Methods for detecting free and bound antibody in tlnps and uses thereof

Abstract

The application belongs to the technical field of drug detection, and provides a method for detecting free antibodies and bound antibodies in tLNP and application thereof. The method can simultaneously detect the contents of free antibodies and bound antibodies in tLNP, has high sensitivity, high accuracy, high reproducibility and good durability, and is convenient for detecting the contents of free antibodies and bound antibodies in tLNP and quality monitoring.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a method for detecting free antibodies and bound antibodies in tLNP and its application. Background Technology

[0002] Liposome nanoparticles (LNPs) are nanoscale vesicles formed by the self-assembly of lipid molecules, and are currently an important carrier for the in vivo delivery of biopharmaceutical active ingredients such as nucleic acids (e.g., mRNA), proteins, peptides, and antibodies. LNPs offer advantages such as protecting active ingredients, efficient cellular entry, scalability, and good biocompatibility; however, their targeting is primarily concentrated in the liver. Conjugating targeting ligands (such as antibodies, peptides, aptamers, and glycosyl groups) to the surface of LNPs to prepare targeted liposome nanoparticles (tLNPs) enables precise delivery to specific tissues or cells, thereby reducing off-target effects, lowering systemic toxicity, and increasing local drug concentrations. tLNPs can overcome the liver-biased nature of LNPs, achieving targeting of the lungs, spleen, tumors, and immune cells (such as T cells). tLNPs are increasingly being applied in areas such as tumor-targeted mRNA vaccines, in vivo CAR-T / M preparation, and organ-specific protein replacement therapy.

[0003] Currently, antibodies commonly used in the preparation of tLNPs include IgG and VHH (Variable Domain of Heavy Chain of Heavy-Chain Antibody). VHH, also known as nanobodies, is a single variable domain derived from natural heavy chain antibodies (HcAb, lacking light chains) of the camel family. With a molecular weight of only 12-15 kDa, it is the smallest known antibody fragment capable of completely and specifically binding to antigens. VHH is often used as a targeting ligand for tLNPs (e.g., conjugated to the end of PEG lipids) to achieve precise delivery of bioactive components to tumor cells, T cells, etc. However, due to the fact that antibodies are generally added in excess during the conjugation process, the tLNP product may contain both free and bound antibodies due to incomplete purification, affecting the quality of the tLNP product.

[0004] In the research and quality control of tLNPs, free antibodies and bound antibodies are two core indicators that directly determine targeting efficiency, immunogenicity, and batch stability. Currently, the main methods for detecting free and bound antibodies in tLNPs include size exclusion chromatography (SEC), enzyme-linked immunosorbent assay (ELISA), gel electrophoresis (SDS-PAGE), and the BCA method. Size exclusion chromatography (SEC) with fluorescence / UV quantification utilizes the difference in molecular size to separate Ab-LNPs (larger molecules, eluted first) from free antibodies (smaller molecules, eluted later), quantifying by peak area integration. However, this method is not suitable for small-molecule VHH antibodies, as free antibodies are not easily distinguished from solvent peaks. ELISA and BCA methods cannot distinguish between free and bound antibodies. Gel electrophoresis has low sensitivity and moderate separation ability, and is often used for qualitative tests. Moreover, most current methods cannot simultaneously detect free and bound antibodies, especially when using small-molecule VHH antibodies as LNP conjugates, where free antibody and solvent peaks are often indistinguishable, making it difficult to determine the free antibody content in quality control assays. Therefore, existing methods for detecting free and bound antibodies in tLNPs fail to meet current production and technological needs. Summary of the Invention

[0005] This invention provides a method for detecting free and bound antibodies in tLNP and its application. The method of this invention can accurately detect free and bound antibodies in tLNP, facilitating accurate detection and quality control of these antibodies. Furthermore, the method of this invention can simultaneously detect free and bound antibodies in tLNP samples, improving detection efficiency and avoiding errors caused by different batches, thus making the detection results more accurate and reliable.

[0006] Currently, the main antibodies used in the preparation of tLNP include IgG and VHH. The inventors of this invention discovered that while VHH offers advantages such as convenient coupling and minimal impact on the structure and function of LNP nanoparticles due to its smaller molecular weight, it presents a challenge in distinguishing it from solvent peaks during detection. This makes it difficult to simultaneously detect free and bound antibodies in the quality control testing of tLNP prepared using VHH, resulting in low detection efficiency and sensitivity that fails to meet production and technical requirements. To address this issue, the inventors, through extensive research, discovered that by employing a specific mobile phase in liquid chromatography, even when detecting tLNP products prepared using the smaller molecular weight VHH, simultaneous detection of free and bound antibodies in the sample can be achieved, improving both accuracy and efficiency.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for detecting free antibodies and bound antibodies in a sample containing targeted liposome nanoparticles, the method comprising the following steps: (1) Provide sample solution and control solution, wherein the sample solution includes the sample to be tested and diluent A, and the control solution includes antibody standard and diluent B, wherein diluent A and diluent B are the same; (2) Perform liquid chromatography detection on the sample solution and the reference solution respectively, and calculate the content of free antibody and bound antibody according to the external standard method based on the detection results; The mobile phase for the liquid chromatography detection includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of trifluoroacetic acid and mobile phase B is a mixed solution of trifluoroacetic acid, water and acetonitrile.

[0008] In this invention, "free antibody" refers to an antibody that is not coupled to the surface of liposome nanoparticles and is free in solution; "bound antibody" refers to an antibody that has been bound to liposome nanoparticles by chemical methods.

[0009] In this invention, "targeted liposome nanoparticles (tLNPs)" refers to liposome nanoparticles (LNPs) formed by binding with a targeting antibody. "Targeting antibody" refers to an antibody capable of conferring a "preferential" target organ / cell onto the LNP; any antibody possessing this characteristic and suitable for tLNP preparation is applicable to this invention. For example, the targeting antibody may include any one of VHH, IgG, or a combination of at least two. In some preferred embodiments, the targeting antibody included in the tLNP is VHH.

[0010] In this invention, "diluent" refers to a solvent that can dissolve / dilute a sample to form a concentration suitable for liquid chromatography detection. Any solvent in the art that can achieve the above-mentioned purpose is applicable to this invention. According to some preferred embodiments, in step (1), the diluent A includes DPBS and / or PBS.

[0011] According to some preferred embodiments, in step (1), the diluent B includes DPBS and / or PBS.

[0012] In some particularly preferred embodiments, both diluent A and diluent B are DPBS (preferably free of calcium and magnesium ions).

[0013] According to a preferred embodiment of the present invention, in step (2), the liquid chromatography detection is performed using a gradient elution method, and the specific elution gradient is as follows: In the first 1 minute, the volume fraction of mobile phase A was 65%, and the remainder was mobile phase B. From 1 to 16 minutes, the volume fraction of mobile phase A changed uniformly from 65% to 50%, with the remainder being mobile phase B; Between 16 and 25 minutes, the volume fraction of mobile phase A changed uniformly from 50% to 10%, with the remainder being mobile phase B. Between 25 and 35 minutes, the volume fraction of mobile phase A changed uniformly from 10% to 0, with the remainder being mobile phase B. From 35 to 35.1 min, the volume fraction of mobile phase A changed uniformly from 0 to 65%, with the remainder being mobile phase B; After 35.1 minutes, the volume fraction of mobile phase A was 65%, with the remainder being mobile phase B.

[0014] In some particularly preferred embodiments, in step (2), the chromatographic column used in the liquid chromatography detection is a reversed-phase column.

[0015] Preferably, the chromatographic column has a length of 30-100 mm, an inner diameter of 2-5 mm, a packing particle size of 2-10 μm, and a stationary phase of monodisperse polymer microspheres.

[0016] More preferably, the chromatographic column has a length of 50 mm, an inner diameter of 3 mm, a packing particle size of 5 μm, and a stationary phase of monodisperse polystyrene-divinylbenzene microspheres.

[0017] Any chromatographic column possessing the aforementioned characteristics can be used in this invention. This invention does not impose any particular restrictions on the specific source of the chromatographic column; it can be prepared using existing technology or obtained directly through commercial purchase. For example, the method of this invention can be performed using a commercially available BioCore RP-1000 chromatographic column.

[0018] According to a preferred embodiment of the present invention, in step (2), the concentration of trifluoroacetic acid in the mobile phase A is 0.08-0.12 vol% (i.e., mobile phase A can be an aqueous solution of trifluoroacetic acid with a concentration of 0.08-0.12 vol%). For example, the concentration of trifluoroacetic acid in mobile phase A can be 0.08 vol%, 0.085 vol%, 0.09 vol%, 0.095 vol%, 0.1 vol%, 0.105 vol%, 0.11 vol%, 0.115 vol%, 0.12 vol%, or it can be a range consisting of any two of the above values, or any intermediate value within that range.

[0019] According to a preferred embodiment of the present invention, in step (2), the concentration of trifluoroacetic acid in the mobile phase B is 0.08-0.12 vol%, and the concentration of acetonitrile is 85-95 vol%. For example, the concentration of trifluoroacetic acid in the mobile phase B can be 0.08 vol%, 0.085 vol%, 0.09 vol%, 0.095 vol%, 0.1 vol%, 0.105 vol%, 0.11 vol%, 0.115 vol%, 0.12 vol%, or it can be a range consisting of any two of the above values, or any intermediate value within that range. For example, the concentration of acetonitrile in the mobile phase B can be 85 vol%, 86 vol%, 87 vol%, 88 vol%, 89 vol%, 90 vol%, 91 vol%, 92 vol%, 93 vol%, 94 vol%, 95 vol%, or it can be a range consisting of any two of the above values, or any intermediate value within that range.

[0020] Any method capable of achieving the aforementioned final concentrations of trifluoroacetic acid and acetonitrile can be used to prepare mobile phase B in this invention. In some preferred embodiments, the preparation method of mobile phase B includes: mixing an aqueous trifluoroacetic acid solution with acetonitrile, wherein the concentration of the aqueous trifluoroacetic acid solution is 0.8-1.2 vol% (e.g., 0.8 vol%, 0.85 vol%, 0.9 vol%, 0.95 vol%, 1 vol%, 1.05 vol%, 1.1 vol%, 1.15 vol%, 1.2 vol%, or a range consisting of any two of the above values, or any intermediate value within that range), and mixing the aqueous trifluoroacetic acid solution with acetonitrile... The volume ratio of nitriles is 1:8-10 (e.g., 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, 1:10, or it can be a range of any two of the above ratios, or any intermediate ratio within that range).

[0021] In some preferred embodiments, in step (2), the conditions for liquid chromatography detection include: column temperature of 55-65℃; mobile phase flow rate of 0.5-0.7 mL / min; injection volume of 2-25 μL; and detection wavelength of 210-220 nm.

[0022] For example, the column temperature can be 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, or 65℃, or it can be any range consisting of any two of the above ratios, or any intermediate ratio within that range.

[0023] For example, the mobile phase rate can be 0.5 mL / min, 0.52 mL / min, 0.55 mL / min, 0.58 mL / min, 0.6 mL / min, 0.62 mL / min, 0.65 mL / min, 0.68 mL / min, or 0.7 mL / min, or it can be a range consisting of any two of the above ratios, or any intermediate ratio within that range.

[0024] For example, the injection volume can be 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, 20 μL, 21 μL, 22 μL, 23 μL, 24 μL, or 25 μL, or it can be a range consisting of any two of the above ratios, or any intermediate ratio within that range.

[0025] For example, the detection wavelength can be 210nm, 211nm, 212nm, 213nm, 214nm, 215nm, 216nm, 217nm, 218nm, 219nm, or 220nm, or it can be any range consisting of any two of the above ratios, or any intermediate ratio within that range.

[0026] In some particularly preferred embodiments, the conditions for liquid chromatography detection include: column temperature of 55-65°C; mobile phase flow rate of 0.6 mL / min; injection volume of 20 μL; and detection wavelength of 215 nm.

[0027] According to some preferred embodiments of the present invention, the method is used to simultaneously detect free antibodies and bound antibodies.

[0028] According to some preferred embodiments of the present invention, the method is used to detect either free antibody or bound antibody alone.

[0029] In a second aspect, the present invention provides the application of the method described in the first aspect in the quality control of targeted liposome nanoparticles.

[0030] In this invention, the quality control of targeted liposome nanoparticles may include quality inspection of the produced or obtained tLNP products to determine whether their quality meets the standards.

[0031] Compared with the prior art, the present invention has at least the following beneficial effects: 1) It can simultaneously and accurately detect the content of free antibodies and bound antibodies in tLNP samples; 2) The detection method has high sensitivity, high accuracy, high reproducibility and good robustness, and can accurately detect and monitor the quality of free antibodies and bound antibodies in tLNP samples. Attached Figure Description

[0032] Figure 1 This is a diagram showing the specificity detection results in Example 1; Figure 2 It is the chromatogram of "Method 1" in Example 2; Figure 3 This is the chromatogram of "Method 2" in Example 2; Figure 4 It is the chromatogram of "Method 1" in Example 3; Figure 5 This is the chromatogram of "Method 2" in Example 3. Detailed Implementation

[0033] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely exemplary and intended to aid in understanding the present invention, and should not be considered as specific limitations of the present invention.

[0034] In the following examples, unless otherwise specified, the VHH antibodies are all heavy chain antibodies purchased from Suzhou Levorotatory Star Biotechnology Co., Ltd., catalog number LS-Ab-008 (product name: anti-hCD84(VHH)), with a single variable domain and a molecular weight of 15kDa.

[0035] Example 1 This embodiment provides a method for simultaneously detecting free antibodies and bound antibodies in tLNP, including the following steps: The tLNP sample used in this embodiment is a lyophilized product (powder) formed by modifying LNP (components: cationic lipid, cholesterol, distearate, polyethylene glycol ester, polyethylene glycol maleimide ester) with VHH antibody and then freeze-drying it.

[0036] (1) Solution preparation Blank solvent: DPBS (calcium and magnesium ion-free) buffer; Sample solution: Accurately weigh tLNP lyophilized product (total antibody content is approximately 500 μg), place it in a 10 mL volumetric flask, add solvent (DPBS, free of calcium and magnesium ions) to dissolve and dilute to the mark, shake well, and the solution is ready; Reference solution: Take free antibody (VHH), dilute with solvent (DPBS, free of calcium and magnesium ions) to a concentration of approximately 50 μg / mL to obtain the reference solution; prepare two parallel solutions. Localization solutions: Take free antibody (VHH), conjugated antibody (VHH-polyethylene glycol maleimide ester), cationic lipid (YK-009, preparation method refers to CN114044741A), cholesterol (Chol), polyethylene glycol ester (PEG2000-DMG), polyethylene glycol maleimide ester, and distearate phosphatidylcholine (DSPC), respectively, dissolve and dilute them to 500 μg / mL in solvent (DPBS, free of calcium and magnesium ions) to prepare localization solutions for free antibody, conjugated antibody, and each lipid.

[0037] (2) Liquid chromatography conditions Mobile phase A: 0.1 v / v trifluoroacetic acid aqueous solution; Mobile phase B: 1 vol% trifluoroacetic acid aqueous solution - acetonitrile (prepared at a volume ratio of 1 vol% trifluoroacetic acid aqueous solution to acetonitrile of 1:9). Flow rate: 0.6 mL / min; Detection wavelength: 215 nm; Column: BioCore RP-1000, 3.0 × 50 mm, 5 μm; Injection volume: 20 μL; Column temperature: 60 ℃; The gradient elution procedure is shown in Table 1 below: Table 1

[0038] Exclusivity Accurately measure 20 μL each of the blank solvent, free antibody localization solution, each lipid localization solution, bound antibody localization solution, and sample solution prepared in (1) and inject them into the high-performance liquid chromatograph. Perform the detection as described above. See the results below. Figure 1 And Table 2.

[0039] Table 2

[0040] Conclusion: High-performance liquid chromatography (HPLC) was used to separate and detect the above samples. The results show that under these chromatographic conditions, both free and bound antibodies can be detected simultaneously. The bound antibody is a conjugate of the antibody and polyethylene glycol maleimide ester. No peak was observed for the polyethylene glycol ester. The blank solvent and other lipid components did not interfere with the detection of free and bound antibodies. The theoretical plate numbers for both the free and bound antibody peaks were greater than 3000, indicating good method specificity. Figure 1 .

[0041] linear Linear solution: Accurately weigh an appropriate amount of antibody reference standard, add diluent (DPBS, free of calcium and magnesium ions) to prepare a reference standard stock solution. Referring to the sample solution concentration (50 μg / mL), accurately measure the reference standard stock solution and dilute it with diluent to a series of concentration gradients (refer to Table 3 for specific concentrations). This is called a linear solution.

[0042] Experimental steps and conclusions: Accurately measure 20 μL of the above linear solutions (each concentration) and inject them into the high performance liquid chromatograph. Test them using the aforementioned method. The results are shown in Table 3 below.

[0043] Table 3

[0044] Conclusion: Within the range of 5.001-80.014 μg / mL, the linear equation for the antibody is y = 20052x + 848.68, r = 0.9996, which is greater than 0.990, indicating a good linear relationship.

[0045] Recovery rate Solution preparation: Blank solvent: DPBS (calcium and magnesium ion-free) buffer; Antibody stock solution: Take free antibody (VHH) and dilute it with solvent (DPBS, free of calcium and magnesium ions) to a concentration of approximately 500 μg / mL; Antibody control solution: Take the antibody stock solution and dilute it with solvent (DPBS, free of calcium and magnesium ions) to a concentration of approximately 50 μg / mL to obtain the control solution; prepare two parallel solutions. Background solution: Take liposome nanoparticles (non-targeted, unconjugated with antibody), add solvent (DPBS, free of calcium and magnesium ions) and dilute to a cholesterol content of approximately 25 μg / mL. The preparation methods for spiking solutions at different levels are as follows: R1-50% solution: Take liposome nanoparticles (containing approximately 250 μg of cholesterol), place them in a 10 mL volumetric flask, accurately add 0.5 mL of antibody stock solution, dilute to the mark with solvent, shake well, and three parallel portions are prepared. R2-100% solution: Take liposome nanoparticles (containing approximately 250 μg of cholesterol) and place them in a 10 mL volumetric flask. Accurately add 1 mL of antibody stock solution, dilute to the mark with solvent, and shake well. Prepare three parallel portions. R3-150% solution: Take liposome nanoparticles (containing approximately 250 μg of cholesterol) and place them in a 10 mL volumetric flask. Accurately add 1.5 mL of antibody stock solution, dilute to the mark with solvent, and shake well. Prepare three parallel portions.

[0046] Experimental steps and conclusions: Accurately measure 20 μL of each of the above blank solvents, antibody reference solutions, background solutions, and spiked solutions at different levels, and inject them into the high-performance liquid chromatograph. Perform detection according to the aforementioned method, record the chromatograms and results, and calculate the recovery rate using the external standard method. The results are shown in Table 4 below.

[0047] Table 4

[0048] Conclusion: At the three concentration levels of 50%, 100%, and 150%, the recoveries of free antibody calculated using the external standard method were all between 90% and 110%. The RSD value was 4.4%, and an RSD less than 10.0% indicates that the method has good accuracy.

[0049] Durability Solution preparation: Blank solvent: DPBS buffer; Sample solution: Take an appropriate amount of tLNP lyophilized product (containing approximately 500 μg of antibody), accurately weigh it, place it in a 10 mL volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and the solution is ready; Reference solution: Take an appropriate amount of antibody, dilute it with solvent to a concentration of about 50 μg / mL to obtain the reference solution; prepare two parallel solutions.

[0050] Experimental steps and conclusions: Accurately measure 20 μL of each of the above solutions and inject them into the liquid chromatograph. Perform the detection according to the aforementioned method and record the chromatogram. The results are shown in Table 5 below.

[0051] Table 5

[0052] Conclusions: Under the chromatographic conditions, with column temperature adjusted within ±5℃, the RSDs of both free and bound antibody contents in the sample solution were less than 2.0%, and the resolution between bound antibody and adjacent peaks was at least 1.57, all greater than 1.5, indicating a good system. With the flow rate adjusted within ±0.1 mL / min, the RSDs of both free and bound antibody contents in the sample solution were less than 2.0%, and the resolution between bound antibody and adjacent peaks was at least 1.51, all greater than 1.5, indicating a good system. With the detection wavelength adjusted within ±5 nm, the RSDs of both free and bound antibody contents in the sample solution were less than 2.0%, and the resolution between antibody and adjacent peaks was at least 1.56, all greater than 1.5, indicating a good system. Robustness: After adjusting column temperature, flow rate, and detection wavelength, there was no significant difference in antibody results compared to normal conditions. In summary, the column temperature, flow rate, and detection wavelength showed good robustness under the chromatographic conditions.

[0053] Example 2 Selection of Chromatographic Separation Mode Based on Example 1, the type of chromatographic column was changed, and different separation modes (ion exchange, hydrophobic interaction) were selected to detect free and bound antibodies, as detailed in Table 6. 20 μL of the sample solution prepared in Example 1 was accurately measured and injected into the high-performance liquid chromatograph, and the results were recorded, as detailed in Table 6. Figure 2 and Figure 3 .

[0054] Table 6

[0055] Conclusion: High-performance liquid chromatography (HPLC) analysis of the separation of free and bound antibodies using columns with the two different separation modes revealed that the chromatographic column using method 1 (ion exchange separation mode) produced a more efficient chromatogram. Figure 2 The free antibody peak was broad, resulting in poor sensitivity, and no obvious response peak was observed for the bound antibody. Using a column in the hydrophobic separation mode of Method 2, the obtained spectrum ( Figure 3 In the original text, the free antibody peak almost overlapped with the solvent peak, and this could not be improved by adjusting the chromatographic parameters. In contrast, the chromatogram obtained using the reversed-phase column (BioCore RP-1000, 3.0 × 50 mm, 5 μm) in Example 1 was significantly better. Figure 1 It can be clearly seen from the results that both free antibodies and bound antibodies can be effectively detected, with good peak shape and satisfactory separation, enabling simultaneous detection of both free and bound antibodies.

[0056] Example 3 Selection of elution gradient Based on the chromatographic conditions of Example 1, the elution gradient was changed (see Table 7 for details), while other chromatographic conditions remained unchanged. Free and bound antibodies were then detected. 20 μL of the sample solution prepared in Example 1 was accurately measured and injected into the high-performance liquid chromatograph (HPLC). The results were recorded. Figure 4 and Figure 5 .

[0057] Table 7

[0058] Conclusion: Based on the chromatographic conditions of Example 1, changing the elution gradient while keeping other chromatographic conditions unchanged, it was found that using the elution gradient in Method 1 resulted in a better chromatogram ( Figure 4 In the chromatogram, the separation between the bound antibody peak and adjacent peaks is less than 1.0, and the separation between the bound antibody peak and adjacent unknown impurity peaks is poor. Using the elution gradient in Method 2, the obtained spectrum ( Figure 5 The resolution between the bound antibody and adjacent peaks in the chromatogram was less than 1.5, and the resolution between the bound antibody peak and adjacent unknown impurity peaks was poor. Using the chromatographic conditions of Example 1, the obtained chromatogram ( Figure 1The separation degree between the bound antibody and the adjacent peak was 1.62, which is greater than 1.5, indicating good separation between the bound antibody peak and the adjacent unknown impurity peak.

[0059] The above embodiments are merely illustrative examples, and the present invention is not limited to these embodiments, meaning that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of raw materials, additions of auxiliary components, and selection of specific methods, without departing from the spirit of the present invention, all fall within the protection and disclosure scope of the present invention.

[0060] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for detecting free antibodies and bound antibodies in a sample containing targeted liposome nanoparticles, characterized in that, The method includes the following steps: (1) Provide sample solution and control solution, wherein the sample solution includes the sample to be tested and diluent A, and the control solution includes antibody standard and diluent B, wherein diluent A and diluent B are the same; (2) Perform liquid chromatography detection on the sample solution and the reference solution respectively, and calculate the content of free antibody and bound antibody according to the external standard method based on the detection results; The mobile phase for the liquid chromatography detection includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of trifluoroacetic acid and mobile phase B is a mixed solution of trifluoroacetic acid, water and acetonitrile.

2. The method according to claim 1, wherein, In step (1), the diluent A includes DPBS and / or PBS; And / or, in step (1), the diluent B includes DPBS and / or PBS.

3. The method according to claim 1, wherein, In step (2), the liquid chromatography detection is performed using a gradient elution method, and the specific elution gradient is as follows: In the first 1 minute, the volume fraction of mobile phase A was 65%, and the remainder was mobile phase B. From 1 to 16 minutes, the volume fraction of mobile phase A changed uniformly from 65% to 50%, with the remainder being mobile phase B; Between 16 and 25 minutes, the volume fraction of mobile phase A changed uniformly from 50% to 10%, with the remainder being mobile phase B. Between 25 and 35 minutes, the volume fraction of mobile phase A changed uniformly from 10% to 0, with the remainder being mobile phase B. From 35 to 35.1 min, the volume fraction of mobile phase A changed uniformly from 0 to 65%, with the remainder being mobile phase B; After 35.1 minutes, the volume fraction of mobile phase A was 65%, with the remainder being mobile phase B.

4. The method according to claim 1, wherein, In step (2), the chromatographic column used in the liquid chromatography detection is a reversed-phase chromatographic column.

5. The method according to claim 4, wherein, The chromatographic column has a length of 30-100 mm, an inner diameter of 2-5 mm, a packing particle size of 2-10 μm, and a stationary phase of monodisperse polymer microspheres.

6. The method according to claim 5, wherein, The chromatographic column has a length of 50 mm, an inner diameter of 3 mm, a packing particle size of 5 μm, and a stationary phase of monodisperse polystyrene divinylbenzene microspheres.

7. The method according to claim 1, wherein, In step (2), the concentration of trifluoroacetic acid in the mobile phase A is 0.08-0.12% by volume. And / or, in the mobile phase B, the concentration of trifluoroacetic acid is 0.08-0.12% by volume, and the concentration of acetonitrile is 85-95% by volume. Preferably, the preparation method of the mobile phase B includes: mixing an aqueous trifluoroacetic acid solution with acetonitrile, wherein the concentration of the aqueous trifluoroacetic acid solution is 0.8-1.2% by volume, and the volume ratio of the aqueous trifluoroacetic acid solution to acetonitrile is 1:8-10.

8. The method according to claim 1, wherein, In step (2), the conditions for liquid chromatography detection include: column temperature of 55-65℃; mobile phase flow rate of 0.5-0.7 mL / min, preferably 0.6 mL / min; injection volume of 2-25 μL, preferably 20 μL; and detection wavelength of 210-220 nm, preferably 215 nm.

9. The method according to claim 8, wherein, The method is used to simultaneously detect free antibodies and bound antibodies; And / or, the method is used to detect either the free antibody or the bound antibody alone.

10. The application of the method according to any one of claims 1-9 in the quality control of targeted liposome nanoparticles.

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