Patent Application
20200384435
The present invention relates to a device for parallel oligomer synthesis based on parallel centrifugal processing of reactors, a method of parallel oligomer synthesis and use thereof.
Oligomer (peptide, oligonucleotide, carbohydrate) synthesis is based on the repetition of similar organic reactions resulting in connecting building units (amino acids, nucleotides, sugars) to form macromolecular organic compounds (peptides, oligonucleotides, carbohydrates). There are various automated synthesizers designed for synthesis of these oligomers based on multitude of technologies. Examples of such automated synthesizers are briefly discussed below.
The system described for instance in the U.S. Pat. No. 8,731,721 or 7,914,739 is capable of continuously synthesizing molecules by providing an array of reaction sites and an array of stations for carrying out synthetic manipulations. The reaction sites and the stations are placed such that the two arrays can be moved relative to each other such that the stations carry out desired steps of a reaction scheme at each reaction site.
The system contains a rotary table with plate modules located along the outer circumference of the table. The plate modules contain plate holders, which support microtiter plates. During operation, the microtiter plates pass under dispensing stations, such that the movement of the table and placement of the stations allows all microtiter plates to receive reactants and incubate the reactants according the predetermined reaction scheme, wherein each well of the microtiter plate is assigned a different oligonucleotide to be synthesized based on predetermined requirements. Based on this assignment, the program instructs each dispenser to output the proper reaction solutions into the proper well.
However, there are many laboratories synthesizing these compounds manually, requiring performance of repetitive processes—addition of reagents, incubation and separation of liquid-phases, which are prone to the human errors resulting from momentary attention lapses. One of the popular techniques for oligomer synthesis utilizes simple disposable plastic syringes. Major advantage of this technique pioneered by Krchnak et al. (Tetrahedron Letters, Volume 28, Issue 38, 1987, Pages 4469-4472, Continuous-flow solid-phase peptide synthesis, Viktor Krchn̆ák, Josef Vágner, Martin Flegel, Otakar Mach, http://dx.doi.org/10.1016/S0040-4039(00)96541-9) is the fact that the syringe is capable of handling majority of organic reagents and can be used under various conditions in enclosed environment. This allows combination of the routine building of the oligomer with very sensitive reactions requiring special attention and conditions.
For instance, the U.S. Pat. No. 8,394,923 discloses a centrifugation based apparatus and method for separating layers of immiscible or partially miscible liquids of different density by removal of lighter layers of liquids by creation of “pockets” from which material cannot be removed by centrifugal force.
In this way, the liquid phase can be eliminated from reaction vessels, where the synthesis occurs. The reaction vessels are held in a tilted position with a tilt towards the axis of rotation. The rotor is then spun at a speed that expels the liquid from the vessels, keeping at the same time the solid support in “pockets” created by centrifugation.
Further, a U.S. Pat. No. 8,022,013 discloses a method of synthesis, wherein a solid-phase support is provided, for instance macrobeads or microbeads, a particle is dispensed in the reaction vessel, permanently bonded in the substrate within the reaction vessel, and functionalized to covalently attach an intermediate compound of a synthetic reaction, a liquid including a reagent is dispensed to the solid-phase support to effect the synthetic reaction, and the liquid is removed from the solid-phase support by centrifugation, whereby the intermediate compound remains attached to the substrate by the particle. The substrate and support are capable of withstanding centrifugal forces generated during high-throughput synthesis.
The solid-phase beads may be held within the vessel during centrifugation by tilting the reaction vessel to form pockets from which the solid-phase cannot be removed by centrifugal force, as described above. Alternatively, mesh or other frit materials can be employed.
All described technologies are limited in the scale applicable to the synthesis and relatively complicated hardware. Tilted centrifugation is good for the synthesis using non-swelling carrier forming the same volume of the solid support throughout the whole synthesis. This type of carrier is suitable for synthesis of oligonucleotides, but it is not usable for peptide synthesis where the carrier can grow substantially during the synthesis. All methods using mesh or frit material to retain solid support must solve a problem of leakage of the liquid through the supporting material during incubation periods.
The first set of available background art documents constitute automatized arrays for performing syntheses as described above. In all of the above described methods of oligomer synthesis, a human intervention is needed. For example, in the methods using centrifugation, each removal of a reagent solution from the reaction vessel after centrifugation is done manually, which incorporates potential errors into the oligomer synthesis, and causes delays, which slow down the synthetical process. Another disadvantage lies in the fact that not complete removal of the liquid from the centrifugation tube after centrifugation can be performed by aspiration of the liquid. There is still certain amount of liquid present in the solid sample even after its careful aspiration, which introduces potential errors into the oligomer synthesis.
The presented invention allows the operator to run long oligomer (peptide, oligonucleotide, carbohydrate) synthesis in parallel automatically, i.e. without human interaction, therefore overcoming the problems of the background art. In any step of the synthesis, however, the operator may remove an individual reactor and perform a special operation requiring special conditions or utilizing very sensitive or special reactant manually. After that step, the reactor can be returned to the device for continuation of the oligomer assembly. Such procedure enables the operator to parallelly synthesize a number of oligomers either in a fully automatic way, or, upon his choice, to interrupt the automated synthetical procedure at any stage in order to perform one or several consecutive synthetical steps manually, and then to return back to the automated synthetical procedure. The device according to the present invention therefore enables the synthesis of oligomers containing even very sensitive, costly or valuable monomeric units, which need special treatment and conditions. Moreover, the siphon based outflows present in the device according to the present invention (as described below) allow for a complete removal of liquids during centrifugation, which overcomes the other above discussed disadvantages of the background art.
The concept of the liquid reactant delivery for the parallel oligomer synthesis is based on parallel centrifugal processing of reactors. A device for parallel oligomer synthesis comprises the following subsystems: centrifugation and outflow subsystem and dosing subsystem, which can be supplied by selection subsystem and storage. All of them may be retained by a boxing subsystem. They are controlled by a control subsystem.
The synthesis takes place in reactors. Each of the reactors has an upper end and a lower end. The upper end is an opened end and the lower end comprises a nozzle. The reactors may be constituted by syringes, preferably made of plastic. However, reactors may be made of any other suitable material in any suitable form in order to stay resistant against the solvents used during the synthesis. A person skilled in the art would be able to decide, which material should/should not be contacted with which solvent.
Inside each reactor, a filter may be provided at the lower end close to (above) the nozzle to provide a semipermeable membrane. As such, it provides a stopper for the solid phase (e.g. resin beads provided with functional groups suitable for immobilization of amino acids used for peptide synthesis or building blocks used in the synthesis of other biooligomers (nucleic acids, carbohydrates)), which is placed in the reactors at the beginning of the synthesis, while it allows the reagents and/or solvents to be washed out of the reactor after completion of each step of the oligomer synthesis. The filter suitable for being placed inside the reactor is, for instance, a sintered glass, plastic (polypropylene, Teflon) or metal mesh.
The nozzle of each reactor is connected to a siphon based outflow. The siphon based outflow comprises a first end and a second end. The first end of the siphon based outflow is connected to the nozzle of the lower end of the reactor. The siphon based outflow provides an outflow for reagents to be removed during the synthesis and works on a siphoning principle, i.e. the second end of the outflow, when placed in the device, is positioned higher than the level of contents of the reactor. Thus, depending on the speed of rotation, the liquid in reactor is either not capable to overcome the gravity based resistance of the siphon at low speed, or it is removed completely from the reactor at high speed rotation by centrifugal forces. The low speed of rotation can be defined by up to 50 rpm, more preferably up to 40 rpm, more preferably from 5 to 30 rpm, even more preferably from 10 to 20 rpm, most preferably about 10 rpm. The medium speed of rotation can be defined by an interval from 50 to 200 rpm, more preferably from 80 to 150 rpm, more preferably from 100 to 130 rpm, most preferably about 120 rpm. The high speed of rotation can be defined by more than 200 rpm, more preferably from 250 to 6000 rpm, more preferably from 500 to 3000 rpm, even more preferably from 800 to 2000 rpm, most preferably about 1000 rpm.
In one embodiment, the siphon based outflow may be a tube in S-shape—hereinafter referred to as an S-trap outflow. The first end of the S-trap outflow is connected to the nozzle of the lower end of the reactor and the second end of the S-trap outflow, when placed in the device, is positioned higher than the level of contents of the reactor.
The second end of the S-trap outflow may also comprise a nozzle. The second end of the S-trap outflow or the nozzle of the S-trap outflow is positioned so that liquids removed from the reactor through the second end of the S-trap outflow or the nozzle of the S-trap outflow flow into a circular outflow channel, which will be described in more details later.
In another embodiment, the siphon based outflow may comprise a vertical tube—hereinafter referred to as an I-trap outflow. The I-trap outflow comprises a channel connected to the nozzle of the reactor syringe, the channel, when in operating position, extending vertically from the nozzle, a peek tubing having a first open end and a second open end, the first open end being connected to the vertical channel, the peek tubing being vertically extended, again in operating position, such that the second open end of the peek tubing is positioned higher than the level of contents of the reactor and a cover, e.g. and inverted syringe, comprising a first open end, side walls, and a second end with a top, covering the peek tubing and creating an interspace between the peek tubing and the cover, the interspace being provided between the second end of the peek tubing and the top of the cover, as well as between the peek tubing and the side walls of the cover.
As described previously, the liquid from the reactor cannot overcome the gravity based resistance of the S-trap or the I-trap at the low speed of rotation (e.g. during liquid transfer from pre-activation compartments or during a synthesis shaking), or it is removed completely from the reactor at high speed rotation by centrifugal forces.
The reactors and the siphon based outflows are positioned in a centrifugation subsystem comprising a centrifuge. The centrifuge is a circular disk, made of inert material, preferably metal, for example steel, aluminum, solvent resistant plastic, glass, etc. and seated on a center axle.
The centrifuge is driven by a driving motor seated on the same common axle as the centrifuge. The driving motor allows the centrifuge to rotate at different speeds, from low (of about 10 rpm), through medium (of about 50 to 200 rpm), to high (of about 1000 rpm) speed of rotation. It also allows a stepped motion.
The centrifuge comprises reactor holders, which are configured to retain the reactors tilted towards the vertical axle of the centrifuge at an angle. The reactors may be positioned at an angle of 30 to 85 degrees, preferentially at the angle of 45 degrees. The holders may be constituted by fixation slots, in which at least parts of the reactors are fixed, such that the reactors are securely fixed during the synthesis, but freely removable when the centrifuge is stopped.
In one embodiment, the centrifuge may comprise two circular disks—an upper disk and a lower disk, both of the disks being made of an inert material and seated on a common axle in the center of mass. Vertically, the upper and the lower disks may be positioned at a distance from each other. The distance between the upper and the lower disk is determined by the size of the reactors. Even though the disks are positioned at a distance, they are physically connected (e.g. with use of screws and supporting brackets) to each other. The first and the second disk may be manufactured as a one construct combining the function of disks and siphon based outflows by e.g. additive manufacturing.
In the above-mentioned embodiment, the upper disk is provided with reactor holder openings at its inner circumference and with siphon based outflow openings at its outer circumference. The fixation slots for the nozzles of the reactors may be provided integrally with the lower disk or they may be formed as a separate element or elements fixed to the lower disk. The fixation slots are positioned at the lower disk's outer circumference. The reactor may then be inserted through the opening in the upper disk into the fixation slot of the lower disk. As the opening is provided at the inner circumference and the fixation slot is provided at the outer circumference, the reactor is positioned at an angle with respect to the central vertical axle of the centrifuge, e.g. at an angle of 45 degrees. The reactors are securely fixed during centrifugation, but they can be easily removed from reactor holders when the centrifuge is static. The reactors can be removed by hand.
The reactors and the siphon based outflows are positioned such that the content of liquids in the reactor reaches a level which is vertically lower than the second end of the siphon based outflow, i.e. the second end of the S-trap outflow or the second end of the peek tubing of the I-trap outflow. The outflow thus operates on a siphoning principle: when the rotation speed of the centrifuge is low, the liquid in the reactor is not capable of overcoming the gravity based resistance of the siphon based outflow, S-trap outflow or I-trap outflow, and remains fully inside the reactor. When the rotation speed of the centrifuge increases, the liquid from the reactor enters the siphon based outflow from the nozzle of the lower end of the reactor and continues through the siphon based outflow, and, depending on the speed of rotation, the liquid may be removed from the reactor through the siphon based outflow partially or completely. This enables the oligomer synthesis to run in parallel and in full cycle, including several partial or complete washing cycles. Thus, removal of liquids using centrifugation and the siphon base outflow is significantly more efficient than a simple aspiration of liquids. The rotation speed of the centrifuge required for the liquid from the reactor to overcome the siphon based outflow may partially depend on the solvent, particularly on the density and viscosity of the solvent. A person skilled in the art would be able to decide, which speed of the centrifuge should be used with a particular solvent.
In order to ensure the outflow of the liquid, a circular outflow channel, preferably in a form of a continuous groove, is positioned at the outer circumference of the centrifuge, the outflow channel arranged so that the siphon based outflows empty into said outflow channel. The circular outflow channel comprises outflow openings positioned on its bottom. The outflow openings of the outflow channel are connected to outflow pipes, which provide an outflow system of the device and drain away the liquid removed from the reactors during the synthesis.
The liquids are removed from the S-trap outflow through the second end or the nozzle of the S-trap outflow and flow into the circular outflow channel, which is positioned on the perimeter of the centrifuge and aligned with the second ends or the nozzles of S-trap outflows such that the second ends or the nozzles of the S-trap outflows are positioned in the circular outflow channel to drain away the liquid as described above.
The liquids are removed from the I-trap outflow through a channel system that is positioned in a channel holder. The channel holder may form part of the lower disc of the centrifuge and/or of the fixation slots, or it can be formed as a separate element attached to the lower disc and/or the fixation slots. The cover of the peek tubing is connected to the channel holder. The channel holder comprises a funnel arranged at one end of the channel system so that the channel system is through the funnel connected with the interspace created between the peek tubing and the cover. The channel system may further comprise at least one channel positioned horizontally, i.e. in parallel with the planes of the lower and the upper discs and/or at least one stopcock, positioned preferably close to the end of the channel system that is opposite to the end with the funnel. The circular outflow channel is arranged so that the channel system empties into the outflow channel to drain away the liquid as described above.
The described arrangement advantageously allows the operator to remove the liquid from the reactors by the succession of fast centrifugation, in which the liquid is transferred from the reactors through the vertical channel and the peek tubing into the interspace created between the peek tubing and the cover, slow rotation, during which the liquid is flowing out of the interspace, through the funnel into the channel system and out of the stopcock, and finally short fast centrifugation, which removes the liquid from the channel system completely, preventing thus any potential crystallization of reagents in solutions.
Above the centrifugation disk, a distribution rotor is positioned. It has a shape of a circular disk and is made of a suitable inert material, e.g. of the same material as the centrifuge. The distribution rotor and the centrifuge have the same common axis, however, the distribution rotor is seated on its own axle and it is connected to a positioning device, which comprises a driving motor to allow positioning of the distribution rotor independently from the centrifuge. The motor driving the distribution rotor allows the distribution rotor to rotate at different speeds and in a stepped motion. The independent movement of the distribution rotor, in particular the stepped motion, allows each of the individual pre-activation compartments (will be described later) to be filled in separately, one by one in an automated way.
The distribution rotor is further movable in vertical direction between a lower position and an upper position. The vertical movement is provided by a lifting device, which may be e.g. another motor or a gas operated piston. The vertical movement is provided in order to allow the distribution rotor to be in contact with the centrifuge in the lower position to ensure a synchronized rotation of the centrifuge and the distribution rotor.
Both the positioning device and the lifting device are positioned above the distribution rotor, outside of a centrifugation drum, in which the centrifuge and the distribution rotor are placed. The distribution rotor is driven by its own driving motor only when it is positioned in the upper position. In the upper position, it is detached from the centrifuge, which can then perform independent operations with the reactors (shaking, rotation, centrifugation). When the distribution rotor is positioned in the lower position, the motor driving the distribution rotor is disengaged from the distribution rotor and all actuation of the distribution rotor is performed by the motor driving the centrifuge so that the synchronized rotation of the centrifuge and the distribution rotor is ensured.
The distribution rotor comprises pre-activation compartments. Reagents are delivered into the pre-activation compartments such that the pre-activation of non-activated building blocks can be achieved. The reagents may be delivered into the pre-activation compartments by a dosing subsystem, which will be described later. The pre-activation compartments are positioned at the outer circumference of the distribution rotor and are provided with grooves directed radially outwards, towards the outer edge of the distribution rotor. At the stage of no rotation or a slow rotation (of about 10 rpm), the liquids contained in the pre-activation compartments cannot overcome the tilt of the grooves, and therefore stay in the pre-activation compartments. These grooves enable the connection between the pre-activation compartments of the distribution rotor and centrifuge when the distribution rotor is in the lower position such that the contents of the compartments may be transferred to the reactors upon synchronized rotation of the distribution rotor and the centrifuge at medium rotation speed (from ca 50 to ca 200 rpm). At the medium speed, the liquids are transferred from each of the pre-activation compartments into the corresponding reactor through the corresponding groove, however, they remain inside the reactor because the speed of rotation is not high enough to overcome the resistance of the siphon based outflow, and the liquids are therefore prevented from flowing out of the reactor.
As the rotation of the centrifuge and the distribution rotor is synchronized, this transfer may be provided for all pre-activation compartments of the distribution rotor and the corresponding reactors in parallel.
The pre-activation compartments can be filled in directly or via a dosing device. In one embodiment, the dosing device is enabled in the system. The dosing device may comprise a distribution valve and a dosing pump, the dosing pump being connected to the distribution valve. The dosing pump may be configured to measure the exact quantity of liquid reagents to be dosed into individual pre-activation compartments. The dosing pump may be a programmable syringe pump.
The distribution valve comprises a plurality of ports. The distribution valve comprises at least one outtake port which is via tubing connected to the centrifugation subsystem and through which the individual pre-activation compartments may be filled in. The other ports of the distribution valve are intake ports and provide for a connection with stock solutions of reactants and/or solvents and/or activating reagents and/or coupling reagents stored in storage containers.
The dosing pump may be connected to the desired reactant via one of the ports of the distribution valve (intake connection; the other ports are closed) and a desired quantity may be sucked by the dosing pump inside the dosing pump. Subsequently, the intake connection may be closed and an outtake connection between the dosing pump and the distribution rotor may be opened.
In one embodiment, a distribution device may be enabled in the system. It connects the dosing pump via the distribution valve to either the selector valve or to the pre-activation compartments of the distribution rotor. The distribution device may be a solenoid valve with three ports switching between the connection of the dosing pump with the outtake port of selector and the connection between the dosing pump and the pre-activation compartments of the distribution rotor (through the distribution valve).
Depending on whether the distribution device is or is not enabled in the system, the distribution valve may comprise up to 12 ports. For example, the distribution valve may have 10 ports when there is no distribution device enabled in the system and 12 ports when there is the distribution device enabled in the system. Alternatively, a multiport rotary valve may be enabled in the system instead of the three-way solenoid valve.
In one embodiment, it is possible to place a tubing with a predefined volume (which is larger than the volume being delivered to the reactor) between the dosing pump and inlet opening of the pre-activation compartment to avoid the reagents entering the dosing pump inner volume. In this arrangement, the distribution device must be placed between selector valve and inlet port of the centrifuge assembly. The distribution device (e.g. the three-way solenoid) is opened in the direction to the dosing pump, liquid is sucked from the distribution valve into the tubing, solenoid is opened in direction of delivery to the particular reactor and liquid is expelled into the reactor, without entering the syringe of the dosing pump and contaminating the liquid in there.
In any case, the desired quantity of the reactant is transferred via tubing and one of the pre-activation compartments is filled in. This process is repeated with either the same reactant or other reactants until the desired number of pre-activation compartments is filled in.
The building blocks, for example amino-acids, for the oligomer synthesis may be stored in storage containers. The storage containers may either be connected directly to the distribution valve of the dosing device or they may be connected through a selector device.
In one embodiment, one of the intake ports of the distribution valve may be used for connection to a selection device. The selection device is a valve configured to select the desired building block solution (e.g. solution of a particular amino-acid in oligopeptide synthesis), which may then be transferred through the selector device, the distribution valve and the dosing pump into the individual pre-activation compartments of the distribution rotor.
The selector device comprises a plurality of ports. Preferably, it may comprise up to 28 intake ports. The intake ports are connected via tubing with the storage containers containing various building block solutions (e.g. amino-acid solutions).
The selector device further comprises an outtake port, which may be connected via tubing with one of the intake ports of the distribution valve of the dosing device. In order to retrieve the desired amino-acid, the connection between the dosing pump, one of the intake ports of the distribution valve, the outtake port of the selector device and one of the intake ports of the selector device is opened and the desired quantity of the desired amino-acid is sucked from the storage container into the dosing pump. The desired quantity of the desired amino-acid is then transferred from the dosing pump through the outtake port of the distribution valve into one of the pre-activation compartments.
In one embodiment, one of the output ports of the dosing pump may be selected to be used exclusively for washing, i.e. for providing a connection to washing solvent only, as it is likely that larger volumes of washing solvents are used during the entire synthesis, or, alternatively, washing solvents can be delivered by separate dosing pump.
In one embodiment, the selector device may comprise coplanar layers of graphite pressed together by spring. Two graphite layers touching each other have extraordinary sealing properties, while at the same time having a very low friction coefficient. Using of graphite for the selector device is especially advantageous as there is no need for further sealing additives or friction reducing additives. The selector device may comprise two coplanar, preferably circular, graphite layers. The upper layer comprises a plurality of holes, or the intake ports, respectively. Preferably, the intake ports are located close to the outer circumference of the upper graphite layer. The lower graphite layer comprises a single hole, or the outtake port, respectively, and an elongated groove. The outtake port is located in the middle of the circular graphite layer. The elongated groove has a first end and a second end. The position of the first end of the groove is identical with the position of the outtake port. The groove is elongated radially outwards form the position of the first end of the groove such that the position of the second end of the elongated groove is identical with position of one of the intake ports in the upper graphite layer. Thus, a connection between the intake port, the elongated groove and the outtake port is established. As the intake ports are positioned in a circular configuration along the outer circumference of the upper graphite layer, the elongated groove allows to establish a connection between the outtake port and any one of the intake ports by rotating of one of the two coplanar graphite layers relatively to the other one. In order to ensure the non-crosscontamination of the desired reactant by the reactant from any other unselected port, this coplanar arrangement selector port should preferably be used in underpressure (suction) regimen, when the sealing of the two coplanar surfaces can be guaranteed.
The solutions of particular building blocks (e.g. amino-acids) may be stored in storage containers. The storage containers may have various sizes and may be made of any suitable materials resistant to the solvent used, preferably plastic. The number of storage containers may correspond to the number of intake ports of the selector device. Thus, it may contain up to 28 storage containers. In one embodiment, the storage containers may have unified shape, for example cylindrical shape and may be disposably placed in a holder. The holder may have openings into which the storage containers may be placed. In this way, all the storage containers are securely placed in one holder.
The centrifugation subsystem and the distribution rotor may be placed in a centrifugation drum. The centrifugation drum may have a cylindrical shape with diameter larger than the diameter of the centrifuge, such that the centrifuge fits in the centrifugation drum.
The centrifugation drum may be placed in a box. The box has an upper part with a circular opening in this upper part, into which the centrifugation drum may be placed. Preferably, in order to minimize the space required for the device, the upper part may further serve as a storage place for the rest of the equipment: the dosing device, the selector device and the holder with the storage containers.
Part of the outflow system may also be placed in the box. As was discussed above, the circular outflow channel comprises outflow openings, which are connected to outflow pipes, which provide an outflow system of the device and drain away the liquid removed from the reactors during the synthesis. These outflow pipes may be combined into one outflow tube, which is positioned within the box.
The centrifuge and the distribution rotor may be sealed within the centrifugation drum. The sealing may be provided by a cover of the centrifugation drum.
The cover may be rotatably attached to the upper part of the box and may rotatably move from an open position to a closed position and vice versa. Preferably, the cover is placed between the distribution rotor and the positioning device (and the lifting device), as both of them are placed above the distribution rotor and outside of the centrifugation drum. The cover may be operated manually or automatically by the control subsystem. The positioning device and the lifting device are then operated together with the cover. The sealing shall be activated in the closed position of the cover, such that the environment within the centrifugation drum is insulated from the space outside the centrifugation drum. The cover may be made of an inert material, for example the same material may be used as is used for the manufacture of the centrifuge. Alternatively, the material may be transparent, for instance plexiglass protected by a sheet of inert material or glass. The transparent material has an advantage that the operator may watch the operation of the device directly.
In one embodiment, the cover may comprise two concentric disks—the inner disk and the outer disk. In this embodiment, the inner disk is preferably made of steel, but may be made of any suitable inert material and the outer disk is preferably made of glass, but may also be made of any suitable inert material. To ensure the safety of the operation, it may be provided that the rotation of both rotors can be enabled only when the seal is in place. There is at least one opening—inlet port in the cover, through which the centrifugation subsystem may be connected to the dosing device.
The sealed cover may especially be important when certain conditions need to be provided inside the centrifugation drum. For instance, in order to provide such conditions inside the centrifugation drum, a temperature control device may be provided inside the centrifugation drum in one embodiment. The temperature control device may comprise for example an infrared radiator and a microwave radiator. Such a temperature control device may provide temperatures up to 90 degrees Celsius. The temperature control device may further comprise a sensor with a feedback control and may be controlled automatically by the control subsystem.
In one embodiment, the device may further comprise a first plurality of magnets, preferably at least two, which may be attached statically to the inner side of the centrifugation drum at the level of the reactors. In this embodiment, a second plurality of magnets is further positioned inside the reactors (i.e. preferably one magnet in each of the reactors). The size of the magnets placed inside the reactors is smaller than the size of the magnets attached to the centrifugation drum. Because of the interaction of the magnets inside the reactors with magnets attached to the centrifugation drum, the resin inside the reactors is thoroughly stirred during a slow rotation (from ca 10 rpm to ca 50 rpm) of the centrifuge in the synthetic process. The addition of magnets is especially advantageous for larger quantities of contents inside the reactor or for large reactor sizes suitable for large volumes of reagents, when simple rotation and/or shaking of the centrifuge is not sufficient to stir the contents to a desired extent.
Every part of the system is controlled by the timing protocol of the program specifically constructed for the presented device. The input data comprise a list of oligomers (e.g. peptides) to be synthetized with specific individual sequences of monomer building blocks (e.g. aminoacids). The device according to the present invention is capable of parallel synthesis of a plurality of different oligomers (e.g. oligopeptides with different aminoacid sequences). The timing protocol of the whole process is then based on the input data. The program allows to check spots of difficult coupling and suggests the best protocol, such that every step may have different protocol (different coupling time, reagent volume etc.). The software also allows the operator to interrupt the process at any time and perform the particular step manually, if needed.
Reagents shall be prepared in storage containers, which shall be connected to the device via the dosing device and/or selector device. When the device is prepared, the control device retrieves the sequences of the particular oligomers (e.g. peptides) to be synthesized and determines the sequence specific timing protocol. The principal use of this method is in the peptide synthesis.
The peptide synthesis performed by the above-described device follows a sequence specific timing protocol and comprises the following steps:
In one preferred embodiment, wherein the volume of the reactors is greater than ca 10 ml, the reactors content in step k) is being stirred by a slow rotation under assembly of magnets when small magnets are placed in each reactor. This ensures a rigorous stirring of larger volumes of reactor contents.
In another preferred embodiment, wherein the volume of the reactors is smaller than ca 10 ml, the reactors content in step k) is being stirred by repetitive back and forth motion of the rotor. Such movement is sufficient to stir smaller volumes of reactor contents and no magnet/magnetic stirrer needs to be placed into the reactors.
Another object of the present invention is the use of the device for parallel oligomer synthesis and/or the method according to the present invention in peptide synthesis. The device according to the present invention is further suitable for use in oligonucleotide or carbohydrate synthesis.
In the following example an embodiment device for parallel oligomer synthesis will be described in the embodiment in which the device comprises all sub-systems in accordance with
The synthesis itself takes place in reactors (
The nozzles 104 of the reactors 101 are connected to S-trap outflows 201. Each of the S-trap outflows 201 has a tubular shape and comprises a first end 202 and a second end 203. The first end 202 of the S-trap outflow 201 is connected to the nozzle 104 of the lower end 103 of the reactor 101. The S-trap outflow 201 works on a siphoning principle, i.e. the second end 203 of the S-trap outflow 201 is positioned higher than the level of content in the reactor 101. The second end 203 of the S-trap outflow 201 also comprises a nozzle. The nozzle of the S-trap outflow 201 is positioned so that a liquid removed from the reactor 101 through the second end nozzle of the S-trap outflow 201 flows into a circular outflow channel 205.
As can be seen in
The centrifuge 301 shown in this example comprises two circular disks—a lower disk 301a and an upper disk 301b, both made of steel and seated on a common axle 402 in the center of mass. The two disks 301a, 301b are positioned vertically at a distance, but physically connected by screws 308. The upper disk 301b comprises reactor holders—openings 106, which are configured to retain the reactors 101 at an angle. The reactors 101 are positioned at an angle of 45 degrees in this example. The lower disk 301a comprises fixation slots 107, in which the nozzles 104 of the lower ends 103 of the reactors 101 are fixed. The reactors 101 are inserted from above, through the reactor holders 106 into the fixation slots 107.
The upper disk 301b is further provided with openings (not shown) for the second ends 203 of the S-trap outflows 201 to be retained in such a way that the nozzles of the second ends 203 of the S-trap outflows 201 are positioned in the circular outflow channel 205 and such that the content of the liquid in the reactor 101 reaches a level which is lower than the second end 203 of the S-trap outflow 201. In this way, the S-trap outflows 201 work on the siphoning principle, such that depending on the speed of rotation, the liquid may stay inside the reactor 101 or may be removed from it partially or completely. The liquid is removed from the reactor 101 through the nozzle of the S-trap outflow 201 and flows into the circular outflow channel 205, which is constituted by a groove positioned on the perimeter of the centrifuge 301 and aligned with the nozzles of S-trap outflows 201, such that the nozzles of S-trap outflows 201 are positioned in the circular outflow channel 205. The circular outflow channel 205 comprises outflow openings positioned on its bottom. The outflow openings of the outflow channel 205 are connected to outflow pipes, which provide an outflow system of the device 1 and drain away the liquid removed from the reactors 101 during the synthesis.
Above the centrifuge 301, a circular disk of the distribution rotor 302 made of plastic is positioned (
The distribution rotor 302 comprises pre-activation compartments 306, which are positioned at the outer circumference of the distribution rotor 302, and which are provided with grooves 307 directed radially outwards, towards the outer edge of the distribution rotor 302. These grooves 307 enable the connection between the pre-activation compartments 306 of the distribution rotor 302 and centrifuge 301 when the distribution rotor 302 is in the lower position such that the contents of the pre-activation compartments 306 is transferred to the reactors 101 upon synchronized rotation of the distribution rotor 302 and the centrifuge 301 at medium rotation speed.
In this example, the pre-activation compartments 306 are filled in via a dosing device 501 (shown schematically in
The distribution valve 502 comprises a plurality of ports, 12 ports in this example. The distribution valve 502 comprises at least one outtake port which is via tubing (not shown in figures) connected to the centrifugation subsystem (distribution rotor 302) and through which the individual pre-activation compartments 306 are filled in through the inlet port 709. The other ports of the distribution valve 502 are intake ports and provide for a connection with stock solutions of reactants and/or solvents and/or activating reagents and/or coupling reagents stored in storage containers placed in locations 710.
The dosing pump 504 is connected to the desired reactant via one of the ports of the distribution valve 502 to make the intake connection (while the other ports are closed) and a desired quantity may be sucked by the dosing pump 504 inside the dosing pump 504. Subsequently, the intake connection may be closed and an outtake connection between the dosing pump 504 and the distribution rotor 302 may be filled through the inlet port 709. In this way, the desired quantity of the reactant is transferred via tubing, and one of the pre-activation compartments 306 is filled in. This process is repeated with either the same reactant or other reactants until the desired number of pre-activation compartments 306 is filled in.
In this example, one of the intake ports of the distribution valve 502 is connected to a selection device 601 (
In this example, the selection device 601 comprises two coplanar, circular layers (604,605) of graphite pressed together by spring 606. The upper layer 604 comprises the plurality of intake ports 602, located close to the outer circumference of the upper layer. The lower graphite layer 605 comprises the outtake port 603 and an elongated groove 607. The outtake port 603 is located in the middle of the circular graphite layer 605. The elongated groove 607 has a first end 608 and a second end 609. The position of the first end 608 of the groove 607 is identical with the position of the outtake port 603. The groove 607 is elongated radially outwards from the position of the first end 608 of the groove 607 such that the position of the second end 609 of the elongated groove 607 is identical with position of one of the intake ports 602 in the upper graphite layer 604. As the intake ports 602 are positioned in a circular configuration along the outer circumference of the upper graphite layer 604, the elongated groove 607 allows to establish a connection between the outtake port 603 and any one of the intake ports 602 by rotating of one of the two coplanar graphite layers (604, 605) relatively to the other one. In order to ensure the non-crosscontamination of the desired reactant by the reactant from any other unselected port, this coplanar arrangement selector port is used in underpressure (suction) regimen, when the sealing of the two coplanar surfaces can be guaranteed. The detailed view of the coplanar structure of the selection device 601 is shown in
The solutions of amino-acids are in this example stored in storage containers 701. The number of storage containers corresponds to the number of intake ports 602 of the selector device 601, i.e. 28 in this example. The storage containers 701 have unified cylindrical shape and are placed in a holder 702. The holder 702 has means for holding the storage containers 701.
As shown in
The centrifugation subsystem is covered in the centrifugation drum 704 by a cover 708, which is rotatably attached to the upper part 706 of the box 705 and moves rotatably from an open position to a closed position and vice versa. The cover 708 is in this example comprises two concentric disks—the inner disk 708a is made of polypropylene and the outer disk 708b is made of glass to allow the operator to watch the operation of the device directly. The inner disk 708a of the cover is placed between the distribution rotor 302 and the positioning device 304 (and the lifting device 305), as both of them are placed above the distribution rotor 302 and outside of the centrifugation drum 704. There is at least one opening—inlet port 709 in the inner disk 708a of the cover 708, through which the distribution rotor 302 of the centrifugation subsystem may be connected to the dosing device 501.
The system is controlled by a timing protocol of the program constructed for operation of the device 1, such that all sub-devices are connected to that control subsystem and operated by the timing protocol of the control subsystem.
In another embodiment, with reference to
The liquid from the reactor 101 cannot overcome the gravity based resistance of the peek tubing 1002 at the low speed of rotation (e.g. during liquid transfer from pre-activation compartments or during a synthesis shaking), or it is removed completely from the reactor 101 at high speed rotation by centrifugal forces. The liquid to be removed flows from the reactors 101 through the I-trap outflow and through a channel system 1005 that is positioned in a channel holder 1006. The channel holder 1006 and the fixation slots 107 are provided integrally. They form a separate element that is attached to the lower disc 301a. The cover 1003 of the peek tubing 1002 is connected to the channel holder 1006. The channel holder 1006 comprises a funnel 1007 arranged at one end of the channel system 1005 so that the channel system 1005 is through the funnel 1007 connected with the interspace 1004 created between the peek tubing 1002 and the cover 1003. The channel system 1005 further comprises at least one channel positioned horizontally 1010, i.e. in parallel with the planes of the lower 301a and the upper 301b, discs and at least one stopcock 1008, positioned close to the end of the channel system 1005 that is opposite to the end with the funnel 1007. The stopcock 1008 may be in closed position, when the liquid is retained in the system, or in open position, when the outflow of the liquid from the system is allowed. The liquid can be removed from the reactors 101 by the succession of fast centrifugation, in which the liquid is transferred from the reactors 101 through the vertical channel 1001 and the peek tubing 1002 into the interspace 1004 created between the peek tubing 1002 and the cover 1003, slow rotation, during which the liquid is flowing out of the interspace 1004, through the funnel 1007 into the channel system 1005 and out of the stopcock 1008, and finally short fast centrifugation, which removes the liquid from the channel system 1005 completely, preventing thus any potential crystallization of reagents in solutions.
This arrangement is advantageous because the operator may decide that he/she wants to collect and use the eluent collected from individual reactors 101—for example for measuring absorbance or spectra of the solutions. Thus, this arrangement is particularly advantageous for the collection of solutions created by cleaving the finished synthetic product by cleaving reagents, for example by trifluoroacetic acid. After incubation of washed resin with cleaving reagent, the stopcock 1008 is turned into closed position and liquid is transferred by centrifugation through the peek tubing 1002 to the interspace 1004 created between the peek tubing 1002 and the inverted syringe as the cover 1003. Rotor may then be taken out of the machine, placed on the assembly of receiving flasks (e.g. Falcon tubes) arranged in a circular fashion and stopcocks 1008 are opened to let the liquid flow into the receiving flanks That solution may then be worked-up in a common procedure (precipitation, evaporation, lyophilization).
In another embodiment, the device 1 for parallel oligomer synthesis comprises a distribution device (not shown) in addition to the components as described in Example 1 or Example 2. The distribution device connects the dosing pump 504 via the distribution valve 502 to either the selector device 601 or to the pre-activation compartments 306 of the distribution rotor 302. The distribution device is a solenoid valve with three ports switching between the connection of the dosing pump 504 with the outtake port of selector device 601 and the connection between the dosing pump 504 and the pre-activation compartments 306 of the distribution rotor 302 (through the distribution valve 502).
In this example, when the distribution device is enabled in the system, the distribution valve 502 comprises 12 ports for connection with the distribution device.
It is also possible to place a tubing with a predefined volume (which is larger than the volume being delivered to the reactor) between the dosing pump 504 and the inlet port 709 of the pre-activation compartments 306 to avoid the reagents entering the dosing pump 504 inner volume. In this arrangement, the distribution device is placed between selector device 601 and inlet port 709 of the centrifuge assembly. The distribution device is first opened in the direction to the dosing pump 504, the liquid is sucked from the distribution valve 502 into the tubing, the distribution device is opened in direction of delivery to one of the reactors 101 and the liquid is expelled into the reactor, without entering the syringe of the dosing pump 504 and contaminating the liquid in there. This schematic arrangement of this example can be seen in
In this example, the device 1 may further (in addition to the components described in the examples above) comprise a temperature control device, provided inside the centrifugation drum 704 (not shown in the figures). The temperature control device in this example comprises an infrared radiator and a microwave radiator, and a sensor with a feedback control. Such a temperature control device may provide temperatures up to 90 degrees Celsius and is controlled automatically by the control subsystem.
In another example (not shown in the figures), the device 1 may further (in addition to the components described in the examples above) comprise a first plurality of magnets which are attached statically to the inner side of the centrifugation drum 704 above the reactors 101, such that they are distributed along the circumference of the centrifugation drum 704. Further, a second plurality of magnets is positioned inside each of the reactors 101. The size of the magnets placed inside the reactors 101 is smaller than the size of the magnets attached to the centrifugation drum 704. Because of the interaction of the magnets inside the reactors 101 with magnets attached to the centrifugation drum 704, the resin inside the reactors 101 is enabled to be mixed more thoroughly during the slow rotation of the centrifuge 301 in the synthetic process. The addition of magnets is especially advantageous for larger quantities of contents inside the reactor, when rotation and/or shaking of the centrifuge is not sufficient to mix the content to a desired extent.
Synthesis of ValA10-insulin A-chain S-sulfonate (Mw 2687.8116 and 2689.9100) Sequence (21 residues): Gly-Ile-Val-Glu-Gln-Cys(SSO3H)-Cys-Thr-Ser-Ile-Cys(SSO3H)-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys(SSO3H)-Asn was synthesised using the device according to the present invention and the method according to the present invention. Preloaded Fmoc-Asn(Trt)-Wang-LL-resin (0.29 mmol/g), 100 μmol scale, was used. The resin was purchased from IRIS Biotech GmbH. Resin was placed to two PP syringes of the 10 ml volume with frits (2×50 μmol of the Fmoc groups).
The control device retrieved the sequence of Gly-Ile-Val-Glu-Gln-Cys(SSO3H)-Cys-Thr-Ser-Ile-Cys(SSO3H)-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys(SSO3H)-Asn and determined the sequence specific timing protocol. Two couplings per position were used (each condensation was performed twice for each step). The first coupling (45 min) was done with 1 ml of 0.5 M amino acid and 0.5 ml of 0.5M DIC in DMF. The second coupling (45 min) was done with 0.5 ml of 0.5 M amino acid and 0.5 ml of 0.5M DIC in DMF.
The dosing device dispensed 1 ml of first N-protected 0.5 M amino acid solution through the lifting and positioning device to the first one of the pre-activating compartments of the distribution rotor, the distribution rotor turned to enable the second pre-activation compartment a reception of contents of the dosing device through lifting and positioning device. The dosing device dispensed 0.5 ml of 0.5 M solution of diisopropylcarbodiimide (DIC) through the lifting and positioning device to the first one of the pre-activating compartments of the distribution rotor, the distribution rotor turned to enable the second pre-activation compartment a reception of contents of the dosing device through lifting and positioning device and the steps repeated to ensure the reception of 0.5 ml of 0.5 M solution of diisopropylcarbodiimide (DIC) to the second one of the pre-activating compartments of the distribution rotor according to the timing protocol.
The distribution rotor was lowered to position in which it makes contact with reactors in the centrifuge, and rotated with a speed of 100 rpm in a synchronized rotation with the centrifuge, causing the contents of the pre-activation compartments to transfer by a centrifugal force via grooves into the reactors. Then the distribution rotor detached from reactors by lifting mechanism and reactors content were stirred by repetitive back and forth motion for 45 minutes in order to complete the condensation reaction.
Then the centrifuge with reactors rotated with the speed of 1000 rpm so that the liquid from the reactors was transferred by a centrifugal force via the S-trap outflows out of the reactors. The dosing device dispensed 1 ml of DMF through the lifting and positioning device to each one of the reactors of the centrifuge, and the centrifuge with reactors rotated with the speed of 1000 rpm so that the liquid from the reactors was transferred by a centrifugal force via the S-trap outflows out of the reactors. This washing step was repeated 5 times.
In the next step, the dosing device dispensed 2 ml of 20% piperidine/2% DBU in DMF through the lifting and positioning device to each one of the reactors of the centrifuge, where a deprotection of N-protected end of the amino acids took place for 2 minutes before the centrifuge with reactors rotated so that the liquid from the reactors was transferred by a centrifugal force via the S-trap outflows out of the reactors. Then washing step occurred using 5 times 1 ml DMF as described above.
The above described procedure was repeated for the second and the following amino acids in the synthesized peptide sequence, until the desired peptide sequence was completed. The final peptide was cleaved from the resin using TFA/scavengers and precipitated in a cold diethyl ether. The precipitate, crude peptide in SH form, was immediately converted to S-sulfonate derivative using a protocol described in Kosinova et al. Biochemistry 2014, 53, 3392-3402. HPLC trace of the crude S-sulfonate product (178 mg) is shown in
The identity of the product main in the main peak was confirmed by MS (negative mode, see
Sequences (24× YGGFL mutants): YGGFL, GGYFL, GGYLF, GGFYL, GGFLY, GGLYF, GGLFY, GYGFL, GYGLF, GYFGL, GYFLG, GYLGF, GYLFG, GFGYL, GFGLY, GFYGL, GFYLG, GFLGY, GFLYG, GLGYF, GLGFY, GLYGF, GLYFG, YGGFL were synthesised using the device according to the present invention and the method according to the present invention.
Fmoc-Rink amide resin s=0.5 mmol/g, 24×20 mg was placed into the PP syringes with fits (24×20 mg) and swollen in DMF. The resin was purchased from IRIS Biotech GmbH. The control device retrieved the sequence of first of the sequences above and determined the sequence specific timing protocol. Single coupling was applied using oxyma/DIC activation. The dosing device dispensed 0.4 ml of first N-protected 0.5 M amino acid solution through the lifting and positioning device to the first one of the pre-activating compartments of the distribution rotor, the distribution rotor turned to enable the second pre-activation compartment a reception of contents of the dosing device through lifting and positioning device. The dosing device dispensed 0.24 ml of 1.0 M solution of diisopropylcarbodiimide (DIC) through the lifting and positioning device to the first one of the pre-activating compartments of the distribution rotor, the distribution rotor turned to enable the second pre-activation compartment a reception of contents of the dosing device through lifting and positioning device and the steps repeated to ensure the reception of 0.24 ml of 1.0 M solution of diisopropylcarbodiimide (DIC) to the second one of the pre-activating compartments of the distribution rotor according to the timing protocol.
The distribution rotor was lowered to position in which it makes contact with reactors in the centrifuge, and rotated with a speed of 100 rpm in a synchronized rotation with the centrifuge, causing the contents of the pre-activation compartments to transfer by a centrifugal force via grooves into the reactors. Then the distribution rotor detached from reactors by lifting mechanism and reactors content were stirred by repetitive back and forth motion for 30 minutes in order to complete the condensation reaction.
Then the centrifuge with reactors rotated with the speed of 1000 rpm for 20 s so that the liquid from the reactors was transferred by a centrifugal force via the S-trap outflows out of the reactors.
The dosing device dispensed 1 ml of DMF through the lifting and positioning device to each one of the reactors of the centrifuge, and the centrifuge with reactors rotated with the speed of 1000 rpm so that the liquid from the reactors was transferred by a centrifugal force via the S-trap outflows out of the reactors. This washing step was repeated 2 times. In the next step, the dosing device dispensed 1 ml of 20% piperidine/2% DBU in DMF through the lifting and positioning device to each one of the reactors of the centrifuge, where a deprotection of N-protected end of the amino acids took place for 2 minutes before the centrifuge with reactors rotated so that the liquid from the reactors was transferred by a centrifugal force via the S-trap outflows out of the reactors. Then washing step occurred using 5 times 1 ml DMF as described above.
The above described procedure was repeated for the second and the following aminoacids in the synthesised peptide sequence, until the desired peptide sequence was completed. After completion of the synthesis, the resin was washed with methanol (2×) and dried. The final peptide was cleaved from the resin by manual addition of 95%TFA/5% H2O (200 μL per reactor) for 2 hrs. HPLC analysis was performed after dilution of the sample with 800 μL of water.
An example of HPLC trace of the crude product is shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 17206537.7 | Dec 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/084329 | 12/11/2018 | WO | 00 |
