Quality control method of lysosome product

Abstract

The invention discloses a quality control method of a lysosome product. The quality control method comprises the following steps: 1) selecting a quality control substance, namely selecting an ADC (Analog to Digital Converter) or AOC (Alpha-Oxidase) substance with a lysosome enzymolysis acting site on an l inner or antibody, or oligonucleotide with a nuclease acting site; (2) carrying out enzymolysis treatment on multiple batches of lysosome products: carrying out enzymolysis treatment on the modal ADC / AOC / oligonucleotide determined in the step (1) by utilizing the multiple batches of lysosome products; (3) collecting a spectrogram in a non-targeted manner through LC-HRMS and identifying the structure of a metabolite; and 4) quantitatively detecting the metabolites by an LC-QqQMS method and establishing a quality control domain. According to the scheme, selected ADC / AOC / oligonucleotide is subjected to incubation and enzymolysis on lysosome, an LC-HRMS is used for identifying metabolites, an LC-QqQMS method is further constructed by utilizing an identification result, and a quality control domain of the metabolites of selected quality control substances in multiple batches of lysosome products is analyzed, so that the quality control method for the specific enzyme activity of the lysosome products is established.

Description

Technical Field

[0001] The present invention relates to the technical field of lysosome, and more particularly to a quality control method for lysosome products. Background Art

[0002] Lysosome Overview: Lysosomes are organelles that decompose biological macromolecules such as proteins, nucleic acids, and polysaccharides. In the 1950s, lysosomes were discovered and established as the central organelles for degradation and metabolism in cells. Lysosomes are single-membrane, dynamic, heterogeneous organelles that vary in location, morphology, size, enzyme content, and substrates. The lysosomal membrane contains hundreds of peripheral membrane proteins, including a variety of transporters and ion channels, and the acidic lysosomal cavity is maintained by Vacuolar ATPases. This low pH (4.5-5.5) value can activate a variety of intralysosomal hydrolases that digest macromolecules including proteins, nucleic acids, lipids, and carbohydrates. Lysosomes accept and digest small molecules and endocytic substances, engulf large particles such as apoptotic cell corpses and pathogenic bacteria, or autophagocytose cytoplasmic contents, including damaged mitochondria, endoplasmic reticulum, and lysosomes. Commercialized lysosome products are widely used in drug development.

[0003] Purification and identification of lysosomal products: Commonly used lysosomal separation and purification techniques include centrifugal separation and purification, fluorescence-assisted organelle sorting, affinity immunoseparation and purification, magnetic nanoparticle separation and purification, Lyso-IP, etc. The identification methods of lysosomal products include: a) acid phosphatase microscopy; b) electron microscopy; c) cell immunofluorescence, etc. None of the above methods can directly determine whether specific enzymes and their activities in lysosomes can serve as an effective substitute for the lysosomal system in vivo.

[0004] Lysosomes play a key role in the development of new biopharmaceuticals: Antibody-Drug Conjugate (ADC) and Antibody-Oligonucleotide Conjugates (AOC) are a new type of biopharmaceuticals that couple small molecule compounds or oligonucleotide molecules to targeted antibodies or antibody fragments through linkers. They can enhance drug targeting and stability, reduce clinical toxicity and side effects, and improve the therapeutic index. They have both the killing effect of traditional small molecule drugs or the protein silencing effect of oligonucleotides and the targeting of antibody drugs, and are mainly used in targeted treatment of anti-tumor or other rare diseases. After entering the body, ADC or AOC drugs can bind to antigens on the surface of target cells through the guidance of antibodies, and further transfer into target cells. ADC or AOC molecules that enter the cells (mainly in lysosomes) can release small molecule toxins / toxin analogs / oligonucleotides (i.e., effector molecules, payloads) through chemical or enzymatic action to "kill" target cells or silence genes to inhibit the expression of pathogenic proteins.

[0005] In the development process of ADC or AOC, it is particularly important whether the payload can be decomposed and released in the lysosome. The released payload permeates or transports out of the lysosome through the lysosomal membrane, interacts with molecular targets in the cytoplasm or nucleus to achieve drug treatment effects. Cleavable ADC / AOC will break the chemical bond between the linker and the payload to release small molecule toxins / oligonucleotides, while non-cleavable ADC / AOC releases the payload through hydrolysis of part of the amino acid sequence of the antibody. This process is often examined in in vitro lysosome experiments, which can evaluate whether the linker can be effectively cut by the lysosome to release the carried payload, providing an in vitro evaluation tool for the design of ADC / AOC linkers.

[0006] Lysosomes play a vital role in the degradation of ADC / AOC drugs and the release of payloads. The main enzyme is cathepsin B, which is a cysteine ​​protease that is mainly located in the lysosomes in the cell and is most active in an acidic environment (pH about 4.5-5.0). Cathepsin B is particularly important in the design of ADC / AOC drugs and is responsible for cutting the linker and releasing the payload. In addition, there are other types of enzymes in the lysosome that can participate in the degradation of ADC / AOC. Cathepsin L is also a cysteine ​​protease in the lysosome. It has similar substrate specificity to cathepsin B, participates in antigen processing and presentation, and also plays a role in protein degradation. Cathepsin D is an aspartic acid protease that can degrade a variety of proteins in the acidic environment of the lysosome. Cathepsin S: A cysteine ​​protease that has similar substrate specificity to cathepsin L and participates in antigen processing and presentation. Lysosomal membrane proteins: such as late endosomal / lysosomal adaptor protein and MAPK and MTOR activating protein 1 (LAMTOR1), may mediate lysosomal degradation of some drugs. Lysosomal receptor proteins: such as SQSTM1 / p62, are known lysosomal receptor proteins that may be involved in the lysosomal degradation of some drugs. Sulfatases: Sulfatases are overexpressed in several cancer types and show potential selectivity. ADCs containing sulfatase-cleaved linkers showed similar potency to the classic cleavable Val-Cit and Val-Ala linkers against Her2+ cell lines in vitro. β-Glucuronidase: This is a glycosidase that catalyzes the hydrolysis of β-glucuronic acid residues and is highly expressed in lysosomes and tumor stroma. ADCs containing β-glucuronic acid linkers showed low levels of aggregation, high plasma stability, and strong in vivo efficacy. Legumain: An aspartic endopeptidase that is overexpressed in multiple cancers and is known to be present in lysosomes. The legumain-cleavable ADC linker is cleaved faster in lysosomal extracts and plasma than traditional Val-Cit-type linkers, while maintaining excellent stability in plasma.

[0007] In addition, after being released, oligonucleotide drugs need to exist stably in lysosomes to exert their efficacy. However, in order to evaluate the stability of oligonucleotide drugs through lysosomal in vitro tests, it is necessary to prove the presence of nucleases in lysosomes. Therefore, verification of nuclease activity is also an important part of lysosomal product quality control.

[0008] The current status and needs of lysosomal product quality control: At present, although there are some technical means for the identification of lysosomal processing and enzymatic products of ADC / AOC drugs, such as LC-HRMS (liquid chromatography-high resolution) mass spectrometry for non-targeted identification of lysosomal processing ADC / AOC enzymatic products, it can accurately measure the mass-to-charge ratio of ions, realize accurate identification and analysis of molecules, and find unique metabolite components by comparing blank control samples in non-targeted analysis mode. It can also search for related compounds based on payload characteristic fragment ions to identify metabolites and metabolic pathways; LC-QqQMS (liquid chromatography-triple quadrupole) mass spectrometry for targeted detection of enzymatic products after lysosomal processing of specific ADC / AOC has high selectivity and high sensitivity, which can be used for quantitative analysis of payload and its metabolites, and provide accurate and reproducible quantitative data for drug metabolism research and pharmacokinetic evaluation. However, the quality control method for specific enzyme activity of commercial lysosomal products is still blank. With the widespread application of lysosomal products in many fields such as drug development, it is urgent to establish an effective quality control method for lysosomal products to ensure the quality and performance of lysosomal products in related applications. Summary of the invention

[0009] The purpose of the present invention is to solve the problem that the quality control methods currently used for commercial lysosomal products cannot directly characterize the enzymatic activity of specific enzymes in the lysosomes on ADC / AOC, the release capacity of the payload and the enzymatic activity of the nuclease, and to propose a quality control method for lysosomal products.

[0010] To solve the above problems, the present invention adopts the following technical solutions:

[0011] A quality control method for a lysosomal product comprises the following steps:

[0012] 1) Selection of quality control substances: Choose an ADC or AOC substance with a lysosomal enzymatic site on the linker or antibody, or an oligonucleotide with a nuclease site;

[0013] 2) Enzymatic treatment of multiple batches of lysosomal products: enzymatic treatment of the model ADC / AOC / oligonucleotide determined in step 1) using multiple batches of lysosomal products;

[0014] 3) LC-HRMS non-targeted acquisition spectra and metabolite identification: Use the non-targeted acquisition mode of LC-HRMS to perform HRMS spectra acquisition and structural identification of the payload and its analogs released by ADC / AOC after lysosomal product treatment, or the metabolites of oligonucleotides;

[0015] 4) LC-QqQMS targeted confirmation and quantification of metabolites and establishment of quality control domain: According to the identification results of step 3), an LC-QqQMS method is established for the identified metabolites, and the metabolites in the enzymatic hydrolysis samples of step 2) are targeted for confirmation and quantification, and the quality control domain of lysosomal products for the metabolites of the selected drugs is constructed;

[0016] 5) Determination of the effectiveness of lysosomal enzyme activity: Based on the quantitative results of the metabolites of the selected quality control substances (model ADC / AOC / oligonucleotides) by lysosomal enzymatic hydrolysis using the LC-QqQMS method, it is determined whether the lysosome has the ability to enzymatically hydrolyze the linker of the ADC / AOC and release the payload, or the ability to cleave the oligonucleotide, thereby confirming the effectiveness of the lysosome for the specific cleavage enzyme or nuclease activity of the model ADC / AOC drug.

[0017] As a further embodiment of the present invention: the lysosomal enzymatic degradation site in the model ADC / AOC determined in step 1) is determined based on known enzyme degradation sites in the lysosome.

[0018] As a further embodiment of the present invention: the nuclease action site of the oligonucleotide determined in step 1) is determined based on the characteristics of the action of the nuclease on the oligonucleotide in the lysosome.

[0019] As a further solution of the present invention: in the step 2), the processing conditions of each batch of lysosome products are kept consistent.

[0020] As a further embodiment of the present invention: the known enzymes in the lysosome include but are not limited to cathepsin B, cathepsin L, cathepsin D, cathepsin S and legumain.

[0021] As a further embodiment of the present invention: the treatment conditions include but are not limited to the treatment temperature, time, lysosomal product formula, and the amount of quality control substance (model ADC / AOC / oligonucleotide) added.

[0022] Compared with the prior art, the advantages of the present invention are:

[0023] In this protocol, the selected ADC / AOC / oligonucleotides were incubated with lysosomes for enzymatic hydrolysis, and the metabolites were identified using LC-HRMS. The identification results were further used to construct an LC-QqQMS method. The quality control domains of the metabolites of the selected quality control substances in multiple batches of lysosomal products were analyzed to establish a quality control method for the specific enzymatic activity of lysosomal products. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the mass spectrum of enzymatic hydrolysis product 1;

[0025] Figure 2 is the chemical molecular structure diagram of the enzymatic hydrolysis product 1;

[0026] Figure 3 It is the time-dependent trend diagram of enzymatic hydrolysis product 1;

[0027] Figure 4 is the mass spectrum of enzymatic hydrolysis product 2;

[0028] Figure 5 is the chemical molecular structure diagram of the enzymatic hydrolysis product 2;

[0029] Figure 6 is the time-dependent trend diagram of enzymatic hydrolysis product 2;

[0030] Figure 7 is the mass spectrum of enzymatic hydrolysis product 3;

[0031] Figure 8 is the chemical molecular structure diagram of the enzymatic hydrolysis product 3;

[0032] Fig. 9 is a time-dependent trend diagram of enzymatic hydrolysis product 3;

[0033] Fig.10 is the mass spectrum of enzymatic hydrolysis product 4;

[0034] Fig.11 is the chemical molecular structure diagram of the enzymatic hydrolysis product 4;

[0035] Fig.12 is a time-dependent trend diagram of enzymatic hydrolysis product 4;

[0036] Fig.13 is the mass spectrum of enzymatic hydrolysis product 5;

[0037] Fig.14 is the chemical molecular structure diagram of the enzymatic hydrolysis product 5;

[0038] Fig.15 is a time-dependent trend diagram of enzymatic hydrolysis product 5;

[0039] Fig.16 is the mass spectrum of enzymatic hydrolysis product 6;

[0040] Fig.17 is a time-dependent trend diagram of enzymatic hydrolysis product 6;

[0041] Fig.18 is the mass spectrum of enzymatic hydrolysis product 7;

[0042] Fig.19 is a time-dependent trend diagram of enzymatic hydrolysis product 7;

[0043] Fig. 20 is the mass spectrum of enzymatic hydrolysis product 8;

[0044] Fig.21is a time-dependent trend diagram of enzymatic hydrolysis product 8;

[0045] Fig. 22 is the mass spectrum of enzymatic hydrolysis product 9;

[0046] Fig.23 is a time-dependent trend diagram of enzymatic hydrolysis product 9;

[0047] Fig.24 This is the first batch result diagram;

[0048] Fig.25 This is the second batch of result graphs;

[0049] Fig.26 This is the result graph of the third batch;

[0050] Fig. 27 This is the fourth batch of result graphs;

[0051] Fig.28 This is the fifth batch of result graphs;

[0052] Fig.29 This is the sixth batch of results. DETAILED DESCRIPTION

[0053] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention; it is obvious that the described embodiments are only part of the embodiments of the present invention, not all of the embodiments, and all other embodiments obtained by ordinary technicians in this field without creative work based on the embodiments of the present invention are within the scope of protection of the present invention. In addition, the specific experimental methods not mentioned in the following embodiments are all carried out according to conventional experimental methods.

[0054] A quality control method for a lysosomal product comprises the following steps:

[0055] 1) Selection of quality control materials: Select an ADC or AOC material with a lysosomal enzymatic degradation site on the linker or antibody, or an oligonucleotide with a nuclease degradation site; wherein the lysosomal enzymatic degradation site in the ADC / AOC is determined based on the known enzyme degradation sites in the lysosome, and the known enzymes in the lysosome include but are not limited to cathepsin B, cathepsin L, cathepsin D, cathepsin S and legumain. The nuclease degradation site of the oligonucleotide is determined based on the characteristics of the nuclease in the lysosome on the oligonucleotide.

[0056] 2) Enzymatic hydrolysis of multiple batches of lysosomal products: enzymatic hydrolysis of the model ADC / AOC / oligonucleotide determined in step 1) is performed using multiple batches of lysosomal products; wherein the treatment conditions of each batch of lysosomal products are kept consistent, and the treatment conditions include but are not limited to the treatment temperature, time, lysosomal product formula, and the amount of quality control substance (model ADC / AOC / oligonucleotide) added.

[0057] 3) LC-HRMS non-targeted acquisition spectra and metabolite identification: Use the non-targeted acquisition mode of LC-HRMS to perform HRMS spectra acquisition and structural identification on the payload and its analogs released by ADC / AOC after treatment with lysosomal products, or the metabolites of oligonucleotides.

[0058] 4) LC-QqQMS targeted confirmation and quantification of metabolites and establishment of quality control domain: Based on the identification results of step 3), an LC-QqQMS method is established for the identified metabolites, and the metabolites in the enzymatic hydrolysis samples of step 2) are targeted for confirmation and quantification, and the quality control domain of lysosomal products for metabolites of the selected drugs is constructed.

[0059] 5) Determination of the effectiveness of lysosomal enzyme activity: Based on the quantitative results of the metabolites of the selected quality control substances (model ADC / AOC / oligonucleotides) by lysosomal enzymatic hydrolysis using the LC-QqQMS method, it is determined whether the lysosome has the ability to enzymatically hydrolyze the linker of the ADC / AOC and release the payload, or the ability to cleave the oligonucleotide, thereby confirming the effectiveness of the lysosome for the specific cleavage enzyme or nuclease activity of the model ADC / AOC drug.

[0060] Based on the above scheme: This method determines the model ADC / AOC / oligonucleotide with specific enzymatic action sites, uses multiple batches of lysosomal products for enzymatic treatment, and combines LC-HRMS and LC-QqQMS technology to accurately determine the lysosome's ability to enzymatically hydrolyze the linker and release the payload of the ADC / AOC or to cleave the oligonucleotide, thereby effectively controlling the quality of the lysosomal product.

[0061] This method fully utilizes the advantages of the two mass spectrometry technologies by first performing non-targeted acquisition and identification by LC-HRMS and then performing targeted confirmation and quantification by LC-QqQMS, thereby improving the accuracy and reliability of enzyme activity detection of lysosomal products and overcoming the defect in the prior art that it is impossible to directly determine whether specific enzymes and their activities in lysosomes can serve as an effective substitute for the in vivo lysosomal system.

[0062] Examples

[0063] Instruments, materials and reagents:

[0064] Unless otherwise specified, the instruments and reagents used in the examples are commercially available and can be used directly without further purification.

[0065] MS: AB SCIEX Triple TOF 5600+, AB SCIEX QTRAP 4500;

[0066] HPLC: Shimadzu LC20, Agilent 1290;

[0067] HPLC-TOF / MS:HPLC: Chromatographic column: Agela Technologies Venusil ASB C18; Mobile phase A: Water (containing 0.1% formic acid); Mobile phase B: Acetonitrile (containing 0.1% formic acid); Flow rate: 0.8 ml / min; Acquisition time: 15 min; Injection volume: 5 μL; Column temperature: 40 °C; Sample box temperature: 8 °C.MS / MS: Ionization mode: ESI; Ion detection mode: Positive ion;

[0068] Scanning mode: IDA; TOF MS: 200-1500Da; CUR: 20; GS1: 50; GS2: 50; ISVF: 5000; TEM: 650℃; DP: 150; CE: 10; TOF MS: 50-1500Da; CUR: 20; GS1: 50; GS2: 50; ISVF: 5000; TEM: 650℃; DP: 150; CE: 50; CES: 15.

[0069] HPLC-QqQMS: HPLC: Column: Agela Technologies Venusil ASB C18; Mobile phase A: Water (containing 0.1% formic acid); Mobile phase B: Acetonitrile (containing 0.1% formic acid); Flow rate: 0.8 ml / min; Acquisition time: 15 min; Injection volume: 5 μL; Column temperature: N / A; Sample box temperature: 8 °C. MS / MS: Ionization mode: ESI; Ion detection mode: Positive ion;

[0070] Scanning method: MRM; m / z 886.4→886.4, DP: 150, CE: 10, CXP: 10; m / z 871.4→871.4, DP: 150, CE: 10, CXP: 10; m / z 857.9→857.9, DP: 150, CE: 10, CXP: 10; m / z 888.1→888.1, DP: 150, CE: 10, CXP: 10; m / z 733.3→733.3, DP: 150, CE: 10, CXP: 10; m / z 1028.4→1028.4, DP: 150, CE: 10, CXP: 10; m / z 925.6→925.6, DP: 150, CE: 10, CXP: 10; m / z 540.1→540.1, DP: 150, CE: 10, other parameters: CXP: 10; EP: 10, CUR: 11, CAD: 4, IS: 5500, TEM: 550℃, GAS1:50, GAS2:50.

[0071] Lysosomal products were obtained from Huizhi Heyuan Biotechnology (Suzhou) Co., Ltd.; ADC drugs were obtained from Qide Pharmaceutical Technology (Suzhou) Co., Ltd.

[0072] (1) Lysosome incubation experiment

[0073] The experiment was performed at six time points: 0h, 24h, 48h, 72h, 5d, and 7d.

[0074] Prepare IPHASE Catabolic buffer reaction solution as needed: IPHASE Catabolic buffer-A: IPHASE Catabolic buffer-B = 100:1;

[0075] The reaction system is: 50ul liver lysosome (2mg / ml), 2ul ADC drug (2mg / ml), 148ul IPHASECatabolic buffer reaction solution, the total volume is 200ul;

[0076] After pre-incubation in a 37°C water bath for 5 minutes, add (human) liver lysosomes to each group, mix well, and immediately take out 30ul and add it to the 150ul pre-cooled acetonitrile prepared in advance to terminate, shake and mix well, and take it as 0min; the remaining samples continue to be placed in a 37°C water bath, and samples are taken at the same time points as 0min;

[0077] After all time points were collected, the samples were shaken on an oscillator for 3 min, centrifuged at 13,000 rpm for 3-5 min, and 100 ul of the supernatant was pipetted into the inner liner tube for subsequent machine testing.

[0078] (2) High-resolution mass spectrometry mining of enzymatic hydrolysis products

[0079] The data was collected by high-resolution mass spectrometry and processed by AB Sciex's analysis software PeakView to obtain the primary and secondary spectra of the enzymatic hydrolysis products. Statistical analysis was performed based on whether the products contained characteristic fragments (main characteristic fragments of the Payload, more than two were selected) and combined with the ADC drug structure to make a judgment. A total of 9 enzymatic hydrolysis products were found. Among them, LC-TOF / MS obtained Figure 1-23 The enzymatic products are shown.

[0080] (3) Triple quadrupole mass spectrometry (MRM mode) product detection

[0081] The MRM mode of QqQMS was used to establish a quantitative analysis method for the 9 degradation products found, which was used to construct a model for detecting and evaluating the efficiency of enzymatic hydrolysis, thereby achieving product quality control. The selection of 9 degradation products can cover the ability of various enzyme systems in lysosomes to degrade ADC. In the degradation model of the ADC drug by different batches of lysosomal products, MRM data were collected for the above 9 degradation products. A threshold range of the response intensity of each batch of lysosomal products to the decomposition ability of the ADC at a specific time point was formed. The response intensity of the degradation product at a specific incubation time point represents the ability of the lysosomal product to degrade the ADC drug. Under normal production processes, the degradation of the ADC by multiple batches of lysosomes and / or lysosomal products from different species should be maintained within a specific response intensity threshold range at a specific time point.

[0082] the following Figure 24-29 Results of 9 degradation products detected by MRM in 6 batches of human lysosomal product for a specific ADC drug.

[0083] (4) Data analysis

[0084] The MRM data results of multiple batches of lysosomal degraded ADC samples are shown in the table below, based on which the threshold ranges of 9 ADC degradation products at specific time points are summarized.

[0085]

[0086] Lysosomes act on different sites of ADC drugs to produce different products. The quality of lysosome products can be evaluated from three different aspects: the length of enzymatic hydrolysis time, the type of enzymatic hydrolysis products, and the amount generated. Whether it is the optimization of pretreatment conditions or the comparison of different batches of products, it has very good reference value and application prospects.

[0087] The above is only a preferred specific implementation of the present invention; however, the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solution and its improved conception within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for quality control of lysosomal products, characterized in that: The following steps are involved: 1) Selection of quality control substances: Choose an ADC or AOC substance with a lysosomal enzymatic site on the linker or antibody, or an oligonucleotide with a nuclease site; 2) Enzymatic treatment of multiple batches of lysosomal products: enzymatic treatment of the model ADC / AOC / oligonucleotide determined in step 1) using multiple batches of lysosomal products; 3) LC-HRMS non-targeted acquisition spectra and metabolite identification: Use the non-targeted acquisition mode of LC-HRMS to perform HRMS spectra acquisition and structural identification of the payload and its analogs released by ADC / AOC after lysosomal product treatment, or the metabolites of oligonucleotides; 4) LC-QqQMS targeted confirmation and quantification of metabolites and establishment of quality control domain: According to the identification results of step 3), an LC-QqQMS method is established for the identified metabolites, and the metabolites in the enzymatic hydrolysis samples of step 2) are targeted for confirmation and quantification, and the quality control domain of lysosomal products for the metabolites of the selected drugs is constructed; 5) Determination of the effectiveness of lysosomal enzyme activity: Based on the quantitative results of the metabolites of the selected quality control substances (modal ADC / AOC / oligonucleotides) by lysosomal enzymatic hydrolysis by the LC-QqQMS method, it is determined whether the lysosome has the ability to enzymatically hydrolyze the linker of the ADC / AOC and release the payload, or the ability to cleave the oligonucleotide, thereby confirming the effectiveness of the lysosome for the specific cleavage enzyme or nuclease activity of the modal ADC / AOC drug; The lysosomal enzymatic degradation sites in the model ADC / AOC determined in step 1) are determined based on known enzyme degradation sites in lysosomes. The nuclease action site of the oligonucleotide determined in step 1) is determined based on the characteristics of the action of the nuclease in the lysosome on the oligonucleotide; The processing conditions of each batch of lysosome products in step 2) are kept consistent; Known enzymes in the lysosome include cathepsin B, cathepsin L, cathepsin D, cathepsin S and legumain.

2. The method for quality control of a lysosomal product according to claim 1, characterized in that: The treatment conditions include the treatment temperature, time, lysosomal product formula and the amount of quality control material (model ADC / AOC / oligonucleotide) added.

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