Patent Description:
Scientific and technological advances of the recent years have made biomolecules promising candidates for a variety of uses, including diagnostic applications, and therapeutic products, like vaccines.

Propelled by major science-technology-medicine advancements, the rise of new therapies that target the genetic machinery, such as gene replacement, correction or modulation, protein expression, is enabling new opportunities for innovative treatment, but also emergency responses in epidemic crisis situations. In that context, various approaches have been developed for mRNA production at scale. Most current process utilize in vitro enzymatic reaction to synthesize mRNA from self-replicating DNA templates, then encapsulate the total RNA into a lipid nanoparticle as immiscible delivery vehicle.

These processes are costly, require highly skilled personnel, produce hazardous waste streams that must be mediated, while the production rate is severely dependent on the performance of the enzymatic reactions, the ability to remove process and product related impurities and the control and performance of the encapsulation into lipid nanoparticles.

In addition, the current practice is to produce mRNA in batch mode, which give little flexibility in terms of changing the scale of the production: for each scale, the process has to be significantly adapted and changes have to be approved by regulatory authorities.

The recent pandemic has shown that there is a need to accelerate the development of new vaccines and their availability to the population.

In this context, <CIT> has described a novel process to produce mRNA in an in vitro system using a continuous flow production.

The system describes in <CIT> has a reaction chamber in which in vitro transcription is performed to continuously produce mRNA.

The reaction chamber operates continuously such that it may be infused with new input material while producing mRNA.

This system is particularly adapted for producing a large amount of mRNA, such as millions of doses in the context of a pandemic, but will not be adapted to produce small quantity of mRNA, for example thousands of doses for early clinical trials, without wasting a large quantity of enzymes and buffers.

Indeed, in the context of changing scale-up in mRNA production, the production of small quantity would generally be done in a smaller batch reactor with a volume adapted to the desired amount of output.

Thus, the system described in <CIT> is not adapted to rapidly elaborate and manufacture different quantity of mRNA with a reduced cost.

The objective of the invention is therefore to solve the above-mentioned problems. The claimed subject-matter of the invention is a method indicated in method claims <NUM>-<NUM>.

To this end, the invention relates to a system for continuous manufacture of biomolecules comprising feed tanks suitable for storing products and a reaction chamber designed to be fed with products from said feed tanks which form in the reaction chamber a reaction phase, said reaction chamber being designed to manufacture said biomolecules from said reaction phase, characterized in that said system comprises a scale tank in fluid communication to the reaction chamber and suitable for storing an immiscible phase which is not miscible with the reaction phase, said system comprising a monitoring and control unit set to control the injection flow rate of the immiscible phase into the reaction chamber in order to maintain a certain filling level of the reaction chamber according to the amount of reaction phase to be injected into the reaction chamber to manufacture a certain amount of biomolecules, so that the lower the amount of reaction phase to be injected into the reaction chamber and the higher the amount of immiscible phase to be injected.

The idea behind the invention is to use a single system adapted to manufacture from small quantity of biomolecules to a large quantity of biomolecules, or the reverse.

The idea consists more particularly in dimensioning the system for the manufacture of biomolecules on a large scale while allowing the manufacture of biomolecules on a small scale by preserving the same conditions of reaction and thus the same qualities results (concentration, purity, time of manufacture, etc.. ), i.e. that the reaction phase must always have the same conditions of reaction.

To this end, an immiscible phase is injected to complete the volume not used by the reaction phase in the reaction chamber in case of small quantity to be manufactured even for scale lower than what the reaction chamber has been typically designed for.

The two phases being immiscible, the reaction phase running in the reaction chamber is always contained in a volume adapted for a stable and effective reaction.

The system of the present invention is thus advantageously adapted to produce different quantity of biomolecules, for example at different stages of vaccine clinical development.

The idea is also to use a single system to produce a chosen quantity of biomolecules in a certain constant residence time. This means that the system according to the invention is designed to produce small or large quantity of biomolecules in the same residence time.

The system according to the invention may also have the following features:.

The invention also extends to a process for continuous manufacture of biomolecules with the system of the invention wherein the manufactured biomolecules are RNA, such as mRNA, or Proteins or DNA and wherein said manufactured biomolecules could be a therapeutic agent, such as a vaccine.

The present invention will be better understood and other advantages will become apparent from the detailed description of the embodiment taken as a non-limiting example and illustrated by the attached drawings, in which:.

The system <NUM> for the continuous manufacture of biomolecules of the invention, as shown in <FIG>, is particularly adapted for producing different quantity and different type of biomolecules, such as RNA (Ribonucleic acid), DNA (Deoxyribonucleic acid) or proteins.

Those manufactured biomolecules could be used as a therapeutic agent, such as vaccine.

The system <NUM> of the invention works continuously notably thanks to a monitoring and control unit <NUM> which is set to enforce reaction conditions based on input parameterization and on a real time monitoring of said reaction conditions via a plurality of sensors distributed over the whole system for a feedback control of the reaction conditions according to the input parameterization.

In order to obtain the required quantity and quality of biomolecules, the reaction conditions that can be controlled in a non-exhaustive way are the temperature, the pH, the flow rate, the residence time, and the quantity of product to be injected for the reaction.

In the example as shown in <FIG>, the system of the invention could be divided in several parts each having a specific function in the manufacture of the biomolecules.

A first part 1P is dedicated to the production of the biomolecules.

The first part 1P comprises two feed tanks <NUM> suitable for storing products and a reaction chamber <NUM> designed to be fed with products from said feed tanks <NUM> which form in the reaction chamber <NUM> a reaction phase.

The reaction chamber <NUM>, also called reactor or bioreactor, is here designed to produce said biomolecules from the reaction phase.

More particularly, the system <NUM> may comprises a mixing chamber <NUM> in fluid communication between the feed tanks <NUM> and the reaction chamber <NUM>. In that case, the mixing chamber <NUM> is designed to be fed with products from said feed tanks <NUM> and to perform a mixing of the products to form said reaction phase and to feed in turn said reaction phase to the reaction chamber <NUM>. The injection flow rate of the products from each feed tank is control in real time by a monitoring and control unit <NUM> set in order to maintain a constant ratio between the products in the mixing chamber <NUM>. This allows to keep a constant reaction in spite of the change of the quantity of biomolecules to be manufactured. Indeed, the goal is to maintain a stable and reproducible reaction no matter the quantity of biomolecules to produce.

As shown in the <FIG>, the system <NUM> comprises a scale tank <NUM> in fluid communication to the reaction chamber <NUM> and suitable for storing an immiscible phase, which is not miscible with the reaction phase.

The immiscible phase used in the system of the present invention can be liquid, such as an organic phase, solid or gaseous.

The monitoring and control unit <NUM> is thus set to control the injection flow rate of the immiscible phase into the reaction chamber <NUM> in order to keep constant a certain filling level of the reaction chamber <NUM> according to the amount of reaction phase to be injected into the reaction chamber <NUM> to manufacture a certain amount of biomolecules, so that the lower the amount of reaction phase to be injected into the reaction chamber <NUM> and the higher the amount of immiscible phase to be injected.

Moreover, in order to separate the immiscible phase from the reaction phase, the system could comprise a valve <NUM> at the outlet 4a of the reaction chamber <NUM>. The monitoring and control unit <NUM> is thus set to receive a data representative of the presence of the reaction phase at the outlet 4a of the reaction chamber <NUM> and to control in turn said valve <NUM>.

As for example, said data representative of the presence of the reaction phase at the outlet 4a of the reaction chamber <NUM> is calculated based on a duration of a certain residence time of the reaction phase in the reaction chamber <NUM>.

The reaction phase is thus injected into an intermediate tank IT and the immiscible phase is injected into a waste tank WT.

Without restricting the scope of the invention, said data representative of the presence of the reaction phase at the outlet 4a of the reaction chamber <NUM> can also be determined by UV sensors or RAMAN.

In order to maintain the reaction phase substantially at a predetermined temperature in the reaction chamber <NUM> according to the input parameterization, the system <NUM> may comprises, as shown in <FIG>, a temperature sensor <NUM> design to measure the temperature of the reaction phase and a thermostat <NUM> arranged in contact with the reaction chamber <NUM> controlled by the monitoring and control unit <NUM> as a function of the measured temperature.

The system may also comprises a pH sensor <NUM> designed to measure the pH of the reaction phase. The system comprises in that case at least one buffer tank <NUM> fluidly connected to the reaction chamber <NUM> suitable to store a pH corrector product. The monitoring and control unit <NUM> is thus set to control the injection flow rate of the pH corrector product as a function of the measured pH level so as to maintain the reaction phase substantially at a predetermined pH level in the reaction chamber <NUM>.

The system may also comprises mass flow meters <NUM> design to measure the flow rate of each product to be injected in the reaction chamber <NUM>. The system <NUM> comprises in that case a mass flow controller <NUM> design to control the injection flow rate of these products in the reaction chamber <NUM>. The monitoring and control unit <NUM> is thus set to control the mass flow controller <NUM> to control the injection flow rate of the reaction phase as a function of the measured flow rate so as to maintain the reaction phase at a certain flow rate in the reaction chamber <NUM>.

The system <NUM> of the invention may also comprises means MC for measuring the concentration of the biomolecules at the outlet 4a of the reaction chamber <NUM> obtained after a preset residence time in the input parameterization and in controlling in return the injection of the products into the reaction chamber <NUM> to increase the quantity of biomolecules produced to obtained the desired quantity of biomolecules.

The system <NUM> of the invention may also comprises a purification system <NUM>, here as example a filtration system, arranged at the outlet 4a of the reaction chamber <NUM> designed to separate the manufactured biomolecules from the rest of the reaction phase. In that case, the system may comprises means for measuring a certain concentration of each product in said rest of the reaction phase and said monitoring and control unit is thus set to control the injection of said rest of the reaction phase in the mixing chamber <NUM>.

A second part 2P of the system <NUM> is dedicated to the purification of the biomolecules, as shown in <FIG>.

In that purpose, the system of the invention comprises a first chromatography device <NUM> comprising a first column <NUM> in fluid communication between the reaction chamber <NUM>, and more particularly the intermediate tank IT, and a collection tank <NUM> and, a second column <NUM> in fluid communication between the first column <NUM> and said collection tank <NUM>. The first chromatography device <NUM> further comprises a first temporary storage tank <NUM> in fluid communication between the first column <NUM> and the second column <NUM>. The first chromatography device <NUM> further comprises a first outlet valve <NUM> at the outlet 16a of the first column <NUM> designed to switch the fluid communication from the first column <NUM> to the first temporary storage tank <NUM> or from the first column <NUM> to the collection tank <NUM>, and an first inlet valve <NUM> at the inlet 18b of the second column <NUM> designed to open or close the fluid communication between the first temporary storage tank <NUM> and the second column <NUM>.

More particularly, the first chromatography device <NUM> may comprises a second temporary storage tank <NUM> in fluid communication between the second column <NUM> and the first column <NUM>. In that case, the first chromatography device <NUM> comprises a second outlet valve <NUM> at the outlet 18a of the second column <NUM> designed to switch the fluid communication from the second column <NUM> to the second temporary storage tank <NUM> or from the second column <NUM> to the collection tank <NUM>, and a second inlet valve <NUM> at the inlet 16b of the first column <NUM> designed to open or close the fluid communication from the second temporary storage tank <NUM> to the first column <NUM>.

The first chromatography device <NUM> may further comprises means for measuring the concentration of biomolecules at the outlet 16a, 18a of the first or second columns <NUM>, <NUM> and the monitoring and control unit <NUM> is set to control the outlet valve <NUM>,<NUM> at the outlet 16a,18a of the first or second columns <NUM>,<NUM> in order to feed respectively the first or second temporary storage tanks <NUM>,<NUM> when the concentration of biomolecules in pre and post fractions of an eluted product fraction from the first or second column <NUM>,<NUM> is above a certain predetermined concentration threshold or to feed a waste tank <NUM> when the concentration of biomolecules is below said certain predetermined concentration threshold.

A second chromatography device <NUM> can be used to improve the purification of the first chromatography device <NUM>. As for example, a reverse chromatography could be used.

A third part 3P is dedicated to the filtration of the purified biomolecules, as shown in <FIG>, and a fourth part 4P is dedicated to the lipidic nanoparticles formation.

In this regard, the system <NUM> comprises an inline diafiltration system <NUM> in fluid communication between the first or second purification device <NUM>, <NUM> and a lipid nanoparticles formulation system <NUM>, said diafiltration system <NUM> comprising several stages <NUM> designed to pass the reaction phase with an exchange buffer stored in a buffer tank <NUM>.

The monitoring and control unit <NUM> is thus set to receive data representative of a certain concentration of biomolecules to be used by the lipid nanoparticles formulation system <NUM> to form the lipid nanoparticles and a data of a real time concentration of the biomolecules measured by a concentration sensor <NUM> at the outlet of the last stage <NUM>. The monitoring and control unit <NUM> is thus set to control the injection of the exchange buffer in the last stage <NUM> in order to dilute the biomolecules at said certain concentration of biomolecules and to inject said biomolecule at said certain concentration in an intermediate tank <NUM> before directing said biomolecules to the lipid nanoparticles formulation system <NUM>.

The invention further relates to a process for manufacturing biomolecules, here for example the manufacturing of mRNA (Messenger ribonucleic acid) for use as a vaccine.

A first step consists in an in vitro transcription of mRNA. In this way, a first master mix comprising linearized specific DNA, coding for at least a part of one protein, is stored in the first feeding tank <NUM> and the second master mix comprising RNA polymerase and nucleotides, is stored in the second feeding tank <NUM>. The first and second master mix are mixed in a chamber <NUM> at a specific ratio to form a reaction phase.

In order to maintain a constant ratio between both master mix in the mixing chamber <NUM>, the injection flow rate of the products from each feed tank <NUM> is controlled in real time by the monitoring and control unit <NUM>.

The reaction phase is then injected in the reaction chamber <NUM> to realize the in vitro transcription of the mRNA according to the reaction conditions imposed by an input parameterization of the monitoring and control unit <NUM>.

This means that the reaction chamber <NUM> comprises all the necessary products and is able to provide all the necessary reaction conditions for the manufacture of mRNA.

The reaction chamber is also fed with an immiscible phase which is non miscible with the reaction phase, because of the reaction phase is an aqueous phase.

The monitoring and control unit <NUM> is thus set to control the injection flow rate of the immiscible phase into the reaction chamber in order to maintain a certain filling level of the reaction chamber <NUM> according to the amount of reaction phase to be injected into the reaction chamber <NUM> to manufacture a certain amount of biomolecules.

More particularly, the lower the amount of reaction phase to be injected into the reaction chamber <NUM> and the higher the amount of immiscible phase to be injected.

It follows that it is necessary to separate the immiscible phase from the reaction phase to enable quick and reliable purification of the mRNA produced.

To this way, the process of the invention consists in determining by means of the monitoring and control unit <NUM> and based on a certain duration of a certain residence time of the reaction phase in the reaction chamber <NUM>, the presence or absence of the reaction phase at the outlet 4a of the reaction chamber <NUM>, and controlling by means of the monitoring and control unit <NUM> the separation of the reaction phase from the immiscible phase.

There is also the need to reuse the products used in the reaction phase, such as enzymes and nucleotides, in order to limit production costs. For this purpose the process consist in separating the manufactured biomolecules from the rest of the reaction phase at the outlet 4a of the reaction chamber <NUM> by diafiltration.

An additional step consists in determining the concentration of each product of said rest of the reaction phase and of controlling in turn the injection of said rest of the reaction phase in the mixing chamber <NUM>. The monitoring and control unit <NUM> is thus set to adjust the concentration of each master mix to be added from the feed tank <NUM> after the rest of the reaction phase to maintain a constant ratio.

The separated reaction phase is then purified in a chromatography step consisting in separating a product fraction eluted from a first column <NUM> to a pre and post fractions of said product fraction eluted from said first column <NUM>, directing said pre and post fractions from the first column <NUM> to a temporary storage tank <NUM>, and directing said pre and post fraction from said temporary storage tank <NUM> to a second column <NUM> once both pre and post fractions are tanked in the temporary storage tank <NUM>.

The product fraction eluted from the second column <NUM> can thus be directed to a collection tank or. purified again.

If the production fraction needed to be purified again, the process comprises an additional step consisting in separating the product fraction eluted from the second column <NUM> to pre and post fractions of said product fraction eluted from said second column <NUM>, directing said pre and post fractions from the second column <NUM> to a second temporary storage tank <NUM>, and by directing said pre and post fraction from said second other temporary storage tank <NUM> to the first column <NUM> once both pre and post fractions are tanked in the second temporary storage tank <NUM>.

The idea is to elute a maximum of mRNA from the column by recovering the post and pre fraction without wasting time.

In that case, it is possible to measure the concentration of biomolecules at the outlet of the first or second column <NUM>,<NUM> by way of measuring means MM, such as UV sensors, in order to direct the pre and post fraction from the first or the second columns respectively to the first or second temporary storage tank <NUM>,<NUM> if the concentration is above a certain predetermined concentration threshold or to a waste tank <NUM> if the concentration is below said certain predetermined concentration threshold.

There is also the need to purify different amounts of biomolecules with the purification device <NUM> of the system <NUM> according to the invention depending on the amount of biomolecules to be manufactured.

The idea of the invention is thus to control the injection flow rate of a completing buffer in the first column <NUM> when the reaction phase passes in the first column <NUM> in order to maintain a certain constant level of filling in the first column <NUM>.

In this situation, one single first column <NUM> can be used and dimensioned for the large quantity of biomolecules to be produced.

This allows continuous process by limiting the use of consumables and human intervention at the time of the change of scale of production.

By change of consumable, it should be understood the change of column according to the quantity of biomolecules to produce.

Thus, the monitoring and control unit <NUM> is set to control the injection flow rate of the completing buffer from a scale buffer tank BT of the chromatography device <NUM> into the first column <NUM> in order to maintain this certain filling level of the first column <NUM> according to the amount of reaction phase to be injected into the first column <NUM>.

More specifically, this means that the lower the amount of reaction phase to be injected into the first column <NUM> and the higher the amount of buffer to be injected into the first column <NUM>.

As for an example, the completing buffer used could be a neutral solution that does not modify the pH or the integrity and quality of the biomolecules produced.

It is to be understood, that the monitoring and control unit can be set to control the injection flow rate of the completing buffer from the scale buffer tank BT into the second column <NUM> to maintain a certain filling level.

A reverse chromatography could then be used in order to upgrade the quality of the purification.

A third step consists in filtering the mRNA purified by the chromatography step.

This step consists in filtering the mRNA of the reaction phase by passing the reaction phase with an exchange buffer in several stages <NUM> of a diafiltration system <NUM> and by injecting a certain amount of the exchange buffer in the last stage <NUM> in order to dilute the mRNA at a certain concentration ready to be used for the lipid nanoparticles formulation.

A fourth step consists in the lipidic nanoparticles formation.

Claim 1:
Process (<NUM>) for continuous manufacturing of biomolecules comprising the following steps:
- feeding a reaction chamber (<NUM>) with products from feed tanks, said products forming in the reaction chamber a reaction phase,
- manufacturing said biomolecules from said reaction phase in the reaction chamber, characterized in that said process further comprises the steps of:
- storing an immiscible phase which is not miscible with the reaction phase in a scale tank (<NUM>) in fluid communication to the reaction chamber,
- controlling with a monitoring and control unit (<NUM>) the injection flow rate of the immiscible phase into the reaction chamber in order to maintain a certain filling level of the reaction chamber according to the amount of reaction phase to be injected into the reaction chamber to manufacture a certain amount of biomolecules, so that the lower the amount of reaction phase to be injected into the reaction chamber and the higher the amount of immiscible phase to be injected.