Systems and methods for performing solid phase oligonucleotide synthesis

Abstract

Aspects disclosed herein relate to methods and systems for performing Solid Phase Oligonucleotide Synthesis (SPOS). In some embodiments, a system configured for use in SPOS may include an obstruction (e.g., a bar) that is configured to disrupt cohesion of a resin cake during SPOS to promote fluidization and mixing of the resin with a reagent. In some embodiments, a system configured for use in SPOS may include one or more spray devices (e.g., spray nozzles and / or spray balls), where the spray devices may be configured to provide solvents and / or reagents to the resin. The spray devices may also be configured to provide wash solvent to wash the walls of the reactor vessel.

Description

[0001] SYSTEMS AND METHODS FOR PERFORMING SOLID PHASE OLIGONUCLEOTIDE SYNTHESIS RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. 119(e) of U.S. provisional application serial number 63 / 711,374, filed on October 24, 2024, entitled “SYSTEMS AND METHODS FOR PERFORMING SOLID PHASE OLIGONUCLEOTIDE SYNTHESIS”, the entire contents of which are incorporated by reference herein.

[0003] FIELD

[0004] Disclosed embodiments are directed to systems and associated methods for Solid Phase Oligonucleotide Synthesis.

[0005] BACKGROUND

[0006] Solid Phase Oligonucleotide Synthesis (SPOS) is a method used to synthesize oligonucleotides. SPOS is implemented on a solid phase media which is generally a solid support which are generally made of controlled pore glass (CPG) or macroporous polystyrene (MPPS) spheres. SPOS is a solid-phase synthesis of oligonucleotides using building blocks which are various nucleoside derivatives, the most common of which are phosphoramidites. Specifically, a starting phosphoramidite building block is attached to a solid phase and then each nucleoside (phosphoramidite) is added and coupled to the phosphoramidite building block in a sequential manner until the desired molecule is obtained. Once the oligonucleotide sequence is obtained, the molecule is then cleaved from the solid support and globally deprotected to yield the desired oligonucleotide.

[0007] SUMMARY

[0008] According to some embodiments, a system for SPOS is provided. The system may comprise a reactor vessel configured to receive resin, and the reactor vessel may include a gas inlet. The system may include a gas source configured to supply gas to the reactor vessel via the gas inlet. In some embodiments, the system further comprises a filter disposed within the reactor vessel positioned above the gas inlet. The system may also include an obstruction positioned within the reactor vessel. In some embodiments, the obstruction may be configured to disrupt cohesion of the resin forming a resin cake in the reactor vessel to initiate or increase fluidization of the resin.

[0009] According to some embodiments, a method of promoting fluidization of resin during SPOS is provided. The method may include positioning resin within a reactor vessel of a solid phase oligonucleotide synthesis system, and at least one oligonucleotide may be attached to the resin. The method may further include introducing gas into the reactor vessel through a filter, causing a resin cake formed within the reactor vessel to move within the reactor vessel and contract an obstruction positioned within the reactor vessel. In some embodiments, contact of the resin cake with the obstruction may disrupt cohesion of the resin cake and initiate or increase fluidization of the resin.

[0010] According to some embodiments, a method of promoting fluidization during SPOS is provided. The method many include positioning resin within a reactor vessel of a solid phase oligonucleotide synthesis system, and the at least one oligonucleotide may be attached to the resin. The method may further include delivering a reagent into the reactor vessel to cause a sidewall of a resin cake formed within the reactor vessel to move toward a sidewall of the reactor vessel, thereby decreasing a size of a channel formed between the sidewall of the resin cake and the sidewall of the reactor vessel (e.g., using a spray nozzle to deliver reagent in a manner that flattens the resin cake).

[0011] BRIEF DESCRIPTION OF DRAWINGS

[0012] Non-limiting embodiments that incorporate one or more aspects of the invention will be described by way of example with reference to the accompanying figures, which are not necessarily drawn to scale. For purposes of clarity, not every component may be labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures: FIG. 1A is a schematic diagram of a system including a reactor vessel configured for use in SPOS, according to some embodiments;

[0013] FIG. IB is a schematic diagram of the reactor vessel of the system of FIG. 1 A, where a resin cake is shown to rise through a reactor vessel, according to some embodiments;

[0014] FIG. 1C is a schematic diagram of the reactor vessel of the system of FIGs. 1 A-1B, where cohesion of the resin cake is disrupted of FIG. IB, according to some embodiments;

[0015] FIG. 2 is a flow diagram of a method for performing one cycle of SPOS, according to some embodiments;

[0016] FIG. 3A is a schematic diagram of a system for promoting fluidization of resin during SPOS, according to some embodiments;

[0017] FIG. 3B is a schematic diagram of a system for promoting fluidization of resin during SPOS, according to some embodiments;

[0018] FIG. 3C is a schematic diagram of a system for promoting fluidization of resin during SPOS, according to some embodiments;

[0019] FIG. 4A is a schematic diagram of a system for reducing “channeling” of a resin cake during SPOS, according to some embodiments;

[0020] FIG. 4B is a schematic diagram of a system for reducing “channeling” of a resin cake during SPOS, according to some embodiments; and

[0021] FIG. 5 is a schematic diagram of a system for SPOS comprising a reactor vessel, according to some embodiments.

[0022] DETAILED DESCRIPTION

[0023] Solid Phase Oligonucleotide Synthesis (SPOS) is a method of synthesizing oligonucleotides. In the SPOS process, there are generally four chemical reactions that occur in order to add a single phosphoramidite to the chain. As will be described in greater detail herein, these four steps generally include a first “de-blocking” or “detritylation” step, a second “coupling” step, a third “oxidation” step, and a fourth “capping” step. In addition, one or more washing steps may be conducted before or after any of the four steps detailed above. For example, a washing step may be conducted after each one of the four steps above to wash away residual reagent used in the SPOS process. The four steps constitute one cycle. One cycle is used to add each nucleoside building block. According to the embodiments disclosed herein, an SPOS system may include a reactor vessel in which the oligonucleotide synthesis occurs. Examples of SPOS systems have been previously described in U.S. Publication No. 2023 / 0340007 (hereinafter referred to as the ’007 application), which is incorporated herein by reference in its entirety. The ’007 application generally describes a method of adding a phosphonamidite to a growing oligonucleotide on a solid phase resin within a bed reactor in which a protecting group is removed from the 5’ position of an oligonucleotide and an activated amidite solution is coupled to the unprotected group, where the activated amidite solution comprises an amidite and the activated amidite solution and the solid phase resin are fluidized in the reactor. Fluidization is the thorough mixing of the solid phase resin and a solution 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 an inert gas through the mixture (e.g., bubbling). When inert gas is used for fluidization, the gas (e.g., nitrogen) may be introduced at a constant rate to ensure complete fluidization. When an activated amidite solution and the solid phase resin are fluidized in the reactor, the amidite reacts at the 5’ position of the oligonucleotide. In addition to the coupling reaction, reagent solutions for deblocking, oxidizing, and capping may be provided. The fluidization may be followed by a flow -through reaction with reagent flow in the downward direction through the resin bed. An example of an SPOS system is shown in reference to FIG. 5, which depicts a diagram for introducing various reagents and solutions into a feed zone, and subsequently introducing the reagents and solutions from the feed zone into a reactor vessel to conduct the fluidization and flow-through steps.

[0024] Accordingly, SPOS systems disclosed herein may generally include two modes hereinafter referred to as a “flow-through” mode and a “fluidization mode”. In a flow-through mode, liquid (e.g., wash solvents and / or reagents) may enter the reactor vessel and be distributed to a resin (e.g., via a spray nozzle). The nozzle itself may be a single nozzle, a family of spray nozzles arranged together, or a plate with a large number of orifices so that the liquid can evenly rain down on the resin bed. The height of the spray nozzle in the reactor may be adjusted upwards throughout the course of a synthesis as the resin bed grows taller in the reactor. After the liquid is distributed to the resin, the reactor vessel may then be emptied of the introduced liquid. In some embodiments, the flow-through mode may be predominantly used for the de- blocking and washing steps of the SPOS process. In some embodiments, fluidization may be caused by an inert gas (e.g., N2 gas) introduced from a gas source into the reactor vessel where the gas causes the solid phase resin and reagent liquid to thoroughly mix, thus resulting in a slurry. The introduction of the inert gas may be maintained at a rate to facilitate mixing of the solid phase resin and reagent liquid while also decreasing the amount of resin that may splash onto the walls of the reactor vessel. The liquid may then be emptied from the reactor vessel at the end of a given fluidization step, which may result in the resin returning to an unfluidized packed configuration forming a resin cake. In some embodiments, the fluidization mode may be predominantly used for the coupling, oxidation, and capping steps detailed herein. In some embodiments, the fluidization mode may be used for the first portion of the deblocking steps detailed herein.

[0025] The SPOS process may be implemented on a solid phase media, for example, by building oligonucleotides on a resin as disclosed herein. In some embodiments, a “resin” as detailed herein may refer to a solid support including insoluble particles to which the oligonucleotide may be bound during synthesis. At certain points in the SPOS process, the resin may be fluidized as discussed above to promote contact between the resin and corresponding reagents or solvents. For example, during fluidization, N2 gas may be provided to a bottom of the reactor vessel to fluidize the resin. The inventors have recognized that in some instances, factors such as the geometry of the reactor vessel, the solvent or change in solvent applied to the resin, and / or the fluidization or plug-flow processes may cause the resin to form into a resin cake (also referred to as a slug). The inventors have found that the resulting resin cake may rise through the reactor vessel without fluidizing the resin or promote mixing of the resin with reagent. In particular, the resin cake may extend across the reactor vessel such that the resin cake inhibits passage of fluids past the resin cake, thus reducing fluidization.

[0026] In view of the above, the inventors have identified a need to disrupt cohesion of resin cakes that may form in the reactor vessel to promote fluidization, to increase the efficiency and reduce the synthesizing time of the SPOS process, and to improve the purity and yield of the resulting synthesized oligonucleotides.

[0027] According to one aspect of the invention, the inventors have recognized that an obstruction may be positioned within the reactor vessel such that the obstruction may be configured to disrupt cohesion of resin cakes formed in the vessel, which may increase fluidization of the resin and improve contact between the resin and the reagent and / or solvent. In some embodiments, the obstruction may be a stationary bar that contacts the resin cake as the resin cake rises through the reactor vessel as will be discussed in greater detail herein.

[0028] The inventors have also appreciated that existing SPOS systems may not sufficiently remove residual reagents between steps of the SPOS process, and that it may be desirable to remove residual reagents as they may negatively impact purity and yield of resulting synthesized oligonucleotides. For example, residual acid may remain in the reactor vessel following a detritylation reaction during the de-blocking step. In such an example, the inventors have found that even very low residual acid amounts can cause a significant decrease in purity and yield of the resulting oligonucleotides.

[0029] In view of the above, the inventors have identified a need to efficiently introduce washing solvents into the reactor vessel to wash unreacted reagents away while also minimizing disruption of the resin to improve the purity and yield of the synthesized oligonucleotides. The inventors have also recognized a need for reducing washing solvent usage and waste as large amounts of wash solvent may be traditionally used to effectively remove unreacted reagents from the reactor vessel.

[0030] According to another aspect of the invention, the inventors have recognized that spray devices (e.g., spray nozzles and / or spray balls) may be employed to wash the walls of the reactor vessel and / or a feed zone vessel. The inventors have also recognized that such spray devices may be employed to efficiently apply wash solvent or reagent to the resin itself. For example, in some embodiments, a spray ball may be employed to wash the walls of the feed zone vessel and / or the reactor vessel while a spray nozzle may be used to distribute wash solvent and / or reagent to the resin so as to not disrupt the resin and / or to re-flatten the resin bed.

[0031] As used herein, the “reactor vessel” refers to the vessel in which the synthetization of the oligonucleotides occurs while the “feed zone vessel” refers to a vessel that may contain solvents and / or reagents which may be introduced into the reactor vessel.

[0032] The inventors have also appreciated that the resin cake during SPOS may shrink and swell when exposed to reagents and / or solvents, thus forming channels between the sidewall of the resin cake and the sidewall of the reactor vessel itself or within the resin bed. The inventors have recognized that such “channeling” as it is referred to as herein is undesirable as it may cause a larger amount of liquid to flow along the channels rather than into the resin cake to promote efficient liquid solid contacting. For example, following a first wash of a resin cake after a deblocking step using an acetonitrile (ACN) solution, the resin cake may shrink and pull away from the walls or form gaps or cracks, thus forming the channeling, and form channels and voids throughout the resin bed. The inventors have appreciated that in some such embodiments, a second wash may be provided to cause the sidewall of the resin cake to move toward the sidewall of the reactor vessel (e.g. outwardly in a radial direction), thereby removing the “channeling”. This may occur due to the resin cake flattening in response to the second wash, and the resin cake being redistributed towards the sidewalls of the reactor vessel and fill in channels and voids throughout the resin bed. That is, if only one wash was provided to the resin cake, the wall channels may remain and the efficiency of the SPOS process may be reduced. Accordingly, the inventors have appreciated that it may be desirable to conduct multiple washes of the resin cake to reduce or prevent disturbance of the resin cake and resulting channeling and to promote fluidization of the resin cake.

[0033] Turning to the figures, specific non-limiting embodiments are described in further detail. It should be understood that the various systems, components, features, and methods described relative to these embodiments may be used either individually and / or in any desired combination as the disclosure is not limited to only the specific embodiments described herein.

[0034] FIGs. 1A-1C show schematic diagrams of a system configured for use in SPOS. The system includes a reactor vessel 100 and a feed zone vessel 110. The reactor vessel 100 may be configured to receive a resin 150 within a bottom of the vessel. The reactor vessel 100 may also include one or more inlets 140 which may be configured to receive gas (e.g., N2 gas) from a gas source 144. The reactor vessel 100 may also may be configured to receive gas (e.g., N2 gas) from a gas source 144 into piping 124 and flow back into the bottom of the reactor through the liquid outlet piping (not shown in the figure). In some embodiments, the reactor vessel 100 further comprises a filter 170 which is configured to evenly distribute the gas (e.g., N2 gas) from the gas source 144 across the entire cross-section of the reactor vessel 100 and fluidize the resin 150. The one or more inlets 140 and the filter 170 may be located along any suitable part of the reactor vessel 100 such as, for example, the bottom of the vessel as shown in FIGs. 1A-1C. As discussed herein, during fluidization of the resin 150, gas (e.g., N2 gas) may bubble up through a resin cake 154 along direction U within the reactor vessel 100 as shown in FIG. IB. To disrupt cohesion of the resin cake to promote fluidization, an obstruction in the form of a bar 300 may be positioned within the reactor vessel such that the resin cake makes contact with the bar 300 as the resin cake moves upwardly through the reactor vessel, thereby disrupting cohesion of the resin cake 154. In some embodiments, disrupting cohesion of the resin cake may promote mixing within the reactor vessel during the SPOS process (e.g., mixing between the resin and a reagent). As shown in FIG. 1C, following the disruption of cohesion of the resin cake 154 due to contact with the bar 300, the broken-up resin 150 may move downwardly within the reactor vessel 100 such that the resin 150 re-settles near the bottom of the vessel.

[0035] The bar 300 may include a first bar portion 302 and a second bar portion 304. In some embodiments, at least a portion of the bar 300 (e.g., first bar portion 302) may be oriented parallel to a longitudinal axis 102 of the reactor vessel 100. In some embodiments, the first bar portion 302 may be oriented vertically. Likewise, in some embodiments, at least a portion of the bar 300 (e.g., second bar portion 304) may be oriented transverse (e.g., perpendicular) to the longitudinal axis 102. In some embodiments, the second bar portion 304 may be oriented horizontally. As shown in FIG. 1 A, the bar 300 may include a bend such that the first bar portion 302 is angled relative to the second bar portion 304. As the resin cake 154 rises through the reactor vessel 100, the resin cake may make contact with the bar 300, causing the resin cake to break up. As disclosed herein, the inventors have appreciated that breaking up the resin cake may promote fluidization, increase the efficiency and reduce the synthesizing time of the SPOS process, and improve the purity and yield of the resulting synthesized oligonucleotides. In some embodiments, the bar 300 may be suspended from the top of the reactor vessel as shown in FIG.

[0036] 1A. In some embodiments, the bar 300 is spaced from the bottom of the reactor vessel 100.

[0037] The feed zone vessel 110 may receive liquid (e.g., reagent and / or solvent) from a first feed zone reservoir 116 that supplies the liquid to the feed zone vessel 110 via supply line 112. The liquid may be stored in the feed zone vessel 110 until it is desirable to supply the liquid to the reactor vessel 100 via supply line 122. The liquid may be supplied from supply line 122 through spray nozzle 164 to distribute the liquid (e.g., reagent and / or solvent) along the resin 150. As disclosed herein, the inventors have recognized that the resin 150 may shrink and swell when exposed to reagents and / or solvents, thus forming undesirable channels between the sidewall of the resin cake and the sidewall of the reactor vessel itself, and channels and voids throughout the resin bed, and that such channels may cause a larger amount of liquid to flow along the channels rather than into the resin cake to promote efficient contacting. To reduce the occurrence of such “channeling” during the distribution of reagent in the reactor vessel 100, the inventors have found that it may be desirable to provide two washes within the reactor vessel. In some embodiments, a first wash of the resin cake may include providing an acetonitrile (ACN) solution to the resin cake, which may cause the resin cake to shrink and pull away from the walls or form voids throughout the cake, thus forming the channeling. In some embodiments, a second wash of the resin cake may then be provided to cause the sidewall of the resin cake to move back towards the sidewall of the reactor vessel, and the channels and voids throughout the middle of the resin bed to fill in, thereby removing the “channeling”. In some embodiments, the second wash of the resin cake may cause the resin cake to flatten, which may redistribute the resin cake such that the resin cake sidewall moves outwardly towards the sidewall of the reactor vessel and fills in voids and channels throughout the middle of the resin bed.

[0038] Each of the reactor vessel 100 and the feed zone vessel 110 may include spray balls 160, 114, respectively (or any other suitable spray device). The spray balls 160, 114 may be configured to receive a wash solvent from respective reservoirs 162, 118, and then may be configured to distribute the wash solvent along the walls of the respective vessels to wash away excess or unused reagent. The spray balls 160, 114 may each represent more than one spray ball so that all surfaces are contacted with the wash. Each of the reactor vessel 100 and feed zone vessel 110 may also include respective waste lines 124, 120, leading to respective waste reservoirs 132, 130. The waste reservoirs may be configured to receive undesirable synthesis byproducts, excess reagent, or any other suitable waste product as the disclosure is not so limited.

[0039] The system of FIG. 1 may also include one or more outlets 142 which may be configured to receive or extract the synthesized oligonucleotides or other end products. The one or more outlets 142 may be located along any suitable part of the reactor vessel 100 such as, for example, the top of the vessel as shown in FIG 1.

[0040] FIG. 2 shows a flow diagram of a method 200 for performing SPOS. The first step is the “de-blocking” step 210, which is generally a detritylation reaction. Specifically, the nucleotide has its 5'- hydroxyl group protected by an acid-labile protection group such as DMT (4,4'-dimethoxytrityl). This protection group is removed during a continuous flow of the acid solution or via an addition of an acid in a solvent. The acid may be for example, trichloroacetic acid (TCA) dichloroacetic acid (DCA) or some other acid that is carried in an inert solvent such as toluene or dichloromethane or other solvents. In some embodiments, 2% TCA, 3% DCA, or 10% DCA is used with toluene. For a DMT protection group, during this “de-blocking” reaction, an orange-colored DMT cation formed is washed out continuously during the flow-through reaction and via addition of a washing solution, e.g., during a washing step 220. Accordingly, this step results in the solid support-bound oligonucleotide precursor bearing a free 5'-terminal hydroxyl group.

[0041] Once the de-blocking step 210 and wash 220 occurs, the “coupling” step 230 is then performed. This coupling involves adding a solution of activated phosphoramidite in a solvent (such as, for example, a 0.02-0.2 M solution of phosphoramidite in acetonitrile (ACN) (or anhydrous ACN)). 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 an 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. Upon the completion of the coupling, any unbound reagents and by-products are removed by washing, e.g. during a washing step 240.

[0042] After the coupling step 230, the next step in the SPOS is the “oxidation” step 250. In the oxidation step, the coupled phosphoramidite that reacted to the 5 ’-terminal OH group results in a phosphite triester linkage (e.g., in which the P atom 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. 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 (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 (IS)- (+)-(10-camphorsulfonyl)-oxaziridine (CSO). In other embodiments, sulfurization to a phosphothiolate linker is done instead of oxidation.

[0043] Once the “oxidation” step 250 occurs, the next step in SPOS is “capping” 270. Capping may be omitted from some or all of the cycles. Capping is performed because a small percentage of the solid support-bound 5'-OH groups (0.1 to 1% or greater) 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. The unreacted 5'-hydroxy groups are, to a large extent, acetylated by the capping mixture. By capping these unreacted OH groups, these truncation impurities can be more readily chromatographically separated out from the desired product. Likewise, if the coupling reaction created other, non-desired products (such as a reaction of an O in the guanosine base or other chemical entities), these non-desired products are also blocked (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. Other capping reagents may also be used.

[0044] In addition to the above four steps of “de-blocking”, “coupling”, “oxidation”, and “capping”, additional washing steps may be conducted. In some embodiments, as indicated by the flow diagram of FIG. 2, washing steps 220, 240, 260, and 280 may be conducted after each of the four respective steps for SPOS detailed above. During a washing step, a spray nozzle or a spray ball may supply a washing solvent to the reactor vessel and / or the resin to wash unreacted reagents away. The inventors have recognized that residual reagents may negatively impact purity and yield of the resulting synthesized oligonucleotides, and thus providing washing steps may be beneficial to increase purity and yield of the resulting product.

[0045] Once these four steps are completed (de-blocking, coupling, either oxidation or sulfurization, and the optional 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 position. Thus, the process may then be repeated and another phosphoramidite moiety added until the chain reaches its desired length.

[0046] 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. FIGs. 3A-3C show schematic diagrams of embodiments of a system for promoting fluidization of resin during SPOS. In particular, FIG. 3 A shows an arrangement where an obstruction in the form of bar 320 includes a first bar portion 322 and a second bar portion 324, where the second bar portion 324 is angled at an obtuse angle 326 relative to the first bar portion 322. By comparison, FIG. 3B shows an arrangement where a bar 340 includes a first bar portion 342 and a second bar portion 344, where the second bar portion 344 is angled at an acute angle 346 relative to the first bar portion 342. Although such exemplary arrangements are disclosed, an obstruction configured for use in disrupting cohesion of a resin cake as disclosed herein may be oriented at any suitable angle as the disclosure is not so limited. In some embodiments, a suitable bend angle between two or more portions of an obstruction may be greater than or equal to 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°, 90°, 95°, 100°, 105°, 110°, 115°, 120°, 125°, 130°, 135°, 140°, 145°, 150°, or greater. In some embodiments, a suitable bend angle between two or more portions of an obstruction may be lesser than or equal to 180° (i.e., no bend), 175°, 170°, 165°, 160°, 155°, 150°, 145°, 140°, 135°, 130°, 125°, 120°, 115°, 110°, 105°, 100°, 95°, 90°, 85°, 80°, 75°, 70°, 65°, 60°, or lesser. Suitable ranges of the foregoing are also contemplated. For example, in some embodiments, a suitable bend angle may be between or equal to 85° and 95°. In some embodiments, a suitable bend angle is 90° such as the bar 300 shown in FIG. 1A.

[0047] In some embodiments, any suitable number of bends may be provided in an obstruction including, but not limited to, 1, 2, 3, 4, 5, or more bends. In some embodiments, any suitable number of obstructions may be provided in a reactor vessel to disrupt cohesion of a resin cake and promote fluidization. In some embodiments, a suitable number of obstructions is greater than or equal to 1, 2, 3, 4, 5, or more obstructions. The obstructions may also be of any suitable shape. For example, the obstructions may be comprise a bar that extends into the reactor vessel as disclosed herein, and the bar may have an “L-shape”, a “T-shape”, or any other suitable shape as the disclosure is not so limited. In addition or alternatively, the bar may have a plurality of secondary bar portions extending from a primary shaft of the bar. For example, the bar 300 of FIG. 1 may include a singular first bar portion 302 but may include a plurality of second bar portions 304 extending outwards from the first bar portion. In such an example, the bar may be of a “pinwheel shape”. FIG. 3C shows an arrangement where an obstruction in the form of bar 360 is positioned within the reactor vessel 100. As shown in FIG. 3C, the bar 360 may not include any bends and may extend horizontally across the vessel 100. In some embodiments, the bar may extend entirely across the vessel 100, as shown in FIG. 3C. In other embodiments, however, the bar 360 may only extend partially across the vessel 100.

[0048] The obstructions may also be of any suitable size to be accommodated within the reactor vessel and to disrupt cohesion of the resin cake as the disclosure is not so limited. In some embodiments, the obstruction may extend downwards (e.g., along a longitudinal axis 102 of the reactor vessel 100) at a length of greater than or equal to 3 inches, 4 inches, 5 inches, 6 inches, 7 inches, 8 inches, 9 inches, 10 inches, 11 inches, 12 inches, 13 inches, 14 inches, 15 inches, 16 inches, 17 inches, 18 inches, 19 inches, 20 inches, or greater. In embodiments where the obstruction may include one or more bends, the bent portions of the obstruction may be of any suitable length as well including, but not limited to, greater than equal to 1 inch, 2 inches, 3 inches, 4 inches, 5 inches, 6 inches, 7 inches, 8 inches, 9 inches, 10 inches, or greater. The obstructions may also be of any suitable transverse dimension including, but not limited to, greater than or equal to 0.25 inches, 0.5 inches, 0.75 inches, 1 inch, 1.5 inches, 2 inches, 2.5 inches, 3 inches, or greater. For example, the obstruction may comprise a bar including first and second bar portions as shown in FIG. 1 and FIGs. 3A-3B, and these bar portions may be constructed and arranged to have suitable dimensional parameters from the foregoing.

[0049] In some embodiments, the size of the obstructions may be quantified as a ratio relative to the size of the vessel itself. For example, the portion of an obstruction which extends downwards (e.g., along a longitudinal axis 102 of the reactor vessel 100) may be of a suitable percentage relative to a length of the reactor vessel (e.g., a height of the vessel along longitudinal axis 102) of greater than or equal to 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or greater. In another example, in embodiments where the obstruction may include one or more bends, the bent portions of the obstruction may be of a suitable percentage relative to a transverse dimension (e.g., diameter) of the reactor vessel of greater than or equal to 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or greater. Of course, percentages greater or lesser than the foregoing are also contemplated. Embodiments described herein are primarily discussed in reference to a stationary obstruction, i.e., the obstruction does not move and disrupts cohesion of the resin cake as a result of the resin cake rising within the reactor vessel to contact the obstruction. However, in some embodiments, at least a portion of the obstruction may be configured to move relative to the reactor vessel to disrupt cohesion of the resin cake. That is, in some embodiments, the obstruction may comprise an agitator, and the agitator may be configured to move in any suitable fashion. The agitator may be configured to translate along a direction parallel and / or transverse to the longitudinal axis of the reactor vessel as the disclosure is not so limited. In some embodiments, the obstruction may be configured to rotate relative to the reactor vessel, e.g., the obstruction may rotate circumferentially around the longitudinal axis of the reactor vessel to disrupt cohesion of the resin cake.

[0050] The obstructions disclosed herein may also be constructed of any suitable material as the disclosure is not so limited. In some embodiments, the obstructions may be constructed out of metal such as steel (e.g., carbon steel, stainless steel), Hastelloy, aluminum, tungsten, iron, brass, copper, titanium, alloys thereof, or plastics, or any other suitable material as the disclosure is not so limited. In some embodiments, the obstructions may be constructed and arranged to be used for multiple SPOS processes. In other embodiments, however, the obstructions may be constructed and arranged to be disposable and replaceable such that a used obstruction may be discarded and a new obstruction may be positioned in the reactor vessel.

[0051] While a variety of examples have been provided in reference to the shape, size, number, and other characteristics of the obstructions disclosed herein, it should be appreciated that the obstructions may be of any suitable characteristic to disrupt cohesion of a resin cake formed in a reactor vessel to increase fluidization of the resin and promote mixing as the disclosure is not so limited.

[0052] FIGs. 4A-4B show schematic diagrams of embodiments of a system for promoting fluidization of resin during SPOS. As disclosed herein, the inventors have recognized that the resin cake positioned within the reactor vessel may shrink and swell when exposed to reagents and / or solvents, thus forming undesirable channels between the sidewall of the resin cake and the sidewall of the reactor vessel itself, and voids within the cake itself. The inventors have recognized that such channels may cause a larger amount of liquid to flow along the channels rather than into the resin cake, thus potentially resulting in less effective contacting during flow-through mode.

[0053] As shown in FIG. 4A, a sidewall 152 of the resin cake 154 may be spaced from the sidewall 180 of the reactor vessel 100 such that one or more channels 182 may be formed between the vessel and the resin cake. In some embodiments, the configuration of FIG. 4A may be the result of a first wash of the resin cake 154 which may cause the resin cake 154 to shrink and pull away from the walls, thus forming the one or more channels 182 as shown in FIG. 4 A. To reduce the occurrence of the “channeling” shown in FIG. 4A, the inventors have found that it may be desirable to split the wash up into multiple segments within the reactor vessel as disclosed herein. In particular, a second wash of the resin cake 154 may cause the sidewall 152 of the resin cake 154 to move back towards the sidewall 180 of the reactor vessel 100, and causing the voids and channels in the middle of the bed to fill back in, thereby removing or reducing the size of the one or more channels 182. In some embodiments, the second wash of the resin cake may cause the resin cake to flatten, which may redistribute the resin cake such that the sidewall 152 of the resin cake 154 moves outwardly towards the sidewall 180 of the reactor vessel 100 that any voids in the middle of the resin cake fill in. In some embodiments, the channel 182 may be a continuous circumferential channel extending completely around or at least partially around the resin cake 154. Any suitable number, shape, or size of channels may be formed and subsequently removed as the disclosure is not so limited. For example, only a portion of the resin cake 154 may shrink and pull away from the sidewalls of the reactor vessel following a first wash such that portions of the resin cake remain in contact with the sidewall of the vessel while other portions of the resin cake are spaced from the sidewall of the vessel.

[0054] The reactor vessel itself may also be of any suitable size, shape, and / or other characteristic to perform oligonucleotide synthesis. In some embodiments, a suitable shape of the reactor vessel may be cylindrical, a rectangular prism, pyramidal, or any other suitable shape as the disclosure is not so limited. In some embodiments, a suitable transverse dimension of the reactor vessel (e.g., an inner diameter in a cylindrical vessel), may be greater than or equal to 3 inches, 4 inches, 5 inches, 6 inches, 7 inches, 8 inches, 9 inches, 10 inches, 11 inches, 12 inches, 13 inches, 14 inches, 15 inches, 16 inches, 17 inches, 18 inches, 19 inches, 20 inches or greater. In some embodiments, a suitable longitudinal dimension of the reactor vessel may be greater than or equal to 5 inches, 10 inches, 15 inches, 20 inches, 25 inches, 30 inches, 35 inches, 40 inches, 45 inches, 50 inches, or greater. Of course, transverse and longitudinal dimensions of the reactor vessel both greater and lesser than the foregoing are also contemplated. In some embodiments, the inside dimension in the horizontal direction may gradually change at different heights, for example it may be advantageous for the reactor to be wider at the top than at the bottom. In some embodiments, the size of the reactor vessel may be quantified in terms of a reaction volume (i.e., an interior volume of the reactor vessel). In some embodiments, a suitable reaction volume of the vessel may be greater than or equal to 1 liter (L), 2 L, 5 L, 10 L, 20 L, 100 L, 200 L, 500 L, 1000 L, or any other suitable volume.

[0055] Any suitable type of resin may be positioned within the reactor vessel as the disclosure is not so limited. In some embodiments, a suitable resin that may be used for oligonucleotide synthesis may include, but is not limited to, controlled pore glass (CPG), polystyrene, or any other suitable type of resin as the disclosure is not so limited. In addition, any suitable types of wash solvents and / or reagents may be employed including, but not limited to, acetonitrile (ACN), toluene, pyridine, and / or lutidine.

[0056] While spray devices have been discussed herein to include spray nozzles and spray balls, the use of spray devices are not limited herein to such examples. As discussed above, in some embodiments, spray nozzles may be employed to distribute wash solvents and / or reagents to the resin and spray balls may be employed to wash the reactor vessel to wash away residual reagents. However, in some embodiments, spray balls may be employed to distribute wash solvents and / or reagents to the resin and spray nozzles may be employed to wash the reactor vessel as the disclosure is not so limited. In addition, any suitable spray pattern type may be used to distribute the wash solvent and / or reagent including, but not limited to a cone spray, a fan spray, a misting spray, or any other suitable spray pattern type. The nozzle itself may be a single nozzle, a family of spray nozzles arranged together, or a plate with a large number of orifices so that the liquid can evenly rain down on the resin bed.

[0057] The embodiments disclosed herein may be combined in any suitable arrangement as the disclosure is not limited in this regard. The embodiments disclosed herein may also be embodied as a method. While the present teachings have been described in conjunction with various embodiments and examples, it is not intended that the present teachings be limited to such embodiments or examples. On the contrary, the present teachings encompass various alternatives, modifications, or equivalents, as will be appreciated by those of skill in the art. Accordingly, the foregoing description is by way of example only.

[0058] EQUIVALENTS

[0059] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0060] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0061] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0062] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0063] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0064] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0065] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims

CLAIMS1. A system for solid phase oligonucleotide synthesis (SPOS), the system comprising:a reactor vessel configured to receive resin, the reactor vessel having a gas inlet; a gas source configured to supply gas to the reactor vessel via the gas inlet; and an obstruction positioned within the reactor vessel, wherein the obstruction is configured to disrupt cohesion of a resin cake in the reactor vessel to increase fluidization of the resin.

2. The system of claim 1, wherein the obstruction comprises a bar that extends into the reactor vessel.

3. The system of claim 2, wherein at least a portion of the bar is oriented parallel to a longitudinal axis of the reactor vessel.

4. The system of claim 3, wherein at least a portion of the bar is oriented transverse to the longitudinal axis of the reactor vessel.

5. The system of any one of claims 2-4, wherein the bar includes at least one bend.

6. The system of claim 5, wherein the at least one bend is 85-95 degrees.

7. The system of any one of claims 1-6, wherein the obstruction is stationary relative to the reactor vessel.

8. The system of any one of claims 1-7, wherein the obstruction extends from a top portion of the reactor vessel such that the obstruction is spaced from a base portion of the reactor vessel.

9. The system of claim 8, wherein the gas inlet is at the base portion of the reactor vessel.

10. The system of claim 9, wherein the reactor vessel has an outlet at the top portion of the reactor vessel.

11. The system of claim 1, wherein the obstruction comprises an agitator that is configured to move relative to the reactor vessel to disrupt cohesion of the resin cake.

12. The system of claim 1, wherein the obstruction is one of a plurality of obstructions configured to disrupt cohesion of the resin cake to increase fluidization of the resin.

13. The system of claim 1, further comprising a filter disposed within the reactor vessel positioned above the gas inlet.

14. A method of promoting fluidization of resin during solid phase oligonucleotide synthesis (SPOS), the method comprising:positioning resin within a reactor vessel of a solid phase oligonucleotide synthesis (SPOS) system, wherein at least one oligonucleotide is attached to the resin; and introducing gas into the reactor vessel and passing the gas through a filter, causing a resin cake formed within the reactor vessel to move within the reactor vessel and contact an obstruction positioned within the reactor vessel,wherein contact of the resin cake with the obstruction disrupts cohesion of the resin cake and increases fluidization of the resin.

15. The method of claim 14, wherein the obstruction remains stationary relative to the reactor vessel.

16. The method of claim 14, wherein the gas increases a pressure underneath the resin cake, causing the resin cake to move upwards within the reactor vessel and contact the obstruction.

17. The method of any one of claims 14-16, further comprising mixing within the reactor vessel after cohesion of the resin cake is disrupted.

18. The method of any one of claims 14-17, wherein the obstruction comprises a bar that extends into the reactor vessel.

19. The method of claim 18, wherein the bar includes at least one bend.

20. The method of claim 19, wherein the at least one bend is 85-95 degrees.

21. The method of claim 14, wherein the obstruction comprises a mechanical agitator that moves relative to the reactor vessel to disrupt cohesion of the resin cake.

22. A method of promoting fluidization of resin during solid phase oligonucleotide synthesis (SPOS), the method comprising:positioning resin within a reactor vessel of a solid phase oligonucleotide synthesis (SPOS) system, wherein at least one oligonucleotide is attached to the resin; and delivering a reagent into the reactor vessel to cause a sidewall of a resin cake formed within the reactor vessel to move toward a sidewall of the reactor vessel, thereby decreasing a size of a channel between the sidewall of the resin cake and the sidewall of the reactor vessel.

23. The method of claim 22, wherein delivering the reagent or wash solvent into the reactor vessel causes the resin cake to flatten, thereby causing the sidewall of the resin cake to move toward the sidewall of the reactor vessel.

24. The method of claim 22, wherein delivering the reagent or wash solvent into the reactor vessel includes delivering a first reagent during a first wash step and delivering a second reagent during a second wash step.

25. The method of claim 22, wherein the step of delivering the reagent into the reactor vessel comprises spraying the resin cake with the reagent.

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