CN110240649A - A kind of Mixed-Modechromatography method of the separation antibody from human blood - Google Patents
A kind of Mixed-Modechromatography method of the separation antibody from human blood Download PDFInfo
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- C07K1/16—Extraction; Separation; Purification by chromatography
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Abstract
The Mixed-Modechromatography method of the invention discloses a kind of from human blood separation antibody.Specific step is as follows: 1) pre-processing, human blood sample adds anti-coagulants, and centrifuging and taking supernatant obtains blood plasma；2) chromatography uses sulfapryidine for the mixed mode medium of aglucon, and fixed bed chromatographs separated plasma, loading pH value 6.0~9.0, dilution rate 0~3.0, elution pH value 3.0~5.0, collection eluting peak；3) desalination and drying, elute collection liquid desalination, and freeze-drying obtains the antibody that purity is greater than 95%.Characteristic of the invention is that the antibody of high-purity can be directly isolated to obtain from human blood, the key of method is to use the Mixed-Modechromatography medium using sulfapryidine as aglucon, without adsorbing antibody under the conditions of adjustment blood ion intensity and pH, it is eluted under solutions of weak acidity, has the characteristics that selectivity is strong, elution requirement is mild, antibody yield and purity is high, simple process, low in cost.
Description
Technical field
The invention belongs to protein stripping technique fields, are related to a kind of Mixed-Modechromatography of separation antibody from human blood
Method.
Background technique
Antibody is that bone-marrow-derived lymphocyte is generated by external antigen stimulation, can be with the immunoglobulin in conjunction with antigentic specificity
(immunoglobulin, Ig) is mainly derived from animal blood, colostrum and yolk, and wherein antibodies in blood content is high, and source is rich
Richness is the primary raw material for preparing immunoglobulin.The extracting method of immunoglobulin includes salting out method (such as ammonium sulfate in blood
Analysis method, sodium polyphosphate flocculence), organic solvent precipitation method (such as cold ethanol precipitation partition method), the polymer precipitation method, become
Property precipitation method etc., using more for ammonium sulfate salting-out process and cold ethanol precipitation method.But salting out method and organic solvent precipitation method are deposited
In time-consuming, product purity and the more low limitation of the rate of recovery, new Crafts of Extracting Immunoglobulin is developed with important and applies valence
Value.
Mixed-Modechromatography is a kind of novel bioseparation technology, and aglucon has multiple functions group concurrently, can be with target
Albumen generates a variety of interactions, mainly hydrophobic and electrostatic interaction.The ligand density of mixed mode medium is usually higher,
Adsorption capacity is big, has salt tolerant characterization of adsorption, elution requirement is mild, particularly suitable for isolating and purifying on a large scale, in antibody
It is applied in the isolating and purifying of equal albumen.Mixed-Modechromatography is reported from Pezziniet in 2011 etc.
(J.Chromatogr.A, 2011,1218:216), antibody isolation technics is continuously available improvement.Patent (US Patent 8,802,
448；US Patent 7,691,980；US Patent 0,264,685) report Mixed-Modechromatography antibody separation in answer
With mixed mode medium can effectively remove non-antibody albuminoid, antibody aggregates and other big molecular impurities.Patent (CN
101899110B；CN101948535A；CN101402671B Mixed-Modechromatography) is reported for immune globulin in livestock and poultry blood
White separation has the characteristics that step is simple, separative efficiency is high, low in cost.Patent (CN107138139A) reports one kind
Mixed mode immunosorbent and preparation method thereof for removing blood plasma pathogenic antibody, the treatment as autoimmune disease
Means.Luo etc. (J.Chromatogr.A, 2018,1533:77) is using the two step layers for being filled with ABI-4FF and MMI-4FF medium
Analysis separates IgM in serum, but a small amount of HSA can also be integrated to chromatography media, influence the purity of separation antibody.Wang etc.
(J.Chromatogr.B, 2013,936:33) uses mixed mode medium Bestarose Diamond MMA, Bestarose
Diamond MMC, MEP HyperCel and PPA HyperCel medium separate IgG from simulation serum, wherein Bestarose
Diamond MMA medium adsorbs IgG and HSA simultaneously, and adsorptive selectivity is poor；Bestarose Diamond MMC and PPA
HyperCel medium is unfavorable for actual separation application there are antibody elution difficulty；MEP HyperCel medium primary attachment IgG,
But still with a small amount of HSA, the selectivity of IgG is general.Therefore, novel Mixed-Modechromatography medium is researched and developed, realizes the special of antibody
Property combine, for antibody large-scale separation purifying have great significance.
It is separated for antibody, establishes the small molecule mixing of pyridines, imidazoles, indoles and multi-aromatic ring class compound
Mode aglucon library has carried out the high flux screening of aglucon and IgG compatibility, finds sulfapryidine class compound to IgG compatibility
Height, salt tolerance are strong, and low to HSA compatibility, can be used as the separation ligand of blood antibody.Using hydrophilic porous microballoon as matrix,
Coupling sulfapryidine is functional ligand, and dielectric structure is shown in Fig. 1.The present invention utilizes the high-affinity of antibody and sulfapryidine, selects
Take sulfapryidine as the mixed mode medium of main functional ligand, directly captures antibody from human blood sample, develop one kind
The new method of Mixed-Modechromatography separation human immunoglobulin(HIg).
Summary of the invention
The Mixed-Modechromatography method of the object of the present invention is to provide a kind of from human blood separation antibody, specifically include as
Lower step:
1) blood sample containing immunoglobulin is taken, by centrifuging and taking supernatant, obtains loading crude product solution；
2) pH to 6.0~9.0 of adjusting loading crude product solution, dilution rate to 0~3.0, after 0.45 μm of membrane filtration, on
Sample is to filled with using sulfapryidine as in the chromatographic column of the mixed mode medium of aglucon, Equilibration buffer wash, elution buffer
Elution collects the corresponding efflux of eluting peak, obtains human antibody solution；
3) by human antibody solution desalination, freeze-drying obtains the human antibody that purity is greater than 95%；
The blood sample containing human immunoglobulin(HIg) is human blood, perhaps removes the blood plasma of haemocyte or goes
Except the serum of haemocyte and fibrinogen etc..
The mixed mode medium is the chromatography media for being coupled sulfapryidine as aglucon；
The sulfapryidine is 4- amino-N- (2- pyridyl group) benzsulfamide or 3- amino-N-pyridin -3- methylbenzene sulphur
Amide.
When sulfapryidine is 4- amino-N- (2- pyridyl group) benzsulfamide, the structure composition of chromatography media are as follows:
When sulfapryidine is 3- amino-N-pyridin -3- methyl benzenesulfonamide, the structure composition of chromatography media are as follows:
The equilibration buffer is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 6.0~8.0) or Tris- hydrochloric acid
Buffer (pH 7.0~9.0)；
The elution buffer is acetic acid-sodium acetate buffer solution (pH 3.0~5.0), citric acid-sodium citrate buffers
Liquid (pH 3.0~5.0) or glycine-HCI buffer (pH 3.0~5.0), the elution buffer are added with 0-0.1M
NaCl；
It is that acetic acid or citric acid are molten that the pH of human immunoglobulin(HIg) crude product solution, which adjusts acid solution used, in the step 2)
Liquid, aqueous slkali used are sodium hydroxide solution.
The dilution rate of loading crude product solution adjusts solution used and is and has adjusted the loading crude product of pH molten in the step 2)
For liquid phase with the buffer solution of pH, buffer solution is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 6.0~8.0) or Tris-
Hydrochloride buffer (pH 7.0~9.0).
The present invention is directed to the feature of human blood sample component complexity, directly handles human blood sample using mixed mode medium
Product make full use of the advantages such as the adsorption capacity of Mixed-Modechromatography is big, selectivity is good, salt-tolerant trait is strong, elution requirement is mild, mention
High separating efficiency simplifies separating step, forms a kind of new method for being directly separated antibody from human blood.Advantages of the present invention exists
In: 1) antibody adsorption capacity big, and processing capacity is strong, and the dynamic bind carrying capacity of human IgG is greater than 25mg/ml humid medium；2) antibody selects
Selecting property is strong, good separating effect, and the dynamic bind carrying capacity of human serum albumin is less than 2mg/ml humid medium；3) adsorption conditions range is wide,
Salt tolerance is strong, and in very wide pH (6.0~9.0) and conductivity range (0-150mS/cm), adsorption capacity is held essentially constant,
It is particularly suitable for human blood physiological condition, separating step can be reduced with Direct Acquisition antibody；4) elution is convenient, molten by adjusting
Nearby complete elution can be realized in liquid pH to 4, avoids peracid, crosses alkali or with high salt etc. have an adverse effect to protein structure or activity
Loss.
Detailed description of the invention
Attached drawing 1 is shown using 4- amino-N- (2- pyridyl group) benzsulfamide as the structure of the mixed mode medium of functional ligand
It is intended to.
Attached drawing 2 is shown by the structure of the mixed mode medium of functional ligand of 3- amino-N-pyridin -3- methyl benzenesulfonamide
It is intended to.
Attached drawing 3 is the chromatography spectrogram of the Mixed-Modechromatography isolating human antibodies of embodiment 2.
Attached drawing 4 is the feed liquid of the Mixed-Modechromatography separation of embodiment 2 and the efficient liquid phase chromatographic analysis figure of elution fraction.
Specific embodiment
The present invention provides a kind of method of separation antibody from human blood.The blood sample containing human immunoglobulin(HIg) is taken,
Centrifuge separation, takes supernatant；Supernatant is subjected to post separation by Mixed-Modechromatography, obtains human antibody solution.Side of the present invention
The human antibody purity that method obtains is greater than 95%.
The method of isolating human antibodies includes the following steps: from human blood
1) blood sample containing human immunoglobulin(HIg) is taken, centrifuging and taking supernatant obtains loading crude product solution；
2) pH to 6.0~9.0 of loading crude product solution is adjusted, dilution rate is 0~3.0, after 0.45 μm of membrane filtration, on
Sample is put down to filled with using sulfapryidine as in the chromatographic column of the mixed mode medium of aglucon, dielectric structure is as depicted in figs. 1 and 2
The buffer that weighs rinses, and elution buffer elution collects the corresponding efflux of eluting peak, obtains human antibody solution；
3) by human antibody solution desalination, freeze-drying obtains the human antibody that purity is greater than 95%；
The blood sample containing human immunoglobulin(HIg) is human blood, perhaps removes the blood plasma of haemocyte or goes
Except the serum of haemocyte and fibrinogen etc..
The mixed mode medium is the chromatography media for being coupled sulfapryidine amino as aglucon；
The sulfapryidine is 4- amino-N- (2- pyridyl group) benzsulfamide or 3- amino-N-pyridin -3- methylbenzene sulphur
Amide.
The equilibration buffer is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, and pH value is 6.0~8.0；Or
Tris- hydrochloride buffer, pH value are pH 7.0~9.0；
The elution buffer is acetic acid-sodium acetate buffer solution (pH 3.0~5.0), citric acid-sodium citrate buffers
Liquid (pH 3.0~5.0) or glycine-HCI buffer (pH 3.0~5.0) add 0-0.1M NaCl；
It is acetic acid or citric acid solution, alkali used that the pH of loading crude product solution, which adjusts acid solution used, in the step 2)
Solution is sodium hydroxide solution.
The dilution rate of loading crude product solution adjusts the buffer solution that solution used is identical pH, phosphoric acid in the step 2)
Sodium dihydrogen-disodium hydrogen phosphate buffer (pH 6.0~8.0) and Tris- hydrochloride buffer (pH 7.0~9.0).
Embodiment 1
Human blood is taken, pH value 7.4, conductivity about 15mS/cm, antibody concentration is about 15mg/ml, human serum albumin concentration
About 60mg/ml.Add anti-coagulants, centrifuging and taking supernatant obtains crude product solution.Using 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate
Buffer (pH 6.0) is diluted, dilution rate 3.0, and adjusting pH value is 6.0,0.45 μm of membrane filtration, obtains chromatography
Loading sample.Coupling 4- amino-N- (2- pyridyl group) benzsulfamide of filling 2ml, which is used as, in chromatographic column (internal diameter 0.5cm) matches
The chromatography media of base, equilibration buffer are 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 6.0), and equilibrium volume is
10ml；Sample loading volume is 4ml, using 10ml Equilibration buffer wash bed, until UV is responded close to baseline；Using 20mM
Acetic acid-sodium acetate buffer solution (pH 4.0) is eluted, effluent volume 10ml；The cleaning of medium uses 0.1M NaOH solution,
Volume is 6ml.Elution fraction, desalination are collected, freeze-drying obtains human antibody, and HPLC purity assay is 96.6%, and yield is
86.0%, human serum albumin removal rate is 99.2%.
Embodiment 2
Human blood is taken, pH value 7.4, conductivity about 15mS/cm, antibody concentration is about 15mg/ml, human serum albumin concentration
About 60mg/ml.Add anti-coagulants, centrifuging and taking supernatant obtains crude product solution.Using 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate
Buffer (pH 7.0) is diluted, dilution rate 3.0, and adjusting pH value is 7.0,0.45 μm of membrane filtration, obtains chromatography
Loading sample.Coupling 4- amino-N- (2- pyridyl group) benzsulfamide of filling 2ml, which is used as, in chromatographic column (internal diameter 0.5cm) matches
The chromatography media of base, equilibration buffer are 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 7.0), and equilibrium volume is
10ml；Sample loading volume is 4ml, using 10ml Equilibration buffer wash bed, until UV is responded close to baseline；Using 20mM
Citric acid-sodium citrate buffer solution (pH 4.0) is eluted, effluent volume 10ml；The cleaning of medium uses 0.1M NaOH
Solution, volume 6ml.Elution fraction, desalination are collected, freeze-drying obtains human antibody, and HPLC purity assay is 98.6%, receives
Rate is 88.0%, and human serum albumin removal rate is 98.8%.Chromatography spectrogram such as Fig. 3 of Mixed-Modechromatography isolating human antibodies
Shown, the efficient liquid phase chromatographic analysis figure of feed liquid and elution fraction is as shown in Figure 4.
Embodiment 3
Human blood is taken, pH value 7.4, conductivity about 15mS/cm, antibody concentration is about 15mg/ml, human serum albumin concentration
About 60mg/ml.Add anti-coagulants, centrifuging and taking supernatant obtains crude product solution.Using 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate
Buffer (pH 7.4) is diluted, dilution rate 3.0, does not adjust pH, and 0.45 μm of membrane filtration obtains the loading of chromatography
Sample.Layer of coupling 4- amino-N- (2- pyridyl group) benzsulfamide as aglucon of filling 2ml in chromatographic column (internal diameter 0.5cm)
Medium is analysed, equilibration buffer is 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 7.4), equilibrium volume 10ml；Sample
Product loading volume is 4ml, using 10ml Equilibration buffer wash bed, until UV is responded close to baseline；Using 20mM glycine-
Hydrochloride buffer (pH 4.0) is eluted, effluent volume 10ml；The cleaning of medium uses 0.1M NaOH solution, and volume is
6ml.Elution fraction, desalination are collected, freeze-drying obtains human antibody, and HPLC purity assay is 97.6%, yield 87.4%,
Human serum albumin removal rate is 98.2%.
Embodiment 4
Human blood is taken, pH value 7.4, conductivity about 15mS/cm, antibody concentration is about 15mg/ml, human serum albumin concentration
About 60mg/ml.Add anti-coagulants, centrifuging and taking supernatant obtains crude product solution.The lower 0.45 μm of membrane filtration of physiological condition, obtains
The loading sample of chromatography.The coupling 3- amino-N-pyridin -3- Methyl benzenesulfonyl of filling 2ml in chromatographic column (internal diameter 0.5cm)
Chromatography media of the amine as aglucon, equilibration buffer are 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 7.4,0.15M
NaCl), equilibrium volume 10ml；Sample loading volume is 1ml, using 10ml Equilibration buffer wash bed, until UV response connects
It is bordering on baseline；It is eluted using 20mM acetic acid-sodium acetate buffer solution (pH 4.0), effluent volume 10ml；The cleaning of medium
Using 0.1M NaOH solution, volume 6ml.Elution fraction, desalination are collected, freeze-drying obtains human antibody, and HPLC analysis is pure
Degree is 98.9%, yield 88.0%, and human serum albumin removal rate is 99.6%.
Embodiment 5
Human plasma is taken, pH value 7.4, conductivity about 15mS/cm, antibody concentration is about 15mg/ml, human serum albumin concentration
About 60mg/ml.Centrifuging and taking supernatant, obtains crude product solution.The lower 0.45 μm of membrane filtration of physiological condition, obtains chromatography
Loading sample.Coupling 4- amino-N- (2- pyridyl group) benzsulfamide of filling 8ml is as aglucon in chromatographic column (internal diameter 0.5cm)
Chromatography media, equilibration buffer be 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 7.4,0.15M NaCl), put down
Weighing apparatus volume is 40ml；Sample loading volume is 4ml, using 40ml Equilibration buffer wash bed, until UV is responded close to baseline；
It is eluted using 20mM acetic acid-sodium acetate buffer solution (pH 4.0), effluent volume 40ml；The cleaning of medium uses 0.1M
NaOH solution, volume 25ml.Elution fraction, desalination are collected, freeze-drying obtains human antibody, HPLC purity assay is
98.7%, yield 85.2%, human serum albumin removal rate is 98.9%.
Embodiment 6
Human plasma is taken, pH value 7.4, conductivity about 15mS/cm, antibody concentration is about 15mg/ml, human serum albumin concentration
About 60mg/ml.Centrifuging and taking supernatant, obtains crude product solution.It is carried out using 50mM Tris- hydrochloride buffer (pH 8.0) dilute
It releases, dilution rate 2.0, adjusts pH to 8.0,0.45 μm of membrane filtration obtains the loading sample of chromatography.Chromatographic column (internal diameter
Chromatography media of the coupling 3- amino-N-pyridin -3- methyl benzenesulfonamide of filling 2ml as aglucon, equalizing and buffering in 0.5cm)
Liquid is 50mM Tris- hydrochloride buffer (pH 8.0), equilibrium volume 10ml；Sample loading volume is 3ml, flat using 10ml
The buffer that weighs rinses bed, until UV is responded close to baseline；Using 20mM acetic acid-sodium acetate buffer solution (pH 5.0,0.1M
NaCl it) is eluted, effluent volume 10ml；The cleaning of medium uses 0.1M NaOH solution, volume 6ml.Collect elution group
Point, desalination, freeze-drying obtains human antibody, and HPLC purity assay is 96.6%, yield 88.0%, human serum albumin removal
Rate is 99.1%.
Embodiment 7
Human plasma is taken, pH value 7.4, conductivity about 15mS/cm, antibody concentration is about 15mg/ml, human serum albumin concentration
About 60mg/ml.Centrifuging and taking supernatant, obtains crude product solution.It is carried out using 50mM Tris- hydrochloride buffer (pH 9.0) dilute
It releases, dilution rate 1.0, adjusts pH to 9.0,0.45 μm of membrane filtration obtains the loading sample of chromatography.Chromatographic column (internal diameter
Chromatography media of the coupling 3- amino-N-pyridin -3- methyl benzenesulfonamide of filling 2ml as aglucon, equalizing and buffering in 0.5cm)
Liquid is 50mM Tris- hydrochloride buffer (pH 9.0,0.1M NaCl), equilibrium volume 10ml；Sample loading volume is 2ml,
Using 10ml Equilibration buffer wash bed, until UV is responded close to baseline；Using 20mM glycine-HCI buffer (pH
4.0) it is eluted, effluent volume 10ml；The cleaning of medium uses 0.1M NaOH solution, volume 6ml.Collect elution group
Point, desalination, freeze-drying obtains human antibody, and HPLC purity assay is 97.6%, yield 86.5%, human serum albumin removal
Rate is 98.2%.
Embodiment 8
Human serum is taken, pH value 7.4, conductivity about 15mS/cm, antibody concentration is about 15mg/ml, human serum albumin concentration
About 60mg/ml.Centrifuging and taking supernatant, obtains crude product solution.The lower 0.45 μm of membrane filtration of physiological condition, obtains chromatography
Loading sample.Coupling 4- amino-N- (2- pyridyl group) the base benzsulfamide of filling 8ml, which is used as, in chromatographic column (internal diameter 0.5cm) matches
The chromatography media of base, equilibration buffer are 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 7.4,0.15M NaCl),
Equilibrium volume is 40ml；Sample loading volume is 4ml, using 40ml Equilibration buffer wash bed, until UV is responded close to base
Line；It is eluted, effluent volume 40ml using 20mM acetic acid-sodium acetate buffer solution (pH 5.0,0.1M NaCl)；Medium
Regeneration uses 0.1M NaOH solution, volume 25ml.Elution fraction, desalination are collected, freeze-drying obtains human antibody, and HPLC divides
Analysing purity is 97.9%, yield 86.2%, and human serum albumin removal rate is 99.5%.
Embodiment 9
Human serum is taken, pH value 7.4, conductivity about 15mS/cm, antibody concentration is about 15mg/ml, human serum albumin concentration
About 60mg/ml.Centrifuging and taking supernatant, obtains crude product solution.Using 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH
7.4) it is diluted, dilution rate 3.0, adjusts pH to 7.4,0.45 μm of membrane filtration obtains the loading sample of chromatography.Layer
Chromatography media of the coupling 3- amino-N-pyridin -3- methyl benzenesulfonamide of filling 2ml in column (internal diameter 0.5cm) as aglucon is analysed,
Equilibration buffer is 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 7.4), equilibrium volume 10ml；Sample loading body
Product is 4ml, using 10ml Equilibration buffer wash bed, until UV is responded close to baseline；It is buffered using 20mM glycine-HCI
Liquid (pH 4.0) is eluted, effluent volume 10ml；The cleaning of medium uses 0.1M NaOH solution, volume 6ml.It collects
Elution fraction, desalination, freeze-drying obtain human antibody, and HPLC purity assay is 98.6%, yield 87.9%, the white egg of people's blood
White removal rate is 98.9%.
Embodiment 10
Human serum is taken, pH value 7.4, conductivity about 15mS/cm, antibody concentration is about 15mg/ml, human serum albumin concentration
About 60mg/ml.Centrifuging and taking supernatant, obtains crude product solution.The lower 0.45 μm of membrane filtration of physiological condition, obtains chromatography
Loading sample.Coupling 4- amino-N- (2- pyridyl group) benzsulfamide of filling 2ml is as aglucon in chromatographic column (internal diameter 0.5cm)
Chromatography media, equilibration buffer be 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 7.4,0.15M NaCl), put down
Weighing apparatus volume is 10ml；Sample loading volume is 1ml, using 40ml Equilibration buffer wash bed, until UV is responded close to baseline；
It is eluted, effluent volume 40ml using 20mM acetic acid-sodium acetate buffer solution (pH 5.0,0.15M NaCl)；Medium is again
It is raw to use 0.1M NaOH solution, volume 25ml.Elution fraction, desalination are collected, freeze-drying obtains human antibody, HPLC analysis
Purity is 99.1%, yield 88.2%, and human serum albumin removal rate is 98.9%.
Claims (6)
1. a kind of Mixed-Modechromatography method of the separation antibody from human blood, it is characterised in that include the following steps:
1) it pre-processes, the blood sample containing immunoglobulin adds anti-coagulants, and centrifuging and taking supernatant obtains loading crude product solution；
2) chromatography adjusts the pH to 6.0~9.0 of loading crude product solution, and dilution rate is 0~3.0, with 0.45 μm of filter membrane mistake
Filter is loaded to filled with using sulfapryidine as in the chromatographic column of the mixed mode medium of aglucon, Equilibration buffer wash, elution is delayed
Fliud flushing elution collects the corresponding efflux of eluting peak, obtains human antibody solution；
3) desalination and drying, by human antibody solution desalination, freeze-drying obtains the human antibody that purity is greater than 95%；
The mixed mode medium is chromatography media of the sulfapryidine as aglucon；
The sulfapryidine is 4- amino-N- (2- pyridyl group) benzsulfamide or 3- amino-N-pyridin -3- Methyl benzenesulfonyl
Amine.
The equilibration buffer pH value is 6.0~9.0；
The elution buffer pH value is 3.0~5.0.
2. a kind of Mixed-Modechromatography method of the separation antibody from human blood as described in claim 1, it is characterised in that institute
The blood sample containing human immunoglobulin(HIg) stated be human blood, perhaps remove haemocyte blood plasma or removal haemocyte and
The serum of fibrinogen.
3. a kind of Mixed-Modechromatography method of the separation antibody from human blood as described in claim 1, it is characterised in that institute
The equilibration buffer stated is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer or Tris- hydrochloride buffer.
4. a kind of Mixed-Modechromatography method of the separation antibody from human blood as described in claim 1, the elution are slow
Fliud flushing is acetic acid-sodium acetate buffer solution, citric acid-sodium citrate buffer solution or glycine-HCI buffer, the elution
0-0.1 M NaCl is added in buffer.
5. a kind of Mixed-Modechromatography method of the separation antibody from blood as described in claim 1, it is characterised in that described
Step 2) in the pH of loading crude product solution to adjust acid solution used be acetic acid or citric acid solution, aqueous slkali used is hydroxide
Sodium solution.
6. a kind of Mixed-Modechromatography method of the separation antibody from blood as described in claim 1, it is characterised in that described
Step 2) in the dilution rate of loading crude product solution to adjust solution used be pH identical as the loading crude product solution after pH has been adjusted
Buffer solution, the buffer solution be sodium dihydrogen phosphate-disodium hydrogen phosphate buffer or Tris- hydrochloride buffer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20220305460A1 (en) * | 2021-03-17 | 2022-09-29 | Bio-Rad Laboratories, Inc. | Mixed mode cation exchange chromatography ligands based on 1,3-dioxoisoindolin-2-yl structures |
| CN116693659A (en) * | 2023-07-11 | 2023-09-05 | 浙江大学 | Two-step mixed mode chromatography method for separating recombinant human serum albumin |
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| US11731108B2 (en) * | 2021-03-17 | 2023-08-22 | Bio-Rad Laboratories, Inc. | Mixed mode cation exchange chromatography ligands based on 1,3-dioxoisoindolin-2-yl structures |
| CN116693659A (en) * | 2023-07-11 | 2023-09-05 | 浙江大学 | Two-step mixed mode chromatography method for separating recombinant human serum albumin |
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