CN107630037A - A kind of purifying process for obtaining high-purity gland relevant viral vector - Google Patents
A kind of purifying process for obtaining high-purity gland relevant viral vector Download PDFInfo
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- CN107630037A CN107630037A CN201710974674.5A CN201710974674A CN107630037A CN 107630037 A CN107630037 A CN 107630037A CN 201710974674 A CN201710974674 A CN 201710974674A CN 107630037 A CN107630037 A CN 107630037A
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- viral vector
- relevant viral
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Abstract
The present invention provides a kind of process for obtaining high-purity gland relevant viral vector, including following steps：A）Gland relevant viral vector culture supernatant is collected, is filtered off except cell fragment, the sample after clarification is used for chromatographic purifying；B）Purified by cation seperation column；C）Purified by attractive gel Filter column；D）Purified by anion-exchange column；E）Concentrated by milipore filter.The present invention purifies since cells and supernatant, greatly simplify the harvest process of gland relevant viral vector；It is not related to the unit operation that cracking, centrifugation, precipitation, ultracentrifugation etc. are difficult to amplify in technique, has great importance to the clinical practice for mass producing and promoting adeno-associated virus.
Description
Technical field
The present invention relates to biological technical field and gene therapy, more particularly to a kind of extensive 2 type glands for obtaining high-purity are related
The purification technique of viral vector.
Background technology
Gene therapy is a kind of method imported foreign gene with security and stability in patient body.Into intracellular, genetic fragment
The product that expression needs realizes therapeutic effect.The key of gene therapy is to select suitable genophore.Recombinate gland related diseases
Poisonous carrier（Recombinant adeno-associated viral, rAAV）It is security reliability gene therapy generally acknowledged at present
Carrier, can mediate foreign gene express steadily in the long term in vivo, and immunogenicity is very low, has broad application prospects.
However, one of obstacle that limitation rAAV is used is the purifying process that exploitation can amplify.Traditional laboratory scale
RAAV purification process is ultracentrifugation, and the maximum drawback of this method is to be difficult to be amplified to production scale.Chromatographic technique and film point
From representative of the technology as modern purification process, bio-pharmaceuticals large-scale production is widely used to.Have at present some documents and
Patent report purifies rAAV using the method for chromatography, but technologies of these reports are primarily present following two problems：1）Chromatography
Technology still contains ultracentrifugation step as ultracentrifugal supplement, whole technique, it is difficult to amplifies；2）RAAV harvest liquids are thin
Cellular lysate liquid, it is meant that rAAV harvest technology is complex, it is necessary to including a series of unit operations such as cracking, centrifugation, filtering.
If the direct purification rAAV from rAAV culture supernatant, rAAV harvest technology will be greatly simplified, it is only necessary to one
Walk filter operation.In addition, not being related to the unit operation that ultracentrifugation etc. is difficult to amplify in technique, also have to large-scale production
There is important meaning.
The content of the invention
In order to solve the above technical problems, the invention provides it is a kind of based on chromatography and membrane filtration technique, from Virus culture
The process of direct purification gland relevant viral vector in clear, including following steps：
Step A：The culture supernatant of gland relevant viral vector is collected, is filtered off except cell fragment, the sample after clarification is used for layer
Analysis purifying；
Step B：Gland relevant viral vector is captured by cation seperation column and preliminary purification；
Step C：Moderate purifying is carried out by attractive gel Filter column；
Step D：Carried out with anion-exchange column consummate；
Step E：The gland relevant viral vector of collection is concentrated by ultrafiltration.
Preferably, 0.1-20 μm of the filtering of deep bed filter step by step is selected in step A, then by 0.22 μm or 0.45 μ
M filters filter, and more preferably select 0.22 μm of filter aseptic filtration.
Preferably, the aglucon of the cation exchange medium selected by step B is Sulphopropyl (SP) strong cation
Cation exchange groups, such as SP Sepharose XL, SP Sepharose FF, Capto SP and Capto SP ImpRes etc..
Preferably, need at least to dilute one times with water before the sample treatment in step B, conductance is adjusted to 5-10 mS/cm, pH
5-7 is adjusted to, it is highly preferred that pH is adjusted to 5.5-6.2.
Preferably, the loading volume in step B is no more than 30 times chromatography column volumes.
Preferably, the level pad in step B is 50-100 mM sodium chloride solutions, pH5.5-6.2.
Preferably, the elution buffer in step B is 20 mM Tris, 50-400 mM sodium chloride solutions, pH6.0-
9.0。
Preferably, step B operating process is, first with Equilibration buffer wash chromatographic column until conductance and ultraviolet absorbing groups
Line stabilization；Then sample is flowed through into chromatographic column；Again with Equilibration buffer wash chromatographic column until conductance and UV absorption baseline are steady
It is fixed；Elution buffer elution chromatography post is finally used, ultraviolet absorption value is monitored, sample is collected after appearance, collect 2 column volumes.
Preferably, the attractive gel filter medium selected by step C is Capto Core 700.
Preferably, the level pad in step C is 20 mM Tris, 50-400 mM sodium chloride solutions, pH6.0-
9.0。
Preferably, step C operating process is, first with Equilibration buffer wash chromatographic column until conductance and ultraviolet absorbing groups
Line stabilization；Then the sample collected in step B is flowed through into chromatographic column, monitors ultraviolet absorption value, starting collection after appearance flows through liquid；
Again with Equilibration buffer wash chromatographic column until conductance and UV absorption baseline stability, stop collecting sample；Finally with 0.5 M
Sodium hydroxide and 27% aqueous isopropanol processing chromatographic column.
Preferably, the samples with water collected in step C is adjusted to conductance 5-10 mS/cm.
Preferably, the aglucon of the anionic exchange medium selected by step D is the strong of Quaternary ammonium (Q)
Anion exchange groups, for example, Q Sepharose XL, Q Sepharose FF, Capto Q, Capto Q ImpRes,
Source 30Q, Source 15Q etc..
Preferably, the level pad in step D is 20 mM Tris, 50-100 mM sodium chloride solutions, pH7.0-
9.0。
Preferably, the elution buffer in step D is 20 mM Tris, 0.5-1.0 M sodium chloride solutions, pH7.0-
9.0。
Preferably, step D operating process is, first with Equilibration buffer wash chromatographic column until conductance and ultraviolet absorbing groups
Line stabilization；Then the sample obtained in step C is flowed through into chromatographic column；Again with Equilibration buffer wash chromatographic column until conductance and purple
Outer absorption baseline stability；Ultraviolet suction is finally monitored with isocratic elution or linear elution pattern elution chromatography post with elution buffer
Receipts value, sample is collected after appearance, collect 2 column volumes.
Preferably, the ultrafiltration concentration pattern in step E is centrifugal concentrating and tangential flow filtration.
Preferably, the milipore filter retention size in step E is 10-300 kD.
The adeno-associated virus purifying process of the present invention is easy to amplify, and can handle cell factory or bioreactor life on a large scale
A large amount of viral vector nutrient solutions of production, the wilderness demand of viral vector can be met.
Brief description of the drawings
Fig. 1 is the chromatography collection of illustrative plates of cation exchange
Fig. 2 is the chromatography collection of illustrative plates of attractive gel filtering
Fig. 3 is the chromatography collection of illustrative plates of anion exchange
Fig. 4 is the viral vector after concentration through SDS-PAGE results：Band 1 is culture supernatant, band 2 is cation-exchange chromatography
Collection liquid, band 3 are attractive gel filtration chromatography collection liquid, band 4 is sample after the concentration of anion-exchange chromatography collection liquid.
Embodiment
Below in conjunction with the accompanying drawings, the preferred embodiment of the present invention is described in further detail：
Step A：The microcarrier carrier culture supernatant of 150 ml adeno-associated viruses is collected, after 10000 rpm centrifuge 10 min, is used
0.22 μm of filter filtering.
Step B：Cation-exchange chromatography purifies（Fig. 1）
1）Clarified solution in step A is diluted to 300 ml with ultra-pure water, pH is adjusted to 5.5-6.2, for cation-exchange chromatography
Loading；
2）First with 50 ml 100 mM sodium chloride buffers（pH6.0）Balance the SP Sepharose that column volume is 10 ml
XL chromatographic columns, the ml/min of flow velocity 10, until conductance, pH and ultraviolet absorption value baseline stability；
3）Adeno-associated virus clarified solution loading after being adjusted with 300 ml, the ml/min of flow velocity 5；
4）After end of the sample, with the mM sodium chloride of 30 ml 100（pH6.0）Wash buffer chromatographic column, wash away be not bound with
And combine weaker impurity；
5）With 20 mM Tris, 200 mM sodium chloride buffers（pH7.5）Elution chromatography post, flow velocity 5 ml/min, 280 nm
20 ml are collected after ultraviolet absorption value appearance, stop collecting；
6）Chromatographic column, the ml/min of regeneration velocity 5 are regenerated with 2 M sodium chloride solutions.
Step C：Attractive gel Purification by filtration（Fig. 2）
1) first with 10 ml 20 mM Tris, 200 mM sodium chloride buffers（pH7.5）It is 1.6 ml's to balance column volume
The chromatographic columns of Capto Core 700, the ml/min of flow velocity 2, until conductance, pH and ultraviolet absorption value baseline stability；
2) loading is carried out with the collection virus liquid afforded in step B, loading flow velocity is 0.8 ml/min, the ultraviolet suctions of 280 nm
Collected after receipts value appearance and flow through liquid；
3) after end of the sample, the 20 mM Tris with 10 ml, 200 mM sodium chloride buffers are continued（pH7.5）Rinse chromatography
Post, until 280 nm ultraviolet absorption values stop collecting when being down to baseline.
Step D：Anion-exchange chromatography purifies（Fig. 3）
1) first with 40 ml 20 mM Tris, 100 mM sodium chloride buffers（pH7.5）It is 7.5 ml's to balance column volume
Capto Q ImPres chromatographic columns, the ml/min of flow velocity 3.5；
2) loading, the ml/ of flow velocity 3.5 are carried out after the collection virus liquid of gained in step C being diluted into 2 times of volumes with ultra-pure water
min；
3) after end of the sample, the 20 mM Tris with 40 ml, 100 mM sodium chloride are continued（pH7.5）Rinse chromatographic column, flow velocity
3.5 ml/min；
4) with 20 mM Tris, 100 mM sodium chloride buffers（pH7.5）As A phases, 20 mM Tris, 500 mM sodium chloride
Buffer solution（pH7.5）As B phases, linear gradient elution, gradient slope 0- are carried out>100%B, elution volume are 75 ml, are treated out
The UV260 at mouth end starts to collect when being more than UV280, and UV260 stops collecting when being less than UV280；
5) chromatographic column is regenerated with 2 M sodium chloride solutions.
Step E：It is concentrated by ultrafiltration
The collection virus liquid of gained in step D is subjected to centrifugal concentrating with 100 kD ultra-filtration centrifuge tube, 4000 rpm, is concentrated into
0.5 ml, sample is then subjected to SDS-PAGE detections（Fig. 4）；As a result show, gland relevant viral vector purifying side of the invention
Method has on the clearance of foreign protein and had great advantage, can by the electrophoresis purity of the gland relevant viral vector prepared by the present invention
Up to more than 95%.
The preferable case study on implementation of the present invention is the foregoing is only, is not intended to limit the invention, it is impossible to think to be embodied
These explanations are confined to, for any modification, replacement or improvement for being made within the principle of the present invention etc., should be included in this
In the protection domain of invention.
Claims (16)
1. a kind of extensive process for obtaining high-purity gland relevant viral vector, it is characterised in that comprise the following steps：
Step A：The cells and supernatant of gland relevant viral vector is collected, removes cell fragment, the sample after clarification is used to chromatograph
Purifying；
Step B：Gland relevant viral vector is captured by cation seperation column and preliminary purification；
Step C：Moderate purifying is carried out by attractive gel Filter column；
Step D：Carried out with anion-exchange column consummate；
Step E：The gland relevant viral vector liquid of collection is added into ultrafiltration system to be concentrated.
2. the method for claim 1, it is characterised in that the cells and supernatant in the step A includes micro-carriers cell culture
Supernatant, adhere-wall culture supernatant suspension cell culture supernatant.
3. the method for claim 1, it is characterised in that removed in the step A using centrifugation or 20-0.1 μm of in-depth filtration
Cell fragment, then filtered with 0.22-0.45 μm of filter.
4. the method for claim 1, it is characterised in that in the step B, the aglucon of cation-exchange chromatography medium is
Sulphopropyl (SP) strong cation exchange group group.
5. the method for claim 1, it is characterised in that in the step B, the level pad of cation-exchange chromatography is conductance
In the range of 5-10 mS/cm, pH 5-7.
6. the method for claim 1, it is characterised in that in the step B, the elution buffer of cation-exchange chromatography is conductance
In the range of 5-40 mS/cm, pH 5-9.
7. the method for claim 1, it is characterised in that in the step C, chromatography media has volume exclusion and adsorption dual work(
The multi-modal dielectric of energy.
8. the method for claim 1, it is characterised in that in the step C, the volume exclusion size of chromatography media in 50 kDa extremely
Between 700 kD.
9. the method for claim 1, it is characterised in that in the step C, level pad is conductance in 5-40 mS/cm, pH
In the range of 5-9.
10. the method for claim 1, it is characterised in that in the step D, the aglucon of chromatography media is Quaternary
Ammonium (Q) strong anion cation exchange groups.
11. the method for claim 1, it is characterised in that in the step D, the level pad of anion-exchange chromatography is conductance
In the range of 5-10 mS/cm, pH 7-9.
12. the method for claim 1, it is characterised in that in the step D, the elution buffer of anion-exchange chromatography is conductance
In the range of 10-100 mS/cm, pH 7-9.
13. the method for claim 1, it is characterised in that in the step E, the exclusion size of milipore filter is in 30 kDa to 300 kD
Between.
14. the method for claim 1, it is characterised in that in the step E, ultrafiltration pattern includes centrifugal concentrating and tangentially flowed through
Filter.
15. the method for claim 1, it is characterised in that in the step E, the form of milipore filter include hollow fiber ultrafiltration membrane and
Flat plate ultrafiltration membrane.
16. any one of preceding claims method, it is used to purify 2 type gland relevant viral vectors.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111304176A (en) * | 2020-03-07 | 2020-06-19 | 北京工业大学 | Adenovirus vector large-scale purification method based on Q-6XL and 4FF |
| CN111876393A (en) * | 2020-06-30 | 2020-11-03 | 恒瑞源正（上海）生物科技有限公司 | Method for large-scale rapid production of high-purity high-activity lentiviral vector |
| CN112226418A (en) * | 2020-09-25 | 2021-01-15 | 阜外华中心血管病医院 | Recombinant adeno-associated virus purification method |
| JP2021518757A (en) * | 2018-03-28 | 2021-08-05 | セルラー・バイオメディシン・グループ・エイチケー・リミテッド | Methods for large-scale preparation of purified preparations of recombinant wrench-will vector in GMP grade |
| CN114317464A (en) * | 2021-12-27 | 2022-04-12 | 武汉汇研生物科技股份有限公司 | Separation and purification method of adeno-associated virus rAAV9 |
| US11845962B2 (en) | 2018-03-29 | 2023-12-19 | Shanghai Cellular Biopharmaceutical Group Ltd. | Method for large-scale production of lentivirus by using GMP-level serum-free suspension cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2021518757A (en) * | 2018-03-28 | 2021-08-05 | セルラー・バイオメディシン・グループ・エイチケー・リミテッド | Methods for large-scale preparation of purified preparations of recombinant wrench-will vector in GMP grade |
| US11845962B2 (en) | 2018-03-29 | 2023-12-19 | Shanghai Cellular Biopharmaceutical Group Ltd. | Method for large-scale production of lentivirus by using GMP-level serum-free suspension cells |
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| CN112226418A (en) * | 2020-09-25 | 2021-01-15 | 阜外华中心血管病医院 | Recombinant adeno-associated virus purification method |
| CN114317464A (en) * | 2021-12-27 | 2022-04-12 | 武汉汇研生物科技股份有限公司 | Separation and purification method of adeno-associated virus rAAV9 |
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