Cross-reference of oligonucleotide extraction methods and kits related applications

Abstract

The present technology relates to methods and kits for extracting oligonucleotides using solid phase extraction. Oligonucleotides are extracted from a biological sample by using a protease-assisted sample pretreatment step followed by a weak anion exchange solid phase extraction process.

Description

[0001] This application claims the benefits of U.S. Provisional Application No. 63 / 505,307, filed May 31, 2023; U.S. Provisional Application No. 63 / 517,951, filed August 7, 2023; and U.S. Provisional Application No. 63 / 586,737, filed September 29, 2023, the full text of which is incorporated herein by reference. Technical Field

[0002] This invention relates to methods and kits for solid-phase extraction of oligonucleotides from biological samples. The invention utilizes a protease-assisted sample pretreatment step followed by a weak anion-exchange solid-phase extraction process to extract oligonucleotides from samples. Background Technology

[0003] Oligonucleotides are typically fragments of nucleic acids, such as intermediate degradation products of DNA and RNA, or microRNAs, that regulate processes in biological systems. Their expression can be dysregulated as disease progresses. Therefore, oligonucleotides have been proposed as diagnostic and prognostic tools for various diseases. Oligonucleotides have also been developed as therapeutic agents for a variety of disease conditions. Therefore, extracting oligonucleotides from complex samples is useful in both research and clinical diagnostic applications.

[0004] However, the biosample extraction of oligonucleotides from complex biological matrices, such as plasma, blood, urine, and tissue samples, remains a significant challenge in developing quantitative analytical methods for oligonucleotides. The polyanionic nature of oligonucleotides ensures that these compounds bind strongly to plasma proteins in addition to binding to other matrix components. Successful bioanalytical sample preparation depends on the difficult process of isolating oligonucleotides from the matrix. Summary of the Invention

[0005] Current techniques employ protocols and kits for extracting oligonucleotides from biological matrices, offering improved results compared to other oligonucleotide extraction techniques, including protein precipitation, protein digestion, liquid-liquid extraction, reversed-phase solid-phase extraction (SPE), strong anion exchange SPE, or combinations thereof. Furthermore, the present invention exhibits unexpectedly excellent recoveries and overcomes potential problems for chromatographic applications caused by the presence of large amounts of matrix material in the extraction solution.

[0006] The present invention includes methods and kits for solid-phase extraction of oligonucleotides. In particular, the present invention relates to a scheme for extracting one or more oligonucleotides from biological samples, which, in one aspect, is detergent-free. In another aspect, the present invention utilizes a protease-assisted sample pretreatment step, and a subsequent weak anion-exchange solid-phase extraction process using an adsorbent material comprising porous particles, wherein the surface of the porous particles is modified with a ligand having a protonable group having a pKa value between 7 and 12, preferably between 8 and 11, and more preferably between about 8 and about 10.

[0007] One of the objectives of this invention is to provide a scheme and kit for effectively extracting a variety of modified oligonucleotides (including lipid-modified oligonucleotides) without the need for adjustments.

[0008] According to some aspects, this disclosure relates to the combined use of protease digestion with weak anion exchange solid-phase extraction.

[0009] In one aspect, this technology relates to a method for extracting one or more oligonucleotides from a biological sample.

[0010] The extraction method may include one or more of the following steps: (1) hydrolyzing the sample protein, (2) loading the digested sample onto an adsorbent, (3) washing to remove any non-oligonucleotide components, and (4) releasing the desired oligonucleotides from the adsorbent by passing the eluent through the adsorbent to obtain an eluent.

[0011] The above-described method steps can be advantageously implemented without the use of detergents in the digestion mixture (i.e., "detergent-free").

[0012] More specifically, the method steps include (1) digesting the biological sample in a detergent-free mixture by combining it with a protease solution, (2) loading the digested sample onto an adsorbent material composed of porous particles, wherein the surface of the porous particles is modified with a ligand having a protonable group having a pKa value between 7 and 12, preferably between 8 and 11, and more preferably between about 8 and about 10, (3) passing one or more washing solutions through the adsorbent material to remove one or more non-target components from the adsorbent material while retaining one or more target analytes on the adsorbent material, and (4) passing an elution solution with a pH range of about 10 to about 12 through the adsorbent material to release the retained one or more target analytes from the adsorbent material into the elution solution (i.e., the eluent). In some examples, the protease solution is proteinase K. In some examples, the detergent-free mixture may contain guanidine.

[0013] In an alternative aspect, the method steps include (1) digesting the sample protein by combining the biological sample with a protease solution (e.g., a proteinase K solution) and guanidine, (2) loading the digested sample onto an adsorbent material comprising porous particles, wherein the surface of the porous particles is modified with a ligand having a protonable group having a pKa value between 7 and 12, preferably between 8 and 11, and more preferably between about 8 and about 10, (3) passing one or more washing solutions through the adsorbent material to remove one or more non-target components from the adsorbent material while retaining one or more target analytes on the adsorbent material, and (4) passing an elution solution with a pH range of about 10 to about 12 through the adsorbent material to release the retained one or more target analytes from the adsorbent material into the elution solution (i.e., the eluent).

[0014] The above aspects may include one or more of the following characteristics. In some examples, the porous particles have a size greater than 5 μm and less than 100 μm.

[0015] In some examples, the method also includes subjecting the eluent solution to analytical techniques selected from liquid chromatography, mass spectrometry (MS), ultraviolet-visible spectroscopy, and combinations thereof.

[0016] In some examples, the one or more washing solutions include a partially aqueous organic solvent solution containing methanol, ethanol, tetrahydrofuran (“THF”), acetonitrile, or combinations thereof.

[0017] In some examples, the elution solution contains a base selected from organic amines, ammonium bicarbonate, ammonium hydroxide, ammonium acetate, or combinations thereof.

[0018] In some examples, the one or more eluents contain the organic amine triethylamine (TEA). For example, 50 mM TEA in 50% MeOH. In some examples, the one or more eluents contain a combination of TEA and ammonium bicarbonate, ammonium hydroxide, or ammonium acetate. In some examples, the one or more eluents contain a combination of TEA and ammonium hydroxide. For example, the one or more eluents may be formed from 100 mM TEA in 50% MeOH with 0.30% NH4OH or 50 mM TEA in 50% MeOH with 0.15% NH4OH.

[0019] While the eluent may contain triethylamine as an organic amine, this technique is not limited to the use of TEA. For example, other organic amines may be used in the eluent of this invention. Other alternatives include, but are not limited to, dimethylamine, trimethylamine, ethanolamine, diethylamine, butylamine, dibutylamine, diisopropylamine, dimethylbutylamine, tripropylamine, diisopropylethylamine, hexylamine, octylamine, dicyclohexylamine, tributylamine, and dihexylamine. The organic amine may be combined with ammonium bicarbonate, ammonium hydroxide, or ammonium acetate to form the eluent. Generally, the concentration of the organic amine can be in the range of 2 mM to 500 mM, more particularly in the range of 5 mM to 200 mM.

[0020] In some examples, the biological sample is a biological fluid. In preferred aspects, the biological fluid includes whole blood samples, plasma samples, serum samples, oral fluid, cerebrospinal fluid, fecal samples, nasal samples, and urine.

[0021] In some examples, the biological sample is biological tissue. In preferred aspects, biological tissue includes liver, kidney, and brain tissue, tissue homogenates, cells, and cell culture supernatants. In embodiments characterized by tissue homogenates, the surfactant-free tissue homogenization step can be performed before or simultaneously with the hydrolytic digestion of the sample proteins. When tissue homogenization is performed prior to digestion, the resulting tissue homogenate can be diluted 2 to 4 times with an aqueous solution before incubation with a protease. In some embodiments, tissue homogenization includes applying an organic solvent at a concentration greater than 20% by volume (e.g., 50% by volume or more) of the biological sample.

[0022] In some examples, the one or more oligonucleotides are selected from double-stranded RNA, single-stranded RNA, single-stranded DNA, double-stranded DNA, double-stranded RNA / DNA hybrids, synthetic RNA, synthetic DNA, or combinations thereof, wherein the size of the one or more oligonucleotides ranges from 10 to 200 units.

[0023] In some examples, the eluent solution also contains an organic solvent at a concentration between 10% and 70% by volume.

[0024] In some examples, the above method may include diluting the elution solution containing the one or more oligonucleotides with an equal volume of water.

[0025] In some examples, this technology relates to kits containing the protease solution, adsorbent material, and elution solution as described above.

[0026] The technique and related extraction scheme of this invention can also be used to facilitate the analysis of protein oligonucleotide conjugates. By using a proteolytic digestion step, the oligonucleotide conjugate is converted into a peptide oligonucleotide conjugate. The peptide component becomes a substitute for the larger protein component, and the remaining amino residues provide sequence positioning information. Ideally, the peptide component produced by proteolysis is between 3 and 5 residues in length, but its length can also be between 1 and 20 residues. In the peptide oligonucleotide form, the SPE scheme described herein can be readily applied to efficiently enrich oligonucleotide analytes for sensitive LC-UV or LC-MS quantification. Attached Figure Description

[0027] The present technology will be more fully understood through the following detailed description taken in conjunction with the accompanying drawings, in which: Figure 1A A flowchart illustrating an example of a method for extracting oligonucleotides is shown.

[0028] Figure 1B A flowchart of another method for extracting oligonucleotides according to the present invention is shown.

[0029] Figure 2 The oligonucleotide recovery rates of three samples using the technology of this invention are shown.

[0030] Figure 3 The comparison using CLARITY with the same sample is shown. ® OTX ™ The results of oligonucleotide plasma recovery using the technique of this invention are compared to those of the oligonucleotide extraction protocol (Phenomenex, Inc. Torrance, CA).

[0031] Figure 4 Showing with CLARITY ® OTX ™ (Extraction protocols and products are commercially available from Phenomenex, Inc.) Compared to the eluent (right), the oligonucleotide IP-RPLC-MS direct injection sample eluent compatibility of the sample (left) prepared with the eluent from the present invention exhibits minimal oligonucleotide (GEM 91) penetration and linear MS response with increasing injection volume.

[0032] Figure 5The invention demonstrates the IP-RPLC-MS analytical performance (retention time, peak intensity, peak shape) of an oligonucleotide performance standard mixture (unextracted plasma sample) injected before (injection #1) and after (injection #4 and injection #5) into plasma containing oligodeoxythymidine (15-35T), GEM 91, and GEM 132 oligonucleotides, using the technique of the present invention.

[0033] Figure 6 Showing a contrast to CLARITY ® OTX ™ Lysis pretreatment and SPE extraction were used to analyze the oligonucleotide IP-RPLC-MS performance (retention time, peak intensity, peak shape) of the LC-MS oligonucleotide performance standard mixture (unextracted plasma sample) injected before (injection #1) and after (injection #4 and injection #5) of plasma containing oligodeoxythymidine (15-35T), GEM 91 and GEM 132 oligonucleotides.

[0034] Figure 7 The robustness of a representative oligonucleotide IP-RP LC / MS system is demonstrated, showing a stable MS% peak height response.

[0035] Figure 8 The consistent retention times of the reference samples 15-35T after protein digestion of oligodeoxythymidine are shown.

[0036] Figure 9 The flexible / scalable method performance was demonstrated by illustrating the linear GEM 91 oligonucleotide MS response for digested and extracted plasma sample volumes ranging from 12.5 μL to 300 μL.

[0037] Figure 10 The cleanliness of extracted oligonucleotide plasma samples, as measured by targeted (MRM) and untargeted full-scan TIC and EIC (total ion count and extracted ion count) IP-RP LC high-resolution MS analysis using the techniques of this invention, is shown.

[0038] Figure 11 This shows the contrast between CLARITY and CLARITY. ® OTX ™ The cleanliness of the extracted oligonucleotide plasma sample was determined by targeted (MRM) and untargeted full-scan TIC and EIC (total ion count and extracted ion count) IP-RP LC high-resolution MS analysis.

[0039] Figure 12 It shows CLARITY ® OTX ™Comparison between the scheme and the present invention: an example of oligonucleotide plasma recovery, wherein the modified SPE sample loading (top loading on water).

[0040] Figure 13 Comparative oligonucleotide recovery results of urine-doped samples using the technique of the present invention are shown for three different elution solutions, with modified SPE sample loading.

[0041] Figure 14 Comparative oligonucleotide recovery results of plasma-doped samples using the technique of the present invention are shown for three different elution solutions, with modified SPE sample loading. Detailed Implementation

[0042] Definitions As used herein, the terms “about” or “approximately” mean + / - 10% of the value stated.

[0043] As used herein, the term “including” means including but not limited to, and the term “comprising” means including but not limited to.

[0044] As used herein, the term "adsorbent" or "adsorbent material" refers to a material to which one or more components of a sample (e.g., oligonucleotides) are adsorbed. In some embodiments, the adsorbent material of the present invention comprises solid particles, preferably solid porous particles, such as solid silica, polymeric or hybrid particles.

[0045] When used with respect to two or more parts, the terms “connection,” “joining,” “attachment,” “coupling,” “couplement,” and “tethering” mean that these parts are physically associated or connected to each other, directly or via one or more other parts acting as a connecting agent, to form a structure that is stable enough that the parts remain physically associated under the conditions of using the structure.

[0046] As used herein, the term "eluent" refers to the carrier portion of the mobile phase, such as a solvent or solvent mixture, which can be used to deliver the sample during chromatography.

[0047] As used herein, the term "eluent" refers to a substance that appears or is eluted from a chromatographic process. Using an "eluting solution" to "elute" molecules (e.g., oligonucleotides or impurities of interest) from an adsorbent means removing the molecule from the adsorbent by altering the solution conditions so that the buffer competes with the molecule of interest for binding to the adsorbent. A non-limiting example is eluting molecules from an adsorbent by altering the pH of the buffer surrounding the adsorbent.

[0048] As used herein, the term “oligonucleotide” (or “OGN”) refers to a polymeric sequence of two or more nucleotides, including RNA, DNA, and their analogues, including those with base modifications, sugar modifications, or linkers for modifying bioavailability. Oligonucleotides are broadly defined, including but not limited to nucleotides derived from double-stranded RNA, single-stranded RNA, single-stranded DNA, double-stranded DNA, dual-standard RNA / DNA hybrids, synthetic RNA, synthetic DNA, and combinations thereof. Examples of OGN modifications include 2'-O-methoxyethyl, 2'-fluoro, phosphate thioester, and / or GalNAc modifications. Other examples of OGN include antisense oligonucleotides (ASO), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), messenger RNA (mRNA), and / or plasmids.

[0049] The size of the "oligonucleotide" used in the present invention is not limited; preferably, the size of the one or more nucleotides ranges from 10 to 200 units.

[0050] As used herein, the term "stationary phase" refers to a phase or portion that is held in place or stationary during chromatographic processes, such as the solid material within the column through which the mobile phase passes.

[0051] As used herein, the term "mobile phase" refers to the phase or portion that moves in a chromatographic method, such as through a column, and it includes both the sample and the eluent.

[0052] As used herein, the terms “functionalization,” “modification,” or “chemical modification” refer to the altered state or structure of molecules in this technique. Molecules can be modified in many ways, including chemically, structurally, and functionally.

[0053] As used herein, and as interchangeable herein, the terms “extraction,” “separation,” or “isolation” refer to increasing the purity of a target molecule (i.e., one or more OGNs), a solution containing the target molecule, and one or more impurities. Typically, the purity of the target molecule is increased by removing impurities from the composition (completely or partially).

[0054] As used herein, unless otherwise indicated, the terms “sample” or “sample matrix” refer to any composition or mixture containing one or more oligonucleotides of interest. Samples can be derived from biological or other sources. Biological sources include eukaryotic and prokaryotic sources, such as plant and animal cells, tissues, and organs. Samples may also include diluents, buffers, detergents, contaminants, debris, etc., found to be mixed with one or more oligonucleotides of interest. Samples may be “partially purified” (i.e., have undergone one or more purification steps, such as filtration) or may be obtained directly from the host cells or organisms that produced one or more oligonucleotides of interest (e.g., samples may contain harvested cell culture media). For example, the sample matrix is ​​clarified. That is, the sample has undergone a clarification step prior to solid-phase extraction. The sample matrix may be in solution form. Solutions may be heterogeneous or homogeneous.

[0055] As used herein, the term "detergent-free" means, for example, in lysis buffers (e.g., TRITON). ™ X-100 (polyoxyethylene octylphenyl ether, available commercially from Dow Chemical Company) and TWEEN ® The protein solubilizing / cell membrane rupture detergent (e.g., surfactant) present in 20 (polyoxyethylene (20) dehydrated sorbitol monolaurate, commercially available from Kroda Americas, LLC) is not present in the compositions or mixtures described herein.

[0056] Oligonucleotides (or OGNs) have a wide range of applications, including research, disease diagnosis, and treatment. OGNs used as therapeutic agents have high growth potential. They are used as initiators for polymerase chain reactions, in gene expression studies, or as probes for DNA sequencing, characterization, and tracking nucleic acids in biological systems. They have undergone clinical trials as potential therapeutic agents for a variety of diseases. Therefore, significant breakthroughs have been made in this field in the past few years.

[0057] Therefore, OGN analysis is important for impurity determination, degradation, or biotransformation product analysis. Medical applications of OGN also require further analysis, such as in disease diagnosis, and the quantitative and qualitative determination of oligonucleotides is a key aspect of their potential drug applications in clinical research.

[0058] One of the objectives of this invention is to provide a scheme and kit for effectively extracting a variety of modified oligonucleotides (including lipid-modified oligonucleotides) without the need for adjustments.

[0059] This invention relates to the combined use of direct protease digestion with weak anion exchange solid-phase extraction. That is, there is no intermediate step between digestion and extraction.

[0060] The SPE adsorbent of the present invention has high pH stability, which allows the elution process to be carried out with a high pH (e.g., >9, preferably about 10 to about 12) eluent, preferably an eluent with a pH of about 10 to about 12.

[0061] Another objective of this invention is to improve the analyte recovery and / or reproducibility of oligonucleotides extracted from complex biofluid matrices. This invention allows for the selective extraction of oligonucleotides from complex biofluid matrices, including tissue homogenates.

[0062] In one example, the present invention relates to solid-phase extraction using certain adsorbent materials. In some examples, the SPE adsorbent employs a weak anion exchange (WAX) mechanism. In a preferred example, the adsorbent exhibits a pKa of surface ligands between 8 and 11, and more preferably between about 8 and about 10.

[0063] In some examples, the SPE adsorbent contains a base particle with a hydrophilic-lipophilic balance, ensuring that hydrophobically modified oligonucleotides can be eluted more easily.

[0064] The adsorbent material according to this technology preferably contains porous particles.

[0065] The size of the porous particles in the adsorbent material is not limited. In a preferred example, the average particle size of the porous particles is greater than 5 μm and less than 100 μm. The surface of the porous particles may be modified with a ligand having a protonable group, the pKa value of which is between 7 and 12, preferably between 8 and 11, and more preferably between about 8 and about 10.

[0066] The ion exchange capacity of the adsorbent is not limited. In some examples, the ion exchange capacity of the adsorbent is preferably between 0.1 mEq and 1 mEq ions / g adsorbent. In some examples, the ion exchange capacity is preferably between 0.2 mEq / g and 0.8 mEq / g.

[0067] In another example, the present invention includes a solid-phase extraction apparatus or kit containing an adsorbent material according to the foregoing aspects and embodiments of the present disclosure. The kit may also contain a protease for rapidly digesting the protein composition of a sample prior to sample loading.

[0068] The specific protease used in this invention is not particularly limited, as long as it can be used for protein hydrolysis of biological samples and preferably does not require the use of detergents. In some examples, the selected protease is proteinase K (e.g., Candida albicans (T. album) proteinase K).

[0069] In some examples, the protein hydrolysis pretreatment step is incubated at above room temperature for more than 5 minutes.

[0070] In some examples, the protein hydrolysis pretreatment step involves the addition of a denaturing agent (e.g., guanidine) and optionally a reducing agent. The amount and type of reducing agent are not limited. Some examples of reducing agents include dithiothreitol or tris(2-carboxyethyl)phosphine.

[0071] In some preferred examples, the purity of proteinase K is greater than 80%. In some preferred examples, the purity of proteinase K is greater than 90%.

[0072] In another preferred example, the activity of proteinase K is at least 40 units / mg of enzyme, wherein one unit of proteinase K hydrolyzes urea-denatured hemoglobin at 37°C and pH 7.5 to produce a color equivalent of 1 μmol of tyrosine per minute.

[0073] In another preferred example, proteinase K is recombinantly expressed, purified, and purified from Pichia pastoris or Escherichia coli.

[0074] In some examples, the nucleic acids of OGN have sizes ranging from 10 to 200 polymers. In some examples, OGN includes N-acetylgalactosamine, modified and lipid-modified single-stranded and double-stranded ribonucleic acid, and deoxyribonucleic acid oligomers or combinations thereof.

[0075] In another example, the present invention relates to a method for pretreating a sample with a protease and then performing solid-phase extraction as follows: (1) mixing the sample with guanidine and a protease; (2) loading a sample fluid containing one or more target oligonucleotides and non-target components onto an adsorbent material having a pKa between 7 and 12, preferably between 8 and 11, and more preferably between about 8 and about 10; (3) passing one or more washing solutions through the adsorbent material, wherein the washing solutions remove the non-target components from the adsorbent material while leaving the target oligonucleotides retained on the adsorbent material; and (4) passing one or more elution solutions with a pH of about 8 to about 10 through the adsorbent material, wherein the target oligonucleotides retained on the adsorbent material are released into one or more elution solutions.

[0076] In some examples, after the eluent or one eluent solution is passed through the adsorbent material, the eluent from the elution is subjected to analytical tools, techniques, or devices. Examples include chromatographic techniques (such as liquid chromatography) and detectors such as mass spectrometry (MS), ultraviolet-visible spectroscopy, or any combination thereof. In some examples, the eluent can be directly injected (i.e., without evaporation) for ion-pairing reversed-phase separation.

[0077] In some examples, the one or more washing solutions comprise a partially aqueous organic solvent solution containing methanol, ethanol, propanol, acetonitrile, or tetrahydrofuran.

[0078] In some examples, the one or more elution solutions have a pH ranging from 9 to 12.

[0079] In some examples, the one or more elution solutions contain anions such as divalent, trivalent, or tetravalent acids.

[0080] In some examples, the one or more elution solutions contain one or more bases selected from organic amines, ammonium bicarbonate, ammonium hydroxide, or ammonium acetate. In a preferred example, the one or more elution solutions contain the organic amine triethylamine (TEA).

[0081] Biological samples are not limited, but should be types of samples that can be digested by protein hydrolysis. In some examples, samples contain biological fluids selected from whole blood samples, plasma samples, serum samples, oral fluid, cerebrospinal fluid, fecal samples, nasal samples, and urine. In some examples, samples contain biological tissues, such as liver, kidney, and brain tissue, tissue homogenates, cells, or cell culture supernatants.

[0082] In some examples, one or more OGNs include double-stranded RNA, single-stranded RNA, single-stranded DNA, double-stranded DNA, double-stranded RNA / DNA hybrids, synthetic RNA, synthetic DNA, or combinations thereof.

[0083] There are no particular restrictions on the size of the OGN. In some examples, the OGN ranges in size from 10 to 200 units.

[0084] In one example, the method of the present invention includes the following steps: (1) digesting the sample by combining it with a protease solution in a detergent-free mixture; (2) loading the digested sample onto an adsorbent material composed of porous particles, the surface of which is modified with a ligand having a protonable group having a pKa value between 7 and 12, preferably between 8 and 11, and more preferably between about 8 and about 10; (3) passing one or more washing solutions through the adsorbent material to remove one or more non-target components from the adsorbent material while retaining one or more target analytes on the adsorbent material; and (4) passing an elution solution with a pH range of about 10 to about 12 through the adsorbent material to release the retained one or more target analytes from the adsorbent material into the elution solution (i.e., the eluent).

[0085] The resulting eluent can be further diluted with water to optimize the analysis. In a preferred example, the dilution is an equal volume of water to the resulting eluent containing the released OGN.

[0086] In some examples, the method may also include top-loading the digested sample into water-containing SPE wells to improve OGN recovery and dilute the amount of guanidine used in the digestion. In a preferred example, the top-loading step is used to improve the retention / binding of the phosphodiester backbone-containing OGN to the SPE adsorbent, subsequently improving oligonucleotide recovery.

[0087] In another example, the present invention relates to a kit comprising the components described herein. In some examples, the components include the protease described herein, an SPE device described herein containing an adsorbent (i.e., an SPE adsorbent containing a ligand with a pKa between 7 and 12, preferably between 8 and 11, and more preferably between about 8 and about 10), and an SPE eluent described herein with a pH > 9, preferably between about 10 and about 12. In some examples, the SPE eluent may optionally contain an organic solvent at a concentration between 10% and 70% by volume.

[0088] Examples Example 1 - Illustrative example of a method for extracting oligonucleotides Illustrative embodiments of the present invention are as follows: Figure 1A As shown in the flowchart. Figure 1A As shown, OGN can be extracted or separated from a desired biological sample using four steps. The biological sample itself is not limited, as long as it contains the desired OGN to be extracted.

[0089] Step 1 is a pretreatment step in which the sample is digested using a protease. In this step, the sample is combined with a protease solution (such as a proteinase K solution) to form a digestion mixture.

[0090] In this embodiment, the pretreatment step further includes adding a reducing agent, such as dithiothreitol or tris(2-carboxyethyl)phosphine. In this embodiment, the pretreatment step includes dithiothreitol.

[0091] In some examples, the mixture may also contain guanidine (e.g., guanidine hydrochloride) to aid in the digestion of the sample matrix. Preferably and advantageously, the mixture is detergent-free.

[0092] After digestion, step 2 involves loading the digested sample onto the SPE adsorbent material. The SPE adsorbent material contains porous particles and utilizes a WAX mechanism because the porous particles are modified with ligands carrying protonable groups, which have pKa values ​​between 7 and 12, preferably between 8 and 11, and more preferably between about 8 and about 10. Both OGN and non-OGN components will be retained on the adsorbent.

[0093] After the loading step, step 3 is to pass one or more washing solutions through the adsorbent material, so that one or more non-OGN components are removed from the adsorbent material, while the one or more desired OGNs remain on the adsorbent material.

[0094] After the washing step, step 4 is to pass an elution solution with a pH range of about 10 to about 12 through the adsorbent material, so that one or more OGNs retained are released from the adsorbent material into the elution solution (i.e., the eluent).

[0095] Although Figure 1A An embodiment of the extraction process according to the present invention is illustrated, but... Figure 1B Another embodiment within the scope of this technology is illustrated. Specifically, Figure 1B A specific sample preparation and extraction protocol according to the present invention is illustrated. The plasma sample pretreatment is detergent-free and includes protein hydrolysis of the plasma sample by applying proteinase K solution and guanidine. The plasma sample is incubated at approximately 55°C and 600 rpm for 40 minutes. Next, the pretreated sample is loaded onto an elution plate (OligoWorks WAX SPE micro-elution plate, available from Corporation, Milford, MA) containing a weak anion exchange adsorbent. To load the pretreated sample, water is first applied, followed by sample loading. A first wash (200 µL of 50 mM NH4OAc) and a second wash (200 µL of 10% MeOH) are applied to ensure sample cleanliness without partial elution of oligonucleotides via a reverse-phase mechanism. Finally, elution is completed with 2 × 50 µL of 50 mM TEA and 50% MeOH at pH 11.5. The eluent can be diluted with 100 µL of water.

[0096] Without being bound by theory, it is believed that the pH of the SPE adsorbent and the eluent solution is the driving mechanism for the desired OGN retention and elution. Combined with the pretreatment step, this approach provides extraction and analytical results superior to comparative techniques, as will be further demonstrated by the following examples.

[0097] Example 2 - Hydrophilic-lipophilic polymer WAX SPE for extracting oligonucleotides from biological fluids The application extracts several different modified oligonucleotides from biological fluids. This sample is produced by mixing 100 μL of rat plasma containing 1 μg / mL TRECOVIRSEN (which is GEM 91 (gene expression regulator 91), GEM 132 (a 20-meric oligonucleotide targeting the HCMV UL36 gene)) and lipid-modified oligonucleotide A. TRECOVIRSEN (GEM 91) is a 25-meric antisense oligodeoxynucleotide phosphate-thiolated molecule targeting HIV GAG RNA. GEM 132 is a fully phosphate-thiolated antisense oligonucleotide with a 2' methoxy cap, used to treat cytomegalovirus retinitis. Lipid-modified oligonucleotide A is synthesized as a negative control spacer antisense oligonucleotide with a 5' palmitate modification, a phosphate-thioester backbone, and a terminal methoxyethyl modification. Lipid modification, along with the oligonucleotide therapeutics, is used to enhance their drug binding and endocytosis.

[0098] Lipid-modified oligonucleotide A 100 μL of doped rat plasma was diluted with a mixture containing 20 μL of 6M guanidine hydrochloride in 60 mM pH 7.5 Tris buffer (i.e., non-lysis / detergent-free buffer), 20 μL of 100 mM dithiothreitol (20 mM final concentration), and 50 μL of proteinase K 20 mg / mL solution (Qiagen, product number 19131). The mixture was vortexed and then incubated at 65 °C for 15 minutes.

[0099] Next, use a solution containing 2mg of OASIS. ® Solid-phase extraction (SPE) was performed using 96-well micro-elution plates and a vacuum manifold with WAX 30µm adsorbent (a commercially available polymeric reversed-phase weak anion exchange mixed-mode adsorbent from Waters Corporation, Milford, MA). Each adsorbent bed was first treated with two 200µL aliquots of methanol, followed by equilibration with two 200µL aliquots of 50mM ammonium acetate pH 5.5 buffer. Plasma digestion samples were loaded onto these treated beds and washed with two additional 200µL aliquots of 50mM ammonium acetate pH 5.5 buffer and one 200µL aliquot of 30% methanol. Finally, the adsorbed and purified analytes were eluted from the SPE wells using two 50µL aliquots of eluent consisting of 50mM triethylamine (pH 11.5) in 50% methanol. The resulting samples were diluted with an equal volume of water and then directly injected for ion-paired reversed-phase chromatography and detected by triple quadrupole mass spectrometry. The experimental conditions used for these experiments are described below. Figure 2 The recovery rate for each oligonucleotide is shown.

[0100] Table 1: Instrumental method for IP-RPLC-MS UHPLC system ACQUITY ™ PREMIER UPLC ™ (Waters Corporation) Mobile phase A 1% HFIP (hexafluoro-2-propanol) 0.1% DIPEA (N,N-diisopropylethylamine) in H20 Mobile phase B 0.75% HFIP (hexafluoro-2-propanol), 0.0375% DIPEA (N,N-diisopropylethylamine), 65% ACN 35% H2O Column ACQUITY ® PREMIER ® Oligonucleotide BEH C18 1.7 μm 2.1 mm x 50 mm (WATERS PN 186009484) Column temperature 50℃ Sample temperature 8℃ Injection volume 10µL Purge solvent 10:90 MeOH: H20 Wash solvent 10:90 MeOH: H20 MS Xevo ® TQ-XS (Waters Corporation) Capillary (kV) 2.0 Desolvation temperature 500℃ Desolvation flow rate 1000 L / Hr Conespray gas flow rate 150 L / Hr Table 2: IP-RP LC gradient parameters: 5 minute analysis time Time (min) Flow (mL / min) % A % B Curve 0.0 95 5 6 3.25 0.6 77 23 6 3.75 0.6 10 90 6 4.1 0.6 10 90 6 4.25 0.6 95 5 6 Table 1 shows the instrumentation methods and protocols used for IP-RPLC-MS. Table 2 shows the percentage recovery rate during a 5-minute run using the same flow rate. It can be seen that a recovery rate exceeding 90% occurs in less than 5 minutes.

[0101] like Figure 2 As shown, GEM 91 and GEM 132 samples exhibited recoveries of over 95%, and lipid-modified oligonucleotide A showed recoveries of over 60%, which is excellent considering the difficulty in separating OGN from the sample matrix using comparative methods that showed lower results.

[0102] Example 3 - Comparison between OGN extraction methods The effectiveness of the proposed solution was tested and compared with alternative solutions. Specifically, using the same sample set as described in Example 2, the solution based on CLARITY was tested using the proposed solution. ® OTX ™ The comparative scheme (a solid-phase extraction scheme requiring lysis-loading buffer and a mixed-mode anion exchange adsorbent) was compared with that of the comparative scheme. Surprisingly, and unlike the comparative scheme, excellent results were achieved without the need for a washing agent in the pretreatment mixture.

[0103] CLARITY ® OTX ™ The sample preparation solution relies on pretreatment of biological samples containing one or more target oligonucleotides with lysis-loading buffer, followed by solid-phase extraction as follows: (a) the biological sample containing one or more target oligonucleotides is mixed with guanidine hydrochloride and TRITON. ™ (b) Mix the X-100 (detergent) loading lysis buffer at a 1:1 ratio, and load the mixed lysis buffer containing the oligonucleotide biological sample onto the AX adsorbent material (CLARITY). ® OTX ™ (c) Two washing solutions are passed through the adsorbent material, wherein the washing solutions remove endogenous biological interference from the adsorbent material while leaving the target oligonucleotides retained on the adsorbent material; and (c) An elution solution consisting of (ammonium bicarbonate, pH 9.5, acetonitrile, and tetrahydrofuran) is passed through the material.

[0104] CLARITY ® OTX ™The main components and extraction procedures are shown in Tables 3 and 4 below. CLARITY ® OTX ™ Other components present in the lysis-loading buffer may include cysteine, TCEP, and sodium phosphate.

[0105] Table 3: CLARITY as reported in the manufacturer's safety data sheet ® OTX ™ in the lysis-loading buffer Major components Lysis buffer components % Guanidine hydrochloride 56-58 TRITON ™ X-100 (detergent) 1-3 Table 4: CLARITY from biological fluids using AX adsorbents thereof ® OTX ™ AX SPE extraction procedure Step Description Sample pre-treatment: Mix lysis buffer and oligonucleotide / serum at a 1:1 ratio and vortex for 1 minute SPE sample loading Load pre-treated lysis:plasma sample Wash 1: Wash 2 times with ammonium acetate equilibration buffer (pH 5.5) Wash 2: Wash 2 times with ammonium acetate buffer containing acetonitrile (pH 5.5) Elution: Elution solution reagent: ammonium bicarbonate (pH 9.5), acetonitrile, and tetrahydrofuran Figure 3 The same sample was shown using CLARITY. ® OTX ™ The results of this method are compared to the oligonucleotide plasma recovery rate results of Example 2. For example... Figure 3 As can be seen from the image, for the sample, the use of detergent (lysis buffer, i.e., TRITON) is necessary. ™ X-100) and conventional schemes for anion exchange adsorbents (i.e., CLARITY) ® OTX ™ Compared to the previous method, the method of the present invention increases the oligonucleotide recovery rate of GEM 91 and GEM 132 samples by about 2 times and the recovery rate of lipid-modified oligonucleotide A samples by 3 times.

[0106] Example 4 - Demonstrated oligonucleotide IP-RPLC-MS method robustness, injection with protocol from the present technology Prepared sample containing prepared and extracted biological fluid oligonucleotides .

[0107] Another objective of this invention is to ensure that the prepared and extracted biological samples are suitable for IP-RPLC-MS analysis and to achieve robust LC-MS system performance within and between days of analyzing extracted oligonucleotide plasma samples with LC column life (approximately 1000 injections).

[0108] Using the technique of this invention, the resulting sample eluent must have sufficient cleanliness so that repeated LC-MS injections of these samples over a long period do not impede chromatographic separation and UV or MS detection. To this end, an oligonucleotide LC-MS performance standard mixture containing LC-MS Gem 91, GEM 132, and oligodeoxythymidine reference standards was prepared and injected each day of analysis of extracted plasma samples. The results were used as a measure of the performance of the chromatographic and MS methods analyzed over multiple days.

[0109] Unbound by theoretical constraints, this invention utilizes a SPE extraction eluent containing methanol, a solvent with weak elution strength. This not only provides high oligonucleotide recovery but also allows for direct injection into LC-UV and / or MS instruments for analysis, exhibiting a linear response with increasing injection volume and minimizing oligonucleotide vacuolar volume penetration. ® OTX ™ This is not the case for elution solutions, which rely on the stronger elution power of acetonitrile and tetrahydrofuran.

[0110] Furthermore, the eluent composition of this disclosure facilitates direct sample injection of various injection volumes. This capability allows for higher sample injection volumes to increase oligonucleotide LC-MS detection and quantification with minimal impact on LC-MS performance. (Compared to CLARITY) ® OTX ™ Compared to solutions, this is particularly advantageous for sample preparation time. (CLARITY from Phenomenex) ® OTX ™ The product's strong eluting solvents (i.e., acetonitrile and tetrahydrofuran) are severely limited in terms of injection volume due to the nature of their elution composition. This limitation restricts the overall analytical method sensitivity and necessitates additional protocol steps for evaporation and sample reconstruction in more suitable LC-MS solutions.

[0111] Example 5 - LC-MS compatible extraction eluate Samples were generated by preparing a solution containing 1 μg / mL GEM 91 oligonucleotides and adding an equal volume of the elution solution of this disclosure (pH 11.5) consisting of 50 mM triethylamine in 50% methanol. Samples were generated by adding an equal volume of the 1 μg / mL GEM 91 oligonucleotide solution to a CLARITY solution consisting of 100 mM ammonium bicarbonate (pH 9.5), 40% acetonitrile, and 10% tetrahydrofuran. ® OTX ™ Comparative samples were prepared in the elution solution. The resulting elution solution containing representative oligonucleotides (i.e., the sample and comparative sample of this disclosure) was diluted 1:1 with water and then directly injected for ion-paired reversed-phase chromatography, and detected by triple quadrupole mass spectrometry using injection volumes of 2.5 μL, 5 μL, and 10 μL.

[0112] Figure 4 Demonstrates and utilizes CLARITY ® OTX ™Compared to the eluent sample (right), the sample (left) prepared with the eluent from the present invention exhibits minimal oligonucleotide (GEM 91) penetration and a linear MS response with increasing injection volume in the direct injection of the oligonucleotide IP-RPLC-MS eluent. The comparative sample shows significant oligonucleotide void volume penetration and a reduced oligonucleotide response with increasing injection volume.

[0113] Example 6 - Consistent LC-MS system performance for injection prepared and extracted plasma samples .

[0114] Using current technology (Example 2) and in comparison with CLARITY ® The OTX protocol generates a mixture of LC-MS oligonucleotide performance standards. The resulting LC-MS oligonucleotide performance standards and extracted plasma samples are then directly injected (10 μL) for ion-paired reversed-phase chromatography and detected by triple quadrupole mass spectrometry.

[0115] Table 5: Mass spectrometer multiple reaction monitor (MRM) oligonucleotide transitions for oligodeoxythymidine reference characterization Oligodeoxythymidine reference standard (OST) Parent ion (m / z) Daughter ion (m / z) Cone (V) Collision (eV) OST 15T 642.00 303.00 50 25 OST 20T 646.40 303.00 50 25 OST 25T 668.10 303.00 50 25 OST 30T 684.70 303.00 50 25 OST 35T 704.60 303.00 50 25 Figure 5 and Figure 6 This disclosure illustrates ( Figure 5 ) and CLARITY ® OTX ™ Pyrolysis pretreatment and SPE extraction ( Figure 6 Compared to the LC-MS oligonucleotide performance (retention time, peak intensity, peak shape) of the LC-MS oligonucleotide performance standard mixture (unextracted plasma sample) injected before and after digestion pretreatment and SPE extraction of plasma containing oligodeoxythymidine (15-35T), GEM 91 and GEM 132 oligonucleotides, the LC-MS performance of the oligonucleotides was significantly different from that of the pure elution solution standard injection standards (#4 and #5) in terms of oligonucleotide peak distortion and signal loss.

[0116] Example 7 - Consistent LC-MS system performance column longevity Using current technology (Example 2) and in comparison with CLARITY ® OTX ™ An extraction protocol was used to prepare LC-MS oligonucleotide performance standard mixtures. These representative LC-MS performance standard mixture samples (i.e., samples and control samples according to the present invention) were injected before and after the injection of the prepared and extracted oligonucleotide-containing plasma samples. As described, the protocol according to Example 2 (the present invention) or CLARITY was used. ® OTX ™The protocol involves preparing and extracting 100 μL of plasma sample containing 1 μg / mL GEM 91 and 1 μg / mL GEM 132. The resulting LC-MS oligonucleotide performance standards and the extracted plasma sample were then directly injected (2 μL to 10 μL) for ion-paired reversed-phase chromatography and detected by triple quadrupole mass spectrometry. Samples were prepared and analyzed over a minimum of 5 individual days, with approximately 175 extracted plasma samples injected, including LC-MS oligonucleotide performance standards injected at the start, middle, and end of each run. The experimental conditions used for these experiments are provided in the above examples.

[0117] Figure 7 and Figure 8 This application illustrates the use of MASSPREP injected into samples that have undergone protease digestion and SPE extraction during a six-day analysis. ® (A protein digestion standard mixture, commercially available from Waters Corporation, Milford, MA) The robustness of the representative oligonucleotide IP-RP LC / MS system for oligodeoxythymidine reference samples 15-35T demonstrated a stable MS% peak height response. Figure 7 ) and consistent retention time ( Figure 8 ), and column life (>950 plasma extract samples).

[0118] Example 8 - Demonstrated flexible and scalable extraction performance Using current technology (Example 2) and in comparison with CLARITY ® OTX ™ The protocol involves preparing a mixture of LC-MS oligonucleotide performance standards. The resulting LC-MS oligonucleotide performance standards and extracted plasma samples are then directly injected (2 μL to 10 μL) for ion-paired reversed-phase chromatography and detected by triple quadrupole mass spectrometry. Samples are prepared and analyzed over a minimum of 5 individual days, with approximately 175 extracted plasma samples injected, including LC-MS oligonucleotide performance standards injected at the start, middle, and end of each run. Experimental conditions for performing these experiments are provided in the above examples. For plasma collections extracted from the present invention and using CLARITY... ® OTX ™ The extracted plasma sample set generated by the protocol was processed using a new LC column.

[0119] As described, plasma samples containing 1 μg / mL GEM 91 and 1 μg / mL GEM 132 were prepared and extracted using the protocol in Example 2, with modifications made to the sample digestion steps and extraction performed using a microelution 96-well SPE format. Then, LC-MS oligonucleotide performance standards and the extracted plasma samples were directly injected (10 μL) for ion-paired reversed-phase chromatography and detected by triple quadrupole mass spectrometry.

[0120] The results are presented in Table 6 below.

[0121] Table 6: Plasma and digestion reagent volumes for evaluating scalability and extraction performance of plasma digestion pre-treatment Oligonucleotide containing plasma starting volume (μL) Guanidine volume (µL) Dithiothreitol volume (µL) Proteinase K (μL) Final digestion sample (μL) 12.5 1.25 1.25 2.5 17.5 25.0 2.50 2.50 5.0 35 50.0 5.0 5.0 10.0 70 100.0 10.0 10.0 20.0 140 200.0 20.0 20.0 30.0 250 300.0 30.0 30.0 40.0 400 Figure 9 The flexible / scalable method performance was demonstrated by illustrating the linear GEM 91 oligonucleotide MS response for digested and extracted plasma sample volumes ranging from 12.5 μL to 300 μL.

[0122] Example 9 - Minimal MS matrix artifacts produced from biological matrix and reagent artifacts in extracted eluate samples .

[0123] Another objective of this invention is to ensure that the prepared and extracted biological samples (prepared as described in this disclosure) are sufficiently clean and suitable for IP-RPLC-MS, so as to minimize MS matrix artifacts caused by biological matrix and reagent artifacts in the extracted eluent sample.

[0124] Unbound by theory, the present invention allows for the elution of oligonucleotide analytes using an eluent composed of a solvent with relatively weak elution strength. In one example, the eluent contains 50% (v / v) methanol. This ensures that reasonably high injection volumes can be directly injected for ion-pairing reversed-phase separation without breakthrough. The eluent can be diluted 2-fold with water to further promote high volumetric loading. Consequently, evaporation of the eluent can be avoided for faster turnaround. ® OTX ™ The acetonitrile and THF content in the eluent results in significantly stronger solvent elution, which can impair direct injection.

[0125] This invention relates to a sample preparation method that involves pretreating oligonucleotide-containing biofluid samples with a protease followed by solid-phase extraction. The resulting eluent has sufficient cleanliness to minimize matrix interference from sample pretreatment and extraction reagents, as well as the biological matrix. Reducing these interferences improves the selectivity and specificity of the overall method.

[0126] As described, the scheme according to Embodiment 2 or the CLARITY scheme can be used. ® OTX ™The protocol involves preparing and extracting 100 μL of plasma samples containing 0.1 µg / mL–5 µg / mL GEM 91 and 1 μg / mL GEM 132. The resulting LC-MS oligonucleotide performance standards and the extracted plasma samples were then directly injected (2 μL to 10 μL) for ion-paired reversed-phase chromatography and detected by triple quadrupole and HRMS mass spectrometry. The experimental conditions used for these experiments are provided in the above examples.

[0127] This disclosure ( Figure 10 ) and CLARITY ® OTX ™ Eluent ( Figure 11 Compared to this, an example comparison of the cleanliness of extracted oligonucleotide plasma samples as determined by targeted (MRM) and untargeted full-scan TIC and EIC (total ion count and extracted ion count) IP-RP LC high-resolution MS analysis shows that CLARITY ® OTX ™ The eluent showed significant oligonucleotide void volume penetration and reduced oligonucleotide response with increasing injection volume.

[0128] Example 10 - Comparison to CLARITY ® OTX ™ Improved oligonucleotide recovery compared to CLARITY® shows that when loaded to the top, there is Recovery of oligonucleotides containing phosphodiester backbones increased when SPE cartridge was conditioned with water .

[0129] Another objective of this invention is to ensure that the prepared and extracted biological samples (prepared as described in this disclosure) are sufficiently clean and suitable for IP-RPLC-MS, minimizing MS matrix artifacts caused by the biological matrix and reagent artifacts in the extracted eluent sample. To achieve high oligonucleotide recoveries from the extracted biological samples, efforts have focused on ensuring complete digestion of the plasma sample and appropriate SPE sample loading and elution conditions to achieve complete digestion. It has been observed that SPE recovery of oligonucleotides containing the phosphodiester backbone is improved when the SPE protocol described in the above examples is modified by loading the top of the digested sample into SPE wells containing water.

[0130] Unbound by theory, it is believed that the guanidine hydrochloride reagent used for sample digestion interferes with the binding of oligonucleotides to weak anion exchange adsorbents. Therefore, the digested sample is diluted with water during SPE loading to reduce the guanidine concentration. This reduction in concentration improves the binding or retention of oligonucleotides to the adsorbent, resulting in higher oligonucleotide recovery.

[0131] The preparation of this example is as follows: a standard mixture of oligodeoxythymidine (15-mer, 20-mer, 25-mer, 30-mer, and 35-mer) and ssDNA (20-mer) was added to rat plasma to obtain a final concentration of 0.01 μmol / µL (oligodeoxythymidine) and 1 µg / mL ssDNA.

[0132] 100 μL of rat plasma containing oligodeoxythymidine (15-35 NT) polymers and ssDNA was diluted with 20 μL of 6M guanidine hydrochloride in 60 mM pH 7.5 Tris buffer (0.66 M final concentration), 20 μL of 300 mM tris(2-carboxyethyl)phosphine (30 mM final concentration), and 50 μL of proteinase K 20 mg / mL solution (Qiagen, product number 19131). The mixture was vortexed and then incubated at 55 °C for 60 min.

[0133] Next, use a solution containing 2mg of OASIS. ® Solid-phase extraction (SPE) was performed using a 96-well micro-elution plate and vacuum manifold with WAX 30µm adsorbent (a polymeric reversed-phase weak anion exchange mixed-mode adsorbent commercially available from Waters Corporation, Milford, MA). Each adsorbent bed was first treated with two 200µL volumes of methanol, followed by equilibration with two 200µL volumes of 50mM ammonium acetate pH 5.5 buffer. A full volume of digested plasma sample (190µL) was loaded directly onto the treated plate or onto the top of 100µL of water pre-loaded into the SPE wells. These samples were then slowly drained from the SPE wells. Water-top loading reduced the guanidine concentration in the digested plasma sample from 0.63M to 0.41M after SPE loading. The sample was then washed with two additional 200µL volumes of 50mM ammonium acetate pH 5.5 buffer and one 200µL volume of 10% methanol. Finally, the adsorbed and purified analyte was eluted from the SPE wells using two 50 µL eluents consisting of 50 mM triethylamine in 50% methanol (pH 11.5). The resulting samples were diluted with an equal volume of water and then directly injected for ion-paired reversed-phase chromatography and detected by triple quadrupole mass spectrometry.

[0134] Figure 12 This demonstrates the recovery rate and CLARITY of each oligonucleotide using the sample preparation and extraction methods presented in the above examples, including direct plasma sample loading onto SPE and top loading onto SPE wells containing water. ® OTX ™ Results of the plan.

[0135] like Figure 12 As can be seen, top loading significantly improves the recovery rate of the method used in the above embodiments, and is superior to CLARITY. ® OTX ™ More than twice the size of the original plan.

[0136] Example 11 - Use of a detergent-free protocol for biological samples formed from tissue homogenates .

[0137] The extraction method of this invention can be used for organ tissues. To produce a sample, the organ tissue is homogenized before or simultaneously with protein hydrolysis. The following examples illustrate an embodiment in which digestion is performed after homogenization.

[0138] Thirty (30) mg of organ tissue was added to zirconia, ceramic, glass, or steel beads along with 70 μL of methanol, 20 μL of 6M guanidine denaturant solution, and 10 μL of 0.5M TCEP reducing agent solution. The tissue was then lysed using a tissue homogenizer (Precellys homogenizer, Bertin Corporation, Rockville Maryland) via bead destruction. Solutions with this composition can also be used with sonication and pressure / shear-based destruction. In this step, a tissue weight / solvent volume greater than 1 mg / 2 μL solvent but less than 1 mg / 10 μL solvent is advantageous. Optionally, a guanidine solution was added from a stock solution consisting of 3M to 6M guanidine. The guanidine counterion can be, but is not limited to, chloride or thiocyanate. The organic solvent can be methanol or another proton or aprotic solvent. Methanol and ethanol are preferred. For bead-based destruction, the procedure can be performed using homogenizing beads with a diameter ranging from 0.1 mm to 2 mm.

[0139] After collecting the homogenate, add 50 μL of water to the tube, followed by 50 μL of 20 mg / mL proteinase K. Incubate the sample at 55 °C for 40 minutes, or at 40 °C for 1 hour if analyzing double-stranded oligonucleotides. The proteinase K concentration and aliquot volume can be optimized along with the incubation time and temperature to achieve the desired digestion completeness.

[0140] In one alternative embodiment, proteinase K is added to the tissue sample before or during the homogenization step (i.e., digestion and homogenization occur simultaneously). In some embodiments, a proteolytic enzyme other than proteinase K is applied.

[0141] In any optional step of the protocol, the tissue homogenate digested with proteinase K can be clarified for particulate matter using procedures including, but not limited to, high-speed centrifugation or filtration. The supernatant or filtered solution will be further processed using the solid-phase extraction techniques outlined above (e.g., Figure 1A or Figure 1B ).

[0142] Example 12 - Comparison of elution solutions for oligonucleotide solid phase extraction methods In this embodiment, three different eluent solutions (i.e., eluents) are used in the solid-phase extraction method according to the present invention, and the recovery rates of oligonucleotide extraction are compared. Each of the three eluents contains triethylamine (TEA), and some eluents also contain ammonium hydroxide. Specifically, eluent 1 contains 50 mM TEA and 50% MeOH. Eluent 2 contains 50 mM TEA, 50% MeOH, and 0.15% NH4OH. Eluent 3 contains 100 mM TEA, 50% MeOH, and 0.3% NH4OH.

[0143] To compare recovery results based on elution solution type (i.e., eluent 1, eluent 2, or eluent 3), a stock sample was prepared as follows: The following components were added to and mixed with a lyophilized sample of 20-mer ssDNA promoter (10 μg, 1.6 nmol): 1.74 μL tyrosine (Tyr, 10 mg / mL) (9.6 nmol); 100 µL 100% MeOH; 17.5 µL Milli-Q water; 2000 µL 200 mM ammonium acetate at pH 5.5 to achieve a final volume of 4000 μL.

[0144] Then, using a 10 mg 1 cc column, solid-phase extraction was performed on the prepared stock sample using the following protocol. The column was treated with 1000 μL of 100% MeOH to run through the column to the waste. The treated column was then equilibrated with 1000 μL of 100 mM ammonium acetate (pH 5.5). Vacuum-based suction was applied and the fluid was directed to the waste. The prepared stock sample (1000 μL) was loaded onto the column and suction was applied. A first wash of 600 μL of 100 mM ammonium acetate (pH 5.5) was applied and allowed to pass through the column to the waste. A second wash of 600 μL of 50% MeOH was added to the column and allowed to pass through the column to the waste. To elute the extract from the column, 200 μL of one of the eluent solutions (i.e., eluent 1, eluent 2, or eluent 3) was added to the column to elute the bound analyte into the collection tube. A specific elution solution was added three times in total to collect the combined volume from the column. For example, 200 μL of eluent 1 was added to the column three times in total to collect the combined volume eluted with eluent 1 (combined sample 1). 200 μL of eluent 2 was added to the column three times in total to collect the combined volume eluted with eluent 2 (combined sample 2). And 200 μL of eluent 3 was added to the column three times in total to collect the combined volume eluted with eluent 3 (combined sample 3).

[0145] The pooled samples (i.e., pooled sample 1, pooled sample 2, and pooled sample 3) were diluted with equal volumes of 100 mM ammonium acetate and Milli-Q water and analyzed using LC-UV injection.

[0146] Table 7: LC-UV data acquisition details UHPLC system ACQUITY ™ PREMIER UPLC ™ (Waters Corporation) Column ACQUITY ™ PREMIER BEH C18 OST column (p / n 186009484, Waters Corporation), 2.1 x 50 mm Solvent line A 0.1 M TEAA (100) Solvent line B 0.1 M TEAA / acetonitrile (50 / 50) Column temperature 60℃ Sample temperature 4℃ Injection volume 5.0µL Injection type Full loop, needle overflow Weak wash 100% Milli-Q water (600 μL) Strong wash 10% acetonitrile / 90% HPLC grade water (200 μL) Seal wash 20 / 80 ACN / H2O Detector TUV Wavelength 260 nm Filter None Flow rate 0.6 mL / min Sampling rate 20 points / sec Table 8: LC-UV gradient parameters Time (min) Flow rate (mL / min) % A % B Curve 0.0 0.6 99.9 0.1 15 0.6 0 100 6 15.1 0.6 0 100 1 18.0 0.6 99.9 0.1 1 Using the procedure described above, the recoveries of analytes were tested with three different SPE eluents (eluent 1, eluent 2, and eluent 3). The results are presented in Table 9 below. Higher recoveries were observed when the SPE eluent contained more than 50 mM TEA and at least 0.3% ammonium hydroxide. Regardless of the eluent concentration, the pH of the SPE eluent remained constant, indicating that the ionic strength of TEA and ammonium hydroxide affected the recovery of bound analytes (i.e., 20-mer ssDNA and lipid ASO).

[0147] Table 9: Effect of SPE eluent composition on oligonucleotide recovery Eluent Composition of eluent 20-mer ssDNA Lipid ASO 1 50 mM TEA, 50% MeOH 84.5±7.4% 72.5±0.9% 2 50 mM TEA, 50% MeOH, 0.15% NH4OH 84.9±7.8% 70.0±0.8% 3 100 mM TEA, 50% MeOH, 0.3% NH4OH 93.4±2.4% 90.4±0.9% Example 13 - Method and elution solution for oligonucleotide urine and plasma WAX SPE recovery using the present technology on samples containing oligodeoxythymidine (15-35T), Gem91, Gem132 oligonucleotides Figure 13 .

[0148] In this embodiment, the oligonucleotide recovery rate using the method of the present invention was evaluated for three elution solutions from Example 12. Two different oligonucleotide samples were analyzed: a urine sample and a plasma sample. Each of the urine and plasma samples was doped with four different oligonucleotide-containing solutions: oligodeoxythymidine (15, 20, 25, 30, and 35-ODT), GEM91, GEM132, and lipid-modified oligonucleotides, wherein the final concentrations of GEM91 and GEM132 were 1 μg / mL, and the final concentrations of oligodeoxythymidine and lipid-modified oligonucleotides were 0.1 pmol / µL.

[0149] Doped urine and plasma samples were prepared separately for use by diluting 100 μL of doped (plasma or urine) sample with 20 μL of 6M guanidine hydrochloride in 60 mM pH 7.5 Tris buffer (0.66 M final concentration), 10 μL of 500 mM tris(2-carboxyethyl)phosphine (approximately 28 mM final concentration), and 50 μL of proteinase K 20 mg / mL solution (Qiagen, product number 19131). The processed samples were vortexed and then incubated at 55 °C for 60 min.

[0150] After incubation, solid-phase extraction was performed using a 96-well microelution plate and a vacuum manifold filled with 2 mg of Oasis WAX 30 µm adsorbent. Each adsorbent bed was first treated with two 200 µL aliquots of methanol, followed by equilibration with two 200 µL aliquots of 50 mM ammonium acetate pH 5.5 buffer. A full volume of digested plasma sample (180 µL) was loaded onto the top of 100 µL of water pre-loaded into the SPE wells. These samples were then slowly expelled from the SPE wells. The samples were then washed with two additional 200 µL aliquots of 50 mM ammonium acetate pH 5.5 buffer and one 200 µL aliquot of 10% methanol. Finally, the adsorbed and purified analytes were eluted from the SPE wells using two 25 μL volumes of one of the three eluent solutions (eluent 1: consisting of 50 mM triethylamine (TEA) in 50% methanol; eluent 2: consisting of 50 mM TEA in 50% methanol with 0.15% ammonium hydroxide (NH4OH); or eluent 3: consisting of 100 mM TEA in 50% methanol with 0.3% ammonium hydroxide (NH4OH)).

[0151] The obtained samples were diluted with an equal volume of water and then directly injected for ion-paired reversed-phase chromatography and detected by triple quadrupole mass spectrometry. Specifically, six samples were injected and analyzed by triple quadrupole mass spectrometry: urine eluted with eluent 1 was doped with oligonucleotides (… Figure 13 (Left side bar); urine eluted with eluent 2 was doped with oligonucleotides ( Figure 13 (Central stripe); urine eluted with eluent 3 was doped with oligonucleotides ( Figure 14 (Right bar); plasma doped with oligonucleotides eluted with eluent 1 ( Figure 14 (Left side bar); plasma doped with oligonucleotides eluted with eluent 2 ( Figure 14 (intermediate strip); and plasma-doped oligonucleotides eluted with eluent 3 ( Figure 13 (Right side bar).

[0152] Figure 14 and ​ The data shown indicate that while eluent 1 provided the recovery of oligonucleotides, eluents 2 and 3 (which consist of a combination of organic amines and ammonium hydroxide) provided improved or optimized recovery, and in some cases provided an almost 1.5-fold increase in recovery (as a result of lipid modification).

[0153] The volume of water was determined, and then directly injected for ion-paired reversed-phase chromatography and detected by triple quadrupole mass spectrometry.

[0154] While this disclosure has been specifically shown and described with reference to its exemplary embodiments, those skilled in the art will understand that various changes in form and detail may be made without departing from the scope of the technology as covered by the appended claims.

Claims

1. A method for extracting one or more oligonucleotides from a biological sample, the method comprising: The biological sample is combined with a protease solution, allowing the sample to undergo protein hydrolysis and digestion in a detergent-free mixture. The digested sample is loaded onto an adsorbent material containing porous particles, wherein the surface of the porous particles is modified with a ligand having a protonable group and the ligand having a pKa value between about 8 and about 11. One or more washing solutions are passed through the adsorbent material, causing one or more non-oligonucleotide components to be removed from the adsorbent material, while the one or more oligonucleotides remain on the adsorbent material; and An elution solution with a pH range of about 10 to about 12 is passed through the adsorbent material, causing one or more oligonucleotides retained from the adsorbent material to be released into the elution solution, thereby obtaining an eluent.

2. The method of claim 1, wherein the protease solution comprises proteinase K.

3. The method according to any one of claims 1 to 2, wherein the detergent-free mixture further comprises guanidine.

4. The method according to any one of claims 1 to 3, wherein the porous particles have a size greater than 5 μm and less than 100 μm.

5. The method according to any one of claims 1 to 4, wherein the method further comprises subjecting the eluent to liquid chromatography, mass spectrometry (MS), ultraviolet-visible spectroscopy, or a combination thereof.

6. The method according to any one of claims 1 to 5, wherein the method further comprises directly injecting the eluent without evaporation to perform ion pairing reverse phase separation.

7. The method according to any one of claims 1 to 6, wherein the one or more washing solutions comprise a partially aqueous organic solvent solution containing methanol or ethanol, or combinations thereof.

8. The method according to any one of claims 1 to 7, wherein the elution solution comprises a base selected from organic amines, ammonium bicarbonate, ammonium hydroxide, ammonium acetate, or combinations thereof.

9. The method according to any one of claims 1 to 8, wherein the one or more elution solutions comprise the organic amine triethylamine (TEA).

10. The method according to any one of claims 1 to 9, wherein the biological sample comprises a biological fluid selected from the group consisting of whole blood samples, plasma samples, serum samples, oral fluid, cerebrospinal fluid, fecal samples, nasal samples, and urine.

11. The method according to any one of claims 1 to 10, wherein the biological sample comprises biological tissue selected from the group consisting of: liver, kidney and brain tissue, tissue homogenate, cells and cell culture supernatant.

12. The method of claim 11, wherein the biological sample comprises a tissue homogenate, and the tissue homogenization step without surfactant is performed simultaneously with the hydrolytic digestion of the sample proteins.

13. The method of claim 11, wherein the biological sample comprises a tissue homogenate, and the surfactant-free tissue homogenization step is performed before the sample proteins are hydrolyzed and digested.

14. The method of claim 12 or claim 13, wherein an organic solvent is added at a concentration greater than 20% by volume (e.g., 50% or more) during the tissue homogenization step.

15. The method according to any one of claims 13 to 14, wherein after the tissue homogenization step, the tissue homogenate is diluted 2 to 4 times with an aqueous solution and then incubated with a protease to hydrolyze and digest the sample protein.

16. The method according to any one of claims 1 to 15, wherein the one or more oligonucleotides are selected from double-stranded RNA, single-stranded RNA, single-stranded DNA, double-stranded DNA, double-stranded RNA / DNA hybrids, synthetic RNA, synthetic DNA, or combinations thereof, wherein the size range of the one or more nucleotides is from 10 to 200 units.

17. The method according to any one of claims 1 to 16, wherein the eluent solution further comprises an organic solvent at a concentration between 10% and 70% by volume.

18. The method according to any one of claims 1 to 17, the method further comprising diluting the eluent with an equal volume of water.

19. The method according to any one of claims 1 to 18, wherein the protease solution, the adsorbent material, and the elution solution are included in the kit.

20. A method for extracting one or more oligonucleotides from a biological sample, the method comprising: The biological sample is hydrolyzed and digested by combining it with proteinase K solution and guanidine. The digested sample is loaded onto an adsorbent material containing porous particles, wherein the surface of the porous particles is modified with a ligand having a protonable group having a pKa value between about 8 and about 11 (e.g., between about 8 and about 10). One or more washing solutions are passed through the adsorbent material, causing one or more non-oligonucleotide components to be removed from the adsorbent material, while the one or more oligonucleotides remain on the adsorbent material; and An elution solution with a pH range of about 10 to about 12 is passed through the adsorbent material, causing one or more oligonucleotides retained from the adsorbent material to be released into the elution solution, thereby obtaining an eluent.

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