Continuous in-vitro transcription system

Abstract

The single use system for continuous in-vitro transcription of RNA biomolecules from an immobilized DNA template includes a pair of recirculation loops, each recirculation loop powered by at least one low shear pump controlled by a singular use back pressure control valve. The single use system for continuous in-vitro transcription of RNA biomolecules from an immobilized DNA template further includes a single-use sensor controlling flow and the pressure within each recirculation loop. The single use system for continuous in-vitro transcription of RNA biomolecules from an immobilized DNA template includes a bypass valve opening on each recirculation loop permitting a stream from each loop releases to join in a residence spool, a mixture will preheat to a target temperature of a reactor.

Description

CONTINUOUS IN-VITRO TRANSCRIPTION SYSTEMFIELD OF THE INVENTION

[0001] The present invention pertains a Continuous In-Vitro Transcription System with an immobilized DNA template.BACKGROUND

[0002] In-Vitro Transcription (IVT) is a widely used laboratory technique to synthesize messenger ribonucleic acid (mRNA) molecules from a deoxyribonucleic acid (DNA) plasmid template via enzymatic reactions outside of a living cell. This technique allows for the generation of custom designed mRNA sequences that are critical for research applications and for the production of mRNA therapeutics.

[0003] In the production of mRNA therapeutics for clinical and commercial stage applications, the reaction is typically done batchwise with all buffers, reactants, and enzymes added to a heated vessel, typically single-use plastic or glass, such as a shaker flask in an incubator or a rocking bioreactor, and mixed until the reaction has completed. The reaction is then quenched with an appropriate buffer and the product pool is collected and sent to downstream processing. This setup presents challenges as the process scales larger, with many companies opting to scale out instead of scale up.

[0004] An unwanted byproduct of the IVT process is double stranded RNA (dsRNA). dsRNA is a major impurity that can result in lower mRNA expression and produce an immunogenic response in the patient. Data suggests that immobilizing the plasmid DNA template on a substrate can lead to lower amount of dsRNA impurity generation.

[0005] With a need to transition existing RNA synthesis processes to larger scales for commercial manufacturing while limiting the formation of potentially harmful byproducts, a new system is needed to enable the end-user to seamlessly scale their production workflows as they progress from pre-clinical research to cGMP manufacturing.

[0006] A multi-configurable single-use system design optimized for the batch, continuous / semi -continuous in-vitro transcription of RNA molecules utilizing a looped pipe reactor with ability to contain or not contain an immobilized DNA template, and devices for circularization of113401472.1the RNA molecules post-transcription when reconfigured can additionally be used for amplification, manufacture, and linearization of DNA plasmids.SUMMARY

[0007] The single use system for continuous in-vitro transcription of RNA biomolecules from an immobilized DNA template includes a pair of recirculation loops, each recirculation loop powered by at least one low shear pump controlled by a singular use back pressure control valve. The single use system for continuous in-vitro transcription of RNA biomolecules from an immobilized DNA template further includes a single-use sensor controlling flow and the pressure within each recirculation loop. The single use system for continuous in-vitro transcription of RNA biomolecules from an immobilized DNA template includes a bypass valve opening on each recirculation loop permitting a stream from each loop releases to join in a residence spool, a mixture will preheat to a target temperature of a reactor.BRIEF DESCRIPTION OF DRAWINGS

[0008] In the following, the present invention is described in more detail with references to the drawings in which:

[0009] Figure 1 illustrates a schematic diagram for a Single-Use Continuous IVT Skid;

[0010] Figure 2 illustrates a schematic diagram for a Single-Use Continuous IVT Skid with NTP Pump Block;

[0011] Figure 3 illustrates a schematic diagram for a Single-Use Continuous IVT System with Circularization Loop; and

[0012] Figure 4 illustrates a schematic diagram for a Continuous DNA Amplification with Linearization loop.DETAILED DESCRIPTION OF THE EMBODIMENTS

[0013] The following invention is a single-use system for continuous in-vitro transcription of RNA biomolecules from an immobilized DNA template.213401472.1

[0014] The single use design is designed to allow the user to replicate the modularity and multi operational functionality of the device across tubing inner diameter scales from 1 / 8” - 1 / 2” tubing scales.

[0015] Figure 1 displays the proposed diagram of the system while Figures 2-3 display the system with additional add-on features.

[0016] Nucleotide Triphosphate (NTP) reagents and enzyme buffers in single use bags or mixers flow through each recirculation loop powered by a low shear pump and controlled by a single use back pressure control valve until they reach the target flow rate determined by the end user.

[0017] Flow and pressure in the loops are monitored by single-use sensors. Once each loop’s target flow rate has been met, the bypass valve is opened on each loop allowing the two streams to join in a residence spool, where the mixture will be preheated to the target temperature of the reactor. The spool temperature is monitored by two temperature sensors in the spool and the flow of the pre-heated fluid is controlled via a single-use fluid control valve.

[0018] Once target temperature is met, the fluid mixture is sent to a reactor in the form of a looped pipe containing the immobilized DNA template. Residence time in the looped pipe is controlled by a low shear pump on the loop's outlet that will recirculate the mixture.

[0019] Once the reaction time or other sensing criteria are met, the 3 way bypass valve opens allowing the collection of the mRNA mixture. The reaction mixture is quenched with the appropriate buffer as determined by the end user as it is collected into a single-use mixer or bag or sent further downstream for circularization.

[0020] In the instance of the device with the NTP pump block, the end user is able to individually adjust the concentration of the NTPs in the reactor based on sensing or other recipe criteria.

[0021] In the instance of the device with the circularization loop add-on, the contents from the IVT looped pipe reactor are mixed in-line with a circularization buffer before being heated in the circularization looped pipe reactor to the target temperature for the target time as determined by the end user. Once reaction criteria have been met, the 3 -way valve opens to the collection vessel.

[0022] The looped pipe reactor is designed to use single-use sensors to facilitate in-process monitoring and control. The sensor types include but are not limited to UV-Vis, Raman, Conductivity, pH, Pressure, and Temperature, Conductivity and other sensors.313401472.1

[0023] The looped pipe reactor is made of a biocompatible ceramic for heat transfer, examples include but are not limited to alumina and zirconia.

[0024] The DNA template is immobilized on a resin made of agarose, or a similar polysaccharide or synthetic polymer.

[0025] The device is designed to be configurable to fit a typical manufacturing facility and workflow, and can be configured to sit on a lab bench, in a pharmaceutical isolator, or as an independent mobile system on castors.

[0026] The device is designed to be fully autonomous and driven by process equipment and control modules, phases, recipes, operations, and unit operations in accordance with general system control standards.

[0027] In a separate instance of the device designed for DNA amplification, the amplification enzyme is immobilized on a resin made from agarose or similar polysaccharide or synthetic polymer, while the DNA template and dNTPs and primer sequence molecules flow over the resin bed in the looped pipe reactor, replicating the target DNA template. An additional loop housing having a linearization enzyme can also be configured to linearize a supercoiled plasmid DNA construct post amplification. This instance is depicted in Figure 4.

[0028] The automated control of the device may be driven either through a locally based PLC or via a cloud driven communication protocol.

[0029] The automated control of the device may incorporate and control outside subsystems such as mixing vessels, weigh scales, reactors, pumps, mechanical drives, sensing transmitters which are not local to the device but necessary to complete the specified unit operation. This control may either be facilitated via hardware signal communication or cloud driven signal communication protocols.

[0030] The above description is only for the specific embodiment of the present invention and is not intended to limit the scope of the present invention. The equivalent changes or modifications made by the structures, features and principles of the present invention should be included in Within the scope of the patent application of the present invention.413401472.1

Claims

WHAT IS CLAIMED IS:

1. A single use system for continuous in-vitro transcription of RNA biomolecules from an immobilized DNA template, comprises: a pair of recirculation loops, each recirculation loop powered by at least one low shear pump controlled by a singular use back pressure control valve; a single-use sensor controlling flow and the pressure within each recirculation loop; and a bypass valve opening on each recirculation loop permitting a stream from each loop releases to join in a residence spool, a mixture will preheat to a target temperature of a reactor.

2. The single use system for continuous in-vitro transcription of RNA biomolecules from the immobilized DNA template of claim 1, includes a pair of two temperature sensors in the mixture and a flow of the mixture is controlled by a single-use fluid control valve.

3. The single use system for continuous in-vitro transcription of RNA biomolecules from the immobilized DNA template of claim 2, includes the mixture is distributed to a looped pipe once the target temperature is met.

4. The single use system for continuous in-vitro transcription of RNA biomolecules from the immobilized DNA template of claim 3, includes the looped pipe containing the immobilized DNA template.

5. The single use system for continuous in-vitro transcription of RNA biomolecules from the immobilized DNA template of claim 4, includes residence time in the looped pipe is controlled by a low shear pump on a looped pipe outlet that will recirculate the mixture.

6. The single use system for continuous in-vitro transcription of RNA biomolecules from the immobilized DNA template of claim 5, wherein when a sensing criteria is achieved, a bypass valve opens allowing a collection of the mixture.513401472.

17. The single use system for continuous in-vitro transcription of RNA biomolecules from the immobilized DNA template of claim 6, includes a buffer quenches the mixture and collected in a single-use mixer or continues downstream for circularization.

8. The single use system for continuous in-vitro transcription of RNA biomolecules from the immobilized DNA template of claim 7, includes an NTP pump block, the NPT pump block individually adjust a concentration of NTPs in the reactor.

9. The single use system for continuous in-vitro transcription of RNA biomolecules from the immobilized DNA template of claim 8, includes a circularization loop add-on, a content from a IVT looped pipe reactor are mixed in-line with a circularization buffer before being heated in the IVT looped pipe reactor to the target temperature for a target time.

10. The single use system for continuous in-vitro transcription of RNA biomolecules from the immobilized DNA template of claim 9, includes a 3-way valve to open to a collection vessel.

11. The single use system for continuous in-vitro transcription of RNA biomolecules from the immobilized DNA template of claim 10, includes a looped pipe reactor is designed to use singleuse sensors to facilitate in-process monitoring and control.

12. The single use system for continuous in-vitro transcription of RNA biomolecules from the immobilized DNA template of claim 11, wherein a sensor type includes a UV-Vis sensor , a Raman sensor , a Conductivity sensor ,a pH sensor, a Pressure, and Temperature sensor.

13. The single use system for continuous in-vitro transcription of RNA biomolecules from the immobilized DNA template of claim 12, wherein the looped pipe reactor is made of a biocompatible ceramic for heat transfer.

14. The single use system for continuous in-vitro transcription of RNA biomolecules from the immobilized DNA template of claim 13, wherein the biocompatible ceramic is composed from a group of alumina and zirconia.613401472.

115. The single use system for continuous in-vitro transcription of RNA biomolecules from the immobilized DNA template of claim 14, wherein the immobilized DNA template is positioned on a resin made of agarose, or a similar polysaccharide or synthetic polymer.

16. The single use system for continuous in-vitro transcription of RNA biomolecules from the immobilized DNA template of claim 15, wherein the system is configurable to sit on a lab bench, in a pharmaceutical isolator, or as an independent mobile system on castors.713401472.1

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