CN107192828A - For the bio-evaluation method of biopharmaceutical composition - Google Patents
For the bio-evaluation method of biopharmaceutical composition Download PDFInfo
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- CN107192828A CN107192828A CN201710351589.3A CN201710351589A CN107192828A CN 107192828 A CN107192828 A CN 107192828A CN 201710351589 A CN201710351589 A CN 201710351589A CN 107192828 A CN107192828 A CN 107192828A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
Abstract
The present invention relates to a kind of bio-evaluation method for biopharmaceutical composition, including step：It is primarily based on ELISA sandwich methods principle and sets up the quantitative approach for being directed to each protein component of biopharmaceutical composition；Confirm the common biological action mechanism of each protein component of biopharmaceutical composition and bio-evaluation method is set up based on the mechanism of action；The biological activity of the biopharmaceutical composition of different proportion mixing is detected using set up bio-evaluation method, on the basis of certain one-component protein concentration, the biological activity testing result and the EC of 4 parameter curve fits of the biopharmaceutical composition under the conditions of record different mixing proportion50Value.The inventive method can be according to biological activity testing result or the EC of 4 parameter curve fits50The change of value and judge whether certain component of biopharmaceutical composition inactivates, activity research and estimation of stability available for biopharmaceutical composition.
Description
Technical field
The present invention relates to biological technical field, more particularly to bio-evaluation method is specifically referred to a kind of for life
The bio-evaluation method of medicine composition.
Background technology
With the continuous improvement of biotechnology level, including gene clone technology, cell culture and purification technique, biology point
Analysis technology etc. grows continuously and fast, and biopharmaceutical macromolecular drug research and development progressively replace chemical drug small-molecule drug and turn into pharmaceutical field
Study hotspot.It is biological medicament to have 8 in ten medicine before global marketing ranking in 2015, and research and development are in clinic to listing stage
Biological medicament has more than 4500, is that market is powerful, researches and develops hot.At home, bio-pharmaceuticals is listed in national medical industry " ten
Three or five " the key breakthrough field of development plan.And " biological industry doubles and Health China action plan " is then included into country ten
Three or five planning outlines.
Biotech drug mainly includes biochemical the blood product such as clotting factor, albumin for extracting class medicine such as people source
Deng, and genetic engineering class medicine includes vaccine, monoclonal antibody, fusion protein, polypeptide drug etc..Wherein monoclonal antibodies
Immense success and potentiality of the medicine with its security and validity and in antitumor, autoimmune disease field, in biological skill
It is leading in the big development tide of art medicine.There are 6 in ten 8 biotech drugs before global drug product in 2015
Monoclonal antibody drug, and occupy global drug product champion throughout the year be Abbott Laboratories adalimumab.
There is traditional chemical drug small molecule class medicine preferable antitumor cell to act on, but its poor, toxic side effect of selectivity
It is larger, also there is killing to normal cell in patient's body, strongly limit the application and development of chemical drug small molecule class medicine.Biological medicament
1 prominent distinguishing feature and advantage of thing particularly monoclonal antibodies medicine are targeting, with for breast cancer cell table
Exemplified by the Trastuzumab monoclonal antibody of face HER2 acceptors, the medicine in vivo can specific effect incited somebody to action in tumor cell surface HER2 acceptors
Tumour cell kills or suppressed growth of tumour cell, reaches the purpose for the treatment of patient with breast cancer.But the application of bio-pharmaceutical
There is its limitation, equally by taking Trastuzumab as an example, and all high expression in breast cancer tumor cells surface of not all patient with breast cancer
The HER2 receptor proteins that HER2 receptor proteins are expressed in other words all show preferable affinity with Trastuzumab.Clinical research shows
In fact only about 30%-40% patient with breast cancer, which receives the treatment of Trastuzumab monoclonal antibody, can show good therapeutic effect, and
Therapeutic effect of the most of patient therein when continuing to receive Trastuzumab treatment can progressively weaken.Similar case has many bags
Burning hot immunologic test point PD1, PDL1, CTLA4 monoclonal antibody medicine in research and development market etc. at present is included, the list of single action target spot is only relied on
Antiradiation drug can not meet increasingly complicated clinical needs.Current most bio-pharmaceutical particularly anti-tumor monoclonal antibody
Medicine also and is introduced into treatment fiest-tire medication ranks, but as the supplement of chemical drug small-molecule drug and radiation therapy,
To improve the quality of life of cancer patient.
Under such overall background, in order to overcome the problem of single target spot of bio-pharmaceutical acts on limitation, biotechnology medicine
Many new directions, including antibody coupling small-molecule drug ADC class medicines is presented in the research of thing, granted with Amgen 2013
Kadcyla (T-DM1) is representative；Bispecific antibody Bispecific monoclonal antibody class medicines, with
Blinatumomab (BiTE) granted Amgen2014 is representative；And multiple bio-pharmaceutical drug combinations
(Combination Cocktail) or bio-pharmaceutical and small-molecule drug drug combination, are such as directed to patient with breast cancer's HER2 target spots
2 monoclonal antibody drug Trastuzumab and Pertuzumab drug combinations, for 2 lists of NHL
Clonal antibody medicine epratuzumab and rituximab drug combination etc., the connection of the substantial amounts of bio-pharmaceutical for different target spots
The clinical trial of medicine is shared in each big Clinical Research Center is actively developed.The development of domestic biotech drug is more external
Although starting late, growth momentum is swift and violent, immediately following international paces, since the biological similar medicine research and development of traditional target spot, to new
Immunologic test point target spot innovation biological medicament, then to antibody coupling small-molecule drug ADC, bispecific antibody, bio-pharmaceutical
Composition etc., has dozens of development project to be in preclinical or clinical research different phase, and the Biopharmaceutical Enterprises being related to surpass
Various schools of thinkers is crossed, country bio-pharmaceutical IND, which declares quantity and also risen year by year, in recent years reaches that nearly hundred IND declare/year.2016, on
Extra large medicine is based on treatment patient with breast cancer in the world using 2 monoclonal antibody drug Trastuzumab and Pertuzumab connection
Share the Research Thinking of medicine, it is initiative by this 2 antibody molecules according to 1:1 mass ratio is mixed to form antibody compositions, obtains
To a kind of novel compositions medicine, for treating breast cancer.Trastuzumab (T components) and Pertuzumab (P components) this 2
Monoclonal antibody molecule targets breast cancer cell surface growth factor receptors HER2, acts on the different epitopes of HER2 extracellular regions, suppresses
Growth of tumour cell, with synergy.Preclinical pharmacology result of study also indicates that the novel antibodies composition of medicine has and is better than T
The drug effect of component or P components administered alones.The relatively conventional monoclonal antibody medicine progress of the novel compositions medicine is obvious, with wider
Application prospect.In addition, the bio-pharmaceutical for adjusting class medicine for VEGF monoclonal antibody, PCSK9 monoclonal antibodies, adalimumab, autophagy is combined
The research of thing etc. also appears in the newspapers end repeatly, such medicine by multiple different mechanism of action, different action target spot drug regimens and play each
From effect, the defect for overcoming the action target spot of foregoing single target spot bio-pharmaceutical single reaches better healing effect.
The expansion and progress of biotech drug research direction certainly will inspire the continuous change of bio-pharmaceutical quality research means
Leather, the final products listing of biotech drug be unable to do without the guarantee of quality research.Even if the quality of single bio-pharmaceutical component
Research meanses have perfected, and cannot guarantee that after multiple bio-pharmaceutical formation compositions, and the ripe means of tradition before are also applicable.
By taking the biopharmaceutical composition that 2 kinds of monoclonal antibody drugs are combined as an example, product purity, activity are paid close attention in quality research
And impurity content.Wherein impurity content detection and conventional monoclonal antibody medicine is basically identical；Product purity detection is then needed to composition
2 core components of monoclonal antibody are made a distinction, and the separating degree of impurity peaks need to be considered incessantly in purity analysis method development process,
The separating effect of 2 core components of consideration is needed, development difficulty is higher than conventional monoclonal antibody medicine；And for the work of the antibody compositions
Property evaluation is then most difficult：First, the target spot of 2 core monoclonal antibody components is different, and biological assessment means are also likely to difference,
2 have respective biological evaluation method as traditional monoclonal antibody medicine, are detecting single using respective Biological Activity Methods
Synergy is likely to occur during anti-composition, it is also possible to mutually hinder to cause testing result to be difficult to evaluate；Secondly, 2 albumen
Concentration needs to detect confirmation respectively, it is impossible to simply with biological evaluation of the protein concentration after 2 mixing as composition
Benchmark, because the mechanism of action of the two may be different；Further in stability study, it is desirable to pass through the biology of said composition
Learn Activity determination and can distinguish and judge whether each component of composition activity decrease occurs.So in brief, for biology
The Biological Activity Methods exploitation difficult point of pharmaceutical composition is how to set up and can evaluate life using a kind of Biological Activity Methods
Medicine composition as overall newtype drug biological activity, while can also characterize and chase after using the Biological Activity Methods
The change of the bioactivity of each single-activity component of track biopharmaceutical composition.
Carry out by it is multiple for different target spot epitope bio-pharmaceuticals combine obtained by biopharmaceutical composition biology work
Property method exploitation, particularly the exploitation of cell in vitro Biological Activity Methods, it is necessary to overcome it is above-mentioned for biopharmaceutical composition life
The problem of thing Activity determination.The problem of respective Activity determination means being there may be for different single-activity components, as far as possible
Find based on to setting up cytoactive method based on each active component all related mechanism of action, can so realize life
Medicine composition is considered as an entirety and evaluated with a kind of Biological Activity Methods, and the Biological Activity Methods can be used in evaluating
The biological activity of each one-component in biopharmaceutical composition；Determine for the albumen of different single-activity protein components
Amount, conventional method is difficult to certainly measure extract of protein different being sufficiently separated for protein component based on HPLC chromatogram method,
The present invention is realized using the method for the sandwich standard measures of ELISA to be carried out accurately determining to each one-component of biopharmaceutical composition
Amount；, will not by the way of data acquisition for the tracking of the activity change situation of biopharmaceutical composition each one-component
Mixed, and detected with for overall Biological Activity Methods according to various ratios with single-activity protein component, by
It can be used in evaluating each one-component in the Biological Activity Methods, therefore calculating and recording the combination of different proportion bio-pharmaceutical
During the biological activity of thing, the detection knot when the ratio of other components changes is recorded on the basis of certain one-component respectively
Change (including the activity assay, the EC of Activity determination 4- or 5- parameter curve of fruit50Value etc.).It can be converged according to the change
Must be to assuming that certain one-component is constant and Activity determination situation during other components change, being collected by this actually should
This is utilized to judge the bio-pharmaceutical group under stability study or other conditions for overall bioactivity testing result with middle
The activity change of each component of compound.Based on above-mentioned thinking, the bio-evaluation method of biopharmaceutical composition is carried out
Exploitation, completes the present invention, can be realized to the biological activity of biopharmaceutical composition each component using the inventive method
Analyse in depth, activity research and estimation of stability for biopharmaceutical composition.
The content of the invention
The invention aims to overcome drawbacks described above and deficiency to be realized there is provided a kind of to biopharmaceutical composition
Bioactivity is analysed in depth and studied, biopharmaceutical composition can be considered as to the life of the overall evaluation biopharmaceutical composition
Thing activity, while the life of the same analysis of biological activity method to each component of biopharmaceutical composition can also be utilized
Thing activity carries out analysis tracking, can realize what each component may occur in the stability study of biopharmaceutical composition
The bio-evaluation method for biopharmaceutical composition that bioactivity degraded situation is monitored.
To achieve these goals, the invention provides a kind of bio-evaluation for biopharmaceutical composition
Method, is characterized in, comprises the following steps：
Step (1)：The protein quantification method for each protein component being directed in biopharmaceutical composition is set up, for accurate
Detect the protein concentration of each protein component of biopharmaceutical composition；
Step (2)：Confirm the common biological action mechanism of each protein component in biopharmaceutical composition, and base
Bio-evaluation method is set up in the mechanism of action, the bio-evaluation method, which is met, can evaluate bio-pharmaceutical group
The biological activity of each single protein component of compound；
Step (3)：Each component of biopharmaceutical composition is mixed in the case of concentration known according to different proportion, profit
The biological activity for the biopharmaceutical composition that different proportion is mixed is detected with set up bio-evaluation method, with certain list
On the basis of one component protein concentration, the biological activity detection knot of the biopharmaceutical composition under the conditions of record different mixing proportion
Really.
Preferably, the quantitative approach for biopharmaceutical composition each protein component is the albumen based on ELISA
Quantitative approach.
Preferably, described biopharmaceutical composition includes at least two components, described component is selected from monoclonal antibody
Medicine, fusion protein, polypeptide drug.
Preferably, described biopharmaceutical composition is monoclonal antibody combination, if described bio-pharmaceutical combination
Each protein component of thing is the different epitopes based on same target spot, then foundation is for the biological action mechanism of same target spot
Bio-evaluation method；If the target spot of each protein component of described biopharmaceutical composition is different, pin is set up
Bio-evaluation method to Fc sections of common biological functions of monoclonal antibody finds the cell for including each different target spot
Strain, sets up the biological activity method that described cell line is directed to different component.
Preferably, described biopharmaceutical composition is the bio-pharmaceutical group that each component is directed to same target spot different epitopes
Compound, same target spot different epitopes biopharmaceutical composition exemplarily include EGFR target drugs, HER2 target drugs,
VEGF/VEGFR target drugs, PCSK9 target drugs, TNFa target drugs, CD20 target drugs, autophagy regulation class medicine etc.
Biopharmaceutical composition.
Preferably, described biopharmaceutical composition is mixed according to a certain percentage by the bio-pharmaceutical of 2 or more than 2
Form.
Preferably, bio-evaluation method described in step (2) includes ELISA binding activity method, cell in vitro
Activity methods, interior animal experiment activity methods.
Preferably, biological activity testing result described in step (3) include activity assay, dose-effect relationship 4- parameters or
5- parameter curve fits EC50Value.
Preferably, the bio-evaluation method in described step (2) specifically includes following steps：
Step (21)：Take the logarithm growth period cell digested through pancreatin after adjust certain density and spread 96 porocyte plates, put
Put a period of time and treat cell attachment；
Step (22)：Take out testing protein medicine or the dilution of composition culture medium obtains the sample of serial dilutions simultaneously
Add and be incubated a period of time in cell plates jointly with cell, the propagation of cell is suppressed in the presence of medicine, add cell
Dyeing liquor MTS detects cell inhibitory effect situation；
Step (23)：4- parameter fittings are carried out according to the dose-effect relationship of drug concentration and staining signals value, calculating is treated
Survey drug concentration EC50 values when protein drug signal value suppresses half, protein drug reference material EC50 values divided by testing protein medicine
Thing EC50 values calculate and obtain biological activity value of the testing protein medicine with respect to reference material.
Preferably, the quantitative approach in described step (1) is to specifically include following steps：
Step (11)：The specific antigen taken for protein drug is coated with 96 hole elisa plates, plus BSA closings；
Step (12)：The protein drug standard protein of concentration known is taken to carry out being serially diluted the standard for obtaining series concentration song
Line sample is added to be combined in elisa plate with envelope antigen, at the same time adds internal control sample and testing sample；
Step (13)：It is incubated and the ELIAS secondary antibody that elisa plate adds coupling HRP marks is taken out after a period of time, is incubated one section
Time followed by adds TMB and carries out chromogenic reaction；
Step (14)：4- ginsengs are carried out according to the dose-effect relationship of the sample concentration of standard curve sample well and chromogenic reaction OD values
Curve fit, draws the quantitation curves for being directed to protein drug A or B respectively, by the colour developing in internal control sample well and testing sample hole
Reaction OD values bring the sample concentration for being calculated in standard curve and obtaining internal control sample and testing sample into, to the method degree of accuracy, essence
The quantitative detection of density domination and testing sample.
Beneficial effects of the present invention are characterized in particular in：Using comprehensive to bio-pharmaceutical group method of the invention, it is possible to system
The biological activity of compound is evaluated, and is built based on the collective effect mechanism using each single protein component in composition
Vertical biological evaluation method, is considered as entirety, using unified method to biopharmaceutical composition by biopharmaceutical composition
Biological activity is evaluated；Based on ELISA quantitative means, to composition, each single protein component carries out accurate protein quantification,
By set up unified biological activity method can be individually used for the biological activity detection of each single protein component, because
This can carry out the calculating of biological activity testing result, including active matched curve EC based on single protein component50Value or activity
Detected value, and judge whether other protein components occur active degradation according to the change of these testing results.It is unified based on this
Activity determination means and method can realize the comprehensive analysis to the biological activity of biopharmaceutical composition.
The scope of application of biopharmaceutical composition biological activity assays based on Research Thinking of the present invention is quite varied,
, only need to the collective effect mechanism based on composition including monoclonal antibody drug, fusion protein, polypeptide protein medicinal composition
Biological activity method exploitation, and protein quantification are carried out, comprehensive bio-pharmaceutical combination is can be realized as with reference to data analysis
The bio-evaluation of thing.Involved biological activity method includes cytoactive, binding activity and animal activity in vivo
Experiment etc..The inventive method is strong supplement and the support of the pharmacy part research researched and developed to biopharmaceutical composition, can
Activity research, product clearance, stability study for such product etc..
Brief description of the drawings
Fig. 1 a are that 50%, 75% monoclonal antibody component A biological activity detects collection of illustrative plates.
Fig. 1 b are that 125%, 150% monoclonal antibody component A biological activity detects collection of illustrative plates.
Fig. 2 a are that 50%, 75% monoclonal antibody B component biological activity detects collection of illustrative plates.
Fig. 2 b are that 125%, 150% monoclonal antibody B component biological activity detects collection of illustrative plates.
Fig. 3 a are that 50%, 75% monoclonal antibody medicine composition P biological activities detect collection of illustrative plates.
Fig. 3 b are that 125%, 150% monoclonal antibody medicine composition P biological activities detect collection of illustrative plates.
Fig. 4 is that monoclonal antibody component A protein quantification detects collection of illustrative plates.
Fig. 5 is that monoclonal antibody B component protein quantification detects collection of illustrative plates.
Fig. 6 a are by the monoclonal antibody medicine composition P (A+B), P (A*+B) biological activity testing result destroyed.
Fig. 6 b are by the monoclonal antibody medicine composition P (A+B*), P (A*+B*) biological activity testing result destroyed.
Fig. 7 a are to be detected on the basis of the protein concentration of monoclonal antibody medicine component A by the monoclonal antibody medicine composition P (A+ destroyed
B), P (A*+B) biological activity testing result.
Fig. 7 b are to be detected on the basis of the protein concentration of monoclonal antibody medicine component A by the monoclonal antibody medicine composition P (A+ destroyed
B*), P (A*+B*) biological activity testing result.
Fig. 8 a are to be detected on the basis of the protein concentration of monoclonal antibody medicine B component by the monoclonal antibody medicine composition P (A+ destroyed
B), P (A*+B) biological activity testing result.
Fig. 8 b are to be detected on the basis of the protein concentration of monoclonal antibody medicine B component by the monoclonal antibody medicine composition P (A+ destroyed
B*), P (A*+B*) biological activity testing result.
Fig. 9 is the general flow chart of the bio-evaluation method for biopharmaceutical composition of the present invention.
Embodiment
Below in conjunction with specific embodiment, the present invention is expanded on further.These embodiments be merely to illustrate the present invention and without
In limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition or is pressed
According to the condition proposed by manufacturer.Unless otherwise defined, all specialties used in text are ripe with this area with scientific words
Practice meaning known to personnel identical.In addition, any method similar or impartial to described content and material all can be applied to
In the inventive method.Preferable implementation described in text only presents a demonstration with material to be used.
In order to the clearer technology contents for understanding the present invention, described in detail especially exemplified by following examples.
First, material and reagent
Material：The monoclonal antibody medicine A and monoclonal antibody medicine B of certain target spot different epitopes are directed to simultaneously according to 1:1 mass concentration ratio
Example combination (A monoclonal antibodies component and B monoclonal antibody concentration of component be 5mg/mL) is into obtaining being combined by 2 monoclonal antibody protein drugs
Biopharmaceutical composition P (or it is designated as P (A+B) or P100).Entirety and biopharmaceutical composition reference material are regarded as, it is raw
Thing activity regards 100% as；P is considered as entirety, is diluted using Formulation Buffer Buffer and obtains different titer levels
The biopharmaceutical composition P of (50%, 75%, 125%, 150% titer level)50、P75、P125、P150.By monoclonal antibody medicine A life
Thing activity regards 100% as and (is designated as A100), i.e. monoclonal antibody medicine A reference materials are diluted using Formulation Buffer Buffer and obtained
The monoclonal antibody medicine A of different titer levels (50%, 75%, 125%, 150% titer level)50、A75、A125、A150.By monoclonal antibody medicine
Thing B biological activity regards 100% as and (is designated as B100), i.e. monoclonal antibody medicine B reference materials utilize Formulation Buffer Buffer to carry out
Dilution obtains the monoclonal antibody medicine B of different titer levels (50%, 75%, 125%, 150% titer level)50、B75、B125、B150。
Monoclonal antibody medicine A and monoclonal antibody medicine B are mixed to get component A containing monoclonal antibody medicine and monoclonal antibody medicine according to different quality concentration ratio
Biopharmaceutical composition P (3A+B), P (2A+B), P (A+2B), the P (A+3B) of thing B component different proportion.
Reagent：Cytoactive experiment includes cell culture medium, dyeing liquor MTS, pancreatin etc. with reagent；ELISA quantitative experiments
Include specificity for the detection antigen of monoclonal antibody medicine component A and monoclonal antibody medicine B component, the enzyme mark of coupling HRP marks with reagent
Secondary antibody, TMB colour reagents etc..
Cell line：For monoclonal antibody medicine component A and monoclonal antibody medicine B component cell inhibitory effect Activity determination experiment cell
Strain.
Consumptive material：Cytoactive is tested and ELISA quantitative experiments 96 orifice plates, suction pipe etc..
2nd, experimental method
1st, cytoactive is tested：Proliferation inhibition activity test experience based on experiment cell line,
Experimentation is as follows：
Take the logarithm growth period cell digested through pancreatin after adjust certain density and spread 96 porocyte plates, place a period of time
Treat cell attachment；
Then take out monoclonal antibody medicine to be measured or the dilution of composition culture medium obtains sample and the addition of serial dilutions
It is incubated a period of time in cell plates jointly with cell, the propagation of cell is suppressed in the presence of medicine, adds cell dyeing
Liquid MTS detects cell inhibitory effect situation.
It can be found that dose-effect relationship is presented with the change of drug concentration in Proliferation Ability degree, according to drug concentration and dye
The dose-effect relationship of chrominance signal value carries out 4- parameter fittings, can calculate the medicine obtained when monoclonal antibody medicine signal value to be measured suppresses half
Thing concentration EC50 values.Monoclonal antibody medicine reference material EC50 values divided by monoclonal antibody medicine EC50 value calculating to be measured obtain monoclonal antibody medicine phase to be measured
To the biological activity value of reference material.
2nd, ELISA quantitative experiments：Monoclonal antibody medicine A and monoclonal antibody medicine B detection antigen is directed to using specificity, is based on
ELISA sandwich methods experimental principle sets up the ELISA protein quantification methods for this 2 components, for monoclonal antibody medicine A and monoclonal antibody medicine
Thing B ELISA quantitative approach, in addition to coating specific antigen is different, remaining step is basically identical.
Experimentation is as follows：
The specific antigen taken for monoclonal antibody medicine A or B is coated with 96 hole elisa plates, plus BSA closings；
Then the monoclonal antibody medicine A or B standard albumen of concentration known is taken to carry out being serially diluted the standard for obtaining series concentration song
Line sample is added to be combined in elisa plate with envelope antigen, at the same time adds internal control sample and testing sample；
It is incubated and the ELIAS secondary antibody that elisa plate adds coupling HRP marks is taken out after a period of time, is incubated a period of time followed by
Add TMB and carry out chromogenic reaction.
4- parameter curve plans are carried out according to the dose-effect relationship of the sample concentration of standard curve sample well and chromogenic reaction OD values
Close, the quantitation curves for being directed to monoclonal antibody medicine A or B respectively are drawn, by internal control sample well and the chromogenic reaction OD values in testing sample hole
The sample concentration for obtaining internal control sample and testing sample can be calculated to the method degree of accuracy, precision control by bringing into standard curve
System, and testing sample quantitative detection.
3rd, the specific steps of the bio-evaluation method for biopharmaceutical composition of the invention
1st, monoclonal antibody medicine A and monoclonal antibody medicine B is directed to the different epitopes of the same target spot of tumour cell, 2 component independent roles
The inhibited proliferation to tumour cell is respectively provided with, by monoclonal antibody medicine A and B according to mass ratio 1:1 is mixed to form new bio medicine
When compositions are acted on tumour cell, monoclonal antibody medicine component A and B component are combined with tumor targets different epitopes, can be played
Cooperative effect, it is more obvious to the proliferation inhibiting effect of tumour cell.The common mechanism of action based on monoclonal antibody medicine A or B,
Screening determines that the tumor cell line of high expression target is used for the cell inhibitory effect activity methods exploitation for A or B component,
Screen obtained experiment and sensitivity is showed to monoclonal antibody A or B component with tumor cell line.Proliferation Ability life is set up based on the cell line
Thing activity test method, available for monoclonal antibody A or monoclonal antibody B or the biology of monoclonal antibody medicine composition (the two combination is considered as into entirety)
Learn activity rating.Method Of Accomplishment is developed, and monoclonal antibody A or monoclonal antibody B or monoclonal antibody medicine group are detected using the biological cell activity methods
The typical collection of illustrative plates of the biological activity of compound is shown in Fig. 1 a~Fig. 3 b.To the biological activity method respectively to monoclonal antibody A or monoclonal antibody B or
Monoclonal antibody medicine composition carries out the checking and confirmation of activity test method, and the high spot reviews method degree of accuracy is shown in Table 1~table 3 respectively,
It can be found that detecting the biological activity of monoclonal antibody A, monoclonal antibody B or monoclonal antibody medicine composition respectively using the biological activity method
When, the method accuracy in detection in the range of active titer level 50%~150% is met in the range of 75%~125%,
Complete confirmation for the biological cell activity methods of monoclonal antibody medicine composition to this.On the basis of method confirmation, enter one
Step completes the Method validation of the cytoactive method.
The monoclonal antibody component A biological activity accuracy in detection of table 1. investigates result
| Sample ID | Testing result | The rate of recovery |
| A50 | 54% | 108% |
| A75 | 84% | 112% |
| A125 | 131% | 105% |
| A150 | 149% | 99% |
The monoclonal antibody B component biological activity accuracy in detection of table 2. investigates result
| Sample ID | Testing result | The rate of recovery |
| B50 | 49% | 98% |
| B75 | 66% | 88% |
| B125 | 154% | 123% |
| B150 | 147% | 98% |
The monoclonal antibody composition P biological activities of table 3. detection precision investigates result summary sheet
| Sample ID | Testing result | The rate of recovery |
| P50 | 59% | 118% |
| P75 | 87% | 116% |
| P125 | 127% | 102% |
| P150 | 150% | 100% |
2nd, the biological cell activity methods for monoclonal antibody medicine composition are set up and after Method Of Accomplishment confirmation and checking,
Set up the ELISA protein quantification methods respectively for said composition monoclonal antibody medicine component A and monoclonal antibody medicine B component.ELISA albumen
The key that quantitative approach is set up, which is to screen, to be confirmed available for the exploitation of medicine quantitative ELISA methods respectively for single
The specific antigen of antiradiation drug component A and monoclonal antibody medicine B component.Screening determines the monoclonal antibody for being directed to monoclonal antibody medicine composition respectively
After the specific antigen of component A and monoclonal antibody B component, ELISA quantitative approach is set up for monoclonal antibody component A and monoclonal antibody B component respectively,
This method, which is met, specific to carry out protein quantification, and phase to the monoclonal antibody component A in monoclonal antibody medicine composition or monoclonal antibody B component
Do not interfered between mutually.Method Of Accomplishment develop, for monoclonal antibody component A and monoclonal antibody B component protein quantification standard curve see Fig. 4~
Fig. 5.
Carry out method validation and confirmation, precision of the high spot reviews for the actually detected result of quantitative approach of certain monoclonal antibody component
Degree and the degree of accuracy, and do not disturbed by another monoclonal antibody component presence or absence.With the ELISA quantitative approach for monoclonal antibody component A
Method validation and confirmation exemplified by, preparing 3 samples includes A+B (A and B is according to mass ratio 1:1 mixing, A concentration keeps 5mg/
ML), (A and B are according to mass ratio 1 by A+3B:3 mixing, wherein A concentration keeps 5mg/mL), (A and B are according to mass ratio 3 by A+1/3B:
1 mixing, wherein A concentration keeps 5mg/mL), 6 independences of the plate arrangement mode of different samples are carried out for this 3 samples
ELISA quantitatively detects, every time detection do 3 it is parallel, detection data summarization is shown in Table 4, and 18 detections are respectively obtained for 3 samples
As a result and statistical analysis is carried out, the RSD of monoclonal antibody component A concentration mensuration result is met<10%, the rate of recovery meets 90%~
In the range of 110%.By that analogy, the method validation for the ELISA quantitative approach of monoclonal antibody B component collects with confirmation result is shown in Table
5, respectively obtain 18 testing results for 3 samples and carry out statistical analysis, the RSD of monoclonal antibody B component concentration mensuration result
Meet<10%, the rate of recovery is met in the range of 90%~110%.Confirm that set up ELISA is quantified by these experimental results
Method can be accurately to monoclonal antibody medicine composition component A and B component realize protein quantification, and do not interfere with each other.
The monoclonal antibody component A protein quantification accuracy in detection of table 4. and precision investigate result summary sheet
The monoclonal antibody B component protein quantification accuracy in detection of table 5. and precision investigate result summary sheet
3rd, complete respectively for monoclonal antibody component A and component B ELISA quantitatively detect after, further by monoclonal antibody component A and
Monoclonal antibody component B is mixed according to different mass ratios, the monoclonal antibody medicine composition of serial mixed proportion is obtained, with for monoclonal antibody medicine
The monoclonal antibody medicine composition of the serial mixed proportion of biological cell activity methods detection of compositions, is recorded in different proportion condition
Under active 4- parameter curve fits EC50 values, and relative monoclonal antibody medicine component A and B component are according to 1:1 mass ratio
The relative biological activity of monoclonal antibody medicine composition (P (A+B)).Calculated and obtained containing not homogeneity on the basis of monoclonal antibody medicine component A
The biological cell Activity determination of the monoclonal antibody medicine composition of the component A of amount ratio the results are shown in Table 6, using monoclonal antibody medicine B component as base
The biological cell Activity determination that standard calculates the monoclonal antibody medicine composition of the B component for the ratio containing different quality that obtains the results are shown in Table 7.
Table 6. calculates serial mixed proportion monoclonal antibody medicine composition biological activity on the basis of monoclonal antibody component A
Table 7. calculates serial mixed proportion monoclonal antibody medicine composition biological activity on the basis of monoclonal antibody B component
When the biology that monoclonal antibody medicine composition is calculated on the basis of monoclonal antibody component A it can be seen from the result of table 6 and table 7
Learn activity when, with the continuous decline of monoclonal antibody component A weight/mass percentage composition be monoclonal antibody B component weight/mass percentage composition it is continuous on
Rise, the EC of Activity determination result50Value constantly declines, and composition is with respect to monoclonal antibody component A and B according to 1:P (the A of 1 mass ratio mixing
+ B) bioactivity of monoclonal antibody medicine composition then constantly rises, and illustrates that monoclonal antibody B component has folk prescription to the biological function of component A
To collaboration facilitation.And when calculating the biological activity of monoclonal antibody medicine composition on the basis of monoclonal antibody component B, same hair
Now as the decline of B component weight/mass percentage composition is the rising of component A weight/mass percentage composition, the EC of Activity determination result50Value is not
It is disconnected to decline, and composition with respect to monoclonal antibody component A and B according to 1:The life of P (A+B) monoclonal antibody medicine composition of 1 mass ratio mixing
Thing activity constantly rises, and same explanation monoclonal antibody component A has unidirectional collaboration facilitation to the biological function of B component.
By the above results it can be found that monoclonal antibody component A and B component mutually have collaboration facilitation on antitumor action,
By the two be combined into new monoclonal antibody medicine composition treat tumour in terms of have positive effect.At the same time, according to
The experimental result of table 6 and table 7 can obtain the Relative biological under the different quality ratio mixing condition of monoclonal antibody component A and B component
Activity value and EC50The situation of change of value, in actual applications can be according to the relative activity value or EC of monoclonal antibody composition50Value
Change judge monoclonal antibody component A and B component ratio change, with reference to ELISA concentration quantitative results can interpolate that monoclonal antibody component A or
Whether B component there is activity change, realizes in-depth analysis and research to monoclonal antibody medicine composition P biological activity.4th, originally
Invention practical application
Monoclonal antibody medicine A and monoclonal antibody medicine B are taken according to 1:1 mass percent is mixed to get P (A+B), and monoclonal antibody medicine A and
B concentration is 2.5mg/mL；2.5mg/mL monoclonal antibody medicine A is placed in 80 DEG C of high temperature and destroyed within 30 minutes, and is assumed
Its concentration does not change, by the monoclonal antibody medicine A Jing Guo high-temperature process and the monoclonal antibody medicine B component without destruction according to matter
Amount compares 1:1 is mixed to get P (A*+B), and monoclonal antibody medicine B concentration is 2.5mg/mL；By 2.5mg/mL monoclonal antibody medicine B at 80 DEG C
High temperature is placed and destroyed for 30 minutes, and assumes that its concentration does not change, by the monoclonal antibody medicine B Jing Guo high-temperature process and
Monoclonal antibody medicine component A without destruction is according to mass ratio 1:1 is mixed to get P (A+B*), and monoclonal antibody medicine A concentration is 2.5mg/
mL；The monoclonal antibody medicine A destroyed for 30 minutes and monoclonal antibody medicine B component will be placed through 80 DEG C of high temperature, it is assumed that its concentration is not sent out
Changing, according to mass ratio 1:1 is mixed, and obtains P (A*+B*).Obtain after above-mentioned 4 samples, utilize foregoing biology
Cytoactive method and ELISA protein quantifications method detect the biological activity and each self-corresponding monoclonal antibody of this 4 samples respectively
The protein concentration of medicine component A and B component, and according to biological activity of the testing result to 4 monoclonal antibody medicine composition samples
Analyzed.
1st step is analyzed, and 4 samples are accordingly to be regarded as into entirety, directly utilizes UV methods, the monoclonal antibody that 4 warps are destroyed in various degree
Medicine A and B component directly carry out protein concentration analysis, respectively obtain 4 samples as overall protein concentration testing result point
8 are not shown in Table, it can be found that detecting that the protein concentration of 4 samples does not observe the notable change of protein concentration by direct UV methods
Change.UV methods carry out protein quantification based on the overall amino acid composition of protein composition, it is impossible to distinguish composition each component
Change in concentration.
Table 8. is by the monoclonal antibody medicine composition protein concentration UV method protein concentration testing result summary sheets destroyed
| Sample ID | Monoclonal antibody medicine composition monoclonal antibody medicine protein concentration (mg/mL) |
| P(A+B) | 5.0 |
| P(A*+B) | 4.8 |
| P(A+B*) | 5.1 |
| P(A*+B*) | 5.3 |
Obtained protein concentration result is directly detected according to the UV methods of monoclonal antibody medicine composition, monoclonal antibody medicine composition is regarded
For entirety, with monoclonal antibody medicine A and monoclonal antibody medicine B according to 1:The reference material of 1 combination is standard, and P (A+B), P (A*+ are detected respectively
B), P (A+B*), 4 biological activities by the monoclonal antibody composition sample destroyed of P (A*+B*), testing result are shown in Fig. 6 a~6b
(on the basis of monoclonal antibody medicine composition overall density) and table 9.
Table 9. is by the monoclonal antibody medicine composition biological activity testing result summary sheet destroyed
It is can be seen that by above-mentioned experimental result when being considered as overall with monoclonal antibody medicine composition, the combination detected based on UV methods
The protein concentration of thing, monoclonal antibody medicine component A or monoclonal antibody medicine component B can be sensitively detected using the Biological Activity Methods
Active change when being damaged, when monoclonal antibody component A or monoclonal antibody component B is not damaged, P (A+B) cytoactive with
The biological activity of reference material has no significant difference, and when stability destruction occurs for the A or B of monoclonal antibody medicine composition, P (A*+
B), P (A+B*), P (A*+B*) cytoactive are remarkably decreased.When stability destruction occurs for component A and B component,
P (A*+B*) cytoactive declines the most notable.
P (A*+B), P (A+B*), P (A*+B*) this 3 samples are obtained although testing by above-mentioned 1st step and can detect
Cytoactive declines, it can be difficult to judging overall caused by being due to A active components or being destroyed due to B activity component
The cytoactive of sample declines, and the experimental analysis of follow-up 2nd step and the 3rd step need to be carried out for the query.2nd step analysis and utilization point
Safety pin it is unknown to above-mentioned 4 to monoclonal antibody medicine component A and monoclonal antibody medicine component B ELISA quantitative approach through high-temperature stability
The protein concentration of each component of the monoclonal antibody medicine composition of destruction carries out detection determination.The analysis of 2nd step determines protein concentration inspection
Survey the results are shown in Table 10.
Table 10. is by the monoclonal antibody medicine composition protein concentration ELISA testing result summary sheets destroyed
It is can be found that by the protein quantification testing result of table 10 and utilizes that is set up to be directed to monoclonal antibody component A and monoclonal antibody component respectively
B ELISA protein quantifications method can specific recognition and detect monoclonal antibody medicine composition component A and component B " effective "
Protein concentration, when by monoclonal antibody medicine component A or component B through high temperature, " effective " egg obtained based on the ELISA quantitative approach
White concentration is remarkably decreased, with being expected unanimously.Accurate ELISA protein quantification results are follow-up accurate judgement monoclonal antibody medicine groups
Whether the component A or component B of compound occur the basis of bioactivity change.Quantitatively detect unknown to 4 by the 2nd step ELISA
The monoclonal antibody medicine composition through high temperature component A and B component carry out protein quantification detection respectively, according to protein quantification knot
Really, it can tentatively show that the component A and B component of P (A+B) sample are not damaged；The component A of P (A*+B) sample is destroyed,
B component is normal；The component A of P (A+B*) sample is normal, and B component is destroyed；The component A and B component of P (A*+B*) sample by
To destruction.But the conclusion be based only on it is that ELISA protein quantifications are drawn, it is necessary to by cytoactive detection it is further really
Card, that is, carry out being divided based on ELISA protein quantifications result the cell bio-activity of 4 protein medicinal compositions for the 3rd step
Analysis detection.
The component A or B component of monoclonal antibody medicine composition are determined in the protein ELISA quantitative experiment analyzed by the 2nd step
Accurately after " effective " protein concentration, biological cell activity experiment that can simultaneously for monoclonal antibody medicine component A or B component is utilized
Analysis of biological activity is carried out to 4 monoclonal antibody medicine composition samples destroyed by different degrees of stability, respectively with monoclonal antibody medicine
The protein concentration of thing component A or carried out on the basis of the protein concentration of monoclonal antibody medicine B component relative monoclonal antibody medicine composition (not by
The A of destruction, B component are according to 1:1 mixing) reference material analysis of biological activity.And by testing result and complete before table 6,
Activity Results data of the table 7 under the conditions of different A, B component mixed proportion are compared, and A and B component are speculated according to Activity Results
Portfolio ratio, and be compared with reality by the ELISA A quantitatively obtained and B component portfolio ratio, to based on ELISA
The component A and the relative combinations ratio of B component quantitatively obtained is confirmed, and infers whether are the component A in composition or B component
Destroyed.
Detect that the monoclonal antibody medicine composition sample of stability destruction is biological on the basis of the protein concentration of monoclonal antibody medicine component A
Learn cytoactive result and see Fig. 7 a~7b (on the basis of monoclonal antibody medicine component A " effective " concentration) and table 11.
Table 11. (" is had by the monoclonal antibody medicine composition biological activity result summary sheet destroyed with monoclonal antibody medicine component A
On the basis of effect " concentration)
By the above results it can be seen that based on being destroyed in various degree to 4 warps on the basis of monoclonal antibody medicine A " effective " concentration
The activity of monoclonal antibody medicine composition is detected, according to testing result, with reference to table 6 obtain according to different proportion monoclonal antibody A or B group
Point Activity determination result, the A and B of the monoclonal antibody medicine composition destroyed in various degree to 4 warps relative scale speculates,
It can be found that the component A and the ratio of B component that are speculated according to Activity Results and the component A and B that are obtained by the 2nd step protein quantification
The actually detected ratio of component is basically identical.For P (A+B*) sample, because the protein concentration of A protein components does not become substantially
Change, it can be deduced that the inference that B protein components are destroyed in said composition.For P (A*+B) sample, because B protein components
Protein concentration does not change substantially, it can be deduced that the inference that A protein components are destroyed in said composition.For P (A*+B*) sample
Product, the protein quantification result based on albumin A component and B component is relatively low, and the sample is with respect to P (A+B) protein composition mark
The cytoactive of quasi- product is not consistent, it can be deduced that what A protein components and B protein components were destroyed in said composition pushes away
By.
Detect that the monoclonal antibody medicine composition sample of stability destruction is biological on the basis of the protein concentration of monoclonal antibody medicine B component
Learn cytoactive result and see Fig. 8 a~8b (on the basis of monoclonal antibody medicine component B " effective " concentration) and table 12.
Table 12. is by the monoclonal antibody medicine composition biological activity testing result summary sheet destroyed (with monoclonal antibody medicine component B
" effective "
On the basis of concentration)
By the above results it can be seen that based on being destroyed in various degree to 4 on the basis of monoclonal antibody medicine B " effective " concentration
The activity of monoclonal antibody medicine composition is detected, according to testing result, with reference to table 7 obtain according to different proportion monoclonal antibody A or B group
Point Activity determination result, the A and B of the monoclonal antibody medicine composition destroyed in various degree to 4 warps relative scale speculates,
It can be found that the component A and the ratio of B component that are speculated according to Activity Results and the component A and B that are obtained by the 2nd step protein quantification
The actually detected ratio of component is also basically identical.Also according to the result to the A of 4 monoclonal antibody medicine compositions and the phase of B component
Comparative example can be confirmed, and obtain what is destroyed for the component A or B component in 4 monoclonal antibody medicine composition samples
Conclusion.
Using the experiment of above-mentioned 3 steps, the unknown monoclonal antibody medicine destroyed in various degree of 4 simulations is combined
The activity of cell biology of thing is analyzed, it can be found that the cell bio-activity of the monoclonal antibody medicine composition containing destruction component
There is different degrees of decline, while the monoclonal antibody medicine declined based on ELISA protein quantifications and activity confirmation to cell bio-activity
The relative scale of the component of composition has carried out accurate analysis, can conclude that component A or B component hair in monoclonal antibody medicine composition
Breakoff phenomenon is given birth to.
In summary, the present invention establishes a kind of bio-evaluation method for biopharmaceutical composition, the party
Method is constantly to research and develop progress in biotech drug, and the quality of the particularly drug discovery process of new bio pharmaceutical composition is ground
Study carefully the expansion in field.Conventional needle can not be met to a variety of bio-pharmaceuticals to the biological evaluation method of single creature drug component
The need for the bioactivity of the composition of formation is analysed in depth.The present invention combines Biological Activity Methods exploitation, ELISA protein quantifications
Analysis method is developed, and by the Activity determination interpretation of result to different mixing proportion sample, can be realized by a kind of biology
Activity methods realize that the activity change situation that each component of complicated biopharmaceutical composition may occur is analyzed.Should
Method is clear, exploitation and, easily popularization moderate using difficulty, disclosure satisfy that and the biological function of biopharmaceutical composition is commented
The need for valency, product clearance, stability study etc..
Beneficial effects of the present invention are characterized in particular in：Using comprehensive to bio-pharmaceutical group method of the invention, it is possible to system
The biological activity of compound is evaluated, and is built based on the collective effect mechanism using each single protein component in composition
Vertical biological evaluation method, is considered as entirety, using unified method to biopharmaceutical composition by biopharmaceutical composition
Biological activity is evaluated；Based on ELISA quantitative means, to composition, each single protein component carries out accurate protein quantification,
By set up unified biological activity method can be individually used for the biological activity detection of each single protein component, because
This can carry out the calculating of biological activity testing result, including active matched curve EC based on single protein component50Value or activity
Detected value, and judge whether other protein components occur active degradation according to the change of these testing results.It is unified based on this
Activity determination means and method can realize the comprehensive analysis to the biological activity of biopharmaceutical composition.
The scope of application of biopharmaceutical composition biological activity assays based on Research Thinking of the present invention is quite varied,
, only need to the collective effect mechanism based on composition including monoclonal antibody drug, fusion protein, polypeptide protein medicinal composition
Biological activity method exploitation, and protein quantification are carried out, comprehensive bio-pharmaceutical combination is can be realized as with reference to data analysis
The bio-evaluation of thing.Involved biological activity method includes cytoactive, binding activity and animal activity in vivo
Experiment etc..The inventive method is strong supplement and the support of the pharmacy part research researched and developed to biopharmaceutical composition, can
Activity research, product clearance, stability study for such product etc..
In this description, the present invention is described with reference to its specific embodiment.But it is clear that can still make
Various modifications and alterations are without departing from the spirit and scope of the present invention.Therefore, specification and drawings are considered as illustrative
And it is nonrestrictive.
Claims (10)
1. a kind of bio-evaluation method for biopharmaceutical composition, it is characterised in that comprise the following steps：
Step (1)：The protein quantification method for each protein component being directed in biopharmaceutical composition is set up, for accurately detecting
The protein concentration of each protein component in biopharmaceutical composition；
Step (2)：Confirm the common biological action mechanism of each protein component in biopharmaceutical composition, and based on institute
The biological action mechanism stated sets up bio-evaluation method, and described bio-evaluation method, which can be used for evaluating, gives birth to
The biological activity of each single protein component in medicine composition；
Step (3)：Each protein component of biopharmaceutical composition is mixed in the case of concentration known according to different proportion, profit
The biological activity for the biopharmaceutical composition that different proportion is mixed is detected with set up bio-evaluation method, with certain list
On the basis of one component protein concentration, the biological activity detection knot of the biopharmaceutical composition under the conditions of record different mixing proportion
Really.
2. the bio-evaluation method according to claim 1 for biopharmaceutical composition, it is characterised in that institute
Quantitative approach in the step of stating (1) is the protein quantification method based on ELISA.
3. the bio-evaluation method according to claim 1 for biopharmaceutical composition, it is characterised in that institute
The biopharmaceutical composition stated includes at least two components, and described component is selected from monoclonal antibody drug, fusion protein, polypeptide
Class medicine.
4. the bio-evaluation method according to claim 1 for biopharmaceutical composition, it is characterised in that institute
The biopharmaceutical composition stated is monoclonal antibody combination, if each protein component of described biopharmaceutical composition is
Based on the different epitopes of same target spot, then the bio-evaluation side of the biological action mechanism for same target spot is set up
Method；If the target spot of each protein component of described biopharmaceutical composition is different, set up for Fc section of monoclonal antibody jointly
The bio-evaluation method of biological function finds the cell line for including each different target spot, sets up described cell
Strain is directed to the biological activity method of different component.
5. the bio-evaluation method according to claim 1 for biopharmaceutical composition, it is characterised in that institute
The biopharmaceutical composition stated is the biopharmaceutical composition that each component is directed to same target spot different epitopes.
6. the bio-evaluation method according to claim 1 for biopharmaceutical composition, it is characterised in that institute
Component in the biopharmaceutical composition stated is mixed according to preset ratio.
7. the bio-evaluation method according to claim 1 for biopharmaceutical composition, it is characterised in that institute
Bio-evaluation method in the step of stating (2) includes ELISA binding activity method, cell in vitro activity methods, in vivo
Zoopery activity methods.
8. the bio-evaluation method according to claim 1 for biopharmaceutical composition, it is characterised in that institute
It is bent that biological activity testing result in the step of stating (3) includes activity assay, dose-effect relationship 4- parameters or 5- parameter fittings
Line EC50Value.
9. the bio-evaluation method according to claim 1 for biopharmaceutical composition, it is characterised in that institute
Bio-evaluation method in the step of stating (2) specifically includes following steps：
Step (21)：Take the logarithm growth period cell digested through pancreatin after adjust certain density and spread 96 porocyte plates, place one
The section time treats cell attachment；
Step (22)：Take out testing protein medicine or the dilution of composition culture medium obtains sample and the addition of serial dilutions
It is incubated a period of time in cell plates jointly with cell, the propagation of cell is suppressed in the presence of medicine, adds cell dyeing
Liquid MTS detects cell inhibitory effect situation；
Step (23)：4- parameter fittings are carried out according to the dose-effect relationship of drug concentration and staining signals value, calculating obtains egg to be measured
Baiyao thing signal value suppresses drug concentration EC50 values during half, protein drug reference material EC50 values divided by testing protein medicine
EC50 values calculate and obtain biological activity value of the testing protein medicine with respect to reference material.
10. the bio-evaluation method according to claim 1 for biopharmaceutical composition, it is characterised in that
Quantitative approach in described step (1) is to specifically include following steps：
Step (11)：The specific antigen taken for protein drug is coated with 96 hole elisa plates, plus BSA closings；
Step (12)：The protein drug standard protein of concentration known is taken be serially diluted the standard curve sample for obtaining series concentration
Product are added to be combined in elisa plate with envelope antigen, at the same time adds internal control sample and testing sample；
Step (13)：It is incubated and the ELIAS secondary antibody that elisa plate adds coupling HRP marks is taken out after a period of time, is incubated a period of time
Followed by add TMB and carry out chromogenic reaction；
Step (14)：4- parameters are carried out according to the dose-effect relationship of the sample concentration of standard curve sample well and chromogenic reaction OD values bent
Line is fitted, and the quantitation curves for being directed to protein drug A or B respectively is drawn, by internal control sample well and the chromogenic reaction in testing sample hole
OD values bring the sample concentration for being calculated in standard curve and obtaining internal control sample and testing sample into, to the method degree of accuracy, precision
Control and the quantitative detection of testing sample.
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