Long oligonucleotide isolation

Abstract

A method for separating an oligonucleotide or nucleic acid biopolymer for analysis includes loading a sample containing a plurality of oligonucleotides to a chromatographic column; flowing a mobile phase formed by combining the first solution and the second solution through the chromatographic column to elute the plurality of oligonucleotides; at least one of the first oligonucleotide and the second oligonucleotide is analyzed using a mass spectrometer. The plurality of oligonucleotides includes a first oligonucleotide and a second oligonucleotide. The first solution includes an ion pair reagent. The ion pair reagent comprises a primary amine or a secondary amine. The ratio of the first solution and the second solution in the mobile phase varies over time to separate the first oligonucleotide from the second oligonucleotide.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. non-provisional application No. 63 / 482,151, filed on January 30, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates generally to the fields of chromatography and mass spectrometry, including methods for separating long oligonucleotides. Background Art

[0004] Due to recent events, particularly the COVID pandemic, mRNA has been widely used as a useful therapeutic, such as various mRNA-based COVID vaccines. The production of non-natural transcripts by in vitro transcription requires analytical workup to determine the reaction outcome and yield. Ion-pair reversed-phase chromatography is widely used for the characterization of proteins and nucleic acids, for example for pharmaceuticals. Ion-pair reversed-phase chromatography can be coupled with UV and mass spectrometry to characterize the main product as well as impurities of pharmaceuticals. However, nucleic acids consist of many structurally and chemically related variants. These variants are often not well resolved. As can be seen from the above, there is a need for improved separation methods for long oligonucleotides. Summary of the Invention

[0005] In a first aspect, a method for separating and analyzing oligonucleotides or nucleic acid biopolymers may include loading a sample containing a plurality of oligonucleotides onto a chromatographic column. The plurality of oligonucleotides may include a first oligonucleotide and a second oligonucleotide. The method may further include passing a mobile phase composed of a first solution and a second solution through the chromatographic column to elute the plurality of oligonucleotides. The first solution may include an ion pair reagent. The ion pair reagent may include a primary amine or a secondary amine. The ratio of the first solution and the second solution in the mobile phase may vary over time to separate the first oligonucleotide from the second oligonucleotide. The method may further include analyzing at least one of the first oligonucleotide and the second oligonucleotide using a mass spectrometer.

[0006] In various embodiments of the first aspect, the sample can include a plurality of oligonucleotides dissolved in the first solution, the second solution, or any combination thereof.

[0007] In various embodiments of the first aspect, the second solution can include an ion-pairing agent.

[0008] In various embodiments of the first aspect, the ion pair reagent can include at least 4 carbon atoms. In specific embodiments, the primary amine of the ion pair reagent can include an alkyl chain having at least 4 carbon atoms, such as pentylamine or hexylamine. In specific embodiments, the secondary amine of the ion pair reagent can include two alkyl chains, at least one of which includes at least 3 carbon atoms, such as N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentylamine, or dihexylamine.

[0009] In various embodiments of the first aspect, the first solution can include a fluoroalcohol, such as hexafluoroisopropanol (HFIP).

[0010] In various embodiments of the first aspect, the first solution can include formic acid or a formates salt, acetic acid or an acetate salt, or any combination thereof.

[0011] In various embodiments of the first aspect, the first solution can include a first organic solvent, such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

[0012] In various embodiments of the first aspect, the second solution can include a second organic solvent, such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

[0013] In various embodiments of the first aspect, the chromatographic column may comprise a stationary phase comprising divinylbenzene or a derivative thereof. In certain specific embodiments, the stationary phase may be porous with an average pore size of about peace treaty and the first and second species may have a molecular weight of less than 10 kDa. In certain embodiments, the stationary phase may be porous with an average pore size of about peace treaty between, and

[0014] The first and second can have a length of no greater than about 300 nt. In certain embodiments, the stationary phase can be porous with an average pore size of about peace treaty and the first and second may have a length greater than 300 nt.

[0015] In various embodiments of the first aspect, the first species and the second species can have a length of no greater than about 15,000 nt.

[0016] In various embodiments of the first aspect, the multiple oligonucleotides can be ribonucleic acid (RNA) oligonucleotides. In multiple specific embodiments, the ribonucleic acid (RNA) oligonucleotides can contain modifications. In multiple specific embodiments, the ribonucleic acid (RNA) oligonucleotides can be produced in vitro. In multiple specific embodiments, the ribonucleic acid (RNA) oligonucleotides can be produced in vivo. In multiple specific embodiments, the ribonucleic acid (RNA) oligonucleotides can be encapsulated RNA oligonucleotides.

[0017] In a second aspect, a method for separating encapsulated oligonucleotides or nucleic acid biopolymers for analysis may include dissolving a sample containing a plurality of encapsulated oligonucleotides. The plurality of encapsulated oligonucleotides may include a first encapsulated oligonucleotide containing a first oligonucleotide and a second encapsulated oligonucleotide containing a second oligonucleotide. Dissolving the sample may include releasing the first and second oligonucleotides. The method may also include loading the dissolved sample onto a chromatographic column; and passing a mobile phase composed of a combination of the first solution and the second solution through the chromatographic column to elute the first and second oligonucleotides. The first solution may include an ion pair reagent. The ion pair reagent may include a primary amine or a secondary amine. The ratio of the first solution and the second solution in the mobile phase may be varied over time to separate the first oligonucleotide from the second oligonucleotide. The method may further include analyzing at least one of the first oligonucleotide and the second oligonucleotide using a mass spectrometer.

[0018] In various embodiments of the second aspect, the sample can include a plurality of oligonucleotides dissolved in the first solution, the second solution, or any combination thereof.

[0019] In various embodiments of the second aspect, the second solution can include an ion-pairing agent.

[0020] In various embodiments of the second aspect, the ion pair reagent can include at least 4 carbon atoms. In specific embodiments, the primary amine of the ion pair reagent can include an alkyl chain having at least 4 carbon atoms, such as pentylamine or hexylamine. In specific embodiments, the secondary amine of the ion pair reagent can include two alkyl chains, at least one of which includes at least 3 carbon atoms, such as N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentylamine, or dihexylamine.

[0021] In various embodiments of the second aspect, the first solution can include a fluoroalcohol, such as hexafluoroisopropanol (HFIP).

[0022] In various embodiments of the second aspect, the first solution can include formic acid or a formates salt, acetic acid or an acetate salt, or any combination thereof.

[0023] In various embodiments of the second aspect, the first solution can include a first organic solvent, such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

[0024] In various embodiments of the second aspect, the second solution can include a second organic solvent, such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

[0025] In various embodiments of the second aspect, the chromatographic column can include a stationary phase comprising divinylbenzene or a derivative thereof. In certain specific embodiments, the stationary phase can be porous with an average pore size of about peace treaty and the first and second species may have a molecular weight of less than 10 kDa. In certain embodiments, the stationary phase may be porous with an average pore size of about peace treaty between, and

[0026] The first and second can have a length of no greater than about 300 nt. In certain embodiments, the stationary phase can be porous with an average pore size of about peace treaty and the first and second may have a length greater than 300 nt.

[0027] In various embodiments of the second aspect, the first species and the second species can have a length of no greater than about 15,000 nt.

[0028] In various embodiments of the second aspect, the first and second oligonucleotides can be ribonucleic acid (RNA) oligonucleotides.

[0029] In various embodiments of the second aspect, the first and second oligonucleotides can be deoxyribonucleic acid (DNA) oligonucleotides.

[0030] In various embodiments of the first aspect, the first and second oligonucleotides may comprise modifications.

[0031] In various embodiments of the second aspect, the first and second oligonucleotides can be produced in vitro.

[0032] In various embodiments of the second aspect, the first and second oligonucleotides can be produced in vivo.

[0033] In a third aspect, a separation method for analyzing viral particles may include dissolving a sample containing a plurality of viral particles. The plurality of viral particles may include a first viral particle containing a first oligonucleotide and a second viral particle containing a second oligonucleotide. Dissolving the sample may include releasing the first and second oligonucleotides. The method may also include loading the dissolved sample onto a chromatographic column; and passing a mobile phase composed of a combination of the first solution and the second solution through the chromatographic column to elute the first and second oligonucleotides. The first solution may include an ion pair reagent. The ion pair reagent may include a primary amine or a secondary amine. The ratio of the first solution and the second solution in the mobile phase may be varied over time to separate the first oligonucleotide from the second oligonucleotide. The method may further include analyzing at least one of the first oligonucleotide and the second oligonucleotide using a mass spectrometer.

[0034] In various embodiments of the third aspect, the sample can include a plurality of oligonucleotides dissolved in the first solution, the second solution, or any combination thereof.

[0035] In various embodiments of the third aspect, the second solution may include an ion-pairing agent.

[0036] In various embodiments of the third aspect, the ion pair reagent can include at least 4 carbon atoms.

[0037] In certain embodiments, the primary amine of the ion pair reagent can comprise an alkyl chain having at least 4 carbon atoms, such as pentylamine or hexylamine.

[0038] In certain embodiments, the secondary amine of the ion pair reagent may include two alkyl chains, at least one of which includes at least 3 carbon atoms, such as N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentylamine, or dihexylamine.

[0039] In various embodiments of the third aspect, the first solution can include a fluoroalcohol, such as hexafluoroisopropanol (HFIP).

[0040] In various embodiments of the third aspect, the first solution may include formic acid or a formates salt, acetic acid or an acetate salt, or any combination thereof.

[0041] In various embodiments of the third aspect, the first solution can include a first organic solvent, such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

[0042] In various embodiments of the third aspect, the second solution can include a second organic solvent, such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

[0043] In various embodiments of the third aspect, the chromatographic column can include a stationary phase comprising divinylbenzene or a derivative thereof. In certain specific embodiments, the stationary phase can be porous with an average pore size of about peace treaty and the first and second species may have a molecular weight of less than 10 kDa. In certain embodiments, the stationary phase may be porous with an average pore size of about peace treaty between, and

[0044] The first and second can have a length of no greater than about 300 nt. In certain embodiments, the stationary phase can be porous with an average pore size of about peace treaty and the first and second may have a length greater than 300 nt.

[0045] In various embodiments of the third aspect, the first species and the second species may have a length of no greater than about 15,000 nt.

[0046] In various embodiments of the third aspect, the first and second oligonucleotides can be ribonucleic acid (RNA) oligonucleotides.

[0047] In various embodiments of the third aspect, the first and second oligonucleotides can be deoxyribonucleic acid (DNA) oligonucleotides.

[0048] In various embodiments of the third aspect, the first and second oligonucleotides may comprise modifications. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] For a more complete understanding of the principles and advantages disclosed herein, reference is now made to the following description taken in conjunction with the accompanying drawings, in which:

[0050] Figure 1A 、 1B , 1C and 1D show the chemical structures of RNA nucleosides A, G, C and U and the positions of potential hydrogen bond donors and hydrogen bond acceptors, respectively.

[0051] Figure 2A and 2B The chemical structures of exemplary tertiary amines triethylamine and diisopropylethylamine are shown, along with the positions of potential hydrogen bond donors and hydrogen bond acceptors, respectively.

[0052] Figure 3A 、 3B The chemical structures of exemplary secondary amines diethylamine, dipropylamine, and dibutylamine and the positions of potential hydrogen bond donors and hydrogen bond acceptors are shown in 3C and 3D, respectively.

[0053] Figure 4A and4B The chemical structures of exemplary primary amines pentylamine and hexylamine are shown, along with the positions of potential hydrogen bond donors and hydrogen bond acceptors, respectively.

[0054] Figure 5A 、 5B 5C and 5C are flow charts of methods of isolating and analyzing oligonucleotides according to various embodiments.

[0055] Figure 6 is a block diagram of an exemplary chromatography system according to various embodiments.

[0056] Figure 7 is a block diagram of an exemplary mass spectrometry system in accordance with various embodiments.

[0057] Figure 8 Shown are analysis results of viral particles according to various embodiments.

[0058] It should be understood that the drawings are not necessarily drawn to scale, nor are the relationships between objects in the drawings necessarily drawn to scale. The drawings are intended to provide clarity and understanding of the various embodiments of the devices, systems, and methods disclosed herein. Wherever possible, the same reference numerals will be used throughout the drawings to refer to the same or similar parts. Furthermore, it should be understood that the drawings are not intended to limit the scope of the present teachings in any way. DETAILED DESCRIPTION

[0059] Described herein are various embodiments of methods for separating long oligonucleotides.

[0060] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

[0061] In the detailed description of various embodiments, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will appreciate that these various embodiments may be practiced with or without these specific details. In other examples, structures and devices are shown in block diagram form. Furthermore, those skilled in the art will readily recognize that the specific order in which the methods are presented and executed is exemplary, and it is contemplated that this order may be varied and still remain within the spirit and scope of the various embodiments disclosed herein.

[0062] All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, treatises, and internet web pages, are expressly incorporated by reference in their entirety for any purpose. 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 the various embodiments described herein belong.

[0063] It should be understood that there is an implicit "about" before the temperatures, concentrations, times, pressures, flow rates, cross-sectional areas, etc. discussed in this teaching, so that slight and insubstantial deviations are within the scope of this teaching. In this application, unless otherwise specifically provided, the use of the singular includes the plural. In addition, the use of "including," "comprising," and "containing" is not intended to be limiting. It should be understood that both the foregoing general description and the following detailed description are exemplary and illustrative only and are not limitations of this teaching.

[0064] As used herein, "a" or "an" may also mean "at least one" or "one or more." Furthermore, the use of "or" is inclusive, such that the phrase "A or B" is true when "A" is true, "B" is true, or both "A" and "B" are true. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0065] "System" describes a set of real or abstract components comprising a whole, in which each component interacts with or relates to at least one other component within the whole.

[0066] Ion-pair chromatography (IPC) is an effective reversed-phase liquid chromatography (RPLC) technique for separating organic ions and partially ionized organic analytes. This technique uses the same types of stationary and mobile phases as RPLC; the key feature of IPC is the inclusion of one or more ion-pairing agents in the mobile phase.

[0067] The purpose of adding an ion-pairing agent to the mobile phase is generally to alter the retention of ionic analytes. By varying the mobile phase concentration of the ion-pairing agent, the retention factor of oppositely charged analytes can be consistently increased by 10-20 times compared to the value in the absence of the ion-pairing agent. Correspondingly, the retention factor of similarly charged analytes may be consistently decreased by 10-20 times. The retention factor of uncharged analytes is generally somewhat unaffected by the presence of the ion-pairing agent.

[0068] IPC has been applied to nearly all areas of analytical chemistry where chromatography is used. In IPC, rich aqueous mobile phases can be used with a variety of buffers and ionic and non-ionic additives, making the technique suitable for separating important classes of biomolecules, particularly amino acids, peptides, proteins, and nucleic acids.

[0069] RNA, or ribonucleic acid, is a type of nucleic acid made up of long chains of nucleotides, each composed of a sugar, a phosphate group, and a base. RNA has four different types of bases: adenine, guanine, cytosine, and uracil. Figures 1A-1D Adenine ( Figure 1A ), guanine ( Figure 1B ), cytosine ( Figure 1C ) and uracil ( Figure 1D ) Chemical structure of a nucleoside (ribose and base, excluding the phosphate group). Groups that can act as hydrogen bond donors are labeled D, and groups that can act as hydrogen bond acceptors are labeled A. Some groups can act as both donors and acceptors and are labeled D and A. As can be seen from these figures, a large number of groups can act as hydrogen bond acceptors, while a smaller number can act as hydrogen bond donors.

[0070] Figure 2A and 2B The structures of exemplary tertiary amines triethylamine (TEA) and diisopropylethylamine (DIPEA) and the positions of potential hydrogen bond donors and hydrogen bond acceptors are shown, respectively. TEA and DIPEA are commonly used ion pair reagents. However, as Figure 2A and 2B As shown, tertiary amines can only act as hydrogen bond acceptors.

[0071] Figure 3A 、 3B 3C and 3D respectively show the chemical structures of exemplary secondary amines diethylamine (DEA), dipropylamine (DPA) and dibutylamine (DBA) and the positions of potential hydrogen bond donors and hydrogen bond acceptors. Figure 3A 、 3B As shown in Figure 3C, secondary amines can act as both hydrogen bond donors and hydrogen bond acceptors.

[0072] Figure 4A and 4B The chemical structures of exemplary primary amines pentylamine and hexylamine are shown, along with the positions of potential hydrogen bond donors and hydrogen bond acceptors. Figure 4A and 4B As shown, primary amines can act as both hydrogen bond donors and hydrogen bond acceptors.

[0073] The use of ion-pairing agents that act as hydrogen bond donors can increase interactions with nucleic acid bases, particularly at the Watson-Crick interface. This hydrogen-bonding interaction with the bases can improve retention time. Because the number of hydrogen bond acceptor sites increases with nucleic acid length, retention time can become length-dependent, thereby improving the separation of large oligonucleotides. Furthermore, because the number and arrangement of hydrogen bond acceptor groups vary between bases, differential retention may occur depending on the sequence of the oligonucleotide.

[0074] Figure 5A An exemplary method 500 for analyzing a sample containing a plurality of oligonucleotides is shown. In various embodiments, the oligonucleotide species can include ribonucleic acid (RNA) oligonucleotides or deoxyribonucleic acid (DNA) oligonucleotides. The oligonucleotides can contain natural or non-natural modifications. The oligonucleotides can be produced in vitro or in vivo. In various embodiments, the oligonucleotides can be encapsulated RNA oligonucleotides, which are encapsulated in a structure containing lipids, proteins, or any combination thereof.

[0075] At 502, an ion pairing reagent can be combined with a sample. In various embodiments, the sample can be dissolved or resuspended in a first solution, a second solution, or any combination thereof. The ion pairing reagent can include a primary amine or a secondary amine. In various embodiments, the ion pairing reagent can include at least about 4 carbon atoms, but typically no more than about 16 carbon atoms.

[0076] The primary amine ion pair reagent can include an alkyl chain having at least 4 carbon atoms. In certain embodiments, the primary amine ion pair reagent can include pentylamine or hexylamine.

[0077] The secondary amine ion pair reagent may comprise two alkyl chains, at least one of which comprises at least 3 carbon atoms, but typically less than 8 carbon atoms, such as N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentylamine, or dihexylamine.

[0078] At 504, the sample and ion pair reagent may be loaded onto a chromatography column. In various embodiments, the chromatography column may include a stationary phase comprising divinylbenzene or a derivative thereof. In various embodiments, the stationary phase may be porous, for example, having an average pore size of at least about For example, the average pore size is at least about Even the average pore size is at least about The choice of average pore size may be influenced by the size of the oligonucleotides to be separated. to about The stationary phase can be used to separate oligonucleotides with a size less than 10 kDa. The average pore size is about to about The stationary phase can be used to separate oligonucleotides with a length of no more than about 300 nt. to about Stationary phases can be used to separate oligonucleotides greater than about 300 nt in length, but typically not greater than about 15,000 nt in length.

[0079] At 506, an eluent can be applied to a chromatographic column to elute a plurality of oligonucleotides. Typically, the sample solution conditions when applied to the chromatographic column are preferably close to the starting conditions of the eluent gradient. In various embodiments, the eluent can include a combination of a first solution and a second solution. In a plurality of specific embodiments, the ratio of the first solution to the second solution can be varied to form gradient solvent conditions, thereby causing the separation of various oligonucleotides based on differential retention within the chromatographic column, such as the separation of a first oligonucleotide from a second oligonucleotide.

[0080] In various embodiments, only the concentration of the ion-pairing agent in the first solution and the second solution varies, and the gradient solvent conditions include a gradient in the concentration of the ion-pairing agent. For example, the second solution may contain more or less ion-pairing agent than the first solution, or even only the first solution may contain the ion-pairing agent while the second solution does not contain the ion-pairing agent. In other embodiments, the first and second solutions may contain the same concentration of the ion-pairing agent, such that the concentration of the ion-pairing agent remains substantially constant throughout the separation process. In various embodiments, the concentration of the ion-pairing agent may be between about 1 mM and about 500 mM, such as between about 5 mM and about 500 mM.

[0081] In various embodiments, the first solution may include a fluoroalcohol, such as hexafluoroisopropanol (HFIP). In various embodiments, the concentration of the fluoroalcohol may be between about 5mM and about 500mM. In a plurality of alternative embodiments, the fluoroalcohol may not be included. In various embodiments, the first solution may include formic acid or formate, acetic acid or acetate, or any combination thereof, to achieve a pH in the range of, for example, about 2 to about 11, such as about 4 to 10. In a plurality of alternative embodiments, the first solution may not include formic acid or formate, acetic acid or acetate. In various embodiments, the first solution may include a first organic solvent, such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof. In various embodiments, the concentration of the organic solvent may be between about 0% and about 100%. When less than about 100%, the remaining solution may include water.

[0082] In various embodiments, the second solution can include similar or identical components as the first solution at different concentrations. For example, the second solution can include a fluoroalcohol, formic acid, a formate, acetic acid, an acetate, and / or an organic solvent. In other embodiments, the second solution can include at least some components that are different from the first solution.

[0083] The oligonucleotide species can be determined using a detector (eg, a UV / VIS detector or a mass spectrometer) at 508. Additionally, the sample can be analyzed based on the signal and retention time observed by the detector to identify the oligonucleotide species and / or quantify the oligonucleotide.

[0084] Figure 5A An exemplary method 530 for analyzing a sample containing a plurality of encapsulated oligonucleotides is shown. In various embodiments, the encapsulated RNA oligonucleotide can be encapsulated in a structure containing a lipid, a protein, or any combination thereof. In various embodiments, the encapsulated oligonucleotide species can include ribonucleic acid (RNA) oligonucleotides or deoxyribonucleic acid (DNA) oligonucleotides. The oligonucleotides can contain natural or non-natural modifications. The oligonucleotides can be produced in vitro or in vivo.

[0085] At 532, an ion-pairing reagent can be combined with the sample. In various embodiments, the sample can be dissolved or resuspended in the first solution, the second solution, or any combination thereof. In various embodiments, dissolving or resuspending the sample can dissolve lipids and / or denature proteins of the encapsulated structure, thereby releasing the oligonucleotides contained therein into solution.

[0086] The ion pair reagent can include a primary amine or a secondary amine. In various embodiments, the ion pair reagent can include at least about 4 carbon atoms, but typically no more than about 16 carbon atoms.

[0087] The primary amine ion pair reagent can include an alkyl chain having at least 4 carbon atoms. In certain embodiments, the primary amine ion pair reagent can include pentylamine or hexylamine.

[0088] The secondary amine ion pair reagent may comprise two alkyl chains, at least one of which comprises at least 3 carbon atoms, but typically less than 8 carbon atoms, such as N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentylamine, or dihexylamine.

[0089] At 534, the sample and ion pair reagent can be loaded onto the chromatography column. In various embodiments, the chromatography column can include a stationary phase comprising divinylbenzene or a derivative thereof. In various embodiments, the stationary phase can be porous, for example, having an average pore size of at least about For example, the average pore size is at least about Even the average pore size is at least about The choice of average pore size may be influenced by the size of the oligonucleotides to be separated. to about The stationary phase can be used to separate oligonucleotides with a size less than 10 kDa. The average pore size is about to about The stationary phase can be used to separate oligonucleotides with a length of no more than about 300 nt. to about Stationary phases can be used to separate oligonucleotides greater than about 300 nt in length, but typically not greater than about 15,000 nt in length.

[0090] At 536, an eluent can be applied to the chromatographic column to elute the plurality of oligonucleotides. Typically, the sample solution conditions when applied to the chromatographic column are preferably close to the starting conditions of the eluent gradient. In various embodiments, the eluent can include a combination of a first solution and a second solution. In particular embodiments, the ratio of the first solution to the second solution can be varied to form gradient solvent conditions, thereby causing the separation of the various oligonucleotides based on differential retention within the chromatographic column, e.g., separation of a first oligonucleotide from a second oligonucleotide.

[0091] In various embodiments, the concentration of the ion-pairing agent in the first and second solutions can vary, and the gradient solvent conditions can include a gradient in the concentration of the ion-pairing agent. For example, the second solution can contain more or less ion-pairing agent than the first solution, or even the first solution can contain the ion-pairing agent while the second solution does not. In other embodiments, the first and second solutions can contain the same concentration of the ion-pairing agent, such that the concentration of the ion-pairing agent remains substantially constant throughout the separation process. In various embodiments, the concentration of the ion-pairing agent can be between about 5 mM and about 500 mM.

[0092] In various embodiments, the first solution may include a fluoroalcohol, such as hexafluoroisopropanol (HFIP). In various embodiments, the concentration of the fluoroalcohol may be between about 5 mM and about 500 mM. In various embodiments, the first solution may include formic acid or a formate, acetic acid or an acetate, or any combination thereof, to achieve a pH in the range of, for example, about 2 to about 11, such as about 4 to 10. In various embodiments, the first solution may include a first organic solvent, such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof. In various embodiments, the concentration of the organic solvent may be between about 0% and about 100%. When less than about 100%, the remaining solution may include water.

[0093] In various embodiments, the second solution can include similar or identical components at different concentrations as the first solution. For example, the second solution can include a fluoroalcohol, formic acid, a formate salt, acetic acid, an acetate salt, and / or an organic solvent.

[0094] In other embodiments, the second solution may contain at least some different components than the first solution.

[0095] The oligonucleotide species can be determined using a detector (e.g., a UV / VIS detector or a mass spectrometer) at 538. Additionally, the sample can be analyzed based on the signal and retention time observed by the detector to identify the oligonucleotide species and / or quantify the oligonucleotide.

[0096] Figure 5C An exemplary method 560 for analyzing a sample containing a plurality of viral particles is shown. The viral particles can include a lipid and / or protein structure that encapsulates an oligonucleotide. In various embodiments, the oligonucleotide species can include ribonucleic acid (RNA) oligonucleotides or deoxyribonucleic acid (DNA) oligonucleotides. The oligonucleotides can contain natural or non-natural modifications. The oligonucleotides can be produced in vitro or in vivo.

[0097] At 562, an ion-pairing reagent can be combined with the sample. In various embodiments, the sample can be dissolved or resuspended in the first solution, the second solution, or any combination thereof. In various embodiments, dissolving or resuspending the sample can solubilize the viral particles by dissolving lipids and / or denaturing proteins of the viral particles, thereby releasing oligonucleotides contained within the viral particles into solution.

[0098] The ion pair reagent can include a primary amine or a secondary amine. In various embodiments, the ion pair reagent can include at least about 4 carbon atoms, but typically no more than about 16 carbon atoms.

[0099] The primary amine ion pair reagent can include an alkyl chain having at least 4 carbon atoms. In certain embodiments, the primary amine ion pair reagent can include pentylamine or hexylamine.

[0100] The secondary amine ion pair reagent may comprise two alkyl chains, at least one of which comprises at least 3 carbon atoms, but typically less than 8 carbon atoms, such as N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentylamine, or dihexylamine.

[0101] At 564, the sample and ion pair reagent can be loaded onto the chromatography column. In various embodiments, the chromatography column can include a stationary phase comprising divinylbenzene or a derivative thereof. In various embodiments, the stationary phase can be porous, for example, having an average pore size of at least about For example, the average pore size is at least about Even the average pore size is at least about The choice of average pore size may be influenced by the size of the oligonucleotides to be separated. to about The stationary phase can be used to separate oligonucleotides with a size less than 10 kDa. The average pore size is about to about The stationary phase can be used to separate oligonucleotides with a length of no more than about 300 nt. to about Stationary phases can be used to separate oligonucleotides greater than about 300 nt in length, but typically not greater than about 15,000 nt in length.

[0102] At 566, an eluent can be applied to the chromatographic column to elute the plurality of oligonucleotides. Typically, the sample solution conditions when applied to the chromatographic column are preferably close to the starting conditions of the eluent gradient. In various embodiments, the eluent can include a combination of a first solution and a second solution. In particular embodiments, the ratio of the first solution to the second solution can be varied to form gradient solvent conditions, thereby causing the separation of the various oligonucleotides based on differential retention within the chromatographic column, e.g., separation of a first oligonucleotide from a second oligonucleotide.

[0103] In various embodiments, only the concentration of the ion-pairing agent in the first solution and the second solution varies, and the gradient solvent conditions include a gradient in the concentration of the ion-pairing agent. For example, the second solution may contain more or less ion-pairing agent than the first solution, or even only the first solution may contain the ion-pairing agent while the second solution does not contain the ion-pairing agent. In other embodiments, the first and second solutions may contain the same concentration of the ion-pairing agent, such that the concentration of the ion-pairing agent remains substantially constant throughout the separation process. In various embodiments, the concentration of the ion-pairing agent may be between about 5 mM and about 500 mM.

[0104] In various embodiments, the first solution may include a fluoroalcohol, such as hexafluoroisopropanol (HFIP). In various embodiments, the concentration of the fluoroalcohol may be between about 5 mM and about 500 mM. In various embodiments, the first solution may include formic acid or a formate, acetic acid or an acetate, or any combination thereof, to achieve a pH in the range of, for example, about 2 to about 10. In various embodiments, the first solution may include a first organic solvent, such as methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof. In various embodiments, the concentration of the organic solvent may be between about 0% and about 100%. When less than about 100%, the remaining solution may include water.

[0105] In various embodiments, the second solution can include similar or identical components as the first solution at different concentrations. For example, the second solution can include a fluoroalcohol, formic acid, a formate, acetic acid, an acetate, and / or an organic solvent. In other embodiments, the second solution can include at least some components that are different from the first solution.

[0106] The oligonucleotide species can be determined using a detector (e.g., a UV / VIS detector or a mass spectrometer) at 568. Additionally, the sample can be analyzed based on the signal and retention time observed by the detector to identify the oligonucleotide species and / or quantify the oligonucleotide.

[0107] Figure 6 A liquid chromatography system 600 according to one aspect of the present invention is depicted. The liquid chromatography system 600 includes an analytical pump 602 to pump a solvent through the system 600. The system 600 includes a sample reservoir 604 containing a sample to be analyzed. The system 600 also includes a separation column 606 and a detector 608. The system 600 also includes a controller 610.

[0108] The liquid chromatography system 600 is adapted to extract a sample from a sample reservoir 604. The sample can then be introduced into the system.

[0109] The liquid chromatography system 1000 is also adapted to introduce a sample into the separation column 606 .

[0110] The system 600 is further adapted to inject the sample into the separation column 606 via an analytical flow. This can be accomplished by directing the sample via the analytical pump 602. The separation column 606 can separate the sample into component substances based on retention time within the separation column 606. After the sample is separated by the separation column 606, the separated components can be detected by a detector 608. In some embodiments, the detector 608 can be an optical detector, such as an absorption detector, a refractive index detector, a fluorescence detector, or the like. In other embodiments, the detector 608 can be a conductivity detector or an electrochemical detector. In other embodiments, the detector 608 can be a mass spectrometer.

[0111] In various embodiments, the separation column 606 is typically composed of a tube filled with a stationary phase medium. The stationary phase medium can affect the time (retention time) it takes for a compound to travel through the column. This effect can be different for different compounds, making it possible to separate individual components of a sample based on their respective retention times. There are a variety of stationary phase media, including porous materials, ionic materials, polar materials, non-polar materials, and the like. Based on the size of the molecule and the ability of the molecule to enter the porous material, the porous material can affect the retention time. Based on the charge attraction or repulsion between the ionic material and the compound, the ionic material can affect the retention time. Based on the hydrophobicity or hydrophilicity of the compound, polar and non-polar materials can affect the retention time.

[0112] In various embodiments, reverse phase separation can be used to separate nucleotides and nucleosides, wherein hydrophobic non-polar stationary phase material is used together with mobile phases having different hydrophobicities depending on the ratio of polar solvent to organic solvent. For example, a C18 column can be used together with ammonium acetate or ammonium formate buffer systems to separate nucleosides. In a plurality of specific embodiments, an aqueous mobile phase of 5mM ammonium acetate at a pH of about 5 can be used, and a gradient of acetonitrile (up to about 40%) or methanol (up to about 50%) of increasing concentration can be used to separate nucleosides. Suitable columns and buffer systems will be apparent to those skilled in the art, and are within the scope of the present disclosure.

[0113] In other embodiments, nucleotides and nucleosides can be separated using hydrophilic interaction liquid chromatography (HILIC) using a hydrophilic stationary phase material and a hydrophobic mobile phase, such as acetonitrile. Suitable columns and buffer systems will be apparent to those skilled in the art and are within the scope of this disclosure.

[0114] Various embodiments of the mass spectrometry platform 700 may include, for example, Figure 7 In various embodiments, the mass spectrometry platform 700 can operate as the detector 608 of the system 600. In various embodiments, Figure 7The elements of may be incorporated into a mass spectrometry platform 700. According to various embodiments, the mass spectrometer 700 may include an ion source 702, a mass analyzer 704, an ion detector 706, and a controller 708.

[0115] In various embodiments, the ion source 702 generates a plurality of ions from the sample. The ion source may include, but is not limited to, a matrix-assisted laser desorption / ionization (MALDI) source, an electrospray ionization (ESI) source, an atmospheric pressure chemical ionization (APCI) source, an atmospheric pressure photoionization (APPI) source, an inductively coupled plasma (ICP) source, an electron beam ionization source, a chemical ionization source, a photoionization source, a glow discharge ionization source, a thermal spray ionization source, and the like.

[0116] In various embodiments, the mass analyzer 704 may separate ions based on their mass-to-charge ratio. For example, the mass analyzer 704 may include a quadrupole mass filter analyzer, a quadrupole ion trap analyzer, a time-of-flight (TOF) analyzer, an electrostatic trap (e.g., an orbitrap) mass analyzer, a Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer, etc. In various embodiments, the mass analyzer 704 may also be configured to fragment ions using collision-induced dissociation (CID), electron transfer dissociation (ETD), electron capture dissociation (ECD), photoinduced dissociation (PID), surface-induced dissociation (SID), etc., and further separate the fragmented ions based on their mass-to-charge ratio.

[0117] In various embodiments, the ion detector 706 can detect ions. For example, the ion detector 706 can include an electron multiplier, a Faraday cup, or the like. Ions leaving the mass analyzer can be detected by the ion detector. In various embodiments, the ion detector can be quantitative, so that an accurate count of ions can be determined. In various embodiments, such as for an electrostatic trap (e.g., an orbitrap) mass analyzer, the mass analyzer 704 and the ion detector 706 can be combined into a single device.

[0118] In various embodiments, the controller 708 can communicate with the ion source 702, the mass analyzer 704, and the ion detector 706. For example, the controller 708 can configure the ion source or enable / disable the ion source. Furthermore, the controller 708 can configure the mass analyzer 704 to select a specific mass range to be detected. Furthermore, the controller 708 can adjust the sensitivity of the ion detector 706, for example by adjusting the gain. Furthermore, the controller 708 can adjust the polarity of the ion detector 706 based on the polarity of the detected ions. For example, the ion detector 706 can be configured to detect positive ions or negative ions.

[0119] While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be understood by those skilled in the art.

[0120] In addition, in describing various embodiments, this specification may present the method and / or process as steps in a specific order. However, insofar as the method or process does not rely on the specific order of the steps set forth herein, the method or process should not be limited to the specific sequence of steps described. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of the steps set forth in the specification should not be interpreted as a limitation to the claims. In addition, claims for the method and / or process should not be limited to performing their steps in the order written, and it will be readily understood by those skilled in the art that the order can be varied and still remain within the spirit and scope of the various embodiments.

[0121] Example 1 is a nucleic acid for analysis encapsulated in bacteriophage. Figure A is a blank injection. Figure 8 B shows the results of injection of RNA packaged within phage. Figure 8 C shows the injection results of phage extracted and purified RNA.

Claims

1. A method for separating oligonucleotides or nucleic acid biopolymers for analysis, comprising: loading a sample containing a plurality of oligonucleotides onto a chromatography column, the plurality of oligonucleotides comprising a first oligonucleotide and a second oligonucleotide; passing a mobile phase composed of a first solution and a second solution through a chromatography column to elute the plurality of oligonucleotides, the first solution comprising an ion pair reagent comprising a primary or secondary amine, wherein a ratio of the first solution to the second solution in the mobile phase varies over time, thereby separating the first oligonucleotide from the second oligonucleotide; and At least one of the first oligonucleotide and the second oligonucleotide is analyzed using a mass spectrometer. 2 . The method of claim 1 , wherein the sample comprises a plurality of oligonucleotides dissolved in the first solution, the second solution, or any combination thereof. The method of claim 1 , wherein the second solution comprises an ion-pairing agent. The method of claim 1 , wherein the ion pair reagent comprises at least 4 carbon atoms.

5. The method of claim 4, wherein the primary amine of the ion pair reagent comprises an alkyl chain containing at least 4 carbon atoms.

6. The method of claim 5, wherein the primary amine comprises pentylamine or hexylamine.

7. The method of claim 4, wherein the secondary amine of the ion pair reagent comprises two alkyl chains, at least one of which contains at least 3 carbon atoms.

8. The method of claim 7, wherein the secondary amine comprises N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentylamine, or dihexylamine.

9. The method of claim 1, wherein the first solution comprises a fluoroalcohol.

10. The method of claim 9, wherein the fluoroalcohol comprises hexafluoroisopropanol (HFIP).

11. The method of claim 1, wherein the first solution comprises formic acid or a formates salt, acetic acid or an acetate salt, or any combination thereof.

12. The method of claim 1, wherein the first solution comprises a first organic solvent.

13. The method according to claim 12, wherein the first organic solvent comprises methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol or any combination thereof. The method of claim 1 , wherein the second solution comprises a second organic solvent.

15. The method according to claim 14, wherein the second organic solvent comprises methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

16. The method of claim 1, wherein the chromatography column comprises a stationary phase comprising divinylbenzene or a derivative thereof.

17. The method of claim 16, wherein the stationary phase is porous with an average pore size of about to about And the size of the first species and the second species is less than 10 kDa.

18. The method of claim 16, wherein the stationary phase is porous with an average pore size of about to about And the length of the first species and the second species is no greater than about 300 nt.

19. The method of claim 16, wherein the stationary phase is porous with an average pore size of about to about The lengths of the first and second species are greater than 300 nt.

20. The method of claim 1, wherein the first and second species are no greater than about 15,000 nt in length.

21. The method of claim 1, wherein the plurality of oligonucleotides are ribonucleic acid (RNA) oligonucleotides.

22. The method of claim 21, wherein the ribonucleic acid (RNA) oligonucleotide contains a modification.

23. The method of claim 21, wherein the RNA oligonucleotide is produced in vitro.

24. The method of claim 21, wherein the RNA oligonucleotide is produced in vivo.

25. The method of claim 21, wherein the ribonucleic acid (RNA) oligonucleotide is a packaging RNA oligonucleotide.

26. A method for separating encapsulated oligonucleotides or nucleic acid biopolymers for analysis, comprising: dissolving a sample comprising a plurality of encapsulating oligonucleotides, the plurality of encapsulating oligonucleotides comprising a first encapsulating oligonucleotide comprising a first oligonucleotide and a second encapsulating oligonucleotide comprising a second oligonucleotide, wherein dissolving the sample comprises releasing the first and second oligonucleotides; The dissolved sample is loaded onto the column; passing a mobile phase composed of a first solution and a second solution through a chromatography column to elute the first and second oligonucleotides, the first solution comprising an ion pair reagent comprising a primary or secondary amine, wherein a ratio of the first solution to the second solution in the mobile phase varies over time, thereby separating the first oligonucleotide from the second oligonucleotide; and At least one of the first oligonucleotide and the second oligonucleotide is analyzed using a mass spectrometer.

27. The method of claim 26, wherein the sample comprises a plurality of oligonucleotides dissolved in the first solution, the second solution, or any combination thereof.

28. The method of claim 26, wherein the second solution comprises an ion-pairing agent.

29. The method of claim 26, wherein the ion pair reagent comprises at least 4 carbon atoms.

30. The method of claim 29, wherein the primary amine of the ion pair reagent comprises an alkyl chain containing at least 4 carbon atoms.

31. The method of claim 30, wherein the primary amine comprises pentylamine or hexylamine.

32. The method of claim 29, wherein the secondary amine of the ion pair reagent comprises two alkyl chains, at least one of which contains at least 3 carbon atoms.

33. The method of claim 32, wherein the secondary amine comprises N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentylamine or dihexylamine.

34. The method of claim 26, wherein the first solution comprises a fluoroalcohol.

35. The method of claim 34, wherein the fluoroalcohol comprises hexafluoroisopropanol (HFIP).

36. The method of claim 26, wherein the first solution comprises formic acid or a formates salt, acetic acid or an acetate salt, or any combination thereof.

37. The method of claim 26, wherein the first solution comprises a first organic solvent.

38. The method of claim 37, wherein the first organic solvent comprises methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

39. The method of claim 26, wherein the second solution comprises a second organic solvent.

40. The method of claim 39, wherein the second organic solvent comprises methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

41. The method of claim 26, wherein the chromatography column comprises a stationary phase comprising divinylbenzene or a derivative thereof.

42. The method of claim 41, wherein the stationary phase is porous with an average pore size of about to about And the size of the first species and the second species is less than 10 kDa.

43. The method of claim 41, wherein the stationary phase is porous with an average pore size of about to about And the length of the first species and the second species is no greater than about 300 nt.

44. The method of claim 41, wherein the stationary phase is porous with an average pore size of about to about The lengths of the first and second species are greater than 300 nt.

45. The method of claim 26, wherein the first and second species are no greater than about 15,000 nt in length.

46. ​​The method of claim 26, wherein the first and second oligonucleotides are ribonucleic acid (RNA) oligonucleotides.

47. The method of claim 26, wherein the first and second oligonucleotides are deoxyribonucleic acid (DNA) oligonucleotides.

48. The method of claim 26, wherein the first and second oligonucleotides contain modifications.

49. The method of claim 26, wherein the first and second oligonucleotides are produced in vitro.

50. The method of claim 26, wherein the first and second oligonucleotides are produced in vivo.

51. A method for isolating viral particles for analysis, comprising: Lysing a sample comprising a plurality of viral particles, the plurality of viral particles comprising a first viral particle comprising a first oligonucleotide and a second viral particle comprising a second oligonucleotide, wherein lysing the sample comprises releasing the first and second oligonucleotides; The dissolved sample is loaded onto the column; passing a mobile phase composed of a first solution and a second solution through a chromatography column to elute the first and second oligonucleotides, the first solution comprising an ion pair reagent comprising a primary or secondary amine, wherein a ratio of the first solution to the second solution in the mobile phase varies over time, thereby separating the first oligonucleotide from the second oligonucleotide; and At least one of the first oligonucleotide and the second oligonucleotide is analyzed using a mass spectrometer.

52. The method of claim 51, wherein the sample comprises a plurality of oligonucleotides dissolved in the first solution, the second solution, or any combination thereof.

53. The method of claim 51, wherein the second solution comprises an ion-pairing agent.

54. The method of claim 51, wherein the ion pair reagent comprises at least 4 carbon atoms.

55. The method of claim 54, wherein the primary amine of the ion pair reagent comprises an alkyl chain containing at least 4 carbon atoms.

56. The method of claim 55, wherein the primary amine comprises pentylamine or hexylamine.

57. The method of claim 54, wherein the secondary amine of the ion pair reagent comprises two alkyl chains, at least one of which contains at least 3 carbon atoms.

58. The method of claim 57, wherein the secondary amine comprises N-ethylpropylamine, dipropylamine (DPA), N-ethylbutylamine, N-propylbutylamine, dibutylamine (DBA), dipentylamine or dihexylamine.

59. The method of claim 51, wherein the first solution comprises a fluoroalcohol.

60. The method of claim 59, wherein the fluoroalcohol comprises hexafluoroisopropanol (HFIP).

61. The method of claim 51, wherein the first solution comprises formic acid or a formates salt, acetic acid or an acetate salt, or any combination thereof.

62. The method of claim 51, wherein the first solution comprises a first organic solvent.

63. The method of claim 62, wherein the first organic solvent comprises methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

64. The method of claim 51, wherein the second solution comprises a second organic solvent.

65. The method of claim 64, wherein the second organic solvent comprises methanol, ethanol, acetone, acetonitrile, tetrahydrofuran, isopropanol, or any combination thereof.

66. The method of claim 51, wherein the chromatography column comprises a stationary phase comprising divinylbenzene or a derivative thereof.

67. The method of claim 66, wherein the stationary phase is porous with an average pore size of about to about And the size of the first species and the second species is less than 10 kDa.

68. The method of claim 66, wherein the stationary phase is porous with an average pore size of about to about And the length of the first species and the second species is no greater than about 300 nt.

69. The method of claim 66, wherein the stationary phase is porous with an average pore size of about to about The lengths of the first and second species are greater than 300 nt.

70. The method of claim 51, wherein the first and second species are no greater than about 15,000 nt in length.

71. The method of claim 51, wherein the first and second oligonucleotides are ribonucleic acid (RNA) oligonucleotides.

72. The method of claim 51, wherein the first and second oligonucleotides are deoxyribonucleic acid (DNA) oligonucleotides.

73. The method of claim 51, wherein the first and second oligonucleotides contain modifications.

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