Spectral monitoring of in vitro transcription

Abstract

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

Description

Technical Field

[0001] The present invention relates to a method of monitoring an in vitro transcription (IVT) reaction for producing RNA (e.g., mRNA) by obtaining a spectrum of a reactant or product during the IVT reaction. The spectrum can then be compared to a predetermined reference spectrum of the reactant or product of the IVT reaction. A change in the amount of the reactant or product is represented by the difference between the obtained spectrum and the predetermined reference spectrum. Background Art

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

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

[0004] Furthermore, current methods for monitoring IVT reactions typically require removing an aliquot of the reaction mixture from the reaction vessel and processing it before the RNA yield can be determined. Typically, such methods are limited to determining the amount of a single product or reactant of the IVT reaction (e.g., the amount of RNA). Furthermore, the presence of enzyme components can interfere with the accurate determination of the amount of RNA in the reaction vessel.

[0005] Therefore, there is a need for a method that can monitor the amount of reactants or products of an IVT reaction within a reaction vessel without the need to obtain aliquots or remove enzyme components. Ideally, such a method can monitor multiple reactants and products of an IVT reaction simultaneously and can be used for process optimization and process control during large-scale RNA production. Summary of the invention

[0006] The present invention particularly relates to a method for monitoring an in vitro transcription (IVT) reaction for producing RNA, particularly messenger RNA (mRNA), in a reaction vessel using a spectroscopic probe.

[0007] When incident light (e.g., laser light) strikes a molecule, it is scattered inelastically, resulting in a change in the initial wavelength. The inventors have discovered that when an IVT reaction occurs in a reaction vessel, the resulting shift between the incident and scattered light frequencies can provide a molecular fingerprint of one or more reactants and / or products. The spectrum obtained during the reaction can be automatically converted to the concentrations of the major products and reactants (i.e., RNA, NTP, and H2PO4 - molar concentration). This online approach can increase the throughput of experiments during process development and can provide valuable process and quality data during large-scale manufacturing of RNA-based therapeutics (particularly mRNA-based therapeutics, including mRNA-based vaccines). These data can be used to optimize reaction conditions or determine whether a batch of RNA can proceed to downstream manufacturing steps, thereby reducing the time and cost 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 producing RNA (e.g., mRNA) within a reaction vessel, the method comprising (i) obtaining a spectrum of a reactant or product during the IVT reaction; and (ii) comparing the spectrum obtained in step (i) with a predetermined reference spectrum of the reactant or product of the IVT reaction, wherein the difference between the spectrum obtained in step (i) and the predetermined reference spectrum indicates a change in the amount of the reactant or product.

[0009] In some embodiments, the reactant or product is selected from RNA, pyrophosphate (PPi), H + , inorganic phosphate (Pi) and ribonucleotide (NTP). In some embodiments, the product is RNA or Pi. In some embodiments, the reactant is one or more ribonucleotides (NTP).

[0010] In some embodiments, the method determines a change in the amount of more than one reactant or more than one product of an IVT reaction. In some embodiments, the method determines a change in the amount of more than one reactant and more than one product of an IVT reaction. In some embodiments, the more than one reactant is adenosine triphosphate (ATP) and guanosine triphosphate (GTP). In some embodiments, the more than one reactant is cytidine triphosphate (CTP) and uridine triphosphate (UTP). In some embodiments, the more than one product is 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 obtaining a series of spectra during an IVT reaction process. In some embodiments, each spectrum in the series is obtained within a time period of 10-60 seconds. In some embodiments, the series of spectra comprises a group of at least 3, at least 5, or at least 9 spectra. In some embodiments, multiple series of spectra are obtained during an IVT reaction process.

[0013] In some embodiments, each spectrum in the series is preprocessed prior to step (ii). In some embodiments, the spectra in the series are acquired continuously, and the preprocessing comprises smoothing the spectra by applying a digital filter that fits the continuously acquired spectra with a low-order polynomial by a linear least squares method.

[0014] In some embodiments, prior to step (ii), the spectrum or spectral series obtained in step (i) is normalized relative to a wavelength region with reduced or no background noise. In some embodiments, prior to any pre-processing step, the spectral series is normalized relative to a wavelength region with reduced or no background noise.

[0015] In some embodiments, step (ii) comprises performing a qualitative spectral comparison of the series of spectra obtained in step (i) with 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 that there is no significant difference between the spectrum obtained in step (i) and the predetermined reference spectrum. In some embodiments, a WSD value of more than one standard deviation indicates that there is a significant difference between the spectrum obtained in step (i) and the predetermined reference spectrum.

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

[0017] In some embodiments, the spectrum, series of spectra, and predetermined reference spectrum / predetermined reference spectrum series (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 nephelometer. In a specific embodiment, the spectrometer is a Raman spectrometer.

[0018] In some embodiments, the spectrum obtained in step (i) spans a wavelength region 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 region includes 300 cm -1 Up to 3000cm -1 , and the spectra were obtained using a Raman spectrometer.

[0019] In some embodiments, the spectrum obtained 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 includes 801 cm -1 Up to 831cm -1 , and the spectra were obtained using a Raman spectrometer.

[0021] In some embodiments, the product is Pi. In some embodiments, the wavelength region includes 875 cm -1 Up to 900cm -1 , and the spectra were obtained using a Raman spectrometer.

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

[0023] In some embodiments, the spectrum obtained in step (i) detects the turbidity of the solution in which the IVT reaction occurs. In some embodiments, the turbidity indicates the accumulation of insoluble precipitates. In some embodiments, the insoluble precipitate is Mg2PPi. In some embodiments, the turbidity is measured using a turbidimeter, a UV spectrometer, or a nephelometer. In some embodiments, the turbidity is measured using a UV spectrometer at a wavelength in the region of 290nm to 410nm (e.g., 300nm to 350nm), for example, at 310nm, 320nm, 330nm, 340nm, or 350nm.

[0024] In some aspects, the present invention also relates to a method for making RNA, such as mRNA, using an in vitro transcription (IVT) reaction, the method comprising: (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 comprising RNA polymerase and necessary reactants to initiate the IVT reaction; and (c) monitoring one or more of the reactants or products of the IVT reaction using a method of the present invention as listed above.

[0025] In some embodiments, step (c) comprises obtaining a series of spectra, wherein the spectra span a wavelength region suitable for monitoring the DNA template and / or one or more enzyme components (eg, RNA polymerase) during (or after completion of) the IVT reaction.

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

[0027] In some embodiments, the spectrum obtained 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 obtained in step (c) comprises or consists of: 780 cm -1 Up to 1200cm -1 and / or 1430cm -1 Up to 1510cm -1 to determine the amount of SP6 RNA polymerase to be added.

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

[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 a nuclease. In some embodiments, the spectrum obtained in step (c) is used to monitor the amount or addition of the protease. In some embodiments, the wavelength region obtained in step (c) comprises or consists of: 505 cm -1 Up to 610cm -1 (For example, 550cm -1 Up to 600cm -1 )、715cm -1 Up to 775cm -1 and / or 1385cm -1 Up to 1395cm -1 , to determine the amount or addition of protease.

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

[0031] In some embodiments, if the RNA does not reach the target amount at one or more specified time points, the IVT reaction is terminated. In some embodiments, the RNA batch produced by the IVT reaction is discarded.

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

[0033] In some embodiments, the two or more designated time points are equally spaced apart throughout the IVT reaction, In 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 if the change in the amount of RNA between at least two or more time points is about zero. In some embodiments, 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 (A) a predetermined reference spectrum of the reactants or products, or (B) a kinetic model previously determined for an IVT reaction using similar or identical conditions and reactants.

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

[0037] In some aspects, the present invention also relates to a method for making 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) obtaining a spectrum of the RNA during the IVT reaction to determine a first value; and (ii) comparing the first value obtained in step (i) with a second value derived from a predetermined reference spectrum of the RNA; and (b) purifying the RNA if the first value is equal to or exceeds the second value.

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

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

[0040] In some embodiments, a first value and a second value 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, the reactants in the IVT reaction include magnesium (Mg 2+ ) and NTP. In some embodiments, the IVT reaction is replenished with NTP at least once during the process of making the RNA. In some embodiments, the IVT reaction is replenished with NTP periodically during the process of making the RNA.

[0043] In some embodiments, the IVT reaction replenishes NTPs when the NTP concentration approaches depletion. In some embodiments, depletion is approached when the NTP concentration is no more than 5% of the NTP concentration present when the IVT reaction is initiated. In some embodiments, depletion is approached when the NTP concentration is no more than 5 mM. In some embodiments, depletion is approached when the NTP concentration is no more than 3 mM.

[0044] In some embodiments, the IVT reaction is continuously replenished with NTPs during the RNA manufacturing process.

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

[0046] In some embodiments, the total NTP concentration in the IVT reaction is maintained above a lower limit of 2 mM. In 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 a method for making RNA using an in vitro transcription (IVT) reaction, the method comprising (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 comprising RNA polymerase and necessary reactants to initiate the IVT reaction; and (c) monitoring the IVT reaction by obtaining a series of spectra, wherein the spectra span a wavelength region suitable for monitoring the DNA template and / or the RNA polymerase during the IVT reaction.

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

[0049] In some embodiments, the spectrum obtained 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 obtained in step (c) comprises or consists of: 780 cm -1 Up to 1200cm -1 and / or 1430cm -1 Up to 1510cm -1 to determine the amount of SP6 RNA polymerase to be added.

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

[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 obtained in step (c) is used to monitor the amount or addition of the protease. In some embodiments, the wavelength region obtained in step (c) comprises or consists of: 505 cm -1 Up to 610cm -1 (For example, 550cm -1 Up to 600cm -1 )、715cm -1 Up to 775cm -1 and / or 1385cm -1 Up to 1395cm -1 , to determine the amount or addition of protease. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The embodiments of the present invention will be described below with reference to the accompanying drawings, in which:

[0053] Figure 1 During the IVT reaction, the -1 Illustration of the spectral properties of a Raman spectrum in a selected wavelength region. The dotted areas below the spectrum, outlined by dashed lines, represent areas where RNA, NTPs (including CTP and UTP), or the reaction byproduct H2PO4 can be detected. - The wavelength region where the amount of Pi changes is shown in the figure. The peaks are labeled to indicate the representative wavelengths for detecting these reactants and products by Raman spectroscopy. The dashed line represents the first spectrum recorded 8 minutes after the start of the IVT reaction. The dark gray solid line is the last spectrum of the IVT reaction recorded after 90 minutes. The light gray line represents the intermediate spectrum recorded during the IVT reaction.

[0054] Figure 2 The evolution of the weighted spectral difference (WSD) values ​​calculated for the following spectral series obtained using a Raman spectrometer is shown: (a) 801-831 cm -1 Wavelength region (RNA) and (b) 1107-1146 cm -1 wavelength region (NTP). Spectra were obtained during IVT reactions performed at 37°C. Results obtained at 2 mL scale are shown as solid lines (mean of n=3) ± one standard deviation (shown as dashed lines). Results obtained at 250 mL scale ( The results of an illustrative experiment conducted with 250 cells (n=1) are shown as a solid line with open circles.

[0055] Figure 3Shown are RNA concentrations during the IVT reaction, determined online by a univariate PLS model based on Raman spectroscopy data obtained from within the reaction vessel (dotted line) or offline by RiboGreen assay of aliquots removed from the reaction vessel (dashed line).

[0056] Figure 4 Shown are the real-time concentrations of purine-based NTPs (ATP-GTP), pyrimidine-based NTPs (CTP-UTP), and inorganic phosphate (Pi), as determined by a univariate PLS model based on a series of Raman spectra acquired during a representative IVT reaction.

[0057] Figure 5 Shown are the concentration profiles of reactants (ATP-GTP, CTP-UTP) and products (RNA, Pi, and PPi) of the IVT reaction based on a series of Raman spectra acquired during a representative IVT reaction (filled circles), with superimposed kinetic model predictions (solid lines). RNA concentrations were determined by Raman spectroscopy (open circles) and RiboGreen assay (open triangles).

[0058] Figure 6 Predictions for turbidity progression during IVT reactions conducted at 31°C, 37°C, and 42°C are shown. In panel (a), turbidity predictions are shown as lines up to 5 hours at 31°C, 37°C, and 42°C, with prediction bands representing 95% prediction intervals represented by dashed lines. Turbidity data (absorbance: 320 nm) used for kinetic modeling are shown as solid circles. Additional experimental data obtained after 1.5 hours are shown as open circles and were not used for kinetic modeling. Panel (b) shows a time-temperature-conversion (TTT) diagram covering a range of 1%-99% isoconversion. The gray filled area shows the time-temperature domain, which prevents significant turbidity from appearing during the IVT reaction.

[0059] Figure 7 RNA concentrations obtained during three different batch size IVT reactions are shown. Specifically, the IVT reactions produced 150 mg, 1 g, or 20 g of mRNA, represented by open circles, closed squares, and open triangles, respectively. Concentrations were determined by a univariate PLS model based on Raman spectroscopy, as described in Example 3. As shown, the IVT reactions were monitored for 2 hours.

[0060] Figure 8 Shown are the concentrations of the products RNA and PPi and two of the nucleotide reactants (GTP and ATP) during IVT reactions performed at different batch sizes. Figure 8Figures (a)-(c) 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). As shown, the reaction was monitored for about 4 hours. The IVT reactions were performed with mRNA batch sizes of 1 g or 20 g, as represented by open circles or filled squares, respectively. The numbered lines indicate the reaction stages. 1 is the IVT reaction stage, 2 is the termination stage (started by adding DNase I, followed by proteinase K), and 3 is the quenching stage (started by adding DTT).

[0061] Fig. 9 The RNA concentrations obtained during an IVT reaction to produce 1 g of mRNA, measured online by Raman spectroscopy or offline by RiboGreen assay (depicted by solid circles or open squares, respectively), are shown. The numbered lines indicate the reaction phases. 1 is the IVT reaction phase, 2 is the termination phase (initiated by addition of DNase I followed by proteinase K), and 3 is the quenching phase (initiated by addition of DTT). As shown, the reaction was monitored for approximately 4 hours.

[0062] Fig.10 is a graphic representation of the spectral properties of ATP and GTP obtained from a Kaiser Raman spectrometer. Fig.10 In panels (a) and (b) of Figure 1, four different concentrations of ATP and GTP were tested in the spike experiment to identify the wavelength region in which ATP and GTP can be identified. Panels (a) and (b) represent the wavelength region for 1550 cm -1 Up to 1600cm -1 and 1550cm -1 to about 1605cm -1 The spectrum obtained in the wavelength region. Fig.10 In the sub-figures (c) and (d), the regions where ATP and GTP have been identified are superimposed. -1 Up to 1590cm -1 The spectra obtained in the wavelength region of about 640 cm -1 To about 775cm -1 The spectrum obtained in the wavelength region.

[0063] Fig.11 is a graphic representation of the spectral properties of plasmid DNA determined by Raman spectroscopy. Fig.11 As shown in the sub-figure (a), the span is 400cm -1 Up to 3000cm -1The wavelength regions of the IVT reaction mixture 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. Regions A to C are boxed and shown in Fig.11 Figure (b)-(d) shows the 505cm -1 Up to 705cm -1 The spectrum obtained in the wavelength region of 1325 cm -1 Up to 1370cm -1 The spectrum obtained in the wavelength region of 1585cm -1 Up to 1720cm -1 The spectrum obtained in the wavelength region.

[0064] Fig.12 is a graphic representation of the spectral properties of DNase I determined by Raman spectroscopy. Fig.12 As shown in the sub-figure (a), the span is 400cm -1 Up to 3000cm -1 The wavelength regions of the IVT reaction mixture 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. Regions A to C are boxed and shown in Fig.12 Figure (b)-(d) shows the -1 Up to 515cm -1 Spectra obtained in the wavelength region. Sub-figure (c) shows the spectrum at about 1005 cm -1 To about 1085cm -1 The spectrum obtained in the wavelength region of 2915 cm -1 Up to 3000cm -1 The spectrum obtained in the wavelength region.

[0065] Fig.13 is a graphic representation of the spectral properties of SP6 RNA polymerase determined by Raman spectroscopy. Fig.11 As shown in the sub-figure (a), the span is 350cm -1 Up to 1700cm -1 The wavelength regions of the IVT reaction mixture 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 were monitored. Regions A and B are boxed and shown in Fig.13 Figure (b) and (c) show that the -1Up to 1140cm -1 The spectrum obtained in the wavelength region of 1430cm -1 Up to 1500cm -1 The spectrum obtained in the wavelength region. definition

[0066] In order to make the present invention more easily understood, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification.

[0067] Unless the context clearly dictates otherwise, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, and plural terms include singular referents. For example, "spectrum" is understood to mean one or more spectra. Thus, the terms "a or an", "one or more", and "at least one" may be used interchangeably herein.

[0068] Unless otherwise specified or obvious from the context, as used herein, the term "or" is to be understood as inclusive and encompasses "or" and "and". In addition, when used herein, "and / or" is considered to be a specific disclosure of the presence or absence of each of the two specified features or components. Therefore, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following aspects: 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).

[0069] It should be understood that wherever aspects are described herein with the language "comprising," similar aspects described in the form of "consisting of" and / or "consisting essentially of" are also provided.

[0070] As used herein, the term "about" refers to an accuracy interval that will be understood by those skilled in the art to still ensure the technical effect of the feature in question. The term indicates a deviation of ±10% from the value shown. In some embodiments, the deviation is ±5% of the value shown. In some embodiments, the deviation is ±1% of the value shown.

[0071] The term "reaction vessel" refers to any container suitable for conducting an IVT reaction. The reaction vessel contains the reactants, enzyme components, and any additional components (e.g., buffer reagents, etc.) required to conduct the IVT reaction. The volume of the reaction vessel and / or its configuration can depend on the scale of the IVT reaction and / or the application for preparing RNA. Suitable reaction vessels can be made of glass, plastic, or stainless steel. In some embodiments, the reaction vessel can be sterilized and sealed to avoid contamination (e.g., a disposable sterilizable and sealable plastic bag).

[0072] As used herein, the term "bioreactor" refers to a reaction vessel that may include means for heating and / or providing agitation for the reaction mixture or is adapted to be operably connected to such means. Typically, the bioreactor also includes ports for adding reactants and / or for inserting probes. In some embodiments, the bioreactor may include a bypass for inserting a spectroscopic probe.

[0073] 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 having a length of at least 100 ribonucleotides (e.g., at least 200 ribonucleotides or at least 400 ribonucleotides). In addition to messenger RNA (mRNA), this may also include ribosomal RNA, ribozymes, riboswitches and / or other long non-coding RNAs (lncRNAs), e.g., Kcnq1ot1, Xlsirt, Xist and HOTAIR.

[0074] As used herein, the term "mRNA" refers to a polyribonucleotide encoding at least one polypeptide. mRNA may contain one or more coding regions and non-coding regions (e.g., 5' untranslated region and 3' untranslated region). mRNA as used herein encompasses both modified RNA and unmodified RNA. For example, mRNA may include one or more nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. Unless otherwise indicated, the mRNA sequence is presented in a 5' to 3' direction. 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 certain embodiments, the tail structure is a poly (C) tail. More typically, the tail structure is a poly (A) tail.

[0075] As used herein, the term "sequence optimized" is used to describe a nucleotide sequence modified relative to a naturally occurring or wild-type nucleic acid. Such modifications may include, for example, codon optimization and / or the use of 5'UTR and 3'UTR that are not usually associated with a naturally occurring or wild-type nucleic acid. As used herein, the terms "codon optimized" 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, but do not change its amino acid sequence, thereby improving the protein expression of the nucleic acid. In the context of the present invention, "codon optimized" may also refer to the following process: by removing suboptimal nucleotide sequences from a nucleotide sequence list with a filter, for example, filtering by the presence of guanine-cytosine content, codon adaptation index, unstable nucleic acid sequence or motif and / or pause sites and / or terminator signals, thereby obtaining one or more optimized nucleotide sequences.

[0076] As used herein, the term "spectrum" refers to a plurality of detected signals recorded with a spectrometer as light emission or absorption from a sample at more than one time point across one wavelength or at one or more time points across two or more wavelengths. For example, a spectrum can be acquired at a single wavelength, such as over a period of time for the duration of an IVT reaction. In some embodiments, more than one spectrum is acquired at more than one wavelength. In typical embodiments, more than one spectrum is acquired at more than one wavelength at more than one time point during an IVT reaction.

[0077] As used herein, the term "template DNA" (or "DNA template") relates to a DNA molecule comprising 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 an RNA transcript encoded by the template DNA. The template DNA comprises all elements required for IVT, in particular a promoter element for binding a DNA-dependent RNA polymerase (such as, for example, T3, T7 or SP6 RNA polymerase), which is operably linked to a DNA sequence encoding the desired RNA transcript. In addition, the template DNA may contain primer binding sites 5' and / or 3' to the DNA sequence encoding the RNA transcript, in order to determine the identity of the DNA sequence encoding the RNA transcript, for example by PCR or DNA sequencing. The "template DNA" in the context of the present invention 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, which comprises a nucleotide sequence encoding the desired RNA transcript.

[0078] Unless otherwise defined herein, technical terms and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs and commonly used in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, exemplary methods and materials are described below. In the event of a conflict, the present specification, including definitions, shall prevail.

[0079] Generally, nomenclatures used in connection with the 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, and techniques thereof, 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.

[0080] Throughout the present specification and examples, the words “have” and “comprise” or variations such as “has / having”, “comprises / comprising”, will be understood to imply the inclusion of the stated integer or groups of integers but not the exclusion of any other integer or groups of integers.

[0081] All publications and other references cited herein are hereby incorporated by reference in their entirety.Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. DETAILED DESCRIPTION

[0082] The present invention relates to a method for monitoring an in vitro transcription (IVT) reaction for producing RNA, particularly messenger RNA (mRNA), in a reaction vessel, the method comprising (i) obtaining a spectrum of a reactant or product during the IVT reaction; and (ii) comparing the spectrum obtained in step (i) with a predetermined reference spectrum of the reactant or product of the IVT reaction. The difference between the spectrum obtained in step (i) and the predetermined reference spectrum indicates a change in the amount of the reactant or product.

[0083] The methods of the present invention are particularly advantageous because the amount of reactants or products can be monitored within the reaction vessel. Unlike some prior art methods, the methods of the present invention do not require the removal of aliquots or removal of enzyme components from the IVT reaction prior to obtaining the spectrum. In addition, information about the amount of reactants or products in the IVT reaction can be provided very quickly. In some embodiments, the amount of RNA in the reaction vessel can be monitored in essentially real time. Another advantage of the present invention is that it allows for the simultaneous monitoring of multiple reactants and / or products. In some embodiments, the spectrum obtained in step (i) provides information about one or more reactants (e.g., NTPs) as well as the primary product (RNA) and byproducts (e.g., PPi, Pi, and / or H) of the IVT reaction. + ) quantity information. In vitro transcription

[0084] "In vitro transcription" or "IVT" refers to a process in which transcription occurs in vitro (i.e., in an artificial environment such as a reaction vessel rather than in an organism) to produce a synthetic RNA product. An IVT reaction is typically performed in the presence of an RNA polymerase and a template. The template is typically a DNA template, e.g., a linearized plasmid or a circular plasmid. In some embodiments, the template (e.g., a DNA template) is monitored during the IVT reaction. In some embodiments, the amount of the template (e.g., a DNA template) in the reaction vessel is monitored by acquiring a spectrum from which information about the amount of the template can be obtained.

[0085] 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, an RNA polymerase, such as an SP6, T7, or T3 RNA polymerase, is monitored during the IVT reaction. In some embodiments, the amount of RNA polymerase in the reaction vessel is monitored by acquiring a spectrum from which information about the amount of template can be obtained.

[0086] In some embodiments, the DNA template can be optimized to facilitate more efficient transcription and / or downstream translation. For example, the DNA template can be optimized with respect to the following items: cis-regulatory elements (e.g., TATA boxes, termination signals, and protein binding sites), artificial recombination sites, x sites, CpG dinucleotide content, negative CpG islands, GC content, polymerase slippage sites, and / or other elements associated with transcription; the DNA template can be optimized with respect to the following items: cryptic splicing sites, secondary structures of RNA transcripts, stable free energy of RNA transcripts, repetitive sequences, unstable motifs, and / or other elements associated with RNA processing and stability; the DNA template can be optimized with respect to the following items: codon usage bias, codon adaptability, internal x sites, ribosome binding sites (e.g., IRES), premature poly (A) sites, Shine-Dalgarno (SD) sequences, and / or other elements associated with translation; and / or the DNA template can be optimized with respect to the following items: codon background, codon-anticodon interactions, translation pause sites, and / or other elements associated with protein folding. Optimization methods known in the art can be used in the present invention, such as those described in WO 2021 / 226461, or GeneOptimizer and OptimumGene described in US 2011 / 0081708 by Thermo Fisher (ThermoFisher), which are incorporated herein by reference in their entirety. TM .

[0087] In the reaction process, RNA polymerase synthesizes RNA in a template-dependent manner. In some embodiments, RNA polymerase is SP6 RNA polymerase. In other embodiments, RNA polymerase is T7 RNA polymerase. In some embodiments, RNA polymerase is T3 RNA polymerase.

[0088] When RNA polymerase transcribes the nucleotide sequence of the template into RNA, it incorporates ribonucleotides (NTPs) into the nascent RNA transcript. During the NTP incorporation process, pyrophosphate (PPi) is released.

[0089] In some embodiments, the IVT reaction further comprises a pyrophosphatase. The pyrophosphatase hydrolyzes PPi into inorganic phosphate (Pi).

[0090] In order to function efficiently, RNA polymerase requires divalent cations as cofactors. Suitable divalent cations include magnesium (Mg 2+ ) or manganese (Mn 2+ In some embodiments, the IVT reaction includes Mg 2+ or Mn 2+ . Reactants

[0091] In some embodiments, the methods of the invention are used to monitor changes in the amount of one reactant. In some embodiments, changes in the amount of more than one reactant are monitored.

[0092] The main reactant of the IVT reaction is NTP. NTP is used to form RNA, the main product of the IVT reaction. The progress of the IVT reaction can be monitored by determining the change in the amount of NTP. In fact, the level of NTP will decrease as they are incorporated into RNA. Therefore, in some embodiments, the method of the present invention monitors the change in the amount of one or more NTPs. In some embodiments, the change in the amount of one, two or three NTPs is monitored simultaneously. For example, depending on the spectroscopic method selected for obtaining the spectrum in step (i) of the method of the present invention, it may be possible to monitor the change in the amount of a single NTP of interest (e.g., ATP, CTP, GTP or UTP). In some embodiments, the method of the present invention is used to monitor the change in the amount of ATP. In some embodiments, the method of the present invention is used to monitor the change in the amount of CTP. In some embodiments, the method of the present invention is used to monitor the change in the amount of GTP. In some embodiments, the method of the present invention is used to monitor the change in the amount of UTP. In some embodiments, the method of the present invention is used to monitor the change in the amount of: ATP and UTP, ATP and CTP, ATP and GTP, GTP and CTP, GTP and UTP, UTP and CTP, ATP, CTP and GTP, ATP, CTP and UTP, or GTP, UTP and CTP.

[0093] RNA can be synthesized from NTPs comprising naturally occurring nucleosides (also referred to herein as "unmodified nucleosides"; i.e., adenosine, guanosine, cytidine, and uridine). Thus, the NTPs in the IVT reaction can be adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP).

[0094] In some embodiments, RNA can be synthesized by including one or more modified nucleosides in an IVT reaction. Thus, one or more NTPs in an 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 a modified UTP (also referred to herein as UTPm), e.g., a uridine analog such as N1-methyl pseudouridine.

[0095] In some embodiments, the one or more modified NTPs comprise a nucleoside analog selected from the group consisting of 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 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.

[0096] In some embodiments, the modified NTP comprises a nucleoside analog selected from the group consisting of pseudouridine, N1-methyl pseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the modified NTP comprises the nucleoside analog N1-methyl pseudouridine.

[0097] In some embodiments, the modified NTP comprises a nucleoside analog selected from the group consisting of 5-azacytidine, 6-azacytidine, 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-thiopseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-cytidine. -pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thiozebularine, 2-thiozebularine, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxypseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysine, 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 ,N4 , 2'-O-trimethylcytidine, 1-thio-cytidine, 2'-F-arabino-cytidine, 2'-F cytidine and 2'-OH-arabino-cytidine. In some embodiments, the modified NTP comprises the nucleoside analog 5-methylcytidine.

[0098] In some embodiments, the modified NTP comprises a nucleoside selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and any combination thereof.

[0099] In some embodiments, the IVT reaction comprises 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-methyl pseudouridine triphosphate.

[0100] In some embodiments, changes in the amount of one or more unmodified NTPs are monitored during the IVT reaction. The modified NTPs are detected substantially the same as the unmodified NTPs. Typically, no or minimal adjustments are required to adapt the monitoring methods of the present invention to one or more modified NTPs. Thus, in some embodiments, changes in the amount of one or more modified NTPs are monitored during the IVT reaction. In particular embodiments, changes in the amount of one or more unmodified NTPs and one or more modified NTPs are monitored using the methods of the present invention.

[0101] Depending on the method used to obtain the spectrum during the IVT reaction, it may 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, because their structures are similar, thus resulting in similar spectral characteristics. Therefore, 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 obtained to monitor changes in all NTPs in the IVT reaction. Specifically, in some embodiments, changes in ATP, UTP, GTP, and CTP are each monitored separately. product

[0102] The main products of IVT reaction can include RNA, inorganic pyrophosphate (PPi), H +and inorganic phosphate (Pi). During the course of the IVT reaction, the amount of RNA increases. As reactants are consumed during the IVT reaction, the amount of RNA may stabilize. The methods of the present invention can 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 the operator to terminate the reaction early, for example, because the amount of RNA does not increase in a predicted manner during the initial stages of the IVT reaction. In addition, monitoring the amount of RNA as the IVT reaction proceeds enables the operator to stop the IVT reaction when the amount of RNA begins to stabilize, thereby potentially shortening the time required for the IVT reaction. Both of these interventions can be used to increase the yield of RNA in the manufacturing process. Therefore, in some embodiments, the methods of the present invention monitor changes in the amount of RNA throughout the IVT reaction.

[0103] During NTP incorporation, PPi and H + The amount of PPi and / or H may be generated as a byproduct of the IVT reaction. The progress of the IVT reaction may be monitored by determining changes in the amount of such byproducts. In some embodiments, PPi and / or H may be monitored during the IVT reaction. + The change in quantity.

[0104] As a byproduct of the IVT reaction, PPi can form an insoluble precipitate with magnesium, which is typically included in the reagent. The incorporation of magnesium into the Mg2PPi insoluble precipitate means that Mg is available as a cofactor for RNA polymerase. 2+ Thus, the presence of PPi in an IVT reaction is associated with reduced transcription efficiency. Thus, in some embodiments, changes in the amount of PPi are monitored during an IVT reaction.

[0105] In some embodiments, the IVT reaction includes a pyrophosphatase. The pyrophosphatase can hydrolyze PPi to Pi. Thus, 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.

[0106] Due to the generation of H + , pH typically increases during an IVT reaction. An increase in pH can negatively impact the efficiency of an IVT reaction. Therefore, in some embodiments, pH is monitored during an IVT reaction. + The change in quantity.

[0107] In some embodiments, the change in the amount of a product (e.g., RNA) is monitored. In some embodiments, the change in the amount of more than one product is monitored. For example, the change in the amount of RNA can be monitored while the change in the amount of other products is monitored. In some embodiments, the amount of RNA and the amount of PPi and / or Pi in the reaction vessel are monitored by acquiring a spectrum, from which information about the amount of these products can be obtained.

[0108] In some embodiments, changes in the amount of one or more reactants (e.g., NTPs) and one product (e.g., RNA) are monitored. For example, the same spectrum can provide information about the amount of NTPs (e.g., NTPs) in the IVT reaction in addition to the amount of RNA. 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.

[0109] In some embodiments, the amount of RNA and NTP (e.g., purine and / or pyrimidine nucleotides) is monitored throughout the IVT reaction. In some embodiments, the amount of RNA and PPi and / or Pi is monitored throughout the IVT reaction. In some embodiments, the amount of (i) RNA, (ii) NTP (e.g., purine and / or pyrimidine nucleotides), and (iii) PPi and / or Pi is monitored throughout the IVT reaction. In some embodiments, the amount of (i) RNA, (ii) NTP (e.g., purine and / or pyrimidine nucleotides), (iii) PPi and / or Pi, and optionally (iv) H is monitored throughout the IVT reaction. + . Additional components

[0110] The IVT reaction typically includes a buffer, such as Tris, HEPES, citrate, acetate, or phosphate. In some embodiments, the buffer is selected based on the fact that it does not interfere with the spectroscopic readings of the reactants and / or the desired product in the IVT reaction. A suitable buffer is, for example, Tris. In some embodiments, the concentration of Tris in the IVT reaction is 10 mM-100 mM. In some embodiments, the concentration of Tris in the IVT reaction is 15 mM-35 mM. In some embodiments, the concentration of Tris in the IVT reaction is 25 mM.

[0111] In some embodiments, the IVT reaction further comprises one or more salts, such as sodium chloride and magnesium chloride. Magnesium chloride can be used to provide divalent cations, which can act as a cofactor to increase the efficiency of RNA polymerase. In some embodiments, the IVT reaction further comprises an RNase inhibitor. In some embodiments, the IVT reaction further comprises DTT. termination

[0112] The IVT reaction can be terminated, for example, by adding a nuclease (e.g., DNase I) to digest the template (e.g., DNA template). The 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) in the reaction vessel is monitored by acquiring a spectrum from which information about the amount of the template can be obtained. This information can be used to monitor digestion of the template (e.g., DNA template) after the addition of a nuclease (e.g., DNase I). The ability to monitor the termination of an IVT reaction is useful, for example, to confirm that a batch of mRNA is ready for the next manufacturing step (e.g., purification).

[0113] Alternatively, the template can be removed from the solution without degradation. For example, the template can be immobilized on magnetic beads. Monitoring the amount of the template (e.g., DNA plasmid) will indicate the removal of the template, for example, by removing the beads using a magnet.

[0114] A protease (e.g., proteinase K) may be added to inactivate any enzyme components (e.g., RNA polymerase and nuclease, if present). DTT may also be added to quench the reaction, if necessary. The concentration of the protease (e.g., proteinase K) and / or other enzyme components may be monitored throughout the IVT reaction, or only during the termination phase.

[0115] In some embodiments, the presence and / or amount of one or more of the following in a reaction vessel is monitored by acquiring a spectrum, from which information about the amount of one or more of the template, the nuclease, the protease, and the RNA polymerase can be obtained. For example, information about the presence of a nuclease or protease can be used in an automated process to confirm that an enzyme component is added to a reaction vessel at the appropriate time (i.e., during the termination phase after the IVT reaction is complete) and / or to confirm that the protease has degraded the enzyme component.

[0116] In some embodiments, digestion of the template and the presence of a nuclease (i.e., DNase I) are monitored. In some embodiments, digestion of the template (e.g., by DNase I) and digestion of an RNA polymerase (e.g., by proteinase K) are monitored. In some embodiments, digestion of the template is monitored, followed by digestion of a protease (e.g., proteinase K) RNA polymerase and a nuclease (e.g., DNase I). In some embodiments, self-digestion of a protease (e.g., proteinase K) is monitored. Spectrometer

[0117] In order to obtain the spectrum in step (i) of the method of the present invention and the predetermined reference spectrum in step (ii), various spectrometers can be used. For example, in contrast to the absorbance-based method, a light scattering (LS)-based technique can be used to implement the claimed method. As shown in the examples of the present application, a Raman spectrometer or a turbidimeter can be used in the implementation of the present invention. Therefore, in some embodiments, the spectrum in step (i) of the method of the present invention and the reference spectrum in step (ii) are obtained using LS-based techniques.

[0118] Suitable LS-based techniques include Raman spectroscopy. Thus, in some embodiments, the spectrometer used in the methods of the present invention is a Raman spectrometer.

[0119] LS-based methods can be complex in terms of equipment and required data analysis. A relatively simple method of implementing the methods of the present invention is to use a turbidity meter. During the IVT reaction, an insoluble Mg2PPi precipitate may be formed as a byproduct. As the IVT reaction proceeds and this byproduct increases, the turbidity increases. The rate of appearance of turbidity provides an indirect indication of RNA production because the formation of the Mg2PPi precipitate depends on the byproducts from the IVT reaction. Therefore, in some embodiments, the spectrometer used in the methods of the present invention is a turbidity meter. In some embodiments, assessing turbidity can provide a complementary method for monitoring IVT reactions.

[0120] In some embodiments, vibrational techniques can be used to implement the claimed methods. Vibrational spectroscopy is based on periodic changes in polarizability (Raman) or dipole moment (infrared) caused by molecular vibrations of molecules or groups of atoms within a molecule, as well as discrete energy transitions and frequency changes during scattering or absorption of electromagnetic radiation. The advantage of using vibrational techniques such as Raman or infrared (IR) spectroscopy is the ability to monitor multiple components, such as reactants and / or products, during an IVT reaction.

[0121] As shown in one of the exemplary embodiments of the present invention, wavelength regions corresponding to the primary reactants (e.g., NTPs) and products (e.g., mRNA, PPi, and / or Pi) of an IVT reaction can be identified in the vibrational spectrum. In fact, software-based comparison with a predetermined reference spectrum allows not only quantitative analysis of reactants or products, but also quantitative determination of specific reactants or products within a specified time range when an IVT reaction is performed in a reaction vessel.

[0122] Quantitative analysis can include determining a change in the amount of one or more reactants or products relative to a baseline - for example, a baseline spectrum before the IVT reaction is initiated (e.g., by adding RNA polymerase). Quantitative determination can include monitoring the concentration of one or more reactants or products during the IVT reaction.

[0123] Advantageously, the reactants and / or products of an IVT reaction can be monitored using vibrational techniques such as Raman or IR spectroscopy without the need to label the reactants or products or to obtain aliquots from the reaction vessel.

[0124] Thus, in some embodiments, the spectrometer used in the inventive method is a Raman spectrometer. In some embodiments, the spectrometer used in the inventive method is an IR spectrometer. In some embodiments, the spectrometer used in the inventive method is a near IR (NIR) spectrometer. In some embodiments, the spectrometer used in the inventive method is a mid-IR (MIR) spectrometer.

[0125] In some embodiments, it may be advantageous to use more than one spectroscopic method to monitor the IVT reaction. In particular, a Raman spectrometer, an IR spectrometer (e.g., a NIR or MIR spectrometer), and a turbidity meter, or any combination of two or more thereof, may be used to monitor the IVT reaction. In some embodiments, a Raman spectrometer and a turbidity meter are used to monitor the IVT reaction. In other embodiments, an IR spectrometer and a turbidity meter are used to monitor the IVT reaction. In some embodiments, a Raman spectrometer and an IR spectrometer are used to monitor the IVT reaction. Acquire spectrum

[0126] The present invention relates to obtaining at least one spectrum during an IVT reaction process to monitor the amount of reactants and / or products within a reaction vessel. In some embodiments, the present invention also relates to obtaining at least one spectrum during an IVT reaction process to monitor the amount of RNA polymerase and / or template (e.g., DNA template) within a reaction vessel. In some embodiments, the present invention also relates to obtaining at least one spectrum during an IVT reaction process to monitor the addition of a nuclease (e.g., DNase I) or a protease (Protease K) within a reaction vessel to quench or terminate the IVT reaction.

[0127] In some embodiments, a spectrum containing information about one or more reactants (eg, one or more NTPs) is obtained during the IVT reaction process. In some embodiments, a spectrum of one or more products (eg, RNA) is obtained during the IVT reaction process.

[0128] The acquisition time of the spectrum is related to the width of the wavelength region, that is, it takes longer to obtain a spectrum in a wider wavelength region than to obtain a spectrum in a narrower wavelength region. In order to reduce the acquisition time of the spectrum, it may be preferable to limit the step of obtaining the spectrum to a wavelength region specific to a particular reactant or product. For example, in some embodiments, the spectrum obtained in step (i) of the method of the present invention is limited to a wavelength region that provides information about the amount of RNA present in the reaction vessel. Reducing the acquisition time by reducing the wavelength region allows the reactants or products to be monitored in essentially real time. Therefore, in some embodiments, a spectrum including 600cm -1 Up to 1300cm -1 In some embodiments, a Raman spectrum including 800 cm -1 Up to 1250cm -1 Raman spectra in the wavelength region.

[0129] In some embodiments, it may be desirable to obtain a spectrum covering multiple reactants and products of an IVT reaction. Thus, in some embodiments, the spectrum obtained in step (i) spans a wavelength region suitable for monitoring the overall evolution of multiple reactants and products during an IVT reaction. In some embodiments, a wavelength region is selected that provides information about the amounts of: (i) RNA, (ii) NTPs (e.g., purine and / or pyrimidine nucleotides), (iii) PPi and / or Pi, and optionally (iv) H+ in an IVT reaction. For example, the inventors have found that obtaining a spectrum containing 150 cm -1 Up to 4000cm -1 A Raman spectrum of the wavelength region takes 50 seconds and provides information about all the above-mentioned products and reactants.

[0130] In some embodiments, each spectrum in the series is obtained over a period of less than one minute. In some embodiments, each spectrum in the series is obtained 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 series is obtained over a period of 1 to 60 seconds (e.g., 10-50 seconds or 5-20 seconds).

[0131] Acquiring one or more spectra during the course of an IVT reaction can provide qualitative and / or quantitative data to inform a user of the progress of the reaction. The term "information" may be used to describe such qualitative and / or quantitative data acquired. 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, it may be advantageous to obtain spectra across a wider wavelength region that include information about multiple reactants and products of an IVT reaction, particularly when optimizing reaction conditions for producing a particular RNA (e.g., taking into account the nucleotide composition and / or length of the RNA).

[0132] In some embodiments, a spectrum series is obtained. By obtaining multiple spectra, data can be merged to reduce background noise. The optimization of the signal-to-noise ratio helps to better distinguish the signal from the background noise associated with any detection method. In some embodiments, the spectrum series includes a set with at least 3, at least 5, or at least 9 spectra. In some embodiments, a series with 3 to 20 spectra is obtained. In some embodiments, a series with 5 to 15 spectra is obtained. In some embodiments, a series with 8 to 12 spectra is obtained.

[0133] An important consideration when acquiring spectra is the effect of acquisition on spectral resolution. While acquiring fewer spectra can reduce acquisition time, this may result in a loss of quality. For example, the noise of the spectrum may not be easily distinguished from the signal and may therefore affect spectral interpretation and analysis. The number of spectra that can be acquired to obtain a viable data set may also depend on the specific spectroscopic method and / or specific spectrometer used to acquire the spectra in step (i) of the method of the present invention. For example, the inventors have found that acquiring a series of 10 Raman spectra reduces background noise. Therefore, in a specific embodiment, a series of 10 spectra is acquired. In some embodiments, each spectrum in the series is acquired within a time period of 1 to 60 seconds (e.g., 10-50 seconds).

[0134] After initiating an IVT reaction, the amount of one or more reactants and one or more products may vary throughout the reaction until the reaction has been terminated, for example, by the addition of a nuclease (e.g., DNase I) to digest the template. Thus, in some embodiments, a series of spectra is acquired over the course of an IVT reaction. It may be desirable to acquire spectra at intervals during the course of an IVT reaction, for example to determine the evolution of the amount of one or more reactants and / or one or more products. In some embodiments, a series of spectra is acquired at intervals that are typically equally spaced over the course of an IVT reaction. In some embodiments, multiple series of spectra are acquired over the course of an IVT reaction to reduce the signal-to-noise ratio of a single data point.

[0135] In some embodiments, a spectrum or a series of spectra is obtained at intervals of 1-30 minutes during the IVT reaction process. In some embodiments, a spectrum or a series of spectra is obtained at intervals of 30 minutes. In some embodiments, a spectrum or a series of spectra is obtained at intervals of 15 minutes. In some embodiments, a spectrum or a series of spectra is obtained at intervals of 10 minutes. In some embodiments, a spectrum or a series of spectra is obtained at intervals of 5 minutes. In some embodiments, a spectrum or a series of spectra is obtained at intervals of 4 minutes. In some embodiments, a spectrum or a series of spectra is obtained at intervals of 3 minutes. In some embodiments, a spectrum or a series of spectra is obtained at intervals of 2 minutes. In some embodiments, a spectrum or a series of spectra is obtained at intervals of 1 minute.

[0136] In some embodiments, a spectrum or series of spectra is acquired at intervals of less than 1 minute (e.g., at intervals of 50 seconds or less, 40 seconds or less, 30 seconds or less, 20 seconds or less, or 10 seconds or less).

[0137] In some embodiments, spectra are acquired continuously over the course of the IVT reaction. The acquired spectra can be combined into a spectrum set as described above to improve the signal-to-noise ratio of each data point. For example, 3, 4, 5, 6, 7, 8, 9, or 10 spectra can be acquired and combined into a set. Although the spectrum set represents a series of spectra acquired over a certain period of time (e.g., over a period of 1, 2, 3, 5, 6, 7, 8, 9, or 10 minutes), they can also be represented as a single data point. Monitoring of reactants and / or products during IVT reactions

[0138] In some embodiments, a spectrum is acquired over a wavelength region to monitor the overall evolution of the amount of at least one reactant (eg, one or more NTPs) and at least one product (eg, RNA) of an IVT reaction.

[0139] In some embodiments, the wavelength region provides information on all major products and reactants of the IVT reaction, including RNA, PPi / Pi, and NTPs. Suitable wavelength regions for monitoring these products and reactants using a Raman spectrometer include 300 cm -1 Up to 3000cm -1 In some embodiments, the wavelength region includes 600 cm -1 Up to 1300cm -1 In other embodiments, the wavelength region includes 800 cm -1 Up to 1250cm -1 .

[0140] In some embodiments, the spectrum obtained in step (i) of the methods of the present invention spans a wavelength region specific to a single product or reactant of an IVT reaction.

[0141] In some embodiments, the product is RNA (e.g., mRNA). In some embodiments, the amount of RNA is monitored using a Raman spectrometer. Suitable wavelength regions for monitoring the amount of RNA in an IVT reaction using a Raman spectrometer include 801 cm -1 Up to 831cm -1 In some embodiments, at about 810 cm -1 The amount of RNA in the IVT reaction was monitored at a wavelength of 1.

[0142] In some embodiments, the product is Pi. In some embodiments, the amount of Pi is monitored using Raman spectroscopy. Suitable wavelength regions for monitoring the amount of Pi in an IVT reaction using Raman spectroscopy include 875 cm -1 Up to 900cm -1 .

[0143] In some embodiments, the reactant is one or more NTPs (e.g., one, two, or three individual NTPs). In some embodiments, the reactant includes 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. Suitable wavelength regions for monitoring the amount of NTPs in an IVT reaction using a Raman spectrometer include 600 cm -1 Up to 1300cm -1 , including, for example, 700cm -1 Up to 800cm -1 In some embodiments, a narrower wavelength region is used to monitor the amount of NTP in an IVT reaction.

[0144] In some embodiments, suitable wavelength regions for monitoring the amount of NTP in an IVT reaction using Raman spectroscopy include 1100 cm -1 Up to 1120cm -1 or 1107cm -1 Up to 1146cm -1 .1113cm -1 Up to 1115cm -1 The wavelength region specifically corresponds to the PO2 - Therefore, in some embodiments, the suitable wavelength region for monitoring the amount of NTP in an IVT reaction using a Raman spectrometer includes 1113 cm -1 Up to 1115cm -1 (For example, 1115cm -1 ).

[0145] NTPs can be classified according to the presence of purine or pyrimidine bases. In some embodiments, wavelength regions are selected that allow for the detection of purine nucleotides (e.g., ATP and GTP) and pyrimidine nucleotides (e.g., CTP and UTP), respectively.

[0146] 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, the amount of purine nucleotides can also be monitored in a wavelength range of 1300 cm -1 Up to 1600cm -1 Therefore, in some embodiments, the suitable wavelength region for monitoring the amount of purine nucleotides in an IVT reaction using a Raman spectrometer includes 1300 cm -1 Up to 1600cm -1 In some embodiments, suitable wavelength regions for monitoring the amount of GTP and ATP in an IVT reaction using Raman spectroscopy include 1580 cm -1 .1580cm -1 The wavelength 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 includes 1580 cm -1 .

[0147] 1560cm -1 Up to 1580cm -1 The main Raman shift in the wavelength range is due to the amount of GTP in the IVT reaction. Therefore, in some embodiments, a suitable wavelength region for monitoring the amount of GTP in the IVT reaction using a Raman spectrometer includes 1560 cm -1 Up to 1600cm -1 , for example 1560cm -1 Up to 1590cm -1 or 1560cm -1 Up to 1580cm -1 .

[0148] In addition, in Raman spectroscopy, the -1 Up to 750cm -1 The wavelength range of 650 cm-1 is used to identify distinct peaks of GTP and ATP. The appropriate wavelength region for monitoring the amount of GTP in an IVT reaction using a Raman spectrometer includes 650 cm-1. -1 Up to 700cm -1 The appropriate wavelength region for monitoring the amount of ATP in an IVT reaction using Raman spectroscopy includes 700 cm -1 Up to 750cm -1 In some embodiments, a Raman spectrometer is used to monitor the wavelengths of 650 cm -1 Up to 750cm -1 The wavelength region includes 1560cm -1 Up to 1580cm -1 The wavelength region is used to determine the amount of ATP and GTP.

[0149] In some embodiments, the reactants are pyrimidine nucleotides. In some embodiments, the amount of pyrimidine nucleotides is monitored using a Raman spectrometer. Using a Raman spectrometer, the amount of pyrimidine nucleotides can be monitored using a Raman spectrometer. -1 Up to 785cm -1 At (e.g. at 780cm -1 CTP is detected at 785 cm due to the stretching of the C=C bonds corresponding to carbons 5 and 6 of uracil. -1 Up to 810cm -1 (For example, 786cm -1 Up to 789cm -1 ) at a wavelength region of 780 cm . In one embodiment, a suitable wavelength region for monitoring the amount of pyrimidine nucleotides in an IVT reaction using a Raman spectrometer includes 780 cm -1 Up to 789cm -1 .

[0150] Alternatively or additionally, due to the stretching of the C=N bond, it is also possible to -1 Up to 1250cm -1 (For example, 1230cm -1 Up to 1245cm -1 ) to detect CTP-UTP. Therefore, in some embodiments, the suitable wavelength region for monitoring the amount of pyrimidine nucleotides in an IVT reaction using a Raman spectrometer includes 1230 cm -1 Up to 1245cm -1 .

[0151] Using Raman spectroscopy, the -1 and 1290cm -1 CTP is detected by monitoring at 1240cm -1 The wavelength corresponds to the CN stretching of unsaturated amines. Therefore, in some embodiments, a suitable wavelength region for monitoring the amount of CTP in an IVT reaction using Raman spectroscopy includes 1240 cm -1 and 1290cm -1 .

[0152] In some embodiments, the spectrum obtained in step (i) of the method of the present invention is measured at a wavelength specific to a single product or reactant of the IVT reaction. For example, using a Raman spectrometer, a wavelength of 633 cm -1 ATP is detected at 1670 cm (which corresponds to the vibration of the purine ring). Using Raman spectroscopy, the appropriate wavelength region for monitoring the amount of UTP includes 1670 cm -1, which is associated with the carbon 4 ketone function. Therefore, in some embodiments, the appropriate wavelengths for monitoring the amount of ATP and UTP in an IVT reaction using a Raman spectrometer are 633 cm -1 and 1670cm -1 .

[0153] Mg2PPi is a byproduct 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, UV spectrometer or nephelometer. It can be measured within a wavelength range including 290nm to 410nm, for example 300nm to 350nm. Exemplary wavelengths for measuring turbidity using a UV spectrometer are 310nm, 320nm, 330nm, 340nm or 350nm. For example, using a UV spectrometer, turbidity can be measured at an absorbance of 320nm. A turbidimeter can also be used to measure the presence of insoluble components (measurements are typically given in nephelometer turbidity units [NTU]). Monitoring the termination of IVT reactions

[0154] In some embodiments, spectra of a wavelength region are obtained to monitor template and RNA polymerase during an IVT reaction. For example, changes in the amount of template or RNA polymerase may indicate contamination of the IVT reaction.

[0155] In some embodiments, the template (e.g., DNA template) is monitored throughout the IVT reaction. In some embodiments, the template is monitored only during the termination phase, e.g., to confirm the destruction or removal of the template. In some embodiments, the template is monitored using a Raman spectrometer. A suitable wavelength region for monitoring the template using a Raman spectrometer is 505 cm -1 Up to 705cm -1 In some embodiments, a suitable wavelength region for monitoring the template using a Raman spectrometer is 1325 cm -1 Up to 1365cm -1 or 1585cm -1 Up to 1720cm -1 .

[0156] In some embodiments, RNA polymerase (e.g., SP6 RNA polymerase) is monitored throughout the IVT reaction. In some embodiments, RNA polymerase is monitored during the termination phase only to confirm destruction of the enzyme (e.g., by protease digestion). In some embodiments, RNA polymerase is monitored using Raman spectroscopy. A suitable wavelength region for monitoring RNA polymerases such as SP6 RNA polymerase using Raman spectroscopy is 780 cm -1 Up to 1200cm -1In some embodiments, a suitable wavelength region for monitoring RNA polymerase (e.g., SP6 RNA polymerase) using Raman spectroscopy is 1200 cm -1 or 1430cm -1 Up to 1510cm -1 .

[0157] In some embodiments, a spectrum of a wavelength region is obtained to monitor the addition of a component (eg, an enzyme component) to terminate an IVT reaction. In an automated system, it may be useful to confirm that the enzyme component is added at the correct time point in the IVT reaction.

[0158] In some embodiments, the addition or amount of a nuclease (e.g., DNase I) is monitored. In some embodiments, the amount of a nuclease (e.g., DNase I) is monitored during the termination phase only to confirm the destruction of the enzyme (e.g., by proteinase K digestion). In some embodiments, the addition or amount of a nuclease (e.g., DNase I) is monitored using a Raman spectrometer. A suitable wavelength region for monitoring nucleases (e.g., DNase I) using a Raman spectrometer is 450 cm -1 Up to 520cm -1 In some embodiments, a suitable wavelength region for monitoring DNase I using Raman spectroscopy is 1000 cm -1 Up to 1090cm -1 or 2915cm -1 Up to 3000cm -1 .

[0159] 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 region for monitoring a protease such as proteinase K using a Raman spectrometer is 505 cm -1 Up to 610cm -1 , for example 550cm -1 Up to 600cm -1 In some embodiments, a suitable wavelength region for monitoring proteases such as proteinase K using Raman spectroscopy is 715 cm -1 Up to 775cm -1 or 1385cm -1 Up to 1395cm -1 . Preprocessing spectra

[0160] The raw spectral data obtained in step (i) of the method of the present invention can be processed to improve the signal-to-noise ratio. Therefore, in some embodiments, the spectrum obtained in step (i) is preprocessed before step (ii). In some embodiments, each spectrum obtained in the series is preprocessed before step (ii). Preprocessing can facilitate comparative analysis of spectra. For example, the wavelength region of the spectrum obtained in step (i) can be normalized relative to a wavelength region with low background noise or no background noise. Using a Raman spectrometer, a suitable wavelength region with low background noise includes 3100cm -1 and 3600cm -1 Normalization is typically accomplished using a suitable algorithm (e.g., the Standard Normal Variable (SNV) algorithm). In some embodiments, the spectrum or series of spectra obtained in step (i) is normalized to a suitable wavelength region. In one embodiment, the spectrum or series of spectra is acquired using a Raman spectrometer, and the 3100 cm -1 Up to 3600cm -1 The wavelength region is used for normalization.

[0161] Alternatively, or in addition, pre-processing can apply digital filters to smooth data using spectral series. Applying such filters can improve the accuracy of data without distorting signal trends. In some embodiments, the digital filter fits the spectrum containing adjacent data points continuously acquired by linear least squares method with a low-order polynomial. In a typical embodiment, the spectral series is obtained at intervals with equal spacing. Since the data points are therefore equally spaced, the analytical solution of the least squares equation can be found in the form of a single "convolution coefficient" set, which can be applied to all spectra in the series to provide a smoothed data set. For example, a suitable digital filter can adopt the Savitzky-Golay (SG) algorithm. Compare the spectrum to a reference spectrum

[0162] 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).

[0163] In some embodiments, the predetermined reference spectrum is a first spectrum or a first series of spectra obtained 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 amounts of reactants or target products reflect the progress of the IVT reaction.

[0164] In some embodiments, the predetermined reference spectrum is a spectrum or series of spectra obtained during an IVT reaction using known conditions and reactants. In some embodiments, the predetermined reference spectrum is a spectrum or series of spectra of a reactant or product of the IVT reaction, typically obtained with the reactant or product dissolved or suspended in a reaction buffer for the IVT reaction. In either scenario, the concentrations of the reactants and / or products are known for the predetermined reference spectrum and can therefore be used to infer the concentrations of the reactants and / or products of the spectrum or series of spectra obtained in step (i) of the method of the invention.

[0165] Thus, in some embodiments, a predetermined reference spectrum is associated with a particular concentration of a reactant or product. In some embodiments, a 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, wherein each spectrum in the series is associated with a different concentration.

[0166] In some embodiments, determining the concentration of a reactant or product comprises a linear regression analysis. In some embodiments, the concentration of a reactant or product is determined from a predetermined series of reference spectra using a partial least squares (PLS) model, wherein each spectrum in the series is associated with a different concentration of a reactant or product.

[0167] In some embodiments, the concentration of reactants or products is determined using linear regression analysis. In one embodiment, the concentration of reactants or products is determined using a PLS model. PLS finds a linear regression model by projecting the predictor and observable variables into a new space. The basic relationships between the variables are set in this new space and then projected back into the original space. PLS regression is widely used in chemometrics and related fields.

[0168] In some embodiments, step (ii) comprises performing a qualitative spectral comparison of the spectrum or spectral series obtained in step (i) with a predetermined reference spectrum. In some embodiments, the qualitative spectral comparison comprises calculating a weighted spectral difference (WSD) value between the spectrum or spectral series obtained in step (i) and the predetermined reference spectrum.

[0169] In some embodiments, WSD values ​​within a range of one standard deviation or less indicate a high degree of similarity between the spectrum obtained in step (i) and the predetermined reference spectrum, e.g., no significant difference. Thus, in these embodiments, where applicable, the amount of reactants or products in the spectrum obtained in step (i) is considered to be the same as the amount of reactants or products in the predetermined reference spectrum, or the amount of reactants or products in the spectrum obtained in step (i) is considered to remain unchanged relative to the amount of reactants or products in the predetermined reference spectrum.

[0170] In some embodiments, a WSD value of more than one standard deviation (e.g., two standard deviations) indicates a low similarity, e.g., a significant difference, between the spectrum obtained in step (i) and the predetermined reference spectrum. Thus, in these embodiments, the amount of reactant or product in the spectrum obtained in step (i) is considered to be greater or less than the amount of reactant or product in the reference spectrum, where applicable. Process Optimization

[0171] 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, a spectral series obtained by monitoring an IVT reaction can be used to create a kinetic model of one or more reactants and / or products. The kinetic model can be used to predict or simulate changes in the amount of one or more products under different conditions (e.g., at different 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 the optimal set of conditions for producing RNA using an IVT reaction.

[0172] In some embodiments, the kinetic model is used to predict the yield of RNA produced by the IVT reaction. In some embodiments, the kinetic model is used to simulate the IVT reaction to predict the consumption of reactants. In certain embodiments, the kinetic model is used to predict the consumption of NTPs during the IVT reaction.

[0173] The kinetic model can also be used as a reference to compare spectra obtained during an IVT reaction to determine whether the IVT reaction is proceeding at an expected level of efficiency. Typically, the kinetic model is created using a series of spectra obtained from an IVT reaction conducted 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 an IVT reaction. Making RNA

[0174] The monitoring methods of the present invention can also be used as process control or quality control means during RNA manufacturing (particularly during large-scale production of RNA). For example, the monitoring methods of the present invention may be particularly suitable for large-scale production of RNA, such as mRNA, for therapeutic applications.

[0175] Thus, the present invention also relates to a method for making RNA (e.g., mRNA) using an in vitro transcription (IVT) reaction, the method comprising: (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 comprising an RNA polymerase and necessary reactants to initiate an IVT reaction; and (c) monitoring one or more reactants or products of the IVT reaction using the methods for monitoring the IVT reaction described herein. In some embodiments, the one or more reactants or products are RNA.

[0176] 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) obtaining a spectrum of the RNA during the IVT reaction to determine a first value, and (ii) comparing the first value obtained in step (i) with a second value derived from a predetermined reference spectrum of the RNA; and (b) purifying the RNA if the first value equals or exceeds the second value. Quality Control

[0177] Monitoring of an IVT reaction by a spectroscopic method (e.g., Raman spectroscopy) as described herein can be used to determine whether an IVT reaction during the manufacture of a batch of RNA (e.g., mRNA) meets one or more predetermined parameters. The decision to continue or terminate an IVT reaction may depend on one or more predetermined parameters.

[0178] In some embodiments, one or more of the reactants or products are monitored throughout the IVT reaction during the production of RNA (e.g., mRNA). In some embodiments, one or more reactants are NTPs. In some embodiments, one or more products can be RNA, PPi, and / or H + In some embodiments, one or more reactants and one or more products are monitored during the RNA manufacturing process.

[0179] For example, the spectrum of one or more reactants or products obtained in step (i) of the monitoring method of the present invention can be compared with a predetermined reference spectrum of one or more of the reactants or products. If the obtained spectrum sufficiently corresponds to the predetermined reference spectrum, the IVT reaction can be continued. If not, it can be terminated.

[0180] Comparing one or more spectra obtained 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 predetermined set of 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 obtained at one or more specified time points during the IVT reaction, wherein the series of spectra is compared to a predetermined reference spectrum. Such comparison between series of spectra between one or more specified time points can be used, for example, to determine the amount of RNA or a change in the amount of RNA.

[0181] Monitoring multiple time points throughout the reaction can provide insight into how the IVT reaction is progressing. For example, it can be used to confirm whether the amount of RNA is increasing as expected at various intervals during the IVT reaction. In some embodiments, the two or more designated time points are equally spaced apart 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.

[0182] In some embodiments, the RNA production rate during the IVT reaction can be measured at different time points. For example, in some embodiments, the RNA production rate is measured in the early stages of the reaction, i.e., within the first 20 minutes. In some embodiments, the RNA production rate is measured in the later stages 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 IVT reaction and terminate the reaction when the RNA production rate is about zero (meaning that the amount of RNA in the IVT reaction has stabilized).

[0183] In some embodiments, spectral comparison can be sufficient to determine whether an IVT reaction during the production of a batch of RNA (e.g., mRNA) is proceeding as expected. For example, if the spectrum obtained during the IVT reaction deviates significantly from a predetermined reference spectrum, the IVT reaction may not be proceeding effectively, and the operator may decide to terminate it. If the spectrum of the IVT reaction is sufficiently similar to the predetermined reference spectrum, the operator may continue the production run.

[0184] In some embodiments, the spectrum obtained from the IVT reaction deviates significantly from a predetermined reference spectrum, e.g., the obtained spectrum deviates from the predetermined reference spectrum by more than one standard deviation. In such embodiments, the operator may determine that the batch of RNA (e.g., mRNA) produced by the IVT reaction is to be discarded.

[0185] In some embodiments, the spectrum from the IVT reaction deviates significantly from a predetermined reference spectrum, for example, the obtained spectrum deviates from the predetermined reference spectrum by more than one standard deviation. In such embodiments, the operator may determine that the reaction is to be terminated.

[0186] In some embodiments, the spectrum obtained from the IVT reaction is compared to a kinetic model determined for a previous IVT reaction using similar or identical conditions and reactants. In some embodiments, the spectrum from the IVT reaction is sufficiently similar to the kinetic model, e.g., the obtained spectrum deviates from the kinetic model by less than one standard deviation. In such embodiments, an operator can determine that the IVT reaction can proceed and / or release the RNA (e.g., mRNA) batch for subsequent production steps, such as RNA purification. In some embodiments, the spectrum from the IVT reaction deviates significantly from the kinetic model, e.g., the obtained spectrum deviates from the kinetic model by more than one standard deviation. In such embodiments, an operator can determine that the reaction will be terminated and the RNA (e.g., mRNA) batch will not proceed to subsequent production steps.

[0187] In some embodiments, a qualitative spectral comparison is used to assess the deviation from a predetermined reference spectrum. In 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) of the method of the present invention and a predetermined reference spectrum.

[0188] In some embodiments, a WSD value within one standard deviation or less indicates that there is no significant difference between the spectrum obtained in step (i) and the predetermined reference spectrum. In some embodiments, a WSD value of more than one standard deviation (e.g., two standard deviations) indicates that there is a significant difference between the spectrum obtained in step (i) and the predetermined reference spectrum.

[0189] In some embodiments, the spectrum obtained in step (i) of the methods of the present invention is used to calculate the amount of RNA present in the reaction vessel 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 specified time points during the IVT reaction, and step (ii) comprises determining the amount of RNA or the change in the amount of RNA between the one or more specified time points by comparing the series of spectra to a predetermined reference spectrum.

[0190] In some embodiments, if the RNA does not reach the target amount at one or more designated time points, the IVT reaction can be terminated.

[0191] In some embodiments, monitoring the change in the amount of RNA may be more advantageous than estimating the amount (or concentration) of RNA in the reaction vessel. In some embodiments, if the change in the amount of RNA is less than a predetermined value at two or more specified time points, the IVT reaction may be terminated. Termination in this context may mean that the IVT reaction is interrupted prematurely, or that the IVT reaction is completed.

[0192] For example, in some embodiments, a batch of RNA (e.g., mRNA) produced by an IVT reaction can be discarded if the change in the amount of RNA is less than a predetermined value at two or more specified time points. Typically, these time points are early in the IVT reaction, for example, within the first 5-30 minutes of a production run. In some embodiments, a batch of RNA produced by 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. In some embodiments, a batch of RNA produced by 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, and the at least two or more time points are at least 5, 10, 15, 20, or 25 minutes apart, for example, 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.

[0193] For example, the two or more designated time points may be during the first 10 minutes, first 20 minutes, or first 30 minutes of the IVT reaction. The predetermined value may correspond to an expected fold increase in the amount of RNA during the first 10 minutes, first 20 minutes, or first 30 minutes of the IVT reaction, for example, a 2-fold increase between the first 5 minutes and first 20 minutes of the IVT reaction, or a 3-fold increase between the first 5 minutes and first 30 minutes of the IVT reaction.

[0194] In some embodiments, a batch of RNA (e.g., mRNA) produced by an IVT reaction can be discarded if the RNA does not reach a target amount or concentration at one or more specified time points. For example, the one or more specified time points can be during the first 10 minutes, the first 20 minutes, or the first 30 minutes of the IVT reaction. The predetermined value can correspond to the expected concentration of RNA during the first 10 minutes, the first 20 minutes, or the first 30 minutes of the IVT reaction, for example, 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 specified time points can be during the last third of normal production, for example, between 60 minutes and 90 minutes after the start of the IVT reaction. The predetermined value can correspond to the expected concentration of RNA during that time period, for example, 4-5 g / L.

[0195] Spectral data obtained during the IVT reaction can also be used to determine when the RNA amount has stabilized. In some embodiments, this information can be used to terminate the IVT reaction and proceed to the next production step (e.g., purification).

[0196] In some embodiments, the IVT reaction can be terminated if the change in the amount of RNA between at least two or more time points is about zero. Typically, these time points are late in the IVT reaction, e.g., within 60-90 minutes of a production run. In some embodiments, the IVT reaction can be terminated if the change in the amount of RNA between at least two or more time points is about zero, the at least two or more time points being 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, if there is no change in the amount of RNA between 65 minutes and 70 minutes after initiation of production, the IVT reaction can be terminated (e.g., by adding RNA polymerase).

[0197] The present invention also 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) obtaining a spectrum of the RNA during the IVT reaction to determine a first value and (ii) comparing the first value obtained in step (i) with a second value derived from a predetermined reference spectrum of the RNA; and (b) purifying the RNA if the first value is equal to or exceeds the second value.

[0198] 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, at least 8 g / L.

[0199] Acquiring a spectrum corresponding to the RNA at multiple time intervals and comparing it to a predetermined reference spectrum can provide information about how the RNA concentration increases during the IVT reaction. This information can be used to determine 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). Therefore, in some embodiments, a spectrum of the RNA is repeatedly acquired at equally spaced intervals to determine a first value, and the first value is compared to a second value derived from a predetermined reference spectrum. In some embodiments, the first value is considered equal to the second value derived from the 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 obtained at each interval equals or exceeds the second value at the corresponding interval derived from the predetermined reference spectrum.

[0200] In some embodiments, the first and second values ​​are compared using a qualitative spectral comparison. In some embodiments, the qualitative spectral comparison comprises calculating a weighted spectral difference (WSD) value between the RNA spectrum obtained during the IVT reaction and a predetermined reference spectrum.

[0201] In some embodiments, a WSD value within one standard deviation or less indicates that there is no significant difference between the spectrum obtained in step (i) and the predetermined reference spectrum. In some embodiments, a WSD value of more than one standard deviation (e.g., two standard deviations) indicates that there is a significant difference between the spectrum obtained in step (i) and the predetermined reference spectrum.

[0202] If the first value does not equal or exceed the second value, the operator can determine that the RNA (eg, mRNA) batch is not to proceed with purification. Process Control

[0203] In some embodiments, information obtained from monitoring the reactants and / or products of an IVT reaction can be used to replenish the reactants in the reaction vessel. For example, during the course of an IVT reaction, NTPs are consumed. 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 an operator or an operating system of the reaction vessel when the one or more NTPs are about to be depleted. Thus, the one or more NTPs can be replenished to the IVT reaction as needed to maintain a suitable concentration of each NTP. In some embodiments, when the concentration of one or more NTPs is close to depletion, the one or more NTPs are replenished to the IVT reaction.

[0204] In some embodiments, the IVT reaction is supplemented with one or more NTPs at least once during the process of making RNA (e.g., mRNA). In some embodiments, the IVT reaction is supplemented with one or more NTPs periodically during the process of making RNA. In some embodiments, the IVT reaction is continuously supplemented with one or more NTPs during the process of making RNA (e.g., during fed-batch operation of an appropriately configured reaction vessel).

[0205] In some embodiments, the IVT reaction is supplemented with NTPs to maintain the concentration of NTPs present when the IVT reaction is initiated. In some embodiments, the IVT reaction is supplemented with NTPs to restore the concentration of NTPs to the concentration present when the IVT reaction is initiated. In some embodiments, the concentration of each NTP is maintained within a range of 20-100%, 20-75%, or 25%-50% of that present when the IVT reaction is initiated.

[0206] In some embodiments, the IVT reaction is supplemented with NTPs when the concentration of NTPs in the reaction is no more than 5% of the initial concentration of NTPs at the start of the IVT reaction. In some embodiments, the IVT reaction is supplemented with one or more NTPs when the concentration of NTPs in the reaction is about 5 mM or less. In some embodiments, the IVT reaction is supplemented with one or more NTPs when the concentration of NTPs in the reaction is about 3 mM or less.

[0207] In some embodiments, when the IVT reaction is initiated, the concentration of each NTP is 1 mM-10 mM, 1 mM-6 mM, 2 mM-6 mM, or 3 mM-6 mM. In some embodiments, the total NTP concentration in the IVT reaction is maintained above the lower limit of 0.5 mM. In some embodiments, the total NTP concentration in the IVT reaction is maintained at 10 mM to 20 mM.

[0208] In some embodiments, a single NTP may be present at a specific concentration. For example, when each NTP is present at a concentration of 1 mM-10 mM, each NTP may be present at a concentration of 4 mM. In some embodiments, the concentration of each NTP is proportional to the number of times the NTP appears in the RNA transcript.

[0209] In some embodiments, a Raman spectrometer is used to monitor the amount of one or more NTPs in a reaction vessel. Raman spectrometers have defined limits of detection (LoD) and limits of quantitation (LoQ). The LoD and LoQ values ​​of the Raman spectrometer are shown in Table 1. Therefore, in some embodiments, the LoD and / or LoQ of the Raman spectrometer are considered when selecting the concentration of one or more NTPs to supplement the reaction vessel. Table 1. Detection limit and quantification limit of Raman spectrometer

[0210] In some embodiments, a Raman spectrometer is used to monitor the amount of template and / or RNA polymerase during an IVT reaction. For example, a change in the amount of template or RNA polymerase may indicate contamination of the IVT reaction.

[0211] In some embodiments, Raman spectroscopy is used to monitor the addition of one or more enzyme components, such as nucleases (eg, DNase I) and / or proteases (eg, proteinase K), to terminate the IVT reaction.

[0212] In some embodiments, Raman spectroscopy is used to monitor the amount of template to confirm the destruction of the reagent during the termination phase of the IVT reaction.

[0213] In some embodiments, Raman spectroscopy is used to monitor the amount of RNA polymerase and / or nuclease (eg, DNase I) to confirm the destruction of these enzymes during the termination phase of the IVT reaction. Batch size

[0214] The method of the present invention can be used for optimizing reaction conditions before large-scale manufacturing of RNA (e.g., mRNA). Alternatively, the method of the present invention can be used as a process and / or quality control means during large-scale manufacturing of RNA (e.g., mRNA).

[0215] Large-scale manufacturing typically involves producing 100 mg or more RNA (e.g., mRNA) batches. Thus, in some embodiments, at least 100 mg RNA is synthesized in a single batch. In some embodiments, at least 200 mg RNA is synthesized in a single batch. In some embodiments, at least 300 mg RNA is synthesized in a single batch. In some embodiments, at least 400 mg RNA is synthesized in a single batch. In some embodiments, at least 500 mg RNA is synthesized in a single batch. In some embodiments, at least 600 mg RNA is synthesized in a single batch. In some embodiments, at least 700 mg RNA is synthesized in a single batch. In some embodiments, at least 800 mg RNA is synthesized in a single batch. In some embodiments, at least 900 mg RNA is synthesized in a single batch.

[0216] In some embodiments, the term "large-scale manufacturing" refers to the production of batches of at least 1g of RNA (e.g., mRNA). In some embodiments, at least 1g of mRNA is synthesized in a single batch. In some embodiments, at least 5g of RNA is synthesized in a single batch. In some embodiments, at least 10g of RNA is synthesized in a single batch. In some embodiments, at least 25g of RNA is synthesized in a single batch. In some embodiments, at least 50g of RNA is synthesized in a single batch. In some embodiments, at least 75g of RNA is synthesized in a single batch. In some embodiments, at least 100g of RNA is synthesized in a single batch. In some embodiments, at least 150g of RNA is synthesized in a single batch. In some embodiments, at least 200g of RNA is synthesized in a single batch. In some embodiments, at least 250g of RNA is synthesized in a single batch. In some embodiments, at least 500g of RNA is synthesized in a single batch. In some embodiments, at least 750g of RNA is synthesized in a single batch.

[0217] In some embodiments, the term "large-scale manufacturing" refers to the production of batches 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. Reaction vessel

[0218] The IVT reaction is carried out in a suitable reaction vessel, such as a bioreactor. To facilitate monitoring of the IVT reaction, a spectroscopic probe is typically inserted into the reaction vessel. The use of the spectroscopic probe allows the amount of one or more reactants and / or one or more products to be directly monitored during the IVT reaction process 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 an embodiment, 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. Therefore, 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 online or in situ.

[0219] In some embodiments, it may be more convenient to incorporate the spectroscopic probe into a bypass branching off from the reaction vessel. Placing the spectroscopic probe in the bypass may be beneficial to avoid, for example, background noise or other interference associated with the stirring means provided in the reaction vessel.

[0220] In some embodiments, the reaction vessel further comprises an access port for adding reactants during the IVT reaction.

[0221] In some embodiments, the reaction vessel is configured for fed-batch or continuous operation.

[0222] Suitable reaction vessel can be made of glass, plastic or stainless steel. In certain embodiments, reaction vessel can be sterilized and sealed to avoid contamination (e.g., disposable sterilizable and sealable plastic bag). In certain embodiments, reaction vessel can be heatable. In certain embodiments, reaction vessel is a bioreactor. Reaction volume

[0223] IVT reactions can be performed on a small scale, for example, during optimization of an IVT reaction with a specific RNA of interest (eg, mRNA for therapeutic use). In some embodiments, the volume of the reaction vessel is at least 2 mL.

[0224] More typically, the reaction vessel (e.g., bioreactor) is selected to be optimized to allow for scaling up of the IVT reaction to accommodate larger batches (e.g., for commercial production of mRNA). In some embodiments, the volume of the reaction vessel is at least 200 mL. For large-scale manufacturing, larger volumes may be selected. In some embodiments, the volume of the reaction vessel is 12.5 L to 2000 L. In some embodiments, the volume of the reaction vessel is 500 L, 1000 L, or 2000 L or more. Means for heating

[0225] Typically, IVT reactions occur at temperatures above room temperature, for example, between 32°C and 42°C (e.g., 35°C-39°C, or about 35°C, 36°C, 37°C, 38°C, or 39°C). Thus, in some embodiments, the reaction vessel (e.g., bioreactor) includes means for heating. Means for stirring the IVT reaction

[0226] In some embodiments, the IVT reaction occurs without stirring. In other embodiments, the IVT reaction is stirred, for example, at 100 rpm to 400 rpm.

[0227] Thus, in some embodiments, the reaction vessel (e.g., a bioreactor) includes means for providing agitation of the reaction mixture contained therein. For example, the reaction vessel can be programmable to provide agitation during the IVT reaction process. Thus, in some embodiments, the reaction vessel is configured to provide agitation. In some embodiments, the agitation is 100 rpm to 400 rpm. Examples

[0228] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention. Example 1. Quantitative monitoring of RNA, NTPs and Pi by spectroscopy

[0229] This example demonstrates the use of online spectroscopy to monitor products and reactants during an IVT reaction.

[0230] IVT reactions were performed using 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 MgCl2, 5 mM NaCl). UTP is a modified UTP in which uridine is replaced by a uridine analog (e.g., N1-methyl pseudouridine).

[0231] The coding region of the template encoded an exemplary approximately 1.9 kb mRNA, whose nucleotide sequence contained 504 A, 490 G, 568 C, and 379 U. The reaction was started by adding 1.1 ng / μL pyrophosphatase and 100 ng / μL RNA polymerase, followed by incubation at 37°C for 90 minutes. IVT reactions were either performed in a graduated cylinder with a total reaction volume of 2 mL without stirring, or in a bioreactor system with a reaction volume of 250 mL ( The mixture was carried out in a Sartorius reactor with stirring at 250 rpm.

[0232] Using ProCellics TMThe IVT reaction was analyzed online (i.e., in the reaction vessel) using a 785 nm excitation laser source with a power of 350 mW at the probe tip and a high sensitivity spectrometer with a peak at 150 cm -1 Up to 4000cm -1 Raman shift bandwidth (Stokes signal) sampling step 1 cm -1 , using a back-thinned charge-coupled device (CCD) detector. Immerse the spectrometer probe directly into a 25 mL glass test tube containing 2 mL of IVT reaction or a 250 mL The Raman measurements were isolated from any external light using an aluminum foil layer to ensure the integrity of the analysis. The integration time for each acquisition was 50 seconds (average of 10 spectra). The pre-processing step was performed using ProCellics TM Software (Resolution Spectroscopy Systems, Inc.) was used for the preprocessing. The first step was to generate derivatives (1st order, 15 cm -1 Step size, 2nd order polynomial); Step 2, calculate 3,100cm -1 Up to 3,600cm -1 A custom standard normal variate (SNV) between .

[0233] Empirical characteristic 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 is assigned to the phosphodiester bond (OPO symmetric stretching), and the peak at 1095 cm -1 The peak at is designated as PO2 - Symmetrical expansion (OPO - ), which is consistent with published literature. The intensity ratio of these two peaks gives structural information about RNA, namely the ratio between ordered and disordered structures. Since phosphodiester bonds are unique to RNA, the same characteristic frequency (810cm -1 During the IVT reaction, the -1 A new peak appeared at , and its area seemed to be related to RNA production. This peak was designated as H2PO4 - .

[0234] NTP can be divided into two groups, one containing purine bases (ATP and GTP) and one containing pyrimidine bases (CTP and UTP). Adenine is a 6-monosubstituted purine. It consists of a 6-monosubstituted purine at 633 cm -1The weak band observed at 780 cm-1 is indicated and can be attributed to the out-of-plane bending vibration of CH. Guanine is 6-oxo-2-aminopurine. CTP is indicated at 780 cm-1. -1 The medium intensity band at 786-789 cm is caused by the vibration of the cytosine ring. In the UTP spectrum, it can also be seen at about 786-789 cm due to the stretching of the C=C bond (carbons 5 and 6 of uracil). -1 After the IVT reaction, 1113 cm -1 -1115cm -1 The peak near the decrease is attributed to NTP consumption. This peak may be related to the PO2 - Retraction related.

[0235] Figure 1 Shows coverage of 800cm -1 Up to 1300cm -1 Figure 2. Example Raman spectra of the region. The dashed line represents the first spectrum recorded 500 seconds after the start of the IVT reaction. The solid dark grey line is the last spectrum of the IVT reaction recorded after 90 minutes. It can be seen that the spectral region corresponding to RNA and the reaction byproduct Pi shows an increase in intensity after 90 minutes, while the spectral region corresponding to NTP shows a decrease in intensity after 90 minutes.

[0236] This example demonstrates that online spectroscopy can be used to identify various components of an IVT reaction. "Online" in this context means that the spectroscopic probe is immersed in the solution in which the IVT reaction occurs (e.g., in the reaction vessel or in a bypass of the reaction vessel). Online spectroscopy can be used to monitor the production of RNA and the formation of the byproduct Pi. It can also be used to monitor the consumption of NTPs during the IVT reaction. The methods described herein are not limited to Raman spectroscopy, but can also be adapted to use other spectroscopic methods (particularly light scattering-based methods) to identify appropriate wavelength regions in a spectrum or series of spectra corresponding to the reactants or products of interest. Example 2. Monitoring changes in products and reactants using reference spectra

[0237] This example demonstrates that spectra acquired during an IVT reaction can be compared to predetermined reference spectra of reactants and products of the IVT reaction to monitor changes in those reactants and products over time.

[0238] To monitor changes in reactants and products in the reaction vessel, the spectrum obtained during the IVT reaction is compared to the predetermined spectrum of the reactant or product of interest using a percent weighted spectral difference (WSD) calculation. For quantification of spectral differences, the WSD calculation provides a weighting function based on relative signal amplitudes. WSD values ​​close to zero indicate a high similarity to the reference spectrum, while high WSD values ​​indicate a large difference between the obtained spectrum and the predetermined spectrum. The WSD value can be calculated using the following formula: A i and B i are the intensities of spectra A and B at wavelength i, respectively.

[0239] Using Raman spectroscopy as described in Example 1, 300 cm -1 Up to 3000cm -1 The wavelength region was selected to monitor the overall evolution of the spectrum during the IVT reaction. For the specific monitoring of RNA (product) and NTP (reactant), 801 cm -1 -831cm -1 Wavelength region and 1107cm -1 -1146cm -1 Wavelength region.

[0240] Figure 2 The 801 cm-1 region in panel (a) is shown during an IVT reaction performed as described in Example 1. -1 -831cm -1 RNA region and 1107 cm in sub-image (b) -1 -1146cm -1 Evolution of WSD values ​​in the NTP region. At 2 mL and 250 mL scales, the amount of RNA increased throughout the reaction and reached a plateau toward the end of the reaction time ( Figure 2 , sub-figure (a). This is accompanied by a steady decline in NTP over the same period ( Figure 2 , sub-figure (b)).

[0241] This example demonstrates that obtaining a series of spectra during an IVT reaction and comparing those spectra to predetermined reference spectra of reactants and products of the IVT reaction can be used to determine changes in the concentrations of reactants and products over the course of the IVT reaction. Example 3. Construction of Partial Least Squares (PLS) Model

[0242] This example demonstrates the construction of a partial least squares (PLS) model using predetermined spectra of reactants and products of an IVT reaction at specific concentrations. The PLS model can be used as a predictive dataset to determine the respective concentrations of reactants and products based on the spectra obtained during the IVT reaction.

[0243] Like linear regression, the partial least squares (PLS) model aims to calculate the set of parameters that connect the response variable (Y matrix) to the independent variables (X matrix). PLS finds a linear regression model by projecting the response and independent variables into a new space. The underlying relationships between the variables are set in this new space and then projected back into the original space. PLS regression can be used to predict the response variable Y from a large set of independent variables X by reducing the set of X variables into a smaller set of uncorrelated components. Least squares regression is performed on these components, thereby reducing multicollinearity between the X values.

[0244] PLS is particularly useful for finding patterns in spectral features when relative chemical data are available. The software (version 16) was used to construct PLS models of Raman spectroscopy data of 42 different mixtures containing different concentrations of NTP, H2PO4 - Each of the 42 mixtures was constructed as a D-optimal design using JMP software (version 14). The D-optimal design was created by arranging the maximum and minimum concentrations of each component (see Table 2). The spectra of each of the 42 mixtures were recorded as described in Example 1. Table 2: Concentrations of products and reactants

[0245] Based on the spectra of the 42 mixtures, a PLS model was constructed for each of the ATP-GTP, CTP-UTP, Pi and RNA parameters. Due to the high correlation of these parameters, the Raman features of adenine and guanine bases overlap and cannot be distinguished in the PLS model. The same situation was observed for cytosine and uracil bases. Therefore, 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. The PLS model was then used as a prediction data set to calculate the respective concentrations of reactants and products with respect to time. The PLS SIMCA file was imported into the ProCellics software of the Raman spectrometer to monitor the concentrations of reactants and products in the reaction vessel during the IVT reaction, as shown in Example 4. Example 4. Monitoring RNA production in a reaction vessel

[0246] This example demonstrates that spectroscopy can be used to determine RNA concentration within a reaction vessel during an IVT reaction.

[0247] At the beginning of the IVT reaction (t=0 minutes), the RNA concentration was zero and increased over time after the addition of RNA polymerase to initiate transcription, as described in Example 1. Using online Raman spectroscopy and the PLS model described in Example 3, the RNA concentration at 18.3 minutes was determined to be 2.8 g / L. The concentration increased to 4.1 g / L at 64.1 minutes. These values ​​were compared to offline quantification results using the RiboGreen assay. Aliquots were removed from the IVT reaction at different time points. The concentration calculated from the RiboGreen assay data was 2.0 g / L at 16 minutes and 3.5 g / L at 62 minutes. Figure 3 The concentration values ​​obtained using Raman spectroscopy are plotted relative to the concentration values ​​obtained using the RiboGreen assay.

[0248] For the RiboGreen assay, Reagent (Thermo Fisher Scientific) was diluted 200 times in TE buffer, and the reagent solution was added to an equal volume of RNA in TE. The samples in the microplate were incubated in the dark at room temperature for 5-30 min. The sample volume for microplate determination was 200 mL. Microplate determination was performed using a CytoFluor II fluorescence microplate reader (SoftMax Pro 6.5.1). The sample was excited at 495 nm and fluorescence was measured at 521 nm. A graph of the relationship between the integrated fluorescence emission intensity and the RNA concentration was drawn without subtracting background fluorescence. The relative error of the RiboGreen determination was 12%, while the relative error of the PLS model (as described in Example 3) was 11%.

[0249] This example demonstrates that spectroscopy can be used to determine RNA concentration during an IVT reaction in a reaction vessel. The online spectroscopy method produces concentrations comparable to standard offline methods (e.g., RiboGreen assay). Example 5. Monitoring NTP consumption and Pi production in a reaction vessel

[0250] This example demonstrates that spectroscopy can be used to monitor NTP consumption and inorganic phosphate (Pi) accumulation during an IVT reaction within a reaction vessel.

[0251] Magnesium (Mg 2+) is an essential cofactor for RNA polymerase and has a direct impact on the transcription rate and therefore the production of RNA. It can combine with pyrophosphate (PPi) and lead to the formation of an insoluble precipitate that affects the RNA yield during production. Pyrophosphatase is added to the IVT reaction to hydrolyze PPi and increase RNA yield. This results in the production of inorganic phosphate (Pi). At the beginning of the IVT reaction (t=0 minutes), the Pi concentration is zero and increases over time after the addition of RNA polymerase to initiate transcription, as described in Example 1. As ribonucleotides (NTPs) are incorporated into RNA, their concentration within the reaction vessel decreases.

[0252] The concentrations of ATP-GTP, CTP-UTP, and Pi within the reaction vessel were monitored using online Raman spectroscopy and the PLS model described in Example 3. As expected, the Raman spectral intensities in the selected spectral regions decreased and increased, respectively, indicating that the concentrations of ATP-GTP and CTP-UTP decreased and the concentration of Pi increased during the IVT reaction. Representative spectra are shown in Figure 2. Figure 4 The relative errors of the calculated values ​​were 14% and 13% for ATP-GTP and CTP-UTP concentrations, respectively, and 4% for Pi concentration.

[0253] This example demonstrates that spectroscopy can be used to monitor NTP and pyrophosphate concentrations during an IVT reaction in a reaction vessel. The on-line information provided by spectroscopy is valuable in determining whether NTP concentrations need to be adjusted. Example 6. Establishment of a kinetic model for IVT reactions

[0254] This example demonstrates how the kinetic model for the IVT reaction described in Example 1 is consistent with experimental observations using, inter alia, the spectroscopic methods described herein.

[0255] The reaction stoichiometry for the net synthesis of an RNA transcript consisting of n nucleotides is given by: 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 in each copy of the fully transcribed RNA, respectively, where the total number of nucleotides is equal to n(=nA+nG+nC+nU). Using the RNA encoded by the template plasmid described in Example 1, Reaction 1 adopts the following mechanism scheme: 504ATP+490GTP+568CTP+379UTP→1RNA+1940PPi+1940H + H +It can be represented alone, as present in solution, or considered as bound to PPi. The presence of pyrophosphate (PPi) was shown to inhibit the RNA synthesis reaction. This is why a second reaction is required to hydrolyze PPi to Pi with the help of pyrophosphatase. Reaction 2 adopts the following mechanistic scheme: 1940PPi+1940H2O→3880Pi

[0256] By using the NTP and Pi concentrations determined using the PLS model as described in Example 3, and assuming that reactions 1 and 2 occur during the IVT reaction, the best data fit resulted in predicted RNA and PPi concentrations as Figure 5 Based on this analysis, the following kinetic model can be formulated: r1=(4.795×10 -4 )×[ATP+GTP] 1 ×[CTP+UTP] 1 as well as r2=(1.805×10 -5 )×[PPi] 1 ×[H2O] 1 4.795×10 -4 and 1.805×10 -5 are the rate constants of r1 and r2, respectively, in g / (s×mol).

[0257] Using this empirical model, ATP-GTP, CTP-UTP, Pi and RNA concentrations were predicted. The RNA kinetic profile was compared with the concentration set calculated by the PLS model (indicated by open circles) and the RiboGreen quantitative values ​​described in Example 4 (indicated by open triangles) (see Figure 5 ). The observed agreement between the predictions (kinetic model) and the experimental data (Raman-PLS and RiboGreen) confirms the ability of spectroscopy combined with the PLS method described in Example 3 to quantify online RNA production.

[0258] In addition to estimating RNA content, the kinetic model also estimates the progression of other reactants and products, including PPi (see Figure 5 Figure 2 (e) shows that PPi increases first and then decreases slowly. This behavior seems to be consistent with reaction 1 (PPi production) and reaction 2 (PPi degradation by pyrophosphatase). + increase (see Figure 5 Panel (d)) is consistent with the drop in pH during the IVT reaction as observed previously. Example 7. Monitoring and Modeling the Occurrence of Turbidity

[0259] This example demonstrates that spectroscopy can be used to monitor the appearance of turbidity during an IVT reaction, which is caused by the accumulation of insoluble components such as Mg2PPi precipitates as a byproduct of the reaction.

[0260] The appearance of turbidity was monitored during the IVT reaction at three different temperatures (31°C, 37°C, 42°C). Absorbance measurements were performed during a 90-minute run time with a 2-minute interval between measurements. The SpectraMax M5 UV-Visible spectrophotometer (Molecular ) Turbidity was measured at 320 nm. The presence of insoluble components (e.g. Mg2PPi precipitate) was estimated by measuring the absorbance at 320 nm.

[0261] A kinetic model was developed by combining advanced kinetics and statistical analysis of stability data obtained at 31°C, 37°C, and 42°C. The rate of turbidity appearance is temperature dependent, allowing for Arrhenius-based modeling. The higher the temperature at which the IVT reaction is conducted, the faster the turbidity appears. Advanced kinetic modeling was applied by fitting the collected data to form a two-step kinetic model that describes the progression of the reaction as a function of time and temperature.

[0262] The model was used to predict long-term turbidity levels for up to 5 hours (see Figure 6 It is also used to estimate the safety zone defining the time-temperature domain in which the occurrence of turbidity can be prevented (see Figure 6 Component (b)).

[0263] This example demonstrates that spectroscopic measurement of turbidity during an IVT reaction can be used as a method to monitor the formation of reaction byproducts (e.g., Mg2PPi precipitates). Example 8. Scalability

[0264] This example demonstrates that the progress of an IVT reaction can be reproducibly monitored using the methods disclosed in the previous examples as the batch size is increased or the IVT reaction is performed using a fed-batch system.

[0265] RNA was quantitatively monitored during the IVT reaction using online Raman spectroscopy and the PLS model described in Example 3. The IVT reaction was performed as described in Example 1 for 2 hours, but using a different DNA template encoding an approximately 2 kb mRNA with a nucleotide sequence containing 609 A, 449 C, 522 G, and 392 U. As measured by the increase in RNA concentration, the RNA concentration increased significantly regardless of the batch size (150 mg, 1 g, or 20 g; see Figure 7 ) regardless of the IVT response progression.

[0266] The scalability of the monitoring method disclosed herein was also observed using another DNA template encoding approximately 2 kb of mRNA with a nucleotide sequence containing 554 A, 492 C, 517 G, and 406 U (see Figure 8 ). Comparable results between the two different batch sizes (1 g and 20 g) were obtained when monitoring the product RNA and PPi as well as the nucleotide reactants, as shown by the amount of GTP-ATP. For the 20 g batch, the IVT reaction was performed in a 5000 L reaction vessel.

[0267] Consistent with Example 4, RNA concentrations determined by online quantification (Raman) and offline quantification (RiboGreen assay) were comparable. Fig. 9 As shown, mRNA increases as the IVT reaction proceeds, and the IVT reaction is stopped after the addition of DNase I (to digest the template). After template digestion, proteinase K is added to digest the enzyme components, including RNA polymerase and DNase I. After adding DTT to quench the reaction, a decrease in the amount of mRNA is observed. This is because the volumetric DTT dilutes the sample, rather than the mRNA product being lost.

[0268] Furthermore, the applicability of the modeling system described in Example 3 was tested in IVT reactions with four different DNA templates. The spectra obtained could be fitted to the previously determined mathematical model parameters for predicting the progress of IVT reactions.

[0269] In addition, IVT reactions were also performed in reaction vessels using fed-batch operation. The monitoring methods described in the previous examples were also effective under these conditions.

[0270] This example demonstrates that the monitoring methods described in the previous examples can be used when the batch size is increased or when a fed-batch system is used for IVT reactions. Example 9. Monitoring a single NTP

[0271] This example demonstrates that spectroscopy can be used to monitor a single NTP.

[0272] Different concentrations of ATP and GTP were tested by spiking the reaction buffer to identify the reactant region. Raman spectra were acquired using a Kaiser Raman Rxn2 analyzer (Endress+Hauser). Using this system, Raman spectra were obtained at 1550 cm -1 -1600cm -1 and 650cm -1 -750cm -1 The wavelength range of ATP and GTP is determined as Fig.10 As shown. At 650cm -1 -750cm -1The superimposed spectra of ATP and GTP obtained in the range show discrete regions corresponding to each nucleotide, such as Fig.10 shown. Example 10. Monitoring the termination of a reaction in a reaction vessel

[0273] This example demonstrates that the spectroscopic methods described in the previous examples can also be used to monitor the termination of an IVT reaction.

[0274] The feasibility of using spectroscopy to monitor the termination of IVT reactions was investigated. Different concentrations of template (DNA plasmid), nuclease (DNase I), or RNA polymerase (SP6 RNA polymerase) were spiked into the reaction buffer to identify wavelengths suitable for monitoring these components.

[0275] Fig.11 Illustrative spectra obtained when testing DNA plasmids at concentrations of 0 mg / mL, 0.075 mg / mL, 0.15 mg / mL, 0.3 mg / mL, and 0.6 mg / mL are shown. The reaction buffer did not include RNA polymerase to avoid progression of the IVT reaction. A suitable wavelength region for monitoring DNA plasmids was identified as 505 cm -1 Up to 710cm -1 Other suitable wavelength regions for monitoring DNA plasmid quantity include 1325 cm -1 Up to 1365cm -1 and 1585cm -1 Up to 1720cm -1 Monitoring changes in DNA plasmid concentration can be used to monitor termination of the IVT reaction, for example, by the addition of a nuclease (eg, DNase I).

[0276] Fig.12 Illustrative spectra obtained when testing DNA enzyme I at concentrations of 0 ku / mL, 0.063 ku / mL, 0.125 ku / mL, 0.25 ku / mL, and 0.5 ku / mL are shown. The reaction buffer does not include a DNA template. A suitable wavelength region for monitoring nucleases such as DNA enzyme I is identified as including 450 cm -1 Up to 520cm -1 Other suitable wavelength regions for monitoring nuclease levels include 1000 cm -1 Up to 1090cm -1 and 2915cm -1 Up to 3000cm -1 The spectra obtained by monitoring DNase I can be used to determine whether it has been added, but can also detect its removal, for example, by digestion with a protease such as proteinase K.

[0277] Fig.13Illustrative spectra obtained when testing 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 are shown. The reaction buffer did not include a DNA template to prevent the progression of the IVT reaction. A suitable wavelength region for monitoring SP6 RNA polymerase was identified as 780 cm -1 Up to 1200cm -1 Another suitable wavelength region for monitoring RNA polymerase levels includes 1430 cm -1 Up to 1510cm -1 Monitoring 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 (eg, proteinase K).

[0278] Similar spiking experiments were also performed to identify wavelength regions for detecting proteinase K, which is added to digest RNA polymerase to terminate the IVT reaction or to digest DNase I and terminate the DNase I reaction. In some embodiments, the spectrum obtained in step (c) is used to monitor the amount or addition of proteinase. Suitable wavelength regions for monitoring proteinase K were identified to include 505 cm -1 Up to 610cm -1 , for example 550cm -1 Up to 600cm -1 Other suitable wavelength regions for monitoring protease levels include 715 cm -1 Up to 775cm -1 and 1385cm -1 Up to 1395cm -1 .

[0279] This example demonstrates that the spectroscopic method used to monitor IVT reactions can also be used to monitor the removal of DNA plasmids and enzyme components used to terminate the reaction.

Claims

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

2. The method of claim 1, wherein the reactant or the product is selected from RNA, pyrophosphate (PPi), H + , inorganic phosphate (Pi) and ribonucleotides (NTPs).

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

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

5. The method of any one of the preceding claims, wherein the method determines changes in the amount of more than one reactant or more than one product of the IVT reaction.

6. The method of claim 5, wherein the method determines changes in the amounts of more than one reactant and more than one product of the IVT reaction.

7. The method of claim 5 or 6, wherein the more than one reactant is adenosine triphosphate (ATP) and guanosine triphosphate (GTP).

8. The method of claim 5 or 6, wherein the more than one reactant is cytidine triphosphate (CTP) and uridine triphosphate (UTP).

9. The method of any one of claims 5-8, wherein the more than one product is RNA and Pi.

10. The method of any one of the preceding claims, wherein the reaction vessel is a bioreactor.

11. A method as claimed in any preceding claim, wherein the reaction vessel has an access port or bypass for inserting a spectroscopic probe.

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

13. A method as claimed in any preceding claim, wherein step (i) comprises obtaining a series of spectra over the course of the IVT reaction.

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

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. The method of any one of claims 13-15, wherein a plurality of spectral series are obtained over the course of the IVT reaction.

17. A method as claimed in any one of claims 13 to 16, wherein each spectrum in the series is pre-processed prior to step (ii).

18. A method as claimed in claim 17, wherein the spectra in the series are acquired continuously and the pre-processing comprises smoothing the spectra by applying a digital filter which fits the continuously acquired spectra with a low-degree polynomial by a linear least squares method.

19. A method as claimed in any one of the preceding claims, wherein prior to step (ii), the spectrum or series of spectra obtained in step (i) is normalized relative to a wavelength region with reduced or no background noise.

20. The method of claim 17 or 18, wherein prior to any pre-processing step, the spectral series is normalized relative to a wavelength region with reduced or no background noise.

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

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

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

25. The method of any one of the preceding claims, wherein the predetermined reference spectrum is associated with a specific concentration of the reactant or the product.

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

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

28. A method as claimed in claim 26 or 27, wherein the concentration of the reactant or the product is determined from a predetermined series of reference spectra using a partial least squares (PLS) model, wherein each spectrum in the series is associated with a different concentration of the reactant or the product.

29. The method as claimed in any one of the preceding claims, wherein the spectrum, the series of spectra and the predetermined reference spectrum / the series of predetermined reference spectra, where applicable, are obtained using a spectrometer for vibrational spectroscopy.

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

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

32. The method of any one of the preceding claims, wherein the spectrum obtained in step (i) spans a wavelength region suitable for monitoring the overall evolution of reactants and products during the IVT reaction.

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

34. The method of claim 32 or 33, wherein the wavelength region includes 300 cm -1 Up to 3000cm -1 , and the spectrum was obtained using a Raman spectrometer.

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

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

37. The method of claim 36, wherein the wavelength region includes 801 cm -1 Up to 831cm -1 , and the spectrum was obtained using a Raman spectrometer.

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

39. The method of claim 38, wherein the wavelength region includes 875 cm -1 Up to 900cm -1 , and the spectrum was obtained using a Raman spectrometer.

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

41. The method of claim 40, wherein the wavelength region comprises 600 cm -1 Up to 1,300cm -1 , and the spectrum was obtained using a Raman spectrometer.

42. The method of claim 40 or 41, wherein the wavelength region comprises or consists of: 1107cm -1 Up to 1146cm -1 or 1113cm -1 Up to 1115cm -1 , to determine the amount of NTP.

43. The method of any one of claims 40-42, wherein the wavelength region comprises or consists of: 633cm -1 , to determine the amount of ATP.

44. The method of any one of claims 40-42, wherein the wavelength region comprises or consists of: 1300 cm -1 Up to 1600cm -1 , to determine the amount of ATP and GTP.

45. The method of any one of claims 40-42, wherein the wavelength region comprises or consists of: 780cm -1 , to determine the amount of CTP.

46. ​​The method of any one of claims 40-42, wherein the wavelength region comprises or consists of: 786cm -1 Up to 789cm -1 , to determine the amount of UTP.

47. The method of any one of claims 40-42, wherein the wavelength region comprises or consists of: 1230cm -1 Up to 1245cm -1 to determine the amount of CTP and UTP.

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

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

50. The method of claim 49, wherein the insoluble precipitate is Mg2PPi.

51. The method of claims 48-50, wherein turbidity is measured using a turbidimeter, UV spectrometer, or nephelometer.

52. The method of claim 51, wherein the turbidity is measured using a UV spectrometer at a wavelength in the region of 290 nm to 410 nm, for example, at 310 nm, 320 nm, 330 nm, 340 nm or 350 nm.

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

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

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

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

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. The method of claim 57, wherein the RNA batch produced by the IVT reaction is discarded.

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

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

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

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

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

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

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

67. The method of any one of claims 54-66, wherein the spectra obtained in step (c) are used to monitor the amount of DNA template.

68. The method of claim 67, wherein the wavelength region obtained in step (c) comprises or consists of: 500 cm -1 Up to 710cm -1 , to determine the amount of DNA template.

69. The method of any one of claims 54-68, wherein the spectra obtained in step (c) are used to monitor the amount of RNA polymerase, optionally wherein the RNA polymerase is SP6 RNA polymerase.

70. The method of claim 69, wherein the wavelength region obtained in step (c) comprises or consists of: 780cm -1 Up to 1200cm -1 or 1430cm -1 Up to 1510cm -1 , to determine the amount of RNA polymerase.

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

72. The method of claim 71, wherein the spectra obtained in step (c) are used to monitor the amount or addition of the nuclease.

73. The method of claim 72, wherein the wavelength region obtained in step (c) comprises or consists of: 450 cm -1 Up to 520cm -1 to determine the amount or addition of the nuclease.

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

75. The method of claim 74, wherein the spectra obtained in step (c) are used to monitor the amount or addition of the protease.

76. The method of claim 75, wherein the wavelength region obtained in step (c) comprises or consists of: 505cm -1 Up to 610cm -1 , optional 550cm -1 Up to 600cm -1 to determine the amount or addition of the protease.

77. A method for producing RNA using an in vitro transcription (IVT) reaction, the method comprising: a) monitoring the production of the RNA in the reaction vessel by: (i) obtaining a spectrum of the RNA during the IVT reaction to determine a first value; and (ii) comparing the first value obtained in step (i) with a second value derived from a predetermined reference spectrum of RNA; and b) purifying the RNA if the first value equals or exceeds the second value.

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

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

80. A method as claimed in claim 77, wherein steps (i) and (ii) are repeated at equally spaced intervals, and if the first value obtained at each interval is equal to or exceeds the second value at the corresponding interval derived from the predetermined reference spectrum, the RNA is purified.

81. The method of any one of claims 77-80, wherein the first value and the second value are considered equal if they are within one standard deviation of each other.

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

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

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

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

86. The method of any one of claims 53-85, wherein the reactants in the IVT reaction include magnesium (Mg 2 + ) and NTP.

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

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

89. The method of claim 87 or 88, wherein the IVT reaction replenishes NTPs when NTP concentrations approach depletion.

90. The method of claim 89, wherein depletion is approached when the concentration of the NTPs is no more than 5% of the concentration of NTPs present when the IVT reaction is initiated.

91. The method of claim 89, wherein depletion is approached when the concentration of NTP does not exceed 5 mM.

92. The method of claim 91, wherein depletion is approached when the concentration of NTP does not exceed 3 mM.

93. The method of claim 86, wherein the IVT reaction continuously replenishes NTPs during the process of producing the RNA.

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

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

96. 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 a range of 20%-100%, 20%-75%, or 25%-50% of that present at initiation of the IVT reaction.

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

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

100. The method of claim 99, wherein the spectra obtained in step (c) are used to monitor the amount of the DNA template.

101. The method of claim 100, wherein the wavelength region obtained in step (c) comprises or consists of: 500 cm -1 Up to 710cm -1 , to determine the amount of the DNA template.

102. The method of any one of claims 99-101, wherein the spectra obtained in step (c) are used to monitor the amount of RNA polymerase, optionally wherein the RNA polymerase is SP6 RNA polymerase.

103. The method of claim 102, wherein the wavelength region obtained in step (c) comprises or consists of: 780cm -1 Up to 1200cm -1 or 1430cm -1 Up to 1510cm -1 , to determine the amount of RNA polymerase.

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

105. The method of claim 104, wherein the spectra obtained in step (c) are used to monitor the amount or addition of the nuclease.

106. The method of claim 105, wherein the wavelength region obtained in step (c) comprises or consists of: 450 cm -1 Up to 520cm -1 to determine the amount or addition of the nuclease.

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

108. The method of claim 107, wherein the spectra obtained in step (c) are used to monitor the amount or addition of the protease.

109. The method of claim 108, wherein the wavelength region obtained in step (c) comprises or consists of: 505cm -1 Up to 610cm -1 , optional 550cm -1 Up to 600cm -1 to determine the amount or addition of the protease.

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