CN105316296A

CN105316296A - Method of purifying adenovirus granules

Info

Publication number: CN105316296A
Application number: CN201410490317.8A
Authority: CN
Inventors: 戴东升; 杨仁佳; 周鹏; 焦进安; 王星星; 陈婧; 王斐
Original assignee: YABAO GROUP TAIYUAN PHARMACEUTICAL CO Ltd
Current assignee: YABAO GROUP TAIYUAN PHARMACEUTICAL CO Ltd
Priority date: 2014-06-13
Filing date: 2014-09-24
Publication date: 2016-02-10

Abstract

The invention aims to provide a method of removing host DNA from a high-concentration cell suspension, and a suitable method of purifying adenovirus. One simple, feasible method of removing host DNA from the high-concentration cell suspension is obtained by screening, the aggregation and precipitation problem of adenovirus granules caused by the addition of a cationic detergent is avoided, and the impact of the cationic detergent upon following purification is also avoided.

Description

A kind of method of adenovirus particles

Technical field

The present invention relates to the method for a kind of adenovirus particles of viral preparation field.

Background technology

Vaccine preparation field progress in recent years creates the demand to scale operation, and the method being badly in need of stability and high efficiency provides enough vaccines.Vaccine for some diseases can based on recombinant adenovirus particle.To carry out a large amount of effort at present to optimize the adenovirus preparation method based on cell.Cell is carried out high-density culture, with postoperative infection to obtain higher total virus yield.This common virion of method (VP) yield obtaining the results viral solution of high virus concentration in single bio-reactor is about 1.5*1012VP/ml.

Accumulation cell debris and host DNA is easy to the method for high-density culturing cell.Must be removed these further in purge process.US7326555 discloses a kind of method removing host cell DNA from harvested cell culture.This method can optionally precipitate host cell DNA and do not precipitate adenovirus particles from cell culture.But this method is only applicable to the Adenovirus Purification in low cell density nutrient solution, has no the application of precipitate virus particle in high-cell-density cultivation liquid.Because virion number is higher, the method uses cationic detergent can make a large amount of aggregate and precipitate of adenovirus in a large number, affects yield.The main reason of nowadays influence Adenovirus Purification is that complex steps, yield is low.Therefore the method filtering out purification of adenoviral in a kind of easy, quick, efficient high-density cells suspension is particularly important.

Summary of the invention

The object of the present invention is to provide a kind of method removing host DNA in high-density cells suspension, and the method for suitable purification of adenoviral.The present invention filters out the method removing host DNA in the high-density cells suspension of a set of simple possible, avoid add cationic detergent and the aggregate and precipitate problem of the adenovirus particles caused and cationic detergent on the impact of subsequent purification.

The invention provides a kind of from comprising 5*10 ⁶-150*1 ⁰the method of adenovirus particles in the cell suspension of 6 cells/ml, described method comprises: a) by the lysis in described cell suspension and by centrifugal segregation host cell debris; B) cross-flow ultrafiltration system concentrating virus suspension, and remove related substances in cell culture medium, replacing system is the suitableeest work system of nuclease; C) wide spectrum nuclease BENZONASE is added ^tMdigestion host DNA; D) SepharoseG25 desalting column removes nuclease non-digestion fragment host DNA, the host DNA in high-density cells suspension can be removed and reach more than 80% in b), c) and d) 3 steps.

The present invention relates to from the method for high-cell density suspension purifying from the adenovirus particles of cell lysate.Cell cultures to the method for high-cell density by cell cultures being obtained to high-cell density, is that those skilled in the art are known by described high-cell density suspension.The concrete grammar obtaining high-cell-density cultivation thing discloses and WO2004/099396 and WO2005/095578.Infect high-cell-density cultivation thing with adenovirus particles to breed at cell suspension to allow described adenovirus.The method infecting high-cell-density cultivation thing is also well known by persons skilled in the art.The concrete grammar obtaining the high-cell-density cultivation thing of high virus concentration is disclosed in EP08168181.9.According to the present invention, once adenovirus is bred and killed most cells in cell culture, then from high-cell density suspension adenovirus particles.

Cracking and clarification

The described method the first step comprises lysis in cell suspension.Wherein the step of lysing cell film allows from the high-cell density suspension harvested cell infected relevant (in born of the same parents) relevant with acellular (born of the same parents) adenovirus.In the present invention, cleavage method is mainly physical method, and this is ordinary method.Cell suspension is carried out mechanical shearing to discharge a large amount of adenovirus by carrying out Hollow Fiber Ultrafiltration after multigelation 3 times.

A large amount of host cell debris and host protein precipitation is had after cracking.Owing to not adding the step of cationic detergent precipitation host DNA in step of the present invention, therefore cell pyrolysis liquid clarification steps is carried out after cracking.Clarification steps can use dead-end filtration, and tangential flow filtration, micro-filtration and centrifugal etc. carries out.The present invention considers simplicity and economy problems, and the cross-flow ultrafiltration used in subsequent step, therefore selects centrifugal method.Cell pyrolysis liquid is carried out centrifugal, can be good at place to go cell debris and albumen precipitation.

Concentrated

In the present invention, the adenovirus particles suspension of clarification can pass through uf processing.Ultrafiltration is used for concentrating virus suspension, removes the related substances in cell culture medium, removes small portion host DNA, and replaceable damping fluid is lower step ferment treatment prepares.Those skilled in the art understand buffer-exchanged occur condition and which kind of damping fluid be applicable to this step.Adenovirus particles suspension can concentrate 5-20 doubly.

Selected ultra-filtration membrane pore size should be enough little of to retain adenovirus particles, and enough large with effective removal of contamination on this basis.This depends on manufacturer and film type.The ultrafiltration of tangential flow pattern is first-selected.The present invention adopts QuixStand tangential flow filtration system (GE), and ultrafiltration and concentration and the exchange buffering liquid of adenovirus particles suspension are carried out in 500KD molecular weight cut-off ultrafiltration post (GE).The damping fluid changed is 20mMTris-HClpH7.6,10mMMgCl ₂, 1mMDTT, and the existence strictly forbidding stain remover and EDTA in damping fluid.

Ferment treatment

The present invention need add nuclease in the adenovirus particles suspension after uf processing.The present invention is adding wide spectrum nuclease BENZONASE ^tMbefore also to add Mixed Circumscription restriction endonuclease (EcoRI+BamHI+EcoRII+Sau3AI).Due to the different recognition site of Mixed Circumscription restriction endonuclease and the recognition sequence of the shortest 4 bases, ensure that host DNA can by fragmentation, the digestion of nuclease after being convenient to, even if wide spectrum nuclease decomposes not exclusively also be convenient to follow-up SepharoseG25 removal.The present invention uses wide spectrum nuclease BENZONASE ^tM(Merck), this enzyme can dissociate DNA and RNA (strand, double-strand, linear and ring-type) efficiently, and product is 5 '-monophosphic acid nucleotide of oligonucleotide.

SepharoseG25 removes residual DNA fragment

The present invention introduces desalting column first and removes DNA not digested in ferment treatment step before adenovirus particles purifying.Due to the existence of restriction enzyme, host DNA is by fragmentation, and molecular weight mostly is 200-500bp, and available SepharoseG25 removes.PS-80 should added before SepharoseG25.It is highly preferred for there is 0.1%PS-80 in damping fluid for the low residue DNA level realized in product, combines and adenovirus aggregation because this can weaken adenovirus/DNA.After this step, host DNA residual quantity is less than 1%.

Following purification steps

According to the present invention, later step can be anion-exchange chromatography step.In described step, adenovirus particles is bonded on positively charged column material.The present invention's anionic exchange medium used is Source30Q (GE), and it has carrying capacity large, easily the advantage such as process.Wash-out subsequently allows from impurity and remaining host cell DNA separating viral particles.Use anion-exchange chromatography to carry out Adenovirus Purification extensively to be described, therefore this is on the one hand also within the limit of power of those skilled in the art.The present invention in the laggard stepping row space exclusion chromatography step of anion-exchange chromatography with the robustness of increase method.Many different chromatography matrix may be used for Adenovirus Purification, and are suitable, and those skilled in the art can be easy to the optimum anion-exchange material finding purification of adenoviral.Optional down stream processing steps can be added in described method.These steps can comprise, such as steric exclusion chromatography step, reverse phase absorption step, hydrophobic chromatography step, hydroxylapatite chromatography step.More details visible WO03/097797, WO02/44348 of these steps.

The technique of the Mixed Circumscription restriction endonuclease that the technical program is used and the coupling of wide spectrum nuclease is convenient and swift, avoid add cationic detergent cause subsequent purification difficulty and cause adenovirus particles to precipitate.Carry out SepharoseG25 removal DNA fragmentation after enzymolysis and host DNA clearance can be brought up to more than 99%.Greatly facilitate subsequent purification.Through test of many times, the technical program has simple to operate, convenient and swift, removes DNA thorough, can meet the requirement that industrialization is produced.

Embodiment

Embodiment 1: lysis and clarification in cell suspension

1) collect 1L high-density 293 cell suspension, in-30 DEG C of refrigerators, place 2h, after it freezes, room temperature is placed and is made it melt, 3 times repeatedly.The cell suspension crossed by multigelation, through 500KD ultrafiltration post ultrafiltration 20min, makes cytoclasis more complete, for convenience of operation, pours ultrafiltration peritoneal effluent into former container.

2) cell pyrolysis liquid is put 8000rpm in whizzer, 4 DEG C of centrifugal 1h.Collecting centrifugal supernatant is that adenovirus particles suspension is about 1L.Detect virion number and host DNA content.

Embodiment 2: the replacing of the concentrated and enzyme cutting buffering liquid of adenovirus particles suspension

1) assemble QuixStand tangential flow filtration system, change 500KD molecular weight cut-off ultrafiltration post (dividing use with the ultrafiltration post in cell destruction step).

2) put in tangential flow filtration system by 1L adenovirus particles suspension, ultrafiltration and concentration is to 100ml.

3) add 100ml ultrapure water at every turn, again add 100ml ultrapure water when being concentrated into 100ml, 6 times so repeatedly, now adenovirus particles suspension should be colourless.

4) enzyme cutting buffering liquid 1L is prepared, containing 20mMTris-HClpH7.6,10mMMgCl in damping fluid ₂, 1mMDTT.Add 100ml enzyme cutting buffering liquid at every turn, when being concentrated into 100ml, again add 100ml enzyme cutting buffering liquid, 6 times so repeatedly, collect adenovirus particles suspension 50ml.Measure ultrafiltration exchange buffering liquid and terminate rear virion number and host DNA content.

Embodiment 3: the enzymolysis of adenovirus particles

1) Mixed Circumscription restriction endonuclease (EcoRI+BamHI+EcoRII+Sau3AI) 500ul (4U/ul) is added, 37 DEG C of water-bath 4h.

2) wide spectrum nuclease BENZONASE is added ^tM, the enzyme that spends the night is cut.Measure virion number and host DNA content.

Embodiment 4:SepharoseG25 removes residual DNA fragment

PS-800.05ml is added in 50ml ferment treatment adenovirus particles suspension.It is highly preferred for there is 0.1%PS-80 in damping fluid for the low residue DNA level realized in product, combines and adenovirus aggregation because this can weaken adenovirus/DNA.Through SepharoseG25, collecting peak 1 is adenovirus particles peak, and peak 2 is DNA fragmentation peak.After this step, host DNA residual quantity is less than 1%.Collect adenovirus particles peak 90ml.

Embodiment 5: adenovirus particles following purification steps

Ion-exchange purification:

Level pad A:Tris-HCl (0.05M) pH8.0

Elution buffer B (1MNaCl) pH7.5

1.XK26 post, gets 100mLSOUCE30Q filler dress post, balance, loading, elution flow rate 10ml/min.

2. balance: balance pillar with buffer A Tris-HClpH8.0 and be about 500ml.

3. loading: the direct loading 90ml of viral concentration liquid.

4. wash baseline: buffer A is washed baseline and is about 200ml.

5. wash: 30%B wash-out foreign protein.

6. wash-out: 30%-60%B linear elution object virus.

7.100%B process pillar, purified water is cleaned.

A liquid: 0.05MTris-Hcl, pH8.0

B liquid: 1MNaCl.

Claims

1. a method for adenovirus particles, is characterized in that described method comprises:

A) by the lysis in described cell suspension and by centrifugal segregation host cell debris;

B) cross-flow ultrafiltration system concentrating virus suspension, and remove related substances in cell culture medium, replacing system is the suitableeest work system of nuclease;

C) wide spectrum nuclease BENZONASE is added ^tMdigestion host DNA;

D) SepharoseG25 desalting column removes nuclease non-digestion fragment host DNA, the host DNA in high-density cells suspension can be removed and reach more than 80% in b), c) and d) 3 steps.