Method for continuous production of biomolecules

Abstract

The invention relates to a system (1) for the continuous production of biomolecules, comprising a feed tank (2) adapted to store products and a reaction chamber (4) designed to feed products from said feed tank, the products forming a reaction phase in the reaction chamber, said reaction chamber being designed to produce said biomolecules from said reaction phase. The system of the invention comprises a metering tank (6) in fluid communication with the reaction chamber and adapted to store an immiscible phase, and a monitoring and control unit (2) arranged to control the injection flow rate of the immiscible phase into the reaction chamber, so as to maintain a specific filling level of the reaction chamber constant depending on the amount of the reaction phase to be injected into the reaction chamber to produce a specific amount of biomolecules.

Description

Technical Field

[0001] The present invention relates generally to the field of in vitro production of biomolecules, and in particular to the production of mRNA into lipid nanoparticles for therapeutic applications such as vaccines. Background Art

[0002] Scientific and technological advances in recent years have made biomolecules promising candidates for a variety of uses, including diagnostic applications and therapeutic products such as vaccines.

[0003] Driven by major scientific-technological-medical advances, the rise of new therapies targeting genetic mechanisms, such as gene replacement, correction or regulation, protein expression, provides new opportunities for innovative treatments as well as emergency response in epidemic crisis situations. In this context, various methods have been developed to produce mRNA on a large scale. Most current methods utilize in vitro enzymatic reactions to synthesize mRNA from self-replicating DNA templates, followed by encapsulation of total RNA into lipid nanoparticles as immiscible delivery vehicles.

[0004] These methods are costly, require highly skilled personnel, generate hazardous waste streams that must be disposed of, and productivity is critically dependent on the performance of the enzymatic reactions, the ability to remove process and product related impurities, and the control and performance of encapsulation into lipid nanoparticles.

[0005] Furthermore, current practice is to produce mRNA in batch mode, which leaves little flexibility in changing the scale of production: for each scale, the process must be significantly adapted and the changes must be approved by regulators.

[0006] The recent pandemic has demonstrated the need to accelerate the development of new vaccines and make them available to the general public.

[0007] In this context, WO2021212034 describes a new method for producing mRNA in an in vitro system using continuous flow production.

[0008] The system described in WO2021212034 has a reaction chamber in which in vitro transcription is performed to continuously produce mRNA.

[0009] The reaction chamber runs continuously so that new input material can be injected while mRNA is being produced.

[0010] The system is particularly suitable for producing large quantities of mRNA, such as millions of doses in a pandemic situation, but would not be suitable for producing small quantities of mRNA without wasting large amounts of enzymes and buffers, such as thousands of doses in early clinical trials.

[0011] In practice, as mRNA production is scaled up, small quantities will often be produced in smaller batch reactors with volumes appropriate for the desired yield.

[0012] Therefore, the system described in WO2021212034 is not suitable for quickly processing and manufacturing different quantities of mRNA at reduced costs. Summary of the invention

[0013] SUMMARY OF THE INVENTION Therefore, an object of the present invention is to solve the above-mentioned problems.

[0014] To this end, the present invention relates to a system for the continuous manufacture of biomolecules, the system comprising a feed tank suitable for storing a product and a reaction chamber designed to be fed with the product from the feed tank, the product forming a reaction phase in the reaction chamber, the reaction chamber being designed to manufacture the biomolecule from the reaction phase, characterised in that the system comprises a metering tank fluidically connected to the reaction chamber and suitable for storing an immiscible phase that is immiscible with the reaction phase, the system comprising a monitoring and control unit, the monitoring and control unit being arranged to control the injection flow rate of the immiscible phase into the reaction chamber so as to maintain a specific filling level of the reaction chamber according to the amount of reaction phase to be injected into the reaction chamber to manufacture a specific amount of biomolecules, such that the lower the amount of reaction phase to be injected into the reaction chamber, the higher the amount of immiscible phase to be injected.

[0015] The idea behind the present invention is to use a single system that is suitable for the production of small to large quantities of biomolecules, or vice versa.

[0016] The idea is more specifically to dimension the system for large-scale manufacturing of biomolecules while allowing small-scale production of biomolecules by maintaining the same reaction conditions and therefore the same quality results (concentration, purity, production time, etc.), i.e. the reaction phases must always have the same reaction conditions.

[0017] For this reason, in the case of small production runs, even on a scale lower than that for which the reaction chamber is normally designed, the immiscible phase is injected to fill the unused volume of the reaction phase in the reaction chamber.

[0018] Since the two phases are immiscible, the reaction phase operating in the reaction chamber is always contained in a volume suitable for a stable and efficient reaction.

[0019] Thus, the system of the present invention is advantageously suitable for producing different quantities of biomolecules, for example at different stages of clinical development of a vaccine.

[0020] The idea is also to use a single system to produce a selected amount of biomolecules within a specific constant residence time. This means that the system according to the invention is designed to produce small or large amounts of biomolecules within the same residence time.

[0021] The system according to the present invention may also have the following features:

[0022] the system comprises a valve at the outlet of the reaction chamber, and the monitoring and control unit is arranged to receive data indicative of the presence of the reaction phase at the outlet of the reaction chamber and to subsequently control the valve to separate the immiscible phase from the reaction phase.

[0023] Reaction phase separation;

[0024] - the data representing the presence of the reactive phase at the outlet of the reaction chamber are based on the presence of the reactive phase in the reaction chamber.

[0025] The duration of stay is calculated;

[0026] - the system comprises a mixing chamber in fluid communication between the feed tank and the reaction chamber, the mixing chamber being designed to be fed with the products from the feed tank and to mix the products to form the reaction phase and then feed the reaction phase to the reaction chamber, the monitoring and control unit being arranged to control in real time the injection flow rate of the products from each feed tank so as to maintain a constant ratio between the products in the mixing chamber;

[0027] - the system comprises a first chromatographic device, the first chromatographic device comprising a first column in fluid communication between a reaction chamber and a collection tank and a second column in fluid communication between the first column and the collection tank, the first chromatographic device comprising a first temporary storage tank in fluid communication between the first column and the second column, the first chromatographic device further comprising a first outlet valve at the outlet of the first column, the first outlet valve being designed to switch the fluid communication from the first column to the first temporary storage tank or from the first column to the collection tank, and the first chromatographic device comprising a first inlet valve at the inlet of the second column, the first inlet valve being designed to open or close

[0028] fluid communication between the first temporary storage tank and the second column;

[0029] the first chromatographic device comprises a second temporary tank in fluid communication between the second column and the first column, the first chromatographic device comprises a second outlet valve at the outlet of the second column, the second outlet valve being designed to switch the fluid communication from the second column to the second temporary tank or from the second column to the collection tank, the first chromatographic device also comprises a second inlet valve at the inlet of the first column, the second inlet valve being designed to open or

[0030] closing fluid communication from the second temporary storage tank to the first column;

[0031] the first chromatography device comprises means for measuring the concentration of biomolecules at the outlet of the first column or the second column, and wherein the monitoring and control unit is arranged to control the outlet valve at the outlet of the first column or the second column so as to feed the first temporary storage tank or the second temporary storage tank respectively when the concentration of biomolecules in the front fraction and the back fraction of the eluted product fraction from the first column or the second column is above a certain predetermined concentration threshold, or when

[0032] feeding the waste tank when the concentration of the biomolecule is below the specific predetermined concentration threshold;

[0033] - the system comprises a metering buffer tank suitable for storing a replenishing buffer, the metering buffer tank being fluidically connected to the first column, and wherein the monitoring and control unit is arranged to control the injection flow rate of the replenishing buffer from the metering buffer tank to the first column so as to maintain a specific filling level of the column according to the amount of reaction phase to be injected into the column, such that the lower the amount of reaction phase to be injected into the first column, the higher the buffer to be injected into the first column

[0034] The higher the amount of liquid;

[0035] - the system comprises an online diafiltration system in fluid communication between the purification device and the lipid nanoparticle formulation system, the diafiltration system comprising several stages designed to pass a reaction phase with an exchange buffer, the monitoring and control unit being configured to receive data representing a concentration of a specific biomolecule to be used by the lipid nanoparticle formulation system to form lipid nanoparticles, the monitoring and control unit being configured to control the concentration of the reaction phase in the final stage

[0036] injection of an exchange buffer to dilute the biomolecule at the specified biomolecule concentration;

[0037] The system comprises a purification system arranged at the outlet of the reaction chamber, the purification system being designed to

[0038] Separation of the manufactured biomolecules from the rest of the reaction phase;

[0039] - said system comprises means for measuring the specific concentration of each product in said remaining part of the reaction phase, and said monitoring and control unit is arranged to control the injection of said remaining part of the reaction phase in the mixing chamber.

[0040] The invention also extends to a method for continuously manufacturing a biomolecule using the system of the invention, wherein the produced biomolecule is RNA, such as mRNA or a protein or DNA, and wherein the manufactured biomolecule may be a therapeutic agent, such as a vaccine.

[0041] More specifically, the method for continuous production of biomolecules of the present invention comprises the following steps:

[0042] - feeding the reaction chamber with a product from a feed tank, said product forming a reaction phase in the reaction chamber, said biomolecule being manufactured from said reaction phase in the reaction chamber, storing an immiscible phase immiscible with the reaction phase in a metering tank fluidically connected to the reaction chamber, and controlling the injection flow rate of the immiscible phase into the reaction chamber by means of a monitoring and control unit so as to maintain a specific filling level of the reaction chamber according to the amount of reaction phase to be injected into the reaction chamber to manufacture a specific amount of biomolecules, such that the lower the amount of reaction phase to be injected into the reaction chamber, the higher the amount of immiscible phase to be injected.

[0043] The method of the present invention may further comprise the following steps:

[0044] - receiving in the monitoring and control unit data indicating the presence of the reaction phase at the outlet of the reaction chamber and subsequently controlling with the monitoring and control unit a valve at said outlet of the reaction chamber in order to separate the immiscible phase from the reaction phase;

[0045] - the data representing the presence of the reactive phase at the outlet of the reaction chamber are based on the presence of the reactive phase in the reaction chamber.

[0046] The duration of stay is calculated;

[0047] - feeding the product from the feed tank to a mixing chamber in fluid communication between the feed tank and the reaction chamber, mixing the products to form the reaction phase, and then feeding the reaction phase to the reaction chamber, and controlling the injection flow rate of the product from each feed tank in real time with the monitoring and control unit so as to maintain the mixing chamber

[0048] A constant ratio between the products in

[0049] - performing a chromatography step comprising separating the product fraction eluted from the first column into a front fraction and a rear fraction of the product fraction eluted from the first column;

[0050] directing the front fraction and the rear fraction from the first column to a first temporary storage tank, and once the front fraction and the rear fraction are both stored in the temporary storage tank, directing the front fraction and the rear fraction from the first temporary storage tank to a second column;

[0051] - separating the product fraction eluted from the second column into a front fraction and a rear fraction of the product fraction eluted from the second column, directing the front fraction and the rear fraction from the second column to a second temporary storage tank, and once both the front fraction and the rear fraction are stored in the second temporary storage tank,

[0052] The latter fraction is directed from the second temporary storage tank to the first column;

[0053] - measuring the concentration of the biomolecule at the outlet of the first column or the second column, and controlling the outlet valve at the outlet of the first column or the second column with the monitoring and control unit so as to feed the first temporary storage tank or the second temporary storage tank respectively when the concentration of the biomolecule in the front fraction and the rear fraction of the eluted product fraction from the first column or the second column is higher than a specific predetermined concentration threshold, or when the concentration of the biomolecule is lower than the specific predetermined concentration threshold

[0054] Feed the waste tank when

[0055] - storing the buffer in a metering buffer tank fluidically connected to the first column and controlling the injection flow rate of the buffer from the metering buffer tank to the first column with the monitoring and control unit so as to maintain a specific filling level of the column according to the amount of reaction phase to be injected into the column, so that the lower the amount of reaction phase to be injected into the first column, the higher the amount of buffer to be injected into the first column;

[0056] - passing the reaction phase with an exchange buffer in several stages of an online diafiltration system in fluid communication between the purification device and the lipid nanoparticle formulation system, receiving data representing a specific biomolecule concentration to be used by the lipid nanoparticle formulation system for forming lipid nanoparticles in a monitoring and control unit, and controlling the injection of the exchange buffer in the final stage with the monitoring and control unit so as to obtain the lipid nanoparticles at the specific biomolecule concentration.

[0057] dilution of biomolecules;

[0058] - A purification system arranged at the outlet of the reaction chamber is used to separate the produced biomolecules from the rest of the reaction phase

[0059] separation;

[0060] - measuring the specific concentration of each product in said remaining part of the reaction phase and controlling the injection of said remaining part of the reaction phase in the mixing chamber with said monitoring and control unit. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The invention will be better understood and other advantages will become apparent from the detailed description of an embodiment thereof, given as a non-limiting example and illustrated by the accompanying drawings, in which:

[0062] [ Figure 1 ]-[ Figure 1 ] is a schematic diagram of the system of the present invention;

[0063] [ Figure 2 ]-[ Figure 2 ] is a schematic diagram of a first portion of a system of the present invention for producing biomolecules;

[0064] [ Figure 3 ]-[ Figure 3] is a schematic diagram of the second part of the system of the present invention for purifying molecules;

[0065] [ Figure 4 ]-[ Figure 4 ] is a schematic diagram of the third part of the system of the present invention for filtering biomolecules. DETAILED DESCRIPTION

[0066] like[ Figure 1 ] As shown in FIG. 1 , the system 1 for continuously manufacturing biomolecules of the present invention is particularly suitable for producing different quantities and different types of biomolecules, such as RNA (ribonucleic acid), DNA (deoxyribonucleic acid) or proteins.

[0067] Those produced biomolecules can be used as therapeutic agents, such as vaccines.

[0068] The system 1 of the invention works continuously in particular thanks to a monitoring and control unit 2 which is arranged to implement the reaction conditions based on input parameterization and based on real-time monitoring of the reaction conditions via a plurality of sensors distributed throughout the system for feedback control of the reaction conditions according to the input parameterization.

[0069] Reaction conditions that can be controlled in a non-exhaustive manner in order to obtain the desired amount and quality of biomolecules are temperature, pH, flow rate, residence time and the amount of product injected into the reaction.

[0070] exist[ Figure 1 ] In the example shown in FIG. 1 , the system of the present invention can be divided into several parts, each of which has a specific function in the manufacture of biomolecules.

[0071] The first part 1P is dedicated to the production of biomolecules.

[0072] The first part 1P comprises two feed tanks 3 suitable for storing products and a reaction chamber 4 designed to be fed with products from said feed tanks 3 , said products forming a reaction phase in the reaction chamber 4 .

[0073] The reaction chamber 4 , also referred to as a reactor or bioreactor, is designed here to produce the biomolecules from a reaction phase.

[0074] More specifically, the system 1 may comprise a mixing chamber 5 in fluid communication between the feed tank 3 and the reaction chamber 4. In this case, the mixing chamber 5 is designed to be fed with the product from the feed tank 3 and to perform mixing of the product to form the reaction phase and to subsequently feed the reaction phase to the reaction chamber 4. The injection flow rate of the product from each feed tank is controlled in real time by a monitoring and control unit 2, which is arranged to maintain a constant ratio between the products in the mixing chamber 4. This allows to maintain a constant reaction despite changes in the amount of biomolecules to be produced. In fact, the goal is to maintain a stable and reproducible reaction no matter how many biomolecules are produced.

[0075] like[ Figure 1 ] As shown in FIG. 1 , the system 1 includes a metering tank 6 which is fluidically connected to the reaction chamber 4 and is suitable for storing an immiscible phase which is immiscible with the reaction phase.

[0076] The immiscible phase used in the system of the present invention may be a liquid such as an organic phase, a solid or a gas.

[0077] The monitoring and control unit 2 is therefore arranged to control the injection flow rate of the immiscible phase into the reaction chamber 4 so as to keep a specific filling level of the reaction chamber 4 constant according to the amount of reaction phase to be injected into the reaction chamber 4 to produce a specific amount of biomolecules, such that the amount of reaction phase to be injected into the reaction chamber 4 is lower, and the amount of immiscible phase to be injected is higher.

[0078] Furthermore, in order to separate the immiscible phase from the reactive phase, the system may comprise a valve 7 at the outlet 4a of the reaction chamber 4. The monitoring and control unit 2 is therefore arranged to receive data representing the presence of the reactive phase at the outlet 4a of the reaction chamber 4 and to control said valve 7 in turn.

[0079] For example, the data representative of the presence of the reaction phase at the outlet 4 a of the reaction chamber 4 are calculated based on the duration of a specific residence time of the reaction phase in the reaction chamber 4 .

[0080] The reactive phase is thus injected into the intermediate tank IT and the immiscible phase into the waste tank WT.

[0081] Without limiting the scope of the invention, said data representative of the presence of the reactive phase at the outlet 4a of the reaction chamber 4 can also be determined by means of a UY sensor or a RAMAN.

[0082] In order to keep the reaction phase in the reaction chamber 4 substantially at a predetermined temperature according to the input parameterization, such as [ Figure 2 ] As shown in FIG. 1 , the system 1 may include a temperature sensor 8 designed to measure the temperature of the reaction phase and a thermostat 9 arranged to be in contact with the reaction chamber 4, which is controlled by the monitoring and control unit 2 according to the measured temperature.

[0083] The system may also include a pH sensor 10 designed to measure the pH of the reaction phase. In this case, the system includes at least one buffer tank 11, which is fluidically connected to the reaction chamber 4 and is suitable for storing a pH regulator product. The monitoring and control unit 2 is therefore configured to control the injection flow rate of the pH regulator product according to the measured pH level so as to substantially maintain the reaction phase at a predetermined pH level in the reaction chamber 4.

[0084] The system may also comprise a mass flow meter 12 designed to measure the flow rate of each product to be injected into the reaction chamber 4. In this case, the system 1 comprises a mass flow controller 13 designed to control the injection flow rate of these products in the reaction chamber 4. The monitoring and control unit 2 is therefore arranged to control the mass flow controller 13 to control the injection flow rate of the reaction phase according to the measured flow rate to keep the reaction phase at a specific flow rate in the reaction chamber 4.

[0085] The system 1 of the present invention may also include a device MC for measuring the concentration of biomolecules at the outlet 4a of the reaction chamber 4 obtained after a preset residence time in the input parameterization, and then controlling the injection of products into the reaction chamber 4 to increase the amount of biomolecules produced to obtain the desired amount of biomolecules.

[0086] The system 1 of the invention may also comprise a purification system 14, here as an example of a filtration system, arranged at the outlet 4a of the reaction chamber 4, which purification system is designed to separate the produced biomolecules from the rest of the reaction phase. In this case, the system may comprise means for measuring the specific concentration of each product in the rest of the reaction phase, and the monitoring and control unit is therefore arranged to control the injection of the rest of the reaction phase in the mixing chamber 5.

[0087] The second part of system 1, 2P, is dedicated to the purification of biomolecules, such as [ Figure 3 ] as shown.

[0088] To this end, the system of the present invention includes a first chromatographic device 15, which includes a first column 16 that is fluidically connected between the reaction chamber 4 (and more specifically the intermediate tank IT) and the collection tank 17, and a second column 18 that is fluidically connected between the first column 16 and the collection tank 17. The first chromatographic device 15 also includes a first temporary storage tank 19 that is fluidically connected between the first column 16 and the second column 18. The first chromatographic device 15 also includes a first outlet valve 20 at the outlet 16a of the first column 16 and a first inlet valve 21 at the inlet 18b of the second column 18, the first outlet valve being designed to switch the fluid connection from the first column 16 to the first temporary storage tank 19 or from the first column 16 to the collection tank 17, and the first inlet valve being designed to open or close the fluid connection between the first temporary storage tank 19 and the second column 18.

[0089] More specifically, the first chromatographic device 15 may include a second temporary storage tank 22 in fluid communication between the second column 18 and the first column 16. In this case, the first chromatographic device 1 includes a second outlet valve 23 at the outlet 18a of the second column 18 and a second inlet valve 24 at the inlet 16b of the first column 16, the second outlet valve being designed to switch the fluid communication from the second column 18 to the second temporary storage tank 22 or from the second column 18 to the collection tank 17, and the second inlet valve being designed to open or close the fluid communication from the second temporary storage tank 22 to the first column 16.

[0090] The first chromatographic device 1 may also include a device for measuring the concentration of biomolecules at the outlet 16a, 18a of the first column 16 or the second column 18, and the monitoring and control unit 2 is configured to control the outlet valves 20, 23 at the outlet 16a, 18a of the first column 16 or the second column 18 so as to feed the first temporary storage tank 19 or the second temporary storage tank 22 respectively when the concentration of biomolecules in the front fraction and the rear fraction of the eluted product fraction from the first column 16 or the second column 18 is higher than a specific predetermined concentration threshold, or to feed the waste tank 25 when the concentration of biomolecules is lower than the specific predetermined concentration threshold.

[0091] The second chromatography device 26 can be used to improve the purification of the first chromatography device 1. For example, reverse phase chromatography can be used.

[0092] The third part 3P is dedicated to filtering the purified biomolecules, such as [ Figure 4 ] as shown in FIG, the fourth part 4P is dedicated to the formation of lipid nanoparticles.

[0093] In this regard, the system 1 includes an online diafiltration system 27 in fluid communication between the first purification device 1 or the second purification device 26 and the lipid nanoparticle formulation system 28 , the diafiltration system including several stages 29 designed to pass the reaction phase with an exchange buffer stored in a buffer tank 30 .

[0094] The monitoring and control unit 2 is therefore configured to receive data representing a specific biomolecule concentration to be used by the lipid nanoparticle preparation system 28 to form lipid nanoparticles and data of the real-time concentration of the biomolecule measured by the concentration sensor 31 at the outlet of the final stage 29. The monitoring and control unit 2 is therefore configured to control the injection of the exchange buffer in the final stage 29 so as to dilute the biomolecule at the specific biomolecule concentration and to inject the biomolecule into the intermediate tank 32 at the specific concentration before directing the biomolecule to the lipid nanoparticle preparation system 28.

[0095] The invention furthermore relates to a method for producing biomolecules, here for example mRNA (messenger RNA) for use as a vaccine.

[0096] The first step consists in in vitro transcription of mRNA. In this way, a first master mix containing linearized specific DNA encoding at least a portion of a protein is stored in a first feed tank 3, and a second master mix containing RNA polymerase and nucleotides is stored in a second feed tank 3. The first master mix and the second master mix are mixed in a specific ratio in chamber 5 to form a reaction phase.

[0097] In order to maintain a constant ratio between the two main mixtures in the mixing chamber 5 , the injection flow rate of the product from each feed tank 3 is controlled in real time by the monitoring and control unit 2 .

[0098] The reaction phase is then injected into the reaction chamber 4 to achieve in vitro transcription of mRNA according to the reaction conditions imposed by the input parameterization of the monitoring and control unit 2 .

[0099] This means that reaction chamber 4 contains all necessary products and is able to provide all necessary reaction conditions for the manufacture of mRNA.

[0100] Since the reaction phase is aqueous, the reaction chamber is also fed with an immiscible phase that is immiscible with the reaction phase.

[0101] The monitoring and control unit 2 is therefore arranged to control the injection flow rate of the immiscible phase into the reaction chamber in order to maintain a specific filling level of the reaction chamber 4 depending on the amount of reaction phase to be injected into the reaction chamber 4 to produce a specific amount of biomolecules.

[0102] More specifically, the amount of the reactive phase to be injected into the reaction chamber 4 is lower, while the amount of the immiscible phase to be injected is higher.

[0103] Therefore, it is necessary to separate the immiscible phase from the reaction phase to enable rapid and reliable purification of the produced mRNA.

[0104] To this end, the method of the present invention consists in determining the presence or absence of the reaction phase at the outlet 4a of the reaction chamber 4 by means of a monitoring and control unit 2 and based on a specific duration of a specific residence time of the reaction phase in the reaction chamber 4, and in controlling the separation of the reaction phase from the immiscible phase by means of the monitoring and control unit 2.

[0105] It is also necessary to reuse the products used in the reaction phase, such as enzymes and nucleotides, in order to limit production costs. To this end, the method consists in separating the manufactured biomolecules from the rest of the reaction phase by diafiltration at the outlet 4a of the reaction chamber 4 .

[0106] Another step consists in determining the concentration of each product of said remaining part of the reaction phase and then controlling the injection of said remaining part of the reaction phase in the mixing chamber 5. The monitoring and control unit 2 is therefore arranged to adjust the concentration of each main mixture added from the feed tank 2 after the remaining part of the reaction phase in order to maintain a constant ratio.

[0107] The separated reaction phase is then purified in a chromatography step, which includes separating the product fraction eluted from the first column 16 into a front fraction and a rear fraction of the product fraction eluted from the first column 16, directing the front fraction and the rear fraction from the first column 16 to a temporary storage tank 19, and once both the front fraction and the rear fraction are stored in the temporary storage tank 19, directing the front fraction and the rear fraction from the temporary storage tank 19 to a second column 18.

[0108] The product fraction eluted from the second column 18 can thus be directed to a collection tank or purified again.

[0109] If the production fraction needs to be purified again, the method includes an additional step, which includes separating the product fraction eluted from the second column 18 into a front fraction and a rear fraction of the product fraction eluted from the second column 18, directing the front fraction and the rear fraction from the second column 18 to a second temporary storage tank 22, and once both the front fraction and the rear fraction are stored in the second temporary storage tank 22, directing the front fraction and the rear fraction from the second other temporary storage tank 22 to the first column 16.

[0110] The idea is to elute the most mRNA from the column by recovering the late and early fractions without wasting time.

[0111] In this case, the concentration of the biomolecules at the outlet of the first column 16 or the second column 18 can be measured by a measuring device MM such as a UV sensor, so that if the concentration is higher than a specific predetermined concentration threshold, the front fraction and the rear fraction from the first column or the second column are respectively directed to the first temporary storage tank 19 or the second temporary storage tank 22, or if the concentration is lower than the specific predetermined concentration threshold, the front fraction and the rear fraction from the first column or the second column are directed to the waste tank 30.

[0112] Depending on the amount of biomolecules to be produced, different amounts of biomolecules need to be purified using the purification device 15 of the system 1 according to the invention.

[0113] Therefore, the idea of ​​the present invention is to control the injection flow rate of the make-up buffer in the first column 16 while the reaction phase passes through the first column 16 in order to maintain a certain constant filling level in the first column 16 .

[0114] In this case, a single first column 16 may be used and sized to be suitable for producing large quantities of biomolecules.

[0115] This allows a continuous process by limiting the use of consumables and human intervention when production scales change.

[0116] A change in consumables should be understood as changing the chromatography column depending on the amount of biomolecules produced.

[0117] Therefore, the monitoring and control unit 2 is arranged to control the injection flow rate of the making-up buffer from the metering buffer tank BT of the chromatographic device 15 into the first column 16 in order to maintain this specific filling level of the first column 16 according to the amount of reaction phase to be injected into the first column 16.

[0118] More specifically, this means that the amount of the reaction phase to be injected into the first column 16 is lower, while the amount of the buffer to be injected into the first column 16 is higher.

[0119] For example, the complementation buffer used can be a neutral solution that does not alter the pH or the integrity and quality of the biomolecules produced.

[0120] It will be appreciated that the monitoring and control unit may be arranged to control the injection flow rate of the make-up buffer from the metering buffer tank BT into the second column 18 in order to maintain a certain filling level.

[0121] Reverse phase chromatography can then be used to improve the quality of the purification.

[0122] The third step consists in filtering the mRNA purified by the chromatography step.

[0123] This step consists in filtering the mRNA of the reaction phase by passing it through several stages 29 of a diafiltration system 27 with an exchange buffer and by injecting at the final stage 29 a quantity of exchange buffer in order to dilute the mRNA at a specific concentration to be used for the formulation of lipid nanoparticles.

[0124] The fourth step consists in the formation of lipid nanoparticles.

[0125] This step is completely known, but the object of the present invention is to be able to use a specific concentration of mRNA that is particularly suitable for forming nanoparticles. This concentration is determined by the input parameters and the dilution is carried out during the diafiltration process.

Claims

1. A method for the continuous production of biomolecules (1), comprising the following steps: - feeding the reaction chamber (4) with the product from the feed tank, said product forming a reaction phase in said reaction chamber, - producing said biomolecule from said reaction phase in said reaction chamber, characterised in that said method further comprises the following steps: - storing an immiscible phase that is immiscible with the reaction phase in a metering tank (6) in fluid communication with the reaction chamber, - controlling the injection flow rate of the immiscible phase into the reaction chamber using a monitoring and control unit (2), So as to maintain a specific filling level of the reaction chamber according to the amount of the reaction phase to be injected into the reaction chamber to produce a specific amount of biomolecules, so that the lower the amount of the reaction phase to be injected into the reaction chamber, the higher the amount of the immiscible phase to be injected.

2. The method for continuous production of biomolecules according to claim 1, characterized in that The method comprises the following steps: - receiving in the monitoring and control unit data representative of the presence of the reaction phase at the outlet of the reaction chamber, and - The valve (7) at the outlet (4a) of the reaction chamber (4) is then controlled by the monitoring and control unit to separate the immiscible phase from the reactive phase.

3. The method for continuous production of biomolecules according to claim 2, characterized in that The data representative of the presence of the reaction phase at the outlet of the reaction chamber are calculated based on the duration of the residence time of the reaction phase in the reaction chamber.

4. A method for the continuous production of biomolecules according to any one of the preceding claims, characterised in that The method comprises the following steps: - feeding the product from the feed tank to a mixing chamber (5) in fluid communication between the feed tank and the reaction chamber, - mixing of the products to form the reaction phase and subsequently feeding the reaction phase to the reaction chamber, and - Controlling in real time the injection flow rate of said products from each feed tank with said monitoring and control unit in order to maintain a constant ratio between said products in said mixing chamber.

5. A method for the continuous production of biomolecules according to any one of the preceding claims, characterised in that The method comprises a chromatography step comprising: - separating the product fraction eluted from the first column (16) into a front fraction and a rear fraction of the product fraction eluted from the first column, - directing the front fraction and the rear fraction from the first column to a first temporary storage tank (19), and - Once both the front fraction and the back fraction are stored in the temporary storage tank, the front fraction and the back fraction are directed from the first temporary storage tank to a second column (18).

6. The method for continuous production of biomolecules according to claim 5, characterized in that The method comprises the following steps: - separating the product fraction eluted from the second column into a front fraction and a rear fraction of the product fraction eluted from the second column, and - directing the front fraction and the rear fraction from the second column to a second temporary storage tank (22), and - Once both the front fraction and the back fraction are stored in the second temporary storage tank, directing the front fraction and the back fraction from the second temporary storage tank to the first column.

7. The method for continuous production of biomolecules according to claim 6, characterized in that The method comprises the following steps: - measuring the concentration of the biomolecule at the outlet of the first column or the second column, and - Controlling the outlet valve at the outlet of the first column or the second column with the monitoring and control unit so as to feed the first temporary storage tank or the second temporary storage tank respectively when the concentration of biomolecules in the front fraction and the rear fraction of the eluted product fraction from the first column or the second column is higher than a specific predetermined concentration threshold, or to feed the waste tank (25) when the concentration of biomolecules is lower than the specific predetermined concentration threshold.

8. The method for the continuous production of biomolecules according to any one of claims 5 to 7, characterized in that The method comprises the following steps: - storing the buffer in a metering buffer tank (BT) fluidically connected to the first column, and - controlling the injection flow rate of the buffer from the metering buffer tank into the first column with the monitoring and control unit so as to maintain a specific filling level of the column according to the amount of the reaction phase to be injected into the column, so that the lower the amount of the reaction phase to be injected into the first column, the higher the amount of the buffer to be injected into the first column.

9. A method for the continuous production of biomolecules according to any one of the preceding claims, characterised in that The method comprises the following steps: - passing the reaction phase with an exchange buffer in several stages (29) of an online diafiltration (27) system in fluid communication between the purification device and the lipid nanoparticle formulation system (28), - receiving in said monitoring and control unit data representing the concentration of a specific biomolecule to be used by said lipid nanoparticle formulation system to form lipid nanoparticles, - controlling the injection of said exchange buffer in the final stage with said monitoring and control unit in order to dilute said biomolecules at said specific biomolecule concentration.

10. The method for the continuous production of biomolecules according to any one of the preceding claims, characterized in that The method comprises the step of separating the produced biomolecules from the rest of the reaction phase using a purification system (14) arranged at the outlet of the reaction chamber.

11. The method for the continuous production of biomolecules according to claims 4 and 10, characterized in that The method comprises the following steps: - measuring the specific concentration of each product in said remaining portion of said reaction phase, and - controlling the injection of said remaining portion of said reaction phase in said mixing chamber with said monitoring and control unit.

12. The method for continuous production of biomolecules according to any one of claims 1 to 11, wherein the produced biomolecule is RNA.

13. The method for continuous production of biomolecules according to claim 12, wherein the RNA produced is mRNA.

14. The method for continuous production of biomolecules according to any one of claims 1 to 11, wherein the produced biomolecules are proteins.

15. The method for continuous production of biomolecules according to any one of claims 1 to 11, wherein the produced biomolecule is DNA.

16. A method for the continuous production of biomolecules according to any one of claims 11 to 15, wherein the produced biomolecules are therapeutic agents.

17. The method for continuous production of biomolecules according to claim 16, wherein the therapeutic agent is a vaccine.

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