CN109593133A - A kind of charge isomer separation method of anti human nerve growth factor antibodies - Google Patents
A kind of charge isomer separation method of anti human nerve growth factor antibodies Download PDFInfo
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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Abstract
The invention discloses a kind of charge isomer separation methods of anti human nerve growth factor antibodies.Including first passing through affinity chromatography, the pH for the eluting peak being collected into is adjusted to 5.0-5.50；Conductance is adjusted to 4.90-5.10ms/cm；It is to appear to penetrate peak and when A280 absorption value reaches 20mAU using anion exchange chromatography, start to collect and flow through liquid, the liquid that flows through being collected into is the anti human nerve growth factor removed after charge isomer.Using the charge isomer high income for the anti human nerve growth factor antibodies that method of the invention obtains, activity is good.
Description
Technical field
The present invention relates to the separation field of antibody more particularly to a kind of charge isomers of anti human nerve growth factor antibodies
Separation method.
Background technique
The pain transmission path that anti human nerve growth factor antibodies can be mediated with block nerves growth factor, thus in nerve
Property the painful diseases such as pain, fracture pain, osteoarthritis, rheumatoid arthritis, gout arthralgia in terms of have and widely control
Treatment effect.During production, extraction and storage, antibody can after a series of translations modification, including glycosylate, gather
The effects of closing, is broken, deamidated can make antibody show a plurality of types of inhomogeneities, and then generate different types of
Variant, they have differences in the physicochemical properties such as molecular weight, hydrophobicity, charge.Charge isomer usually according to its relative to
The isoelectric point of main peak is divided into acid or alkali isomerization body.Generally, heterogeneous acidic body has the opposite lower isoelectric point of main peak,
And alkali isomerization body then has the opposite higher isoelectric point of main peak.
The method of preparative separation antibody charge isomer usually uses cation-exchange chromatography technology, this method at present
Using absorb-elute mode, acid peak and alkaline peak are removed, to obtain main peak.The side of analytic type separation antibody charge isomer
Method has ion-exchange chromatography, reversed-phase liquid chromatography, hydrophobic chromatography, size exclusion chromatograph, SDS- polyacrylamide gel electrophoresis, hair
Capillary isoelectrofocusing, capillary zone electrophoresis, mass spectrum etc., wherein capillary isoelectric focusing is used for the analysis of antibody charge isomer
Have it is efficient, quick, separating degree is high, the economic and high advantage of automation.
Summary of the invention
The purpose of the present invention is to provide a kind of high income, the charge isomeries of the good anti human nerve growth factor antibodies of activity
Body separation method.
To achieve the above object, the present invention provides a kind of charge isomer separation side of anti human nerve growth factor antibodies
Method, which is characterized in that include the following steps,
Affinity chromatography: the cell fermentation liquid containing anti human nerve growth factor antibodies is chromatographed through affinity column, A280
Collection obtains eluting peak；The pH for the eluting peak being collected into is adjusted to 5.0-5.50；Conductance is adjusted to 4.90-5.10ms/cm；
Anion-exchange chromatography: to appear to penetrate peak and A280 absorption value reaches using anion exchange chromatography
When 20mAU, start collection and flow through liquid, the liquid that flows through being collected into is the anti human nerve growth factor removed after charge isomer.
Further, the cell fermentation liquid containing anti human nerve growth factor antibodies be recombination anti human nerve growth because
The fermentation liquid of sub- antibody Chinese hamster ovary celI.
Further, the pH adjusting is adjusted using sodium-acetate buffer；Preferably, pH is adjusted to 5.2.
Further, the conductance is adjusted to adjust using the sodium-acetate buffer of 5.0-5.50.
Further, the anion-exchange chromatography step is to be balanced with the sodium-acetate buffer of 25mM pH5.00-5.50
15-25 column volume of anion exchange chromatography；It is preferred that 20 column volumes；To regulate the feed liquid loading of pH and conductance to yin from
Sub- displacement chromatography column, it is to appear to penetrate peak and when A280 absorption value reaches 15-25mAU；It is preferred that starting collection when 20mAU and flowing through
Liquid；Chromatographic column is rinsed after completion of the sample, then with the sodium-acetate buffer of 25mM pH5.00-5.50, is declined to A280 absorption value
When to 15-25mAU；It is preferred that stopping collection when 20mAU and flowing through liquid, the liquid that flows through collected is after removing charge isomer
Anti human nerve growth factor.
Anti human nerve growth factor antibodies feed liquid after taking affinity chromatography, with 2M Tris-Cl buffer (pH9.0) by pH
It is adjusted within the scope of 5.0-5.50.When pH is higher than 5.50, when loading anti human nerve growth factor wholly or largely hang over yin from
On sub- displacement chromatography column, including main peak, alkaline peak and acid peak, it is seldom to penetrate peak, does not form separating effect.PH is lower than 5.0,
All or most penetrates again for main peak, alkaline peak and acid peak, and does not have separating effect.The present invention in pH5.0-5.50 and
Within the scope of conductance, main peak penetration ratio is more, and alkaline peak and acid peak penetrate fewer, Capto Q albumen yield 75%- after purification
85%, if albumen yield is lower than this range, purifying loses industrial value, if albumen yield is higher than this range, alkalinity
It will be increased with acidic charge content of isomer ratio.
Conductance is adjusted within the scope of 4.90-5.10ms/cm with 25mM sodium-acetate buffer (pH5.0-5.50).
With 25mM sodium-acetate buffer (pH5.00-5.50) balance anion displacement chromatography 20 column volumes of column.
The feed liquid loading of pH and conductance will be regulated to anion exchange chromatography, it is to appear to penetrate peak and A280 absorption value
When reaching 20mAU, starts collection and flow through liquid.
Chromatographic column is rinsed after completion of the sample, then with 25mM sodium-acetate buffer (pH5.00-5.50), to A280 absorption value
When dropping to 20mAU, stop collection and flow through liquid, the liquid that flows through collected is the anti human nerve life after removing charge isomer
The long factor.
Anion-exchange chromatography provided by the invention penetrates the anti human nerve growth factor after modal cutoff charge isomer
Detect through capillary isoelectric focusing (cIEF): main peak isoelectric point (pI) is the isoelectric point one behind 6.81, with cation-exchange chromatography
It causes；Main peak area percentage 65.77% is improved than cation-exchange chromatography main peak area percentage (48.06%)
36.85%.It is 50.3ng/ml through cytoactive detection IC50, than cation-exchange chromatography cell activity, (IC50 is 78.9ng/
Ml), activity improves 36.3%.
The invention discloses a kind of anion-exchange chromatographies to penetrate mode in anti human nerve growth factor antibodies charge isomery
Application in body separation, this method make anti human nerve growth factor by the pH and conductance condition of adjusting anion-exchange chromatography
Main peak penetrates chromatographic column, and alkalinity and heterogeneous acidic body are adsorbed on chromatographic column.Pass through absorption-elution with cation-exchange chromatography
Modal cutoff antibody charge isomer method is compared, and has easy method, alkalinity and the removal of heterogeneous acidic body more thorough, acquisition
The higher advantage of anti human nerve growth factor antibodies biological activity.According to capillary isoelectric focusing as a result, affinity chromatography,
Sample main peak isoelectric point is 6.81 after Capto Q, Capto S, main peak area percentage is respectively 35.01%, 65.77%,
48.06%.Cytoactive detection (inhibiting the activity of nerve growth factor) result: sample after affinity chromatography, Capto Q, Capto S
The IC50 of product is respectively 102.4ng/mL, 50.3ng/mL, 78.9ng/mL, implies that the nerve growth factor for inhibiting half quantity,
Sample aequum lacks 28.6ng/mL (36.3%) after sample ratio Capto S after Capto Q.
Detailed description of the invention
Fig. 1 is the refined solution phasor of anti human nerve growth factor fermentation liquid affinity chromatography.
Fig. 2 is the refined solution that anti human nerve growth factor uses Capto Q anion-exchange chromatography again after affinity chromatography
Phasor.
Fig. 3 is the refined solution that anti human nerve growth factor uses Capto S cation-exchange chromatography again after affinity chromatography
Phasor.
Fig. 4 is that the capillary isoelectric focusing after anti human nerve growth factor fermentation liquid affinity chromatography detects figure.
Fig. 5 be anti human nerve growth factor fermentation liquid affinity chromatography after using sample after Capto Q anion-exchange chromatography
The capillary isoelectric focusing of product detects figure.
Fig. 6 be anti human nerve growth factor fermentation liquid affinity chromatography after using sample after Capto S cation-exchange chromatography
The capillary isoelectric focusing of product detects figure.
After Fig. 7 anti human nerve growth factor antibodies affinity chromatography and further sample after Capto Q, Capto S chromatography
The activity of cell biology of product detects figure.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.Embodiment
In particular technique or condition person is not specified, described technology or conditions or according to the description of product according to the literature in the art
Book carries out.Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.
Recombinant anti human Anti-NGF Antibody Chinese hamster ovary celI used in following embodiment: anti human nerve growth factor antibodies
Sequence of heavy chain as shown in SEQ ID NO:1, sequence of light chain, can be artificial synthesized as shown in SEQ ID NO:2；By the anti human nerve
Growth factor antibodies sequence of heavy chain connect to obtain heavy chain recombinant vector with pEE6.4 carrier by double digestion (III/EcoR of Hind I),
The anti human nerve growth factor antibodies sequence of light chain is connect by double digestion (III/EcoR of Hind I) with pEE12.4 carrier
To light chain recombinant vector, then after the heavy chain expression frame double digestion (I/Sal of Not I) on heavy chain recombinant vector, will be connected to same
The light chain recombinant vector of double digestion (I/Sal of Not I) is transferred to Chinese hamster ovary celI again and obtains the life of recombinant anti human nerve after the completion of building
Long factor antibody Chinese hamster ovary celI.
Embodiment 1: the affinity chromatography of anti human nerve growth factor antibodies
Affinity column: Hitrap Mabselect 5ml prepacked column is purchased from GE Healthcare company, and tomographic system is
AKTA Purifier。
Recombinant anti human Anti-NGF Antibody Chinese hamster ovary celI is in 200mL CD FortiCHO culture medium, with Preliminary fermentation
Concentration 3 × 106Cell/ml, at 37 DEG C of temperature, 5%CO2In incubator after the culture of 50rpm shaking flask 7 days, culture solution at 4 DEG C,
It is centrifuged 10min under the conditions of 5000rpm, collects fermented liquid supernatant, then with 0.45 μm of film filtering fermentating liquid supernatant；With 30ml phosphate
Buffer (pH7.0) balances affinity column Hitrap Mabselect 5ml prepacked column, then by above-mentioned fermented liquid supernatant
35ml loading is to chromatographic column；Chromatographic column is rinsed with 30ml phosphate buffer (pH7.0) again, finally uses acetate buffer
(pH3.2) affinity column is eluted, A280 absorption value starts to collect eluting peak when reaching 20mAU, be down to A280 absorption value
Stop collecting when 20mAU.The result is shown in Figure 1 (the refined solution phasor that Fig. 1 is anti human nerve growth factor fermentation liquid affinity chromatography).From
It is that fermentation liquid penetrates peak that Fig. 1, which can be seen that first peak, comprising substances such as CHO host protein, pigments, gives up the part.Second
A peak is anti human nerve growth factor eluting peak, collects the part that A280 absorption value is higher than 20mAU.
Obtained solution will be collected with 2M Tris-Cl buffer (pH9.0) and be adjusted to required pH value (Capto Q chromatography tune
It saves to pH5.2；5.0) Capto S chromatography is adjusted to, freeze spare in -20 DEG C.
Embodiment 2: the Capto Q anion-exchange chromatography of anti human nerve growth factor antibodies
Anion exchange chromatography: Hitrap Capto Q 5ml prepacked column is purchased from GE Healthcare company, chromatography system
System is AKTA Purifier.
Anti human nerve growth factor antibodies feed liquid after the resulting pH of embodiment 1 to be adjusted to 5.2 affinity chromatography is used
25mM sodium-acetate buffer (pH5.2) adjusts feed liquid conductance to 5.2ms/cm；With the 25mM sodium-acetate buffer of 100ml
(pH5.2) Capto Q 5ml prepacked column is balanced；The feed liquid 5ml loading of pH and conductance will be regulated to Capto Q chromatographic column, to
Appearance penetrates peak and when A280 absorption value reaches 20mAU, starts to collect and flows through liquid；After completion of the sample, then with 25mM sodium acetate
Buffer (pH5.2) rinses chromatographic column, when A280 absorption value drops to 20mAU, stops collection and flows through liquid；Then with containing 1M
The 25mM sodium-acetate buffer (pH5.2) of NaCl cleans chromatographic column；As a result see that (Fig. 2 is anti human nerve growth factor by parent to Fig. 2
With the refined solution phasor for using Capto Q anion-exchange chromatography after chromatography again).Figure it is seen that first peak is anti-human mind
Peak is penetrated through growth factor, collects the part that A280 absorption value is higher than 20mAU.The 25mM acetic acid that second peak is the NaCl containing 1M
The eluting peak that sodium buffer (pH5.2) cleaning pillar obtains (ingredient does not detect).
Collect obtain flow through liquid freeze it is spare in -20 DEG C.
Adjusting pH obtained by embodiment 1 is respectively 4.8,4.9,5.0,5.1,5.3,5.5,5.6 by verifying by applicant,
When 5.7, discovery main peak area is respectively 41.63%, 45.22%, 50.38%, 65.77%, 68.21%, 72.62%,
73.16%, 73.89%；The total protein rate of recovery is respectively 89.50%, 88.12%, 86.0%, 84.28%, 80.32%,
76.57%, 63.93%, 50.27%.As can be seen that main peak area and the total protein rate of recovery are comprehensively considered, when pH is 5.0-5.5
When, effect is best.
Embodiment 3: the Capto S cation-exchange chromatography of anti human nerve growth factor antibodies
Cation-exchange chromatography post: Hitrap Capto S 5ml prepacked column is purchased from GE Healthcare company, chromatography system
System is AKTA Purifier.
Anti human nerve growth factor antibodies feed liquid after the resulting pH of embodiment 1 to be adjusted to 5.0 affinity chromatography is used
25mM sodium-acetate buffer (pH5.0) adjusts feed liquid conductance to 5.0ms/cm；With the 25mM sodium-acetate buffer of 100ml
(pH5.0) Hitrap Capto S 5ml prepacked column is balanced；The feed liquid 5ml loading of pH and conductance will be regulated to S layers of Capto
Analyse column；After completion of the sample, chromatographic column is rinsed with the 25mM sodium-acetate buffer (pH5.0) of 30ml, is walked to A280 absorption value flat
Afterwards, be 25mM sodium-acetate buffer (pH5.0) with A liquid, the 25mM sodium-acetate buffer (pH5.0) that B liquid is the NaCl containing 0.5M into
Row gradient elution, 100%A liquid to 100%B liquid, gradient elution 100min start to collect when A280 absorption value reaches 20mAU
Eluting peak stops collecting when A280 absorption value drops to 20mAU；As a result see that (Fig. 3 is anti human nerve growth factor process to Fig. 3
The refined solution phasor of Capto S cation-exchange chromatography is used after affinity chromatography again).From figure 3, it can be seen that occurring when gradient elution
Anti human nerve growth factor eluting peak collects the part that A280 absorption value is higher than 20mAU.
The sample that collection obtains freezes spare in -20 DEG C.
Embodiment 4: capillary isoelectric focusing (cIEF) detection of anti human nerve growth factor antibodies
Capillary electrophoresis manufacturer be AB Sciex, model P ACE MDQ Plus；Coatings capillary pipe factory
Family is Agilent；Ampholytes producer is GE Healthcare；CIEF Peptide Marker producer is Genscript,
Isoelectric point scope is 10.0-4.1；10KD ultrafiltration membrane producer is Millipore.
Feed liquid (1 gained of embodiment), Capto Q penetrate liquid (2 gained of embodiment) and Capto S eluent after affinity chromatography
(3 gained of embodiment) sample concentration and desalting processing: taking 10KD ultrafiltration membrane, 400uL 20mM Tris (pH8.0) solution be added,
Revolving speed 14000rpm is centrifuged 15min at 15 DEG C, discards lower layer's filtrate, repeat above step until being surveyed with ultraviolet specrophotometer
Obtaining sample concentration is 2.5mg/mL；10KD ultrafiltration membrane upper layer is added in sample after taking 60ug to be concentrated, and 400uL 10mM Tris is added
(pH8.0) solution, revolving speed 14000rpm are centrifuged 15min at 15 DEG C, discard lower layer's filtrate；10KD ultrafiltration membrane upper layer is added
400uL 20mM Tris (pH8.0) solution, revolving speed 14000rpm are centrifuged 15min at 15 DEG C, discard lower layer's filtrate, repeat two
It is secondary, film upper layer all samples are taken out, for use.
CIEF sample preparation: by 200ul 3M Urea-cIEF Gel, 12uL ampholytes, 20uL cathode stabilization liquid
(500mM arginine), 2.0ul anodic stabilization liquid (20mM phosphoric acid), each 2.0uL of pI Markers standard items are sufficiently mixed: being added
Sample after above-mentioned desalination, after being sufficiently mixed, loading.CIEF separation process is divided into two steps: focusing and migrates.Focusing
In, apply voltage at capillary both ends and forms pH gradient in capillary under the action of ampholytes, after gradient is formed,
PI markers and isolated sample are detected by the chemical transport method of acetic acid, according to the linear relationship of pH gradient measurement to
The pI value of sample, is detected using the UV detector of 280nm wavelength, with 32Karat software data processing.
It the results are shown in Table 1 and Fig. 4-6.Wherein Fig. 4 is the capillary etc. after anti human nerve growth factor fermentation liquid affinity chromatography
Electrofocusing detects figure.Fig. 5 be anti human nerve growth factor fermentation liquid affinity chromatography after using Capto Q anion-exchange chromatography
The capillary isoelectric focusing of sample detects figure afterwards.Fig. 6 be anti human nerve growth factor fermentation liquid affinity chromatography after using Capto
The capillary isoelectric focusing of sample detects figure after S cation-exchange chromatography.From table 1 and Fig. 4-6 as can be seen that using different separation
The isoelectric point of purifying substance obtained by method is 6.81, is only 35.01% by affinity chromatography on main peak area, affine layer
It is 65.77% using the highest of anion-exchange chromatography after analysis, is using cation-exchange chromatography after affinity chromatography
48.06%, it can be seen that separative efficiency of the anti human nerve growth factor fermentation liquid Jing Guo affinity chromatography and anion-exchange chromatography
Highest, up to 65.77% are far longer than the effect of cation-exchange chromatography.
The sample isoelectric point and main peak area percentage table of table 1cIEF measurement
Embodiment 5: anti human nerve growth factor antibodies activity of cell biology detection
TF-1 cell strain (being purchased from Shanghai Bai Li Biotechnology Co., Ltd) is trained with the RPMI1640 containing 10% fetal calf serum
It supports based on being cultivated under 37 DEG C, 5% carbon dioxide conditions, control cell concentration is every 1ml containing 1.0 × 10E5~7.0 × 10E5
Cell is used for Determination of biological activity in 24~36 hours after passage.
It is anti-human for the above-mentioned affinity chromatography of 1000ng/ml, Capto Q, Capto S that initial concentration is separately added into 96 orifice plates
Anti-NGF Antibody, and by 1:3 volume ratio etc. than gradient in basal medium (containing 10% fetal calf serum
RPMI1640 dilution in), keeping every hole culture volume is 100 μ l, and 8ng recombinant human nerve is then added in each test hole
Growth factor makes its final concentration of 40ng/ml.Mixing is placed on spare in incubator.
It is centrifuged after taking out suitable TF-1 cell by 5*10E4, every hole cell, and thin three times with RPMI1640 resuspension cleaning
Born of the same parents are to substitute original serum-containing media.It cleans every hole in 96 orifice plates added with test sample backward and adds 100 μ l containing 5*10E4
The culture medium of TF-1 cell, mixing are placed on 37 DEG C, cultivate 72 hours under 5% carbon dioxide conditions.After 72 hours, every hole is added
20 μ l of MTS solution reacts 2 hours under 37 DEG C, 5% carbon dioxide conditions, and absorbance, note are then measured at wavelength 490nm
Record measurement result.Test data is handled using four parametric regression calculating methods, data analysis result affinity chromatography as shown in Figure 7
Cytoactive detection curve (the y=0.14549+ (1.46313-0.14549)/(1+ (x/102.38897) ^ of sample afterwards
1.68317),R2=0.9985.Cytoactive detection curve (y=0.13+ (the 1.43986- of sample after Capto Q chromatography
0.13)/(1+(x/50.35025)^2.18332),R2=0.9977.The cytoactive detection curve of sample after Capto S chromatography
(y=0.13799+ (1.44771-0.13799)/(1+ (x/78.94306) ^1.63991), R2=0.9996.It is computed affine
The cell activity IC50 of anti human nerve growth factor antibodies is 102.4ng/ml after chromatography, after Capto Q anion-exchange chromatography
The cell activity IC50 of anti human nerve growth factor antibodies is 50.3ng/ml, anti human nerve after Capto S cation-exchange chromatography
The cell activity IC50 of growth factor antibodies is 78.9ng/ml.As can be seen that using Capto Q anion exchange layer of the present invention
The cell activity highest of anti human nerve growth factor antibodies after analysis, significantly larger than by Capto S cation-exchange chromatography or not
By the cell activity of affinity chromatography.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art are not departing from the principle of the present invention and objective
In the case where can make changes, modifications, alterations, and variations to the above described embodiments within the scope of the invention.
SEQUENCE LISTING
<110>Wei Ming biological medicine Co., Ltd
<120>a kind of charge isomer separation method of anti human nerve growth factor antibodies
<130> WMSW-18007-CNI
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1391
<212> DNA
<213>artificial synthesized
<400> 1
aagcttatgc ccctgctgct gctgctgcct ctgctgtggg ctggcgctct ggctcaggtg 60
cagctggtgc agtccggcgc tgaggtgaag aagcctggcg cctccgtgaa ggtgtcctgc 120
aaggtgtcgg cttcaccctg accgagctgt ccatccactg ggtgaggcag gcccctggaa 180
agggcctgga gtggatgggc ggcttcgatc ccgaggatgg cgagaccatc tacgcccaga 240
agttccaggg caggtgacca tgaccgagga cacctccacc gacaccgcct acatggagct 300
gacctccctg aggtccgagg acaccgccgt gtactactgc tccaccatct tcggcgtggt 360
gaccaacttc gacaactggg gcagggcaca ctggtgaccg tgtcctccgc ctccaccaag 420
ggcccttccg tgttccctct ggccccttgc tccaggtcca cctccgagtc caccgctgct 480
ctgggctgcc tggtgaagga ctacttcccg agcccgtgac cgtgtcctgg aactccggcg 540
ctctgacctc cggagtgcac accttccctg ccgtgctgca gtcctccggc ctgtactccc 600
tgtcctccgt ggtgaccgtg ccctcctcct ccctggcacc aagacctaca cctgcaacgt 660
ggaccacaag ccctccaaca ccaaggtgga caagagggtg gagtccaagt acggccctcc 720
ttgccctcct tgtcctgccc ctgagttcct gggcggcccc tcgtgttcct gttccccccc 780
aagcccaagg acaccctgat gatctccagg acccctgagg tgacctgcgt ggtggtggac 840
gtgtcccagg aggaccccga ggtgcagttc aactggtacg tggacggcgg gaggtgcaca 900
acgccaagac caagcccagg gaggagcagt tcaactccac ctacagggtg gtgtccgtgc 960
tgaccgtgct gcaccaggac tggctgaacg gcaaggagta caagtgcaag gtgtccaaca 1020
agggcctgcc ctcctccatc agaagaccat ctccaaggcc aagggccagc ccagggaacc 1080
tcaggtgtac accctgcccc cctcccagga ggagatgacc aagaaccagg tgtccctgac 1140
ctgcctggtg aagggcttct acccctcgac atcgctgtgg agtgggagtc caacggccag 1200
cccgagaaca actacaagac cacccccccc gtgctggact ccgacggctc cttcttcctg 1260
tactccaggc tgaccgtgga caagtccagg tggcggaggg caacgtgttc tcctgctccg 1320
tgatgcacga ggccctgcac aaccactaca cccagaaatc cctgtccctg tccctgggca 1380
agtgagaatt c 1391
<210> 2
<211> 700
<212> DNA
<213>artificial synthesized
<400> 2
aagcttatgc ctctgctgct gctgctgccc ctgctgtggg ctggagctct ggctgacatc 60
cagatgaccc agtccccttc cagcctgtcc gctagcgctg gcgatagggt gacaatcacc 120
tgcagggctc ccaggccatc aggaacgacc tgggctggta ccagcagaag cccggaaagg 180
cccccaagcg gctgatctac gctgccttta atctgcagag cggcgtgcct tccaggttct 240
ccggcagcgg atccggcacc gagttcaccc tgaccatctc ctccctgcag cccgaggacc 300
tggctagcta tactgccagc aatacaaccg gtacccctgg accttcggcc agggcaccaa 360
ggtggagatc aaacggaccg tggccgcccc cagcgtgttc atcttccctc cttccgacga 420
gcagctgaag tccggcacgc ttccgtggtg tgcctgctga acaactttta cccccgggag 480
gccaaggtgc agtggaaggt ggacaacgct ctgcagtccg gcaacagcca ggagtccgtg 540
acagagcagg acagcaagga ctcccctaca gcctgagctc caccctgacc ctgagcaagg 600
ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc ctgagcagcc 660
ctgtgaccaa gtcttttaac aggggcgagt gtgagaattc 700
Claims (5)
1. a kind of charge isomer separation method of anti human nerve growth factor antibodies, which is characterized in that include the following steps,
Affinity chromatography: the cell fermentation liquid containing anti human nerve growth factor antibodies is chromatographed through affinity column, and A280 is collected
Obtain eluting peak；The pH for the eluting peak being collected into is adjusted to 5.0-5.50；Conductance is adjusted to 4.90-5.10ms/cm；
Anion-exchange chromatography: to appear to penetrate peak and A280 absorption value reaches 20mAU using anion exchange chromatography
When, start collection and flow through liquid, the liquid that flows through being collected into is the anti human nerve growth factor removed after charge isomer.
2. the charge isomer separation method of anti human nerve growth factor antibodies as described in claim 1, which is characterized in that described
Cell fermentation liquid containing anti human nerve growth factor antibodies is the fermentation for recombinating anti human nerve growth factor antibodies Chinese hamster ovary celI
Liquid.
3. the charge isomer separation method of anti human nerve growth factor antibodies as described in claim 1, which is characterized in that described
PH adjusting is adjusted using sodium-acetate buffer；Preferably, pH is adjusted to 5.2.
4. the charge isomer separation method of anti human nerve growth factor antibodies as described in claim 1, which is characterized in that described
Conductance is adjusted to adjust using the sodium-acetate buffer of 5.0-5.50.
5. the charge isomer separation method of anti human nerve growth factor antibodies as described in claim 1, which is characterized in that described
Anion-exchange chromatography step is, with the sodium-acetate buffer balance anion displacement chromatography column 15- of 25mM pH5.00-5.50
25 column volumes；It is preferred that 20 column volumes；The feed liquid loading of pH and conductance will be regulated to anion exchange chromatography, it is to appear
Penetrate peak and when A280 absorption value reaches 15-25mAU；It is preferred that starting collection when 20mAU and flowing through liquid；After completion of the sample, then use
The sodium-acetate buffer of 25mM pH5.00-5.50 rinses chromatographic column, when A280 absorption value drops to 15-25mAU；It is preferred that
When 20mAU, stop collect flow through liquid, collect flow through liquid be remove charge isomer after anti human nerve growth because
Son.
Priority Applications (1)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111606968A (en) * | 2020-06-02 | 2020-09-01 | 兴盟生物医药(苏州)有限公司 | Method for reducing content of acidic charge heteroplasmon of Fc fusion protein |
| CN114544839A (en) * | 2022-01-20 | 2022-05-27 | 未名生物医药有限公司 | Charge variant detection method for anti-human nerve growth factor antibody |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101928341A (en) * | 2010-06-30 | 2010-12-29 | 深圳科兴生物工程有限公司 | Purifying process for separating isomers of recombined human interferon alpha1b and detection method thereof |
| CN105175548A (en) * | 2015-08-13 | 2015-12-23 | 齐鲁制药有限公司 | Purification method of recombinant human vascular endothelial growth factor receptor-antibody fusion protein |
| CN106198780A (en) * | 2016-06-24 | 2016-12-07 | 华南师范大学 | Protein modified open tubular column and the application in monoclonal antibody charge isomer separates |
| CN108059676A (en) * | 2017-12-28 | 2018-05-22 | 未名生物医药有限公司 | A kind of anti human nerve growth factor scFv antibody and preparation method |
-
2018
- 2018-12-18 CN CN201811553816.1A patent/CN109593133B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101928341A (en) * | 2010-06-30 | 2010-12-29 | 深圳科兴生物工程有限公司 | Purifying process for separating isomers of recombined human interferon alpha1b and detection method thereof |
| CN105175548A (en) * | 2015-08-13 | 2015-12-23 | 齐鲁制药有限公司 | Purification method of recombinant human vascular endothelial growth factor receptor-antibody fusion protein |
| CN106198780A (en) * | 2016-06-24 | 2016-12-07 | 华南师范大学 | Protein modified open tubular column and the application in monoclonal antibody charge isomer separates |
| CN108059676A (en) * | 2017-12-28 | 2018-05-22 | 未名生物医药有限公司 | A kind of anti human nerve growth factor scFv antibody and preparation method |
Non-Patent Citations (1)
| Title |
|---|
| B HOUSTON: "Purification and biological activity of a single charge isomer of pituitary-derived chicken growth hormone", 《J ENDOCRINOL》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111606968A (en) * | 2020-06-02 | 2020-09-01 | 兴盟生物医药(苏州)有限公司 | Method for reducing content of acidic charge heteroplasmon of Fc fusion protein |
| CN114544839A (en) * | 2022-01-20 | 2022-05-27 | 未名生物医药有限公司 | Charge variant detection method for anti-human nerve growth factor antibody |
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