired from the IVT reaction are compared to a predetermined kinetic model for an IVT reaction using similar or identical conditions and reactants. In some embodiments, the spectra from the IVT reaction are sufficiently similar to the kinetic model, e.g., the acquired spectra deviate from the kinetic model by less than one standard deviation. In such embodiments, an operator may determine whether the IVT reaction can proceed to a subsequent production step, e.g., RNA purification, and / or release the batch of RNA (e.g., mRNA). In some embodiments, the spectra from the IVT reaction deviate significantly from the kinetic model, e.g., the acquired spectra deviate from the kinetic model by more than one standard deviation. In such embodiments, an operator may determine that the reaction should be terminated and that the batch of RNA (e.g., mRNA) should not proceed to a subsequent production step.

[0175] In some embodiments, the deviation from a predetermined reference spectrum is assessed using a qualitative spectral comparison, which in some embodiments comprises calculating a weighted spectral difference (WSD) value between the spectrum or sequence of spectra obtained in step (i) of the method of the invention and the predetermined reference spectrum.

[0176] In some embodiments, a WSD value within one standard deviation or less indicates no significant difference between the spectrum obtained in step (i) and the predetermined reference spectrum, while in some embodiments, a WSD value greater than one standard deviation (e.g., two standard deviations) indicates a significant difference between the spectrum obtained in step (i) and the predetermined reference spectrum.

[0177] In some embodiments, the amount of RNA present in the reaction vessel is calculated using the spectrum obtained in step (i) of the methods of the invention as part of the comparison with a predetermined reference spectrum in step (ii). In some embodiments, step (i) comprises obtaining a series of spectra at one or more designated time points during the IVT reaction, and step (ii) comprises comparing the series of spectra with a predetermined reference spectrum to determine the amount or change in amount of RNA between the one or more designated time points.

[0178] In some embodiments, the IVT reaction may be terminated if the target amount of RNA is not reached at one or more designated time points.

[0179] In some embodiments, monitoring the change in the amount of RNA may be advantageous rather than estimating the amount (or concentration) of RNA in a reaction vessel. In some embodiments, the IVT reaction may be terminated if the change in the amount of RNA is less than a predetermined value at two or more specified time points. In this context, termination may mean either stopping the IVT reaction early or completing the IVT reaction.

[0180] For example, in some embodiments, a batch of RNA (e.g., mRNA) resulting from an IVT reaction can be discarded if the change in the amount of RNA is less than a predetermined value at two or more designated time points. Typically, these time points are early in the IVT reaction, e.g., within the first 5-30 minutes of a production run. In some embodiments, a batch of RNA resulting from an IVT reaction can be discarded if the change in the amount of RNA is about zero between at least two or more time points that are at least 5, 10, 15, 20, or 25 minutes apart, e.g., about 6 minutes apart, about 7 minutes apart, about 8 minutes apart, about 9 minutes apart, about 10 minutes apart, about 15 minutes apart, about 20 minutes apart, or about 25 minutes apart.

[0181] For example, the two or more specified time points can be in the first 10, 20, or 30 minutes of the IVT reaction. The predetermined value can correspond to an expected fold increase in the amount of RNA in the first 10, 20, or 30 minutes of the IVT reaction, such as a 2-fold increase from the first 5 to the first 20 minutes of the IVT reaction, or a 3-fold increase from the first 5 to the first 30 minutes of the IVT reaction.

[0182] In some embodiments, a batch of RNA (e.g., mRNA) resulting from an IVT reaction can be discarded if the RNA does not reach a target amount or concentration at one or more designated time points. For example, the one or more designated time points can be during the first 10, 20, or 30 minutes of the IVT reaction. The predetermined value can correspond to an expected concentration of RNA during the first 10, 20, or 30 minutes of the IVT reaction, e.g., a concentration of 1 g / L after the first 5 or 10 minutes, a concentration of 2 g / L after the first 10-20 minutes, or a concentration of 3 g / L after the first 25-30 minutes. Alternatively, the one or more designated time points can be during the last third of normal production, e.g., 60-90 minutes after the start of the IVT reaction. The predetermined value can correspond to an expected concentration of RNA during that time, e.g., 4-5 g / L.

[0183] Spectral data acquired during the IVT reaction can also be used to determine when the amount of RNA has plateaued, and in some embodiments, this information can be used to terminate the IVT reaction and proceed to the next production step (e.g., purification).

[0184] In some embodiments, an IVT reaction can be terminated when the change in RNA quantity is about zero between at least two or more time points. Typically, these time points are late in the IVT reaction, e.g., within 60-90 minutes of a production run. In some embodiments, an IVT reaction can be terminated when the change in RNA quantity is about zero between at least two or more time points at least about 5 minutes apart, e.g., about 6 minutes apart, about 7 minutes apart, about 8 minutes apart, about 9 minutes apart, or about 10 minutes apart. For example, an IVT reaction can be terminated when there is no change in RNA quantity 65-70 minutes after production has been initiated (e.g., by adding RNA polymerase).

[0185] The present invention also relates to a method for producing RNA (e.g., mRNA) using an in vitro transcription (IVT) reaction, comprising: (a) monitoring the production of RNA in a reaction vessel by (i) acquiring a spectrum of the RNA during the IVT reaction to determine a first value; and (ii) comparing the first value acquired in step (i) to a second value derived from a predetermined reference spectrum of the RNA; and (b) purifying the RNA if the first value is greater than or equal to the second value.

[0186] To determine RNA yield, a predetermined reference value can be derived from a predetermined reference spectrum of known concentration. In some embodiments, the second value corresponds to a target concentration. In some embodiments, the target concentration of RNA is at least 3 g / L, at least 3.5 g / L, at least 4 g / L, at least 4.5 g / L, at least 5 g / L, at least 5.5 g / L, at least 6 g / L, at least 6.5 g / L, at least 7 g / L, at least 7.5 g / L, or at least 8 g / L.

[0187] Acquiring a spectrum corresponding to RNA at multiple time intervals and comparing it to a predetermined reference spectrum can provide information about the extent to which the concentration of RNA is increasing during the IVT reaction. This information can be useful in determining when to terminate the reaction and purify the RNA (e.g., mRNA for use in therapeutic applications) for downstream production steps (e.g., formulation, lyophilization, and / or encapsulation in lipid nanoparticles). Thus, in some embodiments, acquiring a spectrum of RNA to determine a first value and comparing the first value to a second value derived from a predetermined reference spectrum are repeated at equal intervals. In some embodiments, the first value is considered equal to the second value derived from a predetermined reference spectrum of the RNA if the values ​​are within one standard deviation of each other. In some embodiments, the RNA is purified if the first value acquired at each interval is equal to or greater than the second value derived from the predetermined reference spectrum at the corresponding interval.

[0188] In some embodiments, the first and second values ​​are compared using a qualitative spectral comparison, which in some embodiments includes calculating a weighted spectral difference (WSD) value between the spectrum of the RNA obtained during the IVT reaction and a predetermined reference spectrum.

[0189] In some embodiments, a WSD value within one standard deviation or less indicates no significant difference between the spectrum obtained in step (i) and the predetermined reference spectrum, while in some embodiments, a WSD value greater than one standard deviation (e.g., two standard deviations) indicates a significant difference between the spectrum obtained in step (i) and the predetermined reference spectrum.

[0190] If the first value does not equal or exceed the second value, the operator may decide not to proceed with that batch of RNA (eg, mRNA) for purification.

[0191] Process Management In some embodiments, information obtained from monitoring the reactants and / or products of an IVT reaction can be used to replenish reactants in a reaction vessel. For example, NTPs are consumed during the course of an IVT reaction. If the amount of one or more NTPs is too low, the IVT reaction cannot proceed. For example, monitoring the amount of one or more NTPs during the course of an IVT reaction can be used to alert a reaction vessel operator or operating system when the NTPs are nearing depletion. Thus, one or more NTPs can be replenished in the IVT reaction as needed, thereby maintaining an appropriate concentration of each NTP. In some embodiments, when the concentration of one or more NTPs approaches depletion, the NTPs are replenished in the IVT reaction.

[0192] In some embodiments, the IVT reaction is supplemented with one or more NTPs at least once during the process of producing RNA (e.g., mRNA). In some embodiments, the IVT reaction is supplemented with one or more NTPs periodically during the process of producing RNA. In some embodiments, the IVT reaction is supplemented with one or more NTPs continuously during the process of producing RNA (e.g., during fed-batch operation of a suitably configured reaction vessel).

[0193] In some embodiments, the IVT reaction is supplemented with NTPs to maintain the concentration of NTPs present at the start of the IVT reaction. In some embodiments, the IVT reaction is supplemented with NTPs to return the concentration of NTPs to the concentration present at the start of the IVT reaction. In some embodiments, the concentration of each NTP is maintained within 20-100%, 20-75%, or 25%-50% of the concentration present at the start of the IVT reaction.

[0194] In some embodiments, when the concentration of NTP in reaction is 5% or less of the initial concentration of NTP at the beginning of IVT reaction, IVT reaction is supplemented with NTP.In some embodiments, when the concentration of NTP in reaction is about 5mM or less, IVT reaction is supplemented with one or more NTP.In some embodiments, when the concentration of NTP in reaction is about 3mM or less, IVT reaction is supplemented with one or more NTP.

[0195] In some embodiments, each NTP is present at a concentration of 1-10 mM, 1-6 mM, 2-6 mM, or 3-6 mM at the start of the IVT reaction. In some embodiments, the total NTP concentration in the IVT reaction is maintained above a lower limit of 0.5 mM. In some embodiments, the total NTP concentration in the IVT reaction is maintained between 10 mM and 20 mM.

[0196] In some embodiments, each NTP may be present at a specific concentration. For example, when each NTP is present at a concentration of 1 to 10 mM, each NTP may be present at a concentration of 4 mM. In some embodiments, each NTP is present at a concentration proportional to the number of occurrences of the NTP in the RNA transcript.

[0197] In some embodiments, a Raman spectrometer is used to monitor the amount of one or more NTPs in the reaction vessel. The Raman spectrometer has a specified limit of detection (LoD) and limit of quantitation (LoQ). The LoD and LoQ values ​​of the Raman spectrometer are shown in Table 1. Therefore, in some embodiments, the concentration at which the reaction vessel is to be replenished with one or more NTPs is selected taking into account the LoD and / or LoQ of the Raman spectrometer.

[0198] [Table 1]

[0199] In some embodiments, a Raman spectrometer is used to monitor the amount of template and / or RNA polymerase in the IVT reaction. For example, changes in the amount of template or RNA polymerase can indicate contamination of the IVT reaction.

[0200] In some embodiments, a Raman spectrometer is used to monitor the completion of the IVT reaction by the addition of one or more enzymatic components, such as a nuclease (e.g., DNase I) and / or a protease (e.g., proteinase K).

[0201] In some embodiments, the amount of template is monitored using a Raman spectrometer to confirm destruction of this reagent during the termination phase of the IVT reaction.

[0202] In some embodiments, Raman spectroscopy is used to monitor the amount of RNA polymerase and / or nuclease (e.g., DNase I) to confirm destruction of these enzymes during the termination phase of the IVT reaction.

[0203] Batch Size The methods of the invention can be used to optimize reaction conditions prior to large-scale production of RNA (e.g., mRNA). Alternatively, the methods of the invention can be used as a process and / or quality control measure during large-scale production of RNA (e.g., mRNA).

[0204] Large-scale manufacturing typically involves producing batches of RNA (e.g., mRNA) of 100 mg or more. Thus, in some embodiments, at least 100 mg of RNA is synthesized in a single batch. In some embodiments, at least 200 mg of RNA is synthesized in a single batch. In some embodiments, at least 300 mg of RNA is synthesized in a single batch. In some embodiments, at least 400 mg of RNA is synthesized in a single batch. In some embodiments, at least 500 mg of RNA is synthesized in a single batch. In some embodiments, at least 600 mg of RNA is synthesized in a single batch. In some embodiments, at least 700 mg of RNA is synthesized in a single batch. In some embodiments, at least 800 mg of RNA is synthesized in a single batch. In some embodiments, at least 900 mg of RNA is synthesized in a single batch.

[0205] In some embodiments, the term "large-scale manufacturing" refers to the production of a batch of at least 1 g of RNA (e.g., mRNA). In some embodiments, at least 1 g of mRNA is synthesized in a single batch. In some embodiments, at least 5 g of RNA is synthesized in a single batch. In some embodiments, at least 10 g of RNA is synthesized in a single batch. In some embodiments, at least 25 g of RNA is synthesized in a single batch. In some embodiments, at least 50 g of RNA is synthesized in a single batch. In some embodiments, at least 75 g of RNA is synthesized in a single batch. In some embodiments, at least 100 g of RNA is synthesized in a single batch. In some embodiments, at least 150 g of RNA is synthesized in a single batch. In some embodiments, at least 200 g of RNA is synthesized in a single batch. In some embodiments, at least 250 g of RNA is synthesized in a single batch. In some embodiments, at least 500 g of RNA is synthesized in a single batch. In some embodiments, at least 750 g of RNA is synthesized in a single batch.

[0206] In some embodiments, the term "large-scale manufacturing" refers to the production of a batch of at least 1 kg of RNA (e.g., mRNA). In some embodiments, at least 1 kg of RNA is synthesized in a single batch. In some embodiments, at least 5 kg of RNA is synthesized in a single batch. In some embodiments, at least 10 kg of RNA is synthesized in a single batch. In some embodiments, at least 10 kg of RNA is synthesized in a single batch. In some embodiments, at least 100 kg of RNA is synthesized in a single batch. In some embodiments, at least 1000 kg of RNA is synthesized in a single batch.

[0207] reaction vessel The IVT reaction is carried out in a suitable reaction vessel, e.g., a bioreactor. To facilitate monitoring of the IVT reaction, a spectroscopic probe is typically inserted into the reaction vessel. The use of a spectroscopic probe allows for direct monitoring of the amount of one or more reactants and / or one or more products during the course of the IVT reaction, without interrupting the reaction, e.g., for sample extraction. In some embodiments, the reaction vessel has a dedicated access port for inserting the spectroscopic probe. In such embodiments, the spectroscopic probe is directly immersed in the solution in which the IVT reaction occurs. Direct separation of the spectroscopic probe from the solution can reduce the risk of contamination of the IVT reaction. Thus, in some embodiments, the spectroscopic probe is not directly immersed in the solution in which the IVT reaction occurs; for example, the spectroscopic probe can be separated from the IVT reaction by a barrier that does not interfere with detection. In some embodiments, the spectroscopic probe is positioned in-line or in situ.

[0208] In some embodiments, it may be further advantageous to incorporate the spectroscopic probe in a bypass branching off from the reaction vessel. Placing the spectroscopic probe in the bypass may be advantageous to avoid interference, e.g., background noise, associated with stirring means provided within the reaction vessel.

[0209] In some embodiments, the reaction vessel additionally comprises an access port for adding reactants during the IVT reaction.

[0210] In some embodiments, the reactor vessel is configured for fed-batch or continuous operation.

[0211] Suitable reaction vessels may be made of glass, plastic, or stainless steel. In some embodiments, the reaction vessel may be sterilized and sealed to prevent contamination (e.g., a disposable, sterilizable, and sealable plastic bag). In some embodiments, the reaction vessel may be heatable. In some embodiments, the reaction vessel is a bioreactor.

[0212] Reaction volume IVT reactions can be performed on a small scale, for example, while optimizing an IVT reaction with a particular RNA of interest (e.g., mRNA for therapeutic use). In some embodiments, the reaction vessel has a volume of at least 2 mL.

[0213] More typically, for optimization, a reaction vessel (e.g., a bioreactor) is selected that allows for scaling up of the IVT reaction to accommodate larger batches (e.g., for commercial production of mRNA). In some embodiments, the reaction vessel has a volume of at least 200 mL. For large-scale production, larger volumes may be selected. In some embodiments, the reaction vessel has a volume of 12.5 L to 2000 L. In some embodiments, the reaction vessel has a volume of 500 L, 1000 L, or 2000 L or more.

[0214] heating means Typically, IVT reactions are carried out at temperatures above room temperature, for example, 32°C to 42°C (e.g., 35°C to 39°C or about 35°C, 36°C, 37°C, 38°C, or 39°C). Accordingly, in some embodiments, the reaction vessel (e.g., bioreactor) comprises a heating means.

[0215] Stirring means for IVT reactions In some embodiments, the IVT reaction is carried out without stirring, while in other embodiments, the IVT reaction is stirred, for example, at 100 rpm to 400 rpm.

[0216] Thus, in some embodiments, the reaction vessel (e.g., bioreactor) includes a means for providing agitation of the reaction mixture contained therein. For example, the reaction vessel may be programmable to provide agitation during the course of the IVT reaction. Thus, in some embodiments, the reaction vessel is configured to provide agitation. In some embodiments, the agitation is between 100 rpm and 400 rpm. [Example]

[0217] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0218] Example 1. Quantitative monitoring of RNA, NTP, and Pi by spectroscopy This example illustrates the monitoring of products and reactants during an IVT reaction using in-line spectroscopy.

[0219] IVT reactions were performed with 0.05 mg / mL linear template plasmid in the presence of NTPs and RNA polymerase in Tris reaction buffer (25 mM Tris pH 7.5, 2 mM spermidine, 25 mM MgCl, 5 mM NaCl). The UTP was modified UTP in which uridine was replaced with a uridine analog such as N1-methylpseudouridine.

[0220] The coding region of the template encoded an exemplary approximately 1.9 kb mRNA with a nucleotide sequence containing 504 As, 490 Gs, 568 Cs, and 379 Us, respectively. Reactions were initiated by adding 1.1 ng / μL pyrophosphatase and 100 ng / μL RNA polymerase and then incubated at 37° C. for 90 minutes. IVT reactions were performed either without stirring in a 2 mL total reaction volume in a graduated cylinder or with stirring at 250 rpm in a 250 mL reaction volume in a bioreactor system (Ambr250®, Sartorius).

[0221] IVT reactions were analyzed in-line (i.e., within the reaction vessel) using a ProCellics™ system (RESOLUTION Spectra Systems-Merck Millipore) for collection of Raman spectra. This system uses a 785 nm excitation laser source with 350 mW of power at the probe tip and a 150 cm back-thinned charge-coupled device (CCD) detector. -1 ~4000cm -1 Raman shift bandwidth (Stokes signal) of 1 cm -1A highly sensitive spectrometer was used for the sampling step. The spectroscopic probe was directly immersed in a 25 mL glass test tube filled with 2 mL of IVT reaction solution or in an Ambr250® bioreactor filled to 250 mL. To ensure analytical integrity, an aluminum foil layer was used to isolate the Raman measurements from any external light. A 50-second integration time (average of 10 spectra) was used for each collection. The preprocessing step was performed using ProCellics™ software (RESOLUTION Spectra Systems). The preprocessing consisted of the creation of derivatives (order 1, 15 cm) according to the Savitzky-Golay (SG) algorithm as a first step. -1 Step, polynomial degree 2) and the second step, 3,100 cm -1 ~3,600cm -1 This included the calculation of customized standard normal variates (SNV) in the

[0222] Empirical characteristic vibrational frequencies of chemical functional groups were used for spectral interpretation and compared with the molecular structures of the IVT components. -1 The peak at 1095 cm was assigned to the phosphodiester bond (OPO symmetric stretching). -1 Peak PO2 - Symmetric Stretching (OPO - The intensity ratio of these two peaks provides structural information about the RNA, i.e., the ratio between ordered and disordered structures. Because the phosphodiester bond is specific to RNA, it has the same characteristic frequency (810 cm) regardless of the polyribonucleotide sequence. -1 ) can be monitored during the IVT reaction. -1 A new peak appeared at 1000 kJ / min, the area of ​​which seemed to correlate with the production of RNA. - was assigned to.

[0223] NTPs can be divided into two groups: those with purine bases (ATP and GTP) and those with pyrimidine bases (CTP and UTP). Adenine is a 6-monosubstituted purine. It has a 633 cm -1This is represented by a weak band at 780 cm, which can be attributed to the CH out-of-plane bending vibration. Guanine is 6-oxo-2-aminopurine. CTP has a weak band at 780 cm, which is caused by the vibration of the cytosine ring. -1 In the UTP spectrum, a band of medium intensity appears at approximately 786-789 cm due to the stretching of the C=C bond (carbon positions 5 and 6 of uracil). -1 A band can also be observed at approximately 1113-1115 cm -1 The peak decreased over the course of the IVT reaction and was attributed to NTP consumption. This peak was due to the PO2 triphosphate group. - May be related to stretching.

[0224] 800cm -1 ~1300cm -1 An illustrative Raman spectrum covering the region of NTP is shown in Figure 1. The dashed line represents the first recorded spectrum, 500 seconds after the start of the IVT reaction. The dark gray solid line is the spectrum recorded at the end of the IVT reaction, after 90 minutes. As can be seen, the spectral region corresponding to RNA and the reaction by-product Pi shows an increase in intensity after 90 minutes, while the spectral region corresponding to NTP shows a decrease in intensity after 90 minutes.

[0225] This example demonstrates that in-line spectroscopy can be used to identify various components of an IVT reaction. In this context, "in-line" means that a spectroscopic probe is immersed in the solution in which the IVT reaction occurs (e.g., in the reaction vessel or in the bypass of the reaction vessel). In-line spectroscopy can be used to monitor the production of RNA and the formation of Pi as a by-product. It can also be used to monitor the consumption of NTPs during an IVT reaction. The methods described herein are not limited to Raman spectroscopy and can be applied to identifying suitable wavelength regions corresponding to reactants or products of interest in a spectrum or series of spectra using other spectroscopic methods, particularly light-scattering-based methods.

[0226] Example 2. Monitoring product and reactant changes using reference spectra This example illustrates that by comparing spectra acquired during an IVT reaction with predetermined reference spectra of the reactants and products of the IVT reaction, changes in those reactants and products over time can be monitored.

[0227] To monitor changes in reactants and products present in the reaction vessel, spectra acquired during the IVT reaction were compared to the predetermined spectrum of the reactant or product of interest using a percent weighted spectral difference (WSD) calculation. For quantifying spectral differences, the WSD calculation provides a weighting function based on relative signal magnitude. A WSD value close to 0 indicates a high degree of similarity with the reference spectrum, while a high WSD value indicates a large difference between the acquired spectrum and the predetermined spectrum. WSD values ​​can be calculated using the following formula:

number

[0228] Raman spectroscopy as described in Example 1 was used to monitor global spectral changes during the IVT reaction at 300 cm. -1 ~3000cm -1 The wavelength ranges of 801–831 cm were selected for specific monitoring of RNA (product) and NTP (reactant), respectively. -1 and 1107-1146 cm -1 The wavelength range was selected.

[0229] FIG. 2 shows the peaks at 801-831 cm in panel (a) during the IVT reaction carried out as described in Example 1. -1 RNA region and panel (b) 1107–1146 cm -1The change in WSD values ​​for the NTP region is illustrated. At both the 2 mL and 250 mL scales, the amount of RNA increased throughout the reaction, reaching a plateau toward the end of the reaction time (Figure 2, panel (a)). This was accompanied by a steady decrease in NTPs over the same period (Figure 2, panel (b)).

[0230] This example demonstrates that acquiring a series of spectra during an IVT reaction and comparing those spectra to predetermined reference spectra of the reactants and products of the IVT reaction can be used to determine changes in the concentrations of the reactants and products over the course of the IVT reaction.

[0231] Example 3. Building a Partial Least Squares (PLS) Model This example illustrates the construction of a partial least squares (PLS) model using given spectra of reactants and products of an IVT reaction at specified concentrations, which can be used as a predictive data set to determine the respective concentrations of reactants and products from spectra acquired during the IVT reaction.

[0232] Similar to linear regression, a partial least squares (PLS) model aims to calculate a set of parameters linking a response variable (the Y matrix) with independent variables (the X matrix). In PLS, a linear regression model is developed by projecting the response and independent variables into a new space. This new space establishes the underlying relationships between the variables, which are then projected back into the original space. PLS regression is useful for predicting a response variable Y from a large set of independent variables X by reducing the set of X variables to a smaller set of uncorrelated components. Performing least squares regression on these components thus reduces multicollinearity among the X values.

[0233] PLS is particularly useful for uncovering patterns in spectral signatures when relative chemical data are available. SIMCA® software (version 16) was used to analyze the ATP-dependent ATP synthesis of NTP, H2PO4, and ATP in a buffer solution constructed as a D-optimal design using JMP software (version 14). -PLS models were constructed for the Raman spectral data of 42 different mixtures containing various concentrations of each of ATP and RNA. D-optimal designs were created by swapping the highest and lowest concentrations of each component (see Table 2). Spectra for each of these 42 mixtures were recorded as described in Example 1.

[0234] [Table 2]

[0235] [Table 3]

[0236] Based on the spectra of the 42 mixtures, PLS models were constructed for each of the parameters ATP-GTP, CTP-UTP, Pi, and RNA. The Raman signatures of adenine and guanine bases overlapped and were indistinguishable in the PLS models due to the high correlation between these parameters. The same was observed for cytosine and uracil bases. As a result, ATP and GTP, as well as CTP and UTP, were quantified by summing their concentrations, and the two identified parameters were ATP-GTP and CTP-UTP, respectively. The PLS models were then used as a predictive dataset to calculate the respective concentrations of reactants and products over time. The PLS SIMCA files were imported into the Raman spectrometer's ProCellics software to monitor the concentrations of reactants and products in the reaction vessel during the IVT reaction, as exemplified in Example 4.

[0237] Example 4. Monitoring RNA production in a reaction vessel This example illustrates that spectroscopy can be used to determine the RNA concentration in a reaction vessel during an IVT reaction.

[0238] The RNA concentration is 0 at the beginning of the IVT reaction (t = 0 min) and increases over time after transcription is initiated by the addition of RNA polymerase as described in Example 1. Using in-line Raman spectroscopy and PLS modeling as described in Example 3, the RNA concentration at 18.3 min was determined to be 2.8 g / L. This concentration increased to 4.1 g / L at 64.1 min. These values ​​were compared with offline quantification using the RiboGreen assay. Aliquots were removed from the IVT reaction at various time points. Concentrations calculated from the RiboGreen assay data were 2.0 g / L at 16 min and 3.5 g / L at 62 min. Figure 3 plots the concentration values ​​obtained by Raman spectroscopy against those obtained by the RiboGreen assay.

[0239] For the RiboGreen assay, RiboGreen® reagent (ThermoFisher) was diluted 200-fold with TE buffer, and this reagent solution was added to an equal volume of RNA in TE. Samples in the microplate were incubated at room temperature for 5–30 minutes, protected from light. The sample volume for the microplate assay was 200 mL. The microplate assay was performed using a CytoFluor II fluorescence microplate reader (Soft Max Pro 6.5.1). Samples were excited at 495 nm, and fluorescence was measured at 521 nm. Integrated fluorescence emission intensity was plotted against RNA concentration without subtraction of background fluorescence. The relative error of the RiboGreen assay was 12%, while the relative error of the PLS model (described in Example 3) was 11%.

[0240] This example demonstrates that spectroscopy can be used to determine RNA concentration during an IVT reaction in a reaction vessel. The in-line spectroscopic method yielded concentrations comparable to standard offline methods such as the RiboGreen assay.

[0241] Example 5. Monitoring NTP consumption and Pi production in reaction vessels This example demonstrates that spectroscopy can be used to monitor NTP consumption and inorganic phosphate (Pi) accumulation during an IVT reaction in a reaction vessel.

[0242] Magnesium (Mg 2+ ) is an essential cofactor for RNA polymerase and has a direct effect on the transcription rate and, consequently, the amount of RNA produced. In combination with pyrophosphate (PPi), it can lead to the formation of an insoluble precipitate, which can affect the RNA yield during production. Adding pyrophosphatase to an IVT reaction hydrolyzes PPi, enhancing RNA yield. This results in the production of inorganic phosphate (Pi). The concentration of Pi is zero at the beginning of the IVT reaction (t = 0 min) and increases over time after transcription is initiated by the addition of RNA polymerase, as described in Example 1. As ribonucleotides (NTPs) are incorporated into RNA, their concentration in the reaction vessel decreases.

[0243] The concentrations of ATP-GTP, CTP-UTP, and Pi in the reaction vessel were monitored using in-line Raman spectroscopy and the PLS model described in Example 3. As expected, the intensity of the Raman spectra in selected spectral regions decreased and increased, respectively, indicating a decrease in the concentrations of ATP-GTP and CTP-UTP and an increase in the Pi concentration during the IVT reaction. Representative spectra are shown in Figure 4. The relative errors of the calculations were 14% and 13% for the ATP-GTP and CTP-UTP concentrations, respectively, and 4% for the Pi concentration.

[0244] This example demonstrates that spectroscopy can be used to monitor NTP and pyrophosphate concentrations during an IVT reaction in a reaction vessel. The in-line information provided by spectroscopy can be useful in determining whether the NTP concentration needs to be adjusted.

[0245] Example 6. Building a kinetic model of the IVT reaction This example illustrates how the kinetic model of the IVT reaction described in Example 1 is consistent with experimental observations made using, inter alia, the spectroscopic methods described herein.

[0246] The reaction stoichiometry for the total synthesis of an RNA transcript from nucleotides is given by the following equation: nA ATP+nG GTP+nC CTP+nU UTP+DNA→RNAn+(n-1)PPi+DNA nA, nG, nC, and nU represent the number of adenine, guanine, cytosine, and uracil bases, respectively, in each copy of the fully transcribed RNA, and the sum of nucleotides equals n (=nA+nG+nC+nU). Using RNA encoded by the template plasmid described in Example 1, the following mechanistic scheme was employed for Reaction 1. 504ATP+490GTP+568CTP+379UTP→1RNA+1940PPi+1940H + H + can be either expressed alone as in solution or considered bound to PPi. The presence of pyrophosphate (PPi) has been shown to inhibit the RNA synthesis reaction. This is why a second reaction is required to hydrolyze PPi to Pi with the aid of pyrophosphatase. The following mechanistic scheme was adopted for reaction 2: 1940PPi+1940H2O→3880Pi

[0247] Using the concentrations of NTP and Pi determined using the PLS model as described in Example 3, and assuming that reactions 1 and 2 occur during the IVT reaction, the best fit of the data led to the prediction of the concentrations of RNA and PPi, as shown in Figure 5. Based on this analysis, the kinetic model can be expressed as follows: r1=(4.795×10 -4 ) × [ATP + GTP] 1 ×[CTP+UTP] 1 , and r2=(1.805×10 -5 )×[PPi]1 ×[H2O] 1 4.795×10 -4 and 1.805 x 10 -5 are the rate constants in g / (s×mol) for r1 and r2, respectively.

[0248] This empirical model was used to predict ATP-GTP, CTP-UTP, Pi, and RNA concentrations. The RNA kinetic profile was compared to a set of concentrations (represented by open circles) and RiboGreen quantification values ​​(represented by open triangles) calculated using the PLS model described in Example 4 (see panel (a) of Figure 5). The agreement observed between the predictions (kinetic model) and experimental data (Raman-PLS and RiboGreen) confirmed the ability of the spectroscopy method combined with the PLS method described in Example 3 to quantify RNA production in-line.

[0249] Beyond estimating RNA content, the kinetic model estimated the progression of other reactants and products, including PPi (see panel (e) of Figure 5), which showed that PPi first increased and then slowly decreased. This behavior appears consistent with reaction 1 (production of PPi) and reaction 2 (degradation of PPi by pyrophosphatase). Furthermore, the modeled H + The increase in pH (see panel (d) of Figure 5) was consistent with the decrease in pH during the IVT reaction as previously observed.

[0250] Example 7. Monitoring and modeling the appearance of opacity This example illustrates that spectroscopy can be used to monitor the appearance of turbidity during an IVT reaction caused by the buildup of insoluble components such as Mg2PPi precipitate as a reaction by-product.

[0251] During the IVT reaction, the appearance of turbidity was monitored at three different temperatures (31°C, 37°C, and 42°C). Absorbance measurements were taken at 2-minute intervals during the 90-minute run time. Turbidity was measured with a SpectraMax M5 UV-Visible spectrophotometer (Molecular Devices®). The presence of insoluble components, such as Mg2PPi precipitate, was estimated by measuring absorbance at 320 nm.

[0252] A kinetic model was developed by combining advanced kinetics and statistical analysis of stability data acquired at 31°C, 37°C, and 42°C. The rate of turbidity appearance was temperature dependent, allowing for Arrhenius-based modeling. The higher the temperature at which the IVT reaction was performed, the more rapidly turbidity appeared. Advanced kinetic modeling was applied by fitting the collected data, leading to a two-stage kinetic model that describes the progress of the reaction as a function of time and temperature.

[0253] This model was used to predict turbidity levels for longer periods up to 5 hours (see Figure 6, panel (a)), and was also used to estimate a safety region that bounds the time-temperature window where turbidity appearance can be prevented (see Figure 6, panel (b)).

[0254] This example demonstrates that spectroscopic measurement of turbidity during an IVT reaction can be used as a method to monitor the formation of reaction by-products such as Mg2PPi precipitate.

[0255] Example 8. Scalability This example illustrates that the progress of an IVT reaction can be reproducibly monitored by the methods disclosed in the previous examples when batch sizes are increased or when the IVT reaction is performed using a fed-batch system.

[0256] Quantitative monitoring of RNA during IVT reactions was performed using in-line Raman spectroscopy and PLS modeling as described in Example 3. IVT reactions were performed over a 2-hour period as described in Example 1, but with a different DNA template encoding a ∼2 kb mRNA whose nucleotide sequence contained 609 As, 449 Cs, 522 Gs, and 392 Us. The progress of the IVT reactions, as measured by increasing RNA concentrations, was comparable regardless of RNA batch size (150 mg, 1 g, or 20 g; see Figure 7).

[0257] The scalability of the monitoring method disclosed herein was also observed with another DNA template encoding a ∼2 kb mRNA whose nucleotide sequence contained 554 A, 492 C, 517 G, and 406 U (see Figure 8). Comparable results were obtained between two different batch sizes (1 g and 20 g) when monitoring product RNA and PPi and nucleotide reactants, as indicated by the amount of GTP-ATP. For the 20 g batch, the IVT reaction was performed in a 5000 L reaction vessel.

[0258] Consistent with Example 4, RNA concentrations determined by in-line quantification (Raman) and offline quantification (RiboGreen assay) were comparable. As shown in Figure 9, mRNA increased as the IVT reaction progressed and was halted by the addition of DNase I (thereby digesting the template). After template digestion, proteinase K was added to digest the enzymatic components, including the RNA polymerase and DNase I. After adding DTT to quench the reaction, a drop in the amount of mRNA was observed. This was attributed to dilution of the sample by adding DTT, rather than loss of mRNA product.

[0259] Additionally, the applicability of the modeling system described in Example 3 was tested in IVT reactions with four different DNA templates. The acquired spectra could be fitted within predetermined mathematical model parameters to predict the progress of the IVT reaction.

[0260] Additionally, IVT reactions were also performed in reactors using fed-batch operation, and the monitoring methods described in the previous examples worked equally well under these conditions.

[0261] This example demonstrates that the monitoring methods described in the previous examples can also be used when batch sizes are increased or when IVT reactions are performed using a fed-batch system.

[0262] Example 9. Monitoring of individual NTPs This example illustrates that spectroscopy can be used to monitor individual NTPs.

[0263] Various concentrations of ATP and GTP were tested by spiking them into the reaction buffer to identify the reaction interval. Raman spectra were collected using a Kaiser Raman Rxn2 analyzer (Endress+Hauser). Using this system, ATP and GTP were detected at 1550–1600 cm, respectively, as shown in Figure 10. -1 and 650-750cm -1 It was possible to identify the wavelength range of 650-750cm. -1 Overlaying the spectra acquired for ATP and GTP in this region revealed distinct regions corresponding to each nucleotide, as shown in FIG.

[0264] Example 10. Monitoring the completion of a reaction in a reaction vessel This example demonstrates that the spectroscopic methods described in the previous examples can also be used to monitor the completion of an IVT reaction.

[0265] The feasibility of using spectroscopy to monitor the completion of an IVT reaction was investigated by spiking various concentrations of template (DNA plasmid), nuclease (DNase I), or RNA polymerase (SP6 RNA polymerase) into the reaction buffer to identify suitable wavelengths for monitoring these components.

[0266] Figure 11 shows illustrative spectra acquired when DNA plasmids were tested at concentrations of 0 mg / mL, 0.075 mg / mL, 0.15 mg / mL, 0.3 mg / mL, and 0.6 mg / mL. The absence of RNA polymerase in the reaction buffer prevented the IVT reaction from proceeding. The preferred wavelength region for monitoring DNA plasmids is 505 cm. -1 ~710cm -1 Another wavelength region suitable for monitoring the amount of DNA plasmids is 1325 cm -1 ~1365cm -1 and 1585 cm -1 ~1720cm -1 Monitoring changes in DNA plasmid concentration can be used to monitor the completion of the IVT reaction, for example, by adding a nuclease such as DNase I.

[0267] Figure 12 shows illustrative spectra acquired when testing DNase I at concentrations of 0 ku / mL, 0.063 ku / mL, 0.125 ku / mL, 0.25 ku / mL, and 0.5 ku / mL. The reaction buffer did not contain DNA template. A suitable wavelength region for monitoring nucleases such as DNase I is 450 cm. -1 ~520cm -1 Other wavelength regions suitable for monitoring the amount of nucleases include 1000 cm -1 ~1090cm -1 and 2915 cm -1 ~3000cm -1 The spectra obtained from monitoring DNase I can be used to determine whether it has been added, but also to detect its removal by digestion with a protease, for example, proteinase K.

[0268] Figure 13 shows illustrative spectra acquired when SP6 RNA polymerase concentrations of 0 mg / mL, 0.045 mg / mL, 0.09 mg / mL, 0.18 mg / mL, and 0.36 mg / mL were tested. The inclusion of DNA template in the reaction buffer prevented the IVT reaction from proceeding. The preferred wavelength region for monitoring SP6 RNA polymerase is 780 cm. -1 ~1200cm -1 Another wavelength region suitable for monitoring the amount of RNA polymerase was identified as 1430 cm -1 ~1510cm -1 Monitoring the changes in SP6 RNA polymerase allows the user to detect its removal during termination of the IVT reaction, for example by digestion with a protease such as proteinase K.

[0269] Similar spiking experiments were also performed to identify the wavelength region for detecting proteinase K, which is added to digest RNA polymerase to terminate the IVT reaction or DNase I to terminate the DNase I reaction. In some embodiments, the spectrum acquired in step (c) is used to monitor the amount or addition of proteases. A suitable wavelength region for monitoring proteinase K is 505 cm. -1 ~610cm -1 , for example 550cm -1 ~600cm -1 Other wavelength regions suitable for monitoring the amount of proteases include 715 cm -1 ~775cm -1 and 1385 cm -1 ~1395cm -1 It included.

[0270] This example demonstrates that the spectroscopic methods used to monitor the IVT reaction can also be used to monitor the removal of the DNA plasmid and enzyme components used to terminate the reaction.

Claims

1. 1. A method for monitoring an in vitro transcription (IVT) reaction for the production of RNA in a reaction vessel, comprising: (i) acquiring a spectrum of a reactant or product during the IVT reaction; (ii) comparing the spectrum obtained in step (i) with predetermined reference spectra of the reactants or products of the IVT reaction; wherein a difference between the spectrum acquired in step (i) and the predetermined reference spectrum indicates a change in the amount of the reactant or product.

2. The reactants or products include RNA, pyrophosphate (PPi), H ＋ , inorganic phosphate (Pi) and ribonucleotides (NTP).

3. 3. The method of claim 2, wherein the product is RNA or Pi, and optionally the RNA is mRNA.

4. 3. The method of claim 2, wherein the reactant is one or more ribonucleotides (NTPs).

5. The method of any one of claims 1 to 4, wherein the change in amount of two or more reactants or two or more products of the IVT reaction is determined.

6. 6. The method of claim 5, wherein the changes in the amounts of two or more reactants and two or more products of the IVT reaction are determined.

7. 7. The method of claim 5 or 6, wherein the two or more reactants are adenosine triphosphate (ATP) and guanosine triphosphate (GTP).

8. 7. The method of claim 5 or 6, wherein the two or more reactants are cytidine triphosphate (CTP) and uridine triphosphate (UTP).

9. The method of any one of claims 5 to 8, wherein the two or more products are RNA and Pi.

10. The method according to any one of claims 1 to 9, wherein the reaction vessel is a bioreactor.

11. The method of any one of claims 1 to 10, wherein the reaction vessel has an access port or bypass for inserting a spectroscopic probe.

12. 12. The method of claim 11, wherein the spectroscopic probe is (a) immersed in a solution in which the IVT reaction occurs, or (b) not immersed in the solution in which the IVT reaction occurs, and optionally the spectroscopic probe is separated from the solution by a barrier that does not interfere with detection.

13. 13. The method of any one of claims 1 to 12, wherein step (i) comprises acquiring a series of spectra over the course of the IVT reaction.

14. The method of claim 13, wherein each spectrum in the sequence is acquired over a period of 10 to 60 seconds.

15. 15. The method of claim 13 or 14, wherein the series of spectra comprises a set of at least 3, at least 5 or at least 9 spectra.

16. 16. The method of any one of claims 13 to 15, wherein a series of multiple spectra are acquired over the course of the IVT reaction.

17. A method according to any one of claims 13 to 16, wherein each of the spectra in the series is pre-processed before step (ii).

18. 18. The method of claim 17, wherein the spectra in the sequence are acquired successively, and preprocessing comprises smoothing the spectra by applying a digital filter that fits a low-order polynomial to the successively acquired spectra by linear least squares.

19. 19. The method of any one of claims 1 to 18, wherein the spectrum or series of spectra obtained in step (i) is normalized to a wavelength region in which background noise is reduced or absent prior to step (ii).

20. 19. The method of claim 17 or 18, wherein the series of spectra is normalized to a wavelength region in which background noise is reduced or absent before any pre-processing steps.

21. 18. The method of claim 17, wherein step (ii) comprises a qualitative spectral comparison between the series of spectra obtained in step (i) and the predetermined reference spectrum.

22. 22. The method of claim 21, wherein the qualitative spectral comparison comprises calculating a weighted spectral difference (WSD) value.

23. 23. The method of claim 22, wherein a WSD value of one standard deviation or less indicates no significant difference between the spectrum obtained in step (i) and the predetermined reference spectrum.

24. 23. The method of claim 22, wherein a WSD value greater than one standard deviation indicates a significant difference between the spectrum obtained in step (i) and the predetermined reference spectrum.

25. 25. The method of any one of claims 1 to 24, wherein the predetermined reference spectrum correlates with a specified concentration of the reactant or product.

26. 26. The method of claim 25, wherein step (ii) further comprises determining the concentration of the reactant or product.

27. 27. The method of claim 26, wherein determining the reactant or product concentration comprises linear regression analysis.

28. 28. The method of claim 26 or 27, wherein a partial least squares (PLS) model is used to determine the concentration of the reactant or product from a series of predetermined reference spectra, each spectrum in the series correlating with a different concentration of the reactant or product.

29. 29. The method of any one of claims 1 to 28, wherein the spectrum, sequence of spectra and the predetermined reference spectrum / sequence of predetermined reference spectra are obtained using a spectrometer for vibrational spectroscopy, if applicable.

30. 30. The method of claim 29, wherein the spectrometer is a Raman spectrometer.

31. 30. The method of claim 29, wherein the spectrometer is an infrared (IR) spectrometer.

32. 32. The method of any one of claims 1 to 31, wherein the spectrum acquired in step (i) spans a wavelength range suitable for monitoring the overall evolution of multiple reactants and products during the IVT reaction.

33. 33. The method of claim 32, wherein the plurality of reactants and products comprises RNA, inorganic phosphate (Pi), and one or more ribonucleotides (NTP).

34. The wavelength range is 300 cm －１ ~3000cm －１ and the spectrum is obtained using a Raman spectrometer.

35. 32. The method of any one of claims 1 to 31, wherein the spectrum acquired in step (i) spans a wavelength region specific to one product or reactant of the IVT reaction.

36. 36. The method of claim 35, wherein the product is RNA, and optionally the product is mRNA.

37. The wavelength range is 801 cm －１ ~831cm －１ and the spectrum is obtained using a Raman spectrometer.

38. 36. The method of claim 35, wherein the product is Pi.

39. The wavelength range is 875 cm －１ ~900cm －１ and the spectrum is obtained using a Raman spectrometer.

40. 36. The method of claim 35, wherein the reactant is one or more ribonucleotides (NTPs).

41. The wavelength range is 600 cm －１ ~1,300cm －１ and the spectrum is obtained using a Raman spectrometer.

42. The wavelength range is 1107 cm to determine the amount of NTP. －１ ~1146cm －１ or 1113 cm －１ ~1115cm －１ 42. The method of claim 40 or 41, comprising or consisting of:

43. The wavelength range is 633 cm to determine the amount of ATP. －１ 43. The method of any one of claims 40 to 42, comprising or consisting of:

44. The wavelength range is 1300 cm to determine the amount of ATP and GTP. －１ ~1600cm －１ 43. The method of any one of claims 40 to 42, comprising or consisting of:

45. The wavelength range is 780 cm to determine the amount of CTP. －１ 43. The method of any one of claims 40 to 42, comprising or consisting of:

46. The wavelength range is 786 cm to determine the amount of UTP. －１ ~789cm －１ 43. The method of any one of claims 40 to 42, comprising or consisting of:

47. The wavelength range is 1230 cm to determine the amount of CTP and UTP. －１ ~1245cm －１ 43. The method of any one of claims 40 to 42, comprising or consisting of:

48. 29. The method of any one of claims 1 to 28, wherein the spectrum acquired in step (i) detects the turbidity of the solution in which the IVT reaction occurs.

49. 49. The method of claim 48, wherein the turbidity indicates an accumulation of insoluble precipitates.

50. The insoluble precipitate is Mg ２ 50. The method of claim 49, wherein the compound is PPi.

51. 51. The method of any one of claims 48 to 50, wherein the turbidity is measured using a turbidimeter, an ultraviolet spectrometer or a nephelometer.

52. 52. The method of claim 51, wherein the turbidity is measured using an ultraviolet spectrometer at a wavelength in the range of 290 nm to 410 nm, such as 310 nm, 320 nm, 330 nm, 340 nm or 350 nm.

53. 1. A method for producing RNA using an in vitro transcription (IVT) reaction, comprising: a) providing a DNA template comprising the nucleotide sequence of said RNA operably linked to an RNA polymerase promoter; b) adding the DNA template to a reaction vessel containing an RNA polymerase and reactants necessary to initiate the IVT reaction; c) monitoring one or more of the reactants or products of the IVT reaction using the method of any one of claims 1 to 52; A method comprising:

54. 54. The method of claim 53, wherein step (c) further comprises acquiring a series of spectra over a wavelength range suitable for monitoring one or more enzymatic components, such as the DNA template and / or the RNA polymerase, during and optionally after completion of the IVT reaction.

55. 55. The method of claim 53 or 54, wherein the one or more products comprise RNA, and optionally the one or more products comprise mRNA.

56. 56. The method of claim 55, wherein step (i) comprises obtaining a series of spectra at one or more designated time points during the IVT reaction, and step (ii) comprises determining the amount or change in amount of RNA between the one or more designated time points by comparing the series of spectra with the predetermined reference spectrum.

57. 57. The method of claim 56, wherein the IVT reaction is terminated if the RNA does not reach a target amount at the one or more designated time points.

58. 58. The method of claim 57, wherein the batch of RNA resulting from the IVT reaction is discarded.

59. 57. The method of claim 56, wherein the IVT reaction is terminated if the change in amount of RNA is less than a predetermined value at two or more designated time points.

60. 60. The method of claim 59, wherein the batch of RNA resulting from the IVT reaction is discarded.

61. 61. The method of claim 59 or 60, wherein the two or more designated time points are equally spaced throughout the IVT response.

62. 62. The method of claim 61, wherein each interval is 10 minutes or less, 5 minutes or less, 1 minute or less, or 30 seconds or less.

63. 57. The method of claim 56, wherein the IVT reaction is terminated when the change in the amount of RNA is about zero between at least two or more time points.

64. 64. The method of claim 63, wherein the at least two or more time points are at least 5 minutes apart.

65. 55. The method of claim 53 or 54, wherein the IVT reaction is terminated if the spectrum of the IVT reaction acquired in step (i) deviates by more than one standard deviation from either (A) the predetermined reference spectrum of the reactant or product, or (B) a predetermined kinetic model for an IVT reaction using similar or identical conditions and reactants.

66. 55. The method of claim 53 or 54, wherein a batch of RNA resulting from the IVT reaction is discarded if the spectrum of the IVT reaction obtained in step (i) deviates by more than one standard deviation from either (A) the predetermined reference spectrum of the reactants or products, or (B) a predetermined kinetic model for an IVT reaction using similar or identical conditions and reactants.

67. 67. The method of any one of claims 54 to 66, wherein the spectrum acquired in step (c) is used to monitor the amount of DNA template.

68. The wavelength range obtained in step (c) is determined to be 500 cm －１ ~710cm －１ 68. The method of claim 67, comprising or consisting of:

69. 69. The method of any one of claims 54 to 68, wherein the spectrum acquired in step (c) is used to monitor the amount of RNA polymerase, and optionally the RNA polymerase is SP6 RNA polymerase.

70. The wavelength region obtained in step (c) is used to determine the amount of RNA polymerase. －１ ~1200cm －１ or 1430 cm －１ ~1510cm －１ 70. The method of claim 69, comprising or consisting of:

71. 71. The method of any one of claims 54 to 70, comprising adding a nuclease (e.g., DNase I) to terminate the IVT reaction.

72. 72. The method of claim 71, wherein the spectrum acquired in step (c) is used to monitor the amount or addition of the nuclease.

73. The wavelength region obtained in step (c) may be selected from the group consisting of 450 cm and 450 cm to determine the amount or addition of the nuclease. －１ ~520cm －１ 73. The method of claim 72, comprising or consisting of:

74. 74. The method of any one of claims 54 to 73, comprising adding a protease (e.g., proteinase K) to terminate the IVT reaction or nuclease activity.

75. 75. The method of claim 74, wherein the spectrum acquired in step (c) is used to monitor the amount or addition of the protease.

76. The wavelength region obtained in step (c) may be selected from the group consisting of 505 cm, 506 cm, 508 cm, 509 cm, 510 cm, 511 cm, 512 cm, 513 cm, 514 cm, 515 cm, 516 cm, 517 cm, 518 cm, 519 cm, 519 cm, 520 cm, 521 cm, 522 cm, 5 －１ ~610cm －１ , optionally 550 cm －１ ~600cm －１ 76. The method of claim 75, comprising or consisting of:

77. 1. A method for producing RNA using an in vitro transcription (IVT) reaction, comprising: a) producing said RNA in a reaction vessel, (i) acquiring a spectrum of the RNA in the IVT reaction to determine a first value; (ii) comparing said first value obtained in step (i) with a second value derived from a predetermined reference spectrum of RNA; and monitoring by b) purifying the RNA if the first value is greater than or equal to the second value; A method comprising:

78. 78. The method of claim 77, wherein the second value corresponds to a target concentration.

79. 79. The method of claim 78, wherein the target concentration is at least 3 g / L.

80. 78. The method of claim 77, wherein steps (i) and (ii) are repeated at equal intervals, and the RNA is purified if the first value obtained at each interval is greater than or equal to the second value at the corresponding interval derived from the predetermined reference spectrum.

81. A method according to any one of claims 77 to 80, wherein the first and second values ​​are considered to be equal if they are within one standard deviation of each other.

82. 82. The method of any one of claims 53 to 81, wherein the reaction vessel is a bioreactor.

83. A method according to any one of claims 53 to 82, wherein the reaction vessel has an access port or bypass for inserting a spectroscopic probe.

84. 84. The method of claim 83, wherein the spectroscopic probe is (a) immersed in a solution in which the IVT reaction occurs, or (b) not immersed in the solution in which the IVT reaction occurs, and optionally the spectroscopic probe is separated from the solution by a barrier that does not interfere with detection.

85. 85. The method of any one of claims 53 to 84, wherein the reaction vessel has an access port for adding reactants during the IVT reaction.

86. The reactant in the IVT reaction is magnesium (Mg ２＋ 86. The method of any one of claims 53 to 85, comprising:

87. 87. The method of claim 86, wherein the IVT reaction is supplemented with an NTP at least once during the process of producing the RNA.

88. 88. The method of claim 87, wherein the IVT reaction is periodically replenished with NTPs during the process of producing the RNA.

89. 89. The method of claim 87 or 88, wherein the IVT reaction is replenished with an NTP when the concentration of the NTP is approaching depletion.

90. 90. The method of claim 89, wherein depletion is approached when the NTP is at a concentration of 5% or less of the concentration of NTP that was present when the IVT reaction was initiated.

91. 90. The method of claim 89, wherein depletion is approached when the NTP is at a concentration of 5 mM or less.

92. 92. The method of claim 91, wherein depletion is approached when the NTP is at a concentration of 3 mM or less.

93. 87. The method of claim 86, wherein the IVT reaction is continuously replenished with NTPs during the process of producing the RNA.

94. 94. The method of any one of claims 86-93, wherein each NTP is present at a concentration of 1-10 mM, 1-6 mM, 2-6 mM, or 3-6 mM when the IVT reaction is initiated.

95. 95. The method of any one of claims 86-94, wherein the IVT reaction is supplemented with an NTP to maintain or return the concentration to the concentration of NTP that was present when the IVT reaction was initiated.

96. 95. The method of any one of claims 86-94, wherein the IVT reaction is supplemented with NTPs to maintain the concentration of each NTP within the range of 20% to 100%, 20% to 75%, or 25% to 50% of that present when the IVT reaction was initiated.

97. 95. The method of any one of claims 86-94, wherein the total NTP concentration in the IVT reaction is maintained above a lower limit of 2 mM.

98. 98. The method of claim 97, wherein the total NTP concentration in the IVT reaction is maintained between 10 mM and 20 mM.

99. 1. A method for producing RNA using an in vitro transcription (IVT) reaction, comprising: a) providing a DNA template comprising the nucleotide sequence of said RNA operably linked to an RNA polymerase promoter; b) adding the DNA template to a reaction vessel containing an RNA polymerase and reactants necessary to initiate the IVT reaction; c) monitoring the IVT reaction by acquiring a series of spectra, the spectra spanning a wavelength range suitable for monitoring the DNA template and / or the RNA polymerase during the IVT reaction; A method comprising:

100. 100. The method of claim 99, wherein the spectrum acquired in step (c) is used to monitor the amount of DNA template.

101. The wavelength range obtained in step (c) is determined to be 500 cm －１ ~710cm －１ 101. The method of claim 100, comprising or consisting of:

102. 102. The method of any one of claims 99 to 101, wherein the spectrum acquired in step (c) is used to monitor the amount of RNA polymerase, and optionally, the RNA polymerase is SP6 RNA polymerase.

103. The wavelength region obtained in step (c) is used to determine the amount of RNA polymerase. －１ ~1200cm －１ or 1430 cm －１ ~1510cm －１ 103. The method of claim 102, comprising or consisting of:

104. 104. The method of any one of claims 99 to 103, comprising adding a nuclease (e.g., DNase I) to terminate the IVT reaction.

105. 105. The method of claim 104, wherein the spectrum acquired in step (c) is used to monitor the amount or addition of the nuclease.

106. The wavelength region obtained in step (c) may be selected from the group consisting of 450 cm and 450 cm to determine the amount or addition of the nuclease. －１ ~520cm －１ 106. The method of claim 105, comprising or consisting of:

107. 107. The method of any one of claims 99 to 106, comprising adding a protease (e.g., proteinase K) to terminate the IVT reaction.

108. 108. The method of claim 107, wherein the spectrum acquired in step (c) is used to monitor the amount or addition of the protease.

109. The wavelength region obtained in step (c) may be selected from the group consisting of 505 cm, 506 cm, 508 cm, 509 cm, 510 cm, 511 cm, 512 cm, 513 cm, 514 cm, 515 cm, 516 cm, 517 cm, 518 cm, 519 cm, 519 cm, 520 cm, 521 cm, 522 cm, 5 －１ ~610cm －１ , optionally 550 cm －１ ~600cm －１ 109. The method of claim 108, comprising or consisting of:

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