CN102262155A - Method for measuring biological activity of recombined human epidermal growth factor and application of method - Google Patents
Method for measuring biological activity of recombined human epidermal growth factor and application of method Download PDFInfo
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Abstract
The invention discloses a method for measuring the biological activity of a recombined human epidermal growth factor (rhEGF) and application of the method. The method comprises the following steps of: blocking the combination between the EGF and the EGFR by an antibody to indirectly measure the biological activity of EGF and EGF receptor (EGFR) combination by a competitiveness-restrained flow cytometry; processing experimental data by adopting a computer program (four-parameter regression) to acquire half effective inhibition concentration (EC50) of an EGF standard substance and an EGF sample respectively and thus obtaining the relative biological activity of the EGF sample; and calculating the biological activity of the EGF sample according to the biological activity (IU/mL) of the EGF standard substance. In the method, the test period is only one day from sample injection except cell pre-culturing; a germfree environment is not needed during test; and compared with the biological activity measurement method for the rhEGF in the Chinese Pharmacopoeia, the method has the advantages that: the method is more convenient to realize and the quality is more controllable.
Description
Technical field
The present invention relates to the Determination of biological activity field of Porcine HGF, more specifically to the Determination of biological activity method and the application thereof of recombinant human epidermal growth factor (EGF).
Background technology
HEGF (EGF) is the polypeptide that contains 53 amino acid residues, and molecular weight is 6045 dalton.In vivo with experiment in vitro in EGF (EGF-R ELISA EGFR) stimulates epithelium and mesenchymal cell to grow by the acceptor on the cell membrane.
At present general recombinant human epidermal growth factor Determination of biological activity method adopts cell proliferation method/MTT colourimetry (" Chinese pharmacopoeia, version was three ones in 2010, the 74th page of appendix), this method has spread effect according to recombinant human epidermal growth factor to the growth of mouse embryo fibroblasts (Balb/c 3T3 cell), the upgrowth situation of Balb/c 3T3 cell is different because of the difference of recombinant human epidermal growth factor biologic activity, detects the biologic activity of recombinant human epidermal growth factor with this.Its determination method is:
In 37 ℃, the cultivation of 5% carbon dioxide, the control cell concentration is that every 1ml contains 1.0 * 10 to Balb/c 3T3 cell line with complete culture solution
5～5.0 * 10
5Individual cell, the back of going down to posterity was used for Determination of biological activity in 24～36 hours.Discard the nutrient solution in the culture flask, digestion and collecting cell are made into every 1ml with complete culture solution and contain 5.0 * 10
4～8.0 * 10
4The cell suspension of individual cell is inoculated in the 96 porocyte culture plates, every hole 100 μ l.Under 37 ℃, 5% carbon dioxide conditions, cultivate.Change into after 24 hours and keep nutrient solution, put under 37 ℃, 5% carbon dioxide conditions and cultivated 24 hours.The Tissue Culture Plate of preparation discards and keeps liquid, adds standard solution and need testing solution, and every hole 100 μ l put under 37 ℃, 5% carbon dioxide conditions and cultivated 64～72 hours.Every hole adds MTT solution 20 μ l, cultivates 5 hours under 37 ℃, 5% carbon dioxide conditions.More than operate under the aseptic condition and carry out.After discarding the liquid in the culture plate, adding dimethyl sulfoxide (DMSO) (DMSO) 100 μ l in every hole, on microplate reader, is reference wavelength with 630nm behind the mixing, measures absorbance in wavelength 570nm place, the record measurement result.Test figure adopts computer program or four parametric regression computing methods to handle.
As far back as 1963, Slater just found that the dehydrogenasa in the mitochondria (as succinate dehydrogenase, diaphorase) can open the azoles ring of tetramethyl azo azoles salt (MTT), generated blue product first
(formazan).Around this principle, Mosmann (Mosmann T.Rapid colorimetric assay for celluar growth and survival:application to proliferation and cytotoxicity assays.J Immunol Methods, 1983,65:55) founded the MTT detection method in nineteen eighty-three, this method is very effective aspect the survival of measuring cell and propagation, because this change in color only occurs over just in the cell alive, and first
Formation amount and viable count and functional status be proportional.At present the MTT determination method has been widely used in the testing of multiple growth factor such as epidermal growth factor, TNF, interleukin 2 etc.
The MTT colourimetry is subjected to than multifactor impact, as cell number, MTT concentration and retention time, lysate consumption, and have sometimes that susceptibility is on the low side, some problems such as solubleness that organic solvent produces protein precipitation and product is on the low side.The MTT colourimetry adds from sample, and the test period was at least 4 days, needs to keep gnotobasis in the test.
, experimental period loaded down with trivial details in view of bioassay method length, poor accuracy, error big (Liu Lan etc., the Chinese biological goods are learned magazine, 2002; 15 (3): 175-176), expend a large amount of human and material resources, therefore in drug standard from now on biologicall test will be gradually by advanced person's physico-chemical method, maybe can guarantee clinical drug safety effectively other method replace, or only be present in the production conceptual phase.
Summary of the invention
The present invention at above shortcomings in traditional MTT colourimetry, develops the competitive fluidic cell method that suppresses on the basis of existing technology, and it is active with combining of EGF-R ELISA (EGFR) to be used for detecting epidermal growth factor (EGF).Except that cultured cell in advance, add from sample, the test period is 1 day only, need not gnotobasis in the process of the test, is its test principle below:
The monoclonal antibody of EGF and anti-epidermal growth factor receptor can be competitive in conjunction with EGFR, the biologic activity that the combination by antibody blocking EGF and its acceptor comes indirect determination EGF to combine with EGFR.
Anti-human epidermal growth factor acceptor monoclonal antibody fluidic cell method Determination of biological activity principle: in detection architecture, add second monoclonal antibody of fluorescein-labeled isothiocyanic acid, measure the fluorescence intensity of second monoclonal antibody-former monoclonal antibody-EGFR bond by indirect immunofluorescence.The high low reaction of fluorescence intensity the variation of antibody concentration, result's calculating can be passed through four parametric line analyses (sigmoid curve), obtains half effectively in conjunction with concentration (EC
50), compare the number percent of the relative biologic activity of calculating antibody concentration (according to " Chinese pharmacopoeia with reference material, the sigmoid curve that quantitative response parallel lines principle is inferred sample and reference material in version in 2010, two appendix biologic assays has identical maximal value, minimum value and slope).
Pacing items: Guava Easycyte Mini Flow Cytometer flow cytometer.Adopt the Guava ExpressPlus program in CytoSoft 4.1 softwares.Selected specific cells group (EGFR expresses cell mass preferably) is as the counting object.
Active somatic cell does not take place process of the test apoptosis or propagation suppress, and the mensuration process does not need gnotobasis, compares with immunofluorescence, can carry out quantitative measurement (EC
50).
According to above-mentioned monoclonal antibody Determination of biological activity principle, the monoclonal antibody that can design the anti-human epidermal growth factor acceptor that adopts saturation concentration is with the competitive EGFR in conjunction with the tumor cell membrane surface of the recombinant human epidermal growth factor of variable concentrations, adding fluorescein-labeled second monoclonal antibody then combines with above-mentioned monoclonal antibody on the surface of cell membrane, on flow cytometer, adopt specific program and select specific cell compartment to obtain the average fluorescent strength value of each sample, test figure adopts computer program GraphPad Prism software (four parametric regressions) to handle, and obtains the half effective inhibition concentration (EC of EGF standard items and sample respectively
50), thereby obtain the relative biologic activity of EGF sample with respect to the EGF standard items.According to the biologic activity (IU/mL) of EGF standard items, can calculate the biologic activity of EGF sample.
The present invention is with " the recombinant human epidermal growth factor Determination of biological activity method in the Chinese pharmacopoeia is compared, and that assay method of the present invention is implemented is more convenient (except that cultured cell in advance, can finish test in one day, need not gnotobasis in the process of the test; Official method needs four days at least, and process of the test needs gnotobasis), quality is more controlled.
The Determination of biological activity method that the purpose of this invention is to provide a kind of recombinant human epidermal growth factor, adopt anti-human epidermal growth factor acceptor monoclonal antibody to combine the EGF-R ELISA of the surface of cell membrane of specific tumors clone with the potpourri competitiveness of the recombinant human epidermal growth factor of variable concentrations, on flow cytometer, adopt specific program and select specific cell compartment to obtain the average fluorescent strength value of each sample.
The present invention realizes by following technical scheme:
A kind of Determination of biological activity method of recombinant human epidermal growth factor, its step comprises:
1), under the condition that is fit to, cultivates specific tumors cell (detection cell);
2), EGF sample and EGF standard items are diluted in the monoclonal antibody of anti-human epidermal growth factor acceptor of saturation concentration, with 1) in the specific tumors cell effect;
3), the centrifugal supernatant of outwelling, suspension cell adds fluorescein-labeled second monoclonal antibody again, reacts 15-60 minute;
4), detect cell average fluorescent strength, by data processing calculating EGF sample biologic activity.
Specific tumors cell in the described step 1) is moderate or people's cell of highly expressing EGF-R ELISA, and described specific tumors cell is 7X10 with the concentration of the adjusted suspension of PBS
6Individual/mL.
Described specific tumors cell is preferably any one of human lung adenocarcinoma cell H-125 clone, A431, DiFi; Human lung adenocarcinoma cell H-125 clone more preferably.
The monoclonal antibody of the anti-human epidermal growth factor acceptor described step 2) can be Buddhist nun's trastuzumab or Cetuximab; The antibody concentration that is adopted is 50ug/mL, and the EGF dilution of variable concentrations is 10 dilutabilitys of the downward doubling dilution of 25ug/mL.
Reaction time in the described step 3) is preferably 30 minutes.
Described step 4) is the centrifugal supernatant of outwelling, again suspension cell, add a certain amount of 1% paraformaldehyde PBS, on flow cytometer, adopt specific program and select specific cell compartment to obtain the average fluorescent strength value of each sample, test figure adopts computer program GraphPad Prism software (four parametric regressions) to handle, and obtains the half effective inhibition concentration (EC of EGF standard items and sample respectively
50), thereby obtain the relative biologic activity of EGF sample with respect to the EGF standard items.According to the biologic activity (IU/mL) of EGF standard items, can obtain the biologic activity (IU/mL) of EGF sample.
The present invention provides the application of Determination of biological activity method in the Quality Control of recombinant human epidermal growth factor or derivatives thereof of recombinant human epidermal growth factor simultaneously.
The recombinant product biologic activity is a detection index important in the Quality Control, has stable biologically active to guarantee product.
The present invention can combine EGFR with the monoclonal antibody competitiveness of anti-human epidermal growth factor acceptor according to recombinant human epidermal growth factor (EGF), under the prerequisite of monoclonal antibody with saturation concentration, the competitive monoclonal antibody that suppresses of EGF combines with EGFR, be dose dependent (four parametric regressions, anti-sigmoid curve), according to half effective inhibition concentration (EC
50) comparison, can calculate the biologic activity of EGF sample with respect to the EGF standard items.
The biologic activity that the fluidic cell method that the present invention adopts competition to suppress is measured recombinant human epidermal growth factor has advantages such as step is easy, accuracy is high, quality is more controlled.
Description of drawings
The amount effect curve of Fig. 1, EGF sample and EGF standard items
Four parametric lines (Buddhist nun's trastuzumab) are tested in Fig. 2, the checking of 70%R-100%R-150%R fluidic cell method for the first time
The amount effect curve of Fig. 3, EGF component 1 and EGF component 2
Embodiment
Below be non-limiting examples of the present invention, will help those of ordinary skill in the art and further understand the present invention.
The method that adopts human lung adenocarcinoma cell H-125 clone (from Cuba molecular immunology center) to measure the EGF activity comprises the steps:
(1) Cell Culture Lab is cultivated certain amount of H-125 cell, and cell viability generally should be not less than 70%.Under 4 ℃, 1100rpm centrifugal 5 minutes.Carefully outwell supernatant, utilize remaining small amount of liquid that cell precipitation is dissolved, slowly add 10mL left and right sides PBS then, mixing.Under 4 ℃, 1100rpm centrifugal 5 minutes once more, carefully outwell supernatant, utilize remaining small amount of liquid that cell precipitation is dissolved, slowly add 10mL left and right sides PBS, mixing.After centrifugal 5 minutes, carefully outwell supernatant under 4 ℃, 1100rpm, utilize remaining small amount of liquid that cell precipitation is dissolved, cell suspension to the concentration of adjusting H-125 clone with PBS is 5X10
6～10X10
6Individual/mL.
(2) Buddhist nun's trastuzumab solution of preparation 50 μ g/mL and 100 μ g/mL, with Buddhist nun's trastuzumab solution of 100 μ g/mL EGF sample or the standard items of 50 μ g/mL are diluted to 25 μ g/mL EGF (containing 50ug/mL Buddhist nun's trastuzumab), use 10 dilutabilitys of the downward doubling dilution of 50ug/mL Buddhist nun's trastuzumab solution then.
(3) each centrifuge tube adds the corresponding sample solution of 20 μ L: 50ug/mL Buddhist nun's trastuzumab solution, 25 μ g/mL EGF (containing 50ug/mL Buddhist nun's trastuzumab) with and 10 downward doubling dilution liquid.Under 4 ℃, 1100rpm centrifugal 1 minute, 4 ℃ stand-by (the stand-by time was less than 4 hours) down.
(4) in the centrifuge tube of existing 20 μ L sample solutions, add 25 μ L cell suspension, get mixing with nose heave the reverting to take drugs of rifle.4 ℃ are incubated 30 minutes down.
(5) in each centrifuge tube, add 700 μ LPBS, under 4 ℃, 1100rpm centrifugal 4 minutes.
(6) carefully remove supernatant, vibration gently on the vortex oscillator.In each centrifuge tube, add the anti-people FITC of 20 μ L (DAKO F0056) dilute solutions (dilution ratio: 1: 30), get mixing with nose heave the reverting to take drugs of rifle.4 ℃ are incubated 30 minutes down.
(7) in each centrifuge tube, add 700 μ LPBS, under 4 ℃, 1100rpm centrifugal 4 minutes.
(8) carefully remove supernatant, vibration gently on the vortex oscillator.In each pipe, add 500 μ L, 1% paraformaldehyde PBS then.Read the data (average fluorescent strength in selected zone) of sample in the flow cytometer with the specific program (Guava Express Plus) of H-125 clone.
(9) data processing: adopt computer program (four parametric regressions) to handle, obtain the half effective inhibition concentration (EC of EGF standard items and sample then respectively
50), thereby obtain the relative biologic activity of EGF sample with respect to the EGF standard items.Its amount effect curve is seen Fig. 1.
Preferably the concentration of cell suspension is 7x10 in the step (1)
6Individual/mL.
Further preferably step (3) is 50 μ g/mL with initial saturated Buddhist nun's trastuzumab concentration that the EGF competition suppresses EGFR.
Further preferably, comprise in the step (9) and utilize GraphPad Prism software (four parametric regressions) to handle, infer the EGF sample according to quantitative response parallel lines principle in two appendix biologic assays of version Chinese Pharmacopoeia in 2010 and have identical maximal value, minimum value and slope, obtain the half effective inhibition concentration (EC of EGF standard items and sample then respectively with the EGF standard items
50) (EC
50Be inversely proportional to biologic activity), thus the relative biologic activity of EGF sample obtained with respect to the EGF standard items, according to the biologic activity (IU/mL) of EGF standard items, can calculate the biologic activity (IU/mL) of EGF sample.
Use identical fluidic cell method to measure in the methodology checking of the relative biology of Buddhist nun's trastuzumab in conjunction with activity:
The method that adopts human lung adenocarcinoma cell H-125 clone (from Cuba Centro De Inmunologia Molecular) to measure the relative biologic activity of Buddhist nun's trastuzumab comprises the steps:
(1) preparation 50 μ g/mL Buddhist nun trastuzumab reference material dilution and sample diluting liquids dilute 8 dilutability to 0.050 μ g/mL downwards with the monoclonal antibody dilution then;
(2) each centrifuge tube add 20 μ L variable concentrations reference material dilution and sample diluting liquid, subsequent step is with embodiment 1;
(3) data processing: adopt computer program (four parametric regressions) to handle, the half that obtains Buddhist nun's trastuzumab reference material and sample then respectively is effectively in conjunction with concentration (EC
50), thereby obtain the relative biologic activity of Buddhist nun's trastuzumab sample with respect to Buddhist nun's trastuzumab reference material.
Measure in the methodology checking of biologic activity in this flow cytometer method:
(1) accuracy: use reference material certainly as contrast, with respect to the biologic activity of reference material R, the relative error absolute value should be not more than 10% when measuring 70%R, 150%R and 100%R.
Annotate: 100%R means that the reference material sample in like manner dilutes 2 parts of serial dilutions, and portion is a reference material self, and portion is 100%R in addition; 70%R and 150%R mean that the contained reference material concentration of this sample series dilution is 70% or 150% of corresponding reference material serial dilutions, but indicate concentration still with reference to corresponding reference material serial dilutions concentration; Follow-up 50%R and 200%R are in like manner.
(2) precision:
Middle precision: need to use under two different concentration known this method to measure the biologic activity of test sample with respect to reference material, replication 2 times is undertaken by 2 different operators in 2 different tests skies; The coefficient of variation between the replication of same sample must not be greater than 15%.
Repeatability: in this research, need to measure the biologic activity of same test sample with respect to reference material by same analyst.Test needs to repeat more than 2 times; The coefficient of variation of repeatability must not be greater than 15%.
Cells were tested by flow cytometry biologic activity methodology checking result:
(1) accuracy
Accuracy testing is carried out three times altogether, and preceding twice test undertaken by a biologic activity assistant director, tests for the third time by another assistant director and is undertaken, and four parametric plots of test are seen Fig. 2 for the first time.
Table 1 accuracy testing for the first time
Calculate relative error (down together) according to formula (1-1):
When test sample is 70% reference material: the ÷ 70% * 100%=8.3% of relative error=(75.8%-70%)
When test sample is 150% reference material: relative error=-8.5%
Table 2 accuracy testing for the second time
When test sample is 100% reference material: relative error=-6.5%
Table 3 is accuracy testing for the third time
When test sample is 70% reference material: relative error=1.3%
When test sample is 150% reference material: relative error=-5.6%
Above-mentioned test findings shows that in 70%～150% reference material biology relative activity scope this method is (the relative error absolute value is not more than 10%) accurately.
(2) precision
The precision test bay coefficient of variation in the middle of the table 4
| Sample | Result-1 | Result-2 | | SD | CV | |
| 70％R | 75.8％ | 70.9％ | 73.35％ | 0.034648 | 4.8％ | |
| 150％R | 137.2％ | 141.6％ | 139.40％ | 0.031113 | 2.3％ |
Above-mentioned test findings show this method meet in the middle of precision requirement (coefficient of variation is not more than 15%), raw data sees Table 1 and table 2.
The table 5 replica test coefficient of variation:
Above-mentioned test findings shows that this method meets repeatability and requires (coefficient of variation is not more than 15%).
Measure the relative biology of Buddhist nun's trastuzumab from this fluidic cell method and verify that in conjunction with the methodology of activity the result can draw inference, relative error is no more than 10% when using this fluidic cell method to measure the EGF biologic activity, and the methodological coefficient of variation is not more than 15%.
Step by embodiment 1 is carried out the EGF Determination of biological activity, and test figure sees Table 6.
The different rEGF biologic activity of table 6 relatively
| |
|
|
| Minimum value | 10.00 | 10.00 |
| Maximal value | 279.1 | 279.1 |
| ?LogED 50 | -0.5096 | -0.4883 |
| Slope (Hillslope) | -0.7530 | -0.7530 |
| ?ED 50(ug/mL) | 0.3093 | 0.3249 |
| Curve fitting degree R 2 | 0.9987 | 0.9976 |
The result shows:
ED
50(EGF component 1)=0.3093ug/mL
ED
50(EGF component 2)=0.3249ug/mL
The comparison of flow cytometer method and pharmacopeia mtt assay in the embodiment 4 recombinant human epidermal growth factor Determination of biological activity
By inference, the result with fluidic cell method (EGF with Buddhist nun's trastuzumab competition in conjunction with EGFR) and pharmacopeia mtt assay mensuration recombinant human epidermal growth factor biologic activity should have comparability: the relative biologic activity that the EGF sample that two kinds of detection methods obtain is compared with the EGF standard items is answered basically identical.But compare with the pharmacopeia mtt assay, the fluidic cell method has advantages such as step is easy, accuracy is high, quality is more controlled; These two kinds of methods can complement one another, and the fluidic cell method can be used as conventional quantitatively use, and the pharmacopeia mtt assay can be reduced to the sxemiquantitative discrimination method and use (every batch is only carried out once in quality control is let pass).
Claims (8)
1. recombinant human epidermal growth factor Determination of biological activity method, adopt anti-human epidermal growth factor acceptor monoclonal antibody with the EGF-R ELISA of recombinant human epidermal growth factor competitiveness in conjunction with the surface of cell membrane of specific cells, on flow cytometer, adopt specific program and select specific cell compartment to obtain the average fluorescent strength value of each sample, it is characterized in that this method comprises following steps:
1), under the condition that is fit to, cultivates the specific tumors cell;
2), EGF sample and EGF standard items are diluted in the monoclonal antibody of anti-human epidermal growth factor acceptor of saturation concentration, with 1) in cell effect;
3), the centrifugal supernatant of outwelling, suspension cell adds fluorescein-labeled second monoclonal antibody again, reacts 15-60 minute;
4), detect cell average fluorescent strength, by data processing calculating EGF sample biologic activity.
2. recombinant human epidermal growth factor Determination of biological activity method according to claim 1 is characterized in that, the specific tumors cell in the described step 1) is moderate or people's cell of highly expressing EGF-R ELISA; Described specific tumors cell is 7X10 with the concentration of the adjusted suspension of PBS
6Individual/mL.
3. the Determination of biological activity method of recombinant human epidermal growth factor according to claim 2 is characterized in that, described specific tumors cell is preferably any one of human lung adenocarcinoma cell H-125 clone, A431, DiFi.
4. the Determination of biological activity method of recombinant human epidermal growth factor according to claim 3 is characterized in that, described specific tumors cell is human lung adenocarcinoma cell H-125 clone more preferably.
5. the Determination of biological activity method of recombinant human epidermal growth factor according to claim 1 is characterized in that, described step 2) in the monoclonal antibody of anti-human epidermal growth factor acceptor can be Buddhist nun's trastuzumab or Cetuximab; The Buddhist nun's trastuzumab antibody concentration that is adopted is preferably 50ug/mL.
6. the Determination of biological activity method of recombinant human epidermal growth factor according to claim 1 is characterized in that, the reaction time in the described step 3) is preferably 30 minutes.
7. recombinant human epidermal growth factor Determination of biological activity method according to claim 1, it is characterized in that: described step 4) is the centrifugal supernatant of outwelling, again suspension cell, add a certain amount of 1% paraformaldehyde PBS, on flow cytometer, adopt specific program and select specific cell compartment to obtain the average fluorescent strength value of each sample, test figure adopts computer program GraphPad Prism software (four parametric regressions) to handle, and obtains the medium effective concentration (EC of EGF standard items and sample respectively
50), thereby obtain the relative biologic activity of EGF sample with respect to the EGF standard items, according to the biologic activity (IU/mL) of EGF standard items, can obtain the biologic activity (IU/mL) of EGF sample.
8. the application of the Determination of biological activity method of the arbitrary described recombinant human epidermal growth factor of claim 1-7 in the Quality Control of recombinant human epidermal growth factor or derivatives thereof.
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