Aptamer-based affinity capture method for selective enrichment of Fc domain of human immunoglobulin

Abstract

The invention relates to an aptamer-based affinity capture method for selective enrichment of a human immunoglobulin Fc domain. The invention provides a method for capturing a human immunoglobulin Fc domain in a biological fluid sample. The method includes providing an affinity capture device. The affinity capture device comprises a surface having an aptamer that is immobilized to the surface of the affinity capture device and is at least 80% identical to SEQ ID NO 1. The biological fluid sample is diluted with a binding buffer. The binding buffer solution comprises (A) tris (hydroxymethyl) aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 [mu] M and about 20 mM; and (C) a total monovalent cation concentration of 0 to not more than 100 mM. The human immunoglobulin Fc domain in the biological fluid sample is adsorbed to the aptamer with the binding buffer.

Description

[0001] This application is a divisional application. The application date of the original application is 2020.06.26, the application number is 202080046686.1 (PCT / IB2020 / 056089), and the name of the invention is “Appatamer-based affinity capture method for selective enrichment of human immunoglobulin FC domain”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This patent application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 866,830, filed on June 26, 2019, entitled “Aptamer Based Affinity Capture Methods for the Selective Enrichment of Human Immunoglobulin FcDomains,” the entire contents of which are hereby incorporated by reference.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on June 24, 2020 is named W-4126-US02_(102994_1005_3)_SL.txt and is 5 kilobytes in size. Technical Field

[0006] The present disclosure relates to methods and kits for selectively capturing human Fc domains found in immunoglobulins or contained within humanized monoclonal antibodies using aptamers. More specifically, the present technology relates to improved selectivity and binding of aptamer ligands to more effectively capture human Fc domains found in immunoglobulins. Background Art

[0007] In the past few decades, the market of immunotherapy has grown exponentially. Until 2018, more than 80 kinds of antibody therapeutic agents have been approved by FDA, and hundreds of candidates have undergone clinical trials. Human monoclonal antibodies (mAbs) represent one of the most commonly used antibody treatments, and provide a significantly reduced immunogenicity risk. About 80% of the FDA-approved antibody drugs are fully human or humanized antibodies with more than 90% human sequence origin. In order to keep up with this rapid growth in demand for human antibody therapeutic agents, high throughput, selective purification of target proteins from cell culture and biofluid samples is required. Summary of the invention

[0008] Methods and kits are needed that use aptamers to selectively capture human Fc domains found in immunoglobulins or contained within humanized monoclonal antibodies. Due to the rapid growth in demand for human antibody therapeutics, the methods of the present technology are high-throughput and selective purification of target proteins from cell culture and biofluid samples. The present technology is robust and facilitates human Fc capture, enabling detailed and accurate analysis to be performed during pharmacokinetic, pharmacodynamic, and biotransformation studies.

[0009] Surprisingly, it was found that there is competition between monovalent and divalent cations in the binding buffer used to bind the aptamer to the substrate, and surprisingly, the binding buffer is susceptible to the influence of different cations. These surprising findings lead to the exclusion of common buffers from the present method, as described in detail below. One benefit of the present technology is that complex matrices (e.g., biological fluids) can be reduced at their monovalent cation concentrations, and the negative effects of endogenous monovalent ions are minimized, which provides improved binding capacity of the aptamer without loss of selectivity.

[0010] In one aspect, the present technology relates to a method for capturing a human immunoglobulin Fc domain in a biological fluid sample. The method includes providing an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 1. The method also includes diluting the biological fluid sample with a binding buffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The human immunoglobulin Fc domain in the biological fluid sample is adsorbed to the aptamer with a binding buffer. SEQ ID NO 1 is shown below. The method may include one or more of the embodiments described herein.

[0011] SEQ ID NO 1:

[0012] 5' end - G GrArGrG[i2FU]rG C[i2FU]CC GAAArGrGAA[i2FC][i2FU]CC – 3' end

[0013] Where A, C, G, U represent the four nucleobases. Capital letters correspond to deoxyribonucleotides, and rX indicates ribonucleotides. [i2FC] and [i2FU] represent 2'-fluoro substituted pyrimidine nucleotides

[0014] In another aspect, the present technology relates to a method for capturing a human immunoglobulin Fc domain in a biological fluid sample. The method includes providing an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 2. The method also includes diluting the biological fluid sample with a binding buffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The human immunoglobulin Fc domain in the biological fluid sample is adsorbed to the aptamer with a binding buffer. SEQ ID NO 2 is shown below. The method may include one or more of the embodiments described herein.

[0015] SEQ ID NO 2:

[0016] 5' end – G GrArGrG[i2FU]rG[i2FC][i2FU][i2FC][i2FC]GAAArGrG rA rA[i2FC][i2FU]CC – 3' end

[0017] Where A, C, G, U represent the four nucleobases. Capital letters correspond to deoxyribonucleotides, and rX indicates ribonucleotides. [i2FC] and [i2FU] represent 2'-fluoro substituted pyrimidine nucleotides

[0018] In another aspect, the present technology relates to a method for capturing a human immunoglobulin Fc domain in a biological fluid sample. The method includes providing an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 3. The method also includes diluting the biological fluid sample with a binding buffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The human immunoglobulin Fc domain in the biological fluid sample is adsorbed to the aptamer with a binding buffer. SEQ ID NO 3 is shown below. The method may include one or more of the embodiments described herein.

[0019] SEQ ID NO 3:

[0020] 5’ end–GG rA rG rG[i2FU]rG[i2FC][i2FU][i2FC]CGAAA rG rG rA rA[i2FC][i2FU]CC–3’ end

[0021] Where A, C, G, U represent the four nucleobases. Capital letters correspond to deoxyribonucleotides, and rX indicates ribonucleotides. [i2FC] and [i2FU] represent 2'-fluoro substituted pyrimidine nucleotides

[0022] In another aspect, the present technology relates to a method for capturing a human immunoglobulin Fc domain in a biological fluid sample. The method includes providing an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 4. The method also includes diluting the biological fluid sample with a binding buffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The human immunoglobulin Fc domain in the biological fluid sample is adsorbed to the aptamer with the binding buffer. SEQ ID NO 4 is shown below. The method may include one or more of the embodiments described herein.

[0023] SEQ ID NO 4:

[0024] 5’ end–G(OMe)G(OMe)rA rG rG[i2FU]rG[i2FC][i2FU][i2FC][i2FC]G(OMe)A(OMe)A(OMe)A(OMe)rGrGrArA[i2FC][i2FU]C(OMe)C(OMe)–3’ end

[0025] Wherein A, C, G, U represent four nucleobases. Capital letters correspond to deoxyribonucleotides, and rX indicates ribonucleotides. [i2FC] and [i2FU] represent 2'-fluoro substituted pyrimidine nucleotides. C(OMe), G(OMe) and A(OMe) represent 2'-methoxy substituted nucleotides.

[0026] In another aspect, the present technology relates to a method for capturing a human immunoglobulin Fc domain in a biological fluid sample. The method includes providing an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 5. The method also includes diluting the biological fluid sample with a binding buffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The human immunoglobulin Fc domain in the biological fluid sample is adsorbed to the aptamer with the binding buffer. SEQ ID NO 5 is shown below. The method may include one or more of the embodiments described herein.

[0027] SEQ ID NO 5:

[0028] 5' end–GG rA rG rG[i2FU]rG CU(OMe)CCGAAA rG rG AA[i2FC][i2FU]CC–3' end

[0029] Wherein A, C, G, U represent four nucleobases. Capital letters correspond to deoxyribonucleotides, and rX indicates ribonucleotides. [i2FC] and [i2FU] represent 2'-fluoro substituted pyrimidine nucleotides. U(OMe) represents 2'-methoxy substituted nucleotides.

[0030] In another aspect, the present technology relates to a method for capturing a human immunoglobulin Fc domain in a biological fluid sample. The method includes providing an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 6. The method also includes diluting the biological fluid sample with a binding buffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The human immunoglobulin Fc domain in the biological fluid sample is adsorbed to the aptamer with the binding buffer. SEQ ID NO 6 is shown below. The method may include one or more of the embodiments described herein.

[0031] SEQ ID NO 6:

[0032] 5’ end–G(OMe)G(OMe)rA rG rG[i2FU]rG[i2FC]U(OMe)[i2FC][i2FC]G(OMe)A(OMe)A(OMe)A(OMe)rGrGrArA[i2FC][i2FU]C(OMe)C(OMe)–3’ end

[0033] Wherein A, C, G, U represent four nucleobases. Capital letters correspond to deoxyribonucleotides, and rX indicates ribonucleotides. [i2FC] and [i2FU] represent 2'-fluoro substituted pyrimidine nucleotides. U(OMe), C(OMe), G(OMe) and A(OMe) represent 2'-methoxy substituted nucleotides.

[0034] In another aspect, the present technology relates to a method for capturing a human immunoglobulin Fc domain in a biological fluid sample. The method includes providing an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 7. The method also includes diluting the biological fluid sample with a binding buffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The human immunoglobulin Fc domain in the biological fluid sample is adsorbed to the aptamer with the binding buffer. SEQ ID NO 7 is shown below. The method may include one or more of the embodiments described herein.

[0035] SEQ ID NO 7:

[0036] 5’ end–G(OMe)G(OMe)rA rG rG[i2FU]rG[i2FC][i2FU]C(OMe)[i2FC]G(OMe)A(OMe)A(OMe)A(OMe)rGrGrArA[i2FC][i2FU]C(OMe)C(OMe)–3’ end

[0037] Wherein A, C, G, U represent four nucleobases. Capital letters correspond to deoxyribonucleotides, and rX indicates ribonucleotides. [i2FC] and [i2FU] represent 2'-fluoro substituted pyrimidine nucleotides. C(OMe), G(OMe) and A(OMe) represent 2'-methoxy substituted nucleotides.

[0038] In another aspect, the present technology relates to a method for capturing a human immunoglobulin Fc domain in a biological fluid sample. The method includes providing an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 8. The method also includes diluting the biological fluid sample with a binding buffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The human immunoglobulin Fc domain in the biological fluid sample is adsorbed to the aptamer with the binding buffer. SEQ ID NO 8 is shown below. The method may include one or more of the embodiments described herein.

[0039] SEQ ID NO 8:

[0040] 5’ end–G(OMe)G(OMe)rA rG rG[i2FU]rG[i2FC][i2FU][i2FC]C(OMe)G(OMe)A(OMe)A(OMe)A(OMe)rGrGrArA[i2FC][i2FU]C(OMe)C(OMe)–3’ end

[0041] Wherein A, C, G, U represent four nucleobases. Capital letters correspond to deoxyribonucleotides, and rX indicates ribonucleotides. [i2FC] and [i2FU] represent 2'-fluoro substituted pyrimidine nucleotides. C(OMe), G(OMe) and A(OMe) represent 2'-methoxy substituted nucleotides.

[0042] In another aspect, the present technology relates to a method for capturing a human immunoglobulin Fc domain in a biological fluid sample. The method includes providing an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 9. The method also includes diluting the biological fluid sample with a binding buffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The human immunoglobulin Fc domain in the biological fluid sample is adsorbed to the aptamer with the binding buffer. SEQ ID NO 9 is shown below. The method may include one or more of the embodiments described herein.

[0043] SEQ ID NO 9:

[0044] 5’ end–GG rA rG[i2FG][i2FU]rG C[i2FU]CCGAAA rG rG AA[i2FC][i2FU]CC–3’ end

[0045] Where A, C, G, U represent the four nucleobases. Capital letters correspond to deoxyribonucleotides, and rX indicates ribonucleotides. [i2FC] and [i2FU] represent 2'-fluoro substituted pyrimidine nucleotides

[0046] In another aspect, the present technology relates to a method for capturing a human immunoglobulin Fc domain in a biological fluid sample. The method includes providing an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 10. The method also includes diluting the biological fluid sample with a binding buffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The human immunoglobulin Fc domain in the biological fluid sample is adsorbed to the aptamer with the binding buffer. SEQ ID NO 10 is shown below. The method may include one or more of the embodiments described herein.

[0047] SEQ ID NO 10:

[0048] 5’ end–GG rA rG rG[i2FU][i2FG]C[i2FU]CCGAAA rG rG AA[i2FC][i2FU]CC–3’ end

[0049] Where A, C, G, U represent the four nucleobases. Capital letters correspond to deoxyribonucleotides, and rX indicates ribonucleotides. [i2FC] and [i2FU] represent 2'-fluoro substituted pyrimidine nucleotides

[0050] In another aspect, the present technology relates to a method for capturing a human immunoglobulin Fc domain in a biological fluid sample. The method includes providing an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 11. The method also includes diluting the biological fluid sample with a binding buffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The human immunoglobulin Fc domain in the biological fluid sample is adsorbed to the aptamer with the binding buffer. SEQ ID NO 11 is shown below. The method may include one or more of the embodiments described herein.

[0051] SEQ ID NO 11:

[0052] 5' end – GGrArGrG[i2FU]rGrC[i2FU]CC GAAArGrGAA[i2FC][i2FU]CC – 3' end

[0053] Where A, C, G, U represent the four nucleobases. Capital letters correspond to deoxyribonucleotides, and rX indicates ribonucleotides. [i2FC] and [i2FU] represent 2'-fluoro substituted pyrimidine nucleotides

[0054] In another aspect, the present technology relates to a method for capturing a human immunoglobulin Fc domain in a biological fluid sample. The method includes providing an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 12. The method also includes diluting the biological fluid sample with a binding buffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The human immunoglobulin Fc domain in the biological fluid sample is adsorbed to the aptamer with the binding buffer. SEQ ID NO 12 is shown below. The method may include one or more of the embodiments described herein.

[0055] SEQ ID NO 12:

[0056] 5' end – G GrA rG rG[i2FU]rG C rU CC GAAArG rGA A[i2FC][i2FU]CC – 3' end

[0057] Where A, C, G, U represent the four nucleobases. Capital letters correspond to deoxyribonucleotides, and rX indicates ribonucleotides. [i2FC] and [i2FU] represent 2'-fluoro substituted pyrimidine nucleotides

[0058] In another aspect, the present technology relates to a method for capturing a human immunoglobulin Fc domain in a biological fluid sample. The method includes providing an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 13. The method also includes diluting the biological fluid sample with a binding buffer. The binding buffer includes (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The human immunoglobulin Fc domain in the biological fluid sample is adsorbed to the aptamer with the binding buffer. SEQ ID NO 13 is shown below. The method may include one or more of the embodiments described herein.

[0059] SEQ ID NO 13:

[0060] 5' end–GG rA rG rG[i2FU]rG CU(OMe)CCGAAA rG rG AA[i2FC][i2FU]CC–3' end

[0061] Wherein A, C, G, U represent four nucleobases. Capital letters correspond to deoxyribonucleotides, and rX indicates ribonucleotides. [i2FC] and [i2FU] represent 2'-fluoro substituted pyrimidine nucleotides. U(OMe) represents 2'-methoxy substituted nucleotides.

[0062] In some embodiments, the aptamer is at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). The aptamer can, for example, be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). These percentages can be used to form a range, for example, the aptamer can be 85% to 99% identical or 90% to 95% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). In some embodiments, the aptamer is a 23-nucleotide aptamer. The aptamer can be a 23-nucleotide aptamer with an extended sequence or a truncated sequence of about 3 residues. The aptamer can be a 23-nucleotide aptamer with an extended sequence or a truncated sequence of about 5 residues. The aptamer can be covalently immobilized or non-covalently immobilized to the surface of the affinity capture device.

[0063] In some embodiments, the binding buffer has a monovalent cation concentration of less than about 50 mM. The binding buffer may have a monovalent cation concentration of less than about 30 mM. In some embodiments, the pH of the binding buffer is between about 5 and about 9. The pH of the binding buffer may be between about 6 and about 8. The pH of the binding buffer may be about 7.2. For example, the pH of the binding buffer can be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. These pH values ​​can be used to form a range, for example, from about 7.0 to about 7.4, or from about 7.1 to about 7.3.

[0064] The biological fluid sample can be diluted. For example, the biological fluid sample can be diluted by a factor of 2, 10, or 20. In some embodiments, the biological fluid sample is diluted by a factor of 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. These dilution factors can be used to form a range, such as about 2 to about 20, or about 2 to about 10, or about 10 to about 20. In some embodiments, the biological fluid sample is diluted to obtain a total monovalent cation concentration of the biological fluid sample of no more than 100mM, no more than 50mM, or no more than 30mM. The total monovalent cation concentration of the diluted biological fluid sample can be no more than 100mM, 90mM, 80mM, 70mM, 60mM, 50mM, 40mM, 30mM, 20mM, or 10mM.

[0065] In some embodiments, the method further comprises eluting the adsorbed human immunoglobulin Fc domain from the fixed aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM. The ammonium is presented in the form of tetramethylammonium, triethylammonium, ammonium formate or ammonium acetate. In some embodiments, the ammonium concentration is between about 50 mM and about 500 mM. The pH of the eluent may be between about 6.5 and about 8.0. The pH of the eluent may be about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0. These pH values ​​can be used to form a range, for example, between about 7.0 and about 8.0, or between about 6.5 and about 7.5.

[0066] In some embodiments, the method further comprises washing the adsorbed human immunoglobulin Fc domain with a binding buffer. The method may further comprise washing the adsorbed human immunoglobulin Fc domain with a binding buffer comprising Ca + The adsorbed human immunoglobulin Fc domain was washed with a buffer.

[0067] In some embodiments, the concentration of magnesium cations is between about 50 μM and about 1 mM. The concentration of magnesium cations can be, for example, 50 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM, 500 μM, 550 μM, 600 μM, 650 μM, 700 μM, 750 μM, 800 μM, 850 μM, 900 μM, 950 μM or 1000 μM. These concentrations can be used to form a range, for example, between about 50 μM and about 500 μM, or between about 50 μM and about 200 μM.

[0068] In some embodiments, the method further comprises analyzing the eluted human immunoglobulin Fc domain with a detector. The detector can be a sandwich enzyme-linked immunosorbent assay or a mass spectrometer.

[0069] In some embodiments, the human immunoglobulin Fc domain is contained within a humanized monoclonal antibody.The method may also include purifying the humanized monoclonal antibody.

[0070] In another aspect, the present technology relates to a method for eluting a human immunoglobulin Fc domain from an immobilized aptamer. The method includes providing an affinity capture device having a surface containing an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 1. The immobilized aptamer has an adsorbed human immunoglobulin Fc domain. The method also includes eluting the adsorbed human immunoglobulin Fc domain from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM. The ammonium is presented in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The method may include one or more of the embodiments described herein.

[0071] In another aspect, the present technology relates to a method for eluting a human immunoglobulin Fc domain from an immobilized aptamer. The method includes providing an affinity capture device having a surface containing an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO2. The immobilized aptamer has an adsorbed human immunoglobulin Fc domain. The method also includes eluting the adsorbed human immunoglobulin Fc domain from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM. The ammonium is presented in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The method may include one or more of the embodiments described herein.

[0072] In another aspect, the present technology relates to a method for eluting a human immunoglobulin Fc domain from an immobilized aptamer. The method includes providing an affinity capture device having a surface containing an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 3. The immobilized aptamer has an adsorbed human immunoglobulin Fc domain. The method also includes eluting the adsorbed human immunoglobulin Fc domain from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM. The ammonium is presented in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The method may include one or more of the embodiments described herein.

[0073] In another aspect, the present technology relates to a method for eluting a human immunoglobulin Fc domain from an immobilized aptamer. The method includes providing an affinity capture device having a surface containing an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO4. The immobilized aptamer has an adsorbed human immunoglobulin Fc domain. The method also includes eluting the adsorbed human immunoglobulin Fc domain from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM. The ammonium is presented in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The method may include one or more of the embodiments described herein.

[0074] In another aspect, the present technology relates to a method for eluting a human immunoglobulin Fc domain from an immobilized aptamer. The method includes providing an affinity capture device having a surface containing an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 5. The immobilized aptamer has an adsorbed human immunoglobulin Fc domain. The method also includes eluting the adsorbed human immunoglobulin Fc domain from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM. The ammonium is presented in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The method may include one or more of the embodiments described herein.

[0075] In another aspect, the present technology relates to a method for eluting a human immunoglobulin Fc domain from an immobilized aptamer. The method includes providing an affinity capture device having a surface containing an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 6. The immobilized aptamer has an adsorbed human immunoglobulin Fc domain. The method also includes eluting the adsorbed human immunoglobulin Fc domain from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM. The ammonium is presented in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The method may include one or more of the embodiments described herein.

[0076] In another aspect, the present technology relates to a method for eluting a human immunoglobulin Fc domain from an immobilized aptamer. The method includes providing an affinity capture device having a surface containing an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 7. The immobilized aptamer has an adsorbed human immunoglobulin Fc domain. The method also includes eluting the adsorbed human immunoglobulin Fc domain from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM. The ammonium is presented in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The method may include one or more of the embodiments described herein.

[0077] In another aspect, the present technology relates to a method for eluting a human immunoglobulin Fc domain from an immobilized aptamer. The method includes providing an affinity capture device having a surface containing an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 8. The immobilized aptamer has an adsorbed human immunoglobulin Fc domain. The method also includes eluting the adsorbed human immunoglobulin Fc domain from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM. The ammonium is presented in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The method may include one or more of the embodiments described herein.

[0078] In another aspect, the present technology relates to a method for eluting a human immunoglobulin Fc domain from an immobilized aptamer. The method includes providing an affinity capture device having a surface containing an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 9. The immobilized aptamer has an adsorbed human immunoglobulin Fc domain. The method also includes eluting the adsorbed human immunoglobulin Fc domain from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM. The ammonium is presented in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The method may include one or more of the embodiments described herein.

[0079] In another aspect, the present technology relates to a method for eluting a human immunoglobulin Fc domain from an immobilized aptamer. The method includes providing an affinity capture device having a surface containing an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 10. The immobilized aptamer has an adsorbed human immunoglobulin Fc domain. The method also includes eluting the adsorbed human immunoglobulin Fc domain from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM. The ammonium is presented in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The method may include one or more of the embodiments described herein.

[0080] In another aspect, the present technology relates to a method for eluting a human immunoglobulin Fc domain from an immobilized aptamer. The method includes providing an affinity capture device having a surface containing an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 11. The immobilized aptamer has an adsorbed human immunoglobulin Fc domain. The method also includes eluting the adsorbed human immunoglobulin Fc domain from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM. The ammonium is presented in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The method may include one or more of the embodiments described herein.

[0081] In another aspect, the present technology relates to a method for eluting a human immunoglobulin Fc domain from an immobilized aptamer. The method includes providing an affinity capture device having a surface containing an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 12. The immobilized aptamer has an adsorbed human immunoglobulin Fc domain. The method also includes eluting the adsorbed human immunoglobulin Fc domain from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM. The ammonium is presented in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The method may include one or more of the embodiments described herein.

[0082] In another aspect, the present technology relates to a method for eluting a human immunoglobulin Fc domain from an immobilized aptamer. The method includes providing an affinity capture device having a surface containing an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 13. The immobilized aptamer has an adsorbed human immunoglobulin Fc domain. The method also includes eluting the adsorbed human immunoglobulin Fc domain from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM. The ammonium is presented in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The method may include one or more of the embodiments described herein.

[0083] In some embodiments, the aptamer is at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). The aptamer can, for example, be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). These percentages can be used to form a range, for example, the aptamer can be 85% to 99% identical or 90% to 95% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). In some embodiments, the aptamer is a 23-nucleotide aptamer. The aptamer can be a 23-nucleotide aptamer with an extended sequence or a truncated sequence of about 3 residues. The aptamer can be a 23-nucleotide aptamer with an extended sequence or a truncated sequence of about 5 residues. The aptamer can be covalently immobilized or non-covalently immobilized to the surface of the affinity capture device.

[0084] In some embodiments, the ammonium concentration is between about 50 mM and about 500 mM. For example, the ammonium concentration can be about 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM. These values ​​can be used to form a range, for example, between about 200 mM and about 400 mM.

[0085] In some embodiments, the eluent has a pH between about 6.5 and about 8.0. For example, the pH of the eluent can be about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. These pH values ​​can be used to form a range, such as between about 7.0 and 8.0, or between about 6.5 and about 7.0.

[0086] The method may further comprise analyzing the eluted human immunoglobulin Fc domain using a detector. The detector may be a sandwich enzyme-linked immunosorbent assay or a mass spectrometer.

[0087] In some embodiments, the human immunoglobulin Fc domain is contained within a humanized monoclonal antibody.The method may also include purifying the humanized monoclonal antibody.

[0088] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device having a surface containing an aptamer that is immobilized on the surface of the affinity capture device and is at least 80% identical to SEQ ID NO 1. The kit also includes a bottle of binding buffer. The binding buffer includes (A) tris (hydroxymethyl) aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The kit may include one or more of the embodiments described herein.

[0089] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device having a surface containing an aptamer, which is immobilized on the surface of the affinity capture device and is at least 80% identical to SEQ ID NO 2. The kit also includes a bottle of binding buffer. The binding buffer includes (A) tris (hydroxymethyl) aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The kit may include one or more of the embodiments described herein.

[0090] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device having a surface containing an aptamer, which is immobilized on the surface of the affinity capture device and is at least 80% identical to SEQ ID NO 3. The kit also includes a bottle of binding buffer. The binding buffer includes (A) tris (hydroxymethyl) aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The kit may include one or more of the embodiments described herein.

[0091] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device having a surface containing an aptamer, which is immobilized on the surface of the affinity capture device and is at least 80% identical to SEQ ID NO4. The kit also includes a bottle of binding buffer. The binding buffer includes (A) tris (hydroxymethyl) aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The kit may include one or more of the embodiments described herein.

[0092] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device having a surface containing an aptamer, which is immobilized on the surface of the affinity capture device and is at least 80% identical to SEQ ID NO 5. The kit also includes a bottle of binding buffer. The binding buffer includes (A) tris (hydroxymethyl) aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The kit may include one or more of the embodiments described herein.

[0093] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device having a surface containing an aptamer, which is immobilized on the surface of the affinity capture device and is at least 80% identical to SEQ ID NO 6. The kit also includes a bottle of binding buffer. The binding buffer includes (A) tris (hydroxymethyl) aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The kit may include one or more of the embodiments described herein.

[0094] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device having a surface containing an aptamer, which is immobilized on the surface of the affinity capture device and is at least 80% identical to SEQ ID NO 7. The kit also includes a bottle of binding buffer. The binding buffer includes (A) tris (hydroxymethyl) aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The kit may include one or more of the embodiments described herein.

[0095] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device having a surface containing an aptamer, which is immobilized on the surface of the affinity capture device and is at least 80% identical to SEQ ID NO 8. The kit also includes a bottle of binding buffer. The binding buffer includes (A) tris (hydroxymethyl) aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The kit may include one or more of the embodiments described herein.

[0096] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device having a surface containing an aptamer that is immobilized on the surface of the affinity capture device and is at least 80% identical to SEQ ID NO 9. The kit also includes a bottle of binding buffer. The binding buffer includes (A) tris (hydroxymethyl) aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The kit may include one or more of the embodiments described herein.

[0097] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device having a surface containing an aptamer that is immobilized on the surface of the affinity capture device and is at least 80% identical to SEQ ID NO 10. The kit also includes a bottle of binding buffer. The binding buffer includes (A) tris (hydroxymethyl) aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The kit may include one or more of the embodiments described herein.

[0098] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device having a surface containing an aptamer that is immobilized on the surface of the affinity capture device and is at least 80% identical to SEQ ID NO 11. The kit also includes a bottle of binding buffer. The binding buffer includes (A) tris (hydroxymethyl) aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The kit may include one or more of the embodiments described herein.

[0099] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device having a surface containing an aptamer that is immobilized on the surface of the affinity capture device and is at least 80% identical to SEQ ID NO 12. The kit also includes a bottle of binding buffer. The binding buffer includes (A) tris (hydroxymethyl) aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The kit may include one or more of the embodiments described herein.

[0100] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device having a surface containing an aptamer that is immobilized on the surface of the affinity capture device and is at least 80% identical to SEQ ID NO 13. The kit also includes a bottle of binding buffer. The binding buffer includes (A) tris (hydroxymethyl) aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The kit may include one or more of the embodiments described herein.

[0101] In some embodiments, the aptamer is at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). The aptamer can, for example, be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). These percentages can be used to form a range, for example, the aptamer can be 85% to 99% identical or 90% to 95% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). In some embodiments, the aptamer is a 23-nucleotide aptamer. The aptamer can be a 23-nucleotide aptamer with an extended sequence or a truncated sequence of about 3 residues. The aptamer can be a 23-nucleotide aptamer with an extended sequence or a truncated sequence of about 5 residues. The aptamer can be covalently immobilized or non-covalently immobilized to the surface of the affinity capture device.

[0102] In some embodiments, the binding buffer has a monovalent cation concentration of less than about 50 mM. The binding buffer may have a monovalent cation concentration of less than about 30 mM. In some embodiments, the pH of the binding buffer is between about 5 and about 9. The pH of the binding buffer may be between about 6 and about 8. The pH of the binding buffer may be about 7.2. For example, the pH of the binding buffer can be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. These pH values ​​can be used to form a range, for example, from about 7.0 to about 7.4, or from about 7.1 to about 7.3.

[0103] In some embodiments, the concentration of magnesium cations is between about 50 μM and about 1 mM. The concentration of magnesium cations can be, for example, 50 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM, 500 μM, 550 μM, 600 μM, 650 μM, 700 μM, 750 μM, 800 μM, 850 μM, 900 μM, 950 μM or 1000 μM. These concentrations can be used to form a range, for example, between about 50 μM and about 500 μM, or between about 50 μM and about 200 μM.

[0104] In some embodiments, the kit further comprises an ammonium bottled eluent having an ammonium concentration between about 10 mM and about 1000 mM. The ammonium can be present in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate.

[0105] In some embodiments, the kit further comprises a + The buffer may be concentrated, and the kit may further include instructions for diluting the concentrated buffer to a working concentration using water. For example, the working concentration of the buffer may be between about 0.5 mM and 20 mM, and the concentrated concentration may be between about 20 mM and 500 mM.

[0106] In some embodiments, the bottle buffer comprises 10 mM Tris, 5 mM CaCl2, pH 7.2.

[0107] In some embodiments, the kit further comprises instructions for performing any of the methods described herein.

[0108] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer immobilized on the surface of the affinity capture device and at least 80% identical to SEQ ID NO 1. The kit also includes a bottle of eluent, the eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The kit may include one or more of the embodiments described herein.

[0109] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer immobilized on the surface of the affinity capture device and at least 80% identical to SEQ ID NO 2. The kit also includes a bottle of eluent, the eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The kit may include one or more of the embodiments described herein.

[0110] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer immobilized on the surface of the affinity capture device and at least 80% identical to SEQ ID NO 3. The kit also includes a bottle of eluent, the eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The kit may include one or more of the embodiments described herein.

[0111] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer immobilized on the surface of the affinity capture device and at least 80% identical to SEQ ID NO 4. The kit also includes a bottle of eluent, the eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The kit may include one or more of the embodiments described herein.

[0112] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer immobilized on the surface of the affinity capture device and at least 80% identical to SEQ ID NO 5. The kit also includes a bottle of eluent, the eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The kit may include one or more of the embodiments described herein.

[0113] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer immobilized on the surface of the affinity capture device and at least 80% identical to SEQ ID NO 6. The kit also includes a bottle of eluent, the eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The kit may include one or more of the embodiments described herein.

[0114] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer immobilized on the surface of the affinity capture device and at least 80% identical to SEQ ID NO 7. The kit also includes a bottle of eluent, the eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The kit may include one or more of the embodiments described herein.

[0115] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer immobilized on the surface of the affinity capture device and at least 80% identical to SEQ ID NO 8. The kit also includes a bottle of eluent, the eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The kit may include one or more of the embodiments described herein.

[0116] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer immobilized on the surface of the affinity capture device and at least 80% identical to SEQ ID NO 9. The kit also includes a bottle of eluent, the eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The kit may include one or more of the embodiments described herein.

[0117] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer immobilized on the surface of the affinity capture device and at least 80% identical to SEQ ID NO 10. The kit also includes a bottle of eluent, the eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The kit may include one or more of the embodiments described herein.

[0118] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer immobilized on the surface of the affinity capture device and at least 80% identical to SEQ ID NO 11. The kit also includes a bottle of eluent, the eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The kit may include one or more of the embodiments described herein.

[0119] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer immobilized on the surface of the affinity capture device and at least 80% identical to SEQ ID NO 12. The kit also includes a bottle of eluent, the eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The kit may include one or more of the embodiments described herein.

[0120] In another aspect, the present technology relates to a kit. The kit includes an affinity capture device, the affinity capture device including a surface having an aptamer, the aptamer immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 13. The kit also includes a bottle of eluent, the eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The kit may include one or more of the embodiments described herein.

[0121] In some embodiments, the aptamer is at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). The aptamer can, for example, be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12 or SEQ ID NO 13). These percentages can be used to form a range, for example, the aptamer can be 85% to 99% identical or 90% to 95% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). In some embodiments, the aptamer is a 23-nucleotide aptamer. The aptamer can be a 23-nucleotide aptamer with an extended sequence or a truncated sequence of about 3 residues. The aptamer can be a 23-nucleotide aptamer with an extended sequence or a truncated sequence of about 5 residues. The aptamer can be covalently immobilized or non-covalently immobilized to the surface of the affinity capture device.

[0122] In some embodiments, the ammonium concentration is between about 50 mM and about 500 mM. For example, the ammonium concentration can be about 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM. These values ​​can be used to form a range, for example, between about 200 mM and about 400 mM.

[0123] In some embodiments, the eluent has a pH between about 6.5 and about 8.0. For example, the pH of the eluent can be about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. These pH values ​​can be used to form a range, such as between about 7.0 and 8.0, or between about 6.5 and about 7.0.

[0124] The kit may also include instructions for performing any of the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 is a flow chart of a method for capturing a human immunoglobulin Fc domain in a biological fluid sample according to an exemplary embodiment of the present technology.

[0126] Figure 2A is a graph showing the effect of divalent cations on hIgG recovered in the eluate for various binding buffers according to exemplary embodiments of the present technology. Figure 2A The effect of divalent cations in the binding buffer is shown, i.e., the recovery of 5 μg of aptamer immobilized on 40 μL of streptavidin resin when loaded with 100 μg of human IgG. Buffer A contains: 10 mM Tris HCl, pH 7.2; Buffer B contains: 10 mM Tris HCl, 5 mM MgCl2, pH 7.2; Buffer C contains: 10 mM Tris HCl, 5 mM CaCl2; Buffer D contains: 10 mM Tris HCl, 5 mM MnCl2; Buffer E contains: 10 mM Tris HCl, 5 mM zinc acetate. The buffer proposed by Nakamura et al. contains: 145 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 20 mM Tris HCl, pH 7.6. Experimental details can be found in Example 1.

[0127] Figure 2B is a graph showing the effect of divalent cations on penetrated hIgG for various binding buffers according to exemplary embodiments of the present technology. Figure 2BThe effect of divalent cations in the binding buffer is shown, i.e., the penetration of 5 μg of aptamer immobilized on 40 μL of streptavidin resin when loaded with 100 μg of human IgG. Buffer A contains: 10 mM Tris HCl, pH 7.2; Buffer B contains: 10 mM Tris HCl, 5 mM MgCl2, pH 7.2; Buffer C contains: 10 mM Tris HCl, 5 mM CaCl2; Buffer D contains: 10 mM Tris HCl, 5 mM MnCl2; Buffer E contains: 10 mM Tris HCl, 5 mM zinc acetate. The buffer proposed by Nakamura et al. contains: 145 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 20 mM Tris HCl, pH 7.6. Experimental details can be found in Example 1.

[0128] Figure 3A is a graph showing the effect of monovalent cations on hIgG recovered in the eluate for various binding buffers according to exemplary embodiments of the present technology. Figure 3A The effect of monovalent cations in the binding buffer is shown, i.e., the recovery of 5 μg of aptamer immobilized on 40 μL streptavidin resin when loaded with 100 μg human IgG. Buffer A contains: 10 mM TrisHCl, 150 mM NaCl, pH 7.2; Buffer B contains: 10 mM TrisHCl, 150 mM NaCl, 5 mM KCl, pH 7.2; Buffer C contains: 10 mM TrisHCl, 150 mM NaCl, 5 mM MnCl2, pH 7.2; Buffer D contains: 10 mM TrisHCl, 150 mM NaCl, 5 mM CaCl2, pH 7.2; Buffer E contains: 10 mM Tris HCl, 150 mM NaCl, 5 mM MnCl2, pH 7.2; Buffer F contains: 10 mM Tris HCl, 150 mM NaCl, 5 mM zinc acetate, pH 7.2. Buffer G contains: 10 mM Tris HCl, 5 mM MgCl2, pH 7.2; Buffer H contains: 10 mM Tris HCl, 5 mM CaCl2, pH 7.2; Buffer I contains: 10 mM Tris HCl, 5 mM MnCl2, pH 7.2: Buffer J contains: 10 mM Tris HCl, 5 mM zinc acetate, pH 7.2. Experimental details can be found in Example 1.

[0129] Figure 3B is a graph showing the effect of monovalent cations on penetrated hIgG for various binding buffers according to exemplary embodiments of the present technology. Figure 3BThe effect of monovalent cations in the binding buffer is shown, i.e., the penetration of 5 μg of aptamer immobilized on 40 μL streptavidin resin when loaded with 100 μg of human IgG. Buffer A contains: 10 mM Tris HCl, 150 mM NaCl, pH 7.2; Buffer B contains: 10 mM Tris HCl, 150 mM NaCl, 5 mM KCl, pH 7.2; Buffer C contains: 10 mM Tris HCl, 150 mM NaCl, 5 mM MnCl2, pH 7.2; Buffer D contains: 10 mM Tris HCl, 150 mM NaCl, 5 mM CaCl2, pH 7.2; Buffer E contains: 10 mM Tris HCl, 150 mM NaCl, 5 mM MnCl2, pH 7.2; Buffer F contains: 10 mM Tris HCl, 150 mM NaCl, 5 mM zinc acetate, pH 7.2. Buffer G contains: 10 mM Tris HCl, 5 mM MgCl2, pH 7.2; Buffer H contains: 10 mM Tris HCl, 5 mM CaCl2, pH 7.2; Buffer I contains: 10 mM Tris HCl, 5 mM MnCl2, pH 7.2: Buffer J contains: 10 mM Tris HCl, 5 mM zinc acetate, pH 7.2. Experimental details can be found in Example 1.

[0130] Figure 4A is a graph showing the effect of pH buffer composition (tris vs. phosphate) on hIgG recovered in the eluate according to exemplary embodiments of the present technology. Figure 4A The effect of pH buffer composition (Tris vs. phosphate) is shown for the recovery of 5 μg of aptamer immobilized on 40 μL streptavidin resin when loaded with 100 μg human IgG. Buffer A contains: 20 mM sodium phosphate, pH 7.0; Buffer B contains: 20 mM sodium phosphate, 150 mM NaCl, pH 7.0; Buffer C contains: 20 mM sodium phosphate, 5 mM MgCl2, pH 7.0; Buffer D contains: 10 mM Tris HCl, pH 7.2; Buffer E contains: 10 mM Tris HCl, 150 mM NaCl, pH 7.2; Buffer F contains: 10 mM Tris HCl, 5 mM MgCl2, pH 7.2. Experimental details can be found in Example 1.

[0131] Figure 4B is a graph showing the effect of pH buffer composition (tris vs. phosphate) on penetrated hIgG according to exemplary embodiments of the present technology. Figure 4BThe effect of pH buffer composition (Tris vs. phosphate) is shown for the penetration of 5 μg of aptamer immobilized on 40 μL streptavidin resin when loaded with 100 μg of human IgG. Buffer A contains: 20 mM sodium phosphate, pH 7.0; Buffer B contains: 20 mM sodium phosphate, 150 mM NaCl, pH 7.0; Buffer C contains: 20 mM sodium phosphate, 5 mM MgCl2, pH 7.0; Buffer D contains: 10 mM Tris HCl, pH 7.2; Buffer E contains: 10 mM Tris HCl, 150 mM NaCl, pH 7.2; Buffer F contains: 10 mM Tris HCl, 5 mM MgCl2, pH 7.2. Experimental details can be found in Example 1.

[0132] Figure 5 is a graph showing non-specific binding studies using different binding buffers according to an exemplary embodiment of the present technology. Figure 5 Non-specific binding studies using different binding buffers are shown for the recovery and breakthrough of 5 μg of aptamer immobilized on 40 L streptavidin resin when loaded with 100 μg of human IgG. Buffer A contains: 10mM Tris HCl, 150mM NaCl, 5mM MeCl2, pH 7.2; buffer B contains: 10mM Tris HCl, 150mM NaCl, 5mM CaCl2, pH 7.2; buffer C contains: 10mM TrisHCl, 150mM NaCl, 5mM MnCl2, pH 7.2; buffer D contains: 10mM Tis HCl, 150mM NaCl, 5mM zinc acetate, pH7.2, buffer E contains: 10mM Tris HCl, 5mM MgCl2, pH 7.2; buffer F contains: 10mM Tris HCl, 5mM CaCl2, pH 7.2; buffer G contains: 10mM Tris HCl, 5mM MnCl2, pH 7.2: buffer H contains: 10mM Tris HCl, 5mM zinc acetate, pH 7.2. Experimental details can be found in Example 2.

[0133] Figure 6 is a graph illustrating the effect of magnesium concentration in a binding buffer and the choice of elution buffer according to an exemplary embodiment of the present technology. Figure 6The effect of Mg concentration in the binding buffer and the choice of elution buffer are shown for the recovery and breakthrough of 5 μg of aptamer immobilized on 40 μL streptavidin resin when loaded with 100 μg human IgG. The binding buffer contained 10 mM Tris HCl, pH 7.2, with MgCl2 concentration ranging from 1 μM to 200 mM. The captured hIgG was eluted with 200 mM ammonium acetate or 200 mM EDTA. Experimental details can be found in Example 2.

[0134] Figure 7 is a diagram showing aptamer versus single domain antibody V according to an exemplary embodiment of the present technology HH Graph of the binding capacity. Figure 7 Display of aptamers versus single domain antibodies V HH When loaded with 500 μg of human IgG, the binding capacity of the aptamer or V HH ) of 40 μL streptavidin resin. Experimental details can be found in Example 3.

[0135] Fig. 8A is a LC-UV curve of a plasma sample according to an exemplary embodiment of the present technology.

[0136] Figure 8B is an LC-UV curve of aptamer flow-through according to an exemplary embodiment of the present technology.

[0137] Figure 8C is an LC-UV curve of the eluate fractions according to an exemplary embodiment of the present technology.

[0138] Fig.8D is an LCUV overlay of eluate fractions recovered from 40 μg of streptavidin resin with 5 μg, 12.5 μg, and 25 μg of immobilized aptamer according to an exemplary embodiment of the present technology.

[0139] Fig. 8E is a table showing calculated loading and recovery of NIST mAbs and plasma proteins according to an exemplary embodiment of the present technology.

[0140] Fig.9A is an aptamer 2 using wash buffers A to F according to an exemplary embodiment of the present technology ndOverlay of LC-UV curves of wash fractions. Buffer A contained: 10 mM Tris HCl, 0.5 mM MgCl2, pH 7.2 (binding buffer); Buffer B contained: 10 mM Tris HCl, 5 mM CaCl2, pH 7.2; Buffer C contained: 145 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 20 mM Tris HCl, pH 7.6 (Nakamura et al.); Buffer D contained: 20 mM phosphate, pH 7.0; Buffer E contained: 0.1 M glycine HCl, pH 2.7; Buffer F contained: 150 mM NaCl. Experimental details can be found in Example 4.

[0141] Fig. 9B is a method of washing the sample in a washing buffer solution A to F according to an exemplary embodiment of the present technology. nd Overlay of LC-UV curves of aptamer eluate fractions after the washing step. Buffer A contained: 10 mM Tris HCl, 0.5 mM MgCl2, pH 7.2 (binding buffer); Buffer B contained: 10 mM Tris HCl, 5 mM CaCl2, pH 7.2; Buffer C contained: 145 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 20 mM Tris HCl, pH 7.6 (Nakamura et al.); Buffer D contained: 20 mM phosphate, pH 7.0; Buffer E contained: 0.1 M glycine HCl, pH 2.7; Buffer F contained: 150 mM NaCl. Experimental details can be found in Example 4.

[0142] Fig. 9C is the use of an exemplary embodiment according to the present technology 2 nd Wash buffers A to F at 2 nd Calculated recovery of NIST mAb and plasma proteins in wash and eluate fractions. Buffer A contained: 10 mM Tris HCl, 0.5 mM MgCl2, pH 7.2 (binding buffer); Buffer B contained: 10 mM Tris HCl, 5 mM CaCl2, pH 7.2; Buffer C contained: 145 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 20 mM Tris HCl, pH 7.6 (Nakamura et al.); Buffer D contained: 20 mM phosphate, pH 7.0; Buffer E contained: 0.1 M glycine HCl, pH 2.7; Buffer F contained: 150 mM NaCl. Experimental details can be found in Example 4. DETAILED DESCRIPTION

[0143] The present technology includes methods and kits for selectively capturing human Fc domains found in immunoglobulins using aptamers. Using the present technology, the selectivity and binding capacity of certain aptamer ligands have been significantly improved, making them more effective for capturing human IgG (hIgG). Together with specific embodiments of aptamer-fixed resins and the form factor of affinity capture devices, powerful and robust techniques for anti-human Fc capture have been designed. In an exemplary embodiment and in an optimized workflow, the fixed aptamer resin is contained in a pipette tip and is used to quickly and selectively capture humanized Fc domains contained in monoclonal antibody (mAb) therapeutics from biological fluid samples obtained during preclinical rodent or monkey trials.

[0144] Affinity capture methods are some of the most powerful technologies used to promote antibody purification, antibody characterization, and clinical diagnostic development. If appropriately designed affinity consumables are available, the enrichment of target antibodies can be simplified to a one-step process. It has been proposed that aptamers (i.e., a group of short single-stranded oligonucleotides with unique 3D conformations) are novel affinity ligands for high capacity and high selectivity enrichment. The size of these chemically synthesized molecules is generally less than 10 kDa, and their high affinity can be achieved by targeted screening amplified via SELEX (systematic evolution of ligands for exponential enrichment). Due to its relatively small size, aptamers can be controlled to achieve high surface coverage on solid supports, minimize any possible steric hindrance, and ensure a high yield of target proteins with significantly reduced sample preparation time. In addition, since they are oligonucleotides synthesized in vitro, aptamers are generally highly stable, and they can be efficiently and economically manufactured, with potentially better batch-to-batch reproducibility and protein-based ligands.

[0145] Anti-human Fc aptamer ligands consisting of 40 or less nucleotides are shown in U.S. Patent No. 8,637,656 (incorporated herein in its entirety by reference) by Nakamura et al. as a protein A substitute for purified antibodies and biotherapeutics. Previous studies involving these aptamers have shown that the GGUGCU protrusion motif is essential for selective interaction with human IgG Fc domains. The two stem structures at both ends of the protrusion motif form the critical affinity motif of the molecule together with 3 or 4 nucleotide rings. It is also applied to incorporate 2'-fluoro groups in selected pyrimidine nucleotide residues to improve the stability and selectivity of the aptamer. In the crystallographic study of human IgG aptamer complexes, it was found that the optimized aptamer interacts with several conserved residues of human IgG Fc domains mainly via van der Waals contacts and hydrogen bonds, rather than electrostatic forces. In addition, it is suggested that the binding structure of the aptamer can be stabilized by calcium ions, and the binding can be reversed by calcium chelation. However, despite these valuable basic studies of anti-human Fc aptamers, there remains a need for robust methods that facilitate human Fc capture so that more detailed and accurate analyses can be performed during pharmacokinetic, pharmacodynamic, and biotransformation studies.

[0146] Using the present technology, a novel, aptamer-based sample preparation method is provided for selectively capturing human immunoglobulin Fc domains. A 23-nucleotide aptamer having any one of SEQ ID NO 1 to SEQ ID NO 13 can be used. In addition, other sequences described in U.S. Patent No. 8,637,656 can be used, and the entire text is incorporated herein by reference. That is, SEQ ID NO 2 to SEQ ID NO 13 have been shown to have the ability to selectively capture human immunoglobulins, and are equivalent in their applicability to SEQ ID NO 1 relative to the present technology. Some sequences described in U.S. Patent No. 8,637,656 may lack the species selectivity required for selective capture of human Fc domains from animals and preclinical biological fluids. These aptamer sequences may mimic the relatively broad specificity of protein A. However, it is believed that they will also be suitable for the method of the present invention, possibly as an alternative method for purifying monoclonal antibodies from cell culture media. Each of SEQ ID No 1 to SEQ ID No 13 is shown below.

[0147] SEQ ID NO 1:

[0148] 5' end - G GrArGrG[i2FU]rG C[i2FU]CC GAAArGrGAA[i2FC][i2FU]CC – 3' end

[0149] SEQ ID NO 2:

[0150] 5' end–G GrArGrG[i2FU]rG[i2FC][i2FU][i2FC][i2FC]GAArGrG rA rA[i2FC][i2FU]CC–3' end

[0151] SEQ ID NO: 3:

[0152] 5' end–GG rA rG rG[i2FU]rG[i2FC][i2FU][i2FC]CGAAA rG rG rA rA[i2FC][i2FU]CC–3' end

[0153] SEQ ID NO: 4:

[0154] 5' end–G(OMe)G(OMe)rA rG rG[i2FU]rG[i2FC][i2FU][i2FC][i2FC]G(OMe)A(OMe)A(OMe)A(OMe)rGrGrArA[i2FC][i2FU]C(OMe)C(OMe)–3' end

[0155] SEQ ID NO: 5:

[0156] 5' end–GG rA rG rG[i2FU]rG CU(OMe)CCGAAA rG rG AA[i2FC][i2FU]CC–3' end

[0157] SEQ ID NO: 6:

[0158] 5' end–G(OMe)G(OMe)rA rG rG[i2FU]rG[i2FC]U(OMe)[i2FC][i2FC]G(OMe)A(OMe)A(OMe)A(OMe)rGrGrArA[i2FC][i2FU]C(OMe)C(OMe)–3' end

[0159] SEQ ID NO: 7:

[0160] 5' end–G(OMe)G(OMe)rA rG rG[i2FU]rG[i2FC][i2FU]C(OMe)[i2FC]G(OMe)A(OMe)A(OMe)A(OMe)rGrGrArA[i2FC][i2FU]C(OMe)C(OMe)–3' end

[0161] SEQ ID NO: 8:

[0162] 5’ end–G(OMe)G(OMe)rA rG rG[i2FU]rG[i2FC][i2FU][i2FC]C(OMe)G(OMe)A(OMe)A(OMe)A(OMe)rGrGrArA[i2FC][i2FU]C(OMe)C(OMe)–3’ end

[0163] SEQ ID NO 9:

[0164] 5’ end–GG rA rG[i2FG][i2FU]rG C[i2FU]CCGAAA rG rG AA[i2FC][i2FU]CC–3’ end

[0165] SEQ ID NO 10:

[0166] 5’ end–GG rA rG rG[i2FU][i2FG]C[i2FU]CCGAAA rG rG AA[i2FC][i2FU]CC–3’ end

[0167] SEQ ID NO 11:

[0168] 5' end – GGrArGrG[i2FU]rGrC[i2FU]CC GAAArGrGAA[i2FC][i2FU]CC – 3' end

[0169] SEQ ID NO 12:

[0170] 5' end – G GrA rG rG[i2FU]rG C rU CC GAAArG rGA A[i2FC][i2FU]CC – 3' end

[0171] SEQ ID NO 13:

[0172] 5' end - GG rA rG rG [i2FU] rG CU (OMe) CCGAAA rG rG AA [i2FC] [i2FU] CC - 3' end Wherein A, C, G, U represent the four nucleobases. Capital letters correspond to deoxyribonucleotides, and rX indicates a ribonucleotide. [i2FC] and [i2FU] represent 2'-fluoro substituted pyrimidine nucleotides. U (OMe), C (OMe), G (OMe) and A (OMe) represent 2'-methoxy substituted nucleotides.

[0173] In all embodiments of the present technology, the aptamer ligand is immobilized to a substrate suitable for performing affinity capture and sample enrichment. Substrates include, but are not limited to, surfaces of microtiter plates, pipette tips, and labware, as well as porous and non-porous resins composed of polymers, silica, and organosilica.

[0174] When the aptamer is immobilized on the resin, the resin can be immobilized on a consumable (e.g., a microplate, a pipette tip, and / or other labware). In this way, the resin can be used to increase the surface area and / or amount of aptamer bound to a surface. In other embodiments, the resin with the immobilized aptamer can be used as a stationary phase in a chromatographic column.

[0175] The sequence of the aptamer can be modified from its 5'- or 3'-end or one of its internal residues. The sequence modification can confer a chemical handle and an optional spacer arm portion, including but not limited to a hydrophilic molecular composition, such as 2 to 50 repeating units of polyethylene glycol or polyethylene oxide. In addition, the sequence modification can be used to achieve directional fixation. For example, in the case of adding a primary amino group to the 5' or 3' end, the aptamer can be modified via a nucleophilic / electrophilic reaction scheme to produce a single point connection with another molecule, surface or resin that also confers limited directionality. In some embodiments, the above-mentioned chemical handle may include biotin, amine, carboxyl or thiol groups. During fixation and storage, the fixed aptamer can be enhanced with one or more nuclease inhibitors to improve its shelf life. Nucleases can also be added to the storage solution and working buffer to extend the stability of the fixed aptamer.

[0176] Figure 1A method 100 of capturing a human immunoglobulin Fc domain in a biological fluid sample is shown. The method includes providing an affinity capture device 105. The affinity capture device has a surface containing an aptamer that is immobilized on the surface of the affinity capture device and is at least 80% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13). The aptamer may be 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO 1 (or SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 11, SEQ ID NO 12 or SEQ ID NO 13). The aptamer may be a 23 nucleotide aptamer. The aptamer may be a 23 nucleotide aptamer having an extended sequence of about 3 residues or about 5 residues or a truncated sequence. The aptamer may be non-covalently or covalently immobilized on the surface of the affinity capture device.

[0177] The method 100 also includes diluting the biological fluid sample 110 with a binding buffer. The binding buffer includes (A) tris (hydroxymethyl) aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The method 100 also includes adsorbing the human immunoglobulin Fc domain in the biological fluid sample to the aptamer 115 with the binding buffer.

[0178] The binding properties of aptamers are highly dependent on the composition of the buffer system used (see Figures 2 to Figure 5 Based on the research work, it has been found that aptamers operate in a metal-dependent mechanism. When using aptamers, it has been observed that the presence of appropriately selected divalent ions is essential for maintaining high affinity and selectivity (Figure 2, Figure 5). Nakamura and coworkers proposed that calcium ions are the most critical component in forming the affinity-capacity motif of the aptamer. Herein, magnesium ions are shown to be more effective in increasing the binding capacity of the aptamer without introducing any significant amount of nonspecific binding to other non-targeted proteins. The concentration of magnesium cations can be between 50 μM and about 1 mM.

[0179] As used herein, the term "about" means that the numerical value is approximate and that minor variations will not significantly affect the practice of the disclosed embodiments. Where numerical limitations are used, unless the context indicates otherwise, "about" means that the numerical value may vary by ±10% and still be within the scope of the disclosed embodiments.

[0180] At the same time, monovalent ions have been found to act as interferents of affinity binding motifs, thereby serving as potential alternative eluents to chelators as proposed by Nakamura and coworkers. Therefore, in some embodiments of the present technology, monovalent cation solutions, including but not limited to ammonium, sodium, and potassium solutions, may be used in the form of elution buffers to aid in the recovery of target proteins / analytes at neutral pH (Figures 3, 4, and Figure 6 ). In addition, the aptamers of the present invention have been found to work more advantageously in Tris buffer compared to phosphate buffer as proposed by Nakamura and coworkers. In fact, when used with the same ionic additives, Tris buffer was found to produce better binding capacity than phosphate buffer. Triethylamine (TEA), MES (2-ethanesulfonic acid) and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) can confer advantages over phosphate buffer.

[0181] In some embodiments, the binding buffer has a monovalent cation concentration of less than about 50 mM. The binding buffer may have a monovalent cation concentration of less than about 30 mM. In some embodiments, the pH of the binding buffer is between about 5 and about 9. The pH of the binding buffer may be between about 6 and about 8. The pH of the binding buffer may be about 7.2. For example, the pH of the binding buffer can be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. These pH values ​​can be used to form a range, for example, from about 7.0 to about 7.4, or from about 7.1 to about 7.3.

[0182] The biological fluid sample can be diluted. For example, the biological fluid sample can be diluted by a factor of 2, 10, or 20. In some embodiments, the biological fluid sample is diluted by a factor of 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. These dilution factors can be used to form a range, such as about 2 to about 20, or about 2 to about 10, or about 10 to about 20. In some embodiments, the biological fluid sample is diluted to obtain a total monovalent cation concentration of the biological fluid sample of no more than 100mM, no more than 50mM, or no more than 30mM. The total monovalent cation concentration of the diluted biological fluid sample can be no more than 100mM, 90mM, 80mM, 70mM, 60mM, 50mM, 40mM, 30mM, 20mM, or 10mM.

[0183] refer to Figure 1 , the method 100 may also include using an eluent to elute the adsorbed human immunoglobulin Fc domain 120 from the immobilized aptamer. The ammonium concentration of the eluent is between about 10 mM and about 1000 mM. The ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The ammonium concentration may be, for example, between about 50 mM and about 500 mM. The pH of the eluent may be between about 6.5 and about 8.0.

[0184] Method 100 may further include washing the adsorbed human immunoglobulin Fc domain 125 with a binding buffer. + The wash is completed with buffer.

[0185] The method 100 may further include using a detector to analyze the eluted human immunoglobulin Fc domain 130. The detector may be a sandwich ELISA or a mass spectrometer.

[0186] In some embodiments, the human immunoglobulin Fc domain is contained within a humanized monoclonal antibody, and the method further comprises purifying the humanized monoclonal antibody (not shown).

[0187] The present technology also relates to a method for eluting a human immunoglobulin Fc domain from an immobilized aptamer. The method includes providing an affinity capture device having a surface containing an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 1. The immobilized aptamer has an adsorbed human immunoglobulin Fc domain. An eluent having an ammonium concentration between about 10 mM and about 1000 mM is used to elute the adsorbed human immunoglobulin Fc domain from the immobilized aptamer. The ammonium is presented in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The method may include any one of the embodiments described herein.

[0188] The present technology also relates to a kit. The kit may include an affinity capture device comprising a surface having an aptamer that is immobilized on the surface of the affinity capture device and is at least 80% identical to SEQ ID NO 1. The kit may also include a bottled binding buffer comprising (A) tris(hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) a magnesium cation at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no more than 100 mM. The kit may include any one of the embodiments described herein. The kit may also include a bottled eluent having an ammonium concentration between about 10 mM and about 1000 mM. Ammonium can be presented in the form of tetramethylammonium, triethylammonium, ammonium formate or ammonium acetate. The kit may also include a Ca + The buffer provided in the kit can be concentrated, and the kit can include instructions for diluting the concentrated buffer to a working concentration. Water can be used to dilute the buffer. The kit can also include instructions for performing any of the methods described herein.

[0189] The present technology relates to a kit comprising an affinity capture device comprising a surface having an aptamer immobilized on the surface of the affinity capture device and at least 80% identical to SEQ ID NO 1 and a bottle of eluent. The ammonium concentration of the eluent is between about 10 mM and about 1000 mM. The ammonium can be present in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate. The kit may include any of the embodiments described herein. The kit may include instructions for performing any of the methods described herein.

[0190] In practice, anti-human Fc aptamers can be used with carefully defined protocol steps (i.e., binding, washing, and elution steps). Specifically, for the binding step, the aptamer can be used with Tris, TEA, MES, or HEPES binding buffers (composed of Mg in a concentration range of 10 μM to 20 mM or more ideally 50 μM to 1 mM). The monovalent cation concentration is intentionally kept to a minimum in the binding buffer, which is less than 50 mM, more ideally less than 30 mM. In addition, the pH of the solution is designed to be between 5 and 9, more ideally 6 to 8. The use of this formulation and method improves the binding ability of the aptamer without losing selectivity (see Figure 5 and Figure 6). This is in contrast to the example presented by Nakamura and coworkers, which describes a buffer of 145 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 20 mM Tris, pH 7.6. Because it is critical to minimize the presence of any monovalent ions during the binding step of the aptamer capture protocol, certain embodiments of the present invention require a 2-fold to 20-fold sample dilution. In this way, the complex matrix (e.g., biological fluid) is reduced at its monovalent cation concentration and the negative effects of endogenous monovalent ions are minimized.

[0191] (Figures 8 and 9).

[0192] In an exemplary embodiment, the method includes a washing step consisting of two procedures. The first washing procedure is performed exclusively with a binding buffer to wash the adsorbed analyte and the resin. The second washing procedure is preferentially performed with a binding buffer containing Ca 2+ The enrichment was performed using a buffer of 5% HCl as it has been observed to significantly reduce non-specific binding of aptamer ligands and improve the purity of captured / enriched hIgG in the complex sample matrix ( FIG. 9 ).

[0193] In the final step of the method, the captured Fc domains can optionally be eluted by using a neutral pH buffer (pH between 6.5 and 8.0) with a chelating agent, including those described by Nakamura and co-workers (such as 10 mM to 200 mM EDTA, malate, citrate, and porphine), or with concentrated monovalent cations (such as K + 、Na + 、Ag + and Cs +). Alternatively, the Fc domain containing the analyte can be eluted with a novel buffer system as disclosed herein (the novel buffer system is based on bisphosphonates, including but not limited to methylenephosphonate and etidronate) or with MS compatible volatile monovalent cations such as ammonium, tetramethylammonium or triethylammonium. In one embodiment, an elution buffer consisting of 10mM to 1000mM or more ideally 50mM to 500mM ammonium formate or ammonium acetate is used. Using this type of eluent, an online immunoaffinity MS assay can be performed. Similarly, common protein A elution buffers with acidic pH (pH≤4.0), such as 0.1M glycine HCl, 0.1M citric acid, 5% acetic acid or formic acid, are another option for eluting captured Fc domain analytes. The choice of these elution conditions extends the compatibility of aptamer affinity capture with many downstream processes. In some embodiments, anti-human Fc aptamers and corresponding devices are employed to perform sample preparation for subsequent LC or LCMS-based assays such as reverse phase chromatography, ion exchange, hydrophilic interaction liquid chromatography, hydrophobic interaction chromatography, protein A affinity chromatography, subunit analysis, peptide mapping, glycan profiling, and intact mass analysis. Analysis facilitated by the present technology may also include online immunoaffinity capture, capillary electrophoresis, and mass spectrometry. Additionally, the methods described herein may be applied to enzyme-linked immunosorbent assays (Example 7). In another embodiment, heat-induced denaturation of the aptamer may be applied to elute the captured human Fc analyte without introducing any elution buffer.

[0194] In summary, the present technology specifies a novel sample preparation method for selective capture of human Fc domains. Since it is based on the use of smaller affinity ligands, it shows favorable binding capacity compared to methods relying on full-sized IgG antibody ligands, and a clear improvement over the existing technology established by Nakamura and colleagues. ( Figure 7 ).

[0195] Example 1: Capture of hIgG using different binding buffers

[0196] The aptamer with SEQ ID NO 1 was adapted from US 8,637,656 B2 and contained a biotin tag at its 5'-end. The synthesis of the aptamer was performed by Integrated DNA Technologies (Coralville, IA). Each 5 μg portion of the biotinylated aptamer was immobilized to 40 μL of high capacity streptavidin resin (commercially available from Thermo Fisher Scientific, San Jose, CA) in a 96-well filter plate (commercially available from MilliporeSigma, Burlington, MA) via incubation at room temperature for 30 minutes. A positive pressure manifold (commercially available from Waters Technologies Corporation, Milford, MA) was used to drive the flow through the filter plate. The amount of immobilized aptamer was determined by absorbance at 260 nm using a DS-11 spectrophotometer (commercially available from DeNovix Inc., Wilmington, DE). The non-covalently immobilized aptamer affinity resin was used to perform affinity capture of 100 μg of human IgG (IgG from human serum, commercially available from Sigma-Aldrich (now MilliporeSigma), St. Louis, MO) contained in 200 μL of several different binding buffers (specified below). After mixing for 1 hour, the resin was washed with two replicates of 200 μL volume to remove any unbound protein. The combined hIgG flow-through and wash fractions were saved to determine the amount of hIgG penetration. The captured human IgG was eluted with two replicates of 200 μL volume of elution buffer (200 mM EDTA, 10 mM tris, pH 7.2). The eluate was saved for use with a fluorescence plate reader (Ex. 280 nm, Em. 370 nm; Gemini TM XPS plate reader, commercially available from Molecular Devices, San Jose, CA).

[0197] The binding ability of the aptamers incorporated herein by reference is highly dependent on the composition of the binding buffer. In order to study the effect of divalent ions on the binding properties of aptamers, a series of Tris buffers containing different divalent cations were screened. The binding buffers tested were: 10mM Tris buffer, pH 7.2 with 5mM MgCl2; 10mM Tris buffer, pH 7.2 with 5mM CaCl2; 10mM Tris buffer, pH 7.2 with 5mM MnCl2, and 10mM Tris buffer, pH 7.2 with 5mM zinc acetate. Tris buffer (10mM Tris / Tris HCl, pH 7.2) without added cations was used as a control and compared with the buffer system proposed by Nakamura and colleagues (145mM NaCl, 5.4mM KCl, 1.8mM CaCl2, 0.8mM MgCl2, 20mM Tris, pH 7.6). As shown in Figure 2, divalent cations (especially Zn 2+ and Mg 2+ ) resulted in a significant improvement in the capture and recovery of hIgG. These two buffers serve as exemplary embodiments of their recovery and selectivity. On the other hand, surprisingly, when the buffer proposed by Nakamura and co-workers was adopted, only 20% of the loaded hIgG was captured (Comparative Example), which means that the presence of concentrated sodium chloride (NaCl) in the buffer and the combination of various divalent / monovalent cations inhibited the binding ability of the aptamer.

[0198] Thus, by using the inclusion and exclusion of Na + or K +The binding buffers of cations were used to compare binding capacity and protein recovery, which again demonstrated the negative impact of capturing hIgG from the presence of monovalent cations. The binding buffers explored in this particular study include a) buffers without 150mM NaCl; 10mM Tris buffer, pH 7.2 with 5mM MgCl2; 10mMTris buffer, pH 7.2 with 5mM CaCl2; 10mMTris buffer, pH 7.2 with 5mM MnCl2, and 10mM Tris buffer, pH 7.2 with 5mM zinc acetate b) was made into a matching buffer also containing 150mM NaCl and 10mM Tris buffer, pH 7.2 with 5mM KCl, 150mM NaCl. The Tris buffer containing 150mM NaCl was also used as a control. The results of this study are summarized in Figure 3. Based on these data, it appears that the presence of 150 mM NaCl in the binding buffer containing divalent cations is disruptive to the adsorption of hIgG to the immobilized aptamer, as reflected by a significant decrease in aptamer binding capacity and hIgG recovery. Adding potassium cations (K) to the binding buffer containing sodium chloride (NaCl) had no effect on improving recovery. This result is consistent with the observed binding properties of the aptamer when using the binding buffer proposed by Nakamura and co-workers, as it contains relatively high concentrations of monovalent cations, i.e., concentrations greater than 10 mM.

[0199] The type of aptamer binding buffer is another factor that affects the capture efficiency. Phosphate-based buffers are used here as an alternative to the binding step. Three phosphate buffers were applied in this study, including: 20mM Na2HPO3, pH 7.0 without added NaCl; 20mM Na2HPO3, pH 7.0 with 150mM NaCl; and 20mM Na2HPO3, pH 7.0 with 5mM MgCl2. 10mMTris buffer with the same ionic composition was used as a control. As shown in Figure 4, when used with the same ionic additives, Tris buffer provides better binding capacity than phosphate buffer. When the aptamer is used with a buffer containing only phosphate, it shows almost no affinity for IgG, while its affinity can be increased by adding 5mM MgCl2. 2+ Based on the reported crystal structure of the hIgG aptamer complex associated with the present technology, it is reasonable to propose that phosphates may interrupt the folding of the aptamer into its active affinity motif.

[0200] In general, divalent cations (such as Zn 2+ or Mg 2+) as an exemplary embodiment of the present technology. Additional experiments have been performed to demonstrate the usability of these buffers, and the results are detailed below.

[0201] Example 2: Effect of binding and elution buffers on aptamer selective capture

[0202] The presence of divalent cations in the binding buffer has the potential to promote the activation of the affinity motif of the aptamer, but can also promote non-specific binding. Therefore, we also aim to address the non-specific binding of the aptamer. The biotinylated aptamer was immobilized as described in Example 1 and subsequently used for hIgG capture. Tris binding buffers containing different divalent cations (Mg, Ca, Mn, Zn) were prepared with and without 150 mM NaCl and then used in this study. The composition of these binding and elution buffers was listed earlier in Example 1. Rabbit IgG (rIgG) (available from Sigma-Aldrich (now MilliporeSigma), St.Louis, MO) and bovine serum albumin (BSA) (available from Sigma-Aldrich (now MilliporeSigma), St.Louis, MO) were used as two negative controls to study the non-specific binding of the aptamer. 100 μg of hIgG, rIgG and BSA were loaded onto the non-covalently fixed aptamer resin using 200 μL volumes of various binding buffers. Following binding and washing steps, the captured proteins were eluted with elution buffer, and protein recovery in each eluate was measured using a fluorescence plate reader.

[0203] The results of protein recovery are summarized in Figure 5 Interestingly, the inclusion of Zn 2+ Buffers containing Zn resulted in high levels of nonspecific binding to all proteins tested, especially when NaCl was not present. Therefore, binding buffers containing Zn were considered unsuitable for this method. 2+ The same problem was observed with the buffer of , which also produced high levels of non-specific binding to rIgG. In accordance with these results, it should be noted that some NaCl can be included in the wash buffer in order to attenuate some types of non-specific binding.

[0204] Further analysis of the results showed that Mg 2+ The buffer composed of MgCl2 was the most promising in terms of binding, as it provided efficient capture with negligible nonspecific binding. However, it can be shown that having different concentrations of MgCl2 in the binding buffer 2+ May have an effect on binding capacity. Figure 6 The summed up, Mg 2+Binding buffers with concentrations between 50 μM and 1 mM resulted in the highest hIgG capture and recovery under all conditions tested. Thus, it appears that using buffers with concentrations greater than 20 mM or less than 10 μM Mg 2+ Binding buffers with poor binding may result in suboptimal hIgG binding.

[0205] In another study, we chose to investigate the use of concentrated ammonium acetate buffer for hIgG elution ( Figure 6 ). Since our preliminary results indicated that binding was highly dependent on the presence of certain divalent cations, we aimed to understand whether competing cations could be used to displace divalent cations from the hIgG aptamer complex, thereby eluting the captured hIgG. As proposed by Nakamura, EDTA buffer can be used as an elution buffer. However, this non-volatile chelator may cause problems for downstream mass spectrometry (MS) analysis. In contrast, buffers with competing volatile monovalent cations such as ammonium acetate (NH4OAc), ammonium formate, or ammonium bicarbonate may be more MS-friendly elution buffers. In this regard, 200 mM ammonium acetate, pH 7.2 was used to elute the captured hIgG from the aptamer, and the mass of this eluate was compared to that obtained with 200 mM EDTA elution buffer. Figure 6 As shown, the elution efficiency of concentrated NH4OAc is highly dependent on the Mg content in the binding buffer. 2 + For concentrations ranging from 50 μM to 10 mM Mg 2+ With a binding buffer composed of NH4OAc, hIgG recovery can be 80% to 90% of that obtained using EDTA elution buffer.

[0206] In summary, Mg 2+ The 10 mM Tris binding buffer can minimize undesirable nonspecific binding and provide exemplary binding capacity. The Mg content in the ideal binding buffer is 2+ The concentration is between 10 μM and 10 mM, more ideally 50 μM to 1 mM. Importantly, using this selected binding buffer, concentrated ammonium buffers (eg, 200 mM ammonium acetate) can be used to achieve higher recoveries compared to MS-friendly elution buffers.

[0207] Example 3: Evaluation of aptamer binding ability

[0208] To determine the binding capacity of the aptamers, a protocol established by the present technology was used to perform sample preparation involving amounts of hIgG exceeding 500 μg. 5 μg, 12.5 μg, 25 μg, 37.5 μg, 50 μg, and 100 μg of biotinylated aptamers were immobilized on 40 μL of streptavidin resin. The procedures for aptamer immobilization and hIgG capture were the same as those described in the previous examples. The recovery of hIgG was plotted against the amount of immobilized aptamer to estimate the maximum binding capacity of the aptamer ( Figure 7 ). 10mM Tris, 0.5mM Mg, pH 7.2 and 200mM EDTA, 10mM Tris, pH 7.2 were used for binding and elution, respectively. According to the hIgG recovery curve, it can be seen that by using the optimized buffer and protocol of the present technology, the maximum binding capacity of the aptamer is close to 6.5μg hIgG / μg immobilized aptamer, which is about 20 times higher than the maximum binding capacity estimated using an anti-human Fc antibody based on full-length IgG. It is also worth noting that the surface coverage of the aptamer is constrained in this test by the limitation of non-covalent fixation to the streptavidin resin. A higher binding capacity of the aptamer prepared by direct fixation can be predicted. The coverage of the immobilized aptamer can be about 1nmol / m 2 to about 1000nmol / m 2 To minimize cost, lower coverage immobilization may be preferred. However, to achieve high binding capacity surfaces, higher coverage is desired.

[0209] In yet another example, the ability of this aptamer-based affinity capture has been compared with commercial nanobody ligands for human Fc capture. H Nanobodies to the H domain have been previously developed and are now available from Thermo-Fisher Scientific In this study, anti-human Fc V H The H ligand was recombinantly expressed (GenScript, Piscataway, NJ) from a sequence adapted from US 9,040,666 B2 (incorporated herein by reference in its entirety) and was designed with a C-terminal extension consisting of an additional cysteine ​​residue, a TEV protease cleavage site, and a His tag. The resulting Nanobody was treated with ProTEV (commercially available from Promega, Madison, WI) to remove its His tag and then PCR amplified with EZ-Link TM , iodoacetyl-PEG2 biotin (commercially available from Thermo-Fisher Scientific, San Jose, CA). HThe purity of H was assessed by LCMS and determined to be greater than 80% biotinylated. H V was non-covalently immobilized to 40 μL of streptavidin resin slurry and used to capture hIgG using the same procedure as the aptamer. Immobilized V was measured by absorbance at 280 nm using a DS-11 spectrophotometer (commercially available from DeNovix Inc., Wilmington, DE). H The amounts of HO. 20 mM phosphate, pH 7.0 and 0.1 M glycine HCl, pH 2.7 were recommended by the CaptureSelect literature from Thermo Fisher Scientific as appropriate binding and elution buffers. Figure 7 As shown, based on the V H The maximum binding capacity of H ligand (5 μg hIgG / μg immobilized V H H) is lower than the maximum binding capacity of the aptamer.

[0210] Example 4: Capture of hIgG from biological fluids

[0211] To investigate the selectivity of purifying human Fc domains from biofluid matrices, different amounts of NIST mAb (NIST Reference Material 8671) were spiked into rat plasma and then affinity captured using non-covalently immobilized aptamers using the protocol described in this invention. A BEH300C4 column (1.7 μm, 2.1×100 nm) (commercially available from Waters Technologies Corporation, Milford, MA) was used. H-ClassBio (available from Waters Technologies Corporation, Milford, MA) was used to analyze the purity of the flow-through, wash, and eluate fractions. The protein concentration of each fraction was determined using a fluorescence plate reader (Gemini XPS (available from Molecular Devices, LLC, San Jose, CA); Ex. 280 nm, Em. 370 nm); and recalculated based on the chromatographic purity assessment. Figure 8A-8C Exemplary data from the case where 12.5 μg of immobilized aptamer was used to purify 100 μg of NIST mAb from 20 μL rat plasma matrix are shown. After purification, approximately 85% of the incorporated NIST mAb was recovered, with purity increasing from 10.6% to 72.4%.

[0212] A minor portion of the impurities in the purified NIST mAb eluate was determined to be nonspecific binding of endogenous proteins of rat plasma to the aptamer. The binding competition between NIST mAb and plasma proteins was studied by loading 20 μL of rat plasma containing 50 μg of NISTmAb onto increasing amounts of immobilized aptamer (5 μg, 12.5 μg, 25 μg). The results of this experiment are summarized in Figure 8D-Figure 8E Thus, it can be observed that the binding ratio of NIST mAb to plasma proteins for the current experimental method is close to 7:3 (from UV measurement). These data suggest that nonspecific binding can be further reduced.

[0213] Thus, an additional washing step (second wash) was applied before the elution step. To test this optional protocol step, 20 μL of plasma and 25 μg of NIST mAb were diluted to 200 μL with binding buffer and then loaded onto 40 μL of streptavidin resin, to which 5 μg of aptamer was non-covalently immobilized. After the first wash with binding buffer, two 200 μL volumes of each potential second wash buffer were applied, including the combination binding buffer proposed by Nakamura and colleagues (10 mM Tris, 5 mM CaCl2, pH 7.2); 20 mM phosphate, pH 7.0; 0.1 M glycine HCl, pH 2.7; 150 mM NaCl and binding buffer (control). Elution was then performed with EDTA buffer. In this article, the second wash and elution fractions were collected and analyzed by LC-UV analysis. As Fig.9A As shown in Figure 2, the binding buffer proposed by Nakamura et al. and 20 mM phosphate, 0.1 M glycine, and 150 mM NaCl were found to be not suitable secondary wash buffers. In both cases, the captured NISTmAb was eluted together with plasma impurities. On the other hand, Tris buffer containing 5 mM CaCl2 was able to selectively wash impurities from the aptamer. Fig. 9B and Fig. 9C The immobilized aptamer showed the best NIST mAb recovery, together with the highest purity after applying a secondary wash step based on a tris buffer containing 5 mM CaCl2. 2+ A buffer of is recommended, however optionally, a secondary wash buffer is used in an optimized protocol for capturing human Fc domains from biological fluid samples. Exemplary embodiments of the invention are outlined in the following schemes.

[0214] Example 5: Methods for capturing humanized mAb-based therapeutic proteins from preclinical-type biofluid samples Exemplary Schemes

[0215] This exemplary protocol is intended for use with immobilized aptamer materials or devices.

[0216] Buffer used:

[0217] ○Combination / 1 st Wash buffer: 10 mM Tris, 0.5 mM MgCl2, pH 7.2

[0218] ○2 nd (Secondary) Wash Buffer: 10 mM Tris, 5 mM CaCl2, pH 7.2

[0219] ○ Elution buffer: 10mM Tris, 200mM EDTA, pH 7.2 or 200mM ammonium acetate, pH 7.2 (for MS analysis)

[0220] Biofluid sample preparation:

[0221] o It is recommended to dilute the biofluid sample at least ten-fold with binding buffer before loading the sample onto the immobilized aptamer. For example, dilute 10 μL of biofluid sample with 90 μL of binding buffer to make a final volume of 100 μL.

[0222] Combination steps:

[0223] ○ Use binding buffer (compared to the volume of affinity resin / bed, about 10 column volumes (CV))

[0224] To equilibrate the immobilized aptamer resin / device

[0225] ○ Load the diluted biofluid sample onto the immobilized aptamer

[0226] ○ Incubate the device at room temperature for 1 hour with mixing. (e.g., plate shaker at 5-600 rpm)

[0227] ○Remove flow-through solution from resin / device

[0228] Washing steps:

[0229] ○ Perform 1 using binding buffer (approximately 10 CV of resin used) st Washing steps

[0230] ○ Remove 1 from resin / device st Washing solution

[0231] ○ Perform 2 washes using secondary wash buffer (approximately 10 CV of resin used) nd Washing steps.

[0232] ○Remove secondary wash solution from resin / unit

[0233] Elution steps:

[0234] o Elute captured human Fc analyte with desired elution buffer (approximately 10 CV of resin may be used, although the volume of elution buffer may be varied as desired)

[0235] ○ Save fractions as eluate for downstream analysis

[0236] Example 6: Online Immunoaffinity Capture MS Analysis Example

[0237] Immobilized anti-human Fc aptamers can be used for online immunoaffinity capture MS to facilitate the identification and quantification of humanized biotherapeutics. In some embodiments, the immunoaffinity column is filled with immobilized aptamer resin and directly coupled to LCMS for analysis. A solvent management system can be used in the present application to deliver binding buffer, secondary wash buffer, and MS-compatible elution buffer. The elution buffer described herein includes, but is not limited to, volatile monovalent salts such as ammonium formate, ammonium acetate, ammonium bicarbonate, tetramethylammonium or triethylammonium; volatile acids such as formic acid, acetic acid, TFA or DFA; volatile solvents such as acetonitrile and methanol. To avoid MS contamination, the LC flow can be diverted to waste for binding and washing steps and then switched back to MS for elution steps. Depending on the LC settings, the first 30 seconds to 2 minutes of the effluent from the immunoaffinity column can also be directed to waste. Alternatively, an MS-compatible wash buffer can be used. When eluting and detecting by MS, retention time, peak area, and m / z values ​​are used together to characterize the components of the sample. In another embodiment, the packed aptamer immunoaffinity column can be applied to a two-dimensional (2D) LCMS system for more comprehensive analysis. The secondary analytical column involved in such a 2D-LCMS system can be equipped with a variety of techniques, including but not limited to normal phase separation, reverse phase separation, size exclusion separation, ion exchange separation, HILIC separation, HIC separation, affinity capture or enzyme digestion.

[0238] Example 7: Anti-human Fc ELISA Example

[0239] As anti-human Fc affinity ligands, the aptamer sequences incorporated herein by reference can be used together with various aspects of the present invention to establish a sensitive enzyme-linked immunosorbent assay (ELISA) for accurately quantifying human IgG or humanized therapeutic agents with Fc domains. In some embodiments, the anti-human Fc aptamer is coated on the surface of the ELISA plate and used to capture the target IgG from a biological fluid. The above-mentioned selected binding buffer is applied to ensure that the desired binding capacity and specificity are achieved. Then one or more washing buffers in the washing buffer are used to wash unbound impurities from the plate. In a typical embodiment, a secondary detection ligand with an enzyme tag (such as horseradish peroxidase) is used to form a sandwich immunoassay. The detection ligand is combined with human IgG at one or more domains (including but not limited to CH1, light chain κ, light chain λ, VH or VL). After the binding of the detection ligand, a substrate (usually TMB) is added to the ELISA plate and catalyzed by the enzyme tag to produce a colored product whose concentration can be measured using a UV plate reader. In an alternative embodiment, the aptamer ligand is free in solution but modified to include an enzyme tag. Direct ELISA measurements can then be performed using the binding schemes described in the present technology. In another embodiment, the aptamer ligand is modified to include the enzyme tag itself to facilitate indirect ELISA measurements.

[0240] Those skilled in the art will recognize or be able to determine using only routine experimentation many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the present technology and are covered by the following claims. The contents of all references, issued patents, and published patent applications cited throughout this application are hereby incorporated by reference.

[0241] Sequence Listing

[0242]

[0243]

[0244]

[0245] This application relates to the following implementation schemes:

[0246] 1. A method for capturing a human immunoglobulin Fc domain in a biological fluid sample, the method comprising:

[0247] providing an affinity capture device comprising a surface having an aptamer, the aptamer being immobilized on the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 1;

[0248] The biological fluid sample is diluted with a binding buffer, the binding buffer comprising:

[0249] (A) Tris (hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES);

[0250] (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and

[0251] (C) a total monovalent cation concentration of 0 to no greater than 100 mM; and

[0252] The human immunoglobulin Fc domain in the biological fluid sample is adsorbed to the aptamer using the binding buffer.

[0253] 2. The method according to embodiment 1, wherein the aptamer is at least 85% identical to SEQ ID NO 1.

[0254] 3. The method according to embodiment 1, wherein the aptamer is at least 90% identical to SEQ ID NO 1.

[0255] 4. The method according to embodiment 1, wherein the aptamer is at least 95% identical to SEQ ID NO 1.

[0256] 5. The method according to embodiment 1, wherein the aptamer is at least 98% identical to SEQ ID NO 1.

[0257] 6. The method according to embodiment 1, wherein the aptamer is at least 99% identical to SEQ ID NO 1.

[0258] 7. The method according to embodiment 1, wherein the aptamer is a 23-nucleotide aptamer.

[0259] 8. The method according to embodiment 1, wherein the aptamer is a 23-nucleotide aptamer having an extended sequence or a truncated sequence of about 3 residues.

[0260] 9. The method according to embodiment 1, wherein the aptamer is a 23-nucleotide aptamer having an extended sequence or a truncated sequence of about 5 residues.

[0261] 10. The method of any one of embodiments 1 to 9, wherein the aptamer is non-covalently immobilized to the surface of the affinity capture device.

[0262] 11. The method of any one of embodiments 1 to 9, wherein the aptamer is covalently immobilized to the surface of the affinity capture device.

[0263] 12. The method of any one of embodiments 1 to 11, wherein the binding buffer has a monovalent cation concentration of less than about 50 mM.

[0264] 13. The method of any one of embodiments 1 to 12, wherein the binding buffer has a monovalent cation concentration of less than about 30 mM.

[0265] 14. The method of any one of embodiments 1 to 13, wherein the pH of the binding buffer is between about 5 and about 9.

[0266] 15. The method of any one of embodiments 1 to 14, wherein the pH of the binding buffer is between about 6 and about 8.

[0267] 16. The method of any one of embodiments 1 to 15, wherein the pH of the binding buffer is about 7.2.

[0268] 17. The method according to any one of embodiments 1 to 16, wherein the biological fluid sample is diluted by a factor of 2.

[0269] 18. The method of any one of embodiments 1 to 17, wherein the biological fluid sample is diluted by a factor of 10.

[0270] 19. The method of any one of embodiments 1 to 18, wherein the biological fluid sample is diluted by a factor of 20.

[0271] 20. The method of any one of embodiments 1 to 16, wherein the biological fluid sample is diluted to obtain a total monovalent cation concentration of the biological fluid sample of no greater than 100 mM.

[0272] 21. The method of embodiment 20, wherein the biological fluid sample is diluted to obtain a total monovalent cation concentration of the biological fluid sample of no greater than 50 mM.

[0273] 22. The method of embodiment 20, wherein the biological fluid sample is diluted to obtain a total monovalent cation concentration of the biological fluid sample of no greater than 30 mM.

[0274] 23. The method according to any one of embodiments 1 to 22 further comprises eluting the adsorbed human immunoglobulin Fc domain from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate or ammonium acetate.

[0275] 24. A method according to embodiment 23, wherein the ammonium concentration is between about 50 mM and about 500 mM.

[0276] 25. A method according to embodiment 23 or 24, wherein the eluent has a pH between about 6.5 and about 8.0.

[0277] 26. The method according to any one of embodiments 1 to 25, further comprising washing the adsorbed human immunoglobulin Fc domain with the binding buffer.

[0278] 27. The method according to any one of embodiments 1 to 26, further comprising treating with a + The adsorbed human immunoglobulin Fc domain is washed with a buffer.

[0279] 28. The method of any one of embodiments 1 to 27, wherein the concentration of the magnesium cation is between about 50 μM and about 1 mM.

[0280] 29. The method according to embodiment 23 also includes using a detector to analyze the eluted human immunoglobulin Fc domain.

[0281] 30. A method according to embodiment 29, wherein the detector is a sandwich enzyme-linked immunosorbent assay.

[0282] 31. A method according to embodiment 29, wherein the detector is a mass spectrometer.

[0283] 32. A method according to embodiment 1, wherein the human immunoglobulin Fc domain is contained within a humanized monoclonal antibody.

[0284] 33. The method according to embodiment 32 also includes purifying the humanized monoclonal antibody.

[0285] 34. A method for eluting a human immunoglobulin Fc domain from an immobilized aptamer, the method comprising:

[0286] providing an affinity capture device comprising a surface having an aptamer immobilized to the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 1, wherein the immobilized aptamer has an adsorbed human immunoglobulin Fc domain;

[0287] The adsorbed human immunoglobulin Fc domain is eluted from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate or ammonium acetate.

[0288] 35. A method according to embodiment 34, wherein the ammonium concentration is between about 50 mM and about 500 mM.

[0289] 36. A method according to embodiment 34 or 35, wherein the eluent has a pH between about 6.5 and about 8.0.

[0290] 37. The method according to any one of embodiments 34 to 36 further comprises analyzing the eluted human immunoglobulin Fc domain with a detector.

[0291] 38. A method according to embodiment 37, wherein the detector is a sandwich enzyme-linked immunosorbent assay.

[0292] 39. A method according to embodiment 37, wherein the detector is a mass spectrometer.

[0293] 40. A method according to embodiment 34, wherein the human immunoglobulin Fc domain is contained within a humanized monoclonal antibody.

[0294] 41. The method according to embodiment 40 also includes purifying the humanized monoclonal antibody.

[0295] 42. A kit, comprising:

[0296] an affinity capture device comprising a surface having an aptamer immobilized to the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 1; and

[0297] Bottled binding buffer, the binding buffer comprising:

[0298] (A) Tris (hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES);

[0299] (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and

[0300] (C) Total monovalent cation concentration of 0 to no greater than 100 mM.

[0301] 43. A kit according to embodiment 42, wherein the binding buffer has a monovalent cation concentration of less than about 50 mM.

[0302] 44. A kit according to embodiment 42 or 43, wherein the binding buffer has a monovalent cation concentration of less than about 30 mM.

[0303] 45. A kit according to any one of embodiments 42 to 44, wherein the pH of the binding buffer is between about 5 and about 9.

[0304] 46. ​​A kit according to any one of embodiments 42 to 45, wherein the pH of the binding buffer is between about 6 and about 8.

[0305] 47. The kit of any one of embodiments 42 to 46, wherein the pH of the binding buffer is about 7.2.

[0306] 48. The kit of any one of embodiments 42 to 47, wherein the concentration of the magnesium cation is between about 50 μM and about 1 mM.

[0307] 49. The kit of any one of embodiments 42 to 47, further comprising a bottle of eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is in the form of tetramethylammonium, triethylammonium, ammonium formate, or ammonium acetate.

[0308] 50. The kit according to any one of embodiments 42 to 49, further comprising Ca + of buffer in bottles.

[0309] 51. A kit according to embodiment 50, wherein the buffer is concentrated and the kit further comprises instructions for diluting the concentrated buffer to a working concentration using water.

[0310] 52. The kit of any one of embodiments 42 to 51, further comprising instructions for performing the method of any one of embodiments 1 to 25.

[0311] 53. A kit, comprising:

[0312] an affinity capture device comprising a surface having an aptamer immobilized to the surface of the affinity capture device and being at least 80% identical to SEQ ID NO 1; and

[0313] The bottle contains an eluent having an ammonium concentration between about 10 mM and about 1000 mM, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate or ammonium acetate.

[0314] 54. A kit according to embodiment 53, wherein the ammonium concentration is between about 50 mM and about 500 mM.

[0315] 55. The kit of embodiment 53 or 54, wherein the eluent has a pH between about 6.5 and about 8.0.

[0316] 56. The kit of any one of embodiments 53 to 55, further comprising instructions for performing the method of any one of embodiments 13 to 25.

Claims

1. A method for capturing a human Fc domain present in a biological fluid sample, the method comprising: providing an affinity capture device comprising a surface having an anti-human Fc aptamer; The biological fluid sample is diluted with a binding buffer, the binding buffer comprising: (A) Tris (hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no greater than 100 mM; and The human Fc domain in the biological fluid sample is adsorbed to the aptamer using the binding buffer. The method according to claim 1 , wherein the aptamer is a 23-nucleotide aptamer. The method according to claim 1 , wherein the aptamer has a bulge motif and a loop structure.

4. The method of claim 1, wherein the aptamer is non-covalently immobilized to the surface of the affinity capture device.

5. The method of claim 1, wherein the binding buffer has a monovalent cation concentration of less than about 50 mM. The method of claim 1 , wherein the binding buffer has a monovalent cation concentration of less than about 30 mM. The method of claim 1 , wherein the pH of the binding buffer is between about 5 and about 9.

8. The method of claim 7, wherein the pH of the binding buffer is between about 6 and about 8.

9. The method of claim 8, wherein the pH of the binding buffer is about 7.

2.

10. The method of claim 1, wherein the biological fluid sample is diluted by a factor of 2.

11. The method of claim 1, wherein the biological fluid sample is diluted by a factor of 10.

12. The method of claim 1, wherein the biological fluid sample is diluted by a factor of 20.

13. The method of claim 1, wherein the biological fluid sample is diluted to obtain a total monovalent cation concentration of the biological fluid sample of no greater than 100 mM.

14. The method of claim 1, wherein the biological fluid sample is diluted to obtain a total monovalent cation concentration of the biological fluid sample of no greater than 50 mM.

15. The method of claim 1, wherein the biological fluid sample is diluted to obtain a total monovalent cation concentration of the biological fluid sample of no greater than 30 mM.

16. The method according to any one of claims 1 to 15, further comprising eluting the adsorbed human immunoglobulin Fc domain from the immobilized aptamer using an eluent having an ammonium concentration between about 10 mM and about 1000 mM.

17. The method of claim 16, wherein the ammonium is present in the form of tetramethylammonium, triethylammonium, ammonium formate or ammonium acetate.

18. The method of claim 16, wherein the ammonium concentration is between about 50 mM and about 500 mM.

19. The method of claim 16, wherein the eluent has a pH between about 6.5 and about 8.

0.

20. The method according to any one of claims 1 to 15, further comprising washing the adsorbed human immunoglobulin Fc domain with the binding buffer.

21. The method according to any one of claims 1 to 15, further comprising using a + The adsorbed human immunoglobulin Fc domain is washed with a buffer.

22. The method of any one of claims 1 to 15, wherein the concentration of the magnesium cation is between about 50 mM and about 1 mM.

23. The method of claim 16, further comprising analyzing the eluted human immunoglobulin Fc domain with a detector.

24. The method of claim 23, wherein the detector is a sandwich ELISA.

25. The method of claim 23, wherein the detector is a mass spectrometer.

26. A kit, comprising: an affinity capture device comprising a surface having an anti-human Fc aptamer; and Bottled binding buffer, the binding buffer comprising: (A) Tris (hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) Total monovalent cation concentration of 0 to no greater than 100 mM.

27. The kit according to claim 26, wherein the aptamer is a 23-nucleotide aptamer.

28. The kit of claim 26, wherein the aptamer has a bulge motif and a loop structure.

29. The kit of claim 26, wherein the binding buffer has a monovalent cation concentration of less than about 50 mM.

30. The kit of claim 26, wherein the binding buffer has a monovalent cation concentration of less than about 30 mM.

31. The kit of claim 26, wherein the pH of the binding buffer is between about 5 and about 9.

32. The kit of claim 31, wherein the pH of the binding buffer is between about 6 and about 8.

33. The kit of claim 32, wherein the pH of the binding buffer is about 7.

2.

34. The kit of claim 26, wherein the concentration of the magnesium cation is between about 50 mM and about 1 mM.

35. The kit of any one of claims 26 to 34, further comprising a bottle of eluent having an ammonium concentration of between about 10 mM and about 1000 mM.

36. The kit of claim 35, wherein the ammonium is in the form of tetramethylammonium, triethylammonium, ammonium formate or ammonium acetate.

37. according to each described test kit in claim 26 to 34, also comprise comprising Ca + of buffer in bottles.

38. The kit of claim 37, wherein the buffer is concentrated and the kit further comprises instructions for diluting the concentrated buffer to a working concentration using water.

39. The kit of claim 36, further comprising instructions for performing the method of claim 1.

40. A method for capturing a human Fc domain present in a biological fluid sample, the method comprising: providing an affinity capture device comprising a surface having an aptamer bound to an Fc region; The biological fluid sample is diluted with a binding buffer, wherein the binding buffer comprises: (A) Tris (hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no greater than 100 mM; and The human Fc domain in the biological fluid sample is adsorbed to the aptamer using the binding buffer.

41. A method for capturing a human Fc domain present in a biological fluid sample, the method comprising: providing an affinity capture device comprising a surface having a metal-dependent aptamer that binds to an Fc domain; The biological fluid sample is diluted with a binding buffer, wherein the binding buffer comprises: (A) Tris (hydroxymethyl)aminomethane (Tris), trimethylamine (TES), 2-ethanesulfonic acid (MES), or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); (B) magnesium cations at a concentration between about 10 μM and about 20 mM; and (C) a total monovalent cation concentration of 0 to no greater than 100 mM; and The human Fc domain in the biological fluid sample is adsorbed to the aptamer using the binding buffer.

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