CN103792200A - Biology activity determination method of monoclonal antibody - Google Patents

Biology activity determination method of monoclonal antibody Download PDF

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CN103792200A
CN103792200A CN201410064974.6A CN201410064974A CN103792200A CN 103792200 A CN103792200 A CN 103792200A CN 201410064974 A CN201410064974 A CN 201410064974A CN 103792200 A CN103792200 A CN 103792200A
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monoclonal antibody
elisa
cell
anti human
human egf
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CN103792200B (en
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何伟
刘永清
喻志爱
白先宏
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Biotech Pharmaceuticals Co Ltd
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Abstract

The invention discloses a biology activity determination method of a monoclonal antibody of a recombined anti-human epidermal growth factor receptor. The method comprises the following steps: incubating human lung cancer-lymph node metastasis cells H292 and anti-EGFR monoclonal antibody with different concentrations; adding a cell counting dye reagent CCK-8; determining the light absorption value of stained vital cells through a microplate reader; processing experiment data through a computer program; calculating the biology activity of an anti-EGFR sample relative to an anti-EGFR standard. According to the invention, a cell strain with the detected cells H292 is applicable to antibodies with different affinities, the cell culture time is short, and the needed cell quantity is less; staining fluid of an MTT colorimetric method is improved, CCK-8 staining fluid is used, the product solubility is good, further solution treatment is not needed, the detection result can be detected at different time after the staining fluid is added, the developing result is stable, and the quality is controllable. The biology activity determined by the method is related to clinical effects, thus meeting the relevant technical requirement of FDA.

Description

A kind of Determination of biological activity method of monoclonal antibody
Technical field
The present invention relates to the Determination of biological activity technical field of antibody, more specifically to the Determination of biological activity method of the monoclonal antibody of recombinant anti human EGF-R ELISA.
Background technology
EGF-R ELISA (Epithelial growth factor receptor, EGFR) be a kind of glycoprotein, belong to tyrosine kinase receptor, be distributed widely in the cell surfaces such as mammal epithelial cell, fibroblast, spongiocyte, horn cell, its signal path (Hanley SC that plays an important role in the physiology courses such as growth, propagation and the differentiation of cell, Assouline-Thomas B, Makhlin J, et al.J Endocrinol, 2011,211(3): 231-239).Research shows, EGFR is relevant with propagation, Angiogenesis, tumor invasion, transfer and the apoptotic inhibition of tumour cell, in many entity tumors, there is high expressed or the unconventionality expression of EGFR, therefore target spot (Kim H that can be using EGFR as oncotherapy, Kim SH, Kim MJ, et al.J Immunother, 2011,34(4): 372-381; Oh M, Lee JY, Shin DH, et al.Cancer Sci, 2011,102(3): 597-604).
The Determination of biological activity of monoclonal antibody is the important evaluating of restructuring biological products quality control, is related to the result for the treatment of of monoclonal antibody.The general biologic activity that body outer cell proliferation inhibition method is recorded is called tiring of monoclonal antibody.
As far back as 1963, Slater just found that the dehydrogenasa in mitochondria (as succinate dehydrogenase, diaphorase) can open the azoles ring of tetramethyl azo azoles salt (MTT), generates blue product first
Figure BDA0000469556580000011
(formazan).Around this principle, Mosmann(Mosmann T.J Immunol Methods, 1983,65:55) found MTT detection method in nineteen eighty-three, this method measure aspect the survival of cell and propagation very effective because the variation of this color only occurs over just in cell alive, and first formation volume be proportional to viable count and functional status.At present, MTT determination method has been widely used in multiple growth factor as in the testing of epidermal growth factor, TNF, interleukin 2 etc.
The assay method of at present general recombinant anti human EGFR antibody biologic activity, is the propagation/WST colourimetry that adopts DiFi cell or MDA-MB-468 cell, all adopts the method for improved MTT dyeing.This method is inhibited to the growth of these cells according to anti-EFGR antibody, and the upgrowth situation of DiFi cell or MDA-MB-468 cell line is different because of the difference of the biologic activity of restructuring anti-egfr antibodies, detects the biologic activity of anti-egfr antibodies with this.Its determination method is respectively:
(1) DiFi cell proliferation colourimetry: DiFi cell line uses complete culture solution in 37 ℃, 5% carbon dioxide cultivation, 1640 complete medium re-suspended cells, the blue dyeing of placenta, counts under mirror, adjusts cell concentration to 2 × 10 5individual/ml, 100 μ l/ holes, oscillator mixed after 5 minutes, and 96 orifice plates are put to 37 ℃, 5%CO 2incubator is hatched 90-102 hour, hatch the PMS in CellTiter96Aqueous Non-Radioactive Cell Proliferation Assay MTS kit and MTS are melted before finishing and balance to room temperature, getting 100ul PMS adds in 2mlMTS, mix, add Tissue Culture Plate, 20ul/ hole, 37 ℃, 5%CO 2hatch 2 hours.Hatch after end, add 10%SDS15ul/ hole color development stopping, 490nm wavelength is measured absorbance, result is used Softmax5.0 software analysis (Zhang Feng etc., DiFi cell inhibitory effect method is measured the biologic activity of anti-epidermal growth factor receptor monoclonal antibody, the academic Conference Papers compilation of the 5th national immunodiagnosis and vaccine [C], 2011).
(2) MDA-MB-468 cell proliferation/WST colourimetry: MDA-MB-468 dilution for cell (containing the F12/DMEM nutrient culture media of 0.1g/L PF-68) is adjusted to cell concentration (5～8) × 10 4individual/mL, adds 96 orifice plates, every hole 100 μ L, and blank adds 100 μ L dilutions.Sample and reference material are diluted to 50 μ g/mL in advance with sample diluting liquid (containing the F12/DMEM nutrient culture media of 0.2%FBS), 3 times of gradient dilution to 0.023 μ g/mL again, add 96 orifice plates, every hole 50 μ L, add 40 μ LF12/DMEM nutrient culture media (containing 5%FBS), cultivate 5 days for 37 ℃, add 30 μ L WST-8 nitrite ions, continue to hatch 4h, take 630nm as reference wavelength, measure absorbance (A) value in wavelength 450nm place.Take protein concentration as horizontal ordinate, A450 value is ordinate, carries out four parameter fittings, obtains anti-" S " type curve, with medium effective concentration (EC 50) expression biologic activity.The sample of 50 μ g/mL and reference material equal-volume are mixed, by same way dilution application of sample, measure the recovery, be calculated as follows Relative Biological activity and the recovery (Tao Lei etc., the foundation of humanization anti-epidermal growth factor receptor monoclonal antibody quality control method, Products in China is learned magazine, 2013 01 month the 26th the 1st phase of volume).
Sample Relative Biological activity (%)=reference material EC 50/ sample EC 50× 100%
Recovery sample Relative Biological activity (%)=reference material EC 50/ recovery sample EC 50× 100%
Figure BDA0000469556580000021
Above-mentioned two kinds of methods have all adopted two kinds of different cell lines, and have used the reagent of two kinds of different improved MTT colourimetrys: PMS/MTS and WST-8 nitrite ion.But these two kinds of cells are not strong for the applicability of different affinity anti-egfr antibodies, be only applicable to the anti-egfr antibodies of high-affinity, cause susceptibility on the low side, owing to producing some problems such as the solubleness of protein precipitation and product is on the low side after dyeing, need 10%SDS or 10% dmso solution step, strengthened the situation of variation testing result.
The present invention is on the basis of existing MTT decoration method, and adopting another to detect cell line-people lung cancer lymphatic metastasis cell is H292 cell line, by (6.0～10.0) × 10 4the cell suspension of individual cell, is inoculated in 96 porocyte culture plates, and 100 μ l/ holes, put 37 ℃, 5%CO by 96 orifice plates 2incubator is hatched 18～24 hours.According to the compatibility of anti-egfr antibodies, sample and reference material are diluted to (300-1000) μ g/mL in advance with sample diluting liquid (containing 1640 nutrient culture media of 1%FBS), then 5 times or 4 times of gradient dilutions, be diluted to 5 -7or 4 -7initial concentration, add 96 orifice plates, every hole 200 μ L, cultivate 68～72 hours for 37 ℃, add the CCK-8 nitrite ion after 30 μ L dilutions, continue to hatch 2～4h, take 630nm as reference wavelength, measure absorbance (A) be worth in wavelength 450nm place.
Detection cell H292 cell line of the present invention is all suitable for for the antibody of different affinity, and cell incubation time is short, and the cell quantity needing is less; And the present invention improved the dyeing liquor of MTT colourimetry, use CCK-8 dyeing liquor instead, the solubleness of product is better, does not need further dissolution process, and adding can be in different time detecting result after dyeing liquor, and colour developing result is stable, and quality is more controlled.
Summary of the invention
The object of this invention is to provide a kind of Determination of biological activity method of the monoclonal antibody that is applicable to recombinant anti human EGF-R ELISA.
The present invention adopts the recombinant anti human EGF-R ELISA monoclonal antibody of variable concentrations, is combined with the EGF-R ELISA of the surface of cell membrane of specific tumors cell, and the propagation of specific tumors cell is suppressed, and the method comprises the steps:
(1) prepare specific tumors cell suspension; The cell membrane energy overexpression EGF-R ELISA (EGFR) of described specific tumors cell;
(2) prepare the monoclonal antibody solution of the recombinant anti human EGF-R ELISA (EGFR) of variable concentrations;
(3) monoclonal antibody of the recombinant anti human EGF-R ELISA of specific tumors cell and variable concentrations is hatched 68-72 hour, adds cell count staining reagent CCK-8 concussion to mix, 37 ℃ of incubation 2-4 hour;
(4) in microplate reader take 630nm as reference wavelength, in wavelength 450nm place measure absorbance, calculate the biologic activity of anti-EGFR sample with respect to anti-EGFR standard items by data processing.
Specific tumors cell behaviour lung cancer lymphatic metastasis cell H292 in described step (1), this cell is sensitiveer, is applicable to the anti-egfr antibodies of high-affinity and medium affinity, and the density of described specific tumors cell suspension is (6-10) × 10 4individual/mL, preferred density is 8 × 10 4individual/mL.
The monoclonal antibody of the recombinant anti human EGF-R ELISA (EGFR) described in described step (2) is the antibody from EGFR with different affinity, (Ben-Fillippo et al.J Pharmacokine Pharmacodyn (2012) 39:125-139) as shown in table 1, be specially any one of Buddhist nun's trastuzumab, Cetuximab (Cetuximab), Victibix (Panitumumab) or Zalutumumab, be preferably Buddhist nun's trastuzumab; The monoclonal antibody solution example of described recombinant anti human EGF-R ELISA (EGFR) or standard items concentration dilution be to (300-1000) μ g/mL, then downward 5 times or 7 dilutabilitys of 4 times of doubling dilutions, and preferably initial concentration is 300 μ g/mL.
Affinity and the hypotype of table 1, different anti-EGFR monoclonal antibody
Antibody Affinity/Avidity(M) Isotype
Panitumumab 5X10 -11 IgG2
Cetuximab 4X10 -10 IgG1
IMC-11F8（Nectitumumab） 3X10 -10 IgG1
Nimotuzumab 1X10 -9 IgG1
Zalutumumab 7X10 -9 IgG1
The data processing of described step (4) is to use GraphPad Prism software to carry out data regression, according to the F value between the sigmoid curve of the inhibiting rate computing reference product under different pairs concentration and sample, and the median effective dose concentration IC of computing reference product and sample 50rand IC 50s.
The data processing of described step (4) is further for to utilize four parametric regressions of GraphPad Prism software to process, when the matched curve collimation hypothesis of reference material and sample is confirmed after (P value >0.05), and curve coefficients R 2>0.9025(is R>0.95), use curve maximal value, minimum value, the slope of constrained, the median effective dose concentration IC of computing reference product and sample 50rand IC 50s.
The application of the Determination of biological activity method that the present invention also provides a kind of recombinant anti human EGF-R ELISA (EGFR) monoclonal antibody in the Quality Control of anti-recombinant human epidermal growth factor acceptor (EGFR) monoclonal antibody.
The present invention can be combined with the EGFR of H292 cell surface according to the monoclonal antibody of recombinant anti human EGF-R ELISA (EGFR), Buddhist nun's trastuzumab of variable concentrations is combined with EGFR, be dose dependent (four parametric regressions, inverse sigmoid curve), according to half effective inhibition concentration (IC 50) comparison, can calculate the biologic activity of anti-EGFR monoclonal antibody sample with respect to anti-EGFR monoclonal antibody standard items.
The in-vitro multiplication inhibition method that the present invention adopts is measured the biologic activity of recombinant anti human EGFR monoclonal antibody, has the advantages such as applicability is wide, accuracy is high, quality is more controlled.
Accompanying drawing explanation
Fig. 1. the anti-EGFR monoclonal antibody of variable concentrations is to H292 cell proliferation inhibition rate
Fig. 2. the specificity of Buddhist nun's trastuzumab Determination of biological activity
Fig. 3: the repeatability of Buddhist nun's trastuzumab Determination of biological activity
Fig. 4: the middle precision of Buddhist nun's trastuzumab Determination of biological activity
Embodiment
Embodiment of the present invention illustrate by the following example.But, should be appreciated that embodiment of the present invention are not limited to the specific detail of these embodiment, because for the person of ordinary skill of the art, other variation is known, or is apparent according to direct disclosed content and appended claims.Therefore, all technology realizing based on foregoing of the present invention all belong to scope of the present invention.The list of references of quoting is herein incorporated to herein in full by reference with it.
Experimental technique described in following embodiment, if no special instructions, is conventional method; Described reagent and biomaterial, if no special instructions, all can obtain from commercial channels.
Embodiment 1: in-vitro multiplication inhibition method is measured the biologic activity of the monoclonal antibody of recombinant anti human EGF-R ELISA
The method that adopts people's lung cancer lymphatic metastasis cell H292 cell line (purchased from Centro De Inmunologia Molecular of Cuba) to measure anti-egfr antibodies activity comprises the steps:
(1) prepare H292 cell suspension: H292 cell suspension cell viability should be not less than 85%, cell count.In clean bench, being made into density with complete culture solution is (6～10) × 10 4the cell suspension of individual/mL.
(2) cell suspension inoculation is in 96 orifice plates, and 100 μ L/ holes (blank hole adds 100 μ L complete culture solutions) are placed in 37 ℃ of CO 2in incubator, cultivate.
(3) prepare Buddhist nun's trastuzumab solution of variable concentrations, Buddhist nun's trastuzumab solution example or standard items are diluted to (300-1000) μ g/mL, then downward 5 times or 7 dilutabilitys of 4 times of doubling dilutions with RPMI1640 nutrient solution/1% hyclone (FBS).
(4) H292 cell is abandoned supernatant after 96 orifice plates are cultivated 18-20 hour, washes plate once with 180 μ L maintain liquid, abandons supernatant, and according to the form below adds sample and the reference material after 200 μ L dilutions, and each concentration is done 2 multiple holes and can be selected C/D behavior sample capable; E/F behavior is with reference to conduct, and positive hole and blank well all add 200 μ L maintain liquid, and 96 orifice plates are placed in 37 ℃ of CO 2in incubator, cultivate.
Figure BDA0000469556580000051
Figure BDA0000469556580000061
(5) cultivate after 68-72 hour, every hole adds CCK-8 dilution 30 μ L, and in 37 ℃ of enzyme mark incubation shaking tables, 300rpm concussion mixes 10 minutes, then in 37 ℃, CO 2incubation 2～4 hours (from the complete calculating of CCK-8 dilution application of sample) in incubator.In microplate reader, wavelength 450nm place (reference wavelength 630nm) measures absorbance.
Calculate according to the following formula the inhibiting rate in every hole:
Inhibiting rate %=(positive control hole mean light absorbency-test hole absorbance)/(mean light absorbency-blank hole, positive control hole mean light absorbency) × 100%
The anti-EGFR monoclonal antibody of variable concentrations to H292 cell inhibitory rate as shown in Figure 1.
(6) data processing: use the log[agonist under Sigmoidal dose-response – Stimulation in the non-linear regression (Nonlinear Regression (curve fit)) of GraphPad Prism software (version5.01)] vs.response--variable slope carries out data regression.According to the F value between the sigmoid curve of the inhibiting rate computing reference product under different pairs concentration and sample (i.e. four parametric lines), the median effective dose concentration IC of computing reference product and sample 50rand IC 50s.
In described step (1), H292 cell density is preferably 8x10 4individual/mL.
The initial saturated Buddhist nun's trastuzumab concentration that suppresses EGFR in described step (3) is preferably 300 μ g/mL.
In described step (6), further utilize four parametric regressions of GraphPad Prism software to process, when the matched curve collimation hypothesis of reference material and sample is confirmed after (P value >0.05), and curve coefficients R 2>0.9025(is R>0.95), use curve maximal value, minimum value, the slope of constrained, the median effective dose concentration IC of computing reference product and sample 50rand IC 50s.
Embodiment 2: body outer cell proliferation inhibition method is measured the method validation of anti-EGFR monoclonal antibody biologic activity
Adopt lung cancer lymphatic metastasis cell H292 cell line (purchased from Centro De Inmunologia Molecular of Cuba) to measure the method validation of Buddhist nun's trastuzumab biologic activity, comprise the steps:
(1) specificity: Buddhist nun's trastuzumab is human IgG1's subclass κ type immunoglobulin (Ig), has been used to belong to human IgG1's subclass κ type immunoglobulin (Ig) together and contrast as homotype, and test at least repeats 2 times.Acceptable standard: the non-EGFR monoclonal antibody of homotype should with almost no cross reaction of H292 cell, do not present similar response curve.
As shown in Figure 2, homotype control antibodies T1h monoclonal antibody and almost no cross reaction of H292 cell, do not present similar response curve to result, illustrates that T1h monoclonal antibody is in the time of Cmax, do not suppress H292 cell enlargement yet.
(2) repeatability: with by same analyst, same test sample being tested from the parallel dilution of initial concentration in single test, and carry out at different tests plate.Test at least repeats 3 times.Acceptable standard: the coefficient of variation (relative standard deviation) between the replication of same sample must not be greater than 50%.
As shown in Figure 3, data result is as follows for the four repeated parametric line analysis charts of the Determination of biological activity of Buddhist nun's trastuzumab:
Figure BDA0000469556580000071
Upper table shows that the relative activity of twice replication result of same sample is respectively 119% and 120%, and the coefficient of variation (relative standard deviation) is 0.6%, is less than 50%, meets repeated requirement.
(3) precision in the middle of: same test sample need not measured on the same day or be measured by different analysts by same analyst, and test repeats 2-3 time.Acceptable standard: same sample not on the same day the coefficient of variation between replication (relative standard deviation) must not be greater than 50%.
To the assessment of same sample precision in the middle of different time carries out, as shown in Figure 4, data result is as follows for four parametric line analysis charts:
Figure BDA0000469556580000072
The coefficient of variation between the replication of the above results demonstration Determination of biological activity is respectively 6.5% and 23%, the variation that shows Determination of biological activity is larger, this is relevant with the complicacy of biological products based on activity of cell biology assay method, but the coefficient of variation is less than 50%, meet acceptable standard, in the middle of the stage of method optimization at present, precision is acceptable.
(4) accuracy: carry out the recovery of determination methods by known activity (reference material 100%) the mensuration accuracy of test sample.The recovery=biology relative activity measured value/biology relative activity given value.Acceptable standard: its accuracy (50%～200%) in measurement range, the coefficient of variation (CV%<50%) should meet the requirements.
The sample of known organism being learned to active (reference material 100%) has carried out respectively 2 times, 3 times tests, and result is as shown in the table:
Figure BDA0000469556580000081
Upper table demonstration, the recovery of twice test is respectively 121% and 177%, meets the measurement range requirement of 50%-200%, and the coefficient of variation is 27%, is less than 50%, so this Determination of biological activity method meets the requirement of accuracy.
Can draw inference from the party's science of law the result, use body outer cell proliferation inhibition method to measure repeatability better, the methodological coefficient of variation is not more than 30%.
Embodiment 3: the biologic activity comparison of the anti-EGFR monoclonal antibody of different restructuring
High-affinity antibody: with EGFR affinity 10 -10～10 -11
Medium affinity antibodies: with EGFR affinity 10 -8～10 -9
The high-affinity antibody adopting in the present embodiment is Cetuximab, and medium affinity antibodies is Buddhist nun's trastuzumab.Carry out anti-EGFR monoclonal antibody Determination of biological activity according to the step of embodiment 1, test figure is as shown in the table:
? High-affinity antibody Medium affinity antibodies
Minimum value ～-194.2 -0.1674
Maximal value 49.3 41.64
Log?IC 50 ～-3.583 0.2132
Slope (Hillslope) 0.8181 0.9383
IC 50（ug/mL） ～0.0002611 1.634
Curve degree R 2 0.909 0.9248
Learn the IC of high-affinity antibody from upper table data 50value is about 0.0002611, the IC of medium affinity antibodies 50value is 1.634, show that H292 cell line is all more responsive to different affinity antibodies, be specially adapted to the low Determination of biological activity to medium affinity antibodies, solved the low antibody to medium affinity in insensitive problems of conventional cell line such as A431 cell line, DiFi cell line, MDA-MB-468 cell lines.The IC of high-affinity anti-egfr antibodies 50be worth lowlyer, only need will after the sample initial concentration adjustment in embodiment 1, can obtain better matched curve, therefore, the anti-egfr antibodies of different affinity all can carry out biologic activity detection by this cell line.

Claims (7)

1. a Determination of biological activity method for recombinant anti human EGF-R ELISA monoclonal antibody, is characterized in that, the method comprises following steps:
(1) prepare specific tumors cell suspension; The cell membrane overexpression EGF-R ELISA of described specific tumors cell;
(2) prepare the monoclonal antibody solution of the recombinant anti human EGF-R ELISA of variable concentrations;
(3) monoclonal antibody of the recombinant anti human EGF-R ELISA of specific tumors cell and variable concentrations is hatched 68-72 hour, adds cell count staining reagent CCK-8 concussion to mix, 37 ℃ of incubation 2-4 hour;
(4) in microplate reader take 630nm as reference wavelength, in wavelength 450nm place measure absorbance, calculate the biologic activity of anti-EGFR sample with respect to anti-EGFR standard items by data processing.
2. the Determination of biological activity method of recombinant anti human EGF-R ELISA monoclonal antibody according to claim 1, it is characterized in that, specific tumors cell behaviour lung cancer lymphatic metastasis cell H292 in described step (1), the density of described specific tumors cell suspension is 6 × 10 4-10 × 10 4individual/mL, preferred density is 8 × 10 4individual/mL.
3. the Determination of biological activity method of recombinant anti human EGF-R ELISA monoclonal antibody according to claim 1, it is characterized in that, the monoclonal antibody of the recombinant anti human EGF-R ELISA in described step (2) is any one of Buddhist nun's trastuzumab, Cetuximab, Victibix or Zalutumumab, the monoclonal antibody solution example of described recombinant anti human EGF-R ELISA or standard items concentration dilution are to 300-1000 μ g/mL, preferred concentration is 300 μ g/mL, then downward 5 times or 7 dilutabilitys of 4 times of doubling dilutions.
4. the Determination of biological activity method of recombinant anti human EGF-R ELISA monoclonal antibody according to claim 3, is characterized in that, the monoclonal antibody of described recombinant anti human EGF-R ELISA is Buddhist nun's trastuzumab.
5. the Determination of biological activity method of recombinant anti human EGF-R ELISA monoclonal antibody according to claim 1, it is characterized in that, the data processing of described step (4) is for being used GraphPad Prism software to carry out data regression, according to the F value between the sigmoid curve of the inhibiting rate computing reference product under different pairs concentration and sample, the median effective dose concentration IC of computing reference product and sample 50rand IC 50s.
6. the Determination of biological activity method of recombinant anti human EGF-R ELISA monoclonal antibody according to claim 1, it is characterized in that, the data processing of described step (4) is to utilize four parametric regressions of GraphPad Prism software to process, when the matched curve collimation hypothesis of reference material and sample is confirmed after (P value >0.05), and curve coefficients R 2>0.9025(is R>0.95), use the curve maximal value of constrained, minimum value, slope, the median effective dose concentration IC of computing reference product and sample 50rand IC 50s.
7. the application of the recombinant anti human EGF-R ELISA monoclonal antibody Determination of biological activity method described in claim 1-5 in the Quality Control of recombinant anti human EGF-R ELISA monoclonal antibody.
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