Oligonucleotide synthesizing device and method for producing the same

Abstract

The present invention provides an apparatus and method for synthesizing oligonucleotides and related compounds. In particular, the present invention allows for the efficient manufacture of reagents supplied in an apparatus for synthesizing such oligomers.

Description

[Technical Field]

[0001] The present invention relates generally to the field of oligomer synthesis on an industrial or laboratory scale. Improved methods and apparatus for synthesizing oligomers, particularly oligonucleotides and related polymers, are disclosed. In particular, the present invention allows for the efficient production of reagents to be supplied to apparatus for synthesizing such oligomers. [Background technology]

[0002] Oligonucleotides and related oligomers are produced by a variety of synthetic strategies, including the commonly used phosphoramidite method. Synthetic strategies generally involve the stepwise addition of monomeric or oligomeric building blocks, e.g., nucleoside phosphoramidites, to a growing oligomer chain. Typically, the growing oligomer chain is composed of nucleotides or their analogs and is anchored to a solid support, e.g., via the hydroxyl groups of the ribose moiety. Reactive groups on the oligomer chain are blocked with appropriate protecting groups to prevent unintended reactions. For example, a growing oligonucleotide chain can be anchored to a solid support via the 3' hydroxyl group and carry protecting groups, such as those listed in Table 1, to block the hydroxyl groups of the ribose moiety and the exocyclic amino groups of the base moiety from reaction.

[0003] [Table 1]

[0004] Generally, chemical oligonucleotide synthesis relies on the use of a first type of protecting group for functional groups located on bases / base analogs and ribose / ribose analog moieties that are not involved in chain elongation, and a second type of temporary protecting group to control backbone elongation. The temporary protecting groups are placed on the building blocks to be added to prevent double insertion of building blocks or multimerization of building block sites. The first and second types of protecting groups are orthogonal to each other, meaning that one type can be removed under conditions that do not affect the other type of protecting group. Generally, the first type of protecting group is "permanent" in that it is cleavable under alkaline conditions and is only removed once chain assembly is complete. The second type of temporary protecting group is cleavable under acidic conditions and is removed once per synthesis cycle. Thus, a coupling cycle involves linking a protected building block to the unprotected end of the oligonucleotide backbone, followed by deprotection of the extended oligonucleotide in preparation for the subsequent coupling cycle.

[0005] For phosphoramidite oligonucleotide synthesis, the synthesis cycle typically begins with selective deprotection of the 5' hydroxyl group. This is accomplished by incubating the solid support with a deprotecting reagent, DR, typically a detritylation reagent capable of removing the DMT group. The growing oligonucleotide chain is then incubated with a coupling reagent, CR, containing an appropriately protected nucleoside phosphoramidite and an activating reagent. The tricoordinate phosphite triester resulting from the coupling reaction is then oxidized using an oxidation reagent, OR, to generate a phosphate triester. For phosphorothioate or phosphorodithioate synthesis, sulfurization is performed instead of oxidation using a sulfurizing reagent, SR, to generate a phosphorothioate triester. The capping step is accomplished by incubating the solid support with a capping reagent, also known as a blocking reagent, BR, to remove any unreacted groups on the resin. This includes a step of blocking to prevent sequence deletions in subsequent coupling steps. In other cases, capping can be performed before or after oxidation of the tricoordinate phosphite triester. Between each step, the resin can be washed with a processing solvent, typically acetonitrile or toluene. The growing oligonucleotide chain is anchored to the solid support by any conceivable method, for example, via an exocyclic amine or via the hydroxyl group of the ribose / ribose-like moiety. Also disclosed are variations on phosphoramidite synthesis, such as reverse 5'-3' oligonucleotide synthesis, in which a 5'-phosphoramidite is added.

[0006] For phosphonate oligonucleotide synthesis, nucleoside H-phosphonate monoesters bearing temporary acid-sensitive protecting groups such as DMT are used. The internucleoside H-phosphonate diester bond is oxidized at the end of chain assembly. Depending on the reaction conditions used, phosphodiester linkages, phosphorothioate linkages, phosphoroselenoate, or phosphoramidate analogs can be generated in this process.

[0007] Depending on the exact procedure used, the composition of the above reagents may vary. Table 2 provides a non-limiting summary of commonly used compositions.

[0008] [Table 2]

[0009] Patent Documents 1 and 2 disclose oligonucleotide production apparatuses. Commercially available automated oligonucleotide synthesizers are configured so that deprotection solution DS, oxidation solution OS, and sulfurization solution SS are pre-prepared and provided in tanks and delivered to the synthesis reactor by one or more pump circuits. Blocking solution BS is prepared by mixing two pre-prepared solutions in-line. Coupling solution CS is mixed within the synthesizer by combining a solution of phosphoramidite in acetonitrile with a solution of activating agent. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] US2008 / 0058512 [Patent Document 2] US5,681,534 Summary of the Invention [Problem to be solved by the invention]

[0011] However, this design has the disadvantage that it is not possible to "fine-tune" the reagent composition for a particular coupling cycle, for example, by changing the concentration of the deprotecting agent. Furthermore, large tanks are required, and scale-up of the synthesis is limited by the tank capacity. To overcome these problems, the present invention provides an improved oligonucleotide synthesis apparatus that can produce all reagents on demand through in-line mixing. Pure liquid reagents can be fed into the system directly from their respective storage containers. Non-liquid reagents can be fed into the system as concentrated stock solutions. As a further improvement, the present invention provides an oligonucleotide synthesis apparatus that includes a batch reactor instead of a column bed reactor, thereby providing additional options for running and controlling the synthesis method. [Means for solving the problem]

[0012] In one specific embodiment, the present invention provides a method for "fine-tuning" the composition of deprotection reagents by defining the composition for each coupling cycle. It has previously been reported that the deprotection step requires maintaining a delicate balance between avoiding acid-catalyzed degradation of oligonucleotide intermediates, e.g., due to depurination, and achieving efficient removal of protecting groups. To address this challenge, various deprotection compositions have been compared; for example, Septak teaches the use of 15% DCA rather than 3% DCA or 3% TCA (Nucleic Acids Research, 1996, Vol. 24, No. 15, pp. 3053-3058). However, to the inventors' knowledge, all automated oligonucleotide synthesis methods rely on the use of the same deprotection agent in all cycles of a given synthesis. In contrast, the present invention provides an apparatus and method for individually adjusting the composition of deprotection reagents for each coupling cycle.

[0013] Accordingly, one embodiment of the present invention is an automated oligonucleotide synthesizer comprising: a) a reaction vessel connected to a waste container via a liquid conduit; b) a liquid supply for delivering liquid reagents to the reaction vessel; c) a bypass conduit for transferring the liquid flow from the liquid supply into a waste container without passing through the reaction vessel; d) Control unit and the liquid supply comprising: b-1) at least one mixing device; b-2) at least two liquid supply lines connected to at least one mixing device, each liquid supply line including at least one liquid conduit having n liquid inlets, where n is an integer between 1 and 25, and at least one liquid pump; The present invention relates to an automated oligonucleotide synthesizer comprising:

[0014] Accordingly, another aspect of the present invention is an oligomer synthesis apparatus comprising: a) a reaction vessel connected to a waste container via a liquid conduit; b) a liquid supply for delivering liquid reagents to the reaction vessel; c) a bypass conduit for transferring the liquid flow from the liquid supply into a waste container without passing through the reaction vessel; d) Control unit and the liquid supply comprising: b-1) at least one mixing device; b-2) at least four upstream liquid supply lines connected to at least one mixing device, each upstream liquid supply line including one liquid conduit having n liquid inlets, where n is an integer between 1 and 25, and one liquid pump; b-3) n sensors, where n is an integer greater than or equal to 1, positioned downstream of the mixer and determining at least one property of the liquid exiting the mixer, and at least one measurement provided by the sensor is used as a feedback signal for adjusting the activity of one or more liquid pumps.

[0015] Preferably, the device is used for the solid-phase synthesis of oligonucleotides, particularly ribonucleic acids, 2'-deoxyribonucleic acids, oligonucleotide phosphorothioates, xenonucleic acids, and related molecules. In some embodiments, the device is used for solid-phase synthesis methods using the phosphoramidite method.

[0016] The following figures and their descriptions are provided for illustrative purposes only and are not to be construed as limiting the scope of the claims. [Brief explanation of the drawings]

[0017] [Figure 1] 1 is a basic embodiment of an automated oligomer synthesizer with four liquid supply lines. [Figure 2] 10 is a further embodiment of the device additionally including a recirculation conduit. [Figure 3]A further embodiment of the device in which one of the liquid supply lines functions as a recirculation circuit. [Figure 4] 10 is a further embodiment of a device having a reservoir. [Figure 5] 10 is another embodiment of an apparatus including a manifold. [Figure 6] 1 is another embodiment of an apparatus including two mixing devices and a manifold. [Figure 7] 10 is a further embodiment of a device having two liquid supply lines. [Figure 8] 10 is another embodiment of the device having two liquid supply lines and a recirculation circuit. [Figure 9] 8 is an embodiment of the device of FIG. 7 in which an additional sensor is located downstream of the mixing device. [Figure 10] 4 is an embodiment of the device of FIG. 3 having only two liquid supply lines. [Figure 11] 3 is an embodiment of the device of FIG. 2 having only three liquid supply lines. [Figure 12] 1 is another embodiment of an apparatus having three liquid supply lines, one of which functions as a recirculation circuit. [Figure 13] 4 is a further embodiment of the device of FIG. 3, having only three liquid supply lines. DETAILED DESCRIPTION OF THE INVENTION

[0018] The term "oligonucleotide" as used herein is used in the most general sense to refer to any oligomer that comprises at least two nucleotide units linked by phosphorodiester moieties or similar structures, such as found in phosphorothioates, phosphorodithioates, diastereomerically pure phosphorothioates, phosphoramidates, phosphorodiamidates, arsenic diesters, and phosphoroselenoates.Natural nucleoside units usually comprise ribose or 2'-deoxyribose moieties, and nucleic acid bases selected from adenine, guanine, cytosine, thymine, and uracil.The term "nucleoside unit" as used herein encompasses not only natural nucleosides, but also artificial compounds. The latter can carry a substituent in the ribose / ribose analog moiety, such as -F, -OMe (-O-CH), or methoxyethyl (-O-CH-CH-O-CH, also known as MOE, -O-methoxyethyl) substituent, or the ribose is modified with an additional methylene bridge connecting the 2' hydroxyl group and the 4' carbon, or the ribose moiety is replaced by another cyclic monosaccharide such as a pentose (e.g., arabinose) or hexose, by an acyclic monosaccharide (e.g., threose), or by an alternative structure such as cyclohexene, threoninol, serinol, or glycol. All of these substances are referred to herein as "ribose analogs." Similarly, artificial nucleosides can exhibit non-standard bases, artificial analogs of nucleobases, or abasic sites. Non-limiting examples of oligonucleotides other than deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are locked nucleic acids (LNA), amino LNA, constrained ethyl nucleic acid analogs (cET), bridged nucleic acids (BNA), tricyclo DNA, unlocked nucleic acids (UNA), phosphoramidite morpholino oligonucleotides (PMO), iRNA, dsRNA, and oligonucleotide phosphorothioates, and derivatives and analogs thereof.

[0019] As used herein, the term "liquid supply" is used to mean a component of the device of the present invention that functions to provide liquid reagents to be introduced into a reactor (reaction vessel). In particular, the liquid supply may include one or more liquid inlets, reservoirs or tanks, liquid conduits, pumps, valves, liquid mixing devices, heat exchangers, manifolds, and sensors.

[0020] As used herein, the term "liquid supply line" refers to a liquid conduit including one or more liquid inlets connected to a liquid pump and one or more liquid containers. Valves can regulate fluid communication between each liquid inlet and the conduit. Thus, the liquid supply line can provide controlled liquid flow from one or more liquid inlets / containers to downstream devices. The pumps and valves are controlled by a local controller and / or a central controller. In some embodiments, each liquid supply line has between 1 and 25 inlets, preferably between 3 and 10 inlets. Such one or more inlets are connected to branching liquid conduits, which are then connected to multiple reservoirs. The outermost inlet of each liquid supply line is connected to a source of process solvent and liquid nitrogen, allowing the line to be vented between processing steps.

[0021] In some embodiments, the liquid supply of the apparatus further comprises a third liquid supply line connected to the mixing device, the third liquid supply line comprising at least one liquid conduit having n liquid inlets and at least one liquid pump, where n is an integer between 1 and 25.

[0022] As used throughout this document, two components of the device may be said to be "connected" if they are fed by one another. For example, the components may be joined by a fluid conduit that allows fluid to pass from one component to the other.

[0023] The liquid inlet is connected to a reservoir configured to hold its contents in an inert gas. For example, the reservoir can include a controllable valve that can be connected to a vacuum source and a second controllable valve that can be connected to a source of inert gas, such as nitrogen. In the case of automatic inerting, the reservoir can further include an electronic or mechanical pressure control device, allowing the valve to be operated automatically. In the case of manual inerting, the reservoir can include a pressure measuring device and a manually controlled valve.

[0024] The reservoir can be made of any suitable material, for example, metal, enamel, or a polymer such as polypropylene, polyethylene, polyvinyl chloride, polystyrene, and polyetheretherketone. Preferably, a material that is essentially inert to the reagents to which the reservoir is exposed is selected. Depending on the contents, various reservoirs can be made of different materials. Preferably, the material complies with regulations applicable to the production of pharmaceuticals, cosmetics, and / or food and beverages, i.e., the material complies with good manufacturing practices (GMP). Furthermore, conductive materials can be used to minimize the risk of electrostatic ignition. In some embodiments, the reservoir is made of stainless steel or HASTELLOY® alloy, or a metal coated with a conductive polymer. The size of the reservoir can be selected according to the intended synthesis scale, and depending on the contents, reservoirs of different sizes can be used simultaneously. In some embodiments, the reservoirs can have an internal volume of about 1 liter (L) to 40 cubic meters, e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 550, 770, 990, 1120, 1300, or 1500 L. In some embodiments, some reservoirs can have an internal volume of about 10 to 40 liters. In some embodiments, reservoirs for process solvents, e.g., acetonitrile and toluene, can be vessels with volumes of 20 to 40 cubic meters. Other reagents, such as solutions of certain protected nucleoside phosphoramidites, are provided in much smaller reservoirs with volumes of about 1 or 5 liters.

[0025] Those skilled in the art will understand that any reaction vessel can be used with the aforementioned liquid supply. The reaction vessel can be made of any suitable material, such as metal, glass, enamel, or polymers such as polypropylene, polyethylene, polyvinyl chloride, polystyrene, and polyetheretherketone. Preferably, materials are selected that are essentially inert to the reagents to which the reaction vessel is exposed and that comply with Good Manufacturing Practice (GMP). Furthermore, conductive materials can be used to minimize the risk of static ignition. The size and dimensions of the reaction vessel can be selected according to the intended scale of synthesis. For example, column reactors with a maximum internal volume of 5, 10, 30, 40, 50, 60, 70, 75, 80, 90, 100, 150, or 200 liters can be used. As a further example, a batch reactor having a maximum internal volume of 5, 10, 30, 40, 50, 60, 70, 75, 80, 90, 100, 150, 200, 250, 300, 450, 600, or 650 liters can be used. As a further example, a reactor having an internal volume of 100 or 200 ml can be used. In some embodiments of the present invention, the internal volume of the reactor is about 30 to 250 liters, preferably about 40 to 200 liters, and most preferably about 40 to 150 liters. As used herein, the term "about" indicates that a deviation of plus or minus 10% from the given value is possible.

[0026] For example, solid-phase oligonucleotide synthesis is typically carried out using a column reactor packed with a solid support onto which the growing oligonucleotide chain is confined. Typically, the column reactor may include a bottom frit and a top frit, between which the solid support is packed. Solid supports with or without an optional first protected nucleotide attached to the support are available from a variety of commercial suppliers.

[0027] Particularly when using a column reactor, it may be useful to use a pump to circulate the reagent mixture through the column. Thus, in some embodiments, the device further includes a recirculation circuit that uses a pump to move a flow from a liquid conduit connecting the reaction vessel to a waste container back into the reaction vessel. The recirculation circuit is integrated into one of the liquid supply lines of the device. In this case, the recirculation circuit is included in one of the liquid supply lines connected to the liquid conduit via one of its inlets and a multi-way valve, and the pump can drive the recirculation of fluid from the liquid conduit connecting the reaction vessel to the waste container back into the reaction vessel via the mixing device. The recirculation circuit is arranged to allow the direction of liquid flow through the column to be reversed. For example, a column reactor is connected to the liquid supply via a valve assembly that allows liquid to flow forward and backward through the reactor, as well as bypass the reactor completely.

[0028] Alternatively, the inventors have found that a batch reactor can be used. This reactor can include a bottom frit or filter cloth for retaining the support within the reactor, a mixing device, and one or more spray balls or nozzles for rinsing the interior surface of the reactor. Rotating or stationary spray balls or nozzles can be used, with the spray ball or nozzle located at the top of the reactor and connected to a liquid supply. Those skilled in the art will routinely select a mixing device depending on the materials being mixed and, for example, the physical robustness of the support used. For example, a stirrer including a rotating impeller can be used. Such an impeller can be a turbulent mixer that generates axial, mixed, or radial flow of the liquid within the reactor. Known impellers include ship-type propellers, pitched-blade turbines, flat-blade turbines, and flat-blade paddles. The use of baffle blades is useful for improving mixing. Alternatively, or in addition, mixing can be achieved by bubbling a gas through the liquid. Alternatively, or in addition, mixing can be achieved by circulating the liquid using, for example, a pump circuit. Those skilled in the art will typically select mixing means to efficiently distribute materials within the reaction medium while avoiding foaming. When a solid support is used in the reaction vessel, the reaction vessel and stirring blades are preferably designed to minimize shear forces. Preferably, the operation of the mixing device is controlled by a controller. The batch reactor is preferably configured to operate in a protective atmosphere. For example, the reaction vessel of the batch reactor can include a first controllable valve that can be connected to a vacuum source, a second controllable valve that can be connected to a source of inert gas such as nitrogen, and an electronic or mechanical pressure control device. Preferably, the valves are automatically operated and controlled by the controller. The batch reactor can be a jacketed reactor to facilitate temperature control. The use of a batch reactor has several advantages over a packed column reactor, including the ability to sample solid materials and more uniform reaction conditions throughout the reactor.

[0029] Accordingly, another embodiment of the present invention relates to an apparatus for solid-phase oligonucleotide synthesis, characterized in that synthesis is carried out in a batch reactor, which in some embodiments includes a mixing device, one or more spray balls or nozzles for rinsing the interior surfaces of the reactor, and is configured to operate in a protective gas.

[0030] Those skilled in the art will appreciate that various valves, preferably automated valves, can be used to control liquid flow throughout the apparatus. For example, liquid flow into or out of a reactor can be controlled by valves integrated into the inlet and outlet liquid conduits.

[0031] Those skilled in the art will understand that any mixing device capable of mixing at least two input liquids, e.g., three or four input liquids, into a single homogeneous solution can be used in the present invention. Therefore, those skilled in the art will select a mixing device to suit the specific device circumstances in terms of mixing effectiveness, size, and pressure drop. The mixing device may consist of or include a series of mixing tees or mixing crosses that combine at least two input streams in series. The mixing device may include one or more mixing tees or T-valves and a heat exchanger. The mixing device may include a static mixer. The mixing device may consist of or include a static mixer having at least two inlets and one outlet. In some embodiments, the mixing device may consist of or include a multi-way valve, e.g., a T-valve and a static mixer. The multi-way valve and static mixer are arranged so that several streams are combined by the valve and then directed to the static mixer. The mixing device consists of or includes a manifold that mixes the liquid streams of at least two liquid supply lines and transfers the mixed streams into a static mixer or heat exchanger. The mixing device can be shrouded to allow for heating or cooling of the mixed liquid.

[0032] In one embodiment, at least the inner surface of the mixing device and the inner surface of one of the liquid supply lines are made of an acid-resistant material. This means that at least the inner surface of one liquid supply line, from at least one of the liquid inlets through the pump to the mixing device, is made of or coated with an acid-resistant material. As used herein, "acid-resistant" means that the material is recognized as resistant to or essentially inert to acidic solutions, for example, a solution of 50% DCA (dichloroacetic acid) in toluene and 200 ppm water. Acid resistance can also be evaluated using a 100% DCA solution containing 200 ppm water. The acid resistance of materials can be tested using well-established procedures, such as those described in DIN 50905-4 (March 2018 and later). Metallic materials that show no signs of localized corrosion and experience material loss below 0.01 mm / year when exposed to acidic liquids can be classified as acid-resistant. Metallic materials that do not release significant amounts of leachables or extractables in the acidic solution to which they are exposed can be classified as acid-resistant. The amount of leachables or extractables is considered significant if it causes detectable contamination of the synthesized oligomer that would be unacceptable as a pharmaceutical ingredient according to guidelines established by regulatory authorities. A material can be considered acid-resistant, e.g., resistant to a solution of 50% DCA (dichloroacetic acid) in toluene containing 200 ppm water, or resistant to 100% DCA, if it does not release detectable amounts of leachables or extractables into the synthesized oligonucleotide. In one embodiment, at least the inner surface of the mixing device and one of the liquid supply lines are made of an acid-resistant alloy. Furthermore, at least the inner surface of the mixing device and one of the liquid supply lines can include an acid-resistant polymer coating or glass coating. For example, the acid-resistant material can be selected from the group consisting of highly resistant nickel-based alloys, polymer coatings, and glass linings. Suitable polymer coatings include polytetrafluoroethylene (PTFE), perfluoroalkoxyalkane (PFA), ethylene tetrafluoroethylene (ETFE), or ethylene chlorotrifluoroethylene (ECTFE).Suitable nickel-based alloys include, for example, HASTELLOY® C-4, C22, C-2000, and C276 alloys, which are identified by the material numbers / Unified Numbering System for Metals and Alloys (UNS) as 2.4610 / N06455, 2.4602 / N06022, 2.4675 / N06200, and 2.4819 / N10276, respectively.

[0033] The device of the present invention may further include n sensors, where n is an integer greater than or equal to 1, positioned downstream of the mixer and determining at least one property of the liquid exiting the mixer. The n sensors can help analyze the composition of the liquid exiting the mixing device and thereby send a signal to the control unit. For example, at least one measurement provided by at least one of the sensors can be used as a feedback signal to adjust the activity of one or more pumps included in the liquid supply unit. This allows for adjusting the flow from the liquid pump to the mixing device to achieve and maintain a given set point. Thus, the sensors can be part of a feedback loop controlling the pumps of each liquid supply line. Additionally or alternatively, the device can include at least one flow sensor integrated into each liquid supply line upstream of the mixing device, e.g., positioned between the pump and the mixing device, and at least one measurement provided by the sensor is used as a feedback signal to adjust the activity of one or more pumps. Furthermore, at least one measurement provided by the at least one sensor is used to control whether the liquid flow is transferred into the reaction vessel or transferred to a waste container without passing through the reaction vessel. In some embodiments, the number n of sensors is selected from the group consisting of 1, 2, 3, and 4. Those skilled in the art will recognize that a variety of sensors are suitable and will select an appropriate sensor depending on the envisioned synthesis chemistry. In some embodiments, the n sensors are independently selected from the group consisting of an infrared spectrophotometer, a density sensor, a refractometer, a conductivity sensor, a temperature sensor, an impedance sensor, and a UV / Vis absorption sensor. In some embodiments, the n sensors are independently selected from the group consisting of an infrared spectrophotometer, a refractometer, a density sensor, a conductivity sensor, a temperature sensor, an impedance sensor, and a UV / Vis absorption sensor, and at least one of the n sensors is an infrared spectrophotometer. In some embodiments, the n sensors are independently selected from the group consisting of an infrared spectrophotometer, a refractometer, a density sensor, a conductivity sensor, a temperature sensor, an impedance sensor, and a UV / Vis absorption sensor, and at least one of the n sensors is a density sensor. Additional sensors can be integrated into the liquid conduit connecting the reaction vessel to the waste container.The sensor is selected from the group consisting of an infrared spectrophotometer, a density sensor, a refractometer, a conductivity sensor, a temperature sensor, an impedance sensor, and a UV / Vis absorption sensor. In one embodiment, the device can include a conductivity sensor, a temperature sensor, and a UV absorption sensor, which are positioned downstream of the mixing device and determine at least one property of the liquid exiting the mixing device.

[0034] The liquid supply may further include at least one heat exchanger, which may be located between the mixing device and the first of the n sensors (s1 to sn), or the heat exchanger may be part of the mixing device.

[0035] Preferably, the liquid pumps integrated into the liquid supply lines are designed so that the surfaces exposed to the reagents are inert. It is preferred that at least one of the pumps has a corrosion-resistant surface (and thus, in particular, an "acid-resistant" surface as defined above), such as a HASTELLOY alloy or a polytetrafluoroethylene surface. Liquid pumps particularly suitable for use in liquid supply lines provide a constant flow with minimal pulsation, allowing for smooth fluctuations in the liquid flow rate. For example, diaphragm pumps can be used. In some embodiments of the present invention, each pump can provide a maximum flow rate of up to 50 L / min, 80 L / min, 100 L / min, 200 L / min, or 300 L / min. In some embodiments, one pump can provide a maximum flow rate of 100 L / min, while the other pump can provide a maximum flow rate of 60 L / min. In one embodiment, pumps p1 and p2 of the first and second liquid supply lines provide 10 ml / min, and pump p3 of the third liquid supply line has a pumping force of approximately 50 ml / min.

[0036] The controller, among other things, controls the operation of the equipment's pumps and automated valves and can receive signals from the system's sensors (n sensors and / or additional sensors) as well as from operators. The controller can include multiple devices forming each hierarchical level of control, as in the case of a supervisory control and data acquisition (SCADA) control system architecture. For example, the controller can include one or more remote monitoring computers that collect data from and send control commands to the peripheral devices, as well as one or more peripheral devices, such as remote terminal units (RTUs), programmable logic controllers (PLCs), and user interfaces, such as GUI panels. The PLCs and supervisory SCADA software can receive inputs from field sensors, such as temperature, pressure, level, weight, position, or concentration sensors, among others. One or more SCADA supervisory computing platforms can additionally interact with a manufacturing execution system (MES), which in turn can interact with an enterprise resource planning (ERP) system. Additionally, one or more SCADA monitoring computing platforms can perform logging tasks by transmitting sensor data related to specific process parameters, such as the composition of the deprotection reagent, to a dedicated database. In some embodiments, the control unit includes at least one SCADA system and at least one PLC, along with sensors and actors that control the operation of pumps and valves. Alternatively, the local control unit can rely on signals received from sensors downstream of the mixing unit to regulate the function of the pump. Thus, the local control unit can be under the supervision of the central control unit or independent.

[0037] Preferably, the controller is configured to control the execution of a synthesis procedure that repeats a coupling cycle at least two times, the procedure defining a separate composition of the acidic deprotection reagent for each coupling cycle, and the controller accordingly directing the flow of reagents through at least one liquid supply line, the inner surface of which is made of an acid-resistant material. The controller allows a user to define the sequence of process steps, and in particular the composition of the coupling reagent for each coupling cycle. When executing this synthesis procedure, the controller can manage the operation of valves and pumps to achieve a specific composition of the deprotection reagent in each coupling cycle, and can control the composition via signals obtained from a field sensor, for example, a flow sensor upstream of the mixer or a sensor positioned between the mixer and the reaction vessel.

[0038] In some embodiments, the device of the present invention, or at least its exposed surface, is made of a material that is essentially inert to the reagents to which it is exposed. In some embodiments, the device, or at least its exposed surface, is made of stainless steel, HASTELLOY® alloy, or polymer-coated metal. Preferably, the material complies with regulations applicable to the production of pharmaceuticals, cosmetics, and / or food and beverages, i.e., the material complies with Good Manufacturing Practice (GMP). In some embodiments, at least the exposed surface of the mixing device and one line of the device, i.e., one liquid conduit including one or more liquid inlets, valves, and pumps, ending at the inlet of the mixing device, is made of an acid-resistant material, such as an acid-resistant alloy or an acid-resistant polymer coating, and the other parts of the device are made of stainless steel.

[0039] Operation of the apparatus of the present invention can include the following steps. For illustrative purposes, the steps are described with reference to Figure 4, but should not be construed as limiting the teachings to the particular apparatus of Figure 4. Furthermore, it should be understood that other reagents can be used depending on the synthetic strategy. Thus, the following sequence of steps is merely one possible routine example for one particular coupling cycle.

[0040] 1) Install the liquid supply line [Table 3] Note that all baths contain pure liquids unless they contain reagents that are solid at room temperature and must be supplied as a stock solution.

[0041] 2) Prepare the detritylation solution The liquid flows from vessels v1-3 and v2-2 to a mixer 5. The flow is first transferred to a waste container 7, and once the desired mixture is obtained, the flow is transferred to a reaction vessel (6). 3) Rinse lines 1 and 2, the mixing chamber, and the reaction vessel with acetonitrile from vessels v1-1 and v2-1. 4) Prepare a coupling solution with protected nucleoside phosphoramidite 1 Liquid flows from vessels v3-2 and v4-3 to mixer 5. The stream can be first transferred to waste container 7, and once the desired mixture is obtained, the stream is transferred to reaction vessel 6. The coupling solution is recycled from liquid conduit 9 through line 4 and mixer 5 to reaction vessel 6. 5) Rinse lines 2, 3 and 4, the mixing chamber, and the reaction vessel with acetonitrile from vessels v2-1, v3-1 and v4-1. 6) Prepare the oxidizing solution Liquid flows from vessels v1-4, v4-5 and v3-n to mixer 5. The flow is first transferred to waste container 7 and then transferred to reaction vessel 6 once the desired mixture is obtained. Alternatively: Prepare a sulfurized solution The liquid flows from vessels v4-4 and v3-n to mixer 5. The flow is first transferred to waste container 7, and once the desired mixture is obtained, the flow is transferred to reaction vessel 6. During the reaction, recirculation takes place via line 2. 7) Rinse lines 2 and 1, 4, 3 (oxidation) or 4, 3 (sulfurization), the mixing chamber, and the reaction vessel with acetonitrile from vessels v2-1, v1-1, v4-1, and v3-1 (oxidation) or v2-1, v4-1, and v3-1 (sulfurization). 8) Prepare the blocking (also known as capping) solution Liquid flows from vessels v1-2, v4-2, v3-n and v2-1 to mixer 5. The stream is first transferred to waste container 7, and once the desired mixture is obtained, the stream is transferred to reaction vessel 6. Recirculation occurs during the reaction via line 2. 9) Rinse lines 1, 2, 3 and 4, the mixing chamber, and the reaction vessel with acetonitrile from vessels v1-1, v2-1, v3-1 and v4-1.

[0042] As can be seen from the above process, all reagents can be mixed from pure liquids, except in cases where the starting materials are not liquid and need to be supplied as solutions. Apart from procedural advantages, this increases the stability of the reagents used and therefore the stability of the process.

[0043] The present specification further discloses an advantageous method for synthesizing oligomers using the apparatus detailed above, in which at least one solution independently selected from the group consisting of a deprotection solution, an oxidation solution, and a sulfurization solution is produced by in-line mixing. In a further advantageous method for synthesizing oligomers using the apparatus detailed above, at least two solutions independently selected from the group consisting of a deprotection solution, a blocking solution, an oxidation solution, and a sulfurization solution are produced by in-line mixing. In another embodiment, at least three solutions independently selected from the group consisting of a deprotection solution, a blocking solution, an oxidation solution, and a sulfurization solution are produced by in-line mixing.

[0044] The apparatus of the present invention can be used to carry out an oligonucleotide synthesis procedure that involves at least two repeated coupling cycles, the procedure defining a separate composition of acidic deprotection reagent for each coupling cycle.

[0045] The explanations and definitions given above with respect to the apparatus of the present invention are equally applicable with respect to the method of the present invention, and vice versa. Preferably, the method is performed automatically.

[0046] As used herein, the term "automatically" describes a method that is performed mechanically without human intervention and constant human control. This may mean that the steps of the synthesis method are managed by a controller, as detailed above. However, the device and / or controller may be configured to allow or even require human intervention under certain circumstances. This may occur, for example, when unpredictable events occur, such as when a processing parameter falls outside a certain predetermined range. Furthermore, it may be advantageous to perform a certain number of coupling cycles automatically, for example, under human control and / or with human intervention to perform certain critical steps.

[0047] The steps of the method detailed herein can be carried out in the order exactly as described. However, those skilled in the art will understand that the order of the steps may vary in some cases. As used herein, the phrase "the following steps 1 to x are carried out" refers to a method in which each step from step 1 to x is carried out, although not necessarily in the order shown.

[0048] As used herein, the terms "coupling cycle," "building block addition cycle," "iterative polymer synthesis process cycle," "extension cycle," and "synthesis cycle" are synonymous and refer to the steps required to extend a polymer chain, such as an oligonucleotide chain, by one building block during synthesis. Typically, one synthesis cycle includes at least the steps of providing an unprotected polymer chain for coupling and coupling the building block to the polymer chain. The step of providing an unprotected polymer chain includes removal of temporary protecting groups, separation, and washing of the polymer chain.

[0049] In one preferred embodiment, the present invention provides a method for automatically assembling an oligonucleotide chain by repeating building block coupling cycles, wherein in each building block coupling cycle, steps 1 to 6 of the following are performed: 1. providing a nmer oligonucleotide bound to a solid support, the oligonucleotide comprising a first reactive group capable of extending the oligonucleotide backbone by reacting with a second reactive group contained in an incorporated building block, where n is an integer greater than or equal to 1; 2. providing a building block to be incorporated, the building block comprising a second reactive group capable of reacting with a first reactive group of a nmer oligonucleotide, the first reactive group further comprising the first reactive group blocked with a temporary acid-sensitive protecting group; 3. contacting the nmer oligonucleotide with the building block to be incorporated under conditions that allow the first reactive group of the nmer oligonucleotide to bind to the second reactive group of the building block to be incorporated, to produce a protected and extended nmer oligonucleotide that is blocked from further extension by a temporary acid-sensitive protecting group; 4. Producing an acidic deprotection reagent by mixing at least two liquid compositions; 5. contacting the protected and extended nmer oligonucleotide of step 3 with the acidic deprotection reagent of step 4, thereby cleaving the temporary acid-sensitive protecting group from the extended nmer oligonucleotide; and 6. Removing the deprotection reagent and soluble cleavage products from the extended nmer oligonucleotide and using them as the nmer oligonucleotide in step 1 for subsequent recycling. This concerns the method by which this will be carried out again.

[0050] The above phrase "providing an nmer oligonucleotide" is understood in the broadest possible sense. Initially, step 1 can involve providing a nucleoside immobilized on a solid support and bearing a temporary, acid-sensitive protecting group that blocks backbone extension. Some such immobilized nucleosides are commercially available. The temporary protecting group can then be removed by incubation with an acidic deprotection reagent, followed by a dehydration / washing step to obtain the nmer oligonucleotide. In other cases, assembly of the oligonucleotide chain may already have begun with a longer oligonucleotide bound to the solid support. Between each coupling cycle, step 1 can partially overlap with steps 5 and 6 of the preceding coupling cycle, i.e., cleavage of the temporary protecting group from the extended nmer oligonucleotide, and can further include a rinsing step to condition the nmer oligonucleotide deprotected and extended in the preceding coupling cycle for use in the subsequent coupling step.

[0051] Those skilled in the art will appreciate that numerous solid supports can be used for oligonucleotide synthesis, including supports made from controlled pore glass (CPG) and cross-linked polystyrene beads. Linkers for anchoring the first oligonucleotide to the solid support are also well known in the art. When synthesizing oligonucleotides having backbones derived from monosaccharide moieties linked by phosphodiester bonds, the first reactive group can be a hydroxyl group, preferably a primary hydroxyl group, such as the 5' hydroxyl group of a ribose moiety, although the first reactive group can be any reactive group involved in backbone formation of the given oligonucleotide structure.

[0052] As used herein, the term "oligonucleotide backbone" refers to a repeating chain of phosphodiester / phosphodiester-like bonds and ribose / ribose-like moieties that provides the backbone to which the nucleobases / nucleobase analogs are linked. Typically, a first reactive group is located at one end of the oligonucleotide backbone and a second reactive group is located at the other end of the oligonucleotide.

[0053] The phrase "providing a building block" is understood in the broadest possible sense. The building block to be incorporated can include any nucleoside or nucleoside analogue incorporated into a nucleotide chain. The building block can include an abasic site, a nucleobase, or a nucleobase analogue linked via a glycosidic bond or an analogue of such a bond. The building block can be a monomer or an oligomer, i.e., the building block can be an oligonucleotide itself. The building block can further include a non-nucleoside modification such as cholesterol. The building block includes a second reactive group capable of reacting with the first reactive group of a nmer oligonucleotide. The second reactive group can be a phosphoramidite group, such as a 3'-O-(N,N-diisopropylphosphoramidite) group or an H-phosphonate monoester. Furthermore, the building block includes a first reactive group blocked from reaction by a temporary protecting group. The first reactive group contained in the building block is identical to the chemical moiety present at the corresponding position on the nmer oligonucleotide. The acid-sensitive protecting group blocking the first reactive group of the building block can be a trityl-type protecting group, such as triphenylmethyl (trityl), 4-monomethoxytrityl (MMT), 4,4'-dimethoxytrityl (DMT), 9-phenylxanthen-9-yl (pixyl), or 9-(p-methoxyphenyl)xanthin-9-yl (MOX).

[0054] In some embodiments, the first reactive group is a hydroxyl group, preferably a primary hydroxyl group, such as the 5' hydroxyl group of a ribose / ribose-analogous moiety, and the second reactive group is a phosphoramidite group, such as a 3'-O-(N,N-diisopropylphosphoramidite) group. In further embodiments, the first reactive group is a hydroxyl group, preferably a primary hydroxyl group, such as the 5' hydroxyl group of a ribose / ribose-analogous moiety, and the second reactive group is a phosphonate monoester group.

[0055] Those skilled in the art will be familiar with the conditions required to react the first reactive group of a nmer oligonucleotide with the second reactive group of a building block. For example, an acidic azole catalyst such as 1H-tetrazole or 5-ethylthio-1H-tetrazole can be used to induce the formation of a phosphite triester group by contacting the nmer oligonucleotide with the building block in an anhydrous solvent, typically acetonitrile. As is common in the art, the protected and extended nmer oligonucleotide is then protected by exposing the phosphite triester group to a sulfurizing or oxidizing reagent. As a further example, when the second reactive group is a phosphonate monoester group, pivaloyl chloride, 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl), and other compounds can be used as activating agents.

[0056] This method envisions preparing a new acidic deprotection reagent for each coupling cycle by mixing at least two liquid compositions. Therefore, the composition of the deprotection reagent can be individually determined for each coupling cycle. This allows, for example, the acid content to be adapted to the specific conditions occurring in each coupling cycle. For example, purine bases are more easily cleaved from oligonucleotides than pyrimidine bases, leaving abasic sites. So-called depurination occurs more easily at the ends of oligonucleotide chains than in the middle. Therefore, it is advantageous to be able to optimize the conditions for removing temporary protecting groups at each step. This allows the procedure to minimize unwanted side reactions and maximize the purity of the crude oligonucleotide chain assembled during synthesis.

[0057] In some methods of the present invention, the composition of the deprotection reagent is different between at least two coupling cycles. The composition of the deprotection reagent may vary, particularly with respect to the acid content. For example, the acid concentration may increase with the number of cycles. In one embodiment, the acid content in the deprotection reagent is selected from the range of 0.1% (w / w) to 50% (w / w) of acid for each coupling cycle individually. For example, the concentration of the acid in the deprotection reagent can be 0.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50% (w / w). In many cases, the concentration of the acid in the deprotection reagent can be selected from DCA in the range of 0.5 to 20%. The acid contained in the deprotection reagent can be selected from DCA and TCA, and preferably DCA. The acid content in the deprotection reagent can be selected individually for each coupling cycle from a range of 0.1% (w / w) to 50% (w / w) DCA in a solvent. The acid content in the deprotection reagent can be selected individually for each coupling cycle from a range of 0.1% (w / w) to 20% (w / w) TCA in a solvent. The preferred solvent is toluene.

[0058] The deprotection reagent can be produced by combining a pure DCA stream and a solvent stream in the mixing device of the present invention. Instead of pure acids, concentrated solutions of each acid in a solvent can be used and diluted sequentially by mixing with a liquid, e.g., a solvent. For example, a 50% solution of DCA in toluene can be supplied as a stock solution and then combined with toluene to achieve the desired concentration. Alternatively, a TCA solution can be used. The deprotection solution is typically made by diluting an acid, such as DCA, in a solvent, such as toluene, although the deprotection reagent can contain additional components, such as a cation scavenger. The deprotection reagent is removed from the solid support after the deprotection reaction is complete by draining and / or by a washing step. The deprotection reagent is replaced by rinsing with a processing solvent, e.g., acetonitrile.

[0059] The following aspects of the invention are also disclosed: 1. An oligomer synthesis apparatus comprising: a) a reaction vessel connected to a waste container via a liquid conduit; b) a liquid supply for delivering liquid reagents to the reaction vessel; c) a bypass conduit for transferring the liquid flow from the liquid supply into a waste container without passing through the reaction vessel; d) Main control unit and the liquid supply comprising: b-1) at least one mixing device; b-2) at least four upstream liquid lines connected to at least one mixing device, each upstream liquid line including one liquid conduit having n liquid inlets, where n is an integer between 1 and 25, and one liquid pump; and b-3) n sensors, where n is an integer greater than or equal to 1, positioned downstream of the mixer and determining at least one property of the liquid exiting the mixer, wherein at least one measurement provided by the sensor is used as a feedback signal for adjusting the activity of one or more liquid pumps. 2. The apparatus of embodiment 1, wherein the master controller receives inputs from the n sensors and regulates the flow of liquid from the one or more pumps. 3. The device of aspect 1 or 2, wherein the pump is controlled by a local controller that receives input from the sensor. 4. The apparatus of any one of aspects 1 to 3, wherein the mixing device is connected to the reaction vessel via a liquid conduit. 5. The apparatus of any one of aspects 1 to 3, wherein the mixing device is connected to a manifold via a liquid conduit, and the manifold is connected to the reaction vessel via a liquid conduit. 6. The apparatus of aspect 5, wherein the manifold is further connected to at least one liquid conduit that supplies incoming liquid from the liquid inlet by a liquid pump. 7. The apparatus of any one of aspects 1 to 6, wherein the liquid supply section further includes at least one heat exchanger, and preferably at least one of the heat exchangers is positioned between the mixing section and the first sensor of the n sensors. 8. The apparatus of any one of aspects 1 to 7, wherein the reaction vessel is a packed column reactor or a batch reactor. 9. The apparatus of any one of aspects 1 to 8, wherein the inner surface of the mixing apparatus and at least one liquid line are made from a corrosion-resistant material, preferably HASTELLOY alloy or polytetrafluoroethylene. 10. The apparatus of any one of aspects 1 to 9, wherein the n sensors are independently selected from the group consisting of an infrared spectrophotometer, a density sensor, a refractometer, a conductivity sensor, a temperature sensor, an impedance sensor, and a UV / Vis absorption sensor. 11. The apparatus of any one of aspects 1 to 10, further comprising a recirculation circuit that moves flow from the liquid conduit through the mixing section and the n sensors by means of a pump and into the reaction vessel. 12. The apparatus of any one of aspects 1 to 10, further comprising a recirculation circuit that moves flow from the liquid conduit through the mixing section and the n sensors by a pump and into the reaction vessel, wherein one of the liquid lines, together with the pump for that liquid line, is connected to the liquid conduit through one of its inlets and a three-way valve to function as a recirculation line. 13. An oligonucleotide solid phase synthesis apparatus, the oligonucleotide solid phase synthesis apparatus comprising a batch reactor for carrying out synthesis within the batch reactor. 14. A method for synthesizing oligonucleotides, comprising using an apparatus according to any one of aspects 1 to 13. 15. The method of embodiment 14, wherein at least two solutions independently selected from the group consisting of a deprotection solution, a blocking solution, an oxidation solution, and a sulfurization solution are produced in a liquid line by in-line mixing.

[0060] Text description of the illustration image022.gif. FIG. 1 shows a basic embodiment of an apparatus according to the present invention. The liquid supply section 19 of the apparatus includes a mixing device 5 connected to four liquid supply lines: a first liquid supply line 1, a second liquid supply line 2, a third liquid supply line 3, and a fourth liquid supply line 4. Each liquid supply line includes a pump (p1, p2, p3, or p4, respectively), a liquid conduit (l1, l2, l3, or l4, respectively), and n inlets (i1-1 to i1-n, i2-1 to i2-n, i3-1 to i3-n, and i4-1 to i4-n, respectively), where n is an integer greater than or equal to 1. The fluid connection between each liquid inlet and the conduit can be adjusted by a valve 10. Liquid supply lines 1, 2, 3, and 4 are highlighted with dotted boxes for clarity. It is understood that the entire length of the liquid conduit (l1, l2, l3, or l4, respectively) leading to the inlet of the mixer 5 is considered to belong to each liquid supply line. The mixer 5 combines the flows from any of the liquid supply lines 1, 2, 3, or 4 and mixes them to produce a single homogeneous solution. The homogeneous solution is directed into the reaction vessel 6 through a liquid conduit 20 containing n sensors (s1 to sn), where n is an integer equal to or greater than 1. The sensors can send feedback signals related to the composition of the solution to the control unit 8. The control unit 8 uses the feedback signals to adjust the flow of the line pumps (p1, p2, p3, or p4, respectively). Unless the composition of the solution passing through the sensors is unstable at a given set interval, the solution is not directed into the reaction vessel but is instead directed into the waste container 7 via the automatic three-way valve 12 and the bypass conduit 11. In addition to pumps p1, p2, p3, and p4, controller 8 controls fluid flow through conduits 11, 20 and liquid flow through liquid conduit 9 connecting the reaction vessels to waste container 7. This allows controller 8 to determine the liquid to incubate with the contents of the reaction vessels and for how long.

[0061] Figure 2 shows a further embodiment of the device of the invention, and all remarks regarding Figure 1 apply to Figure 2. In addition to the components mentioned above, a liquid recirculation conduit 16 with a pump 17 is connected to the liquid conduit 9 via a three-way valve 12. This arrangement makes it possible to circulate a given solution emerging from the reaction vessel 6 via the mixing device 5 and a liquid conduit 20 with n sensors (s1 to sn) back to the reaction vessel 6. The operation of the three-way valve 12 and the pump 17 is controlled by the control unit 8. An additional liquid inlet 18 can be provided in the recirculation conduit 16 to provide flexibility.

[0062] FIG. 3 shows a further embodiment of the device of the present invention, and all remarks regarding FIG. 1 apply to FIG. 3. In addition to the components described above, a heat exchanger 13 is integrated into the liquid conduit 20 between the mixer 5 and the first sensor s1. This allows for adjusting the temperature of the solution to obtain more reliable measurements from the sensor and for adjusting the temperature of the reagents in the reaction vessel 6. Furthermore, a further sensor 23 is integrated into the liquid conduit 9 downstream of the reaction vessel. This sensor also sends a signal to the control unit that correlates with events inside the reaction vessel. In this embodiment, the second liquid supply line 2 performs a dual function, with one of its inlets connected to the liquid conduit 9 via a three-way valve 12 positioned between the sensor 23 and the waste container 7. This arrangement allows for a given liquid emerging from the reaction vessel 6 to be circulated back to the reaction vessel 6 via the liquid conduit 12, the mixer 5, and the liquid conduit 20 with the n sensors (s1 to sn) without the need for a dedicated recirculation conduit and pump as shown in FIG. 2. The operation of the three-way valve 12 and the pump p2 is controlled by the control unit 8.

[0063] Figure 4 shows a further embodiment of the apparatus of the present invention, and all remarks regarding Figure 3 apply to Figure 4. Three liquid supply lines 1, 3, 4 are shown, each with four reservoirs (v1-1 to v1-4, v3-1 to v3-4, and v4-1 to v4-4, respectively) and n-4 inlets (i1-5 to i1-n, i3-5 to i3-n, and i4-5 to i4-n, respectively), where n is an integer equal to or greater than 7. As explained with reference to Figure 3 above, the second liquid supply line 2 performs the dual function of liquid supply line and recirculation conduit. The second liquid supply line 2 is shown with two reservoirs (v2-1), (v2-2) and n-2 inlets (i2-3 to i2-n), where n is an integer equal to or greater than 5. The liquid supply unit 19 includes a heat exchanger 13 and two sensors (s1) and (s2).

[0064] FIG. 5 illustrates a further embodiment of the apparatus of the present invention. The liquid supply section 19 of the apparatus can include a mixing device 5 connected to four liquid supply lines 1, 2, 3, and 4. Each liquid supply line includes a pump ((p1), (p2), (p3), or (p4), respectively), a liquid conduit ((l1), (l2), (l3), or (l4), respectively), and n inlets ((i1-1 to i1-n), (i2-1 to i2-n), (i3-1 to i3-n), and (i4-1 to i4-n), respectively), where n is an integer greater than or equal to 1. The fluid connection between each liquid inlet and the conduit can be adjusted by a valve 10. The mixed solution leaving the mixing device 5 is sent to a manifold 14 via a liquid conduit 21. A heat exchanger 13 and a sensor (s1) are integrated into the liquid conduit 21. Sensor (s1) sends a signal to local control 15 which adjusts the operation of pumps (p1), (p2), (p3) or (p4) in the liquid supply line. Local control 15 may be controlled by control unit 8 or may be independent. Manifold 14 is further connected to four additional liquid conduits, each containing a pump 17 and two liquid inlets 18. The liquid flow exiting the manifold is sent to reaction vessel 6 through liquid conduit 22. An additional heat exchanger 13 and sensor 23 are integrated into liquid conduit 22. All other components are positioned relative to FIG. 3.

[0065] Figure 6 shows a further embodiment of the device of the invention similar to Figure 5. A manifold 14 receives inputs from two mixing devices 5 each connected to four liquid supply lines 1, 2, 3, 4.

[0066] Figure 7 shows a further embodiment of an apparatus having two liquid supply lines. The apparatus of Figure 7 essentially corresponds to the apparatus described with reference to Figure 1, but includes only two liquid supply lines 1, 2 and does not include a sensor located downstream of the mixing device 5.

[0067] Figure 8 shows another embodiment of an apparatus having two liquid supply lines and a recirculation circuit. The apparatus of Figure 8 essentially corresponds to the apparatus described with respect to Figure 7, but further includes a recirculation circuit including a liquid recirculation conduit 16 and a liquid pump 17. The recirculation circuit shown in Figure 8 also includes an additional liquid inlet 18.

[0068] Furthermore, the embodiment of FIG. 1 includes a liquid flow sensor 24 located in each of the two liquid supply lines 1, 2 located downstream of each liquid pump p1, p2 and upstream of the mixing device 5.

[0069] Figure 9 shows an embodiment of the device of Figure 1 having only two liquid supply lines 1, 2. The explanations given with respect to Figure 1 also apply to the embodiment shown in Figure 9, except that the two further liquid lines 3, 4 are omitted.

[0070] FIG. 10 shows an embodiment of the apparatus described with respect to FIG. 3, but with only two liquid supply lines.

[0071] Figure 11 shows an embodiment of the device of Figure 2 having only three liquid supply lines. The comments made regarding the embodiment of Figure 2 also apply to the embodiment of Figure 11, except that the number of liquid supply lines 1, 2 is reduced.

[0072] Figure 12 shows another embodiment of an apparatus having three liquid supply lines, one of which functions as a recirculation circuit. The embodiment of Figure 12 is similar to the embodiment described with respect to Figure 3, except that the sensor and the fourth liquid supply line are omitted.

[0073] Figure 13 shows a further embodiment of the device of Figure 3, which has only three liquid supply lines. Unlike the embodiment of Figure 12, a sensor 23 is provided in addition to sensors s1 to sn. [Explanation of symbols]

[0074] 1 Liquid supply line 1 i1-1 Line 1 Entrance No. 1 i1-2 Line 1 Entrance No. 2 i1-3 Line 1 Entrance No. 3 i1-4 Line 1 Entrance No. 4 i1-n Line 1 entrance number n p1 Line 1 liquid pump l1 Line 1 liquid conduit 2 Liquid supply line 2 i2-1 Line 2 Entrance No. 1 i2-2 Line 2 Entrance No. 2 i2-3 Line 2 Entrance No. 3 i2-4 Line 2 Entrance No. 4 i2-n Line 2 entrance number n p2 Line 2 liquid pump l2 Line 2 liquid conduit 3 Liquid supply line 3 i3-1 Line 3 Entrance No. 1 i3-2 Line 3 Entrance No. 2 i3-3 Line 3 Entrance No. 3 i3-4 Line 3 Entrance No. 4 i3-n Line 3 entrance number n p3 Line 3 liquid pump l3 Line 3 liquid conduit 4 Liquid supply line 4 i4-1 Line 4 Entrance No. 1 i4-2 Line 4 Entrance No. 2 i4-3 Line 4 entrance number 3 i4-4 Line 4 entrance number 4 i4-n Line 4 entrance number n p4 Line 4 liquid pump l4 Line 4 liquid conduit v1-n Line 1 storage tank number n v2-n Line 2 storage tank number n v3-n Line 3 storage tank number n v4-n Line 4 storage tank number n 5 Mixing device 6 Reactor 7. Waste container 8 Control Unit 9 Controllable Liquid Conduits 10 valves 11 Bypass conduit s1 Sensor 1 s2 Sensor 2 sn Sensor n 12 Three-way valve 13 Heat exchanger 14 Manifold 15 Local control unit 16 Fluid recirculation conduit 17 Liquid Pump 18 Liquid inlet 19 Liquid supply section 20 Liquid conduit between the mixer and the reactor 21 Liquid conduit between mixer and manifold 22 Liquid conduit between manifold and reactor 23 Sensors 24 Flow sensor

Claims

1. 1. An automated oligonucleotide synthesizer for individually adjusting the composition of an acidic deprotection reagent for each coupling cycle, comprising: a) a reaction vessel (6) connected to a waste container (7) via a liquid conduit (9); b) a liquid supply (19) for delivering liquid reagents to the reaction vessel (6); c) a bypass conduit (11) for transferring the liquid flow from the liquid supply (19) into the waste container (7) without passing through the reaction vessel (6); d) a control unit (8); and the liquid supply (19) comprises: b-1) at least one mixing device (5); b-2) at least two liquid supply lines (1, 2) connected to the at least one mixing device, each liquid supply line including at least one liquid conduit (l1, l2) having n liquid inlets (il-n, i2-n), where n is an integer between 1 and 25, and at least one pump (p1, p2); Including, a control unit (8) that allows the user to define the composition of the coupling reagent for each coupling cycle; the composition of the acidic deprotection reagent differs between at least two coupling cycles; The automated oligonucleotide synthesizer.

2. 2. The apparatus according to claim 1, wherein the inner surfaces of at least the at least one mixing device (5) and one of the at least two liquid supply lines (1, 2) are made from an acid-resistant material and / or comprise an acid-resistant coating.

3. 3. The apparatus according to claim 2, wherein the inner surfaces of at least the at least one mixing device (5) and one of the at least two liquid supply lines (1, 2) are made from an acid-resistant alloy and / or comprise an acid-resistant polymeric coating.

4. The liquid supply (19) further comprises a third liquid supply line (3) connected to at least one mixing device (5), said third liquid supply line (3) having n liquid inlets (i3 4. The device according to claim 1, further comprising at least one liquid conduit (l3) having a plurality of pumps (p3) and at least one liquid pump (p3) with a plurality of pumps (p4) and a plurality of pumps (p5) having a plurality of pumps (p6) and a plurality of pumps (p7) and a plurality of pumps (p8) and a plurality of pumps (p9) and a plurality of pumps (p10) and a plurality of pumps (p11) and a plurality of pumps (p12) and a plurality of pumps (p13) and a plurality of pumps (p14) and a plurality of pumps (p15) and a plurality of pumps (p16) and a plurality of pumps (p17) and a plurality of pumps (p18) and a plurality of pumps (p19) and a plurality of pumps (p21) and a plurality of pumps (p19) and a plurality of pumps (p22) and a plurality of pumps (p

5. The apparatus according to any one of claims 1 to 4, wherein the at least one mixing device (5) comprises a static mixer.

6. 6. The device according to any one of claims 1 to 5, further comprising n sensors (s1 to sn) positioned downstream of the at least one mixer (5) and for determining at least one property of the liquid emerging from the mixer (5), wherein n is an integer greater than or equal to 1.

7. 7. The device according to claim 6, wherein at least one measurement value provided by at least one of the sensors (s1-sn) is used as a feedback signal for adjusting the activity of one or more pumps comprised in the liquid supply (19).

8. 8. Apparatus according to claim 6 or 7, wherein at least one measurement provided by at least one of the sensors (s1-sn) is used to control whether the liquid flow is transferred into the reaction vessel (6) or into a waste container (7) without passing through the reaction vessel (6).

9. 9. The device according to any one of claims 1 to 8, further comprising at least one flow sensor (24) integrated into each of the at least two liquid supply lines (1, 2) upstream of the at least one mixing device (5), wherein at least one measurement provided by said flow sensor is used as a feedback signal for adjusting the activity of one or more of the at least one pumps (p1, p2).

10. Apparatus according to any one of the preceding claims, wherein the liquid supply (19) further comprises at least one heat exchanger (13).

11. The apparatus of any one of claims 6 to 10, comprising a conductivity sensor, a temperature sensor, and a UV absorption sensor.

12. The apparatus according to any one of claims 1 to 11, wherein the reaction vessel (6) is a packed column reactor or a batch reactor.

13. 13. The apparatus according to any one of the preceding claims, further comprising a recirculation circuit in which the flow from the liquid conduit (9) connecting the reactor to the waste container is transferred back to the reactor (6) by a pump (17).

14. 14. Apparatus according to claim 13, wherein the recirculation circuit is comprised in one of at least two liquid supply lines (1, 2) connected to a liquid conduit (9) via one of its inlets (i1-n, i2-n) and a multi-way valve (12), the pumps (p1, p2) of which are capable of driving the recirculation of fluid from the liquid conduit (9) connecting the reaction vessel with a waste container, via at least one mixing device (5) and back into the reaction vessel.

15. A method for automatically assembling an oligonucleotide chain by repeating building block coupling cycles, wherein in each building block coupling cycle, the following steps 1 to 6 are performed:

1. A nmer oligonucleotide bound to a solid support, which reacts with a second reactive group contained in the building block to be incorporated to form the oligonucleotide backbone. providing an oligonucleotide comprising a first reactive group capable of extending 2. Providing a building block to be incorporated, the building block comprising a second reactive group capable of reacting with a first reactive group of the nmer oligonucleotide, and further comprising the first reactive group blocked with a temporary acid-sensitive protecting group; 3. contacting the nmer oligonucleotide with the building block to be incorporated under conditions that allow the first reactive group of the nmer oligonucleotide to bond to the second reactive group of the building block to be incorporated, to produce a protected and extended nmer oligonucleotide blocked from further extension by a temporary acid-sensitive protecting group; 4. Producing an acidic deprotection reagent by mixing at least two liquid compositions, the composition of the acidic deprotection reagent being different for at least two coupling cycles and being individually determined for each coupling cycle; 5. contacting the protected and extended nmer oligonucleotide of step 3 with the acidic deprotection reagent of step 4, thereby cleaving the temporary acid-sensitive protecting group from the extended nmer oligonucleotide; and 6. Removing the deprotection reagent and soluble cleavage products from the extended nmer oligonucleotide and using them as nmer oligonucleotides in step 1 of the subsequent cup recycling. The method, wherein the above is performed again.

16. 16. The method of claim 15, wherein the acid content in the deprotection reagent is selected from the range of 0.1% (w / w) to 50% (w / w) acid individually for each coupling cycle.

17. The method according to any one of claims 15 to 16, wherein the acid-sensitive temporary protecting group is a trityl-type protecting group.

18. The method of any one of claims 15 to 17, wherein the first reactive group is a hydroxyl group.

19. 19. The method of claim 18, wherein the second reactive group is selected from the group consisting of a phosphoramidite group and an H-phosphonate monoester group.

20. 20. The method of claim 19, wherein step 3 comprises contacting the building block with an nmer oligonucleotide under conditions that allow for the formation of a phosphite triester group, and mixing the resulting compound with an oxidation reagent or a sulfurization reagent to convert the phosphite triester group to a phosphate triester group or a thiophosphate triester group, respectively.

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