CN109470806A - By Two-dimensional Liquid with respect to the method that monoclonal antibody class product cell strain carries out high flux screening - Google Patents
By Two-dimensional Liquid with respect to the method that monoclonal antibody class product cell strain carries out high flux screening Download PDFInfo
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- CN109470806A CN109470806A CN201811222166.2A CN201811222166A CN109470806A CN 109470806 A CN109470806 A CN 109470806A CN 201811222166 A CN201811222166 A CN 201811222166A CN 109470806 A CN109470806 A CN 109470806A
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- monoclonal antibody
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- dimensional liquid
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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
Abstract
The present invention relates to a kind of by Two-dimensional Liquid with respect to the method that monoclonal antibody class product cell strain carries out high flux screening, including being detected by Two-dimensional Liquid with respect to monoclonal antibody class product, the monoclonal antibody class product is Avastin, specifically includes the following steps: (1) first dimension liquid chromatogram is separated；(2) two-dimensional liquid chromatographies are detected: the first dimension liquid chromatogram being cut resulting fraction according to retention time and drains into two-dimensional liquid chromatography, is detected.The present invention improves the efficiency of screening, saves manpower, and due to avoiding interminable sample treatment and storing step, avoids the possibility that sample is degraded, improve precision of analysis.
Description
Technical field
The present invention relates to a kind of by Two-dimensional Liquid with respect to the method that monoclonal antibody class product cell strain carries out high flux screening, belongs to
Antibody class product includes monoclonal antibody and fusion protein analysis technical field.
Background technique
Nowadays, monoclonal antibody (referred to as " monoclonal antibody ") has obtained large-scale production and has been widely applied, especially therapeutic
Monoclonal antibody, and the sufficiently stable monoclonal antibody of physicochemical properties can be secreted by how filtering out before antibody process exploitation
Cell strain, become the pivotal start point of process exploitation.At this stage, in antibody class research and development of products and manufacturing enterprise, it is desirable to
To the polymer content data of monoclonal antibody secreted by certain cell strain, it is necessary to successively successively complete the experiment of two steps by hand: collect first
The monoclonal antibody in cell culture fluid is captured, and then the monoclonal antibody that will be captured obtains reaction using other chromatography as sample
The analysis of monoclonal antibody physicochemical property is as a result, the time of whole process time-consuming a couple of days, and along with coordinating in a large amount of Manpower And Organizations
Demand, it is time-consuming and laborious, and due to interminable sample treatment and storing step, the probability that sample is degraded is increased, is reduced
Precision of analysis.
Summary of the invention
In order to solve above-mentioned problems of the prior art, the purpose of the present invention is to provide one kind to pass through two-dimentional liquid phase
To the method that monoclonal antibody class product cell strain carries out high flux screening, the efficiency of screening is improved, saves manpower, and due to avoiding
Interminable sample treatment and storing step, avoid the possibility that sample is degraded, improve precision of analysis.
In order to achieve the above objectives, the invention provides the following technical scheme: by Two-dimensional Liquid with respect to monoclonal antibody class product cell strain
The method for carrying out high flux screening, including detected by Two-dimensional Liquid with respect to monoclonal antibody class product, the monoclonal antibody class product is shellfish
Pearl monoclonal antibody is cut down, specifically includes the following steps:
(1) first dimension liquid chromatogram is separated:
Chromatographic condition are as follows: use affinity chromatographic column, using added with eluant, eluent pH value for 6.3-7.3 buffer solution as stream
Dynamic phase A, using pH value for 2.5-3.5 buffer solution as Mobile phase B, flow velocity 0.3-0.5mL/min, Detection wavelength 280nm,
Condition of gradient elution are as follows: 0~3min, 0%B；3~3.1min, 0%~100%B；3.1~6min, 100%B；6~6.1min,
100%~0%B；6.1~10min, 0%B.
(2) two-dimensional liquid chromatographies are detected: the first dimension liquid chromatogram is cut resulting fraction according to retention time
Two-dimensional liquid chromatography is drained into, is detected:
Chromatographic condition are as follows: size exclusion chromatography post is used, to there is buffer solution of the pH value of additive for 5.5-6.5 as stream
Dynamic phase, flow velocity 0.5-1.0mL/min, Detection wavelength 280nm, isocratic elution.
Further, in the step (2), the specification of the size exclusion chromatography post is 2.1 × 150mm, fills partial size
It is 1.7 μm.Make the separating effect that sample has had.
Further, in the step (2), the specification of the size exclusion chromatography post is 4.6 × 250mm, fills partial size
It is 2.0 μm.Keep sample separating effect preferable.
Further, in the step (2), the additive is the NaCl of 90-110mmol/L.Improve the separation of sample
Effect.
Further, in the step (2), the buffer solution of the mobile phase is pH=6.0, concentration is 50mmol/L's
Na2HPO4Buffer solution.Keep the peak shape of sample preferable.
Further, in the step (1), the specification of the affinity chromatographic column is 2.1 × 50mm, and filling partial size is 1.7-
12μm.Keep antibody separating effect preferable.
Further, in the step (1), the buffer solution of the mobile phase A is pH=6.8, concentration 50mmol/L
Tris-HCl buffer solution.Keep peak shape preferable, to have good separating effect.
Further, in the step (1), the eluant, eluent is the NaCl of 40-60mmol/L.
Further, in the step (1), the buffer solution of the Mobile phase B is the buffer solution of pH=3.0, by
The glycine of 0.1mol/L is added HCl and is made.Make the separating effect that antibody has had.
The beneficial effects of the present invention are: by Two-dimensional Liquid with respect to the method that monoclonal antibody class product is detected, make entirely to examine
Survey process foreshortens to dozens of minutes, significantly improves the efficiency of analysis detection, saves a large amount of manpowers, and tediously long due to avoiding
Sample treatment and storing step, avoid the possibility that sample is degraded, improve precision of analysis, and then improve
The efficiency of subsequent monoclonal antibody class product cell strain screening, realizes the mesh that high flux screening is carried out to monoclonal antibody class product cell strain
's.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention and the accompanying drawings.
Detailed description of the invention
Fig. 1 is Two-dimensional Liquid in embodiment three with respect to the first dimension liquid chromatogram that Avastin is detected；
Fig. 2 is Two-dimensional Liquid in embodiment three with respect to the second dimension liquid chromatogram that Avastin is detected.
Specific embodiment
With reference to the accompanying drawings and examples, specific embodiments of the present invention will be described in further detail.Implement below
Example is not intended to limit the scope of the invention for illustrating the present invention.
Embodiment one
The cell strain of Avastin uses culture medium in I I culture medium of SFM for the glutamic acid for being added to 4mmol/L
0.2 μm of filter membrane filters out cell and other biggish particles, and sample to be tested is made.
It is detected by Two-dimensional Liquid with respect to Avastin, to carry out high flux screening to Avastin cell strain,
The condition that first dimension liquid chromatogram is separated are as follows: affinity chromatographic column is used, to be added with the pH of 40mmol/L NaCl eluant, eluent
The Tris-HCl buffer solution that value is 6.3 is mobile phase A, and as Mobile phase B, flow velocity is the buffer solution for being 2.5 using pH value
0.3mL/min, column temperature are 25 DEG C, Detection wavelength 280nm, condition of gradient elution are as follows: 0~3min, 0%B；3~3.1min,
0%~100%B；3.1~6min, 100%B；6~6.1min, 100%~0%B；6.1~10min, 0%B；Second dimension liquid phase
The condition that chromatography is detected are as follows: use size exclusion chromatography post, be 5.5 Na to there is the pH value of additive2HPO4Buffer solution
For mobile phase, flow velocity 0.5mL/min, Detection wavelength 280nm, column temperature is 25 DEG C, isocratic elution.By the first dimension liquid chromatogram
Sample introduction valve injection, sample volume be 10 μ g, when first dimension liquid chromatogram retention time reach 6.00min when, pass through switching valve, will
Fraction drains into two-dimensional liquid chromatography and is detected, and when the first dimension liquid chromatogram retention time reaches 9.00min, will switch
Valve switches back to the first dimension liquid chromatogram again.
The specification of size exclusion chromatography post is 2.1 × 150mm, and filling partial size is 1.7 μm.
Additive is the NaCl of 90mmol/L.
The specification of affinity chromatographic column is 2.1 × 50mm, and filling partial size is 1.7 μm.
In the condition of first dimension liquid chromatogram separation, HCl is added in the glycine that the buffer solution of Mobile phase B is 0.1mol/L
It is made.
The results showed that Avastin separating effect in the second dimension size exclusion chromatography post is preferable.
Embodiment two
The cell strain of Avastin uses culture medium in I I culture medium of SFM for the glutamic acid for being added to 4mmol/L
0.2 μm of filter membrane filters out cell and other biggish particles, and sample to be tested is made.
It is detected by Two-dimensional Liquid with respect to Avastin, to carry out high flux screening to Avastin cell strain,
The condition that first dimension liquid chromatogram is separated are as follows: affinity chromatographic column is used, to be added with the pH of 60mmol/L NaCl eluant, eluent
The Tris-HCl buffer solution that value is 7.3 is mobile phase A, and as Mobile phase B, flow velocity is the buffer solution for being 3.5 using pH value
0.5mL/min, Detection wavelength 280nm, column temperature are 25 DEG C, condition of gradient elution are as follows: 0~3min, 0%B；3~3.1min,
0%~100%B；3.1~6min, 100%B；6~6.1min, 100%~0%B；6.1~10min, 0%B；Second dimension liquid phase
The condition that chromatography is detected are as follows: use size exclusion chromatography post, be 6.5 Na to there is the pH value of additive2HPO4Buffer solution
For mobile phase, flow velocity 1.0mL/min, Detection wavelength 280nm, column temperature is 25 DEG C, isocratic elution.By the first dimension liquid chromatogram
Sample introduction valve injection, sample volume be 20 μ g, when first dimension liquid chromatogram retention time reach 6.00min when, pass through switching valve, will
Fraction drains into two-dimensional liquid chromatography and is detected, and when the first dimension liquid chromatogram retention time reaches 9.00min, will switch
Valve switches back to the first dimension liquid chromatogram again.
The specification of size exclusion chromatography post is 4.6 × 250mm, and filling partial size is 2.0 μm.
Additive is the NaCl of 110mmol/L.
The specification of affinity chromatographic column is 2.1 × 50mm, and filling partial size is 5 μm.
In the condition of first dimension liquid chromatogram separation, HCl is added in the glycine that the buffer solution of Mobile phase B is 0.1mol/L
It is made.
The results showed that Avastin separating effect in the second dimension size exclusion chromatography post is preferable.
Embodiment three
The cell strain of Avastin uses culture medium in I I culture medium of SFM for the glutamic acid for being added to 4mmol/L
0.2 μm of filter membrane filters out cell and other biggish particles, and sample to be tested is made.
It is detected by Two-dimensional Liquid with respect to Avastin, to carry out high flux screening to Avastin cell strain,
The condition that first dimension liquid chromatogram is separated are as follows: affinity chromatographic column is used, to be added with the pH of 50mmol/L NaCl eluant, eluent
The Tris-HCl buffer solution that value is 6.8 is mobile phase A, and as Mobile phase B, flow velocity is the buffer solution for being 3.0 using pH value
0.5mL/min, Detection wavelength 280nm, column temperature are 25 DEG C, condition of gradient elution are as follows: 0~3min, 0%B；3~3.1min,
0%~100%B；3.1~6min, 100%B；6~6.1min, 100%~0%B；6.1~10min, 0%B；Second dimension liquid phase
The condition that chromatography is detected are as follows: use size exclusion chromatography post, be 6.0 Na to there is the pH value of additive2HPO4Buffer solution
For mobile phase, flow velocity 1.0mL/min, Detection wavelength 280nm, column temperature is 25 DEG C, isocratic elution.
The specification of size exclusion chromatography post is 4.6 × 250mm, and filling partial size is 2.0 μm.
Additive is the NaCl of 100mmol/L.
In the condition that two-dimensional liquid chromatography is detected, Na2HPO4The concentration of buffer solution is 50mmol/L.
The specification of affinity chromatographic column is 2.1 × 50mm, and filling partial size is 12 μm.
In the condition of first dimension liquid chromatogram separation, the buffer solution of mobile phase A is the Tris- that concentration is 50mmol/L
HCl buffer solution, the glycine that the buffer solution of Mobile phase B is 0.1mol/L are added HCl and are made.
By the sample introduction valve injection of the first dimension liquid chromatogram, sample volume is 20 μ g, when the first dimension liquid chromatogram retention time reaches
When to 7.00min, by switching valve, fraction is drained into two-dimensional liquid chromatography and is detected, when the first dimension liquid chromatogram is protected
When the time being stayed to reach 8.50min, switching valve is switched back to the first dimension liquid chromatogram, the first dimension liquid chromatogram spectrogram and second again
Dimension liquid chromatogram spectrogram is shown in Fig. 1 and Fig. 2 respectively
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (9)
1. by Two-dimensional Liquid with respect to the method that monoclonal antibody class product cell strain carries out high flux screening, which is characterized in that including passing through
Two-dimensional Liquid is detected with respect to monoclonal antibody class product, and the monoclonal antibody class product is Avastin, specifically includes the following steps:
(1) first dimension liquid chromatogram is separated:
Chromatographic condition are as follows: use affinity chromatographic column, using added with eluant, eluent pH value for 6.3-7.3 buffer solution as mobile phase
A, using pH value for 2.5-3.5 buffer solution as Mobile phase B, flow velocity 0.3-0.5mL/min, Detection wavelength 280nm, gradient
Elution requirement are as follows: 0~3min, 0%B；3~3.1min, 0%~100%B；3.1~6min, 100%B；6~6.1min,
100%~0%B；6.1~10min, 0%B；
(2) two-dimensional liquid chromatographies are detected: the first dimension liquid chromatogram being cut resulting fraction according to retention time and is drained
To two-dimensional liquid chromatography, detected:
Chromatographic condition are as follows: size exclusion chromatography post is used, to there is buffer solution of the pH value of additive for 5.5-6.5 as flowing
Phase, flow velocity 0.5-1.0mL/min, Detection wavelength 280nm, isocratic elution.
2. the method according to claim 1 by Two-dimensional Liquid with respect to monoclonal antibody class product cell strain progress high flux screening,
It is characterized in that, the specification of the size exclusion chromatography post is 2.1 × 150mm in the step (2), filling partial size is 1.7 μ
m。
3. the method according to claim 1 by Two-dimensional Liquid with respect to monoclonal antibody class product cell strain progress high flux screening,
It is characterized in that, the specification of the size exclusion chromatography post is 4.6 × 250mm in the step (2), filling partial size is 2.0 μ
m。
4. the method according to claim 1 by Two-dimensional Liquid with respect to monoclonal antibody class product cell strain progress high flux screening,
It is characterized in that, the additive is the NaCl of 90-110mmol/L in the step (2).
5. the method according to claim 1 by Two-dimensional Liquid with respect to monoclonal antibody class product cell strain progress high flux screening,
It is characterized in that, in the step (2), the buffer solution of the mobile phase is pH=6.0, concentration is 50mmol/L Na2HPO4
Buffer solution.
6. the method according to claim 1 by Two-dimensional Liquid with respect to monoclonal antibody class product cell strain progress high flux screening,
It is characterized in that, the specification of the affinity chromatographic column is 2.1 × 50mm in the step (1), filling partial size is 1.7-12 μm.
7. the method according to claim 1 by Two-dimensional Liquid with respect to monoclonal antibody class product cell strain progress high flux screening,
It is characterized in that, in the step (1), the buffer solution of the mobile phase A is pH=6.8, concentration is 50mmol/L Tris-
HCl buffer solution.
8. the method according to claim 1 by Two-dimensional Liquid with respect to monoclonal antibody class product cell strain progress high flux screening,
It is characterized in that, the eluant, eluent is the NaCl of 40-60mmol/L in the step (1).
9. the method according to claim 1 by Two-dimensional Liquid with respect to monoclonal antibody class product cell strain progress high flux screening,
It is characterized in that, the buffer solution of the Mobile phase B is the buffer solution of pH=3.0, by 0.1mol/ in the step (1)
The glycine of L is added HCl and is made.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101705480B1 (en) * | 2015-08-19 | 2017-02-09 | 고려대학교 산학협력단 | Ultra-high Sensitivity Biosensor based on 2-Dimensional Chromatography |
| CN106399432A (en) * | 2015-07-31 | 2017-02-15 | 中国科学院大连化学物理研究所 | Method for preparing N-linked glycopeptide from monoclonal antibody and N-linked glycopeptide |
| CN108715615A (en) * | 2013-09-13 | 2018-10-30 | 百济神州有限公司 | Anti- PD1 antibody and its purposes as therapeutic agent and diagnosticum |
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2018
- 2018-10-19 CN CN201811222166.2A patent/CN109470806A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108715615A (en) * | 2013-09-13 | 2018-10-30 | 百济神州有限公司 | Anti- PD1 antibody and its purposes as therapeutic agent and diagnosticum |
| CN106399432A (en) * | 2015-07-31 | 2017-02-15 | 中国科学院大连化学物理研究所 | Method for preparing N-linked glycopeptide from monoclonal antibody and N-linked glycopeptide |
| KR101705480B1 (en) * | 2015-08-19 | 2017-02-09 | 고려대학교 산학협력단 | Ultra-high Sensitivity Biosensor based on 2-Dimensional Chromatography |
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| KOEN SANDRA 等: "The opportunities of 2D-LC in the analysis of monoclonal antibodies", 《BIOANALYSIS》 * |
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Application publication date: 20190315 |