Oxidation / sulfurization step and wash after oxidation / sulfurization

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 a step of oxidation reaction solution recycling. In some embodiments, recycling the oxidation reaction solution requires a significantly reduced amount of oxidation solution during the entire SPOS process relative to SPOS processes where the oxidation step of each cycle is carried out using fresh oxidation solution. In some embodiments, oligonucleotides produced using systems and methods of the disclosure have higher purity than oligonucleotides produced using conventional SPOS techniques.

Description

[0001] OXIDATION / SULFURIZATION STEP AND WASH AFTER OXIDATION / SULFURIZATION

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. 119(e) of the filing date of U.S. provisional application serial number 63 / 711,313, filed October 24, 2024, entitled “OXIDATION / SULFURIZATION STEP AND WASH AFTER OXIDATION / SULFURIZATION”, the entire contents 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 solid phase oligonucleotide synthesis (SPOS). The disclosure is based, in part, on SPOS systems and methods comprising a step of oxidation reaction solution recycling. In some embodiments, recycling the oxidation reaction solution requires a significantly reduced amount of oxidation solution during the entire SPOS process relative to SPOS processes where the oxidation step of each cycle is carried out using only fresh oxidation solution. In some embodiments, oligonucleotides produced using systems and methods of the disclosure have higher purity than oligonucleotides produced using conventional SPOS techniques. In some embodiments, oxidation 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 reactor; a feed zone vessel connected to the reactor; a fresh oxidation solution feed vessel connected to the feed zone vessel; a reuse oxidation solution vessel connected to both the reactor and the feed zone vessel; and a post-oxidation / sulfurization (post-O / S) solution vessel connected to the reactor and the feed zone vessel.

[0009] In some embodiments, the system further comprises a sulfurization solution feed vessel connected to the feed zone vessel.

[0010] In some embodiments, the system further comprises a waste module.

[0011] In some embodiments, the oxidation solution feed vessel comprises an oxidation solution comprising iodine, pyridine, and water.

[0012] In some embodiments, the reuse oxidation solution vessel comprises an oxidation solution that has previously been used in at least one solid phase oligonucleotide synthesis (SPOS) cycle.

[0013] In some embodiments, the post-O / S solution vessel comprises a coupling solution that has previously been used in at least one SPOS cycle, and the solvent wash after coupling. In some embodiments, the coupling solution and the solvent wash after coupling comprises the activated phosphoramidite, and acetonitrile (ACN), and optionally, toluene or another cosolvent.

[0014] In some embodiments, the reactor contains a solid substrate. In some embodiments, the solid substrate comprises a resin.

[0015] In some embodiments, the system further comprises an acetonitrile (ACN) feed vessel. In some embodiments, the system further comprises one or more pumps.

[0016] In some embodiments, the system further comprises no pumps (e.g., relies on a pressure differential to move fluid from one location to another).

[0017] In some aspects, the disclosure provides a method for recycling oxidation solution during an oligonucleotide synthesis, the method comprising during a first cycle of solid phase oligonucleotide synthesis (SPOS) that uses oxidizer, contacting an oligonucleotide linked to a solid support in a reactor with a volume of a first oxidation solution introduced into the reactor; moving the oxidation solution from the reactor to an oxidation solution reuse vessel to produce a recycled oxidation solution; completing the first cycle of SPOS that uses oxidizer and beginning a second cycle of SPOS that uses oxidizer; and during the second cycle of SPOS that uses oxidizer, contacting the oligonucleotide linked to the solid support in the reactor with a volume of the recycled oxidation solution. In some embodiments, the first oxidation solution comprises iodine in a solvent comprising pyridine and water. In some embodiments, the first oxidation solution comprises iodine: solvent at a concentration ranging from 0.01 to 0.1 M.

[0018] In some embodiments, during the first cycle of SPOS that uses oxidizer, the first oxidation solution and solid support are fluidized. In some embodiments, during the second cycle of SPOS that uses oxidizer, the first recycled oxidation solution and solid support are fluidized.

[0019] In some embodiments, step (iii) further comprises using a solvent wash to chase residual oxidizer solution from the reactor to the first oxidation solution reuse vessel.

[0020] In some embodiments, the first SPOS cycle of step (iii) comprises reusing coupling solution from the same SPOS cycle for washing the reactor and solid support after oxidation or thiolation.

[0021] In some embodiments, the second SPOS cycle of step (iii) comprises reusing coupling wash solution from the same SPOS cycle for washing the reactor and solid support after oxidation or thiolation.

[0022] BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows a representative process flow diagram for a fluidized bed reactor (FBR), according to some aspects of the technology.

[0023] FIGs 2A-2B show representative process flow diagrams for material flow in oxidation / sulfurization reactions, according to some aspects of the technology.

[0024] FIG. 3 shows a representative schematic diagram for a FBR configured for material reuse, according to some aspects of the technology.

[0025] FIG. 4 shows a representative schematic diagram for a FBR configured for acetonitrile (ACN) reuse, according to some aspects of the technology.

[0026] FIG. 5 shows a representative schematic diagram for a FBR comprising a distillation unit, according to some aspects of the technology.

[0027] FIG. 6 shows a representative schematic diagram for a FBR configured for no material reuse, according to some aspects of the technology.

[0028] FIG. 7 shows a representative process flow diagram for a fully fluidized, reuse SPOS process in an FBR, according to some aspects of the technology. DETAILED DESCRIPTION

[0029] 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.

[0030] 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 (DCA). In some embodiments, the acid (e.g., TCA, DCA, 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 “de-blocking” 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.

[0031] 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”.

[0032] 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.

[0033] 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”.

[0034] 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.

[0035] 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.

[0036] 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 (e.g., “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”). 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.

[0037] 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, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 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, 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.

[0038] 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.

[0039] 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 (DCA) 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. 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.

[0040] Fluidized Bed Reactor

[0041] 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).

[0042] 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.

[0043] 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 (e.g., pressure differentials). 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 connected to 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 direct 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.

[0044] 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 (13A), 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 in fluid communication with 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 DCA solution to be reused in additional detritylation steps on other cycles.

[0045] 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.

[0046] 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 is 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 (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 (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.

[0047] 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) are 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.

[0048] 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”.

[0049] 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.

[0050] 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 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.

[0051] FBR systems comprising capping modules as described herein provide several advantages over 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.

[0052] In some embodiments, use of FBR systems described by the disclosure for SPOS results in higher crude purity and yield of oligonucleotides.

[0053] 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. 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] In some embodiments, use of FBR systems described by the disclosure for SPOS produce higher batch to batch consistency relative to PBRs.

[0059] Oxidation / Sulfiirization Solution Reuse

[0060] Aspects of the disclosure relate to FBRs comprising a modified oxidation / sulfurization module. In some embodiments, the reuse methods described herein significantly reduce the amount of oxidation / sulfurization solution needed to perform synthesis of an entire oligonucleotide using SPOS. In the embodiments, the systems and methods reduce the amount of oxidation / sulfurization solution used during SPOS by between about 1.1-fold and about 1.5-fold. In the embodiments, the systems and methods reduce the amount of oxidation / sulfurization solution used during SPOS by at least 1.1-, at least 1.2-, at least 1.3-, at least 1.4-, or at least 1.5-fold relative to previously described SPOS reactions using FBRs or PBRs. In the embodiments, the reused oxidation solution serves as the wash after coupling reaction and therefore less or no fresh wash solvent is needed to rinse the resin after coupling. In some aspects, the oxidation / sulfurization modules described herein further comprise a post-oxidation / sulfurization (post-O / S) solution vessel, which contains coupling reaction solution that has been used during the same SPOS cycle.

[0061] FIG. 2A shows one embodiment of a FBR system comprising an oxidation / sulfurization module configured for coupling reaction solution reuse. The FBR comprises one or more oxidation solution feed vessels (also referred to as oxidizer feed vessels) (15), a reuse wash vessel (24), a fresh acetonitrile (ACN) vessel (19), and / or one or more sulfurization feed vessels (16; not shown in FIG. 2A). In some embodiments, the one or more oxidizer feed vessel (15) is connected to a feed zone vessel (29). In some embodiments, the one or more sulfurization feed vessels (16; not shown in FIG. 2A) is connected to a feed zone vessel (29). In some embodiments, the feed vessel (29) is in fluid communication with the reactor (30). In some embodiments, the one or more oxidizer feed vessels (15) comprise an oxidizer solution comprising iodine and pyridine. The amount of oxidizer (e.g., iodine) in an oxidizer solution charged to the reactor per cycle may vary. In some embodiments, the amount of iodine in an oxidizer solution charged to the reactor per cycle ranges from about 1.0 equivalents and 5 equivalents (e.g., 1, 2, 3, 4, or 5 equivalents). In some embodiments, the concentration of iodine in an oxidizer solution ranges from about 0.02 M to about 0.1 M (e.g., about 0.02, 0.04, 0.05, 0.06, 0.08, or 0.1 M).

[0062] 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 in a sulfurization solution that is charged to the reactor in a single cycle ranges from about 1.0 equivalents and 12 equivalents (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 equivalents). In some embodiments, the concentration of xanthane hydride in a sulfurization solution ranges from about 0.1 M to about 0.5 M (e.g., about 0.1, 0.2, 0.3, 0.4, or 0.5 M).

[0063] In some embodiments, the oxidation / sulfurization module further comprises an oxidation solution reuse vessel (23; shown in FIG. 2B), which is connected to the feed zone (29). In some embodiments, the oxidation solution reuse vessel (23) is configured to store oxidation solution after it is used in an oxidation reaction so that it can be reused on the next cycle. In some embodiments, an oxidation / sulfurization module further comprises a reactor (30) connected to the feed zone vessel (29). In some embodiments, the oxidation solution feed vessel (15) and the oxidation solution reuse vessel (23) are connected. In some embodiments, the oxidation solution reuse vessel (23) is connected to a waste module.

[0064] The following is an illustrative example describing the function of oxidation / sulfurization modules. SPOS methods described by the disclosure comprise a step of performing an oxidation step of the first SPOS cycle in the synthesis that uses the oxidizer, comprising charging a volume of unused oxidation solution from an oxidation solution feed vessel (15) to the reactor (30) via the feed zone vessel (29). At the completion of the oxidation step of the first SPOS cycle in the synthesis that uses the oxidizer, the used oxidation solution is pushed from the reactor (30) to the oxidation solution reuse vessel (23). The process is repeated n-1 times, where N is the number of nucleotides in the oligonucleotide being synthesized by the SPOS reaction that use the oxidizer.

[0065] Although the foregoing example describes oxidation solutions, the skilled artisan will recognize that the same processes and systems may be utilized for sulfurization solutions and reactions.

[0066] In some embodiments, the reactor (30) is connected to a reuse wash vessel (24; shown in FIG. 2B), which is configured to store the coupling reaction and wash after coupling. The rationale for the reuse of the coupling reaction solution for rinsing of the solid support bound growing oligonucleotide chain post oxidation / sulfurization is that it comprises high amounts of dehydrating agent (e.g., ACN and residual activated phosphoramidites), which is an excellent way to wash and remove residual water from the post-oxidation packed bed in the reactor. Excess phosphoramidite in this used coupling solution serves as a dehydrating agent, and the composition of this reaction rinse does not contain species that would further react with the resin bound oligonucleotides. In some embodiments, the oxidation / sulfurization module is configured to work under fluidization conditions. In some embodiments, nothing is recycled, recovered, or reused from one synthesis batch to another. Using this approach for the oxidation reactions significantly reduces waste and cost for these reactions relative to other SPOS systems such as PBRs and previously described OFBR systems.

[0067] Additional embodiments of FBR oxidation / sulfurization modules configured for oxidation / sulfurization solution reuse are shown in FIGs. 2A-2B.

[0068] In some embodiments, an FBR machine is configured to reuse acetonitrile (ACN). FIG.

[0069] 4 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.

[0070] Aspects of the disclosure relate to FBR machines, and methods of using FBR machines, comprising a distillation unit. FIG. 5 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.

[0071] No Reuse SPOS Processes

[0072] 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. 6 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.

[0073] Fully fluidized SPOS in FBR

[0074] 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. 7 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 deblock reaction, first, the material in vessel 33 is added to the fluidized bed reactor in a first portion, fluidized with the solid support, then removed via the filter at the bottom of the reactor. Then, 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 in vessel 21 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.

[0075] EXAMPLES

[0076] FBR with reuse oxidizer, and reuse of coupling reaction material for the wash after oxidation and thiolation. 5 mmol scale.

[0077] This example describes SPOS that uses 2.7 equivalent of oxidizer on the first cycle that uses oxidation, then subsequently 2.2 equivalent of oxidizer per cycle and the remaining cycles that use oxidation (Table 1). Attempts were not made to reduce it further. First, the used oxidizer from the previous cycle was charged to the reactor, fluidized with the resin, and then pushed out of the reactor. Destination was waste. Then the fresh oxidizer solution was pushed into the reactor, fluidized with the resin, and then pushed out of the reactor. Destination was the holding vessel for used oxidizer solution that would be reused on the next cycle. Then, the resin bed was washed with coupling reagents solution and coupling reaction wash material from earlier in the cycle. Tables 2-7 show representative data for this SPOS process.

[0078] Table 1. BCJ-013. BCJ-D00084-013 Lpa SS, reuse oxidizer

[0079] destination when push out of material source reaction mode reactor

[0080] reuse acid reuse DCA tank fluidize Waste

[0081] reuse acid reuse DCA tank flow-through waste

[0082] new acid, 6% 6% acid feed tank flow-through reuse acid tank

[0083] reuse solvent wash WI_DCAtank 1 flow-through Waste

[0084] reuse solvent wash WI_DCAtank 2 flow-through WI_DCAtank 1

[0085] new solvent chase ACN feed tank Flow-through WI_DCAtank 1

[0086] reuse solvent wash WI_DCAtank3 flow-through WI_DCAtank 2

[0087] reuse solvent wash WI_DCAtank4 flow-through WI_DCAtank3

[0088] reuse solvent wash WI_DCAtank5 flow-through WI_DCAtank4

[0089] reuse solvent wash WI_DCAtank6 flow-through WI_DCAtank5

[0090] new solvent reactor

[0091] wall wash ACN feed tank Flow-through WI_DCAtank5

[0092] new solvent washes ACN feed tank flow-through WI_DCAtank6

[0093] new amidite, post O / S tank (to be used for the amidite + activator activator fluidize wash after oxidation)

[0094] new solvent chase post O / S tank (to be used for the and washes ACN feed tank flow-through wash after oxidation)

[0095] Reuse oxidizer Reuse OXID tank fluidize Waste

[0096] oxidizer oxidizer feed tank fluidize Reuse OXID tank

[0097] New solvent chase ACN feed tank Flow-through Reuse OXID tank

[0098] post O / S wash post O / S tank flow-through Waste

[0099] Reuse capping

[0100] reaction solution Reuse CAP tank flow-through Waste

[0101] New Capping Capping A feed

[0102] reagents and tank, Capping B

[0103] dilution solvent for feed tank, DIL_CAP

[0104] capping reaction tank fluidize Reuse CAP tank

[0105] reuse solvent wash Wl CAP tank flow-through DIL_CAP tank

[0106] new solvent chase ACN feed tank flow-through Wl CAP tank

[0107]

[0108] new solvent wash Toluene feed tank flow-through Wl CAP tank

[0109] Table 2. total Fresh ACN wash total after toluene deblocking used in includingthe Neat deblock + amount DCA chase / wash total DCA prefilled to added to after used in the counter 6% DCA reuse deblock deblock current wash Cycle (mL) (mL) (mL)l (mL)l vessels(mL)2 1 626 0.0 589 37.6 2423 2 353 0.0 332 21.2 369 3 378 0.0 355 22.7 385 4 394 0.0 370 23.6 379 5 413 0.0 388 24.8 375 6 433 0.0 407 26.0 377 7 453 0.0 426 27.2 527 8 491 0.0 462 29.5 525 9 499 0.0 469 29.9 531 10 521 0.0 490 31.3 533 11 534 0.0 502 32.0 520 12 562 0.0 528 33.7 522 13 584 0.0 549 35.0 664 14 609 0.0 572 36.5 667 15 628 0.0 590 37.7 658 16 642 0.0 604 38.5 665 17 669 0.0 629 40.1 667 18 695 0.0 653 41.7 686 19 714 0.0 671 42.8 835 20 728 0.0 684 43.7 822 21 751 0.0 706 45.0 831 22 771 0.0 724 46.2 832 23 794 0.0 747 47.7 821 24 803 0.0 755 48.2 817 25 831 0.0 781 49.9 971 26 855 0.0 803 51.3 1001 27 868 0.0 816 52.1 966 28 893 0.0 839 53.6 957 29 909 0.0 855 54.6 967 30 933 0.0 877 56.0 962 31 952 0.0 895 57.1 1103 32 987 0.0 927 59.2 1101 33 1004 0.0 944 60.3 1113 34 1028 0.0 966 61.7 1116 35-1 1046 0.0 983 62.8 1131 35-2 1072 1007 64.3 1116 Totals 23894 1525 28932 Normalized

[0110] per mmol

[0111]

[0112] (L / mmol) 4.78 0.305 5.79 Table 3.

[0113] Amidite, note that

[0114] FBR was

[0115] 0.1 M and

[0116] PBR was

[0117] 0.2 M,

[0118] FBR used less

[0119] pyridine in Acid molar amidite wash after contact equiv equivalent deblocking time Amidite overall versus Cycle (mL) (minutes) Amidite (g) (mL) resin 1 0 13.5 MG 73 89 1.77 2 0 9.8 MU 77 94 1.88 3 0 9.9 MC 72 88 1.76 4 0 9.9 MG 73 89 1.78 5 0 9.9 MG 73 89 1.77 6 0 9.9 ADEMA 84 103 2.05 7 0 10.2 ADEMA 82 100 2.00 8 0 10.3 ADEMA 82 100 2.00 9 0 10.5 MG 73 89 1.77 10 0 10.5 MC 73 89 1.77 11 0 10.8 MC 73 89 1.78 12 0 11.0 MG 73 89 1.79 13 0 10.6 MA 76 93 1.85 14 0 10.8 MC 72 88 1.77 15 0 10.9 MG 73 90 1.79 16 0 11.2 MA 75 91 1.82 17 0 11.5 MU 77 94 1.89 18 0 11.8 MC 73 90 1.79 19 0 11.5 MU 77 94 1.89 20 0 11.8 MA 76 92 1.85 21 0 11.8 MC 73 89 1.78 22 0 12.1 MU 77 94 1.88 23 0 12.3 MG 73 89 1.78 24 0 11.9 MG 81 99 1.98 25 0 12.2 FU 83 101 2.01 26 0 12.3 FU 83 101 2.01 27 0 12.4 FC 82 100 2.00 28 0 12.4 FG 82 99 1.99 29 0 12.7 MA 84 102 2.05 30 0 12.7 MA 84 103 2.06 31 0 12.8 MC 82 100 1.99 32 0 12.9 MC 80 98 1.96 33 0 13.0 MG 81 99 1.98 34 0 13.1 MU 86 105 2.10 35-1 0 13.3 MUS 86 105 2.10

[0120]

[0121] 35-2 0 13.2 Totals 0 2724 3322

[0122] Normalized

[0123] per mmol

[0124]

[0125] (L / mmol) 0.000 0.66

[0126] Table 4.

[0127] Fresh

[0128] ACN

[0129] activator Coupling wash

[0130] equivalent contact after

[0131] Activator Activator versus time coupling Oxidizer Oxidizer Cycle (g) (mL) resin (minutes) (mL) (g) (mL) 1 76 94 9.4 10 75 266 271 2 76 94 9.4 10 75 217 221 3 78 96 9.6 10 75 217 221 4 77 95 9.5 10 75 217 221 5 77 95 9.5 10 75 217 221 6 83 102 10.2 15 75 217 221 7 84 104 10.4 15 75 217 221 8 85 105 10.5 15 75 217 221 9 77 95 9.5 10 75 217 221 10 77 95 9.5 10 75 217 221 11 77 95 9.5 10 75 217 221 12 76 94 9.4 10 75 217 221 13 77 95 9.5 10 75 217 221 14 77 95 9.5 10 75 217 221 15 76 94 9.4 10 75 217 221 16 77 95 9.5 10 75 216 220 17 76 94 9.4 10 75 217 221 18 77 95 9.5 10 75 217 221 19 75 93 9.3 10 75 217 221 20 77 95 9.5 10 75 217 221 21 77 95 9.5 10 75 217 221 22 76 94 9.4 10 75 217 221 23 77 95 9.5 10 75 217 221 24 83 102 10.2 10 75 217 221 25 83 102 10.2 15 75 217 221 26 83 102 10.2 15 75 217 221 27 86 106 10.6 15 75 217 221 28 86.0 106 10.6 15 75 217 221 29 83.0 102 10.2 10 75 217 221 30 86 106 10.6 10 75 217 221 31 85 105 10.5 10 75 217 221 32 84 104 10.4 10 75 217 221 33 86 106 10.6 10 75 217 221

[0132]

[0133] 34 85 105 10.5 10 75 217 221 35-1 85.2 105 10.5 10 75

[0134] 35-2

[0135] Totals 3457 2625 7426 7562 Normalized

[0136] per mmol

[0137]

[0138] (L / mmol) 0.69 0.53 1.5

[0139] Table 5.

[0140] Oxidation or Fresh AON

[0141] sulfurization wash after

[0142] Xanthane Xanthane contact oxidation or Diluted Diluted Diluted hydride hydride time sulfurization Capping Capping Capping Cycle (g) (mL) (minutes) (mL) A(g) A(mL) B (g) 1 9 25 41 52 39 2 9 25 41 52 36 3 9 25 42 53 37 4 9 25 41 52 40 5 9 25 41 52 38 6 9 25 40 51 39 7 9 25 43 54 36 8 9 25 41 52 38 9 9 25 40 51 39 10 9 25 41 52 38 11 9 25 41 52 38 12 9 25 43 54 37 13 9 25 42 53 37 14 9 25 41 52 37 15 9 25 41 52 38 16 9 25 49 62 30 17 9 25 37 47 36 18 9 25 39 49 39 19 9 25 41 52 38 20 9 25 41 52 38 21 9 25 41 52 39 22 9 25 42 53 38 23 9 25 41 52 37 24 9 25 41 52 38 25 9 25 41 52 38 26 9 25 38 48 39 27 9 25 40 51 39 28 9 25 41 52 38 29 9 25 40 51 42 30 9 25 38 48 38 31 9 25 42 53 37

[0143]

[0144] 32 9 25 41 52 37 33 9 25 42 53 38 34 9 25 41 52 37

[0145] 35-1 265 265 11 965

[0146] 35-2

[0147] Totals 265 265 1815

[0148] Normalized

[0149] per mmol

[0150]

[0151] (L / mmol) 0.053 0.363

[0152] Table 6.

[0153] Standard

[0154] Capping B

[0155] Standard reagent Fresh Fresh

[0156] CappingA (concentration ACN ACN Toluene reagent is 20vol% used for Capping used for used for (concentration acetic diluting reaction wash wash total Diluted is 20 vol% 1 anhydride, 30 capping contact after after materials Capping methylimidizole) vol% lutidine) reagents time capping capping charged to Cycle B(mL) (mL) (mL) (mL) (minutes)4 (mL)2 (mL) synthesizer 1 49 10 10 155 5.3 160 50

[0157] 2 45 10 9 77 5.3 90 50

[0158] 3 46 11 9 79 5.3 90 50

[0159] 4 50 10 10 81 5.3 90 50

[0160] 5 47 10 9 79 5.3 90 50

[0161] 6 49 10 10 79 5.3 90 50

[0162] 7 45 11 9 80 5.3 90 50

[0163] 8 47 10 9 79 5.3 90 50

[0164] 9 49 10 10 79 5.3 90 50

[0165] 10 47 10 9 79 5.3 90 50

[0166] 11 47 10 9 79 5.3 90 80

[0167] 12 46 11 9 80 5.3 90 80

[0168] 13 46 11 9 79 5.3 90 80

[0169] 14 46 10 9 78 5.3 90 80

[0170] 15 47 10 9 79 5.3 90 80

[0171] 16 37 12 7 80 5.3 90 80

[0172] 17 45 9 9 74 5.3 90 80

[0173] 18 49 10 10 78 5.3 90 80

[0174] 19 47 10 9 79 5.3 90 80

[0175] 20 47 10 9 79 5.3 90 80

[0176] 21 49 10 10 80 5.3 90 80

[0177] 22 47 11 9 80 5.3 90 110

[0178] 23 46 10 9 78 5.3 90 110

[0179] 24 47 10 9 79 5.3 90 110

[0180] 25 47 10 9 79 5.3 90 110

[0181] 26 49 10 10 77 5.3 90 110

[0182] 27 49 10 10 79 5.3 90 110

[0183] 28 47 10 9 79 5.3 90 110

[0184]

[0185] 29 52 10 10 82 5.3 90 110 30 48 10 10 77 5.3 90 110 31 46 11 9 80 5.3 90 110

[0186] 32 46 10 9 78 5.3 90 110

[0187] 33 47 11 9 80 5.3 90 110

[0188] 34 46 10 9 79 5.3 90 110

[0189] 35-1

[0190] 35-2

[0191] Totals 353 320 2767 3130 2810 16.6 Normalized

[0192] per mmol

[0193]

[0194] (L / mmol) 0.071 0.064 0.553 0.626 0.562 16.6

[0195] 1 Toluene and DCA volumes were calculated based on the DCA solution being 6 vol% DCA in toluene

[0196] 2 The large amount of ACN used for cycle 1 includes the initial volume of ACN used to fill the integrated wash feed bottles.

[0197] 3 Assumed 75 g for the missed data point, and included it in the total calculation at the bottom of the column.

[0198] 4 Does not include contact time with reuse capping reagents.

[0199] Material Composition:

[0200] DCA was 6 vol% in toluene

[0201] Amidites were 0.1M solutions in ACN (mU solvent was 10vol% toluene in ACN) Activator was 0.5 M ETT in ACN

[0202] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent)

[0203] Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[0204] Cap A was 4 vol% 1 methylimidizole in ACN

[0205] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in CAN

[0206] Table 7. BCJ-D00084- eLN 013

[0207] Strand Lpa Sense

[0208] mass of initial resin whole batch (g) 19.6100

[0209] resin loading (umol / g) 255

[0210] Synthesis scale (umol) 5,001

[0211] mass of final resin whole batch (g) 90.1000

[0212] mass gain (g) 70.49

[0213] Mass gain per mmol scale 14.10

[0214] Crude mass yield (by weighing) 0.92

[0215]

[0216] FLP% (homogenized sample)* 0.80

[0217] Mass of resin taken for C&D (g) 0.1483

[0218] Fraction of the whole batch 0.16%

[0219] Mass of oligo in C&D resin sample (g) 0.1160

[0220] 30 wt% NH4OH solutin mass (g) 2.1248

[0221] NH4OH plus oligo mass (g) 2.2408

[0222] Aliquot mass of C&D sol'n for OD (g) 0.1802

[0223] DI water added for dilution (g) 20.44

[0224] NanoDrop measured A260 #1 7.96

[0225] NanoDrop measured A260 #2 7.99

[0226] NanoDrop measured A260 #3 7.99

[0227] NanoDrop measured A260 AVE 7.98

[0228] OD dilution factor 114.46

[0229] Total OD for whole batch 1,243,452

[0230] Mass Na Salt calculated from total OD 53.97

[0231] OD / umol 249

[0232] crude % yield by OD 77%

[0233]

[0234] Purity corrected yield by OD 62%

[0235] BXW- 167-5. PBR comparison. 230 umol scale. This example did not reuse oxidizer, or reuse of coupling reaction material for the wash after oxidation and thiolation. This example uses 2.7 equivalent of oxidizer per cycle. Tables 8-12 show representative data for this SPOS reaction.

[0236] Table 8.

[0237] total toluene

[0238] used in

[0239] Toluene deblock + total DCA ACN in pyridine in wash after chase / wash used in wash after wash after

[0240] 3% DCA deblocking after deblock deblock deblocking deblocking

[0241] Cycle (mL) (mL) (mL)l (mL)l (mL)l (mL)

[0242] 1 53.02 10.6 63.62 1.5906 21.2 10.6

[0243]

[0244] 2 55.14 10.6 65.74 1.6542 21.2 10.6 3 58.32 10.6 68.92 1.7496 21.2 10.6 4 60.43 10.6 71.03 1.8129 21.2 10.6 5 46.98 10.6 57.58 1.4094 21.2 10.6 6 48.57 10.6 59.17 1.4571 21.2 10.6 7 50.89 10.6 61.49 1.5267 21.2 10.6 8 52.48 10.6 63.08 1.5744 21.2 10.6 9 54.08 10.6 64.68 1.6224 21.2 10.6 10 56.51 10.6 67.11 1.6953 21.2 10.6 11 58.1 10.6 68.7 1.743 21.2 10.6 12 59.7 10.6 70.3 1.791 21.2 10.6 13 61.3 10.6 71.9 1.839 21.2 10.6 14 63.62 10.6 74.22 1.9086 21.2 10.6 15 65.2 10.6 75.8 1.956 21.2 10.6 16 66.79 10.6 77.39 2.0037 21.2 10.6 17 69.25 10.6 79.85 2.0775 21.2 10.6 18 70.83 10.6 81.43 2.1249 21.2 10.6 19 72.43 10.6 83.03 2.1729 21.2 10.6 20 74.02 10.6 84.62 2.2206 21.2 10.6 21 76.33 10.6 86.93 2.2899 21.2 10.6 22 77.93 10.6 88.53 2.3379 21.2 10.6 23 79.52 10.6 90.12 2.3856 21.2 10.6 24 81.95 10.6 92.55 2.4585 21.2 10.6 25 83.55 10.6 94.15 2.5065 21.2 10.6 26 85.14 10.6 95.74 2.5542 21.2 10.6 27 86.73 10.6 97.33 2.6019 21.2 10.6 28 89.07 10.6 99.67 2.6721 21.2 10.6 29 90.64 10.6 101.24 2.7192 21.2 10.6 30 92.24 10.6 102.84 2.7672 21.2 10.6 31 94.67 10.6 105.27 2.8401 21.2 10.6 32 96.27 10.6 106.87 2.8881 21.2 10.6 33 97.85 10.6 108.45 2.9355 21.2 10.6 34 99.45 10.6 110.05 2.9835 21.2 10.6 35 101.77 10.6 112.37 3.0531 21.2 10.6 Totals 2531 371 2825.8469 75.9231 742 371

[0245] Normalized

[0246] per mmol

[0247] (L / mmol or

[0248]

[0249] kg / mmol) 12.23 0.328 3.171 1.6

[0250] Table 9.

[0251] ACN in Acid Coupling wash contact contact after time Amidite Activator time coupling Cycle (minutes) Amidite (mL) (mL) (minutes)2 (mL)

[0252]

[0253] 1 6.05 MG 2.34 4.68 10 21.2 2 6.28 MU 2.34 4.68 10 21.2 3 6.64 MC 2.34 4.68 10 21.2 4 6.88 MG 2.34 4.68 10 21.2 5 5.35 MG 2.34 4.68 10 31.2 6 5.53 ADEMA 2.34 4.68 10 31.2 7 5.8 ADEMA 2.34 4.68 10 31.2 8 5.98 ADEMA 2.34 4.68 10 31.2 9 6.16 MG 2.34 4.68 10 31.2 10 6.43 MC 2.34 4.68 10 31.2 11 6.61 MC 2.34 4.68 10 31.2 12 6.79 MG 2.34 4.68 10 31.2 13 6.97 MA 2.34 4.68 10 31.2 14 7.24 MC 2.34 4.68 10 31.2 15 7.41 MG 2.34 4.68 10 31.2 16 7.6 MA 2.34 4.68 10 31.2 17 7.88 MU 2.34 4.68 10 31.2 18 8.05 MC 2.34 4.68 10 31.2 19 8.23 MU 2.34 4.68 10 31.2 20 8.41 MA 2.34 4.68 10 31.2 21 8.67 MC 2.34 4.68 10 31.2 22 8.86 MU 2.34 4.68 10 31.2 23 9.03 MG 2.34 4.68 10 31.2 24 9.31 MG 2.34 4.68 10 31.2 25 9.5 FU 2.34 4.68 10 31.2 26 9.67 FU 2.34 4.68 10 31.2 27 9.85 FC 2.34 4.68 10 31.2 28 10.12 FG 2.34 4.68 10 31.2 29 10.29 MA 2.34 4.68 10 31.2 30 10.47 MA 2.34 4.68 10 31.2 31 10.75 MC 2.34 4.68 10 31.2 32 10.93 MC 2.34 4.68 10 31.2 33 11.11 MG 2.34 4.68 10 31.2 34 11.29 MU 2.34 4.68 10 31.2 35 11.55 MUS 2.34 4.68 10 31.2 Totals 82 164 1052

[0254] Normalized

[0255] per mmol

[0256] (L / mmol or

[0257]

[0258] kg / mmol) 0.35 0.7 4.496 Table 10.

[0259] Oxidation or AON in

[0260] sulfurization wash after

[0261] Xanthane contact oxidation or

[0262] Oxidizer hydride time sulfurization

[0263]

[0264] Cycle (mL) (mL) (minutes) (mL)3 1 12.72 0 3.25 31.8

[0265] 2 12.72 0 3.25 31.8

[0266] 3 12.72 0 3.25 31.8

[0267] 4 12.72 0 3.25 31.8

[0268] 5 12.72 0 3.25 31.8

[0269] 6 12.72 0 3.25 31.8

[0270] 7 12.72 0 3.25 31.8

[0271] 8 12.72 0 3.25 31.8

[0272] 9 12.72 0 3.25 31.8

[0273] 10 12.72 0 3.25 31.8

[0274] 11 12.72 0 3.25 31.8

[0275] 12 12.72 0 3.25 31.8

[0276] 13 12.72 0 3.25 31.8

[0277] 14 12.72 0 3.25 31.8

[0278] 15 12.72 0 3.25 31.8

[0279] 16 12.72 0 3.25 31.8

[0280] 17 12.72 0 3.25 31.8

[0281] 18 12.72 0 3.25 31.8

[0282] 19 12.72 0 3.25 31.8

[0283] 20 12.72 0 3.25 31.8

[0284] 21 12.72 0 3.25 31.8

[0285] 22 12.72 0 3.25 31.8

[0286] 23 12.72 0 3.25 31.8

[0287] 24 12.72 0 3.25 31.8

[0288] 25 12.72 0 3.25 31.8

[0289] 26 12.72 0 3.25 31.8

[0290] 27 12.72 0 3.25 31.8

[0291] 28 12.72 0 3.25 31.8

[0292] 29 12.72 0 3.25 31.8

[0293] 30 12.72 0 3.25 31.8

[0294] 31 12.72 0 3.25 31.8

[0295] 32 12.72 0 3.25 31.8

[0296] 33 12.72 0 3.25 31.8

[0297] 34 12.72 0 3.25 31.8

[0298] 35 12.72 11.66 10.4 31.8

[0299] Totals 445.2 11.66 1113

[0300] Normalized

[0301] per mmol

[0302] (L / mmol or

[0303]

[0304] kg / mmol) 1.903 0.05 4.756

[0305] Table 11.

[0306] Capping ACN used

[0307] reaction in wash total contact after materials CappingA Capping B time capping charged to

[0308]

[0309] Cycle (mL) (mL) (minutes) (mL)3 synthesizer 1 2.65 2.65 2.31 31.8

[0310] 2 2.65 2.65 2.31 31.8

[0311] 3 2.65 2.65 2.31 31.8

[0312] 4 2.65 2.65 2.31 31.8

[0313] 5 2.65 2.65 2.31 31.8

[0314] 6 2.65 2.65 2.31 31.8

[0315] 7 2.65 2.65 2.31 31.8

[0316] 8 2.65 2.65 2.31 31.8

[0317] 9 2.65 2.65 2.31 31.8

[0318] 10 2.65 2.65 2.31 31.8

[0319] 11 2.65 2.65 2.31 31.8

[0320] 12 2.65 2.65 2.31 31.8

[0321] 13 2.65 2.65 2.31 31.8

[0322] 14 2.65 2.65 2.31 31.8

[0323] 15 2.65 2.65 2.31 31.8

[0324] 16 2.65 2.65 2.31 31.8

[0325] 17 2.65 2.65 2.31 31.8

[0326] 18 2.65 2.65 2.31 31.8

[0327] 19 2.65 2.65 2.31 31.8

[0328] 20 2.65 2.65 2.31 31.8

[0329] 21 2.65 2.65 2.31 31.8

[0330] 22 2.65 2.65 2.31 31.8

[0331] 23 2.65 2.65 2.31 31.8

[0332] 24 2.65 2.65 2.31 31.8

[0333] 25 2.65 2.65 2.31 31.8

[0334] 26 2.65 2.65 2.31 31.8

[0335] 27 2.65 2.65 2.31 31.8

[0336] 28 2.65 2.65 2.31 31.8

[0337] 29 2.65 2.65 2.31 31.8

[0338] 30 2.65 2.65 2.31 31.8

[0339] 31 2.65 2.65 2.31 31.8

[0340] 32 2.65 2.65 2.31 31.8

[0341] 33 2.65 2.65 2.31 31.8

[0342] 34 2.65 2.65 2.31 31.8

[0343] 35 2.65 2.65 2.31 31.8

[0344] Totals 92.75 92.75 1113

[0345] Normalized

[0346] per mmol

[0347] (L / mmol or

[0348]

[0349] kg / mmol) 0.396 0.396 4.756 35.133

[0350] 1 This wash includes ACN used for coupling push

[0351] 2 Recycle time

[0352] 3 includes push volume Material Composition:

[0353] DCA solution: 3 volume% DCA in toluene

[0354] Amidites solutions:

[0355] Activator was 0.5 M ETT

[0356] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent) Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[0357] Cap A was 20 vol% 1 methylimidizole in ACN

[0358] Cap B was 20vol% acetic anhydride and 30vol% lutidine in ACN Table 12.

[0359] eLN BXW-D00302- 167-5 Strand ss

[0360] mass of initial resin whole batch (g) 0.9026 resin loading (umol / g) 255 Synthesis scale (umol) 230 mass of final resin whole batch (g) 4.0601 mass gain (g) 3.16 Mass gain per mmol scale 13.72 Crude mass yield (by weighing) 0.90

[0361]

[0362] FLP% (homogenized sample)* 0.75

[0363] Mass of resin taken for C&D (g) 0.0375 Fraction of the whole batch 0.92% Mass of oligo in C&D resin sample (g) 0.0292 30wt% NH4OH solutin mass (g) 0.8450 NH4OH plus oligo mass (g) 0.8742 Aliquot mass of C&D sol’n for OD (g) 0.1992

[0364] DI water added for dilution (g) 20.42 NanoDrop measured A260S1 5.67 NanoDrop measured A26082 5.67 NanoDrop measured A26083 5.71 NanoDrop measured A260 AVEr5.68

[0365] OD dilution factor 103.52 Total OD for whole batch 55,681 Mass Na Salt calculated from total OD 2.42 OD / umol 242 crude % yield by OD 75% Purity corrected yield by OD

[0366]

[0367] 57%

[0368] MW of Na Salt (g / mol) 13986.6 Absorption Factor (OD / mg) 23.04 Theoretical OD / umol (Na Salt) 322.25 ug Na Salt / OD 43.40 theoretical on-resin mass gain / mmol

[0369]

[0370] (DEA treated, DMT-OFF) 15.29 Compare BCJ-013 and REO-059 versus BXW- 167-5 and CWO-006. BCJ-013 and REO-059 implemented the reuse oxidation reaction step. Implementing reused oxidizer reagent solution from cycle to cycle used 20-25% less oxidizer solution. Compare BCJ-013 and REO-059 versus BXW- 167-5. The FBR with reuse oxidation reaction solution used 6X to 13X less fresh ACN than the PBR for washing after oxidation and thiolation reactions. Purity corrected crude yield was higher for FBR (Tables 13-16). Example CWO-006 does not have any reuse steps. It fluidizes the oxidation reaction then pushes the used oxidation reaction solution to waste. Then it washes with fresh solvent and pushes the solvent wash to waste. This embodiment uses more oxidizer solution and it uses more solvent for washing compared to REO-059, for example, but it does have several advantages. It has shorter cycle time, smaller plant footprint because it does not have the extra vessels for the reuse oxidizer material or material for the reuse wash, and less capital cost.

[0371] Table 13.

[0372] total Fresh ACN wash

[0373] after deblocking including

[0374] the amount prefilled to

[0375] total toluene total DCA the counter current wash

[0376] used in used in vessels, and includingthe Total toluene scale deblock deblock amount used to dilute the used in the wash Example synthesizer (mmol) (L / mmol) (L / mmol) pyridine (L / mmol) after deblock BXW-167-5 PBR 0.23 12.2 0.33 3.2 - BCJ-013 FBR 5 4.8 0.31 5.8 - REO-059 FBR 5 2.4 0.27 2.7 0

[0377]

[0378] CWO-006 FBR 5 8.229 0.525 7.9564 2.04666

[0379] Table 14.

[0380] Amidite, note that

[0381] FBR was 0.1 M in

[0382] pyridine the lab and PBR average average Fresh

[0383] used in was 0.2 M, FBR amidite activator ACN wash

[0384] wash after used slightly less equivalent equivalent after

[0385] deblocking molar equiv versus Activator versus coupling Oxidizer

[0386]

[0387] Example (L / mmol) overall (L / mmol) resin (L / mmol) resin (L / mmol) (L / mmol) BXW-167- 5 1.60 0.35 2.0 0.70 10.0 4.50 1.90 BCJ-013 0.00 0.66 1.9 0.69 10.1 0.53 1.51 REO-059 0.02 0.67 1.91 0.69 9.86 0.54 1.51

[0388]

[0389] CWO-006 0.068222 0.666 1.9 0.695 9.93 0.739 1.811

[0390] Table 15.

[0391] Standard Fresh

[0392] Standard Capping B ACN used Fresh Fresh ACN Capping A reagent 20vol% for ACN used wash after reagent 20 vol% acetic diluting for wash Xanthane oxidation or 1 anhydride, 30 capping after hydride sulfurization methylimidizole vol% lutidine reaction capping Example (L / mmol) (L / mmol) (L / mmol) (L / mmol) (L / mmol) (L / mmol) BXW-167- 5 0.050 4.76 0.40 0.40 0.00 4.76 BCJ-013 0.053 0.36 0.07 0.06 0.55 0.63 REO-059 0.043 0.77 0.06 0.06 1.02 0.82

[0393]

[0394] CWO-006 0.043 3.607 0.133 0.13 1.564 6.364

[0395] Table 16.

[0396] total ACN used in

[0397] total the process

[0398] materials including all purity

[0399] charged to reagent solutions crude crude corrected

[0400] synthesizer and washes purity, yield, yield by

[0401] Example (L / mmol) (L / mmol) %FLP OD / umol OD, %

[0402] BXW- 167-5 35.1 18.7 75.2 242.0 57

[0403] BCJ-013 16.6 8.6 80.0 249.0 62

[0404] REO-059 11.6 6.2 81.47 261 66

[0405]

[0406] CWO-006 34.6 20.2 84.9 259 68

[0407] FBR with reuse oxidizer, and reuse of coupling reaction material for the wash after oxidation and thiolation. 0.09 mmol scale.

[0408] A set of 36mer siRNA sense strand oligonucleotide syntheses was performed at a 0.09 mmol scale on a FBR system configured for oxidation / sulfurization solution reuse. Table 17 describes one example of the SPOS integration sequence that was used for the syntheses. This group of experiments was executed identically except for the oxidation steps.

[0409] In Experiment 1, at each oxidation step in the synthesis, 2.2 equivalence of oxidation reagent was used. In nucleotide addition cycle 1, at the end of the oxidation step, the excess oxidation reagent was drained from the reactor and temporarily stored in a dedicated vessel named “reuse ox”. In later nucleotide addition cycles, the material in reuse ox was first fully charged to the reactor to fluidize and mix with the solid phase. The excess was drained and sent to waste. Then 2.2 equivalence of oxidation reagent was charged to the reactor, fluidized and mixed with the solid phase. The excess then sent to the “reuse ox” vessel to be used in the next synthesis cycle.

[0410] In Experiment 2, the oxidation steps were executed the same way as in Experiment 1, except that only 1.8 equivalence of oxidation reagent was used.

[0411] In Experiment 3, the oxidation steps were executed the same way as in Experiment 2, except for one difference. The solid phase was rinsed by 1 mL of fresh ACN at the end of the oxidation step. This portion of ACN can displace some oxidation reagent absorbed by the solid phase, and it was combined with the material in the “reuse ox” vessel.

[0412] In Experiment 4, the oxidation steps were executed the same way as in Experiment 3, except that 2.0 equivalence of oxidation reagent was used.

[0413] In Experiment 5, the oxidation steps doesn’t contain a reuse oxidation step. 2.0 equivalence of oxidation reagent was used each time, mixing with the solid phase for the same duration as the whole oxidation step in Experiment 4, then drained from the reactor and sent to waste.

[0414] Tables 18-20 provide summaries of the experiments and data. The comparison of Experiment 1 and Experiment 2 shows that decreasing fresh oxidation reagent equivalence from 2.2 to 1.8 causes measurable negative impacts on the purity and yield. The comparison of Experiment 3 and Experiment 2 shows that doing a ACN rinse after oxidation step and combining this material with the “reuse ox” material is beneficial. This is due to the solid phase absorbs a significant amount of oxidation reagent which can be partially recovered via a ACN rinse. The comparison of Experiment 4 and Experiment 1 shows that 2.0 equivalence of fresh oxidation reagents can be used to generate good purity and yield. The comparison of Experiment 5 and Experiment 4 shows that without the oxidation reuse approach, 2.0 equivalence of fresh oxidation reagent gives much lower purity and yield. Table 17: summary of the involved experiments and data.

[0415] Reuse ACN Fresh 12 Purity corrected Crude OD Mass gain ELN FLP RRT < 1 RRT > 1

[0416] OX? chase? Equiv. yield by OD (OD / umol) (g / mmol) Experiment

[0417] Yes Yes 2.0 50.8% 28.8% 20.4% 38.8% 246 14.08 5

[0418] Experiment

[0419] No Yes 2.0 77.7% 15.8% 6.5% 59.3% 246 14.32 4

[0420] Experiment

[0421] Yes Yes 1.8 72.7% 20.1% 7.2% 56.2% 249 14.73 3

[0422] Experiment

[0423] No No 1.8 64.5% 29.7% 5.8% 50.4% 252 14.48 2

[0424] Experiment

[0425] Yes No 2.2 77.9% 15.8% 6.3% 61.0% 252 14.53 1

[0426]

[0427] Table 18: Summary of detritylation conditions used in the 0.09 mmol scale experiments.

[0428] Cycle 6% DCA total total total Fresh pyridine in Acid

[0429] (mL) toluene DCA ACN wash wash after contact used in used in after deblocking time deblock + deblock deblocking (mL) (minutes) chase / wash (mL)1 includingthe

[0430] after amount

[0431] deblock prefilled to

[0432] (mL)1 the counter

[0433] current wash

[0434] vessels(mL)2

[0435] 1 10.9 10.2 0.7 46.2 0 14.50

[0436] 2 5.8 5.5 0.3 6.6 0 14.50

[0437] 3 6.2 5.8 0.4 6.6 0 13.97

[0438] 4 6.6 6.2 0.4 6.6 0 14.00

[0439] 5 6.9 6.5 0.4 6.6 0 14.48

[0440] 6 7.3 6.9 0.4 6.6 0 14.50

[0441] 7 7.7 7.3 0.5 9.0 0 14.50

[0442] 8 8.1 7.6 0.5 9.0 0 14.50

[0443] 9 8.5 8.0 0.5 9.0 0 14.98

[0444] 10 8.9 8.3 0.5 9.0 0 14.97

[0445] 11 9.2 8.7 0.6 9.0 0 15.00

[0446] 12 9.6 9.0 0.6 9.0 0 15.48

[0447] 13 10.0 9.4 0.6 11.4 0 15.50

[0448] 14 10.4 9.7 0.6 11.4 0 15.47

[0449] 15 10.8 10.1 0.6 11.4 0 15.50

[0450] 16 11.1 10.5 0.7 11.4 0 16.50

[0451] 17 11.5 10.8 0.7 11.4 0 16.00

[0452] 18 11.9 11.2 0.7 11.4 0 16.50

[0453] 19 12.3 11.5 0.7 13.8 0 17.00

[0454] 20 12.7 11.9 0.8 13.8 0 16.48

[0455]

[0456] 21 13.0 12.3 0.8 13.8 0 16.98

[0457] 22 13.4 12.6 0.8 13.8 0 17.00

[0458] 23 13.8 13.0 0.8 13.8 0 17.00

[0459] 24 14.2 13.3 0.9 13.8 0 17.00

[0460] 25 14.6 13.7 0.9 16.2 0 17.48

[0461] 26 14.9 14.0 0.9 16.2 0 17.48

[0462] 27 15.3 14.4 0.9 16.2 0 18.00

[0463] 28 15.7 14.8 0.9 16.2 0 18.00

[0464] 29 16.1 15.1 1.0 16.2 0 18.00

[0465] 30 16.5 15.5 1.0 16.2 0 18.48

[0466] 31 16.8 15.8 1.0 18.6 0 18.48

[0467] 32 17.2 16.2 1.0 18.6 0 18.48

[0468] 33 17.6 16.5 1.1 18.6 0 19.00

[0469] 34 18.0 16.9 1.1 18.6 0 19.50

[0470] 35 18.4 17.3 1.1 18.6 0 19.50

[0471] 36 18.7 17.6 1.1 18.6 0 19.47 Totals 414 26 493 0

[0472] Normalized 4.60 0.29 5.48 0.00

[0473] per mmol

[0474]

[0475] (L / mmol)

[0476] Table 19: Summary of cou pling conditions used in the 0.09 mmo scale experiments.

[0477] Cycle Amid Amid Amidite, note amidit Activ Activa activat Coupl Fresh ite ite (g) that FBR was e ator tor or ing ACN 0.1 M and equiva (g) (mL) equiva conta wash PBR was 0.2 lent lent Ct after M, FBR used versus versus time coupl less molar resin resin (minu ing equiv overall tes) (mL) (mL)

[0478] 1 MG 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 2 MU 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 3 MC 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 4 MG 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 5 MG 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 6 ADE 1.5 1.8 2.00 1.5 1.8 10.00 10 0.5

[0479] MA

[0480] 7 ADE 1.5 1.8 2.00 1.5 1.8 10.00 10 0.5

[0481] MA

[0482] 8 ADE 1.5 1.8 2.00 1.5 1.8 10.00 15 0.5

[0483] MA

[0484] 9 MG 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 10 MC 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 11 MC 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 12 MG 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5

[0485]

[0486] 13 MA 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 14 MC 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 15 MG 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 16 MA 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 17 MU 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 18 MC 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 19 MU 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 20 MA 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 21 MC 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 22 MU 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 23 MG 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 24 MG 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 25 FU 1.5 1.8 2.00 1.5 1.8 10.00 15 0.5 26 FU 1.5 1.8 2.00 1.5 1.8 10.00 15 0.5 27 FC 1.5 1.8 2.00 1.5 1.8 10.00 15 0.5 28 FG 1.3 1.6 1.80 1.3 1.6 9.00 15 0.5 29 MA 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 30 MA 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 31 MC 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 32 MC 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 33 MG 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 34 MU 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 35 MUS 1.3 1.6 1.80 1.3 1.6 9.00 10 0.5 36

[0487] Totals 47 58 58 77.5 Normalized per 0.64 0.64 0.19 mmol (L / mmol)

[0488]

[0489] Table 20: Summary of capping conditions used in the 0.09 mmol scale experiments.

[0490] Cycle Diluted Diluted Diluted Diluted Capping Fresh Capping Capping Capping Capping reaction ACN A(g) A(mL) B (g) B (mL) contact used for time wash (minutes) after capping (mL)2

[0491] 1 0.7 0.9 0.7 0.9 4 3.8

[0492] 2 0.7 0.9 0.7 0.9 4 3.6

[0493] 3 0.7 0.9 0.7 0.9 4 3.5

[0494] 4 0.7 0.9 0.7 0.9 4 3.4

[0495] 5 0.7 0.9 0.7 0.9 4 3.2

[0496] 6 0.7 0.9 0.7 0.9 4 2.9

[0497] 7 0.7 0.9 0.7 0.9 4 2.7

[0498]

[0499] 8 0.7 0.9 0.7 0.9 4 2.4

[0500] 9 0.7 0.9 0.7 0.9 4 2.3

[0501] 10 0.7 0.9 0.7 0.9 4 2.2

[0502] 11 0.7 0.9 0.7 0.9 4 2.7

[0503] 12 0.7 0.9 0.7 0.9 4 2.7

[0504] 13 0.7 0.9 0.7 0.9 4 2.7

[0505] 14 0.7 0.9 0.7 0.9 4 2.7

[0506] 15 0.7 0.9 0.7 0.9 4 2.7

[0507] 16 0.7 0.9 0.7 0.9 4 2.7

[0508] 17 0.7 0.9 0.7 0.9 4 2.7

[0509] 18 0.7 0.9 0.7 0.9 4 2.7

[0510] 19 0.7 0.9 0.7 0.9 4 2.7

[0511] 20 0.7 0.9 0.7 0.9 4 2.7

[0512] 21 0.7 0.9 0.7 0.9 4 2.7

[0513] 22 0.7 0.9 0.7 0.9 4 3.2

[0514] 23 0.7 0.9 0.7 0.9 4 3.2

[0515] 24 0.7 0.9 0.7 0.9 4 3.2

[0516] 25 0.7 0.9 0.7 0.9 4 3.2

[0517] 26 0.7 0.9 0.7 0.9 4 3.2

[0518] 27 0.7 0.9 0.7 0.9 4 3.2

[0519] 28 0.7 0.9 0.7 0.9 4 3.2

[0520] 29 0.7 0.9 0.7 0.9 4 3.2

[0521] 30 0.7 0.9 0.7 0.9 4 3.2

[0522] 31 0.7 0.9 0.7 0.9 4 3.2

[0523] 32 0.7 0.9 0.7 0.9 4 3.2

[0524] 33 0.7 0.9 0.7 0.9 4 3.2

[0525] 34 0.7 0.9 0.7 0.9 4 3.2

[0526] 35

[0527] 36

[0528] Totals 101.7539 Normalized per mmol 1.13

[0529] (L / mmol)

[0530]

[0531] FBR with reuse oxidizer, and reuse of coupling reaction material for the wash after oxidation and thiolation. 5 mmol scale. Example RE0-D00084-059.

[0532] This example describes SPOS that uses 2.7 equivalent of oxidizer on the first cycle that uses oxidation, then subsequently 2.2 equivalent of oxidizer per cycle and the remaining cycles that use oxidation (Table xx). Attempts were not made to reduce it further. First, the used oxidizer from the previous cycle was charged to the reactor, fluidized with the resin, and then pushed out of the reactor. Destination was waste. Then the fresh oxidizer solution was pushed into the reactor, fluidized with the resin, and then pushed out of the reactor. Destination was the holding vessel for used oxidizer solution that would be reused on the next cycle. Then, the resin bed was washed with coupling reagents solution and coupling reaction wash material from earlier in the cycle. Tables 21-27 show representative data for this SPOS process.

[0533] Table 21: RE0-D00084-059

[0534] reaction destination when push Used material Source

[0535] mode out of reactor in reuse first part of wash after base reuse DCA tank 2 fluidize Waste

[0536] last detrit 2-36 base new acid, 10% 10% acid feed tank fluidize Waste

[0537] 1 flow- base reuse acid reuse DCA tank 1 Waste

[0538] through 2-36 flow- base new acid, 10% 10% acid feed tank reuse DCA tank 1

[0539] through 1-36 flow- base reuse solvent wash WI_DCAtank 1 reuse DCA tank 2

[0540] through 1-36 flow- base reuse solvent wash WI_DCAtank 1 Waste

[0541] through 1-36 base reuse solvent wash WI_DCAtank2 fluidize WI_DCAtank 1

[0542] 1-36 base new solvent chase ACN feed tank fluidize WI_DCAtank 1

[0543] 1-36 new solvent feed zone wall base ACN feed tank fluidize WI_DCAtank 2

[0544] wash 1-36 base reuse solvent wash WI_DCAtank3 fluidize WI_DCAtank 2

[0545] 1-36 base new solvent reactor wall wash ACN feed tank fluidize WI_DCAtank 2

[0546] 1-36 base pyridine washes pyridine feed tank fluidize WI_DCAtank3

[0547] 1-36 new solvent feed zone wall flow- base ACN feed tank WI_DCAtank3

[0548] and resin washes through 1-36 post O / S tank (to be amidite + activator new amidite, activator fluidize used for the wash after base ox / sulf) 1-35 post O / S tank (to be new solvent chase and amidite flow- ACN feed tank used for the wash after base zone wash through

[0549] ox / sulf) 1-35 base Reuse oxidizer Reuse oxidizer tank fluidize Waste

[0550] 1-35 base oxidizer oxidizer feed tank fluidize Reuse oxidizer tank

[0551]

[0552] 2-34 base sulfurization reagent sulfurization feed tank fluidize Waste

[0553] 35 Flow- base New solvent chase ACN feed tank Reuse oxidizer tank through 1-35 flow- base post O / S wash post O / S tank Waste

[0554] through 1-35 Reuse capping reaction flow- base Reuse CAP tank Waste

[0555] solution through 1-34 New Capping reagents and Capping A feed tank,

[0556] dilution solvent for capping Capping B feed tank, fluidize Reuse CAP tank base reaction DIL_CAP tank 1-34 flow- base reuse solvent wash Wl CAP tank DIL_CAP tank

[0557] through 1-34 flow- base new solvent reactor wall wash ACN feed tank Wl CAP tank

[0558] through 1-34 new solvent feed zone wall flow- base ACN feed tank Wl CAP tank

[0559]

[0560] and resin wash through 1-34 note: reuse solvent wash tanks (WI_DCA, Wl CAP, reuse CAP) filled with ACN to begin synthesis

[0561] Table 22: Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse.

[0562] total Fresh ACN wash

[0563] after deblocking

[0564] total

[0565] total DCA including the amount Pyridine pyridine in 10% DCA Neat DCA toluene used in prefilled to the wash after wash after Cycle added to used in

[0566] (mL) deblock counter current wash deblock deblocking reuse (mL) deblock

[0567] (mL)l vessels and pre-run (mL) (mL) (mL)l

[0568] fresh acn wash (mL)

[0569] 2

[0570] 1 342 0.0 308 34 2170 184 0.83

[0571] 2 176 0.0 159 18 238 186 0.84

[0572] 3 186 0.0 167 19 240 183 0.82

[0573] 4 194 0.0 174 19 242 183 0.82

[0574] 5 199 0.0 179 20 243 184 0.83

[0575] 6 230 0.0 207 23 242 183 0.82

[0576] 7 260 0.0 234 26 286 279 1.25

[0577] 8 314 0.0 283 31 276 282 1.27

[0578] 9 312 0.0 281 31 275 280 1.26

[0579] 10 322 0.0 290 32 279 281 1.27

[0580]

[0581] 11 325 0.0 293 33 271 281 1.27 12 337 0.0 303 34 277 282 1.27 13 353 0.0 318 35 314 369 1.66 14 357 0.0 321 36 338 435 1.96 15 352 0.0 317 35 345 436 1.96 16 360 0.0 324 36 351 435 1.96 17 369 0.0 332 37 347 435 1.96 18 380 0.0 342 38 360 435 3.99 19 381 0.0 343 38 347 436 4.00 20 390 0.0 351 39 356 434 3.97 21 390 0.0 351 39 341 435 3.99 22 399 0.0 359 40 344 435 3.99 23 409 0.0 368 41 363 433 3.96 24 426 0.0 383 43 359 434 3.97 25 430 0.0 387 43 363 438 4.01 26 434 0.0 391 43 363 435 3.99 27 449 0.0 404 45 363 435 3.99 28 452 0.0 407 45 374 436 4.00 29 457 0.0 411 46 366 436 4.00 30 471 0.0 424 47 368 435 3.99 31 476 0.0 428 48 368 435 3.99 32 480 0.0 432 48 358 436 4.00 33 482 0.0 434 48 361 434 3.97 34 493 0.0 444 49 361 436 4.00 35-1 494 0.0 445 49 363 434 3.97 35-2 503 0.0 452 50 356 435 3.99 Totals 13385 12047 1339 13567 13169 98

[0582] Normalized

[0583] per mmol

[0584]

[0585] (L / mmol) 2.409 0.268 2.713 2.634 0.020

[0586] Table 23: Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse.

[0587] Amidite,

[0588] note that

[0589] FBRwas 0.1

[0590] Acid amidite

[0591] M and PBR

[0592] contact Amidite equivalent

[0593] Cycle Amidite was 0.2 M,

[0594] time (g)5 FBRused versus

[0595] (minutes) resin

[0596] less molar

[0597] equiv

[0598] overall (mL)

[0599]

[0600] 1 12.1 MG 75.1 92 1.83 2 14.3 MU 76.3 93 1.86 3 14.3 MC 74.4 91 1.81 4 14.2 MG 76.7 94 1.87 5 14.4 MG 75.8 92 1.85 6 14.5 ADEMA 82.5 101 2.01 7 15.0 ADEMA 83.0 101 2.02 8 15.1 ADEMA 82.7 101 2.02 9 15.0 MG 75.6 92 1.84 10 15.0 MC 72.8 89 1.78 11 15.1 MC 72.1 88 1.76 12 15.3 MG 75.9 93 1.85 13 15.3 MA 76.1 93 1.86 14 15.4 MC 71.8 88 1.75 15 15.4 MG 76.1 93 1.86 16 15.6 MA 76.0 93 1.85 17 15.5 MU 76.0 93 1.85 18 15.7 MC 74.7 91 1.82 19 15.8 MU 76.6 93 1.87 20 15.8 MA 76.4 93 1.86 21 15.9 MC 71.8 88 1.75 22 16.1 MU 77.3 94 1.89 23 16.2 MG 74.1 90 1.81 24 16.3 MG 82.2 100 2.00 25 16.5 FU 82.7 101 2.02 26 16.5 FU 83.3 102 2.03 27 16.5 FC 85.7 105 2.09 28 16.5 FG 82.0 100 2.00 29 16.7 MA 85.0 104 2.07 30 16.8 MA 81.4 99 1.99 31 16.8 MC 80.9 99 1.97 32 17.0 MC 81.0 99 1.98 33 17.1 MG 81.7 100 1.99 34 17.2 MU 86.3 105 2.10 35-1 17.2 MUS 85.0 104 2.07

[0601]

[0602] 35-2 17.3

[0603] Totals 2747 3350

[0604] Normalized

[0605] per mmol

[0606] (L / mmol) 0.670 Table 24: Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse.

[0607] activator Coupling Fresh AC N

[0608] Activator Activator equivalent contact wash after Oxidizer Oxidizer Cycle

[0609] (g)5 (mL) versus time coupling (g) (mL)

[0610] resin (minutes) (mL)

[0611] 1 78 96 9.6 10 73.8 266 271

[0612] 2 77 95 9.5 10 75.1 217 221

[0613] 3 77 95 9.5 10 73.8 217 221

[0614] 4 77 95 9.5 10 78.9 217 221

[0615] 5 77 95 9.5 10 75.1 217 221

[0616] 6 83 102 10.2 15 73.8 217 221

[0617] 7 83 102 10.2 15 73.8 217 221

[0618] 8 83 102 10.2 15 80.2 217 221

[0619] 9 78 96 9.6 10 76.3 217 221

[0620] 10 78 96 9.6 10 73.8 217 221

[0621] 11 77 95 9.5 10 75.1 217 221

[0622] 12 78 96 9.6 10 73.8 218 222

[0623] 13 78 96 9.6 10 77.6 217 221

[0624] 14 77 95 9.5 10 73.8 217 221

[0625] 15 77 95 9.5 10 80.2 217 221

[0626] 16 78 96 9.6 10 75.1 217 221

[0627] 17 79 98 9.8 10 77.6 217 221

[0628] 18 77 95 9.5 10 85.2 217 221

[0629] 19 77 95 9.5 10 78.9 217 221

[0630] 20 75 93 9.3 10 76.3 217 221

[0631] 21 77 95 9.5 10 78.9 217 221

[0632] 22 78 96 9.6 10 76.3 217 221

[0633] 23 77 95 9.5 10 80.2 217 221

[0634] 24 83 102 10.2 10 75.1 217 221

[0635] 25 83 102 10.2 15 84.0 217 221

[0636] 26 84 104 10.4 15 73.8 217 221

[0637] 27 83 102 10.2 15 73.8 217 221

[0638] 28 84 104 10.4 15 75.1 217 221

[0639] 29 82 101 10.1 10 73.8 217 221

[0640] 30 83 102 10.2 10 72.5 218 222

[0641]

[0642] 31 83 102 10.2 10 72.5 217 221 32 83 102 10.2 10 82.7 217 221 33 84 104 10.4 10 77.6 217 221

[0643] 34 83 102 10.2 10 76.3 217 221

[0644] 35-1 83 102 10.2 10 75.1

[0645]

[0646] 35-2

[0647] Totals 3449 2675.5725 7430.87 7567.1

[0648] Normalized

[0649] per mmol

[0650] (L / mmol) 0.690 0.535 1.513

[0651] Table 25: Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse.

[0652] Oxidation or

[0653] Xanthane sulfurization Fresh ACN wash after Diluted Diluted Diluted Xanthane

[0654] Cycle hydride contact oxidation or sulfurization CappingA CappingA Capping B hydride (g)

[0655] (mL) time (mL) (g) (mL) (g) (minutes) 4

[0656] 1 9 49.6 37 46 38 2 9 49.6 37 46 36 3 9 50.9 37 47 34 4 9 48.3 38 48 36 5 9 49.6 37 47 36 6 9 49.6 37 47 36 7 9 49.6 37 47 37 8 9 48.3 37 47 37 9 9 49.6 37 47 37 10 9 49.6 37 47 37 11 9 49.6 36 46 38 12 9 49.6 36 46 41 13 9 50.9 37 47 36 14 9 49.6 37 47 36 15 9 104.3 38 48 35 16 9 98.0 38 48 37 17 9 98.0 38 48 36 18 9 96.7 37 47 36

[0657]

[0658] 19 9 100.5 38 48 36 20 9 99.2 37 47 38 21 9 99.2 37 47 37 22 9 100.5 37 47 35 23 9 100.5 38 48 37 24 9 105.6 38 48 36 25 9 100.5 37 47 37 26 9 100.5 38 48 36 27 9 100.5 38 48 35 28 9 100.5 37 47 38 29 9 98.0 38 48 37 30 9 99.2 38 48 35 31 9 100.5 38 48 36 32 9 99.2 37 47 37 33 9 103.1 37 47 37 34 9 98.0 37 47 37 35-1 213.34 213.34 11 1132.3

[0659]

[0660] 35-2

[0661] Totals 213.34 213.34 3829.516539

[0662] Normalized

[0663] per mmol

[0664] (L / mmol) 0.043 0.766

[0665] Table 26: Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse.

[0666] Standard

[0667] Standard Capping B Fresh

[0668] CappingA reagent ACN used Capping Fresh ACN

[0669] Diluted reagent (concentration for reaction used for Cycle Capping (concentration is 20vol% diluting contact wash after

[0670] B(mL) is 20 vol% 1 acetic capping time capping methylimidizole) anhydride, 30 reagents (minutes)4 (mL)2

[0671] (mL) vol%lutidine) (mL)

[0672] (mL)

[0673] 1 48 9.3 9.5 150 7.4 173.0

[0674] 2 45 9.3 9.0 148 7.4 95.4

[0675] 3 42 9.4 8.5 147 7.4 86.5

[0676] 4 45 9.6 9.0 149 7.4 81.4

[0677] 5 45 9.4 9.0 148 7.4 94.1

[0678] 6 45 9.4 9.0 148 7.4 81.4

[0679]

[0680] 7 46 9.4 9.2 149 7.4 82.7 8 46 9.4 9.2 149 7.4 98.0 9 46 9.4 9.2 149 7.4 96.7

[0681] 10 46 9.4 9.2 149 7.4 95.4

[0682] 11 47 9.1 9.4 149 7.4 94.1

[0683] 12 51 9.1 10.2 152 7.4 113.2

[0684] 13 45 9.4 9.0 148 7.4 112.0

[0685] 14 45 9.4 9.0 148 7.4 108.1

[0686] 15 44 9.6 8.7 148 7.4 114.5

[0687] 16 46 9.6 9.2 150 7.4 117.0

[0688] 17 45 9.6 9.0 149 7.4 117.0

[0689] 18 45 9.4 9.0 148 7.4 112.0

[0690] 19 45 9.6 9.0 150 7.4 136.1

[0691] 20 47 9.4 9.5 150 7.4 109.4

[0692] 21 46 9.4 9.2 149 7.4 120.9

[0693] 22 44 9.4 8.7 147 7.4 141.2

[0694] 23 46 9.6 9.2 150 7.4 145.0

[0695] 24 45 9.6 9.0 149 7.4 143.8

[0696] 25 46 9.4 9.2 149 7.4 153.9

[0697] 26 45 9.6 9.0 149 7.4 146.3

[0698] 27 44 9.6 8.7 148 7.4 109.4

[0699] 28 47 9.4 9.5 150 7.4 147.6

[0700] 29 46 9.6 9.2 150 7.4 160.3

[0701] 30 44 9.6 8.8 149 7.4 146.3

[0702] 31 45 9.6 9.0 149 7.4 151.4

[0703] 32 46 9.4 9.2 149 7.4 150.1

[0704] 33 46 9.4 9.2 149 7.4 138.7

[0705] 34 46 9.4 9.2 149 7.4 142.5

[0706] 35-1

[0707] 35-2

[0708] Totals 320 310 5074 4115.7761

[0709] Normalized

[0710] per mmol

[0711]

[0712] (L / mmol) 0.064 0.062 1.015 0.823

[0713] Table 27: Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse. total Fresh

[0714] AC N wash

[0715] after total ACN deblocking used in Fresh including the Fresh ACN total ACN Fresh ACN the ACN used Fresh ACN ACN amount ACN used wash after used in used for process in wash after post

[0716] Cycle prefilled to in amidite oxidation or capping wash after including activator coupling DEA the counter solution sulfurization reagents capping all reagent solution (mL) Wash current wash (mL) (mL) (mL) soutions (mL) vessels and and the acn from washes the pyridine

[0717] wash (mL)2

[0718] 1 2354.1 82 87 74 50 12 173.0 2831.9 2 422.8 84 86 75 50 12 95.4 824.1 3 422.8 82 86 74 51 12 86.5 813.0 4 424.1 84 86 79 48 12 81.4 814.6 5 426.7 83 86 75 50 12 94.1 826.2 6 424.1 91 92 74 50 12 81.4 823.7 7 563.6 91 92 74 50 12 82.7 965.2 8 557.3 91 92 80 48 12 98.0 978.8 9 553.4 83 87 76 50 12 96.7 957.8 10 558.5 80 87 74 50 12 95.4 956.0 11 550.9 79 86 75 50 12 94.1 946.4 12 558.5 83 87 74 50 12 113.2 977.5 13 681.5 84 87 78 51 12 112.0 1104.2 14 771.6 79 86 74 50 12 108.1 1179.5 15 779.2 84 86 80 104 12 114.5 1259.3 16 784.3 83 87 75 98 12 117.0 1256.8 17 780.5 83 88 78 98 12 117.0 1256.5 18 791.2 82 86 85 97 12 112.0 1264.6 19 779.7 84 86 79 101 12 136.1 1277.1 20 786.1 84 83 76 99 12 109.4 1250.5 21 772.1 79 86 79 99 12 120.9 1247.5 22 774.6 85 87 76 101 12 141.2 1276.1 23 791.2 81 86 80 101 12 145.0 1296.1 24 788.6 90 92 75 106 12 143.8 1307.7 25 796.2 91 92 84 101 12 153.9 1329.8 26 793.7 91 93 74 101 12 146.3 1311.3 27 793.7 94 92 74 101 12 109.4 1275.8 28 806.4 90 93 75 101 12 147.6 1325.2 29 798.8 93 91 74 98 12 160.3 1327.6 30 798.8 89 92 73 99 12 146.3 1310.5 31 798.8 89 92 73 101 12 151.4 1316.4 32 789.9 89 92 83 99 12 150.1 1315.2 33 791.2 90 93 78 103 12 138.7 1305.6

[0719]

[0720] 34 793.7 95 92 76 98 12 142.5 1309.6 35-1 792.5 93 92 75 1132 0 0.0 2185.4 35-2 787.4 0 0 0 0 0 0.0 1913 2700.4 Totals 26639 3015 3104 2676 3830 412 4116 1913 45704

[0721] Normalized

[0722] per mmol

[0723]

[0724] (L / mmol) 5.328 0.603 0.621 0.535 0.766 0.082 0.823 0.383 9.14

[0725] 1 Toluene and DC A volumes were calculated based on the DCA solution being 10 vol% DCA in toluene

[0726] 2 The large amount of ACN used for cycle 1 includes the initial volume of ACN used to fill the integrated wash feed bottles.

[0727] 4 Does not include contact time with reuse reagents.

[0728] 5 These values were corrected to remove the extra added to compensate for the dilution cart sample.

[0729] Material Composition:

[0730] DCA was 10 vol% in toluene

[0731] Amidites were 0.1M solutions in ACN (mU solvent was 10vol% toluene in ACN) Activator was 0.5 M ETT in ACN

[0732] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent)

[0733] Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[0734] Cap A was 4 vol% 1 methylimidizole in ACN

[0735] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in CAN M0<toXmO59(UO M0009034.059

[0736] wta ttowema

[0737] WMJ ww mm totoi to

[0738] 245 245 5,005 5.005 gsf toat mm wtxea to 92.5000 92.5000

[0739] 7245 72.25 1443 14.43 0.94 0.94

[0740] aaw

[0741]

[0742] HP* MWW*

[0743] MMt tom* to C&O (g> 04401 0.1401 Haetom of me mtoto totes 045* 045% of m COO mm ww* to 0.1093 03093 30 mt% W4W tobto <gj 2.0750 2.0230 $to togo WI to X1M3 2x1553 «w bf C&O tor CO to AJilS 0.2135 ;£$ warn totod to ttotoxs to 20.43 20.43 wwl>w amtoed 4t2CO »1 9.50 9.55

[0744] me&wmf 4250 «2 04? 9.57 totfto> w mameed A2$0 »3 9.54 9.54 tomBw meetoml A26Q AW 945 9.55 GOOtotoMeeto 94.52 94.52 to$ GO to totoe touh 1..305,M3 1.305.353 MMI W toh totoewS torn* toto GO $05 55.55 oo / wto 251 251

[0745] 01% 31%

[0746]

[0747] to^to eeemteO ytod to 00 w% 53%

[0748] FBR without reuse oxidizer, and without reuse of coupling reaction material for the wash after oxidation and thiolation. 5 mmol scale. All single pass to waste, experiment CWQ-D00084-006.

[0749] This example describes SPOS that uses 2.6 equivalent of oxidizer. Attempts were not made to reduce it further. Tables 28-34 show representative data for this SPOS process.

[0750] Table 28: Example CWO-D00084-006

[0751] destination reaction when push Used material Source

[0752] mode out of in

[0753]

[0754] reactor base new acid, 6% 6% acid feed tank Fluidize Waste

[0755] 1-36 flow- base new acid, 6% 6% acid feed tank Waste through 1-36 solvent wash, acetonitrile, toluene, flow- base Solvent feed tank Waste and pyridine through 1-36 flow- base solvent chase ACN feed tank Waste through 1-36 solvent wash, acetonitrile, toluene, base Solvent feed tank Fluidize Waste and pyridine 1-36 solvent wash, acetonitrile, toluene, flow- base Solvent feed tank Waste and pyridine through 6-36 flow- base solvent washes ACN feed tank Waste through 1-36 base amidite + activator new amidite, activator Fluidize Waste

[0756] 1-35 flow- base solvent chase and washes ACN feed tank Waste through 1-35 base oxidizer oxidizer feed tank Fluidize Waste

[0757] 1-34 base sulfurization sulfurization feed tank Fluidize Waste

[0758] 35 Flow- base solvent chase ACN feed tank Waste through 1-34 Capping reagents and dilution solvent Capping A feed tank, Capping B base Fluidize Waste for capping reaction feed tank, solvent tank 1-34 flow- base solvent chase and wash ACN feed tank Waste

[0759]

[0760] through 1-34

[0761] Table 29. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[0762] total Neat

[0763] ACN wash

[0764] 1 vol% ACN in after

[0765] total deblocking

[0766] total DCA Pyridine in pyridine

[0767] toluene including

[0768] 6 vol% used in 70 / 30 solution

[0769] Cycle DCA(mL) used in the predeblock ACN / Toluene wash after

[0770] deblock run

[0771] (mL)l (mL)l wash after deblocking washing

[0772] deblock (mL) (mL)

[0773] and post

[0774] DEA wash

[0775] (mL)2

[0776] 1 790 742 47 432 302.61 1195

[0777]

[0778] 2 802 754 48 461 322.61 281 3 828 778 50 491 343.48 288 4 851 800 51 520 364.35 298 5 872 820 52 548 383.48 291 6 902 847 54 579 405.22 304 7 926 871 56 605 423.48 309 8 954 897 57 634 443.48 318 9 978 919 59 661 462.61 313 10 1008 947 60 706 493.91 323 11 1031 969 62 729 510.43 314 12 1053 989 63 754 527.83 323 13 1082 1017 65 783 547.83 333 14 1101 1035 66 819 573.04 341 15 1131 1063 68 841 588.70 338 16 1158 1088 69 867 606.96 356 17 1174 1104 70 902 631.30 359 18 1210 1138 73 924 646.96 359 19 1227 1153 74 966 676.52 360 20 1258 1183 76 999 699.13 369 21 1283 1206 77 1022 715.65 382 22 1298 1220 78 1055 738.26 382 23 1326 1246 80 1083 758.26 384 24 1348 1267 81 1114 780.00 385 25 1375 1292 82 1139 797.39 397 26 1395 1311 84 1165 815.65 399 27 1424 1339 85 1193 834.78 405 28 1454 1367 87 1226 858.26 413 29 1468 1380 88 1267 886.96 420 30 1502 1412 90 1287 900.87 425 31 1525 1433 91 1314 920.00 430 32 1557 1464 93 1352 946.09 425 33 1587 1492 95 1373 960.87 435 34 1606 1510 96 1398 978.26 439 35-1 1630 1532 98 1435 1004.35 449

[0779]

[0780] 35-2 1661 1561 100 1468 1027.83 2361 Totals 43773 41147 2626 34111 23877 15905

[0781] Normalized

[0782] per mmol

[0783] (L / mmol) 8.229 0.525 6.822 4.775 3.181 Table 30. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[0784] Amidite,

[0785] note that

[0786] FBR was

[0787] 0.1 M and

[0788] Acid PBRwas amidite

[0789] Cycle contact Amidite Amidite 0.2 M, equivalent

[0790] time (g)5 FBR used versus

[0791] (minutes) less resin

[0792] molar

[0793] equiv

[0794] overall

[0795] (mL)

[0796] 1 6.8 MG 76.5 93 1.87

[0797] 2 6.7 MU 70.8 86 1.73

[0798] 3 6.8 MC 73.2 89 1.79

[0799] 4 6.8 MG 73.1 89 1.78

[0800] 5 6.5 MG 73.9 90 1.80

[0801] 6 6.5 ADEMA 82.1 100 2.00

[0802] 7 6.7 ADEMA 81.8 100 2.00

[0803] 8 6.8 ADEMA 81.7 100 1.99

[0804] 9 6.9 MG 75.5 92 1.84

[0805] 10 6.9 MC 73.8 90 1.80

[0806] 11 7.0 MC 73.2 89 1.79

[0807] 12 7.1 MG 74.1 90 1.81

[0808] 13 7.2 MA 74.9 91 1.83

[0809] 14 7.3 MC 72.5 88 1.77

[0810] 15 7.4 MG 74.0 90 1.80

[0811] 16 7.5 MA 74.8 91 1.82

[0812] 17 7.6 MU 77.8 95 1.90

[0813] 18 7.7 MC 73.1 89 1.78

[0814] 19 7.8 MU 77.6 95 1.89

[0815] 20 7.9 MA 76.8 94 1.87

[0816] 21 8.0 MC 73.5 90 1.79

[0817] 22 8.2 MU 77.8 95 1.90

[0818] 23 8.2 MG 73.8 90 1.80

[0819] 24 8.3 MG 82.9 101 2.02

[0820] 25 8.4 FU 82.6 101 2.01

[0821] 26 8.5 FU 83.4 102 2.03

[0822] 27 8.6 FC 86.1 105 2.10

[0823] 28 8.7 FG 74.7 91 1.82

[0824] 29 8.8 MA 84.0 102 2.05

[0825] 30 8.9 MA 84.8 103 2.07

[0826]

[0827] 31 9.0 MC 81.5 99 1.99 32 9.1 MC 80.8 99 1.97

[0828] 33 9.2 MG 82.2 100 2.00

[0829] 34 9.3 MU 85.2 104 2.08

[0830] 35-1 9.4 MU(S) 86.0 105 2.10

[0831]

[0832] 35-2 9.5

[0833] Totals 2731 3330

[0834] Normalized

[0835] per mmol

[0836] (L / mmol) 0.666

[0837] Table 31. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[0838] Fresh

[0839] activator Coupling

[0840] AC N wash

[0841] Activator Activator equivalent contact Oxidizer Oxidizer Cycle after

[0842] (g)5 (mL) versus time (mL)

[0843] coupling (g)

[0844] resin (minutes)

[0845] (mL)

[0846] 1 77 95 9.5 10 101.8 261 266

[0847] 2 78 96 9.6 10 104.3 261 266

[0848] 3 81 100 10.0 10 105.6 261 266

[0849] 4 78 96 9.6 10 106.9 262 267

[0850] 5 79 98 9.8 10 106.9 262 267

[0851] 6 85 105 10.5 15 104.3 262 267

[0852] 7 84 104 10.4 15 103.1 262 267

[0853] 8 83 102 10.2 15 104.3 262 267

[0854] 9 78 96 9.6 10 108.1 262 267

[0855] 10 78 96 9.6 10 113.2 262 267

[0856] 11 78 96 9.6 10 100.5 262 267

[0857] 12 78 96 9.6 10 104.3 262 267

[0858] 13 78 96 9.6 10 106.9 262 267

[0859] 14 78 96 9.6 10 101.8 262 267

[0860] 15 77 95 9.5 10 100.5 262 267

[0861] 16 77 95 9.5 10 101.8 262 267

[0862] 17 77 95 9.5 10 103.1 262 267

[0863]

[0864] 18 77 95 9.5 10 113.2 261 266 19 77 95 9.5 10 112.0 262 267

[0865] 20 78 96 9.6 10 110.7 259 264

[0866] 21 79 98 9.8 10 100.5 262 267

[0867] 22 77 95 9.5 10 105.6 262 267

[0868] 23 76 94 9.4 10 113.2 261 266

[0869] 24 83 102 10.2 10 103.1 261 266

[0870] 25 83 102 10.2 15 106.9 262 267

[0871] 26 84 104 10.4 15 103.1 262 267

[0872] 27 84 104 10.4 15 101.8 262 267

[0873] 28 83 102 10.2 15 106.9 263 268

[0874] 29 85 105 10.5 10 109.4 262 267

[0875] 30 84 104 10.4 10 103.1 262 267

[0876] 31 84 104 10.4 10 106.9 263 268

[0877] 32 85 105 10.5 10 100.5 254 259

[0878] 33 84 104 10.4 10 104.3 263 268

[0879] 34 83 102 10.2 10 106.9 262 267

[0880] 35-1 86 106 10.6 10 109.4

[0881]

[0882] 35-2

[0883] Totals 3477 3694.656 8894 9057.0

[0884] Normalized

[0885] per mmol

[0886] (L / mmol) 0.695 0.739 1.811

[0887] Table 32. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[0888] Oxidation or Fresh AC N

[0889] Xanthane Xanthane sulfurization wash after Diluted Diluted Diluted Cycle hydride hydride contact oxidation or Capping Capping Capping (g) (mL) time sulfurization A(g) A(mL) B(g)

[0890] (minutes) 4 (mL)

[0891] 1 — — 10 484.7 79 100 77

[0892] 2 — — 10 489.8 78 99 77

[0893] 3 — — 10 488.5 72 91 78

[0894] 4 — — 10 478.4 78 99 77

[0895] 5 — — 10 870.2 78 99 77

[0896]

[0897] 6 — — 10 493.6 78 99 77 7 — — 10 478.4 78 99 77 8 — — 10 489.8 79 100 76 9 — — 10 489.8 79 100 77 10 — — 10 482.2 78 99 77 11 — — 10 477.1 79 100 77 12 — — 10 479.6 79 100 77 13 — — 10 478.4 79 100 77 14 — — 10 483.5 78 99 77 15 — — 10 478.4 78 99 77 16 — — 10 475.8 79 100 77 17 — — 10 473.3 79 100 77 18 — — 10 487.3 82 104 72 19 — — 10 493.6 79 100 75 20 — — 10 491.1 77 97 77 21 — — 10 494.9 77 97 77 22 — — 10 483.5 76 96 77 23 — — 10 487.3 76 96 77 24 — — 10 482.2 76 96 77 25 — — 10 482.2 76 96 77 26 — — 10 486.0 76 96 77 27 — — 10 488.5 76 96 77 28 — — 10 482.2 76 96 77 29 — — 10 493.6 77 97 77 30 — — 10 487.3 76 96 77 31 — — 10 489.8 76 96 76 32 — — 10 489.8 76 96 77 33 — — 10 488.5 75 95 77 34 — — 10 475.8 76 96 77 35-1 212.6 212.6 12 1159.0 —

[0898] --

[0899]

[0900] 35-2

[0901] Totals 212.6 212.6 18034.35

[0902] Normalized

[0903] per mmol

[0904] (L / mmol) 0.043 3.607 Table 33. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[0905] Standard

[0906] Standard Capping B Fresh

[0907] Fresh

[0908] Capping A reagent ACN Capping

[0909] ACN used

[0910] Diluted reagent (concentration used for reaction

[0911] Cycle Capping (concentration is20vol% diluting contact for wash

[0912] after

[0913] B(mL) is20vol%l acetic capping time

[0914] capping methylimidizole) anhydride, 30 reagents (minutes)4

[0915] (mL)

[0916] (mL) vol%lutidine) (mL)

[0917] (mL)

[0918] 1 96 20.0 19.2 232 7.5 461.8

[0919] 2 96 19.7 19.2 231 7.5 494.9

[0920] 3 97 18.2 19.5 226 7.5 521.6

[0921] 4 96 19.7 19.2 231 7.5 549.6

[0922] 5 96 19.7 19.2 231 7.5 570.0

[0923] 6 96 19.7 19.2 231 7.5 606.9

[0924] 7 96 19.7 19.2 231 7.5 636.1

[0925] 8 95 20.0 19.0 231 7.5 653.9

[0926] 9 96 20.0 19.2 232 7.5 685.8

[0927] 10 96 19.7 19.2 231 7.5 707.4

[0928] 11 96 20.0 19.2 232 7.5 751.9

[0929] 12 96 20.0 19.2 232 7.5 785.0

[0930] 13 96 20.0 19.2 232 7.5 801.5

[0931] 14 96 19.7 19.2 231 7.5 825.7

[0932] 15 96 19.7 19.2 231 7.5 863.9

[0933] 16 96 20.0 19.2 232 7.5 900.8

[0934] 17 96 20.0 19.2 232 7.5 917.3

[0935] 18 90 20.7 18.0 230 7.5 944.0

[0936] 19 94 20.0 18.7 230 7.5 988.5

[0937] 20 96 19.5 19.2 230 7.5 1006.4

[0938] 21 96 19.5 19.2 230 7.5 1031.8

[0939] 22 96 19.2 19.2 229 7.5 1064.9

[0940] 23 96 19.2 19.2 229 7.5 1110.7

[0941] 24 96 19.2 19.2 229 7.5 1127.2

[0942] 25 96 19.2 19.2 229 7.5 1155.2

[0943] 26 96 19.2 19.2 229 7.5 1185.8

[0944] 27 96 19.2 19.2 229 7.5 1204.8

[0945] 28 96 19.2 19.2 229 7.5 1248.1

[0946] 29 96 19.5 19.2 230 7.5 1251.9

[0947] 30 96 19.2 19.2 229 7.5 1297.7

[0948]

[0949] 31 95 19.2 19.0 228 7.5 1324.4 32 96 19.2 19.2 229 7.5 1343.5 33 96 19.0 19.2 228 7.5 1388.0

[0950] 34 96 19.2 19.2 229 7.5 1410.9

[0951] 35-1 — — — —

[0952]

[0953] 35-2 - -- - -- Totals 665 652 7818 31818.07

[0954] Normalized

[0955] per mmol

[0956] (L / mmol) 0.133 0.130 1.564 6.364

[0957] Table 34. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[0958] total ACN

[0959] wash after total ACN deblocking used in including Fresh Fresh the Fresh ACN total ACN

[0960] the preACN used ACN ACN used process ACN used wash after used in

[0961] run in wash for wash including Cycle in amidite oxidation or capping

[0962] washing, activator after after all solution sulfurization reagents

[0963] post DEA solution coupling capping reagent (mL) (mL)

[0964] wash and (mL) (mL) soutions acnfrom and pyridine washes wash (mL)

[0965] 1 1497.3 84 86 102 485 26 461.8 2740.7 2 603.8 78 87 104 490 25 494.9 1882.6 3 631.0 80 90 106 489 24 521.6 1941.4 4 662.1 80 87 107 478 25 549.6 1989.2 5 674.8 81 88 107 870 25 570.0 2416.2 6 709.3 90 94 104 494 25 606.9 2124.1 7 732.6 90 93 103 478 25 636.1 2158.7 8 761.5 90 92 104 490 25 653.9 2217.0 9 775.6 83 87 108 490 26 685.8 2254.4 10 817.1 81 87 113 482 25 707.4 2312.9 11 824.7 80 87 101 477 26 751.9 2346.8 12 851.0 81 87 104 480 26 785.0 2413.5 13 881.2 82 87 107 478 26 801.5 2462.4 14 914.0 80 87 102 483 25 825.7 2516.6 15 927.1 81 86 101 478 25 863.9 2562.0 16 963.2 82 86 102 476 26 900.8 2634.8 17 990.1 85 86 103 473 26 917.3 2680.3

[0966]

[0967] 18 1005.7 80 86 113 487 26 944.0 2741.6 19 1036.6 85 86 112 494 25 988.5 2826.8 20 1068.1 84 87 111 491 25 1006.4 2872.4 21 1097.3 81 88 101 495 25 1031.8 2918.2 22 1119.9 85 86 106 483 25 1064.9 2969.8 23 1142.5 81 84 113 487 25 1110.7 3044.1 24 1165.5 91 92 103 482 25 1127.2 3086.2 25 1194.3 91 92 107 482 25 1155.2 3146.5 26 1215.1 92 93 103 486 25 1185.8 3199.8 27 1239.4 95 93 102 489 25 1204.8 3247.3 28 1271.7 82 92 107 482 25 1248.1 3308.1 29 1306.8 92 94 109 494 25 1251.9 3373.6 30 1325.8 93 93 103 487 25 1297.7 3425.2 31 1350.0 89 93 107 490 25 1324.4 3478.8 32 1371.0 89 94 101 490 25 1343.5 3513.0 33 1396.0 90 93 104 489 25 1388.0 3585.2 34 1417.2 94 92 107 476 25 1410.9 3621.6 35-1 1453.5 94 96 109 1159 0 0.0 2911.9 35-2 3389.1 0 0 0 0 0 0.0 3389.1 Totals 39782 2997 3129 3695 18034 858 31818 100313

[0968] Normalized

[0969] per mmol

[0970]

[0971] (L / mmol) 7.956 0.599 0.626 0.739 3.607 0.172 6.364 20.06

[0972] 1 Toluene and DCA volumes were calculated based on the DCA solution being 6 vol% DCA in toluene

[0973] 2 Fresh ACN includes ACN from chase wash, wall wash, fresh ACN washes, and ACN from the pyridine solutions

[0974] 4 Does not include contact time with reuse reagents.

[0975] 5 These values were corrected to remove the extra added to compensate for the dilution cart sample.

[0976] Material Composition:

[0977] DCA was 6 vol% in toluene

[0978] Amidites were 0.1M solutions in ACN (mU solvent was 10vol% toluene in ACN) Activator was 0.5 M ETT in ACN

[0979] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent) Sulfurization reagent was 0.2 M xanthane hydride in pyridine Cap A was 4 vol% 1 methylimidizole in ACN

[0980] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in ACN

[0981] Table 35.

[0982] CW^OOOOOAMIM mci (htowiMC) mMS Of WtW bAUh (g> iwoo 19,9000 bAdWg f . Bl MA& WmaQ W ww TW

[0983] M® gw* UM 14.M ww m$SA w®* < W WwWI 09

[0984]

[0985] awi

[0986] O mo» Ufegn &x CO (g> aim aim FiMtw of tM wMO WW* OvMW ai4% MMI » £&0 mA* wr$e (gj 0400 aion 30 ' wi% 33H4OH mm (g> om 1WB »O pto <$ge mw W44 2.0344

[0987] CW by CO (g> aim aim (X *ww aOOeO (g$ B.M M.B ABO »l I3.4A 13.44 W»0©W mwed ABO Al 13.49 13.49

[0988] ABO A3 1349 1140 W»cOw mbAW^O A3§0 AW 13M 11.40 OOOAvtwfMtb^ B40 T<S^ O0 «Ksf b®t€h 3,mcm

[0989] MM$ m m OHOXMAO <x> MB M.B OO / wmot IBj 259 ery^^WMbyCXS . HO59 SOI!

[0990]

[0991] RAT Ay €WA?t«4 WOO

Claims

CLAIMSWhat is claimed is:

1. A system for synthesizing oligonucleotides, the system comprising:a reactor;an optional feed zone vessel connected to the reactor;a fresh oxidation solution feed vessel;a reuse oxidation solution vessel;a fresh solvent feed vessel; anda post-oxidation / sulfurization (post-O / S) solution vessel.

2. The system of claim 1 further comprising a sulfurization solution feed vessel connected to the feed zone vessel.

3. The system of claim 1 or 2 further comprising a waste module.

4. The system of any one of claims 1 to 3, wherein the oxidation solution feed vessel comprises an oxidation solution comprising iodine, pyridine, and water.

5. The system of any one of claims 1 to 4, wherein the reuse oxidation solution vessel comprises an oxidation solution that has previously been used in at least one solid phase oligonucleotide synthesis (SPOS) cycle.

6. The system of any one of claims 1 to 5, wherein the post-O / S solution vessel comprises a coupling solution that has previously been used in at least one SPOS cycle.

7. The system of claim 6, wherein the coupling solution comprises activated phosphoramidite with excess activator, and solvents acetonitrile (ACN) and, optionally toluene or another co- solvent.

8. The system of any one of claims 1 to 7, wherein the reactor contains a solid substrate, optionally a resin.

9. The system of any one of claims 1 to 8 further comprising an acetonitrile (ACN) feed vessel.

10. The system of any one of claims 1 to 9 further comprising one or more pumps, or no pumps at all.

11. A method for recycling oxidation solution during an oligonucleotide synthesis, the method comprising:(i) during a first cycle of solid phase oligonucleotide synthesis (SPOS) that uses oxidizer, contacting an oligonucleotide linked to a solid support in a reactor with a volume of a first oxidation solution introduced into the reactor;(ii) moving the oxidation solution from the reactor to an oxidation solution reuse vessel to produce a recycled oxidation solution;(iii) completing the first cycle of SPOS that uses oxidizer and beginning a second cycle of SPOS that uses oxidizer; and(iv) during the second cycle of SPOS that uses oxidizer, contacting the oligonucleotide linked to the solid support in the reactor with a volume of the recycled oxidation solution.

12. The method of claim 11, wherein the first oxidation solution comprises iodine in a solvent comprising pyridine and water.

13. The method of claim 12, wherein the oxidation solution comprises iodine solvent at a concentration ranging from about 0.01 M to about 0.1 M.

14. The method of claim 13, wherein the oxidation solution has not previously been introduced into the reactor.

15. The method of any one of claims 11 to 14, wherein during step (i), the oxidation solution and solid support are fluidized.

16. The method of any one of claims 11 to 15, wherein during the second cycle of SPOS that uses oxidizer, the recycled oxidation solution and solid support are fluidized.

17. The method of any one of claims 11 to 16, wherein step (iii) further comprises using a solvent wash to chase residual oxidizer solution from the reactor to the oxidation solution reuse vessel.

18. The method of any one of claims 11 to 17, wherein the second SPOS cycle of step (iii) comprises reusing coupling solution from the same SPOS cycle for washing the reactor and solid support after oxidation or thiolation.

19. The method of any one of claims 11 to 17, wherein the second SPOS cycle of step (iii) comprises reusing coupling wash solution from the same SPOS cycle for washing the reactor and solid support after oxidation or thiolation.

Images