Process and system for flow-through oligonucleotide synthesis

Abstract

The present invention relates to a process and system for flow-through solid-phase oligonucleotide synthesis. The process includes the steps of detritylation, coupling, oxidation, and capping. The process includes at least one step of recompacting the synthesis resin, which is performed after the detritylation step. The step of recompacting the synthesis resin includes an initial fluidization step followed by a repacking step. In the fluidization step, the solvent flows from the bottom to the top of the column, and in the repacking step, the solvent flows from the top to the bottom of the column.

Description

[Technical field]

[0001] The present invention relates to processes, methods and systems for solid phase oligonucleotide synthesis. [Background technology]

[0002] Oligonucleotides in nature are DNA or RNA molecules. They have attracted great interest due to their use in drug discovery, therapeutics, molecular diagnostics, etc. To date, there are more than 10 FDA-approved oligonucleotide drugs, most of which have been approved since 2016. Oligonucleotides can be synthesized in solution or in a flow-through process, where a synthetic resin is provided to a column. A protected nucleoside or nucleotide is attached to the resin via a linker. In the first step of the reaction, the protected nucleoside or nucleotide is deprotected by a deprotecting agent. A second nucleotide dissolved in a solvent is added to the column to attach the second nucleotide to the first nucleotide or nucleoside. These steps can be repeated to produce an oligonucleotide chain or sequence. After the desired oligonucleotide sequence is synthesized, the oligonucleotide is separated from the resin.

[0003] The synthesis resin may shrink and expand during the process, depending on the type of resin, the ratio of the amount of resin to the amount of growing oligonucleotide, and the solvent used in the process. The expansion and contraction of the resin may increase the phase heterogeneity of the resin during oligonucleotide synthesis. Phase heterogeneity may result in non-optimal flow through the resin, i.e., the front may not be horizontal or vertical to the column wall, and therefore the flow may not be uniform throughout the resin. This will result in a negative impact on the synthesis and the quality of the synthesized oligomer.

[0004] No. 5,641,459 discloses a machine for synthesizing oligonucleotides having individual moduli for connecting each of a large number of different monomers and other fluids used in the synthesis process.

[0005] No. 6,469,157 B1 discloses an apparatus for preparing polynucleotides on a solid support in a reactor comprising a column containing an immobilized solid support functionalized for polynucleotide synthesis.

[0006] EP3650455A1 discloses a new solvent that can be used as an alternative to toluene in the deprotection step in solid-phase nucleic acid synthesis methods, and a method in which the removal of protecting groups from protected nucleoside phosphoramidites is carried out in a solution containing an acid with a pKa of 0.2-0.8 and acetonitrile.

[0007] There is a need in the art for improved processes and systems for oligonucleotide synthesis that overcome the problems of the prior art. There is also a need for processes and systems for oligonucleotide synthesis that prevent or minimize the adverse effects of resin phase heterogeneity. Summary of the Invention

[0008] The object of the present invention is to provide a process and a system for oligonucleotide synthesis that overcomes the drawbacks of the prior art systems and processes. This is achieved by the process defined in claim 1, the system defined in claim 16 and the method defined in claim 22.

[0009] According to one aspect of the present invention, there is a flow-through process for solid phase oligonucleotide synthesis, in which a column is provided with a first hydroxyl-protected sequence unit attached to a synthesis resin via a chemical bond. The process comprises the following steps (i) to (iii), which are repeated at least once as a cycle: i. deprotecting the hydroxyl group of the first sequence unit bound to the synthesis resin by detritylation: ii. providing a liquid reaction solution comprising at least one second sequence unit; iii. passing the reaction solution over the synthetic resin having at least one first sequence unit bound thereto, whereby at least one second sequence unit binds to the resin-bound first sequence unit, and the reaction solution compacts the resin to a more compact state in the column provided in step (i); Including, Within that process, a step of removing the protecting group from the first sequence unit bound to the synthetic resin is carried out in an acidic solution, and the process includes at least one step of recompacting the resin.

[0010] In one aspect of the invention, there is a system for flow-through solid phase oligonucleotide synthesis. The system includes at least one column arranged to be filled with synthesis resin; at least a first pump; at least a first reservoir for monomers and reagents; at least one detector; and at least one processor. A tube connects the first reservoir to the column, and the first pump and a valve are arranged to direct flow through the system. The processor is in communication with at least the first pump, the valve, and the detector. The system is arranged to provide a fluidization flow path configured to direct a flow of solvent from the first reservoir to the bottom of the column, such that the solvent passes through the column from the bottom to the top of the column. The system is arranged to provide a refill flow path configured to direct a flow of solvent from the first reservoir to the top of the column, such that the solvent passes through the column from the top of the column. The system is arranged to first flow the solvent through the fluidization flow path and subsequently flow the solvent through the refill flow path during one synthesis cycle.

[0011] In one aspect of the invention, there is a method for performing flow-through solid-phase oligonucleotide synthesis using a system for flow-through solid-phase oligonucleotide synthesis. The system includes at least one column arranged to fill with synthetic resin; at least a first pump; at least a first reservoir for monomers and reagents; at least one detector; and at least one processor. A tube connects the first reservoir to the column. The first pump and a valve are arranged to direct the flow in the system. The processor is in communication with at least the first pump, the valve, and the detector. The synthetic resin is placed in the column, and a first sequence unit is bonded to the synthetic resin via a chemical bond. The method includes at least one step of recompacting, during which the system is configured to first fluidize the synthetic resin placed in the column by flowing a solvent from the bottom to the top of the column through a fluidization channel in a fluidization step. The fluidization step is followed by a refilling step in which the system uses a refilling channel to flow a solvent from the top to the bottom of the column to refill the synthetic resin placed in the column. [Brief description of the drawings]

[0012] [Figure 1A] 2 is a flow chart of a process according to the present invention. [Figure 1B] 2 is a flow chart of a process according to the present invention. [Figure 2A] 1 is a schematic diagram of a system according to the present invention; [Figure 2B] 1 is a schematic diagram of a system according to the present invention; [Diagram 3] 1 is a schematic diagram of a system according to the present invention; [Figure 4] 1 is a schematic diagram of a system according to the present invention; [Diagram 5] This is a block scheme according to the present invention. [Figure 6A] HPLC chromatograms from examples according to the present invention. [Figure 6B] HPLC chromatogram from a comparative example.

[0013] definition Terms used herein have their ordinary meaning in the art unless otherwise specified.

[0014] The term "flow-through" is used to describe a process in which solutions, including reagents, and / or solvents, and / or additives, etc., are passed over / through a column, and the term includes both single pass (i.e., the solution(s) are passed over the column once) and recirculation (i.e., the solution(s) are recirculated through the system and passed through the column at least one more time).

[0015] The term "sequence unit" refers to a compound used as a unit in a sequence. A sequence unit can store / carry information. Examples of sequence units include nucleoside(s), nucleotides, phosphorodiamidate morpholino oligomers (PMO), bicyclic / tricyclic nucleosides, and nucleotide analogs such as LNA, cEt, ENA, GNA, TNA, FNA, etc. Examples of sequences include DNA, RNA, and analogs of 2' modifications such as MOE, OMe, F, etc. A sequence unit can also be any non-nucleotide, non-nucleoside based compound in a sequence together with DNA, RNA, or their analogs. The first sequence unit can also be part of a universal support.

[0016] The term "nucleotide" refers to a nucleic acid subunit, which includes a sugar group, a base, and a phosphate group or any other phosphate analog, with phosphorothioate being one of the most common.

[0017] The term "nucleoside" refers to a compound that includes a sugar and a base.

[0018] The term "base" refers to a nitrogenous base. There are many different bases that can be used. The five most common in DNA and RNA are adenine, cytosine, guanine, and thymine / uracil (abbreviated A, C, G, and T / U). Other examples are methyluracil (MeU) and methylcytosine (MeC), or any other functional group(s) attached or substituted / included in the base structure.

[0019] The term "nucleic acid" includes both DNA and RNA, and analogs of DNA and RNA that differ in backbone structure from DNA or RNA.

[0020] The term "synthetic resin" includes organic compounds or solid phases capable of forming a packed bed in a column, and synthetic resins include, for example, polymeric materials based on polystyrene or divinylbenzene, but also other solid phases or combinations of solid phases having any degree of cross-linking or pore structure.

[0021] The term "reagent" refers to any compound, sequence unit, reagent, or solvent used to generate the nucleotide sequence grown in the flow-through system.

[0022] As used herein, the term "about" should be understood to encompass variations of ±10%, or ±5%, or ±1%.

[0023] The term "fluidization" refers to the time when more than about 1% of the surface area of ​​the synthetic resin is no longer in contact with the bottom of the column. This stage can be obtained, for example, by applying a flow from the bottom or the top of the column.

[0024] Throughout this specification, the terms "oligonucleotide", "polynucleotide" and "oligomer" are used interchangeably and refer to a linear or branched chain or sequence of sequence units, i.e., a sequence, e.g., a nucleic acid such as DNA / RNA or analogs thereof.

[0025] As used herein, unless otherwise specified, "sequence units" such as "nucleoside(s)," "nucleotide(s)," "oligonucleotide(s)," and "polynucleotide(s)" refer to those having activated and / or protecting groups, as appropriate.

[0026] The term "detritylation" or "detritylation" refers to the removal of a protecting group(s) to allow for the addition of additional sequence units. Detritylation includes the removal of dimethoxytrityl or monomethoxytrityl or any other protecting group to allow for the addition and coupling of additional sequence units.

[0027] The term "linearly scalable" refers to a synthesis process in which smaller or larger scale synthesis conditions can be transferred to scales of alternative sizes that yield similar or identical synthesis results.

[0028] The term "conditional threshold" is used to mean a predefined value of at least one parameter set in the control software to terminate the previous step(s) and initiate the next step.

[0029] "ACN" is an abbreviation for acetonitrile and is used with its standard meaning in the art.

[0030] "DCA" and "TCA" are abbreviations for dichloroacetic acid and trichloroacetic acid, respectively, and are used with their standard meaning in the art.

[0031] "PAT" is an abbreviation for Process Analytical Technology and is used in the art with its standard meaning.

[0032] "CPP" is an abbreviation for Critical Process Parameters and is used in the art with its standard meaning.

[0033] "CQA" is an abbreviation for Critical Quality Attributes and is used in the art with its standard meaning.

[0034] "NIR" is an abbreviation for Near Infrared and is used in the art with its standard meaning.

[0035] "UV / Vis" is an abbreviation for Ultraviolet / Visible and is used in the art with its standard meaning. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Terms such as "top", "bottom", "upper", "lower", etc. are used only to refer to the geometric shapes of the embodiments of the invention illustrated in the drawings and are not intended to limit the invention in any way.

[0037] Methods and processes for synthesizing oligonucleotides include phosphoramidite, phosphotriester, and H-phosphonate methods, as well as other similar methods, all of which are commonly known in the art. Although the systems and processes described herein are described in relation to phosphoramidite methods, it is understood that the processes and systems can also be utilized using other synthesis methods.

[0038] As discussed in the background, oligonucleotides or oligomers can be synthesized in a flow-through process, where the growing oligonucleotide is immobilized on a resin arranged in a column. A flow-through system includes a column(s), a valve(s), and a pump(s), all in fluid communication with each other using tubing. The pump(s) are arranged to pump liquid through the system onto the columns. Before synthesis begins, the column is loaded with a resin to which the first nucleotide or nucleoside is attached, either directly via a covalent bond or via a linker unit, also called a universal support. The linker unit may be the sequence unit that remains on the resin after cleavage, and therefore is not part of the final sequence unit after cleavage.

[0039] In a first aspect of the present invention, there is a flow-through process for solid phase oligonucleotide synthesis in which a column is provided with a first hydroxyl-protected sequence unit attached to a synthesis resin via a chemical bond. The process comprises the following steps (i) to (iii) which are repeated at least once as a cycle: i. deprotecting the hydroxyl group of the first sequence unit bound to the synthesis resin by detritylation: ii. providing a liquid reaction solution comprising at least one second sequence unit; iii. passing the reaction solution over the synthetic resin having at least one first sequence unit bound thereto, whereby at least one second sequence unit binds to the resin-bound first sequence unit, and the reaction solution compacts the resin to a more compact state in the column provided in step (i); Includes.

[0040] The second sequence unit is bound to the first sequence unit bound to the synthetic resin. The step of removing the protecting group from the first sequence unit bound to the synthetic resin, i.e., the detritylation step, is carried out in an acidic solution. The process includes at least one step of recompacting the synthetic resin.

[0041] As one of skill in the art will appreciate, in the case of a cyclic process, the term "first sequence unit" refers to the last added sequence unit.

[0042] The term "reaction solution" encompasses any reagent, reagents, array unit(s), solvents, additives, etc. required for a growing array.

[0043] The step of recompacting the synthetic resin includes a step of fluidizing the synthetic resin by flowing a solvent from the bottom of the column in an upward or countercurrent direction, and reloading the synthetic resin. The step of reloading the resin follows the step of fluidizing the synthetic resin. In the step of reloading the synthetic resin, the solvent is flowed from the top of the column in a downward or countercurrent direction.

[0044] In other words, in a first aspect of the present invention, there is a flow-through process for solid phase oligonucleotide synthesis, in which a first hydroxyl-protected sequence unit is attached to a resin via a chemical bond. The process comprises the following steps (i)-(iii), which are repeated at least once as a cycle: i. deprotecting the hydroxyl group of the first sequence unit bound to the synthesis resin by detritylation: ii. providing a liquid reaction solution comprising at least one second sequence unit; iii. passing the reaction solution over the synthetic resin having at least one first sequence unit bound thereto, whereby at least one second sequence unit binds to the resin-bound first sequence unit, and the reaction solution compacts the resin to a more compact state in the column provided in step (i); Includes.

[0045] The second sequence unit is bound to the first sequence unit bound to the synthetic resin. The step of removing the protecting group from the first sequence unit bound to the synthetic resin, i.e., the detritylation step, is carried out in an acidic solution. The process includes at least one step of fluidizing the resin by flowing a solvent from the bottom of the column in an upward or countercurrent direction, followed by reloading the resin. In the reloading step, the solvent is flowed from the top of the column downward, i.e., in the flow direction.

[0046] In one embodiment of the present invention, the re-compacting step includes fluidizing the resin.

[0047] In one embodiment of the invention, the sequence unit is a nucleotide(s) or a nucleoside(s).

[0048] The method according to the first aspect may be used to synthesize oligonucleotides, oligomers, DNA molecules, RNA molecules, analogues of DNA / RNA, or any other macromolecule comprising sequence units, e.g. sequences or strands of nucleic acids. All embodiments, aspects and variations described herein may relate to all types of macromolecules mentioned above.

[0049] Before starting oligonucleotide synthesis, a synthetic resin is provided in the column. As described above, the first sequence unit, or nucleic acid, or monomer is bound to the synthetic resin directly or through a linker unit. The synthetic resin is a polymer or polymeric material that has a particulate appearance with different degrees of pore structure development, and typically has some degree of cross-linking, such as 70% or less, or 50% or less, or 30% or less, or 10% or less. The synthetic resin may be any type of synthetic resin used in the art, such as polystyrene (PS), divinylbenzene (DVB), or a mixture of PS-DVB, etc. The synthetic resin may have any shape or form, such as plate-like, particulate, fibrous, etc. In one embodiment, the resin is cross-linked polystyrene, such as NittoPhase or Primer Support 5G or similar. The synthetic resin may expand and / or contract when contacted with various solvents, such as oligonucleotide synthesis or processing. The expansion and contraction of the synthetic resin placed in the column may result in the formation of non-uniform resin regions in the column, or the formation of non-uniformity within the resin. Resin heterogeneity can occur in other ways as well, and other types of resins, such as mixtures of polymers and other materials, can experience heterogeneity in the oligonucleotide synthesis process.

[0050] A flow chart of a general oligonucleotide process or synthesis according to the invention is shown in FIG. 1a. In a first step, the first sequence unit is deprotected by detritylation 301. In the detritylation step 301, the 5'-dimethoxytrityl protecting group is removed from the first sequence unit. As a result, a hydroxyl group becomes available for coupling. If the first sequence unit is more than one sequence unit (i.e., a chain or sequence is attached to a resin), then for example the hydroxyl group of the last sequence unit in the sequence becomes available for coupling. In some cases, functional groups ready for further functionalization, such as hydroxyl, amine, etc., may be available at any position other than the last sequence unit, allowing, for example, branched oligonucleotides, oligonucleotide derivatives, other sequences, other sequence units, other polymers.

[0051] Following detritylation, there is a first wash step 302 during which solvent is passed through the column. The amount of wash required depends on physical parameters such as column length and diameter, bed height, diffusion, etc., and can be determined by one of skill in the art. The amount of wash required can also be determined by satisfying a conditional threshold, as determined by one of skill in the art.

[0052] After the detritylation step 301, the synthetic resin in the column may shrink and / or become inhomogeneous and / or contain inhomogeneities, as described above. Thus, following the first wash step 302, there is a first recompacting step 303. The first recompacting step 303 comprises two steps: a first step 303' of fluidizing the resin, followed by a step 303'' of recharging the resin. In the fluidizing step 303', a solvent, for example ACN, is first passed through the column in a countercurrent or countercurrent direction, i.e. from the bottom of the column to the top. The solvent is passed through the column in a countercurrent direction until the synthetic resin or resin bed is fluidized. Thus, until a fluidized bed is formed in the column. Once the fluidized bed is formed, a recharging step 303'' follows. In the recharging step 303'' the solvent is passed through the column in the opposite direction, i.e. in the direction of flow, or from the top of the column to the bottom.

[0053] The process or synthesis of oligonucleotides may further comprise an optional recompacting step 303'''', as shown in Figure lb. Such optional recompacting step 303''' is performed prior to the detritylation step 301. The optional recompacting step 303''' comprises a fluidization step 303' followed by a refilling step 303'', i.e., the recompacting step 303 described above.

[0054] In one embodiment, a first step 303 of recompacting the synthetic resin is performed after detritylation 301. As described above, the first recompacting step 303 includes a first step 303' of fluidizing the resin, where a solvent passes from the bottom to the top of the column, followed by a step 303'' of repacking the resin, during which a solvent passes from the top to the bottom of the column. A second recompacting step may be performed after a fourth washing step 309, discussed below. Additional recompacting steps can be performed after any of the washing steps 302; 305; 307; 309 in the synthesis (all washing steps are discussed further below).

[0055] Following the first recompacting step 303 is a reaction step 320. The reaction step 320 includes a coupling step 304 followed by a second washing step 305. In the coupling step 304, at least one second sequence unit is passed over the resin (through the column). During the second washing step 305, a solvent is passed through the column, such as acetonitrile (ACN), propionitrile, dimethylformamide (DMF), followed by ACN or any other solvent or combination of solvents suitable for facilitating the removal of reagents, by-products, excess sequence units, additives, etc. The coupling step 304 can be repeated once or several times so that the at least one second nucleotide or nucleoside can be recirculated over the resin. Prior to the coupling 304, the at least one second nucleotide or nucleoside can be activated by an activating agent, such as BTT, ETT, activating agent 42. In the coupling step 304, the phosphorus in the second sequence unit forms a covalent bond with the hydroxyl group in the resin-bound first sequence unit. It is advantageous to have anhydrous reaction conditions during the coupling step 304. Anhydrous conditions may result in a higher yield and / or higher purity of the formed oligonucleotide or oligomer.

[0056] Oxidation / thiolation 330 follows the reaction step 320. Oxidation / thiolation step 330 includes an oxidation step 306 and a third wash step 307. In the oxidation step 306, an oxidizing agent / thiolation agent / sulfurizing agent is added to the column 304. The oxidizing agent / thiolation agent / sulfurizing agent converts the newly formed trivalent phosphorus to a pentavalent phosphorus (e.g., phosphorothioate, phosphodiester). Secondly, the remaining oxidizing agent / thiolation agent / sulfurizing agent is washed away in a third wash step 307. In the third wash step 307, a solvent is passed through the column.

[0057] After the oxidizing / thiolation / sulfurizing agents are washed away, a full capping step 340 follows. The full capping step 340 includes a capping step 308 and a fourth wash step 309. In the capping step 308, a capping agent is added to the column. The capping agent blocks unreacted hydroxyl groups and prevents them from reacting in the next step. The introduction of the capping agent is followed by a fourth wash step 309. In the fourth wash step 309, a solvent is passed through the column. The capping step may be optional. A capping step may also be included in the oxidation / thiolation step.

[0058] The steps of detritylation 301, recompaction 303, reaction 320, oxidation / thiolation 330, and full capping 340 are repeated until an oligomer or oligonucleotide or polynucleotide containing the desired nucleic acid sequence is synthesized. After the desired sequence is synthesized, the newly formed sequence (e.g., the newly formed oligonucleotide or polynucleotide) is removed from the resin in a cleavage step 310. The desired sequence can be removed from the resin by any standard technique available. The oligonucleotide or polynucleotide can contain up to 25, or up to 40-50, or up to 70-80, or up to 100 or more sequence units, or nucleotides or nucleosides.

[0059] An exemplary oligonucleotide synthesis cycle includes repetition of the above steps: detritylation 301, first wash step 302, first recompaction 303, reaction 320, oxidation 330, and capping 340. After the desired sequence has been synthesized, it is removed from the resin in a cleavage step 310. Any protecting group or groups can also be cleaved from the oligomer or oligonucleotide before, during, or immediately after removal from the resin.

[0060] By using a glass (or any other suitable transparent material) column, it is possible to monitor the first re-compacting step and visually monitor the resin placed in the column. After a successful first re-compacting step 303, the resin appears more uniform or homogenous within the column. In one embodiment, the first re-compacting step 303 is performed such that the flow rate during the fluidization step is 25-75 cm / h, the fluidization step lasts for 0.25-0.75, or 0.25-1 column volumes, and the flow rate during the re-packing step is 100-200 cm / h, the re-packing lasts for 0.25-0.75, or 0.25-1 or more column volumes.

[0061] In the processes described herein, which include two or more synthesis cycles and a second recompacting step performed after full capping 340 (or after the fourth wash step 309), it is possible to monitor the second recompacting step by following the red front that forms in the next / subsequent detritylation step 301. In the case of a proper second recompacting step, the red front or line that forms during the detritylation step 301 appears as an approximately straight line perpendicular to the column wall. This is in contrast to the case where the second recompacting step 303 was not performed, in which case the line is not straight or perpendicular, or the front does not form a line.

[0062] As described above, the nucleoside(s) or nucleotide(s) are activated prior to the reaction step 320. In one embodiment, the sequence unit(s) (e.g., nucleoside(s) or nucleotide(s)) are activated in a separately defined mixing chamber. In one embodiment, the sequence unit(s) (e.g., nucleoside(s) or nucleotide(s)) are activated in a tube of the system. Activation of the nucleoside(s) or nucleotide(s) in a tube of the system is sometimes referred to as in-line activation. In-line activation can occur before reaching the column or in the column.

[0063] Any suitable solvent or solvents can be used in the processes described herein. Typically, acetonitrile (ACN) is used as a solvent in the washing steps 302; 305; 307; 309, and an acidic solution is used in the detritylation step 301, where the protecting group is removed from the first nucleoside(s) or nucleotide(s) bound to the resin. In one embodiment, the pKa of the acidic solution used in the detritylation step is such that the protecting group can be released, e.g., the pKa value of DCA or TCA relative to dimethoxytrityl is, e.g., 1.35 for DCA and 0.66 for TCA. In one embodiment, the pKa of the acidic solution used in the detritylation step is 0.2-3, or 0.2-2, or 0.2-1.5, or 0.2-0.8.

[0064] In one embodiment, the reaction solution is passed over the resin at a flow rate and volume that allows the second sequence units in the reaction solution to contact at least 99%, or 98%, or 97%, or 96%, or 95%, or 90% of the immobilized sequence units.

[0065] In the flow-through method for solid phase synthesis of oligomers or oligonucleotides described herein, a solution containing a second sequence unit is passed through a column containing at least one immobilized first sequence unit. The solution may be passed through the column once or may be recirculated and passed through the column multiple times, for example, two times, or three times, or more. In the case of recirculation, different reagents and / or additives may be added to the recirculated solution. Those skilled in the art can determine the appropriate number of cycles depending on the type of oligonucleotide to be synthesized, the desired result, the type of reagents, etc.

[0066] In one embodiment, heat is applied to the at least one second array unit prior to passing over the synthetic resin. The at least one second array unit may be heated by at least 1° C. prior to passing over the synthetic resin.

[0067] In this context, heating may refer to heating of 1°C, or 3°C, or 5°C or more. Those skilled in the art can determine the heating level depending on the reaction, the type(s) of sequence unit(s), or nucleic acid(s) / nucleotide(s) / nucleoside(s), type(s) of reagent, etc. Heating the sequence unit(s) or nucleoside(s) or nucleotide(s) and possible other reagents such as capping agent(s), oxidizing agent(s) is advantageous in that it may increase the reaction rate. Heating can be used in the process of oligonucleotide synthesis to increase the reaction rate of one or more reaction steps.

[0068] The different steps of the process described herein can be monitored using one, two or several detectors. In one embodiment, the progress of at least one step of the process is monitored by at least two detectors. In one embodiment, the progress of the process is monitored continuously. In one embodiment, the progress of the process is monitored by spectroscopy, preferably near infrared (NIR) spectroscopy.

[0069] As mentioned above, one, some or all parts / steps of the process described herein can be monitored in a continuous or discontinuous manner. Thus, the detritylation 301, the first recompaction 303, the reaction step 320, the oxidation / thiolation step 330, the full capping step 340, and / or the different washing steps 302;305;307;309 can be monitored. The detectors can be NIR and / or UV / Vis detectors. Continuous monitoring can provide information about the progress of the different steps, such as the detritylation 301, the recompaction 303, the coupling 304, the oxidation 306, the capping 308, the different washing steps 302;305;307;309. Such information can be, for example, when the currently ongoing step is finished and the next step is started, i.e., when a conditional threshold is reached. Information from the monitoring can also be used to modify the synthesis protocol depending on the output from the continuous monitoring. The process can be monitored in various ways, such as in-line, online, or both. The process may be monitored in a manner that allows for continuous monitoring.

[0070] As described above, activation of sequence unit(s) or nucleoside(s) or nucleotide(s) may be carried out in a separately defined mixing chamber. In such a case, the mixing chamber may be emptied and washed between cycles using dilution with a solvent, and the progress of the washing may be monitored using a detector such as a UV / Vis detector or a NIR detector, or any other type of detector as determined to be appropriate by the skilled artisan. Emptying and washing the mixing chamber may be carried out either by performing the two processes independently and simultaneously while washing the resin in the column, or by passing the washing solvent first through one of these units and then through the other unit (i.e., first through the mixing chamber and then over the column, or vice versa).

[0071] Washing of the mixing chamber can be combined with washing of the column, for example by passing a wash solvent, such as ACN, sequentially first through the mixing chamber and then through the column.

[0072] In another embodiment, the outflow of the mixing chamber can be monitored using an air bubble detector.

[0073] The different washing steps 302; 305; 307; 309 can be performed using displacement techniques. The washing of the column can be monitored in-line and in real time and can continue until a conditional threshold is reached. Such a threshold is determined by the skilled person. The monitor can be a UV / Vis or NIR monitor / detector, or any other suitable type of detector.

[0074] In one embodiment, the process uses software-controlled real-time conditional monitoring, enabling the use of process analytical technology (PAT) to measure critical process parameters (CPPs) that affect critical quality attributes (CQAs).

[0075] Process Analytical Engineering (PAT) is an important tool for quality assurance in the biotechnology and pharmaceutical manufacturing industries. It is an advantage for a process to be PAT enabled, as PAT is important for meeting the requirements of various regulatory agencies, for example the US Food and Drug Administration (FDA). The use of UV / Vis and / or NIR spectroscopy to monitor the different steps of a reaction can enable the use of PAT.

[0076] With PAT, the user can select parameters, such as time, absorbance of light at a particular wavelength, temperature, presence or absence or both of particular intermediates, by-products, etc., that will control the process. The defined parameters can then be documented and recorded in the control software in accordance with 21 CFR part 11 compliance.

[0077] In one embodiment, the process is linearly scalable. The process described herein is linearly scalable, i.e., the liquid flow rate (cm / h) in the column is the same regardless of the size / ratio of the column, and therefore the same linear flow rate can be used at all scales, e.g., μmol, mmol, mol, etc. This means that the process described herein can be scaled up (or down) using the same relative excess of sequence unit(s) or nucleoside(s) or nucleotide(s) at smaller scales (e.g., μmol) as at larger scales (e.g., mmol), or vice versa (i.e., transfer of conditions from larger to smaller scales), since the process uses the same linear liquid flow rates at various steps. Thus, the process can be optimized on a small scale before scaling up or down, all saving time, reagents, solvents, and reducing waste. The process can produce similar or identical results, e.g., yield and purity, in the scaled version as well as in the scale where the parameters were optimized. For a process to be linearly scalable, it will be understood by one of ordinary skill in the art that not all process parameters (e.g., pressure, temperature, etc.) and not all synthesis components (e.g., additives, solvents, etc.) must have a linear relationship between different synthesis scales.

[0078] In a second aspect of the invention, there is a system 100 for flow-through solid phase oligonucleotide synthesis. The system 100 includes at least one column 101 arranged to be filled with synthesis resin, at least a first pump 102a, at least a first reservoir 104a for solvents and reagents, at least one detector 106, and at least one processor 114. A tube 108 connects the first reservoir 104a to the column 101. The first pump 102a and valves 109a-d are arranged to direct flow within the system 100. The processor 114 is in communication with at least the first pump 102a, the valves 109a-d, and the detector 106. The system 100 is arranged to provide a fluidization channel 200 arranged to direct a flow of solvent from the first reservoir 104a to the bottom 101' of the column and to pass the solvent through the column 101 from the bottom 101' to the top 101'' of the column, and a recharge channel 210 arranged to direct a flow of solvent from the first reservoir 104a to the top 101'' of the column and to pass the solvent through the column 101 from the top 101'' to the bottom 101'. The system 100 is arranged to first pass the solvent through the fluidization channel 200 and subsequently pass the solvent through the recharge channel 210 during one synthesis cycle. Such a system is shown diagrammatically in FIG. 2a.

[0079] In one embodiment, a third valve 109c is positioned to provide a waste outlet, i.e., drain waste from the system 100, when solvent passes from the bottom 101' to the top 101'' of the column. Similarly, a fourth valve 109d is positioned to provide a waste outlet, i.e., drain waste from the system 100, when solvent passes from the top 101'' to the bottom 101' of the column.

[0080] In one embodiment, as shown in FIG. 2a, the second valve 109b is positioned downstream of the first reservoir 104a and upstream of the column 101. The second valve 109b may be positioned to direct flow into the column 101 from either the top 101′ or the bottom 101″. In this manner, the second valve 109b can facilitate the fluidization channel 200 and the recharge channel 210 by directing the flow.

[0081] In one embodiment, as shown in Fig. 2a, the first valve 109a is disposed downstream of the first reservoir 104a and upstream of the first pump 102a. In such a configuration, the first valve 109a may be disposed to select the type of liquid entering the system 100 and the first pump 102a from the first reservoir 104a, particularly if the first reservoir 104a is realized by multiple reservoirs. In other embodiments, the functionality of selecting the type of liquid is realized by the first reservoir 104a, such that the first pump 109a is realized by or incorporated in the first reservoir 104a.

[0082] In one embodiment, the system 100 is arranged to provide a recirculation flow path 220. The recirculation flow path 220 is arranged to recirculate the solvent(s), reagents, etc., on the column 101 and through the system 100. Such a system 100 is shown diagrammatically in FIG. 2b. In other embodiments, the recirculation flow path 220 may be realized by the tube 108 and / or the fluidization flow path 200 and the recharge flow path 210. The second pump 102b may be arranged in fluid communication with the recirculation flow path 220, as shown in FIG. 2b. In such a configuration, the second pump 102b may be arranged to regulate the flow in the recirculation flow path 220.

[0083] The components used in the system 100 according to the present invention are standard components used in the art and therefore well known to those skilled in the art.

[0084] The column 101 may be any type of column, such as a glass column, a column made from stainless steel, etc. The column 101 is arranged substantially straight in the direction of gravity within the system 100, such that the column top 101'' faces substantially upward (towards the sky) and the column bottom 101' faces substantially downward (towards the earth). In this manner, liquid flows from the top of the column 101'' to the bottom 101' in the direction of gravity. The column 101 is arranged to be filled with a synthetic resin.

[0085] The system 100 may include two or more reservoirs 104a, such as two reservoirs (first 104a and second 104b) as shown in Figures 2-4. In such a case, the first reservoir 104a may include the solvent(s), the detritylation agent(s), and the capping agent(s), and the second reservoir 104b may include the solvent(s), the sequence unit or nucleic acid(s), and the capping agent(s), or vice versa, or in any other combination.

[0086] The system 100 may include two or more pumps 102a, such as two or more pumps, a first pump 102a and a second pump 102b, as shown in Figures 2-4. Each pump 102a, 102b may be in fluid communication with at least one of the reservoirs 104(s) in the system 100, with the first pump 102a disposed downstream and in fluid communication with the first reservoir 104a and the second pump 102b disposed downstream and in fluid communication with the second reservoir 104b, as shown in Figures 2-4. The pump(s) 102 may be any device that can be used to drive the flow of liquid in the system 100, such as hydraulic pump(s), pneumatic pump(s), etc.

[0087] In one embodiment, the fluidization channel 200 and the recharge channel 210 are realized by the tube 108, so that no extra tubes are required. Alternatively, the system 100 is arranged to use the tube 108 to first flow liquid from the bottom 101' of the column to the top 101'' of the column or in a countercurrent direction, and subsequently flow liquid from the top 101' to the bottom 101'' of the column. Additionally, the recirculation channel 220 can also be realized by the tube 108. Such a system is shown diagrammatically in FIG. 3.

[0088] The system 100 may further include a column bypass 112 disposed in fluid communication with the tube 108 and the column 101. The column bypass 112 is shown diagrammatically in the system 100 in FIG. 4. A solvent inlet and a waste outlet may be disposed in fluid communication with the column 101 and the fluid bypass 112. The solvent inlet may be disposed upstream of the column 101 and the waste outlet may be disposed downstream of the column 101, or vice versa. The column bypass 112 may be configured to recirculate liquid(s) in the system 100 without passing through the column 101, for example, during activation, washing, etc.

[0089] The system 100 shown in any of Figures 2-4 may further include pressure sensor(s) 111a; 111b disposed downstream and in fluid communication with the reservoir(s) 104a; 104b. The system 100 may further include flow diverting valves 120a; 120b in fluid communication with the first pump 102a and the second pump 102b. The flow diverting valves 120a; 120b may be positioned to direct flow into or out of the respective pumps 102a; 102b. The flow diverting valves 120a; 120b may be rotary valves, or solenoid valves, or another suitable type of valve.

[0090] The pressure may vary during the synthesis process due to, for example, different flow rates, expansion of resins, use of particular solvents, size of equipment components such as tubing, etc. The liquid flow rates are arranged to be adjusted and regulated during use of the system 100 by pumps 102a; 102b in communication with the processor 114. Regulation can be based on pressure (in addition to other regulation parameters) such that the pressure within the system 100 is kept within predetermined limits. Such predetermined limits will depend on the process and components of the system 100 and should be determined by one of ordinary skill in the art.

[0091] The system 100 may further comprise filters, for example in the form of inlet filter(s) 121 and in-line filter(s) 122. The inlet 121 and in-line 122 filters may be arranged as shown diagrammatically in FIG. 4, although other arrangements are possible. The inlet filter 121 may be arranged at the tip(s) of the inlet(s) to the system 100 to prevent undesired particles and the like from entering the system 100. The in-line filter(s) 122 may be arranged to filter out undesired components, such as precipitates formed during the process. The in-line filter 122 may be arranged, for example, upstream or downstream of the pump(s) 102a; 102b, or in the column bypass 112, or in any other suitable location of the system 100.

[0092] Valves 109a-f may be any type of valve that one of skill in the art would deem suitable for a flow-through oligonucleotide synthesis system, for example, rotary valves, or solenoid valves, or another suitable type of valve.

[0093] The valves 109a-f and / or flow diverter valves 120a-b in the system 100 are positioned to direct flow in the system 100 and are in fluid communication with the tube 108 and the reservoir(s) 104a; 104b. The valves 109a-f and / or flow diverter valves 120a-b are controlled by and in communicative communication with the processor 114. The valves 109a-f and / or flow diverter valves 120a-b in the system 100 are positioned to be automated and controlled by the processor 114. The valves 109a-f and / or flow diverter valves 120a-b can be adjusted in response to feedback from a monitor to the system 100. Such adjustments can be provided by the processor 114, for example, in a computer in communication with the system 100.

[0094] The detector 106 may be any type of detector, such as a UV / Vis detector, a fluorescence detector, a NIR detector, etc. In one embodiment, the detector 106 is a spectroscopic detector, preferably a NIR detector. The detector 106 is disposed downstream of the column 101. The detector 106 is in communicative communication with the processor 114. The system 100 may include two or more detectors 106.

[0095] The system 100 can include more than one column 101, for example at least two columns 101 that can be connected in parallel. The use of more than one column 101 facilitates automated sequential synthesis of other sequences, synthesis at different scales, optimization of parameters for process development, and the like.

[0096] Although two reservoir(s) 104a; 104b are shown in Figures 2-4, it should be understood that multiple reservoirs, e.g., each containing a different type of solvent, sequence unit, monomer, nucleic acid(s), different drugs, reagents, etc., can be used instead. It should further be understood that different components of system 100, such as detector 106, bubble detector 107, pumps 102a; 102b, temperature device 103, etc., can be located at different locations in system 100. Note that Figures 2-4 merely show some examples of possible configurations of components in system 100.

[0097] In one embodiment of the present invention, the system 100 further comprises: - a temperature regulation device 103 arranged upstream of the column 101 and downstream of at least the first reservoir 104a; - a first temperature sensor 105a; a second temperature sensor 105b; Includes.

[0098] As shown in FIGS. 2 to 4, the first temperature sensor 105a is disposed upstream of the column 101 and downstream of the temperature adjustment device 103, and the second temperature sensor 105b is disposed downstream of the column 101.

[0099] The temperature regulation device 103 can be arranged to heat or cool, or both, liquids, liquid mixtures or synthetic liquid mixtures etc. in the system 100. The liquids may include solvent(s), and / or reagents, and / or nucleic acid(s), and / or capping agents etc. The temperature regulation device 103 is arranged upstream of the column 101 and downstream of the reservoirs 104a; 104b to allow the liquids to be heated before they reach the column 101.

[0100] System 100 may further include one or more flow paths (not shown) realized by tubing arranged to flow liquids having different temperatures through system 100 and onto column 101. Such flow paths are in fluid communication with at least temperature regulator 103, column 101, and reservoir(s) 104a-b.

[0101] In one embodiment of the invention, the system 100 further comprises a temperature regulator bypass 123, as shown generally in Figure 4. The temperature regulator bypass 123 is disposed in fluid communication with the temperature regulator 103. The temperature regulator bypass 123 may be disposed to bypass the composition fluid from the temperature regulator 103 such that the composition fluid does not pass through the temperature regulator 103.

[0102] In one embodiment of the present invention, the system 100 further includes a separately defined mixing chamber 113 disposed in fluid communication with at least the first reservoir 104a, and an air bubble detector 107 disposed upstream of the separately defined mixing chamber 113, as shown in Figures 2-4.

[0103] The separately defined mixing chamber 113 can be used to activate the nucleoside(s) or nucleotide(s) in the system 100. It is disposed in fluid communication with at least the first reservoir 104a such that the system 100 can be configured to flow the nucleoside(s) or nucleotide(s) and at least one activating agent(s) from the at least the first reservoir 104a into the separately defined mixing chamber 113 by opening at least the first valve 109a and the fifth valve 109e.

[0104] The air bubble detector 107 may be positioned upstream of the separately defined mixing chamber 113. The air bubble detector 107 is in communicative communication with the processor 114. The air bubble detector 107 may be positioned to detect the presence of an air bubble as an indication that the mixing chamber is approaching or has been emptied. The air bubble detector 107 may be positioned upstream of the separately defined mixing chamber 113 and downstream of a second fifth valve 109e′ that is in fluid communication with the remainder of the system 100.

[0105] In one embodiment of the invention, detector 106 is selected from, for example, a UV / Vis photodetector, a fluorescence detector, and a NIR detector. System 100 may be configured to continuously monitor the progress of the oligonucleotide synthesis process or to monitor particular reaction steps, such as at particular time points, during use of system 100.

[0106] In a third aspect, there is a method for performing flow-through solid-phase oligonucleotide synthesis 300 using a system 100 for flow-through solid-phase oligonucleotide synthesis. The system 100 includes at least one column 101 arranged to be filled with a synthesis resin, at least a first pump 102a, at least a first reservoir 104a for monomers and reagents, at least one detector 106, and at least one processor 114, with a tube 108 connecting the first reservoir 104a to the column 101. The first pump 102a and valves 109a-d are arranged to direct the flow in the system 100. The processor 114 is in communication with at least the first pump 102a, the valves 109a-d, and the detector 114. A synthesis resin is placed in the column 101, and a first sequence unit is attached to the synthesis resin via a chemical bond. The method 300 includes at least one step of reconsolidation 303, during which the system 100 is configured to first fluidize the synthetic resin disposed within the column 101 by flowing a solvent through the fluidization channel 200 from the bottom 101' to the top 101'' of the column in a fluidization step 303'. Following the fluidization step 303', a recharge step 303'' is performed in which the system 100 uses the recharge channel 210 to flow a solvent from the top 101'' to the bottom 101' of the column to recharge the synthetic resin disposed within the column 101.

[0107] In one embodiment of the present invention, there is a method 300 for performing flow-through solid-phase oligonucleotide synthesis using the system for flow-through oligonucleotide synthesis 100. The method 300 includes the following steps: - Detritylation step 301: the system 100 is arranged to flow an acidic solution containing a detritylation agent from the first reservoir 104a through the tube 108 into the column 101 by opening at least the first valve 109a, the second valve 109b and the third valve 109c; - a first wash step 302: the system 100 is arranged to flow the solvent from the first reservoir 104a through the tube 108 to the column 101 by opening at least the first valve 109a, the second valve 109b and the third valve 109c; - a first reconsolidation step 303: the system 100 is configured to initially fluidize the synthetic resin placed in the column 101 by flowing a solvent through the fluidization channel 200 from the bottom 101' to the top 101'' of the column in a fluidization step 303'. Following the fluidization step 303', a recharging step 303'' is performed in which the system 100 uses the recharging channel 210 to flow a solvent from the top 101'' to the bottom 101' of the column to recharge the synthetic resin placed in the column 101; - reacting step 320: the system 100 is arranged to flow at least one sequence unit from the first reservoir 104a through the tube 108 to the column 101 by opening at least the first valve 109a, the second valve 109b and the third valve 109c. The second part of the reacting step 320 is a second washing step 305, in which the system 100 is arranged to flow the solvent from the first reservoir 104a through the tube 108 to the column 101 by opening at least the first valve 109a, the second valve 109b and the third valve 109c; - an oxidation / thiolation step 330: the system 100 is arranged to flow the oxidizing or thiolation agent from the first reservoir 104a through the tube 108 to the column 101 by opening at least the first valve 109a, the second valve 109b and the third valve 109c. The first part of the oxidation / thiolation is followed by a third wash step 307, in which the system 100 is arranged to flow the solvent from the first reservoir 104a through the tube 108 to the column 101 by opening at least the first valve 109a, the second valve 109b and the third valve 109c; - Full capping step 309: the system 100 is arranged to flow capping agent from the first reservoir 104a through the tube 108 to the column 101 by opening at least the first valve 109a, the second valve 109b and the third valve 109c. A first part of the capping step 309 is followed by a fourth wash step 307, in which the system 100 is arranged to flow solvent from the first reservoir 104a through the tube 108 to the column 101 by opening at least the first valve 109a, the second valve 109b and the third valve 109c.

[0108] As noted above, the system 100 may have more than one reservoir 104a, such as two reservoirs 104a; 104b, as shown in Figures 2-4. In such cases, the first reservoir 104a may be replaced with the second reservoir 104b (or vice versa) in any step of the method 300 described herein.

[0109] In one embodiment, a second re-compacting step is performed after capping 340 (or after the fourth washing step 309). Additional re-compacting steps can be performed at any time in the method 300 according to the invention, for example after any of the washing steps 302; 305; 307; 309.

[0110] In one embodiment of the invention, the flow rate during the fluidization step 303' is between 25 and 75 cm / h and the fluidization step 303' lasts for 0.25 to 0.75, or 0.25 to 1, or more column volumes. In one embodiment of the invention, the flow rate during the refilling step 303'' is between 100 and 200 cm / h and the refilling step 303'' lasts for 0.25 to 0.75, or 0.25 to 1, or more column volumes.

[0111] The flow rate and time of the re-compacting step 303 depends on the type of reaction and / or the desired result and / or the size (length and diameter) or volume and type of column 101, etc. In the method 300 according to the present invention, the system 100 is configured to perform the re-compacting step 303 for a predetermined period of time using a predetermined flow rate provided by the processor 114 determined based on the above parameters.

[0112] In one embodiment of the present invention, the progress of the method 300 is monitored by at least one detector 106 positioned downstream of the column 101 .

[0113] In one embodiment of the present invention, the method 300 includes an activation step prior to the reaction step 320. In the activation step, the system 100 is configured to activate at least one nucleotide or nucleoside by providing an activating agent to at least one sequence unit. The activation can be performed in the tube 108 of the system 100 or in a separately defined mixing chamber 113. The separately defined mixing chamber 113 is disposed in fluid communication with the first reservoir 104a. In one embodiment, the system 100 is configured to monitor the departure of the activated at least one sequence unit from the separately defined mixing chamber 113 using an air bubble detector 107 disposed upstream of the separately defined mixing chamber 113.

[0114] In one embodiment of the invention, the method 300 includes an additional step prior to the reacting step 320, in which the solution containing at least one second sequence unit, or nucleotide or nucleoside, is heated before entering the column 101. In the heating step, the system 100 is arranged to flow the solution containing at least one second sequence unit, or nucleotide or nucleoside, through a temperature adjustment device 103. The temperature adjustment device 103 is arranged upstream of the column 101 and downstream of the first reservoir 104a.

[0115] A typical flow-through oligonucleotide synthesis using the system 100 according to the present invention is shown in the block scheme of Figure 5 and in the flow chart of Figure 1a. As mentioned before, the synthesis can be divided into the following steps: - Detritylation 301 The synthesis is started by arranging the system 100 to provide at least one detritylation agent from the first reservoir 104a to the column 101, using at least the first valve 109a and the first pump 102a. After the detritylation step 301 is finished, for example as determined by a conditional threshold detected by the detector 106, a solvent is passed through the system 100 to wash away all residues from the detritylation step 301. The system 100 is configured to provide a solvent from the first reservoir 104a (or the second reservoir 104b) by using at least the first valve 109a and the first pump 102a. After the first washing step 302 is finished, for example as determined by a conditional threshold detected by the detector 106, a recompaction step 303 follows.

[0116] - Re-compaction 303 In the re-compaction step 303, the solvent is first passed countercurrently through the column 101, i.e. from the bottom 101'' to the top 101' of the column. The solvent is passed countercurrently through the column 101, i.e. from the top 101' to the bottom 101'' of the column, until the synthetic resin or resin bed is at least partially fluidized, for example 90%, or 95%, or 99% fluidized, or until at least 1% of the resin is not in contact with the bottom of the column 101''. The synthetic resin is fluidized when it is not in contact with the bottom 101'' of the column, and therefore the resin is fluidized when there is a space between the synthetic resin and the bottom 101'' of the column that is not filled with resin (for example a gap of more than 1%). Thus, until a fluidized bed is arranged in the column 101. In other words, in the re-compacting step 303, the system 100 is arranged to provide solvent from the first reservoir 104a to the column 101 using first the fluidization channel 200 and then the refill channel 210. After the re-compacting step 303 is finished, the resin is uniformly packed into the column 101. If a transparent column 101 is used in the system 100, this can be observed visually.

[0117] - Reaction step 320 The reaction step comprises a coupling step 304 followed by a second washing step 305. In the coupling step 304, the system 100 is arranged to pass at least one sequence unit, or nucleoside or nucleotide, from the second reservoir 104b to the column 101 by using at least the seventh valve 109g and the second pump 102b. After the coupling step 304 is finished, as determined by a conditional threshold detected, for example, by the detector 106, a second washing step 305 follows. During the second washing step 305, a solvent passes through the system 100 in order to wash away all residues from the coupling 304. The system 100 is configured to provide a solvent from the second reservoir 104a (or the first reservoir 104a) by using at least the seventh valve 109g (or the first valve 109a) and the second pump 102b (or the first pump 102a). After the second washing step 305 is finished, for example as determined by a conditional threshold detected by the detector 106, an oxidation step 330 follows. The coupling step 304 can be repeated one or several times such that at least one sequence unit, or nucleotide or nucleoside, can be recirculated through the column 101.

[0118] - Oxidation / thiolation step 330 The oxidation or thiolation step 330 follows the coupling step 320. The system 100 is configured to provide an oxidizing / thiolation agent from the first reservoir 104a to the column 101 by using at least the first valve 109a and the first pump 102a. After the oxidation step 306 is finished, for example as determined by a conditional threshold detected by the detector 106, a solvent passes through the system 100 to wash away all residues from the oxidation in a third washing step 307. The system 100 is configured to provide a solvent from the first reservoir 104a (or the second reservoir 104b) by using at least the first valve 109a (or the seventh valve 109g) and the first pump 102a (or the second pump 102b). After the third washing step 307 is finished, for example as determined by a conditional threshold detected by the detector 106, a capping step 340 follows.

[0119] - Capping 340 In the capping step 340, the system 100 is configured to provide a capping agent from the second reservoir 104b to the column 101 by using at least the seventh valve 109g and the second pump 102b. After the capping step 308 is finished, for example as determined by a conditional threshold detected by the detector 106, a solvent passes through the system 100 in a fourth wash step 309 to wash away any residue from the capping step 308. The system 100 is configured to provide a solvent from the first reservoir 104a (or the second reservoir 104b) by using at least the first valve 109a (or the seventh valve 109g) and the first pump 102a (or the second pump 102b).

[0120] In one embodiment of the present invention, the reaction step 320 is preceded by an activation step. During the activation step, the system 100 is configured to activate at least one nucleoside or nucleotide by providing an activating agent. In one embodiment, the system 100 is configured to activate at least one sequence unit, or nucleoside or nucleotide, in the tube 108 of the system 100 by simultaneously flowing the activating agent and the at least one sequence unit, or nucleoside or nucleotide through the tube 108 of the system 100. In one embodiment, the system 100 is configured to activate at least one sequence unit, or nucleoside or nucleotide, in a separately defined mixing chamber 113, and such activation can be monitored by a detector. Already activated sequence units, or nucleoside(s) or nucleoside(s) can also be used in the method 300 according to the present invention, in which case an activation step is not necessary.

[0121] The present invention is not limited to the above-mentioned embodiments. Various alternatives, modifications, and equivalents can be used. Therefore, the above-mentioned embodiments should not be interpreted as limiting the scope of the present invention defined by the appended claims. Moreover, all embodiments, aspects, and examples can be combined with each other unless otherwise specified. EXAMPLES

[0122] HPLC analytical method and conditions. Column: Phenomenex Aeris Peptide XB-C18, 2.6 μm, 100 Å, 150 × 2.1 mm. Mobile phase buffer A: 100 mM hexylammonium acetate, pH 7, buffer B: 50% acetonitrile in A. Gradient: 0% B for 4 min, 0-30% B in 2 min, 30-80% B in 30 min, 80-100% B in 2 min, 100% B for 2 min. Flow rate: 0.25 ml / min. Column temperature: 50 °C. UV absorbance was recorded at 260 nm.

[0123] Example 1: Recompaction before the coupling step Oligonucleotide synthesis was carried out using standard conditions for the phosphoramidite synthesis of the Test-13 oligonucleotide sequence (SL Beaucage, MH Caruthers, Tetrahedron Lett, Vol 22, Issue 20, pp 1859-1862, 1981) using commercially available polystyrene / DVB crosslinked resin pre-derivatized with T. A recompaction (fluidization and repacking) step was performed prior to the coupling step.

[0124] Specific conditions: - 1.25 equivalents of "amidite" - Coupling time 2 minutes result: - Total yield based on weight gain (after thorough drying), 84%. - Total yield (based on partial cleavage from resin and deprotection, A260 units), 77%. - Purity by HPLC: 84%. - The HPLC chromatogram of the obtained sequence is shown in Figure 6a.

[0125] Example 2: Carried out without reconsolidation (Comparative Example) Oligonucleotide synthesis was carried out using standard conditions for phosphoramidite synthesis of the Test-13 oligonucleotide sequence (SL Beaucage, MH Caruthers, Tetrahedron Lett, Vol 22, Issue 20, pp 1859-1862, 1981) using commercially available polystyrene / DVB crosslinked resin pre-derivatized with T. No recompaction step was performed prior to the coupling step.

[0126] Specific conditions: - 1.25 equivalents of "amidite" - Coupling time 2 minutes result: - Total yield based on weight gain (after thorough drying), 66%. - Purity by HPLC: 84%. - The HPLC chromatogram of the obtained sequence is shown in Figure 6b.

Claims

1. 1. A system (100) for flow-through solid phase oligonucleotide synthesis, said system (100) comprising: At least one column (101) arranged to be filled with a synthetic resin; at least a first pump (102a); at least a first reservoir (104a) for monomers and reagents; at least one detector (106); at least one processor (114); Including, A tube (108) connects the first reservoir (104a) to the column (101); the first pump (102a) and valves (109a-d) are arranged to direct flow within the system (100); the processor (114) is in communication with at least the first pump (102a), the valves (109a-d), and the detector (114); the system (100) is arranged to provide a fluidisation channel (200) arranged to direct a flow of solvent from the first reservoir (104a) to the bottom (101′) of the column so that the solvent passes through the column (101) from the bottom (101′) to the top (101″) of the column, the system (100) is arranged to provide a recharge path (210) arranged to direct a flow of solvent from the first reservoir (104a) to the top (101'') of the column (101) so that the solvent passes through the column (101) from the top (101'') to the bottom (101') of the column; The system (100) is configured to first flow a solvent through the fluidization channel (200) and then flow a solvent through the recharge channel (210) in one synthesis cycle.

2. the third valve (109c) is arranged to provide a waste outlet when solvent passes from the bottom (101') of the column to the top (101''); 2. The system (100) of claim 1, wherein the fourth valve (109d) is positioned to provide a waste outlet when solvent passes from the top (101'') to the bottom (101') of the column.

3. The system (100) of claim 1 or 2, wherein the fluidization channel (200) and the recharge channel (210) are realized by the tube (108).

4. 4. The system (100) of claim 1, further configured to provide a recirculation flow path (220), the recirculation flow path (220) being configured to recirculate solvents and reagents over the column (101).

5. The system (100) comprises: a temperature control device (103) arranged upstream of the column (101) and downstream of the at least first reservoir (104a); a first temperature sensor (105a) disposed upstream of the column (101) and downstream of the temperature control device (103); a second temperature sensor (105b) located downstream of the column (101); The system (100) of any one of claims 1 to 3, comprising:

6. The system (100) comprises: a separately defined mixing chamber (113) disposed in fluid communication with said at least first reservoir (104a); an air bubble detector (107) located upstream of said separately defined mixing chamber (113); The system (100) of any one of claims 1 to 3, further comprising:

7. A method for performing flow-through solid-phase oligonucleotide synthesis (300) using the system (100) of any one of claims 1 to 3, wherein a synthetic resin is disposed in the column (101), a first sequence unit is attached to the synthetic resin via a chemical bond, and the method (300) comprises: the system (100) causing a solvent to flow from the bottom (101′) of the column to the top (101″) through the fluidization channel (200) in a fluidization step (303′). the system (100) is configured to initially fluidize the synthetic resin disposed in the column (101) by a re-compaction flow path (210) and, following the fluidization step (303′), a re-packing step (303″) is performed in which the system (100) uses the re-packing flow path (210) to flow a solvent from the top (101″) of the column to the bottom (101′) to re-pack the synthetic resin disposed in the column (101).

8. 8. The method (300) of claim 7, wherein the method (300) comprises the following steps: Detritylation step (301): The system 100 is configured to flow an acidic solution containing a detritylation agent from the first reservoir (104a) through the tube (108) into the column (101) by opening at least a first valve (109a), a second valve (109b), and a third valve (109c); First washing step (302): the system (100) is configured to allow solvent to flow from the first reservoir (104a) through the tube (108) to the column (101) by opening at least the first valve (109a), the second valve (109b), and the third valve (109c); First re-compaction step (303): the system (100) is configured to initially fluidize the synthetic resin disposed in the column (101) by flowing a solvent from the bottom (101') to the top (101'') of the column through the fluidization channel (200) in a fluidization step (303'), and following the fluidization step (303'), the system (100) performs a re-packing step (303'') in which the system (100) uses the re-packing channel (210) to flow a solvent from the top (101'') to the bottom (101') of the column to re-pack the synthetic resin disposed in the column (101); Reaction step (320): the system (100) is configured to flow at least one sequence unit from the first reservoir (104a) to the column (101) through the tube (108) by opening at least the first valve (109a), the second valve (109b), and the third valve (109c), followed by a second washing step (305) in which the system (100) is configured to flow a solvent from the first reservoir (104a) to the column (101) through the tube (108) by opening at least the first valve (109a), the second valve (109b), and the third valve (109c); Oxidation / thiolation step (330): the system (100) is configured to allow an oxidizing agent or a thiolation agent to flow from the first reservoir (104a) to the column (101) through the tube (108) by opening at least the first valve (109a), the second valve (109b), and the third valve (109c), followed by a third washing step (307), in which the system (100) is configured to allow a solvent to flow from the first reservoir (104a) to the column (101) through the tube (108) by opening at least the first valve (109a), the second valve (109b), and the third valve (109c); Capping step (309): the system (100) is configured to allow a capping agent to flow from the first reservoir (104a) to the column (101) through the tube (108) by opening at least the first valve (109a), the second valve (109b), and the third valve (109c), followed by a fourth washing step (307), in which the system (100) is configured to allow a solvent to flow from the first reservoir (104a) to the column (101) through the tube (108) by opening at least the first valve (109a), the second valve (109b), and the third valve (109c).

9. 9. The method (300) according to claim 7 or 8, wherein the progress of the synthesis carried out according to the method (300) is continuously monitored by at least one detector (106) arranged downstream of the column (101).

10. The process described in claim 9, wherein the process uses software-controlled real-time conditional monitoring that enables the use of process analytical engineering (PAT) to measure critical process parameters (CPPs) that affect critical quality attributes (CQAs).

11. The flow rate during the fluidization step (303') is 25 to 75 cm / h; the fluidization step (303') lasts for 0.25 to 1 column volume; the flow rate during the refilling step (303'') is between 100 and 200 cm / h; the repacking step (303'') lasts for 0.25 to 1 column volume; The method (300) according to any one of claims 7 to 10.

12. 11. The method (300) of any one of claims 7 to 10, wherein the method (300) comprises an activation step prior to the reacting step (320), and during the activation step, the system (100) is configured to activate the at least one sequence unit by providing an activating agent to the at least one sequence unit.

13. 13. The method of claim 12, wherein the system (100) is configured to activate the at least one alignment unit in the tube (108) of the system (100) by simultaneously flowing the activator and the at least one alignment unit through the tube (108) of the system (100).

14. 13. The method (300) of claim 12, wherein the at least one array unit is activated in a separately defined mixing chamber (113).

15. 15. The method (300) of claim 14, wherein the activated at least one array unit exiting the separately defined mixing chamber (113) is monitored by an air bubble detector (107).

16. 11. The method (300) of any one of claims 7 to 10, wherein the method (300) comprises an additional step before the reacting step (320), wherein the solution containing at least one second sequence unit is heated before entering the column (101), and wherein the heating step is configured such that the system (100) passes the solution containing the at least one second sequence unit through a temperature control device (103) located upstream of the column (101).

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