Automated synthesis reactor system with a recirculation loop

Abstract

An automated system of reactors carries out a solid-phase peptide synthesis, and more particularly a solid-phase peptide synthesizer which is automated, by means of a reactor with a liquid-recirculation loop making it possible to measure, in real time, chemical species in the reactor via measuring cells. This system includes inlet pipes, namely: pipes dedicated to the introduction of resin, pipes dedicated to the introduction of the synthesis and washing solvent, pipes dedicated to the introduction of the agent for deprotecting the amino acid introduced, pipes dedicated to the introduction of the reagents, and includes an assembly reactor and a loop for recirculation of the liquid of the reactor.

Claims

1. A reactor system for performing a solid-phase peptide synthesis, the reactor system comprising: an inlet pipe dedicated to the introduction of resin; an inlet pipe dedicated to the introduction of a synthesis and washing solvent; an inlet pipe dedicated to the introduction of a deprotecting agent of an amino acid supplied; an inlet pipe dedicated to the introduction of reagents; an assembly reactor; a recirculation loop of a liquid in the assembly reactor comprising at least one measuring cell for indirect quantification of the progress of the reaction on the solid phase; and a level sensor to determine a height of the liquid in the assembly reactor with respect to a height of a bed of the resin.

2. The reactor system for performing a solid-phase peptide synthesis according to claim 1, wherein the level sensor is a radar sensor.

Description

DESCRIPTION OF THE FIGURES

(1) The present invention will be better understood from the detailed description below and the appended drawings which are given as an illustration, and therefore, are not limiting of the present invention, and wherein:

(2) FIG. 1 is a schematic view showing a synthesis reactor system (14) to achieve a solid-phase peptide synthesis with a liquid-recirculation loop (10) of the reactor making it possible to measure in real time, the evolution of the chemical species in the peptide automatic synthesizer of the present invention.

(3) The numeric references of FIG. 1 designate the following elements: An inlet pipe dedicated to the introduction of the resin (1), an inlet pipe dedicated to the introduction of the synthesis solvent (2), optionally introduced by a self-priming pump. According to a preferred embodiment, a pre-activation reactor (5) of the amino acids and/or dissolution of the powders and connected to the assembly reactor (9) by an inlet pipe (6), an inlet pipe dedicated to the introduction of the deprotection agent of the amino acid (3) introduced by a self-priming pump and an inlet pipe dedicated to the introduction of the solvent (4) are connected to the assembly reactor (9), an additional inlet pipe dedicated to the introduction of reagents (8) and an inlet pipe dedicated to the introduction of the solvent are connected to the pre-activation reactor (5). The liquid-recirculation loop (10) of the reactor comprising at least one detector (11), preferably a near-infrared spectrophotometric measuring cell, a command module (13) controlled by a software making it possible for the automatisation of the reactor system, thanks to the advanced online control in the recirculation loop. A three-way valve (12) at the outlet of the sensors on the recirculation loop of the assembly reactor (9) enables, according to the needs, to pass into draining mode or not.

(4) FIG. 2 represents a calibration curve of the piperidine by UV (390 nm) for the quantification by infrared.

(5) FIG. 3 represents the calibration curve of the piperidine between 0.01% and 35% v.

(6) FIG. 4 represents the calibration curve of the piperidine between 0.01% and 1% v.

(7) FIG. 5 represents the monitoring in the liquid phase and over time of the coupling of histidine on a peptide-resin. Observation of the disappearance of the reagents and the formation of a product (DICU).

(8) FIG. 6 represents the monitoring of a deprotection step of histidine.

(9) FIG. 7 represents the washings of the piperidine after deprotection of histidine.

(10) FIG. 8 represents the disappearance of the deprotection agent over time for a fixed percolation flow rate.

DETAILED DESCRIPTION

(11) Below, preferred embodiments and implementations of the system and of the method according to the invention are described. This description is made also in reference to the appended figures.

(12) The synthesis reactor is a stainless steel reactor with a capacity of 25 litres. A filtration device is placed at the bottom of the reactor in order to retain the resin and discharge the solvents. This filtration device is formed of a sintered stainless steel material but, could consist of a filtering sheet or any other filtration system known to a person skilled in the art.

(13) The reactor has a stirring blade in order to mix, as best as possible, the resin and the liquid. This stirring blade can rotate in both rotating directions. On the top of the reactor, several inlets are present, of which one inlet is dedicated to the introduction of the resin and one inlet is dedicated to the introduction of the synthesis solvent (DMF), synthesis solvent, introduced by a self-priming pump. The introduction flow rate, as well as the volume introduced are measured and quantified by a mass flow sensor. The flow rates can range from 35 l/h to 600 l/h. The solvent can be heated or cooled as needed before entry into the reactor via a heat exchanger. In order to clean the reactor correctly between each step of the synthesis, a device for dispersing the solvent is located at the end of the line, at the level of the reactor. This device operates correctly between 20 and 1000 l/h. An inlet pipe dedicated to the introduction of the agent for deprotecting the amino acid can also be placed on the top of the assembly reactor. It is done thanks to a self-priming pump, of which the flow rate is 20 to 1000 l/h. The introduction flow rate, as well as the volume introduced are measured and quantified by a mass flow sensor. The deprotection agent can be premixed or not with the synthesis solvent before introduction into the reactor. An inlet pipe for additional solvent, of which the volume and the flow rate are controlled by a mass flow sensor, can also be placed at the top of the assembly reactor.

(14) According to an embodiment of the invention, a nitrogen inlet pipe, with the aim of making the reactor inert or of flushing by nitrogen the reactor, is placed on the top of the assembly reactor.

(15) According to another embodiment of the invention, the assembly reactor can be equipped with different sensors: A near-infrared spectroscopy sensor, A level sensor (of radar-type), to measure the resin or liquid level in the reactor, A pressure sensor, A conductivity cell at the bottom of the reactor to measure the conductivity at any moment of the synthesis in the solid-liquid phase, A pH-meter at the bottom of the reactor to measure the pH at any moment of the synthesis in the solid-liquid phase.

(16) A liquid-recirculation loop of the assembly reactor enables a real-time measurement of the evolution of the chemical species in the reactor via measuring cells which are, a conductivity cell, a near-infrared cell (from 1 mm to 30 mm of optical path) and a UV cell (from 0.5 mm to 10 mm of optical path). For a given step, the flow of the liquid into the loop occurs numerous times and the flow rate of the recirculation can be adjusted according to the steps, if the reactions are slow or rapid.

(17) When a step is finished, the liquid from the reactor is drained via the sintered material, by passing through the measuring cells of the recirculation loop, which can thus give information about the liquid phase at the outlet of the reactor.

(18) According to an embodiment of the invention, the assembly reactor is connected to another reactor which could be used to dissolve powders or to pre-activate the amino acids before introduction into the reactor. This reactor is a glass double-envelope reactor with a capacity of 10 l. It is provided with a stirrer making it possible to dissolve powders. It optionally has a conductivity probe and a pressure sensor. There is an inlet on the top, in order to introduce the powders. An inlet pipe for solvent enables, as for the assembly reactor, to introduce the solvent, thanks to a self-priming pump, ranging between 20 and 1000 l/h. The flow rate and the introduction volume are measured by a mass flow sensor. A last inlet pipe is present to introduce a solvent or a coupling agent.

(19) The assembly is controlled by a software making it possible for the automation of the installation thanks to the advanced online control.

Example 1. Operation of the Installation

(20) During a solid-phase synthesis, this starts by introducing the resin into the reactor via the dedicated inlet. A predefined volume of synthesis solvent is added at a predefined flow rate via the dedicated inlet. The resin-solvent mixture is stirred, then, once the resin is inflated, the solvent is drained via the outlet at the reactor bottom. The operation is restarted several times. At the same time, the amino acid or the linker is dissolved/or pre-activated in the dissolution reactor in the DMF. This is stirred. Either the coupling agent is added or not. If so, the pre-activation step is followed, preferably thanks to a conductivity cell.

(21) Once the pre-activation is finished, the mixture is introduced in the assembly reactor. The stirring is started in the assembly reactor and the recirculation loop is started. The infrared measuring in the recirculation loop enables to quantify the chemical species of the mixture. The concentration of the reagents decrease and those of the sub-products of the reaction increase in the liquid phase until a stabilization. Once the stabilization is reached, the step is finished, then the assembly reactor is drained via the recirculation loop which, thanks to a three-way valve at the outlet of the sensors, enables to pass into draining mode.

(22) With the coupling step being finished, the synthesis solvent is introduced in the reactor at a predefined volume and flow rate. The solvent-resin mixture is stirred and the recirculation loop is started. When the signals of conductivities are stable in the reactor, and/or the signals in the recirculation loop, the draining of the reactor is started. This step is carried out several times, preferably at least three times until reaching the desired residual concentrations in the reactor.

(23) Once this step of washing the resin is finished, the deprotection step starts. The synthesis solvent and the deprotection agent are introduced in the reactor via the dedicated inlet pipes. The stirring in the reactor is then started, as well as the recirculation loop. In real time, the deprotection step is monitored in the reactor thanks to the online measuring cells.

(24) The conductivity increases in the reactor up to a stabilization indicating the end of the reaction. The quantification of the species in the reactor can be done, preferably, thanks to an infrared cell. In this case, the monitoring of the formation of the dibenzofulvene and the consumption of the deprotection agent, here piperidine, is possible.

(25) The increase of the UV absorbance reveals the formation of the dibenzofulvene and its stabilization indicates the end of the deprotection step. The quantification of this species by UV can be done if a stabilization of the UV is not sufficient for interpreting the signals.

(26) Once the step has finished, a draining of the reactor occurs. A step of washing the deprotection agent ensues. This washing step can be done in two ways.

(27) According to an embodiment of the invention, the washing step is carried out in successive batches of introducing the washing solvent, here the DMF, stirring and recirculation loop, then draining. In each batch, an infrared cell measures the concentration of piperidine in the reactor. When the infrared measurement gives the piperidine threshold reached, a last draining is carried out.

(28) According to another preferred embodiment, the washing step can also be done, and surprisingly, by percolation thanks to a system comprising: A distribution of homogenous washing solvent on the resin bed, A measuring of the level of liquid and an adjustment of the flow rate based on the measurement of level of liquid by a suitable sensor.

(29) The optimization of the volumes of washing with DMF is achieved. The flow rate of introducing the DMF is equal, preferably, to the draining flow rate of the reactor thus keeping the constant level of liquid within the reactor, and closest to the level of the resin bed. The measuring of concentration of the deprotection agent, for example piperidine, is measured in real time at the outlet of the reactor. Once the threshold of the deprotection agent is reached, the introduction of the washing solvent is stopped and the draining is finished.

(30) Once the resin is washed, the same steps are restarted up to the end of the assembly of the peptide namely, pre-activation or dissolution of the amino acids, coupling of the amino acid on the resin, washing of the resin, deprotecting of the amino acid, washing of the resin.

(31) The use of a recirculation loop on the assembly reactor comprising a measuring cell, in particular near-infrared, has numerous advantages with respect to the solutions of the prior art. In particular, the following chemical species can be quantified, directly and non-indirectly, according to the step wherein the assembly of the peptide is found.

(32) During the coupling and washing after coupling, the system enables to quantify the amino acids protected by an Fmoc-type group (Fmoc-aa) whatever their activation status, Fmoc-aa-OH, Fmoc-aa-OBt, Fmoc-aa-Oxyma or Fmoc-aa-DIC and valid whatever the amino acid, in the presence or not of HOBt, Oxyma, DIC, DICU, or water, diisopropylurea (DICU) in the presence of the coupling agents, for example HOBt, Oxyma, DIC, Fmoc-aa, the sum of HOBt (1-hydroxybenzotriazole)+Fmoc-aa-OBt in the presence of DIC, DICU and Fmoc-aa; the sum of Oxyma+Fmoc-aa-oxyma in the presence of DIC, DICU and Fmoc-aa; the sum of DIC (N,N′-diisopropylcarbodiimide)+Fmoc-aa-DIC.

(33) The evolution can also be monitored, without quantification if needed, of all species present or not (creation, disappearance or stabilization of the concentration of species) in the presence of other coupling agents in the medium: DIC (N′N′-diisopropylcarbodiimide)+Fmoc-aa-OH, Fmoc-aa-Obt, Fmoc-aa-Oxyma, Fmoc-aa-DIC.

(34) Calibration of Piperidine for Quantification By Infrared

(35) A calibration curve must be made before being able to create a method of quantification by infrared.

(36) The inventors have selected to arbitrarily use an already-existing quantification method which is well-known to a person skilled in the art for piperidine, namely a method of quantification by UV spectrometer.

(37) It is an indirect method where piperidine in the DMF is derivatised with DNFB then analyzed by UV spectrometry at 390 nm after 30 minutes of derivatisation. As a non-limiting example, a spectrophotometer usable according to the invention is that of the company Thermo Scientific of reference Genesys 10S UV-Vis.

(38) After 30 minutes of derivatisation, the concentration of piperidine is measured at 390 nm.

(39) The calibration curve of piperidine is represented in FIG. 2.

(40) Once the calibration curve on UV is achieved, samples of piperidine are prepared at different concentrations known between 0.01% and 35% by volume of piperidine in the DMF. To have an increased precision for low values of piperidine, numerous samples have been prepared between 0.01% and 1% of piperidine.

(41) These samples have then been passed in the measuring cell of the infrared spectrometer (Manufacturer Bruker under the commercial reference Matrix-F) identical or not, to that of the system according to the invention.

(42) In order to have a method for correct quantification of piperidine by infrared, real samples must also be passed over the cell, i.e. samples coming from steps of deprotection and washing after deprotection. These contain, in particular, dibenzofulvenes, reagent traces (Fmoc-aa, DIC, DICU, HOBt, Oxyma) in addition to piperidine in the DMF.

(43) As quantification by infrared is a quantification based on spectral bands and not a highly precise wavelength, it is useful during the calibration, to have samples close to real synthesis solutions.

(44) The near-infrared quantification is a multivariable calibration making use of a matrix resolution and statistical methods. These methods are directly integrated with the software for controlling the infrared spectrometer.

(45) Real samples of deprotection and washing after deprotection have been passed over the infrared cell and have been analyzed at the same by UV spectrometry in order to determine the real concentration of piperidine in the samples.

(46) Once the samples have passed over the NIR (NEAR-INFRARED) cell at known concentrations of piperidine, the multivariable analysis can start and determine the best quantification method proposed by the software. This method must then be tested and proven by passing other samples at known concentrations and see if the calibration curve proposed actually enables to quantify the piperidine in the desired margin of error.

(47) In the case of piperidine, two calibration curves have been produced as the measuring range is very broad (0.01% to 35% of piperidine) and that the precision with a low piperidine content must be important.

(48) Samples at known concentrations of piperidine have been analyzed by the infrared quantification method.

(49) FIG. 3 represents the calibration curve where samples at known concentrations of piperidine have been analyzed by the infrared quantification method between 0.01% and 35% of piperidine.

(50) FIG. 4 represents the calibration curve of piperidine between 0.01% and 1% of piperidine.

(51) The results enable to validate the calibration curves of piperidine by infrared.

(52) Monitoring Different Species Present in the System

(53) The invention proposes a non-limiting example of monitoring and of quantifications by infrared of the different species during different steps of the peptide synthesis.

(54) Monitoring of a Step of Coupling an Histidine on a Given Peptide-Resin

(55) The amino acid and the HOBT are dissolved in the dissolution reactor. They are then introduced in the assembly reactor, as well as the DIC. The stirring is started and the recirculation loop is started up.

(56) The infrared analysis over time can be seen in FIG. 7.

(57) The stabilization of the signals occurs after around 2 hours. The final concentration of the species and in particular, Fmoc-aa-* (corresponding to histidine), is close to the expected concentration. The coupling is finished. The draining of the reactor can occur.

(58) DMF is introduced in the assembly reactor. The stirring is thus started and the recirculation loop is started up. Thus, the quantity of piperidine necessary for the deprotection is added. The measuring of the signals by infrared in the recirculation loop can be seen in FIG. 8.

(59) This shows a consumption of piperidine and a release of dibenzofulvenes in the assembly reactor. The deprotection occurs. Once the stabilization of the signals is observed, the reaction no longer develops. The reactor is then drained.

(60) Once the deprotections are finished, the evolution of the concentration of piperidine can be monitored during the batch washing.

(61) The results are presented in FIG. 9.

(62) For this, a predefined quantity of DMF is introduced in the assembly reactor. The stirring is started, as well as the recirculation loop. By infrared measuring, the quantity of piperidine present in the reactor is quantified. At each stabilization of the concentration of piperidine, the reactor is drained. As long as the obtained value of piperidine is not sufficiently low, a washing is restarted until obtaining the desired concentration.

(63) Optimization of the Washing Time of the System By Percolation

(64) According to a preferred embodiment of the invention, the chemical synthesis reactor system is connected to a percolation system, in order to also optimize the washing time and the quantities of washing solvent used.

(65) In the sense of the present invention, percolation means that the solvent, in particular washing solvent, is passed through a fixed bed, such as a resin to carry out an extraction.

(66) The percolation is carried out on a resin, for example a 4-methylbenzhydrylamine hydrochloride resin or any other resin known by a person skilled in the art (to be confirmed or give another example), simply deposited on a fixed bed on the filtration system and distributed homogenously and horizontally on its surface in order to avoid any preferably path of the washing solvent through the resin bed.

(67) The washing solvent, such as DMF, is introduced thanks to a distribution system which enables to avoid disturbing the resin bed, such that it remains horizontal on its surface.

(68) The liquid level above the resin is controlled as close as possible to the resin bed without disturbing it, in order to decrease the quantity of washing solvent used by limiting the remixing phenomena.

(69) The washing solvent flow rate is optimized thanks to the determination of the transfer kinetics of the species to be removed between the solid phase (resin) and the liquid phase (washing solvent).

(70) Thus, an excessively high flow rate would not enable for the species to diffuse and would involve an overconsumption of the washing solvent and a flow rate which is excessively low would enable for the species to diffuse, but would involve a washing time which is too long.

(71) The following example shows that according to the washing flow rate, the effectiveness of the washings is not the same.

(72) The experiment is carried out in a glass reactor of 10 cm in diameter equipped with a sintered material containing a resin bed, 10 cm high, on which a peptide is coupled. It is sought to reduce the concentration of the deprotection agent by carrying out a percolation with the washing solvent. The resin bed is homogenous and horizontal, there is no preferential path and the washing solvent is distributed such that the resin bed remains horizontal on the surface.

(73) In the following table, the impact of the washing solvent on the effectiveness of washing by percolation can be seen.

(74) TABLE-US-00001 Washing solvent ml of Washing flow rate (ml/min) washing solvent time (min) 50 930 20 80 1000 12 190 1050 6 300 1200 4

(75) At 50 ml/min of percolation flow rate, to reach an optimum washing, 930 ml of washing solvent is needed, that is 20 minutes of washing as a maximum.

(76) At 300 ml/min of percolation flow rate, to reach the same optimum washing, 1200 ml of washing solvent is needed (+20% of additional solvent with respect to a flow rate of 50 ml/min), that is 4 minutes of washing (−79% of time with respect to a flow rate of 50 l/h).

(77) It is seen that at a flow rate that is excessively high, more washing solvent is needed to reach the same washing threshold, but a shorter washing time.

(78) Results of the Percolation on the System According to the Invention

(79) The following example shows a washing by percolation on the reactor of the invention with a resin bed height of 5.6 cm.

(80) An online analysis on the recirculation loop enables to precisely quantify the removal of the deprotection agent, here piperidine.

(81) FIG. 8 shows the disappearance of the deprotection agent over time for a fixed percolation flow rate.

(82) The present invention thus being described, it is clear that the same thing can be modified in numerous ways. Such variations must not be considered as a departure from the sense and scope of the invention, and all the modifications which would be clear for a person skilled in the art are intended to be included in the scope of the following claims.