Nucleotide extension strand verification

Abstract

Methods and systems for synthesizing oligonucleotides in a reaction chamber comprising a solid support. Nucleotide monomers are selected for addition to oligonucleotides. An inlet stream comprising selected monomers is fed into a reaction chamber (column) of the reaction chamber and conditions for oligonucleotide synthesis are provided in the reaction chamber. An outlet stream from the reaction chamber may be split and directed to a waste discharge port and / or an analysis station in which the eluate is analyzed by mass spectrometry (MS) to confirm the identity of the monomers fed to the reaction chamber through the inlet. A determination is made as to whether the outlet stream contains the selected nucleotide monomer or derivative thereof based on results by the MS analysis.

Description

Cross - Reference to Related Applications

[0001] None. Technical Field

[0002] The present disclosure relates to methods and systems for synthesizing oligonucleotides such as RNA and DNA and for monitoring such synthesis. Background Art

[0003] Solid-phase synthesis is a valuable tool that can be used to prepare oligonucleotide products for many uses. The chemical synthesis of oligonucleotides typically occurs in a sequential manner, where one end of the growing chain of nucleotides is attached to a solid surface, and reactive nucleotide monomers are sequentially condensed using repetitive synthesis cycles. The first step of the synthesis cycle is typically the reaction of a protected nucleotide monomer having a hydroxyl group on the surface with a nucleoside attached to the surface. After the initial coupling of the reactive phosphorus group with the hydroxyl group on the surface, subsequent steps typically include capping of the unreacted hydroxyl groups and subsequent oxidation of the reactive phosphorus intermediate. The last step of the cycle is typically the deprotection of the hydroxyl group of the added nucleotide such that the nucleotide has a hydroxyl group that will couple with the next protected nucleotide monomer to be added. After completion of the cycle of adding nucleotides, the desired oligonucleotide product is released from the solid phase, deprotected, and used for further biological applications.

[0004] Desired oligonucleotide synthesis would provide the desired oligonucleotide product in high yield and high purity such that the completed synthesis composition contains only the desired oligonucleotide product. It is also desirable to be able to monitor oligonucleotide synthesis in real time or near real time. Such technology would be advantageous for current products and is known not to exist currently in the industry.

[0005] Alternative techniques for confirming the sequence of the desired oligonucleotide product after synthesis is completed in a synthesizer can analyze the product removed from the synthesis column after cleavage and deprotection by high-resolution mass spectrometry such as (MS-MS) or by next-generation sequencing (NGS). However, although MS allows confirmation of the sufficient molecular weight of the full-length synthesized oligonucleotide, in most cases it cannot confirm the correct sequence, i.e., the order of monomer composition in the oligonucleotide full-length product. The length of the oligonucleotide (not greater than 30-mers) and the chemical modification of some monomers greatly limit the acquisition of sequence information of the desired oligonucleotide product. In addition, separating the desired oligonucleotide from shorter oligonucleotides or other impurities prior to these methods typically requires laborious and time-consuming chromatographic conditions. Moreover, neither of these methods is performed in real time. The MS-MS technique is limited by the length of the oligonucleotide sequence and suffers from severe signal-to-noise ratio erosion that varies with sequence length. In the case of NGS, it may not have the ability to "read" chemically modified nucleotides or identify modified nucleotides due to polymerase function and requirements. For some oligonucleotide therapeutics and other products, the inclusion of modified nucleotides is required.

[0006] Another method uses on-line infrared (IR) analysis to identify sequences during synthesis. However, this technique has the limitation that it cannot easily distinguish the differences between modified and unmodified nucleosides. IR spectral comparison would require in-depth visual comparison because the infrared spectral differences between single-atom changes (such as those in 2'-modified nucleotides) are very small. In contrast, the difference between single-atom changes in the same 2'-modified nucleotide is directly measured by molecular weight, and even a single mass unit difference is within the mass accuracy of the detector.

[0007] Accordingly, there is a need to monitor (on-line) the synthesis to verify the production of the validated oligonucleotide sequence. There is also a need for methods and systems for real-time monitoring of oligonucleotide synthesis. SUMMARY OF THE INVENTION

[0008] As an aspect of the present invention, there is provided a method for synthesizing an oligonucleotide and real-time monitoring of the synthesis. The method includes selecting nucleotide monomers to be added to the oligonucleotide; feeding an inlet stream to a reaction chamber for oligonucleotide synthesis; providing conditions for oligonucleotide synthesis in the reaction chamber; diverting an outlet stream from the reaction chamber to an analysis station; analyzing the outlet stream or a sample thereof by mass spectrometry (MS) at the analysis station; and determining whether the outlet stream contains the selected nucleotide monomer or its derivative based on the results of the MS analysis.

[0009] As another aspect of the present invention, there is provided a system for synthesizing an oligonucleotide. The system includes a reaction chamber that includes an inlet, a solid support for oligonucleotide synthesis, and an outlet; and a mass spectrometry (MS) instrument fluidly connected to the outlet of the reaction chamber.

[0010] These and other features and advantages of the methods and systems of the present invention will become apparent from the following detailed description in conjunction with the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Shows a typical chemical synthesis of an oligonucleotide on a solid support.

[0012] Figure 2 Illustrates an embodiment of the system of the present invention.

[0013] Figure 3 Is a flow chart of an embodiment of the method of the present invention.

[0014] Figures 4 to 11 Shows mass spectrometry data for samples 1-1 to 1-8 from Example 1, respectively.

[0015] Figures 12 to 18The mass spectrometry data of Samples 2-1 to 2-7 from Example 2 are shown respectively. Detailed Description

[0016] Before describing the various embodiments, it should be understood that the teachings of this disclosure are not limited to the specific embodiments described, as these can of course vary. It should also be understood that since the scope of the teachings of the present invention will be limited only by the appended claims, the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this teaching, some exemplary methods and materials will now be described.

[0017] The methods and systems of the present invention can be used to confirm in real time the order in which each nucleotide monomer is added to a growing oligonucleotide by using on-line mass spectrometry for analyzing the effluent from the reaction chamber of an oligonucleotide synthesizer. The methods and systems of the present invention solve several problems and provide several advantages. For example, they provide the first real-time verification during oligonucleotide synthesis and provide the real-time nucleotide monomer addition order by on-line mass spectrometry (MS). They improve the accuracy of sequence verification by evaluating the simple individual nucleotide monomer identities used in each synthesis cycle rather than the entire highly complex final oligonucleotide. The methods and systems of the present invention are independent of oligonucleotide length or oligonucleotide chemical modification. They are also more definitive compared to existing on-line techniques. The methods and systems of the present invention generally do not require high-resolution mass spectrometry.

[0018] The methods and systems of the present invention can be used to synthesize oligonucleotides such as oligoribonucleotides, oligodeoxyribonucleotides, and chimeric oligonucleotides. The desired oligonucleotide can contain ribonucleotides, deoxyribonucleotides, or a mixture thereof, as well as modified nucleotides (e.g., 2'-O-methyl, 2'-fluoro, LNA, 2'-MOE, 2'-CNEt, etc.). In some embodiments, methods for synthesizing an oligonucleotide having a defined sequence or for monitoring its synthesis are provided. The methods include selecting chemical reagents for addition to the oligonucleotide; feeding an inlet stream to a reaction chamber for oligonucleotide synthesis (e.g., a column containing a solid support); providing conditions for oligonucleotide synthesis in the reaction chamber; diverting the effluent from the reaction chamber to an analysis station; analyzing the effluent in the analysis station by mass spectrometry (MS) at a specified time when the effluent contains an activated nucleotide phosphoramidite solution transferred from the synthesizer after coupling; and determining whether the effluent contains the selected activated nucleotide monomer or its derivative based on the results of the MS analysis.

[0019] Figure 1Schematic illustration of a typical chemical synthesis cycle for generating oligonucleotides on a solid support. To complete the synthesis, the first step of the synthesis cycle is typically the reaction of a protected nucleotide monomer with a nucleoside attached to the surface of the solid support (bead or resin), or the reaction of a protected nucleotide monomer with a hydroxyl group on that surface. The hydroxyl group on the surface can be part of a cleavable universal linker (such as a Unylinker or a succinate linker) or an uncleavable surface attachment. After the initial coupling of the reactive phosphorus group with the hydroxyl group, subsequent steps typically include capping of the unreacted hydroxyl groups and subsequent oxidation of the reactive phosphorus intermediate (phosphite triester to phosphate triester). Typically, an oligonucleotide contains internucleotide linkages between the 5'-hydroxyl and 3'-hydroxyl of adjacent nucleotides, which can be a phosphate or a modified phosphorus group (typically a 2-cyanoethyl protected phosphate group). Under certain conditions using certain modified phosphorus groups, oxidation is carried out prior to capping, particularly in cases where the oxidation reagent generates a modified phosphorus group (such as phosphorothioate, boranophosphonate or aminophosphonate). The final step of the synthesis cycle is typically deprotection of the hydroxyl group at the 5'-end of the growing oligonucleotide (e.g., dimethoxytrityl (DMT)) to deblock the hydroxyl group. The deblocked hydroxyl group can then couple with the next incoming protected nucleotide phosphoramidite monomer fed into the reaction chamber under conditions suitable for the coupling reaction. After coupling of the last nucleotide selected for the sequence is completed, the desired oligonucleotide product is released from the solid phase (resin), deprotected, purified and used for further biological applications.

[0020] As another aspect of the present disclosure, a system for synthesizing oligonucleotides or monitoring oligonucleotide synthesis is provided. The system includes an automated oligonucleotide synthesizer, the automated oligonucleotide synthesizer including a reaction chamber, the reaction chamber including: at least one inlet through which reagents are provided to a column for oligonucleotide synthesis; a solid support for oligonucleotide synthesis contained in the column; and an outlet for discharging excess reagents, solvents and washes to waste. The system further includes a mass spectrometry (MS) instrument fluidly connected to the outlet of the reaction chamber. Suitable reaction chambers for solid-phase synthesis of oligonucleotides are described herein and in various publications.

[0021] Figure 2An embodiment of the system 200 of the present invention is shown. The reaction chamber 202 for oligonucleotide synthesis has an inlet 204 and an outlet 206. The reaction chamber 202 contains a solid support suitable for solid-phase synthesis of oligonucleotides. The reaction chamber 202 may define a column containing the solid support such that the components of the inlet stream pass through the column as they move towards the outlet 206. The sample tee 208 receives the outlet stream from the outlet 206 such that the outlet stream can be sent to process waste 210 or for analysis. In some embodiments, some or most of the outlet stream is sent to process waste 210, and a sample of the outlet stream is sent for analysis. The pump 212 provides a solid force to cause the outlet stream to flow from the reaction chamber 202 to the analysis component. For example, an isocratic pump can be used to continuously pump the outlet stream throughout the process. Initially, the outlet stream from the reaction chamber 202 will be sent to waste 214, but at a desired time point, such as during the column wash period of the synthesis, a "start" signal is sent to perform a series of programmed tasks to automatically collect samples for retention and analysis. The programmed tasks may include switching the sample valve 216 such that the flow path from the isocratic pump 212 switches the outlet stream from waste 214 to the analysis component, such as by sending the outlet stream or a sample thereof to a UHPLC-MS instrument.

[0022] Samples are collected from the flowing outlet stream using the UHPLC-MS online sample manager 218. The collected samples can be placed in sample vials for analysis and / or retention.

[0023] In some embodiments, after the sample is created, the system 200 will immediately automatically initiate an analysis run of the sample. In some embodiments, the system also includes a diluent source 220 and a mobile phase pump 222 that are fluidly connected to the online sample manager 218. The system may also include an MS diluent source 224 and a flow rate regulating pump 226 that are fluidly connected to the MS instrument 230. The mobile phase pump 222 can control the flow of the sample from the online sample manager 218 to the mass spectrometry instrument 230. During analysis, the sample can be automatically diluted online to adjust the concentration of the sample using the flow rate regulating pump 226 and the valve 228 before MS detection. The computing device 232 is communicatively connected to the mass spectrometry instrument 230 such that it receives the results of the MS analysis. In certain embodiments, and particularly in large-scale manufacturing, if the results show an incorrect identity of the expected monomer phosphoramidite, demonstrating that the wrong base has been added to the growing oligonucleotide sequence, an automatic shutdown mechanism is implemented to abort the oligonucleotide synthesis.

[0024] Figure 3A flowchart of an embodiment of the method of the present invention is provided. At 301, the online sample manager 218 and the mass spectrometer 230 are started. At 302, the sample pump 212 is started. At 303, the synthesis of the oligonucleotide required in the reaction chamber 202 is started. At 304, after the oligonucleotide synthesis conditions in the reaction chamber 202 have persisted for a period of time, a sampling signal is sent to the online sample manager 218. The sampling signal can be sent by the reaction chamber 202, a sensor, or a computing device communicatively connected to or associated with the reaction chamber 202 or a sensor of the reaction chamber 202. At 305, the online sample manager collects a sample of the effluent stream, including any phosphoramidite. At 306, the online sample manager sends the sample to the mass spectrometer. At 307, the mass spectrometer provides the mass of the phosphoramidite or its derivative in real time. At 308, when the synthesis has ended and the sample collection and analysis are complete, the system can pump idly until the reaction chamber 202 generates another "start" signal. Then, this will start the system to begin another sample collection and analysis cycle.

[0025] After synthesis is complete, the generated sample data is analyzed. Sample / mass identification is assigned. All generated sample data is collected by the computing device 232 and compiled into a report that confirms the sequence of the oligonucleotide product by confirming the order of the nucleotide monomers fed into the reaction chamber. The report can also include a timestamp of the MS analysis results, the feed time of the inlet stream into the reaction chamber, and the association between the timestamp and the feed time, residence time, or other data used for confirmation or verification.

[0026] The methods and systems of the present invention can be used to synthesize any desired oligonucleotide product. In some embodiments, the oligonucleotide is an oligoribonucleotide (RNA). In some embodiments, the oligonucleotide is an oligodeoxyribonucleotide (DNA). In some embodiments, the oligonucleotide is a chimeric oligonucleotide that contains both ribonucleotides and deoxyribonucleotides or modified deoxy / ribonucleotides (including but not limited to 2'-OMe, 2'F, 2'-MOE, LNA, phosphorothioate, PNA, phosphonate linkages). In some embodiments, the length of the oligonucleotide is at least 15 nucleotides. In some embodiments, the length of the oligonucleotide is at least 50 nucleotides. In some embodiments, the length of the oligonucleotide is at least 70 nucleotides. In some embodiments, the length of the oligonucleotide is at least 75 nucleotides. In some embodiments, the length of the oligonucleotide is at least 100 nucleotides. In some embodiments, the length of the oligonucleotide is at least 125 nucleotides. In some embodiments, the length of the oligonucleotide is at least 150 nucleotides. In some embodiments, the length of the oligonucleotide is from about 15 nucleotides to about 500 nucleotides. In some embodiments, the length of the oligonucleotide is from about 40 nucleotides to about 300 nucleotides.

[0027] In some embodiments, phosphoramidite-based methods are used to synthesize the desired oligonucleotides. In some embodiments, the synthesis method includes support-bound nucleosides with 5'-DMT protecting groups. In some embodiments, the synthesis method includes support-bound nucleosides with 3'-DMT protecting groups. In some embodiments, the synthesis method includes support-bound nucleosides with 5'-silyl protecting groups. In some embodiments, the synthesis method includes support-bound nucleosides with oxidatively removable protecting groups. In some embodiments, the synthesis includes the steps of detritylation, coupling of the support-bound nucleoside with a nucleoside phosphoramidite monomer, capping of the unreacted 5'-hydroxyl, and oxidation of the phosphoramidite. In some embodiments, oligonucleotide synthesis is automated. In some embodiments, the oligonucleotide is detritylated prior to performing the methods of the invention.

[0028] In some embodiments, the oligonucleotide comprises a phosphorus protecting group or a nucleobase protecting group. In some embodiments, the oligonucleotide comprises a phosphorus protecting group and a nucleobase protecting group. In some embodiments, the oligonucleotide is an RNA comprising a phosphorus protecting group, a nucleobase protecting group, and a 2'-hydroxyl protecting group.

[0029] In some embodiments, the selected nucleotide monomer is a ribonucleotide having 2'- and 5'-protecting groups. For example, the ribonucleotide can have a 5'-DMT protecting group and a 2'-protecting group selected from: a thiocarbonylaminoformate (TC) protecting group, a bis(2-acetoxyethoxy)methyl (ACE) protecting group, a tert-butyldimethylsilyl (TBDMS) protecting group, a triisopropylsilyloxymethyl (TOM) protecting group, a pivaloyloxymethyl (PivOM) protecting group, and a 2-cyanoethoxymethyl (CEM) protecting group.

[0030] In some embodiments, the selected nucleotide monomer comprises a phosphorus protecting group, whereby the phosphorus moiety of the oligonucleotide is linked to the phosphorus protecting group. In some embodiments, the phosphorus moiety is a phosphate, phosphoramidite, or H-phosphonate group. In some embodiments, the phosphorus protecting group is a methyl or cyanoethyl (e.g., β-cyanoethyl) group. The methyl group can be removed using, for example, benzenethiol or sodium 2-carbamoyl-2-cyanoethylidene-1,1-dithiolate. The cyanoethyl group can be removed using, for example, a non-nucleophilic amine or a hindered amine such as diethylamine, tert-butylamine, or 1,8-diazabicycloundec-7-ene (DBU).

[0031] In some embodiments, the selected nucleotide monomers comprise nucleobase protecting groups. Any nucleobase in the oligonucleotide can comprise a nucleobase protecting group. In some embodiments, the nucleobase protecting group is acetyl, isobutyryl, benzoyl, etc. For example, the protected nucleobase can be N 6 -benzoyl-A, N 6 -isobutyryl-A, N 4 -acetyl-C, N 4 -isobutyryl-C or N 2 -isobutyryl-G (including amidine protecting groups such as dimethylacetamidine, etc.). In some embodiments, the nucleobase protecting group is removed by contacting the oligonucleotide with a polyamine. In some embodiments, the nucleobase protecting group is removed by contacting the oligonucleotide with a diamine such as 1,2-diaminoethane. In some embodiments, exposing the oligonucleotide to 1,2-diaminoethane at room temperature for 2 hours results in deprotection of the nucleobase. In some embodiments, the nucleobase protecting group is benzoyl, isobutyryl, acetyl, phenoxyacetyl, tert-butylphenoxyacetyl, dimethylformamidine, dimethylacetamidine, etc. In some embodiments, the selected nucleotide monomers comprise modified nucleobases, such as 5-alkyl-pyrimidines (such as 5-methyl-C (m 5 C), 5-hydroxymethyl-C (5hmC), 5-methyl-U (m 5 U)), N 6 -alkyl-purines (such as N 6 -methyl-A (m 6 A)), pseudouridine (Φ), 1-methylpseudouridine (m 1 Φ)), 2-thiouridine (2sU), 5-fluoro-U (5FU), 7-deazapurine or any other known modified nucleobase used in oligonucleotide synthesis (see, for example, Hu, B., Zhong, L., Weng, Y. et al. Therapeutic siRNA: state of the art. Sig Transduct Target Ther 5, 101 (2020). https: / / doi.org / 10.1038 / s41392-020-0207-x. and Q. Chen, Y. Zhang, H. Yin. Recent advances in chemical modifications of guide RNA, mRNA and donor template for CRISPR-mediated genome editing. Adv. Drug Deliv. Rev., 168 (2021), pp. 246-258).

[0032] The synthesis of oligonucleotides can be carried out in the 3' to 5' direction, where the final protecting group is removed from the 5'-hydroxyl of the resulting oligonucleotide, or the synthesis can be carried out in the 5' to 3' direction, where the final protecting group is removed from the 3'-hydroxyl of the resulting oligonucleotide.

[0033] The synthesized oligonucleotides can be treated with various basic amines (ammonia, ammonium hydroxide, methylamine, ethylenediamine, etc.) to optionally remove various protecting groups, nucleobase protecting groups, and base-labile 2'-hydroxy protecting groups (if present), such as TC or PivOM, and may cleave the linker, typically a succinate linker or unylinker that attaches the oligonucleotide to the solid support.

[0034] Any suitable conditions can be used to remove the 2'-hydroxy protecting group. In some embodiments, the TC or PivOM protecting group is removed from the 2'-hydroxy by contacting the oligoribonucleotide with a diamine such as 1,2-diaminoethane. In some embodiments, the PivOM protecting group is removed from the oligoribonucleotide by contacting the oligoribonucleotide with ammonia or an alkylamine. In some embodiments, contacting the oligoribonucleotide with a diamine, ammonia, or an alkylamine results in simultaneous deprotection of the 2'-hydroxy and cleavage of the oligoribonucleotide from the solid support. In some embodiments, the ACE protecting group is removed by contacting the oligoribonucleotide with an acid.

[0035] After coupling of the last nucleotide monomer in the desired oligonucleotide product sequence, the synthesized oligonucleotide can be cleaved from the solid support, can be partially (for some oligoribonucleotides that still have 2'-hydroxy protecting groups) or fully deprotected, and then can be eluted from the solid support in a buffer and can exit the reaction chamber through its outlet. A typical buffer can contain 0.2 M sodium phosphate, 0.6 M sodium chloride (pH 7.4), and 10% dimethylformamide (loading buffer). In some embodiments, the oligonucleotide is treated with 1,2-diaminoethane, thereby simultaneously removing the 2'-OH protecting group (such as TC or PivOM), nucleobase amine protecting groups (such as acetyl, benzoyl, isobutyryl, phenoxyacetyl, amidine), and phosphorus protecting groups (i.e., β-cyanoethyl) from the oligonucleotide and cleaving the oligonucleotide from the synthesis solid support prior to elution.

[0036] The nucleotide monomers contain reactive phosphorus groups, such as phosphoramidites, H-phosphonates or other reactive phosphorus or modified groups, such that they can be coupled to a hydroxyl group embedded on a solid surface or on a growing oligonucleotide. The nucleotide monomers are derived from protected 2'-deoxynucleosides (dA, dC, dG, and T), protected ribonucleosides (A, C, G, and U), or protected chemically modified nucleosides. A typical desired oligonucleotide product contains internucleotide linkages between the 5'-hydroxyl and 3'-hydroxyl of adjacent nucleotides. However, it may also be desirable to form unnatural internucleotide linkages between any hydroxyl groups on adjacent nucleotides.

[0037] "Nucleotide" refers to the subunit of an oligonucleotide (whether DNA, RNA, or an analogue thereof) which includes a phosphate group, a sugar group, and a heterocyclic base, as well as analogues of such subunits. Other groups (e.g., protecting groups) may be attached to any component of the nucleotide. "Nucleoside" or "nucleoside moiety" refers to the oligonucleotide subunit that includes a sugar group and a heterocyclic base, as well as analogues of such subunits.

[0038] The terms "nucleoside" and "nucleotide" are intended to include those moieties containing not only the known purine and pyrimidine bases, such as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U), but also other heterocyclic bases that have been modified. Pyrimidines, purines, heterocyclic bases, and modified heterocyclic bases are referred to herein as "nucleobases". Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated heterocycles, or other heterocycles. Such modifications include, for example, diaminopurine and its derivatives, inosine and its derivatives, alkylated purines or pyrimidines, acylated purines or pyrimidines, thiolated purines or pyrimidines, etc., or the addition of protecting groups, such as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl, phenoxyacetyl, dimethylformamidine, dibutylformamidine, N,N-diphenylcarbamate, etc. In addition, the terms "nucleoside" and "nucleotide" include those moieties containing not only conventional ribose and deoxyribose, but also other sugars. Modified nucleosides or nucleotides also include modifications to the sugar moiety, e.g., where one or more hydroxyl groups are replaced by a halogen atom or an aliphatic group, or are functionalized as an ether, an amine, etc. Modified nucleosides or nucleotides also include modifications to the internucleotide linkages or backbone moieties. "Analogue" refers to a molecule having structural features recognized in the literature as mimetics, derivatives, having a similar structure, or other similar terms, and includes, for example, polynucleotides incorporating unnatural (not normally found in nature) nucleotides, unnatural nucleotide mimetics (such as 2'-modified nucleosides), peptide oligonucleotides, oligonucleoside phosphonates, and any polynucleotide having added substituents (such as protecting groups or linking groups).

[0039] "Oligonucleotide" refers to a compound containing multiple nucleoside partial subunits linked by internucleotide linkages. Thus, the term also refers to a compound containing multiple nucleotide partial subunits or residues. Oligonucleotides can contain ribonucleosides or deoxyribonucleosides or mixtures thereof. Oligonucleotides can include natural and / or unnatural nucleosides, nucleoside analogs, and modified nucleosides, and can include natural (phosphodiester) and / or unnatural internucleotide linkages (e.g., phosphorothioates, phosphonates, boranophosphonates, aminophosphates, amide linkages such as peptide nucleic acids (PNAs)).

[0040] The inlet stream can be fed into the reaction chamber by any suitable technique. Generally, the reaction chamber will have one or more inlets connected to sources of reactive nucleotide monomers, other reagents for coupling and deprotection reactions, and one or more diluents. The inlet stream typically contains the selected nucleotide monomer(s) intended to be added to the oligonucleotide being synthesized. However, it is contemplated that the methods and systems of the present invention can reliably and rapidly detect whether a selected nucleotide monomer is likely absent from the inlet stream (when the nucleotide monomer is intended to be present) due to mechanical failure or operator error. In some embodiments, the method further includes stopping the feeding of the inlet stream into the reaction chamber if it is determined that the outlet stream does not contain the selected nucleotide monomer.

[0041] In some embodiments, the inlet stream is fed from one or more reservoirs that are fluidly connected to the inlet of the reaction chamber. The reservoirs can be directly or indirectly connected to the inlet, such as via a series of conduits through which one or more valves are inserted. One of the reservoirs can be a source of the selected nucleotide monomer, and it can be fluidly connected to the inlet stream prior to the feeding step, such as by opening a valve disposed between the reservoir and the inlet. In some embodiments, the step of determining whether the outlet stream contains the selected nucleotide monomer or its derivative is performed prior to the subsequent feeding of nucleotide monomers into the reaction chamber.

[0042] In some embodiments of the methods and systems, oligonucleotides are synthesized in a reaction chamber configured for solid-phase synthesis. In some embodiments, the reaction chamber contains a solid support, such as a planar substrate or beads. In some embodiments, the solid-phase support includes a plurality of discrete resin sheets. In some embodiments, the plurality of discrete resin sheets includes a plurality of resin beads.

[0043] In some embodiments, oligonucleotides are prepared via solid-phase synthesis. In such embodiments, the synthesis is conducted on a solid support maintained between two filters in a column, the filters enabling free passage of reagents and solvents. In some embodiments, the synthesis is conducted on a flat surface. In some embodiments, the synthesis is conducted on a non-planar surface. In some embodiments, the synthesis is conducted on the surface of a support, the surface of the support on which the synthesis occurs resisting diffusion, absorption, or penetration of the reagents and chemicals associated with oligonucleotide synthesis beyond or into the body of the support (as opposed to some polymeric oligonucleotide reaction chamber supports that permit such diffusion and penetration such that oligonucleotide synthesis occurs within the body of the support). In some embodiments, the synthesis is conducted on a substantially smooth surface of a support, the surface on which the synthesis occurs being at most minimally uneven such that the degree of unevenness (if any) will not substantially affect the rate at which reagents can be uniformly applied to the surface, mixed on the surface, or removed from the surface (as opposed to some controlled pore glass oligonucleotide reaction chamber supports that contain pores and unevennesses that slow the application and removal of reagents). The solid, substantially smooth surface need not be flat and can include, for example, flat surfaces, tubes, cylinders, arrays of depressions or pores, and combinations of these elements, as well as other designs presenting surface portions having the above-described properties. For example, a substantially smooth surface includes a surface (or portion of a surface) that can be addressed by an inkjet printhead.

[0044] In some embodiments, the oligonucleotide is attached to a solid support (such as controlled pore glass) or a polymeric support (e.g., a polystyrene (PS) support). Suitable solid supports are polymeric in some cases and can have a variety of forms and compositions and be derived from naturally occurring materials, naturally occurring materials that have been synthetically modified, or synthetic materials. Examples of suitable support materials include, but are not limited to, polysaccharides (such as agarose (e.g., agarose available commercially from Pharmacia as Sepharose and dextran (e.g., those available commercially under the trade names Sephadex and Sephacryl also available commercially from Pharmacia)), polyacrylamide, polystyrene, polyvinyl alcohol, copolymers of hydroxyethyl methacrylate and methyl methacrylate, silica, Teflon, glass, and the like.

[0045] In some embodiments, the synthesis of oligonucleotides is carried out on a solid phase (e.g., CPG), which contains a 3'-terminal nucleotide linked to the solid phase by a base-labile linker (such as a succinate linker or a universal support linker (e.g., Glen Research UnySupport) or UnyLinker). The synthesis cycle includes deprotection of the 5'-OH protecting group (e.g., trityl or DMT), coupling of the phosphoramidite in the presence of an activator, capping of unreacted hydroxyl groups with a base-labile protecting group (e.g., an acetyl protecting group), and oxidation of the phosphite triester linkage to a phosphate triester linkage.

[0046] The reaction chamber can include or be connected to a device that provides multiple features to complete oligonucleotide synthesis, and such features and devices include, but are not limited to, one or more of the following: delivery of multiple reagents in liquid form from reagent reservoirs to the reaction chamber; delivery of multiple reagents to a vessel that can be used to mix the reagents before delivery to the reaction chamber; purging of argon at a steady flow rate with a needle valve to maintain an inert atmosphere in all reservoirs and the reaction chamber; allowing for the delivery of reagents in a controlled metered manner to ensure a specified delivery volume; a mechanism for allowing the resin and internal solvent to be stirred and mixed; a UV detector connected to the outlet to detect the UV absorbance of the outlet stream leaving the reaction chamber; a pressure sensor for detecting the pressure at which the reaction chamber is maintained throughout the synthesis; a pump that connects a solvent reservoir to the reaction chamber and then to a waste line to allow the solvent to be fed into the reaction chamber, flow through the reaction chamber, and go directly to waste; a pump that allows the outlet stream from the reaction chamber to flow back to the inlet of the reaction chamber, thereby allowing the solvent to circulate within the reaction chamber; a specific configuration of pumps and valves, where the delivery of reactive nucleotide monomers to the reaction chamber is carried out through one valve block and pump, and the reagents are delivered to the reaction chamber through a separate valve block and pump to prevent cross-contamination and ensure the ability to clean the valves and lines prior to subsequent reagent and monomer deliveries.

[0047] In addition to feeding the inlet stream into the reaction chamber, the method of the present invention also includes providing conditions for oligonucleotide synthesis in the reaction chamber. Such conditions can be established before, during, or after feeding the inlet stream into the reaction chamber, such that these conditions are present for at least a portion of the time when the contents of the inlet stream are in the reaction chamber. Conditions for oligonucleotide synthesis include the selection and concentration of nucleotide monomers, the selection and concentration of reagents, temperature elevation or reduction, etc. In some embodiments, the conditions are adjusted or selected to complete one or more steps of the method of synthesizing an oligonucleotide. For example, the conditions can cause the method to include one or more activation steps, one or more coupling steps, one or more capping steps, one or more oxidation steps, one or more detritylation steps, one or more cleavage steps, and / or one or more deprotection steps.

[0048] In some embodiments, the synthesis method of the present invention further comprises one or more of the following steps: directly connecting or coupling a first nucleotide to a solid support or indirectly connecting or coupling it to a solid support via the 5'-OH of a nucleoside that has been connected to the solid support via a linker; removing a 5'-protecting group (such as DMT) from the first nucleotide; activating the 3'-phosphoramidite of the incoming selected monomer; coupling the selected nucleotide monomer to the 5'-OH of the first nucleotide, whereby the selected nucleotide monomer becomes the second nucleotide of the growing oligonucleotide, and the second nucleotide is connected to the first nucleotide connected to the solid support via a phosphite triester; oxidizing the phosphite triester to form a phosphate triester; capping any unreacted 5'-OH nucleotides, such as by acetylation.

[0049] The methods and systems of the present invention may also include diverting an effluent stream from the reaction chamber to an analysis station. The timing of diverting the effluent stream may be selected based on the synthesis cycle; typically, the effluent stream will be diverted for analysis before or after the expected completion of the cycle. The effluent stream or a sample of the effluent stream can then be analyzed by mass spectrometry at the analysis station. If it is determined by MS analysis that the effluent stream contains the selected nucleotide monomer or its derivative, it is thereby confirmed that the selected monomer was indeed fed to the reaction chamber in accordance with the defined sequence of the oligonucleotide to be synthesized and in the appropriate order.

[0050] In some embodiments, MS analysis determines the presence of derivatives of the selected monomer in the effluent stream. Derivatives expected to be in the effluent stream include nucleotide monomers that have lost one or more protecting groups such as phosphorus protecting groups (e.g., methyl or cyanoethyl) or are present in their activated form (i.e., diisopropylamine has been replaced by a tetrazole activator or ethylthiotetrazole (ETT)), or the phosphoramidite has been hydrolyzed to the corresponding H-phosphonate.

[0051] In some embodiments, the method includes re-coding one or more time points related to the synthesis of the oligonucleotide or the operation of the reaction chamber or its associated devices. For example, the method may include applying a timestamp to the results from MS analysis and recording the feed times when various inlet streams are fed to the reaction chamber. The method may also include associating the timestamp with the feed times of the inlet streams and using the timestamp and the associated feed times to determine the presence of the selected nucleotide monomer in the inlet streams. In some embodiments, the method includes using the timestamp and the associated feed times to calculate the nucleotide sequence of the oligonucleotide synthesized in the reaction chamber. In some embodiments, the feed times are recorded each time the composition of the inlet stream changes. In some embodiments, the feed times are associated with the timestamp applied to the MS analysis results, such as by associating the timestamp with the feed times when they are separated by a selected time interval. The interval may be predetermined, fixed, or variable.

[0052] In some embodiments, one or more timestamps and one or more feed times are used to determine the presence of selected nucleotide monomers in the inlet stream. In some embodiments, the method further includes a residence time, which corresponds to the measured or estimated time that the reactants are present in the reaction chamber. The association between the timestamp and the feed time can be based on or related to the residence time.

[0053] In some embodiments, timestamps and associated feed times are used to confirm the synthesis of the desired oligonucleotide to calculate the nucleotide sequence of the oligonucleotide synthesized in the reaction chamber.

[0054] In some embodiments, the recorded time points are used as a quality control measure or to verify the oligonucleotide sequence. In some embodiments, the method further includes assigning or approving the synthesized oligonucleotide if the calculated nucleotide sequence matches a predetermined nucleotide sequence. In some embodiments, the method further includes withholding or rejecting the synthesized oligonucleotide if the calculated nucleotide sequence does not match a predetermined nucleotide sequence. In some embodiments, the selected nucleotide monomers in the inlet stream are not analyzed prior to the feed step because the analysis of the outlet stream is sufficient as a quality control measure.

[0055] In some embodiments, the method of the present invention is a current cGMP-compliant manufacturing process, including appropriate quality control for producing the desired oligonucleotide product. In some embodiments, the method of the present invention is used to synthesize the desired oligonucleotide product suitable for use as an active pharmaceutical ingredient (API) and suitable for clinical use. In some embodiments, the present disclosure provides methods and systems for controlling the quality of oligonucleotides produced in solid-phase synthesis, wherein the methods include a cGMP-compliant manufacturing process.

[0056] In some embodiments, the method of the present invention further includes comparing the MS analysis results of the outlet stream or a sample thereof with one or more predetermined MS patterns. In some embodiments, the MS analysis includes evaluating the spectral results of fragments having a mass of 250 - 1000 Da. For example, when the selected nucleotide monomer is 793.3 and if the MS detects a fragment having a mass of 792.3 - 794.3 Da, it is determined that the outlet stream contains the selected nucleotide monomer.

[0057] In some embodiments, the method further comprises one or more steps for purifying the oligonucleotide. For example, the method can include synthesizing the oligonucleotide on a solid support; reacting the oligonucleotide with an orthoester linker to form an oligonucleotide-orthoester linker conjugate, wherein the orthoester linker comprises an affinity tag; cleaving the oligonucleotide-orthoester linker conjugate from the solid support used for synthesis; separating the oligonucleotide-orthoester linker conjugate using chromatography or an affinity capture method; and cleaving the orthoester linker from the oligonucleotide-orthoester linker conjugate to release the purified oligonucleotide. Further details and teachings regarding oligonucleotide purification can be found in U.S. Patent Application Publication No. 2020 / 0181124A1 to Dellinger et al.

[0058] The methods and systems of the present invention also employ or include a mass spectrometry (MS) instrument. An MS instrument generally can include an ion detector, a mass analyzer, and a detector.

[0059] The mass analyzer can be any device configured to separate, sort, or filter analyte ions based on their respective masses (e.g., mass-to-charge ratio or m / z ratio). Examples of mass analyzers include, but are not limited to, multipole electrode structures (e.g., mass filters, ion traps), time-of-flight (TOF) analyzers, electrostatic analyzers (ESA), and sector magnetic fields. The mass analyzer can include a system of more than one mass analyzer, particularly when ion fragmentation is desired. As an example, the mass analyzer can be tandem MS or MS n system. As another example, the mass analyzer can include a mass filter, followed by a collision cell or other ion fragmentation device, followed by another mass filter or analyzer.

[0060] The MS instrument includes a detector configured to analyze a sample by measuring the mass-to-charge ratio of the ions received by the detector. The results can be presented as a mass spectrum, i.e., a plot of intensity versus mass-to-charge ratio. Mass spectrometry can be used to distinguish the components of a complex mixture. In some embodiments, the MS instrument is selected from a time-of-flight mass spectrometer, a single quadrupole (SQ) LC / MSD, a triple quadrupole (TQ) LC / MS, and a quadrupole time-of-flight (TOF / Q-TOF).

[0061] In some embodiments, MS analysis is performed using a low-resolution detector. The instrument can be configured for direct injection mass spectrometry, liquid chromatography / mass spectrometry, gas chromatography / mass spectrometry, ion mobility / mass spectrometry, supercritical fluid chromatography / mass spectrometry, or any combination thereof. In some embodiments, the outlet stream is analyzed by liquid chromatography / mass spectrometry (LC / MS) or by ultra-high pressure liquid chromatography (UHPLC). In some embodiments, the MS instrument is a low-resolution MS instrument. As used herein, "low resolution" refers to mass spectrometry analysis that gives a mass-to-charge ratio to two decimal places.

[0062] In some embodiments, prior to analysis by mass spectrometry, the outlet stream or a sample thereof is purified using high performance liquid chromatography (HPLC). The outlet stream is typically introduced into the HPLC as a mixture containing multiple components. In some embodiments, the methods of the present invention allow for the separation of nucleotide monomers from other components in the outlet stream that may have a similar size. The one or more other components can be of any type. In some embodiments, reverse-phase HPLC is used to purify the outlet stream or a sample thereof, such as by using a reverse-phase column (e.g., a C5 or C18 hydrocarbon column). In some embodiments, the outlet stream or a sample thereof is purified on a normal-phase HPLC column. The HPLC system can include an injector, a pump, and an HPLC column. In some embodiments, the HPLC system is connected to a triple quadrupole LC-MS, an orbitrap LC-MS, an ion trap LC-MS, or a TOF LC-MS. In some embodiments, the methods and systems of the present invention include an ultra-high pressure liquid chromatography mass spectrometer (UHPLC-MS) fluidly connected to the reaction chamber.

[0063] In some embodiments, the systems of the present invention further include an on-line sample manager between the outlet of the reaction chamber and the MS instrument. The on-line sample manager receives the outlet stream from the reaction chamber and delivers the outlet stream or a sample thereof to the MS instrument. The system can also include one or more pumps between the reaction chamber and the on-line sample manager. The system can also include one or more valves between the on-line sample manager and the MS instrument.

[0064] Other components of the systems of the present invention can include one or more computing devices connected to the reaction chamber and / or its associated components. One or more computing devices connected to the MS instrument; it can be the same computing device connected to the reaction chamber, or it can be a separate stand-alone computing device. For example, the present invention can include one or more computing devices communicatively connected to the reaction chamber, the on-line sample manager, and / or the MS instrument, wherein the one or more computing devices include a processor and a storage medium. The processor is configured to execute instructions stored on the storage medium to determine, based on data from the MS instrument, whether the outlet stream from the reaction chamber contains a selected nucleotide monomer or a derivative thereof. The one or more computing devices can also be configured to create, maintain, regulate, or otherwise control the conditions within the reaction chamber; and / or initiate and / or stop feeding the inlet stream to the reaction chamber; and / or activate and / or deactivate the on-line sample manager. In some embodiments, the instructions stored on the computer medium include instructions for various laboratory functions and / or instrument operations, such as for the operation of a liquid chromatography device. Such instructions can be integrated with or separate from instructions for operating the reaction chamber and / or the MS instrument.

[0065] In some embodiments, the system further includes a sensor at the outlet of the reaction chamber, where the sensor detects the presence or absence of nucleotide monomers in the diluent, or the presence or absence of other components of the inlet stream. The sensor can be communicatively coupled to one or more computing devices, and the one or more computing devices can also be configured to direct the outlet stream to an online sample manager and / or activate the online sample manager if the sensor detects the presence or absence of nucleotide monomers or the presence or absence of another component in the outlet stream.

[0066] After reading the present disclosure, it will be apparent to those of ordinary skill in the art that each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with the features of any other several embodiments without departing from the scope or spirit of the present teachings. Any of the methods recited can be performed in the order of events recited or in any other order that is logically possible.

[0067] It should be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. The defined terms supplement the technical and scientific meaning of the defined terms as commonly understood and accepted in the technical field of the present teachings.

[0068] The term "valve" generally encompasses any structure that can be adjusted (such as by switching or turning on or off) to change the flow path into, out of, and / or through the structure. Generally, the valve is substantially fluid-tight to prevent fluid loss from the flow path. Examples of suitable valves (e.g., a first valve, a second valve, a third valve, or a fourth valve) are rotary valves, such as rotary valves including a stator and a rotor. The rotary valve includes a stator and a rotor, where one or both of the stator and the rotor can be rotated to different rotary valve positions. The stator and the rotor have adjacent surfaces, and one or both are configured to rotate relative to the other. In this embodiment, the valve inlet and one or more outlets are channels or through-holes in the stator. The rotor includes switchable fluid paths, which can be grooves in the rotor surface. By rotating the stator and / or the rotor, the fluid path connects different valve inlets to the valve outlets. Other examples of suitable valves are diaphragm valves. Typical valve materials include metallic materials that may or may not be inert. Ideally, the valve has a low dead volume so as to leave a low flushing time and not trap samples.

[0069] The term "duct" generally encompasses any structure configured to define a flow path for fluid to travel from one point (e.g., the inlet of the duct) to another point (e.g., the outlet of the duct), but a duct can also convey fluid to an intermediate point. A duct can be flexible, rigid, or in some degree or part both. A duct can be relatively long or short, and / or linear or non-linear, so long as it provides a flow path from one component (such as a gas source) to another component (such as an exhaust port). For example, a duct can be a long tube, a short fitting, or a manifold with multiple inlets and / or outlets. A duct typically has an inlet and an outlet, but in some embodiments, a duct can have multiple inlets and / or outlets, such as where a duct with two or more inlets converges or joins to one outlet, or where a duct with one inlet bifurcates or splits into two or more outlets. A duct is often described by its length and inner diameter (i.d.), which can be used to calculate the volume of the duct. The geometry of a duct can vary broadly and includes circular, rectangular, square, D-shaped, trapezoidal, or other polygonal cross-sections. A duct can include varying geometries (e.g., a rectangular cross-section at one section and a trapezoidal cross-section at another section).

[0070] The term "connected" means that two components are fluidly connected, physically connected, and / or communicatively connected. The term "fluidly connected" means that two components are in fluid communication and includes both a direct connection between the two components and an indirect connection where one or more other components are in the flow path between the two components. For example, when fluid flows from a first component to a second component, the first and second components are fluidly connected if the outlet of the first component is physically connected to the inlet of the second component, or if a duct connects the first and second components, or if one or more intermediate components (such as valves, pumps, or other structures, etc.) are located between the two components, and vice versa. Components can be physically connected in any suitable manner, such as by using ferrules, brazing, and other means. Generally, for the devices of the present invention, a physical connection that is fluid-tight and / or minimizes dead volume is desired. The term "communicatively connected" means that two components are able to exchange information, for example, by transmitting and receiving communication data. Components can be communicatively connected via a wired communication network or a wireless communication network. A wired network typically includes circuitry for wired communication and complies with known communication standards such as Ethernet, and performs the process of transmitting communication data to and receiving communication data from an external computing device via a network such as the Internet. A wireless network (such as WiFi) typically includes communication via radio or other waves and can be via an antenna. Wireless communication typically complies with known communication standards such as IEEE 802.11 to perform the process of transmitting the transmitted and received communication data to and receiving the communication data from an external component.

[0071] As used herein, the term "linker" refers to a hydrocarbon chain (e.g., (C1-C12) alkylene, (C2-C12) alkenylene, (C2-C12) alkynylene), which is optionally substituted with substituents or interspersed with other atoms, as shown by -(CHR’)a-Wb-(CHR’)c-Vd-(CHR’)e-, where W and V are independently -O-, -S-, or -NR'-; R’ is H or (C1-C6) alkyl; and a, b, c, d, and e are independently integers from 0 to 10, preferably from 0 to 6, or preferably from 0 to 3, and the sum of a, b, c, d, and e is preferably an integer from 2 to 6. The hydrocarbon chain may be interspersed with -O-R”, -O-CO-R”, -NR’-R”, -NR’-CO-R”, -CO-NR’-R”, -CO-R”, or combinations thereof, where R’ and R” are independently H or (C1-C6) hydrocarbon groups.

[0072] "Thiocarbamate protecting group" refers to a protecting group that includes a thiocarbonyl group having a nitrogen and an oxygen bonded to the thiocarbonyl carbon atom: -O-C(S)N-.

[0073] The term "electron-withdrawing group" refers to a chemical group that withdraws electrons from the reaction center. Non-limiting examples of electron-withdrawing groups (EWGs) are halogens (e.g., fluorine and chlorine), haloalkyls (e.g., CH2Cl, CF3, etc.), nitriles (-RCN), carbonyls (-COR), sulfonyls (-SO3R), ammonium (N+R3), and nitro (-NO2).

[0074] The term "chromatography" refers to a method for separating one or more compounds, which involves using a stationary phase and a mobile phase or eluent that moves through or across the stationary phase. Non-limiting examples of chromatography include fluorescence affinity purification, high performance liquid chromatography, and gas chromatography.

[0075] The term "alkyl" refers to a straight-chain or branched-chain alkyl, preferably having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbons. Examples of such alkyls include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl, etc. In some embodiments, the alkyl may be a cycloalkyl.

[0076] The alkyl may be unsubstituted or substituted as defined above. The term "halogen-substituted alkyl" refers to an alkyl substituted with one or more halogen atoms. Non-limiting examples include trifluoromethyl, trifluoroethyl, pentafluoroethyl, 2,2,2-trichloroethyl, chloromethyl, etc. The halogen-substituted alkyl may be unsubstituted or substituted as defined above. The term "fluorine-substituted alkyl" refers to an alkyl substituted with one or more fluorine atoms.

[0077] The term "alkenyl" refers to a straight-chain or branched-chain hydrocarbon having preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 carbons and having one or more carbon-carbon double bonds. Non-limiting examples of alkenyl include vinyl, 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl and 2-butenyl. The alkenyl may be unsubstituted or substituted with one or more suitable substituents as defined above. The term "halogen-substituted alkenyl" refers to an alkenyl substituted with one or more halogen atoms. The term "fluoro-substituted alkenyl" refers to an alkenyl substituted with one or more fluorine atoms.

[0078] The term "alkynyl" refers to a straight-chain or branched-chain hydrocarbon having preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 carbons and having one or more carbon-carbon triple bonds. Alkynyl includes, but is not limited to, ethynyl, propynyl and butynyl. The alkynyl may be unsubstituted or substituted with one or more suitable substituents as defined above. The term "halogen-substituted alkynyl" refers to an alkynyl substituted with one or more halogen atoms. The term "fluoro-substituted alkynyl" refers to an alkynyl substituted with one or more fluorine atoms.

[0079] "Carbocycle" or "carbocyclic group" refers to a saturated ring (i.e., cycloalkyl), a partially unsaturated ring (e.g., cycloalkenyl, cycloalkanedienyl, etc.) or an aromatic ring having 3 to 7 carbon atoms as a monocyclic ring, 7 to 12 carbon atoms as a bicyclic ring and up to about 20 carbon atoms as a polycyclic ring. A monocyclic carbocycle has 3 to 6 ring atoms, more usually 5 or 6 ring atoms. A bicyclic carbocycle has, for example, 7 to 12 ring atoms arranged as a bicyclo[4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms or a spiro-fused ring arranged as a bicyclo[5,6] or [6,6] system. Non-limiting examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl and phenyl. Non-limiting examples of bicyclic carbocycles include naphthyl.

[0080] "Heteroalkyl", "heteroalkenyl", and "heteroalkynyl" respectively refer to alkyl, alkenyl, and alkynyl groups in which one or more carbon atoms have been replaced by heteroatoms such as O, N, or S. Any carbon within the alkyl, alkenyl, or alkynyl group (meaning the first carbon, terminal carbon, or internal carbon) can independently be replaced by a heteroatom (O, N, or S). For example, if the carbon atom of the alkyl group connected to the parent molecule is replaced by a heteroatom (such as O, N, or S), the resulting heteroalkyls are respectively alkoxy groups (such as -OCH3, etc.), aminoalkyl groups (such as -NHCH3, -N(CH3)2, etc.), or thioalkyl groups (such as -SCH3). If a non-terminal carbon atom of the alkyl group not connected to the parent molecule is replaced by a heteroatom (such as O, N, or S), the resulting heteroalkyls are respectively alkyl ethers (such as -CH2CH2-O-CH3, etc.), alkylamines (such as -CH2NHCH3, CH2N(CH3)2, etc.), or thioalkyl ethers (such as -CH2-S-CH3). If the terminal carbon atom of the alkyl group is replaced by a heteroatom (such as O, N, or S), the resulting heteroalkyls are respectively hydroxyalkyl groups (such as -CH2CH2-OH), aminoalkyl groups (such as -CH2NH2), or alkyl mercaptan groups (such as -CH2CH2-SH). Heteroalkyl, heteroalkenyl, or heteroalkynyl groups can have, for example, 1 to 24 carbon atoms. C1-C6 heteroalkyl means a heteroalkyl group having 1 to 6 carbon atoms. "Substituted heteroalkyl", "substituted heteroalkenyl", or "substituted heteroalkynyl" means a heteroalkyl, heteroalkenyl, or heteroalkynyl group as defined herein, in which one or more hydrogen atoms have been replaced by non-hydrogen substituents, as defined in the definition of "substituted".

[0081] The term "aryl" refers to an unsubstituted or substituted aromatic carbocyclic substituent, as commonly understood in the art, which has 3 to 7 carbon atoms as a monocyclic ring, 7 to 12 carbon atoms as a bicyclic ring, and up to about 20 carbon atoms as a polycyclic ring, such as phenyl, naphthyl, anthracenyl, indanyl, etc. It should be understood that according to Hückel's rule, the term aryl applies to planar cyclic substituents containing 4n + 2 electrons. An aryl group can be unsubstituted or substituted by one or more suitable substituents, as defined above. The term "halogen-substituted aryl" refers to an aryl group substituted by one or more halogen atoms or halogen-containing substituents. The term "fluoro-substituted aryl" refers to an aryl group substituted by one or more fluorine atoms or fluorine-containing substituents.

[0082] "Arylalkyl" refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom (usually the terminal carbon atom) has been replaced by an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenyleth-1-yl, naphthylmethyl, 2-naphthyleth-1-yl, naphthobenzyl, 2-naphthophenyleth-1-yl, etc. An arylalkyl group can contain 6 to 24 carbon atoms, for example, the sum of the alkyl carbon atoms and the aryl carbon atoms is at most 6 to 24 carbon atoms. The aryl group can be unsubstituted or substituted by one or more suitable substituents, as defined above.

[0083] The term "carbonyl" refers to a substituent containing a carbon double-bonded to oxygen. Examples of such substituents include aldehydes, ketones, carboxylic acids, esters, amides, carbonates, and carbamates. The carbonyl may be unsubstituted or substituted with one or more suitable substituents as defined above.

[0084] The term "amino" refers to any nitrogen-containing moiety. Non-limiting examples of amino are NH2- (primary), RHN- (secondary), and R2N (tertiary), where R is alkyl, alkenyl, alkynyl, aryl, heterocyclic, or heteroaryl. The RHN- and R2N groups may be unsubstituted or substituted as defined above.

[0085] The term "heteroaryl" refers to a monocyclic or bicyclic 5- or 6-membered ring system, where the heteroaryl is unsaturated and satisfies Hückel's rule. Non-limiting examples of heteroaryl include furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, 1,3,4-oxadiazol-2-yl, 1,2,4-oxadiazol-2-yl, 5-methyl-1,3,4-oxadiazole, 3-methyl-1,2,4-oxadiazole, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuryl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolinyl, benzothiazolinyl, quinazolinyl, etc. The heteroaryl may be unsubstituted or substituted as defined above.

[0086] The term "heterocyclic" or "heterocyclic group" refers to a monocyclic, bicyclic or tricyclic moiety containing 1 to 4 heteroatoms selected from O, N and S. The heterocyclic group optionally contains one or more double bonds. Heterocyclic groups include, but are not limited to, azetidinyl, tetrahydrofuranyl, imidazolidinyl, pyrrolidinyl, piperidinyl, piperazinyl, oxazolidinyl, thiazolidinyl, pyrazolidinyl, thiomorpholinyl, tetrahydrothiazinyl, tetrahydrothiadiazinyl, morpholinyl, oxetanyl, tetrahydrodiazinyl, oxazinyl, oxathiazinyl, indolinyl, isoindolinyl, quinuclidinyl, chromanyl, isochromanyl and benzoxazinyl. Non-limiting examples of monocyclic saturated or partially saturated ring systems are tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, imidazolidin-1-yl, imidazolidin-2-yl, imidazolidin-4-yl, pyrrolidin-1-yl, pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-1-yl, piperidin-2-yl, piperidin-3-yl, piperazin-1-yl, piperazin-2-yl, piperazin-3-yl, 1,3-oxazolidin-3-yl, isothiazolidine, 1,3-thiazol-3-yl, 1,2-pyrazolidin-2-yl, 1,3-pyrazolidin-1-yl, thiomorpholinyl, 1,2-tetrahydrothiazin-2-yl, 1,3-tetrahydrothiazin-3-yl, tetrahydrothiadiazinyl, morpholinyl, 1,2-tetrahydrodiazin-2-yl, 1,3-tetrahydrodiazin-1-yl, 1,4-oxazin-2-yl and 1,2,5-oxathiazin-4-yl. The heterocyclic group may be unsubstituted or substituted with one or more suitable substituents, as defined above.

[0087] "Halogen" or "halo" means fluorine, chlorine, bromine and iodine.

[0088] As used in the specification and the appended claims, and in addition to their ordinary meaning, the terms "substantially" or "essentially" mean within the limits or degree acceptable to a person of ordinary skill in the art. For example, "substantially substituted" means that cancellation is acceptable to a person skilled in the art.

[0089] As used in the specification and the appended claims, and in addition to its ordinary meaning, the terms "substantially" and "about" mean within the limits or amount acceptable to a person of ordinary skill in the art. The term "about" generally means plus or minus 15% of the indicated number. For example, "about 10" may indicate a range from 8.5 to 11.5. For example, "substantially the same" means that a person of ordinary skill in the art would consider the items to be the same after comparison.

[0090] In the present disclosure, numerical ranges include the numbers defining the range. It should be recognized that, for purposes of illustration, chemical structures and chemical formulas may be stretched or enlarged.

[0091] Whenever a range of the number of atoms in a structure is indicated (e.g., C1-C 24 alkyl, C2-C 24 alkenyl, C2-C24 alkynyl, etc.), especially considering that substituents can be described by any carbon atom within the sub-range or by any individual number of carbon atoms falling within the indicated range. For example, using the ranges of listing 1 - 24 carbon atoms (e.g., C1 - C 24 ), 1 - 6 carbon atoms (e.g., C1 - C6), 1 - 4 carbon atoms (e.g., C1 - C4), 1 - 3 carbon atoms (e.g., C1 - C3), or 2 - 24 carbon atoms (e.g., C2 - C 24 ) to describe groups (such as alkyl groups) includes and specifically describes alkyl groups having any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 carbon atoms (as appropriate) and any of its sub-ranges (e.g., 1 - 2 carbon atoms, 1 - 3 carbon atoms, 1 - 4 carbon atoms, 1 - 5 carbon atoms, 1 - 6 carbon atoms, 1 - 7 carbon atoms, 1 - 8 carbon atoms, 1 - 9 carbon atoms, 1 - 10 carbon atoms, 1 - 11 carbon atoms, 1 - 12 carbon atoms, 1 - 13 carbon atoms, 1 - 14 carbon atoms, 1 - 15 carbon atoms, 1 - 24 carbon atoms, 2 - 3 carbon atoms, 2 - 4 carbon atoms, 2 - 5 carbon atoms, 2 - 6 carbon atoms, 2 - 7 carbon atoms, 2 - 8 carbon atoms, 2 - 9 carbon atoms, 2 - 10 carbon atoms, 2 - 11 carbon atoms, 2 - 12 carbon atoms, 2 - 13 carbon atoms, 2 - 14 carbon atoms, 2 - 15 carbon atoms, 2 - 16 carbon atoms, 3 - 4 carbon atoms, 3 - 5 carbon atoms, 3 - 6 carbon atoms, 3 - 7 carbon atoms, 3 - 8 carbon atoms, 3 - 9 carbon atoms, 3 - 10 carbon atoms, 3 - 11 carbon atoms, 3 - 12 carbon atoms, 3 - 13 carbon atoms, 3 - 14 carbon atoms, 3 - 15 carbon atoms, 3 - 16 carbon atoms, 3 - 17 carbon atoms, 3 - 18 carbon atoms, 3 - 19 carbon atoms, 3 - 20 carbon atoms, 3 - 21 carbon atoms, 3 - 22 carbon atoms, 3 - 23 carbon atoms, and / or 3 - 24 carbon atoms, as appropriate).

[0092] As disclosed herein, a plurality of numerical ranges are provided. It should be understood that each intermediate value, to the tenth of a unit of the lower limit (unless the context clearly indicates otherwise), between the upper and lower limits of the range, is also specifically disclosed. Each smaller range between any of the stated values or intermediate values in the range and any other of the stated values or intermediate values in the range is encompassed within the invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range, and each range of the limits that are either both included or both not included in these smaller ranges is also encompassed within the invention, subject to any specifically excluded limits in the range. Where the stated range includes one or both of the limits, ranges excluding either or both of the included limits are also included in the invention.

[0093] All patents and patent publications mentioned herein are hereby incorporated by reference in their entirety.

[0094] As used in the specification and the appended claims, the terms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise. Thus, for example, "a device" includes one device and a plurality of devices.

[0095] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Examples General methods

[0096] DNA and RNA strands are chemically synthesized by sequentially conjugating each nucleotide. With each addition of a nucleotide, a portion of the nucleotide solution is transferred from the column outlet of the synthesizer to an online sample manager or other comparable sample preparation and injection device equipped with a mass spectrometer. Sample concentration and / or ion strength adjustment is performed by programming the online sample manager to automatically dilute the sample either before or after sample injection. This results in real-time sample aliquots being available for identification testing. The sample is then automatically prepared and injected into a mass spectrometry detector (MSD) for mass identification.

[0097] Synthesis is performed using an AKTA Oligopilot having positions for delivering a variety of different amide solutions. The synthesis support is loaded into a 2 cm × 2.54 cm synthesis column to provide a reaction chamber for oligonucleotide synthesis. The amides are prepared at a concentration of 200 mM in ACN.

[0098] A sample stream is continuously withdrawn from the outlet of the reaction chamber for analysis by mass spectrometry to determine the identity of the amides and to verify the order of amide addition.

[0099] A sample tee on the reaction chamber downstream of the outlet splits the outlet flow. One branch of the tee is directed to process waste, while the other branch is directed to a sample pump and then to a sample valve on an online sample manager. The sample tee is located near the outlet of the reaction chamber to minimize longitudinal backmixing.

[0100] The sample pump installed between the sample branch of the tee and the mass spectrometer provides a continuous and stable sample flow to the mass spectrometer during synthesis. The sample pump is located near the sample tee to minimize the pipe length between the sample tee and the pump and to maintain sufficient priming pressure for reliable pump function. The pump moves the sample through the post-pump tube of the narrow-bore tube (with an evaluated length of up to 30 meters), across the laboratory to the mass spectrometer. The narrow-bore pipe between the pump and the mass spectrometer is used to maintain the integrity of the sample by minimizing the retention volume in the pipe (thereby minimizing backmixing caused by long-distance longitudinal diffusion).

[0101] The sample valve on the online sample manager typically directs the flow from the pump to sample waste located on the online sample manager. Upon receiving an electrical signal from the reaction chamber, the sample valve redirects the sample flow to collect the sample. The signal is controlled by a specific command inserted into the reaction chamber method code. The flow rate of the sample pump is used to regulate the arrival time of the expected sample to coincide with the sample signal from the reaction chamber. The collected sample can be analyzed immediately or subsequently by mass spectrometry.

[0102] By providing a stable and continuous sample flow from the point at the outlet of the reaction chamber, samples can be collected at any time to verify the identity of the outlet flow from steps other than coupling. Example 1

[0103] In this example, the nucleotide sequence of an oligonucleotide containing eight nucleotides (including modified nucleotides) was confirmed. An 8-base ribonucleic acid (RNA) oligonucleotide sequence composed of 2'-fluoro and 2'-methoxy RNA nucleotides was synthesized using an NCEV system connected to the reaction chamber. The standard nucleic acid solid-phase synthesis protocol of 1) detritylation, 2) coupling, 3) oxidation, and 4) capping was used. The solid support used was CPG and pre-loaded with 3'-DMT-5'dT. The synthesis reagents used are listed in Table 1. All washing steps were carried out with acetonitrile. Table 1. Input summary of the synthesis steps

[0104] The reaction chamber settings were the same as those listed in the general method information section.

[0105] Sample creation and analysis were performed using a customized Agilent 1290 UHPLC consisting of two (2) analytical pumps (sample and mobile phase), an online sample manager (AS) with an external valve and customized rotor seals, and an Agilent 6135XT single quadrupole MSD. When collecting samples using the AS (before sample injection), the samples were automatically diluted with acetonitrile (ACN). The mobile phase pump solution was 0.1% formic acid + 50% ACN. The instrument parameters used are listed in Table 2. Table 2: UHPLC-MS Method Parameters

[0106] The nucleotides were conjugated in the following order: 3’-fAfAmCfCmGfAfGmA-5’. Each nucleotide addition was collected and analyzed. The experimental data are shown in Table 3 and Figures 4 - 11 in. The chemical species detected were the products of water-quenched activated amides and the formation of H-phosphonate species (Scheme 1). When preparing samples for mass spectrometry measurement, water was added to the synthesis matrix.

[0107] Scheme 1 shows the formation of phosphoramidite quenched species: Table 3: Experimental Data from Example 1

[0108] Figure 4 Mass spectrometry data from sample 1-1 with the theoretical chemical structure (fA) are shown. Figure 5 Mass spectrometry data from sample 1-2 with the theoretical chemical structure (fA) are shown. Figure 6 Mass spectrometry data from sample 1-3 with the theoretical chemical structure (mC) are shown. Figure 7 Mass spectrometry data from sample 1-4 with the theoretical chemical structure (fC) are shown. Figure 8 Mass spectrometry data from sample 1-5 with the theoretical chemical structure (mG) are shown. Figure 9 Mass spectrometry data from sample 1-6 with the theoretical chemical structure (fA) are shown. Figure 10 Mass spectrometry data from sample 1-7 with the theoretical chemical structure (fG) are shown. Figure 11 Mass spectrometry data from sample 1-8 with the theoretical chemical structure (mA) are shown.

[0109] Each position of the 8-mer RNA sequence was confirmed. The nucleotides were confirmed to be in the H-phosphonate protected configuration. Example 2

[0110] In this example, the nucleotide sequence of an oligonucleotide containing seven nucleotides (including modified nucleotides) was confirmed. A 7-base ribonucleic acid (RNA) oligonucleotide sequence composed of 2'-fluoro and 2'-methoxy RNA nucleotides was synthesized using the NCEV system connected to the reaction chamber. Solid-phase synthesis was carried out as described in Example 1. After sample injection, the sample was automatically diluted using a flow regulator.

[0111] The settings of the reaction chamber were the same as those listed in the general method section.

[0112] Sample creation and analysis were performed using a customized Agilent 1290UHPLC consisting of three (3) analytical pumps (sample, mobile phase, and flow regulation pump), a flow regulator, a customized online sample manager (AS) with an external valve, and an Agilent 6135XT single quadrupole MSD. Samples were collected in pure form through the AS. After sample injection, the sample was diluted using 0.1% formic acid + 50% ACN through the flow regulator. The mobile phase pump solution was 0.1% formic acid + 50% ACN. The instrument parameters used are listed in Table 4. Table 4: UHPLC-MS method parameters

[0113] The nucleotides were conjugated in the following order: 3'-fAmCfCmGfAfGmA-5'. Each nucleotide addition was collected and analyzed. The experimental data are shown in Table 5 and Figures 12 - 18 below. Table 5: Experimental data from Example 2

[0114] Figure 12 Show the mass spectrometry data of sample 2-1 with the theoretical chemical structure (fA). Figure 13 Show the mass spectrometry data of sample 2-2 with the theoretical chemical structure (mC). Figure 14 Show the mass spectrometry data of sample 2-3 with the theoretical chemical structure (fC). Figure 15 Show the mass spectrometry data of sample 2-4 with the theoretical chemical structure (mG). Figure 16 Show the mass spectrometry data of sample 2-5 with the theoretical chemical structure (fA). Figure 17 Show the mass spectrometry data of sample 2-6 with the theoretical chemical structure (fG). Figure 18 Show the mass spectrometry data of sample 2-7 with the theoretical chemical structure (mA).

[0115] Identify each position of the heptamer RNA sequence. Confirm that the nucleotides are in the H-phosphonate protected configuration. Exemplary Embodiments

[0116] Before describing various exemplary embodiments, it should be understood that the teachings of the present disclosure are not limited to the specific embodiments described, as these can of course vary. Exemplary embodiments provided in accordance with the presently disclosed subject matter can include, but are not limited to, the following:

[0117] Embodiment 1. A method for synthesizing an oligonucleotide and monitoring the synthesis in real time, the method comprising selecting a nucleotide monomer for addition to the oligonucleotide; feeding an inlet stream to a reaction chamber for oligonucleotide synthesis; providing conditions for oligonucleotide synthesis in the reaction chamber; diverting an outlet stream from the reaction chamber to an analysis station; analyzing the outlet stream or a sample thereof by mass spectrometry (MS) at the analysis station; and determining whether the outlet stream contains the selected nucleotide monomer or a derivative thereof based on the results of the MS analysis.

[0118] Embodiment 2. The method according to Embodiment 1, the method further comprising stopping the feeding of the inlet stream to the reaction chamber if it is determined that the outlet stream does not contain the selected nucleotide monomer.

[0119] Embodiment 3. The method according to Embodiment 1 or Embodiment 2, the method further comprising connecting a source of the selected nucleotide monomer to the inlet stream prior to the feeding step.

[0120] Embodiment 4. The method according to any one of Embodiments 1 to 3, wherein the determination step is performed before feeding a subsequent nucleotide monomer to the reaction chamber.

[0121] Embodiment 5. The method according to any one of Embodiments 1 to 4, the method further comprising applying a timestamp to the results of the MS analysis, correlating the timestamp with the feeding time of the inlet stream into the reaction chamber, and using the timestamp and the associated feeding time to determine the presence of the selected nucleotide monomer in the inlet stream.

[0122] Embodiment 6. The method according to Embodiment 5, the method comprising using the timestamp and the associated feeding time to calculate the nucleotide sequence of the oligonucleotide synthesized in the reaction chamber.

[0123] Embodiment 7. The method according to Embodiment 6, the method further comprising assigning or approving the synthesized oligonucleotide if the calculated nucleotide sequence matches a predetermined nucleotide sequence.

[0124] Embodiment 8. The method according to embodiment 6, the method further comprising withholding or rejecting the synthesized oligonucleotide if the calculated nucleotide sequence does not match the predetermined nucleotide sequence.

[0125] Embodiment 9. The method according to any one of embodiments 1 to 8, wherein the outlet stream or a sample thereof is analyzed by liquid chromatography / mass spectrometry (LC / MS).

[0126] Embodiment 10. The method according to any one of embodiments 1 to 9, wherein the MS analysis is performed with a low-resolution detector.

[0127] Embodiment 11. The method according to any one of embodiments 1 to 10, wherein the selected nucleotide monomers of the inlet stream are not analyzed before the feeding step.

[0128] Embodiment 12. The method according to any one of embodiments 1 to 11, wherein the oligonucleotide comprises ribonucleotides, deoxyribonucleotides, or a mixture thereof.

[0129] Embodiment 13. The method according to embodiment 12, wherein the selected nucleotide monomers are 3'-phosphoramidite ribonucleotides having protecting groups at the 2'-, 5'-, and nucleobase positions.

[0130] Embodiment 14. The method according to embodiment 13, wherein the ribonucleotide has a 5'-dimethoxytrityl protecting group and a 2'-protecting group selected from: a thiocarbonyl carbamate (TC) protecting group, a bis(2-acetoxyethoxy)methyl (ACE) protecting group, a tert-butyldimethylsilyl (TBDMS) protecting group, a triisopropylsilyloxymethyl (TOM) protecting group, a pivaloyloxymethyl (PivOM) protecting group, and a 2-cyanoethoxymethyl (CEM) protecting group.

[0131] Embodiment 15. The method according to any one of embodiments 1 to 14, wherein the selected nucleotide monomers are 793.3 Da, and if the MS detects a fragment with a molecular weight of 792.3 - 794.3 Da, it is determined that the outlet stream contains the selected nucleotide monomers.

[0132] Embodiment 16. The method according to any one of embodiments 1 to 15, wherein the selected nucleotide monomers comprise a modified nucleobase.

[0133] Embodiment 17. A system for synthesizing oligonucleotides, the system comprising a reaction chamber, the reaction chamber comprising an inlet, a solid support for oligonucleotide synthesis, and an outlet; and a mass spectrometry (MS) instrument fluidly connected to the outlet of the reaction chamber.

[0134] Embodiment 18. The system according to Embodiment 17, the system further comprising an on-line sample manager between the outlet of the reaction chamber and the MS instrument, whereby the on-line sample manager receives the outlet stream or a sample thereof from the reaction chamber and delivers the outlet stream or a sample thereof to the MS instrument.

[0135] Embodiment 19. The system according to Embodiment 17 or 18, the system further comprising a sample pump between the reaction chamber and the on-line sample manager.

[0136] Embodiment 20. The system according to any one of Embodiments 17 to 19, the system further comprising a flow regulating valve between the on-line sample manager and the MS instrument.

[0137] Embodiment 21. The system according to any one of Embodiments 17 to 20, wherein the solid support is a planar substrate or a bead.

[0138] Embodiment 22. The system according to any one of Embodiments 17 to 21, the system further comprising one or more computing devices communicatively connected to the reaction chamber, the on-line sample manager, and the MS instrument, wherein the one or more computing devices include a processor and a storage medium, and the processor is configured to execute instructions stored on the storage medium to: determine whether the outlet stream from the reaction chamber contains a selected nucleotide monomer or a derivative thereof based on data from the MS instrument.

[0139] Embodiment 23. The system according to Embodiment 22, wherein the one or more computing devices are further configured to: control the conditions in the reaction chamber; initiate and / or stop feeding the inlet stream to the reaction chamber; and / or activate and / or deactivate the on-line sample manager.

[0140] Embodiment 24. The system according to any one of Embodiments 17 to 23, the system further comprising a sensor at the outlet of the reaction chamber, wherein the sensor detects the presence or absence of a nucleotide monomer in the diluent.

[0141] Embodiment 25. The system according to claim 24, wherein the sensor can be communicatively connected to one or more computing devices, and the one or more computing devices are further configured to direct the outlet stream to the on-line sample manager and / or activate the on-line sample manager if the sensor detects the presence of a nucleotide monomer in the outlet stream.

[0142] Embodiment 26. The system according to any one of embodiments 17 to 25, wherein the MS instrument is a low-resolution MS instrument.

[0143] In view of the present disclosure, it is noted that the methods and systems can be implemented in accordance with this teaching. Additionally, the various components, materials, structures, and parameters are included by way of illustration and example only and are not meant to be limiting. In view of the present disclosure, this teaching can be implemented in other applications, and the components, materials, structures, and devices for implementing these applications can be determined while remaining within the scope of the appended claims.

Claims

1. A method for synthesizing an oligonucleotide and monitoring the synthesis in real time, the method comprising: selecting a nucleotide monomer to be added to the oligonucleotide; feeding an inlet stream into a reaction chamber for oligonucleotide synthesis; providing conditions for oligonucleotide synthesis in the reaction chamber; diverting an outlet stream from the reaction chamber to an analysis station; analyzing the outlet stream or a sample thereof by mass spectrometry (MS) at the analysis station; and determining whether the outlet stream contains the selected nucleotide monomer or a derivative thereof based on the results of the MS analysis.

2. The method according to claim 1, the method further comprising: if it is determined that the outlet stream does not contain the selected nucleotide monomer, stopping feeding the inlet stream into the reaction chamber.

3. The method according to claim 1, the method further comprising connecting a source of the selected nucleotide monomer to the inlet stream before the feeding step.

4. The method according to claim 1, wherein the determining step is performed before feeding a subsequent nucleotide monomer into the reaction chamber.

5. The method according to claim 1, the method further comprising applying a timestamp to the results of the MS analysis, associating the timestamp with the feeding time when the inlet stream enters the reaction chamber, and using the timestamp and the associated feeding time to determine the presence of the selected nucleotide monomer in the inlet stream.

6. The method according to claim 5, the method comprising using the timestamp and the associated feeding time to calculate the nucleotide sequence of the oligonucleotide synthesized in the reaction chamber.

7. The method according to claim 6, the method further comprising assigning or approving the synthesized oligonucleotide if the calculated nucleotide sequence matches a predetermined nucleotide sequence.

8. The method according to claim 6, the method further comprising withholding or rejecting the synthesized oligonucleotide if the calculated nucleotide sequence does not match the predetermined nucleotide sequence.

9. The method according to claim 1, wherein the outlet stream or a sample thereof is analyzed by liquid chromatography / mass spectrometry (LC / MS).

10. The method according to claim 1, wherein the MS analysis is performed using a low-resolution detector.

11. The method according to claim 1, wherein the selected nucleotide monomer of the inlet stream is not analyzed before the feeding step.

12. The method according to claim 1, wherein the oligonucleotide comprises ribonucleotides, deoxyribonucleotides or a mixture thereof.

13. The method according to claim 12, wherein the selected nucleotide monomer is a 3'-phosphoramidite ribonucleotide having protecting groups at the 2'-, 5'- and nucleobase positions.

14. The method according to claim 13, wherein the ribonucleotide has a 5'-dimethoxytrityl protecting group and a 2'-protecting group selected from the group consisting of a thiocarbonylamino formate (TC) protecting group, a bis(2-acetoxyethoxy)methyl (ACE) protecting group, a tert-butyldimethylsilyl (TBDMS) protecting group, a triisopropylsilyloxymethyl (TOM) protecting group, a pivaloyloxymethyl (PivOM) protecting group, and a 2-cyanoethoxymethyl (CEM) protecting group.

15. The method according to claim 1, wherein the selected nucleotide monomer is 793.3 Da, and if the MS detects a fragment with a molecular weight of 792.3 - 794.3 Da, it is determined that the outlet stream contains the selected nucleotide monomer.

16. The method according to claim 1, wherein the selected nucleotide monomer comprises a modified nucleobase.

17. A system for synthesizing oligonucleotides, the system comprising: a reaction chamber, the reaction chamber including an inlet, a solid support for oligonucleotide synthesis, and an outlet; and a mass spectrometry (MS) instrument fluidly connected to the outlet of the reaction chamber.

18. The system according to claim 17, the system further comprising an on-line sample manager between the outlet of the reaction chamber and the MS instrument, whereby the on-line sample manager receives the outlet stream or a sample thereof from the reaction chamber and delivers the outlet stream or a sample thereof to the MS instrument.

19. The system according to claim 17, the system further comprising a sample pump between the reaction chamber and the on-line sample manager.

20. The system according to claim 17, the system further comprising a flow control valve between the on-line sample manager and the MS instrument.

21. The system according to claim 17, wherein the solid support is a planar substrate or a bead.

22. The system according to claim 17, the system further comprising one or more computing devices communicatively connected to the reaction chamber, the on-line sample manager, and the MS instrument, wherein the one or more computing devices include a processor and a storage medium, and the processor is configured to execute instructions stored on the storage medium to: determine whether the outlet stream from the reaction chamber contains a selected nucleotide monomer or a derivative thereof based on data from the MS instrument.

23. The system according to claim 22, wherein the one or more computing devices are further configured to: control the conditions within the reaction chamber; initiate and / or stop feeding the inlet stream to the reaction chamber; and / or activate and / or deactivate the on-line sample manager.

24. The system according to claim 23, the system further comprising a sensor at the outlet of the reaction chamber, wherein the sensor detects the presence or absence of a nucleotide monomer in a diluent.

25. The system according to claim 24, wherein the sensor is communicatively connected to the one or more computing devices, and The one or more computing devices are further configured to direct the outlet stream to the online sample manager and / or activate the online sample manager if the sensor detects the presence of nucleotide monomers in the outlet stream.

26. The system of claim 17, wherein the MS instrument is a low-resolution MS instrument.

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