CN102186875A - Clarification process at higher cell density - Google Patents
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- CN102186875A CN102186875A CN2009801405074A CN200980140507A CN102186875A CN 102186875 A CN102186875 A CN 102186875A CN 2009801405074 A CN2009801405074 A CN 2009801405074A CN 200980140507 A CN200980140507 A CN 200980140507A CN 102186875 A CN102186875 A CN 102186875A
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Abstract
The present invention relates to a method for the clarification of a cell broth containing mammalian cells as well as secreted desired biological substances having an overall positive charge in the cell broth wherein the cells are present at a high cell density by contacting the cell broth with anion exchange material, allowing an adequate incubation time to allow formation of a cell pellet and a supernatant layer, and separating the resulting cell pellet from the supernatant layer. The present invention further relates to a method for the recovery of secreted desired biological substances from a cell broth containing mammalian cells at a high cell density producing the secreted desired biological substance having an overall positive charge in the cell broth by performing the steps above, followed by extracting secreted desired biological substances from the supernatant layer.
Description
The present invention relates to be used to clarify the method for cell culture fluid (broth) and reclaim the method that excretory is expected biological agents from cell culture fluid, described cell culture fluid contains the cell that produces described excretory expectation biological agents.
Fermentation method for producing such as the biological agents of medicine, particularly monoclonal antibody provides complicated cell culture fluid, should separate with purifying biological by many steps from this cell culture fluid and learn material.
The first step is that this step is called clarification from the liquid separation solid material of cell culture fluid such as cell and cell debris.In many cases, biological agents is present in the extracellular and therefore is present in the liquid of cell culture fluid.
Up to now the example of the defecation method of Shi Yonging comprise centrifugal, filter (as micro-filtration, depth type filtration and by absolute aperture membrane filtration) and expanded bed chromatography.Can use flocculation to strengthen any of these defecation method, particularly with the filtration combination.
Known flocculation agent for this purpose can be that simple ionogen is to synthetic polyelectrolyte (as deae dextran, the polymkeric substance based on acryl, polyvinylamine) or inorganic materials (as diatomite or perlite).The progress of this respect is to use chitosan in recent years.
The use shortcoming of many flocculation agents is: they may be in conjunction with the expectation biological agents of being paid close attention to, the expectation biological agents that their may inactivation be paid close attention to, flocculence takes a long time and/or flocculation agent may be difficult to or spend that higher just to be prepared into medical use desired high-quality.In addition, need remove throw out from final purifying molecule, this is to need expensive time-consuming assay determination to confirm the process of removing of this throw out usually.
Method of the present invention does not have any of these shortcoming.
And these existing methods confirm only to realize clarification under low cell density.In addition, these methods of great majority do not confirm to be successfully used to mammalian cell yet, particularly are not successfully used to produce the mammalian cell of excretory expectation biological agents.
An object of the present invention is to provide cost-effective method to be used to clarify the cell culture fluid from the bio-reactor results, this cell culture fluid contains mammalian cell and excretory expectation biological agents.
Another object of the present invention provides and is used to clarify the method that contains than the cell culture fluid of the mammalian cell of high-cell density.In specific embodiment, the present invention relates to be used to clarify the method for cell culture fluid, the excretory expectation biological agents that described cell culture fluid contains mammalian cell and substratum and have total positive charge in described cell culture fluid, the initial cell density of wherein said cell is at least 15 * 10
6Individual cell/ml, described method is carried out as follows:
Described cell culture fluid is contacted with the particulate anion-exchange material,
B. hatch the enough time so that form cell mass (pellet) and supernatant layer, and
C. separating obtained cell mass and described supernatant layer.
U.S. Patent application 2003/170810 has been described this quadrat method, wherein the clarification of granular cell lysate is carried out as follows: with lysate and anionite-exchange resin batch mixed, then by filtration, centrifugal or gravity separation method separatin non-soluble material (comprising resin and and any material of its bonded, cell and cell debris) and soluble material.Different with disclosing of this U.S. Patent application, according to the present invention, with not cleaved before anionite-exchange resin mixes, the expectation biological agents has under the condition of total positive charge and clarifies cell in extra high cell density and cell culture fluid at them.Astoundingly, clarification of the present invention can realize with the cell culture fluid of high-cell density.
In the present invention, " cell culture fluid " finger kind the cell culture of complete mammalian cell, it can also contain substratum and excretory biological agents as hereinafter definition.In the method for the invention, particularly when cell density is very high, also can be desirably the parent material of bio-reactor be diluted to preferred cell density.For purpose of the present invention, so the material of dilution still is encompassed in the scope of term cell culture fluid.
" excretory biological agents " mainly discharges (active or passive) by mammalian cell and goes into the biological agents in the substratum when this paper refers to that it produces.
" expectation " refers to that at this paper biological agents is wittingly by using mammalian cell to produce.
" total positive charge " of excretory expectation biological agents is under this paper refers to solvent condition in cell culture fluid, and the electrostatic contribution of positive charge and negatively charged ions group causes clean positive charge on the biological agents.The total charge of biological agents is based on the pK of acid and alkaline residue
aAnd the pH of solution, be the pH of cell culture fluid in this case.For the biological agents that has clean positive charge in cell culture fluid, the pI of this material (wherein net charge is 0 pH) must be higher than the pH of cell culture fluid.
" substratum " at born of the same parents' external environment of this paper phalangeal cell, nutrition and other compositions that it contains the sustenticular cell growth and produces, but also can contain waste product or host cell proteins (HCP) or from the material of cracked cell.The composition of substratum can change during cell cultures, and can exhaust one or more original composition in the clarification stage.
" contact " refers in cell culture fluid to introduce anion-exchange material and makes cell settlement (for example under gravity or use centrifugal) at this paper.
" anion-exchange material " is a little less than this paper refers to particulate or the reinforcing yin essence ion-exchange chromatography media.Anion-exchange material comprises carrier usually, and it can be the mixture of organic materials or inorganic materials or organic and inorganic materials.Suitable organic materials is based on the medium and the methacrylic ester (metacrylate) of agarose.Suitable inorganic materials is silica, pottery and metal.The preferred size of particle is 15-150 μ m.Their preferred sizes are 15-70 μ m.Particulate density can be suitable for making cell from cell culture fluid sedimentation quickly, but density can not be too high, because too intensive according to observations particle does not cause sedimentation.
" enough incubation times " precipitation of inner cell between this paper refers at this moment causes distinct cell mass volume and supernatant layer.
" separation " as by decantation or extraction supernatant, perhaps for example extracts agglomerate by the relief outlet of bottom out from container in any method that this paper refers to remove from cell mass supernatant.
" supernatant layer " is the liquid that is positioned at the top owing to sedimentation.The supernatant layer can (and usually) still contain cell, and its cell density significantly is lower than initial cell density.
The initial cell density of the inventive method is at least 15 * 10
6Individual cell/ml, more preferably at least 80 * 10
6Individual cell/ml.As actual upper bound, can use up to 175 * 10
6Individual cell/ml, more preferably up to 130 * 10
6The cell density of individual cell/ml carries out method of the present invention.
Cell density can use cell counter such as Vi-CELL
TM(use trypanblue exclusion method) measured, but can use other appropriate method, comprises cytometry, packed cellvolume determines or Coulter counter (using electrical sensing zone method (Electrical Sensing Zone Method)).
If initial cell density is higher than 130 * 10
6Individual cell/ml, then diluting cells nutrient solution at first preferably.In practice, preferably at first dilute initial cell density and be higher than 100 * 10
6The cell culture fluid of individual cell/ml.Dilution can preferably proceed to cell density and be no more than 80 * 10
6Individual cell/ml.Thereby cell culture fluid can not cause the mammalian cell cracking with the solution dilution of not obvious change cellular environment, promptly uses the isotonic solution such as PBS to dilute.
According to another embodiment, the present invention relates to reclaim the method for excretory expectation biological agents from cell culture fluid, described cell culture fluid contains the cell that is created in the excretory expectation biological agents that has total positive charge in the described cell culture fluid, cell in the wherein said cell culture fluid is a mammalian cell, and initial cell density is at least 15 * 10
6Individual cell/ml, described method is carried out as follows:
Described cell culture fluid is contacted with the particulate anion-exchange material,
B. hatch the enough time so that form cell mass and supernatant layer,
C. separating obtained cell mass and described supernatant layer, and
D. extract described excretory expectation biological agents from described supernatant layer.
" recovery " refers to obtain to be substantially free of the expectation product of by product and refuse at this paper.
The invention still further relates to the method that reclaims excretory expectation biological agents from cell culture fluid, as indicated above, described cell culture fluid contains the cell that produces described excretory expectation biological agents, cell in the wherein said cell culture fluid is a mammalian cell, and initial cell density is at least 15 * 10
6Individual cell/ml, wherein further handle the gained cell mass as follows:
E. with gained cell mass resuspending,
F. hatch the enough time so that form cell mass and supernatant layer,
G. separating obtained cell mass and described supernatant layer, and
H. extract described excretory expectation biological agents from described supernatant layer.
In other method of the present invention, the step e to h of aforesaid method is repeated one or many.
In another preferred method of the present invention, thereby gained cell mass resuspending is not caused the mammalian cell cracking in the solution of not obvious change cellular environment, as resuspending in water-based (preferred wait ooze) salts solution, more preferably PBS.
Preferably, collect supernatant layer and extract excretory expectation biological agents from the blended supernatant.
The example of mammalian cell comprises CHO (Chinese hamster ovary) cell, hybridoma, BHK (young hamster kidney) cell, myeloma cell, the people's cell such as the HEK-293 cell, human lymphoblastoid, E1 immortalization HER cell, such as the mouse cell of NS0 cell.More preferably use E1 immortalization HER cell, most preferably the PER.C6 cell.
In preferred embodiments, the cell in the inventive method is an E1-immortalization HER cell, more preferably PER.C6 cell (see United States Patent (USP) 5,994,128, its content is incorporated this paper by reference into).
The PER.C6 cell is an exemplary cells, and it carries out preservation (see for example United States Patent (USP) 5,994,128, EP 0833934 B1, its content is incorporated this paper by reference into) with ECACC preserving number 96022940.
Being used for clarifying cell culture fluid can be by being suitable for realizing at least 15 * 10
6Any cell culture processes of the mammalian cell density of individual cell/ml obtains.Appropriate method in this respect is described in for example WO2005095578, WO2004099396 and WO2008006494.Its content is incorporated this paper by reference into.
The biological agents that can be produced by cell of the present invention (for example by expressing its (reorganization) gene of coding) is for example viral or (reorganization) albumen, particularly acceptor, enzyme, fusion rotein, such as from the proteic blood protein of coagulation cascade, such as the multifunctional protein of erythropoietin, for example be used for the virus or the bacterioprotein of vaccine; Immunoglobulin (Ig) such as IgG or IgM etc.Optimization protein, more preferably immunoglobulin (Ig) or its part are produced by cell.Preferably, the biological agents such as albumen or vaccine that is produced by cell can be as the activeconstituents in the pharmaceutical preparation.In the present invention, term " product " and " biological agents " are used interchangeably.
The appropriate method of extracting excretory expectation biological agents from the supernatant layer is for example to filter (as depth type filtration, micro-filtration, ultrafiltration, diafiltration), chromatography (as size exclusion chromatography, avidity chromatography, cation-exchange chromatography, hydrophobic interaction chromatography, fixing metal avidity chromatography), double water-phase extraction, precipitation or centrifugal.Advantageously, the expectation biological agents can be extracted very effectively by cation-exchange chromatography.In the expectation biological agents is under the situation of immunoglobulin (Ig), and avidity chromatography, particularly albumin A chromatography and cation-exchange chromatography are particularly suitable separation methods.
Description of drawings
Fig. 1: with the Si-PEI initial settlement and use the recovery of product after the PBS washing step subsequently.
(a) for various anion-exchange materials, as the supernatant cell density of the function of time.
(b) for various anion-exchange materials, as the supernatant volume of the function of time.
Embodiment
Symbol:
X
t=total cell density, individual cell/mL.
Si-PEI=Bakerbond Wide-Pore PEI (polymine) Prep LC Packing grafting silica beads (Prep LC Packing grafted silica bead) (JT Baker)
DEAE Hyper D=diethylamino ethyl grafting ceramic bead (diethylaminoethyl grafted ceramic bead) (Pall)
Streamline DEAE=diethylamino ethyl grafting sepharose 4B (diethylaminoethyl grafted agarose bead) (GE Healthcare)
PrA HPLC=analyzing proteins A high pressure liquid chromatography (HPLC) (Analytical Protein A High Pressure Liquid Chromatography)
The PBS=phosphate-buffered saline
According to the described method of WO2008006494, use the PER.C6 of various cell densities
Cell carries out and prepares following clarification and test.PER.C6
Cell produces antibody.The concentration of antibody is measured by PrA HPLC in the supernatant layer.When needs, promptly when cell density very high, when cell is obviously facilitated (contribute) working volume, then to the biomass corrected concentrations.
Embodiment 1: low cell density clarification
The Si-PEI adding of difference amount is contained in each bottle of 10ml cell culture.X
t=4.3 * 10
6Individual cell/ml.Made cell settlement 15 minutes.Only need the Si-PEI of 5% (vol) to come the cell of sedimentation 97%.The cell that adds the Si-PEI sedimentation 99% of 10% (vol).The product rate of recovery is 100%.
Add Si-PEI and obviously shorten the cell settlement required time.In addition, adding Si-PEI compares with contrast (not adding Si-PEI) and produces the more agglomerate of consolidation.
Embodiment 2: medium cell density clarification
The Si-PEI adding of difference amount is contained in each bottle of 5ml cell culture.X
t=63.5 * 10
6Individual cell/ml.Made cell settlement 30 minutes.The cell that adds the Si-PEI sedimentation 87% of 5% (vol).The cell that adds the Si-PEI sedimentation 89% of 10% (vol).The cell that adds the Si-PH sedimentation 85% of 20% (vol).In each case, the gained cell density is lower than 10 * 10
6Individual cell/ml, this is suitable for as depth type filtration.The product rate of recovery is 97%.
Add Si-PEI and obviously shorten the cell settlement required time.In addition, adding Si-PEI compares with contrast (not adding Si-PEI) and produces the more agglomerate of consolidation.
Embodiment 3: the high-cell density clarification
The Si-PEI of 10% (vol) is added in the 345ml cell culture fluid.X
t=123 * 10
6Individual cell/ml.Owing to high-cell density carries out sedimentation in 2 hours.After these two hours, the cell density of gained supernatant is 13.6 * 10
6Individual cell/ml.The agglomerate volume is 53% (not adding in the contrast of Si-PEI is 93%) of cumulative volume.Pour out supernatant, agglomerate is oozed the PBS washed twice with waiting, each washing back sedimentation 1 hour.Twice washing after product rate of recovery is 93%.Overall treatment time is 4 hours.After mixing supernatant, finally handling volume is 600ml, and cell density is 9.9 * 10
6Individual cell/ml.
Embodiment 4: increase by repeated washing and reclaim product
The Si-PEI of 10% (vol) is added in the 345ml cell culture fluid.X
t=78 * 10
6Or 120 * 10
6Individual cell/ml.Pour out about 200ml supernatant layer behind the initial settlement.This volume is replaced with equal-volume PBS, made cell settlement 60 minutes.Pour out about 200ml supernatant layer again, and replace sedimentation subsequently 60 minutes with equal-volume PBS.Pour out 200ml supernatant layer once more.
Product recovery by several washing steps is summarized in Fig. 1.The result shows that by only collecting product behind two washing steps from supernatant, the rate of recovery of product is significantly improved.
Embodiment 5: high-cell density and with the clarification of various anion-exchange materials
Anion-exchange material Si-PEI, DEAE Hyper D or the Streamline DEAE of 10% (vol) are added 20ml cell culture fluid sample, initial X
tBe 123 * 10
6Individual cell/ml.
Fig. 2 a illustrates for every kind of anion-exchange material, as the supernatant cell density of the function of time and the contrast that does not add anion-exchange material.
Fig. 2 b illustrates as function of time supernatant volume.Add anion-exchange material and cause the more cell mass of consolidation, be i.e. supernatant volume of Zeng Jiaing.
Embodiment 6: high cell density clarification
Use X
t=150 * 10
6The cutting of individual cell/ml is clarified.Under this cell density, observe considerably less sedimentation with undiluted cutting.With PBS cell culture is diluted to promote sedimentation at 1: 1.For this reason, it is 20ml that the 10ml substratum is diluted to final volume, and adds 10% (total vol) Si-PEI (2ml).After twice washing, the product rate of recovery is 92.6%.The concentration of HCP reduces by 28%.Overall treatment time is 4 hours.After mixing supernatant, finally handling volume is 48ml (increasing by 4.8 times), and cell density is 6.1 * 10
6Individual cell/ml.
Embodiment 7: the purifying of expectation biological agents
With initial cell density is 175 * 10
6The cell culture harvest thing of individual cell/mL is～75 * 10 with the PBS dilution
6Individual cell/mL (original volume is 1.7L).After the dilution, add Si-PEI chromatography media (cutting of the Si-PEI resin of 0.1L/L dilution) to cutting.Make cell settlement～60 minute.Pour out the supernatant that contains product, and with settled cell PBS washed twice.Initial supernatant is mixed with twice washing to increase the product rate of recovery (～95%).The mixture (pool) that merges contains and is lower than 5 * 10
6Individual cell/ml, and HCP content reduces by 59%.
The product that reclaims after the Si-PEI sedimentation is further purified by depth type filtration.Depth type filtration is made up of elementary filter (being generally 10 or 5 μ tm apertures) and secondary filter subsequently (being generally 3 or 1 μ m aperture), described elementary filter is used for further reducing cell lump (cell mass), and described secondary filter is removed littler particle and prepared clarifying cutting to carry out sterilising filtration by gradient 0.8/0.2 μ m filter usually.Depth type filtration row (train) can be Millipore Millistak+HC filters, and it contains medium, as D0HC (elementary) and X0HC (secondary) subsequently; Perhaps CUNO ZetaPlus filter, it contains for example 10M02 (elementary) and 60ZA05A (secondary) subsequently.Under any circumstance, (Supor Pall) further filters by 0.8/0.2 m filter with clarifying cutting.
In addition, observing 85% HCP by secondary filter during depth type filtration reduces.HCP can be owing to the charged character of these filters by the minimizing of secondary filter, existing report (Yigzaw Y in this former document, Piper R, Tran M, Shukla AA.2006. Exploitation of the Adsorptive Properties of Depth Filters for Host Cell Protein Removal during Mon
Clarifying material is further purified by the cation-exchange chromatography such as GigaCap S (Tosoh).Monoclonal antibody (product) is fixed on the resin, and capacity (capacity) is＞the 95g/L chromatography media.The condition that is used for fixing antibody is slightly acid (pH～5.3), and specific conductivity is～4.5mS/cm.In conjunction with after, antibody is washed with level pad, at last with containing the buffer step wash-out of 100mM sodium-chlor.Obtain HCP content by this step and further reduce (78%).
The antibody of wash-out can be by the single step purification that is combined into of chromatography and filtering technique, until the purity requirement that suits the requirements.
By the CEX step, total minimizing of host cell proteins is as shown in the table in the cell culture harvest thing.
| ?HCP(μg/mg?MAb) | The %HCP clearance rate | |
| The cell culture harvest thing | ?200 | ?0 |
| Behind the PEI cell settlement | ?81 | ?59 |
| After the depth type filtration | ?12 | ?85 |
| After CEX catches | ?2.8 | ?78 |
| Amount to | ?99 |
Claims (11)
1. be used to clarify the method for cell culture fluid, the excretory expectation biological agents that described cell culture fluid contains mammalian cell and have total positive charge in described cell culture fluid, the initial cell density of wherein said cell is at least 15 * 10
6Individual cell/ml, described method is carried out as follows:
Described cell culture fluid is contacted with the particulate anion-exchange material,
B. hatch the enough time so that form cell mass and supernatant layer, and
C. separating obtained cell mass and described supernatant layer.
2. reclaim the method for excretory expectation biological agents from cell culture fluid, described cell culture fluid contains the cell that is created in the excretory expectation biological agents that has total positive charge in the described cell culture fluid, cell in the wherein said cell culture fluid is a mammalian cell, and initial cell density is at least 15 * 10
6Individual cell/ml, described method is carried out as follows:
Described cell culture fluid is contacted with the particulate anion-exchange material,
B. hatch the enough time so that form cell mass and supernatant layer,
C. separating obtained cell mass and described supernatant layer, and
D. extract described excretory expectation biological agents from described supernatant layer.
3. the method for claim 2, wherein the gained cell mass is further handled as follows:
E. with gained cell mass resuspending,
F. hatch the enough time so that form cell mass and supernatant layer,
G. separate once more gained cell mass and described supernatant layer, and
H. extract described excretory expectation biological agents from described supernatant layer.
4. the method for claim 3, wherein repeating step e to h.
5. claim 3 or 4 method wherein merged each supernatant layer before extracting described expectation biological agents.
6. each method among the claim 2-5 wherein uses cation-exchange chromatography to extract described expectation biological agents.
7. claim 3 or 4 method, wherein with gained cell mass resuspending in aqueous saline solution.
8. the method for claim 6, wherein said aqueous saline solution is PBS.
9. each method among the claim 1-8, wherein said initial cell density is lower than 130 * 10
6Individual cell/ml.
10. each method among the claim 1-8, wherein said initial cell density is greater than 100 * 10
6Individual cell/ml, and wherein in step " a " before with described cell dilution for being no more than 80 * 10
6Individual cell/ml.
11. each method in the aforementioned claim, wherein said expectation biological agents are immunoglobulin (Ig) or its part.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US10632008P | 2008-10-17 | 2008-10-17 | |
| US61/106,320 | 2008-10-17 | ||
| EP08167506 | 2008-10-24 | ||
| EP08167506.8 | 2008-10-24 | ||
| PCT/EP2009/063565 WO2010043700A1 (en) | 2008-10-17 | 2009-10-16 | Clarification process at higher cell density |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102186875A true CN102186875A (en) | 2011-09-14 |
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ID=40367660
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009801405074A Pending CN102186875A (en) | 2008-10-17 | 2009-10-16 | Clarification process at higher cell density |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP2346891A1 (en) |
| JP (1) | JP2012505863A (en) |
| KR (1) | KR20110085980A (en) |
| CN (1) | CN102186875A (en) |
| AU (1) | AU2009305343A1 (en) |
| CA (1) | CA2739501A1 (en) |
| WO (1) | WO2010043700A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012030512A1 (en) | 2010-09-03 | 2012-03-08 | Percivia Llc. | Flow-through protein purification process |
| US10421986B2 (en) | 2013-09-30 | 2019-09-24 | Janssen Vaccines & Prevention B.V. | Method for the clarification of high-density crude cell culture harvest |
| US10695744B2 (en) | 2015-06-05 | 2020-06-30 | W. R. Grace & Co.-Conn. | Adsorbent biprocessing clarification agents and methods of making and using the same |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030170810A1 (en) * | 2001-09-18 | 2003-09-11 | Masoud Vedadi | Methods and apparatuses for purification |
| WO2007123961A2 (en) * | 2006-04-20 | 2007-11-01 | Wyeth | Purification processes for isolating purified vesicular stomatitis virus from cell culture |
-
2009
- 2009-10-16 JP JP2011531501A patent/JP2012505863A/en active Pending
- 2009-10-16 KR KR1020117008313A patent/KR20110085980A/en not_active Application Discontinuation
- 2009-10-16 CN CN2009801405074A patent/CN102186875A/en active Pending
- 2009-10-16 AU AU2009305343A patent/AU2009305343A1/en not_active Abandoned
- 2009-10-16 CA CA2739501A patent/CA2739501A1/en not_active Abandoned
- 2009-10-16 EP EP09820285A patent/EP2346891A1/en not_active Withdrawn
- 2009-10-16 WO PCT/EP2009/063565 patent/WO2010043700A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030170810A1 (en) * | 2001-09-18 | 2003-09-11 | Masoud Vedadi | Methods and apparatuses for purification |
| WO2007123961A2 (en) * | 2006-04-20 | 2007-11-01 | Wyeth | Purification processes for isolating purified vesicular stomatitis virus from cell culture |
Non-Patent Citations (1)
| Title |
|---|
| FRANK RISKE ET AL.,: ""The use of chitosan as a flocculant in mammalian cell culture dramatically improves clarification throughput without adversely impacting monoclonal antibody recovery"", 《JOURNAL OF BIOTECHNOLOGY》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2012505863A (en) | 2012-03-08 |
| AU2009305343A1 (en) | 2010-04-22 |
| WO2010043700A1 (en) | 2010-04-22 |
| EP2346891A1 (en) | 2011-07-27 |
| CA2739501A1 (en) | 2010-04-22 |
| KR20110085980A (en) | 2011-07-27 |
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