Stable cell strain, construction method thereof and method for detecting biological activity of recombinant human erythropoietin

Abstract

The invention provides a construction method of a stable cell strain for detecting the biological activity of EPO and PEG-EPO human recombinant proteins. The method comprises the following steps: (1) constructing recombinant lentiviral particles containing EPOR and STAT5-RE-Luciferase-2P sequences; (2) infecting cells by using the recombinant lentivirus particles; and (3) screening positive cells, and carrying out amplification culture to obtain the stable cell strain. The invention further discloses a stable cell strain for detecting the biological activity of the EPO and PEG-EPO human recombinant protein and a method for detecting the biological activity of the EPO and PEG-EPO. The method has the advantages of simple operation, convenience, rapidness, sensitive reaction, strong specificity, high accuracy and good reproducibility.

Description

Technical Field

[0001] The present invention relates to the technical field of biological drug activity detection, and more specifically, to a method for detecting the biological activity of human recombinant EPO and PEG-EPO. Background Art

[0002] Recombinant human erythropoietin (rhEPO) is a glycoprotein efficiently expressed by transferring the human erythropoietin (EPO) gene into mammalian cells using genetic engineering technology. Its relative molecular mass is about 18kDa, which can selectively stimulate the differentiation and proliferation of erythroid precursor cells and promote the development and maturation of red blood cells. In clinical practice, rhEPO is mainly used to treat chronic renal failure (CRF), aplastic anemia, and anemia caused by chemotherapy in cancer patients. It has attracted much attention due to its significant efficacy. In order to increase the long-term stability of protein drugs, activated polyethylene glycol (PEG) is combined with the free amino group of rhEPO molecules to form a stable covalent compound PEG-EPO. By affecting the receptor binding affinity and the in vivo biological activity of the protein, the stability of the protein can be increased and its half-life in the blood can be prolonged, reducing the number of dosing times for patients. This is a strategy of choice to increase the effective pharmacokinetic and pharmacodynamic properties of various therapeutic proteins. Therefore, whether it is rhEPO or PEG-EPO, in order to adapt to the needs of batch production quality control (QC) release testing at different stages of its research and development and clinical products, it is necessary to seek appropriate testing methods or means to meet the relevant requirements of product quality control.

[0003] Biological activity determination is an important indicator for controlling the effectiveness of protein drugs. Among the biological activity methods of EPO and PEG-EPO human recombinant proteins, the commonly used detection methods are reticulocyte counting method and cell proliferation method. The reticulocyte counting method requires the use of mice for experiments, which is complicated to operate, with an experimental cycle of at least 4 days and large variability in results. Although the cell proliferation method is simpler than the animal method, the EPO-dependent cell culture cycle is long, the experimental cycle is long, and the method variability is large, which cannot meet the needs of rapid and accurate release testing of quality control (QC). Therefore, a highly specific, efficient, rapid and accurate biological activity detection method is urgently needed.

[0004] The contents of the background technology section are merely the technologies known to the inventors and do not necessarily represent the prior art in the field. Summary of the invention

[0005] In view of the above research objectives, one of the objectives of the present invention is to provide a method for constructing a stable cell line for detecting the biological activity of EPO and PEG-EPO human recombinant proteins, the method comprising the following steps:

[0006] (1) constructing a recombinant lentiviral particle containing EPOR and STAT5-RE-Luciferase-2P sequences, wherein the EPOR sequence is shown in SEQ ID NO: 1, the STAT5-RE sequence is shown in SEQ ID NO: 2, and the Luciferase-2P sequence is shown in SEQ ID NO: 3;

[0007] (2) using the above-mentioned recombinant lentiviral particles to infect cells;

[0008] (3) Screen positive monoclonal cells and expand the culture to obtain stable cell lines.

[0009] Furthermore, constructing recombinant lentiviral particles containing EPOR and STAT5-RE--Luciferase-2P sequences includes the following steps: synthesizing the EPOR sequence and the STAT5-RE-Luciferase-2P sequence, and subcloning them into lentiviral vectors respectively, then using the lentiviral vectors containing the EPOR sequence and the STAT5-RE-Luciferase-2P sequence to transform Escherichia coli respectively, selecting single clones and extracting plasmids; co-transfecting different cells with the extracted EPOR lentiviral plasmid and STAT5-RE-Luciferase-2P lentiviral plasmid respectively with the auxiliary plasmid; culturing the transfected cells containing the EPOR lentiviral plasmid and the STAT5-RE-Luciferase-2P lentiviral plasmid respectively, taking the cell culture supernatant, and preparing recombinant EPOR lentiviral particles and STAT5-RE-Luciferase-2P lentiviral particles;

[0010] The co-transfected cells are preferably 293T cells;

[0011] And / or, the helper plasmid is a packaging plasmid and an envelope plasmid.

[0012] Furthermore, in step (2), the infected cells are HEK293 cells;

[0013] And / or, in step (3), pressure screening of positive cells is performed by simultaneously adding Puromycin and Hygromycin B to the cell culture medium.

[0014] The second object of the present invention is to provide a stable cell line for detecting the biological activity of EPO and PEG-EPO human recombinant proteins, which is constructed by the above method, and the stable cell line contains an EPOR sequence and a STAT5-RE-Luciferase-2P sequence, wherein the EPOR sequence is shown in SEQ ID NO: 1, the STAT5-RE sequence is shown in SEQ ID NO: 2, and the Luciferase-2P sequence is shown in SEQ ID NO: 3.

[0015] The third object of the present invention is to provide a method for detecting the biological activity of EPO and PEG-EPO, comprising the following steps:

[0016] (A) inoculating and culturing the stable cell line constructed by the construction method or the stable cell line at an inoculation density of 1E4 / well to 10E4 / well (e.g., 1E4 / well, 2E4 / well, 4E4 / well, 6E4 / well, 8E4 / well, 10E4 / well, preferably 4E4 / well to 6E4 / well);

[0017] (B) adding the test substance EPO or PEG-EPO to the stable cell line;

[0018] (C) incubating in an incubator for 4-24 hours (e.g., 4 hours, 6 hours, 8 hours, and 24 hours, preferably 5±1 h);

[0019] (D) Detect the chemiluminescence value and calculate the relative biological activity of the analyte.

[0020] The present invention provides a method for detecting the biological activity of EPO and PEG-EPO human recombinant proteins, which is simple to operate, convenient and fast, sensitive to reaction, strong in specificity, high in accuracy and good in reproducibility, is beneficial to the quality control and clinical application of EPO and PEG-EPO human recombinant proteins, and has far-reaching application value.

[0021] The present invention combines the specific mechanism of action of EPO and the advantages of the reporter gene method, constructs a HEK293 / STAT5-RE-Luc / EPOR genetically engineered cell line, develops a biological activity determination method that is simultaneously applicable to EPO and PEG-EPO human recombinant proteins, and studies the specificity, accuracy, precision, linear range and durability of the method, thereby proving that the method is simple, rapid, accurate, has strong specificity, good precision and accuracy, and is robust, and can be used for the determination of the biological activity of EPO and PEG-EPO human recombinant proteins, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments 1, 2 and 3 of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0023] Figure 1 Shown is a plasmid map of the EPOR lentiviral construction;

[0024] Figure 2 Shown is the plasmid map of STAT5-RE-Luciferase-2P lentiviral construction;

[0025] Figure 3 Shown is the result graph of the target protein expression detected by flow cytometry of the constructed HEK293 / STAT5-RE-Luc / EPOR stable cell line; wherein, Blank is the blank control, and the HEK293 / STAT5-RE-Luc / EPOR stable cell line is used as the blank control; Only Second Antibody refers to the HEK293 / STAT5-RE-Luc / EPOR stable cell line incubated with only the detection antibody (PE Donkey anti-Rabbit IgG) but not the EPOR antibody; IsotypeControl refers to the HEK293 / STAT5-RE-Luc / EPOR stable cell line incubated with other target antibodies (Dupilumab) and the detection antibody but not with the EPOR antibody; EPOR Antibody refers to the HEK293 / STAT5-RE-Luc / EPOR stable cell line incubated with the EPOR antibody (EPOR / EPO ReceptorAntibody) and the detection antibody (PE Donkey anti-Rabbit IgG).

[0026] Figure 4 Shown is the result of functional activity verification of the constructed HEK293 / STAT5-RE-Luc / EPOR stable cell line;

[0027] Figure 5 Shown is the result of mycoplasma detection of the constructed HEK293 / STAT5-RE-Luc / EPOR stable cell line;

[0028] Figure 6 Shown are graphs of the confluence results after overnight cell culture at different serum concentrations (samples without EPO and PEG-EPO added);

[0029] Figure 7 Shown are graphs of the confluence results after overnight cell culture under different assay media and cell seeding density conditions (without the addition of EPO and PEG-EPO samples);

[0030] Figure 8 The graph shows the sample concentration range and dilution results for the functional activity assay parameters; the results of the 2-fold dilution of the EPO sample are shown in Figure 8 -A-1, 2.5 times dilution results see Figure 8 -A-2, 3-fold dilution results see Figure 8 -A-3; the results of the 2-fold dilution of PEG-EPO samples are shown in Figure 8 -B-1, 2.5 times dilution results see Figure 8 -B-2, 3-fold dilution results see Figure 8 -B-3;

[0031] Fig. 9 middle, Fig. 9 A1. Fig. 9 A2. Fig. 9 A3. Fig. 9 A4 shows the concentration range of PEG-EPO samples, dilution method and result graph in the test parameters;

[0032] Fig.10 The display shows the concentration range, dilution method and result graph of PEG-EPO samples in the test parameters; the PEG-EPO sample 300nM starting 3-fold dilution is shown in Fig.10 -A, 150nM starting 2.5-fold dilution see Fig.10 -B, 100 nM starting 2-fold dilution see Fig.10 -C;

[0033] Fig.11 Displayed are the concentration range and dilution method of PEG-EPO samples in the test parameters and the result graph;

[0034] In FIG. 12 , 12-1 shows the detection result of PEG-EPO samples in HEK293 / STAT5-RE-Luc / EPOR cells and HEK293 cells in the specificity confirmation;

[0035] Figure 12-2 Shown is the test result of different samples in HEK293 / STAT5-RE-Luc / EPOR cells in the confirmation of PEG-EPO specificity;

[0036] Figure 12-3 Shown is a graph showing the results of different samples tested in HEK293 / STAT5-RE-Luc / EPOR cells during EPO specificity confirmation;

[0037] Fig.13 Shown is a graph of relative accuracy validation results;

[0038] Fig.14 Shown is a graph of the linearity verification results from the relative accuracy confirmation. DETAILED DESCRIPTION

[0039] In the following description, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0040] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, terms in the singular shall include plural forms, and terms in the plural shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural indicators. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include endpoints and all values ​​between the endpoints.

[0041] definition

[0042] In order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0043] The term "specificity" in the present invention refers to the ability of the analytical method to correctly determine the analyte in the presence of other components (such as impurities, excipients, etc.), also known as specificity.

[0044] The term "relative accuracy" (accuracy) is used to evaluate the degree to which the actual measured value of the analytical method's test results is close to or consistent with the theoretical value. The closer the measured value is to the theoretical value, the higher the accuracy of the method is.

[0045] The term "precision" in the present invention refers to the consistency or closeness between actual measured values, usually expressed as relative standard deviation RSD (%), including intra-day precision, inter-day precision and reproducibility. Intra-day precision refers to the coefficient of variation (CV) between multiple test results on the same day under the same conditions in the same experiment, and inter-day precision refers to the coefficient of variation of the test results of the same experiment on different days and by different operators. Reproducibility is one of the important components of precision determination, which needs to be completed through experiments between different laboratories. It is generally used for analytical method transfer or standardization of analytical methods. In order to verify the precision of this method, intra-day and inter-day repeatability and precision were studied.

[0046] The term "robustness" refers to the degree to which the measurement results are not affected by small changes in the measurement conditions, providing a basis for the established method to be used for routine testing.

[0047] The term "linearity" refers to the ability of a linear test result to be directly proportional to the concentration of the analyte in the sample within the designed range.

[0048] The term "cell seeding density" refers to the density of viable cells seeded, which greatly affects cell growth and survival, and is sometimes referred to as "cell density" in the present invention.

[0049] The term "stable cell line", also known as a stable cell line, refers to a cell line that is obtained by genetic engineering to stably overexpress or interfere with a specific gene. Stable transfection (also known as stable transformation) aims to integrate foreign DNA into the host genome in order to permanently change the genetic composition of the cell. The main advantage of a stable cell line is that it can maintain consistent gene expression for a long time.

[0050] The term "subcloning" refers to the transfer of a fragment of a plasmid into another plasmid that can be used as a vector. The purpose of subcloning includes but is not limited to: the new vector may have specific markers for antibiotic selection or fluorescent expression; transfer a cloned fragment to an expression vector of a more suitable host (such as bacteria, mammals, insects, plants, etc.); place the target gene under a different expression promoter (such as from a constitutive to an inducible promoter); or fuse or label the experimental gene with another protein or marker. Well-known subcloning methods include the use of restriction enzyme digestion and PCR cloning techniques.

[0051] The term "transformation" refers to the direct introduction of exogenous genes into prokaryotic cells (such as bacteria) by a recombinant plasmid (vector) containing exogenous genes, allowing them to acquire new genetic characteristics.

[0052] The term "transfection" refers to the introduction of recombinant plasmid vectors or free nucleotides into eukaryotic cells mediated by liposomes or the like.

[0053] The term "infection" refers to the pathophysiological process caused by the invasion of recombinant viral vectors into recipient cells.

[0054] The term "cell confluence" refers to the probability or proportion of cell fusion under specific conditions. In cell biology, cell confluence can be used to study the mechanisms of cell-cell interactions and cell fusion. For example, when studying how some viruses invade and infect host cells, cell confluence can be used to evaluate the interaction between viruses and cell membranes.

[0055] The term "titer" can also be called "viral titer". It is a term used to express concentration and is usually used in chemistry, pathology and immunology. Viral titer refers to the concentration of the virus suspension, which can also be called the potency of the virus. Lentivirus titer is used to evaluate the amount of virus required to transduce a certain number of target cells, that is, the amount of virus contained in each liter of solution. Transduction unit (TU) is the viral vector genome that can infect and integrate into cells.

[0056] "Detection window" refers to the ratio of the maximum value of the upper platform to the minimum value of the lower platform of the four-parameter fitting curve obtained by the detection of antibodies or other macromolecular therapeutic drugs based on the drug action mechanism in vitro, such as the reporter gene method. The detection window of the reporter gene method is particularly important. In the CMC stage of antibody products, a detection window that is too small cannot guarantee the robustness of the developed activity method, thereby affecting the safety, effectiveness and reliability of batch release and stability test results.

[0057] Recombinant human erythropoietin (rhEPO) is obtained by transferring the human erythropoietin (EPO) gene into mammalian cells for expression. In the present invention, the terms rhEPO and EPO are used interchangeably.

[0058] Method for constructing stable cell lines

[0059] One aspect of the present invention relates to a method for constructing a stable cell line, the method comprising the following steps:

[0060] (1) constructing recombinant lentiviral particles containing an EPOR sequence and a STAT5-RE-Luciferase-2P sequence, respectively, wherein the EPOR sequence is shown in SEQ ID NO: 1, the STAT5-RE sequence is shown in SEQ ID NO: 2, and the Luciferase-2P sequence is shown in SEQ ID NO: 3;

[0061] (2) using the above-mentioned recombinant lentiviral particles to simultaneously infect cells;

[0062] (3) Screening positive cells and expanding the culture to obtain a stable cell line, wherein the positive cells are cells containing the EPOR sequence and the STAT5-RE-Luciferase-2P sequence.

[0063] In some embodiments, in the step (1), constructing a recombinant lentiviral particle containing EPOR and STAT5-RE-Luciferase-2P sequences comprises the following steps: synthesizing the EPOR sequence and the STAT5-RE-Luciferase-2P sequence, and subcloning them into lentiviral vectors respectively, then using the lentiviral vectors containing the EPOR sequence and the STAT5-RE-Luciferase-2P sequence to transform Escherichia coli respectively, selecting single clones and extracting plasmids; co-transfecting different cells with the extracted EPOR lentiviral plasmid and STAT5-RE-Luciferase-2P lentiviral plasmid respectively with the auxiliary plasmid; culturing the transfected cells containing the EPOR lentiviral plasmid and the STAT5-RE-Luciferase-2P lentiviral plasmid respectively, and taking the cell culture supernatant to prepare recombinant EPOR lentiviral particles and STAT5-RE-Luciferase-2P lentiviral particles;

[0064] The co-transfected cells are preferably 293T cells;

[0065] And / or, the helper plasmid is a packaging plasmid and an envelope plasmid.

[0066] In some embodiments, in step (2), the infected cells are HEK293 cells;

[0067] And / or, in step (3), positive cells are screened by simultaneously adding Puromycin and Hygromycin B to the cell culture medium; wherein the preferred concentrations are 1 μg / ml Puromycin and 200 μg / ml Hygromycin B.

[0068] Another aspect of the present invention relates to a stable cell line constructed by the method described above, wherein the stable cell line contains an EPOR sequence and a STAT5-RE-Luciferase-2P sequence, wherein the EPOR sequence is shown in SEQ ID NO: 1, the STAT5-RE sequence is shown in SEQ ID NO: 2, and the Luciferase-2P sequence is shown in SEQ ID NO: 3.

[0069] EPO and PEG-EPO biological activity detection method

[0070] Another aspect of the present invention provides a method for detecting the biological activity of EPO and PEG-EPO, comprising the following steps:

[0071] (A) inoculating the stable cell line constructed by the construction method or the stable cell line at 1E4 / well to 10E4 / well (e.g., 1E4 / well, 2E4 / well, 4E4 / well, 6E4 / well, 8E4 / well, 10E4 / well, preferably 4E4 / well to 6E4 / well)

[0072] density inoculation, cultivation;

[0073] (B) adding the EPO or PEG-EPO to be tested to the above-mentioned cultured stable cell line;

[0074] (C) incubating in an incubator for 3-24 hours (e.g., 4 hours, 6 hours, 8 hours and 24 hours, preferably 5±1 h); preferably 4-16 hours; more preferably 4-6 hours;

[0075] (D) Detect the chemiluminescence value and calculate the relative biological activity of the analyte.

[0076] In some embodiments, in step (A), the culture medium used is supplemented with 0.02%-10% (e.g., 0.02%, 0.1%, 0.5%, 1%, 2%, 5%, 10%) FBS by volume, preferably 0.1%-2%.

[0077] In some embodiments, in step (A), the culture medium used is selected from one of DMEM+1% FBS, phenol red-free DMEM+1% FBS, Opti-MEM+1% FBS, DMEM+1% HI FBS, phenol red-free DMEM+1% HI FBS, and Opti-MEM+1% HI FBS.

[0078] In some embodiments, in step (C), the culture conditions are 36-38° C., 4-6% carbon dioxide;

[0079] Among them, 37°C and 5% carbon dioxide are preferred.

[0080] In some embodiments, the plate reading parameters are 1000-500 ms, and the plate reading height is 5-10 mm, wherein 1000 ms and 5 mm are preferred.

[0081] In some embodiments, in step (B), the substance to be detected is EPO, and an initial concentration of 20-50 nM and 2-3-fold gradient dilutions of 10 to 11 concentration points of EPO are added.

[0082] In some embodiments, in step (B), the substance to be detected is PEG-EPO, and the substance to be detected is PEG-EPO, and PEG-EPO is added at an initial concentration of 30-300 nM and 2-3 times gradient dilutions of 10 to 11 concentration points.

[0083] As an example of an EPO test, the test parameters are:

[0084]

[0085] As an example of a PEG-EPO assay, the assay parameters are:

[0086]

[0087] The present invention constructs HEK293 / STAT5-RE-Luc / EPOR genetic engineering cell strain for the first time, and finds the advantages of the cell strain in detecting EPO and / or PEG-EPO human recombinant protein. In addition, based on the cell strain, a method for determining the biological activity of EPO and / or PEG-EPO human recombinant protein is provided. The method is simple, rapid, accurate, highly specific, and has good precision and accuracy, and the method is robust.

[0088] Example

[0089] The invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. In addition, the experimental methods in the following examples are conventional methods commonly used in the industry unless otherwise specified. The raw materials, reagent materials, etc. used in the following examples are commercially available products unless otherwise specified.

[0090] Example 1: Construction and screening of stable cell line HEK293 / STAT5-RE-Luc / EPOR

[0091] 1.1 Experimental Materials

[0092] HEK293 cells were purchased from ATCC;

[0093] Lenti-X 293T cells were purchased from Clontech;

[0094] F-12K medium, DMEM medium, reduced serum medium (Opti-MEM), DPBS, penicillin-streptomycin (PS), MEM non-essential amino acid solution (100×), 0.4% trypan blue, fetal bovine serum (FBS), heat-inactivated fetal bovine serum (HI.FBS), puromycin, hygromycin B, 0.05% trypsin-EDTA, StemPro TM Accutase TM Cell dissociation medium was purchased from Thermo Company;

[0095] The lentivirus titer rapid test card was purchased from Beijing Biolong Immunotechnology Co., Ltd.;

[0096] EPOR / EPO Receptor Antibody was purchased from LSBio;

[0097] PE Donkey anti-Rabbit IgG (minimal x-reactivity) Antibody was purchased from Biolegend; MycoAlert TM PLUS Mycoplasma Detection Kit was purchased from Lonza;

[0098] ONE-GLO Luciferase Assay Kit (ONE-Glo TM Luciferase Assay) was purchased from Promega;

[0099] EPO: Jialinhao (recombinant human erythropoietin injection, EPO) was obtained from Shandong Ahua Biopharmaceutical Co., Ltd.;

[0100] PEG-EPO: EPO stock solution is modified with polyethylene glycol (PEG) and repurified to obtain polyethylene glycol recombinant human erythropoietin stock solution (hereinafter referred to as PEG-EPO stock solution). PEG-EPO can be prepared by conventional methods in the art. For example, EPO or rhEPO glycoprotein is modified by covalently linking 1 to 3 low alkoxy polyethylene glycol (PEG) groups, and the preparation is filled into PEG-EPO injection solutions of different specifications for use.

[0101] For details of EPO buffer, please refer to the Jialinhao product manual

[0102] PEG-EPO buffer (10 mM sodium dihydrogen phosphate, 40 mM sodium sulfate, 0.01% poloxamer 188, pH adjusted to 6.2 by NaOH) was prepared and stored in this laboratory.

[0103] Dupilumab (1L273A) was purchased from Sanofi

[0104] Anti-Claudin18.2 antibody was from Astellas Pharma Inc.

[0105] 1.2 Plasmid preparation and lentiviral particle preparation

[0106] The nucleotide sequence of EPOR (NM_000121.4) (SEQ ID NO: 1) was downloaded from the official website of NCBI, and a third-party company was commissioned to synthesize the human EPOR target gene and STAT5-RE-Luciferase-2P (also referred to as STAT5-RE-Luc) reporter gene sequence (STAT5-RE sequence is shown in SEQ ID NO: 2, and Luciferase-2P sequence is shown in SEQ ID NO: 3), and the EPOR target gene and STAT5-RE-Luciferase-2P (also referred to as STAT5-RE-Luc) reporter gene were subcloned into different lentiviral vectors (GenScript ProBio). Subsequently, the two lentiviral vectors were used to transform Escherichia coli (GenScript ProBio), single clones were selected and plasmids were extracted, and EPOR and STAT5-RE-Luciferase-2P lentiviral plasmids were obtained, respectively. The plasmid information is as follows: Figure 1 and Figure 2 shown.

[0107] About 1 to 4 million Lenti-X 293T cells were seeded into a 10 cm cell culture dish, and the complete culture medium was DMEM + 10% fetal bovine serum. After overnight incubation, Lenti-X 293T cells were co-transfected with the above-constructed EPOR lentiviral plasmid (10 μg) and packaging plasmid (psPAX2) and envelope plasmid (pMD2G) (12.5 μg) (GenScript ProBio) and Lipofectamine 2000 (Thermo Corporation), and Lenti-X 293T cells were co-transfected with STAT5-RE-Luciferase-2P lentiviral plasmid (10 μg) and packaging plasmid (psPAX2) and envelope plasmid (pMD2G) (12.5 μg) (GenScript ProBio) and Lipofectamine 2000 (Thermo Corporation) to obtain EPOR-transfected Lenti-X 293T cells and STAT5-RE-Luciferase-2P lentiviral plasmid-transfected Lenti-X 293T cells, respectively. After 6 hours of transfection, the culture medium was removed and replaced with fresh complete culture medium (DMEM plus 10% fetal bovine serum). After 48 hours, the supernatant of Lenti-X 293T cells was collected into a sterile 50ml centrifuge tube, and another 10ml of fresh DMEM complete culture medium was added to the above cell culture dish, and then returned to the carbon dioxide incubator for continued culture. After another 24 hours, all the cell supernatants in the Lenti-X 293T cells transfected with EPOR and the Lenti-X 293T cells transfected with STAT5-RE lentiviral plasmid were collected into sterile 50ml centrifuge tubes, and the supernatants were filtered through a 0.45μm filter membrane together with the cell supernatants collected above for 48 hours, and ultracentrifuged, and the supernatant was removed to obtain EPOR recombinant lentiviral particles and STAT5-RE-Luciferase-2P recombinant lentiviral particles, respectively. Then, resuspend the virus particles with an appropriate amount of DPBS, divide them into 200 μl per tube, test the titer with a lentivirus titer rapid test card, and store them in a -80°C refrigerator for later use.

[0108] 1.3 Construction of stable cell line (HEK293 / STAT5-RE-Luc / EPOR)

[0109] About 1 million HEK293 cells were seeded into 6-well cell culture plates, and the complete culture medium was DMEM plus 10% fetal bovine serum. After culturing overnight, HEK293 cells were simultaneously infected with 200 μl EPOR recombinant lentiviral particles and 200 μl STAT5-RE-Luciferase-2P recombinant lentiviral particles. After 24 hours of infection, the complete medium was removed and replaced with fresh complete medium. After continuing to infect for 24 hours, 1 μg / ml Puromycin and 200 μg / ml Hygromycin B were added simultaneously for pressure screening. The concentrations of Puromycin and Hygromycin B were the concentrations in the cell culture system after addition. A pool of positive cells (HEK293 cells simultaneously infected with the above two recombinant lentiviral particles) was obtained. Subsequently, the positive cells were inoculated into a 96-well cell culture plate at a density of one cell per well for monoclonal screening. About 20 monoclonal cells were collected, and the expression amount of the target protein of the monoclonal cells was detected by flow cytometry screening. The positive monoclonal cells obtained by screening were transferred to a 6-well plate, and cultured and expanded to a 10 cm culture dish for expansion culture to obtain a stable cell line HEK293 / STAT5-RE-Luc / EPOR. The expression of the target protein was confirmed by flow cytometry. At the same time, the cells constructed above were inoculated into a white 96-well plate at a density of about 40,000 / well. After overnight, EPO human recombinant protein was added. After incubation for a certain period of time, Bio-Glo TM LuciferaseAssay kit was used to verify the functional activity, see Figure 3 and Figure 4 The results of mycoplasma detection of cells after freezing are shown in Figure 5 shown.

[0110] Example 2: Using the above stable cell lines to detect the biological activity of EPO and PEG-EPO

[0111] 2.1 Cell seeding density

[0112] In the activity method detection experiment, the cell seeding density is one of the key factors. Different cell seeding densities will have a certain degree of influence on the cell growth state in the detection culture plate, and then produce differences in the signals generated after stimulation with EPO and PEG-EPO human recombinant proteins. The present invention optimizes the cell seeding density, which can ensure that the cell density in the subsequent experimental detection culture plate maintains a relatively appropriate confluence rate, thereby obtaining a relatively consistent detection window and relative biological activity.

[0113] Here, E represents a power of 10, for example, 1E4 represents 1×10 4 (Same below).

[0114] Gradient dilution, taking 30nM, 1:2.5 times gradient dilution as an example, that is, 2.5 times gradient dilution, the next concentration point can be calculated as 30nM / 2.5=12nM. (The same below)

[0115] The following concentrations of EPO and PEG-EPO are the concentrations after adding to the cell culture system (ie, adding to the cell culture plate or cell culture well).

[0116] Methods: HEK293 / STAT5-RE-Luc / EPOR cells were seeded into white opaque 96-well plates at 1E4 / well, 2E4 / well, 4E4 / well, 6E4 / well, 8E4 / well, and 10E4 / well seeding densities. Two replicates were set for each seeding density and cultured overnight in complete medium (DMEM + 10% fetal bovine serum). Then, 30nM, 1:2.5-fold gradient dilution of 10 concentration points of EPO (concentration range of 30nM to 0.008nM) and 100nM, 1:2.5-fold gradient dilution of 10 concentration points of PEG-EPO (concentration range of 100nM to 0.026nM) were transferred to the above two replicate cell culture plates, and parallel duplicate wells were set for each concentration point. After culturing for 5 hours at 37℃, 5% carbon dioxide incubator, ONE-GLO luciferase detection kit was added to detect chemiluminescence values.

[0117] Results: The detection window varies with the cell seeding density. The detection window shows a trend from low to high and then decreases. When the cell seeding density is between 4 and 6E4 / well, the detection window is larger and more stable. When the cell culture is overnight and the sample is added, the cell confluence rate is 80%. Therefore, the cell seeding density of 4E4 / well and 6E4 / well was selected for subsequent experiments. On the basis of meeting the detection requirements, the time cost of cell culture can be reduced. The results are shown in Table 1 and Table 2.

[0118] Table 1 Results of activity analysis of EPO at different cell seeding densities

[0119]

[0120]

[0121] Table 2 Activity analysis results of PEG-EPO at different cell seeding densities

[0122]

[0123] 2.2 Detection time

[0124] After determining the cell seeding density and adding EPO and PEG-EPO samples, the curve R of the test results changes with the incubation time of the samples. 2 The results of four parameters such as EC50 and the slope of the curve will also be different accordingly.

[0125] Methods: HEK293 / STAT5-RE-Luc / EPOR cells were seeded into white opaque 96-well plates at a density of 4E4 / well, and two groups were set up for overnight culture. Then, 30nM, 1:2.5 gradient dilution of 10 concentration points of EPO (concentration range of 30nM-0.008nM) and 30nM, 1:2.5 gradient dilution of 10 concentration points of PEG-EPO (concentration range of 30nM-0.008nM) were transferred to the above two groups of cell culture plates separately, and parallel duplicate wells were set for each concentration point, and incubated at 37℃, 5% carbon dioxide incubator for 4 hours, 6 hours, 8 hours and 24 hours, respectively, and then the chemiluminescence value was detected using the ONE-GLO luciferase detection kit.

[0126] Results: Under the condition of cell seeding density of 4E4 cells / well, except for the 24-hour test results, the EC50 of EPO samples was relatively consistent with the extension of the test time; the EC50 of PEG-EPO samples changed relatively little with the test time; the EC50 of EPO and PEG-EPO tended to be consistent with the increase of test time, and the detection window gradually decreased with the increase of test time. Therefore, the detection time of 5±1h was selected for subsequent activity test experiments. The detailed results are shown in Tables 3 and 4.

[0127] Table 3 Activity analysis results of EPO at different incubation times

[0128] Incubation time 4 hours 6 hours 8 hours 24 hours <![CDATA[R 2 ]]> 0.999 0.997 0.999 0.981 <![CDATA[EC 50 (nM)]]> 0.427 0.445 0.467 2.57 CV maximum value (%) 2.1 2.8 6.5 2.8 Detection Window 4.88 4.45 4.18 1.77

[0129] Table 4 Activity analysis results of PEG-EPO at different incubation times

[0130] Incubation time 4 hours 6 hours 8 hours 24 hours <![CDATA[R 2 ]]> 1.000 1.000 0.999 0.993 <![CDATA[EC 50 (nM)]]> 2.165 2.061 1.657 3.077 CV maximum value (%) 2.8 7.2 4.2 6.3 Detection Window 5.38 5.05 3.71 1.96

[0131] 2.3 Detection of FBS ratio in culture medium

[0132] Different concentrations of FBS will affect the growth state of cells, which will ultimately cause corresponding fluctuations in the detection result window, parallelism, CV and relative biological activity results.

[0133] Methods: 7 different volume ratios of FBS were added to DMEM medium as the detection medium, and HEK293 / STAT5-RE-Luc / EPOR cells were seeded into white opaque 96-well plates at a seeding density of 4E4 / well and cultured overnight. Then, 30nM, 1:2.5 gradient dilution of 10 concentration points of EPO (concentration range of 30nM to 0.008nM) and 300nM, 1:3 gradient dilution of 11 concentration points of PEG-EPO (concentration range of 300nM to 0.005nM) were transferred to the above cell culture plates, incubated at 37℃, 5% carbon dioxide incubator for 5 hours, and then the chemiluminescence value was detected using the ONE-GLO luciferase detection kit.

[0134] Results: The activity results of EPO / PEG-EPO were detected by comparing 7 different FBS ratios added to DMEM as the detection medium. The detection window was higher when 0.1% FBS, 0.5% FBS, 1% FBS, and 2% FBS were added. Considering the cell adhesion state after plating, under the detection conditions of 0.1% FBS and 0.5% FBS, the cell extension state was inferior to that under the detection conditions of 1% FBS and 2% FBS. In addition, under the detection conditions of 1% FBS and 2% FBS, there was no significant difference in the cell extension state and confluence rate. Taking all factors into consideration, 1% FBS concentration was selected as the serum concentration in the detection medium, see Table 5-1, 5-2, 5-3 Table 6-1, 6-2, 6-3 and Figure 6 .

[0135] Table 5-1 Activity analysis results of EPO at 1%, 2%, 5%, and 10% FBS concentrations

[0136] FBS concentration (%) 1 2 5 10 <![CDATA[R 2 ]]> 0.994 0.999 0.998 0.998 <![CDATA[EC 50 (nM)]]> 0.269 0.355 0.407 0.548 CV maximum value (%) 1.7 6.5 4.6 2.2 Detection Window 3.24 4.88 4.49 4.57

[0137] Table 5-2 Activity analysis results of EPO at 0.1%, 0.5%, 1%, and 2% FBS concentrations

[0138] FBS concentration (%) 0.1 0.5 1 2 <![CDATA[R 2 ]]> 0.998 0.998 0.998 0.999 <![CDATA[EC 50 (nM)]]> 0.246 0.282 0.267 0.273 CV maximum value (%) 6.7 5.8 3.0 3.5 Detection Window 5.35 5.35 4.78 4.34

[0139] Table 5-3 Activity analysis results of EPO at 0.02%, 0.1%, and 0.5% FBS concentrations

[0140] FBS concentration (%) 0.02 0.1 0.5 <![CDATA[R 2 ]]> 1.000 0.998 0.997 <![CDATA[EC 50 (nM)]]> 0.389 0.289 0.297 CV maximum value (%) 4.4 3.2 5.1 Detection Window 5.06 5.27 5.03

[0141] Table 6-1 Activity analysis results of PEG-EPO at 1%, 2%, 5%, and 10% FBS concentrations

[0142] FBS concentration (%) 1 2 5 10 <![CDATA[R 2 ]]> 0.998 1.000 1.000 1.000 <![CDATA[EC 50 (nM)]]> 1.247 1.413 1.519 1.941 CV maximum value (%) 4.0 8.1 3.2 2.6 Detection Window 3.58 5.32 4.92 4.86

[0143] Table 6-2 Activity analysis results of PEG-EPO at 0.1%, 0.5%, 1%, and 2% FBS concentrations

[0144] FBS concentration (%) 0.1 0.5 1 2 <![CDATA[R 2 ]]> 1.000 0.999 1.000 0.999 <![CDATA[EC 50 (nM)]]> 1.135 0.859 0.735 0.846 CV maximum value (%) 6.5 2.4 5.8 4.3 Detection Window 5.15 5.15 4.81 4.40

[0145] Table 6-3 Activity analysis results of PEG-EPO at 0.02%, 0.1%, and 0.5% FBS concentrations

[0146] FBS concentration (%) 0.02 0.1 0.5 <![CDATA[R 2 ]]> 0.999 1.000 1.000 <![CDATA[EC 50 (nM)]]> 1.283 1.055 1.291 CV maximum value (%) 3.3 7.0 2.9 Detection Window 5.13 5.05 5.22

[0147] 2.4 Testing culture medium composition, FBS serum composition and cell seeding density

[0148] 2.4.1 Method: Prepare the following detection medium: DMEM + 1% FBS, DMEM + 1% FBS without phenol red, Opti-MEM + 1% FBS, DMEM + 1% HI FBS, DMEM + 1% HI FBS without phenol red, Opti-MEM + 1% HI FBS. Use the above detection medium to inoculate HEK293 / STAT5-RE-Luc / EPOR cells into a white opaque 96-well plate at a density of 4E4 / well, and culture them overnight with the above detection medium. Then, 30nM, 1:2.5 gradient dilution of 11 concentration points of EPO (30nM ~ 0.003nM), and 300nM, 1:3 gradient dilution of 11 concentration points of PEG-EPO (300nM ~ 0.005nM) were transferred to the above cell culture plates, incubated at 37°C, 5% carbon dioxide incubator for 5 hours, and then the chemiluminescence value was detected using the ONE-GLO luciferase detection kit.

[0149] Results: As shown in Tables 7 and 8, the detection window was low when DMEM was used as the detection medium. There was no significant difference between 1% FBS and 1% HI FBS when the detection medium was phenol red-free DMEM and Opti-MEM. Therefore, 1% FBS was used as the serum component of the detection medium in subsequent experiments.

[0150] Table 7 EPO activity test results with different test medium components and different serum types

[0151]

[0152] Table 8 Activity test results of different PEG-EPO test medium components and different serum types

[0153]

[0154] 2.4.2 Methods: Phenol red-free DMEM and Opti-MEM were used as the detection medium, and the cell seeding density was 4E4 / well and 6E4 / well for detection.

[0155] Results: As shown in Table 9, Table 10 and Figure 7 As shown in the figure, there is no significant difference in the cell status after plating with the two test media at different cell densities. When Opti-MEM is used as the test medium, the detection window of EPO and PEG-EPO samples is higher, and there is no significant difference in cell extensibility and detection window when the cell density is 4E4 cells / well and 6E4 cells / well.

[0156] Table 9 Activity test results of EPO at different cell densities and different culture medium components

[0157]

[0158] Table 10 Activity test results of PEG-EPO at different cell densities and different culture medium components

[0159]

[0160] In summary, the detection culture medium was Opti-MEM + 1% FBS, and the cell seeding density was 4E4 cells / well.

[0161] 2.5 Optimization of plate reading parameters

[0162] The plate reading parameters of EPO and PEG-EPO samples were different during the detection. 2 The results of four parameters such as EC50 and the slope of the curve will also be different accordingly. In this report, the plate reading parameters of the samples are determined.

[0163] Methods: HEK293 / STAT5-RE-Luc / EPOR cells were seeded into a white opaque 96-well plate at a density of 4E4 / well and cultured overnight. Then, 30nM, 1:2.5 gradient dilution of 11 concentration points of EPO (30nM~0.003nM), and 300nM, 1:3 gradient dilution of 11 concentration points of PEG-EPO (300nM~0.005nM) were transferred to the above cell culture plates, respectively, and incubated at 37℃, 5% carbon dioxide incubator. After 5 hours, the ONE-GLO luciferase detection kit was used to detect the chemiluminescence value in combination with the acquisition time and detection height of each well of the microplate reader.

[0164] Results: EPO and PEG-EPO samples had different detection parameters. 2The EC50 values ​​were relatively stable. Under the plate reading parameters of 1000ms and 5mm, the detection window was relatively large, and the CV value between duplicate wells was relatively small. Compared with other plate reading parameters, under the plate reading parameters of 1000ms and 5mm, the drop of the upper platform was not obvious. Combined with the configuration of different microplate readers in different laboratories in the later stage, the plate reading parameters selected for EPO and PEG-EPO samples were 1000ms and 5mm. The detailed results are shown in Tables 11 and 12.

[0165] Table 11 Activity test results of EPO samples with different plate reading parameters

[0166]

[0167] Table 12 Activity test results of different plate reading parameters for PEG-EPO samples

[0168]

[0169] 2.6 Luciferase Assay Reagent

[0170] Different brands of luciferase detection reagents were used and the results were analyzed.

[0171] Methods: HEK293 / STAT5-RE-Luc / EPOR cells were seeded into a white opaque 96-well plate at a seeding density of 4E4 / well and cultured overnight. Then, 30nM, 1:2.5 gradient dilution of 11 concentration points of EPO (30nM~0.003nM), and 300nM, 1:3 gradient dilution of 11 concentration points of PEG-EPO (300nM~0.005nM) were transferred to the above cell culture plate and incubated at 37℃, 5% carbon dioxide incubator. After 5 hours, Vazyme's Bio-Lite Luciferase AssaySystem (Cat. No.: DD1201-02) and Progema's ONE-GLO fluorescence detection reagent (Cat. No.: E6120) were used to measure the chemiluminescence value.

[0172] Results: Two detection reagents were used to detect EPO and PEG-EPO samples. 2 , detection window, CV and EC50 results between replicate wells showed no significant difference. Considering the delivery time and batch stability of the kit, the ONE-GLO test kit was selected for this method. The detailed results are shown in Table 13.

[0173] Table 13 Luciferase activity test results of different brands

[0174]

[0175] 2.7 Sample concentration range and dilution method

[0176] Due to the different initial concentrations and dilution methods of EPO / PEG-EPO samples, the detected curve R 2 The results of four parameters such as EC50 and the slope of the curve will also be different accordingly. Therefore, the concentration range and dilution method of the samples are determined in this report.

[0177] 2.7.1 Methods: HEK293 / STAT5-RE-Luc / EPOR cells were seeded into a white opaque 96-well plate at a seeding density of 4E4 / well. After overnight culture, EPO and PEG-EPO samples were set starting from 30 nM, and 10 concentration points were made by 2-fold (30 nM-0.059 nM), 2.5-fold (30 nM-0.008 nM), and 3-fold gradient dilution (30 nM-0.002 nM), respectively. The samples were transferred to the above-mentioned cell culture plate and incubated in a 5% carbon dioxide incubator at 37°C for 5 hours. Then, the ONE-GLO luciferase detection kit was added to detect the chemiluminescence value.

[0178] Results: When the starting concentration of EPO sample was 30nM, the distribution of the upper and lower platform points of 2-fold dilution and 3-fold dilution was unreasonable. When it was 2.5-fold dilution, the upper and lower platform points were relatively uniform. The dilution method of EPO sample was set as the starting concentration of 30nM and 2.5-fold dilution. When the starting concentration of PEG-EPO sample was 30nM, the distribution of points on the four-parameter curve measured by the three dilution methods was unreasonable, and there were fewer distribution points on the upper platform. The upper platform needs to be further optimized, see Table 14 and Figure 8 .

[0179] Table 14 Activity test results of EPO / PEG-EPO at different dilution multiples

[0180]

[0181] 2.7.2 Method: Based on the above analysis, the starting concentration of PEG-EPO needs to be increased. The PEG-EPO sample is set to start from 75nM, 2.5-fold dilution (75nM~0.02nM), 100nM starting 3-fold dilution (100nM~0.005nM), 40nM starting 2-fold dilution (40nM~0.078nM), 300nM starting 3-fold dilution (300nM~0.015nM) for a total of 10 concentration points for further optimization.

[0182] Results: When the starting concentrations of PEG-EPO samples were 75nM, 100nM, and 40nM, the distribution of platform points on the curve was unreasonable. When the starting concentration was 300nM, there was a point drop problem on the upper platform. The starting concentration and dilution method of the upper platform need to be further optimized. See Table 15 and Fig. 9 .

[0183] Table 15 Activity test results of different dilution methods of PEG-EPO samples

[0184] Key Parameters 75nM(1:2.5) 100nM(1:3) 40nM(1:2) 300nM(1:3) <![CDATA[R 2 ]]> 0.999 0.999 0.999 0.998 <![CDATA[EC 50 (nM)]]> 1.195 1.187 1.343 1.631 CV maximum value (%) 3.9 4.9 11.1 3.3 window 6.03 5.96 6.00 6.28

[0185] 2.7.3 Method: Set 300nM as the starting point for 3-fold dilution (300nM~0.005nM), 150nM as the starting point for 2.5-fold dilution (150nM~0.02nM), and 100nM as the starting point for 2-fold dilution (100nM~0.1nM), for a total of 11 concentration points to continue optimizing the platform for PEG-EPO samples.

[0186] Results: Since the upper platform still has drop points when the starting dilution is 300nM and 3 times, the points on the curve are mostly concentrated on the slope when the starting dilution is 100nM and 2 times, and the platform under the curve is not complete when the starting dilution is 150nM and 2.5 times, further optimization is needed, see Table 16 and Fig.10 .

[0187] Table 16 Activity test results of different dilution methods of PEG-EPO samples

[0188] Key Parameters 300nM(1:3) 150nM(1:2.5) 100nM(1:2) <![CDATA[R 2 ]]> 0.999 1.000 0.999 <![CDATA[EC 50 (nM)]]> 1.367 1.400 1.468 CV maximum value (%) 2.3 4.6 9.6 window 6.18 6.44 6.67

[0189] 2.7.4 Method: Set 300nM as the starting point for 3-fold dilution (300nM~0.005nM), 175nM as the starting point for 2.7-fold dilution (175nM~0.008nM), 160nM as the starting point for 2.8-fold dilution (160nM~0.005nM), 200nM as the starting point for 2.7-fold dilution (200nM~0.01nM), and 150nM as the starting point for 2.5-fold dilution (150nM~0.016nM), for a total of 11 concentration points for optimization.

[0190] Results: When the starting dilution was 200 nM and 2.7 times, there were still dropouts. When the starting dilution was 150 nM and 2.5 times, the points on the curve were more evenly distributed under the condition of 175 nM and 2.7 times dilution, and the requirements of even distribution of the upper and lower platforms were met. See Table 17 for details. Fig.11 :

[0191] Table 17 Activity test results of different dilution methods of PEG-EPO samples

[0192]

[0193] In summary, the dilution conditions of the EPO sample are: the starting concentration is 30 nM, the dilution factor is 2.5 times, and the dilution conditions of the PEG-EPO sample are: the starting concentration is 175 nM, the dilution factor is 2.7 times.

[0194] Example 3: Detection method validation

[0195] 3.1 Confirmation of Exclusivity

[0196] Specificity refers to the ability of the analytical method to correctly determine the analyte in the presence of other components (such as impurities, excipients, etc.), also known as specificity. In order to evaluate the specificity of the established method, this implementation verified the specificity of HEK293 / STAT5-RE-Luc / EPOR cells based on the complete STAT5 signaling pathway, the specificity of HEK293 / STAT5-RE-Luc / EPOR cells to EPO and PEG-EPO high temperature forced degradation samples, and other components or other target antibodies.

[0197] Under the experimental conditions in Tables 18 and 19, the biological activity of the samples was determined using the method established in this study.

[0198] Table 18 Parameters of the method for detecting the biological activity of EPO human recombinant protein

[0199] EPO experimental parameters Parameter Value Cell plating density 6.67E5cells / mL Cell incubation time 16±5h EPO starting concentration 30nM EPO dilution multiple 2.5 Number of dilution concentration points (total) 11 EPO incubation time 5±1h

[0200] Table 19 Parameter values ​​for the method for detecting the biological activity of PEG-EPO human recombinant protein

[0201] PEG-EPO Experimental Parameters Parameter Value Cell plating density 6.67E5cells / mL Cell incubation time 16±5h PEG-EPO initial concentration 175nM PEG-EPO dilution factor 2.7 Number of dilution concentration points (total) 11 PEG-EPO incubation time 5±1h

[0202] Methods: HEK293 / STAT5-RE-Luc / EPOR cells and HEK293 cells were seeded into white opaque 96-well plates at a seeding density of 4E4 / well and cultured overnight. PEG-EPO was then added to the wells seeded with HEK293 / STAT5-RE-Luc / EPOR cells and HEK293 cells, respectively. The detection parameters and PEG-EPO concentration point settings are shown in Table 19.

[0203] HEK293 / STAT5-RE-Luc / EPOR cells were inoculated into white opaque 96-well plates at a density of 4E4 / well and cultured overnight. EPO, EPO buffer, Dupilumab (1L273A batch) and EPO forced degradation samples denatured at 64°C for 16 hours were then added separately to different wells inoculated with HEK293 / STAT5-RE-Luc / EPOR cells. PEG-EPO, PEG-EPO buffer, anti-Claudin18.2 antibody and PEG-EPO forced degradation samples denatured at 50°C for 8 weeks were then added separately to different wells, and the above samples were transferred to cell culture plates, incubated at 37°C, 5% carbon dioxide, and after 5 hours, the activity results were detected using ONE-GLO fluorescence detection reagent to detect the chemiluminescence value.

[0204] Results: As shown in Table 20, Table 21 and Figure 12, the detection signal value of HEK293 / STAT5-RE-Luc / EPOR cells increased with the increase of EPO and PEG-EPO concentrations, showing a good dose-effect curve. However, HEK293 cells without STAT5-RE-Luc / EPOR signaling pathway failed to show a dose-effect curve, indicating that the HEK293 / STAT5-RE-Luc / EPOR cells constructed in Example 1 have good signaling pathway specificity ( Figure 12-1 ). The active EPO and PEG-EPO were subjected to high temperature forced degradation denaturation treatment, and showed reduced biological activity compared with the undenatured EPO and PEG-EPO samples, and were positively correlated with temperature and reaction time, reflecting the good sensitivity of the detection method. In addition, other different target antibodies such as Dupilumab, Claudin18.2, and EPO and PEG-EPO buffer solutions could not produce corresponding dose-effect curves, indicating that this method has excellent specificity and can specifically detect EPO and PEG-EPO ( Figure 12-2 and Figure 12-3 ).

[0205] Table 20 PEG-EPO sample biological activity detection method specificity study experimental results

[0206]

[0207]

[0208] Table 21 Experimental results of specificity study of biological activity detection method of EPO samples

[0209]

[0210] 3.2 Relative Accuracy

[0211] Accuracy is used to evaluate the degree to which the actual measured value of the analytical method test result is close to or consistent with the theoretical value. The closer the measured value is to the theoretical value, the higher the accuracy of the method is.

[0212] Method: 30nM EPO and 175nM PEG-EPO samples were taken as reference samples for method accuracy confirmation and tested under the experimental conditions of Table 18 and Table 19. Then, 5 samples with different starting concentrations and titer levels were prepared: EPO was 15nM, 22.5nM, 30nM, 37.5nM, and 45nM respectively; PEG-EPO was 87.5nM, 131.25nM, 175nM, 218.75nM, and 262.5nM respectively. The theoretical relative biological activity values ​​corresponding to different molar concentrations were 50%, 75%, 100%, 125%, and 150%. Parallel double wells were set for each sample, and the results were averaged. The experiment was repeated twice, counted as Time1 and Time2 respectively. Under the experimental conditions of Table 18 and Table 19 (the above starting concentrations were selected for EPO and PEG-EPO concentration points, and the dilution concentration points were set according to the dilution multiple and the number of dilution concentration points in the table), the method established in this study was used to perform corresponding tests on the samples at each of the above titer levels, and the relative biological activity and recovery rate of each of the five samples were calculated based on the measured results. The calculation formula is as follows:

[0213] Relative biological activity (%) = EC of reference product 50 / Sample EC 50 ×100%

[0214] Recovery rate (%) = relative biological activity measured value / relative biological activity theoretical value × 100%

[0215] Results: The average values ​​of the relative biological activities of EPO at five different titer levels were 48.40%, 75.73%, 99.06%, 122.08% and 155.99%, respectively, with RSD values ​​less than 10%, and the recovery range was between 96.81% and 103.99%. The average values ​​of the relative biological activities of PEG-EPO at five different titer levels were 45.55%, 68.32%, 97.17%, 126.30% and 157.42%, respectively, with RSD values ​​less than 10%, and the recovery range was between 91.09% and 104.94% (see Table 22, Table 23 and Table 24). Fig.13 ). In addition, the linear fitting parameters R of EPO and PEG-EPOR 2 are 0.9890 and 0.9912 respectively, and the linear fit is good ( Fig.14 ), the measured average value of relative biological activity is in good agreement with the corresponding theoretical value.

[0216] Table 22: Relative biological activity and recovery rate of EPO of different titers

[0217]

[0218] Table 23: Relative biological activity and recovery rate of PEG-EPO with different titers

[0219]

[0220]

[0221] 3.3 Precision evaluation

[0222] Precision refers to the consistency or closeness between actual measured values, usually expressed as relative standard deviation RSD (%), including intra-day precision, inter-day precision and reproducibility. Intra-day precision refers to the coefficient of variation between multiple test results on the same day under the same conditions in the same experiment, and inter-day precision refers to the coefficient of variation between the test results of the same experiment on different days and by different operators. Reproducibility is one of the important components of precision determination, which needs to be completed through experiments between different laboratories and is generally used in the case of analytical method transfer or standardization of analytical methods. Therefore, the intra-day and inter-day repeatability and precision were evaluated based on this detection method.

[0223] Method: Take EPO and PEG-EPO with an initial concentration of 30nM and 175nM as the test samples, respectively. Under the experimental conditions of Table 18 and Table 19, repeatability tests were performed on them within 3 days. Parallel double wells were set for each sample, and the average was taken. Each sample was tested three times a day. According to the test results, the average value and relative standard deviation (RSD) were calculated.

[0224] Results: The mean daily biological activities of three different biological activities of EPO and PEG-EPO were 94.48% and 99.86%, and the RSDs were 6.91% and 5.07%, both less than 10%, indicating that the method of the present invention has good precision (see Tables 24 and 25).

[0225] Table 24: Intra-day and inter-day precision of EPO

[0226]

[0227] Table 25: Intra-day and inter-day precision of PEG-EPO

[0228]

[0229] 3.4 Durability evaluation

[0230] Below, the durability of the detection method was evaluated for different cell generations, different detection times, and different cell seeding densities.

[0231] Method: The detection time (EPO incubation time) was 4 hours, 5 hours, and 6 hours, three different generations of HEK293 / STAT5-RE-Luc / EPOR cells P14 / P23 / P39 and three seeding densities (4E4 / well, 5E4 / well, 6E4 / well), and other conditions were in accordance with the experimental conditions of Table 18 and Table 19 to evaluate the durability of the method involved in the present invention. Cell generations refer to the number of times a cell develops from one state to the next, and are usually used to describe the growth and division process of cells. P14, P23, and P39 refer to the 14th, 23rd, and 39th generations, respectively.

[0232] Results: As shown in Table 26, Table 27 and Table 28, under three different detection time conditions, the relative biological activity RSDs of EPO and PEG-EPO were 3.57% and 5.51%, respectively, less than 10%; under three seeding density conditions, the relative biological activity RSDs of EPO and PEG-EPO were 11.79% and 9.79%, respectively, less than 20%, and under three different cell generations, the relative biological activity RSDs of EPO and PEG-EPO were 14.35% and 12.93%, respectively, less than 20%. The relative biological detection values ​​under all the above conditions were within the range of 70-130%, which means that there was no significant effect on the test results, indicating that the durability of the detection method of the present invention is good.

[0233] Table 26: Activity results at different detection times

[0234]

[0235] Table 27: Activity results of different cell seeding densities

[0236]

[0237] Table 28: Activity results of different cell generations

[0238]

[0239] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments or to replace some of the technical features therein by equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention. In addition, although the elements of the present invention may be described or required in individual form, it is also conceivable to have multiple elements unless explicitly limited to a single element.

Claims

1. A method for constructing a stable cell line, characterized in that: The method comprises the following steps: (1) constructing recombinant lentiviral particles containing an EPOR sequence and a STAT5-RE-Luciferase-2P sequence, respectively, wherein the EPOR sequence is shown in SEQ ID NO: 1, the STAT5-RE sequence is shown in SEQ ID NO: 2, and the Luciferase-2P sequence is shown in SEQ ID NO: 3; (2) using the above-mentioned recombinant lentiviral particles to simultaneously infect cells; (3) Screening positive cells and expanding the culture to obtain a stable cell line, wherein the positive cells are cells containing the EPOR sequence and the STAT5-RE-Luciferase-2P sequence.

2. The construction method according to claim 1, characterized in that: In the step (1), constructing a recombinant lentiviral particle containing EPOR and STAT5-RE-Luciferase-2P sequences comprises the following steps: The EPOR sequence and the STAT5-RE-Luciferase-2P sequence are synthesized and subcloned into lentiviral vectors, respectively. The lentiviral vectors containing the EPOR sequence and the STAT5-RE-Luciferase-2P sequence are then used to transform Escherichia coli, respectively, and single clones are selected and plasmids are extracted; the extracted EPOR lentiviral plasmid and STAT5-RE-Luciferase-2P lentiviral plasmid are co-transfected into different cells with auxiliary plasmids, respectively; the transfected cells containing the EPOR lentiviral plasmid and the STAT5-RE-Luciferase-2P lentiviral plasmid are cultured, and the cell culture supernatant is taken to prepare recombinant EPOR lentiviral particles and STAT5-RE-Luciferase-2P lentiviral particles; The co-transfected cells are preferably 293T cells; And / or, the helper plasmid is a packaging plasmid and an envelope plasmid.

3. The construction method according to claim 1 or 2, characterized in that: In the step (2), the infected cells are HEK293 cells; And / or, in step (3), positive cells are screened by simultaneously adding Puromycin and Hygromycin B to the cell culture medium; wherein the preferred concentrations are 1 μg / ml Puromycin and 200 μg / ml Hygromycin B.

4. A stable cell line, characterized in that It is constructed by the method described in any one of claims 1 to 3, and the stable cell line contains an EPOR sequence and a STAT5-RE-Luciferase-2P sequence, wherein the EPOR sequence is shown in SEQ ID NO: 1, the STAT5-RE sequence is shown in SEQ ID NO: 2, and the Luciferase-2P sequence is shown in SEQ ID NO:

3.

5. A method for detecting the biological activity of recombinant human erythropoietin, characterized in that: The following steps are involved: (A) inoculating the stable cell line according to claim 4 and culturing; (B) adding the EPO or PEG-EPO to be tested to the above-mentioned cultured stable cell line; (C) continuing to incubate for 3-24 hours, preferably 4-16 hours, and more preferably 4-6 hours; (D) Detect the chemiluminescence value and calculate the relative biological activity of the analyte.

6. The detection method according to claim 5, characterized in that: In the step (A), the detection culture medium used contains 0.02%-10% FBS by volume, preferably 0.1%-2% FBS by volume; And / or, in step (A), the inoculation density is a cell density of 1E4 / well-10E4 / well; preferably 4E4 / well-6E4 / well; And / or, in the step (A), the culture medium used is selected from one of DMEM+1% FBS, phenol red-free DMEM+1% FBS, Opti-MEM+1% FBS, DMEM+1% HI FBS, phenol red-free DMEM+1% HI FBS, and Opti-MEM+1% HI FBS; And / or, in step (C), the culture conditions are 36-38° C. and 4-6% carbon dioxide; Among them, 37°C and 5% carbon dioxide are preferred; And / or, in step (C), the plate reading parameters are 1000-500 ms, and the plate reading height is 5-10 mm; 1000 ms and 5 mm are preferred.

7. The detection method according to claim 5, characterized in that: In the step (B), the substance to be detected is EPO, and EPO is added at an initial concentration of 20-50 nM and 2-3 times gradient dilutions of 10 to 11 concentration points.

8. The detection method according to claim 5, characterized in that: In the step (B), the substance to be detected is PEG-EPO, and PEG-EPO is added at an initial concentration of 30-300 nM and 2-3 times gradient dilutions of 10 to 11 concentration points.

9. The detection method according to claim 7, characterized in that: The test parameters for detecting EPO are:

10. The detection method according to claim 8, characterized in that: The test parameters for detecting PEG-EPO are:

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