A method for rapidly quantitatively determining the biological activity of anti-PCSK9 monoclonal antibody drugs

Abstract

The application discloses a method for rapidly and quantitatively determining biological activity of anti-PCSK9 monoclonal antibody drugs, and is based on HepG2 liver cancer cells, and utilizes the principle that specific binding of a series of concentration gradient PCSK9 monoclonal antibodies to a certain concentration of PCSK9 protein indirectly causes a dose response of DiI-LDL absorption to LDL receptors on the surface of liver cells, so that a biological activity detection method of the PCSK9 monoclonal antibody drugs is preliminarily established, and methodological verification proves that the method has strong specificity, good accuracy and high precision, and can be used for release detection of products, and has important significance for process control in a production process and drug quality control.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological drug activity detection, and particularly relates to a method for rapidly and quantitatively determining the biological activity of an anti-PCSK9 monoclonal antibody drug. BACKGROUND

[0002] In clinic, the level of low-density lipoprotein (LDL-C) is often used as an evaluation of atherogenic lipoprotein, and these lipoproteins are also an independent risk factor for cardiovascular disease. At present, statins widely used in clinic for lipid-lowering cannot reduce the serum LDL-C of all people to the ideal level. With the progress of technology and research innovation, PCSK9 monoclonal antibody is the most effective star lipid-lowering target after statins. Up to now, three PCSK9 targeted drugs have been approved globally, and more and more enterprises are optimistic about the PCSK9 targeted drug market and have developed a number of drugs in research, among which lerodalcibep, Bococizumab and other drugs are in phase III clinical trials. It is expected that the PCSK9 targeted drug market will experience explosive growth in the coming years.

[0003] PCSK9 (proprotein convertase subtilisin 9) is a new target in the field of lipid-lowering drug development in recent years, and PCSK9 plays an important role in LDL-C metabolism. Its principle of action is that LDL-C combined with LDL-R is internalized into hepatocytes through vesicles, and then the acidic environment of the endosome causes LDL-C to separate from its receptor. The circulating vesicles send LDL-R back to the cell surface, while the endosome containing LDL-C fuses with lysosomes, leading to the degradation of LDL-C. At the hepatocyte plasma membrane, the catalytic domain of secreted PCSK9 binds to LDL-R and is internalized into the endosome pathway. The low pH in the body enhances the affinity of PCSK9 for LDL-R, preventing the recycling of the receptor to the cell surface. After PCSK9 binds to LDL-R, it binds to LDL-C to form a new complex that enters the lysosome and is degraded together, ultimately leading to a decrease in LDL-R and a decrease in the degradation of LDL-C, thereby increasing the level of LDL-C.

[0004] CN201710816808..0 discloses a novel anti-PCSK9 monoclonal antibody, which has a large molecular weight and a complex structure. The key quality attributes of the product cannot be completely monitored by physical and chemical detection methods, and the titer of the product needs to be detected by corresponding biological activity methods. The present application establishes a biological activity determination method based on fluorescence-labeled chemiluminescence, simulates the principle of anti-PCSK9 monoclonal antibody in vitro, blocks the signal pathway of PCSK9 protein and LDL-C, indicates the biological activity of anti-PCSK9 monoclonal antibody, optimizes the operation steps and parameters, improves the accuracy, precision, durability and the like, and can be used for product release detection. It has important significance for process control and drug quality control in the production process. SUMMARY

[0005] To solve the problems in the prior art, the present application provides a method for rapidly and quantitatively determining the biological activity of anti-PCSK9 monoclonal antibody drugs, which has the following determination principle: human hepatoma cells HepG2 can express LDL receptors, and when PCSK9 protein exists, the combination of PCSK9 protein and LDL receptors can promote the degradation of LDL receptors, thereby inhibiting the absorption of LDL by HepG2 cells. Anti-PCSK9 monoclonal antibody specifically targets PCSK9 protein, which can block the combination of PCSK9 protein and LDL receptors on the surface of HepG2 cells, thereby promoting the internalization of LDL. The method of the present application indicates the biological activity of anti-PCSK9 monoclonal antibody by the absorption of fluorescently labeled LDL by cells.

[0006] The first aspect of the present application provides a method for rapidly and quantitatively determining the biological activity of anti-PCSK9 monoclonal antibody drugs, which comprises the following steps:

[0007] (1) Take the HepG2 cells in the logarithmic growth phase, plate them in a cell culture plate, and culture them for 16-18 hours;

[0008] (2) Dilute the anti-PCSK9 monoclonal antibody drug reference and test samples with an antibody diluent to an initial concentration, and then prepare at least 10 concentration gradients by diluting by a factor of two, wherein the antibody diluent contains human PCSK9 protein;

[0009] (3) Add the reference and test samples of each concentration in step (2) to the cell culture plate, and incubate them with the cells for about 2 hours, respectively;

[0010] (4) After the incubation is completed, add DiI-LDL reagent to the cell culture plate and incubate with the cells for 16-18 hours, measure the fluorescence value in the cells, and fit a four-parameter curve to calculate the relative biological activity of the test product, the light chain variable region of the PCSK9 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2.

[0011] In some embodiments, the cell culture plate is pretreated with poly-D-lysine, which includes coating the cell culture plate with a 0.1 mg / ml poly-D-lysine solution at 100 μL / well for 2 hours, and blowing dry. In some embodiments, the coating is performed in a 37°C, 5% CO2 incubator. In some embodiments, the cell viability after step (1) is no less than 90%.

[0012] In some embodiments, step (1) specifically includes:

[0013] (a) Cell culture: resuscitate HepG2 cells and subculture;

[0014] (b) Cell counting: take HepG2 cells in logarithmic growth phase and in good growth condition, discard the culture medium, wash the cells, and digest and count;

[0015] (c) Cell plating: according to the counting results, adjust the HepG2 cells to a certain cell density with an analysis medium, add to the cell culture plate at 100 μl / well, and culture for 16-18 hours.

[0016] In some embodiments, the subculture uses complete culture medium, which includes DMEM high-glucose culture medium with a final concentration of 10% fetal bovine serum (FBS) and 1% non-essential amino acids (NEAA). In some embodiments, the analysis medium includes DMEM high-glucose culture medium with a final concentration of 2% FBS and 1% NEAA. In some embodiments, the culture condition is 37°C, 5% CO2.

[0017] In some embodiments, the cell plating density is 2.5 x 10 5 -5 x 10 5 5 / ml, preferably 2.5 x 10

[0018] In some embodiments, step (2) specifically includes:

[0019] ​(d) Preparation of reference and test samples: Take the anti-PCSK9 monoclonal antibody reference and test samples, pre-dilute to 150-600 μg / ml as the starting concentration with the antibody diluent, and then prepare at least 10 concentration gradients according to 2-fold dilution.

[0020] In some preferred embodiments, pre-dilute to 300 μg / ml as the starting concentration, and then prepare 10 concentration gradients according to 2-fold dilution.

[0021] In some embodiments, the antibody diluent contains human PCSK9 protein at a final concentration of 30 μg / ml. In some embodiments, the antibody diluent further contains DMEM and NEAA, and the NEAA is at a final concentration of 1%.

[0022] In some embodiments, the step (3) specifically comprises:

[0023] (e) Sample loading: discard the culture medium in the cell culture plate, and add 100 μl / well of each concentration of the reference and test samples to the cell culture for about 2 hours; the culture condition is 37°C, 5% CO2.

[0024] In some embodiments, the step (4) specifically comprises:

[0025] (f) DiI-LDL internalization: add DiI-LDL reagent to the cell culture for 16-18 hours;

[0026] (g) Plate washing: take out the cell culture plate, wash the cells, discard the supernatant, and then add PBS to suspend the cells;

[0027] (h) Plate reading: read the fluorescence value at an excitation wavelength of 490 nm and an emission wavelength of 520 nm;

[0028] (i) Analysis: use the four-parameter model to fit the dose-response curve of the fluorescence value and the concentration of the anti-PCSK9 monoclonal antibody.

[0029] In some embodiments, the relative activity of the test sample is calculated according to the following formula: Relative biological activity (%) of the test sample = EC 50 value of the reference / EC 50 × 100%.

[0030] In some preferred embodiments, the concentration of the DiI-LDL reagent is 25 μg / ml, and the sample loading volume is 20 μl / well.

[0031] In some embodiments, the anti-PCSK9 monoclonal antibody comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 1 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 2. In some embodiments, the anti-PCSK9 monoclonal antibody further comprises a light chain constant region selected from a human kappa light chain constant region, a lambda light chain constant region, or a variant thereof, and a heavy chain constant region selected from a human IgGl, IgG2, IgG3, IgG4 constant region, or a variant thereof. In some preferred embodiments, the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 3 and the heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 4.

[0032] The second aspect of the present application provides use of the method of the first aspect of the present application in determining the biological activity of an anti-PCSK9 monoclonal antibody drug.

[0033] The third aspect of the present application provides use of the method of the first aspect of the present application in quality control of the production process of an anti-PCSK9 monoclonal antibody drug.

[0034] The method for rapidly and quantitatively determining the biological activity of an anti-PCSK9 monoclonal antibody drug provided by the present application has stable and reliable detection results, high accuracy, simplified and optimized operation steps, avoids cell contamination and errors caused by multi-step operations, reduces reagent consumption, saves costs, shortens detection time, and can be used in product release inspection to improve the controllability of production quality.

[0035] Unless otherwise defined, all technical and scientific terms used in the context of the present application have the same meaning as understood by one of ordinary skill in the art. Some terms are explained below:

[0036] The term "proprotein convertase subtilisin / kexin type 9 (PCSK9)," "PCSK9," or "NARC-1" refers to a naturally occurring human proprotein convertase belonging to the K subfamily of the subtilisin family of serine proteases. PCSK9 is synthesized as a zymogen, undergoes autocatalytic intramolecular processing in the endoplasmic reticulum, and is believed to function as a proprotein convertase. When used herein, the term refers to any naturally occurring PCSK9, preferably any native PCSK9 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). A representative amino acid sequence of human PCSK9 and a representative nucleic acid sequence encoding it are disclosed by GenBank Accession Nos. NP_777596.2 and FJ525880.1, respectively. The term "PCSK9" encompasses "full-length," unprocessed PCSK9 as well as any form of PCSK9 that results from processing within the cell, or any fragment thereof. The term also includes naturally occurring variants of PCSK9, e.g., splice variants, derivative variants, substitution variants, deletion variants, and / or insertion variants, or allelic variants, e.g., mutants D374Y, S127R, and F216L.

[0037] The term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that can bind to a particular antigen. A natural, intact antibody comprises two heavy chains and two light chains. Each heavy chain is comprised of one variable region (VH) and three constant regions (CH1, CH2, and CH3), with a hinge region between CH1 and CH2; each light chain is comprised of one variable region (VL) and one constant region (CL). The heavy chains of mammals can be classified as alpha, delta, epsilon, gamma, and mu, and the light chains of mammals can be classified as lambda or kappa. The antibody is in the shape of a "Y", with the neck of the "Y" shape being composed of the CH2 and CH3 of the two heavy chains, which are joined by a disulfide bond of the hinge region; each arm of the "Y" shape includes the variable region and CH1 of one of the heavy chains, which are connected to the variable region and CL of one of the light chains by a disulfide bond. The variable regions of the light and heavy chains determine the binding of the antibody to the antigen. Each variable region of the chain contains three hypervariable regions (hypervariable region, HVR), which are called complementarity-determining regions (Complementarity-Determining Region, CDR), wherein the CDR region of the light chain (L) includes LCDR1, LCDR2, and LCDR3, and the CDR region of the heavy chain (H) includes HCDR1, HCDR2, and HCDR3. The three CDRs are separated by side continuous parts called framework regions (Framework Region, FR), which are more highly conserved than CDRs and form a scaffold to support the hyper-variable loop. Antibodies can be classified into several categories according to the amino acid sequence of the heavy chain constant region, for example, according to whether they contain alpha, delta, epsilon, gamma, and mu heavy chains, antibodies can be classified into five main categories or isomers, respectively: IgA, IgD, IgE, IgG, and IgM. Several main antibody classifications can also be divided into subcategories, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), or IgG4 (γ4 heavy chain), etc.

[0038] The term "LDL" refers to low density lipoprotein (Low Density Lipoprotein, abbreviated as LDL), which is converted by the hydrolysis of lipoprotein esterase to release free fatty acids from the triglycerides in very low density lipoprotein (VLDL).

[0039] The term "DiI-LDL" refers to red fluorescent labeled human low density lipoprotein (Human DiI-LDL), which is LDL labeled with fluorescent probe DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate).

[0040] The term "biological activity" refers to the specific ability or potential of a biological product to achieve a certain biological effect based on biological products, which can be evaluated by the biological effect of a specific cell line to evaluate the corresponding biological activity of the biological product. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The S-curve of each concentration cell plating for detection fitting drawing is shown.

[0042] Figures 2A-2D The concentration gradient optimization results of anti-PCSK9 monoclonal antibody are shown, wherein Figure 2A For mode one, Figure 2B For mode two, Figure 2C For mode three, Figure 2D For mode four.

[0043] Figure 3 The optimization results of PCSK9 protein concentration in diluent are shown.

[0044] Figure 4 The optimization results of DiI-LDL dosage are shown.

[0045] Figure 5 The optimization results of DiI-LDL loading volume are shown.

[0046] Figures 6A-6C The results of HepG2 cell passage (P13, P26, P40) stability study are shown, wherein Figure 6A For cell P13 generation, Figure 6B For cell P26 generation, Figure 6C For cell P40 generation.

[0047] Figure 7 The long-term (2-8℃, 9 months) stability trend of anti-PCSK9 monoclonal antibody finished product is investigated by the method established by the application. DETAILED DESCRIPTION

[0048] The application will be further described below in conjunction with specific examples and drawings. It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application. The experimental methods in the embodiments of the application are not specified, which are usually carried out according to the conventional conditions, or according to the conditions suggested by the manufacturers of raw materials or commodities. The reagents without specific source are the conventional reagents purchased on the market.

[0049] Establishment of the method of example 1

[0050] 1.1 Experimental reagents, materials and equipment

[0051] Human hepatoma cell HepG2 was derived from ATCC; DiI-LDL (Cat. No. L3482) was derived from Invitrogen Corporation; high-sugar medium (DMEM, Cat. No. 11995-065), non-essential amino acids (NEAA, Cat. No. 11140-050) and 0.25% trypsin (Cat. No. 25200-056) were all derived from Gibco; fetal bovine serum (FBS, Cat. No. 35010155) was derived from CORNING; cell culture flasks and cell culture plates were derived from CORNING; CO2incubator was purchased from Thermo Corporation; cell counter was purchased from Countstar Corporation; clean bench was purchased from Suzhou Antai Air Technology Co., Ltd.; multifunctional enzyme label instrument was purchased from Thermo Corporation.

[0052] PCSK9 protein was prepared by Simcere (Suzhou) Pharmaceutical Co., Ltd., specifically, the corresponding PCSK9 protein was prepared according to the amino acid sequence recorded in Genbank Accession No. NP_777596.2 (Update Date 2022-07-10).

[0053] Reference product anti-PCSK9 monoclonal antibody (hereinafter referred to as antibody X) was provided by Simcere (Chengdu) Biotechnology Co., Ltd., and test product antibody X was prepared by Simcere (Suzhou) Pharmaceutical Co., Ltd. The antibody X reference product and test product were screened and prepared using the method for antibody 18.156.8 in Example 1-3 of patent CN201710816808.0, or all the contents in this patent document are incorporated by reference into the present application. The amino acid sequences of the antibody X reference product and test product are as follows:

[0054] > Light chain variable region:

[0055]

[0056] > Heavy chain variable region:

[0057]

[0058] > Light chain constant region:

[0059]

[0060] > Heavy chain constant region:

[0061]

[0062] 1.2 Solution preparation

[0063] Complete medium (DMEM high glucose medium with 10% FBS + 1% NEAA) : Take 500 ml DMEM, add 56.2 ml FBS and 5.62 ml NEAA (100x), mix well to obtain.

[0064] Analysis medium (DMEM high glucose medium with 2% FBS + 1% NEAA) : Take 500 ml DMEM, add 10.3 ml FBS and 5.2 ml NEAA (100x), mix well to obtain.

[0065] Diluent A: Take 500 ml DMEM, add 5 ml NEAA (100x), mix well to obtain.

[0066] 1.3 Cell culture and plating

[0067] Cell culture: Resuscitate HepG2 cells and subculture in a 37°C, 5% CO2 incubator using complete medium;

[0068] Cell counting: Take HepG2 cells in logarithmic growth phase and in good growth condition, discard the culture medium, wash the cells with 5 ml PBS, place in a 37°C, 5% CO2 incubator for 1-5 minutes of trypsin digestion, terminate with complete medium and centrifuge, discard the supernatant, resuspend the cells with analysis medium to prepare a HepG2 cell suspension, stain with trypan blue solution, and count on a cell counter;

[0069] Cell plating: According to the counting results, adjust the HepG2 cell suspension to a certain cell density with analysis medium, add 100 μl / well to the wells of a 96-well cell culture plate, and place in a 37°C, 5% CO2 incubator for 16-18 hours to allow the cells to adhere firmly.

[0070] 1.4 Biological activity detection

[0071] Reference and test sample preparation: Dilute the reference and test samples with diluent A to 200-300 μg / ml as the starting concentration, and then further dilute to 10-11 concentration gradients;

[0072] PCSK9 protein solution preparation: Dilute the PCSK9 protein with diluent A to a certain working concentration;

[0073] Sample addition: Discard the culture medium in the cell culture plate, add 50 μl / well of PCSK9 protein solution, then add 50 μl / well of each concentration gradient of reference and test samples, respectively, set 3 replicate wells for each concentration for detection, gently mix the 96-well cell culture plate after sample addition, and place in a 37°C, 5% CO2 incubator for about 2-4 hours, preferably 2 hours;

[0074] DiI-LDL internalization: DiI-LDL was added to 96-well cell culture plates at 10 μl / well and incubated at 37 °C in a 5% CO2 incubator for 16-18 hours, preferably 16 h;

[0075] Plate washing: the cell culture plates were removed, the supernatant was discarded, 200 μl / well PBS was added to wash the cells, the PBS was discarded, and 100 μl / well PBS was added to resuspend the cells;

[0076] Plate reading: the program was set to moderate shaking, "fluorescence" was added, bottom reading, and the plate was read in multiple-point mode with an excitation wavelength of 490 nm and an emission wavelength of 520 nm to read the fluorescence value (Relative Fluorescence Unit, RFU). The data were analyzed using Origin 2018 software, with the concentration of the reference or test sample as the horizontal coordinate and the RFU corresponding to each concentration as the vertical coordinate. A four-parameter fitting regression model was used to draw an S-curve, and the curve fitting equation was as follows:

[0077] Y = (A1-A2) / (1+(X / x0)p)+A2

[0078] X: the theoretical concentration of the reference or test sample

[0079] Y: the RFU corresponding to the reference or test sample

[0080] A1: the asymptote of the curve

[0081] A2: the asymptote of the curve

[0082] p: the slope of the curve

[0083] x0: the half-effective concentration (EC 50 )

[0084] The calculation formula of the relative biological activity of the test sample was as follows:

[0085] Relative biological activity of test sample (%) = EC 50 value of reference / EC 50 value of test sample x 100%

[0086] Optimization of the method of Example 2

[0087] 2.1 Optimization of cell culture plate pretreatment

[0088] Poly-L-lysine (Biyun Tian, item number 25988-63-0) or poly-D-lysine (Biyun Tian, item number ST508) was diluted to 0.05 mg / ml or 0.1 mg / ml with PBS, respectively. The 96-well cell culture plates were coated with the above poly-lysine solution, 100-200 μL per well, in a 37 °C, 5% CO2 incubator for 2-4 hours, the poly-D-lysine solution was discarded, and the plates were washed with 100 μL / well of PBS, which was discarded. The plates were placed in a biological safety cabinet, and the plates were blown for 1-3 hours with a table-top internal circulating air until there was no liquid in the wells. HepG2 cells were plated at a density of 5 x 10 5 The 96-well cell culture plates were incubated in a 37 °C, 5% CO2 cell culture incubator for 16-18 hours. The growth and adhesion of the cells were observed under a microscope.

[0089] The results showed that the cells plated on the cell culture plates treated with the poly-L-lysine solution were abnormal, the adhesion of the HepG2 cells was poor, the boundaries were blurred, and some cells did not adhere. The cell count showed that the viability of the cells in some wells was less than 90%, which could easily lead to large fluctuations in the test results. The cell culture plates treated with the poly-D-lysine solution at 0.1 mg / ml and 100 μL / well were coated in a 37 °C, 5% CO2 incubator for 2 hours and blown dry for 3 hours. This treatment could promote the adhesion of the cells without affecting the growth of the cells (ensuring that the cell viability was greater than 90%), and it was stable, reliable, and could effectively reduce the relative deviation between the wells in subsequent sample addition and liquid discarding operations, thereby improving the accuracy of the test and enhancing the robustness of the method.

[0090] 2.2 Optimization of the optimal cell plating density

[0091] The method established in Example 1 was used for testing, in which the plating density of the HepG2 cells was 1 x 10 5 , 2.5 x 10 5 , 5 x 10 5 , or 1 x 10 6 per ml, the PCSK9 protein solution was diluted to 60 μg / ml, the reference was diluted to 11 concentrations (300, 150, 41.2, 29.4, 21, 15, 10.7, 7.7, 5.5, 1.5, 0.5) μg / ml with diluent A, and the DiI-LDL was diluted to 100 μg / ml.

[0092] The fitted S-curve is shown in Figure 1 The results showed that there was no obvious upper platform and the response value was too low (R 2 = 0.98143) when the cell density was 1 x 10 5 per ml, and the response value was too low (R 6At a cell density of 1 cell / ml, the plateau concentration distribution is insufficient (R). 2 (0.97388), at a cell plating density of 5×10 5 2.5×10 5 A significant dose-response effect was observed at a concentration of 100 cells / ml, with a correlation coefficient exceeding 0.99 (R0.05). 2 The concentrations were 0.99313 and 0.99529 respectively, with appropriate window values ​​and more uniform distribution at each concentration point. Cell growth density and cell count were observed after 24 hours of culture following cell grafting. The optimal cell grafting density was 2.5 × 10⁻⁶. 5 per ml.

[0093] 2.3 Optimization of the concentration gradient of antibody X

[0094] The test was performed according to the method established in Example 1, wherein HepG2 cells were used at a concentration of 2.5 × 10⁻⁶. 5 PCSK9 protein solution was diluted to 60 μg / ml per well, and DiI-LDL was diluted to 100 μg / ml. The reference standard and antibody X stock solution of one batch were serially diluted with diluent A according to the four methods shown in the table below.

[0095] Table 1 Concentration gradient of anti-PCSK9 monoclonal antibody

[0096]

[0097]

[0098] The dose-response curves for each dilution method are shown in the figure. Figures 2A-2D One of the methods is Figure 2A Method two is Figure 2B Method three is Figure 2C Method four is Figure 2D .

[0099] By comparing the R values ​​of the dose-response curve fitting in four different ways... 2 The adjusted R-squared is shown in the table below:

[0100] Table 2. Fitting results of dose-response curves at different concentration gradients.

[0101]

[0102] Method 2 (2-fold dilution) Fitting curve R 2The adjusted R-square is better than the first, third and fourth methods. According to the S-shaped graph analysis, the upper platform of the first method reference and the antibody X stock solution does not coincide, the concentration point distribution is less, the lower platform concentration point distribution is less, and there is no obvious lower platform. The third S-shaped curve is asymmetric, and there is no lower platform. The fourth curve is not good, the points are concentrated in the lower platform, and the linear interval point distribution is less.

[0103] The second method has a better S-shaped dose-effect curve, the distribution of each concentration point is more uniform, the upper and lower platforms are obvious, the repeatability is good, the 2-fold dilution method is not easy to make mistakes, and therefore the second method is preferred to dilute the antibody X with 300 μg / ml as the starting concentration, 2-fold gradient dilution, and a total of 10 concentration points.

[0104] 2.4 PCSK9 protein concentration optimization

[0105] To simplify the operation and shorten the detection time, the PCSK9 protein is prepared into an antibody diluent, and then the reference and test samples are diluted with the antibody diluent, so that the antigen-antibody mixing can be completed during the antibody dilution, and the step of adding PCSK9 protein is simplified. Therefore, the PCSK9 protein concentration in the antibody diluent is optimized. The specific method is as follows:

[0106] According to the method established in Example 1, the HepG2 cells were plated at 2.5×10 5 cells / ml per well, the DiI-LDL was diluted to 100 μg / ml, the PCSK9 protein was diluted to 60 μg / ml, 30 μg / ml and 20 μg / ml with diluent A respectively to prepare three antibody diluents, the reference was diluted with the above antibody diluents, 300 μg / ml was used as the starting concentration, 2-fold gradient dilution was performed for 10 concentration points, and each concentration of the reference was added to the cell culture plate at 100 μL / well to incubate with the cells.

[0107] The results show that as the PCSK9 protein concentration increases, the number of LDLR on the surface of HepG2 cells decreases, which reduces the Dil-LDL reagent entering the cells, and the fluorescence intensity decreases. When the PCSK9 protein concentration in the antibody diluent is 30 μg / ml, a typical S-shaped curve is shown, the distribution of each concentration point is relatively more uniform, the upper and lower platform trends are obvious, and the fluorescence signal window value is appropriate (ratio > 2 times); when the PCSK9 protein concentration in the antibody diluent is 20 μg / ml, there is no typical S-shaped curve, and there is no lower platform. The upper platform concentration point is more, the window value is smaller, which easily affects the signal ratio of the window during the experiment, and increases the risk of experimental failure; when the PCSK9 protein concentration in the antibody diluent is 60 μg / ml, the concentration points are concentrated in the lower platform, and the linear region has fewer concentration points, which leads to a large fluctuation of the half-effect concentration (EC 50 ) and poor stability and reproducibility of the method (seeFigure 3 Based on the dual consideration of obtaining better method stability and ensuring stimulation effect (suitable window value), the concentration of PCSK9 protein in the antibody dilution is preferably 30 μg / ml.

[0108] 2.5 Optimal DiI-LDL reagent concentration and sample volume study

[0109] The test was performed according to the method established in Example 1, in which the HepG2 cells were plated at 2.5 x 10 5 cells / ml per well, and the reference was diluted in the antibody dilution containing a final concentration of 30 μg / ml PCSK9 protein according to Table 1. The reference at each concentration was added to the cells in the culture plate at 100 μL / well for incubation, and the DiI-LDL reagent was prepared at 100 μg / ml, 50 μg / ml, 25 μg / ml, 20 μg / ml, and 16 μg / ml, and added to the corresponding wells at 10 μl / well. It was detected that the correlation parameters of the four-parameter curve fitted under different DiI-LDL concentrations were shown in Table 3, and the dose-effect curve was shown in Figure 2. Figure 4

[0110] Table 3 Fitted curve parameters of different DiI-LDL concentrations

[0111]

[0112] The results showed that when the DiI-LDL reagent concentration was 25 μg / ml, 50 μg / ml, and 100 μg / ml, respectively, the signal value was enhanced in turn with the increase of the DiI-LDL concentration, the signal window ratio (A2 / A1) was similar (all > 2), a typical S-shaped curve was obtained, and the point distribution and fitting trend were consistent, the difference in X0 (EC 50 ) value was not obvious, indicating that the three DiI-LDL concentrations could be used for detection. When the DiI-LDL concentration was 25 μg / ml and 50 μg / ml, the upper and lower platforms were obvious, the window value and RFU signal value were suitable, and based on the consideration of saving DiI-LDL reagent and signal intensity suitability, the preferred amount of Dil-LDL was 25 μg / ml x 10 μl / well. When the DiI-LDL reagent concentration was 16 μg / ml and 20 μg / ml, A2 / A1 was less than 2, the window was small, and the signal value was poor.

[0113] ​In addition, it was found in the study that adding 10 μl / well (smaller sample volume) easily led to poor sample accuracy, resulting in decreased accuracy of data between duplicate wells. Therefore, the sample volume was further optimized under the condition of determining the optimal dosage. The test was performed according to the foregoing method, wherein the reference was prepared into a sample with an initial concentration of 300 μg / ml, 2-fold gradient dilution, a total of 10 concentrations; the addition of DiI-LDL was divided into two ways, group one: 25 μg / ml x 20 μl / well, group two: 20 μg / ml x 25 μl / well, parallel 3 duplicate wells were detected, and the results are shown in Figure 5 .

[0114]

[0115] The results show that the R-square of group two (25 μg / ml x 20 μl / well) after adjustment is 0.99357, and the linear fitting is optimal; by observing the graph.

[0116] In order to further determine the accuracy of the two ways (group one and group two), the antibody A reference was prepared into samples with initial concentrations of 300 μg / ml, 150 μg / ml and 600 μg / ml (representing 50%, 100%, 200% expected potency samples, respectively) for detection, and the experiment was repeated by 2 inspectors (A and B), and the recovery rate (preset recovery rate should be 80%-120%) was calculated. The recovery rate results are shown in Table 4.

[0117] Table 4 Recovery rate results

[0118]

[0119] The results show that each expected potency sample of the two DiI-LDL sample addition methods has a typical S-shaped dose-effect curve, and the recovery rate meets the requirements. Compared with group two, the deviation of the results of two detections of group one is significantly reduced, the deviation between 3 duplicate wells is smaller, and there is no obvious deviation well phenomenon. From the experimental operation, it is easier to control the accuracy of the sample addition system with 20 μl / well, so the DiI-LDL reagent is preferably added in the system of 25 μg / ml x 20 μl / well.

[0120] 2.6 PCSK9 monoclonal antibody specific binding with LDL research

[0121] LDL proteins include Ox-LDL (oxidized LDL), ac-LDL (acetylated LDL) and several other modified forms. To study the PCSK9 mAb binding specificity to these common modified LDL, we purchased red fluorescent labeled acetylated human low density lipoprotein (Human DiI-Ac-LDL, Cat No: 20606ES76) and red fluorescent labeled oxidized human low density lipoprotein (Human DiI-Ox-LDL, Cat No: 20609ES76) from Yisen Biotech (Shanghai) Co., Ltd. The analysis medium was diluted to 30 μg / ml, and the recommended Ex: 549 nm; Em: 565 nm was used for reading the plate. The specific method is as follows:

[0122] The method established in Example 1 was used for testing, in which the HepG2 cells were plated at 2.5 x 10 5 cells / ml per well, and the DiI-LDL was diluted to 25 μg / ml. The PCSK9 protein was diluted to 30 μg / ml with diluent A, and the reference was diluted with the above antibody diluent. The starting concentration was 300 μg / ml, and 10 concentration points were diluted by 2-fold gradient. The reference at each concentration was added to the cell culture plate at 100 μL / well for incubation with the cells.

[0123] The results showed that the PCSK9 mAb reacted with Human DiI-Ac-LDL and Human DiI-Ox-LDL to show lower fluorescence, but there was no signal gradient with the increase of PCSK9 mAb concentration, and there was no obvious detection window. This indicated that when the lysine residues of LDL apolipoprotein were acetylated and oxidized, the LDL complex no longer bound to the LDL receptor, and the PCSK9 mAb had specific binding to LDL protein, which was not interfered by other similar modifications.

[0124] Example 3 Stability study of HepG2 cell passage

[0125] The state of the cells is a key factor for biological activity detection. The stability of HepG2 cell passage (P13-P40) was studied, and the operation steps were optimized according to the method of Example 2.

[0126] 1. Cell treatment: three different passages of HepG2 cells (P13, P26, P40) were plated, diluted to 2.5 x 10 5 cells / ml with complete medium, 100 μl per well was plated into a 96-well cell culture plate, and cultured for 16-24 h.

[0127] 2. Biological activity detection

[0128] Antibody dilution preparation: PCSK9 protein was diluted to 30 μg / ml with PCSK9 dilution solution to prepare the antibody dilution sample for treatment.

[0129] Reference, test sample (batch YF-1) preparation: the reference and test sample were pre-diluted to 300 μg / ml as the starting concentration, and then 2-fold gradient dilution was performed to obtain 10 concentration gradients.

[0130] The plate was read according to the method of Example 1, and the relative biological activity was analyzed. The experimental results are shown in Table 5 below:

[0131] Table 5 Biological activity results of different cell passages

[0132]

[0133] The results show that the relative biological activity of a batch of test sample was detected using three different cell passages (P13, P26, P40), and the results of the antibody X stock solution are shown in Figure 6A , 6B , 6C, and the results all meet the preset acceptance criteria (activity of 70% to 140%), proving that the HepG2 cell passages are stable at P13 to P40, and can be used for biological activity detection.

[0134] Application of the biological activity detection method of Example 4 in the quality control of PCSK9 monoclonal antibody

[0135] According to the reference regulations and pharmacopoeia requirements, the optimized detection method was used to perform long-term stability test on three batches of antibody X finished products (batches AQ-1, AQ-2, and AQ-3), and after the end of the test, all stability data were summarized and trend analysis was performed using Excel software to evaluate the stability of PCSK9 monoclonal antibody. The stability results are shown in Figure 7 , and the results show good stability.

[0136] Comparative Example 1

[0137] The difference from Example 3 is that the final concentration of the PCSK9 antibody in the comparative experiment is 200 μg / ml, the antibody X is diluted by 2.5 times, there are 10 concentration gradients, and the Dil-LDL concentration is 16 μg / ml. The method of Example 3 was used to evaluate the biological activity of different cell passages (P13-P40), and the results are shown in Table 6.

[0138] Table 6 Biological activity results of different cell passages

[0139]

[0140]

[0141] As shown in Table 6, the relative biological activity of the same batch of test sample was detected using three different cell passages (P13, P26, P40), and the results showed that the three different cell passages had large deviations, which proved that the HepG2 cell passages were unstable in the P13-P40 state and could not be used for biological activity detection. Therefore, the experimental parameters set in the comparative example 1 could not accurately detect the biological activity of the antibody X.

[0142] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A method for rapid quantitative determination of the biological activity of anti-PCSK9 monoclonal antibody drugs, characterized by, The method comprises the following steps: (1) Logarithmic phase HepG2 cells were plated in cell culture plates pretreated with poly-D-lysine at a cell plating density of 2.5 x 10 5 cells / ml and incubated for 16-18 hours. The step (1) specifically comprises: (a) cell culture: resuscitate HepG2 cells and subculture, subculture with complete culture medium, which comprises DMEM high-sugar culture medium with final concentration of 10% FBS and 1% NEAA; (b) cell counting: take HepG2 cells in logarithmic growth phase and in good growth state, discard the culture medium, wash the cells, and digest and count them; (c) cell plating: according to the counting result, adjust the HepG2 cells to a certain cell density with analysis culture medium, and add the cells into the cell culture plate at 100 μl / well, and culture for 16-18 hours, wherein the analysis culture medium comprises DMEM high-sugar culture medium with final concentration of 2% FBS+1% NEAA; (2) preparation of reference and test samples: take anti-PCSK9 monoclonal antibody reference and test samples, dilute the anti-PCSK9 monoclonal antibody drug reference and test samples to 300 μg / ml as the initial concentration with antibody diluent, and then prepare at least 10 concentration gradients according to 2 times of dilution ratio, wherein the antibody diluent comprises human PCSK9 protein with final concentration of 30 μg / ml; (3) add the reference and test samples of each concentration in step (2) into the cell culture plate, and incubate with the cells for 2 hours respectively; (4) after the incubation is completed, add DiI-LDL reagent into the cell culture plate, and incubate with the cells for 16 hours, and then measure the fluorescence value in the cells, and fit the four-parameter curve to calculate the relative biological activity of the test sample, wherein the concentration of the DiI-LDL reagent is 25 μg / ml, and the adding volume is 20 μl / well; The light chain variable region of the anti-PCSK9 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 3, and the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

4.

2. The method of claim 1, wherein, The pretreatment comprises coating the cell culture plate with 0.1 mg / ml poly-D-lysine solution at 100 μL / well for 2 hours, and blowing dry.

3. The method of claim 1, wherein, The antibody diluent further comprises DMEM and NEAA.

4. The method of claim 1, wherein, The step (3) specifically comprises: (e) sample adding: discard the culture medium in the cell culture plate, and add the reference and test samples of each concentration into the cells at 100 μl / well respectively, and incubate for 2 hours.

5. The method of claim 1, wherein, The step (4) specifically comprises: (f) DiI-LDL internalization: add DiI-LDL reagent and culture with the cells for 16 hours; (g) plate washing: take out the cell culture plate, wash the cells, discard the supernatant, and then add PBS to suspend the cells; (h) plate reading: read the fluorescence value; (i) analysis: use the four-parameter model to fit the dose-response curve of the fluorescence value and the anti-PCSK9 monoclonal antibody concentration.

6. The method according to any one of claims 1 to 5, characterized in that, The culture or incubation conditions include 37°C, 5% CO2; the calculation formula of relative biological activity of the test sample is as follows: relative biological activity (%) of the test sample = EC 50 value of the reference product / EC value of the test sample x 100%. 50 ​ 7. Use of the method of any one of claims 1-6 in determining the biological activity of an anti-PCSK9 monoclonal antibody drug or in quality control of a production process of an anti-PCSK9 monoclonal antibody drug, wherein, The light chain variable region of the anti-PCSK9 monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO: 1, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 2, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 3, and the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO:

4. The light chain variable region of the anti-PCSK9 monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO: 1, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 2, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 3, and the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 4.

Images