CN110133241B

CN110133241B - Novel method for measuring biological activity of recombinant human soluble gp130-Fc fusion protein

Info

Publication number: CN110133241B
Application number: CN201910420569.6A
Authority: CN
Inventors: 王军志; 饶春明; 于雷; 贾春翠; 周勇; 姚文荣; 史新昌; 秦玺; 裴德宁
Original assignee: National Institutes for Food and Drug Control
Current assignee: National Institutes for Food and Drug Control
Priority date: 2019-05-21
Filing date: 2019-05-21
Publication date: 2022-05-27
Anticipated expiration: 2039-05-21
Also published as: CN110133241A

Abstract

The invention relates to a novel method for measuring the biological activity of recombinant human soluble gp130-Fc fusion protein. According to the method, a single clone cell strain capable of stably expressing the SIE luciferase is obtained by transfecting CHO-K1 cells with a SIE luciferase reporter gene plasmid and then performing pressure screening. And stimulating and activating luciferase reporter gene expression at the downstream of the SIE reaction element by using IL-6/sIL-6 Ra complex, competitively binding soluble gp130-Fc fusion protein to IL-6/sIL-6 Ra complex, inhibiting luciferase expression in a dose-dependent manner, and fitting a four-parameter curve according to the measured chemiluminescence value to determine the biological activity of the recombinant human soluble gp130-Fc fusion protein after adding a luciferase substrate. The invention establishes a rapid and accurate detection method aiming at the biological activity of the recombinant human soluble gp130-Fc fusion protein, and avoids the problems of unstable factors caused by uncertainty of cell culture, cell pollution caused by long-time incubation and the like.

Description

Novel method for measuring biological activity of recombinant human soluble gp130-Fc fusion protein

Technical Field

The invention relates to the field of recombinant drug biological activity detection, and establishes a method for quickly and accurately determining the biological activity of a recombinant human soluble gp130-Fc fusion protein.

Background

Glycoprotein 130(gp130), with a molecular weight of 130kD, also known as IL-6R β, is an important component of the IL-6 receptor system. After IL-6 Ra forms an IL-6/sIL-6 Ra complex with IL-6, it can bind to membrane-type gp130 molecules, causing signal transduction, known as the trans-signaling pathway, which causes pro-inflammatory effects. Soluble gp130, as a natural inhibitor of trans-signaling, acts selectively on the IL-6/sIL-6 Ra complex and is unable to bind either IL-6 or sIL-6 Ra alone. Two dimers of sgp130 molecules are combined with IgG1-Fc to form recombinant human soluble gp130-Fc fusion protein (sgp130-Fc), which can simulate natural trans-signaling inhibitor sgp130, has higher binding affinity and can inhibit abnormal inflammatory reaction caused by the increase of IL-6/sIL-6R alpha complex.

Ulcerative Colitis (UC) is an intestinal disease mainly caused by nonspecific inflammatory diseases limited to the colonic mucosa and submucosa, has the characteristics of difficult healing, easy recurrence, easy canceration and the like, and is listed as one of the existing difficult and complicated diseases by WHO. IL-6 is a central cytokine involved in the pathobiology of inflammatory bowel disease, and IL-6 and sIL-6R alpha levels in UC patients are increased and trans-signaling is enhanced. sgp130-Fc is an innovative, selective IL-6 transreceptor inhibitor and anti-inflammatory agent for the treatment of UC, and shows very good therapeutic effects in mouse models of human diseases such as arthritis, inflammatory bowel disease and colon-related cancer, and its expected therapeutic effects are similar to those of Tocilizumab (TCZ), a full-effect IL-6 blocker, or anti-Tumor Necrosis Factor (TNF) drugs, but are more concerned about safety, and have now entered phase II/III clinics.

The activity measurement is the measurement of the content, effective components and potency of the drug, and is an important quality control index for ensuring the effectiveness of the drug. The current method for detecting the biological activity of the recombinant human soluble gp130-Fc fusion protein is a BAF3/gp130 cell proliferation inhibition method, and the principle is as follows: the sgp130-Fc fusion protein can inhibit BAF3/gp130 cell proliferation caused by IL-6/sIL-6 Ra complex. However, BAF3/gp130 cells proliferated only partially dependent on IL-6/sIL-6 Ra, if the cultures were too dense, BAF3/gp130 cells lost this dependence and could only be maintained in low density cultures, and growth dependence on IL-6/sIL-6 Ra was monitored periodically (4 to 6 weeks) by parallel cultures of IL-6/sIL-6 Ra-free cells, and once this dependence was lost, it could not be used for activity detection. The uncertainty of cell culture also brings unstable factors to activity detection, and easily causes low signal-to-noise ratio, large variation degree and poor repeatability of experimental data.

Disclosure of Invention

The method comprises the steps of constructing a cell strain for stably expressing the SIE luciferase reporter gene, wherein an IL-6/sIL-6 Ra complex can be combined with cell surface gp130, and activates an SIE reaction element through signal transduction to start the expression of downstream luciferase; sgp130-Fc can competitively bind to the IL-6/sIL-6 Ra complex, inhibiting luciferase expression; after addition of the luciferase substrate, biological activity of sgp130-Fc fusion proteins was determined by fitting a four-parameter curve to chemiluminescence values.

The invention aims to provide a method for rapidly determining the biological activity of sgp130-Fc fusion protein, which comprises the following steps:

(1) transferring the reporter gene plasmid containing the SIE reaction element into cells, and performing pressurized screening to obtain a stably expressed monoclonal cell strain D23;

(2) serial dilution is carried out on the sgp130-Fc fusion protein drug, the sgp130-Fc fusion protein drug and an IL-6/sIL-6R alpha compound are incubated for 1h at 37 ℃, then the sgp130-Fc fusion protein drug and the IL-6/sIL-6R alpha compound are added into the cells of the step (1), and stimulation is carried out for 7h at 37 ℃;

(3) discarding the culture solution of (2), adding luciferase substrate to determine chemiluminescence value, fitting four-parameter curve, and determining IC according to the results₅₀The values determine the relative biological activity of sgp130-Fc fusion proteins.

Chinese hamster ovary CHO cells are immortal, can be passaged for more than one hundred generations, and are cells widely used in bioengineering. The CHO-K1 cell as the transformed cell line is widely used for expressing recombinant protein, and has the advantages of fast growth and easy culture. CHO-K1 cells do not express IL-6R alpha, SIE luciferase reporter gene plasmids are introduced into CHO-K1 cells, IL-6/sIL-6R alpha complexes are combined with membrane type gp130 to induce JAK kinase coupled with the membrane type gp130 to be phosphorylated and to phosphorylate tyrosine residues on the gp130, Src homology domain-2 (SH2) on STAT3 and STAT1 reacts with phosphorylated tyrosine to form dimers, the dimers are separated from the combination sites of the dimers, the dimers are transferred into nucleus after phosphorylation, and SIE reaction elements are recognized and combined to start the expression of SIE downstream luciferase. Sgp130-Fc fusion protein specifically binds to IL-6/sIL-6R complex, prevents its binding to membrane-type gp130, and blocks the above-described trans-signaling pathway.

The amount of luciferase expression in effector cells is therefore inversely proportional to the concentration of sgp130-Fc fusion protein.

In an embodiment of the present invention, the effector cells stably expressing the SIE reporter gene are CHO-K1 cells obtained by antibiotic pressure screening, and are named D23.

In an embodiment of the invention the reporter gene is luciferase.

In an embodiment of the invention, the steps comprise: and (3) transfecting CHO-K1 cells by using a reporter gene plasmid containing the SIE sequence, and adding corresponding antibiotics to screen to obtain a monoclonal cell strain stably expressing the SIE reporter gene.

In an embodiment of the invention, F12K medium was used for the preparation of cell suspensions and for the dilution of the sgp130-Fc fusion protein and the reference, the number of plated cells being 2X 10⁴～4×10⁴One/hole, preferably 3X 10⁴Per well.

In an embodiment of the invention, the reporter gene expression is activated by the addition of IL-6/sIL-6 Ra complex at a concentration of 5. mu.g/mL IL-6, 2.5. mu.g/mL sIL-6 Ra, a pre-diluted concentration of 25000ng/mL for the sgp130-Fc fusion protein, and 8 concentrations for 2-fold dilution.

In an embodiment of the invention, the pre-incubation conditions of the IL-6/sIL-6 Ra complex and the sgp130-Fc fusion protein are 37 ℃ and 0.5-2 hours, preferably 1 hour. The stimulation time by adding the medicine is 5-10 hours, preferably 7 hours.

In an embodiment of the present invention, chemiluminescence values are detected using a luciferase substrate kit, which may be used, for example, Bright-G1o from promega, luciferase from Biovision, Britelite plus from PerkinElmer, preferably Bright-G1 o.

In the embodiment of the invention, chemiluminescence values are read by a microplate reader, an inverted S-shaped four-parameter curve is fitted, and IC of the sgp130-Fc fusion protein and a reference substance is determined₅₀The relative biological activity of the sgp130-Fc fusion protein (relative biological activity ═ reference IC) was determined₅₀Sample IC₅₀×100％)。

The invention has the beneficial effects that: the invention adopts a transgenic cell activity measuring method, has short experimental period, simple and convenient operation and high-efficiency stability, and avoids the possible problems of unstable factors caused by uncertainty of cell culture, cell pollution caused by long-time incubation and the like.

Drawings

FIG. 1 is a graph showing the response of the D23 cell line to different pre-incubation times of the IL-6/sIL-6 Ra complex and sgp130-Fc fusion protein;

FIG. 2 is a graph showing the response of D23 cell line to different concentrations of IL-6/sIL-6 Ra complex;

FIG. 3 is a graph showing the response of D23 cell line to IL-6+ sIL-6 Ra at various ratios;

FIG. 4 is a graph showing the response of D23 cell line to different cell densities;

FIG. 5 is the range of active concentrations of sgp130-Fc fusion protein;

FIG. 6 is a graph showing the response of the D23 cell line to different drug durations;

FIG. 7 is a graph showing the response curves (serum concentrations) of the D23 cell line to reaction solutions containing different serum concentrations;

FIG. 8 is a specific response curve of the D23 cell line;

Detailed Description

The invention is further illustrated and described in the following specific examples, which are intended to be purely exemplary of the invention and are not to be considered as limiting the invention. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or conditions recommended by the manufacturers.

EXAMPLE 1 screening of CHO-K1 cell line stably expressing SIE-luciferase

1. Materials and reagents

CHO-K1 cells were derived from ATCC; pGL4.47[ luc2P/SIE/Hygro]Plasmids were purchased from Promega; ViaFect^TMTransfection reagents were purchased from Promega; F12K medium, Fetal Bovine Serum (FBS) purchased from GIBCO; hygromycin B was purchased from Solebao Biotechnology Ltd; recombinant human IL-6 and sIL-6R α were purchased from peprotech; Bright-G1o luciferase substrate was purchased from Promega; the sgp130-Fc, EPO-Fc, IL15-Fc and GH-Fc fusion proteins are retained in a recombinant drug chamber of the Chinese institute of food and drug assay; TCZ is the monoclonal antibody chamber retention of China institute for food and drug testing; IL-6 antibody was purchased from Chinesia.

2. Test procedure

2.1 transfection of SIE plasmid

CHO-K1 cell concentration was adjusted to 5X 10 with F12K medium containing 10% FBS⁵PermL, 5mL to T25 cell culture flasks were taken. pGL4.47[ luc2P/SIE/Hygro ] was transfected according to the instructions after incubation at 37 ℃ for 24h]And adding 300 mu g/mL hygromycin B into the plasmid after 48 hours for pressurized screening, replacing a new hygromycin B-containing culture medium every three days, and continuously culturing for 2-3 weeks. After cell density and viability were restored, the cells were plated in 96-well plates at a density of 0.8 cells/well. During the incubation period, wells containing single clones were observed and labeled. When the confluence degree of the cells in the monoclonal hole reaches more than 50%, digesting the cells and transferring the cells to a 24-well plate; when the confluency of the cells in the 24-well plate reaches more than 50%, the cells are digested and transferred to a 6-well plate for gradually expanding culture.

2.2 detection of reactivity

Primary screening: the monoclonal cell lines obtained in 2.2 were added to a 96-well plate at 37 ℃ and 5% CO at a density of 2 ten thousand per well₂The culture was carried out for about 24 hours. Preparing culture solution containing 2 μ G/mL recombinant human IL-6 and 1 μ G/mL sIL-6 Ra, diluting in series at 2 times, adding 10 μ L to each well, culturing for 5h, discarding reaction solution, adding 50 μ L Bright-G1o luciferase substrate, detecting chemiluminescence value, and screening out the product with higher reactivity to IL-6/sIL-6 Ra complexThe good cell lines were rescreened. Re-screening: the cell strains left in the primary screening are respectively added into a 96-well plate according to the density of 2 ten thousand per well, the temperature is 37 ℃, and the CO content is 5 percent₂The culture was carried out for about 24 hours. Preparing a complex culture solution containing 2. mu.g/mL of recombinant human IL-6 and 1. mu.g/mL of sIL-6 Ra, diluting the sgp130-Fc fusion protein in a 10-fold serial manner, adding 10. mu.L of the complex and 10. mu.L of the sgp130-Fc fusion protein to each well, culturing for 5 hours, discarding the reaction solution, adding 50. mu.L of Bright-G1o luciferase substrate, detecting chemiluminescence values, fitting a four-parameter curve, and determining the signal-to-noise ratio and IC according to the signal-to-noise ratio and IC₅₀And screening a cell strain with a good inhibitory effect on the sgp130-Fc fusion protein as an experimental cell, and naming the cell strain as D23.

Example 2 methodological optimization of assay of biological Activity of sgp130-Fc fusion proteins

2.1 optimization of the Pre-incubation time of the IL-6/sIL-6 Ra Complex with sgp130-Fc

Since Sgp130-Fc fusion protein blocks the trans-signaling pathway by specifically binding to the IL-6/sIL-6R complex and preventing its binding to membrane-type gp130, the extent of binding of sgp130-Fc to the IL-6/sIL-6 Ra complex determines its inhibitory effect, and the extent of binding is influenced by the time of pre-incubation, so that the pre-incubation time of both needs to be explored.

D23 cells were added to a 96-well plate at a density of 2 ten thousand per well at 37 ℃ with 5% CO₂The culture was carried out for about 24 hours. Mixing the IL-6/sIL-6R alpha compound with serial diluted sgp130-Fc, placing the mixture in an incubator at 37 ℃, carrying out incubation treatment for 2h, 1h and 0h (namely no incubation), adding a luciferase substrate after the cells act for 5h, and detecting the chemiluminescence value. From the response curves (FIG. 1), the signal-to-noise ratio, IC, are combined₅₀And time cost, the pre-incubation time was determined to be 1 h.

2.2 optimization of IL-6/sIL-6 Ra Complex concentration

Solutions of IL-6+ sIL-6 Ra complexes at concentrations of (10+ 5). mu.g/mL, (5+ 2.5). mu.g/mL, (2+ 1). mu.g/mL, (1+ 0.5). mu.g/mL were prepared and pre-incubated with serially diluted sgp130-Fc for 1h, respectively. Adding 20 mu L of the fluorescent enzyme substrate into each hole, reacting for 5 hours, and detecting the chemiluminescence value. From the response curves (FIG. 2), the signal-to-noise ratio, IC, are combined₅₀And reagent cost, after selecting (5+2.5) mu g/mLConcentration of IL-6+ sIL-6R α in the experiments described above.

2.3 optimization of the ratio of IL-6 to sIL-6 Ra

sIL-6 Ra was diluted to 0.25. mu.g/mL, as IL-6: sIL-6R α ═ 3:1, 2:1, 1; 1. four sets of complex solutions were prepared at a concentration ratio of 0.5:1, and pre-incubated with serial dilutions of sgp130-Fc for 1 h. Adding 20 mu L of the fluorescent enzyme substrate into each hole, reacting for 5 hours, and detecting the chemiluminescence value. From the response curves (FIG. 3), the signal-to-noise ratio, IC are combined₅₀And reagent cost, determined to be 2: 1.

2.4 optimization of cell plating Density

Dividing D23 cells into 1 × 10 cells⁴2 x 10 pieces/hole⁴One/hole, 3X 10⁴4 x 10 pieces/hole⁴Adding each well into a white 96-well culture plate, and culturing for 24 h. Pre-incubation for 1h IL-6+ sIL-6 Ra at a concentration of (5+2.5) μ g/mL was pre-incubated with serial dilutions of sgp130-Fc for 1h, and 20 μ L was added per well. After 5h of action, luciferase substrate was added and the chemiluminescence was measured. From the signal-to-noise ratio calculated from the upper and lower platforms (FIG. 4), D23 cell density was determined to be 3X 10⁴Subsequent experiments were performed per well.

2.5 optimization of dilution factor of sgp130-Fc fusion protein

The sgp130-Fc fusion protein was pre-diluted to a concentration of 25000ng/mL, and serial dilutions were performed at 2, 3, and 4 fold ratios, respectively, with 8 gradients. Preincubation with (5+2.5) μ g/mL IL-6+ sIL-6R α for 1h followed by addition of cells for 5h to detect luciferase chemiluminescence and fitting a four parameter curve (FIG. 5). The results show that the 2-fold dilution is uniform at each point on the curve (2, 3 concentration points on the upper and lower plateaus and linear portions, respectively).

2.6 optimization of drug action time

D23 cells were plated at 3.0X 10⁴Perwell add to 96 well plate, 37 ℃ 5% CO₂And culturing for 24 h. The sgp130-Fc fusion protein was prediluted to 25000ng/mL, 2-fold diluted in 8 gradients, and preincubated with (5+2.5) μ g/mL IL-6+ sIL-6 Ra for 1 h. Luciferase signal values were detected after 3h, 4h, 5h, 6h, 7h and 8h respectively and a four parameter curve was fitted (FIG. 6). Bonding IC₅₀And signal-to-noise ratio results, 7h was selected asThe action time of the medicine.

2.7 optimization of serum concentration

D23 cells were adjusted to density of 3.0X 10 in serum-free F12K medium⁴Perwell add to 96 well plate, 37 ℃ 5% CO₂And culturing for 24 h. IL-6, sIL-6 Ra and sgp130-Fc fusion proteins were diluted with F12K medium containing 0.2%, 1%, 5%, 10% serum, respectively, incubated for 1h and luciferase signal values were measured after 7h of cell addition and a four-parameter curve was fitted (FIG. 7). Bonding IC₅₀And signal to noise ratio, serum concentration was determined to be 10%.

Example 3 methodological validation of assay of biological Activity of sgp130-Fc fusion proteins

3.1 specificity

The method is a biological activity method aiming at the sgp130-Fc fusion protein, so that the specificity of the sgp130-Fc fusion protein is verified by adopting different varieties of medicaments, including TCZ, EPO-Fc, IL15-Fc, GH-Fc and IL-6 antibodies. The reactivity of D23 cells to the above-described drug and sgp130-Fc fusion protein was determined according to the test conditions determined in example 2. The results are shown in FIG. 8, and the method is not responsive to other Fc fusion proteins (EPO-Fc, IL15-Fc, GH-Fc) and only partially responsive to IL-6 antibody and IL-6 receptor single antibody (TCZ), indicating that the specificity of the method is better.

3.2 accuracy (recovery)

And (3) taking an sgp130-Fc fusion protein sample and a reference substance, respectively pre-diluting to 25000ng/mL, diluting by 2 times, and carrying out 8-gradient dilution, and then mixing the diluted sample and the reference substance in equal volumes. The spiked recovery test was performed using the experimental conditions determined in example 2. The recovery rate is between 94.1 and 106.2 percent.

3.3 linearity

A sgp130-Fc fusion protein sample and a reference substance are taken, pre-diluted to 25000ng/mL, and five groups of sample solutions with the pre-dilution concentrations of 50%, 75%, 100%, 125% and 150% of the pre-dilution concentration (25000ng/mL) of the sample are prepared. And (3) performing the same operation on the five groups of sample solutions and a reference substance, and diluting the five groups of sample solutions by 2 times and 8 concentration gradients respectively. The assay was carried out 3 times per day for 3 consecutive days according to the experimental conditions defined in example 2. Calculating the phase of each group of samplesFor biological activity, the average of its measured values and the theoretical value were fitted linearly. As a result, R is obtained²A linear fit is better at 0.9917. Indicating that the linearity of the method is good.

Claims

1. A novel method for measuring the biological activity of recombinant human soluble gp130-Fc fusion protein is characterized in that: the method comprises the steps of transfecting CHO-K1 cells with SIE luciferase reporter gene plasmids to obtain cell strains stably expressing the SIE luciferase reporter gene, stimulating and activating the expression of the downstream luciferase reporter gene of an SIE reaction element by using IL-6/sIL-6 alpha complex, blocking a trans-signal channel after adding sgp130-Fc, and inhibiting the expression of the luciferase reporter gene; fitting a four-parameter curve according to the measured fluorescent signal value to determine the biological activity of the recombinant human soluble gp130-Fc fusion protein;

the method comprises the following steps:

step 1, transferring plasmids containing SIE luciferase into cells, and performing pressurized screening to obtain stably expressed monoclonal cell strains;

step 2, serial dilution is carried out on the sgp130-Fc fusion protein drug, the sgp130-Fc fusion protein drug and the IL-6/sIL-6R alpha compound are incubated for 1h at 37 ℃, and then the sP 130-Fc fusion protein drug and the IL-6/sIL-6R alpha compound are added into the cells in the step 1 and stimulated for 7h at 37 ℃;

step 3, discarding the culture solution in the step 2, adding luciferase substrate to measure chemiluminescence value, fitting a four-parameter curve according to IC₅₀The relative biological activity of sgp130-Fc fusion proteins was determined;

the parameter setting and material selection in the above steps are as follows:

(1) preparing a cell suspension by using F12K culture medium containing 10% serum;

(2) the number of plated cells was 2X 10⁴－4×10⁴Per well;

(3) the concentration of IL-6 is 5 mug/mL, the concentration of sIL-6R alpha is 2.5 mug/mL, the pre-dilution concentration of sgp130-Fc is 25000ng/mL, and 8 concentrations are diluted by 2 times;

(4) pre-incubation conditions of sgp130-Fc and IL-6/sIL-6 Ra complex were 37 degrees for 0.5-2 hours; adding medicine for 5-10 hr;

(5) chemiluminescence values were measured using luciferase substrate kits such as Bright-G1o from promega, luciferase from Biovision, or Britelite plus from PerkinElmer;

(6) reading chemiluminescence values by using a microplate reader, and determining IC of sgp130-Fc fusion protein and a reference substance by fitting a four-parameter curve₅₀Values, in relative biological activity: relative biological activity as reference IC₅₀Sample IC₅₀Reflecting the biological activity of the sgp130-Fc fusion protein.

2. The method of claim 1, wherein: wherein the reporter gene is luciferase.

3. The method according to claim 1 or 2, characterized in that: the parameter setting is selected as follows:

(2) the number of plated cells was 3X 10⁴Per well.

4. The method according to claim 1 or 2, characterized in that: the parameter setting is selected as follows:

(4) the pre-incubation condition of sgp130-Fc and IL-6/sIL-6 Ra complex was 1 hour; the stimulation time was 7 hours with drug addition.

5. The method according to claim 1 or 2, characterized in that: wherein the materials are selected as follows:

(5) chemiluminescence was measured using a luciferase substrate kit, Bright-G1o from Promega.

6. The method of any one of claims 1 to 5 for quality control in the manufacture and development of sgp130-Fc fusion protein pharmaceuticals.