CN107192828B - For the bio-evaluation method of biopharmaceutical composition - Google Patents
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Abstract
The present invention relates to a kind of bio-evaluation methods for biopharmaceutical composition, comprising steps of being primarily based on ELISA sandwich method principle establishes 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 established based on the mechanism of action；Utilize the biological activity of the biopharmaceutical composition of established bio-evaluation method detection different proportion mixing, on the basis of certain one-component protein concentration, the biological activity testing result of the biopharmaceutical composition under the conditions of different mixing proportion and the EC of 4- parameter curve fit are recorded50Value.The method of the present invention can be according to biological activity testing result or the EC of 4- parameter curve fit50The variation of value and judge whether certain component of biopharmaceutical composition inactivates, can be used for the activity research and estimation of stability of biopharmaceutical composition.
Description
Technical field
The present invention relates to field of biotechnology, in particular to a kind of bio-evaluation method, in particular to be directed to is given birth to
The bio-evaluation method of medicine composition.
Background technique
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 gradually replace chemical drug small-molecule drug and become pharmaceutical field
Research hotspot.Having 8 in ten drug before global marketing ranking in 2015 is biological medicament, 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 multiplication 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 coagulation factor, albumin for extracting class drug such as people source
Deng and genetic engineering class drug include vaccine, monoclonal antibody, fusion protein, polypeptide drug etc..Wherein monoclonal antibodies
Drug is with its safety and validity and in antitumor, autoimmune disease field immense success and potentiality, in biological skill
It is leading in the big development wave of art drug.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.
Traditional chemical drug small molecule class drug has preferable antitumor cell effect, but its poor, toxic side effect of selectivity
It is larger, also there is killing to patient's body normal cell, strongly limits the application and development of chemical drug small molecule class drug.Biological medicament
The prominent distinguishing feature of 1 of object especially monoclonal antibodies drug and advantage are targeting, to be directed to breast cancer cell table
For the Trastuzumab monoclonal antibody of face HER2 receptor, the medicine in vivo can specific effect incited somebody to action in tumor cell surface HER2 receptor
Tumour cell kills or inhibits growth of tumour cell, achievees the purpose that treat patient with breast cancer.However 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 protein that HER2 receptor protein is expressed in other words all shows preferable affinity with Trastuzumab.Clinical research shows
In fact the patient with breast cancer for only having about 30%-40%, which receives the treatment of Trastuzumab monoclonal antibody, can show good therapeutic effect, and
Therapeutic effect of the major part patient therein when continuing to receive Trastuzumab treatment can gradually weaken.Similar case has many packets
Immunologic test point PD1, PDL1, CTLA4 monoclonal antibody medicine etc. that research and development market is burning hot at present is included, the list of single action target spot is only relied on
Antiradiation drug is unable to satisfy increasingly complicated clinical needs.Current most bio-pharmaceutical especially anti-tumor monoclonal antibody
Drug also and does not enter treatment fiest-tire medication ranks, but the supplement as 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 problems, such as the single target spot effect limitation of bio-pharmaceutical, biotechnology medicine
Many new directions, including antibody coupling small-molecule drug ADC class drug are presented in the research of object, granted with Amgen 2013
Kadcyla (T-DM1) is representative；Bispecific antibody Bispecific monoclonal antibody class drug, with
Amgen2014 granted Blinatumomab (BiTE) 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 HER2 target spot
2 monoclonal antibody drug Trastuzumab and Pertuzumab drug combinations, for 2 lists of non-Hodgkin lymphoma
Clonal antibody drug epratuzumab and rituximab drug combination etc., largely for the connection of the bio-pharmaceutical of 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 world paces, since the similar medicine research and development of the biology of traditional target spot, to newly
Immunologic test point target spot innovation biological medicament, then arrive 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 are super
Various schools of thinkers is crossed, country bio-pharmaceutical IND declared quantity and also rises year by year and reaches nearly hundred IND and declare/year in recent years.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
The Research Thinking of medicine is shared, it is initiative that this 2 antibody molecules are mixed to form antibody compositions according to 1:1 mass ratio, it obtains
To a kind of novel compositions drug, for treating breast cancer.Trastuzumab (T component) and Pertuzumab (P component) this 2
Monoclonal antibody molecule targets breast cancer cell surface growth factor receptors HER2, acts on the different epitopes of HER2 extracellular region, inhibits
Growth of tumour cell has synergistic effect.Preclinical pharmacology result of study also indicates that the novel antibodies composition of medicine has better than T
The drug effect of component or P components administered alone.The relatively traditional monoclonal antibody medicine progress of the novel compositions drug is obvious, has wider
Application prospect.In addition, adjusting the bio-pharmaceutical combination of class drug for VEGF monoclonal antibody, PCSK9 monoclonal antibody, adalimumab, autophagy
The research of object etc. also appears in the newspapers end repeatly, such drug combines multiple and different mechanism of action, different role target drug and plays each
From the effect of, the defect for overcoming the action target spot of aforementioned 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 of biotech drug list the guarantee for be unableing to do without quality research.Even if the quality of single bio-pharmaceutical component
Research means have perfected, and after cannot guarantee that multiple bio-pharmaceuticals form composition, the mature means of tradition before are also applicable in.
By taking the biopharmaceutical composition that 2 kinds of monoclonal antibody drugs are composed 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 are almost the same；Product purity detection is then needed to composition
2 core components of monoclonal antibody distinguish, and need to consider the separating degree of impurity peaks incessantly in purity analysis method development process,
Need to consider that the separating effect of 2 core components, development difficulty are higher than conventional monoclonal antibody medicine；And it is directed to the work of the antibody compositions
Property evaluation be then most difficult: firstly, the target spot of 2 core monoclonal antibody components is different, biological assessment means are also likely to difference,
2 as traditional monoclonal antibody medicine have respective biological evaluation method, single being detected using respective Biological Activity Methods
Synergistic effect is likely to occur when anti-composition, it is also possible to mutually hinder so that testing result is difficult to evaluate；Secondly, 2 albumen
Concentration needs to detect confirmation respectively, cannot simply use 2 mixing after protein concentration as composition biological evaluation
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 the composition
It learns Activity determination and can distinguish and judge whether each component of composition active decline occurs.So in brief, for biology
The Biological Activity Methods exploitation difficult point of pharmaceutical composition is how to establish and can evaluate life using a kind of Biological Activity Methods
The biological activity of medicine composition newtype drug as a whole, while can also characterize and chase after using the Biological Activity Methods
The variation of the bioactivity of each single active ingredient of track biopharmaceutical composition.
It carries out living by multiple biology for being directed to biopharmaceutical composition obtained by different target spot epitope bio-pharmaceuticals combine
Property method exploitation, the especially exploitation of cell in vitro Biological Activity Methods needs to overcome above-mentioned raw for biopharmaceutical composition
The problem of object Activity determination.Different single active ingredients there may be aiming at the problem that respective Activity determination means, as far as possible
It finds based on cell activity method is established based on the mechanism of action all relevant to each active component, is can be realized in this way by life
Medicine composition is considered as an entirety and is evaluated with a kind of Biological Activity Methods, which can be used in evaluating
The biological activity of each one-component in biopharmaceutical composition；It is fixed for the albumen of different single-activity protein components
Amount, conventional method are difficult to realize 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 measure of ELISA and accurately determine to each one-component of biopharmaceutical composition
Amount；It will not by the way of data acquisition for the tracking of the activity change situation of each one-component of biopharmaceutical composition
It is mixed with single-activity protein component according to various ratios, and is detected with for whole Biological Activity Methods, 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
When the biological activity of object, the detection knot when the ratio of other components changes is recorded on the basis of certain one-component respectively
Variation (including activity assay, the EC of Activity determination 4- or 5- parameter curve of fruit50Value etc.).It can be converged according to the variation
Must be to assuming that certain one-component is constant and Activity determination situation when other components change, being summarized by this actually to answer
The bio-pharmaceutical group under stability study or other conditions is judged for whole bioactivity testing result using this with middle
Close the activity change of each component of object.Based on above-mentioned thinking, the bio-evaluation method of biopharmaceutical composition is carried out
Exploitation is completed the present invention, can be realized using the inventive method to the biological activity of each component of biopharmaceutical composition
It analyses in depth, activity research and estimation of stability for biopharmaceutical composition.
Summary of the invention
The purpose of the invention is to overcome drawbacks described above and deficiency, a kind of realize to biopharmaceutical composition is provided
Bioactivity analysed in depth and studied, biopharmaceutical composition can be considered as to the life of the overall evaluation biopharmaceutical composition
Object activity also can use the life of the same analysis of biological activity method to each component of biopharmaceutical composition simultaneously
Object activity carries out analysis tracking, can be realized in the stability study of biopharmaceutical composition each component may occur
The bio-evaluation method for biopharmaceutical composition that bioactivity degradation situation is monitored.
To achieve the goals above, the present invention provides a kind of bio-evaluations for biopharmaceutical composition
Method, its main feature is that, comprising the following steps:
Step (1): establishing the protein quantification method for each protein component in biopharmaceutical composition, for accurate
Detect the protein concentration of each protein component of biopharmaceutical composition；
Step (2): the common biological action mechanism of each protein component in confirmation biopharmaceutical composition, and base
Bio-evaluation method is established in the mechanism of action, bio-evaluation method satisfaction can evaluate bio-pharmaceutical group
Close the biological activity of each single protein component of object；
Step (3): each component of biopharmaceutical composition is mixed in known concentration according to different proportion, benefit
The biological activity of the biopharmaceutical composition of different proportion mixing is detected, with established bio-evaluation method with certain list
On the basis of one component protein concentration, the biological activity for recording the biopharmaceutical composition under the conditions of different mixing proportion detects knot
Fruit.
Preferably, the quantitative approach for each protein component of biopharmaceutical composition is the albumen based on ELISA
Quantitative approach.
Preferably, the biopharmaceutical composition includes at least two components, the group is selected from monoclonal antibody
Drug, fusion protein, polypeptide drug.
Preferably, the biopharmaceutical composition is monoclonal antibody combination, if the bio-pharmaceutical combines
Each protein component of object is the different epitopes based on same target spot, then establishes the biological action mechanism for being directed to same target spot
Bio-evaluation method；If the target spot of each protein component of the biopharmaceutical composition is different, needle is established
Bio-evaluation method to Fc sections of common biological functions of monoclonal antibody finds the cell comprising each different target spots
The biological activity method that the cell strain is directed to different component is established in strain.
Preferably, the biopharmaceutical composition is the bio-pharmaceutical group that each component is directed to same target spot different epitopes
Close object, same target spot different epitopes biopharmaceutical composition illustratively include EGFR target drug, HER2 target drug,
VEGF/VEGFR target drug, PCSK9 target drug, TNFa target drug, CD20 target drug, autophagy adjust class drug etc.
Biopharmaceutical composition.
Preferably, the biopharmaceutical composition is mixed according to a certain percentage by 2 or 2 or more bio-pharmaceuticals
It forms.
Preferably, bio-evaluation method described in step (2) includes ELISA combination activity methods, 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- parameter or
5- parameter curve fit EC50Value.
Preferably, bio-evaluation method in the step (2) specifically includes the following steps:
Step (21): the cell of logarithmic growth phase adjusts certain density after pancreatin digests and spreads 96 porocyte plates, puts
It is adherent to cell to set a period of time；
Step (22): testing protein drug or composition are taken out with culture medium and dilutes to obtain the sample of serial dilutions simultaneously
It is added in cell plates and is incubated for a period of time jointly with cell, the proliferation of cell is suppressed under the action of drug, and cell is added
Dyeing liquor MTS detects cell inhibitory effect situation；
Step (23): carrying out 4- parameter fitting according to the dose-effect relationship of drug concentration and staining signals value, be calculated to
Drug concentration EC50 value when protein drug signal value inhibits half is surveyed, protein drug reference material EC50 value is divided by testing protein medicine
Biological activity value of the testing protein drug with respect to reference material is calculated in object EC50 value.
Preferably, the quantitative approach in the step (1) be specifically includes the following steps:
Step (11): it takes the specific antigen for protein drug to be coated with 96 hole elisa plates, BSA is added to close；
Step (12): the standard for taking the protein drug standard protein of known concentration to be serially diluted to obtain series of concentrations is bent
Line sample is added in elisa plate in conjunction with envelope antigen, and internal control sample and sample to be tested are added at the same time；
Step (13): the ELIAS secondary antibody that coupling HRP label is added in elisa plate is taken out after being incubated for a period of time, is incubated for one section
Time is followed by added TMB and carries out chromogenic reaction；
Step (14): 4- ginseng is carried out according to the dose-effect relationship of the sample concentration of standard curve sample well and chromogenic reaction OD value
Curve fit draws the quantitation curves for being directed to protein drug A or B respectively, by the colour developing of internal control sample well and sample to be tested hole
Reaction OD value brings in standard curve the sample concentration that internal control sample and sample to be tested is calculated into, to method accuracy, essence
The quantitative detection of density domination and sample to be tested.
Beneficial effects of the present invention are characterized in particular in: being used method of the invention, it is possible to system comprehensively to bio-pharmaceutical group
The biological activity for closing object 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 for biopharmaceutical composition, using unified method to biopharmaceutical composition
Biological activity is evaluated；Accurate protein quantification is carried out based on ELISA quantitative means single protein component each to composition,
Since established 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 variation of these testing results.It is unified based on this
Activity determination means and method can be realized 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 very extensive,
It, only need to be based on the collective effect mechanism of composition including monoclonal antibody drug, fusion protein, polypeptide protein medicinal composition
The exploitation of biological activity method, and protein quantification are carried out, combined data analysis can be realized as comprehensive bio-pharmaceutical combination
The bio-evaluation of object.Involved biological activity method includes cell activity, in conjunction with activity and animal activity in vivo
Experiment etc..The inventive method is the strong supplement and support of the pharmacy part research to biopharmaceutical composition research and development, can
For the activity research of such product, product clearance, stability study etc..
Detailed description of the invention
Fig. 1 a is 50%, 75% monoclonal antibody component A biological activity test map.
Fig. 1 b is 125%, 150% monoclonal antibody component A biological activity test map.
Fig. 2 a is 50%, 75% monoclonal antibody B component biological activity test map.
Fig. 2 b is 125%, 150% monoclonal antibody B component biological activity test map.
Fig. 3 a is 50%, 75% monoclonal antibody medicine composition P biological activity test map.
Fig. 3 b is 125%, 150% monoclonal antibody medicine composition P biological activity test map.
Fig. 4 is monoclonal antibody component A protein quantification test map.
Fig. 5 is monoclonal antibody B component protein quantification test map.
Fig. 6 a be by destroy monoclonal antibody medicine composition P (A+B), P (A*+B) biological activity testing result.
Fig. 6 b be by destroy monoclonal antibody medicine composition P (A+B*), P (A*+B*) biological activity testing result.
Fig. 7 a is 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 is 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 is 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 is 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 invention.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is further explained.These embodiments are merely to illustrate the present invention and do not have to
In limiting the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition or presses
According to condition proposed by manufacturer.Unless otherwise defined, all professional and scientific terms as used herein and this field are ripe
It is identical to practice meaning known to personnel.In addition, any method similar to or equal to what is recorded and material all can be applied to
In the method for the present invention.The preferred methods and materials described herein are for illustrative purposes only.
Technology contents of the invention are understood in order to clearer, and spy lifts following embodiment and is described in detail.
One, material and reagent
Material: while for the monoclonal antibody medicine A and monoclonal antibody medicine B of certain target spot different epitopes according to the mass concentration ratio of 1:1
Example combination (A monoclonal antibody component and B monoclonal antibody concentration of component be 5mg/mL) is into obtaining being composed of 2 monoclonal antibody protein drugs
Biopharmaceutical composition P (or it is denoted as P (A+B) or P100).It is regarded as entirety and biopharmaceutical composition reference material, it is raw
Object activity regards 100% as；P is considered as entirety, is diluted to obtain different titer levels using Formulation Buffer Buffer
The biopharmaceutical composition P of (50%, 75%, 125%, 150% titer level)50、P75、P125、P150.By the life of monoclonal antibody medicine A
Object activity regards 100% as and (is denoted as A100), i.e. monoclonal antibody medicine A reference material is diluted to obtain using Formulation Buffer Buffer
The monoclonal antibody medicine A of different titer levels (50%, 75%, 125%, 150% titer level)50、A75、A125、A150.By monoclonal antibody medicine
The biological activity of object B regards 100% as and (is denoted as B100), i.e. monoclonal antibody medicine B reference material utilizes 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 object B component different proportion.
Reagent: cell activity experiment reagent includes cell culture medium, dyeing liquor MTS, pancreatin etc.；ELISA quantitative experiment
It include specificity for the detection antigen of monoclonal antibody medicine component A and monoclonal antibody medicine B component, the enzyme mark of coupling HRP label with reagent
Secondary antibody, TMB colour reagent etc..
Cell strain: for monoclonal antibody medicine component A and monoclonal antibody medicine B component cell inhibitory effect Activity determination experiment cell
Strain.
Consumptive material: cell activity experiment and ELISA quantitative experiment 96 orifice plates, suction pipe etc..
Two, experimental method
1, cell activity is tested: the proliferation inhibition activity test experience based on experiment cell strain,
Experimentation is as follows:
The cell of logarithmic growth phase adjusts certain density after pancreatin digests and spreads 96 porocyte plates, places a period of time
It is adherent to cell；
Then monoclonal antibody medicine or composition to be measured is taken out to be diluted to obtain sample and the addition of serial dilutions with culture medium
It is incubated for a period of time in cell plates jointly with cell, the proliferation of cell is suppressed under the action of drug, and cell dyeing is added
Liquid MTS detects cell inhibitory effect situation.
It can be found that dose-effect relationship is presented with the variation 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 fitting, and medicine when monoclonal antibody medicine signal value to be measured inhibits half can be calculated
Object concentration EC50 value.Monoclonal antibody medicine phase to be measured is calculated divided by monoclonal antibody medicine EC50 value to be measured in monoclonal antibody medicine reference material EC50 value
To the biological activity value of reference material.
2, ELISA quantitative experiment: it is directed to the detection antigen of monoclonal antibody medicine A and monoclonal antibody medicine B using specificity, is based on
ELISA sandwich method experimental principle establishes the ELISA protein quantification method for being directed to this 2 components, for monoclonal antibody medicine A and monoclonal antibody medicine
The ELISA quantitative approach of object B, in addition to coating specific antigen is different, remaining step is almost the same.
Experimentation is as follows:
It takes the specific antigen for monoclonal antibody medicine A or B to be coated with 96 hole elisa plates, BSA is added to close；
Then the monoclonal antibody medicine A of known concentration or B standard albumen is taken to be serially diluted to obtain the standard song of series of concentrations
Line sample is added in elisa plate in conjunction with envelope antigen, and internal control sample and sample to be tested are added at the same time；
The ELIAS secondary antibody that coupling HRP label is added in elisa plate is taken out after being incubated for a period of time, is incubated for a period of time followed by
TMB is added and carries out chromogenic reaction.
It is quasi- that 4- parameter curve is carried out according to the dose-effect relationship of the sample concentration of standard curve sample well and chromogenic reaction OD value
It closes, the quantitation curves for being directed to monoclonal antibody medicine A or B respectively is drawn, by the chromogenic reaction OD value of internal control sample well and sample to be tested hole
The sample concentration that can be calculated internal control sample and sample to be tested is brought in standard curve into method accuracy, precision control
The quantitative detection of system and sample to be tested.
Three, the specific steps of the bio-evaluation method for biopharmaceutical composition of the invention
1, 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 all had, monoclonal antibody medicine A and B is mixed to form new bio medicine according to mass ratio 1:1
When compositions and tumour cell act on, monoclonal antibody medicine component A and B component can play in conjunction with tumor targets different epitopes
Synergistic 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 for the exploitation of the cell inhibitory effect activity methods of A or B component,
It screens obtained experiment and sensitivity is showed to monoclonal antibody A or B component with tumor cell line.It is raw that Proliferation Ability is established based on the cell strain
Object activity test method can be used for the biology of monoclonal antibody A or monoclonal antibody B or monoclonal antibody medicine composition (the two combination is considered as entirety)
Learn activity rating.Method Of Accomplishment exploitation detects monoclonal antibody A or monoclonal antibody B or monoclonal antibody medicine group using the biological cell activity methods
The typical map for closing the biological activity of object 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 verifying and confirmation of activity test method, and high spot reviews method accuracy is shown in Table 1~table 3 respectively,
It can be found that using the biological activity method in the biological activity for detecting monoclonal antibody A, monoclonal antibody B or monoclonal antibody medicine composition respectively
When, in the range of the method accuracy in detection in active 50%~150% range of titer level is met 75%~125%,
Complete the confirmation of the biological cell activity methods to this for monoclonal antibody medicine composition.On the basis of method confirmation, into one
Step completes the methodology validation of the cell activity method.
1. monoclonal antibody component A biological activity accuracy in detection of table investigates result
| Sample ID | Testing result | The rate of recovery |
| A50 | 54% | 108% |
| A75 | 84% | 112% |
| A125 | 131% | 105% |
| A150 | 149% | 99% |
2. monoclonal antibody B component biological activity accuracy in detection of table investigates result
| Sample ID | Testing result | The rate of recovery |
| B50 | 49% | 98% |
| B75 | 66% | 88% |
| B125 | 154% | 123% |
| B150 | 147% | 98% |
3. monoclonal antibody composition P biological activity of table detects precision and investigates result summary sheet
| Sample ID | Testing result | The rate of recovery |
| P50 | 59% | 118% |
| P75 | 87% | 116% |
| P125 | 127% | 102% |
| P150 | 150% | 100% |
2, after establishing simultaneously Method Of Accomplishment confirmation and verifying for the biological cell activity methods of monoclonal antibody medicine composition,
Establish the ELISA protein quantification method for being directed to the composition monoclonal antibody medicine component A and monoclonal antibody medicine B component respectively.ELISA albumen
It is single that the key that quantitative approach is established is that screening confirmation can be used for being directed to respectively for drug quantitative ELISA methods exploitation
The specific antigen of antiradiation drug component A and monoclonal antibody medicine B component.Screening has determined 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 established for monoclonal antibody component A and monoclonal antibody B component respectively,
This method meet can specificity in monoclonal antibody medicine composition monoclonal antibody component A or monoclonal antibody B component carry out protein quantification, and phase
It is not interfered between mutually.Method Of Accomplishment exploitation, 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 accuracy, and not by the interference of another monoclonal antibody component presence or absence.To be directed to the ELISA quantitative approach of monoclonal antibody component A
Method validation and confirmation for, prepare 3 samples include A+B (A is mixed with B according to mass ratio 1:1, A concentration keep 5mg/
ML), A+3B (A is mixed with B according to mass ratio 1:3, wherein A concentration keep 5mg/mL), A+1/3B (A and B are according to mass ratio 3:
1 mixing, wherein the concentration of A keeps 5mg/mL), 6 independences of the plate arrangement mode of different samples are carried out for this 3 samples
ELISA quantitative detection, each detection does 3, and detection data, which summarizes, is shown in Table 4 in parallel, respectively obtains 18 detections for 3 samples
As a result and statistical analysis is carried out, the RSD of monoclonal antibody component A concentration mensuration result meets < 10%, and the rate of recovery meets 90%~
In 110% range.And so on, method validation and confirmation result for the ELISA quantitative approach of monoclonal antibody B component, which summarize, to be shown in Table
5,18 testing results are respectively obtained for 3 samples and carry out statistical analysis, the RSD of monoclonal antibody B component concentration mensuration result
Meet < 10%, the rate of recovery meets in 90%~110% range.Confirm that established ELISA is quantitative by these experimental results
Method accurately the component A to monoclonal antibody medicine composition and B component can realize protein quantification, and not interfere between each other.
4. monoclonal antibody component A protein quantification accuracy in detection of table and precision investigate result summary sheet
5. monoclonal antibody B component protein quantification accuracy in detection of table and precision investigate result summary sheet
3, complete respectively for the ELISA quantitative detection of monoclonal antibody component A and component B after, further by monoclonal antibody component A and
Monoclonal antibody component B is mixed according to different mass ratios, obtains the monoclonal antibody medicine composition of serial mixed proportion, with for monoclonal antibody medicine
The biological cell activity methods of compositions detect the monoclonal antibody medicine composition of serial mixed proportion, are recorded in different proportion condition
Under active 4- parameter curve fit EC50 value, and opposite monoclonal antibody medicine component A and B component are according to made of 1:1 mass ratio
The relative biological activity of monoclonal antibody medicine composition (P (A+B)).It is calculated on the basis of monoclonal antibody medicine component A containing not homogeneity
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 the monoclonal antibody medicine composition of the B component of the ratio containing different quality is calculated in standard 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 for calculating monoclonal antibody medicine composition on the basis of monoclonal antibody component A it can be seen from the result of table 6 and table 7
When learning activity, with the continuous decline of monoclonal antibody component A mass percentage be monoclonal antibody B component mass percentage it is continuous on
It rises, the EC of Activity determination result50Value constantly decline, composition is with respect to the P (A that monoclonal antibody component A and B is mixed according to 1:1 mass ratio
+ B) bioactivity of monoclonal antibody medicine composition then constantly rises, illustrate 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, it is same to send out
Now with the rising that the decline of B component mass percentage is component A mass percentage, the EC of Activity determination result50Value is not
Disconnected decline, and composition is with respect to the life of monoclonal antibody component A and B P (A+B) monoclonal antibody medicine composition mixed according to 1:1 mass ratio
Object activity constantly rises, and equally illustrates that 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,
The two, which is combined into novel monoclonal antibody medicine composition in terms for the treatment of tumour, positive effect.At the same time, according to
Relative biological under the different quality ratio mixing condition of the available monoclonal antibody component A of the experimental result of table 6 and table 7 and B component
Activity value and EC50The situation of change of value, in practical 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 variation, in conjunction with ELISA concentration quantitative result can judge monoclonal antibody component A or
Whether B component there is activity change, realizes in-depth analysis and research to the biological activity of monoclonal antibody medicine composition P.Four, originally
Invention practical application
Take monoclonal antibody medicine A and monoclonal antibody medicine B to be mixed to get P (A+B) according to 1:1 mass percent, and monoclonal antibody medicine A and
The concentration of B is 2.5mg/mL；The monoclonal antibody medicine A of 2.5mg/mL is placed 30 minutes in 80 DEG C of high temperature and is destroyed, and is assumed
There is no variations for its concentration, by the monoclonal antibody medicine A Jing Guo high-temperature process and the monoclonal antibody medicine B component without destruction according to matter
Amount is mixed to get P (A*+B) than 1:1, and the concentration of monoclonal antibody medicine B is 2.5mg/mL；By the monoclonal antibody medicine B of 2.5mg/mL at 80 DEG C
High temperature is placed and is destroyed for 30 minutes, and assumes its concentration there is no variation, by the monoclonal antibody medicine B Jing Guo high-temperature process and
Monoclonal antibody medicine component A without destruction is mixed to get P (A+B*) according to mass ratio 1:1, and the concentration of monoclonal antibody medicine A is 2.5mg/
mL；30 minutes monoclonal antibody medicine A destroyed 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 is mixed according to mass ratio 1:1, obtains P (A*+B*).After obtaining above-mentioned 4 samples, biology above-mentioned is utilized
Cell activity method and ELISA protein quantification method detect respectively this 4 samples biological activity and corresponding monoclonal antibody
The protein concentration of drug component A and B component, and according to testing result to the biological activity of 4 monoclonal antibody medicine composition samples
It is analyzed.
4 samples are accordingly to be regarded as entirety by step 1 analysis, UV method are directly utilized, by 4 monoclonal antibodies through destroying in various degree
Drug A and B component directly carry out protein concentration analysis, respectively obtain the protein concentration testing result of 4 samples as a whole point
8 are not shown in Table, it can be found that not observing the significant change of protein concentration by the protein concentration that direct UV method detects 4 samples
Change.UV method carries out protein quantification based on the amino acid composition of protein composition entirety, and each component of composition cannot be distinguished
Concentration variation.
Table 8. is by the monoclonal antibody medicine composition protein concentration UV method protein concentration testing result summary sheet 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 |
The protein concentration directly detected according to the UV method of monoclonal antibody medicine composition is as a result, monoclonal antibody medicine composition is regarded
For entirety, the reference material combined using monoclonal antibody medicine A and monoclonal antibody medicine B according to 1:1 detects P (A+B), P (A*+ as standard respectively
B), the biological activity of P (A+B*), P (A*+B*) 4 by the monoclonal antibody composition sample destroyed, 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 can be seen that by above-mentioned experimental result when being considered as whole with monoclonal antibody medicine composition, the combination based on the detection of UV method
The protein concentration of object can sensitively detect monoclonal antibody medicine component A or monoclonal antibody medicine component B using the Biological Activity Methods
Active variation when being damaged, when monoclonal antibody component A or monoclonal antibody component B is not damaged, the cell activity of P (A+B) with
The biological activity of reference material has no significant difference, and when stability destruction occurs for the A of monoclonal antibody medicine composition or B, P (A*+
B), P (A+B*), P (A*+B*) cell activity be remarkably decreased.When stability, which occurs, for component A and B component destroys,
The cell activity decline of P (A*+B*) is the most significant.
Although being able to detect to obtain P (A*+B), P (A+B*), P (A*+B*) this 3 samples by the experiment of above-mentioned step 1
Cell activity decline, it can be difficult to judgement be caused by destroying due to A active component or due to B activity component integrally
The cell activity of sample declines, and the experimental analysis of subsequent step 2 and step 3 need to be carried out for the query.Step 2 analysis and utilization point
Safety pin is unknown through high-temperature stability to above-mentioned 4 to the ELISA quantitative approach of monoclonal antibody medicine component A and monoclonal antibody medicine component B
The protein concentration of each component of the monoclonal antibody medicine composition of destruction carries out detection determination.Step 2, which is analyzed, 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 sheet destroyed
It is can be found that by 10 protein quantification testing result of table and utilizes that is established to be directed to monoclonal antibody component A and monoclonal antibody component respectively
The ELISA protein quantification method of B 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, consistent with expection.Accurate ELISA protein quantification is the result is that subsequent accurate judgement monoclonal antibody medicine group
Whether the component A or component B for closing object occur the basis of bioactivity variation.It is unknown to 4 by step 2 ELISA quantitative detection
The monoclonal antibody medicine composition through high temperature component A and B component carry out protein quantification detection respectively, according to protein quantification knot
Fruit can tentatively show that the component A of P (A+B) sample and B component 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.However the conclusion is based only on what ELISA protein quantification obtained, needs by the further true of cytoactive detection
Card carries out dividing based on cell bio-activity of the ELISA protein quantification result to 4 protein medicinal compositions for step 3
Analysis detection.
The component A or B component of monoclonal antibody medicine composition are determined in the protein ELISA quantitative experiment analyzed by step 2
Accurately after " effective " protein concentration, the biological cell activity experiment that can be directed to monoclonal antibody medicine component A or B component simultaneously is utilized
The monoclonal antibody medicine composition sample destroyed to 4 by different degrees of stability carries out analysis of biological activity, respectively with monoclonal antibody medicine
The protein concentration of object component A or carried out on the basis of the protein concentration of monoclonal antibody medicine B component opposite monoclonal antibody medicine composition (not by
The A of destruction, B component are mixed according to 1:1) analysis of biological activity of reference material.And will test result with complete before table 6,
Activity Results data of the table 7 under the conditions of different A, B component mixed proportion are compared, and speculate A and B component according to Activity Results
Portfolio ratio, and be compared by the A that ELISA is quantitatively obtained with the portfolio ratio of B component with practical, to being based on ELISA
The relative combinations ratio of the component A and B component that quantitatively obtain is confirmed, and infers whether are the component A in composition or B component
It is destroyed.
The monoclonal antibody medicine composition sample biology that stability is destroyed is detected on the basis of the protein concentration of monoclonal antibody medicine component A
It learns cell activity result and sees 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 on the basis of monoclonal antibody medicine A " effective " concentration
The activity of monoclonal antibody medicine composition is detected, according to testing result, in conjunction with table 6 obtain according to different proportion monoclonal antibody A or B group
Point Activity determination as a result, speculate to the relative scale of the A and B of 4 monoclonal antibody medicine compositions through destroying in various degree,
It can be found that the ratio of the component A and B component speculated according to Activity Results and the component A and B that are obtained by step 2 protein quantification
The actually detected ratio of component is almost the same.For P (A+B*) sample, because the protein concentration of A protein component does not become substantially
Change, it can be deduced that the inference that B protein component destroys in the composition.For P (A*+B) sample, because B protein component
Protein concentration does not change substantially, it can be deduced that the inference that A protein component destroys in the 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 cell activity of quasi- product is not consistent, it can be deduced that A protein component and B protein component destroyed in the composition pushes away
By.
The monoclonal antibody medicine composition sample biology that stability is destroyed is detected on the basis of the protein concentration of monoclonal antibody medicine B component
It learns cell activity result and sees 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, in conjunction with table 7 obtain according to different proportion monoclonal antibody A or B group
Point Activity determination as a result, speculate to the relative scale of the A and B of 4 monoclonal antibody medicine compositions through destroying in various degree,
It can be found that the ratio of the component A and B component speculated according to Activity Results and the component A and B that are obtained by step 2 protein quantification
The actually detected ratio of component is also almost the same.Also according to the result to the A of 4 monoclonal antibody medicine compositions and the phase of B component
Comparative example is able to carry out confirmation, obtains being directed to what component A or B component in 4 monoclonal antibody medicine composition samples were destroyed
Conclusion.
Using the experiment of above-mentioned 3 steps, the unknown monoclonal antibody medicine destroyed in various degree that 4 are simulated is combined
The activity of cell biology of object is analyzed, it can be found that the cell bio-activity containing the monoclonal antibody medicine composition for destroying component
There are different degrees of decline, while the monoclonal antibody medicine declined based on ELISA protein quantification 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 conclusion 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 especially drug discovery process of new bio pharmaceutical composition is ground
Study carefully the expansion in field.Conventional needle is unable to satisfy to a variety of bio-pharmaceuticals the biological evaluation method of single creature drug component
The needs that the bioactivity of the composition of formation is analysed in depth.The exploitation of present invention combination Biological Activity Methods, ELISA protein quantification
Analysis method exploitation, and by the Activity determination interpretation of result to different mixing proportion sample, can be realized through a kind of biology
The activity change situation that activity methods realize that each component to complicated biopharmaceutical composition may occur is analyzed.It should
Method is clear, develops and moderate using difficulty, easily promotes, can satisfy and comment the biological function of biopharmaceutical composition
The needs of valence, product clearance, stability study etc..
Beneficial effects of the present invention are characterized in particular in: being used method of the invention, it is possible to system comprehensively to bio-pharmaceutical group
The biological activity for closing object 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 for biopharmaceutical composition, using unified method to biopharmaceutical composition
Biological activity is evaluated；Accurate protein quantification is carried out based on ELISA quantitative means single protein component each to composition,
Since established 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 variation of these testing results.It is unified based on this
Activity determination means and method can be realized 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 very extensive,
It, only need to be based on the collective effect mechanism of composition including monoclonal antibody drug, fusion protein, polypeptide protein medicinal composition
The exploitation of biological activity method, and protein quantification are carried out, combined data analysis can be realized as comprehensive bio-pharmaceutical combination
The bio-evaluation of object.Involved biological activity method includes cell activity, in conjunction with activity and animal activity in vivo
Experiment etc..The inventive method is the strong supplement and support of the pharmacy part research to biopharmaceutical composition research and development, can
For the activity research of such product, product clearance, stability study 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 invention.Therefore, the description and the appended drawings should be considered as illustrative
And not restrictive.
Claims (9)
1. a kind of bio-evaluation method for biopharmaceutical composition, which comprises the following steps:
Step (1): establishing the protein quantification method based on ELISA for each protein component in biopharmaceutical composition,
For accurately detecting the protein concentration of each protein component in biopharmaceutical composition；
Step (2): the common biological action mechanism of each protein component in confirmation biopharmaceutical composition, and it is based on institute
The biological action mechanism stated establishes bio-evaluation method, and the bio-evaluation method can be used for evaluating life
The biological activity of each single protein component in medicine composition；
Step (3): each protein component of biopharmaceutical composition is mixed in known concentration according to different proportion, benefit
The biological activity of the biopharmaceutical composition of different proportion mixing is detected, with established bio-evaluation method with certain list
On the basis of one component protein concentration, the biological activity for recording the biopharmaceutical composition under the conditions of different mixing proportion detects knot
Fruit obtains biopharmaceutical composition and biology under the conditions of different mixing proportion according to the biological activity testing result
Learn active relationship；
Step (4): according to passing through the pass of biopharmaceutical composition and biological activity under different mixing proportion in step (3)
System, thus it is speculated that the relative scale of each protein component in pharmaceutical composition will obtain in relative scale relationship and the step (1) that are speculated
The detection protein concentration of each protein component compare and verifying and judge whole biopharmaceutical composition, in pharmaceutical composition
The activity stability of each protein component changes.
2. the bio-evaluation method according to claim 1 for biopharmaceutical composition, which is characterized in that institute
The biopharmaceutical composition stated includes at least two components, and the group is selected from monoclonal antibody drug, fusion protein, polypeptide
Class drug.
3. the bio-evaluation method according to claim 1 for biopharmaceutical composition, which is characterized in that institute
The biopharmaceutical composition stated is monoclonal antibody combination, if each protein component of the 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 established
Method；If the target spot of each protein component of the biopharmaceutical composition is different, establish for monoclonal antibody Fc section jointly
The bio-evaluation method of biological function finds the cell strain comprising each different target spots, establishes the cell
Strain is directed to the biological activity method of different component.
4. the bio-evaluation method according to claim 1 for biopharmaceutical composition, which is characterized in that institute
The biopharmaceutical composition stated is the biopharmaceutical composition that each component is directed to same target spot different epitopes.
5. the bio-evaluation method according to claim 1 for biopharmaceutical composition, which is characterized in that institute
The component in biopharmaceutical composition stated is mixed according to preset ratio.
6. the bio-evaluation method according to claim 1 for biopharmaceutical composition, which is characterized in that institute
Bio-evaluation method in the step of stating (2) include ELISA combination activity methods, cell in vitro activity methods, in vivo
Zoopery activity methods.
7. the bio-evaluation method according to claim 1 for biopharmaceutical composition, which is characterized in that institute
Biological activity testing result in the step of stating (3) includes that activity assay, dose-effect relationship 4- parameter or 5- parameter fitting are bent
Line EC50Value.
8. the bio-evaluation method according to claim 1 for biopharmaceutical composition, which is characterized in that institute
Bio-evaluation method in the step of stating (2) specifically includes the following steps:
Step (21): the cell of logarithmic growth phase adjusts certain density after pancreatin digests and spreads 96 porocyte plates, places one
The section time waits for that cell is adherent；
Step (22): taking out testing protein drug or composition is diluted to obtain sample and the addition of serial dilutions with culture medium
It is incubated for a period of time in cell plates jointly with cell, the proliferation of cell is suppressed under the action of drug, and cell dyeing is added
Liquid MTS detects cell inhibitory effect situation；
Step (23): 4- parameter fitting is carried out according to the dose-effect relationship of drug concentration and staining signals value, egg to be measured is calculated
Baiyao object signal value inhibits drug concentration EC50 value when half, and protein drug reference material EC50 value is divided by testing protein drug
Biological activity value of the testing protein drug with respect to reference material is calculated in EC50 value.
9. the bio-evaluation method according to claim 1 for biopharmaceutical composition, which is characterized in that institute
Quantitative approach in the step of stating (1) be specifically includes the following steps:
Step (11): it takes the specific antigen for protein drug to be coated with 96 hole elisa plates, BSA is added to close；
Step (12): the protein drug standard protein of known concentration is taken to be serially diluted to obtain the standard curve sample of series of concentrations
Product are added in elisa plate in conjunction with envelope antigen, and internal control sample and sample to be tested are added at the same time；
Step (13): the ELIAS secondary antibody that coupling HRP label is added in elisa plate is taken out after being incubated for a period of time, is incubated for a period of time
TMB is followed by added and carries out chromogenic reaction；
Step (14): it is bent that 4- parameter is carried out according to the dose-effect relationship of the sample concentration of standard curve sample well and chromogenic reaction OD value
Line fitting draws the quantitation curves for being directed to protein drug A or B respectively, by the chromogenic reaction of internal control sample well and sample to be tested hole
OD value brings in standard curve the sample concentration that internal control sample and sample to be tested is calculated into, to method accuracy, precision
The quantitative detection of control and sample to be tested.
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| CN105628910A (en) * | 2014-11-07 | 2016-06-01 | 上海中信国健药业股份有限公司 | Biological activity detection method for VEGF targeted therapy drugs |
| CN106405095A (en) * | 2015-07-28 | 2017-02-15 | 南京农业大学 | Detection of animal-derived allergen bovine serum albumin by using indirect competitive ELISA method |
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| CN105628910A (en) * | 2014-11-07 | 2016-06-01 | 上海中信国健药业股份有限公司 | Biological activity detection method for VEGF targeted therapy drugs |
| CN106405095A (en) * | 2015-07-28 | 2017-02-15 | 南京农业大学 | Detection of animal-derived allergen bovine serum albumin by using indirect competitive ELISA method |
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