Method for producing synthetic oligonucleotides and removing agent for removing impurities from organic washing solvents used in oligonucleotide synthesis

Abstract

The present invention provides a method of using a mixed composition of acetonitrile and toluene as an organic cleaning solvent in the production of synthetic oligonucleotides, the mixed composition yielding a higher synthetic oligonucleotide yield than a pure acetonitrile cleaning solvent. The method also provides a process for removing one or more impurities from a cleaning solvent containing acetonitrile and toluene received as a waste liquid stream from an oligonucleotide synthesis process. The process includes adding at least one of an iodine-reactive compound, a sulfur-reactive compound, and / or an acid-reactive compound to the waste stream and fractionating the waste stream. Fractionation produces an overhead fraction and a bottom fraction, the overhead fraction containing acetonitrile and toluene and the bottom fraction containing one or more impurities.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims priority to U.S. Patent Application No. 18 / 225,010, filed on July 21, 2023, and claims the benefit of U.S. Provisional Patent Application No. 63 / 392,441, filed on July 26, 2022, both of which are hereby incorporated by reference in their entirety.

[0002] (Field of the Invention) This technical field generally relates to methods and systems for purifying waste streams containing acetonitrile and toluene, and more specifically, to the addition of one or more reactive compounds to a waste stream that enables the recovery and subsequent recycling of a substantially purified mixture of acetonitrile and toluene. In some cases, the recycled and purified mixture of acetonitrile and toluene, when used as a wash solvent in an oligonucleotide synthesis process, produces higher purity oligonucleotides compared to pure acetonitrile.

Background Art

[0003] Many chemical processes utilize acetonitrile as a solvent or cleaning fluid, resulting in the generation of low-grade acetonitrile waste streams. When these processes are carried out on a manufacturing scale, the volume of the low-grade acetonitrile waste streams generated can be substantial. For example, oligonucleotide (DNA and RNA) synthesis is generally carried out by a four-step cycle (deprotection, activation / coupling, capping, and oxidation), which is repeated for each added nucleotide until the desired sequence is obtained. Between each step, the oligonucleotide bound to the support is typically washed with high-purity (i.e., 100%) acetonitrile to reduce residual reagents from the previous step. This results in the generation of large volumes of acetonitrile waste streams, and when manufacturing approximately one metric ton of oligonucleotide-based active pharmaceutical ingredient (API), approximately 2000 metric tons of acetonitrile are used.

[0004] Synthetic oligonucleotide sequences are promising for therapeutic, diagnostic, and drug target validation applications in the biopharmaceutical industry, and currently, the number of oligonucleotide-based drugs in preclinical or clinical trials is continuing to increase. However, oligonucleotide-based API manufacturers are facing the costs and difficulties in managing both the procurement of large amounts of pure acetonitrile and the disposal of the large volumes of acetonitrile-based waste streams generated. Therefore, there is an urgent need in the industry for methods and systems for recovering and purifying acetonitrile waste streams to produce an acetonitrile-based cleaning solvent suitable for reuse. Further desirable features and characteristics will become apparent from the subsequent detailed description and the appended claims taken in conjunction with the accompanying drawings. SUMMARY OF THE INVENTION

[0005] The present disclosure provides a process and method for removing one or more impurities including iodine-containing compounds, sulfur-containing compounds, and / or basic nitrogen-containing compounds from waste streams generated by oligonucleotide processes. In this case, the waste stream contains not only acetonitrile and toluene in a composition ratio rich in acetonitrile but also one or more impurities. One or more reducing agents / capturing agents including iodine-reactive compounds, sulfur-reactive compounds, and / or acidic-reactive compounds are added to the waste stream. During fractional distillation, the overhead fraction contains acetonitrile and toluene in an azeotropic composition, and the bottom fraction contains one or more impurities. The overhead fraction may be further processed to remove any residual impurities, and the purified mixture of acetonitrile and toluene can be recycled to the oligonucleotide process as a washing solvent.

[0006] In one embodiment, the present disclosure provides a method for treating a waste stream, comprising receiving a waste stream containing acetonitrile, toluene, and one or more iodine-containing compounds, adding an iodine-reactive compound to the waste stream, and fractionating the waste stream to produce an overhead fraction and a bottom fraction. In this case, the overhead fraction contains the acetonitrile and toluene of the waste stream, and the bottom fraction contains one or more iodine-containing compounds of the waste stream.

[0007] In another embodiment, the present disclosure provides a method for treating a waste stream, comprising receiving a waste stream containing acetonitrile, toluene, and one or more sulfur-containing compounds, adding a sulfur-reactive compound to the waste stream, and fractionating the waste stream to produce an overhead fraction and a bottom fraction. In this case, the overhead fraction contains the acetonitrile and toluene of the waste stream, and the bottom fraction contains one or more sulfur-containing compounds of the waste stream.

[0008] In yet another embodiment, the present disclosure provides a method for treating a waste stream, the method comprising receiving a waste stream comprising acetonitrile, toluene, and a basic nitrogen-containing compound, adding an acidic reactive compound to the waste stream, and fractionating the waste stream to produce an overhead fraction and a bottom fraction. In this case, the overhead fraction comprises the acetonitrile and toluene of the waste stream, and the bottom fraction comprises the basic nitrogen-containing compound of the waste stream.

[0009] In a further embodiment, the present disclosure provides a method for producing a synthetic oligonucleotide, the method comprising washing a reaction vessel containing one or more components of an oligonucleotide synthesis process with a mixed washing solution comprising at least acetonitrile and toluene, wherein acetonitrile is at least 70% of the mixed washing solution, and recovering the synthetic oligonucleotide. In this case, the synthetic oligonucleotide is recovered with a minimum oligonucleotide purity.

Brief Description of the Drawings

[0010]

Figure 1

Mode for Carrying Out the Invention

[0011] The following "Mode for Carrying Out the Invention" is essentially merely exemplary and is not intended to limit various embodiments or their applications and uses. Further, it is not intended to be limited by any theory presented in the foregoing Background Art or the following Detailed Description.

[0012] Methods, systems, and processes for purifying oligonucleotide waste streams are provided herein. The processes, methods, and systems of the present invention provide a technically simplified and economically advantageous way to pretreat and recycle waste streams received from oligonucleotide synthesis. As described above, oligonucleotide synthesis processes (e.g., solid-phase synthesis) utilize a significant amount of acetonitrile-based solvents as wash solvents, and during such processes, the acetonitrile-based solvents are contaminated by many impurities.

[0013] Oligonucleotide synthesis processes typically require wash solvents of pure acetonitrile, and thus, acetonitrile-containing waste streams cannot be reused. Therefore, it is necessary to purify the acetonitrile-containing waste streams to an acceptable level for recycling back into the synthesis process. Such synthesis processes have been found to contaminate acetonitrile with a plurality of compounds including toluene (e.g., contaminated by deblocking agents), iodine (e.g., contaminated by oxidation reagents), sulfur-containing compounds such as thiols (e.g., contaminated by sulfidation and activation reagents), and basic nitrogen-containing compounds such as alkylamines (e.g., contaminated by oxidation reagents, capping reagents, and activation reagents). In this case, it is difficult to remove such compounds to an acceptable extent to purify acetonitrile to the purity required for recycling and / or recovery.

[0014] Importantly, it has been found that instead of using pure acetonitrile as the appropriate washing solvent in the oligonucleotide synthesis process, it is possible to utilize a substantially pure mixture of acetonitrile and toluene. Thus, it has been confirmed that in order to reuse a contaminated acetonitrile waste stream that cannot be used in other ways as the washing solvent in the synthesis process, it is necessary to purify the waste stream into a substantially pure mixture of acetonitrile and toluene (i.e., remove other impurities such as iodine, thiols, and alkylamines). Furthermore, surprisingly, it has also been found that in certain compositions, using a purified mixture of acetonitrile and toluene as the washing solvent in the oligonucleotide synthesis process results in a higher purity oligonucleotide compared to washing with pure acetonitrile. Thus, recycling the purified waste stream not only results in a substantial reduction in the amount of acetonitrile-based washing solvent that is discarded (e.g., because the washing solvent is recycled into the oligonucleotide synthesis process rather than discarded), but also the use of the purified waste stream results in a higher purity oligonucleotide.

[0015] As described in connection with U.S. Patent No. 10,336,690, the disclosure of which is hereby incorporated by reference in its entirety, the removal of contaminants from a waste stream can be achieved by fractional distillation in combination with contact with one or more absorbents / adsorbents. In this case, the waste stream can be fed to a fractionating / distillation column where, during fractionation / distillation, the overhead fraction (distillate) contains acetonitrile and low-boiling and / or volatile compounds (e.g., toluene, some water, and most of the iodine, thiols, and alkylamines), while the bottom fraction contains the high-boiling components of the waste stream. Also, as described in connection with U.S. Patent No. 10,336,690, the condensed overhead stream can be sent to a series of absorbers where, upon contact with different absorbents, a purified stream containing acetonitrile can be obtained. However, in this case, since the concentration of contaminants in the overhead fraction is relatively high, the removal of impurities consumes a significant amount of absorbent. Therefore, it has been confirmed that it may be advantageous to remove one or more of these contaminants before contacting the absorbers. This can reduce the cost of the purification process since it can reduce both the size of the column and / or the amount of absorbent / adsorbent utilized.

[0016] As used herein, the term "purify" and variations thereof refer to the reduction in the amount of one or more impurities in a composition. Generally speaking, purification can be achieved via any number of techniques known in the art depending on the nature of the composition being purified and the impurities being reduced. Purification does not necessarily result in a pure product in which all impurities or any particular impurity is completely absent. Further, some of the methods and systems described herein utilize multiple purification steps including the fractionation of an acetonitrile-containing waste stream. In some embodiments, fractionation is the first purification step.

[0017] Referring to FIG. 1, in an exemplary embodiment, the fractionation of waste stream 102 is performed using a system 100 having a fractionation zone 103 that includes a distillation or fractionation column 106. In certain embodiments, the fractionation / distillation column 106 includes internal structures such as packing, trays, sieves, bubble caps, or similar mechanical configurations that can provide a plurality of stages of vapor-liquid flow contact through the column. The number of stages and the type of internal structures vary depending on the specific composition of the acetonitrile-containing waste stream, inlet location, reflux ratio, desired column efficiency, etc. Thus, the internal profile of the fractionation / distillation column 106 can vary for each application.

[0018] The waste stream 102 can be provided by various sources and may contain some impurities having a boiling point higher than that of acetonitrile and some impurities having a boiling point lower than that of acetonitrile. In this regard, some waste stream purification methods rely on taking a "heart cut" of the distillate, in which case two fractionation columns are used to first separate acetonitrile and low-boiling impurities (in the first overhead fraction) from high-boiling impurities (in the first bottoms fraction), and a second fractionation column is used to separate acetonitrile and other similarly boiling species (in the second bottoms fraction) from low-boiling impurities (in the second overhead fraction).

[0019] However, as described herein, such a complex fractionation by two columns is not required. Rather, in some embodiments, the fractionation zone 103 includes a single column 106, and the single overhead fraction 108 is collected up to a single cut point (e.g., just above the boiling point of acetonitrile or an acetonitrile-toluene mixture, such as about 81°C - 82°C at standard pressure (1 ATM)).

[0020] As used herein, the term "overhead" fraction means the fraction withdrawn from the top of a fractionation column. The overhead fraction contains components of the original mixture that have a lower boiling point than the components found in the "bottoms" (i.e., the fraction remaining in and / or removed from the lower part of the fractionation column). This overhead fraction 108 contains acetonitrile and impurities that boil at lower temperatures. In some embodiments, the overhead fraction 108 may also contain some high-boiling impurities such as toluene due to azeotropic behavior with acetonitrile, as well as iodine.

[0021] The methods and systems described herein achieve further purification of the overhead fraction 108 by condensing the overhead fraction 108 and contacting the condensed overhead fraction 108 with one or more adsorbents (e.g., adsorbents 112, 118, and 124 contained within towers 110, 116, and 122, respectively). The nature of the particular adsorbent used in subsequent purification steps is determined by the identity and level of impurities remaining in the overhead fraction 108 and is thus affected by the source of the waste stream 102.

[0022] As noted above, many scientific and manufacturing processes utilize acetonitrile and toluene streams as solvents or as cleaning solutions, generating an acetonitrile waste stream. Various processes may require different degrees of purity of the acetonitrile and toluene streams and / or may require a particular degree of purity with respect to one or more specific impurities.

[0023] One such process that generates particularly large amounts of acetonitrile waste is the manufacture of synthetic oligonucleotides (DNA and RNA). Oligonucleotide synthesis is generally performed by a four-step cycle (deprotection, activation / coupling, capping, and oxidation) that involves washing with pure acetonitrile between each cycle. Since each step in the cycle uses different reagents, the acetonitrile waste streams generated from the washes after each step are likely to be contaminated with different types and levels of impurities.

[0024] For example, each oligonucleotide synthesis cycle typically begins with a blocked nucleotide covalently attached to a support. The blocked nucleotide is typically deblocked in a first step via reaction with an acidic solution such as 3% trichloroacetic acid (TCA) or 3% dichloroacetic acid (DCA) in the presence of toluene or dichloromethane. Once the deblocking reaction is complete, the reagent is drained and the deblocked oligonucleotide (attached to the support) is washed with an acetonitrile-based wash solvent. The impurities found in the resulting acetonitrile waste stream can include unreacted reagent, residual solvent, and / or by-products of the deblocking reaction. For example, the impurities in the deblocked acetonitrile waste stream can include toluene, acetic acid, trichloromethane, and / or propanenitrile. The impurities in the deblocked acetonitrile waste stream typically constitute 0.1 to 30 wt% of the waste stream 102. In an exemplary embodiment, the waste stream 102 includes acetonitrile waste generated during the acetonitrile wash step after the deblocking step of oligonucleotide synthesis.

[0025] The second step of the oligonucleotide synthesis cycle includes activation and coupling of the deblocked oligonucleotide. Activation is performed by contact with any suitable activator known in the art. After activation, the material attached to the support is treated with iodine and water in the presence of a weak base (e.g., pyridine, lutidine, or collidine) that oxidizes the triester phosphite to a tetracoordinate phosphate triester. The deblocked oligonucleotide is coupled (i.e., reacted) with a phosphoramidite to form a phosphite oligomer. The phosphoramidite is variously selected from all available phosphoramidites, and the reaction proceeds as known in the art.

[0026] After coupling, the reagent is discharged, pushed through the column with an acetonitrile-based wash solvent, and the solid support is washed with an acetonitrile-based wash solvent. Again, impurities found in the resulting acetonitrile waste stream can include unreacted reagent, residual solvent, or by-products of the activation and coupling reactions. For example, impurities in the activation / coupling acetonitrile waste stream can include activator molecules, phosphoramidites, iodine, pyridine, propanenitrile, and / or toluene. Impurities in the coupling acetonitrile waste stream typically likewise constitute from 0.1 to 30 wt% of waste stream 102. In an exemplary embodiment, waste stream 102 includes acetonitrile waste generated during an acetonitrile wash step after the activation / coupling step of oligonucleotide synthesis.

[0027] Typically, during the coupling step, only a portion of the deblocked nucleotides react with the phosphoramidite and any unreacted nucleotides must be capped. Capping is performed by contacting the unreacted nucleotides with an amount of acetic anhydride and an amount of N-methylimidazole. After coupling, the reagent is discharged / pushed through the column with an acetonitrile-based wash solvent and the oligonucleotide growing on the solid support is washed with an acetonitrile-based wash solvent. Again, impurities found in the resulting acetonitrile waste stream can include unreacted reagent, residual solvent, or by-products of the capping reaction. For example, impurities in the capping acetonitrile waste stream can include N-methylimidazole, acetic acid, acetic anhydride, lutidine, pyridine, tetrahydrofuran, propanenitrile, and / or toluene. Impurities in the capping acetonitrile waste stream typically likewise constitute from 0.1 to 30 wt% of waste stream 102. In an exemplary embodiment, waste stream 102 includes acetonitrile waste generated during an acetonitrile wash step after the capping step of oligonucleotide synthesis.

[0028] Finally, the phosphite oligomers formed in the activation / coupling step are oxidized or sulfided. Oxidation is achieved with iodine (e.g., via reaction) in the presence of water and pyridine. Sulfidation is achieved by reaction with phenylacetyl disulfide or similar chemicals. After oxidation or sulfidation, the reagent is drained / expelled through the column using a wash acetonitrile solution. The solid support containing the growing oligonucleotide is washed again with the acetonitrile wash stream. The impurities found in the resulting acetonitrile waste stream can include unreacted reagent, residual solvent, or by-products of the oxidation reaction. For example, the impurities in the oxidized acetonitrile waste stream can include organic and inorganic iodine, toluene, tetrahydrofuran, pyridine, propanenitrile, and water. In an exemplary embodiment, waste stream 102 includes the acetonitrile waste generated during the acetonitrile wash step after the oxidation step of oligonucleotide synthesis.

[0029] As will be appreciated, the type and amount of impurities found in the wash streams from each of the above steps can vary. The methods and systems described herein may be used to purify waste stream 102 from the wash step after any particular step, or the waste stream 102 collected and pooled (e.g., within collection tank 104) from the wash steps described in connection with deblocking, coupling, capping, and / or oxidation as described above may be used to purify it.

[0030] The identity and amount of impurities in the overhead fraction 108 may vary depending on the source of waste stream 102, and suitable adsorbents may be selected with respect to these compositions and the final grade (i.e., overall purity) of the desired acetonitrile / toluene end product.

[0031] For example, the overhead fraction obtained by distillation of the collected waste resulting from oligonucleotide synthesis can contain species such as water and toluene, as well as various other impurities, at concentrations up to 30 weight percent of the waste stream. Such impurities may be organic and / or inorganic iodine-containing compounds, sulfur-containing compounds such as thiols (e.g., ethylthiotetrazole (ETT), phenyl acetyl disulfide (PADS)), basic nitrogen-containing compounds (e.g., pyridine, lutidine, picoline, 1-methylimidazole), various alcohols (e.g., methanol), esters (e.g., acetic acid, dichloroacetic acid), nitriles (e.g., propanenitrile), and / or halogenated hydrocarbons (e.g., trichloromethane / dichloromethane).

[0032] Figure 1 shows an exemplary embodiment of a system 100 used to purify a waste stream 102 received from an oligonucleotide synthesis process to a purity acceptable for recycling. In this case, the purification is performed via a system 100 that includes a fractionation zone 103 including a collection tank 104 and a fractionation / distillation column 106, and an adsorbent zone 130 including a first absorption column 110, a second absorption column 116, and a third absorption column 122.

[0033] As shown in Figure 1, the fractionation zone 103 includes a collection tank 104 and a fractionation / distillation column 106. In this case, the waste stream 102 enters the fractionation zone 103 and is collected in the collection tank 104. The collection tank 104 may be used to pool the waste stream 102, such that various forms of the waste liquid stream 102 (e.g., as described above in connection with different compositions of the deblocking, coupling, capping, and / or oxidation steps) are retained within a common volume within the collection tank 104.

[0034] When the waste stream 102 enters the collection tank 104, it may come into contact with one or more reactive compounds (i.e., reducing compounds, scavengers, etc.), but some of the impurities within the waste stream 102 react with the waste stream 102 and then precipitate out of the waste stream 102.

[0035] For example, the first reactive compound 151 can be added to the collection tank 104 and reacted with a part of the components of the waste stream 102. In this case, the first reactive compound 151 may be an iodine-reactive compound that reacts (e.g., reduces) with one or more iodine-containing compounds in the waste liquid stream 102. For example, the iodine-reactive compound may be sodium thiosulfate. On the other hand, the thiosulfate anion reacts with the iodine of the iodine-containing compound, and thus reduces iodine as shown in the following representative reaction, and subsequently precipitates iodine from the waste liquid stream 102. I2 + 2Na2S2O3 → 2NaI + Na2S4O6

[0036] In the second embodiment, the second reactive compound 152 can be added to the collection tank 104 and reacted with a part of the components of the waste stream 102. In this case, the second reactive compound 152 may be a sulfur-reactive compound that reacts (e.g., reduces) with one or more sulfur-containing compounds (e.g., ETT, PADS) such as thiols in the waste stream 102. For example, the sulfur-reactive compound may be silver nitrate that reacts with the sulfur-containing compound as shown in the following representative reaction, and thus reduces the sulfur-containing compound, and subsequently precipitates the sulfur-containing compound from the waste stream 102. 2RS-SR + 2H2O + AgNO3 → 3RS-Ag + RSO2H + 2HNO3

[0037] In the third embodiment, the third reactive compound 153 can be added to the collection tank 104 and reacted with a part of the components of the waste stream 102. In this case, the third reactive compound 153 may be an acidic reactive compound that reacts (e.g., reduces) with one or more basic nitrogen-containing compounds (e.g., pyridine, lutidine, picoline, 1-methylimidazole) such as alkylamines in the waste stream 102. In this case, the acidic reactive compound may be a carboxylic acid (e.g., formic acid) that reacts (e.g., reduces) with the basic nitrogen-containing compound as shown in the following representative reaction, and subsequently precipitates the basic nitrogen-containing compound from the waste stream 102. R1,R2-N-H + HCOOH → R1-R2-N-COH + H2O

[0038] In some cases, two or more of the reactive compounds 151, 152, and / or 153 may be added to the waste stream 102 in the collection tank 104 simultaneously or successively, and each of the reactive compounds 151, 152, and / or 153 may react in combination with the compounds in the waste stream 102.

[0039] For example, the iodine-reactive compound 151 may be added simultaneously with, before, or after the sulfur-reactive compound 152, and both the iodine-reactive compound 151 and the sulfur-reactive compound 152 react with the iodine-containing compound and the sulfur-containing compound in the waste stream 102, respectively. In this case, the sulfur-reactive compound 152 can further react with the reduced iodine-containing compound to further precipitate iodine from the waste liquid stream 102. For example, when the iodine-reactive compound 151 is sodium thiosulfate and the sulfur-reactive compound 152 is silver nitrate, silver nitrate can further reduce the iodide produced by the reaction of sodium thiosulfate and volatile iodine, improving the recovery (e.g., precipitation) of iodine from the waste liquid stream 102. However, it is not necessary to add silver nitrate to reduce and precipitate iodide, and it is possible to sufficiently reduce and precipitate iodine without using silver nitrate.

[0040] In another embodiment, the iodine-reactive compound 151 can be added simultaneously with, before, or after the acidic-reactive compound 153, and both the iodine-reactive compound 151 and the acidic-reactive compound 153 react with the iodine-containing compound and the basic nitrogen-containing compound in the waste liquid stream 102, respectively. In this case, the chemical interaction between the iodine-reactive compound 151 and the acidic-reactive compound 153, as well as the intermediates formed when reducing the iodine-containing compound and the basic nitrogen-containing compound, can act together to reduce the sulfur-containing compound in the waste stream 102. For example, when the iodine-reactive compound 151 is sodium thiosulfate and the acidic-reactive compound 153 is formic acid, the interaction between sodium thiosulfate and formic acid with each of the iodine-containing compound and the basic nitrogen-containing compound can produce a matrix effect, but the intermediates formed by these reactions react with the sulfur-containing compound (e.g., thiol) in the waste stream 102 (e.g., reducing the sulfur-containing compound). In this regard, it may be possible to achieve the desired reduction of all three impurities (e.g., iodine-containing compound, sulfur-containing compound, and basic nitrogen-containing compound) using the iodine-reactive compound 151 and the acidic-reactive compound 153 (e.g., reducing the sulfur-containing compound without using the sulfur-reactive compound 152).

[0041] In yet another embodiment, all three of the reactive compounds 151, 152, and / or 153 can be added to the waste stream 102 in the collection tank 104, and each of the reactive compounds 151, 152, and / or 153 can be reacted simultaneously and / or in combination to reduce each of the iodine-containing compound, the sulfur-containing compound, and the basic nitrogen-containing compound in the waste stream 102. In this case, each of the iodine-containing compound, the sulfur-containing compound, and the basic nitrogen-containing compound can be precipitated from the waste stream 102 by reaction with the reactive compounds 151, 152, and / or 153, as well as the intermediates produced by such reactions.

[0042] FIG. 1 shows each of the reactive compounds 151, 152, and / or 153 being added separately (i.e., by different supply lines) to the collection tank 104, but it is possible to add two or more reactive compounds in combination. For example, reactive compounds 151 and 152 can be added to the collection tank 104 by the same supply line rather than by separate supply lines as shown in FIG. 1. The foregoing also applies to any combination of reactive compounds 151, 152, and 153, and each of the components may be added individually or in any combination. For example, all three reactive compounds 151, 152, and 153 may be added to the collection tank 104 by the same supply line. Alternatively, in some embodiments, any one of the reactive compounds 151, 152, and / or 153 may be added to the feed stream 105 rather than to the collection tank 104. In this case, it may be possible to remove the collection tank 104 from the system 100 and introduce each of the reactive compounds 151, 152, and / or 153 into the waste stream 102 (e.g., when the collection tank 104 is removed from the fractionation zone 103, each of the reactive compounds 151, 152, and / or 153 is directly connected and introduced into the waste stream 102, and when a reactive compound is added to the waste stream 102, the waste stream 102 migrates to the feed stream 105).

[0043] The waste stream 102 and each of the reactive compounds 151, 152, and / or 153 are collected in the collection tank 104 and supplied as a feed stream 105 to the fractionation / distillation column 106. The fractionation / distillation column 106 is used to separate / fractionate the components of the feed stream 105 into an overhead fraction 108 and a bottom fraction 107.

[0044] The overhead fraction 108 contains at least a majority of the acetonitrile originally present in the waste stream 102. For example, the overhead fraction 108 can contain 75% or more, such as 85% or more, such as 95% or more of the acetonitrile originally contained in the waste stream 102. That is, the overhead fraction 108 can contain from about 75% to about 100%, such as from about 85% to about 100%, such as from about 95% to about 100% of the acetonitrile originally contained in the waste stream 102.

[0045] In addition to acetonitrile, the overhead fraction 108 can also contain water, toluene, and other low-boiling impurities. For example, if about 1 weight percent (e.g., 10,000 ppm) of water is present in the waste stream 102, the overhead fraction 108 can contain about 10,000 ppm of water, but more preferably 100 ppm, more preferably 50 ppm, more preferably 30 ppm of water. In this case, the water can form an azeotrope with one of the more components of the overhead fraction 108, such as an azeotrope formed with acetonitrile. In this case, as further described herein, the water can be removed from the overhead fraction 108 by various methods including contact with a desiccant.

[0046] An "azeotrope" (or "azeotropic") composition is a specific combination of two or more components. An azeotrope can be either homogeneous (having one liquid phase) or heterogeneous (having two liquid phases). Azeotropic compositions can be characterized in various ways. For example, at a given pressure, an azeotropic composition boils at a constant characteristic temperature higher than the component with the higher boiling point (maximum-boiling azeotrope) or lower than the component with the lower boiling point (minimum-boiling azeotrope). However, in the case of a heterogeneous azeotrope, the boiling point of the azeotrope is always less than the boiling point of the component with the lower boiling point. In the case of a heterogeneous azeotrope, at this characteristic temperature, the composition of each of the two liquid phases and the gas phase remains constant during boiling. Azeotropic compositions do not fractionate upon boiling or evaporation. Therefore, the components of an azeotropic composition cannot be separated in a phase change.

[0047] An azeotropic composition is also characterized in that at the characteristic azeotropic temperature, the bubble point pressure of the liquid phase is the same as the dew point pressure of the gas phase.

[0048] The behavior of an azeotropic composition is in contrast to that of a non-azeotropic composition in which the liquid composition changes significantly during boiling or evaporation.

[0049] For the purposes of the present disclosure, an azeotropic composition is characterized as a composition that boils at a constant characteristic temperature lower than the boiling points of two or more components (minimum-boiling azeotrope), thereby having the same composition in both the gas phase and the liquid phase.

[0050] However, those skilled in the art will understand that at different pressures, both the composition and the boiling point of an azeotropic composition will change to some extent. Therefore, depending on the temperature and / or pressure, an azeotropic composition can have a variable composition. Thus, those skilled in the art will understand that an azeotropic composition can be defined using a composition range rather than a fixed composition. Furthermore, an azeotropic mixture can also be defined in terms of the exact weight percentages of the components of a composition characterized by a fixed boiling point at a specific pressure.

[0051] Azeotropic or azeotrope-like compositions can be identified using a number of different methods.

[0052] For the purposes of the present disclosure, an azeotropic or azeotrope-like composition is identified experimentally using an ebulliometer (Walas, Phase Equilibria in Chemical Engineering, Butterworth-Heinemann, 1985, 533-544). An ebulliometer is designed to provide a very accurate measurement of the boiling point of a liquid by measuring the vapor-liquid equilibrium temperature.

[0053] The individual boiling points of each of the components are measured at a constant pressure. As will be understood by those skilled in the art, for a binary azeotropic or azeotrope-like composition, the boiling point of one of the components of the composition is measured first. Then, the second component of the composition is added in various amounts, and the boiling point of each of the resulting compositions is measured using an ebulliometer at the said constant pressure. In the case of a ternary azeotropic mixture, the initial composition is composed of a binary blend, and the third component is added in various amounts. The boiling point of each of the resulting ternary compositions is measured using an ebulliometer at the said constant pressure.

[0054] The measured boiling points are plotted against the composition of the composition being tested, for example, in the case of a binary azeotropic mixture, against the amount of the second component added to the composition (expressed in either weight % or mole %). The presence of an azeotropic composition can be identified by observing a maximum boiling temperature higher than the boiling point of any of the individual components or a minimum boiling temperature lower than that.

[0055] As will be understood by those skilled in the art, the identification of an azeotropic or azeotrope-like composition is carried out by comparing the change in the boiling point of the composition when the second component is added to the first component with the boiling point of the first component. Thus, there is no need to calibrate the system to the reported boiling point of a particular component in order to measure the change in boiling point.

[0056] When the top fraction 108 contains toluene, the amount of toluene present in the top fraction 108 can be based on either the azeotropic interaction between acetonitrile and toluene or the composition of toluene present in the feed stream 105. For example, in the case of the azeotropic interaction between acetonitrile and toluene, the relative weight percentages of acetonitrile and toluene in the top fraction 108 can depend on the operating temperature and pressure of the fractionation / distillation column 106. For example, when the fractionation / distillation column 106 is operating at a temperature of 80°C to 86°C and a pressure of 1 ATM, the azeotropic composition contains approximately 76 weight percent acetonitrile and 24 weight percent toluene. In a second embodiment, the relative weight percentage of toluene in the feed stream 105 may be less than the relative weight percentage of toluene in the acetonitrile and toluene azeotropic mixture (e.g., the relative weight percentage of toluene in the feed stream 105 is less than 24 weight percent). In this case, the top fraction 108 may contain substantially the same relative weight percentages of acetonitrile and toluene as the feed stream 105. For example, when the feed stream 105 contains approximately 80 relative weight percent acetonitrile and 20 relative weight percent toluene, the top fraction 108 also contains 80 relative weight percent acetonitrile and 20 relative weight percent toluene (e.g., the relative weight percentage of toluene does not increase to the azeotropic composition of 24 relative weight percent toluene). Thus, the relative weight percentage of toluene in the top fraction 108 can be at most based on the azeotropic interaction between acetonitrile and toluene at the operating temperature and pressure of the fractionation / distillation column 106, or may be lower than the azeotropic composition when the feed stream 105 contains a lower relative weight percentage of toluene than the azeotropic composition. In other words, the relative weight percentage of acetonitrile to toluene may be at least based on the azeotropic composition of acetonitrile and toluene, or may be a higher relative weight percentage than the azeotropic composition when the feed stream 105 contains less toluene than the azeotropic composition.

[0057] However, while it is also possible to operate the fractionation / distillation column 106 at various temperatures and pressures to achieve the desired recovery rate of acetonitrile, the relative composition of acetonitrile and toluene can vary. Further, while a single distillation column 106 is shown, in the case of pressure swing distillation, two or more columns 106 may be used (e.g., two columns 106 in series operating at different temperatures and pressures). Thus, in this regard, while described with respect to an azeotropic composition of 76 weight percent acetonitrile and 24 weight percent toluene, other azeotropic compositions of acetonitrile and toluene may also be possible.

[0058] In addition to acetonitrile and toluene, the overhead fraction 108 may contain one or more impurities such as iodine, sulfur-containing compounds (e.g., ethylthiotetrazole (ETT), BMT, phenylacetyl disulfide (PADS)), basic nitrogen-containing compounds (e.g., pyridine, lutidine, picoline, N-methylimidazole), and esters (e.g., acetic acid, dichloroacetic acid), each of which may be incorporated into the overhead fraction 108 due to the boiling point of the compound (e.g., a boiling point lower than the operating temperature of the fractionation / distillation column 106) and / or due to chemical interactions of the compounds within the waste stream 102 (i.e., as in the case of iodine and water). However, as described above, one or more reactive compounds 151, 152, and / or 153 may be added to the waste stream 102 such that when the feed stream 105 containing such reactive compounds is fractionated, the overhead fraction 108 can contain substantially fewer of these impurities.

[0059] For example, reactive compound 151 is added to waste stream 102 in collection tank 104. When the first reactive compound 151 is an iodine-reactive compound (e.g., sodium thiosulfate), the reaction (e.g., reduction) of the iodine-containing compounds in waste stream 102 via sodium thiosulfate substantially reduces the amount of iodine present in the overhead fraction 108. In this case, rather than iodine remaining in a volatile state (e.g., the iodine that would normally be present in overhead fraction 108 if distilled), the iodine precipitates from feed stream 105 within distillation column 106, and the overhead fraction 108 contains a relatively small amount of iodine. For example, when 5 ppm of iodine is present in feed stream 105, the first reactive compound 151 can reduce the amount of iodine in overhead fraction 108 to less than 2 ppm, in some cases less than 1 ppm, more preferably less than 0.5 ppm, and in some cases to trace amounts (e.g., substantially iodine-free). In these examples, the amount of iodine present in overhead fraction 108 is substantially less than if the first reactive compound 151 had not been added to waste stream 102 prior to fractionation / distillation.

[0060] In the second embodiment, the second reactive compound 152 is a sulfur-reactive compound (e.g., silver nitrate) that is added to the waste stream 102 in the collection tank 104. In this embodiment, the reaction (e.g., reduction) of the sulfur-containing compound (e.g., thiol) in the waste stream 102 via sodium thiosulfate substantially reduces the amount of thiol present in the overhead fraction 108. In this case, rather than the thiol remaining in the volatile state (e.g., the thiol that would normally be present in the overhead fraction 108 if distilled), the thiol precipitates from the feed stream 105 within the distillation column 106, and the overhead fraction 108 contains a relatively small amount of sulfur-containing components. For example, if 200 ppm of a thiol compound is present in the waste stream 102, the second reactive compound 152 can reduce the amount of the thiol compound in the overhead fraction to less than 10 ppm, in some cases less than 5 ppm, more preferably less than 1 ppm, in some cases less than 0.5 ppm, and in yet other cases to trace amounts (e.g., substantially thiol-free). In these embodiments, the amount of thiol present in the overhead fraction 108 is substantially less than if the second reactive compound 152 had not been added to the waste stream 102 prior to fractional distillation.

[0061] In the third embodiment, the third reactive compound 153 is an acidic reactive compound (e.g., formic acid) that is added to the waste stream 102 in the collection tank 104. In this embodiment, the reaction (e.g., reduction) of the basic nitrogen-containing compound (e.g., alkylamine) in the waste stream 102 via formic acid substantially reduces the amount of alkylamine present in the overhead fraction 108. In this case, rather than the alkylamine remaining in the volatile state (e.g., if distilled, it would normally be present in the overhead fraction 108), the alkylamine precipitates from the feed stream 105 in the distillation column 106, and the overhead fraction 108 contains a relatively small amount of basic nitrogen-containing compounds. For example, when there is up to 5 weight percent (e.g., 50,000 ppm) of an alkylamine compound in the waste stream 102, the third reactive compound 152 can reduce the amount of basic nitrogen-containing compounds in the overhead fraction to less than 500 ppm, in some cases less than 200 ppm, more preferably less than 100 ppm. In other embodiments, the concentration of the alkylamine compound can be substantially lower in the waste stream 102, e.g., about 100 ppm. In this case, the third reactive compound 152 can reduce the amount of the alkylamine compound in the overhead fraction to less than 10 ppm, in some cases less than 5 ppm, more preferably less than 1 ppm, in some cases less than 0.5 ppm, and in further additional cases to trace amounts (e.g., substantially free of alkylamine). In these embodiments, the amount of alkylamine compound present in the overhead fraction 108 is substantially less than if the third reactive compound 153 had not been added to the waste stream 102 prior to fractionation / distillation.

[0062] In each of the above cases, the bottom fraction 107 contains either a low-boiling compound (e.g., water) and / or a precipitated compound such as an iodine, sulfur-containing compound, and / or a basic nitrogen-containing compound. In this case, the bottom fraction 107 can be recovered and further processed to remove some of the compounds contained in the bottom fraction 107. For example, when silver nitrate is used as a reducing agent for sulfur-containing compounds and / or iodine, the silver can be recovered via further processing of the bottom fraction 107.

[0063] In an exemplary embodiment, the condensed overhead fraction 108 exits the fractionation zone 103 and is sent to the adsorbent zone 130. In the adsorbent zone 130, the condensed overhead fraction 108 enters a first adsorption tower 110 configured to contain a first adsorbent 112. The first overhead fraction 108 is purified by contacting it with the first adsorbent 112 to produce a purified acetonitrile and toluene stream 114.

[0064] As described above, the amount of iodine-containing species present in the overhead fraction 108 is substantially reduced by the first reactive compound 151, but some iodine-containing components may still be present in the overhead fraction 108. In this case, the overhead fraction 108 can be contacted with an adsorbent 112 selected to reduce the amount of these organic and / or inorganic iodine-containing species, resulting in a purified acetonitrile and toluene stream 114. Suitable adsorbents include silver (Ag)-exchanged zeolites. In some embodiments, the Ag-exchanged zeolite is an Ag-exchanged faujasite zeolite, particularly a faujasite zeolite having a silicon:aluminum (Si:Al) molar ratio of at least about 1.2, for example, a faujasite zeolite having a Si:Al molar ratio of at least about 2.0. In some embodiments utilizing an Ag-exchanged faujasite zeolite, Ag is present at a minimum of about 3 wt%, for example, at least about 15 wt%. In some specific embodiments, Ag is partially ion-exchanged and partially precipitated on the zeolite, and the proportion of Ag that is ion-exchanged depends on the Si:Al molar ratio and wt% Ag. However, typically at least about 10 wt% of Ag is ion-exchanged on the zeolite. This does not mean that the iodine-reducing adsorbent is limited to faujasite zeolites. Rather, any other suitable zeolite can be used.

[0065] In embodiments where one or more Ag-exchanged faujasite zeolites are utilized to reduce organic and / or inorganic iodine-containing species and sulfur-containing species present in the condenser overhead fraction, the adsorbent can be used by contacting the condenser overhead fraction 108 with the adsorbent at an adsorbent:condenser overhead fraction 108 weight ratio of at least about 1:100,000. In some embodiments, the weight ratio of adsorbent:condenser overhead fraction 108 is at least about 1:10,000, 1:1000, or 1:100. The contacting can be carried out by a batch process or a continuous process using suitable equipment, temperature, pressure, and flow rate.

[0066] In some embodiments, the methods provided herein are used to purify waste stream 102 containing organic and / or inorganic iodine-containing compounds, and the resulting purified acetonitrile and toluene streams 114 contain iodine at about 25 ppm or less, such as about 10 ppm or less, such as about 5 ppm or less, such as about 1 ppm or less, such as about 0.5 ppm or less, and in some cases are substantially iodine-free. In some aspects, in some cases, the resulting purified acetonitrile and toluene streams 114 contain iodine from about 0.1 ppm to about 25 ppm, such as from about 0.1 ppm to about 10 ppm, such as from about 0.1 ppm to about 5 ppm, such as from about 0.1 ppm to about 1 ppm, such as from about 0.1 ppm to about 0.5 ppm.

[0067] In some embodiments, the methods provided herein are used to purify waste stream 102 that contains organic and / or inorganic sulfur-containing compounds as impurities. As described above, the amount of sulfur-containing compounds present in the overhead stream 108 is substantially reduced by the second reactive compound 152, but some sulfur-containing components may still be present in the overhead stream 108. In this case, the overhead stream 108 can be contacted with an adsorbent 112 selected to reduce the amount of these sulfur-containing compounds to obtain a purified acetonitrile and toluene stream 114. In some embodiments, the resulting purified acetonitrile and toluene stream 114 contains sulfur at about 10 ppm or less, such as about 6 ppm or less, such as about 5 ppm or less, such as about 4 ppm or less. In some embodiments, the resulting purified acetonitrile and toluene stream 114 contains sulfur from about 0.5 ppm to about 10 ppm, such as from about 0.5 ppm to about 6 ppm, such as from about 0.5 ppm to about 5 ppm, such as from about 0.5 ppm to about 4 ppm.

[0068] In some embodiments, the methods provided herein are used to purify waste stream 102 that contains basic nitrogen-containing compounds such as pyridine, picoline, lutidine, picoline, N-methylimidazole. As described above, the amount of basic nitrogen-containing compounds present in the overhead stream 108 is substantially reduced by the third reactive compound 152, but some basic nitrogen-containing components may still be present in the overhead stream 108. In this case, the purified acetonitrile and toluene stream 114 is passed through a second adsorption tower 116 configured to contain a basic N-reducing adsorbent 118. Similarly in this case, the basic N-reducing adsorbent may be the only adsorbent used. Alternatively, the basic N-reducing adsorbent may be one of a plurality of adsorbents used. When a plurality of adsorbents are used, the adsorbents may be used sequentially. In such embodiments, the basic N-reducing adsorbent can be used at any position in the sequence.

[0069] In a particular exemplary embodiment of FIG. 1, a purified acetonitrile and toluene stream 114 is contacted with a basic N-reducing adsorbent 118 to obtain a purified acetonitrile and toluene stream 120 that contains a reduced amount of basic N-containing compound impurities compared to the waste stream 102. The purified acetonitrile and toluene stream 120 may be collected for use or, if desired, may be subjected to further purification.

[0070] In some embodiments, the basic N adsorbent can include an acidic cation exchange resin. In such embodiments, the relative ratio of adsorbent used per volume of the acetonitrile and toluene stream to be purified (e.g., the condensed overhead fraction 108 or the purified acetonitrile and toluene stream 114) can vary depending on the particular adsorbent, the amount of basic N-containing impurities contained in the acetonitrile and toluene stream, and the desired level of basic N-containing impurities in the resulting purified acetonitrile and toluene stream. In some embodiments, an acidic cation exchange resin such as Amberlyst™-15 is used at a ratio of at least about 1 g of adsorbent per 10 L of acetonitrile and toluene stream, for example, at a ratio of at least about 1 g of adsorbent per 0.1 L of acetonitrile and toluene stream, to reduce basic N-containing impurities in the acetonitrile and toluene stream.

[0071] Accordingly, in some embodiments, the methods provided herein are used to purify a waste stream 102 that contains one or more basic N-containing compounds as impurities. In some embodiments, the purified acetonitrile and toluene streams are produced by removing such impurities from the acetonitrile and toluene streams such that the resulting purified acetonitrile and toluene streams contain a basic N-containing compound of about 100 ppm or less, such as about 50 ppm or less, such as about 25 ppm or less, such as about 10 ppm or less. In some embodiments, the purified acetonitrile and toluene stream 120 is produced by removing such impurities from the acetonitrile and toluene streams such that the resulting purified acetonitrile and toluene streams contain a basic N-containing compound of about 1 ppm to about 100 ppm, such as about 1 ppm to about 50 ppm, such as about 1 ppm to about 25 ppm, such as about 1 ppm to about 10 ppm.

[0072] In some embodiments, the acid exchange resin may also reduce the amount of cations present in the purified acetonitrile and toluene stream 114. For example, impurities such as cationic Fe, Mg, Cr, Ni, Ag, and I2 can be reduced by contacting with a cation exchange resin. In some embodiments, the reduction of cations occurs simultaneously with the basic N reduction by contacting with a basic N reduction adsorbent containing the acidic cation exchange resin described above. In some embodiments, the purified acetonitrile and toluene stream 120 contains, as impurities, less than about 5 ppm, for example, about 0.1 ppm to about 5 ppm of cationic Fe. In some embodiments, the purified acetonitrile and toluene stream 120 contains, as impurities, less than about 5 ppm, for example, about 0.1 ppm to about 5 ppm of cationic Mg. In some embodiments, the purified acetonitrile and toluene stream 120 contains, as impurities, less than about 5 ppm, for example, about 0.1 ppm to about 5 ppm of cationic Cr. In some embodiments, the purified acetonitrile and toluene stream 120 contains, as impurities, less than about 5 ppm, for example, about 0.1 ppm to about 5 ppm of cationic Ni. In some embodiments, the purified acetonitrile and toluene stream 120 contains, as impurities, less than about 5 ppm, for example, about 0.1 ppm to about 5 ppm of cationic Ag. In some aspects, the purified acetonitrile and toluene stream 120 contains, as impurities, less than about 5 ppm, for example, about 0.1 ppm to about 5 ppm of iodine / iodide.

[0073] It may be desirable to reduce the amount of water present in the condensed purified acetonitrile and toluene stream 120. In some embodiments, including the example shown in FIG. 1, the purified acetonitrile and toluene stream 120 proceeds to a third adsorption tower 122. In these embodiments, the third adsorption tower 122 is configured to contain a water-reducing adsorbent (i.e., a desiccant) 124. The purified acetonitrile and toluene stream 120 contacts the water-reducing adsorbent 124 to produce a mixture 126 of purified acetonitrile and toluene, which exits the adsorption zone 130.

[0074] In some embodiments, potassium (K)-exchanged Linde Type A (LTA) type molecular sieves bound with an inorganic binder can be used as the water removal adsorbent 124. Surprisingly, it has been found that porous desiccants exhibit improved performance (i.e., higher water adsorption capacity) with respect to reducing water in the acetonitrile waste stream when they are treated to partially close their pore structures. Thus, in some embodiments, one or more porous desiccants (including one or more K-exchanged LTA type molecular sieves bound with an inorganic binder) may be treated by any suitable process known in the art to partially close the pores of the desiccant prior to use. However, care must be taken not to overly close the pores and limit the water adsorption capacity. The degree of pore closure can be measured by determining the smallest molecule (also known as a plug-gauge molecule) that does not adsorb more than 1 weight % of the molecules when exposed to a partial pressure of 1 atmosphere. In some embodiments, the LTA type adsorbent is treated in a flow such that the pore structure of the desiccant is partially closed to the point where the desiccant has a limited ability to adsorb ethylene, difluoromethane, or both.

[0075] In embodiments utilizing one or more LTA type adsorbents to reduce water in the purified acetonitrile and toluene stream 120 from the waste stream 102, the adsorbent may be used at a weight ratio of at least 5 - 10 mg of desiccant per gram of acetonitrile to be purified. In some embodiments, one or more LTA type adsorbents having a minimum water adsorption capacity of 10 weight % are used. In embodiments utilizing one or more LTA type adsorbents to reduce water in the purified acetonitrile and toluene stream 120, the adsorbent may be used at a weight ratio of at least 5 - 10 mg of desiccant per gram of acetonitrile to be purified. In some embodiments, one or more LTA type adsorbents having a minimum water adsorption capacity of 10 weight % are used. The contact can be carried out by a batch process or a continuous process using appropriate equipment, temperature, pressure, and flow rate.

[0076] In some embodiments, reducing the amount of water present in the condensed purified acetonitrile and toluene stream 120 is effected by a liquid phase process. In these embodiments, the purified acetonitrile and toluene stream 120 is in the liquid phase when contacting the water-reducing adsorbent 124.

[0077]

[0076] Alternatively or additionally, reducing the amount of water present in the purified acetonitrile may be effected by a gas phase process such as pressure-swing adsorption (PSA). In an exemplary embodiment, the purified acetonitrile and toluene stream exiting the fractionation column can be treated to reduce or eliminate condensation of the overhead fraction. Rather, the purified acetonitrile and toluene stream exiting the fractionation column is further heated, for example, superheated at a temperature about 25 °C higher than the fractionation temperature, and the heated vapor is fed to a column containing the reducing adsorbent. In some embodiments, the water-reducing adsorbent is a potassium (K)-exchanged Linde type A (LTA)-type 20 molecular sieve bound with an inorganic binder as described above, which may or may not have partially closed pores. After contact with the water-reducing adsorbent, the vapor is sent to a heat exchanger where it is condensed into the liquid phase of purified acetonitrile and toluene. This liquid phase mixture of purified acetonitrile and toluene may be subjected to further purification, including, if desired, contact with other adsorbents described herein. One of ordinary skill in the art will understand that in the methods described herein, after the adsorbent in the first column is saturated, the mixed vapor of acetonitrile and toluene to be dried is sent to a second column and a plurality of columns containing the water-reducing adsorbent can be utilized in parallel such that the adsorbent in the first column is regenerated. In some embodiments, the regeneration conditions include volatilizing water from the adsorbent by reducing the pressure and condensing the released material as a liquid waste stream.

[0078] In some embodiments, at least a portion of the liquid waste stream may be redirected to the fractionation column for further processing. In some embodiments, the average delta loading of water (i.e., the change in the average water content of the adsorbent from the start to the end of the adsorption process) is about 1 to about 3 kg of water per 100 kg of adsorbent. In these embodiments, the adsorption conditions such as pressure, feed rate, and flow rate may be such that the adsorption process may take about 5 to about 10 minutes.

[0079] Accordingly, in some embodiments, the methods provided herein are used to purify waste stream 102 that contains water as an impurity. In some related embodiments, waste stream 102 is fractionated and the overhead fraction is contacted with a desiccant having a partially closed pore structure. In some embodiments, the purified mixture of acetonitrile and toluene 126, purified by contact with the desiccant, contains water at about 2000 ppm or less, such as about 1000 ppm or less, such as about 500 ppm or less, such as about 150 ppm or less, 100 ppm or less, such as about 50 ppm or less, such as about 25 ppm or less.

[0080] The various embodiments described above describe a waste stream that passes sequentially from a fractionation column, then through an iodine reduction adsorbent, a basic N-containing compound reduction adsorbent, and a water reduction adsorbent, but it will be understood that the scope of this specification is not so limited. Rather, it will be understood that only one or two of the adsorbents may be used in addition to the fractionation column and / or the adsorbents may be used in a different order without limitation. For example, after fractionation and condensation, the condensed overhead fraction 108 can be contacted with a basic N-containing compound reduction adsorbent and the resulting acetonitrile and toluene stream can be contacted with a water reduction adsorbent, or the condensed overhead fraction 108 can be contacted with a water reduction adsorbent and the resulting stream can be contacted with an iodine reduction adsorbent.

[0081] In some embodiments, the methods provided herein are used to purify waste stream 102 from oligonucleotide synthesis to produce a purified mixture 126 of acetonitrile and toluene suitable for reuse in various industrial or manufacturing processes. In some embodiments, the purified mixture 126 of acetonitrile and toluene is suitable for reuse as an inter-step wash solution in oligonucleotide synthesis performed using inter-step acetonitrile washes. In some embodiments, the purified mixture 126 of acetonitrile and toluene has a purity of greater than about 85%, for example, greater than or equal to about 95%, as determined by gas chromatography.

[0082] As described above, in the case of oligonucleotide synthesis processes, it has been found that using a mixed solution of toluene and acetonitrile as a wash solvent is acceptable (e.g., producing oligonucleotides of similar purity compared to a pure acetonitrile wash solvent), and in some cases, surprisingly, it has been found that the purity of the produced oligonucleotides is increased compared to a pure acetonitrile wash solvent.

[0083] For example, in a first series of cases, various wash solvents containing different acetonitrile and toluene purities were tested, and the resulting oligonucleotide purity was obtained based on a first type of support resin and a first type of oligonucleotide test sequence (e.g., test - 20; 20 - mer, etc.). Surprisingly, the mixed solution of toluene and acetonitrile as a wash solvent was found to increase the purity of the first oligonucleotide purity compared to a pure acetonitrile wash solvent.

[0084] For example, when pure acetonitrile is used as a washing solvent in an oligonucleotide synthesis process that utilizes a first type of support resin and a first oligonucleotide test sequence (Test-20), the purity of the resulting synthetic oligonucleotide, as determined by liquid chromatography, can be in the range of 70% to 73%, for example, in the range of 70.2% to 72.2%.

[0085] However, when a substantially pure mixture of 95 wt% acetonitrile and 5 wt% toluene is used as a washing solvent in an oligonucleotide synthesis process that uses a first type of support resin and the Test-20 oligonucleotide sequence, surprisingly, the resulting synthetic oligonucleotide was found to be slightly purer than the synthetic oligonucleotide produced using pure acetonitrile as the washing solvent. For example, the oligonucleotide purity obtained from an oligonucleotide synthesis process using a 95 wt% acetonitrile / 5 wt% toluene blend can have a purity in the range of 70% to 74% purity, more particularly in the range of 70.7% to 73.8% purity. Thus, it can be advantageous to utilize a blend of 95 wt% acetonitrile / 5 wt% toluene as a washing solvent in an oligonucleotide synthesis process as compared to pure acetonitrile.

[0086] In another embodiment, a substantially pure mixture of 85 wt% acetonitrile and 15 wt% toluene is used as a wash solvent in an oligonucleotide synthesis process using the same first support resin and the test - 20 oligonucleotide sequence. In this case, surprisingly, it was found that the resulting synthetic oligonucleotides are slightly purer than the synthetic oligonucleotides produced using pure acetonitrile as the wash solvent. For example, the oligonucleotide purity obtained from an oligonucleotide synthesis process using an 85 wt% acetonitrile / 15 wt% toluene blend can have a purity in the range of 72% - 76% purity, more specifically in the range of 72.5% - 76.4% purity. Thus, compared to pure acetonitrile, it can be advantageous to utilize an 85 wt% acetonitrile / 15 wt% toluene blend as a wash solvent in an oligonucleotide synthesis process.

[0087] As yet another example, when a substantially pure mixture of an azeotropic composition of 76 wt% acetonitrile and 24 wt% toluene is used as a wash solvent in an oligonucleotide synthesis process using the first solid support resin and the test - 20 oligonucleotide sequence, surprisingly, it was found that the resulting synthetic oligonucleotides are slightly purer than the synthetic oligonucleotides produced using pure acetonitrile as the wash solvent. For example, the oligonucleotide purity obtained from an oligonucleotide synthesis process using a 76 wt% acetonitrile / 24 wt% toluene blend can have a purity in the range of 71% - 73% purity, more specifically in the range of 71.3% - 72.2% purity. Thus, compared to pure acetonitrile, it can be advantageous to utilize a 76 wt% acetonitrile / 24 wt% toluene blend as a wash solvent in an oligonucleotide synthesis process.

[0088] In a second series of cases, various washing solvents containing different acetonitrile and toluene purities were tested, and the resulting oligonucleotide purity was obtained based on a second type of support resin and a second type of oligonucleotide test sequence (e.g., test - 18; 18 - mer, etc.). Surprisingly, a mixed solution of toluene and acetonitrile as a washing solvent was also found to enhance the purity of the second oligonucleotide purity compared to a pure acetonitrile washing solvent.

[0089] For example, when a second support resin and a second oligonucleotide test sequence (test - 18) are used, and pure acetonitrile is used as the washing solvent in the oligonucleotide synthesis process, the purity of the resulting synthetic oligonucleotide determined by liquid chromatography can be in the range of 91% - 93%, for example, in the range of 91.2% - 93.1%.

[0090] However, using the same second support resin (second resin) and test oligonucleotide test sequence (test 18), when a substantially pure mixture of 90 wt% acetonitrile and 10 wt% toluene is used as the washing solvent in the oligonucleotide synthesis process, surprisingly, the resulting synthetic oligonucleotide was found to be slightly purer than the synthetic oligonucleotide produced using pure acetonitrile as the washing solvent. For example, the oligonucleotide purity obtained from this oligonucleotide synthesis process using a 90 wt% acetonitrile / 10 wt% toluene blend can have a purity in the range of 92% - 100% purity, more specifically, in the range of 92.6% - 99.8% purity. Therefore, it can be advantageous to utilize a blend of 90 wt% acetonitrile / 10 wt% toluene as the washing solvent in the oligonucleotide synthesis process compared to pure acetonitrile.

[0091] In a second embodiment, using this same second support resin and second test array (Test 18), a substantially pure mixture of 85 wt% acetonitrile and 15 wt% toluene is used as a wash solvent in the oligonucleotide synthesis process. In this case, surprisingly, it was found that the resulting synthetic oligonucleotides are slightly purer than the synthetic oligonucleotides produced using pure acetonitrile as the wash solvent. For example, the oligonucleotide purity obtained from an oligonucleotide synthesis process using an 85 wt% acetonitrile / 15 wt% toluene blend can have a purity in the range of 92% to 96% purity, more particularly in the range of 92.6% to 95.7% purity. Thus, it can be advantageous to utilize an 85 wt% acetonitrile / 15 wt% toluene blend as a wash solvent in the oligonucleotide synthesis process as compared to pure acetonitrile.

[0092] In another embodiment, using this same second support resin and second oligonucleotide test array (Test 18), a substantially pure mixture of 80 wt% acetonitrile and 20 wt% toluene is used as a wash solvent in the oligonucleotide synthesis process. In this case, surprisingly, it was found that the resulting synthetic oligonucleotides are slightly purer than the synthetic oligonucleotides produced using pure acetonitrile as the wash solvent. For example, the oligonucleotide purity obtained from an oligonucleotide synthesis process using an 80 wt% acetonitrile / 20 wt% toluene blend can have a purity in the range of 93% to 97% purity, more particularly in the range of 93.9% to 96.9% purity. Thus, it can be advantageous to utilize an 80 wt% acetonitrile / 20 wt% toluene blend as a wash solvent in the oligonucleotide synthesis process as compared to pure acetonitrile.

[0093] In yet another embodiment, when a substantially pure mixture of an azeotropic composition of 76 wt% acetonitrile and 24 wt% toluene is used as a washing solvent in an oligonucleotide synthesis process using a second support resin and a second oligonucleotide test sequence (T-18), surprisingly, the resulting synthetic oligonucleotide was found to have a higher purity than the synthetic oligonucleotide produced using pure acetonitrile as the washing solvent. For example, the purity of the oligonucleotide obtained from an oligonucleotide synthesis process using a 76 wt% acetonitrile / 24 wt% toluene blend with the second resin and T18 sequence can range from 95% to 97%, and more specifically, have a purity in the range of 95.3% to 96.8%. Thus, it can be advantageous to utilize a blend of 76 wt% acetonitrile / 24 wt% toluene as a washing solvent in an oligonucleotide synthesis process compared to pure acetonitrile.

[0094] Thus, as demonstrated in the foregoing examples, a mixed washing solvent composition of 5 to 24 weight percent toluene and the corresponding 95 to 76 weight percent acetonitrile was found to result in at least the same purity level of the resulting oligonucleotide produced by an oligonucleotide synthesis process, if not a higher purity oligonucleotide than a pure acetonitrile washing solvent. Stated another way, the foregoing examples demonstrate that in an oligonucleotide synthesis process, the use of a mixed washing solvent containing at least about 5 weight percent toluene and at most about 24 weight percent toluene, and at least about 76 weight percent acetonitrile and at most about 95 weight percent acetonitrile (or any range defined by any two of the foregoing values), results in at least an oligonucleotide purity as pure as that of a pure acetonitrile washing solvent, if not purer.

[0095] As described above, the generated purified acetonitrile and toluene mixture 126 may be purified to a substantially pure (e.g., 80% or more, more preferably 95% or more pure) mixture of acetonitrile and toluene, which may be in the state of an azeotropic composition of acetonitrile and toluene, or may be in the state of an acetonitrile-to-toluene ratio containing more acetonitrile than the azeotropic mixture. For example, the generated purified acetonitrile and toluene mixture 126 may be purified to a substantially pure azeotropic composition of about 76 wt% acetonitrile and 24 wt% toluene. In this case, by recycling this mixed composition as a washing solvent in the oligonucleotide synthesis process, a similar oligonucleotide purity as that of pure acetonitrile can be achieved (e.g., recycling of the azeotropic composition is at least acceptable, even if it is considered less preferable compared to pure acetonitrile). However, as described above, in order to target a washing solvent with a more advantageous composition, it may be advantageous to increase the relative weight percentage of acetonitrile in the mixture. For example, in order to achieve a more advantageous blend of acetonitrile and toluene as described above, it may be possible to increase the wt% of acetonitrile from 76 wt% of the azeotropic composition to 80 wt%, more preferably to 85 wt%, in some cases to 90 wt%, and in some examples to 95 wt%.

[0096] An increase in the total weight percentage of acetonitrile can be achieved by adding acetonitrile to the produced purified mixture of acetonitrile and toluene 126 at some point during the process, before using the recycled and purified mixture of acetonitrile and toluene 126 as a washing solvent in the oligonucleotide synthesis process. For example, make-up acetonitrile 154 may be added to the purified mixture of acetonitrile and toluene 126 (e.g., in-line or by any other suitable mixing device) before using the purified mixture of acetonitrile and toluene 126 as a washing solvent blend in the oligonucleotide synthesis process. In this case, the amount of make-up acetonitrile 154 added to the purified mixture of acetonitrile and toluene 126 can be based on the amount of make-up acetonitrile 154 required to achieve the target composition of the blend of acetonitrile and toluene. For example, the amount of make-up acetonitrile 154 added to the purified mixture of acetonitrile and toluene 126 may be the amount required to increase the weight percentage of acetonitrile from the azeotropic composition (e.g., 76 wt%) to 80 wt%, more preferably 85 wt%, in some cases 90 wt%, and in some examples 95 wt% acetonitrile. Alternatively, if the relative weight percentage of acetonitrile to toluene is greater than the azeotropic composition, the amount of make-up acetonitrile 154 added to the purified mixture of acetonitrile and toluene 126 may be the amount required to increase the relative weight percentage of acetonitrile to 80 wt%, more preferably 85 wt%, in some cases 90 wt%, and in some examples 95 wt% acetonitrile.Furthermore, make-up acetonitrile 154 is shown as being added to the purified acetonitrile and toluene mixture 126, but make-up acetonitrile 154 may be added to any one or a combination of the collection tank 104, the feed stream 105, the overhead fraction 108, the purified acetonitrile and toluene stream 114, and / or the purified acetonitrile and toluene stream 120 such that the composition of the acetonitrile and toluene mixture 126 contains any of the above weight percentages of acetonitrile. In any of these cases, the use of the resulting purified acetonitrile and toluene mixture 126, which is recycled to the oligonucleotide synthesis process as a wash solvent, can increase the overall purity of the oligonucleotide synthesis process as compared to when pure acetonitrile is used as the wash solvent.

[0097] The foregoing discussion relates to the purification, possible acetonitrile make-up, and recycling of the recovered waste stream from the oligonucleotide synthesis process for use as a wash solvent, but it should be understood that such discussion is not meant to limit the present invention to only the situation of waste stream recycling. As described above, the use of a blend of acetonitrile and toluene as a wash solvent has been found to be advantageous compared to pure acetonitrile alone. Thus, it may not be necessary to have any of the processing steps related to the purification and make-up of the waste stream 102. Rather, an oligonucleotide synthesis process that utilizes a fresh (e.g., non-recycled) stream of any of the above acetonitrile and toluene compositions may be equally advantageous, if not more advantageous, for use as a wash solvent in the oligonucleotide synthesis process and is thus similarly encompassed by the present disclosure.

[0098] Materials and Methods Examples 1 - 8: In connection with Examples 1-8 described below, an azeotropic or near-azeotropic mixture of acetonitrile and toluene waste containing iodine, thiol, alkylamine and other impurities is treated by adding a reducing agent such as sodium thiosulfate, silver nitrate, and / or formic acid to the feed stream. In the case of sodium thiosulfate, the reducing agent converts all dissolved organic and inorganic iodine compounds into iodides that are not volatile and can be removed through a side outlet without the need for additional equipment.

[0099] Unless otherwise stated, all concentrations are by mass, i.e., percentages are weight percentages and ppm are weight ppm. In all examples, the feed mass in the rectification column was 1 kg of a binary mixture consisting of 76% acetonitrile and 24% toluene.

[0100] Fractional distillation was carried out in a 400 mm packed glass column equipped with a liquid splitter and having a cooling outlet at the top. The column itself consisted of a silver-coated insulated jacket (exhausted to 10-6 mbar), a site strip, and an outer bellows (one bellows packing per 500 mm in length). The packing consisted of corrosion-resistant Raschig rings made of borosilicate glass. The 40 cm packed column was operated at a reflux ratio of RR = 0.33 and a gradually decreasing pressure. Experiments were conducted so that 80% of the initial feed was obtained as the top product. The distillate was divided into three fractions of 200 mL each.

[0101] As used herein, "artificial waste" refers to the compositions shown in Table 1 below.

[0102]

Table 1

[0103] The above-mentioned artificial waste may further contain trace metals of less than 0.2 ppm. The trace metals may include one or more of aluminum, barium, bismuth, cadmium, calcium, chromium, cobalt, copper, iron, lead, lithium, magnesium, manganese, molybdenum, nickel, potassium, silver, sodium, strontium, tin, and zinc.

[0104] Example 9: Test - 20 Oligonucleotides (DNA and RNA) Oligonucleotide (DNA and RNA) synthesis is generally carried out by a four-step cycle (deprotection, activation / coupling, capping, and oxidation / sulfurization), which is repeated for each added nucleotide until the desired sequence is obtained. Between each step, the oligonucleotide bound to the support is washed with an acetonitrile-based washing solvent to reduce residual reagents from the previous step. The washing solvent is usually pure acetonitrile.

[0105] It was hypothesized that a blend of acetonitrile and toluene could be used as the washing solvent instead of pure acetonitrile alone. To test such a hypothesis, various compositions of a substantially pure mixture of acetonitrile and toluene were utilized as the washing solvent in multiple oligonucleotide synthesis reactions, thereby obtaining oligonucleotides of various purities as a result.

[0106] In this case, the following oligonucleotide synthesis test conditions were observed for test 20 (e.g., "20-mer") oligonucleotides.

[0107] [Table 2]

[0108] The oligonucleotide yield was determined by HPLC using UV detection at 260 nm. Furthermore, the identity of the main peak was confirmed by LCMS.

[0109] Example 10: Test - 18 Oligonucleotides (DNA and RNA) Similar to Example 9, oligonucleotide (DNA and RNA) synthesis is generally carried out by a four - step cycle (deprotection, activation / coupling, capping, and oxidation / sulfurization), which is repeated for each added nucleotide until the desired sequence is obtained. Between each step, the oligonucleotide bound to the support is washed with an acetonitrile - based washing solvent to reduce residual reagents from the previous step. The washing solvent is usually pure acetonitrile.

[0110] Similar to Example 9, it was hypothesized that a blend of acetonitrile and toluene, rather than pure acetonitrile alone, could be used as the washing solvent. To test such a hypothesis, various compositions of a substantially pure mixture of acetonitrile and toluene were utilized as the washing solvent in multiple oligonucleotide synthesis reactions, thereby obtaining oligonucleotides of various purities as a result.

[0111] In this case, the following oligonucleotide synthesis test conditions were observed for test 18 (e.g., "18 - mer") oligonucleotides.

[0112] [Table 3]

[0113] The oligonucleotide yield was determined by HPLC using UV detection at 260 nm. Furthermore, the identity of the main peak was confirmed by LCMS. [Example]

[0114] Example 1: Fractional Distillation of Artificial Waste In this example, 1000 mL of artificial waste containing acetonitrile, toluene, tetrahydrofuran (THF), and organic agents from oligonucleotide synthesis as described in Table 01 was introduced into the round-bottom flask of a fractional distillation column. Fractional distillation was carried out without any pretreatment additives or added solvents. The top temperature and bottom temperature were set at 76 °C and 85 °C, respectively. The distillate was condensed again at the top of the column in a condenser and collected into two fractions. The first fraction (cut 1) had the minimum volume. The first fraction contained 1 - 2% water and the components shown in Table 2 below. The main fraction (cut 2) was also collected and analyzed.

[0115] The distillate of the artificially blended rinse solution can be purified by simple fractional distillation. However, various components of the chemicals used in oligonucleotide synthesis can be carried over. Thus, in addition to water, a significant proportion of the alkylamines, acetic acid, dichloroacetic acid, alkylthiols, and iodine used were found in both distillates. The analysis results of the artificial distillation waste liquid are shown in Table 2 together with their specification values ("Spec.").

[0116]

Table 4

[0117] Example 2: Fractional Distillation of Acetonitrile and Toluene with Added Iodine In this example, 200 mg of solid iodine was added to an azeotropic mixture of 361 g of acetonitrile and 114 g of toluene (shown as "azeotropic mixture" in the table below). The mixture was stirred until the solid iodine was completely dissolved and then transferred to the round-bottom flask of a fractional distillation column. The top temperature and bottom temperature were set at 76 °C and 80 °C, respectively. The distillate was condensed in a condenser and collected into equal fractions at the top of the column. It should be noted that the condensate (cuts 1 - 3) was still colored and the smell of iodine could be perceived.

[0118] Analyze the cuts and show the results in Table 3 below together with the standard values ("Spec.").

[0119]

Table 5

[0120] Example 3: Precipitation of iodine from acetonitrile and toluene using sodium thiosulfate In this example, 200 mg of solid iodine and an equimolar amount (259 mg) of solid sodium thiosulfate were added to an azeotropic mixture of 361 g of acetonitrile and 114 g of toluene. The solution was stirred with a magnetic stirrer at 500 rpm until the solids were completely dissolved. Then, distillation was carried out with the top temperature and the bottom temperature set at 81 °C and 85 °C, respectively. The distillate was condensed at the top of the column in a condenser and collected in two equal fractions.

[0121] In this advantageous version, the condensed distillate was colorless. The iodine content was less than 0.1 ppm. The analysis results of the cuts are shown in Table 4 below together with the standard values ("Spec.").

[0122]

Table 6

[0123] Example 4: Precipitation of iodine from artificial waste using sodium thiosulfate [[ID=X]]In this example, 125 mg of iodine and 160 mg of sodium thiosulfate were added to 500 mL of artificial waste. A mixture further containing 65% acetonitrile and 25% toluene was introduced into the round-bottom flask of a fractional distillation column. The top temperature and the bottom temperature were set at 76 °C and 80 °C, respectively. The distillate was condensed again at the top of the column in a condenser and collected in two fractions. The two main fractions were analyzed.

[0124] It should be noted that there seems to be a problem with the content in line 32. The original text has some incorrect expressions. The corrected translation is provided in line 32 above. If you have any other questions, please feel free to let me know.In contrast to Example 3, iodide was found at 1.2 ppm during the analysis of Cut 2. This indicates that higher concentrations of iodine in the oligonucleotide waste require an excess of reducing agent to prevent iodine carry-over. The results of the analysis are shown in Table 5 below together with the specification values ("Spec.").

[0125]

Table 7

[0126] Example 5: Precipitation of Thiol from Acetonitrile and Toluene Using Silver Nitrate In this example, 100 mg of phenylacetyl disulfide (PAD) and 50 mg of 5-ethylthio-1H-tetrazole (ETT) were added to an azeotropic mixture of 380 g of acetonitrile and 120 g of toluene (shown as "azeotropic mixture" in the table below). Finally, 51 mg of silver nitrate was added to this mixture. Then, distillation was carried out with the top temperature and bottom temperature set at 76 °C and 81 °C, respectively. The distillate was condensed at the top of the column in a condenser and collected into two equal fractions.

[0127] In this advantageous version, the condensed distillate was colorless and had no strong odor of sulfur compounds. The alkylthiol content decreased below the detection limit of HPLC. The results of the analysis are shown in Table 6 below together with the specification values ("Spec.").

[0128]

Table 8

[0129] Example 6: Precipitation of Alkylamine from Acetonitrile and Toluene Using Formic Acid In this example, the effectiveness of formic acid as a reducing agent and Lewis acid was investigated to precipitate alkylamine by forming a Lewis adduct. To an azeotropic mixture of 361 g of acetonitrile and 114 g of toluene, 10 g of pyridine, and 5 g each of picoline, 2,6-lutidine, and N-methylimidazole (shown as "azeotropic mixture" in the following table) were added. Finally, 13.24 g of formic acid was added to this mixture. Then, distillation was carried out, and the top and bottom temperatures were set to 81 °C and 85 °C, respectively. The distillate was condensed at the top of the column in a condenser and collected into three equal fractions.

[0130] In this advantageous version, the condensed distillate was colorless. The carry-over of alkylamine was significantly reduced, suggesting that formic acid formed non-volatile salts with the alkylamine base and prevented them from evaporating during distillation. The results of the analysis are shown in Table 7 below together with the standard values ("Spec.").

[0131]

Table 9

[0132] Example 7: Precipitation of iodine from artificial waste using excess sodium thiosulfate In this example, 1000 mL of artificial waste containing 5 ppm of iodine and an equimolar amount of sodium thiosulfate was introduced into the round-bottom flask of a fractional distillation column. 12 mg of sodium thiosulfate was added to reduce the organic iodine compounds in the waste liquid. The top and bottom temperatures were set to 76 °C and 81 °C, respectively. The distillate was condensed again at the top of the column in a condenser and collected into two fractions. The first fraction had a minimum volume. The first fraction contained 1 - 2% water and the components shown in Table 8 below. The main fraction was also collected and analyzed for iodine content. Iodine could not be detected in the distillate within the detection limit of this method. The results of the analysis are shown in Table 8 below together with the standard values ("Spec.").

[0133]

Table 10

[0134] Example 8: Precipitation of Iodine and Alkylamine from Artificial Waste In this example, 500 mL of artificial waste, 10 mg of sodium thiosulfate, and excess formic acid were mixed. A mixture further containing 65% acetonitrile and 25% toluene was introduced into the round-bottom flask of a fractional distillation column. The top temperature and bottom temperature were set to 76 °C and 81 °C, respectively. The distillate was condensed again at the top of the column in a condenser and collected into two fractions.

[0135] The concentrations of all alkylamines tested were below the specification limits, and the amount of iodine in the distillate was less than 0.5 ppm (the detection limit of the HPLC method). The results of the analysis are shown in Table 9 below together with the specification values (“Spec.”).

[0136] [Table 11]

[0137] Example 9: Oligonucleotide Purity Achieved by the Use of Various Washing Solvent Compositions - Test - 20 In this example, four different compositions of acetonitrile-based washing solvents were used to synthesize the same Test - 20 sequence of a 5’ATA CCG ATT AAG CGA AGT TT 3’ 20-mer oligonucleotide four times over under the previously provided experimental conditions. The first test utilized a pure (e.g., 100 wt%) acetonitrile washing solvent, the second test utilized a washing solvent containing 95 wt% acetonitrile and 5 wt% toluene, the third test utilized a washing solvent containing 85 wt% acetonitrile and 15 wt% toluene, and the fourth test utilized a washing solvent containing 76 wt% acetonitrile and 24 wt% toluene (e.g., representative of an azeotropic composition of acetonitrile and toluene). The purity of the resulting 20-mer oligonucleotide was reported based on the HPLC yield, and the results are shown in Table 10 below.

[0138]

Table 12

[0139] Surprisingly, when using an impure acetonitrile washing solvent, it was found that the yield of the test - 20 oligonucleotide product formed during the solid - phase chemical synthesis of oligonucleotides increased slightly. Specifically, it was observed that the use of a blend composition containing 85% acetonitrile and 15% toluene (w / w) washing solvent resulted in the highest oligonucleotide yield. However, in each of the aforementioned cases, the blend composition of the acetonitrile and toluene washing solvents showed at least a similar test - 20 oligonucleotide yield, if not higher, than that of the pure acetonitrile washing solvent. Therefore, this experiment confirmed that in the oligonucleotide synthesis process, it is not only possible to use a mixed washing solvent instead of pure acetonitrile (for example, oligonucleotide synthesis does not necessarily require a pure acetonitrile washing solvent), but it can also be advantageous.

[0140] Example 10: Oligonucleotide purity achieved by using various washing solvent compositions in Test - 18: In another example, five different compositions of an acetonitrile-based wash solvent were used to synthesize the sequence of the test-18 5’TTT TTT TTT TTT TTT TTT 3’ 18-mer oligonucleotide over five test runs under the previously provided experimental conditions. The first test utilized a pure (e.g., 100 wt%) acetonitrile wash solvent, the second test utilized a wash solvent containing 90 wt% acetonitrile and 10 wt% toluene, the third test utilized a wash solvent containing 85 wt% acetonitrile and 15 wt% toluene, the fourth test utilized a wash solvent containing 80 wt% acetonitrile and 20 wt% toluene, and the fifth test utilized a wash solvent containing 76 wt% acetonitrile and 24 wt% toluene (e.g., representative of an azeotropic composition of acetonitrile and toluene). The purity of the resulting 18-mer oligonucleotide was reported based on HPLC yield and the results are shown in Table 11 below.

[0141]

Table 13

[0142] Surprisingly, it was found that when using a non-pure acetonitrile wash solvent, the yield of the test-18 oligonucleotide product formed during the solid-phase chemical synthesis of the oligonucleotide increased slightly. Specifically, it was observed that the use of a blend composition containing 80% acetonitrile and 20% toluene (w / w) wash solvent resulted in the highest oligonucleotide yield. However, in each of the aforementioned cases, the blend composition of the acetonitrile and toluene wash solvent showed at least a similar T-18 oligonucleotide yield, if not higher, than that of the pure acetonitrile wash solvent. Thus, this experiment confirmed that it is possible and potentially advantageous to use a mixed wash solvent rather than pure acetonitrile in the oligonucleotide synthesis process (e.g., oligonucleotide synthesis does not necessarily require a pure acetonitrile wash solvent).

[0143] Aspect: Aspect 1 is a method for treating a waste stream, comprising receiving a waste stream containing acetonitrile, toluene, and one or more iodine-containing compounds, adding an iodine-reactive compound to the waste liquid stream, and fractionating the waste stream to produce an overhead fraction and a bottom fraction, wherein the overhead fraction contains the acetonitrile and toluene of the waste stream, and the bottom fraction contains the one or more iodine-containing compounds of the waste stream.

[0144] Aspect 2 is the method according to Aspect 1, wherein the iodine-reactive compound contains sodium thiosulfate.

[0145] Aspect 3 is the method according to either Aspect 1 or 2, wherein the iodine-reactive compound contains sodium thiosulfate and silver nitrate.

[0146] Aspect 4 is the method according to any one of Aspects 1 to 3, wherein the overhead fraction further contains iodine at a concentration of less than 25 ppm.

[0147] Aspect 5 is the method according to any one of Aspects 1 to 4, wherein the overhead fraction further contains iodine at a concentration of less than 1 ppm.

[0148] Aspect 6 is the method according to any one of Aspects 1 to 5, wherein during the fractionation of the waste stream, the iodine-reactive compound reacts with the iodine of the one or more iodine-containing compounds, and precipitates the iodine of the one or more iodine-containing compounds from the waste stream into the bottom fraction.

[0149] Aspect 7 is the method according to any one of Aspects 1 to 6, further comprising purifying the overhead fraction into a purified overhead fraction consisting essentially of acetonitrile and toluene.

[0150] Aspect 8 is the method according to any one of Aspects 1 to 7, wherein the waste stream further contains one or more sulfur-containing compounds, the method further comprises adding a sulfur-reactive compound to the waste stream, and the bottom fraction further contains one or more sulfur-containing compounds.

[0151] Aspect 9 is the method according to any one of Aspects 1 to 8, wherein the sulfur-reactive compound contains silver nitrate.

[0152] Aspect 10 is the method according to any one of Aspects 1 to 9, wherein the waste stream further contains one or more basic nitrogen-containing compounds, the method further includes adding an acidic-reactive compound to the waste stream, and the bottoms fraction further contains one or more basic nitrogen-containing compounds.

[0153] Aspect 11 is the method according to any one of Aspects 1 to 10, wherein the acidic-reactive compound contains formic acid.

[0154] Aspect 12 is a method for treating a waste stream, comprising receiving a waste stream containing acetonitrile, toluene, and one or more sulfur-containing compounds, adding a sulfur-reactive compound to the waste liquid stream, and fractionating the waste stream to produce a top fraction and a bottoms fraction, wherein the top fraction contains the acetonitrile and toluene of the waste stream, and the bottoms fraction contains one or more sulfur-containing compounds of the waste stream.

[0155] Aspect 13 is the method according to any one of Aspects 1 to 11 or Aspect 12, wherein the top fraction further contains sulfur at a concentration of less than 25 ppm.

[0156] Aspect 14 is the method according to any one of Aspects 1 to 11 or Aspects 12 to 13, wherein the top fraction further contains sulfur at a concentration of less than 1 ppm.

[0157] Aspect 15 is the method according to any one of Aspects 1 to 11 or Aspects 12 to 14, wherein the sulfur-reactive compound contains silver nitrate.

[0158] Aspect 16 is the method according to any one of Aspects 1 to 11 or Aspects 12 to 15, wherein during the fractionation of the waste stream, the sulfur-reactive compound reacts with the sulfur of one or more sulfur-containing compounds to precipitate sulfur from the waste stream into the bottoms fraction.

[0159] Aspect 17 is a method for treating a waste stream, comprising receiving a waste stream containing acetonitrile, toluene, and a basic nitrogen-containing compound, adding an acidic reactive compound to the waste liquid stream, and fractionating the waste stream to produce an overhead fraction and a bottom fraction, wherein the overhead fraction contains the acetonitrile and toluene of the waste stream, and the bottom fraction contains the basic nitrogen-containing compound of the waste stream.

[0160] Aspect 18 is the method according to any one of Aspects 1 to 11, Aspects 12 to 16, or Aspect 17, wherein the overhead fraction further contains one or more nitrogen-containing compounds at a concentration of less than 100 ppm.

[0161] Aspect 19 is the method according to any one of Aspects 1 to 11, Aspects 12 to 16, or Aspects 17 and 18, wherein the acidic reactive compound contains formic acid.

[0162] Aspect 20 is the method according to any one of Aspects 1 to 11, Aspects 12 to 16, or Aspects 17 to 19, wherein during the fractionation of the waste stream, the acidic reactive compound reacts with the basic nitrogen-containing compound to precipitate the basic nitrogen-containing compound from the waste stream into the bottom fraction.

[0163] Aspect 21 is a method for producing a synthetic oligonucleotide, comprising washing a reaction vessel containing one or more components of an oligonucleotide synthesis process with a mixed washing solution containing at least acetonitrile and toluene, wherein acetonitrile constitutes at least 70% of the mixed washing solution, and recovering a synthetic oligonucleotide having a minimum oligonucleotide purity.

[0164] Aspect 22 is the method according to Aspect 21, wherein the mixed washing solution contains a substantially pure mixture of acetonitrile and toluene.

[0165] Aspect 23 is the method according to any one of Aspects 21 and 22, wherein the mixed cleaning solution is received as a purified waste stream recycled from an oligonucleotide synthesis process.

[0166] Aspect 24 is the method according to any one of Aspects 21 to 23, wherein a substantially pure mixture of acetonitrile and toluene contains at least 80% acetonitrile.

[0167] Aspect 25 is the method according to any one of Aspects 21 to 24, wherein a substantially pure mixture of acetonitrile and toluene contains at least 90% acetonitrile.

[0168] Aspect 26 is the method according to any one of Aspects 21 to 24, wherein a substantially pure mixture of acetonitrile and toluene contains an azeotropic mixture of acetonitrile and toluene.

[0169] Aspect 27 is the method according to any one of Aspects 21 to 26, wherein a substantially pure mixture of acetonitrile and toluene contains at least 76% acetonitrile.

[0170] Aspect 28 is the method according to any one of Aspects 21 to 27, wherein a substantially pure mixture of acetonitrile and toluene contains at least 5% toluene.

[0171] Aspect 29 is the method according to any one of Aspects 21 to 28, wherein a substantially pure mixture of acetonitrile and toluene contains at least 15% toluene.

[0172] Aspect 30 is the method according to any one of Aspects 21 to 29, wherein the minimum oligonucleotide purity is 70% or higher.

[0173] Aspect 31 is the method according to any one of Aspects 21 to 30, wherein the minimum oligonucleotide purity is greater than 72%.

[0174] Aspect 32 is the method according to any one of Aspects 21 to 31, wherein the minimum oligonucleotide purity is more than 75%.

[0175] Aspect 33 is the method according to any one of Aspects 21 to 32, wherein the minimum oligonucleotide purity is 90% or more.

[0176] Aspect 34 is the method according to any one of Aspects 21 to 33, wherein the minimum oligonucleotide purity is more than 92%.

[0177] Aspect 35 is the method according to any one of Aspects 21 to 34, wherein the minimum oligonucleotide purity is more than 95%.

[0178] Aspect 36 is the method according to any one of Aspects 21 to 35, wherein the minimum oligonucleotide purity is higher than the oligonucleotide purity produced by washing a reaction vessel containing one or more components of the oligonucleotide synthesis process with a washing solution consisting of acetonitrile.

[0179] Aspect 37 is the method according to any one of Aspects 21 to 36, incorporating any one of the features of any of Aspects 1 to 11, Aspects 12 to 16, or Aspects 17 to 20.

[0180] Aspect 38 is the method according to any one of Aspects 1 to 11, including any one of the features of any of Aspects 21 to 36, Aspects 12 to 16, or Aspects 17 to 20.

[0181] Aspect 39 is the method according to any one of Aspects 12 to 16, including any one of the features of any of Aspects 21 to 36, Aspects 1 to 11, or Aspects 17 to 20.

[0182] Aspect 40 is the method according to any one of Aspects 17 to 20, including any one of the features of any of Aspects 21 to 36, Aspects 1 to 11, or Aspects 12 to 16.

Claims

1. A method for treating a waste stream, comprising: receiving the waste stream comprising acetonitrile, toluene, and one or more iodine-containing compounds; adding an iodine-reactive compound to the waste stream; fractionating the waste stream to produce an overhead fraction and a bottom fraction, wherein the overhead fraction comprises the acetonitrile and the toluene of the waste stream, and the bottom fraction comprises the one or more iodine-containing compounds of the waste stream.

2. The method according to claim 1, wherein the iodine-reactive compound comprises sodium thiosulfate.

3. The method according to claim 1, wherein the iodine-reactive compound comprises sodium thiosulfate and silver nitrate.

4. The method according to claim 1, wherein the overhead fraction further comprises iodine at a concentration of less than 25 ppm.

5. The method according to claim 1, wherein during fractionation of the waste stream, the iodine-reactive compound reacts with iodine of the one or more iodine-containing compounds, and precipitates the iodine of the one or more iodine-containing compounds from the waste stream into the bottom fraction.

6. The method according to claim 1, further comprising purifying the overhead fraction into a purified overhead fraction consisting essentially of acetonitrile and toluene.

7. A method for treating a waste stream, comprising: receiving the waste stream comprising acetonitrile, toluene, and one or more sulfur-containing compounds; adding a sulfur-reactive compound to the waste stream; fractionating the waste stream to produce an overhead fraction and a bottom fraction, wherein the overhead fraction comprises the acetonitrile and the toluene of the waste stream, and the bottom fraction comprises the one or more sulfur-containing compounds of the waste stream.

8. The method according to claim 7, wherein the overhead fraction further comprises sulfur at a concentration of less than 25 ppm.

9. The method according to claim 7, wherein the sulfur-reactive compound comprises silver nitrate.

10. A method for treating a waste stream, comprising: receiving the waste stream comprising acetonitrile, toluene, and a basic nitrogen-containing compound; adding an acidic-reactive compound to the waste stream; Fractionating the waste stream to produce an overhead fraction and a bottom fraction, wherein the overhead fraction contains the acetonitrile and the toluene of the waste stream, and the bottom fraction contains the basic nitrogen-containing compound of the waste stream, and fractionating. A method for treating a waste stream, comprising:

11. The method according to claim 10, wherein the overhead fraction further contains one or more nitrogen-containing compounds at a concentration of less than 100 ppm.

12. The method according to claim 10, wherein the acidic reactive compound contains formic acid.

13. A method for producing a synthetic oligonucleotide, comprising: Washing a reaction vessel containing one or more components of an oligonucleotide synthesis process with a mixed washing solution containing at least acetonitrile and toluene, wherein the acetonitrile constitutes at least 70% of the mixed washing solution; Recovering the synthetic oligonucleotide, the synthetic oligonucleotide comprising a minimum oligonucleotide purity. A method for producing a synthetic oligonucleotide, comprising:

14. The method according to claim 13, wherein the mixed washing solution contains a substantially pure mixture of acetonitrile and toluene.

15. The method according to claim 13, wherein the mixed washing solution is received as a purified waste stream recycled from the oligonucleotide synthesis process.

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