Detritylation step in solid phase oligonucleotide synthesis

Abstract

Aspects of the disclosure relate to methods and systems for solid phase oligonucleotide synthesis (SPOS). In some embodiments, the methods described by the disclosure comprise a step of performing a deblocking step (e.g., a detritylation reaction) during each cycle of the SPOS reaction. In some embodiments, performing the deblocking steps with neat or high concentration DCA addback to the reuse acid solution during deblocking, reuse DCA-rich portion of post-detritylation ACN wash as part of the deblocking process, using the distillation bottoms is used as part of the deblocking process, or deblocking in FBR with thiol scavengers requires a significantly reduced amount of deblocking reagent, such as acid solution, during the entire SPOS process relative to SPOS processes where the deblocking step is carried out at room temperature.

Description

[0001] DETRITYLATION STEP IN SOLID PHASE OLIGONUCLEOTIDE SYNTHESIS RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. 119(e) of the filing date of U.S. provisional application serial number 63 / 711,301, filed October 24, 2024, entitled “DETRITYLATION STEP IN SOLID PHASE OLIGONUCLEOTIDE SYNTHESIS”, the entire contents of which are incorporated by reference herein.

[0003] BACKGROUND

[0004] 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.

[0005] SUMMARY

[0006] Aspects of the disclosure relate to methods and systems for performing SPOS. The disclosure is based, in part, on modifications to deblocking (also referred to as detritylation) reactions during SPOS, for example reuse of deblocking solutions. In some embodiments, the modified deblocking methods and systems are advantageous over previously employed deblocking reactions, for example by requiring less reagent (e.g., acid solutions) and less solvent, and producing oligonucleotides at a higher purity and yield. In some embodiments, deblocking methods described by the disclosure are carried out on a fluidized bed reactor (FBR).

[0007] Accordingly, in some aspects, the disclosure relates to a method comprising contacting a phosphoramidite monomer comprising a protecting group and linked to a solid support in a fluidized bed reactor with a recycled deblocking solution, thereby removing the protecting group from the phosphoramidite monomer to produce a nucleoside.

[0008] In some embodiments, a deblocking solution comprises dichloroacetic acid (DCA) or trichloroacetic acid (TCA).

[0009] In some embodiments, a protecting group comprises 5 ’-DMT (4,4’-dimethoxytityl). In some embodiments, a deblocking solution is charged to the fluidized bed reactor top. In some embodiments, the method further comprises fluidizing a portion of the deblocking solution and solid support within the fluidized bed reactor.

[0010] In some embodiments, the method further comprises charging the recycled deblocking solution in multiple portions into the reactor, and each portion is fluidized with the solid support within the fluidized bed reactor.

[0011] In some embodiments, a fluidized bed reactor comprises an inlet that allows pressurized gas to enter the fluidized bed reactor. In some embodiments, an inlet is positioned at the bottom of the bed reactor.

[0012] In some embodiments, the method further comprises removing the deblocking solution from the fluidized bed reactor after the deblocking solution removes the protecting group from the phosphoramidite monomer.

[0013] In some embodiments, deblocking solution is removed through a filter located at the bottom of the fluidized bed reactor. In some embodiments, a fluidized bed reactor further comprises a filter located at the bottom of the fluidized bed reactor.

[0014] In some embodiments, deblocking solution is added to the fluidized bed reactor comprising a volume between about 50-500 mL per mmol of resin bound oligonucleotide.

[0015] In some embodiments, deblocking solution is added to the fluidized bed reactor in about 1 to 10 separate portions. In some embodiments, each portion comprises a volume between about 10-50 mL per mmol of resin bound oligonucleotide in the fluidized bed reactor.

[0016] In some embodiments, a solid support comprises a resin. In some embodiments, a solid support comprises controlled pore glass. In some embodiments, resin comprises polystyrene.

[0017] In some embodiments, the method further comprises contacting the nucleoside with an activated amidite solution which reacts with the nucleoside to couple a second phosphoramidite monomer to the nucleoside.

[0018] In some embodiments, the method further comprises removing the activated amidite solution from the fluidized bed reactor and contacting the second phosphoramidite monomer with one or more wash solutions. In some embodiments, one or more wash solutions comprise a capping solution comprising one or more of acetic anhydride, N-methylimidazole (NMI), tetrahydrofuran, and pyridine or lutidine. In some embodiments, the method further comprises removing the one or more wash solutions from the fluidized bed reactor and contacting the second phosphoramidite monomer with an oxidation solution.

[0019] In some embodiments, the method further comprises removing the oxidation solution and contacting the nucleoside with a second deblocking solution.

[0020] In some aspects, the disclosure provides a method for recycling deblocking solution, 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); moving a portion of the deblocking solution from the fluidized bed reactor to a holding (e.g., acid reuse) vessel via a filter located at the bottom of the fluidized bed reactor; completing the first cycle of SPOS; optionally, adding a first volume of neat or concentrated DCA to the used deblocking solution in the holding vessel; beginning a second cycle of SPOS, the second cycle comprising contacting a second phosphoramidite monomer linked to the solid support in the fluidized bed reactor with the used deblocking solution, fluidizing the first portion and then pushing the rest through the resin bed in flow through mode; moving a first portion of the used deblocking solution to waste via the filter located at the bottom of the fluidized bed reactor; continuing the deblocking reaction with fresh deblocking solution that passes through the resin bed, exits the reactor, and is collected in the holding vessel to be used on the next cycle; and, completing the second cycle of SPOS.

[0021] In some aspects, the disclosure provides a method for recycling deblocking solution, 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); moving a portion of the deblocking solution from the fluidized bed reactor to a holding (e.g., acid reuse) vessel via a filter located at the bottom of the fluidized bed reactor; completing the first cycle of SPOS; optionally, adding a first volume of neat or concentrated DCA to the used deblocking solution in the holding vessel; beginning a second cycle of SPOS, the second cycle comprising contacting a second phosphoramidite monomer linked to the solid support in the fluidized bed reactor with the used deblocking solution, splitting the used deblocking solution into multiple portions, each portion fluidized with the solid support and then removed from the reactor via a filter located at the bottom of the fluidized bed reactor; continuing the deblocking reaction with fresh deblocking solution that passes through the resin bed, exits the reactor, and is collected in the holding vessel to be used on the next cycle; and, completing the second cycle of SPOS. In some embodiments, the fresh deblock solution is added by multiple portions to the fluidized bed reactor, each portion fluidized with the solid support, then exiting the reactor and collected in the holding vessel to be used on the next cycle.

[0022] In some embodiments, the feed zone is in fluid communication with the fluidized bed reactor.

[0023] In some embodiments, the deblocking solution used to contact the first phosphoramidite monomer comprises a concentration of DCA ranging from about 2% v / v to about 25% v / v.

[0024] In some embodiments, the first volume of neat or high concentration DCA added to the feed zone ranges between 2% and 20% of the portion of deblocking solution in the holding (e.g., acid reuse) vessel. In some embodiments, the first volume of neat or high concentration DCA added to the feed zone is more than 20% of the portion of deblocking solution in the holding (e.g., acid reuse) vessel.

[0025] In some embodiments, each cycle of SPOS further comprises a coupling reaction, oxidation reaction, and a capping reaction.

[0026] In some embodiments, contacting the first or second monomer with the deblocking solution is performed under flow-through conditions.

[0027] In some embodiments, the method is performed n times, where n is the number of phosphoramidite monomers that will be linked to the solid support during the SPOS.

[0028] In some embodiments, the second SPOS cycle comprises contacting the solid substrate with a portion of an acetonitrile (ACN) wash solution that was used to wash the solid substrate after the first phosphoramidite monomer on the solid substrate was contacted with the used deblocking solution.

[0029] In some embodiments, the second SPOS cycle further comprises contacting the solid substrate with a distillation bottoms solution comprising concentrated dichloroacetic acid (DCA), toluene, and acetonitrile prior to contacting the phosphoramidite monomers with the used deblocking solution.

[0030] In some embodiments, deblocking solution (e.g., recycled or reused deblocking solution) comprises one or more thiol scavengers. In some embodiments, one or more thiol scavengers are selected from: 4-(tert-butyl)phenylmethanethiol, 3,6-Dioxaoctane-l,8-dithiol (DODT), and 1-dodecanethiol.

[0031] In some aspects, the disclosure provides a method for recycling deblocking solution, 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 a portion of the deblocking solution from the fluidized bed reactor to a holding vessel via a filter located at the bottom of the fluidized bed reactor; completing the first cycle of SPOS; optionally, adding a first volume of neat or concentrated DCA to the used deblocking solution in the holding vessel; beginning a second cycle of SPOS, the second cycle comprising contacting a second phosphoramidite monomer linked to the solid support in the fluidized bed reactor with the used deblocking solution under fluidizing conditions; moving a first portion of the used deblocking solution to waste via the filter located at the bottom of the fluidized bed reactor; continuing the deblocking reaction with fresh deblocking solution that passes through the resin bed, exits the reactor, and is collected in the holding vessel to be used on the next cycle, and completing the second cycle of SPOS.

[0032] 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 trifluoro acetic 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. In some embodiments, the method further comprises performing one or more washes under fluidizing conditions.

[0033] In some embodiments, none of the deblocking solution, coupling solution, oxidation solution, and the capping solution is reused in an SPOS cycle.

[0034] In some embodiments, method steps are performed “n” times, where “n” is an integer between 19 and 100.

[0035] BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows a representative process flow diagram for a fluidized bed reactor (FBR), according to some aspects of the technology.

[0036] FIGs. 2A-2D show representative process flow diagrams for material flow in detritylation reactions, according to some aspects of the technology.

[0037] FIG. 3 shows a representative schematic diagram for a FBR configured for material reuse, according to some aspects of the technology.

[0038] FIG. 4 shows a representative schematic diagram for a FBR configured for acetonitrile (ACN) reuse, according to some aspects of the technology.

[0039] FIG. 5 shows a representative schematic diagram for a FBR comprising a distillation unit, according to some aspects of the technology.

[0040] FIG. 6 shows a representative schematic diagram for a FBR configured for no material reuse, according to some aspects of the technology.

[0041] FIG. 7 shows a representative process flow diagram for a fully fluidized, reuse SPOS process in an FBR, according to some aspects of the technology.

[0042] DETAILED DESCRIPTION

[0043] 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.

[0044] The first step is the “deblocking” step, which is generally a detritylation reaction. In some embodiments, a nucleotide (e.g., a nucleotide attached to a solid support, for example a resin) has its 5'-hydroxyl group protected by an acid-labile protection group such as the DMT (4,4'-dimethoxytrityl). This protection group may be removed during a continuous flow of an acid solution or via an addition of an acid in a solvent (also referred to as a “deblocking solution”). In some embodiments, the deblocking solution comprises trichloroacetic acid (TCA) or dichloroacetic acid (DCA). In some embodiments, the acid (e.g., TCA, DCA, etc.) is carried in an inert solvent such as toluene, dichloromethane, or another suitable solvent. The concentration or amount of acid in a deblocking solution may vary. In some embodiments, a deblocking solution comprises between 2% and 30% acid. In some embodiments, a deblocking solution comprises 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% DCA in toluene. The amount of deblocking solution used during each deblocking reaction of SPOS may vary. In some embodiments, the amount of deblocking solution used in each deblocking reaction ranges from about 50-500 ml / mmol. In some embodiments, during this “de-blocking” reaction, an orange-colored DMT cation is formed and is washed out continually during the flow-through reaction because the reagent solution is flowing through the resin, and then subsequently after the flow-through reaction is completed by using wash solvents.

[0045] 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”.

[0046] 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.

[0047] 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”.

[0048] In some embodiments, an oxidation step is performed. Before 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 phosphorothioate 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.

[0049] 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.

[0050] In some embodiments, one or more washing steps occurs between of the four SPOS steps. A cycle is defined as the detritylation, coupling, oxidation / thiolation, and capping sequences along with their associated washes. In other words, one nucleotide is added per 4-step “cycle”. In some embodiments, an SPOS cycle comprises one or more washes between deblocking and coupling; one or more washes between coupling and oxidation / sulfurization; one or more washes between oxidation / sulfurization and capping; and, one or more washes between capping and the first step of the next SPOS cycle. In some embodiments, a wash step comprises a multi-stage counter-current wash process. In some embodiments, a bed reactor (e.g., a FBR as described herein) comprises six (6) vessels with wash solvent used for integrated multi-pass washing after deblocking. The first wash step after deblocking is to use the solvent from a first vessel (e.g., vessel “A”) to wash the resin and push to waste. The next step is to use the solvent from a second vessel (e.g., vessel “B”) to push through the resin and push back to refill the first vessel (“A”). Then the solvent from a third vessel (e.g., vessel “C”) washes the resin in the reactor and pushes out to refill the second vessel (“B”), and the process continues with the fourth (“D”), fifth (“E”), and sixth (“F”) vessels. After all six wash vessels are used, fresh solvent wash is used to push through the resin and push back to refill the sixth vessel (“F”). In some embodiments, wash solvent after coupling is only reused for the wash after oxidation or thiolation in the same cycle (e.g., a single nucleotide addition). In some embodiments, wash solvent after capping is only reused for the subsequent wash after capping on the next cycle, not for washing after any other chemistry. In some embodiments, the same wash solvent is used for multiple SPOS cycles. In some embodiments, wash solvent after a deblocking step is only reused for washing after subsequent deblocking steps in the next cycles, and not for washing after any other SPOS steps (e.g., coupling, oxidation / sulfurization, capping). In some embodiments, acetonitrile (ACN) is used for washing the reactor (and the solid substrate, such as resin, contained therein) after the deblocking, coupling, and capping steps.

[0051] 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. In some embodiments, methods of the disclosure comprise performing SPOS cycles “n” times, where “n” is an integer between 3 and 100 (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) times. In some embodiments, the length of an oligonucleotide crated by the methods described herein depends on the number of cycles / times the method is performed.

[0052] 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.

[0053] 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 more abundant starting materials and solvents. The synthesis of oligonucleotides via SPOS requires complex phosphoramidite nucleotides and large quantities of expensive solvents and reagents such as acid solutions (e.g., dichloroacetic acid (DCA) solutions) and acetonitrile (ACN). Furthermore, currently employed SPOS systems and methods are not generally efficient. Kilotons of solvents and starting materials are required per kilogram of oligonucleotide produced.

[0054] 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.

[0055] Fluidized Bed Reactor

[0056] 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). FIG. 3 shows a representative schematic diagram for a FBR configured for material reuse, according to some aspects of the technology.

[0057] 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.

[0058] Turning to the figures, FIG. 1 shows a representative diagram for a fluidized bed reactor (FBR), according to some aspects of the technology. In FIG. 1, small boxes coded by numbers or letters represent material feed vessels (e.g., 1 to 27) or synthesizer operation vessels (e.g., 29 and 30). As used herein, a “feed vessel” refers to a container of suitable volume and geometry for containing a solution used in SPOS. A feed vessel may be made of any suitable material, for example glass, polymers, plastic, metal, etc. In some embodiments, a feed vessel is a carboy. The vessel contents or intended purpose of use are annotated on the diagram. The amidite solutions are contained separately in feed vessels labeled 1-11 and are moved from the vessels into the reactor feed zone by mechanical pumps or by pressure transfers (e.g., pressure differentials). In some embodiments, a FBR comprises between 1 and 20 amidite feed vessels (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 feed vessels) that are 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 difference 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. 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 or high concentration 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 or high concentration 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 or high concentration acid add-back is to reduce overall process volumes. Because the DCA concentration in the reuse DCA solution is higher after adding neat or high concentration 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.

[0059] 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.

[0060] In some embodiments, a FBR comprises a coupling module comprising one or more amidite feed vessels (e.g., (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), and (11) in FIG. 1), an activator feed vessel (12), an acetonitrile (ACN) feed vessel (19), and an amidite feed zone vessel (28). In some embodiments, the one or more amidite feed vessels are in fluid communication with the amidite feed zone vessel (28), which is in fluid communication with the reactor (30). In some embodiments, the activator feed vessel (12) is in fluid communication with the amidite feed zone vessel (28), which is in fluid communication with the reactor (30). The ACN feed vessel (19) may be in fluid communication with both the amidite feed zone vessel (28) and / or the feed zone vessel (29) and / or the reactor (30). In some embodiments, the coupling module is configured to pump or push the specified amidite into the amidite feed zone vessel (28) and chase it in with nitrogen, then pump or push activator solution from the activator feed vessel (12) into the amidite feed vessel (28) and chase in with nitrogen, fluidizing the two together by introducing nitrogen gas into the bottom of the amidite feed vessel (28). In some embodiments, the coupling module is configured to push this mixture into the feed zone vessel (29), and then into the reactor (30) to start the coupling reaction on the resin. In some embodiments, the coupling module is configured to push this mixture from the amidite feed vessel (28) into the reactor (30) to start the coupling reaction on the resin. In some embodiments, the coupling module is configured to operate under fluidization conditions.

[0061] 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 vessels (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; not shown in FIG. 2) 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; not shown in FIG. 2), which is in fluid communication with the feed zone (29). In some embodiments, the oxidation solution reuse vessel (23; not shown in FIG. 2) 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.

[0062] In some embodiments, a FBR comprises a capping module comprising one or more capping solution feed vessels (17, 18). In some embodiments, each of the one or more capping solution feed vessels (17) and (18) is in fluid communication with a feed zone vessel (29). In some embodiments, a capping module further comprises a capping solution reuse vessel (25). In some embodiments, the capping solution reuse vessel (25) is in fluid communication with the reactor (30). In some embodiments, the capping solution reuse vessel (25) is in fluid communication with the feed zone vessel (29). In some embodiments, a capping module further comprises a vessel with reuse wash solvent that is used to dilute the capping reaction on the next cycle (26), which is in fluid communication with the reactor (30) and the feed zone vessel (29). In some embodiments, a capping module further comprises a reuse wash solution vessel (27). In some embodiments, the reuse wash vessel (27) is in fluid communication with the reactor (30) and the feed zone vessel (29). In some embodiments, the capping module is configured to work under fluidization conditions. Capping modules are further described herein in the section entitled “Capping Solution Reuse”.

[0063] 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. 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) then 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.

[0064] In some embodiments, a FBR further comprises a waste module comprising one or more waste containers. In some embodiments, the waste container is in fluid communication with the reactor (30). In some embodiments, a waste module further comprises an evaporator (31) in fluid communication with the reactor (30). In some embodiments, the evaporator is in fluid communication with the waste container. In some embodiments, the evaporator (30) is configured to recover a portion of the acid solution (e.g., deblocking solution) can so that it can be used for solvent washing after deblocking in the next cycle of SPOS. In some embodiments, a waste module 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 recovered from the evaporator (30) 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.

[0065] FBR systems comprising deblocking 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.

[0066] In some embodiments, use of FBR systems and deblocking methods described by the disclosure for SPOS results in higher crude purity and yield of oligonucleotides. As described further in the Examples, SPOS of a 36mer sense strand oligonucleotide using a FBR and deblocking methods described herein resulted in at least 10% higher purity corrected crude yield on average relative to synthesis of the same strand using a packed bed reactor (PBR).

[0067] In some embodiments, use of FBR systems and deblocking methods described by the disclosure for SPOS reduces ACN solvent use by at least a factor of eight relative to previously described SPOS reactions using FBR or PBRs. In some embodiments, dry ACN solvent use is reduced by at least a factor of five relative to previously described SPOS reactions using FBR or PBRs.

[0068] In some embodiments, use of FBR systems and deblocking methods described by the disclosure for SPOS reduces DCA and toluene used for deblocking by at least a factor of two relative to previously described SPOS reactions using FBR or PBRs.

[0069] In some embodiments, use of FBR systems and deblocking methods 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 FBR or PBRs.

[0070] In some embodiments, use of FBR systems and deblocking methods described by the disclosure for SPOS are more amenable to process analytical technologies (PATs) that can derisk larger batches and eliminate oligonucleotide sequencing assays, relative to previously described SPOS reactions using FBR or PBRs.

[0071] In some embodiments, use of FBR systems and deblocking methods described by the disclosure for SPOS are more amenable to different types of resins, with different loading, different swelling and shrinking properties, relative to previously described SPOS reactions using FBR or 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.

[0072] In some embodiments, use of FBR systems and deblocking methods described by the disclosure for SPOS allows for use of less special amidite equivalents relative to previously described SPOS reactions using FBR or PBRs because the systems can charge 100% of the feed tank make-up.

[0073] In some embodiments, use of FBR systems and deblocking methods described by the disclosure for SPOS produce less waste than previously described SPOS reactions using FBR or PBRs. In some embodiments, the systems and methods significantly reduce ACN and recycle toluene for deblocking. For example, FBR-based synthesis of oligonucleotides has demonstrated synthesis process mass intensity (PMI) less than PMI 3000 for a 36mer oligonucleotide sense strand, compared to synthesis PMI 7000 for standard packed bed reactor (PBR).

[0074] In some embodiments, use of FBR systems and deblocking methods described by the disclosure for SPOS produce higher batch to batch consistency relative to previously described SPOS reactions using FBR or PBRs.

[0075] Aspects of the disclosure relate to FBR machines, and methods of using FBR machines, which incorporate reuse of certain materials at one or more steps. FIG. 3 shows a representative schematic diagram for a FBR configured for material reuse, according to some aspects of the technology.

[0076] In some embodiments, an FBR machine is configured to reuse acetonitrile (ACN). FIG.

[0077] 4 shows a representative schematic diagram for a FBR configured for ACN reuse, according to some aspects of the technology. In some embodiments, an FBR configured for ACN reuse comprises one or more (e.g., 1, 2, 3, 4, 5, 6, or more) vessels configured to contain ACN which has been used in one or more SPOS cycles and / or one or more SPOS wash steps. In some embodiments, the one or more vessels is in fluid communication with a feed vessel.

[0078] Neat DCA addback to the reuse acid solution during deblocking

[0079] Aspects of the disclosure relate to FBRs comprising a modified deblocking module. This disclosure is based, in part, on addition (also referred to herein as “add-back”) of new, neat acid (e.g., neat DCA) to reused deblocking solution from a previous SPOS cycle for deblocking reactions in a later SPOS deblocking reaction. In some embodiments, acid add-back methods described herein significantly reduce the amount of toluene needed to perform synthesis of an entire oligonucleotide using SPOS. In the embodiments, the systems and methods reduce the amount of toluene used during SPOS by between about 1.5-fold and about 4-fold. In the embodiments, the systems and methods reduce the amount of toluene used during SPOS by between about 2-fold and about 4-fold. In the embodiments, the systems and methods reduce the amount of acid solution and / or toluene used during SPOS by at least 1.5, at least 2-, at least 3-, or at least 4-fold relative to previously described SPOS reactions using FBR or PBRs.

[0080] FIG. 2A shows an illustration of a representative FBR system configured to recycle acid solutions during subsequent deblocking reactions. Briefly, the FBR system comprises a reactor (30), a feed zone vessel (29), a fresh acid solution (e.g., DCA) vessel (13A), a neat or high concentration acid solution (e.g., neat or high concentration DCA) vessel (13B), and a reuse acid vessel (21). The fresh acid solution vessel (13A), reuse acid solution vessel (21), and the neat acid solution vessel (13B) are connected to the feed zone vessel (29).

[0081] 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) or reuse acid solution vessel (21) 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 1-25 ml / mmol neat DCA is added to the reuse acid solution prior to charging to the reactor. In some embodiments, between about 1%- 10% v / v neat DCA is 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, less fresh DCA solution is needed to complete the reaction.

[0082] 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. One 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. Another purpose of the evaporator is to use a portion of the evaporator bottoms in the deblocking reaction on future cycles of the synthesis.

[0083] 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.

[0084] Reuse DCA-rich portion of post-detritylation ACN wash as part of the deblocking process In some embodiments, the DCA-rich portion of post-deblocking wash is used as part of the deblocking process on future cycles in the synthesis. This is the first material charged to the reactor at the start of the deblocking process steps. The liquid completely fluidizes with the resin for a specific time according to the recipe. Then the liquid pushes out the bottom of the reactor either to waste or to the evaporator.

[0085] FIG. 2B shows an illustration of a representative FBR system configured to reuse acid solutions and the DCA-rich portion of post-deblocking ACN wash during subsequent deblocking reactions. Briefly, the FBR system comprises a reactor (30), a feed zone vessel (29), a fresh acid solution (e.g., DCA) vessel (13 A), a reuse acid vessel (21), and a vessel for reusing the first part of the wash after deblock (vessel 33). The fresh acid solution vessel (13A), reuse acid solution vessel (21), and reuse wash vessel (33) are connected to the feed zone vessel (29).

[0086] Aspects of the disclosure relate to FBRs comprising a modified deblocking module. This disclosure is based, in part, on first charging the DCA-rich portion of post-deblocking wash to the reactor at the start of the deblocking process steps. The amount of this material collected from post-deblocking wash ranges from 1% to 50% of the total amount of solvent used in the post-deblocking wash. The liquid may be combined with additional amount of solvent such as ACN or toluene to increase its volume. The liquid completely fluidizes with the resin for a specific time according to the recipe. Then the liquid pushes out the bottom of the reactor either to waste or to the evaporator. In some embodiments, reuse of DCA wash described herein significantly reduces the amount of DCA needed to perform synthesis of an entire oligonucleotide using SPOS. In the embodiments, the systems and methods reduce the amount of DCA used during SPOS by between about 1.3-fold and about 2-fold relative to previously described SPOS reactions using FBR or PBRs.

[0087] 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, the amount or concentration of reused DCA wash solution charged to the reactor at the start of deblocking may vary. In some embodiments, about 50 ml / mmol reused DCA wash is charged to the reactor and fluidized. In some embodiments, between 10 and 200 ml / mmol reused DCA wash is charged to the reactor. In some embodiments, reused DCA wash charged to the reactor range in concentration between about 1% and 20% DCA. The purpose of reused DCA wash is to reduce overall amount of fresh DCA needed. 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.

[0088] Using the distillation bottoms as part of the deblocking process

[0089] Aspects of the disclosure relate to FBR machines, and methods of using FBR machines, comprising a distillation unit. FIG. 5 shows a representative schematic diagram for a FBR comprising a distillation unit, according to some aspects of the technology. In some embodiments, the distillation bottoms is used as part of the deblocking process. This is an alternative to using the DCA-rich portion of post-deblocking ACN wash as part of the deblocking process. This is the first material charged to the reactor at the start of the deblocking process steps. The liquid completely fluidizes with the resin for a specific time according to the recipe. Then the liquid pushes out the bottom of the reactor either to waste or to the evaporator.

[0090] FIG. 2C shows an illustration of a representative FBR system configured to reuse acid solutions and the distillation bottoms during subsequent deblocking reactions. Briefly, the FBR system comprises a reactor (30), a feed zone vessel (29), a fresh acid solution (e.g., DCA) vessel (13A), a reuse acid vessel (21), and a vessel for reusing the distillation bottoms (vessel 34). The fresh acid solution vessel (13A), reuse acid solution vessel (21), neat or high concentration acid solution vessel (13B), and distillation bottoms vessel (34) are connected to the feed zone vessel (29).

[0091] Aspects of the disclosure relate to FBRs comprising a modified deblocking module. This disclosure is based, in part, on first charging the distillation bottoms to the reactor at the start of the deblocking process steps. The liquid completely fluidizes with the resin for a specific time according to the recipe. Then the liquid pushes out the bottom of the reactor either to waste or to the evaporator. In some embodiments, distillation bottoms described herein significantly reduces the amount of DCA needed to perform synthesis of an entire oligonucleotide using SPOS. In the embodiments, the systems and methods reduce the amount of DCA used during SPOS by between about 1.3-fold and about 2-fold relative to previously described SPOS reactions using FBR or PBRs. FIG. 5 shows a representative schematic diagram for a FBR comprising a distillation unit, according to some aspects of the technology. 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 and distillation bottoms from the previous cycles are used for each step of deblocking. In some embodiments, the amount or concentration of distillation bottoms solution charged to the reactor at the start of deblocking may vary. In some embodiments, about 50 ml / mmol distillation bottoms is charged to the reactor and fluidized. In some embodiments, between 10 and 200 ml / mmol distillation bottoms are charged to the reactor. In some embodiments, distillation bottoms ranging in concentration from about 5% DCA to about 50% DCA are charged to the reactor. The purpose of using distillation bottoms is to reduce overall amount of fresh DCA needed.

[0092] 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.

[0093] Fully fluidized deblocking in FBR

[0094] In some embodiments, all deblocking steps are fluidized. FIG. 7 shows a representative process flow diagram for a fully fluidized, reuse SPOS process in an FBR, according to some aspects of the technology. For example, to perform fully fluidized deblock reaction based on the material reuse scheme depicted in Fig 2B, 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. In some embodiments, each fill-fluidize-drain cycle uses an equal portion of the material in vessel 21, and at the end, vessel 21 is depleted. In some embodiments, the volume of each portion added to the vessel 33 ranges from about 10 mL / mmol (starting resin basis) to about 200 mL / mmol (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mL / mmol). In some embodiments (e.g., embodiments where materials or reagents are not reused), fresh deblocking solution is added to the reactor in 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) portions, each portion fluidized with the solid support, then removed from the reactor and sent to vessel 21. In some embodiments, the volume of each fresh deblocking solution portion ranges from about 10 mL / mmol to about 200 mL / mmol (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mL / mmol). One of the benefits of fully fluidized deblocking 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. The skilled person will understand that, in some embodiments, a fully fluidized SPOS process may be performed without reuse of materials or reagents, according to some aspects of the disclosure.

[0095] Deblocking in FBR with Thiol Scavengers

[0096] FIG. 2D shows an illustration of a representative FBR system configured to use thiol in the deblocking reactions. Briefly, the FBR system comprises a reactor (30), a feed zone vessel (29), a fresh acid solution (e.g., DCA) vessel (13A), and a thiol solution feed vessel (35). The fresh acid solution vessel (13 A) and a thiol solution feed vessel (35) are connected to the feed zone vessel (29).

[0097] Aspects of the disclosure relate to FBRs comprising a modified deblocking module. This disclosure is based, in part, on adding a thiol scavenger to the deblocking reaction solution. The liquid completely fluidizes with the resin for a specific time according to the recipe. Then the liquid pushes out the bottom of the reactor to waste. In some embodiments, thiol scavenger described herein significantly reduces the amount of DCA needed to perform synthesis of an entire oligonucleotide using SPOS. In the embodiments, the systems and methods reduce the amount of DCA used during SPOS by between about 1.5-fold and about 4-fold relative to previously described SPOS reactions using FBR or PBRs.

[0098] In some embodiments, the amount or concentration of thiol solution charged to the reactor at the start of deblocking may vary. In some embodiments, about 50 ml / mmol thiol solution is charged to the reactor along with the acid solution and fluidized. In some embodiments, between 10 and 200 ml / mmol thiol solution is charged to the reactor. In some embodiments, thiol solutions ranging in concentration from about 0.5% to about 5% are charged to the reactor. The purpose of using thiol solutions is to reduce overall amount of fresh DCA needed.

[0099] In some embodiments, the deblocking solution for every step of deblocking is used in excess (e.g., from about 25:1 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.

[0100] No Reuse SPOS Processes

[0101] Aspects of the disclosure relate to FBR machines, and methods of using FBR machines that do not reuse any reagents or materials during SPOS cycles. FIG. 6 shows a representative schematic diagram for a FBR configured for no material reuse, according to some aspects of the technology. In some embodiments, no reuse SPOS processes are fully fluidized. In some embodiments, no reuse SPOS processes reduce plant footprint. In some embodiments, no reuse SPOS processes reduce cycle time. In some embodiments, no reuse FBR processes produce oligonucleotides at higher purity, higher yield, and better scalability relative to previously described SPOS processes, for example the standard PBR process.

[0102] EXAMPLES

[0103] Example 1: Neat DCA addback to the reuse acid solution for Deblocking

[0104] This example describes methods and systems for acid solution reuse during SPOS deblocking reactions, with neat DCA addback to the reuse acid solution. FIG. 2 shows an illustration of the material flow in the deblocking reaction. Briefly, neat DCA or high concentration DCA is added and mixed in with the reuse DCA solution, before the solution pushes into the reactor. The purpose is to reduce overall process volumes. Because the DCA concentration in the reuse DCA solution is higher, less fresh DCA solution is needed to complete the reaction. Also, in Example 3, 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 so that it can be used for solvent washing after detritylation in the next cycle.

[0105] A FBR system comprising a deblocking module configured for DCA add-back was used to synthesize a 36mer sense strand (SS) oligonucleotide 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 acid reuse module. The amount of DCA used in each SPOS cycle for the DCA add-back FBR is shown in Table 2. Data indicate that yield and purity were higher (Table 16) and total deblocking volumes were lower in the DCA add-back FBR system than PBR system (Table 13).

[0106] Table 1. RE0-D00084-045 Lpa SS, with 1% pyridine

[0107] destination when push out of material source reaction mode reactor

[0108] reuse acid +neat

[0109] DCA reuse DCA tank fluidize Waste

[0110] reuse acid + neat

[0111] DCA reuse DCA tank flow-through waste

[0112] new acid, 6% 6% acid feed tank flow-through reuse acid tank

[0113] reuse solvent wash WI_DCAtank 1 flow-through Waste

[0114] reuse solvent wash WI_DCAtank 2 flow-through WI_DCAtank 1

[0115] new solvent chase ACN feed tank Flow-through WI_DCAtank 1

[0116] reuse solvent wash WI_DCAtank3 flow-through WI_DCAtank 2

[0117] reuse solvent wash WI_DCAtank4 flow-through WI_DCAtank3

[0118] reuse solvent wash WI_DCAtank5 flow-through WI_DCAtank4

[0119] reuse solvent wash WI_DCAtank6 flow-through WI_DCAtank5

[0120] new solvent reactor

[0121] wall wash ACN feed tank Flow-through WI_DCAtank5

[0122] pyridine pyridine feed tank flow-through WI_DCAtank6

[0123] new solvent washes ACN feed tank flow-through WI_DCAtank6

[0124] new amidite, post O / S tank (to be used for the amidite + activator activator fluidize wash after oxidation)

[0125] new solvent chase post O / S tank (to be used for the and washes ACN feed tank flow-through wash after oxidation)

[0126] oxidizer oxidizer feed tank fluidize Waste

[0127] New solvent chase ACN feed tank Flow-through Waste

[0128] post O / S wash post O / S tank flow-through Waste

[0129] Reuse capping

[0130] reaction solution Reuse CAP tank flow-through Waste

[0131] New Capping Capping A feed

[0132]

[0133] reagents and tank, Capping B fluidize Reuse CAP tank dilution solvent for feed tank, DIL_CAP

[0134] capping reaction tank

[0135] reuse solvent wash Wl CAP tank flow-through DIL_CAP tank

[0136] new solvent chase

[0137]

[0138] and wash ACN feed tank flow-through Wl CAP tank

[0139] 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 2-7 show representative data for an FBR SPOS reaction as described in this Example.

[0140] Table 2.

[0141] total Fresh

[0142] ACN wash

[0143] total after

[0144] toluene deblocking

[0145] used in includingthe

[0146] deblock + amount

[0147] Neat DCA chase / wash total DCA prefilled to pyridine in added to after used in the counter wash after

[0148] 6% DCA reuse deblock deblock current wash deblocking

[0149] Cycle (mL) (mL) (mL)l (mL)l vessels(mL)2 (mL)

[0150] 1 484 7.4 455 36 2351 3.3

[0151] 2 273 8.3 257 25 372 3.4

[0152] 3 282 8.3 265 25 374 3.3

[0153] 4 298 8.5 280 26 385 3.4

[0154] 5 304 9.4 286 28 389 3.3

[0155] 6 326 10.3 306 30 383 3.3

[0156] 7 350 11.3 329 32 529 4.4

[0157] 8 381 12.3 359 35 528 4.3

[0158] 9 417 12.2 392 37 525 4.3

[0159] 10 427 12.1 402 38 523 4.3

[0160] 11 454 13.3 427 41 514 4.4

[0161] 12 460 13.3 432 41 532 4.4

[0162] 13 472 14.4 444 43 658 5.2

[0163] 14 532 14.3 500 46 649 5.3

[0164] 15 535 14.5 503 47 651 5.3

[0165] 16 534 15.4 502 47 663 5.2

[0166] 17 529 15.3 497 47 655 5.3

[0167] 18 538 16.4 506 49 649 5.2

[0168] 19 547 16.4 514 49 809 6.4

[0169] 20 554 16.4 520 50 819 6.5

[0170] 21 566 17.2 532 51 819 6.5

[0171] 22 578 17.2 544 52 809 6.5

[0172] 23 594 17.2 558 53 804 6.5

[0173]

[0174] 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

[0175] per mmol

[0176]

[0177] (L / mmol) 3.635 0.340 5.657 0.042

[0178] Table 3.

[0179] Amidite,

[0180] note that

[0181] FBR was

[0182] 0.1 M and

[0183] PBR was

[0184] 0.2 M, FBR

[0185] 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 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

[0186]

[0187] 18 15.7 12.03 MC 79 89 1.77 19 19.3 12.22 MU 84 95 1.90

[0188] 20 19.5 12.25 MA 80 90 1.80

[0189] 21 19.6 11.98 MC 80 90 1.80

[0190] 22 19.5 12.15 MU 84 95 1.90

[0191] 23 19.5 12.27 MG 79 88 1.77

[0192] 24 19.7 12.45 MG 88 100 2.00

[0193] 25 22.3 12.45 FU 87 99 1.97

[0194] 26 22.3 12.70 FU 88 100 1.99

[0195] 27 22.3 12.75 FC 90 102 2.04

[0196] 28 22.4 12.88 FG 87 99 1.98

[0197] 29 22.6 12.98 MA 90 102 2.05

[0198] 30 22.4 13.10 MA 90 103 2.06

[0199] 31 25.3 12.78 MC 89 101 2.02

[0200] 32 25.5 12.88 MC 86 98 1.95

[0201] 33 25.2 13.02 MG 87 99 1.97

[0202] 34 25.4 13.20 MU 92 104 2.09

[0203] 35-1 25.6 13.28 MUS 96 109 2.18

[0204] 35-2 25.6 13.32

[0205] Totals 637 2950 3335

[0206] Normalized

[0207] per mmol

[0208]

[0209] (L / mmol) 0.127 0.667

[0210] Table 4.

[0211] activator Coupling Fresh ACN

[0212] equivalent contact wash after

[0213] 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

[0214]

[0215] 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

[0216] 35-2

[0217] Totals 3427 3500 8893 9056.008 Normalized

[0218] per mmol

[0219]

[0220] (L / mmol) 0.685 0.700 1.811

[0221] Table 5.

[0222] Oxidation or Fresh AON

[0223] sulfurization wash after

[0224] Xanthane contact oxidation or Diluted Diluted Diluted Xanthane hydride time sulfurization CappingA CappingA Capping B Cycle hydride (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

[0225]

[0226] 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 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

[0227] 35-2

[0228] Totals 268 268 1815

[0229] Normalized

[0230] per mmol

[0231]

[0232] (L / mmol) 0.054 0.363

[0233] Table 6.

[0234] Standard

[0235] Capping B

[0236] Standard reagent Fresh Fresh CappingA (concentration ACN ACN

[0237] reagent is20vol% used for Capping used for (concentration acetic diluting reaction wash total Diluted is20vol% 1 anhydride, 30 capping contact after materials Capping methylimidizole) vol% lutidine) reagents time capping charged to

[0238]

[0239] Cycle B(mL) (mL) (mL) (mL) (minutes)4 (mL)2 synthesizer 1 46.2 10.2 9.2 153 5.3 210

[0240] 2 44.9 10.3 9.0 77 5.3 90

[0241] 3 46.4 10.3 9.3 78 5.3 90

[0242] 4 46.2 10.2 9.2 78 5.3 90

[0243] 5 46.2 10.3 9.2 78 5.3 90

[0244] 6 45.9 10.2 9.2 78 5.3 90

[0245] 7 46.2 10.3 9.2 78 5.3 90

[0246] 8 46.2 10.4 9.2 78 5.3 90

[0247] 9 44.9 10.3 9.0 77 5.3 90

[0248] 10 46.2 10.2 9.2 78 5.3 90

[0249] 11 46.2 10.3 9.2 78 5.3 90

[0250] 12 46.2 10.3 9.2 78 5.3 120

[0251] 13 46.2 10.2 9.2 78 5.3 120

[0252] 14 43.7 10.3 8.7 76 5.3 120

[0253] 15 46.2 10.3 9.2 78 5.3 120

[0254] 16 46.2 10.2 9.2 78 5.3 120

[0255] 17 45.2 10.3 9.0 77 5.3 120

[0256] 18 44.9 10.3 9.0 77 5.3 120

[0257] 19 46.2 10.2 9.2 78 5.3 120

[0258] 20 46.2 10.3 9.2 78 5.3 120

[0259] 21 45.2 10.2 9.0 77 5.3 120

[0260] 22 44.9 10.3 9.0 77 5.3 150

[0261] 23 46.2 10.3 9.2 78 5.3 150

[0262] 24 44.9 10.3 9.0 77 5.3 150

[0263] 25 44.9 10.3 9.0 77 5.3 150

[0264] 26 44.9 10.2 9.0 77 5.3 150

[0265] 27 46.2 10.2 9.2 78 5.3 150

[0266] 28 44.9 10.4 9.0 78 5.3 150

[0267] 29 44.9 10.2 9.0 77 5.3 150

[0268] 30 45.2 10.3 9.0 78 5.3 150

[0269] 31 46.2 10.1 9.2 77 5.3 150

[0270] 32 46.2 10.3 9.2 78 5.3 150

[0271] 33 46.2 10.3 9.2 78 5.3 150

[0272] 34 46.2 10.3 9.2 78 5.3 150

[0273] 35-1

[0274] 35-2

[0275] Totals 349 310 2715 4260 Normalized

[0276] per mmol

[0277]

[0278] (L / mmol) 0.070 0.062 0.543 0.852 15.6

[0279] Material Composition:

[0280] DCA was 6 vol% in toluene

[0281] Amidites were 0.1M solutions in ACN (mU solvent was 10vol% toluene in ACN)

[0282] Activator was 0.5 M ETT in ACN

[0283] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent) Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[0284] Cap A was 4 vol% 1 methylimidizole in ACN

[0285] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in ACN

[0286] Table 7.

[0287] | R60-D000£4-045 (Lab | RfO-D(XX)84-O45 eLN _ _ UPLC) _ (Release UPLC) Strand [ Lpa Sense Lpa Sense

[0288] mass of initial resin whole batch (g) _ | 19.6000 _ _ 19,6000 resi^ j ™

[0289] Synthesis scale (urnol) _ 1 _ 4,998 _ 4,998 mas^^ 909000 909000 msssgajnlg) ZL30 7190 Mass gain per mmol scale _ 14.27 _ 14.27 Crude mass yield (by weighing! J 0.93 I

[0290]

[0291] FLP% (homogenized sample)* | 0.8311 { 0.79

[0292] Mass of resin taken for C&D (g) _ | _ 0,1613 _ j _ 0.1613 Fraction of the whole batch [ j 015% Mass of oligo in C&D resin sample (g) | 0.1266 j 0.1265 30 wt% NH4OH solatia mass (g) | 2.1993 j 2.1993 NH4OH plus oligo mass (g) | 2.3258 | 2.3258 Aliquot mass of C&D sol’n for OD (g) | 0.3151 | 0.3151 DI water added for dilution (g) | 20.24 | 20.24 NanoDrop measured A26O&1 | 1.4.64 | 14.64 NanoDrop measured A260 #2 _ _ 14.62 _ _ 14.62 NanoDrop measured A260 #3 I 14.55 I 14.55 NanoDrop measured A2.6O AVE [ 14.60 [ 14.60 QD dilution factor _ _ 65.25 _ _ 65.25 TotaTt^^ 1^248314 j 1^248314” Mass Na Salt calculated from total QD [ 54.21 j 54.21 QD / umol 250 250 crude % yield by OD 78% 78%

[0293]

[0294] Purity corrected yield by OD [ 64% j 61% Example 2: PBR comparison without Reuse acid solution and without DCA addback to reuse acid solution. 230 umol scale.

[0295] Tables 8-12 show representative data for a PBR SPOS reaction as described in this Example.

[0296] Table 8.

[0297] totaltoluene

[0298] used in

[0299] Toluene deblock + total DCA ACN in pyridine in

[0300] wash after chase / wash used in wash after wash after

[0301] 3% DCA deblocking after deblock deblock deblocking deblocking

[0302] Cycle (mL) (mL) (mL)l (mL)l (mL)l (mL)

[0303] 1 53.02 10.6 63.62 1.5906 21.2 10.6

[0304] 2 55.14 10.6 65.74 1.6542 21.2 10.6

[0305] 3 58.32 10.6 68.92 1.7496 21.2 10.6

[0306] 4 60.43 10.6 71.03 1.8129 21.2 10.6

[0307] 5 46.98 10.6 57.58 1.4094 21.2 10.6

[0308] 6 48.57 10.6 59.17 1.4571 21.2 10.6

[0309] 7 50.89 10.6 61.49 1.5267 21.2 10.6

[0310] 8 52.48 10.6 63.08 1.5744 21.2 10.6

[0311] 9 54.08 10.6 64.68 1.6224 21.2 10.6

[0312] 10 56.51 10.6 67.11 1.6953 21.2 10.6

[0313] 11 58.1 10.6 68.7 1.743 21.2 10.6

[0314] 12 59.7 10.6 70.3 1.791 21.2 10.6

[0315] 13 61.3 10.6 71.9 1.839 21.2 10.6

[0316] 14 63.62 10.6 74.22 1.9086 21.2 10.6

[0317] 15 65.2 10.6 75.8 1.956 21.2 10.6

[0318] 16 66.79 10.6 77.39 2.0037 21.2 10.6

[0319] 17 69.25 10.6 79.85 2.0775 21.2 10.6

[0320] 18 70.83 10.6 81.43 2.1249 21.2 10.6

[0321] 19 72.43 10.6 83.03 2.1729 21.2 10.6

[0322] 20 74.02 10.6 84.62 2.2206 21.2 10.6

[0323] 21 76.33 10.6 86.93 2.2899 21.2 10.6

[0324] 22 77.93 10.6 88.53 2.3379 21.2 10.6

[0325] 23 79.52 10.6 90.12 2.3856 21.2 10.6

[0326] 24 81.95 10.6 92.55 2.4585 21.2 10.6

[0327] 25 83.55 10.6 94.15 2.5065 21.2 10.6

[0328] 26 85.14 10.6 95.74 2.5542 21.2 10.6

[0329] 27 86.73 10.6 97.33 2.6019 21.2 10.6

[0330] 28 89.07 10.6 99.67 2.6721 21.2 10.6

[0331] 29 90.64 10.6 101.24 2.7192 21.2 10.6

[0332] 30 92.24 10.6 102.84 2.7672 21.2 10.6

[0333] 31 94.67 10.6 105.27 2.8401 21.2 10.6

[0334] 32 96.27 10.6 106.87 2.8881 21.2 10.6

[0335] 33 97.85 10.6 108.45 2.9355 21.2 10.6

[0336] 34 99.45 10.6 110.05 2.9835 21.2 10.6

[0337]

[0338] 35 101.77 10.6 112.37 3.0531 21.2 10.6 Totals 2531 371 2825.8469 75.9231 742 371

[0339] Normalized

[0340] per mmol

[0341] (L / mmol or

[0342]

[0343] kg / mmol) 12.23 0.328 3.171 1.6

[0344] Table 9

[0345] 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

[0346]

[0347] 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

[0348] Normalized

[0349] per mmol

[0350] (L / mmol or

[0351]

[0352] kg / mmol) 0.35 0.7 4.496

[0353] Table 10.

[0354] Oxidation or AON in

[0355] sulfurization wash after

[0356] Xanthane contact oxidation or

[0357] Oxidizer hydride time sulfurization Cycle (mL) (mL) (minutes) (mL)3

[0358] 1 12.72 0 3.25 31.8

[0359] 2 12.72 0 3.25 31.8

[0360] 3 12.72 0 3.25 31.8

[0361] 4 12.72 0 3.25 31.8

[0362] 5 12.72 0 3.25 31.8

[0363] 6 12.72 0 3.25 31.8

[0364] 7 12.72 0 3.25 31.8

[0365] 8 12.72 0 3.25 31.8

[0366] 9 12.72 0 3.25 31.8

[0367] 10 12.72 0 3.25 31.8

[0368] 11 12.72 0 3.25 31.8

[0369] 12 12.72 0 3.25 31.8

[0370] 13 12.72 0 3.25 31.8

[0371] 14 12.72 0 3.25 31.8

[0372] 15 12.72 0 3.25 31.8

[0373] 16 12.72 0 3.25 31.8

[0374] 17 12.72 0 3.25 31.8

[0375] 18 12.72 0 3.25 31.8

[0376] 19 12.72 0 3.25 31.8

[0377] 20 12.72 0 3.25 31.8

[0378] 21 12.72 0 3.25 31.8

[0379] 22 12.72 0 3.25 31.8

[0380] 23 12.72 0 3.25 31.8

[0381] 24 12.72 0 3.25 31.8

[0382] 25 12.72 0 3.25 31.8

[0383] 26 12.72 0 3.25 31.8

[0384] 27 12.72 0 3.25 31.8

[0385] 28 12.72 0 3.25 31.8

[0386] 29 12.72 0 3.25 31.8

[0387] 30 12.72 0 3.25 31.8

[0388] 31 12.72 0 3.25 31.8

[0389] 32 12.72 0 3.25 31.8

[0390]

[0391] 33 12.72 0 3.25 31.8 34 12.72 0 3.25 31.8

[0392] 35 12.72 11.66 10.4 31.8

[0393] Totals 445.2 11.66 1113

[0394] Normalized

[0395] per mmol

[0396] (L / mmol or

[0397]

[0398] kg / mmol) 1.903 0.05 4.756

[0399] Table 11.

[0400] Capping ACN used

[0401] reaction in wash total contact after materials CappingA Capping B time capping charged to Cycle (mL) (mL) (minutes) (mL)3 synthesizer 1 2.65 2.65 2.31 31.8

[0402] 2 2.65 2.65 2.31 31.8

[0403] 3 2.65 2.65 2.31 31.8

[0404] 4 2.65 2.65 2.31 31.8

[0405] 5 2.65 2.65 2.31 31.8

[0406] 6 2.65 2.65 2.31 31.8

[0407] 7 2.65 2.65 2.31 31.8

[0408] 8 2.65 2.65 2.31 31.8

[0409] 9 2.65 2.65 2.31 31.8

[0410] 10 2.65 2.65 2.31 31.8

[0411] 11 2.65 2.65 2.31 31.8

[0412] 12 2.65 2.65 2.31 31.8

[0413] 13 2.65 2.65 2.31 31.8

[0414] 14 2.65 2.65 2.31 31.8

[0415] 15 2.65 2.65 2.31 31.8

[0416] 16 2.65 2.65 2.31 31.8

[0417] 17 2.65 2.65 2.31 31.8

[0418] 18 2.65 2.65 2.31 31.8

[0419] 19 2.65 2.65 2.31 31.8

[0420] 20 2.65 2.65 2.31 31.8

[0421] 21 2.65 2.65 2.31 31.8

[0422] 22 2.65 2.65 2.31 31.8

[0423] 23 2.65 2.65 2.31 31.8

[0424] 24 2.65 2.65 2.31 31.8

[0425] 25 2.65 2.65 2.31 31.8

[0426] 26 2.65 2.65 2.31 31.8

[0427] 27 2.65 2.65 2.31 31.8

[0428] 28 2.65 2.65 2.31 31.8

[0429] 29 2.65 2.65 2.31 31.8

[0430] 30 2.65 2.65 2.31 31.8

[0431] 31 2.65 2.65 2.31 31.8

[0432] 32 2.65 2.65 2.31 31.8

[0433]

[0434] 33 2.65 2.65 2.31 31.8 34 2.65 2.65 2.31 31.8

[0435] 35 2.65 2.65 2.31 31.8

[0436] Totals 92.75 92.75 1113

[0437] Normalized

[0438] per mmol

[0439] (L / mmol or

[0440]

[0441] kg / mmol) 0.396 0.396 4.756 35.133 1 This wash includes ACN used for coupling push

[0442] 2 Recycle time

[0443] 3 includes push volume

[0444] Material Composition:

[0445] DCA solution: 3 volume% DCA in toluene

[0446] Amidites solutions:

[0447] Activator was 0.5 M ETT

[0448] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent) Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[0449] Cap A was 20 vol% 1 methylimidizole in ACN

[0450] Cap B was 20vol% acetic anhydride and 30vol% lutidine in ACN Table 12.

[0451] eLN BXW-D00302- 167-5

[0452] Strand ss

[0453] mass of initial resin whole batch (g) _ 0.9026 _

[0454] resin loading (umol / g) 2S5

[0455] Synthesis scale (umol) 230

[0456] mass of final resin whole batch (g) 4.0601

[0457] mass gain (g) 3.16

[0458] Mass gain per mmol scale 13.72

[0459] Crude mass yield (by weighing) 0.90

[0460]

[0461] FLP% (homogenized sample)* 0.75

[0462] Mass of resin taken for C&D (g) 0.0375

[0463] Fraction of the whole batch 0.92%

[0464] Mass of oligo in C&D resin sample (g) 0.0292

[0465] 30 wt% NH4OH solutin mass (g) 0.8450

[0466] NH4OH plus oligo mass (g) 0.8742

[0467] Aliquot mass of C&D sol*n for OD (g) 0.1992

[0468] DI wateradded for dilution (g) 20.42

[0469] NanoDrop measured A26D&1 5.67

[0470] NanoDrop measured A260#2 5.67

[0471] NanoDrop measured A260#3 5.71

[0472] NanoDrop measured A260AVE * 5.68

[0473] OD dilution factor 103.52

[0474] Total OD for whole batch 55,681

[0475] Mass Na Salt calculated from total OD 2.42

[0476] OD / umol 242

[0477] crude % yield by OD 75%

[0478]

[0479] Purity corrected yield by OD 57%

[0480] MW of Na Salt (g / mol) 13986.6

[0481] Absorption Factor (OD / mg) 23.04

[0482] Theoretical OD / umol (Na Salt) 322.25

[0483] ug Na Salt / OD _ _ 43.40

[0484] theoretical on-resin mass gain / mmol

[0485]

[0486] (DEA treated, DMT-OFF) 15.29

[0487] Tables 13-16 provide representative data comparing FBR and PBR SPOS reactions described in this Example. Table 13.

[0488] total Fresh ACN wash

[0489] after deblocking including

[0490] the amount prefilled to

[0491] total toluene total DCA the counter current wash

[0492] used in used in vessels, and includingthe Total toluene scale deblock deblock amount used to dilute the used in the wash Example synthesizer (mmol) (L / mmol) (L / mmol) pyridine (L / mmol) after deblock BXW-167-5 PBR 0.23 12.2 0.33 3.2 - REO-045 FBR 5 3.6 0.34 5.8 - REO-059 FBR 5 2.4 0.27 2.7 0 REO-063 FBR 5 4.511 0.288 3.008 0.75624

[0493]

[0494] CWO-006 FBR 5 8.229 0.525 7.9564 2.04666

[0495] Table 14.

[0496] Amidite, note that

[0497] FBR was 0.1 M in

[0498] pyridine the lab and PBR average average Fresh

[0499] used in was 0.2 M, FBR amidite activator ACN wash

[0500] wash after used slightly less equivalent equivalent after

[0501] 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 REO-063 0.045 0.67 1.91 0.685 9.79 0.54 1.51

[0502]

[0503] CWO-006 0.068 0.67 1.9 0.695 9.93 0.739 1.811

[0504] Table 15.

[0505] Standard Fresh

[0506] Standard Capping B ACN used Fresh

[0507] Fresh ACN Capping A reagent 20vol% for ACN used

[0508] 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)

[0509] BXW-167- 5 0.050 4.76 0.40 0.40 0.00 4.76

[0510] REO-045 0.054 0.36 0.07 0.06 0.54 0.85

[0511] REO-059 0.043 0.77 0.06 0.06 1.02 0.82

[0512] REO-063 0.043 0.76 0.06 0.06 1.01 1.88

[0513]

[0514] CWO-006 0.043 3.61 0.13 0.13 1.56 6.36 Table 16.

[0515] total ACN used in

[0516] total the process

[0517] materials including all purity

[0518] charged to reagent solutions crude crude corrected

[0519] synthesizer and washes purity, yield, yield by

[0520] Example (L / mmol) (L / mmol) %FLP OD / umol OD, %

[0521] BXW- 167-5 35.1 18.7 75.2 242.0 57

[0522] REO-045 15.6 8.9 83.1 250 64

[0523] REO-059 11.6 6.2 81.5 261 66

[0524] REO-063 15.8 7.6 78.6 263 64

[0525]

[0526] CWO-006 34.6 20.2 84.9 259 68

[0527] Example 3: Reuse DCA-rich portion of post-detritylation wash for pre-detritylation treatment and fully fluidized detritylation.

[0528] This example describes methods and systems for collecting the DCA-rich portion of material during post-detritylation wash, temporarily storing the material, and re-introducing it to mix or flow through the solid phase to start the detritylation reaction. Here, the impact of this approach on SPOS of Lepodisiran (Lpa) siRNA Sense strand was investigated. Data indicate that this approach can effectively reduce the amount of detritylation reagent used in synthesis while maintaining similar levels of purity and yield. The experiments for this example were done in a different laboratory FBR system versus the experimental system used for example one.

[0529] This example also describes methods and systems for performing the detritylation in fully fluidized actions. In typical detritylation steps, the solid support reacts with the DCA-rich portion of the post-detritylation wash, collected from the previous cycle. This used material is added to the fluidized bed reactor in a single portion, fluidized with the solid support, then removed from the reactor and sent to waste. Then the used deblocking reagent was added to the reactor in three portions, each portion fluidized with the solid support, then removed from the reactor and sent to waste. Then the fresh deblocking reagent was added to the reactor in three portions, each portion fluidized with the solid support, then removed from the reactor and sent to the vessel holding the used deblocking reagent. Then in the post-detritylation wash, a portion of the washing solvent was added to the reactor, flowed through the solid support in a plug flow and was collected in a vessel holding the DCA-rich portion of the post-detritylation wash.

[0530] RE0-D00084-063 requires more acid solution compared to RE0-059 because 1) the deblocking steps are fluidized and 2) a lower concentration of the fresh deblocking solution was used. REO- 063 however has the advantage that all reaction steps are fluidized. This example has better scalability compared to other FBR examples described herein. Compared to RE0-059, RE0-063 used 1.9X toluene and 1.07X DCA from the deblocking reagent. RE0-063 also had longer acid-solid support contact time (25 ~ 32 min) than RE0-059 does (12 ~ 17 min), which is a disadvantage because of cycle time and because of generating more reaction byproducts with longer exposure times to the acid solution. Nevertheless, the advantages of better scalability, reliability, consistency batch to batch may still make the fully fluidized deblocking process the desired choice for manufacturing.

[0531] Table 17. RE0-D00084-063 Lpa SS: material source, destination, and reaction mode.

[0532] destination when push out of material source reaction mode reactor

[0533] Pre-DCA (reuse first Pre-DCA tank

[0534] part of wash after last

[0535] detrit) fluidize Waste

[0536] reuse acid reuse DCA tank fluidize Waste

[0537] new acid, 6% 6% acid feed tank fluidize reuse acid tank

[0538] reuse solvent wash WI_DCAtank 1 flow-through Pre-DCA

[0539] reuse solvent wash WI_DCAtank 1 flow-through Waste

[0540] reuse solvent wash WI_DCAtank2 fluidize WI_DCAtank 1

[0541] new solvent chase ACN feed tank Flow-through WI_DCAtank 1

[0542] reuse solvent wash WI_DCAtank3 fluidize WI_DCAtank 2

[0543] reuse solvent wash WI_DCAtank4 fluidize WI_DCAtank3

[0544] new solvent reactor

[0545] wall wash ACN feed tank fluidize WI_DCAtank4

[0546] pyridine pyridine feed tank fluidize WI_DCAtank4

[0547] new solvent washes ACN feed tank flow-through WI_DCAtank4

[0548] new amidite, post O / S tank (to be used for the amidite + activator activator fluidize wash after oxidation)

[0549] new solvent chase post O / S tank (to be used for the

[0550]

[0551] and washes ACN feed tank flow-through wash after oxidation) oxidizer oxidizer feed tank fluidize Waste

[0552] New solvent chase ACN feed tank Flow-through Waste post O / S wash post O / S tank flow-through Waste Reuse capping

[0553] reaction solution Reuse CAP tank flow-through Waste

[0554] New Capping Capping A feed

[0555] reagents and tank, Capping B

[0556] dilution solvent for feed tank, DIL_CAP

[0557] capping reaction tank fluidize Reuse CAP tank reuse solvent wash Wl CAP tank flow-through DIL_CAP tank new solvent chase

[0558]

[0559] and wash ACN feed tank flow-through Wl CAP tank

[0560] Table 2.

[0561] Cycle 6 vol% total total DCA 1.8vol% ACN in total Neat DCA(mL) toluene used in Pyridine in pyridine ACN wash used in deblock 70 / 30 solution after deblock + (mL)l ACN / Toluene wash after deblocking chase / wash wash after deblocking including after deblock (mL) (mL) the predeblock run (mL)l washing and post DEA wash (mL)2 1 883 830 53 188 129.44 1188 2 455 428 27 188 129.44 253 3 465 437 28 190 130.28 258 4 477 448 29 190 130.28 260 5 510 479 31 188 129.44 280 6 521 490 31 191 131.13 258 7 532 500 32 287 197.12 307 8 551 518 33 288 197.96 298 9 563 529 34 288 197.96 302 10 566 532 34 287 197.12 298 11 587 552 35 288 197.96 317 12 607 571 36 287 197.12 324 13 611 574 37 379 260.56 364 14 619 581 37 378 259.72 372 15 633 595 38 378 259.72 351 16 649 610 39 378 259.72 373 17 654 615 39 377 258.87 360 18 671 631 40 379 260.56 365 19 695 653 42 379 260.56 351 20 706 663 42 379 260.56 349 21 718 675 43 377 258.87 359

[0562]

[0563] 22 742 697 44 379 260.56 356 23 747 702 45 377 258.87 361 24 756 711 45 377 258.87 344 25 756 711 45 378 259.72 345 26 755 710 45 377 258.87 347 27 756 711 45 446 306.25 378 28 758 713 46 446 306.25 385 29 756 711 45 443 304.56 391 30 758 713 46 445 305.40 402 31 762 716 46 447 307.09 393 32 756 711 45 446 306.25 398 33 755 710 45 446 306.25 393 34 757 712 45 445 305.40 396 35-1 753 708 45 445 305.40 397 35-2 753 708 45 443 304.56 2170 Totals 23994 22555 1440 12604 8659 15042 Normalized 4.511 0.288 2.521 1.732 3.008 per mmol

[0564] (L / mmol)

[0565]

[0566] Table 3.

[0567] Cycle Acid Amidite Amidite Amidite, amidite

[0568] contact (g)5 note that equivalent time FBRwas versus (minutes) 0.1 M and resin

[0569] PBRwas

[0570] 0.2 M,

[0571] FBRused

[0572] less

[0573] molar

[0574] equiv

[0575] overall

[0576] (mL)

[0577] 1 24.9 MG 80.1 98 1.95

[0578] 2 27.1 MU 79.8 97 1.95

[0579] 3 27.2 MC 77.6 95 1.89

[0580] 4 27.4 MG 81.3 99 1.98

[0581] 5 27.4 MG 81.0 99 1.98

[0582] 6 27.1 ADEMA 88.2 108 2.15

[0583] 7 27.4 ADEMA 85.9 105 2.10

[0584] 8 27.6 ADEMA 87.5 107 2.13

[0585] 9 28.0 MG 81.8 100 2.00

[0586] 10 28.0 MC 77.9 95 1.90

[0587] 11 27.3 MC 78.7 96 1.92

[0588] 12 27.5 MG 81.6 100 1.99

[0589]

[0590] 13 27.7 MA 82.1 100 2.00

[0591] 14 27.8 MC 79.5 97 1.94

[0592] 15 28.2 MG 82.0 100 2.00

[0593] 16 28.4 MA 80.8 99 1.97

[0594] 17 28.6 MU 83.6 102 2.04

[0595] 18 28.6 MC 78.5 96 1.91

[0596] 19 29.0 MU 83.9 102 2.05

[0597] 20 29.2 MA 80.5 98 1.96

[0598] 21 29.4 MC 79.7 97 1.94

[0599] 22 29.5 MU 82.8 101 2.02

[0600] 23 29.8 MG 81.5 99 1.99

[0601] 24 30.1 MG 87.9 107 2.14

[0602] 25 30.3 FU 90.0 110 2.20

[0603] 26 29.5 FU 90.1 110 2.20

[0604] 27 29.6 FC 92.0 112 2.24

[0605] 28 29.9 FG 88.8 108 2.16

[0606] 29 29.9 MA 86.9 106 2.12

[0607] 30 29.8 MA 89.8 110 2.19

[0608] 31 29.9 MC 87.2 106 2.13

[0609] 32 29.9 MC 86.4 105 2.11

[0610] 33 30.1 MG 88.1 107 2.15

[0611] 34 30.2 MU 89.6 109 2.19

[0612] 35-1 30.2 MU(S) 91.9 112 2.24

[0613] 35-2 31.9

[0614] Totals 2945 3591

[0615] Normalized 0.718

[0616] per mmol

[0617] (L / mmol)

[0618]

[0619] Table 4.

[0620] Cycle Activator Activator activator Coupling Fresh Oxidizer Oxidizer (g)5 (mL) equivalent contact ACN (g) (mL) versus time wash

[0621] resin (minutes) after

[0622] coupling

[0623] (mL)

[0624] 1 82 101 10.1 10 80.2 267 271 2 81 100 10.0 10 77.6 217 221 3 81 100 10.0 10 76.3 217 221

[0625]

[0626] 4 83 102 10.2 10 77.6 217 221 5 83 102 10.2 10 73.8 217 221 6 89 110 11.0 15 75.1 216 220 7 89 110 11.0 15 72.5 217 221 8 90 111 11.1 15 80.2 217 221 9 83 102 10.2 10 77.6 217 221 10 81 100 10.0 10 78.9 217 221 11 84 104 10.4 10 81.4 216 220 12 83 102 10.2 10 76.3 217 221 13 82 101 10.1 10 73.8 217 221 14 83 102 10.2 10 80.2 217 221 15 83 102 10.2 10 77.6 217 221 16 84 104 10.4 10 86.5 217 221 17 83 102 10.2 10 75.1 217 221 18 83 102 10.2 10 80.2 217 221 19 83 102 10.2 10 76.3 216 220 20 83 102 10.2 10 77.6 217 221 21 83 102 10.2 10 80.2 217 221 22 82 101 10.1 10 76.3 217 221 23 83 102 10.2 10 73.8 217 221 24 89 110 11.0 10 75.1 217 221 25 87 107 10.7 15 80.2 217 221 26 91 112 11.2 15 81.4 217 221 27 89 110 11.0 15 75.1 217 221 28 89 110 11.0 15 75.1 217 221 29 88 109 10.9 10 76.3 217 221 30 89 110 11.0 10 76.3 217 221 31 88 109 10.9 10 75.1 217 221 32 89 110 11.0 10 75.1 217 221 33 88 109 10.9 10 76.3 218 222 34 89 110 11.0 10 75.1 217 221 35-1 89 110 11.0 10 76.3

[0627] 35-2

[0628] Totals 3686 2702.29 7425 7561.1 Normalized 0.737 0.540 1.512 per mmol

[0629] (L / mmol)

[0630]

[0631] Table 5. Cycle Xanthane Xanthane Oxidation or Fresh AC N Diluted Diluted Diluted Capping B(g) hydride hydride sulfurization wash after Capping Capping

[0632] (g) (mL) contact oxidation or A(g) A(mL)

[0633] time sulfurization

[0634] (minutes) 4 (mL)

[0635] 1 - -- 10 50.9 36 46 36

[0636] 2 - -- 10 50.9 36 46 37

[0637] 3 - -- 10 50.9 36 46 37

[0638] 4 - -- 10 49.6 36 46 37

[0639] 5 - -- 10 50.9 36 46 37

[0640] 6 - -- 10 52.2 36 46 37

[0641] 7 - -- 10 58.5 37 47 37

[0642] 8 - -- 10 54.7 37 47 37

[0643] 9 - -- 10 53.4 36 46 38 10 - -- 10 53.4 36 46 37 11 - -- 10 48.3 36 46 37 12 - -- 10 50.9 36 46 37 13 - -- 10 54.7 37 47 37 14 - -- 10 48.3 36 46 37 15 - -- 10 108.1 36 46 37 16 - -- 10 112.0 36 46 37 17 - -- 10 100.5 36 46 37 18 - -- 10 100.5 36 46 37 19 - -- 10 100.5 36 46 37 20 - -- 10 99.2 37 47 37 21 - -- 10 100.5 36 46 37 22 - -- 10 99.2 36 46 37 23 - -- 10 100.5 36 46 37 24 - -- 10 100.5 36 46 37 25 - -- 10 108.1 37 47 36 26 - -- 10 100.5 36 46 36 27 - -- 10 101.8 36 46 36 28 - -- 10 100.5 37 47 37 29 - -- 10 101.8 36 46 37 30 - -- 10 99.2 36 46 37 31 - -- 10 100.5 36 46 37 32 - -- 10 101.8 36 46 37 33 - -- 10 101.8 37 47 37 34 12 99.2 36 46 37 35-1 213.0 213.0 1029.3 -- -- --

[0644]

[0645] 35-2 -- -- -- Totals 213 213 3793.893

[0646] Normalized 0.043 0.759

[0647] per mmol

[0648] (L / mmol)

[0649]

[0650] Table 6.

[0651] Cycle Diluted Standard Standard Fresh Capping Fresh Capping Capping A Capping B ACN reaction ACN used B(mL) reagent reagent used for contact for wash (concentration (concentration diluting time after is20vol%l is20vol% capping (minutes)4 capping methylimidizole) acetic reagents (mL)

[0652] (mL) anhydride, 30 (mL)

[0653] vol%lutidine)

[0654] (mL)

[0655] 1 45 9.1 9.0 147 7.4 363.9

[0656] 2 46 9.1 9.2 148 7.4 276.1

[0657] 3 46 9.1 9.2 148 7.4 273.5

[0658] 4 46 9.1 9.2 148 7.4 281.2

[0659] 5 46 9.1 9.2 148 7.4 271.0

[0660] 6 46 9.1 9.2 148 7.4 272.3

[0661] 7 46 9.4 9.2 149 7.4 283.7

[0662] 8 46 9.4 9.2 149 7.4 274.8

[0663] 9 47 9.1 9.5 149 7.4 277.4

[0664] 10 46 9.1 9.2 148 7.4 277.4

[0665] 11 46 9.1 9.2 148 7.4 267.2

[0666] 12 46 9.1 9.2 148 7.4 268.4

[0667] 13 46 9.4 9.2 149 7.4 272.3

[0668] 14 46 9.1 9.2 148 7.4 283.7

[0669] 15 46 9.1 9.2 148 7.4 279.9

[0670] 16 46 9.1 9.2 148 7.4 273.5

[0671] 17 46 9.1 9.2 148 7.4 273.5

[0672] 18 46 9.1 9.2 148 7.4 264.6

[0673] 19 46 9.1 9.2 148 7.4 271.0

[0674] 20 46 9.4 9.2 149 7.4 268.4

[0675] 21 46 9.1 9.2 148 7.4 265.9

[0676] 22 46 9.1 9.2 148 7.4 286.3

[0677] 23 46 9.1 9.2 148 7.4 277.4

[0678] 24 46 9.1 9.2 148 7.4 273.5

[0679] 25 45 9.4 9.0 148 7.4 282.4

[0680]

[0681] 26 45 9.1 9.0 147 7.4 279.9 27 45 9.1 9.0 147 7.4 272.3 28 46 9.4 9.2 149 7.4 268.4 29 46 9.1 9.2 148 7.4 267.2 30 46 9.1 9.2 148 7.4 277.4 31 46 9.1 9.2 148 7.4 269.7 32 46 9.1 9.2 148 7.4 267.2 33 46 9.4 9.2 149 7.4 265.9 34 46 9.1 9.2 148 7.4 272.3 35-1 -- -- - - -- -- 35-2 -- -- - - -- -- Totals 311 313 5048 9399.491 Normalized 0.062 0.063 1.010 1.880 per mmol

[0682] (L / mmol)

[0683]

[0684] Material Composition:

[0685] DCA was 6 vol% in toluene

[0686] Amidites were 0.1M solutions in ACN (mU solvent was 10vol% toluene in ACN) Activator was 0.5 M ETT in ACN

[0687] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent)

[0688] Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[0689] Cap A was 4 vol% 1 methylimidizole in ACN

[0690] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in ACN Table 18.

[0691] REO-DQ0084-063 (lab RE9-D00084-063 elN UPLC) (Release UPLC) Strand Lpa Sense Lpa Sense

[0692] mass of initial resin whole batch (g) 20.3200 20.3200 resin loading (umol / g) 246 246 Synthesis scale (umol) 4.999 4,999 mass of final resin whole batch (g) 92.6000 92.6000 mass gain (g) 72.28 72.28 Mass gain per mmol scale 14.46 14.46 Crude mass yield (by weighing) 0.95 0.95

[0693] 0.7857

[0694]

[0695] FLP% (homogenized sample)* 0.8215

[0696] Mass of resin taken for C&D (g) 0.1708 0.1708 Fraction of the whole batch 0.18% 0.18% Mass of oligo In C&D resin sample (g) 0.1333 0.1333 30 wt% NH4OH solutin mass (g) 2.1940 2.1940 NH4OH plus oligo mass (g) 2.3273 2.3273 Aliquot mass of C&D sol’n for 00 (g) 0.1897 0.1897 Di water added for dilution (g) 20.23 20.23 NanoDrop measured A26041 9.69 9.69 NanoDrop measured A260 #2 9.69 9.69 NanoDrop measured A26O #3 9.66 9.66 NanoDrop measured A26O AVE 9.68 9.68 OD dilution factor 107,66 107,66 Total OD for whole batch 1,314,923 1,314,923 Mass Na Salt calculated from total OD 57.07 57.07 OD / umol 263 263 crude % yield by OD 82% 82%

[0697]

[0698] Purity corrected yield by OD 67% 64%

[0699] Example 4: Using the distillation bottoms as part of the deblocking process

[0700] This example describes methods and systems for collecting the distillation bottoms consisting of mostly acetonitrile, toluene, dichloroacetic acid, and dimethoxytrityl-related species. The distillate bottom contains concentrated dichloroacetic acid and the main solvent is toluene and acetonitrile. The distillate bottom materials can be used to substitute for some fresh acid solution used in detritylation reactions. A list of three experiments demonstrates the effects and benefit of using the distillation bottoms as part of the deblocking process. The experiments were done on a smaller scale FBR setup (90 umol scale)

[0701] List of experiments

[0702] • Experiment 024: control, standard baseline served for comparison. Total amount of fresh DCA solution, 4.90 L / mmol.

[0703] • Experiment 026: DCA solution was reduced by 25% from Experiment 024. See details below, Total amount of fresh DCA solution, 3.68 L / mmol.

[0704] • Experiment 040: using the same amount of fresh DCA solution was used as in Experiment 026. Compared to Experiment 026, before each detritylation step, a portion of distillate bottom material was used to mix with solid phase for 3 minutes.

[0705] Table 19: common experiment conditions used in SPOS experiments

[0706] Synthesis sequence Lpa SS 36 mer

[0707] Synthesis scale 0.09 mmol

[0708] Detritylation reagent 6 vol% DCA in toluene

[0709] Detritylation wash Total fresh ACN 5.2 L / mmol. 6 reuse vessels. Fresh ACN amount used in each step:

[0710] Base Volume (mL / mmol) Base Volume (mL / mmol)

[0711] 1 367 19 767

[0712] 2 367 20 767

[0713] 3 367 21 767

[0714] 4 367 22 767

[0715] 5 367 23 767

[0716] 6 367 24 767

[0717] 7 500 25 900

[0718] 8 500 26 900

[0719] 9 500 27 900

[0720] 10 500 28 900

[0721] 11 500 29 900

[0722] 12 500 30 900

[0723] 13 633 31 1033

[0724] 14 633 32 1033

[0725] 15 633 33 1033

[0726] 16 633 34 1033

[0727] 17 633 35 1033

[0728] 18 633 36 1033

[0729] Coupling Amidite solution: 0.1 M in ACN

[0730] Activator solution: 0.5 M ETT in ACN

[0731]

[0732] Amidite amount: 1.8 equivalence for methoxy amidites, 2.0 equivalence for fhiro amidites and AdemA.

[0733] Activator amount: 1:1 volume ratio with respect to amidite solutions.

[0734] Reaction times: 10 mins.

[0735] Coupling wash 25 mL / mmol fresh ACN rinse.

[0736] Oxidation 3 minutes of reaction with reused oxidation solution from the previous cycle.

[0737] Then 5 minutes of reaction with a fresh charge of 0.05 M 12 in pyridine / water (90 / 10), a charge of 2.7 equivalence of 12.

[0738] The material from this step was saved and reused in the next cycle.

[0739] Oxidation wash First, wash with the saved materials from the coupling step and coupling wash step from the current cycle. Then wash with 25 mL / mmol of fresh ACN.

[0740] Capping Cap A reagent: 4% NMI in ACN

[0741] CAP B reagent: 4% acetic anhydride, 6% 2,6-lutidine, 90% ACN.

[0742] 5.6 mL / mmol of CAP A and B each.

[0743] 5.6 mL / mmol of reused capping wash material for dilution.

[0744] Reaction time: 1 minute.

[0745] Capping wash Total fresh ACN 1.1 L / mmol. One reuse vessel. Fresh ACN amount used in each step:

[0746] Base Volume (mL / mmol) Base Volume (mL / mmol)

[0747] 1 24 19 30

[0748] 2 24 20 30

[0749] 3 24 21 30

[0750] 4 24 22 36

[0751] 5 24 23 36

[0752] 6 24 24 36

[0753] 7 24 25 36

[0754] 8 24 26 36

[0755] 9 24 27 36

[0756] 10 24 28 36

[0757] 11 30 29 36

[0758] 12 30 30 36

[0759] 13 30 31 36

[0760] 14 30 32 36

[0761] 15 30 33 36

[0762] 16 30 34 36

[0763] 17 30 35 36

[0764] 18 30

[0765]

[0766] Table 20: Acid solution volume profile (unit mL / mmol) used in Experiments. Profile #1 totalizes to 4,9 L / mmol, Profile #2 totalizes to 3.7 L / mmol, 25% less than Profile #1.

[0767] Profile #1 Profile #2

[0768] Base

[0769] Experiment 024 Experiments 026, 40

[0770] 1 60.4 60.0

[0771] 2 64.6 48.4

[0772] 3 68.8 51.0

[0773] 4 73.0 53.6

[0774] 5 77.2 56.2

[0775] 6 81.4 58.6

[0776] 7 85.8 65.0

[0777] 8 90.0 71.2

[0778] 9 94.2 77.6

[0779] 10 98.4 80.2

[0780] 11 102.6 82.6

[0781] 12 106.8 85.2

[0782] 13 111.0 87.8

[0783] 14 115.2 100.0

[0784] 15 119.6 100.0

[0785] 16 123.8 100.0

[0786] 17 128.1 100.0

[0787] 18 132.3 100.6

[0788] 19 136.5 103.0

[0789] 20 140.7 105.6

[0790] 21 144.9 108.2

[0791] 22 149.1 110.8

[0792] 23 153.3 113.2

[0793] 24 157.7 115.8

[0794] 25 161.9 118.4

[0795] 26 166.1 121.0

[0796] T1 170.3 123.4

[0797] 28 174.5 126.0

[0798] 29 178.7 128.6

[0799] 30 182.9 131.2

[0800]

[0801] 31 187.1 133.6

[0802] 32 191.5 136.2

[0803] 33 195.7 138.8

[0804] 34 199.9 141.4

[0805] 35 204.1 143.8

[0806] 36 207.9 146.4

[0807]

[0808] Results

[0809] Based on results in Table 21, from the comparison of Experiments-024 and 026, it was demonstrated that acid volume profile 2 was not sufficient to generate good purity and yield when the detritylation material reuse approach is unchanged.

[0810] As shown in Table 22 and Figure 3, Experiments-040 showed similar purity and yield as compared to Experment-024, and both experiments were superior to Experiment-026. It was therefore clearly demonstrated the proposed approach 2 and 3 of detritylation material reuse enables the reduction of acid solution required for synthesis.

[0811] Table 19 shows the concentration of DCA in the reuse acid vessel at the end of synthesis. The higher the DCA concentration, the more abundant the acid solution was. The same volume of acid solution was used in the listed experiments. The difference in DCA concentration therefore arise from the different required amount of acid solution and the difference results from the difference in detritylation material reuse approach or the temperature. 040 showed much higher DCA concentrations compared to Experiment-026, which indicates that the material reuse approaches #2 and #3 significantly reduced the required volume of acid solution.

[0812] Table 21: DCA concentration in the reuse vessel at the end of synthesis.

[0813] Experiment Mole% VOL%

[0814] 040 5.20 4.24

[0815] 026 0.48 0.40

[0816]

[0817] Table 22: Summary of purity and yield in SPOS Experiments.

[0818] Experiment FLP RRT < 1 RRT > 1 Crude OD Purity corrected

[0819] (OD / umol) OD yield

[0820] 040 77.61% 17.92% 4.49% 251 60%

[0821] 026 49.05% 47.43% 2.95% 240 37%

[0822]

[0823] 024 77.59% 15.77% 6.31% 252 61% Example 5: Thiol Scavengers

[0824] This example describes SPOS using deblocking using Thiol Scavengers. With escalating interest in therapeutic oligonucleotides, there is a need to address the cost and PMI in the manufacturing process. Current method of deprotection involves a large excess of an acid solution, normally dichloroacetic acid (DCA) in toluene, to remove the dimethoxytrityl protecting group on the 5'-hydroxy. PBR and FBR synthesizers rely on a plug flow or flow-through process because the reaction is equilibrium- driven and requires a continuous flow of acid solution to flush out reaction by-products and shift the equilibrium to product formation. The flow-through process requires a large excess of DCA. Reducing the amount of DCA needed in the detritylation step and increasing reaction robustness will reduce cost, reduce waste, and decrease the environmental impact. Using the fluid bed reactor (FBR) described herein, use of low odor thiol scavengers in the detritylation process was demonstrated to reduce the amount of DCA used during SPOS reactions. These methods also have the opportunity increase robustness for scale up to manufacturing because they make the detritylation reaction a batch reaction instead of flow through a packed bed.

[0825] Detritylation of an alcohol is a reversible reaction. Previously described methods on FBR use a flow-through process to push the reactive DMT cation off the resin using an excess of DCA to ensure reprotection of the alcohol does not occur. For the flow through mode reaction to be efficient, the resin packing should be uniform throughout, without voids or channeling, uniform height across the entire cross section. Furthermore, it needs to achieve uniform distribution of reagents entering the packed bed across the entire radius, and uniform flow in the axial direction throughout the entire reactor. Flow through mode limits batch size due to channeling and back-pressure. In this example, a DMT cation capture method using low odor thiols was developed. This method eliminates the need for flow-through reaction mode and facilitates the reaction to run in completely mixed batch mode in an FBR. By capturing the DMT cation with a thiol, a stable carbon-sulfur bond is formed, preventing the reprotection of the 5’-hydoxy, making the reaction irreversible. The DMT-scavenger adduct is removed along with the DCA and unreacted scavenger in the subsequent washing steps. The amount of DCA needed in the detritylation reaction is reduced. The process is potentially more robust when scaled up in manufacturing. The synthesis conditions for this experiment are summarized below in Table 20 for reference. Table 20: Experiment conditions used in experiment

[0826] Synthesis sequence Lpa SS 36 mer

[0827] Synthesis scale 0.05 mmol

[0828] Scavenger 4-(tert-butyl)phenyl)methanethiol

[0829] Detritylation Solution 10 vol% DCA / 2 vol% Scavenger in toluene

[0830] Detritylation Reagent Total DCA / scavenger in toluene volume 1.8 L / mmol.

[0831] volumes Base Volume Detritylation Solution (mL) Base Volume Detritylation Solution (mL))

[0832] 1 3 19 2.5

[0833] 2 1.8 20 2.5

[0834] 3 1.8 21 2.5

[0835] 4 1.8 22 2.5

[0836] 5 1.8 23 2.5

[0837] 6 1.8 24 3.0

[0838] 7 1.8 25 3.0

[0839] 8 1.8 26 3.0

[0840] 9 1.8 27 3.0

[0841] 10 1.8 28 3.0

[0842] 11 2.2 29 3.0

[0843] 12 2.2 30 3.0

[0844] 13 2.2 31 3.5

[0845] 14 2.2 32 3.5

[0846] 15 2.2 33 3.5

[0847] 16 2.2 34 3.5

[0848] 17 2.2 35 3.5

[0849] 18 2.5 36 3.5 Detritylation Cycle washes 3 mL flow-through toluene wash before detritylation solution addition 2 x 3 mL fluidized toluene wash after detritylation is complete

[0850] 15-35 mL flow-through ACN wash after toluene wash

[0851] Coupling Amidite solution: 0.1 M in ACN

[0852] Activator solution: 0.5 M ETT in ACN

[0853] Amidite amount: 2.0 molar equivalent

[0854] Activator amount: 1:1 volume ratio with respect to amidite solutions.

[0855] Reaction times: 10 mins.

[0856] Coupling wash 15-35 mL ACN wash.

[0857] Oxidation 5 minutes of reaction with 3 mL charge of 0.05 M 12 in pyridine / water (90 / 10) Oxidation wash 2.5 mL of ACN

[0858] Capping Cap A reagent: 4% NMI in ACN

[0859] CAP B reagent: 4% acetic anhydride, 6% 2,6-lutidine, 90% ACN.

[0860] 5.6 mL / mmol of CAP A and B each.

[0861]

[0862] 5.6 mL / mmol of reused capping wash material for dilution.

[0863] Reaction time: 1 minute.

[0864]

[0865] Low odor scavengers tested:

[0866] 4- (tert-butyl)phenylmethanethiol

[0867] 3,6-Dioxaoctane-l,8-dithiol (DODT)

[0868] 1 -dodecanethiol

[0869] 10% Scavenger solution example using 4-(tert-butyl)phenylmethanethiol:

[0870] A solution of 10% DCA and 2% 4-(tert-butyl)phenylmethanethiol in toluene is prepared (detritylation solution). The detritylation step:

[0871] The resin is washed with 3-4 mL of toluene in flow through mode. To the resin is added 1.8-3.0 mL of detritylation solution and fluidized under normal conditions for 7-10 minutes. The detritylation solution is removed and the resin washed with fluidized toluene (2x3mL, 3 fluidizations / wash). The toluene is removed, and the resin is washed with normal volumes of ACN (15-35 mL) using flow-through. Results from completed LPa build are shown in Table 21, and a comparison of 10% FBR Builds with and without Thiol Scavenger is shown in Table 22.

[0872] Table 21.

[0873] 20

[0874] total

[0875] DCA UPLC purity used in crude crude corrected scale deblock purity, yield, yield by Experiment synthesizer (mmol) (L / mmol) %FLP OD / umol OD, % FBR Lpa SS

[0876]

[0877] w / scavenger detrit FBR 0.05 0.18 84.0 274.0 69.0

[0878] TT

[0879] Table 22

[0880] Without With Thiol

[0881] Scavenger, and Scavenger and

[0882] with Flow-through Fluidized

[0883] Detritylation Detritylation

[0884] Scale (mmol) 5 0.05

[0885] 10% DCA solution 3.19 1.83

[0886] DCA 319 183

[0887]

[0888] Mass gain / mmol 13.9 15.7 OD / umol 252 274

[0889] crude yield by OD (%) 78 85

[0890] Purity corrected yield

[0891]

[0892] by OD (%) 63 69

[0893] RE0-D00084-059 data indicates this process has the lowest PMI and DCA consumption.

[0894] Table 23: Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse. No neat DCA addback to reuse. Reuse and fresh acid flow through instead of fluidize.

[0895] reaction destination when push Used material Source

[0896] mode out of reactor in reuse first part of wash after base reuse DCA tank 2 fluidize Waste

[0897] last detrit 2-36 base new acid, 10% 10% acid feed tank fluidize Waste

[0898] 1 flow- base reuse acid reuse DCA tank 1 Waste

[0899] through 2-36 flow- base new acid, 10% 10% acid feed tank reuse DCA tank 1

[0900] through 1-36 flow- base reuse solvent wash WI_DCAtank 1 reuse DCA tank 2

[0901] through 1-36 flow- base reuse solvent wash WI_DCAtank 1 Waste

[0902] through 1-36 base reuse solvent wash WI_DCAtank2 fluidize WI_DCAtank 1

[0903] 1-36 base new solvent chase ACN feed tank fluidize WI_DCAtank 1

[0904] 1-36 new solvent feed zone wall base ACN feed tank fluidize WI_DCAtank 2

[0905] wash 1-36 base reuse solvent wash WI_DCAtank3 fluidize WI_DCAtank 2

[0906] 1-36 base new solvent reactor wall wash ACN feed tank fluidize WI_DCAtank 2

[0907] 1-36 base pyridine washes pyridine feed tank fluidize WI_DCAtank3

[0908] 1-36 new solvent feed zone wall flow- base ACN feed tank WI_DCAtank3

[0909] and resin washes through 1-36 post O / S tank (to be amidite + activator new amidite, activator fluidize used for the wash after base

[0910]

[0911] 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

[0912] ox / sulf) 1-35 base Reuse oxidizer Reuse oxidizer tank fluidize Waste

[0913] 1-35 base oxidizer oxidizer feed tank fluidize Reuse oxidizer tank

[0914] 2-34 base sulfurization reagent sulfurization feed tank fluidize Waste

[0915] 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

[0916] through 1-35 Reuse capping reaction flow- base Reuse CAP tank Waste

[0917] solution through 1-34 New Capping reagents and Capping A feed tank,

[0918] 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

[0919] through 1-34 flow- base new solvent reactor wall wash ACN feed tank Wl CAP tank

[0920] through 1-34 new solvent feed zone wall flow- base ACN feed tank Wl CAP tank

[0921]

[0922] and resin wash through 1-34 note: reuse solvent wash tanks (WI_DCA, Wl CAP, reuse CAP) filled with ACN to begin synthesis

[0923] Table 24: Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse. No neat DCA addback to reuse. Reuse and fresh acid flow through instead of fluidize.

[0924] total Fresh ACN wash

[0925] after deblocking

[0926] total

[0927] Neat DCA toluene total DCA including the amount Pyridine pyridine in 10% DCA used in prefilled to the wash after wash after Cycle added to used in

[0928] (mL) deblock counter current wash deblock deblocking reuse (mL) deblock

[0929] (mL)l vessels and pre-run (mL) (mL) (mL)l

[0930] fresh acn wash (mL)

[0931] 2

[0932] 1 342 0.0 308 34 2170 184 0.83

[0933] 2 176 0.0 159 18 238 186 0.84

[0934] 3 186 0.0 167 19 240 183 0.82

[0935]

[0936] 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 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

[0937]

[0938] 35-2 503 0.0 452 50 356 435 3.99 Totals 13385 12047 1339 13567 13169 98

[0939] Normalized

[0940] per mmol

[0941] (L / mmol) 2.409 0.268 2.713 2.634 0.020

[0942] Table 25: Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse. No neat DCA addback to reuse. Reuse and fresh acid flow through instead of fluidize. Amidite,

[0943] note that FBRwas 0.1

[0944] Acid amidite M and PBR contact Amidite equivalent Cycle Amidite was 0.2 M,

[0945] time (g)5 FBRused versus (minutes) resin less molar equiv

[0946] overall (mL)

[0947] 1 12.1 MG 75.1 92 1.83 2 14.3 MU 76.3 93 1.86 3 14.3 MC 74.4 91 1.81 4 14.2 MG 76.7 94 1.87 5 14.4 MG 75.8 92 1.85 6 14.5 ADEMA 82.5 101 2.01 7 15.0 ADEMA 83.0 101 2.02 8 15.1 ADEMA 82.7 101 2.02 9 15.0 MG 75.6 92 1.84 10 15.0 MC 72.8 89 1.78 11 15.1 MC 72.1 88 1.76 12 15.3 MG 75.9 93 1.85 13 15.3 MA 76.1 93 1.86 14 15.4 MC 71.8 88 1.75 15 15.4 MG 76.1 93 1.86 16 15.6 MA 76.0 93 1.85 17 15.5 MU 76.0 93 1.85 18 15.7 MC 74.7 91 1.82 19 15.8 MU 76.6 93 1.87 20 15.8 MA 76.4 93 1.86 21 15.9 MC 71.8 88 1.75 22 16.1 MU 77.3 94 1.89 23 16.2 MG 74.1 90 1.81 24 16.3 MG 82.2 100 2.00 25 16.5 FU 82.7 101 2.02 26 16.5 FU 83.3 102 2.03 27 16.5 FC 85.7 105 2.09 28 16.5 FG 82.0 100 2.00 29 16.7 MA 85.0 104 2.07 30 16.8 MA 81.4 99 1.99 31 16.8 MC 80.9 99 1.97 32 17.0 MC 81.0 99 1.98 33 17.1 MG 81.7 100 1.99 34 17.2 MU 86.3 105 2.10

[0948]

[0949] 35-1 17.2 MUS 85.0 104 2.07

[0950]

[0951] 35-2 I 17.3 I

[0952] Totals 2747 3350

[0953] Normalized

[0954] per mmol

[0955] (L / mmol) 0.670

[0956] Table 26: Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse. No neat DCA addback to reuse. Reuse and fresh acid flow through instead of fluidize.

[0957] activator Coupling Fresh AC N

[0958] Activator Activator equivalent contact wash after Oxidizer Oxidizer Cycle

[0959] (g)5 (mL) versus time coupling (g) (mL)

[0960] resin (minutes) (mL)

[0961] 1 78 96 9.6 10 73.8 266 271

[0962] 2 77 95 9.5 10 75.1 217 221

[0963] 3 77 95 9.5 10 73.8 217 221

[0964] 4 77 95 9.5 10 78.9 217 221

[0965] 5 77 95 9.5 10 75.1 217 221

[0966] 6 83 102 10.2 15 73.8 217 221

[0967] 7 83 102 10.2 15 73.8 217 221

[0968] 8 83 102 10.2 15 80.2 217 221

[0969] 9 78 96 9.6 10 76.3 217 221

[0970] 10 78 96 9.6 10 73.8 217 221

[0971] 11 77 95 9.5 10 75.1 217 221

[0972] 12 78 96 9.6 10 73.8 218 222

[0973] 13 78 96 9.6 10 77.6 217 221

[0974] 14 77 95 9.5 10 73.8 217 221

[0975] 15 77 95 9.5 10 80.2 217 221

[0976] 16 78 96 9.6 10 75.1 217 221

[0977] 17 79 98 9.8 10 77.6 217 221

[0978] 18 77 95 9.5 10 85.2 217 221

[0979] 19 77 95 9.5 10 78.9 217 221

[0980] 20 75 93 9.3 10 76.3 217 221

[0981] 21 77 95 9.5 10 78.9 217 221

[0982]

[0983] 22 78 96 9.6 10 76.3 217 221 23 77 95 9.5 10 80.2 217 221 24 83 102 10.2 10 75.1 217 221

[0984] 25 83 102 10.2 15 84.0 217 221

[0985] 26 84 104 10.4 15 73.8 217 221

[0986] 27 83 102 10.2 15 73.8 217 221

[0987] 28 84 104 10.4 15 75.1 217 221

[0988] 29 82 101 10.1 10 73.8 217 221

[0989] 30 83 102 10.2 10 72.5 218 222

[0990] 31 83 102 10.2 10 72.5 217 221

[0991] 32 83 102 10.2 10 82.7 217 221

[0992] 33 84 104 10.4 10 77.6 217 221

[0993] 34 83 102 10.2 10 76.3 217 221

[0994] 35-1 83 102 10.2 10 75.1

[0995]

[0996] 35-2

[0997] Totals 3449 2675.5725 7430.87 7567.1

[0998] Normalized

[0999] per mmol

[1000] (L / mmol) 0.690 0.535 1.513

[1001] Table 27: Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse. No neat DCA addback to reuse. Reuse and fresh acid flow through instead of fluidize.

[1002] Oxidation or

[1003] Xanthane sulfurization Fresh ACN wash after Diluted Diluted Diluted Xanthane

[1004] Cycle hydride contact oxidation or sulfurization CappingA CappingA Capping B hydride (g)

[1005] (mL) time (mL) (g) (mL) (g) (minutes) 4

[1006] 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

[1007]

[1008] 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 35-1 213.34 213.34 11 1132.3

[1009]

[1010] 35-2

[1011] Totals 213.34 213.34 3829.516539

[1012] Normalized

[1013] per mmol

[1014] (L / mmol) 0.043 0.766

[1015] Table 28: Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse. No neat DCA addback to reuse. Reuse and fresh acid flow through instead of fluidize. Standard

[1016] Standard Capping B Fresh

[1017] CappingA reagent ACN used Capping Fresh ACN Diluted reagent (concentration for reaction used for Cycle Capping (concentration is 20vol% diluting contact wash after B(mL) is 20 vol% 1 acetic capping time capping methylimidizole) anhydride, 30 reagents (minutes)4 (mL)2 (mL) vol%lutidine) (mL)

[1018] (mL)

[1019] 1 48 9.3 9.5 150 7.4 173.0 2 45 9.3 9.0 148 7.4 95.4 3 42 9.4 8.5 147 7.4 86.5 4 45 9.6 9.0 149 7.4 81.4 5 45 9.4 9.0 148 7.4 94.1 6 45 9.4 9.0 148 7.4 81.4 7 46 9.4 9.2 149 7.4 82.7 8 46 9.4 9.2 149 7.4 98.0 9 46 9.4 9.2 149 7.4 96.7 10 46 9.4 9.2 149 7.4 95.4 11 47 9.1 9.4 149 7.4 94.1 12 51 9.1 10.2 152 7.4 113.2 13 45 9.4 9.0 148 7.4 112.0 14 45 9.4 9.0 148 7.4 108.1 15 44 9.6 8.7 148 7.4 114.5 16 46 9.6 9.2 150 7.4 117.0 17 45 9.6 9.0 149 7.4 117.0 18 45 9.4 9.0 148 7.4 112.0 19 45 9.6 9.0 150 7.4 136.1 20 47 9.4 9.5 150 7.4 109.4 21 46 9.4 9.2 149 7.4 120.9 22 44 9.4 8.7 147 7.4 141.2 23 46 9.6 9.2 150 7.4 145.0 24 45 9.6 9.0 149 7.4 143.8 25 46 9.4 9.2 149 7.4 153.9 26 45 9.6 9.0 149 7.4 146.3 27 44 9.6 8.7 148 7.4 109.4 28 47 9.4 9.5 150 7.4 147.6 29 46 9.6 9.2 150 7.4 160.3 30 44 9.6 8.8 149 7.4 146.3 31 45 9.6 9.0 149 7.4 151.4 32 46 9.4 9.2 149 7.4 150.1 33 46 9.4 9.2 149 7.4 138.7 34 46 9.4 9.2 149 7.4 142.5

[1020]

[1021] 35-1 35-2

[1022]

[1023] Totals 320 310 5074 4115.7761

[1024] Normalized

[1025] per mmol

[1026] (L / mmol) 0.064 0.062 1.015 0.823

[1027] Table 29: Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse. No neat DCA addback to reuse. Reuse and fresh acid flow through instead of fluidize.

[1028] total ACN

[1029] total Fresh ACN

[1030] used wash after

[1031] in the deblocking

[1032] Fresh proces including the Fresh ACN total ACN Fresh ACN

[1033] ACN used Fresh ACN ACN s amount prefilled ACN used wash after used in used for

[1034] in wash after post includi Cycle to the counter in amidite oxidation or capping wash after

[1035] activator coupling DEA ng all current wash solution sulfurization reagents capping

[1036] solution (mL) Wash reagen vessels and the (mL) (mL) (mL)

[1037] (mL) t acn from the

[1038] soutio pyridine wash

[1039] ns and (mL)2

[1040] washe s 2831.

[1041] 1 2354.1 82 87 74 50 12 173.0 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

[1042] 1104.

[1043] 13 681.5 84 87 78 51 12 112.0 2

[1044] 1179.

[1045] 14 771.6 79 86 74 50 12 108.1 5

[1046] 1259.

[1047] 15 779.2 84 86 80 104 12 114.5 3

[1048] 1256.

[1049] 16 784.3 83 87 75 98 12 117.0 8

[1050] 1256.

[1051]

[1052] 17 780.5 83 88 78 98 12 117.0 5 1264.

[1053] 18 791.2 82 86 85 97 12 112.0 6

[1054] 1277.

[1055] 19 779.7 84 86 79 101 12 136.1 1

[1056] 1250.

[1057] 20 786.1 84 83 76 99 12 109.4 5

[1058] 1247.

[1059] 21 772.1 79 86 79 99 12 120.9 5

[1060] 1276.

[1061] 22 774.6 85 87 76 101 12 141.2 1

[1062] 1296.

[1063] 23 791.2 81 86 80 101 12 145.0 1

[1064] 1307.

[1065] 24 788.6 90 92 75 106 12 143.8 7

[1066] 1329.

[1067] 25 796.2 91 92 84 101 12 153.9 8

[1068] 1311.

[1069] 26 793.7 91 93 74 101 12 146.3 3

[1070] 1275.

[1071] 27 793.7 94 92 74 101 12 109.4 8

[1072] 1325.

[1073] 28 806.4 90 93 75 101 12 147.6 2

[1074] 1327.

[1075] 29 798.8 93 91 74 98 12 160.3 6

[1076] 1310.

[1077] 30 798.8 89 92 73 99 12 146.3 5

[1078] 1316.

[1079] 31 798.8 89 92 73 101 12 151.4 4

[1080] 1315.

[1081] 32 789.9 89 92 83 99 12 150.1 2

[1082] 1305.

[1083] 33 791.2 90 93 78 103 12 138.7 6

[1084] 1309.

[1085] 34 793.7 95 92 76 98 12 142.5 6

[1086] 2185.

[1087] 35-1 792.5 93 92 75 1132 0 0.0 4

[1088] 2700.

[1089] 35-2 787.4 0 0 0 0 0 0.0 1913 4 Totals 26639 3015 3104 2676 3830 412 4116 1913 45704 Normal!

[1090] zed per

[1091] mmol

[1092] (L / mmo

[1093]

[1094] I) 5.328 0.603 0.621 0.535 0.766 0.082 0.823 0.383 9.14

[1095] 1 Toluene and DC A volumes were calculated based on the DCA solution being 10 vol% DCA in toluene

[1096] 2 The large amount of ACN used for cycle 1 includes the initial volume of ACN used to fill the integrated wash feed bottles. 4 Does not include contact time with reuse reagents.

[1097] 5 These values were corrected to remove the extra added to compensate for the dilution cart sample.

[1098] Material Composition:

[1099] DC A was 10 vol% in toluene

[1100] Amidites were 0.1M solutions in ACN (mU solvent was 10vol% toluene in ACN) Activator was 0.5 M ETT in ACN

[1101] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent) Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[1102] Cap A was 4 vol% 1 methylimidizole in ACN

[1103] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in CAN Table 30: Example RE0-D00084-059. This is the baseline process with all the reagent and wash solvent reuse. No neat DCA addback to reuse. Reuse and fresh acid flow through instead of fluidize.

[1104] ma twerne ma StrnM toe Seme igeSeme

[1105] mess mO mm whole betoh (gj SOW SOW w SM hew sceie (vmoO

[1106] m»se of frnef mm whole befeh <g> wwa

[1107] SS^SS gem oee ole 14.43 14.43 mm (W weighbwJ 04 cm

[1108]

[1109] MMS of wfem fee awi aio haw* of me whole botch am am of otsgo m O msm wnoH aws 30 wt% rneoe soMm (g> 2^710 2J3770 SMMOe ofes ofege rnM* Xiw X1S43 Ahsmot mms of CW sofo fm OO |g> . aiiB asm wet er eOO fee Worn ig> M43 303 wwW meewW AW »I IM

[1110] Heoooo meeweO ■SMtf foOeo mesomS A2W »3 S S WOop memW AW AW ase cms TO <X> for feeo mm He $eh from to OO M.M $w DO / omef Shi Shi OOe % W £M> _ 8W _ «1%

[1111]

[1112] CM W om wtw W oo 70%

[1113] Example 6: CWO-D00084-006. FBR with no material reuse.

[1114] The CWO-D00084-006 FBR process has no material reuse and therefore allows fastest cycle time among all FBR examples. Compared to other FBR examples with material reuse, CWO-006 uses more solvents, deblocking solution, and capping solution. For example, compared to RE0-059, CWO-006 has 3X total waste, 3X deblocking solution, 1.4X post-coupling washing solvent, 4.7X post-ox / sulf washing solvent, 7.8X post-capping washing solvent, 3.2X total ACN. CWO-006 gives similar purity and yield compared to other FBR examples. Another benefit of this embodiment of the SPOS process is smaller plant footprint. The synthesizer takes up less space in the manufacturing plant because it doesn't have the reuse vessels or the associated valves and other components necessary to transfer materials into and out of the reuse vessels.

[1115] Table 31. The CWO-D00084-006 process is a single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes. Deblocking consists of two steps, in the first one, fresh acid fluidize with the solid support, in the second one, fresh acid flow through the solid support.

[1116] destination reaction when push Used Material Source

[1117] mode out of in reactor

[1118] base new acid, 6% 6% acid feed tank Fluidize Waste

[1119] 1-36 flow- base new acid, 6% 6% acid feed tank Waste through 1-36 solvent wash, acetonitrile, toluene, flow- base Solvent feed tank Waste and pyridine through 1-36 flow- base solvent chase ACN feed tank Waste through 1-36 solvent wash, acetonitrile, toluene, base Solvent feed tank Fluidize Waste and pyridine 1-36 solvent wash, acetonitrile, toluene, flow- base Solvent feed tank Waste and pyridine through 6-36 flow- base solvent washes ACN feed tank Waste through 1-36 base amidite + activator new amidite, activator Fluidize Waste

[1120] 1-35 flow- base solvent chase and washes ACN feed tank Waste through 1-35 base oxidizer oxidizer feed tank Fluidize Waste

[1121] 1-34 base sulfurization sulfurization feed tank Fluidize Waste

[1122] 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

[1123]

[1124] for capping reaction feed tank, solvent tank 1-34 flow- base solvent chase and wash ACN feed tank Waste

[1125]

[1126] through 1-34

[1127] Table 32. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[1128] total Neat

[1129] ACN wash

[1130] after

[1131] 1 vol% ACN in

[1132] total deblocking

[1133] toluene total DCA Pyridine in pyridine including

[1134] 6 vol% used in 70 / 30 solution

[1135] Cycle DCA(mL) used in the predeblock ACN / Toluene wash after

[1136] deblock run

[1137] (mL)l wash after deblocking

[1138] (mL)l washing

[1139] deblock (mL) (mL)

[1140] and post

[1141] DEA wash

[1142] (mL)2

[1143] 1 790 742 47 432 302.61 1195

[1144] 2 802 754 48 461 322.61 281

[1145] 3 828 778 50 491 343.48 288

[1146] 4 851 800 51 520 364.35 298

[1147] 5 872 820 52 548 383.48 291

[1148] 6 902 847 54 579 405.22 304

[1149] 7 926 871 56 605 423.48 309

[1150] 8 954 897 57 634 443.48 318

[1151] 9 978 919 59 661 462.61 313

[1152] 10 1008 947 60 706 493.91 323

[1153] 11 1031 969 62 729 510.43 314

[1154] 12 1053 989 63 754 527.83 323

[1155] 13 1082 1017 65 783 547.83 333

[1156] 14 1101 1035 66 819 573.04 341

[1157] 15 1131 1063 68 841 588.70 338

[1158] 16 1158 1088 69 867 606.96 356

[1159] 17 1174 1104 70 902 631.30 359

[1160] 18 1210 1138 73 924 646.96 359

[1161] 19 1227 1153 74 966 676.52 360

[1162] 20 1258 1183 76 999 699.13 369

[1163] 21 1283 1206 77 1022 715.65 382

[1164] 22 1298 1220 78 1055 738.26 382

[1165] 23 1326 1246 80 1083 758.26 384

[1166] 24 1348 1267 81 1114 780.00 385

[1167] 25 1375 1292 82 1139 797.39 397

[1168] 26 1395 1311 84 1165 815.65 399

[1169] 27 1424 1339 85 1193 834.78 405

[1170]

[1171] 28 1454 1367 87 1226 858.26 413 29 1468 1380 88 1267 886.96 420

[1172] 30 1502 1412 90 1287 900.87 425

[1173] 31 1525 1433 91 1314 920.00 430

[1174] 32 1557 1464 93 1352 946.09 425

[1175] 33 1587 1492 95 1373 960.87 435

[1176] 34 1606 1510 96 1398 978.26 439

[1177] 35-1 1630 1532 98 1435 1004.35 449

[1178]

[1179] 35-2 1661 1561 100 1468 1027.83 2361

[1180] Totals 43773 41147 2626 34111 23877 15905

[1181] Normalized

[1182] per mmol

[1183] (L / mmol) 8.229 0.525 6.822 4.775 3.181

[1184] Table 33. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[1185] Amidite,

[1186] note that

[1187] FBR was

[1188] 0.1 M and

[1189] Acid PBRwas amidite

[1190] Cycle contact Amidite Amidite 0.2 M, equivalent

[1191] time (g)5 FBR used versus

[1192] (minutes) less resin

[1193] molar

[1194] equiv

[1195] overall

[1196] (mL)

[1197] 1 6.8 MG 76.5 93 1.87

[1198] 2 6.7 MU 70.8 86 1.73

[1199] 3 6.8 MC 73.2 89 1.79

[1200] 4 6.8 MG 73.1 89 1.78

[1201] 5 6.5 MG 73.9 90 1.80

[1202] 6 6.5 ADEMA 82.1 100 2.00

[1203] 7 6.7 ADEMA 81.8 100 2.00

[1204] 8 6.8 ADEMA 81.7 100 1.99

[1205] 9 6.9 MG 75.5 92 1.84

[1206] 10 6.9 MC 73.8 90 1.80

[1207] 11 7.0 MC 73.2 89 1.79

[1208] 12 7.1 MG 74.1 90 1.81

[1209] 13 7.2 MA 74.9 91 1.83

[1210] 14 7.3 MC 72.5 88 1.77

[1211] 15 7.4 MG 74.0 90 1.80

[1212]

[1213] 16 7.5 MA 74.8 91 1.82 17 7.6 MU 77.8 95 1.90

[1214] 18 7.7 MC 73.1 89 1.78

[1215] 19 7.8 MU 77.6 95 1.89

[1216] 20 7.9 MA 76.8 94 1.87

[1217] 21 8.0 MC 73.5 90 1.79

[1218] 22 8.2 MU 77.8 95 1.90

[1219] 23 8.2 MG 73.8 90 1.80

[1220] 24 8.3 MG 82.9 101 2.02

[1221] 25 8.4 FU 82.6 101 2.01

[1222] 26 8.5 FU 83.4 102 2.03

[1223] 27 8.6 FC 86.1 105 2.10

[1224] 28 8.7 FG 74.7 91 1.82

[1225] 29 8.8 MA 84.0 102 2.05

[1226] 30 8.9 MA 84.8 103 2.07

[1227] 31 9.0 MC 81.5 99 1.99

[1228] 32 9.1 MC 80.8 99 1.97

[1229] 33 9.2 MG 82.2 100 2.00

[1230] 34 9.3 MU 85.2 104 2.08

[1231] 35-1 9.4 MU(S) 86.0 105 2.10

[1232]

[1233] 35-2 9.5

[1234] Totals 2731 3330

[1235] Normalized

[1236] per mmol

[1237] (L / mmol) 0.666

[1238] Table 34. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[1239] Fresh

[1240] activator Coupling

[1241] AC N wash

[1242] Activator Activator equivalent contact Oxidizer Oxidizer Cycle after

[1243] (g)5 (mL) versus time

[1244] coupling (g) (mL) resin (minutes)

[1245] (mL)

[1246] 1 77 95 9.5 10 101.8 261 266

[1247] 2 78 96 9.6 10 104.3 261 266

[1248] 3 81 100 10.0 10 105.6 261 266

[1249]

[1250] 4 78 96 9.6 10 106.9 262 267 5 79 98 9.8 10 106.9 262 267 6 85 105 10.5 15 104.3 262 267 7 84 104 10.4 15 103.1 262 267 8 83 102 10.2 15 104.3 262 267 9 78 96 9.6 10 108.1 262 267 10 78 96 9.6 10 113.2 262 267 11 78 96 9.6 10 100.5 262 267 12 78 96 9.6 10 104.3 262 267 13 78 96 9.6 10 106.9 262 267 14 78 96 9.6 10 101.8 262 267 15 77 95 9.5 10 100.5 262 267 16 77 95 9.5 10 101.8 262 267 17 77 95 9.5 10 103.1 262 267 18 77 95 9.5 10 113.2 261 266 19 77 95 9.5 10 112.0 262 267 20 78 96 9.6 10 110.7 259 264 21 79 98 9.8 10 100.5 262 267 22 77 95 9.5 10 105.6 262 267 23 76 94 9.4 10 113.2 261 266 24 83 102 10.2 10 103.1 261 266 25 83 102 10.2 15 106.9 262 267 26 84 104 10.4 15 103.1 262 267 27 84 104 10.4 15 101.8 262 267 28 83 102 10.2 15 106.9 263 268 29 85 105 10.5 10 109.4 262 267 30 84 104 10.4 10 103.1 262 267 31 84 104 10.4 10 106.9 263 268 32 85 105 10.5 10 100.5 254 259 33 84 104 10.4 10 104.3 263 268 34 83 102 10.2 10 106.9 262 267 35-1 86 106 10.6 10 109.4

[1251]

[1252] 35-2

[1253] Totals 3477 3694.656 8894 9057.0

[1254] Normalized

[1255] per mmol

[1256] (L / mmol) 0.695 0.739 1.811 Table 35. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[1257] Oxidation or Fresh AC N

[1258] 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)

[1259] (minutes) 4 (mL)

[1260] 1 — — 10 484.7 79 100 77

[1261] 2 — — 10 489.8 78 99 77

[1262] 3 — — 10 488.5 72 91 78

[1263] 4 — — 10 478.4 78 99 77

[1264] 5 — — 10 870.2 78 99 77

[1265] 6 — — 10 493.6 78 99 77

[1266] 7 — — 10 478.4 78 99 77

[1267] 8 — — 10 489.8 79 100 76

[1268] 9 — — 10 489.8 79 100 77

[1269] 10 — — 10 482.2 78 99 77

[1270] 11 — — 10 477.1 79 100 77

[1271] 12 — — 10 479.6 79 100 77

[1272] 13 — — 10 478.4 79 100 77

[1273] 14 — — 10 483.5 78 99 77

[1274] 15 — — 10 478.4 78 99 77

[1275] 16 — — 10 475.8 79 100 77

[1276] 17 — — 10 473.3 79 100 77

[1277] 18 — — 10 487.3 82 104 72

[1278] 19 — — 10 493.6 79 100 75 20 — — 10 491.1 77 97 77 21 — — 10 494.9 77 97 77

[1279] 22 — — 10 483.5 76 96 77

[1280] 23 — — 10 487.3 76 96 77

[1281] 24 — — 10 482.2 76 96 77

[1282] 25 — — 10 482.2 76 96 77

[1283] 26 — — 10 486.0 76 96 77

[1284] 27 — — 10 488.5 76 96 77

[1285] 28 — — 10 482.2 76 96 77

[1286] 29 — — 10 493.6 77 97 77

[1287]

[1288] 30 — — 10 487.3 76 96 77 31 — — 10 489.8 76 96 76

[1289] 32 — — 10 489.8 76 96 77

[1290] 33 — — 10 488.5 75 95 77

[1291] 34 — — 10 475.8 76 96 77

[1292] 35-1 212.6 212.6 12 1159.0 —

[1293]

[1294] 35-2 -- Totals 212.6 212.6 18034.35

[1295] Normalized

[1296] per mmol

[1297] (L / mmol) 0.043 3.607

[1298] Table 36. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[1299] Standard

[1300] Standard Capping B Fresh

[1301] Fresh

[1302] Capping A reagent ACN Capping

[1303] ACN used

[1304] Diluted reagent (concentration used for reaction

[1305] Cycle Capping (concentration is20vol% diluting contact for wash

[1306] after

[1307] B(mL) is20vol%l acetic capping time

[1308] capping methylimidizole) anhydride, 30 reagents (minutes)4

[1309] (mL)

[1310] (mL) vol%lutidine) (mL)

[1311] (mL)

[1312] 1 96 20.0 19.2 232 7.5 461.8

[1313] 2 96 19.7 19.2 231 7.5 494.9

[1314] 3 97 18.2 19.5 226 7.5 521.6

[1315] 4 96 19.7 19.2 231 7.5 549.6

[1316] 5 96 19.7 19.2 231 7.5 570.0

[1317] 6 96 19.7 19.2 231 7.5 606.9

[1318] 7 96 19.7 19.2 231 7.5 636.1

[1319] 8 95 20.0 19.0 231 7.5 653.9

[1320] 9 96 20.0 19.2 232 7.5 685.8

[1321] 10 96 19.7 19.2 231 7.5 707.4

[1322] 11 96 20.0 19.2 232 7.5 751.9

[1323] 12 96 20.0 19.2 232 7.5 785.0

[1324] 13 96 20.0 19.2 232 7.5 801.5

[1325] 14 96 19.7 19.2 231 7.5 825.7

[1326] 15 96 19.7 19.2 231 7.5 863.9

[1327] 16 96 20.0 19.2 232 7.5 900.8

[1328] 17 96 20.0 19.2 232 7.5 917.3

[1329]

[1330] 18 90 20.7 18.0 230 7.5 944.0 19 94 20.0 18.7 230 7.5 988.5 20 96 19.5 19.2 230 7.5 1006.4

[1331] 21 96 19.5 19.2 230 7.5 1031.8

[1332] 22 96 19.2 19.2 229 7.5 1064.9

[1333] 23 96 19.2 19.2 229 7.5 1110.7

[1334] 24 96 19.2 19.2 229 7.5 1127.2

[1335] 25 96 19.2 19.2 229 7.5 1155.2

[1336] 26 96 19.2 19.2 229 7.5 1185.8

[1337] 27 96 19.2 19.2 229 7.5 1204.8

[1338] 28 96 19.2 19.2 229 7.5 1248.1

[1339] 29 96 19.5 19.2 230 7.5 1251.9

[1340] 30 96 19.2 19.2 229 7.5 1297.7

[1341] 31 95 19.2 19.0 228 7.5 1324.4

[1342] 32 96 19.2 19.2 229 7.5 1343.5

[1343] 33 96 19.0 19.2 228 7.5 1388.0

[1344] 34 96 19.2 19.2 229 7.5 1410.9

[1345] 35-1 — — — —

[1346]

[1347] 35-2 - -- - -- Totals 665 652 7818 31818.07

[1348] Normalized

[1349] per mmol

[1350] (L / mmol) 0.133 0.130 1.564 6.364

[1351] Table 37. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[1352] total ACN

[1353] wash after total ACN deblocking used in including Fresh Fresh the Fresh ACN total ACN

[1354] the preACN used ACN ACN used process ACN used wash after used in

[1355] run in wash for wash including Cycle in amidite oxidation or capping

[1356] washing, activator after after all solution sulfurization reagents

[1357] post DEA solution coupling capping reagent (mL) (mL)

[1358] wash and (mL) (mL) soutions acnfrom and pyridine washes wash (mL)

[1359] 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

[1360]

[1361] 6 709.3 90 94 104 494 25 606.9 2124.1 7 732.6 90 93 103 478 25 636.1 2158.7 8 761.5 90 92 104 490 25 653.9 2217.0 9 775.6 83 87 108 490 26 685.8 2254.4 10 817.1 81 87 113 482 25 707.4 2312.9 11 824.7 80 87 101 477 26 751.9 2346.8 12 851.0 81 87 104 480 26 785.0 2413.5 13 881.2 82 87 107 478 26 801.5 2462.4 14 914.0 80 87 102 483 25 825.7 2516.6 15 927.1 81 86 101 478 25 863.9 2562.0 16 963.2 82 86 102 476 26 900.8 2634.8 17 990.1 85 86 103 473 26 917.3 2680.3 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

[1362] Normalized

[1363] per mmol

[1364]

[1365] (L / mmol) 7.956 0.599 0.626 0.739 3.607 0.172 6.364 20.06

[1366] 1 Toluene and DCA volumes were calculated based on the DCA solution being 6 vol% DCA in toluene

[1367] 2 Fresh ACN includes ACN from chase wash, wall wash, fresh ACN washes, and ACN from the pyridine solutions

[1368] 4 Does not include contact time with reuse reagents. 5 These values were corrected to remove the extra added to compensate for the dilution cart sample.

[1369] Material Composition:

[1370] DCA was 6 vol% in toluene

[1371] Amidites were 0.1M solutions in ACN (mU solvent was 10vol% toluene in ACN) Activator was 0.5 M ETT in ACN

[1372] Oxidation solution was 0.05 M iodine (90 / 10 Pyridine / water as solvent) Sulfurization reagent was 0.2 M xanthane hydride in pyridine

[1373] Cap A was 4 vol% 1 methylimidizole in ACN

[1374] Cap B was 4vol% acetic anhydride and 6 vol% lutidine in CAN Table 38. CWO-D00084-006 example. Single pass operation of the FBR reactor. No reuse reagents or reuse solvent washes.

[1375] CWO-D00084-006 (Lab CWO-D00084-006 eLN UPLC) (Release UPLC) Strand Lpa Sense Lpa Sense

[1376] mass of initial resin whole batch (g) 19.9500 19.9500 resin loading (umol / g) 251 251 Synthesis scale (nmol) 5,007 5,007 mass of final resin whole batch (g) 92.7700 92.7700 mass gain (g) 72.82 72.82 Mass gain per mmol scale 14.54 14.54 Crude mass yield (by weighing) 0.95 0.95 FLP% (homogenized sample)* 0.8601 0.8486

[1377]

[1378] Mass of resin taken for C&D (g) 0.1296 0.1296 Fraction of the whole batch 0.14% 0.14% Mass of oligo in C&D resin sample (g) 0.1017 0.1017 30 wt% NH4OH solution mass (g) 1.9927 1.9927 NH4OH plus oligo mass (g) 2.0944 2.0944 Aliquot mass of C&D sol’n for OD (g) 0.2724 0.2724 DI water added for dilution (g) 20.26 20.26 NanoDrop measured A260 #1 11.44 11.44 NanoDrop measured A260 #2 11.49 11.49 NanoDrop measured A260 #3 11.46 11.46 Nanodrop measured A260 AVE 11.46 11.46 OD dilution factor 75.40 75.40 Total OD for whole batch 1,296,083 1,296,083 Mass Na Salt calculated from total OD 56.25 56.25 OD / umol 259 259 crude % yield by OD 80% 80% Purity corrected yield by OD 69% 68%

[1379]

Claims

CLAIMSWhat is claimed is:

1. A method comprising contacting a phosphoramidite monomer comprising a protecting group and linked to a solid support in a fluidized bed reactor with a recycled deblocking solution, thereby removing the protecting group from the phosphoramidite monomer to produce a nucleoside.

2. The method of claim 1, wherein the deblocking solution comprises dichloroacetic acid (DCA) or trichloroacetic acid (TCA) or trifluoroacetic acid (TFA).

3. The method of claim 1 or 2, wherein the protecting group comprises 5’-DMT (4, 4’-dimethoxytityl).

4. The method of any one of claims 1 to 3, wherein the deblocking solution is charged to the fluidized bed reactor top.

5. The method of any one of claims 1 to 4, further comprising fluidizing a portion of the deblocking solution and solid support within the fluidized bed reactor.

6. The method of any one of claims 1 to 5, wherein the fluidized bed reactor comprises an inlet that allows pressurized gas to enter the fluidized bed reactor.

7. The method of claim 6, wherein the inlet is positioned at the bottom of the fluidized bed reactor or the top of the fluidized bed reactor.

8. The method of any one of claims 1 to 7, further comprising removing the deblocking solution from the fluidized bed reactor after the deblocking solution removes the protecting group from the phosphoramidite monomer.

9. The method of any one of claims 1 to 8, wherein the fluidized bed reactor further comprises a filter located at the bottom of the fluidized bed reactor.

10. The method of claim 9, wherein the deblocking solution is removed through a filter located at the bottom of the fluidized bed reactor.

11. The method of any one of claims 1 to 10, wherein the deblocking solution added to the fluidized bed reactor comprises a volume between about 50-500 mL per mmol of stationary phase bound oligonucleotide.

12. The method of any one of claims 1 to 11, wherein the solid support comprises a resin.

13. The method of any one of claims 1 to 11, wherein the solid support comprises controlled pore glass.

14. The method of claim 12, wherein the resin comprises polystyrene.

15. The method of any one of claims 9 to 14, further comprising contacting the nucleoside with an activated amidite solution which reacts with the nucleoside to couple a second phosphoramidite monomer to the nucleoside.

16. The method of claim 15, further comprising removing the activated amidite solution from the fluidized bed reactor and contacting the second phosphoramidite monomer with one or more wash solutions.

17. The method of claim 16, wherein the one or more wash solutions comprise a capping solution comprising one or more of acetic anhydride, acetic acid, N-methylimidazole (NMI), 2,6-lutidine, tetrahydrofuran, and pyridine.

18. The method of claim 16 or 17, further comprising removing the one or more wash solutions from the fluidized bed reactor and contacting the second phosphoramidite monomer with an oxidation solution.

19. The method of claim 18, further comprising removing the oxidation solution and contacting the nucleoside with a second deblocking solution.

20. A method for recycling deblocking solution, the method comprising:(i) 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);(ii) moving a portion of the deblocking solution from the fluidized bed reactor to a holding vessel via a filter located at the bottom of the fluidized bed reactor;(iii) completing the first cycle of SPOS;(iv) optionally, adding a first volume of neat or concentrated DCA to the used deblocking solution in the holding vessel;(v) beginning a second cycle of SPOS, the second cycle comprising contacting a second phosphoramidite monomer linked to the solid support in the fluidized bed reactor with the used deblocking solution, fluidizing the first portion and then pushing the rest through the resin bed in flow through mode;(vi) moving a first portion of the used deblocking solution to waste via the filter located at the bottom of the fluidized bed reactor;(vii) continuing the deblocking reaction with fresh deblocking solution that passes through the resin bed, exits the reactor, and is collected in the holding vessel to be used on the next cycle, and(viii) completing the second cycle of SPOS.

21. The method of claim 20, wherein the feed zone is in fluid communication with the fluidized bed reactor.

22. The method of claim 20 or 21, wherein the deblocking solution used to contact the first phosphoramidite monomer comprises a concentration of DCA ranging from about 2% v / v to about 25% v / v.

23. The method of any one of claims 20 to 22, wherein the first volume of neat or high concentration DCA added to the feed zone ranges between 2% and 20% of the portion of deblocking solution in the holding vessel.

24. The method of any one of claims 20 to 23, wherein each cycle of SPOS further comprises a coupling reaction, oxidation reaction, and a capping reaction.

25. The method of any one of claims 20 to 24, wherein contacting the first or second monomer with the deblocking solution is performed in flow through mode.

26. The method of any one of claims 20 to 25 wherein steps (i)-(vii) are performed n times, where n is the number of phosphoramidite monomers that will be linked to the solid support during the SPOS.

27. The method of any one of claims 20 to 26, wherein step (v) further comprises contacting the solid substrate with a portion of an acetonitrile (ACN) wash solution that was used to wash the solid substrate after the first phosphoramidite monomer on the solid substrate was contacted with the used deblocking solution.

28. The method of any one of claims 20 to 27, wherein step (v) further comprises contacting the solid substrate with a distillation bottoms solution comprising concentrated dichloroacetic acid (DCA), toluene, and acetonitrile prior to contacting the phosphoramidite monomers with the used deblocking solution.

29. The method of any one of claims 20 to 28, wherein the deblocking solution comprises one or more thiol scavengers.

30. The method of claim 29, wherein the one or more thiol scavengers are selected from: 4-(tert-butyl)phenylmethanethiol, 3,6-Dioxaoctane-l,8-dithiol (DODT), and 1 -dodecanethiol.

31. A method for recycling deblocking solution, the method comprising:(i) 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;(ii) moving a portion of the deblocking solution from the fluidized bed reactor to a holding vessel via a filter located at the bottom of the fluidized bed reactor;(iii) completing the first cycle of SPOS;(iv) optionally, adding a first volume of neat or concentrated DCA to the used deblocking solution in the holding vessel;(v) beginning a second cycle of SPOS, the second cycle comprising contacting a second phosphoramidite monomer linked to the solid support in the fluidized bed reactor with the used deblocking solution under fluidizing conditions;(vi) moving a first portion of the used deblocking solution to waste via the filter located at the bottom of the fluidized bed reactor;(vii) continuing the deblocking reaction with fresh deblocking solution that passes through the resin bed, exits the reactor, and is collected in the holding vessel to be used on the next cycle, and(viii) completing the second cycle of SPOS.

32. The method of claim 31, wherein prior to (v) the solid substrate is contacted with a DCA-rich portion of an ACN wash from the first SPOS cycle.

33. A method for recycling deblocking solution, the method comprising:(i) 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;(ii) 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;(iii) contacting the solid support with a coupling solution under fluidizing conditions;(iv) 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;(v) contacting the solid support with an oxidizing solution under fluidizing conditions;(vi) 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;(vii) contacting the solid support with a capping solution under fluidizing conditions, and(viii) 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.

34. The method of claim 33, further comprising performing one or more washes under fluidizing conditions between steps (i) and (ii), (ii) and (iii), (iii) and (iv), (iv) and (v), (v) and (vi), (vi) and (vii), and (vii) and (viii).

35. The method of claim 33 or 34, wherein none of the deblocking solution, coupling solution, oxidation solution, and the capping solution is reused in an SPOS cycle.

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