CN109633053A - The detection method and system of cell culture protein expression quantity and the protein aggregation scale of construction - Google Patents
The detection method and system of cell culture protein expression quantity and the protein aggregation scale of construction Download PDFInfo
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- CN109633053A CN109633053A CN201811558166.XA CN201811558166A CN109633053A CN 109633053 A CN109633053 A CN 109633053A CN 201811558166 A CN201811558166 A CN 201811558166A CN 109633053 A CN109633053 A CN 109633053A
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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
- G01N30/38—Flow patterns
- G01N30/46—Flow patterns using more than one column
- G01N30/461—Flow patterns using more than one column with serial coupling of separation columns
- G01N30/462—Flow patterns using more than one column with serial coupling of separation columns with different eluents or with eluents in different states
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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
Abstract
The present invention relates to technical field of protein detection, more particularly, to the detection method and system of a kind of cell culture protein expression quantity and the protein aggregation scale of construction.The detection method includes the following steps: that affinity chromatographic column and size exclusion chromatograph column of the cell culture through concatenating carry out gradient elution and detection；The mobile phase of gradient elution includes: mobile phase A: 10-200mM sodium dihydrogen phosphate-disodium hydrogen phosphate mixture, 50-500mM sodium chloride, pH 6.8 ± 0.5；Mobile phase B: 10-200mM glycine, 20-300mM sodium chloride, pH 2.5 ± 0.5.Detection method of the invention, affinity chromatographic column and size exclusion chromatograph column are concatenated, gradient elution is carried out using certain mobile phase, protein expression quantity and aggregation percentage in cell culture can be detected simultaneously, it is easy, easy-to-use, low-cost, and result is reliable.
Description
Technical field
The present invention relates to technical field of protein detection, more particularly, to a kind of cell culture protein expression quantity and egg
The detection method and system of the white matter aggregation scale of construction.
Background technique
Modern biological project mostly uses mammalian cell, produces pharmaceutical grade protein, cell culture fluid with Liquid suspension method
In the expression quantity of protein change with incubation time, the phase needs frequently sampling monitoring to find investment and production after incubation
Optimal balance point out；But common affine high effective liquid chromatography for measuring protein expression quantity, the protein expression of measurement
Amount is the addition and value of the morphology of the aggregate comprising protein, morphon and the segment containing Fc, and generally there was only morphon
Protein be only it is required, therefore this method measurement result be not accurate target molecule (morphon) expression
Amount.In addition, the protein molecule of cell secretion due to locating liquid environment, mechanical environment and divides in cell cultivation process
Sub- own physical chemical attribute etc., it may occur that different degrees of aggregation, this is undesired, meeting for the production of protein
The yield and product quality of technique are influenced, therefore the aggregation percentage for monitoring protein in culture is also evaluation culture process
Important parameter.In addition, when carrying out cell strain exploitation, the protein aggregate percentage and expression quantity of different clonal expressions are
Measure the important indicator of screen cell strain, this be also required to a kind of simplicity reliably method to the expression quantity of protein and purity into
Row detection.
Traditional analytical technology is deposited due to the ingredients such as amino acid, vitamin, host protein, DNA in cell culture
, the detection of meeting jamming target albumen, so the aggregation percentage of protein in cell culture can not be directly measured, measurement
Aggregation percentage needs first to purify cells and supernatant sample to be tested using affinity chromatographic column, then is arranged using volume
Resistance-high performance liquid chromatography detection, there are two kinds of disadvantages for this mode: first, existed using a large amount of low pH circulation phase elution of bound
When target protein on affinity chromatographic column, low pH pressure can be easy to cause increasing for sample collection body, although after collecting eluent
PH adjusting can be carried out to gleanings again, but the alkaline solution that pH adjusting uses equally leads to aggregation because topical solutions are inhomogenous
May further it increase, these can all cause test result inaccurate；Second: more labor intensive and precious sample, especially
When cell culture sample size to be measured is more, sample volume is less, this disadvantage is especially prominent, is such as carrying out cell strain
When exploitation.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of cell culture protein expression quantity and the protein aggregation scale of construction
Detection method, to solve existing in the prior art can not accurately detect protein expression quantity and aggregation percentage.
The second object of the present invention is to provide a kind of cell culture protein expression quantity and the protein aggregation scale of construction
Detection system, system structure is simple, easy to operate.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
The detection method of cell culture protein expression quantity and the protein aggregation scale of construction, includes the following steps:
Affinity chromatographic column and size exclusion chromatograph column of the cell culture through concatenating carry out gradient elution and detection；
The mobile phase of gradient elution includes: mobile phase A: 10-200mM sodium dihydrogen phosphate-disodium hydrogen phosphate mixture, 50-
500mM sodium chloride, pH 6.8 ± 0.5；Mobile phase B: 10-200mM glycine, 20-300mM sodium chloride, pH 2.5 ± 0.5.
Two-dimensional liquid chromatography on the market is that one kind can measure protein expression quantity in cell culture simultaneously and gather at present
Effective ways of collective's percentage, but this method must use Two-dimensional Liquid Chromatography, the disadvantage is that equipment price it is high,
It safeguards more common liquid chromatogram complexity etc., leads to that method is not easily controlled and comparison difficulty passes through, therefore these disadvantages limit
It is promoted the use of.
Detection method of the invention concatenates affinity chromatographic column and size exclusion chromatograph column, using certain mobile phase into
Row gradient elution can detect protein expression quantity and aggregation percentage, easy, easy-to-use, expense in cell culture simultaneously
It is cheap, and result is reliable.
Preferably, the mobile phase of gradient elution includes: mobile phase A: 40-60mM sodium dihydrogen phosphate-disodium hydrogen phosphate mixing
Object, 250-350mM sodium chloride, pH 6.8 ± 0.5；Mobile phase B: 50-150mM glycine, 100-200mM sodium chloride, pH 2.5
±0.5。
Preferably, the mobile phase of gradient elution includes: mobile phase A: 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate mixture,
300mM sodium chloride, pH 6.8；Mobile phase B: 100mM glycine, 150mM sodium chloride, pH 2.5.
It is optimized by the flowing phase constituent to gradient elution, target protein monomer and target egg can be further increased
The separating degree of white aggregation, to obtain the percentage of more accurately target protein expression amount and protein aggregate.
Preferably, the mode of concatenation is that affinity chromatographic column and size exclusion chromatograph column directly concatenate.
Preferably, the mode of concatenation is connected to concatenation by six-way valve rotation with size exclusion chromatograph column for affinity chromatographic column.
Affinity chromatographic column is inscribed on the flow path of sample introduction quantitative loop of six-way valve, and under sample introduction state, affinity chromatographic column cell culture is waited for
It surveys liquid to be enriched with through six-way valve feeding affinity chromatographic column, after sample introduction, target protein monomer and its aggregation are enriched in parent
In chromatographic column；After sample introduction, rotation six-way valve is connected to affinity chromatographic column with size exclusion chromatograph column, and gradient elution is by mesh
Mark monomer and its aggregation elution separation of albumen.Compared to direct concatenated mode, further save gradient elution when
Between.
Preferably, when affinity chromatographic column and size exclusion chromatograph column are by the way of directly concatenating, gradient elution program
Are as follows: 0%B 0-60min, 100%B 60.1-90min.
Preferably, when affinity chromatographic column rotates the tandem being connected to using six-way valve with size exclusion chromatograph column, ladder
Spend elution program are as follows: 0%B 0-10min, 100%B 10.1-15.1min, 0%B15.2-40min.It is furthermore preferred that when using
When the tandem of six-way valve rotation connection, after sample introduction, affinity chromatographic column is eluted in advance using ultrapure water in advance.It adopts
Affinity chromatographic column is eluted with ultrapure water, the impurity composition for making to stay in the cell culture supernatant on affinity chromatographic column is logical through six
Valve discharge, is then rotated further by switching six-way valve, is connected to affinity chromatographic column with size exclusion chromatograph column.
Preferably, the flow velocity of gradient elution is 0.2-1mL/min.
Preferably, the sample volume of cell culture is 10-50 μ L.The concentration of protein is 0.5-5mg/ in cell culture
mL。
Preferably, the target protein in cell culture include IgA, IgG (IgG1, IgG2, IgG3, IgG4), IgM,
Any one of Kappa light chain, Lambda light chain, cell factor etc..It is furthermore preferred that the target protein in cell culture is
IgG1。
Preferably, the filler of affinity chromatographic column with base type include specific binding IgA, IgG (IgG1, IgG2,
IgG3, IgG4), IgM, Kappa light chain, Lambda light chain, any one of aglucons such as cell factor.
Preferably, size exclusion chromatograph column includes TOSOH TSK G3000SWXL(5 μm of particles or 3 μm of particles), Waters
Protein BEH2(Or2.5 μm of particles or 1.7 μm of particles) or Agilent AdvanceBio SEC (Any one of 2.7 μm of particles).
Preferably, gradient elution is carried out after cell culture being pre-processed and detection, pretreated method include: to take cell
Culture supernatants, centrifugation take supernatant as sample to be tested again.It is furthermore preferred that the condition of centrifugation are as follows: revolving speed 10000-
16000rpm, time 5-15min, temperature are 5 ± 1 DEG C.It is further preferred that the condition of centrifugation are as follows: revolving speed 13000rpm,
Time is 10min, and temperature is 5 DEG C.
Preferably, it is detected using ultraviolet-visible light.It is furthermore preferred that Detection wavelength is 280nm.
The present invention also provides the detection system of a kind of cell culture protein expression quantity and the protein aggregation scale of construction, packets
Include solvent pump, sample injector six-way valve, affinity chromatographic column, size exclusion chromatograph column, ultraviolet-visible detector；Solvent pump by into
Sample device six-way valve is connected to affinity chromatographic column, and size exclusion chromatograph column is serially connected with affinity chromatographic column, the stream of size exclusion chromatograph column
Outlet is connected to ultraviolet-visible detector.
Preferably, the sample introduction quantitative loop on sample injector six-way valve is arranged between No. two positions of six-way valve and No. five positions, parent
It is inscribed in chromatographic column on the flow path of No. two positions and No. five positions, and is located at the downstream of quantitative loop, solvent pump is connected to sample injector
The No.1 position of six-way valve, size exclusion chromatograph column are external in No. six positions of sample injector six-way valve.
Compared with prior art, the invention has the benefit that
(1) affinity chromatographic column and size exclusion chromatograph column are concatenated, can detect cell simultaneously by detection method of the invention
Protein expression quantity and aggregation percentage in culture；
(2) detection method of the invention, test result is accurate, and the two-dimensional highly effective liquid phase chromatographic without purchasing complex and expensive
Equipment；Also, detection method of the invention, adaptability is good, is not interfered by the impurity composition in cell culture；
(3) detection system of the invention, structure is simple, easy to operate.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the structural schematic diagram for the detection system that the embodiment of the present invention 1 provides；
The chromatogram detected after the gradient elution that Fig. 2 provides for the embodiment of the present invention 1；
Fig. 3 is the partial enlarged view of the chromatogram of Fig. 2；
Fig. 4 is the structural schematic diagram for the detection system that the embodiment of the present invention 2 provides；
The chromatogram detected after the gradient elution that Fig. 5 provides for the embodiment of the present invention 2；
Fig. 6 is the partial enlarged view of the chromatogram of Fig. 5.
Appended drawing reference:
1- solvent pump；2- sample injector six-way valve；3- affinity chromatographic column；
4- size exclusion chromatograph column；5- ultraviolet-visible detector；21- No.1 position；
No. bis- positions 22-；23- third place；No. tetra- positions 24-；
No. five positions 25-；No. six positions 26-；27- quantitative loop；
X- target protein monomer；Y- target protein aggregation；Z- impurity composition；
The variation of L- baseline.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with the drawings and specific embodiments, but
Be it will be understood to those of skill in the art that it is following described embodiments are some of the embodiments of the present invention, rather than it is whole
Embodiment is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.Based on the embodiments of the present invention, ability
Domain those of ordinary skill every other embodiment obtained without making creative work, belongs to guarantor of the present invention
The range of shield.The person that is not specified actual conditions in embodiment, carries out according to conventional conditions or manufacturer's recommended conditions.Agents useful for same
Or production firm person is not specified in instrument, is the conventional products that can be obtained by commercially available purchase.
In the description of the present invention, it should be noted that term " center ", "upper", "lower", "left", "right", "vertical",
The orientation or positional relationship of the instructions such as "horizontal", "inner", "outside" be based on the orientation or positional relationship shown in the drawings, merely to
Convenient for description the present invention and simplify description, rather than the device or element of indication or suggestion meaning must have a particular orientation,
It is constructed and operated in a specific orientation, therefore is not considered as limiting the invention.In addition, term " first ", " second ",
" third " is used for descriptive purposes only and cannot be understood as indicating or suggesting relative importance.
In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, term " installation ", " phase
Even ", " connection " shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or be integrally connected；It can
To be mechanical connection, it is also possible to be electrically connected；It can be directly connected, can also can be indirectly connected through an intermediary
Connection inside two elements.For the ordinary skill in the art, above-mentioned term can be understood at this with concrete condition
Concrete meaning in invention.
The part instrument and reagent information used in the embodiment of the present invention is as follows:
Instrument:
High performance liquid chromatograph (DAD, VWD, MWD or FLR detector): manufacturer, Agilent；Model: Agilent 1260；
The concatenation of the chromatographic column of various embodiments of the present invention is realized in the column oven or sample injector and column oven of HPLC；
Electronic balance: manufacturer, Mei Teletuo benefit；Model, MS204/ML204T；
Refrigerated centrifuge: manufacturer, Ai Bende；Model, 5424R；
Affinity chromatographic column: Life Technologies POROS A/20 (Stainless steel) 2.1mm × 30mm
(1-5024-12)；
Size exclusion chromatograph column: TOSOH TSKgel G3000SWXL, 7.8 × 300mm, 5 μm (008541).
Reagent:
Sodium dihydrogen phosphate dihydrate: pure, traditional Chinese medicines/20040718 are analyzed；
Anhydrous Disodium Phosphate: pure, traditional Chinese medicines/20040618 are analyzed；
Sodium chloride: pure, traditional Chinese medicines/10019318 are analyzed；
Glycine: pure, Sigma/G8790 is analyzed；
Hydrochloric acid: pure, traditional Chinese medicines/10011018 are analyzed.
Embodiment 1
Fig. 1 is the detection of the cell culture protein expression quantity that the embodiment of the present invention 1 provides and the protein aggregation scale of construction
System, as shown in Figure 1, the detection system includes solvent pump 1, sample injector six-way valve 2, affinity chromatographic column 3, size exclusion chromatograph column
4, ultraviolet-visible detector 5.Solvent pump 1 is connected to affinity chromatographic column 3, size exclusion chromatograph column 4 by sample injector six-way valve 2
It is serially connected with affinity chromatographic column 3, the outflow end of size exclusion chromatograph column 3 is connected to ultraviolet-visible detector 5.
Wherein, by affinity chromatographic column 3 use short PEEK pipeline and connector directly connect with size exclusion chromatograph column 4 as
One entirety connects between No. 6 positions of sample injector six-way valve 2 and ultraviolet-visible detector 5.Wherein, affinity chromatographic column 3 exists
Before, size exclusion chromatograph column 4 is in rear, access highly effective liquid phase chromatographic system.
Using the present embodiment direct tandem system to protein expression quantity in cell culture supernatant and egg
White matter aggregation percentage is detected, wherein cell culture is monoclonal antibody, host cell CHO, and culture medium is
Acti-Pro, cell culture IgG1, culture mesostroma are host cell and its albumen and DNA, culture medium, culture addition
Object (soda acid, glucose etc.), cell metabolite etc., take cell culture supernatant, under the revolving speed of 13000rpm/min, 5 DEG C,
It is centrifuged 10min, collects supernatant as sample to be tested, protein concentration is about 2-3mg/mL in sample to be tested.
For sample to be tested through 2 sample introduction of sample injector six-way valve, Autosampler disk temperature is 5 ± 3 DEG C, and sample volume is 30 μ L, into
After sample, mobile phase is pushed to carry out gradient elution by solvent pump 1, gradient elution program such as the following table 1:
1 gradient elution program of table
Wherein, mobile phase A are as follows: 50mM PB (sodium dihydrogen phosphate-disodium hydrogen phosphate mixture), 300mM sodium chloride, pH
6.8；Mobile phase B are as follows: 100mM glycine, 150mM sodium chloride, pH 2.5.The flow velocity of gradient elution are as follows: 0.6mL/min.
After affinity chromatographic column is by target protein monomer and aggregation enrichment, eluted using 100% mobile phase A, Neng Goubao
Demonstrate,prove impurity composition will not interferencing protein aggregation and monomer detection, parent will be enriched in by then reusing 100% Mobile phase B
Protein monomers and its aggregation elution in chromatographic column, and further separate through subsequent size exclusion chromatograph column.
The chromatogram that method through the present embodiment detects is as shown in Figures 2 and 3, as can be known from Fig. 2, by the present embodiment
Detection method, impurity composition Z (include host protein, DNA, nutrient media components) obtains being sufficiently separated elution, while target egg
White aggregation Y and target protein monomer X are efficiently separated, and target protein monomer expression quantity in sample to be tested is calculated and is
2.5mg/mL, protein aggregate percentage are 98.98%.
Embodiment 2
Fig. 4 is the detection of the cell culture protein expression quantity that the embodiment of the present invention 2 provides and the protein aggregation scale of construction
System, as shown in figure 4, the detection system includes solvent pump 1, sample injector six-way valve 2, affinity chromatographic column 3, size exclusion chromatograph column
4, ultraviolet-visible detector 5.Solvent pump 1 is connected to affinity chromatographic column 3, size exclusion chromatograph column 4 by sample injector six-way valve 2
It is serially connected with affinity chromatographic column 3, the outflow end of size exclusion chromatograph column 3 is connected to ultraviolet-visible detector 5.
In the present embodiment, by connecing in the inside of sample injector six-way valve 2 for affinity chromatographic column 3, by affinity chromatographic column 3 connect into
After the sample introduction quantitative loop 27 of sample device six-way valve 2.Specifically, the sample introduction quantitative loop 27 on sample injector six-way valve 2 is arranged in six-way valve
No. two positions 22 and No. five positions 25 between, affinity chromatographic column 3 is inscribed on the flow path of No. two positions 22 and No. five positions 25, and is located at
The downstream of quantitative loop 27, solvent pump 1 are connected to the No.1 position 21 of sample injector six-way valve 2, and size exclusion chromatograph column 4 is external in sample introduction
No. six positions 26 of device six-way valve 2.
When autosampler six-way valve 2 is under Load mode, sample to be tested is injected in quantitative loop 27 by sample introduction needle, cocurrent
Through affinity chromatographic column, in affinity chromatography on-column enrichment, the waste liquids such as impurity are discharged through No. four positions 24 for target protein monomer and aggregation
It collects, after sample introduction, the ultrapure water of one needle of sample introduction or number needle total volume greater than 100 μ L is so as to be trapped on affinity chromatographic column
Impurity composition in cell culture supernatant is expelled to waste liquid via No. 4 positions 24 of six-way valve, to guarantee that impurity composition is not eluted
Into subsequent size exclusion chromatograph column 4 and ultraviolet-visible detector 5, in this way in subsequent gradient elution, only albumen
Matter monomer and its aggregation enter subsequent size exclusion chromatograph column 4 and are further separated.When sample introduction and cleaning step terminate
Afterwards, switching autosampler six-way valve 2 starts solvent pump, by mobile phase through the position of No.1 position 21, two under Inject mode
22, quantitative loop 27 reaches affinity chromatographic column 3, and No. five positions 25 are connected to No. six positions 26, carries out gradient elution.
The system of tandem is connected to protein in cell culture supernatant using the six-way valve rotation of the present embodiment
Expression quantity and protein aggregate percentage are detected, wherein and cell culture is monoclonal antibody, host cell CHO,
Culture medium is Acti-Pro, and cell culture IgG1, cell culture is host cell and its albumen and DNA, culture medium, training
Additive (soda acid, glucose etc.), cell metabolite etc. are supported, cell culture supernatant is taken, in the revolving speed of 13000rpm/min
Under, 5 DEG C, it is centrifuged 10min, collects supernatant as sample to be tested, protein concentration is about 2-3mg/mL in sample to be tested.
For sample to be tested through 2 sample introduction of sample injector six-way valve, Autosampler disk temperature is 5 ± 3 DEG C, under Load mode, two
Number position 22 is connected to third place 23, and No. four positions 24 are connected to No. five positions 25, and No. six positions 26 are connected to No.1 position 21, sample volume 30
μ L after sample introduction, continues 100 μ L ultrapure water of sample introduction, switch sampling device six-way valve 2 to Inject mode, No.1 position 21 and two
Number position 22 is connected to, and third place 23 be connected to No. four positions 24, and No. five positions 25 be connected tos with No. six positions 26, passes through the promotion flowing of solvent pump 1
Gradient elution is mutually carried out, gradient elution program such as the following table 2:
2 gradient elution program of table
Wherein, mobile phase A are as follows: 50mM PB (sodium dihydrogen phosphate-disodium hydrogen phosphate mixture), 300mM sodium chloride, pH
6.8；Mobile phase B are as follows: 100mM glycine, 150mM sodium chloride, pH 2.5.The flow velocity of gradient elution are as follows: 0.6mL/min.
Compared to the mode directly concatenated in embodiment 1, the time of gradient elution is saved.
Chromatogram that method through the present embodiment detects is as it can be seen in figures 5 and 6, as can be known from Fig. 5, by the present embodiment
Detection method, impurity composition Z (include host protein, DNA, nutrient media components) obtains being sufficiently separated elution, while target egg
White aggregation Y and target protein monomer X are efficiently separated, and target protein monomer expression quantity in sample to be tested is calculated and is
2.51mg/mL, protein aggregate percentage are 98.42%.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (10)
1. the detection method of cell culture protein expression quantity and the protein aggregation scale of construction, which is characterized in that including walking as follows
It is rapid:
Affinity chromatographic column and size exclusion chromatograph column of the cell culture through concatenating carry out gradient elution and detection；
The mobile phase of gradient elution includes: mobile phase A: 10-200mM sodium dihydrogen phosphate-disodium hydrogen phosphate mixture, 50-500mM
Sodium chloride, pH 6.8 ± 0.5；Mobile phase B: 10-200mM glycine, 20-300mM sodium chloride, pH 2.5 ± 0.5.
2. the detection method of cell culture protein expression quantity and the protein aggregation scale of construction according to claim 1,
It is characterized in that, the mobile phase of the gradient elution includes: mobile phase A: 40-60mM sodium dihydrogen phosphate-disodium hydrogen phosphate mixture,
250-350mM sodium chloride, pH 6.8 ± 0.5；Mobile phase B: 50-150mM glycine, 100-200mM sodium chloride, pH 2.5 ±
0.5；
Preferably, the mobile phase of the gradient elution includes: mobile phase A: 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate mixture,
300mM sodium chloride, pH 6.8；Mobile phase B: 100mM glycine, 150mM sodium chloride, pH 2.5.
3. the detection method of cell culture protein expression quantity and the protein aggregation scale of construction according to claim 1,
It is characterized in that, the mode of the concatenation is that affinity chromatographic column and size exclusion chromatograph column directly concatenate；
Preferably, when the affinity chromatographic column and the size exclusion chromatograph column are by the way of directly concatenating, gradient elution
Program are as follows: 0%B 0-60min, 100%B 60.1-90min.
4. the detection method of cell culture protein expression quantity and the protein aggregation scale of construction according to claim 1,
It is characterized in that, the mode of the concatenation is connected to concatenation by six-way valve rotation with size exclusion chromatograph column for affinity chromatographic column；
Preferably, the affinity chromatographic column is inscribed on the flow path of sample introduction quantitative loop of six-way valve；
Preferably, when the affinity chromatographic column rotates the tandem being connected to using six-way valve with the size exclusion chromatograph column
When, gradient elution program are as follows: 0%B 0-10min, 100%B 10.1-15.1min, 0%B 15.2-40min.
5. the detection method of cell culture protein expression quantity and the protein aggregation scale of construction according to claim 1,
It is characterized in that, the target protein in the cell culture includes IgA, IgG, IgM, Kappa light chain, Lambda light chain, thin
Any one of intracellular cytokine；
Preferably, the target protein in the cell culture is IgG1.
6. the detection method of cell culture protein expression quantity and the protein aggregation scale of construction according to claim 1,
Be characterized in that, the filler of the affinity chromatographic column with base type include specific binding IgA, IgG, IgM, Kappa light chain,
Any one of Lambda light chain, aglucon of cell factor；
Preferably, the filler of the affinity chromatographic column is Protein A with base type.
7. the detection method of cell culture protein expression quantity and the protein aggregation scale of construction according to claim 1,
It is characterized in that, the size exclusion chromatograph column includes TOSOH TSK G3000SWXL, Waters Protein BEH2 (or
Any one of Agilent AdvanceBio SEC；
Preferably, the size exclusion chromatograph column is TOSOH TSK G3000SWXL。
8. the detection method of cell culture protein expression quantity and the protein aggregation scale of construction according to claim 1,
It is characterized in that, the flow velocity of the gradient elution is 0.2-1mL/min；
Preferably, it is detected using ultraviolet-visible light；
It is furthermore preferred that Detection wavelength is 280nm；
Preferably, gradient elution is carried out after cell culture being pre-processed and detection, pretreated method include: to take cell culture
Object supernatant, centrifugation take supernatant as sample to be tested again；
It is furthermore preferred that the condition of the centrifugation are as follows: revolving speed 10000-16000rpm, time 5-15min, temperature are 5 ± 1
℃。
9. the detection system of cell culture protein expression quantity and the protein aggregation scale of construction, which is characterized in that including solvent pump,
Sample injector six-way valve, affinity chromatographic column, size exclusion chromatograph column, ultraviolet-visible detector；The solvent pump passes through sample injector
Six-way valve is connected to the affinity chromatographic column, and the size exclusion chromatograph column is serially connected with the affinity chromatographic column, the volume row
The outflow end of resistance chromatographic column is connected to the ultraviolet-visible detector.
10. the detection system of cell culture protein expression quantity and the protein aggregation scale of construction according to claim 9,
It is characterized in that, the sample introduction quantitative loop on the sample injector six-way valve is arranged between No. two positions of six-way valve and No. five positions, described
Affinity chromatographic column is inscribed on the flow path of No. two positions and No. five positions, and is located at the downstream of quantitative loop, and the solvent pump is connected to
The No.1 position of the sample injector six-way valve, the size exclusion chromatograph column are external in No. six positions of the sample injector six-way valve.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111879872A (en) * | 2020-07-29 | 2020-11-03 | 纳谱分析技术（苏州）有限公司 | Method for detecting immunoglobulin IgG based on monodisperse polymer and application thereof |
| CN112461943A (en) * | 2020-10-12 | 2021-03-09 | 河南省奶牛生产性能测定有限公司 | High performance liquid chromatography detection method for immunoglobulin igG in cow milk |
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| CN111879872A (en) * | 2020-07-29 | 2020-11-03 | 纳谱分析技术（苏州）有限公司 | Method for detecting immunoglobulin IgG based on monodisperse polymer and application thereof |
| CN111879872B (en) * | 2020-07-29 | 2023-01-17 | 纳谱分析技术（苏州）有限公司 | Method for detecting immunoglobulin IgG based on monodisperse polymer and application thereof |
| CN112461943A (en) * | 2020-10-12 | 2021-03-09 | 河南省奶牛生产性能测定有限公司 | High performance liquid chromatography detection method for immunoglobulin igG in cow milk |
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