Wash after deblock in solid phase oligonucleotide synthesis

Abstract

Aspects of the disclosure relate to methods and systems for solid phase oligonucleotide synthesis (SPOS). In some embodiments, the methods described by the disclosure comprise systems and methods related to washing after deblocking during solid phase oligonucleotide synthesis. In some embodiments, used deblocking solution is combined with a used washing solution and distilled to provide a distillate wash solution that can be used in a later washing step. In some embodiments, wash solutions are heated or pyridine is added for use during countercurrent washing procedures to increase efficiency. In some embodiments, the methods are combined.

Description

[0001] WASH AFTER DEBLOCK IN SOLID PHASE OLIGONUCLEOTIDE SYNTHESIS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. 119(e) of U.S. provisional application serial number 63 / 711,359, filed October 24, 2024, entitled “WASH AFTER DEBLOCK IN SOLID PHASE OLIGONUCLEOTIDE SYNTHESIS”, the entire content of which are incorporated by reference herein.

[0004] BACKGROUND

[0005] Solid Phase Oligonucleotide Synthesis (SPOS) is a technique commonly used to synthesize oligonucleotides. In SPOS, a solid-phase medium is used to facilitate the sequential addition of nucleotides to the growing oligonucleotide bound to the solid-phase medium. The solid-phase medium is typically a solid support made of controlled pore glass (CPG) or macroporous polystyrene (MPPS) spheres, although other solid-phase mediums are known (e.g., PAM resins). This technique allows for the incorporation of a variety of nucleosides and nucleotides, including various nucleoside derivatives, the most common of which are phosphoramidites.

[0006] SUMMARY

[0007] Aspects of the disclosure relate to methods and systems for performing SPOS. The disclosure is based, in part, on modifications to post-deblocking (also referred to as detritylation) wash steps during SPOS, for example distillation of used deblocking solutions and washes after deblocking for recycling as wash solvents, including pyridine in the multi-stage countercurrent washing process, and heating of post-deblocking wash solutions. In some embodiments, the modified post-deblocking wash methods and systems are advantageous over previously employed deblocking wash steps, for example by requiring less solvent (e.g., ACN, toluene, etc.), and producing oligonucleotides at a higher purity and yield. In some embodiments, deblocking methods described by the disclosure are carried out on a fluidized bed reactor (FBR).

[0008] Accordingly, in some aspects, the disclosure provides a system for synthesizing oligonucleotides, the system comprising a feed zone vessel; a reactor in fluid communication with the feed zone vessel; a distillation unit; and a distillate feed vessel in fluid communication with the feed zone vessel; and a wash module comprising one or more wash vessels in fluid communication with the distillate feed vessel. In some embodiments, a distillation unit is in fluid communication with a reactor. In some embodiments, a distillation unit is offline relative to a reactor. In some embodiments, a distillation unit is configured to fill distillate into a feed vessel that feeds the reactor.

[0009] In some embodiments, the system further comprises a heat exchanger operatively associated with an inlet of the reactor. In some embodiments, the system further comprises a heat exchanger operatively associated with an outlet of the reactor. In some embodiments, the feed zone vessel is configured to be heated.

[0010] In some embodiments, the wash module comprises a plurality of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) wash vessels, each wash vessel in fluid communication with the reactor and / or the feed zone vessel. In some embodiments, the wash vessels are arranged in a multi-stage countercurrent arrangement.

[0011] In some embodiments, the system further comprises an acetonitrile (ACN) feed vessel in fluid communication with the feed zone vessel.

[0012] In some embodiments, the distillation unit comprises an evaporator. In some embodiments, the distillation unit comprises a rectification section. In some embodiments, a rectification section is a column with packing or trays, between a boiling evaporator and a condenser, where vapor is repeatedly vaporized and condensed to enrich the vapor with more volatile substances, via liquid refluxing back down the column.

[0013] In some embodiments, the system further comprises one or more pumps.

[0014] In some embodiments, the system used pressure differentials to drive fluid movement without the use of pumps.

[0015] In some aspects, the disclosure provides a method for recycling wash solution during the wash after a solid phase oligonucleotide synthesis (SPOS) deblocking step, the method comprising contacting an oligonucleotide linked to a solid support in a reactor with deblocking solution introduced into the reactor; moving the deblocking solution and solvent wash solution after deblocking from the reactor to a distillation unit; moving a first wash solution through the reactor to the distillation unit; operating the distillation unit to produce a distillate wash solution; moving the distillate wash solution to an integrated wash solution vessel of a wash module or the feed zone or reactor; performing one or more washes by moving one or more wash solutions from wash vessels of the wash module to the reactor and contacting the oligonucleotide on the solid support; and moving the distillate wash solution to the reactor and contacting the oligonucleotide linked to the solid support with the distillate wash solution. In some embodiments, the deblocking solution consists essentially of DCA and toluene prior to contacting the oligonucleotide. In some embodiments, the reused deblocking solution comprises acetonitrile (ACN), toluene, DCA (or TCA or TFA), and trityl byproducts after contacting the oligonucleotide.

[0016] In some embodiments, the distillation unit comprises an evaporator and / or a rectification section.

[0017] In some embodiments, the distillate wash solution comprises ACN, toluene, and DCA (or TCA or TFA). In some embodiments, the distillate wash solution comprises less than 25 uM DCA (or TCA or TFA). In some embodiments, the distillate wash solution comprises no more than 20 uM DCA.

[0018] In some embodiments, the wash module comprises a plurality of (e.g., 2, 3, 4, 5, 6, or more) wash solution vessels in a countercurrent-like arrangement (e.g., the 6th wash on one cycle becomes the 5th wash on the next cycle, and so on, as described further below).

[0019] In some embodiments, the method further comprises moving the distillate wash solution to a wash solution vessel of the wash module after contacting the oligonucleotide.

[0020] In some embodiments, the method further comprises moving a volume of ACN to the reactor and contacting the oligonucleotide linked to the solid substrate. In some embodiments, the method further comprises moving the ACN to the wash solution vessel containing the distillate wash solution after contacting the oligonucleotide linked to the solid substrate.

[0021] In some embodiments, the distillate wash solution and / or the one or more wash solutions from the wash solution vessels are heated prior to contacting the oligonucleotide linked to the solid substrate. In some embodiments, the heated distillate wash solution and the one or more heated wash solutions have a temperature ranging from about 30 °C to about 80 °C.

[0022] In some embodiments, the oligonucleotide is contacted with the wash solution after deblocking under flow-through conditions or fluidized conditions.

[0023] In some embodiments, the oligonucleotide is contacted with the distillate wash solution under flow-through conditions or fluidized conditions.

[0024] In some embodiments, the distillate wash solution comprises about 60-80% ACN and about 20-40% toluene by volume.

[0025] In some aspects, the disclosure provide a method for washing during a solid phase oligonucleotide synthesis (SPOS) deblocking step, the method comprising: contacting an oligonucleotide linked to a solid support in a reactor with deblocking solution introduced into the reactor; and performing one or more countercurrent washes by moving one or more wash solutions from wash vessels of a wash module to the reactor and contacting the oligonucleotide on the solid support, wherein the one or more wash solutions are heated prior to contacting the oligonucleotide linked to the solid support.

[0026] In some embodiments, one or more wash solutions are heated to a temperature ranging from about 30 °C to about 80 °C.

[0027] In some embodiments, deblocking solution consists essentially of DCA (or TCA or TFA) and toluene prior to contacting the oligonucleotide. In some embodiments, deblocking solution comprises acetonitrile (ACN), toluene, DCA (or TCA or TFA), and trityl byproducts after contacting the oligonucleotide.

[0028] In some embodiments, one or more of the wash solutions comprises pyridine.

[0029] In some embodiments, the method further comprises moving a fresh ACN solution lacking pyridine from a vessel to the reactor and contacting the oligonucleotide on the solid support; and moving the fresh ACN solution to a first integrated wash solution vessel of the wash module.

[0030] In some embodiments, the wash module comprises six or more wash solution vessels in a multi-stage countercurrent arrangement. In some embodiments, the wash module comprises five or fewer wash solution vessels in a multi-stage countercurrent arrangement.

[0031] In some embodiments, the oligonucleotide is contacted with the deblocking solution under flow-through conditions. In some embodiments, the oligonucleotide is contacted with the deblocking solution under fluidized conditions. In some embodiments, the oligonucleotide is contacted with the wash solution under flow-through conditions. In some embodiments, the oligonucleotide is contacted with the wash solution under fluidized conditions.

[0032] In some embodiments, the wash solution comprises about 60-80% ACN and about 20- 40% toluene by volume. In some embodiments, the wash solution comprises about 75% ACN and about 25% toluene by volume, or about 70% ACN and about 30% toluene by volume.

[0033] In some aspects, the disclosure provides a method for washing during a solid phase oligonucleotide synthesis (SPOS) deblocking step, the method comprising: contacting an oligonucleotide linked to a solid support in a reactor with deblocking solution introduced into the reactor; and performing one or more countercurrent washes by moving one or more wash solutions from wash vessels of a wash module to the reactor and contacting the oligonucleotide on the solid support, wherein the one or more wash solutions comprise pyridine. In some embodiments, one or more wash solutions comprise about 1% pyridine.

[0034] In some embodiments, deblocking solution consists essentially of DCA and toluene prior to contacting the oligonucleotide.

[0035] In some embodiments, deblocking solution comprises acetonitrile (ACN), toluene, DCA, and trityl byproducts after contacting the oligonucleotide.

[0036] In some embodiments, the wash module comprises six or more wash solution vessels in a multistage countercurrent arrangement. In some embodiments, the wash module comprises five or fewer wash solution vessels in a multistage countercurrent arrangement.

[0037] In some embodiments, one or more wash solutions from the wash solution vessels are heated prior to contacting the oligonucleotide linked to the solid substrate.

[0038] In some embodiments, heated distillate wash solution and one or more heated wash solutions have a temperature ranging from about 30 °C to about 80 °C.

[0039] In some embodiments, the oligonucleotide is contacted with the deblocking solution under flow-through conditions. In some embodiments, the oligonucleotide is contacted with the wash solution under flow-through conditions.

[0040] In some embodiments, wash solution comprises about 70-80% ACN and about 20-30% toluene by volume. In some embodiments, wash solution comprises about 75% ACN and about 25% toluene by volume.

[0041] BRIEF DESCRIPTION OF DRAWINGS

[0042] FIG. 1 shows a representative process flow diagram for a fluidized bed reactor (FBR), according to some aspects of the technology.

[0043] FIG. 2 shows a representative process flow diagram a wash module of a FBR system.

[0044] FIG. 3 shows a representative process flow diagram for three embodiments of heated washing in a FBR system.

[0045] FIG. 4 shows DCA adsorption isotherm of SS-36 at 20°C (green dashed line) and desorption isotherms of SS-36 at 20°C (green line) and 45°C (purple line) equilibrated for one hour.

[0046] FIG. 5 shows HPLC UV chromatograms of 36 mer sense strand synthesis using 10.1 L / mmol of ACN at 20°C (blue line) or 45°C (green line), and 15.7 L / mmol of ACN at 20°C (pink line) or 45°C (brown line) for post-detritylation wash.

[0047] FIG. 6 shows representative data comparing three FBR SPOS reactions. FIG. 7 shows a representative schematic diagram for a FBR configured for material reuse, according to some aspects of the technology.

[0048] FIG. 8 shows a representative schematic diagram for a FBR configured for acetonitrile (ACN) reuse, according to some aspects of the technology.

[0049] FIG. 9 shows a representative schematic diagram for a FBR comprising a distillation unit, according to some aspects of the technology.

[0050] FIG. 10 shows a representative schematic diagram for a FBR configured for no material reuse, according to some aspects of the technology.

[0051] FIG. 11 shows a representative process flow diagram for a fully fluidized, reuse SPOS process in an FBR, according to some aspects of the technology.

[0052] DETAILED DESCRIPTION

[0053] Aspects of the disclosure relate to methods and systems for solid phase oligonucleotide synthesis (SPOS). In the SPOS process, there are generally four chemical reactions that occur in order to add a single phosphoramidite to the chain.

[0054] The first step is the “deblocking” step, which is generally a detritylation reaction. In some embodiments, a nucleotide (e.g., a nucleotide attached to a solid support, for example a resin) has its 5'-hydroxyl group protected by an acid-labile protection group such as the DMT (4,4'-dimethoxytrityl). This protection group may be removed during a continuous flow of an acid solution or via an addition of an acid in a solvent (also referred to as a “deblocking solution”). In some embodiments, the deblocking solution comprises trichloroacetic acid (TCA) or dichloroacetic acid (DC A). In some embodiments, the acid (e.g., TCA, DC A, etc.) is carried in an inert solvent such as toluene, dichloromethane, or another suitable solvent. The concentration or amount of acid in a deblocking solution may vary. In some embodiments, a deblocking solution comprises between 2% and 30% acid. In some embodiments, a deblocking solution comprises 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28, or 30% DCA in toluene. The amount of deblocking solution used during each deblocking reaction of SPOS may vary. In some embodiments, the amount of deblocking solution used in each deblocking reaction ranges from about 50-500 ml / mmol. In some embodiments, during this “deblocking” reaction, an orange-colored DMT cation is formed and is washed out continually during the flow-through reaction because the reagent solution is flowing through the resin, and then subsequently after the flow-through reaction is completed by using wash solvents. Accordingly, this step results in the solid support-bound oligonucleotide precursor bearing a free 5'-terminal hydroxyl group. In some embodiments, the deblocking step of SPOS is performed under flow-through conditions on the packed resin bed, for example as described further herein in the section entitled “Fluidized Bed Reactor”.

[0055] Once the de-blocking step is complete, a “coupling” step is then performed. In some embodiments, a coupling reaction involves contacting the solid support-bound oligonucleotide precursor with a solution of activated phosphoramidite in a solvent. In some embodiments an activated phosphoramidite is dissolved in a solution comprising acetonitrile (ACN) (e.g., anhydrous ACN) and ACN plus toluene to help the solubility of some phosphoramidites. In some embodiments, the phosphoramidite is dissolved at a concentration ranging between 0.02- 0.2 M, for example 0.02 M, 0.05 M, 0.1 M, 0.13 M, 0.16 M, 0.19 M, or 0.2 M, in the solvent (e.g., ACN). The amount of coupling solution used during each coupling reaction of SPOS may vary. In some embodiments, the amount of coupling solution used in each coupling reaction ranges from about 1 to 3 molar equivalents of activated phosphoramidite versus the active sites on the stationary phase. This activated phosphoramidite will react with and couple to the free 5’- terminal hydroxyl group that was previously de-protected. Generally, as is known in the art, the solution of phosphoramidite may be “activated” by the addition of a stoichiometric activating agent that facilitates the coupling reaction. Various activating agents are known to “activate” the phosphoramidite including various azole or imidazole compounds. More than one equivalent of the activating agent is often used, as the acidic nature of the activating agent helps to neutralize the diisopropylamine by-product formed in the coupling. In some embodiments, the coupling step of SPOS is performed under fluidization of the packed resin bed, for example as described further herein in the section entitled “Fluidized Bed Reactor”. Upon the completion of the coupling, some unbound reagents and by-products are removed by washing, and the rest are neutralized during the subsequent oxidation / thiolation step.

[0056] After the coupling step, the next steps in the SPOS are oxidation / thiolation (thiolation is also referred to as “sulfurization”), and “capping”. Capping is performed because a small percentage of the solid support-bound 5'-OH groups (0.1 to 1% or greater) of the oligonucleotide being synthesized remains unreacted and needs to be blocked from further chain elongation to prevent the formation of oligonucleotides with an internal base deletion, commonly referred to as (n-1) deletions. In some embodiments, the unreacted 5'-hydroxy groups are acetylated by the capping mixture, which allows for easier separation of shortmers from the desired product after the oligonucleotides are separated from the solid support, for example by chromatography. Likewise, if other, non-desired products (such as a reaction of an O in the guanosine base or other chemical entities) are created during the coupling step, these non-desired products are also blocked (e.g., capped) from reacting further so that they may be more readily separated out in the subsequent purification steps. In some embodiments, the capping step involves treating the solid support-bound material with a mixture of acetic anhydride and 1 -methylimidazole. The concentration or amount of capping solution (e.g., acetic anhydride and 1 -methylimidazole solution) in a capping solution may vary. In some embodiments, a combined capping solution comprises between 2-20 vol% 1 -Methylimidazole (typically 10 vol%), 2-20 vol% acetic anhydride (typically 10 vol%), 5-30 vol% 2,6-lutidine (typically 15 vol%). The amount of capping solution used during each capping reaction of SPOS may vary. In some embodiments, the amount of capping solution used in each capping reaction ranges from about 5-50 ml / mmol. Other capping reagents may also be used. In some embodiments, the capping step of SPOS is performed under fluidization of the packed resin bed, for example as described further herein in the section entitled “Fluidized Bed Reactor”.

[0057] In some embodiments, an oxidation step is performed. In the oxidation step, the coupled phosphoramidite that reacted to the 5 ’-terminal OH group results in a phosphite triester linkage (e.g., in which the P atom of a phosphate backbone is in an oxidation state of +3). This phosphite triester linkage is not natural and is of limited stability under the conditions of oligonucleotide synthesis and use. Thus, the P atom will be oxidized to a more stable +5 oxidation state via the addition of oxidizers such as iodine and water in the presence of a weak base (e.g., pyridine, lutidine, or collidine). This reaction oxidizes the phosphite triester into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester internucleosidic linkage. Oxidation may be carried out under anhydrous conditions using tert-Butyl hydroperoxide or (lS)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In other embodiments, sulfurization to a phosphothiolate linker is done instead of oxidation. ). In some embodiments, an oxidizer solution comprises iodine and pyridine. In some embodiments, a sulfurization solution comprises xanthane hydride in pyridine. The concentration or amount of oxidizer or sulfurizing agent in an oxidation solution or sulfurization solution may vary. In some embodiments, an oxidization solution comprises between 0.01 to 0.1 M iodine in pyridine / water (typically 0.05 M). The amount of oxidizing solution used during each oxidization reaction of SPOS may vary. In some embodiments, the amount of oxidizing solution used in each oxidization reaction ranges from about 2 to 10 molar equivalents. In some embodiments, a sulfurization solution comprises between 0.1 -0.3 M xanthane hydride (typically 0.2 M) in pyridine / ACN. The amount of sulfurization solution used during each sulfurization reaction of SPOS may vary. In some embodiments, the amount of sulfurization solution used in each sulfurization reaction ranges from about 2 to 10 molar equivalents. Those skilled in the art will appreciate that some embodiments of SPOS may be best designed in which the capping step occurs after this oxidation or sulfurization step, or vice versa. Also, those skilled in the art will appreciate that some embodiments of SPOS may be best designed in which the capping step is omitted from some of the cycles, when high conversion is anticipated.

[0058] Once these four steps are completed (de-blocking, coupling, either oxidation or sulfurization, and capping), the phosphoramidite building block has been added to the growing chain. As will be appreciated, the phosphoramidite building block that was coupled has its own DMT protecting group that is protecting the 5’ -terminal OH group. Thus, the process may then be repeated and another phosphoramidite moiety added until the chain reaches its desired length. Once the chain has reached its desired length the oligonucleotide protecting groups can be removed and the oligonucleotide can be cleaved from the resin and released into solution. In some cases, these protecting groups from the nucleoside amines and the 2-cyanoethyl phosphate protecting groups are globally deprotected in the same base catalyzed hydrolytic cleavage reaction. Aqueous ammonia solutions, mixtures of ammonia and methylamine and others are commonly used for this cleavage / deprotection step. These conditions also efficiently hydrolyze the 3 ’-linker and cleave the oligonucleotide from the resin. In some embodiments the 2- cyanoethyl phosphate protecting groups are first deprotected using DEA.

[0059] In some embodiments, one or more washing steps occurs between of the four SPOS steps. A cycle is defined as the detritylation, coupling, oxidation / thiolation, and capping sequences along with their associated washes. In other words, one nucleotide is added per 4-step “cycle”. In some embodiments, an SPOS cycle comprises one or more washes between deblocking and coupling; one or more washes between coupling and oxidation / sulfurization; one or more washes between oxidation / sulfurization and capping; and, one or more washes between capping and the first step of the next SPOS cycle. In some embodiments, a wash step comprises a multi-stage counter-current wash process. In some embodiments, a bed reactor (e.g., a FBR as described herein) comprises six (6) vessels with wash solvent used for integrated multi-pass washing after deblocking. The first wash step after deblocking is to use the solvent from a first vessel (e.g., vessel “A”) to wash the resin and push to waste. The next step is to use the solvent from a second vessel (e.g., vessel “B”) to push through the resin and push back to refill the first vessel (“A”). Then the solvent from a third vessel “vessel “C”) washes the resin in the reactor and pushes out to refill the second vessel (“B”), and the process continues with the fourth (“D”), fifth (“E”), and sixth (“F”) vessels. After all six wash vessels are used, fresh solvent wash is used to push through the resin and push back to refill the sixth vessel (“F”). FIG. 2 shows a representative process flow diagram a wash module of a FBR system.

[0060] In some embodiments, wash solvent after coupling is only reused for the wash after oxidation or thiolation in the same cycle (e.g., a single nucleotide addition). In some embodiments, wash solvent after capping is only reused for the subsequent wash after capping on the next cycle, not for washing after any other chemistry. In some embodiments, the same wash solvent is used for multiple SPOS cycles. In some embodiments, wash solvent after a deblocking step is only reused for washing after subsequent deblocking steps in the next cycles, and not for washing after any other SPOS steps (e.g., coupling, oxidation / sulfurization, capping). In some embodiments, acetonitrile (ACN) is used for washing the reactor (and the solid substrate, such as resin, contained therein) after the deblocking, coupling, and capping steps.

[0061] The length of an oligonucleotide produced by the SPOS methods and systems described by the specification may vary. In some embodiments, the oligonucleotide ranges from about 3 nucleotides in length to about 100 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

[0062] 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,

[0063] 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,

[0064] 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,

[0065] 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length). In some embodiments, the oligonucleotide is greater than 100 nucleotides in length (e.g., at least 100, 200, 300, etc.). In some embodiments, the oligonucleotide ranges from about 10 nucleotides in length to about 40 nucleotides in length. In some embodiments, the oligonucleotide comprises an oligonucleotide sense strand. In some embodiments, the oligonucleotide comprises an oligonucleotide antisense strand.

[0066] The amount of oligonucleotide produced by SPOS methods and systems described herein may vary. In some embodiments, an SPOS method or system described herein produces between 0.1 to 2.0 mol of oligonucleotide product. In some embodiments, SPOS methods described herein produce between 1 to 20 kg of oligonucleotide product.

[0067] While SPOS is the most popular method of oligonucleotide synthesis, there are many challenges that impede the scale up of oligonucleotides from development to large scale manufacturing. For example, the yield and purity of oligonucleotides generally decreases with increasing strand length due to increasing steric hindrance after the addition of each nucleotide. Additionally, the cost of production of oligonucleotides is significantly greater than not only small molecules, but also peptides, which have much cheaper and abundant starting materials and solvents. The synthesis of oligonucleotides via SPOS requires complex phosphoramidite nucleotides and large quantities of expensive solvents and reagents such as acid solutions (e.g., dichloroacetic acid (DC A) solutions) and acetonitrile (ACN). Furthermore, currently employed SPOS systems and methods are not generally efficient. Kilotons of solvents and starting materials are required per kilogram of oligonucleotide produced.

[0068] Aspects of the disclosure relate to methods and systems for improving efficiency, purity, and yield of SPOS reactions and washes. The disclosure is based, in part, on SPOS methods that comprise modifications to capping reactions and wash after capping. In some embodiments, the modified capping reactions comprise recycling and / or reuse of capping solutions during multiple SPOS cycles, for example using pre-used capping solution on solid substrate (e.g., resin) prior to contacting the resin with new capping solution. The inventors have appreciated and recognized that recycling and reusing capping solutions and washes results in a significant reduction in the amount of capping solutions and ACN used during SPOS (e.g., relative to SPOS without capping solution recycling or SPOS performed on packed bed reactors (PBR)) without sacrificing purity or yield of the oligonucleotides produced using such processes.

[0069] Fluidized Bed Reactor

[0070] Aspects of the disclosure relate to modifications of a fluidized bed reactor (FBR) system for synthesizing oligonucleotides. FBR systems are generally known and are described for example in International Application Serial Number PCT / US2021 / 063185, published on June 23, 2022, as WO2022 / 132681, the entire contents of which are herein incorporated by reference. In some embodiments, SPOS comprising capping and wash steps as described by the disclosure is carried out on a fluidized bed reactor (FBR). The FBR platform generally allows for the use of two distinct modes of operations during the SPOS cycle: flow-through and fluidization. During steps in which there is fluidization of the packed resin bed, the solid substrate (e.g., resin) and reagent liquid are thoroughly mixed to form a slurry. Thorough mixing during fluidization, i.e., slurry formation, can be accomplished by a variety of methods such as mechanical mixing (e.g., stirring or shaking), flowing a liquid up and down through the solid substrate, or moving a gas through the mixture (e.g., bubbling). When gas is used for fluidization, the gas (e.g., nitrogen) may be introduced at a rate to ensure complete fluidization while minimizing the amount of solid substrate (e.g., resin) that splashes up on the reactor walls. The liquid from the reactor empties out the bottom filter at the end of each fluidization step, which results in the solid substrate bed (e.g., resin bed) settling back to an unfluidized packed stage. In some embodiments, fluidization is the preferred operating mode for coupling, oxidation / thiolation, and capping reaction steps of SPOS. During flow-through (e.g., liquid flow down through a packed solid substrate bed, e.g., resin bed) operation, the liquid (e.g., reaction and wash solvents / reagents) enters the reactor through a spray nozzle located above the solid substrate, or other method / device, so that it evenly distributes across the solid substrate to keep the solid substrate flat. The introduced liquid in the reactor then flows down through the solid substrate- flow through the packed solid substrate is similar to what is common in the commercial, unfluidized packed bed reactors (PBR) typically used for SPOS. As compared to a PBR, the FBR can be operated such that the fluid solutions of each step can be removed from the FBR before the next step in either flow-through or fluidization mode. In some embodiments, a combination of fluidization and flow-through is the preferred operating mode for capping and wash steps described by the disclosure.

[0071] Turning to the figures, FIG. 1 shows a representative diagram for a fluidized bed reactor (FBR), according to some aspects of the technology. In FIG. 1, small boxes coded by numbers or letters represent material feed vessels (e.g., 1 to 27) or synthesizer operation vessels (e.g., 29 and 30). As used herein, a “feed vessel” refers to a container of suitable volume and geometry for containing a solution used in SPOS. A feed vessel may be made of any suitable material, for example glass, polymers, plastic, metal, etc. In some embodiments, a feed vessel is a carboy. The vessel contents or intended purpose of use are annotated on the diagram. The amidite solutions are contained separately in feed vessels labeled 1-11 and are moved from the vessels into the reactor feed zone by mechanical pumps or by pressure transfers. In some embodiments, a FBR comprises between 1 and 20 amidite feed vessels (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 feed vessels) that are in fluid communication with the feed zone vessel (28 and or 29), which is in turn in fluid communication with a reactor (30) (also referred to herein as a “bed reactor”). As used herein, “in fluid communication” means fluid flow between two regions (e.g., vessels) via a passageway (e.g., a tube or pipe, such as a feed line) connecting the two regions. In some embodiments, two regions (e.g., vessels) are in direct fluid communication with each other, for example when flow of the fluid between the two regions is unobstructed. In some embodiments, two regions (e.g., vessels) are in indirect fluid communication with each other, for example when flow of the fluid between the two region is controlled by an obstruction, for example a valve, disposed in the passageway between the two regions. Generally, ACN (19), toluene (14), and DCA and toluene (e.g., deblocking solution; 13 and 14) are fed from feed vessels to the feed zone vessel (29) or the reactor (30) via pressure push and controlled with automated flow control valves. In some embodiments, a FBR comprises an activator feed vessel (12) in fluid communication with the feed zone vessel (29) or the reactor (30). In some embodiments, the activator feed vessel (12) comprises ethylthiotetrazole (ETT) in ACN.

[0072] In some embodiments, a FBR comprises a deblocking module comprising a reactor (30), a feed zone vessel (29), a fresh acid solution (e.g., DCA) vessel (I3A), a neat acid solution (e.g., neat DCA) vessel 13B, and a reuse DCA vessel (21). The fresh acid solution vessel (13A), reuse acid solution vessel (21), and the neat acid solution vessel (13B) are connected to the feed zone vessel (29). In some embodiments, the reactor (30) is in fluid communication with the reuse acid solution vessel (21). The system may further comprise an evaporator (31) in fluid communication with the reactor (30) and the reuse acid solution vessel (21). In some embodiments of acid recycling methods described herein, the deprotection solution for the first cycle of an SPOS process comprises only of fresh DCA in toluene, whereas for the subsequent steps of deblocking as a part of nucleotide addition, both fresh deprotection solution and the reused deprotection solution from the previous cycle is used for each step of deblocking. In some embodiments, neat DCA (or high concentration DCA) is added and mixed in with the reuse deblocking solution, before the solution pushes into the reactor (e.g., the neat DCA and reuse deblocking solution are mixed in the feed zone vessel (29) prior to being charged to the reactor (30). The amount or concentration of neat acid solution (e.g., neat DCA or high concentration DCA) added to the reuse deblocking solution may vary. In some embodiments, between about l%-20% v / v neat DCA are added to the reuse acid solution prior to charging to the reactor. The purpose of neat acid add-back is to reduce overall process volumes. Because the DCA concentration in the reuse DCA solution is higher after adding neat DCA, less fresh DCA solution is needed to complete the reaction. In some embodiments, when the reuse acid solution pushes out of the reactor, it does not go to waste; rather, it goes to an evaporator. The purpose of the evaporator is to recover a portion of the solvent can so that it can be used for solvent washing after detritylation in the next cycle, and / or to recover

[0073] DCA solution to be reused in additional detritylation steps on other cycles.

[0074] In some embodiments, the deblocking solution for every step of deblocking is used in excess (e.g., from about 50:1 to 100:1 stoichiometry versus the oligonucleotide on solid substrate) to drive the reaction rapidly to completion with almost complete conversion to the 5'- hydroxyl group of the oligonucleotide.

[0075] In some embodiments, a FBR comprises a coupling module comprising one or more amidite feed vessels (e.g., (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), and (11) in FIG. 1), an activator feed vessel (12), an acetonitrile (ACN) feed vessel (19), and an amidite feed zone vessel (28). In some embodiments, the one or more amidite feed vessels are in fluid communication with the amidite feed zone vessel (28), which in fluid communication with the reactor (30). In some embodiments, the activator feed vessel (12) is in fluid communication with the amidite feed zone vessel (28), which is in fluid communication with the reactor (30). The ACN feed vessel (19) may be in fluid communication with both the amidite feed zone vessel (28) and / or the feed zone vessel (29) and / or the reactor (30). In some embodiments, the coupling module is configured to pump or push the specified amidite into the amidite feed zone vessel

[0076] (28) and chase it in with nitrogen, then pump or push activator solution from the activator feed vessel (12) into the amidite feed vessel (28) and chase in with nitrogen, fluidizing the two together by introducing nitrogen gas into the bottom of the amidite feed vessel (28). In some embodiments, the coupling module is configured to push this mixture into the feed zone vessel

[0077] (29), and then into the reactor (30) to start the coupling reaction on the resin. In some embodiments, the coupling module is configured to push this mixture from the amidite feed vessel (28) into the reactor (30) to start the coupling reaction on the resin. In some embodiments, the coupling module is configured to operate under fluidization conditions.

[0078] In some embodiments, a FBR comprises an oxidation / sulfurization module comprising one or more oxidizer feed vessels (15) and / or one or more sulfurization feed vessels (16). In some embodiments, the one or more oxidizer feed vessel (15) is in fluid communication with a feed zone vessel (29). In some embodiments, the one or more sulfurization feed vessels (16) is in fluid communication with a feed zone vessel (29). In some embodiments, the one or more oxidizer feed vessels (15) comprise an oxidizer solution comprising iodine and pyridine+water. The amount of oxidizer (e.g., iodine) in an oxidizer solution may vary. In some embodiments, the amount of iodine charged to the reactor with an oxidizer solution ranges from about 1.0 equivalents and 10 equivalents (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 equivalents). In some embodiments, the concentration of iodine in an oxidizer solution ranges from about 0.01 M to about 0.1 M (e.g., about 0.01. 0.02. 0.03. 0.04. 0.05. 0.06. 0.07. 0.08. 0.09. 0.1. M). In some embodiments, the one or more sulfurization feed vessels (16) comprises xanthane hydride in pyridine. The amount of sulfurizing agent (e.g., xanthane hydride) in a sulfurization solution may vary. In some embodiments, the amount of xanthane hydride charged to the reactor with a sulfurization solution ranges from about 1.0 equivalents and 10 equivalents (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 equivalents). In some embodiments, the concentration of xanthane hydride in a sulfurization solution ranges from about 0.02 M to about 2 M (e.g., about 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 1.8, 1.9, or 2 M). In some embodiments, the oxidation / sulfurization module further comprises an oxidation solution reuse vessel (23), which is in fluid communication with the feed zone (29). In some embodiments, the oxidation solution reuse vessel (23) is configured to store oxidation solution used during an oxidation reaction. In some embodiments, an oxidation / sulfurization module further comprises a reactor (30) in fluid communication with the feed zone vessel (29). In some embodiments, the oxidation solution feed vessel (15) and the oxidation solution reuse vessel (23) in fluid communication. In some embodiments, the oxidation solution reuse vessel (23) is in fluid communication with a waste module. In some embodiments, the oxidation / sulfurization module is configured to operate under fluidization conditions.

[0079] In some embodiments, a FBR comprises a capping module comprising one or more capping solution feed vessels (17, 18). In some embodiments, each of the one or more capping solution feed vessels (17) and (18) is in fluid communication with a feed zone vessel (29). In some embodiments, a capping module further comprises a capping solution reuse vessel (25). In some embodiments, the capping solution reuse vessel (25) is in fluid communication with the reactor (30). In some embodiments, the capping solution reuse vessel (25) is in fluid communication with the feed zone vessel (29). In some embodiments, a capping module further comprises a vessel with reuse wash solvent that is used to dilute the capping reaction on the next cycle (26), which is in fluid communication with the reactor (30) and the feed zone vessel (29). In some embodiments, a capping module further comprises a reuse wash solution vessel (27). In some embodiments, the reuse wash vessel (27) is in fluid communication with the reactor (30) and the feed zone vessel (29). In some embodiments, the capping module is configured to work under fluidization conditions. Capping modules are further described herein in the section entitled “Capping Solution Reuse”.

[0080] In some embodiments, a FBR comprises one or more wash modules. In some embodiments, a wash module comprises six (6) wash vessels (22A, 22B, 22C, 22D, 22E, 22F) containing wash solvent used for integrated multi-pass washing after deblocking. In some embodiments, each of the six wash vessels, 22A-22F is in fluid communication with both the feed zone vessel (29) and the reactor (30). In some embodiments, each of the six wash vessels, 22A-22F is in fluid communication with the wash vessel immediately adjacent to it. For example, in some embodiments, the solvent from the first vessel (22A) is used to wash the resin in the reactor (30) and pushed to waste; the solvent from the second vessel (22B) is pushed through the resin in the reactor (30) and the pushed back to refill the first vessel (22A); the solvent from the third vessel (22C) the washes the resin in the reactor (30) and is pushed out to refill the second vessel (22B); and the process continues with the fourth (22D), fifth (22E), and sixth (22F) vessels. After each of the six wash vessels are used, fresh solvent wash (e.g., from an ACN feed vessel, (19)) is pushed through the reactor (30) and then pushed back to refill the sixth vessel (22F). Importantly, the FBR designs described herein allow for the use of multistage counter-current wash to minimize solvent use without compromising product quality. In some embodiments, the multi-stage wash with ACN is employed after deprotection, prior to the coupling reaction. In some embodiments, the first six wash steps of ACN used in wash cycles after every detritylation reaction come from wash integration tanks. In some embodiments, after integration wash is complete, the final wash uses the fresh ACN solvent. In some embodiments, a wash module is used exclusively for washing after the detritylation step of SPOS, and not used with any other step in the SPOS cycle.

[0081] In some embodiments, a FBR further comprises an evaporator (31) in fluid communication with the reactor (30). In some embodiments, the evaporator (31) is configured to recover a portion of (or all) solvent from the acid solution (e.g., deblocking solution) so that it can be used for solvent washing after deblocking in the next cycles of SPOS. In some embodiments, the system further comprises a distillate vessel (32) in fluid communication with the evaporator (31) and the feed zone vessel (29). In some embodiments, the portion of solvents recovered from the deblocking solution via the evaporator (31) is pushed to the distillate vessel (32). In some embodiments, the distillate from the distillate vessel is used to fill wash vessel 22F during the last wash step after a deblocking reaction.

[0082] FBR systems comprising capping modules as described herein provide several advantages over previously described fluidized bed reactors and packed bed reactors (PBR) used for SPOS. For example, FBR systems described herein offer higher scalability, flexible batch size, and at least an order of magnitude larger maximum scale.

[0083] In some embodiments, use of FBR systems described by the disclosure for SPOS results in higher crude purity and yield of oligonucleotides.

[0084] In some embodiments, use of FBR systems described by the disclosure for SPOS reduces ACN solvent relative to previously described SPOS reactions using FBR or PBRs.

[0085] In some embodiments, use of FBR systems described by the disclosure for SPOS reduces reduce DCA and toluene used for deblocking by at least a factor of two relative to synthesizers that do not reuse reagents.

[0086] In some embodiments, use of FBR systems described by the disclosure for SPOS allow for better manufacturing control, for example the ability to sample solid substrate (e.g., resin) any time and obtain a representative sample because there is no difference from top to bottom of the resin bed, relative to PBRs.

[0087] In some embodiments, use of FBR systems described by the disclosure for SPOS are more amenable to different types of resins, with different loading, different swelling and shrinking properties, relative to PBRs. This enables higher swelling and higher loading resins. It also gives the possibility of keeping the resin in the reactor and reloading in situ.

[0088] In some embodiments, use of FBR systems described by the disclosure for SPOS allows for use of less equivalents of special amidites relative to PBRs because the systems can charge 100% of the feed tank make-up.

[0089] In some embodiments, use of FBR systems described by the disclosure for SPOS produce less waste than previously described SPOS reactions using FBR or PBRs. In some embodiments, the systems and methods significantly reduce ACN and recycle DCA for deblocking.

[0090] In some embodiments, use of FBR systems described by the disclosure for SPOS produce higher batch to batch consistency relative to PBRs. Capping Solution Reuse

[0091] Aspects of the disclosure relate to FBRs comprising a modified capping module. In some embodiments, the capping solution recycling methods described herein significantly reduce the amount of capping solution and acetonitrile (ACN) needed to perform synthesis of an entire oligonucleotide using SPOS. In the embodiments, the systems and methods reduce the amount of capping solution used during SPOS by between about 2-fold and about 3-fold relative to previously described SPOS reactions using FBR or PBRs. In the embodiments, the systems and methods reduce the amount of ACN used during SPOS by between about 2-fold and about 6- fold relative to previously described SPOS reactions using FBR or PBRs. In the embodiments, the systems and methods reduce the amount of ACN used during SPOS by at least 2-, at least 3-, at least 4-, at least 5-, at least 6-fold relative to previously described SPOS reactions using FBR or PBRs.

[0092] Aspects of the disclosure relate to capping modules configured to reuse a capping reaction solution from the previous cycle at the start of the capping process on the current cycle. FIG. 1 depicts a capping module as described by the disclosure, which comprises one or more capping solution feed vessels. In some embodiments, the first capping solution feed vessel (17) comprises methylimidizole and ACN. The concentration of methylimidizole in a capping solution may vary. In some embodiments, a capping solution comprises between 4 / 96 v / v and 20 / 80 v / v 1 -Methylimidazole / ACN . In some embodiments, the second capping solution feed vessel (18) comprises acetic anhydride, lutidine, and ACN. The concentration of acetic anhydride in a capping solution may vary. In some embodiments, a capping solution comprises between 2 vol% and 40 vol% acetic anhydride. The concentration of lutidine in a capping solution may vary. In some embodiments, a capping solution comprises between 6 vol% and 60 vol% lutidine. Each of the one or more capping solution feed vessels (17) and (18) is connected to a feed zone vessel (29). In some embodiments, the feed zone vessel is connected to a reactor (30).

[0093] In some embodiments, a capping module further comprises a capping solution reuse vessel (25). In some embodiments, the capping solution reuse vessel (25) is connected to the reactor (30) and / or the feed zone vessel (29). In some embodiments, the capping solution reuse vessel is configured to receive a capping solution mixture from the reactor (30) and push the capping solution mixture to the feed zone vessel (29). In some embodiments, a capping module further comprises a vessel with reused wash solvent that is used to dilute a subsequent capping reaction (26), which is connected to the reactor (30) and / or the feed zone vessel (29). In some embodiments, the reuse vessel (26) is configured to receive wash solvent from the reactor (30) and push it into the feed zone vessel (29) for mixing with new capping solutions from the first (17) and second (18) capping solution feed vessels. In some embodiments, the capping solution mixture is pushed to the reactor (30).

[0094] In some embodiments, a capping module further comprises a reuse wash vessel (27). In some embodiments, the reuse wash vessel (27) is connected to the reactor (30) and / or the feed zone vessel (29). In some embodiments, the reuse wash vessel is configured to receive a wash solution (e.g., comprising ACN) from the reactor (30) and push the wash solution to the feed zone vessel (29). In some embodiments, one or more vessels of a wash module are configured to be heated. Examples of techniques for heating wash solutions include using heat exchangers on the inlet or outlet of a reaction vessel, and / or heating the reaction vessel itself. FIG. 3 shows a representative process flow diagram for three embodiments of heated washing in a FBR system. The following is an illustrative example describing the function of capping modules for capping reagent recycling. The process begins during a non-first SPOS cycle (e.g., 2nd, 3rd, 4th, etc. SPOS cycle) with pushing material from the capping solution reuse vessel (25) through the resin in the reactor (30) and out to a waste module. In some embodiments, this step is performed under flow-through conditions. This accomplishes two goals. The primary goal is to rinse out and scavenge any residual water that might still be associated with the solid substrate (e.g., resin) prior to the subsequent capping reaction. Second, it may also accomplish a portion of the subsequent capping reaction. Next, fresh capping solutions from capping solution feed vessel (17) and capping solution feed vessel (18) are pushed to the feed zone vessel. Solvent from feed vessel (26) is also pushed to the feed zone vessel (29) and mixed with the fresh capping solutions to form a capping solution mixture. This is done because the fresh capping solution reaction is intended to be fluidized, but the reagent volume of only the fresh capping solutions from capping solution feed vessel (17) and capping solution feed vessel (18) is insufficient to fluidize the solid substrate (e.g., resin) in the reactor (30). Thus, it is diluted with reuse wash solvent, which comes from vessel (26), rather than using fresh solvent from the ACN feed vessel (19), which is how previously-described FBRs functioned. Thus, this method reduces solvent and reduces waste. At the end of the designated fluidization time, the used capping solution mixture is pushed out of the reactor (30) and collected in vessel 25. Next, a wash solution from the reuse wash vessel (27) is pushed through the reactor (30) via the feed zone vessel (29) to remove residual capping solution mixture. In some embodiments, the integrated wash solution is contacted to the oligonucleotide on the solid substrate (e.g., resin) in the reactor (30) under flow- through conditions. The used integrated wash solution is then pushed from the reactor (30) to the vessel 26, rather than pushing to the waste module. In the final step, any excess capping solution or capping solution mixture is removed from the resin in the reactor (30) by rinsing the support with fresh ACN solvent, which is pushed from the ACN feed vessel (19) to the reactor (30) and pushed through the solid support under flow-through conditions.

[0095] Aspects of the disclosure relate to wash solutions comprising pyridine. In some embodiments, pyridine is present in one or more wash solutions used in countercurrent-like wash steps. In some embodiments, methods described herein further comprise a step of a pyridine wash. In some embodiments, the pyridine wash is performed after the multi-stage countercurrent wash steps. In some embodiments, the pyridine wash is transferred to a final countercurrent wash vessel (e.g., a countercurrent wash vessel that is farthest away from the reactor in the sequence) from when it exits the reactor. In some embodiments, the methods further comprise a fresh solvent (e.g., ACN that does not comprise pyridine) wash step after the pyridine wash. In some embodiments, the fresh solvent wash pushes through the reactor and chases the residual pyridine to the final countercurrent wash vessel when it exits the reactor. Thus, in some embodiments, all of the countercurrent wash stages for the wash after a first deblock step contain pyridine.

[0096] In some embodiments, an FBR machine is configured to reuse acetonitrile (ACN). FIG. 8 shows a representative schematic diagram for a FBR configured for ACN reuse, according to some aspects of the technology. In some embodiments, an FBR configured for ACN reuse comprises one or more (e.g., 1, 2, 3, 4, 5, 6, or more) vessels configured to contain ACN which has been used in one or more SPOS cycles and / or one or more SPOS wash steps. In some embodiments, the one or more vessels is in fluid communication with a feed vessel.

[0097] Aspects of the disclosure relate to FBR machines, and methods of using FBR machines, comprising a distillation unit. A distillation unit may be offline (e.g., not in fluid communication) with respect to a reactor or FBR machine, or inline (e.g., in fluid communication) with respect to a reactor or FBR machine). FIG. 9 shows a representative schematic diagram for a FBR comprising a distillation unit, according to some aspects of the technology. In some embodiments, the distillation bottoms is used as part of the deblocking process.

[0098] No Reuse SPOS Processes

[0099] The skilled person will understand that, in some embodiments, an SPOS process may be performed with or without reuse of materials or reagents, according to some aspects of the disclosure. Aspects of the disclosure relate to FBR machines, and methods of using FBR machines that do not reuse any reagents or materials during SPOS cycles. FIG. 10 shows a representative schematic diagram for a FBR configured for no material reuse, according to some aspects of the technology. In some embodiments, no reuse SPOS processes are fully fluidized. In some embodiments, no reuse SPOS processes reduce plant footprint. In some embodiments, no reuse SPOS processes reduce cycle time. In some embodiments, no reuse FBR processes produce oligonucleotides at higher purity, higher yield, and better scalability relative to previously described SPOS processes, for example the standard PBR process.

[0100] Fully fluidized SPOS in FBR

[0101] Aspects of the disclosure relate to fully fluidized SPOS processes in FBRs. In some aspects, the disclosure provides a method for solid phase oligonucleotide synthesis (SPOS), the method comprising: during a first cycle of solid phase oligonucleotide synthesis (SPOS), contacting a first phosphoramidite monomer linked to a solid support in a fluidized bed reactor with a deblocking solution comprising dichloroacetic acid (DCA) or trichloroacetic acid (TCA) or trifluoroacetic acid (TFA) under fluidizing conditions; moving the deblocking solution from the fluidized bed reactor to a waste vessel via a filter located at the bottom of the fluidized bed reactor; contacting the solid support with a coupling solution under fluidizing conditions; moving the coupling solution from the fluidized bed reactor to a waste vessel via a filter located at the bottom of the fluidized bed reactor; contacting the solid support with an oxidizing solution under fluidizing conditions; moving the oxidizing solution from the fluidized bed reactor to a waste vessel via a filter located at the bottom of the fluidized bed reactor; contacting the solid support with a capping solution under fluidizing conditions, and moving the capping solution from the fluidized bed reactor to a waste vessel via a filter located at the bottom of the fluidized bed reactor. FIG. 11 shows a representative process flow diagram for a fully fluidized, reuse SPOS process in an FBR, according to some aspects of the technology. For example, to perform fully fluidized washing step, between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) repeating fill- fluidize-drain cycles are performed with the material in vessel 21. Each fill-fluidize-drain cycle comprises first adding a portion of the material (e.g., wash solution, reuse wash solution, etc.) in to the fluidized bed reactor, fluidizing the material with solid support, then removing it from the reactor. One of the benefits of fully fluidized SPOS steps are improvements to the reliability and robustness of the process scale up to larger diameter reactors. For example, when using a fully fluidized process, the importance of keeping the solid substrate (e.g., resin bed) flat or to ensure even flow distribution through the solid substrate (e.g., resin bed) is reduced, because the reactions are completely mixed. Thus, in some embodiments, a fully fluidized process is much more forgiving to imperfect resin bed flatness relative to other SPOS methods (e.g., reactions carried out in PBRs). In some embodiments, when all the SPOS reaction steps and all the wash steps are fully fluidized, a mechanical agitator can be used in the reactor instead of inert gas bubbling.

[0102] EXAMPLES

[0103] These Examples describe synthesis of a nucleic acid sense strand using SPOS methods described herein, which comprise distillation of used deblocking solutions and washes after deblocking for recycling as wash solvents, and / or heating of post-deblocking wash solutions, and / or incorporating pyridine in the multi stage countercurrent wash process. Table 1 provides a summary of the Examples.

[0104] Table 1.

[0105] BCJ-0I7. FBR with distillation and use of distillation to wash after deblocking on the next cycles. 5 mmol scale.

[0106] This example describes SPOS comprising recycling of solvents by distillation for subsequent washes using an integrated, multi-step countercurrent wash module. In solid phase oligonucleotide synthesis, sufficient amounts of washing solvents need to be used to wash unreacted reagents away from solid phase. Residual reagents negatively impact purity and yield. Washing solvent, therefore, is a major waste contributor in typical syntheses. The wash after detritylation reaction is especially important. Very low residual acid can cause noticeable decrease of purity and yield. Normally the washes are done at room temperature.

[0107] A FBR system configured for recycling of distillate was used to synthesize a 36mer sense strand (SS) oligonucleotide at 5 mmol scale. Table 3 shows one embodiment of material flow during SPOS. In solid phase oligonucleotide processing, a key processing step is washing after detritylation (deblock) to remove the dichloroacetic acid (DCA) reagent. DCA must be removed to low levels because it interferes with the subsequent coupling reaction. To remove the DCA from the solid phase and reactor, washing with acetonitrile is standard. When these washing materials are sent to waste, they primarily contain acetonitrile (ACN), toluene, DCA, and trityl byproducts of the detritylation reaction. Similarly, the spent detritylation solution contains some mixture of ACN, toluene, DCA, and trityl byproducts. A blend of ACN and toluene with very low DCA content was recovered by distillation and used as the wash solvent on subsequent SPOS cycles. This is an aspect of SPOS that helps reduce process solvent volumes. Tables 2-8 show representative data for BCJ-017.

[0108] Table 2. BCJ-D00084-017 Lpa SS, with distillation

[0109] In parallel to the sequence in the above table, when the deblocking is done, neat DCA is added to the reuse DCA vessel and mixed so that the acid concentration in that vessel is higher when it is used on the next cycle. Also, in parallel to the sequence in the above table, after the reused acid and the wash after acid are pushed into the evaporator, the evaporator distills the solvents and pushes them into the holding tank which is used for washes after deblocking in subsequent cycles.

[0110] Table 3. Table 4.

[0111] Table 5.

[0112] Table 6.

[0113] Table 7.

[0114] 1 Toluene and DC A volumes were calculated based on the DCA solution being 6 vol% DCA in toluene

[0115] 2 The large amount of ACN used for cycle 1 includes the initial volume of ACN used to fill the integrated wash feed bottles. 3 Assumed 75 g for the missed data point, and included it in the total calculation at the bottom of the column.

[0116] 4 Does not include contact time with reuse capping reagents.

[0117] 5 These values were corrected to remove the extra added to compensate for the dilution cart sample.

[0118] Material Composition: DCA was 6 vol% in toluene

[0119] Amidites were 0.1M solutions in ACN (mU solvent was 10vol% toluene in ACN)

[0120] Activator was 0.5 M ETT in ACN

[0121] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent)

[0122] Sulfurization reagent was 0.2 M xanthane hydride in pyridine Cap A was 4 vol% 1 methylimidizole in ACN

[0123] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in ACN

[0124] Table 8.

[0125] BXW-167-5. PBR without distillation. 230 umol scale. Done on an AKTA OPIOO synthesizer

[0126] Tables 9-13 show representative data for BXW-167-5.

[0127] Table 9. Table 10. Table 11.

[0128] Table 12.

[0129] 1 This wash includes ACN used for coupling push

[0130] 2 Recycle time 3 includes push volume

[0131] Material Composition:

[0132] DCA solution: 3 volume% DCA in toluene Amidites solutions:

[0133] Activator was 0.5 M ETT

[0134] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent)

[0135] Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[0136] Cap A was 20 vol% 1 methylimidizole in ACN Cap B was 20vol% acetic anhydride and 30vol% lutidine in ACN

[0137] Table 13. RE0-047. Heated wash.

[0138] Tables 14-21 show representative data for RE0-047.

[0139] Table 14. In parallel to the sequence in the above table, when the deblocking is done, neat DCA is added to the reuse DCA vessel and mixed so that the acid concentration in that vessel is higher when it is used on the next cycle. Table 16.

[0140] Table 17.

[0141] Table 18.

[0142] Table 19.

[0143] Table 20.

[0144] 1 Toluene and DC A volumes were calculated based on the DCA solution being 6 vol% DCA in toluene

[0145] 2 The large amount of ACN used for cycle 1 includes the initial volume of ACN used to fill the integrated wash feed bottles. 3 Assumed 75 g for the missed data point, and included it in the total calculation at the bottom of the column.

[0146] 4 Does not include contact time with reuse capping reagents.

[0147] 5 These values were corrected to remove the extra added to compensate for the dilution cart sample.

[0148] Material Composition: DCA was 6 vol% in toluene

[0149] Amidites were 0.1M solutions in ACN (mU solvent was 10vol% toluene in ACN)

[0150] Activator was 0.5 M ETT in ACN

[0151] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent)

[0152] Sulfurization reagent was 0.2 M xanthane hydride in pyridine Cap A was 4 vol% 1 methylimidizole in ACN

[0153] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in ACN

[0154] Table 21.

[0155] RE0-045. FBR baseline with 1 % pyridine in the wash

[0156] Tables 22-28 show representative data for RE0-045 (RE0-D00084-045 Lpa SS, with 1% pyridine).

[0157] Table 22.

[0158] In parallel to the sequence in the above table, when the deblocking is done, neat DCA is added to the reuse DCA vessel and mixed so that the acid concentration in that vessel is higher when it is used on the next cycle. Table 23.

[0159] Table 24.

[0160] Table 25.

[0161] Table 26.

[0162] Table 27.

[0163] Material Composition:

[0164] DCA was 6 vol% in toluene

[0165] Amidites were 0.1M solutions in ACN (mil solvent was 10vol% toluene in ACN) Activator was 0.5 M ETT in ACN

[0166] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent)

[0167] Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[0168] Cap A was 4 vol% 1 methylimidizole in ACN

[0169] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in ACN

[0170] Table 28. RE0-041. FBR baseline no pyridine.

[0171] Tables 29-35 show representative data for RE0-041 (RE0-D00084-041 Lpa SS, baseline without pyridine).

[0172] Table 29.

[0173] In parallel to the sequence in the above table, when the deblocking is done, neat DCA is added to the reuse DCA vessel and mixed so that the acid concentration in that vessel is higher when it is used on the next cycle. Table 30.

[0174] Table31.

[0175] Table 32.

[0176] Table 33.

[0177] Table 34.

[0178] Material Composition:

[0179] DCA was 6 vol% in toluene

[0180] Amidites were 0.1M solutions in ACN (mil solvent was 10vol% toluene in ACN) Activator was 0.5 M ETT in ACN

[0181] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent)

[0182] Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[0183] Cap A was 4 vol% 1 methylimidizole in ACN

[0184] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in CAN

[0185] Table 35.

[0186] REO-059. Baseline process with all the reagent and wash solvent reuse.

[0187] Tables 36-43 show representative data for REO-059 (RE0-D00084-059 Lpa SS).

[0188] Table 36. note: reuse solvent wash tanks (WI_DCA, Wl CAP, reuse CAP) filled with ACN to begin synthesis

[0189] Table 37.

[0190] Table 38.

[0191] Totals 2747 3350

[0192] Normalized per mmol (L / mmol) 0.670

[0193] Table 39.

[0194] Table 40.

[0195] Table 41.

[0196] Normalized per mmol (L / mmol) 0.064 0.062 1.015 0.823

[0197] Table 42.

[0198] Totals 26639 3015 3104 2676 3830 412 4116 1913 45704

[0199] Normalize d per mmol

[0200] (L / mmol) 5.328 0.603 0.621 0.535 0.766 0.082 0.823 0.383 9.14

[0201] 1 Toluene and DCA volumes were calculated based on the DCA solution being 10 vol% DCA in toluene

[0202] 2 The large amount of ACN used for cycle 1 includes the initial volume of ACN used to fill the integrated wash feed bottles. 4 Does not include contact time with reuse reagents.

[0203] 5 These values were corrected to remove the extra added to compensate for the dilution cart sample.

[0204] Material Composition:

[0205] DCA was 10 vol% in toluene

[0206] Amidites were 0.1M solutions in ACN (mU solvent was 10vol% toluene in ACN) Activator was 0.5 M ETT in ACN

[0207] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent)

[0208] Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[0209] Cap A was 4 vol% 1 methylimidizole in ACN

[0210] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in ACN

[0211] Table 43.

[0212] REO-064. Distillation and using distillate for washing.

[0213] Tables 44-51 show representative data for REO-064 (RE0-D00084-064 Lpa SS).

[0214] Table 44. note: reuse solvent wash tanks (WI_DCA, Wl CAP, reuse CAP) filled with ACN to begin synthesis

[0215] Table 45.

[0216] Table 46.

[0217] Totals 2742 3344

[0218] Normalized per mmol (L / mmol) 0.669

[0219] Table 47.

[0220] Table 48.

[0221] Table 49.

[0222] Totals 312 310 5037 2108.142

[0223] Normalized per mmol (L / mmol) 0.062 0.062 1.007 0.422

[0224] Table 50. | 35-2 | 1676.8 | 0 | 0 | 0 | 0 | 0 | 0.0 | 1676.8 |

[0225] Totals 18506 3009 3091 2729 3784 404 2108 33632

[0226] Normalized per mmol

[0227] (L / mmol) 3.701 0.602 0.618 0.546 0.757 0.081 0.422 6.73

[0228] 1 Toluene and DCA volumes were calculated based on the DCA solution being 6 vol%

[0229] DCA in toluene

[0230] 2 Fresh ACN includes ACN from chase wash, wall wash, fresh ACN washes, and ACN added to the CC cans

[0231] 4 Does not include contact time with reuse reagents.

[0232] 5 These values were corrected to remove the extra added to compensate for the dilution cart sample.

[0233] Material Composition:

[0234] DCA was 10 vol% in toluene

[0235] Amidites were 0.1M solutions in ACN (mU solvent was 10vol% toluene in ACN)

[0236] Activator was 0.5 M ETT in ACN

[0237] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent)

[0238] Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[0239] Cap A was 4 vol% 1 methylimidizole in ACN

[0240] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in CAN

[0241] Table 51.

[0242] CWO-006. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[0243] Tables 52-59 show representative data for CWO-006 (CWO-D00084-006 Lpa SS).

[0244] Table 52.

[0245] Table 53

[0246]

[0247] Table 54.

[0248] Totals 2731 3330

[0249] Normalized per mmol (L / mmol) 0.666

[0250] Table 55.

[0251] Totals 3477 3694.656 8894 9057.0

[0252] Normalized per mmol (L / mmol) 0.695 0.739 1.811

[0253] Table 56.

[0254] Totals 212.6 212.6 18034.35

[0255] Normalized per mmol (L / mmol) 0.043 3.607

[0256] Table 57.

[0257] Table 58. | 35-2 | 3389.1 | 0 | 0 | 0 | 0 | 0 | 0.0 | 3389.1 |

[0258] Totals 39782 2997 3129 3695 18034 858 31818 100313

[0259] Normalized per mmol (L / mmol) 7.956 0.599 0.626 0.739 3.607 0.172 6.364 20.06

[0260] 1 Toluene and DCA volumes were calculated based on the DCA solution being 6 vol% DCA in toluene

[0261] 2 Fresh ACN includes ACN from chase wash, wall wash, fresh ACN washes, and ACN from the pyridine solutions 4 Does not include contact time with reuse reagents.

[0262] 5 These values were corrected to remove the extra added to compensate for the dilution cart sample.

[0263] Material Composition:

[0264] DCA was 6 vol% in toluene Amidites were 0.1M solutions in ACN (mU solvent was 10vol% toluene in ACN)

[0265] Activator was 0.5 M ETT in ACN

[0266] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent)

[0267] Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[0268] Cap A was 4 vol% 1 methylimidizole in ACN Cap B was 4vol% acetic anhydride and 6 vol% lutidine in CAN

[0269] Table 59. Results Tables 60-63 provide comparisons of the previous examples.

[0270] Table 60.

[0271] Table 61. Table 62.

[0272] Table 63. DCA is the most difficult reagent to wash off the resin, necessitating large volumes of washing after the deblock step. Comparing CWO-006 (FBR, no reuse) to BXW-167-5 (PBR, no reuse), the FBR process used 1.6x more total solvent volume of the wash after deblock (2.5x more ACN, 24x less pyridine, 1.3x more toluene), but resulted in higher crude purity (84.9% versus 75.2% FLP) as well as higher crude yield (259 versus 242 OD / umol). Incorporation of countercurrent reuse of the deblock wash into the FBR process (REO-

[0273] 041) allowed for a 1.8x reduction in the total solvent volume of the wash after deblock (5.8 L / mmol vs. 10.2 L / mmol) while maintaining higher crude purity (78.0% FLP) and crude yield (247 OD / umol) compared to the PBR process. In RE0-041, only ACN was used for the wash after deblock. FIG. 6 shows that residual DCA in the final wash after deblocking was about 0.004 mol% on average. RE0-041used 6 countercurrent wash stages. This requires 6 vessels for storing washes between cycles. A benefit of using the six kind of wash steps is that it reduces the amount of solvent needed for washing away the DCA after deblocking. On the other hand, advantages of not using any countercurrent wash stages, and not using any reuse materials at all, like example CWO-006, is that the cycle time is faster, the plant footprint is smaller (less vessels), and the capital cost is lower.

[0274] These examples demonstrate changes to the deblock washing on the FBR that reduce the concentration of residual DCA in the final wash after deblocking while either holding constant or decreasing the volume of fresh ACN used in the deblock wash. In some cases, improvement in the deblock washing is correlated with increased FLP purity and crude yield. Even low levels (-0.005 mol%) of residual DCA remaining on the resin going into coupling may lead to truncation impurities. In all cases, purity and crude yield remain higher in the FBR process than the PBR process.

[0275] 1. In BCJ-017, distillation of solvent waste allowed an increase of the reused wash volumes after deblock while reducing the volume of fresh ACN used by 1.5x (3.8 L / mmol vs. 5.8 L / mmol). The concentration of residual DCA in the final wash after deblocking was only about 0.002 mol% on average. Compared to the baseline (RE0- 041), FLP purity improved by 2% (80.1% vs. 78.0%) and crude yield was comparable (250 OD / umol compared to 247 OD / umol).

[0276] 2. In RE0-047, the washes were heated, reducing the concentration of residual DCA in the final wash to about 0.002 mol% on average. The data suggest that heating the wash solvent makes the washing more efficient. The volume of wash after deblock is comparable to the baseline (5.9 L / mmol).

[0277] 3. In RE0-045, pyridine is added to the deblock washes, targeting approximately 1 vol% in the countercurrent reuse washes. The volume of ACN in the wash after deblock is the same as in the baseline. The addition of this small amount of pyridine (0.04 L / mmol, 40x lower than the amount of pyridine used in the deblock wash in the PBR, BXW-167-5) reduces the concentration of residual DCA in the final wash to below 0.001 mol% on average. FLP purity (81.5%) and yield (250 OD / umol) are comparable to BCJ-017 and higher than the baseline RE0-041. RE0-064 and RE0-059 demonstrate additional changes to deblock washing. Fluidization of the deblock wash improves process robustness by reducing the reliance on the ability to keep the resin bed flat during flow-through washing steps. For example, If the resin bed does not stay flat during the flow through deblocking reaction, and / or if the resin bed does not stay flat during the subsequent washing after deblocking, then each time a wash is fluidized it resets the bed so that it is flat again, and also ensures complete contacting of the resin with the wash because fluidization gives complete mixing. Flow through washing is a more efficient use of the wash solvent because of less back-mixing, if the solvent is evenly distributed and flows through the resin bed without channeling; therefore, it is effective to alternate between fluidizing some of the washes and running some of the washes flow through. The fluidized sections reset the bed flat and channel free so that the subsequent flow through washes are more effective, which is even more important for larger diameter reactors, making the process more scalable. Reduction in the number of countercurrent washes after deblock reduces the cycle time of the synthesis and the amount of ACN used at the beginning of the synthesis to prefill the countercurrent wash vessels. RE0-064 implemented both changes in the distillation version of the process. RE0-064 used three countercurrent wash vessels instead of the six used in the baseline distillation process (BCJ-017). The volume of fresh ACN used in the wash after deblock was comparable to BCJ- 017 (3.7 L / mmol vs. 3.8 L / mmol). Compared to BCJ-017, RE0-064 resulted in lower FLP purity (70.6% vs. 80.1%) and comparable crude yield (249 OD / umol vs. 250 OD / umol).

[0278] RE0-059 implemented fluidized deblock washes and a reduced number of countercurrent wash vessels in the pyridine version of the process. RE0-059 used 3 countercurrent wash vessels as opposed to 6 in RE0-045 (the baseline pyridine process). Additionally, RE0-059 used 8% less ACN (5.3 L / mmol vs. 5.8 L / mmol) and 50% less pyridine (0.02 L / mmol vs. 0.04 L / mmol) in the wash after deblock compared to RE0-045. Crude purity was the same as for RE0-045 (81.5%) while crude yield was higher (261 OD / umol vs. 250 OD / umol), demonstrating no negative impact of the fluidized wash and fewer countercurrent wash vessels. An advantage of using a smaller number of countercurrent wash vessels, for example 3 vessels in RE0-059, is that it reduces cycle time, reduces plant footprint, and reduces capital cost. On the other hand, an advantage of using a larger number of countercurrent wash vessels, for example 6 vessels in RE0-045, is that the process is more forgiving to uneven bed height and uneven washing, if the spray nozzle or other distribution devices are not doing an effective job keeping the resin bed flat and channel free. The FBR process with full reuse and minimized deblock wash volume (RE0-059) utilizes 1.9x less total wash volume for the wash after deblock compared to the FBR process without reuse (CWO-006). Specifically, the process with reuse uses 1.5x less ACN (5.3 L / mmol vs. 8.0 L / mmol), 3.5x less pyridine (0.07 L / mmol vs. 0.02 L / mmol) and no toluene (compared to 2.1 L / mmol in CWO-006). CWO-006 utilizes one fluidized wash after deblock, demonstrating no negative impact on the synthesis. CWO-006 utilized only fresh wash solvent with no reuse, and no distillation, but it used fresh solvent made up at the 70 / 30 ACN / toluene composition, to mimic the distillate. It also included pyridine. Including toluene in the wash solvent is beneficial even if there is no distillation process because the solvent mixture is effective at washing away the DCA, and because toluene is less of a supply chain risk for toluene than ACN. An advantage of adding pyridine to distillate is that it would neutralize any residual amounts of DCA in the distillate. The different versions of wash after deblock described above are summarized in Table 64. Table 64. Summary of different embodiments of wash after deblock demonstrated in the experiments.

Claims

- 96 -CLAIMSWhat is claimed is:

1. A system for synthesizing oligonucleotides, the system comprising: a feed zone vessel; a reactor in fluid communication with the feed zone vessel; a distillation unit; and a distillate collection / feed vessel in fluid communication with the feed zone vessel or reactor so that it can feed distillate back to the process; and a wash module comprising one or more wash vessels in fluid communication with the distillate collection / feed vessel.

2. The system of claim 1 further comprising a heat exchanger operatively associated with an inlet of the reactor.

3. The system of claim 1 further comprising a heat exchanger operatively associated with an outlet of the reactor.

4. The system of any one of claims 1 to 3, wherein the distillation unit is in fluid communication with the reactor.

5. The system of any one of claims 1 to 4, wherein the wash module comprises one or more wash vessels, each wash vessel in fluid communication with the reactor and the feed zone vessel.

6. The system of any one of claims 1 to 5 further comprising an acetonitrile (ACN) feed vessel in fluid communication with the feed zone vessel.

7. The system of claim 5 or 6, wherein the wash module is configured as a countercurrent integrated wash module.

8. The system of any one of claims 1 to 7, wherein the distillation unit comprises an evaporator.- 97 -9. The system of any one of claims 1 to 8, wherein the distillation unit comprises a rectification section providing at least one equilibrium stage of separation above the evaporator.

10. The system of any one of claims 1 to 9 further comprising one or more pumps.

11. The system of any one of claims 1 to 9, wherein pressure differential drives fluid movement.

12. A method for recycling wash solution during a solid phase oligonucleotide synthesis (SPOS) deblocking step, the method comprising:(i) contacting an oligonucleotide linked to a solid support in a reactor with deblocking solution introduced into the reactor;(ii) moving the deblocking solution from the reactor to a distillation unit;(iii) moving a first wash solution through the reactor to the distillation unit;(iv) operating the distillation unit to produce a distillate wash solution;(v) moving the distillate wash solution to a first integrated wash solution vessel of a wash module or to the reactor;(vi) performing one or more washes by moving one or more wash solutions from wash vessels of the wash module to the reactor and contacting the oligonucleotide on the solid support; and(vii) moving the distillate wash solution to the reactor and contacting the oligonucleotide linked to the solid support with the distillate wash solution.

13. The method of claim 12, wherein the deblocking solution consists essentially of DCA or TCA or TFA, and toluene prior to contacting the oligonucleotide.

14. The method of claim 12 or 13, wherein the deblocking solution comprises acetonitrile (ACN), toluene, DCA, and trityl byproducts after contacting the oligonucleotide.- 98 -15. The method of any one of claims 12 to 14, wherein the distillation unit comprises an evaporator and / or a rectification section providing at least one equilibrium stage of separation above the evaporator.

16. The method of any one of claims 12 to 15, wherein the distillation unit comprises an evaporator providing only a single equilibrium stage of separation with no rectification section above the evaporator.

17. The method of any one of claims 12 to 16, wherein the distillate wash solution comprises ACN, toluene, and DCA, TCA, or TFA.

18. The method of claim 17, wherein the distillate wash solution comprises ACN, toluene, and less than 25 uM DCA, TCA, or TFA.

19. The method of claim 17, wherein the distillate wash solution comprises no more than 20 uM DCA, TCA, or TFA.

20. The method of any one of claims 12-19, wherein the first wash solution comprises pyridine.

21. The method of any one of claims 12-20, wherein the one or more of the wash solutions or distillate wash solution of step (vi) or (vii) comprises pyridine.

22. The method of any one of claims 12-21, further comprising:(viii) moving a fresh ACN solution lacking pyridine from a vessel to the reactor and contacting the oligonucleotide on the solid support; and(ix) moving the fresh ACN solution to the first integrated wash solution vessel.

23. The method of any one of claims 12 to 22, wherein the wash module comprises six or more wash solution vessels in a multi-stage countercurrent arrangement.

24. The method of any one of claims 12 to 22, wherein the wash module comprises five or fewer wash solution vessels in a multistage countercurrent arrangement.- 99 -25. The method of any one of claims 12 to 24 further comprising moving the distillate wash solution to a wash solution vessel of the wash module after contacting the oligonucleotide.

26. The method of any one of claims 12 to 25, wherein the distillate wash solution and the one or more wash solutions from the wash solution vessels are heated prior to contacting the oligonucleotide linked to the solid substrate.

27. The method of claim 26, wherein the heated distillate wash solution and the one or more heated wash solutions have a temperature ranging from about 30 °C to about 80 °C.

28. The method of any one of claims 12 to 27, wherein the oligonucleotide is contacted with the deblocking solution under flow-through conditions or fluidized conditions.

29. The method of any one of claims 12 to 28, wherein the oligonucleotide is contacted with the distillate wash solution under flow-through conditions or fluidized conditions.

30. The method of any one of claims 12 to 29, wherein the distillate wash solution comprises about 70-80% ACN and about 20-30% toluene by volume.

31. The method of any one of claims 12 to 30, wherein the distillate wash solution comprises about 75% ACN and about 25% toluene by volume.

32. A method for washing during a solid phase oligonucleotide synthesis (SPOS) deblocking step, the method comprising:(i) contacting an oligonucleotide linked to a solid support in a reactor with deblocking solution introduced into the reactor; and(ii) performing one or more countercurrent washes by moving one or more wash solutions from wash vessels of a wash module to the reactor and contacting the oligonucleotide on the solid support, wherein the one or more wash solutions are heated prior to contacting the oligonucleotide linked to the solid support.- 100 -33. The method of claim 32, wherein the one or more wash solutions are heated to a temperature ranging from about 30 °C to about 80 °C.

34. The method of claim 32 or 33, wherein the deblocking solution consists essentially of DCA or TCA or TFA, and toluene prior to contacting the oligonucleotide.

35. The method of any one of claims 32-34, wherein the deblocking solution comprises acetonitrile (ACN), toluene, DCA or TCA or TFA, and trityl byproducts after contacting the oligonucleotide.

36. The method of any one of claims 32-35, wherein the one or more of the wash solutions comprises pyridine.

37. The method of any one of claims 32-36, further comprising:(viii) moving a fresh ACN solution lacking pyridine from a vessel to the reactor and contacting the oligonucleotide on the solid support; and(ix) moving the fresh ACN solution to a first integrated wash solution vessel of the wash module.

38. The method of any one of claims 32 to 37, wherein the wash module comprises six or more wash solution vessels in a multistage countercurrent arrangement.

39. The method of any one of claims 32 to 38, wherein the wash module comprises five or fewer wash solution vessels in a multistage countercurrent arrangement.

40. The method of any one of claims 32 to 39, wherein the oligonucleotide is contacted with the deblocking solution under flow-through or fluidized conditions.

41. The method of any one of claims 32 to 40, wherein the oligonucleotide is contacted with the wash solution under flow-through or fluidized conditions, or combination of both.- 101 -42. The method of any one of claims 32 to 41, wherein the wash solution comprises about 60-80% ACN and about 20-40% toluene by volume.

43. The method of any one of claims 32 to 41, wherein the wash solution comprises about 75% ACN and about 25% toluene by volume, or about 70% ACN and about 30% toluene by volume.

44. A method for washing during a solid phase oligonucleotide synthesis (SPOS) deblocking step, the method comprising:(i) contacting an oligonucleotide linked to a solid support in a reactor with deblocking solution introduced into the reactor; and(ii) performing one or more countercurrent washes by moving one or more wash solutions from wash vessels of a wash module to the reactor and contacting the oligonucleotide on the solid support, wherein the one or more wash solutions comprise pyridine.

45. The method of claim 44, wherein the one or more wash solutions comprise about 1% pyridine.

46. The method of claim 44 or 45, wherein the deblocking solution consists essentially of DCA or TCA or TFA, and toluene prior to contacting the oligonucleotide.

47. The method of any one of claims 44-46, wherein the deblocking solution comprises acetonitrile (ACN), toluene, DCA or TCA or TFA, and trityl byproducts after contacting the oligonucleotide.

48. The method of any one of claims 44 to 47, wherein the wash module comprises six or more wash solution vessels in a multistage countercurrent arrangement.

49. The method of any one of claims 44 to 48, wherein the wash module comprises five or fewer wash solution vessels in a multistage countercurrent arrangement.- 102 -50. The method of any one of claims 44 to 49, wherein the one or more wash solutions from the wash solution vessels are heated prior to contacting the oligonucleotide linked to the solid substrate.

51. The method of claim 50, wherein the heated distillate wash solution and the one or more heated wash solutions have a temperature ranging from about 30 °C to about 80 °C.

52. The method of any one of claims 44 to 51, wherein the oligonucleotide is contacted with the deblocking solution under flow-through conditions or fluidized conditions.

53. The method of any one of claims 44 to 52, wherein the oligonucleotide is contacted with the wash solution under flow-through conditions or fluidized conditions.

54. The method of any one of claims 44 to 53, wherein the wash solution comprises about 60-80% ACN and about 20-40% toluene by volume.

55. The method of any one of claims 44 to 53, wherein the wash solution comprises about 70% ACN and about 30% toluene by volume.

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