Apparatung and method for producing oligonucleotides including a capping step

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 capping reaction solution recycling. In some embodiments, recycling the capping reaction solution requires a significantly reduced amount of capping solution during the entire SPOS process relative to SPOS processes where the capping 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] APPARATUNG AND METHOD FOR PRODUCING OLIGONUCLEOTIDES INCLUDING A CAPPING STEP

[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 / 711,326, filed October 24, 2024, entitled “CAPPING STEP”, 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 capping reaction solution recycling. In some embodiments, recycling the capping reaction solution requires a significantly reduced amount of certain reagents (e.g., capping solution, acetonitrile (ACN), etc.) during the entire SPOS process relative to SPOS processes where the capping step of each cycle is carried out using only fresh capping solutions. 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, capping methods described by the disclosure are carried out on a fluidized bed reactor (FBR).

[0008] Accordingly, in some aspects, the disclosure provides system for synthesizing oligonucleotides, the system comprising: a reactor; a feed zone vessel in fluid communication with the reactor; a first capping solution feed vessel in fluid communication with the feed zone vessel; a second capping solution feed vessel in fluid communication with the feed zone vessel; a reuse capping solution vessel in fluid communication with both the reactor and the feed zone vessel; a vessel with a solution that is used to dilute the capping reaction in fluid communication with both the feed zone vessel and the reactor; and a reuse wash vessel in fluid communication with both the feed zone vessel and the reactor.

[0009] In some embodiments, the system further comprises an acetonitrile (ACN) solution feed vessel in fluid communication with the feed zone vessel. In some embodiments, the system further comprises a waste module.

[0010] In some embodiments, the first capping solution feed vessel comprises a capping solution comprising methylimidizole and ACN. In some embodiments, the second capping solution feed vessel comprises acetic anhydride, lutidine, and ACN.

[0011] In some embodiments, the reuse capping solution vessel comprises a capping solution that has previously been used in at least one solid phase oligonucleotide synthesis (SPOS) cycle.

[0012] In some embodiments, the vessel with a solution that is used to dilute the capping reaction comprises a wash solution that has previously been used in at least one SPOS cycle wash step. In some embodiments, the dilute solution comprises methylimidizole, acetic anhydride, and lutidine (e.g., because it has been used to wash after capping in a previous cycle), and the solvent ACN.

[0013] In some embodiments, the reuse wash vessel comprises acetonitrile (ACN) that has previously been used in at least one SPOS cycle wash step.

[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 one or more pumps. In some embodiments, the system is configured to move fluids (e.g. reagents) via a pressure differential (e.g., the system does not comprise any pumps).

[0016] In some aspects, the disclosure provides a method for recycling capping solution during an oligonucleotide synthesis, the method comprising performing a first cycle of solid phase oligonucleotide synthesis (SPOS) in a reactor, and moving capping solution used during the first SPOS cycle to a reuse capping solution vessel; performing a second cycle of SPOS comprising a capping step comprising contacting an oligonucleotide linked to a solid support in the reactor with a volume of capping solution introduced into the reactor from the reuse capping solution vessel; moving the capping solution from the reactor to a waste module; moving a first volume of a first new capping solution from a first capping solution feed vessel and a volume of a second new capping solution from a second capping solution feed vessel to a feed zone vessel; moving a volume of solution that is used to dilute the capping reaction from a vessel to the feed zone vessel, and mixing the first new capping solution, second new capping solution, and solution that is used to dilute the capping reaction; moving the capping solution mixture from the feed zone vessel to the reactor and contacting the oligonucleotide linked to the solid support; moving a volume of wash solution from a reuse wash vessel to the reactor and contacting the oligonucleotide linked to the solid support; moving the volume of wash solution from the reactor to the vessel containing the solution that is used to dilute the next capping reaction; moving a volume of acetonitrile (ACN) from an ACN feed vessel to the reactor and contacting the oligonucleotide linked to the solid support; and moving the volume of ACN from the reactor to the reuse wash vessel.

[0017] In some embodiments, the capping agents used in the capping solution (e.g., first capping solution and / or second capping solution) include methylimidazole, acetic anhydride, lutidine, or pyridine. In some embodiments, the capping solution comprises a capping agent(s) and ACN. The capping agents can be used in different combinations and in different orders. In one example, there are two new capping solutions used and the first new capping solution comprises methylimidizole and ACN and the second new capping solution comprises acetic anhydride, lutidine, and ACN. In another example, there are two new capping solutions used and the first new capping solution comprises acetic anhydride and ACN and the second new capping solution comprises methylimidazole and pyridine in ACN. In a further example, there are three new capping solutions used and the first new capping solution comprises methylimidizole and ACN, the second new capping solution comprises acetic anhydride, and the third new capping solution comprises either lutidine or pyridine in ACN.

[0018] In some embodiments, contacting the oligonucleotide with the capping solution introduced into the reactor from the reuse capping solution vessel is performed under flow-through conditions. In this document, “flow-through” means that liquid flows through a packed solid substrate bed, e.g., resin bed supported by a filter. Liquid flows through the packed solid particles and the fdter and out the reactor at a controlled rate.

[0019] In some embodiments, contacting the oligonucleotide with the capping solution mixture is performed under fluidization conditions.

[0020] In some embodiments, contacting of the oligonucleotide with the wash solution from the reuse wash vessel is performed under flow-through conditions. In some embodiments, contacting of the oligonucleotide with the acetonitrile (ACN) is performed under flow-through conditions.

[0021] In some embodiments, the solid substrate comprises a resin.

[0022] In some aspects, the disclosure provides a method for performing a wash step during solid phase oligonucleotide synthesis, the method comprising: in a reactor, contacting a solid phase comprising a linked oligonucleotide with a capping solution; moving the capping solution from the reactor to a waste vessel; and, contacting the solid phase with a distillate wash solution comprising ACN and toluene.

[0023] In some embodiments, the distillate wash solution is produced by distilling waste products of a deblocking reaction and wash that precede step (i). In some embodiments, the distillate wash solution comprises an ACN / toluene azeotrope having a 70% to 30% ACN-to-toluene ratio.

[0024] In some embodiments, the method further comprises (iv) moving the distillate wash solution to a reuse wash vessel.

[0025] BRIEF DESCRIPTION OF THE DRAWING FIG. 1 shows a representative process flow diagram for a fluidized bed reactor (FBR), according to some aspects of the technology.

[0026] FIG. 2 shows a representative schematic diagram for a FBR configured for material reuse, according to some aspects of the technology.

[0027] FIG. 3 shows a representative schematic diagram for a FBR configured for acetonitrile (ACN) reuse, according to some aspects of the technology.

[0028] FIG. 4 shows a representative schematic diagram for a FBR comprising a distillation unit, according to some aspects of the technology.

[0029] FIG. 5 shows a representative schematic diagram for a FBR configured for no material reuse, according to some aspects of the technology.

[0030] FIG. 6 shows a representative process flow diagram for a fully fluidized, reuse SPOS process in an FBR, according to some aspects of the technology. DET AILED DESCRIPTION

[0031] 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.

[0032] The first step is the “deblocking” step, which is generally a detritylation reaction. In some embodiments, a nucleotide (e.g., a nucleotide attached to a solid support, for example a resin) has its 5'-hydroxyl group protected by an acid-labile protection group such as the DMT (4,4'-dimethoxytrityl). This protection group may be removed during a continuous flow of an acid solution or via an addition of an acid in a solvent (also referred to as a “deblocking solution”). In some embodiments, the deblocking solution comprises trichloroacetic acid (TCA) or dichloroacetic acid (DC A). In some embodiments, the acid (e.g., TCA, DC A, etc.) is carried in an inert solvent such as toluene, dichloromethane, or another suitable solvent. The concentration or amount of acid in a deblocking solution may vary. In some embodiments, a deblocking solution comprises between 2% and 30% acid. In some embodiments, a deblocking solution comprises 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% DCA in toluene. The amount of deblocking solution used during each deblocking reaction of SPOS may vary. In some embodiments, the amount of deblocking solution used in each deblocking reaction ranges from about 50-500 ml / mmol. In some embodiments, during this “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.

[0033] 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”.

[0034] 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.

[0035] 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”.

[0036] 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.

[0037] 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.

[0038] In some embodiments, one or more washing steps occurs between of the four SPOS steps. A cycle is defined as the detritylation, coupling, oxidation / thiolation, and capping sequences along with their associated washes. In other words, one nucleotide is added per 4-step “cycle”. In some embodiments, an SPOS cycle comprises one or more washes between deblocking and coupling; one or more washes between coupling and oxidation / sulfurization; one or more washes between oxidation / sulfurization and capping; and, one or more washes between capping and the first step of the next SPOS cycle. In some embodiments, a wash step comprises a multi-stage counter-current wash process. In some embodiments, a bed reactor (e.g., a FBR as described herein) comprises six (6) vessels with wash solvent used for integrated multi-pass washing after deblocking. The first wash step after deblocking is to use the solvent from a first vessel (e.g., vessel “A”) to wash the resin and push to waste. The next step is to use the solvent from a second vessel (e.g., vessel “B”) to push through the resin and push back to refill the first vessel (“A”). Then the solvent from a third vessel “vessel “C”) washes the resin in the reactor and pushes out to refill the second vessel (“B”), and the process continues with the fourth (“D”), fifth (“E”), and sixth (“F”) vessels. After all six wash vessels are used, fresh solvent wash is used to push through the resin and push back to refill the sixth vessel (“F”). 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.

[0039] 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.

[0040] 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.

[0041] While SPOS is the most popular method of oligonucleotide synthesis, there are many challenges that impede the scale up of oligonucleotides from development to large scale manufacturing. For example, the yield and purity of oligonucleotides generally decreases with increasing strand length due to increasing steric hindrance after the addition of each nucleotide. Additionally, the cost of production of oligonucleotides is significantly greater than not only small molecules, but also peptides, which have much cheaper and abundant starting materials and solvents. The synthesis of oligonucleotides via SPOS requires complex phosphoramidite nucleotides and large quantities of expensive solvents and reagents such as acid solutions (e.g., dichloroacetic acid (DC A) solutions) and acetonitrile (ACN). Furthermore, currently employed SPOS systems and methods are not generally efficient. Kilotons of solvents and starting materials are required per kilogram of oligonucleotide produced. 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.

[0042] Fluidized Bed Reactor

[0043] 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).

[0044] 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.

[0045] Turning to the figures, FIG. 1 shows a representative diagram for a fluidized bed reactor (FBR), according to some aspects of the technology. In FIG. 1, small boxes coded by numbers or letters represent material feed vessels (e.g., 1 to 27) or synthesizer operation vessels (e.g., 29 and 30). As used herein, a “feed vessel” refers to a container of suitable volume and geometry for containing a solution used in SPOS. A feed vessel may be made of any suitable material, for example glass, polymers, plastic, metal, etc. In some embodiments, a feed vessel is a carboy. The vessel contents or intended purpose of use are annotated on the diagram. The amidite solutions are contained separately in feed vessels labeled 1-11 and are moved from the vessels into the reactor feed zone by mechanical pumps or by pressure transfers. In some embodiments, a FBR comprises between 1 and 20 amidite feed vessels (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 feed vessels) that are in fluid communication with the feed zone vessel (28 and or 29), which is in turn in fluid communication with a reactor (30) (also referred to herein as a “bed reactor”). As used herein, “in fluid communication” means fluid flow between two regions (e.g., vessels) via a passageway (e.g., a tube or pipe, such as a feed line) connecting the two regions. In some embodiments, two regions (e.g., vessels) are in direct fluid communication with each other, for example when flow of the fluid between the two regions is unobstructed. In some embodiments, two regions (e.g., vessels) are in indirect fluid communication with each other, for example when flow of the fluid between the two region is controlled by an obstruction, for example a valve, disposed in the passageway between the two regions. Generally, ACN (19), toluene (14), and DCA and toluene (e.g., deblocking solution; 13 and 14) are fed from feed vessels to the feed zone vessel (29) or the reactor (30) via pressure push and controlled with automated flow control valves. In some embodiments, a FBR comprises an activator feed vessel (12) in fluid communication with the feed zone vessel (29) or the reactor (30). In some embodiments, the activator feed vessel (12) comprises ethylthiotetrazole (ETT) in ACN.

[0046] 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.

[0047] 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.

[0048] 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 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.

[0049] 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.

[0050] 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 a solution (e.g., 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”.

[0051] 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.

[0052] In some embodiments, a FBR further comprises an evaporator (31) in fluid communication with the reactor (30). In some embodiments, the evaporator is in fluid communication with a waste container. In some embodiments, the evaporator (31) is configured to recover a portion of the acid solution or solvents (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 the deblocking solution or solvents 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.

[0053] FBR systems comprising capping modules as described herein provide several advantages over previously described fluidized bed reactors and packed bed reactors (PBR) used for SPOS. For example, FBR systems described herein offer higher scalability, flexible batch size, and at least an order of magnitude larger maximum scale.

[0054] In some embodiments, use of FBR systems described by the disclosure for SPOS results in higher crude purity and yield of oligonucleotides.

[0055] 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.

[0056] 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 previously described SPOS reactions using OFBR or PBRs.

[0057] 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.

[0058] 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.

[0059] 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 DCA for deblocking.

[0060] In some embodiments, use of FBR systems described by the disclosure for SPOS produce higher batch to batch consistency relative to PBRs.

[0061] Capping Solution Reuse

[0062] Aspects of the disclosure relate to FBRs comprising a modified capping module. In some embodiments, the capping solution recycling methods described herein significantly reduce the amount of capping solution and acetonitrile (ACN) needed to perform synthesis of an entire oligonucleotide using SPOS. In the embodiments, the systems and methods reduce the amount of capping solution used during SPOS by between about 2-fold and about 3-fold relative to previously described SPOS reactions using FBR or PBRs. In the embodiments, the systems and methods reduce the amount of ACN used during SPOS by between about 2-fold and about 6-fold relative to previously described SPOS reactions using FBR or PBRs. In the embodiments, the systems and methods reduce the amount of ACN used during SPOS by at least 2-, at least 3-, at least 4-, at least 5-, at least 6-fold relative to previously described SPOS reactions using FBR or PBRs. Aspects of the disclosure relate to capping modules configured to reuse a capping reaction solution from the previous cycle at the start of the capping process on the current cycle. FIG. 1 depicts a capping module as described by the disclosure, which comprises one or more capping solution feed vessels, depicted as (17) and (18) in FIG. 1 (a further third or more capping solution feed vessel(s) can be included as needed). In some embodiments, the first capping solution feed vessel (17) comprises methylimidizole and ACN. The concentration of methylimidizole in a capping solution may vary. In some embodiments, a capping solution comprises between 4 / 96 v / v and 20 / 80 v / v 1 -Methylimidazole / ACN. In some embodiments, the second capping solution feed vessel (18) comprises acetic anhydride, lutidine, and ACN. The concentration of acetic anhydride in a capping solution may vary. In some embodiments, a capping solution comprises between 2 vol% and 40 vol% acetic anhydride. The concentrations of lutidine and pyridine in a capping solution may vary. In some embodiments, a capping solution comprises between 6 vol% and 60 vol% lutidine. In some embodiments, a capping solution comprises between 6 vol% and 60 vol% pyridine. 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, the feed zone vessel is in fluid communication with a reactor (30). As noted above, example capping agents include methylimidazole, acetic anhydride, lutidine, and pyridine that can be combined in different combinations and capping solutions containing these capping agents and combinations thereof can be added in different orders.

[0063] 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) and / or the feed zone vessel (29). In some embodiments, the capping solution reuse vessel is configured to receive a capping solution mixture from the reactor (30) and push the capping solution mixture to the feed zone vessel (29).

[0064] In some embodiments, a capping module further comprises a vessel with a solution (e.g., reused wash solvent) that is used to dilute a subsequent capping reaction (26), which is in fluid communication with the reactor (30) and / or the feed zone vessel (29). In some embodiments, the vessel (26) is configured to receive wash solvent from the reactor (30) and push it into the feed zone vessel (29) for mixing with new capping solutions from the first (17) and second (18) capping solution feed vessels. In some embodiments, the capping solution mixture is pushed to the reactor (30). In some embodiments, a capping module further comprises a reuse wash vessel (27). In some embodiments, the reuse wash vessel (27) is in fluid communication with the reactor (30) and / or the feed zone vessel (29). In some embodiments, the reuse wash vessel is configured to receive a wash solution (e.g., comprising ACN) from the reactor (30) and push the wash solution to the feed zone vessel (29).

[0065] The following is an illustrative example describing the function of capping modules for capping reagent recycling. The process begins during a non-first SPOS cycle (e.g., 2nd, 3rd, 4th, etc. SPOS cycle) with pushing material from the capping solution reuse vessel (25) through the resin in the reactor (30) and out to a waste module. In some embodiments, this step is performed under flow-through conditions. This accomplishes two goals. The primary goal is to rinse out and scavenge any residual water that might still be associated with the solid substrate (e.g., resin) prior to the subsequent capping reaction. Second, it may also accomplish a portion of the subsequent capping reaction. Next, fresh capping solutions from capping solution feed vessel (17) and capping solution feed vessel (18) are pushed to the feed zone vessel. Solvent from feed vessel (26) is also pushed to the feed zone vessel (29) and mixed with the fresh capping solutions to form a capping solution mixture. In some embodiments, vessel (26) contains wash solvent that has already been used twice in previous cycles. The dilution is done because the fresh capping solution reaction is intended to be fluidized, but the reagent volume of only the fresh capping solutions from capping solution feed vessel (17) and capping solution feed vessel (18) is insufficient to fluidize the solid substrate (e.g., resin) in the reactor (30). Thus, it is diluted with reuse wash solvent, which comes from vessel (26), rather than using fresh solvent from the ACN feed vessel (19), which is how previously-described FBRs functioned. Thus, this method reduces solvent and reduces waste.

[0066] At the end of the designated fluidization time, the used capping solution mixture is pushed out of the reactor (30) and collected in vessel 25. Next, a wash solution from the reuse wash vessel (27) is pushed through the reactor (30) via the feed zone vessel (29) to remove residual capping solution mixture. In some embodiments, the integrated wash solution is contacted to the oligonucleotide on the solid substrate (e.g., resin) in the reactor (30) under flow-through conditions. The used integrated wash solution is then pushed from the reactor (30) to the vessel (26), rather than pushing to the waste module. In the final step, the resin in the reactor (30) is washed again by rinsing the support with fresh ACN solvent, which is pushed from the ACN feed vessel (19) to the reactor (30) and pushed through the solid support under flow-through conditions.

[0067] In some embodiments, an FBR machine is configured to reuse acetonitrile (ACN). FIG.

[0068] 3 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.

[0069] Aspects of the disclosure relate to FBR machines, and methods of using FBR machines, comprising a distillation unit. FIG. 4 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.

[0070] No Reuse SPOS Processes

[0071] 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. FIG. 2 shows a representative schematic diagram for a FBR configured for material reuse, according to some aspects of the technology. 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. 5 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.

[0072] Fully fluidized SPOS in FBR

[0073] 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. 6 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.

[0074] EXAMPLES

[0075] Example 1. RE0-D00084-045.

[0076] This example relates to SPOS reaction integration comprising a reaction with capping solution and ACN recycling. Nothing is recycled, recovered, or reused from one synthesis batch to another. This example describes FBR with reuse of capping reaction material, diluting the capping reaction with solvent that had been used to wash after capping in a previous cycle, washing after capping reaction with wash solvent that had been used to wash after capping in a previous cycle at a 5 mmol scale.

[0077] siRNA manufacturing is typically performed by SPOS in packed bed synthesizers, which are also referred to as packed bed reactors (PBRs). Aspects of this disclosure relate to fluidized bed reactors (FBRs) that enable new SPOS capabilities, for example facilitating integration of a portion of the reagents from each SPOS reaction into the next cycle. Less dilution solvent is required for the SPOS reactions and the required reagent equivalents are reduced by at least 50%. Using this approach for the capping reactions significantly reduces waste and cost for these reactions relative to other SPOS systems such as PBRs and previously described FBR systems.

[0078] An FBR system comprising a capping module configured for reuse of capping solutions and ACN was used to synthesize a 36mer sense strand (SS) oligonucleotide (Lpa) at 5 mmol scale. For comparison, the same 36mer SS oligonucleotide was synthesized using a packed bed reactor (PBR). Table 1 shows one embodiment of material flow during SPOS using the FBR capping solution reuse module.

[0079] Table 1. RE0-D00084-045 Lpa SS:

[0080] destination when push out of material source reaction mode reactor

[0081] reuse acid +neat

[0082] DCA reuse DCA tank fluidize Waste

[0083] reuse acid + neat

[0084] DCA reuse DCA tank flow-through waste

[0085] new acid, 6% 6% acid feed tank flow-through reuse acid tank

[0086] reuse solvent wash WI_DCAtank 1 flow-through Waste

[0087] reuse solvent wash WI_DCAtank 2 flow-through WI_DCAtank 1

[0088] new solvent chase ACN feed tank flow-through WI_DCA tank 1

[0089] reuse solvent wash WI_DCAtank 3 flow-through WI_DCAtank 2

[0090] reuse solvent wash WI_DCAtank 4 flow-through WI_DCAtank 3

[0091] reuse solvent wash WI_DCAtank 5 flow-through WI_DCAtank4

[0092] reuse solvent wash WI_DCAtank 6 flow-through WI_DCAtank 5

[0093] new solvent reactor

[0094] wall wash ACN feed tank flow-through WI_DCAtank 5

[0095] pyridine pyridine feed tank flow-through WI_DCAtank 6

[0096]

[0097] new solvent washes ACN feed tank flow-through WI_DCAtank 6 new amidite, post O / S tank (to be used for the amidite + activator activator fluidize wash after oxidation)

[0098] new solvent chase post O / S tank (to be used for the and washes ACN feed tank flow-through wash after oxidation)

[0099] oxidizer oxidizer feed tank fluidize Waste

[0100] New solvent chase ACN feed tank flow-through Waste

[0101] post O / S wash post O / S tank flow-through Waste

[0102] Reuse capping

[0103] reaction solution Reuse CAP tank flow-through Waste

[0104] New Capping Capping A feed

[0105] reagents and tank, Capping B

[0106] dilution solvent for feed tank, DIL_CAP

[0107] capping reaction tank fluidize Reuse CAP tank

[0108] reuse solvent wash Wl CAP tank flow-through DIL_CAP tank

[0109] new solvent chase

[0110]

[0111] and wash ACN feed tank flow-through Wl CAP tank

[0112] In parallel to the sequence in the above table, when the deblocking is done, neat DCA is added to the reuse DCA vessel and mixed so that the acid concentration in that vessel is higher when it is used on the next cycle. Tables 2-6 below show the amount of reagent used at each step of the SPOS reaction described above. Table 7 provides a description of several characteristics of the SPOS reaction and the resulting synthesized nucleic acid sense strand (“Lpa Sense”) compared to release criteria.

[0113] Table 2.

[0114] total Fresh

[0115] ACN wash

[0116] total after

[0117] toluene deblocking

[0118] used in including the

[0119] deblock + amount

[0120] Neat DCA chase / wash total DCA prefilled to pyridine in added to after used in the counter wash after

[0121] 6% DCA reuse deblock deblock current wash deblocking

[0122] Cycle (mL) (mL) (mL)l (mL)l vessels(mL)2 (mL)

[0123] 1 484 7.4 455 36 2351 3.3

[0124] 2 273 8.3 257 25 372 3.4

[0125] 3 282 8.3 265 25 374 3.3

[0126] 4 298 8.5 280 26 385 3.4

[0127] 5 304 9.4 286 28 389 3.3

[0128] 6 326 10.3 306 30 383 3.3

[0129] 7 350 11.3 329 32 529 4.4

[0130]

[0131] 8 381 12.3 359 35 528 4.3 9 417 12.2 392 37 525 4.3 10 427 12.1 402 38 523 4.3 11 454 13.3 427 41 514 4.4 12 460 13.3 432 41 532 4.4 13 472 14.4 444 43 658 5.2 14 532 14.3 500 46 649 5.3 15 535 14.5 503 47 651 5.3 16 534 15.4 502 47 663 5.2 17 529 15.3 497 47 655 5.3 18 538 16.4 506 49 649 5.2 19 547 16.4 514 49 809 6.4 20 554 16.4 520 50 819 6.5 21 566 17.2 532 51 819 6.5 22 578 17.2 544 52 809 6.5 23 594 17.2 558 53 804 6.5 24 602 18.0 566 54 814 6.6 25 616 18.1 579 55 952 7.4 26 631 18.9 593 57 940 7.4 27 641 19.0 602 57 948 7.4 28 669 18.9 629 59 944 7.5 29 678 20.1 637 61 935 7.5 30 690 20.0 649 61 941 7.5 31 696 20.0 654 62 1083 8.4 32 711 21.1 669 64 1090 8.5 33 720 21.2 677 64 1071 8.4 34 738 22.1 694 66 1053 8.5 35-1 745 22.1 700 67 1062 8.5 35-2 761 0.0 715 46 1060 8.5 Totals 18173 1701 28286 212.4 Normalized

[0132] per mmol

[0133]

[0134] (L / mmol) 3.635 0.340 5.657 0.042

[0135] Table 3.

[0136] Amidite, note that

[0137] FBR was

[0138] 0.1 M and

[0139] PBR was

[0140] 0.2 M, FBR

[0141] Total fresh used less ACN used Acid molar amidite to dilute contact equiv equivalent pyridine time Amidite overall versus Cycle (mL) (minutes) Amidite (g) (mL) resin 1 9.9 9.72 MG 77 87 1.74

[0142]

[0143] 2 10.1 10.27 MU 83 94 1.89 3 10.0 10.18 MC 81 91 1.82 4 10.3 10.18 MG 79 89 1.79 5 10.0 10.47 MG 79 88 1.77 6 10.0 10.52 ADEMA 90 102 2.05 7 13.1 10.83 ADEMA 90 102 2.05 8 12.8 11.37 ADEMA 88 100 1.99 9 12.8 11.72 MG 81 91 1.83 10 13.0 11.48 MC 81 91 1.82 11 13.1 11.02 MC 80 90 1.81 12 13.2 11.20 MG 79 89 1.77 13 15.7 11.32 MA 81 91 1.83 14 15.8 11.80 MC 78 87 1.74 15 15.9 12.08 MG 80 90 1.80 16 15.5 12.03 MA 83 94 1.88 17 15.8 12.03 MU 84 95 1.90 18 15.7 12.03 MC 79 89 1.77 19 19.3 12.22 MU 84 95 1.90 20 19.5 12.25 MA 80 90 1.80 21 19.6 11.98 MC 80 90 1.80 22 19.5 12.15 MU 84 95 1.90 23 19.5 12.27 MG 79 88 1.77 24 19.7 12.45 MG 88 100 2.00 25 22.3 12.45 FU 87 99 1.97 26 22.3 12.70 FU 88 100 1.99 27 22.3 12.75 FC 90 102 2.04 28 22.4 12.88 FG 87 99 1.98 29 22.6 12.98 MA 90 102 2.05 30 22.4 13.10 MA 90 103 2.06 31 25.3 12.78 MC 89 101 2.02 32 25.5 12.88 MC 86 98 1.95 33 25.2 13.02 MG 87 99 1.97 34 25.4 13.20 MU 92 104 2.09 35-1 25.6 13.28 MUS 96 109 2.18 35-2 25.6 13.32

[0144] Totals 637 2950 3335 Normalized

[0145] per mmol

[0146]

[0147] (L / mmol) 0.127 0.667

[0148] Table 4. activator Coupling Fresh ACN

[0149] equivalent contact wash after

[0150] Activator Activator versus time coupling Oxidizer Oxidizer Cycle (g) (mL) resin (minutes) (mL) (g) (mL) 1 83 95 9.46 10 100 261 266 2 82 93 9.35 10 100 262 267 3 83 95 9.52 10 100 261 266 4 82 94 9.42 10 100 262 267 5 82 94 9.37 10 100 262 267 6 88 101 10.10 15 100 261 266 7 88 102 10.16 15 100 261 266 8 90 104 10.39 15 100 260 265 9 82 94 9.41 10 100 261 266 10 83 95 9.48 10 100 261 266 11 83 94 9.45 10 100 262 267 12 83 95 9.51 10 100 261 266 13 83 95 9.46 10 100 262 267 14 83 95 9.48 10 100 262 267 15 81 93 9.30 10 100 261 266 16 82 94 9.39 10 100 262 267 17 82 94 9.39 10 100 262 267 18 81 93 9.30 10 100 262 267 19 81 92 9.23 10 100 262 266 20 83 95 9.46 10 100 262 267 21 82 94 9.41 10 100 262 267 22 82 94 9.42 10 100 262 267 23 82 94 9.42 10 100 261 266 24 91 105 10.50 10 100 261 266 25 91 104 10.42 15 100 262 267 26 91 105 10.51 15 100 262 267 27 90 104 10.41 15 100 261 266 28 88 101 10.13 15 100 261 266 29 89 102 10.20 10 100 262 267 30 88 102 10.15 10 100 262 267 31 88 101 10.13 10 100 262 267 32 91 105 10.51 10 100 262 267 33 89 102 10.20 10 100 262 267 34 91 104 10.45 10 100 262 266 35-1 89 102 10.19 10 100

[0151] 35-2

[0152]

[0153] Totals 3427 3500 8893 9056.008 Normalized

[0154] per mmol

[0155]

[0156] (L / mmol) 0.685 0.700 1.811

[0157] Table 5.

[0158] Oxidation or Fresh ACN

[0159] sulfurization wash after

[0160] Xanthane contact oxidation or Diluted Diluted Diluted Xanthane hydride time sulfurization CappingA CappingA Capping B Cycle hyd ride (g) (mL) (minutes) (mL) (g) (mL) (g) 1 9 25 40 51 37 2 9 25 41 52 36 3 9 25 41 51 37 4 9 25 41 51 37 5 9 25 41 51 37 6 9 25 41 51 37 7 9 25 41 52 37 8 9 25 41 52 37 9 9 25 41 51 36 10 9 25 41 51 37 11 9 25 41 52 37 12 9 25 41 51 37 13 9 25 41 51 37 14 9 25 41 52 35 15 9 25 41 51 37 16 9 25 40 51 37 17 9 25 41 52 36 18 9 25 41 51 36 19 9 25 41 51 37 20 9 25 41 52 37 21 9 25 41 51 36 22 9 25 41 51 36 23 9 25 41 51 37 24 9 25 41 52 36 25 9 25 41 51 36 26 9 25 41 51 36 27 9 25 40 51 37 28 9 25 41 52 36 29 9 25 40 51 36 30 9 25 41 52 36

[0161]

[0162] 31 9 25 40 51 37 32 9 25 41 51 37 33 9 25 41 52 37 34 9 25 41 51 37 35-1 268 268 11 965

[0163] 35-2

[0164] Totals 268 268 1815

[0165] Normalized

[0166] per mmol

[0167]

[0168] (L / mmol) 0.054 0.363

[0169] Table 6.

[0170] Standard

[0171] Capping B

[0172] Standard reagent Fresh Fresh Capping A (concentration ACN ACN

[0173] reagent is 20vol% used for Capping used for (concentration acetic diluting reaction wash total Diluted is 20 vol% 1 anhyd ride, 30 capping contact after materials Capping methylimidizole) vol% lutidine) reagents time capping charged to Cycle B (mL) (mL) (mL) (mL) (minutes)4 (mL)2 synthesizer 1 46.2 10.2 9.2 153 5.3 210

[0174] 2 44.9 10.3 9.0 77 5.3 90

[0175] 3 46.4 10.3 9.3 78 5.3 90

[0176] 4 46.2 10.2 9.2 78 5.3 90

[0177] 5 46.2 10.3 9.2 78 5.3 90

[0178] 6 45.9 10.2 9.2 78 5.3 90

[0179] 7 46.2 10.3 9.2 78 5.3 90

[0180] 8 46.2 10.4 9.2 78 5.3 90

[0181] 9 44.9 10.3 9.0 77 5.3 90

[0182] 10 46.2 10.2 9.2 78 5.3 90

[0183] 11 46.2 10.3 9.2 78 5.3 90

[0184] 12 46.2 10.3 9.2 78 5.3 120

[0185] 13 46.2 10.2 9.2 78 5.3 120

[0186] 14 43.7 10.3 8.7 76 5.3 120

[0187] 15 46.2 10.3 9.2 78 5.3 120

[0188] 16 46.2 10.2 9.2 78 5.3 120

[0189] 17 45.2 10.3 9.0 77 5.3 120

[0190] 18 44.9 10.3 9.0 77 5.3 120

[0191] 19 46.2 10.2 9.2 78 5.3 120

[0192] 20 46.2 10.3 9.2 78 5.3 120

[0193] 21 45.2 10.2 9.0 77 5.3 120

[0194] 22 44.9 10.3 9.0 77 5.3 150

[0195] 23 46.2 10.3 9.2 78 5.3 150

[0196] 24 44.9 10.3 9.0 77 5.3 150

[0197]

[0198] 25 44.9 10.3 9.0 77 5.3 150 26 44.9 10.2 9.0 77 5.3 150

[0199] 27 46.2 10.2 9.2 78 5.3 150

[0200] 28 44.9 10.4 9.0 78 5.3 150

[0201] 29 44.9 10.2 9.0 77 5.3 150

[0202] 30 45.2 10.3 9.0 78 5.3 150

[0203] 31 46.2 10.1 9.2 77 5.3 150

[0204] 32 46.2 10.3 9.2 78 5.3 150

[0205] 33 46.2 10.3 9.2 78 5.3 150

[0206] 34 46.2 10.3 9.2 78 5.3 150

[0207] 35-1

[0208] 35-2

[0209] Totals 349.4 310.8 2715.7 4260 Normalized

[0210] per mmol

[0211]

[0212] (L / mmol) 0.070 0.062 0.543 0.852 15.6

[0213] Material Composition:

[0214] DCA was 6 vol% in toluene

[0215] Amidites were 0.1M solutions in ACN (mU solvent was 10vol% toluene in ACN)

[0216] Activator was 0.5 M ETT in ACN

[0217] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent)

[0218] Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[0219] Cap A was 4 vol% 1 methylimidizole in ACN

[0220] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in ACN

[0221] Table Table 7.

[0222] I REO-D00084-045 (Lab 1 REO-DO00844J45 eLN | UPLC) (Release UPLC) Strand [ Lpa Sense [lpa Sense

[0223] mass of initial resin whole batch (g) | 19.6000 | 19.6000 resin loading (umol / g) 255 255 Synthesis scale (umol} | 4,998 | 4,998 mass of final resin whole batch (g) 90.9000 90.9000 mass gain (g) | 71.30 | 71.30 Mass gain per mmol scale 14.27 14.27 Crude mass yield (by weighing} [ 0.93 | 0.93

[0224]

[0225] FLP% (homogenized sample)* ] 0.8311 ] 0.79

[0226] Mass of resin taken for C&D (g) 0.1613 [ 0.1613 Fraction of the whole batch 0.18% | 0.18% Mass of oligo in C&D resin sample (g) 0.1265 0.1265 30 wt% NH4OH solutln mass (g) 2.1993 | 2.1993 NH4OH plus oligo mass (g) 2.3258 [ 2.3258 Aliquot mass of C&D sol’n for DD (g) 0.3151 | 0.3151 £8 water added for dilution (g) 20.24 | 20.24 t

[0227] NanoDrop measured A250 #1

[0228] NanoDrop measured A26042 14.62 | 14.62 NanoDrop measured A26083r_ 1_4_.55 |r _ 14._55 NanoDrop measured A260 AVf

[0229] OD dilution factor 65.25 | 65.25 Total OD for whole batch 1,248,914 j 1,248,914 Mass Na Sait calculated from total QD 54.21 j 54.21 DD / umol 250 | 250 crude % yield by CD 78% | 78%

[0230]

[0231] Purity corrected yield by OD 64% [ 61%

[0232] Tables 8-11 show reagent amounts used during SPOS of the same sense strand nucleic acid (“Lpa”) using a packed bed reactor (PBR) at a 230 umol scale. Table 12 shows characteristics of the nucleic acid sense strand synthesized using the PBR.

[0233] Table 8.

[0234] totaltoluene

[0235] used in

[0236] Toluene deblock + total DCA ACN in pyridine in wash after chase / wash used in wash after wash after 3% DCA deblocking after deblock deblock deblocking deblocking

[0237]

[0238] Cycle (mL) (mL) (mL)l (mL)l (mL)l (mL) 1 53.02 10.6 63.62 1.5906 21.2 10.6 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

[0239] Normalized

[0240] per mmol

[0241] (L / mmolor

[0242]

[0243] kg / mmol) 12.23 0.328 3.171 1.6

[0244] Table 9. ACN in Acid Coupling wash contact contact after time Amidite Activator time coupling Cycle (minutes) Amidite (mL) (mL) (minutes)2 (mL) 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

[0245] Normalized

[0246] per mmol

[0247] (L / mmolor

[0248]

[0249] kg / mmol) 0.35 0.7 4.496 Table 10.

[0250] Oxidation or AON in sulfurization wash after Xanthane contact oxidation or Oxidizer hyd ride time sulfurization Cycle (mL) (mL) (minutes) (mL)3 1 12.72 0 3.25 31.8 2 12.72 0 3.25 31.8 3 12.72 0 3.25 31.8 4 12.72 0 3.25 31.8 5 12.72 0 3.25 31.8 6 12.72 0 3.25 31.8 7 12.72 0 3.25 31.8 8 12.72 0 3.25 31.8 9 12.72 0 3.25 31.8 10 12.72 0 3.25 31.8 11 12.72 0 3.25 31.8 12 12.72 0 3.25 31.8 13 12.72 0 3.25 31.8 14 12.72 0 3.25 31.8 15 12.72 0 3.25 31.8 16 12.72 0 3.25 31.8 17 12.72 0 3.25 31.8 18 12.72 0 3.25 31.8 19 12.72 0 3.25 31.8 20 12.72 0 3.25 31.8 21 12.72 0 3.25 31.8 22 12.72 0 3.25 31.8 23 12.72 0 3.25 31.8 24 12.72 0 3.25 31.8 25 12.72 0 3.25 31.8 26 12.72 0 3.25 31.8 27 12.72 0 3.25 31.8 28 12.72 0 3.25 31.8 29 12.72 0 3.25 31.8 30 12.72 0 3.25 31.8 31 12.72 0 3.25 31.8 32 12.72 0 3.25 31.8 33 12.72 0 3.25 31.8 34 12.72 0 3.25 31.8 35 12.72 11.66 10.4 31.8 Totals 445.2 11.66 1113

[0251] Normalized

[0252] per mmol

[0253] (L / mmolor

[0254]

[0255] kg / mmol) 1.903 0.05 4.756 Table 11.

[0256] Capping ACN used

[0257] reaction in wash total contact after materials Capping A Capping B time capping charged to Cycle (mL) (mL) (minutes) (mL)3 synthesizer 1 2.65 2.65 2.31 31.8

[0258] 2 2.65 2.65 2.31 31.8

[0259] 3 2.65 2.65 2.31 31.8

[0260] 4 2.65 2.65 2.31 31.8

[0261] 5 2.65 2.65 2.31 31.8

[0262] 6 2.65 2.65 2.31 31.8

[0263] 7 2.65 2.65 2.31 31.8

[0264] 8 2.65 2.65 2.31 31.8

[0265] 9 2.65 2.65 2.31 31.8

[0266] 10 2.65 2.65 2.31 31.8

[0267] 11 2.65 2.65 2.31 31.8

[0268] 12 2.65 2.65 2.31 31.8

[0269] 13 2.65 2.65 2.31 31.8

[0270] 14 2.65 2.65 2.31 31.8

[0271] 15 2.65 2.65 2.31 31.8

[0272] 16 2.65 2.65 2.31 31.8

[0273] 17 2.65 2.65 2.31 31.8

[0274] 18 2.65 2.65 2.31 31.8

[0275] 19 2.65 2.65 2.31 31.8

[0276] 20 2.65 2.65 2.31 31.8

[0277] 21 2.65 2.65 2.31 31.8

[0278] 22 2.65 2.65 2.31 31.8

[0279] 23 2.65 2.65 2.31 31.8

[0280] 24 2.65 2.65 2.31 31.8

[0281] 25 2.65 2.65 2.31 31.8

[0282] 26 2.65 2.65 2.31 31.8

[0283] 27 2.65 2.65 2.31 31.8

[0284] 28 2.65 2.65 2.31 31.8

[0285] 29 2.65 2.65 2.31 31.8

[0286] 30 2.65 2.65 2.31 31.8

[0287] 31 2.65 2.65 2.31 31.8

[0288] 32 2.65 2.65 2.31 31.8

[0289] 33 2.65 2.65 2.31 31.8

[0290] 34 2.65 2.65 2.31 31.8

[0291] 35 2.65 2.65 2.31 31.8

[0292]

[0293] Totals 92.75 92.75 1113 Normalized

[0294] per mmol

[0295] (L / mmolor

[0296]

[0297] kg / mmol) 0.396 0.396 4.756 35.133

[0298] 1 This wash includes ACN used for coupling push

[0299] 2 Recycle time

[0300] 3 includes push volume

[0301] Material Composition:

[0302] DCA solution: 3 volume% DCA in toluene

[0303] Amidites solutions:

[0304] Activator was 0.5 M ETT

[0305] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent) Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[0306] Cap A was 20 vol% 1 methylimidizole in ACN

[0307] Cap B was 20vol% acetic anhydride and 30vol% lutidine in ACN Table 12.

[0308] eLN BXW-D00302-167-5

[0309] Strand SS

[0310] mass of initial resin whole batch (g) 0.9026

[0311] resin loading (umol / g) 25S

[0312] Synthesis scale (umol) 230

[0313] mass of final resin whole batch (g) 4.0601

[0314] mass gain (g) 3.16

[0315] Mass gain per mmol scale 13.72

[0316] Crude mass yield (by weighing) 0.90

[0317]

[0318] FLP% (homogenized sample)* 0.75

[0319] Mass of resin taken for C&D (g) 0.0375

[0320] Fraction of the whole batch 0.92%

[0321] Mass of oligo in C&D resin sample (g) 0.0292

[0322] 30wt% NH4OH solutin mass (g) 0.8450

[0323] NH4OH plus oligo mass (g) 0.8742

[0324] Aliquot mass of C&D sol’n for OD (g) 0.1992

[0325] Di water added for dilution (g) 20.42

[0326] NanoDrop measured A260#l 5.67

[0327] Nano Drop measured A260&2 5.67

[0328] NanoDrop measured A260 S3 5.71

[0329] NanoDrop measured A260 AVE 5.68

[0330] OD dilution factor 103.52

[0331] Total OD for whole batch 55,681

[0332] Mass Na Salt calculated from total OD 2.42

[0333] OD / umol 242

[0334] crude % yield by OD 75%

[0335]

[0336] Purity corrected yield by OD 57%

[0337] MW of Na Salt (g / mol) 13986.6

[0338] Absorption Factor(OD / mg) 23.04

[0339] Theoretical OD / umol (Na Salt) 322.25

[0340] ug Na Salt / OD 43.40

[0341] theoretical on -resin mass gain / mmol

[0342]

[0343] (DEA treated, DMT-OFF) 15.29

[0344] The previous comparison tables (Tables 5, 6, and 11) and the following table 15 show representative data indicating that capping reuse modules and methods used during the SPOS reaction led to a significant reduction in the amount of capping solution reagents and acetonitrile (ACN) used during the capping steps of the synthesis reaction and wash after capping, relative to packed bed reactor (PBR). They also show representative data indicating that capping reuse modules and methods used during the SPOS reaction led to a significant reduction in the amount of total ACN used during the synthesis reaction relative to packed bed reactors (PBRs). Crude purity (%FLP) and crude yield (OD / umol) are higher for 3 FBR syntheses (REO-045 at 83.1%FLP and crude yield 250 OD / umol, REO-059 at 81.5%FLP and crude yield 261 OD / umol, CWO-006 at 84.9%FLP and crude yield 259 OD / umol) than the PBR synthesis (BXW-167-5 at 75.2%FLP and crude yield 242 OD / umol). Fresh ACN used for wash after capping is 5.6-5.8 times lower for FBR with reuse reagents and washes (REO-045, REO-059) than PBR, and 7.5-7.8 times higher for FBR without reuse reagents and washes (CWO-006). Capping A reagent (20 vol% 1 methylimidizole) and Capping B reagent (20vol% acetic anhydride, 30 vol% lutidine) were about 6 times less for FBR with reuse reagent than PBR, and about 2 times less for FBR with reuse reagent than FBR without reuse reagent. On the other hand, advantages of not reusing any reagents or washes at all, like example CWO-006, is that the cycle time is faster, the plant footprint is smaller (less vessels), and the capital cost is lower.

[0345] Example REO-064 uses distillate for the wash after capping. The distillate is generated from the previous cycle. Waste from the deblocking reaction and the wash after deblocking is transferred to a distillation system. The distillate composition is mainly the ACN and toluene azeotrope, which is about 70 / 30 ACN / toluene ratio. This is useful for the wash after capping which requires less fresh solvent. As shown on the table, fresh ACN used for diluting capping reaction and fresh ACN used for wash after capping are lower for this example than for any of the other examples.

[0346] In some embodiments of methods described by the disclosure, the distillate from a deblocking reaction is used for washing the solid substrate (e.g., resin) after the capping reaction of a subsequent SPOS cycle. In some embodiments, the distillate solvents are used 4 times before going to waste. First, the distillate is used as the wash, then it is used as the reuse wash in the next cycle, then it is used as a dilution solvent for the capping reaction on the next cycle, and finally it is used for the reuse capping step on the next cycle, then it goes to waste.

[0347] Table 13. total Fresh ACN wash

[0348] after deblocking including

[0349] the amount prefilled to

[0350] total toluene total DCA the counter current wash

[0351] used in used in vessels, and including the 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 - REO-045 FBR 5 3.6 0.34 5.8 - REO-059 FBR 5 2.4 0.27 2.7 0 CWO-006 FBR 5 8.229 0.525 7.9564 2.04666

[0352]

[0353] REO-064 FBR 5 4.613 0.294 3.701 Used Distillate

[0354] Table 14.

[0355] Amidite, note that

[0356] FBR was 0.1 M in

[0357] pyridine the lab and PBR average average Fresh

[0358] used in was 0.2 M, FBR amidite activator ACN wash

[0359] wash after used slightly less equivalent equivalent after

[0360] deblocking molar equiv versus Activator versus coupling Oxidizer 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 REO-045 0.04 0.67 1.9 0.69 9.8 0.70 1.81 REO-059 0.02 0.67 1.91 0.69 9.86 0.54 1.51 CWO-006 0.068 0.67 1.9 0.695 9.93 0.739 1.811

[0361]

[0362] REO-064 NA 0.67 1.91 0.687 9.81 0.546 1.513

[0363] Table 15.

[0364] Standard Fresh

[0365] Standard CappingB ACN used Fresh

[0366] Fresh ACN CappingA reagent 20vol% for ACN used

[0367] 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)

[0368] BXW-167- 5 0.050 4.76 0.40 0.40 0.00 4.76

[0369] REO-045 0.054 0.36 0.07 0.06 0.54 0.85

[0370] REO-059 0.043 0.77 0.06 0.06 1.02 0.82

[0371] CWO-006 0.043 3.61 0.13 0.13 1.56 6.36

[0372]

[0373] REO-064 0.043 0.76 0.06 0.06 1.01 0.42 Table 16.

[0374] total ACN used in

[0375] total the process

[0376] materials including all purity

[0377] charged to reagent solutions crude crude corrected

[0378] synthesizer and washes purity, yield, yield by

[0379] Example (L / mmol) (L / mmol) %FLP OD / umol OD, %

[0380] BXW-167-5 35.1 18.7 75.2 242.0 57

[0381] REO-045 15.6 8.9 83.1 250 64

[0382] REO-059 11.6 6.2 81.5 261 66

[0383] CWO-006 34.6 20.2 84.9 259 68

[0384]

[0385] REO-064 14.4 6.8 70.6 249 55

[0386] Example 2. RE0-D00084-059.

[0387] This example relates to SPOS reaction integration comprising a reaction with capping solution and ACN recycling. Nothing is recycled, recovered, or reused from one synthesis batch to another. This example describes FBR with reuse of capping reaction material, diluting the capping reaction with solvent that had been used to wash after capping in a previous cycle, washing after capping reaction with wash solvent that had been used to wash after capping in a previous cycle at a 5 mmol scale.

[0388] An FBR system comprising a capping module configured for reuse of capping solutions and ACN was used to synthesize a 36mer sense strand (SS) oligonucleotide at 5 mmol scale.

[0389] Table 17 shows one embodiment of material flow during SPOS using the FBR capping solution reuse module.

[0390] Table 17. RE0-D00084-059 Lpa SS:

[0391] reaction destination when push Used material Source

[0392] mode out of reactor In reuse first part of wash after base reuse DCA tank 2 fluidize Waste

[0393] last detrit 2-36 base new acid, 10% 10% acid feed tank fluidize Waste

[0394] 1 flow- base reuse acid reuse DCA tank 1 Waste

[0395] through 2-36 flow- base new acid, 10% 10% acid feed tank reuse DCA tank 1

[0396]

[0397] through 1-36 flow- base reuse solvent wash WI_DCAtank 1 reuse DCA tank 2

[0398] through 1-36 flow- base reuse solvent wash WI_DCAtank 1 Waste

[0399] through 1-36 base reuse solvent wash WI_DCAtank 2 fluidize WI_DCAtank 1

[0400] 1-36 base new solvent chase ACN feed tank fluidize WI_DCAtank l

[0401] 1-36 new solvent feed zone wall base ACN feed tank fluidize WI_DCAtank 2

[0402] wash 1-36 base reuse solvent wash WI_DCAtank 3 fluidize WI_DCAtank 2

[0403] 1-36 base new solvent reactor wall wash ACN feed tank fluidize WI_DCAtank 2

[0404] 1-36 base pyridine washes pyridine feed tank fluidize WI_DCA tank 3

[0405] 1-36 new solvent feed zone wall flow- base ACN feed tank WI_DCAtank 3

[0406] 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

[0407] ox / sulf) 1-35 base Reuse oxidizer Reuse oxidizer tank fluidize Waste

[0408] 1-35 base oxidizer oxidizer feed tank fluidize Reuse oxidizer tank

[0409] 2-34 base sulfurization reagent sulfurization feed tank fluidize Waste

[0410] 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

[0411] through 1-35 Reuse capping reaction flow- base Reuse CAP tank Waste

[0412] solution through 1-34 New Capping reagents and Capping A feed tank,

[0413] 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

[0414] through 1-34 flow- base new solvent reactor wall wash ACN feed tank Wl CAP tank

[0415] through 1-34 new solvent feed zone wall flow- base ACN feed tank Wl CAP tank

[0416]

[0417] and resin wash through 1-34 note: reuse solvent wash tanks (WI_DCA, Wl CAP, reuse CAP) filled with ACN to begin synthesis Table 18. Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse.

[0418] total Fresh ACN wash

[0419] after deblocking

[0420] total

[0421] total DCA includingthe amount Pyridine pyridine in Neat DCA toluene

[0422] 10% DCA used in prefilled to the wash after wash after Cycle added to used in

[0423] (mL) deblock counter current wash deblock deblocking reuse (mL) deblock

[0424] (mL)l vessels and pre-run (mL) (mL) (mL)l

[0425] fresh acn wash (mL)

[0426] 2

[0427] 1 342 0.0 308 34 2170 184 0.83 2 176 0.0 159 18 238 186 0.84 3 186 0.0 167 19 240 183 0.82 4 194 0.0 174 19 242 183 0.82 5 199 0.0 179 20 243 184 0.83 6 230 0.0 207 23 242 183 0.82 7 260 0.0 234 26 286 279 1.25 8 314 0.0 283 31 276 282 1.27 9 312 0.0 281 31 275 280 1.26 10 322 0.0 290 32 279 281 1.27 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

[0428]

[0429] 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

[0430] Normalized

[0431] per mmol

[0432]

[0433] (L / mmol) 2.409 0.268 2.713 2.634 0.020

[0434] Table 19. Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse.

[0435] Amidite,

[0436] note that

[0437] FBRwasO.l

[0438] Acid amidite

[0439] Mand PBR

[0440] contact Amidite equivalent

[0441] Cycle Amidite was 0.2 M,

[0442] time (g)5 versus

[0443] FBRused

[0444] (minutes) resin

[0445] less molar

[0446] equiv

[0447] overall (mL)

[0448] 1 12.1 MG 75.1 92 1.83

[0449] 2 14.3 MU 76.3 93 1.86

[0450] 3 14.3 MC 74.4 91 1.81

[0451] 4 14.2 MG 76.7 94 1.87

[0452] 5 14.4 MG 75.8 92 1.85

[0453] 6 14.5 ADEMA 82.5 101 2.01

[0454] 7 15.0 ADEMA 83.0 101 2.02

[0455] 8 15.1 ADEMA 82.7 101 2.02

[0456] 9 15.0 MG 75.6 92 1.84

[0457] 10 15.0 MC 72.8 89 1.78

[0458] 11 15.1 MC 72.1 88 1.76

[0459] 12 15.3 MG 75.9 93 1.85

[0460] 13 15.3 MA 76.1 93 1.86

[0461] 14 15.4 MC 71.8 88 1.75

[0462] 15 15.4 MG 76.1 93 1.86

[0463] 16 15.6 MA 76.0 93 1.85

[0464]

[0465] 17 15.5 MU 76.0 93 1.85 18 15.7 MC 74.7 91 1.82

[0466] 19 15.8 MU 76.6 93 1.87

[0467] 20 15.8 MA 76.4 93 1.86

[0468] 21 15.9 MC 71.8 88 1.75

[0469] 22 16.1 MU 77.3 94 1.89

[0470] 23 16.2 MG 74.1 90 1.81

[0471] 24 16.3 MG 82.2 100 2.00

[0472] 25 16.5 FU 82.7 101 2.02

[0473] 26 16.5 FU 83.3 102 2.03

[0474] 27 16.5 FC 85.7 105 2.09

[0475] 28 16.5 FG 82.0 100 2.00

[0476] 29 16.7 MA 85.0 104 2.07

[0477] 30 16.8 MA 81.4 99 1.99

[0478] 31 16.8 MC 80.9 99 1.97

[0479] 32 17.0 MC 81.0 99 1.98

[0480] 33 17.1 MG 81.7 100 1.99

[0481] 34 17.2 MU 86.3 105 2.10

[0482] 35-1 17.2 MUS 85.0 104 2.07

[0483]

[0484] 35-2 17.3

[0485] Totals 2747 3350

[0486] Normalized

[0487] per mmol

[0488] (L / mmol) 0.670

[0489] Table 20. Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse.

[0490] activator Coupling Fresh ACN

[0491] Activator Activator equivalent contact wash after Oxidizer Oxidizer Cycle

[0492] (g)5 (mL) versus time coupling (g) (mL)

[0493] resin (minutes) (mL)

[0494] 1 78 96 9.6 10 73.8 266 271

[0495] 2 77 95 9.5 10 75.1 217 221

[0496] 3 77 95 9.5 10 73.8 217 221

[0497] 4 77 95 9.5 10 78.9 217 221

[0498]

[0499] 5 77 95 9.5 10 75.1 217 221 6 83 102 10.2 15 73.8 217 221

[0500] 7 83 102 10.2 15 73.8 217 221

[0501] 8 83 102 10.2 15 80.2 217 221

[0502] 9 78 96 9.6 10 76.3 217 221

[0503] 10 78 96 9.6 10 73.8 217 221

[0504] 11 77 95 9.5 10 75.1 217 221

[0505] 12 78 96 9.6 10 73.8 218 222

[0506] 13 78 96 9.6 10 77.6 217 221

[0507] 14 77 95 9.5 10 73.8 217 221

[0508] 15 77 95 9.5 10 80.2 217 221

[0509] 16 78 96 9.6 10 75.1 217 221

[0510] 17 79 98 9.8 10 77.6 217 221

[0511] 18 77 95 9.5 10 85.2 217 221

[0512] 19 77 95 9.5 10 78.9 217 221

[0513] 20 75 93 9.3 10 76.3 217 221

[0514] 21 77 95 9.5 10 78.9 217 221

[0515] 22 78 96 9.6 10 76.3 217 221

[0516] 23 77 95 9.5 10 80.2 217 221

[0517] 24 83 102 10.2 10 75.1 217 221

[0518] 25 83 102 10.2 15 84.0 217 221

[0519] 26 84 104 10.4 15 73.8 217 221

[0520] 27 83 102 10.2 15 73.8 217 221

[0521] 28 84 104 10.4 15 75.1 217 221

[0522] 29 82 101 10.1 10 73.8 217 221

[0523] 30 83 102 10.2 10 72.5 218 222

[0524] 31 83 102 10.2 10 72.5 217 221

[0525] 32 83 102 10.2 10 82.7 217 221

[0526] 33 84 104 10.4 10 77.6 217 221

[0527] 34 83 102 10.2 10 76.3 217 221

[0528] 35-1 83 102 10.2 10 75.1

[0529]

[0530] 35-2

[0531] Totals 3449 2675.5725 7430.87 7567.1

[0532] Normalized

[0533] per mmol

[0534] (L / mmol) 0.690 0.535 1.513

[0535] Table 21. Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse. Oxidation or

[0536] Xanthane sulfurization Fresh ACN wash after Diluted Diluted Diluted Cycle Xanthane

[0537] hydride (g) hydride contact oxidation or sulfurization CappingA CappingA Capping B (mL) time (mL) (g) (mL) (g) (minutes)4

[0538] 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 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

[0539] 213.34

[0540]

[0541] 35-1 213.34 11 1132.3 | 35-2 | | | | | | |

[0542]

[0543] Totals 213.34 213.34 3829.516539

[0544] Normalized

[0545] per mmol

[0546] (L / mmol) 0.043 0.766

[0547] Table 22. Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse.

[0548] Standard

[0549] Standard Capping B Fresh

[0550] CappingA reagent ACN used Capping Fresh ACN

[0551] Diluted reagent (concentration for reaction used for

[0552] Cycle Capping (concentration is 20vol% diluting contact wash after

[0553] B(mL) is20vol%l acetic capping time capping

[0554] methyllmldizole) anhydride, 30 reagents (minutes)4 (mL)2

[0555] (mL) vol% lutidine) (mL)

[0556] (mL)

[0557] 1 48 9.3 9.5 150 7.4 173.0

[0558] 2 45 9.3 9.0 148 7.4 95.4

[0559] 3 42 9.4 8.5 147 7.4 86.5

[0560] 4 45 9.6 9.0 149 7.4 81.4

[0561] 5 45 9.4 9.0 148 7.4 94.1

[0562] 6 45 9.4 9.0 148 7.4 81.4

[0563] 7 46 9.4 9.2 149 7.4 82.7

[0564] 8 46 9.4 9.2 149 7.4 98.0

[0565] 9 46 9.4 9.2 149 7.4 96.7

[0566] 10 46 9.4 9.2 149 7.4 95.4

[0567] 11 47 9.1 9.4 149 7.4 94.1

[0568] 12 51 9.1 10.2 152 7.4 113.2

[0569] 13 45 9.4 9.0 148 7.4 112.0

[0570] 14 45 9.4 9.0 148 7.4 108.1

[0571] 15 44 9.6 8.7 148 7.4 114.5

[0572] 16 46 9.6 9.2 150 7.4 117.0

[0573] 17 45 9.6 9.0 149 7.4 117.0

[0574] 18 45 9.4 9.0 148 7.4 112.0

[0575] 19 45 9.6 9.0 150 7.4 136.1

[0576] 20 47 9.4 9.5 150 7.4 109.4

[0577] 21 46 9.4 9.2 149 7.4 120.9

[0578] 22 44 9.4 8.7 147 7.4 141.2

[0579]

[0580] 23 46 9.6 9.2 150 7.4 145.0 24 45 9.6 9.0 149 7.4 143.8

[0581] 25 46 9.4 9.2 149 7.4 153.9

[0582] 26 45 9.6 9.0 149 7.4 146.3

[0583] 27 44 9.6 8.7 148 7.4 109.4

[0584] 28 47 9.4 9.5 150 7.4 147.6

[0585] 29 46 9.6 9.2 150 7.4 160.3

[0586] 30 44 9.6 8.8 149 7.4 146.3

[0587] 31 45 9.6 9.0 149 7.4 151.4

[0588] 32 46 9.4 9.2 149 7.4 150.1

[0589] 33 46 9.4 9.2 149 7.4 138.7

[0590] 34 46 9.4 9.2 149 7.4 142.5

[0591] 35-1

[0592] 35-2

[0593] Totals 320 310 5074 4115.7761

[0594] Normalized

[0595] per mmol

[0596]

[0597] (L / mmol) 0.064 0.062 1.015 0.823

[0598] Table 23. Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse.

[0599] >tal Fresh ACN

[0600] total ACN wash after Fresh

[0601] used in deblocking total ACN Fresh

[0602] Fresh ACN the including the ACN used ACN used Fresh ACN ACN used ACN wash after process ount prefilled to in in wash after used in for post Cycle oxidation or including counter current amidite activator coupling capping wash DEA sulfurization all reagent ish vessels and solution solution (mL) reagents after Wash

[0603] (mL) soutions e acn from the (mL) capping (mL) and idinewash (mL) (mL)

[0604] washes 2

[0605] 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

[0606]

[0607] 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 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

[0608] Normalized

[0609] per mmol

[0610]

[0611] (L / mmol) 5.328 0.603 0.621 0.535 0.766 0.082 0.823 0.383 9.14

[0612] 1 Toluene and DCA volumes were calculated based on the DCA solution being 10 vol% DCA in toluene

[0613] 2 The large amount of ACN used for cycle 1 includes the initial volume of ACN used to 5 fill the integrated wash feed bottles.

[0614] 4 Does not include contact time with reuse reagents.

[0615] 5 These values were corrected to remove the extra added to compensate for the dilution cart sample.

[0616] Material Composition:

[0617] 0 DCA was 10 vol% in toluene

[0618] Amidites were 0.1M solutions in ACN (mU solvent was 10vol% toluene in ACN) Activator was 0.5 M ETT in ACN Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent) Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[0619] Cap A was 4 vol% 1 methylimidizole in ACN

[0620] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in CAN

[0621] Table 24. Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse.

[0622] RE0-DGOQ84 -059 (Lab REODiXX)34O59 etN _ _ UPLC) _ (Release UPLQ Strand _ kpa Sense _ tpa Sense _

[0623] mass of Initial resin whole batch (g) | 20-3.500 2O.35CG resin loading (umnl / g) j 246 246 Synthesis scale (nmol} 5,006 5,006 mass of final resin whole hatch (g) | 92^6000 92J506G mass gain (g) 72.25 72,25 Mass gain per mmol scale | 14.43 14.43 Crude mass (by weighing! | 0.94 0x54

[0624]

[0625] FLP% (homogenized sample)* | 0.3610 0.3348 Mass of resin taken for C&D (g) 0-1401 0.1401 Fraction of the whole batch | 0.15% 0.15% Mass of clsgo in C&D resin sample (g) | 0-1093 0.1093 30 wt% NH4OH solutin mass (g) 2-0770 2.0770 NH4OH plus oligo mass (g) | 2-1863 24363 Aliquot mass of C&D sohn for OD (g) | 0-2123 0.2135 Di water added for dilution (g) | 2043 20.43 NanoDrsp measured A260&1 | 9.56 9.56 NanoDrop measured A26O #2 j 9.57 9.37 NanoDrop measured A260 #3 | 9.54 9.54 NanoDrop measured A26O AVE j 9.56 9.38 DD dilution factor | 94.52 94.52 Total OD for whole batch j 1,305,363 1,305,363 Mass Na Salt calculated from total OD | 55.66 56x86 OD / umol | 281 261 crude % yield by OD j 81% 81%

[0626]

[0627] Purity corrected yield by OD | 70% 88%

[0628] Example 3. CWO-D00084-006. This example relates to SPOS process comprising a reaction with capping solution and ACN washing but no recycling, only single pass of fresh reagents and solvents through the reactor and flowing out to waste. An FBR system comprising a capping module and ACN was used to synthesize a 36mer sense strand (SS) oligonucleotide at 5 mmol scale. Table 25 shows one embodiment of material flow during SPOS using no FBR capping solution reuse module.

[0629] Table 25. CWO-D00084-006 Lpa SS:

[0630] destination reaction when push Used Material Source

[0631] mode out of in reactor

[0632] base new acid, 6% 6% acid feed tank Fluidize Waste

[0633] 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 s-Sa flow- base solvent washes ACN feed tank Waste through 1-36 base amidite + activator new amidite, activator Fluidize Waste

[0634] 1-35 flow- base solvent chase and washes ACN feed tank Waste through 1-35 base oxidizer oxidizer feed tank Fluidize Waste

[0635] 1-34 base sulfurization sulfurization feed tank Fluidize Waste

[0636] 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

[0637]

[0638] through 1-34 Table 26. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[0639] total Neat

[0640] ACN wash

[0641] after

[0642] lvol% ACNin

[0643] total deblocking

[0644] total DCA Pyridine in pyridine

[0645] toluene including

[0646] 6 V0l% used in 70 / 30 solution

[0647] Cycle used in the preDCA (mL) deblock ACN / Toluene wash after

[0648] deblock run

[0649] (mL)l wash after deblocking

[0650] (mL)l washing

[0651] deblock (mL) (mL)

[0652] and post

[0653] DEA wash

[0654] (mL)2

[0655] 1 790 742 47 432 302.61 1195

[0656] 2 802 754 48 461 322.61 281

[0657] 3 828 778 50 491 343.48 288

[0658] 4 851 800 51 520 364.35 298

[0659] 5 872 820 52 548 383.48 291

[0660] 6 902 847 54 579 405.22 304

[0661] 7 926 871 56 605 423.48 309

[0662] 8 954 897 57 634 443.48 318

[0663] 9 978 919 59 661 462.61 313

[0664] 10 1008 947 60 706 493.91 323

[0665] 11 1031 969 62 729 510.43 314

[0666] 12 1053 989 63 754 527.83 323

[0667] 13 1082 1017 65 783 547.83 333

[0668] 14 1101 1035 66 819 573.04 341

[0669] 15 1131 1063 68 841 588.70 338

[0670] 16 1158 1088 69 867 606.96 356

[0671] 17 1174 1104 70 902 631.30 359

[0672] 18 1210 1138 73 924 646.96 359

[0673] 19 1227 1153 74 966 676.52 360

[0674] 20 1258 1183 76 999 699.13 369

[0675] 21 1283 1206 77 1022 715.65 382

[0676] 22 1298 1220 78 1055 738.26 382

[0677] 23 1326 1246 80 1083 758.26 384

[0678] 24 1348 1267 81 1114 780.00 385

[0679] 25 1375 1292 82 1139 797.39 397

[0680] 26 1395 1311 84 1165 815.65 399

[0681] 27 1424 1339 85 1193 834.78 405

[0682] 28 1454 1367 87 1226 858.26 413

[0683] 29 1468 1380 88 1267 886.96 420

[0684] 30 1502 1412 90 1287 900.87 425

[0685] 31 1525 1433 91 1314 920.00 430

[0686]

[0687] 32 1557 1464 93 1352 946.09 425 33 1587 1492 95 1373 960.87 435 34 1606 1510 96 1398 978.26 439

[0688] 35-1 1630 1532 98 1435 1004.35 449

[0689]

[0690] 35-2 1661 1561 100 1468 1027.83 2361

[0691] Totals 43773 41147 2626 34111 23877 15905

[0692] Normalized

[0693] per mmol

[0694] (L / mmol) 8.229 0.525 6.822 4.775 3.181

[0695] Table 27. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[0696] Amidite,

[0697] note that

[0698] FBR was

[0699] 0.1 M and

[0700] Acid PBR was amidite

[0701] contact Amidite 0.2 M, equivalent

[0702] Cycle Amldite

[0703] time (g)5 FBR used versus

[0704] (minutes) less resin

[0705] molar

[0706] equiv

[0707] overall

[0708] (mL)

[0709] 1 6.8 MG 76.5 93 1.87

[0710] 2 6.7 MU 70.8 86 1.73

[0711] 3 6.8 MC 73.2 89 1.79

[0712] 4 6.8 MG 73.1 89 1.78

[0713] 5 6.5 MG 73.9 90 1.80

[0714] 6 6.5 ADEMA 82.1 100 2.00

[0715] 7 6.7 ADEMA 81.8 100 2.00

[0716] 8 6.8 ADEMA 81.7 100 1.99

[0717] 9 6.9 MG 75.5 92 1.84

[0718] 10 6.9 MC 73.8 90 1.80

[0719] 11 7.0 MC 73.2 89 1.79

[0720] 12 7.1 MG 74.1 90 1.81

[0721] 13 7.2 MA 74.9 91 1.83

[0722] 14 7.3 MC 72.5 88 1.77

[0723] 15 7.4 MG 74.0 90 1.80

[0724] 16 7.5 MA 74.8 91 1.82

[0725] 17 7.6 MU 77.8 95 1.90

[0726] 18 7.7 MC 73.1 89 1.78

[0727] 19 7.8 MU 77.6 95 1.89

[0728]

[0729] 20 7.9 MA 76.8 94 1.87 21 8.0 MC 73.5 90 1.79

[0730] 22 8.2 MU 77.8 95 1.90

[0731] 23 8.2 MG 73.8 90 1.80

[0732] 24 8.3 MG 82.9 101 2.02

[0733] 25 8.4 FU 82.6 101 2.01

[0734] 26 8.5 FU 83.4 102 2.03

[0735] 27 8.6 FC 86.1 105 2.10

[0736] 28 8.7 FG 74.7 91 1.82

[0737] 29 8.8 MA 84.0 102 2.05

[0738] 30 8.9 MA 84.8 103 2.07

[0739] 31 9.0 MC 81.5 99 1.99

[0740] 32 9.1 MC 80.8 99 1.97

[0741] 33 9.2 MG 82.2 100 2.00

[0742] 34 9.3 MU 85.2 104 2.08

[0743] 35-1 9.4 MU(S) 86.0 105 2.10

[0744]

[0745] 35-2 9.5

[0746] Totals 2731 3330

[0747] Normalized

[0748] per mmol

[0749] (L / mmol) 0.666

[0750] Table 28. CWQ-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[0751] Fresh

[0752] activator Coupling

[0753] ACN wash

[0754] Activator Activator equivalent contact Oxidizer Oxidizer Cycle after

[0755] (g)5 (mL) versus time (mL)

[0756] coupling (g)

[0757] resin (minutes)

[0758] (mL)

[0759] 1 77 95 9.5 10 101.8 261 266

[0760] 2 78 96 9.6 10 104.3 261 266

[0761] 3 81 100 10.0 10 105.6 261 266

[0762] 4 78 96 9.6 10 106.9 262 267

[0763] 5 79 98 9.8 10 106.9 262 267

[0764] 6 85 105 10.5 15 104.3 262 267

[0765] 7 84 104 10.4 15 103.1 262 267

[0766]

[0767] 8 83 102 10.2 15 104.3 262 267 9 78 96 9.6 10 108.1 262 267

[0768] 10 78 96 9.6 10 113.2 262 267

[0769] 11 78 96 9.6 10 100.5 262 267

[0770] 12 78 96 9.6 10 104.3 262 267

[0771] 13 78 96 9.6 10 106.9 262 267

[0772] 14 78 96 9.6 10 101.8 262 267

[0773] 15 77 95 9.5 10 100.5 262 267

[0774] 16 77 95 9.5 10 101.8 262 267

[0775] 17 77 95 9.5 10 103.1 262 267

[0776] 18 77 95 9.5 10 113.2 261 266

[0777] 19 77 95 9.5 10 112.0 262 267

[0778] 20 78 96 9.6 10 110.7 259 264

[0779] 21 79 98 9.8 10 100.5 262 267

[0780] 22 77 95 9.5 10 105.6 262 267

[0781] 23 76 94 9.4 10 113.2 261 266

[0782] 24 83 102 10.2 10 103.1 261 266

[0783] 25 83 102 10.2 15 106.9 262 267

[0784] 26 84 104 10.4 15 103.1 262 267

[0785] 27 84 104 10.4 15 101.8 262 267

[0786] 28 83 102 10.2 15 106.9 263 268

[0787] 29 85 105 10.5 10 109.4 262 267

[0788] 30 84 104 10.4 10 103.1 262 267

[0789] 31 84 104 10.4 10 106.9 263 268

[0790] 32 85 105 10.5 10 100.5 254 259

[0791] 33 84 104 10.4 10 104.3 263 268

[0792] 34 83 102 10.2 10 106.9 262 267

[0793] 35-1 86 106 10.6 10 109.4

[0794]

[0795] 35-2

[0796] Totals 3477 3694.656 8894 9057.0

[0797] Normalized

[0798] per mmol

[0799] (L / mmol) 0.695 0.739 1.811

[0800] Table 29. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes. Oxidation or Fresh ACN

[0801] 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)

[0802] (minutes) 4 (mL)

[0803] 1 — — 10 484.7 79 100 77 2 — — 10 489.8 78 99 77 3 — — 10 488.5 72 91 78 4 — — 10 478.4 78 99 77 5 — — 10 870.2 78 99 77 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

[0804]

[0805] 35-1 212.6 212.6 12 1159.0 — I 35-2 I I I | I I - I I

[0806]

[0807] Totals 212.6 212.6 18034.35

[0808] Normalized

[0809] per mmol

[0810] (L / mmol) 0.043 3.607

[0811] Table 30. CWQ-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[0812] Standard

[0813] Standard Capping B Fresh

[0814] Fresh

[0815] Capping A reagent ACN Capping

[0816] ACN used Diluted reagent (concentration used for reaction

[0817] forwash

[0818] Cycle Capping (concentration is 20vol% diluting contact

[0819] after

[0820] B (mL) is 20 vol% 1 acetic capping time

[0821] capping methylimidizole) anhydride, 30 reagents (minutes)4

[0822] (mL)

[0823] (mL) vol% lutidine) (mL)

[0824] (mL)

[0825] 1 96 20.0 19.2 232 7.5 461.8

[0826] 2 96 19.7 19.2 231 7.5 494.9

[0827] 3 97 18.2 19.5 226 7.5 521.6

[0828] 4 96 19.7 19.2 231 7.5 549.6

[0829] 5 96 19.7 19.2 231 7.5 570.0

[0830] 6 96 19.7 19.2 231 7.5 606.9

[0831] 7 96 19.7 19.2 231 7.5 636.1

[0832] 8 95 20.0 19.0 231 7.5 653.9

[0833] 9 96 20.0 19.2 232 7.5 685.8

[0834] 10 96 19.7 19.2 231 7.5 707.4

[0835] 11 96 20.0 19.2 232 7.5 751.9

[0836] 12 96 20.0 19.2 232 7.5 785.0

[0837] 13 96 20.0 19.2 232 7.5 801.5

[0838] 14 96 19.7 19.2 231 7.5 825.7

[0839] 15 96 19.7 19.2 231 7.5 863.9

[0840] 16 96 20.0 19.2 232 7.5 900.8

[0841] 17 96 20.0 19.2 232 7.5 917.3

[0842] 18 90 20.7 18.0 230 7.5 944.0

[0843] 19 94 20.0 18.7 230 7.5 988.5

[0844] 20 96 19.5 19.2 230 7.5 1006.4

[0845] 21 96 19.5 19.2 230 7.5 1031.8

[0846] 22 96 19.2 19.2 229 7.5 1064.9

[0847]

[0848] 23 96 19.2 19.2 229 7.5 1110.7 24 96 19.2 19.2 229 7.5 1127.2 25 96 19.2 19.2 229 7.5 1155.2

[0849] 26 96 19.2 19.2 229 7.5 1185.8

[0850] 27 96 19.2 19.2 229 7.5 1204.8

[0851] 28 96 19.2 19.2 229 7.5 1248.1

[0852] 29 96 19.5 19.2 230 7.5 1251.9

[0853] 30 96 19.2 19.2 229 7.5 1297.7

[0854] 31 95 19.2 19.0 228 7.5 1324.4

[0855] 32 96 19.2 19.2 229 7.5 1343.5

[0856] 33 96 19.0 19.2 228 7.5 1388.0

[0857] 34 96 19.2 19.2 229 7.5 1410.9

[0858] 35-1 — — — —

[0859] 35-2 — — — —

[0860] Totals 665 652 7818 31818.07

[0861] Normalized

[0862] per mmol

[0863]

[0864] (L / mmol) 0.133 0.130 1.564 6.364

[0865] Table 31. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[0866] total ACN

[0867] wash after total ACN deblocking used in including Fresh Fresh the Fresh ACN total ACN

[0868] the preACN used ACN ACN used process ACN used wash after used in

[0869] run in wash for wash including Cycle in amidite oxidation or capping

[0870] washing, activator after after all solution sulfurization reagents

[0871] post DEA solution coupling capping reagent (mL) (mL)

[0872] wash and (mL) (mL) soutions acnfrom and pyridine washes wash (mL)

[0873] 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

[0874]

[0875] 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 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

[0876] Normalized

[0877] per mmol

[0878]

[0879] (L / mmol) 7.956 0.599 0.626 0.739 3.607 0.172 6.364 20.06

[0880] 1 Toluene and DCA volumes were calculated based on the DCA solution being 6 vol% DCA in toluene

[0881] 2 Fresh ACN includes ACN from chase wash, wall wash, fresh ACN washes, and ACN from the pyridine solutions

[0882] 4 Does not include contact time with reuse reagents.

[0883] 5 These values were corrected to remove the extra added to compensate for the dilution cart sample.

[0884] Material Composition: DCA was 6 vol% in toluene

[0885] Amidites were 0.1M solutions in ACN (mU solvent was 10vol% toluene in ACN) Activator was 0.5 M ETT in ACN

[0886] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent) Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[0887] Cap A was 4 vol% 1 methylimidizole in ACN

[0888] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in CAN

[0889] Table 32. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes. ewa-txxxm-ood (tab CWO-DO0O84-OOS UPLC) (Release UPLC) strand ipa Seme Ips Seme

[0890] mass erf initial resin whole batch (g) 19.9500 19.9500 resin loading (umol / g) 251 251 Synthesis scale (umd) 5,007 5,007 mass of final resin whole batch (g) 9X7790 9X7700 mass gain (g) 72.82 72.82 Mass gain per mmol scale 1454 1454 Crude m (by weighing) 0x95 0.95

[0891]

[0892] FLP% (homogenized sample)* IX86IH 0.8485 Mass of resin taken for C&EJ (g) 0.1296 0.1296 Fraction erf the whole batch 0.14% 0.14% Mass of chgo in C&D resin sample (g) 0.1017 01017 30 wt% NH4OH sulutin mass (g) 2.9927 3.9927 NH4OH plus oligo mass |gj X1B44 X0944 Aliquot mass of C&D solfo for OD (g) 02724 01724 DI water added for dilution (g) 20.26 2&.2& NanoDrop measured A260 #1 11.44 11.44 NanoDrep measured A260 #2 11.49 11.49 NanoDrep measured A260 #3 11.45 11.45 NanoDrep measured A260 AW 11.45 11.45 OOd Sutton factor 75.40 75x40 Total Of) far whole batch 1,296,033 1,296,033 Mass Na Salt calculated from total OD 56.25 56.25 DO / umol 259 259 crude % yield by0D 88%

[0893]

[0894] Purity corrected yield by OD 59% 58%

[0895] Example 4. REO-064. Distillation and using distillate for washing.

[0896] Tables 33-40 show representative data for REO-064 (RE0-D00084-064 Lpa SS).

[0897] Table 33.

[0898] reaction destination when push Used material Source

[0899] mode out of reactor in reuse first part of wash after base reuse DCA tank 2 fluidize Distillation

[0900]

[0901] last detrit 2-36 base reuse acid reuse DCA tank 1 fluidize Distillation

[0902] 2-36 base reuse acid reuse DCA tank 1 fluidize Distillation

[0903] 2-36 base new acid, 6% 5% acid feed tank fluidize reuse DCA tank 1

[0904] 1-36 flow- base reuse solvent wash WI_DCA tank 1 reuse DCA tank 2

[0905] through 1-36 flow- base reuse solvent wash WI_DCA tank 1 Waste

[0906] through 1-36 flow- base new solvent reactor wall wash ACN feed tank Waste

[0907] through 1-36 flow- base reuse solvent wash WI_DCA tank 2 WI_DCAtank 1

[0908] through 1-36 flow- base new solvent chase ACN feed tank WI_DCAtank 1

[0909] through 1-36 new solvent feed zone wall flow- base ACN feed tank WI_DCAtank 2

[0910] wash through 1-36 flow- base reuse solvent wash WI_DCA tank 3 WI_DCAtank 2

[0911] through 1-36 base Distillate Distillation fluidize WI_DCAtank 3

[0912] 2-36 flow- base new solvent washes ACN feed tank WI_DCAtank 3

[0913] 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 flow- new solvent chase and washes ACN feed tank used for the wash after base through

[0914] ox / sulf) 1-35 base Reuse oxidizer Reuse oxidizer tank fluidize Waste

[0915] 2-34 base oxidizer oxidizer feed tank fluidize Reuse oxidizer tank

[0916] 1-34 base sulfurization reagent sulfurization feed tank fluidize Waste

[0917] 35 flow- base New solvent chase ACN feed tank Waste

[0918] through 1-35 flow- base post 0 / S wash post O / S tank Waste

[0919] through 1-35 Reuse capping reaction flow- base Reuse CAP tank Waste

[0920] solution through 1-34 New Capping reagents and Capping A feed tank,

[0921] 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

[0922]

[0923] through 1-34 flow- base new solvent washes ACN feed tank Wl CAP tank

[0924] through 1-34 flow- base distillate wash Distillation Wl CAP tank

[0925]

[0926] through 2-34 note: reuse solvent wash tanks (WI_DCA, Wl CAP, reuse CAP) filled with ACN to begin synthesis

[0927] Table 34. REO-064 (RE0-D00084-064 Lpa SS).

[0928] total Neat

[0929] ACN wash

[0930] after

[0931] total deblocking

[0932] total DCA

[0933] toluene including

[0934] 6 vol% used in

[0935] Cycle used in the preDCA (mL) deblock

[0936] deblock run

[0937] (mL)l

[0938] (mL)l washing

[0939] and post

[0940] DEA wash

[0941] (mL)2

[0942] 1 877 824 53 2445

[0943] 2 457 430 27 421

[0944] 3 470 442 28 417

[0945] 4 487 457 29 397

[0946] 5 481 452 29 441

[0947] 6 492 463 30 424

[0948] 7 506 475 30 419

[0949] 8 534 502 32 430

[0950] 9 550 517 33 419

[0951] 10 562 528 34 422

[0952] 11 572 537 34 424

[0953] 12 584 549 35 417

[0954] 13 598 563 36 422

[0955] 14 609 572 37 425

[0956] 15 629 591 38 417

[0957] 16 638 599 38 415

[0958] 17 655 616 39 426

[0959] 18 664 625 40 504

[0960] 19 676 635 41 415

[0961] 20 687 646 41 419

[0962] 21 701 659 42 420

[0963] 22 704 661 42 403

[0964] 23 721 678 43 397

[0965] 24 736 692 44 422

[0966] 25 766 720 46 421

[0967] 26 776 730 47 467

[0968]

[0969] 27 786 739 47 427 28 800 752 48 430

[0970] 29 814 765 49 419

[0971] 30 821 772 49 426

[0972] 31 836 785 50 415

[0973] 32 847 796 51 415

[0974] 33 855 803 51 417

[0975] 34 870 818 52 417

[0976] 35-1 889 836 53 413

[0977]

[0978] 35-2 890 837 53 1677

[0979] Totals 24539 23067 1472 18506 Normalized

[0980] per mmol

[0981] (L / mmol) 4.613 0.294 3.701

[0982] Table 35. REO-064 (RE0-D00084-064 Lpa SS)

[0983] Amidite,

[0984] note that

[0985] FBR was

[0986] 0.1 M and

[0987] Acid PBR was amidite contact Amidite 0.2 M, equivalent Cycle Amidite

[0988] time (g)5 FBR used versus (minutes) less resin molar

[0989] equiv

[0990] overall

[0991] (mL)

[0992] 1 23.8 MG 74.6 91 1.82 2 27.1 MU 74.8 91 1.82 3 27.2 MC 71.8 88 1.75 4 27.6 MG 75.6 92 1.84 5 27.6 MG 75.8 92 1.85 6 27.3 ADEMA 81.4 99 1.99 7 27.6 ADEMA 82.4 100 2.01 8 27.9 ADEMA 82.3 100 2.01 9 28.2 MG 75.2 92 1.83 10 28.2 MC 72.0 88 1.76 11 27.6 MC 70.1 85 1.71 12 Z177 MG 74.8 91 1.82 13 28.0 MA 76.5 93 1.87 14 28.1 MC 74.6 91 1.82 15 28.2 MG 75.4 92 1.84 16 28.4 MA 77.3 94 1.89 17 28.8 MU 78.7 96 1.92

[0993]

[0994] 18 28.9 MC 72.8 89 1.78 19 29.1 MU 78.3 95 1.91

[0995] 20 29.4 MA 75.0 91 1.83

[0996] 21 29.6 MC 72.7 89 1.77

[0997] 22 29.8 MU 78.3 95 1.91

[0998] 23 30.0 MG 76.7 94 1.87

[0999] 24 30.2 MG 82.1 100 2.00

[1000] 25 30.0 FU 83.1 101 2.03

[1001] 26 30.0 FU 83.8 102 2.04

[1002] 27 30.2 FC 83.0 101 2.02

[1003] 28 30.4 FG 83.8 102 2.04

[1004] 29 30.6 MA 83.7 102 2.04

[1005] 30 30.8 MA 84.2 103 2.05

[1006] 31 31.1 MC 81.0 99 1.98

[1007] 32 31.3 MC 80.5 98 1.96

[1008] 33 31.6 MG 81.9 100 2.00

[1009] 34 31.5 MU 84.1 103 2.05

[1010] 35-1 31.7 MU(S) 83.5 102 2.04

[1011]

[1012] 35-2 31.9

[1013] Totals 2742 3344

[1014] Normalized

[1015] per mmol

[1016] (L / mmol) 0.669

[1017] Table 36. REO-064 (RE0-D00084-064 Lpa SS)

[1018] Fresh

[1019] activator Coupling

[1020] ACN wash

[1021] Activator Activator equivalent contact Oxidizer Oxidizer Cycle after

[1022] (g)5 (mL) versus time (mL) coupling (g) resin (minutes)

[1023] (mL)

[1024] 1 77 95 9.5 10 75.1 267 271 2 77 95 9.5 10 76.3 217 221 3 77 95 9.5 10 75.1 217 221 4 77 95 9.5 10 75.1 217 221 5 76 94 9.4 10 75.1 217 221 6 83 102 10.2 15 82.7 217 221 7 84 104 10.4 15 77.6 217 221 8 85 105 10.5 15 80.2 217 221

[1025]

[1026] 9 75 93 9.3 10 80.2 217 221 10 78 96 9.6 10 73.8 217 221 11 77 95 9.5 10 91.6 217 221 12 75 93 9.3 10 80.2 217 221 13 75 93 9.3 10 81.4 217 220 14 75 93 9.3 10 73.8 217 221 15 75 93 9.3 10 76.3 217 221 16 78 96 9.6 10 73.8 217 221 17 75 93 9.3 10 75.1 217 221 18 76 94 9.4 10 78.9 217 221 19 75 93 9.3 10 73.8 217 221 20 76 94 9.4 10 75.1 217 221 21 75 93 9.3 10 76.3 217 221 22 75 93 9.3 10 86.5 217 221 23 75 93 9.3 10 72.5 217 221 24 85 105 10.5 10 80.2 217 221 25 84 104 10.4 15 73.8 218 221 26 84 104 10.4 15 80.2 217 221 27 83 102 10.2 15 81.4 217 221 28 85 105 10.5 15 77.6 218 222 29 84 104 10.4 10 76.3 217 221 30 84 104 10.4 10 75.1 217 221 31 85 105 10.5 10 75.1 217 221 32 84 104 10.4 10 77.6 216 220 33 85 105 10.5 10 85.2 217 221 34 84 104 10.4 10 75.1 217 221 35-1 84 104 10.4 10 85.2

[1027] 35-2

[1028] Totals 3435 2729.008 7428.6 7564.8 Normalized

[1029] per mmol

[1030]

[1031] (L / mmol) 0.687 0.546 1.513

[1032] Table 37. REO-064 (RE0-D00084-064 Lpa SS)

[1033] Oxidation or Fresh ACN

[1034] 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)

[1035] (minutes) 4 (mL)

[1036]

[1037] 1 10 48.3 37 47 36 2 — — 10 49.6 36 46 37 3 — — 10 53.4 36 46 36 4 — — 10 56.0 36 46 37 5 — — 10 56.0 37 47 36 6 — — 10 50.9 36 46 36 7 — — 10 56.0 36 46 36 8 — — 10 56.0 36 46 37 9 — — 10 50.9 36 46 36 10 — — 10 52.2 36 46 37 11 — — 10 53.4 36 46 36 12 — — 10 49.6 36 46 37 13 — — 10 54.7 37 47 36 14 — — 10 49.6 36 46 37 15 — — 10 100.5 36 46 36 16 — — 10 103.1 36 46 37 17 — — 10 101.8 36 46 36 18 — — 10 104.3 36 46 37 19 — — 10 101.8 37 47 37 20 — — 10 99.2 36 46 37 21 — — 10 100.5 37 47 37 22 — — 10 101.8 37 47 37 23 — — 10 101.8 36 46 37 24 — — 10 104.3 37 47 37 25 — — 10 100.5 36 46 36 26 — — 10 103.1 36 46 37 27 — — 10 101.8 36 46 37 28 — — 10 104.3 36 46 37 29 — — 10 104.3 36 46 37 30 — — 10 99.2 36 46 36 31 — — 10 100.5 36 46 36 32 — — 10 100.5 36 46 36 33 — — 10 106.9 36 46 37 34 — — 10 100.5 37 47 36 35-1 214.0 214.0 12 1006.4 — — — 35-2 — — — Totals 214 214 3783.715

[1038] Normalized

[1039] per mmol

[1040]

[1041] (L / mmol) 0.043 0.757

[1042] Table 38. REO-064 (RE0-D00084-064 Lpa SS) Standard

[1043] Standard Capping B Fresh

[1044] Fresh Capping A reagent ACN Capping

[1045] ACN used Diluted reagent (concentration used for reaction forwash Cycle Capping (concentration is 20vol% diluting contact

[1046] after B(mL) is 20 vol% 1 acetic capping time capping methylimidizole) anhydride, 30 reagents (minutes)4

[1047] (mL) (mL) vol% lutidine) (mL)

[1048] (mL)

[1049] 1 45 9.4 9.0 148 7.4 355.0 2 46 9.1 9.2 148 7.4 58.5 3 45 9.1 9.0 147 7.4 57.3 4 46 9.1 9.2 148 7.4 44.5 5 45 9.4 9.0 148 7.4 47.1 6 45 9.1 9.0 147 7.4 53.4 7 45 9.1 9.0 147 7.4 47.1 8 46 9.1 9.2 148 7.4 45.8 9 45 9.1 9.0 147 7.4 54.7 10 46 9.1 9.2 148 7.4 47.1 11 45 9.1 9.0 147 7.4 73.8 12 46 9.1 9.2 148 7.4 63.6 13 45 9.4 9.0 148 7.4 54.7 14 46 9.1 9.2 148 7.4 45.8 15 45 9.1 9.0 147 7.4 56.0 16 46 9.1 9.2 148 7.4 45.8 17 45 9.1 9.0 147 7.4 58.5 18 46 9.1 9.2 148 7.4 45.8 19 46 9.4 9.2 149 7.4 66.2 20 46 9.1 9.2 148 7.4 56.0 21 46 9.4 9.2 149 7.4 59.8 22 46 9.4 9.2 149 7.4 59.8 23 46 9.1 9.2 148 7.4 54.7 24 46 9.4 9.2 149 7.4 49.6 25 45 9.1 9.0 147 7.4 44.5 26 46 9.1 9.2 148 7.4 33.1 27 46 9.1 9.2 148 7.4 50.9 28 46 9.1 9.2 148 7.4 47.1 29 46 9.1 9.2 148 7.4 58.5 30 45 9.1 9.0 147 7.4 54.7 31 45 9.1 9.0 147 7.4 48.3 32 45 9.1 9.0 147 7.4 61.1 33 46 9.1 9.2 148 7.4 63.6 34 45 9.4 9.0 148 7.4 45.8

[1050]

[1051] 35-1 - — - — - — 35-2 I - I - I - I - I - I -

[1052]

[1053] Totals 312 310 5037 2108.142 Normalized

[1054] per mmol

[1055] (L / mmol) 0.062 0.062 1.007 0.422

[1056] Table 39. REO-064 (RE0-D00084-064 Lpa SS)

[1057] total ACN

[1058] wash after total ACN deblocking used in including Fresh Fresh the Fresh ACN total ACN

[1059] the preACN used ACN ACN used process ACN used wash after used in

[1060] run in wash for wash including Cycle in amidite oxidation or capping

[1061] washing, activator after after all solution sulfurization reagents

[1062] post DEA solution coupling capping reagent (mL) (mL)

[1063] wash and (mL) (mL) soutions acnfrom and pyridine washes wash (mL)

[1064] 1 2445.3 82 86 75 48 12 355.0 3103.1 2 421.1 82 86 76 50 12 58.5 785.2 3 417.3 79 86 75 53 12 57.3 779.2 4 396.9 83 86 75 56 12 44.5 753.0 5 441.5 83 84 75 56 12 47.1 799.2 6 423.7 89 92 83 51 12 53.4 804.0 7 418.6 90 93 78 56 12 47.1 794.8 8 430.0 90 94 80 56 12 45.8 808.6 9 418.6 83 83 80 51 12 54.7 782.0 10 422.4 79 87 74 52 12 47.1 773.0 11 423.7 77 86 92 53 12 73.8 816.8 12 417.3 82 83 80 50 12 63.6 788.0 13 422.4 84 83 81 55 12 54.7 792.5 14 424.9 82 83 74 50 12 45.8 771.3 15 417.3 83 83 76 101 12 56.0 828.0 16 414.8 85 87 74 103 12 45.8 820.8 17 426.2 86 83 75 102 12 58.5 843.1 18 503.8 80 84 79 104 12 45.8 909.1 19 414.8 86 83 74 102 12 66.2 837.9 20 418.6 82 84 75 99 12 56.0 827.5 21 419.8 80 83 76 101 12 59.8 831.7 22 403.3 86 83 87 102 12 59.8 832.8 23 396.9 84 83 73 102 12 54.7 805.4 24 422.4 90 94 80 104 12 49.6 853.1

[1065]

[1066] 25 421.1 91 93 74 101 12 44.5 836.3 26 466.9 92 93 80 103 12 33.1 880.4 27 427.5 91 92 81 102 12 50.9 856.8 28 430.0 92 94 78 104 12 47.1 857.4 29 418.6 92 93 76 104 12 58.5 854.9 30 426.2 92 93 75 99 12 54.7 852.7 31 414.8 89 94 75 101 12 48.3 833.8 32 414.8 88 93 78 101 12 61.1 847.4 33 417.3 90 94 85 107 12 63.6 869.3 34 417.3 92 93 75 101 12 45.8 836.3 35-1 413.5 92 93 85 1006 0 0.0 1690.1 35-2 1676.8 0 0 0 0 0 0.0 1676.8 Totals 18506 3009 3091 2729 3784 404 2108 33632 Normalized

[1067] per mmol

[1068]

[1069] (L / mmol) 3.701 0.602 0.618 0.546 0.757 0.081 0.422 6.73

[1070] 1 Toluene and DCA volumes were calculated based on the DCA solution being 6 vol% DCA in toluene

[1071] 2 Fresh ACN includes ACN from chase wash, wall wash, fresh ACN washes, and ACN added to the CC cans

[1072] 4 Does not include contact time with reuse reagents.

[1073] 5 These values were corrected to remove the extra added to compensate for the dilution cart sample.

[1074] Material Composition:

[1075] DCA was 10 vol% in toluene

[1076] Amidites were 0.1M solutions in ACN (mU solvent was 10vol% toluene in ACN) Activator was 0.5 M ETT in ACN

[1077] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent) Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[1078] Cap A was 4 vol% 1 methylimidizole in ACN

[1079] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in CAN

[1080] Table 40. REO-064 (RE0-D00084-064 Lpa SS) REO~O0CC84~O64 (Lab R£ODtXMJS4~964 elN UPLC) (Release UPLC) Strand Lpa Seme tpa Sense

[1081] mass of initial resin whole batch (g) 20.36D0 20.3600 resin loading (umul / g) 246 246 Synthesis scale (umtrf) 5,009 5,009 mass of fi nal resin whole batch (g) 90.6000 90-0000 mass gain (g) 70.24 70,24 Mass gain per mmol scale 14,02 14.02 Chide mass yfefcf (by weighing) 0.92 0.92

[1082]

[1083] FI.P% (homogenized sample)* 0.7928 0.7059 Mass rtf resin taken for C&D (g) 0.1134 0,1134 Fraction of the whole hatch 0.13% 0.13% Mass st oligo in C&D resin sample (g) 0.0879 (10879 30 wt% NH4OH solutin mass (g) 2.0525 Z0525 NH40H plus oligo mass (g) 2.14D4 2.1404 Aliquot mass of C&D sol’n for 00 (g| 0.1768 0.1768 Di water ad^for ddutfoa (gj 20.34 20.34 NanoDmp measured A260 #1 6,20 6.26 ffonoDmp measured A26Q #2 6.26 6,26 NanoDrep measured A260 #3 6.31 5.31 mnoDrap measured A260 AW 6,28 5.28 00 dilution factor 116.07 116.07 Total OD for whole batch 1,245,830 1,245,830 Mass Na Salt calculated from total 00 54.07 54.07 DD / umel 249 249 crude % yield by OD s 77%

[1084]

[1085] Punty corrected yield by OD 61% S<%

Claims

CLAIMSWhat is claimed is:

1. A system for synthesizing oligonucleotides, the system comprising:a reactor;a feed zone vessel in fluid communication with the reactor;a first capping solution feed vessel in fluid communication with the feed zone vessel or reactor;a second capping solution feed vessel in fluid communication with the feed zone vessel or reactor;a reuse capping solution vessel in fluid communication with both the reactor and the feed zone vessel or reactor;a vessel with a solution that is used to dilute the capping reaction in fluid communication with the feed zone vessel or reactor; anda reuse wash vessel in fluid communication with the feed zone vessel or reactor.

2. The system of claim 1 further comprising an acetonitrile (ACN) solution feed vessel in fluid communication with the feed zone vessel and / or to the reactor.

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 first and second capping solution feed vessels comprise capping agents comprising methylimidazole, acetic anhydride, lutidine, or pyridine or combinations thereof.

5. The system of claim 4, whereina. the first capping solution feed vessel comprises a capping solution comprising methylimidazole and ACN; andb. the second capping solution feed vessel comprises a capping solution comprising acetic anhydride, lutidine, and ACN.

6. The system of claim 4, whereina. the first capping solution feed vessel comprises a capping solution comprising acetic anhydride and ACN; andb. the second capping solution feed vessel comprises a capping solution comprising methylamidizole, pyridine, and ACN.

7. The system of claim 4, further comprising a third capping solution feed vessel wherein a. the first capping solution feed vessel comprises a capping solution comprising methylamidizole and ACN; andb. the second capping solution feed vessel comprises a capping solution comprising acetic anhydride; andc. the third capping solution feed vessel comprises a capping solution comprising lutidine or pyridine, and ACN.

8. The system of any one of claims 1 to 7, wherein the reuse capping solution vessel comprises a capping solution that has previously been used in at least one solid phase oligonucleotide synthesis (SPOS) cycle.

9. The system of any one of claims 1 to 8, wherein the vessel with the solution that is used to dilute the capping reaction comprises a used wash solution that has previously been used to wash after capping in at least one SPOS cycle wash step.

10. The system of claim 9, wherein the dilute solution comprises methylimidizole, acetic anhydride, lutidine, and ACN because it was used to wash these reagents from the resin in a previous cycle.

11. The system of any one of claims 1 to 10, wherein the reuse wash vessel comprises acetonitrile (ACN) that has previously been used in at least one SPOS cycle wash step.

12. The system of any one of claims 1 to 9, wherein the reactor contains a solid substrate, optionally a resin.

13. The system of any one of claims 1 to 11 further comprising one or more pumps, or no pumps.

14. A method for recycling capping solution during an oligonucleotide synthesis, the method comprising:(i) performing a first cycle of solid phase oligonucleotide synthesis (SPOS) in a reactor, and moving capping solution used during the first SPOS cycle to a reuse capping solution vessel;(ii) performing a second cycle of SPOS comprising a capping step comprising contacting an oligonucleotide linked to a solid support in the reactor with a volume of capping solution introduced into the reactor from the reuse capping solution vessel;(iii) moving the capping solution from the reactor to a waste module;(vi) moving a volume of diluted capping solution from a diluted capping solution feed vessel to the feed zone vessel, and mixing the first new capping solution, second new capping solution, and diluted capping solution;(vii) moving the capping solution mixture from the feed zone vessel to the reactor and contacting the oligonucleotide linked to the solid support;(viii) moving the capping solution mixture to a holding vessel to be used on the next cycle;(ix) moving a volume of wash solution from a reuse wash vessel to the reactor and contacting the oligonucleotide linked to the solid support;(x) moving the volume of wash solution from the reactor to the vessel with material that is used to dilute the next capping reaction;(xi) moving a volume of acetonitrile (ACN) from an ACN feed vessel to the reactor and contacting the oligonucleotide linked to the solid support; and(xii) moving the volume of ACN from the reactor to the reuse wash vessel.

15. The method of claim 14, wherein the first new capping solution comprises methylimidizole and ACN.

16. The method of claim 14 or 15, wherein the second new capping solution comprises acetic anhydride, lutidine, and ACN.

17. The method of any one of claims 14 to 16, wherein the contacting of step (ii) is performed under flow-through conditions.

18. The method of any one of claims 14 to 17, wherein the contacting of step (vii) is performed under fluidization conditions.

19. The method of any one of claims 14 to 18, wherein the contacting of step (ix) is performed under flow-through conditions.

20. The method of any one of claims 14 to 19, wherein the solid substrate comprises a resin.

21. A method for performing a wash step during solid phase oligonucleotide synthesis, the method comprising:(i) in a reactor, contacting a solid phase comprising a linked oligonucleotide with a capping solution;(ii) moving the capping solution from the reactor to a waste vessel; and(iii) contacting the solid phase with a distillate wash solution comprising ACN and toluene.

22. The method of claim 21, wherein the distillate wash solution is produced by distilling waste products of a deblocking reaction and wash that precedes step (i).

23. The method of claim 21 or 22, wherein the distillate wash solution comprises an ACN / toluene azeotrope having a 70% to 30% ACN-to-toluene ratio.

24. The method of any one of claims 21 to 23, further comprising (iv) moving the distillate wash solution to a reuse wash vessel.

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