CN110018253A - The high-efficient liquid phase determining method of Free protein content in a kind of biological products - Google Patents
The high-efficient liquid phase determining method of Free protein content in a kind of biological products Download PDFInfo
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- CN110018253A CN110018253A CN201910300815.4A CN201910300815A CN110018253A CN 110018253 A CN110018253 A CN 110018253A CN 201910300815 A CN201910300815 A CN 201910300815A CN 110018253 A CN110018253 A CN 110018253A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
Abstract
The invention belongs to the detection fields of Free protein content, the high-efficient liquid phase determining method of Free protein content in a kind of specifically disclosed biological products, including chromatographic condition: chromatographic column SRT SEC serial analysis column or TSKgel G3000SW analytical column, 4-30 DEG C of column temperature, mobile phase 0.2M sodium chloride solution, flow velocity 0.2-1.0mL/min, pH 6.8-7.2, Detection wavelength 280nm；The preparation of sample solution: taking sample to be tested appropriate, with flowing phase dilution, obtains sample introduction sample；Assay: sample introduction sample is injected into high performance liquid chromatograph, records chromatogram, and calculate measurement result.The present invention can accurately determine the content of the CRM197 albumen to dissociate in combined vaccine, ensure that the validity and safety of combined vaccine, have the characteristics that low in cost, accuracy is high, it is reproducible, detect it is simple and efficient, convenient for being widely popularized and applied.
Description
Technical field
The invention belongs to the detection technique field of Free protein content, in particular to floating preteins contain in a kind of biological products
The high-efficient liquid phase determining method of amount.
Background technique
Polysaccharide vaccine can provide adult good immanoprotection action, but to children below 2 one full year of life almost without effect, former
Because be CPS be thymus independent antigen (Thymus independent antigen, TI-Ag), studies have shown that by polysaccharide with
Protein carrier coupling prepares combined vaccine, and TI-Ag can be made to be changed into thymus dependent antigen (Thymus dependent
Antigen, TD-Ag), change the antigenic property of CPS, enhance its immunogenicity, induce immunological memory, is 2 years old or less children
Long-term protective effect is provided.
In the preparation process of combined vaccine, there can be a small amount of carrier protein and fail in conjunction with polysaccharide, the load of this part
Body protein becomes floating preteins.Currently used carrier protein include tetanus toxoid (TT), diphtheria toxoid (DT),
Diphtheria is attenuated toxin (CRM197 albumen) and other amino acid fragments that can play carrier protein effect.
Studies have shown that Free protein content is more than that defined limit may reduce the immunogenicity of vaccine or improve secondary anti-
The incidence answered, WHO recommend the Free protein content in bacterial polysaccharide protein combined vaccine to should be less than the 5% of total protein content,
Therefore, in combined vaccine Free protein content be vaccine quality control important indicator.
The method of Free protein content mainly has polyacrylamide gel electrophoresis (referred to as in existing detection combined vaccine
SDS-PAGE method) and high performance liquid chromatography (abbreviation HPLC method).Wherein SDS-PAGE method has the test period long, precision
It is low, poor accuracy, it is less reproducible the disadvantages of.HPLC method is compared to easier, precision is high, accuracy is good, repeated height, still
The method for wanting floating preteins in established HPLC detection combined vaccine, and by the property of combined vaccine, the property of carrier protein
The influence of the various aspects such as matter, the type selecting for testing and analyzing column.
Wherein, the most-often used examination criteria of floating preteins is the HPLC method of 2015 editions " Chinese Pharmacopoeia " records.However, this
Applicant use the detection method detection diphtheria attenuation toxin (CRM197 albumen) as carrier protein and made of combination
When vaccine, the CRM197 albumen to dissociate in the combined vaccine is difficult to accurately detect its content, as a result, the combined vaccine has
Higher security risk.
Summary of the invention
In view of the deficiencies of the prior art, the present invention intends to provide Free protein content in a kind of biological products
High-efficient liquid phase determining method, by combining the property of CRM197 albumen, to chromatographic column, mobile phase and every experiment parameter
Groped, has filtered out a kind of efficient, accurate, accurate high-efficient liquid phase determining method, can accurately measure in combined vaccine
The content of free CRM197 albumen.
To achieve the above object, the present invention provides the following technical scheme that
The high-efficient liquid phase determining method of Free protein content in a kind of biological products, comprising the following steps:
1., chromatographic condition: chromatographic column be SRT SEC serial analysis column or TSKgelG3000SW analytical column, column temperature be 4-30 DEG C,
Mobile phase is 0.2M sodium chloride solution, flow velocity 0.2-1.0mL/min, pH 6.8-7.2, Detection wavelength 280nm；
2., the configuration of sample solution: take sample to be tested appropriate, with flowing phase dilution sample to be tested, obtain sample introduction sample；
3., assay: sample introduction sample is injected into high performance liquid chromatograph, records chromatogram, normalization method, which calculates, according to area surveys
Measure result.
For the present invention by using above-mentioned technical proposal, the CRM197 albumen to dissociate in combined vaccine can be in SRT SEC system
It is eluted out, is made with the flow velocity of 0.2-1.0mL/min with 0.2M sodium chloride in column analytical column or TSKgel G3000SW analytical column
Conjugate in combined vaccine and floating preteins efficiently separate, thus facilitate high performance liquid chromatograph accurately determine it is free
CRM197 albumen content, operator can manage the production of combined vaccine according to its testing result, to guarantee to tie
Close the effective potency and safety of vaccine.Wherein, 0.2M sodium chloride solution component is simple, also can be reduced relative to PBS buffer solution
Cost so that measuring method of the invention effectively reduces testing cost to a certain extent, convenient for being widely popularized and
Using.
Further, step 1. in, the SRT SEC serial analysis column be SRT SEC-300 analytical column, it is described
The specification of TSKgelG3000SW analytical column is 7.5mm × 600mm.
By using above-mentioned technical proposal, it is generally the case that the column length of analytical column is longer, and separating degree is better, but simultaneously
The retention time that will lead to floating preteins increases, so that the appearance of floating preteins is later in chromatogram；In addition, SRT SEC-
Cost is relatively low relative to SRT SEC-600 analytical column and TSKgelG3000SW analytical column for 300 analytical columns.Based on this, SRT SEC
The preferred SRT SEC-300 analytical column of serial analysis column, the specification of TSKgelG3000SW analytical column is preferably 7.5mm × 600mm,
And preferred SRT SEC-300 analytical column in two analytical columns；
Further, step 1. in, the column temperature be 20-26 DEG C.
Further, step 1. in, the column temperature be 23 ± 1 DEG C.
By using above-mentioned technical proposal, 20-26 DEG C be usually laboratory room temperature, column temperature at such a temperature can be accurate
The content of CRM197 albumen is measured, while reduce laboratory for control experimental temperature and column temperature is regulated and controled,
The testing cost of floating preteins is reduced to a certain extent.Wherein, when column temperature is 23 ± 1 DEG C, accuracy highest is detected.
Further, step 1. in, the flow velocity be 0.4-0.75mL/min.
Further, step 1. in, the flow velocity be 0.5mL/min.
By using above-mentioned technical proposal, by lot of experiment validation, when flow velocity is 0.4-0.75mL/min, CRM197
Protein recovery of the albumen in SRT SEC-300 analytical column is higher, wherein optimal when 0.5mL/min.
Further, step 1. in, the pH be 7.0 ± 0.02.
By using above-mentioned technical proposal, when pH is 7.0 ± 0.02, CRM197 albumen is able to maintain the structure of its own
Characteristic improves the accuracy of detection so that the formed structural proteins of CRM197 albumen efficiently separate.
Further, step 2. in, in the sample introduction sample total protein content be 12.5 μ g of >.
Further, step 2. in, in the sample introduction sample total protein content be 20-50 μ g.
It can accurately be surveyed by using above-mentioned technical proposal when the total protein content in sample introduction sample is 12.5 μ g of >
Make the content of the CRM197 albumen in combined vaccine.Wherein, the total protein content of sample introduction sample is controlled in 20-50 μ g, energy
The dosage for enough reducing sample to be tested, to reduce the testing cost of floating preteins to a certain extent.
Further, the assay of the measuring method of the invention free CRM197 albumen suitable for combined vaccine.
In conclusion the invention has the following advantages:
1, the present invention gropes chromatographic column, mobile phase and every experiment parameter, can accurately determine in combined vaccine
The content of free CRM197 albumen ensure that the effective potency and safety of combined vaccine, have low in cost, accuracy
It is high, reproducible, detection it is simple and efficient, convenient for being widely popularized and being applied the characteristics of；
2, the specification and column temperature of the invention by further limiting chromatographic column, the egg in the flow velocity of mobile phase and pH, sample introduction sample
White concentration and protein control product effectively increase the detection accuracy and repeatability of free CRM197 protein content.
Detailed description of the invention
Fig. 1 is the operational flowchart for measuring Free protein content in combined vaccine；
Fig. 2 is the chromatogram of conjugate a；
Fig. 3 is the chromatogram of conjugate b；
Fig. 4 is the chromatogram of conjugate c.
Specific embodiment
Below in conjunction with attached drawing, invention is further described in detail.
One, detection uses equipment and solution
1, equipment and model: 1260 high performance liquid chromatograph of Agilent.
2, chromatographic column:
TOSOH Co., Ltd TSKgelG3000SW (7.5mm × 600mm) analytical column；
TOSOH Co., Ltd TSKgel G5000PWXL (7.8mm × 300mm) analytical column；
Suzhou Sepax Technologies, Inc. SRT SEC-300 (7.8mm × 300mm) analytical column；
Suzhou Sepax Technologies, Inc. SRT SEC-600 (7.8mm × 600mm) analytical column.
3, mobile phase:
Water for injection；
0.45wt% sodium chloride solution, 0.9wt% sodium chloride solution and 0.2M sodium chloride solution use water for injection and chlorination
Sodium chromatographic grade is formulated in proportion；
PBS buffer solution: 50mM PB+0.3mol/L NaCl.
4, albumen:
Protein control product: protein control product used by the application are identical with combined vaccine carrier protein, as CRM197 albumen,
Be by fermentation, purify, saltout, column purification, a series of means such as freeze-drying obtain the freeze-dried powders of purity >=95%, also can be by the
Tripartite, which buys, obtains CRM197 albumen powder.CRM197 albumen relevant information used in this patent are as follows: the self-control of this enterprise, lot number
201810006。
5, sample is measured:
The present invention is with meningococcal polysacharide combined vaccine, b type haemophilus influenzae polysaccharide conjugate vaccine, streptococcus pneumoniae polysaccharides
For combined vaccine, but not merely it is limited to these three combined vaccines, as measurement sample, is successively named as conjugate a, conjugate
B, conjugate c.Conjugate a used in this patent, conjugate b, conjugate c are the self-control of this enterprise, the wherein life of conjugate a
Producing lot number is 201803004, and the product batch number of conjugate b is 201804001, and the product batch number of conjugate c is 201806003.
Two, detection method specific implementation process
The high-efficient liquid phase determining method of Free protein content in a kind of biological products, referring to Fig. 1, comprising the following steps:
1., chromatographic condition: chromatographic column be SRT SEC serial analysis column or TSKgelG3000SW analytical column, column temperature be 4-30 DEG C,
Mobile phase is 0.2M sodium chloride solution, flow velocity 0.2-1.0mL/min, pH 6.8-7.2, Detection wavelength 280nm.
2., the preparation of sample solution: take sample to be tested appropriate, dilute sample to be tested with 0.2M sodium chloride solution, obtain into
All product；
3., assay: sample introduction sample is injected into high performance liquid chromatograph, records chromatogram, normalization method, which calculates, according to area surveys
Measure result.
1, the selection of mobile phase and specificity confirmation
TOSOH Co., Ltd TSKG5000PWXL analytical column (2015 editions Chinese Pharmacopoeias are recommended to use) is selected, in conjugate a, conjugate
B, it is separately added into the CRM197 albumen of 5wt% in conjugate c as sample introduction sample, is infused sample introduction sample using 100 μ l injection annulus
Enter in the analytical column of high performance liquid chromatograph, respectively using water for injection, 0.45wt% sodium chloride, 0.90wt% sodium chloride,
As mobile phase, concurrently setting column temperature as 23 ± 1 DEG C, flow velocity 0.5mL/min, pH is for 0.2M sodium chloride and PBS buffer solution
7.0 ± 0.02, Detection wavelength 280nm.According to the appearance situation and signal-to-noise ratio of chromatogram, analyze what sample introduction sample was suitable for
Mobile phase, each group repeat test 3 times, and measurement result is referring to following table one.
Signal-to-noise ratio when one 4 kinds of mobile phase elution CRM197 albumen of table
Referring to table one, when using water for injection or 0.45wt% sodium chloride as mobile phase, only there is apparent conjugate
Characteristic peak, there can be no apparent CRM197 protein specificity peaks, and signal-to-noise ratio is 0.Use 0.90wt% sodium chloride as flowing
Xiang Shi, 3 tests only have 1 time apparent conjugate characteristic peak and apparent CRM197 protein specificity peak simultaneously.As a result, with note
It penetrates and uses water and 0.45wt% sodium chloride and 0.90wt% sodium chloride as mobile phase, it is difficult to detect or be difficult to stable detection and go out
The characteristic peak of floating preteins in combined vaccine.
When using 0.2M sodium chloride and PBS buffer solution as mobile phase, measurement result can have apparent conjugate simultaneously
Characteristic peak and apparent CRM197 protein specificity peak, and noise is relatively high.But due to PBS buffer solution complicated components and cost compared with
Height, the present invention using the sodium chloride of 0.2M just can reach with testing result similar in PBS buffer solution, effectively reduce detection at
This, therefore preferably 0.2M sodium chloride is as mobile phase.
2, the selection of analytical column
Use 0.2M sodium chloride solution for mobile phase, flow velocity 0.5mL/min, pH are 7.0 ± 0.02, Detection wavelength 280nm,
The CRM197 albumen of 5wt% is added in conjugate a, conjugate b and conjugate c respectively as sample introduction sample, selects not homochromy
Compose column: TOSOH Co., Ltd TSKgel G3000SW (7.5mm × 600mm) analytical column, TOSOH Co., Ltd TSKgel
G5000PWXL (7.8mm × 300mm) analytical column, Suzhou Sepax Technologies, Inc. SRT SEC-300 (7.8mm × 300mm)
Analytical column, Suzhou Sepax Technologies, Inc. SRT SEC-600 (7.8mm × 600mm) analytical column, will using 100 μ l injection annulus
Sample introduction sample injects in the analytical column of high performance liquid chromatograph, and control column temperature is 23 ± 1 DEG C and carried out column analysis.It is tied by evaluation
Close object signal-to-noise ratio and conjugate and CRM197 albumen separating degree, final choice be suitable for analytical column, measurement result referring to
Following table two.
The signal-to-noise ratio and separating degree of two conjugate of table and CRM197 albumen
Referring to table two, when TSKgel G5000PWXL analyzes post separation sample, separating degree, which is slightly less than 1.5 and does not meet separation, to be wanted
Ask, though the analytical column can detect the presence or absence of CRM197 albumen, be difficult to Accurate Determining go out the content of CRM197 albumen because
This, is not suitable under this experiment condition.TSKgel G3000SW analytical column, SRT SEC-300 and SRT SEC-600 analytical column point
It is little from effect gap, all reach the separation requirement of separating degree > 1.5.It is contemplated that TSKgel G3000SW analytical column at
Originally it is apparently higher than the cost of SRT SEC-300 analytical column and SRT SEC-600, and the former signal-to-noise ratio is significantly lower than the latter's
Signal-to-noise ratio, therefore preferentially select SRT SEC serial analysis column as the analytical column of calibrating.
In addition, separating degree is close when using the SRT SEC serial analysis post separation sample that length is 300mm and 600mm
And > 1.90, the separating degree of analytical column are all preferable.In view of " column length is longer, and separating degree is better, but will lead to simultaneously free
The retention time of albumen increases, and the appearance of floating preteins is later in chromatogram " this characteristic, SRT SEC-300 analysis post detection
Want faster compared to SRT SEC-600 analytical column, the last analytical column for preferentially selecting SRT SEC-300 analytical column as calibrating.
3, the determination of detection limit and quantitative limit
To use chromatographic column for SRT SEC-300 (7.8mm × 300mm) analytical column, column temperature be 23 ± 1 DEG C, 0.2M sodium chloride conduct
Mobile phase, flow velocity 0.5mL/min, pH be 7.0 ± 0.02, Detection wavelength 280nm, respectively in conjugate a, conjugate b and
The CRM197 protein control product of different content are added in conjugate c as sample introduction sample, using 100 μ l injection annulus by sample introduction sample
It injects in the analytical column of high performance liquid chromatograph and is analyzed.By evaluate CRM197 albumen signal-to-noise ratio, determine reasonably into
Sample protein content, testing result is referring to following table three.
The signal-to-noise ratio of three various concentration CRM197 albumen of table
Referring to table three, according to testing result, it can determine that whether there is or not the detection of CRM197 albumen limits about in 0.15625 μ g (weight
Measure unit), in conjunction with the regulation of signal-to-noise ratio > 10.0 in professional standard, it can determine that the quantitative limit of CRM197 protein content about exists
0.625 μ g (unit of weight).
According to the quality criteria requirements (should be less than 5%) in the numerical value of quantitative limit and this sample for floating preteins, greatly
Cause can determine that total protein content minimum should be preferred more than 0.625 μ g ÷ 5%=12.5 μ g (unit of weight) in sample introduction sample.
In view of sample cost and the controllability of test, protein sample upper limit of concentration can tolerance range in analytical column according to sample characteristics of for example
It is interior voluntarily to be determined.In the present invention, when total protein content is between 20-50 μ g in sample introduction sample, corresponding signal-to-noise ratio is equal
> 10.0 is not only able to determine the content of CRM197 albumen preferably with this, moreover it is possible to the usage amount of sample be effectively reduced, have
The feature that testing result is accurate, testing cost is low, therefore the total protein content of sample introduction sample is preferably 20-50 μ g.
4, the selection of column temperature
Using chromatographic column for SRT SEC-300 (7.8mm × 300mm), mobile phase is 0.2M sodium chloride solution, flow velocity 0.5mL/
Min, pH are 7.0 ± 0.02, Detection wavelength 280nm, fixed sampling volume is 100 μ l, in conjugate a, conjugate b, are combined
The CRM197 albumen of 5wt% is separately added into object c as test specimen, by signal-to-noise ratio and the separation of evaluating CRM197 albumen
Degree, determines reasonable column temperature, testing result is referring to following table four.
The different column temperatures of table four are to the signal-to-noise ratio of CRM197 albumen and the influence of separating degree
Referring to table four, when column temperature is set in 4-30 DEG C, the equal > 10.0 of the signal-to-noise ratio of CRM197 albumen, conjugate and CRM197
The equal > 1.5 of the separating degree of albumen, therefore can be applicable under this experiment condition.Wherein, when column temperature is at 20-26 DEG C, CRM197 egg
White signal-to-noise ratio and conjugate and the separating degree of CRM197 albumen is higher, further preferably 23 ± 1 DEG C of column temperature.
5, the selection of pH
Using chromatographic column for SRT SEC-300 (7.8mm × 300mm), column temperature is 23 ± 1 DEG C, mobile phase is that 0.2M sodium chloride is molten
Liquid, flow velocity 0.5mL/min, Detection wavelength 280nm, fixed sampling volume are 100 μ l, in conjugate a, conjugate b, are combined
The CRM197 albumen of 5wt% is separately added into object c as test specimen, by signal-to-noise ratio and the separation of evaluating CRM197 albumen
Degree, determines reasonable pH, testing result is referring to following table five.
Five difference pH of table is to the signal-to-noise ratio of CRM197 albumen and the influence of separating degree
Referring to table five, when pH is set in 6.8-7.2, the equal > 350 of the signal-to-noise ratio of CRM197 albumen, conjugate and CRM197
The equal > 1.5 of the separating degree of albumen, therefore can be applicable under this experiment condition.Wherein, when pH is 7.0 ± 0.02, CRM197 egg
White signal-to-noise ratio and conjugate and the separating degree of CRM197 albumen is higher, therefore preferably pH is 7.0 ± 0.02.
6, accuracy and the confirmation of repeatability
The accuracy of this detection method is investigated in this experimental design with sample recovery rate, and each sample detection carries out repeatability three times
Determine.
Chromatographic condition: using chromatographic column for SRT SEC-300 (7.8mm × 300mm), column temperature is 23 ± 1 DEG C, mobile phase is
0.2M sodium chloride solution, flow velocity 0.2-1.0mL/min, pH be 7.0 ± 0.02, Detection wavelength 280nm, fixed sampling volume
For 100 μ l.
Preparation of samples: select conjugate a, conjugate b and conjugate c as sample, extremely with 0.2M sodium chloride dilute sample
Protein concentration is 200 μ g/mL, respectively takes 1mL, is separately added into the CRM197 albumen of 0wt%, 1wt%, 3wt%, 5wt%, detection knot
Fruit is referring to six-table of following table eight, since main distinction point is appearance time difference between chromatogram different in flow rate, with stream
For fast 0.5mL/min, correspondence obtains chromatogram as shown in figs 2-4.
The accuracy and repeatability of the measurement of table six conjugate a
The accuracy and repeatability of the measurement of table seven conjugate b
The accuracy and repeatability of the measurement of table eight conjugate c
Referring to table six to table eight, the present invention is combined with diplococcus meningitidis combined vaccine, Hib combined vaccine, pneumococcus
The corresponding conjugate of vaccine be sample, be added sample in CRM197 albumen the test parameters of setting and under the conditions of, the rate of recovery
All in 80-120% range between, CV% be smaller than 5%, thus indicate that this detection method is accurately credible, repeatability compared with
It is high.Wherein, when flow velocity is 0.4-0.75mL/min, the rate of recovery of CRM197 albumen is between the range of 95-107%
Downward trend after first rising, therefore preferable flow rate is 0.4-0.75mL/min, further preferred flow velocity is 0.5mL/min.
7, it summarizes
To sum up, the present invention in chromatographic column be preferably SRT SEC serial analysis column, it is secondary be selected as TSKgelG3000SW (7.5mm ×
600mm) analytical column, column temperature are 4-30 DEG C, and mobile phase is 0.2M sodium chloride solution, and flow velocity 0.2-1.0mL/min, pH are
6.8-7.2, Detection wavelength 280nm；Total protein content is 12.5 μ g of > in sample introduction sample；Protein control product are CRM197 egg
It is white.With this, the content of the CRM197 albumen to dissociate in combined vaccine can be accurately determined, ensure that effective effect of combined vaccine
Valence and safety, with low in cost, accuracy is high, reproducible, detection is simple and efficient, convenient for what is be widely popularized and applied
Feature.
This specific embodiment is only explanation of the invention, is not limitation of the present invention, those skilled in the art
Member can according to need the modification that not creative contribution is made to the present embodiment after reading this specification, but as long as at this
All by the protection of Patent Law in the scope of the claims of invention.
Claims (10)
1. the high-efficient liquid phase determining method of Free protein content in a kind of biological products, which comprises the following steps:
1., chromatographic condition:
Chromatographic column is SRT SEC serial analysis column or TSKgelG3000SW analytical column, and column temperature is 4-30 DEG C, mobile phase 0.2M
Sodium chloride solution, flow velocity 0.2-1.0mL/min, pH 6.8-7.2, Detection wavelength 280nm；
2., the preparation of sample solution:
It takes sample to be tested appropriate, with flowing phase dilution sample to be tested, obtains sample introduction sample；
3., assay:
Sample introduction sample is injected into high performance liquid chromatograph, records chromatogram, normalization method calculates measurement result according to area.
2. the high-efficient liquid phase determining method of Free protein content, feature in a kind of biological products according to claim 1
Be, step 1. in, the SRT SEC serial analysis column is SRT SEC-300 analytical column, TSKgelG3000SW analysis
The specification of column is 7.5mm × 600mm.
3. the high-efficient liquid phase determining method of Free protein content, feature in a kind of biological products according to claim 1
Be, step 1. in, the column temperature be 20-26 DEG C.
4. the high-efficient liquid phase determining method of Free protein content, feature in a kind of biological products according to claim 3
Be, step 1. in, the column temperature be 23 ± 1 DEG C.
5. the high-efficient liquid phase determining method of Free protein content, feature in a kind of biological products according to claim 1
Be, step 1. in, the flow velocity be 0.4-0.75mL/min.
6. the high-efficient liquid phase determining method of Free protein content, feature in a kind of biological products according to claim 5
Be, step 1. in, the flow velocity be 0.5mL/min.
7. the high-efficient liquid phase determining method of Free protein content, feature in a kind of biological products according to claim 1
Be, step 1. in, the pH be 7.0 ± 0.02.
8. the high-efficient liquid phase determining method of Free protein content, feature in a kind of biological products according to claim 1
Be, step 2. in, in the sample introduction sample total protein content be 12.5 μ g of >.
9. the high-efficient liquid phase determining method of Free protein content, feature in a kind of biological products according to claim 8
Be, step 2. in, in the sample introduction sample total protein content be 20-50 μ g.
10. the high-efficient liquid phase determining method of Free protein content, feature in a kind of biological products according to claim 1
It is, the assay for the CRM197 albumen that dissociates suitable for combined vaccine.
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