Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
  • C07K1/045—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers using devices to improve synthesis, e.g. reactors, special vessels
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J19/24—Stationary reactors without moving elements inside
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J19/24—Stationary reactors without moving elements inside
  • B01J19/2455—Stationary reactors without moving elements inside provoking a loop type movement of the reactants
  • B01J19/2465—Stationary reactors without moving elements inside provoking a loop type movement of the reactants externally, i.e. the mixture leaving the vessel and subsequently re-entering it
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
  • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
  • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
  • G01N21/359—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using near infrared light
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/65—Raman scattering
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/24—Stationary reactors without moving elements inside
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/84—Systems specially adapted for particular applications
  • G01N2021/8411—Application to online plant, process monitoring
  • G01N2021/8416—Application to online plant, process monitoring and process controlling, not otherwise provided for

Definitions

• the present invention relates to an automated reactor system for performing solid phase peptide synthesis, and more particularly to an automated solid phase peptide synthesizer using a liquid recirculation loop reactor for real time measurement of species. in the reactor via measuring cells.
• Peptides are linked amino acid chains that are the building blocks for most living organisms. As a result, the study of peptides and proteins and the ability to synthesize peptides and proteins are of great interest in the biological sciences and medicine.
• Solid phase peptide synthesis was initiated in 1963, when RB Merrifield published the synthesis of a four-amino acid sequence using a solid phase method (RB MERRIFIELD, Solid Phase Peptide Synthesis I. Synthesis of a tetrapeptide, J. Am Chem Soc, 1963, 85 (14), pp 2149-2154).
• the deprotection of the Fmoc group is carried out in a basic medium whereas the deprotection of the Boc group is carried out in an acid medium.
• the Fmoc / tBu strategy is the most used because the final cut of the solid support peptide uses concentrated TFA (trifluoroacetic acid) while the Boc / Bnl strategy requires the use of concentrated hydrofluoric acid, much more dangerous and delicate to manipulate.
• TFA trifluoroacetic acid
• Boc / Bnl strategy requires the use of concentrated hydrofluoric acid, much more dangerous and delicate to manipulate.
• Patent application WO2012056300 protects a method for real-time monitoring of solid phase peptide synthesis (SPPS) under ambient conditions to characterize peptide intermediates or on-line products.
• SPPS solid phase peptide synthesis
• the described technical solution enables real-time monitoring to trace the process of SPPS step reactions by using a light source, an electrospray unit, and a mass spectrometer.
• the use of these analysis techniques have significant disadvantages, namely a sampling of dispersed samples in a solvent which also involves the destruction of said samples, unlike the method according to the invention. We are talking about a destructive method of analysis.
• the solution cited is not quantitative and does not make it possible to know if the resin is washed well between the different steps (washing after coupling or washing the piperidine) but only to know whether the amino acid is coupled to the resin or if the amino acid is deprotected on the resin.
• Patent application WO 2017049128 discloses a system for the control of solid phase peptide synthesis by using detectors which allow the reaction to be subsequently controlled by process modification, such as the removal of the reagent.
• the detectors used measure in the phase liquid through a detection zone in the system, and one or more signals can be generated corresponding to the fluids.
• the patent WO2017049128 uses an electromagnetic radiation detector placed downstream of a reactor to detect a fluid leaving the reactor to produce a signal.
• the parameters can be modulated before or during the solid phase peptide synthesis reaction process.
• the disadvantage of this method is that the control is carried out downstream of the reactor and not in the reactor or in a recirculation loop.
• this method monitors the deprotection but not the monitoring of the coupling and finally, this technique does not allow the analysis in real time multi-pass and, unlike the system according to the invention, this solution involves be in large excess of reagents and solvents, which consumes a lot of reagents and is not at all economically viable.
• Destructive analytical methods of colorimetric or spectrophotometric type, have the disadvantage of being irreversible. In addition, they generate modified products and, more importantly, they lower the yield of the final peptide because they are performed on aliquots of resin-peptide. However, these methods are widely used because they are fast and do not require expensive instrumentation.
• Non-destructive analytical methods can also be used in batch processes. For example, there can be mentioned the monitoring of infrared reactions, based on the appearance or disappearance of functional groups, which can be applied to monitor solid phase chemical synthesis, especially in the field of organic synthesis and to monitor the coupling and deprotection during peptide synthesis. Infrared and Raman spectroscopy are widely used techniques for the detection and characterization of reaction products because they allow products to be analyzed directly on solid supports.
• assembly reactor is a real need, in particular because unlike already existing methods, it would allow a total control of the synthesis and a step-by-step follow-up of the solid phase peptide synthesis for an optimum control of the reaction time, the use of solvents, reagents and a significant cost reduction.
• a new real-time detection method would avoid favoring side reactions by stopping the step as soon as the reaction is complete.
• the present invention provides such a system and methods and overcomes the limitations mentioned above.
• the invention relates to a reactor system for carrying out solid phase peptide synthesis, the reactor system comprising: an inlet pipe (1) dedicated to the introduction of resin, an inlet pipe (2) dedicated to the introduction of the synthesis and washing solvent, an inlet pipe (3) dedicated to the introduction of the deprotecting agent of the supplied amino acid, an inlet pipe (4) dedicated to the introduction of reagents, an assembly reactor (9) and a recirculation loop (10) of the reactor liquid comprising at least one measuring cell (1 1) for indirect quantification of the progress of the reaction on the solid phase .
• the measuring cell (1 1) is a spectrophotometric measuring cell, and preferably the measuring cell (11) is a Raman spectroscopy measuring cell and even more preferably the cell of measurement.
• measurement (1 1) is a measuring cell of near-infrared.
• Near-infrared spectroscopy also abbreviated to SPIR (Near-Infrared Spectroscopy or NIR spectroscopy) is a quantitative and qualitative analysis technique used in chemistry. The technique uses a spectrum extending at wavelengths of 700-2500 nm (1) or between wave numbers 14286 and 4000 cm 1 (v).
• the reactor system for carrying out solid phase peptide synthesis further comprises in the assembly reactor (9) a filtration system.
• a filtration system according to the invention is advantageously a filtration system of sintered stainless material and / or a filter cloth.
• the reactor system for carrying out solid phase peptide synthesis further comprises a reactor (5) for preactivating the amino acids and / or dissolving the powders and an inlet (6) connecting the reactor (5) to the assembly reactor (9).
• the reactor system for performing solid phase peptide synthesis further comprises an additional solvent inlet line (7) and an additional reactant inlet line (8) on the reactor preactivation (5).
• the reactor system for carrying out solid phase peptide synthesis further comprises in the recirculation loop (10) at least one conductivity measuring cell.
• the reactor system for carrying out solid phase peptide synthesis further comprises in the recirculation loop (10) at least one ultraviolet absorbance measuring cell.
• the reactor system for carrying out solid phase peptide synthesis further comprises in the recirculation loop (10) a Raman spectroscopy measuring cell.
• These measuring cells may be additional to the measuring cells already present in the recirculation mouth.
• the reactor system for carrying out solid phase peptide synthesis further comprises a self-priming pump at the inlet line (7) dedicated to the introduction of the synthesis solvent.
• the reactor system for performing solid phase peptide synthesis further comprises a level sensor for measuring the level of resins and / or liquid in the reactor.
• the level sensor allows a continuous level measurement of the liquids present in the reactor and thus to have a real-time monitoring of the washing to optimize the efficiency and reduce the volume of solvent used.
• the reactor system for carrying out solid phase peptide synthesis further comprises in the assembly reactor (9) a pressure sensor.
• the reactor system for carrying out solid phase peptide synthesis further comprises in the assembly reactor (9) a conductivity cell at the bottom of the reactor.
• the reactor system for carrying out solid phase peptide synthesis further comprises in the assembly reactor (9) a pH measuring cell at the bottom of the reactor.
• the reactor system for carrying out solid phase peptide synthesis further comprises a solvent dispersion device located at the end of the line, at the reactor (9).
• Another aspect of the invention is a solid phase peptide synthesis method comprising the following steps:
• the process before the coupling step in the assembly reactor, the process comprises a preliminary step of preactivating the amino acid in a dissolution reactor, the real-time monitoring of the step pre-activation in the reactor by measuring a detector, introducing the preactivated mixture into the assembly reactor.
• the real-time monitoring of the coupling step is done by measuring a detector selected from a near-infrared detector, a conductivity meter, a UV detector, a detector of Raman spectroscopy and / or a pH detector.
• the real-time monitoring of the deprotection step is done by measuring a detector selected from a near-infrared detector, a conductivity meter, a UV detector, a detector of Raman spectroscopy and / or a pH detector.
• the real-time monitoring of the washing step is done by measuring a detector selected from a near-infrared detector, a conductivity meter, a UV detector, a detector of Raman spectroscopy and / or a pH detector.
• the real-time monitoring of the preactivation step is done by measuring a detector selected from a near-infrared detector, a conductivity meter, a UV detector, a detector of Raman spectroscopy and / or a pH detector.
• the washing in step (i) is carried out by percolation.
• the percolation is done with a level control via a radar type sensor.
• the concentration measurement of the deprotection agent is measured in real time at the outlet of the reactor. This measurement is done during percolation to follow its evolution and stop the introduction of the solvent and finish the emptying.
• Another aspect of the invention is the use of a level sensor for determining the height of the liquid in the reactor relative to the height of the resin bed for a percolation step in order to optimize in real time the washing and minimize solvent consumption.
• Another aspect of the invention is the use of a percolation system in the real-time monitoring method according to the invention for the filtration of a solvent.
• the real-time monitoring during the use of a percolation system is via a sensor, including a radar-type sensor, allowing the monitoring of the reaction and the control of the quantities of solvents and reagents in time. to limit the costs.
• the invention also relates to a method and a method for real-time monitoring of a chemical reaction comprising a reactor system according to the invention, a control unit (13) controlled by a software allowing the automation of the control system. reactor through advanced online control in an assembly reactor and in a recirculation loop.
• This real-time monitoring method is advantageously used for peptide synthesis reactions, more preferably solid phase peptide synthesis.
• the invention is a device comprising the computer means for implementing the method of the invention.
• the method according to the invention is at least partly implemented by computer means provided in the system according to the invention.
• the control unit (13) comprises a central control unit of the reactor system according to the invention.
• This plant is advantageously embedded on the system according to the invention.
• this control unit includes a fixed computer terminal (for example, a PC, a Macintosh or a Unix) and / or mobile (for example smartphone / tablet type) provided with one or more software / applications adapted for allow the automatic management of the system control according to the invention following the data provided by the different sensors.
• the software / application triggers control signals within the electronic unit and stopping or not the current stage.
• said appropriate software / applications of the system according to the invention can also collect information on the nature and the quantity of the reagents and solvents used for the peptide synthesis, for the purpose of inventory management of said reagents and solvents.
• the integration by these software / applications adapted to a given future period, can allow to integrate a forecast element for the maintenance of stocks of reagents and solvents to the levels required to ensure a smooth running of all stages of synthesis in the desired times, but not penalizing financially.
• an infrared sensor or an infrared measuring cell is an infrared sensing device that detects infrared wavelengths.
• the device and method of the invention has many advantages over already existing solutions.
• a reactor system and a synthesis method according to the invention also makes it possible to stop the reaction as soon as it is complete by limiting the side reactions and to optimize the purity of the synthesized peptide.
• the monitoring can also be carried out thanks to the conductivity and the temperature in addition to the near-infrared sensors inside the reactor, but it will preferably be carried out on the liquid phase because, thanks to the recirculation loop on the reactor, Many sensors can be placed in addition to the Fourier Transform Near-Infrared Spectroscopy (FTNIR) sensors, such as conductivity and UV sensors.
• FNIR Fourier Transform Near-Infrared Spectroscopy
• Another advantage of the system and method according to the invention is that only one calibration is used for all Fmoc protected natural amino acids.
• the system and method according to the invention also makes it possible to precisely quantify the deprotection agent and the release of the dibenzofulvenes during the deprotection steps of the Fmoc protected amino acids in order to know when the reaction is complete.
• the system and method according to the invention also make it possible to follow the washing and the reduction of the reagent concentrations to a threshold in order to know when the washing is finished. Thanks to this advanced online control, it is possible not only to follow the evolution of the reactions, but also to use them for automation.
• the reactor will be able to operate automatically for several stages depending on the online monitoring results.
• the reactor can operate automatically and thus give rise to a more efficient washing of the resin, operating in a percolation mode.
• the system and method according to the invention uses a percolation process to wash the solvent and optimize the online monitoring by the amount of the reagents and solvents used.
• the liquid is introduced from the top of the reactor to the resin at the same flow rate as the outlet flow of the reactor by means of the control of the height of the liquid. which must remain constant in the reactor during all the washing.
• This washing mode comprises a packed resin bed and a distribution system for the entry of the liquid at the top of the reactor for a good distribution of the liquid on the surface of the resin.
• This washing mode is the most efficient and allows a significant reduction in solvent consumption compared to other existing modes.
• the reactor is also temperature controlled to be able to change temperature between two different stages in a few minutes.
• the present invention reduces the cost required for the purchase of starting materials and the treatment of the residual liquid, considerably reducing the amount of reagents, solvents actually consumed and waste liquid (organic solvent), harmful to the environment.
• FIG. 1 is a schematic view showing a synthesis reactor system (14) for carrying out a solid phase peptide synthesis with a recirculation loop (10) of the reactor liquid making it possible to measure in real time the evolution of the chemical species in the automatic peptide synthesizer of the present invention.
• the numerical references of FIG. 1 denote 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 supplied by a self-priming pump.
• a reactor (5) for preactivating the amino acids and / or dissolving the powders and connected to the assembly reactor (9) via an inlet pipe (6), a dedicated inlet pipe at the introduction of the deprotecting agent of the amino acid (3) fed 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 the reagents (8) and an inlet pipe dedicated to the introduction of the solvent are connected to the preactivation reactor (5).
• the recirculation loop (10) of the reactor liquid comprising at least one detector (1 1), preferably a near-infrared spectrophotometric measuring cell, a control module (13) controlled by a software allowing the automation of the control system. reactor through advanced online control in the recirculation loop.
• a 3-way valve (12) at the output of the sensors on the recirculation loop of the assembly reactor (9) makes it possible, according to the needs, to switch to emptying mode or not.
• Figure 2 shows a calibration curve of piperidine by UV (390nm) for infrared quantization.
• Figure 3 shows the calibration curve of the piperidine between 0.01% and 35% v.
• Figure 4 shows the calibration curve of piperidine between 0.01% and 1% v.
• Figure 5 shows the follow-up in the liquid phase and in time of the coupling of histidine on a peptide-resin. Observation of the disappearance of reagents and the formation of a product (DICU).
• Figure 6 shows a follow-up of a deprotection step of histidine.
• Figure 7 shows washes of piperidine after deprotection of Histidine.
• Figure 8 shows the disappearance of the deprotecting agent over time for a fixed rate of percolation.
• the synthesis reactor is a stainless steel reactor with a capacity of 25 liters.
• a filtration device is placed at the bottom of the reactor to retain the resin and evacuate the solvents.
• This filtration device is formed of a sintered stainless steel material but could consist of a filter cloth or any other filtration system known to those skilled in the art.
• the reactor has a stirring blade in order to mix the resin and the liquid as well as possible. This pale agitation can rotate in both directions of rotation.
• On the top of the reactor are several inputs including an entry dedicated to the introduction of the resin and an entry dedicated to the introduction of synthetic solvent (DMF), synthetic solvent, supplied by a self-priming pump.
• the feed rate and the volume introduced are measured and quantified by a mass flow meter. Flow rates can range from 35 l / h to 600 l / h.
• the solvent may be heated or cooled if necessary before entering the reactor via a heat exchanger.
• a solvent dispersion device is located at the end of the line, at the reactor.
• An inlet line dedicated to the introduction of the deprotecting agent of the amino acid can also be placed on the top of the assembly reactor. It is carried out thanks to a self-priming pump with a flow rate of 20 to 1000 l / h. The feed rate and the volume introduced are measured and quantified by a mass flow meter.
• the deprotection agent may be premixed or not with the synthesis solvent before introduction into the reactor.
• An additional solvent inlet pipe whose volume and flow rate are controlled by a mass flow meter may also be placed at the top of the assembly reactor.
• a nitrogen inlet pipe for the purpose of rendering the reactor inert or flushing with nitrogen the reactor is placed on the top of the assembly reactor.
• the assembly reactor may be provided with different sensors:
• a level sensor (radar type), to measure the level of resin or liquid in the reactor
• a recirculation loop of the liquid of the assembly reactor makes it possible to measure in real time the evolution of the chemical species in the reactor via measurement cells which are, a conductivity cell, a near-infrared cell (from 1 mm to 30mm optical path) and a UV cell (from 0.5mm to 10mm optical path).
• measurement cells which are, a conductivity cell, a near-infrared cell (from 1 mm to 30mm optical path) and a UV cell (from 0.5mm to 10mm optical path).
• the passage of the liquid in the loop takes place many times and the rate of recirculation can be adjusted according to the steps, if the reactions are slow or fast.
• the liquid of the reactor is drained via the sintered material, through the measurement cells of the recirculation loop, which can then give information on the liquid phase at the outlet of the reactor.
• the assembly reactor is connected to another reactor that can be used to dissolve the powders or preactivate the amino acids before introduction into the reactor.
• This reactor is a double-jacketed glass reactor with a capacity of 101. It is equipped with an agitator allowing the dissolution of the powders. It has an optional conductivity sensor and a pressure sensor.
• An inlet exists on the lid to introduce the powders.
• a solvent inlet pipe makes it possible to introduce the solvent by means of a self-priming pump, ranging between 20 and 1000 l / h. The flow rate and the volume of introduction are measured by a mass flow meter. A last inlet line is present to introduce a solvent or a coupling agent.
• the resin is first introduced into the reactor via the dedicated inlet.
• a predefined volume of synthesis solvent is added at a predefined rate via the dedicated input.
• the resin-solvent mixture is stirred and once the resin is swollen, the solvent is drained via the outlet at the bottom of the reactor. We repeat the operation several times.
• the amino acid or the linker in DMF is dissolved or pre-activated in the dissolution reactor. It is stirred. Either add the coupling agent or not. If so, the preactivation step is followed, preferably by means of a conductivity cell.
• the mixture is introduced into the assembly reactor. Agitation is started in the assembly reactor and the recirculation loop is started. The infrared measurement in the recirculation loop makes it possible to quantify the chemical species of the mixture. The concentration of the reagents decreases and those of the byproducts of the reaction increase in the liquid phase until stabilization. Once the stabilization is reached, the step is finished, then the assembly reactor is emptied via the recirculation loop which, thanks to a 3-way valve at the output of the sensors, makes it possible to switch to the emptying mode.
• synthesis solvent is introduced into the reactor at a volume and a predefined flow rate.
• the solvent-resin mixture is stirred and the recirculation loop is started.
• the emptying of the reactor is started. This step is carried out several times, preferably at least 3 times until the desired residual concentrations in the reactor are reached.
• the deprotection step begins.
• the synthesis solvent and the deprotection agent are introduced into the reactor via the dedicated inlet lines.
• the stirring in the reactor is then started as well as the recirculation loop.
• the deprotection step in the reactor is monitored in real time by means of the on-line measurement cells.
• the conductivity increases in the reactor until a stabilization indicating the end of the reaction.
• Quantification of the species in the reactor can be done, preferably, by means of an infrared cell. In this case, the monitoring of the formation of dibenzofulvene and the consumption of the deprotection agent, here piperidine, is possible.
• the increase in UV absorbance reveals the formation of dibenzofulvene and its stabilization indicates the end of the deprotection step.
• the quantification of this species by UV can be carried out if a stabilization of the UV is not sufficient for the interpretation of the signals.
• a washing step of the deprotection agent follows. This washing step can be done in two ways.
• the washing step is carried out in successive batches of introduction of the washing solvent, here the DMF, stirring and recirculation loop, and then emptying.
• the washing solvent here the DMF
• stirring and recirculation loop At each batch, an infrared cell measures the piperidine concentration in the reactor. When the infrared measurement gives the piperidine threshold reached, a last emptying is carried out.
• the washing step can also be done, and surprisingly, by percolation through a system comprising
• the optimization of the DMF washing volumes is achieved.
• the feed rate of the DMF is preferably equal to the discharge rate of the reactor thus keeping the constant level of liquid within the reactor and as close to the level of the resin bed.
• the measurement of the 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 has been reached, the introduction of the washing solvent is stopped and the emptying is finished.
• a recirculation loop on the assembly reactor comprising a measuring cell, particularly near-infrared, has many advantages over the solutions of the prior art.
• the following chemical species can be quantified in a direct and non-indirect manner depending on the stage in which the peptide assembly is located.
• Fmoc-aa an Fmoc-type group
• Fmoc-aa-OFI an activation state
• Fmoc-aa-OBt an activation state
• Fmoc aa-Oxyma an activation state
• Fmoc-aa-DIC an activation state
• a calibration curve must be performed before an infrared quantification method can be created.
• the inventors have arbitrarily chosen to use an already existing quantification method well known to those skilled in the art for piperidine, namely a method of quantification by UV spectrometer.
• a spectrophotometer usable according to the invention is that of the company Thermo Scientific reference Genesys 10S UV-Vis.
• the piperidine concentration is measured at 390 nm.
• piperidine samples are prepared at various known concentrations between 0.01% and 35% by volume of piperidine in DMF. To have increased accuracy for low piperidine values, many samples were prepared between 0.01% and 1% piperidine.
• Quantification in the near infrared is a multi-variable calibration using matrix resolution and statistical methods. These methods are directly integrated into the control software of the infrared spectrometer.
• the multi-variable analysis can be started and the best quantification method proposed by the software can be determined. This method must then be tested and tested by passing other samples at known concentrations and see if the proposed calibration curve effectively quantifies the piperidine in the desired margin of error.
• FIG. 3 represents the calibration curve where samples with known concentrations of piperidine were analyzed by the infrared quantification method between 0.01% and 35% of piperidine.
• Figure 4 shows the piperidine calibration curve between 0.01% and 1% piperidine.
• the invention provides a nonlimiting example of monitoring and infrared quantification of the different species during the different stages of peptide synthesis.
• the amino acid and I ⁇ OBT are dissolved in the dissolution reactor. They are then introduced into the assembly reactor as well as the DIC. The agitation is started and the recirculation loop is started.
• the stabilization of the signals takes place after about 2 hours.
• the final concentration of species, especially Fmoc-aa- * (corresponding to histidine), is close to the expected concentration.
• the coupling is finished.
• the emptying of the reactor can take place.
• a predefined amount of DMF is introduced into the assembly reactor. Stirring is started as well as the recirculation loop.
• the amount of piperidine present in the reactor is quantified by infrared measurement.
• the reactor is drained. As long as the value of piperidine obtained is not sufficiently low, washing is repeated until the desired concentration is obtained. Optimization of the washing time of the system by percolation.
• the chemical synthesis reactor system according to the invention is connected to a percolation system in order to further optimize the washing times and the amounts of washing solvent used.
• percolation means that the solvent, in particular the washing solvent, is passed through a fixed bed, such as a resin, to carry out an extraction.
• the percolation is carried out on a resin, for example a 4-methylbenzhydrylamine hydrochloride resin or any other resin known to those skilled in the art (to be confirmed or give another example), simply deposited in fixed bed on the filtration system and distributed homogeneously and horizontally on its surface to avoid any preferential route of the washing solvent through the resin bed.
• a resin for example a 4-methylbenzhydrylamine hydrochloride resin or any other resin known to those skilled in the art (to be confirmed or give another example
• the washing solvent such as DMF
• the washing solvent is fed through a distribution system that avoids disturbing the resin bed so that it remains horizontal on its surface.
• the liquid level above the resin is controlled closer to the resin bed without disturbing it, in order to reduce the amount of washing solvent used by limiting the remixing phenomena.
• washing solvent is optimized by determining the kinetics of transfer of the species to be removed between the solid phase (resin) and the liquid phase (washing solvent).
• the experiment is carried out in a glass reactor 10 cm in diameter provided with a sintered material containing a bed of resin 10 cm in height on which is coupled a peptide.
• a sintered material containing a bed of resin 10 cm in height on which is coupled a peptide.
• the resin bed is homogeneous and horizontally there is no preferred path and the washing solvent is distributed so that the resin bed remains horizontal on the surface.
• the following example shows a percolation wash on the reactor of the invention with a resin bed height of 5.6 cm.
• Figure 8 shows the disappearance of the deprotection agent over time for a fixed rate of percolation.

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