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US 2025/0369028 A1Published application
Continuous in vitro transcription process and apparatus
AssigneeModernaTX, Inc.
InventorsMatthew Burak, Margaret Franklin, Matthew Heinrich, Tahir Kapoor, Justin Riopel, Yuxin He
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Abstract
Some aspects of the disclosure relate to continuous in vitro transcription (IVT) methods in which Raman spectra are obtained to monitor the progress of an IVT reaction and adapt residence time accordingly. Also provided are continuous IVT reaction apparatuses for monitoring by Raman spectroscopy.
Claims25
An in vitro transcription (IVT) method, the method comprising:
- (i) in a continuous reaction apparatus, incubating an IVT reaction mixture comprising a buffer, magnesium, a DNA, an RNA polymerase, a cap analog, adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP), whereby the RNA polymerase transcribes the DNA to produce an mRNA;
- wherein the IVT reaction mixture is formed by adding a first feed solution to the continuous reaction apparatus at a first input feed rate and a second feed solution to the continuous reaction apparatus at a second input feed rate,
- wherein the IVT reaction mixture is output from the continuous reaction apparatus at a first output flow rate;
- (iii) obtaining Raman spectra from the IVT reaction mixture over time;
- (iv) determining a reaction rate and a target endpoint from the Raman spectra;
- (v) determining a target residence time from the reaction rate and target endpoint;
- (vi) modifying the first input feed rate, second input feed rate, and/or first output flow rate such that a residence time of the IVT reaction mixture in the continuous reaction apparatus is 80% to 120% of the target residence time.
4 more elements
An in vitro transcription method, the method comprising:
- (i)(a) in a preliminary reaction apparatus, incubating a preliminary in vitro transcription (IVT) reaction mixture comprising a buffer, magnesium, a DNA, an RNA polymerase, a cap analog, adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP), whereby the RNA polymerase transcribes the DNA to produce an mRNA;
- (i)(b) obtaining Raman spectra from the preliminary IVT reaction mixture over time;
- (i)(c) determining a reaction rate and a target endpoint from the Raman spectra;
- (i)(d) determining a target residence time from the reaction rate and target endpoint;
- and
- (ii) in a continuous reaction apparatus comprising a plug flow reactor (PFR), incubating an in vitro transcription (IVT) reaction mixture flowing through the PFR at 80% to 120% of the target residence time, the IVT reaction mixture comprising a buffer, magnesium, a DNA, an RNA polymerase, a cap analog, adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP), whereby the RNA polymerase transcribes the DNA to produce the mRNA,
- wherein the IVT reaction mixture is output from the continuous reaction apparatus at a first output flow rate.
4 more elements
An in vitro transcription (IVT) method, the method comprising, in a continuous reaction apparatus comprising a plug flow reactor (PFR):
- (i) incubating an in vitro transcription (IVT) reaction mixture flowing through the PFR with a residence time, the IVT reaction mixture comprising a buffer, magnesium, a DNA, an RNA polymerase, a cap analog, adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP);
- (ii) obtaining Raman spectra from the IVT reaction mixture (a) at two or more points along the PFR separated by a predetermined distance, or (b) outlet location of the PFR, over time;
- (iii) determining a reaction rate and a target endpoint from the Raman spectra;
- (iv) determining a target residence time from the reaction rate and target endpoint;
- and
- (v) modifying the residence time such that the IVT reaction mixture flows through the PFR with 80% to 120% of the target residence time,
- wherein the IVT reaction mixture is output from the continuous reaction apparatus at a first output flow rate.
4 more elements
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An apparatus comprising:
- (i) a first feed solution container;
- (ii) a second feed solution container;
- (iii)(a) a continuous in vitro transcription (IVT) reaction apparatus fluidically coupled downstream of both the first feed solution container and the second feed solution container which is configured to receive a first mixed inlet stream;
- and
- (iii)(b) a Raman sensor coupled to the continuous IVT reaction apparatus.
2 more elements
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-37. (canceled)
Dependencies are inferred from the claim text.