CN101490239A - Improved process for the culturing of cells - Google Patents
Improved process for the culturing of cells Download PDFInfo
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- CN101490239A CN101490239A CNA2007800268102A CN200780026810A CN101490239A CN 101490239 A CN101490239 A CN 101490239A CN A2007800268102 A CNA2007800268102 A CN A2007800268102A CN 200780026810 A CN200780026810 A CN 200780026810A CN 101490239 A CN101490239 A CN 101490239A
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Abstract
The invention relates to a process for the culturing of cells, preferably E1-immortalized HER cells, more preferably PER.C6 cells in a reactor in suspension in a cell culture medium, wherein the cells produce a biological substance, preferably an antibody, wherein at least one cell culture medium component is fed to the cell culture and wherein the cell culture comprising the cells, the biological substance and cell culture medium is circulated over a separation system and wherein the separation system separates the biological substance from substances having a lower molecular weight than the biological substance and wherein the biological substance is retained in or fed back into the reactor. Preferably part of the substances of lower molecular weight is continuously removed from the cell culture.
Description
The present invention relates in reactor in cell culture medium the technology of culturing cell in suspension.
This type of technology is for example known from WO04/099396.Described in this article and how to have improved the cell density of cell culture and the productive rate of the biomaterial of wanting by the growth conditions of optimizing in the fed-batch technology.
In addition, WO05/095578 discloses a kind of technology of culturing cell, it is cultivated and realizes by the cell culture that comprises cell culture medium and cell being carried out continous pouring, wherein, in cell culture, add cell culture medium, cell culture is comprising the filter module cocycle of tubular fibre, cause producing the liquid efflunent lower than cell culture cell density, and the stream of filter module inside is mutual tangential flow (alternating tangential flow), wherein, described cell produces biological substance.Show in the embodiment of WO05/095578, produced 0.9g/L/ days product, this is corresponding to the production concentration of about 0.3g/L in the effluent.
The liquid volume that contains biological substance is big more, and the purifying of biological substance is just required great effort more.In the disclosed technology of WO04/099396 and WO05/095578, the concentration of the biological substance of acquisition is not high.Therefore, be trouble to the processing of the downstream of this biological substance, because need before using the subsequent purification step, be concentrated, perhaps need the biological substance of large volume, lower concentration purifying in addition to biological substance.Therefore, cause volume production power (productivity) lower, therefore given production level is needed bigger and/or more culture vessel and need drop into more thus on equipment with lower cell density culturing cell.
Therefore, an object of the present invention is to provide a kind of technology, wherein, obtain product from cell culture with higher concentration.
Another object of the present invention is to make it possible to culturing cell and production biomaterial in the time period that prolongs.
These purposes by in reactor in cell culture medium in suspension the following technology of culturing cell realize, wherein, described cell produces biological substance, wherein, to at least a cell culture medium component of cell culture feed supplement, and, wherein, the cell culture that comprises cell, biological substance and cell culture medium is in the separation system cocycle, and, wherein, described separation system is separated biological substance and the material lower than described biological substance molecular weight, and wherein, described biological substance is retained in the reactor or by loopback and advances reactor.
For example, the present invention relates in reactor the technology of culturing cell in cell culture medium, wherein, described cell produces biological substance, wherein, to described reactor feed supplement nutrition and/or cell culture medium, and, wherein, the cell culture that comprises cell and cell culture medium is in the aperture or the strainer cocycle of molecular weight separation point between 5 to 500kD.
Have been found that separation system, can in cell culture, accumulate biological substance with greater concn by using the material that biological substance and molecular weight ratio biological substance is low to be separated.
Therefore, the present invention is different from the cell cultures of description of the Prior Art, and its biomaterial that allows to want accumulates with cellular material.
Of the present invention a kind of preferred embodiment in, the part of more low-molecular-weight material continues to shift out from cell culture.
Another advantage of technology of the present invention is, than for example in batches or fed-batch technology, can reach higher viable cell concentration.In addition, than for example in batches or fed-batch technology, the production time (being the time period that cell produces biological substance) can be extended.In addition, than in batches or fed-batch technology, might use littler reactor.It is useful using littler reactor, because this has reduced the equipment input relevant with facility.
In addition, can in the shorter time, obtain the biological substance of greater concn.
We find, can obtain the biological substance of high density in reactor, and can significantly not reduce cell survival and therefore not need the limit production time.Those skilled in the art can expect, and product may take place to be suppressed, that is, and the inhibition that biological substance self is produced biological substance, the perhaps inhibition that causes of other macromole (for example, host cell proteins, enzyme or cell debris) of producing of cell.In addition, we find that the accumulation of the biomaterial of wanting can not damage the function of separation system.
The aspects such as incubation time of technology of the present invention production concentration and prolongation in cell density, cell culture provide sizable benefit than the technology according to WO05/095578 and WO04/099396.Thus, technology of the present invention has caused the improved production to the biomaterial of wanting.
The cell that can be used for producing biological substance is all cells that can produce biologic well known by persons skilled in the art in principle.Described cell can be an eucaryon, for example, a fungi is arranged, for example, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Penicilliumchrysogenum, yeast, Saccharomyces cerevisiae for example, Kluyveromyceslactis, Phaffia rhodozyma, yeast from the Pichia genus, for example, Pichia pastoris, or protokaryon, for example, Escherichia coli, Bacillus sp, for example, B.licheniformis, B.subtilis, B.amyloliquefaciens, B.alkalophilus, Streptomyces sp., Corynebacterium glutamicum, Pseudomonas sp.Eukaryotic example also for example is described in Chu, L., Robinson, D.K., (2001) Curr.Opinion Biotechn., vol.12, p.180-187 in.Preferably, the cell that is used for technology of the present invention is a zooblast, particularly mammalian cell.The example of mammalian cell comprises CHO (Chinese hamster ovary) cell, hybridoma, BHK (young hamster kidney) cell, myeloma cell, people's cell (for example HEK-293 cell, people's lymphoblast), the HER cell of E1 immortalization, mouse cell (for example NSO cell).More preferably, use the HER cell of E1 immortalization, most preferably use the PER.C6 cell.
Can separate primary human embryo retina (HER) cell (Byrd P from fetus, Brown KW, Gallimore PH.1982.Malignant transformation of human embryo retinoblasts bycloned adenovirus 12 DNA.Nature 298:69-71, Byrd PJ, Grand RJA, GallimorePH.1988.Differential transformation of primary human embryo retinal cells byadenovirus E1 regions and combinations of E1A+ras.Oncogene 2:477-484).After cultivating some going down to posterity, primary cell is with death.With regard to purpose of the present invention, the HER cell of E1 immortalization derives from elementary HER cell, and this is by expressing the proteic DNA of encoding adenovirus E 1 A and E1B, being immortalized cell therein.This class can be cultivated above 100 times through the cell of immortalization and be gone down to posterity.The method that is used to obtain E1 immortalization HER cell for example has been described in United States Patent (USP) 5,994,128, be described in Byrd P, Brown KW, Gallimore PH.1982Malignant transformation of human embryo retinoblasts by cloned adenovirus12DNA.Nature 298:69-71, be described in Byrd PJ, Grand RJA, Gallimore PH.1988.Differential transformation of primary human embryo retinal cells by adenovirusE1 regions and combinations of E1A+ras.Oncogene 2:477-484, and be described in Gallimore, P.H., Grand, R.J.A.and Byrd, P.J. (1986) .Transformation of humanembryo retinoblasts with simian virus 40, and adenovirus and ras oncogenes.AntiCancer Res.6 is among the p499-508.For example, by plasmid (being in the control of people's phosphoglyceric kinase (" PGK ") promotor down) the elementary HER cell of transfection with gland-containing virus serotype 5 (Ad5) E1A-and E1B-encoding sequence (Ad5 Nucleotide 459-3510), produce the HER cell of immortalization, comprise that PER.C1, PER.C3, PER.C4, PER.C5, PER.C6, PER.C8 and PER.C9 cell (see, United States Patent (USP) 5,994,128).
A kind of preferred embodiment in, the cell in the technology of the present invention is the HER cell of E1-immortalization, more preferably, is PER.C6 cell (seeing United States Patent (USP) 5,994,128).The example of PER.C6 cell be with the cell of ECACC No.96022940 preservation (see, for example, United States Patent (USP) 5,994,128, EP 0833934 B1).
In technology of the present invention, can in suspension, cultivate any type of cell, for example, as the cell that is fixed, individual cells or in cell cluster or as its combination.Preferably, cell is cultivated as individual cells and/or as a minicell bunch quilt that is no more than 100 cells (more preferably no more than 20 cells).Cell for example can be fixed on the microcarrier, for example, and can be from the commercial microcarrier (Cytodex) that obtains of for example GE Healthcare.
Reactor is defined as comprising the system of cell culture in this article, described cell culture and then comprise cell and cell culture medium.It preferably provides sterile barrier, air filter for example, and with the cell that prevents that other cell contamination from wanting, and it keeps the pair cell advantageous environment by correct culture condition (for example, mixing, temperature, pH, oxygen concn etc.) is provided.
Reactor for example can be more permanent character, and for example, reactor can be stainless steel or glass, perhaps for example can be disposable, and for example, reactor can be Plastic Bottle or plastics bag.The example that is suitable for reactor of the present invention includes but not limited to: steel basin container, air transport be with (airlift) container and disposable container, they can by shake, vibratory movement or stir and mix.Preferred disposable (biology) reactor that uses because they only need low relatively cost of investment, has the turnover time of powerful flexibility of operation, weak point, and is easy at technology it be disposed.Disposable (biology) reactor can commercially obtain, and for example obtains from Hyclone, Sartorius, Applikon or Wave.
Term " separation system " is defined as in the context of the invention and can carries out isolating system based on molecular weight.The separation system that is used for technology of the present invention can be separated the low material of the described biological substance of biological substance and molecular weight ratio.In other words, the molecular weight separation point is selected as: make described molecular weight separation point (MWCO) less than the molecular weight of described biological substance, more preferably with at least 2 the factor, most preferably with at least 3 the factor molecular weight less than described biological substance.Typically, the MWCO of separation system is preferably 5kDa at least, more preferably 10kDa at least, 30kDa at least most preferably, preferred 500kDa at the most, more preferably 300kDa at the most, 100kDa at the most most preferably, but this will depend on the molecular weight of the biological substance that technology of the present invention is produced certainly.For example, be the IgG of 150kDa for molecular weight, having at the most, the separation system of the MWCO of 50kDa is most preferred.
The example of separation system includes but not limited to: strainer, whizzer and water-based two-phase extraction system.
Term " strainer " is represented when using in this article to comprise to have based on molecular weight or size and is come all devices of the ability of separating particles.In principle, in technology of the present invention, any strainer all can use, and low material separates as long as aperture of selecting for use or MWCO can make biological substance and molecular weight ratio biological substance, and typically, this will be MWCO or aperture between 5 to 500kDa.The example that is suitable for strainer of the present invention comprises film filter, porcelain filter and metallic filter.Strainer can use in any form, and strainer can for example be screw winding shape (spiralwound) or piped, perhaps can use with sheet form.Preferably, in technology of the present invention, used strainer is a film filter, preferably, and hollow fiber filter.Term " tubular fibre " expression piped film.The internal diameter of described pipe is 0.1mm at least, and more preferably 0.5mm at least most preferably is 0.75mm at least, and preferably, the internal diameter of pipe is 10mm at the most, more preferably 6mm at the most, most preferably 1mm at the most.The filter module that comprises tubular fibre can obtain from for example General Electric (GE, former Amersham) is commercial.
Cell culture by will comprising biological substance, cell and cell culture medium is in the separation system cocycle, therefore biological substance and cell are stayed in the reactor, and liquid efflunent is than having than lower biological substance content of cell culture and lower cell density.Usually, in technology of the present invention, liquid efflunent does not contain or seldom contains any biological substance and cell.Usually, liquid efflunent only contains the low component of the described biological substance of molecular weight ratio basically.Therefore, basically all cells and basically all biological substances all stay in the reactor usually.
Preferably, the aperture or the MWCO of strainer are selected as: make that the aperture of strainer or MWCO are littler than the diameter or the molecular weight of product, more preferably with at least 2 the factor, most preferably at least 3 the factor is little, to guarantee the high resident of product.Typically, the aperture of separation system or MWCO are preferably 5kDa at least, more preferably 10kDa at least, 30kDa at least most preferably, and/or the preferred 500kDa at the most of the aperture of described strainer/film or MWCO, more preferably 300kDa at the most, 100kDa at the most most preferably, but this will depend on the molecular weight of the biological substance that technology of the present invention is produced certainly.
Molecular weight separation point (MWCO) expression: molecular weight is greater than at least 90% all staying in the separation system in its particle.
Cell culture is represented in separation system (for example strainer) cocycle: cell culture is through separation system (for example strainer), produce liquid efflunent and wherein inclusion be maintained in the reactor or the stream of reactor is advanced in loopback.Wherein the circulation that inclusion is maintained in the reactor or reactor is advanced in loopback often only contains molecular weight basically and equals biological substance or the higher component of molecular weight at least, and therefore described stream will comprise more biological substance than liquid efflunent.
In principle, during technology of the present invention, when cell culture is in separation system cocycle and non-key.The circulation of cell culture can for example directly begin when technology begins, and perhaps just begins when the survivaling cell density of cell reaches certain level.
The circulation of cell culture on strainer can be vertical substantially stream the relative filter surfaces, this is also referred to as the end (dead-end) stream, or it can be the stream substantially parallel with filter surfaces, this is also referred to as tangential flow, for example unidirectional tangential flow (TFF) or cross-stream (cross-flow).A kind of preferred example of cross-stream is mutual tangential flow (ATF), because find when adopting ATF, can (rapidly) generation filter blocks, and even under very high cell density.Usually known, in depth type filtration, need protect last small-bore strainer to avoid blocking by progressive front filter.This practice is based on following common practise: have than aperture or less MWCO the easier obstruction of strainer, limited the production time thus.If use ATF, so with regard to unnecessary use front filter.
Can pass through the migratory cell culture,, perhaps move both, come directed flow perhaps by the moving filter device.For example can come the moving filter device by rotation (rotary filter) or vibration (vibrating type filter).Perhaps, if only come directed flow by the migratory cell culture, strainer is an immobilized so, for example can come the migratory cell culture by pump or pressure.
" mutual tangential flow " expression has the stream with the same direction of filter surfaces (that is, tangential with it), the described reciprocating cutter that flows to, and, also have one with the vertical substantially stream of described filter surfaces direction.Mutual tangential flow can obtain (for example, as US 6,544,424 is described) according to method known to those skilled in the art.
Between the incubation period of pair cell, at least a cell culture medium component, for example, one or more nutrition and/or cell culture medium can be given cell by feed supplement.In technology according to the present invention, part is replenished, perhaps preferably, at least a being good in the nutrition of whole supplement consumed, this is by realizing this nutrition or these nutrition feed supplement to reactors.For example, can be to the complete cell culture medium of reactor feed supplement, this is good, because just need not prepare independent feed supplement respectively more like this.Cell culture medium for example can also more spissated form feed supplement be given cell, and this is good, because smaller volume is easier to operation.In addition, can be to one or more nutrition of reactor feed supplement.For example, carbohydrate, for example glucose or fructose; Amino acid, for example glutamine; And/or peptide advantageously feed supplement to reactor.
Of the present invention a kind of preferred embodiment in, select such cell culture condition for use, make that the ratio productivity of cell growth rate and/or cell is unrestricted, more preferably, make that at least a concentration in the cell culture medium component keeps substantially constant.The example of restrictive cell culture condition is the formation of nutrient restriction and inhibition metabolite (for example ammonia, carbonic acid gas and lactic acid salt).For example, cell culture condition (for example, feed supplement) can be selected as: make cell growth rate unrestricted, this is for example by providing enough nutrition to consume with compensation and/or being avoided the generation of inhibition metabolite (for example as hydrochlorate or ammonia) to realize.For example, can select aeration condition for use, make that carbonic acid gas formation can the restrictive cell growth velocity.Viewpoint from Good Manufacturing Practice (GMP), culturing cell is highly beneficial under non-limiting condition, because, 1) this can provide the constant cell culture environment, constant, good product quality also can be provided under many circumstances, and 2) this can cause high cell survival rate, in some cases, causes cell survival rate to surpass 98%.High cell survival rate has reduced the release of cell contaminants associated (for example host cell proteins), and this helps the purifying to product.In addition, with unrestricted cell cultures speed and/or unrestrictedly come culturing cell to have commercial benefit than productivity, thereby be able in the further shorter time, produce more biological substance, because in described technology, can more early reach higher cell density.
" than the productivity " of cell is the amount of the given biological substance of each cell generation of per time unit, and it is represented as the pg. cell usually
-1My god
-1
The speed (rate of influx or irrigation rate) of adding at least a cell culture medium component (for example nutrition and/or cell culture medium) to cell culture influences the viability and the density of cell.In technology of the present invention, cell culture medium component (for example nutrition and/or cell culture medium) can for example be come feed supplement with successive stream, semi-continuous stream (for example Fen Bu stream) or (staggered) stream at interval.Preferably, cell culture medium component (for example nutrition and/or cell culture medium) is added with successive stream.
The cell culture medium component, for example fully cell culture medium and/or nutrition, in principle can be during described technology whenever feed supplement is to reactor.Preferably, feed supplement reaches to be low to moderate at substrates such as glutamine and glucose and causes starting before the dormant level of cell, or starts before inhibition metabolite (for example lactic acid salt or ammonia) reaches the high level that will stop to growth.During from this, the preferred so that speed feed supplement that reaches the substrate demand of cell culture medium component (for example nutrition and/or fully cell culture medium) is to reactor.
In one embodiment of the invention, cell culture medium adds with the feed rate according to formula (1):
Feed rate=SFR * (total cell culture volume) * (survivaling cell density) (1)
Wherein, feed rate shows with the numerical table that rises of every day, wherein, SFR compares feed rate, promptly, the cell culture medium feed supplement that the culture volume that adds with respect to each survivaling cell with per time unit is represented is to the speed of cell culture, and wherein, survivaling cell density is the quantity of every volume unit survivaling cell.The quantity of survivaling cell can be measured by those skilled in the art, for example, and by trypan blue (Trypan Blue) method for removing.Preferably be selected as between 0.01 to 0.3nL/ cell/sky than feed rate, more preferably between 0.01 to 0.2nL/ cell/sky.
Consider that other parameter may be good when regulating feed rate, for example, to the amount and/or the oxygen uptake rate of the glucose of culture feed supplement.For example, for PER.C6, cell culture medium and/or nutraceutical feed rate preferably are selected as: make glucose concn remain between 3 to 20mmol/L, more preferably between 5 to 15mmol/L.Preferably, glucose concn is 3mmol/L at least, more preferably 5mmol/L at least, preferred 20mmol/L at the most, more preferably 15mmol/L at the most.
In a kind of special embodiment of the present invention, at least once from reactor emigrated cells culture (comprising cell, biological substance and cell culture medium), and in reactor, add liquid (for example, cell culture medium or nutrition feed supplement), with shifting out of compensation cell culture.Shifting out of cell culture can cause more carrying out the longer treatment time under the high-cell density, and makes up high cell survival rate, and this has caused higher productivity.Cell culture can shift out continuously or step by step.
Of the present invention a kind of preferred embodiment in, as long as reach the cell density of wanting, for example, at least 10 * 10
6Individual survivaling cell/ml, preferably at least 20 * 10
6Individual survivaling cell/ml, more preferably at least 30 * 10
6Individual survivaling cell/ml, for example, at the most 200 * 10
6The cell density of individual survivaling cell/ml, emigrated cells culture (comprising cell, biological substance and cell culture medium) from reactor just, and in reactor, add liquid (for example cell culture medium or nutrition feed supplement), with shifting out of compensation cell culture.Preferably, so that the speed that cell density remains in the cell density scope of wanting is come the emigrated cells culture.Than traditional in batches or fed-batch technology, this embodiment of the present invention is highly favourable, because its advantage with technology of the present invention combines with the high viability that can be held the longer time, is achieved further higher cumulative volume productivity.The amount of the biological substance that " volume production power " expression time per unit per unit reactor volume produces, it is represented as g.L usually
-1. day
-1Than traditional instillation process, this embodiment of the present invention is highly beneficial, is grouped together because its advantage with technology of the present invention shifts out stream with the cell culture with high-concentration biological material.The biological substance that cell culture shifts out the stream middle and high concentration makes to have commercial value from wherein gathering in the crops biological substance.Therein in traditional instillation process of emigrated cells culture, cell culture shifts out the biological substance that stream contains and is not sufficient to make the described biological substance of results to have commercial value, so cell culture shifts out circulation and often is considered to waste material.Therefore, in this embodiment of the present invention, can gather in the crops all biological substances of generation with direct, economically feasible and simple mode in theory.
In a kind of particularly preferred embodiment of the present invention, select such cell culture condition for use, make the cell growth rate of cell and/or more unrestricted than productivity, more preferably, also make the concentration of at least a component (for example glucose or glutamine) of cell culture medium keep constant, and, as long as reach the cell density of wanting, at least carry out once operation from reactor emigrated cells culture, and, add liquid (for example cell culture medium) to reactor, with shifting out of compensation cell culture.
Preferably, the speed of effluent is selected as: make the interpolation speed of itself and at least a cell culture medium component (for example nutrition and/or cell culture medium) deduct optional cell culture medium and shift out speed and be equal to substantially.
The cell that produces biological substance for example is the cell that can express the gene of encoding human material.Can be for example by with the gene of the suitable selective marker of the gene that contains the encoding human material and coding () plasmid transfection cell for example, the gene (Neo marker gene) of coding neomycin resistance, thus preparation can be expressed the cell of the gene of encoding human material.Can select cell by selective pressure then through stable transfection, for example, under the situation of Neo marker gene, screen immediately by under the situation that has G418 (genericin), cultivating through cells transfected and at the cell pair cell that shows the biological substance high level expression.Be used to prepare expressing protein E1-immortalization HER cell the clone method and to be used to cultivate this type of cell all be that the technician is known to produce described proteic method, for example can be at US 6,855, find in 544.
Can be by the biological substance of cell generation, for example by expressing the biological substance that coding (reorganization) genes encoding produces, therefore for example be (reorganization) albumen, particularly acceptor, enzyme, fusion rotein, the haemproteins albumen of coagulation cascade (for example from), multifunctional protein (for example erythropoietin), virus or bacterioprotein (for example being used for vaccine), immunoglobulin (Ig) (for example antibody, for example IgG or IgM) etc.; Preferably, produce albumen, more preferably, produce antibody by cell.Preferably, by the biological substance that cell produces, for example albumen or vaccine can be used as the activeconstituents in the pharmaceutical preparation.In the context of the present invention, term " product " and " biological substance " commutative use.
In the context of the present invention, pharmaceutical preparation represents can be used as any preparation of medicine (especially for people's medicine).This similar drug can for example be used for diagnosis or be used to prevent purpose (for example, vaccine) and/or be used for the treatment of purpose, and the enzyme or the albumen that lack of patient for example perhaps is used to kill the antibody of undesired cell.Pharmaceutical preparation also can contain pharmaceutically acceptable carrier or vehicle, and their example is well known to a person skilled in the art.
PER.C6 clone can be used for producing biological substance, and for example the adenovirus of E1-disappearance (sees that for example United States Patent (USP) 6,994,128; Nichols et al, 2002, Propagation of adenoviral vectors:use of PER.C6 cells.In:Curiel D, Douglas JT, editors.Adenoviral vectors forgene therapy.San Diego:Elsevier.p 129-167), other virus (is seen, for example, WO01/38362) or recombinant protein (see, for example United States Patent (USP) 6,855, and 544; Yallop et al, 2005, PER.C6 cells for the manufacture of biopharmaceutical proteins, ModernBiopharmaceuticals:Design, Development and Optimization, 4 Volumes, 779-807
(editor)).
Can be used as that the proteic example of activeconstituents comprises (being trade(brand)name in the bracket) in the pharmaceutical preparation: Tenecteplase (TN Kase
TM), (reorganization) antihemophilic factor (ReFacto
TM), lymphoblast interferon alfa-n1 (Wellferon
TM), (reorganization) thrombin (NovoSeven
TM), Etanercept (Enbrel
TM), Trastuzumab (Herceptin
TM), Infliximab (Remicade
TM), Palivizumab (Synagis
TM), Basiliximab (Simulect
TM), Daclizumab (Zenapaz
TM), Rituximab (Rituxan
TM), (reorganization) plasma thromboplastin component (Benefix
TM) and interferon beta-1a (Avonex
TM).
The example that can be used as the vaccine of activeconstituents in the pharmaceutical preparation comprises: separated proteantigen, its example include but not limited to that live, oral, quaternary Rotavirus Vaccine (RotaShield
TM), Rabies Vaccine (RanAvert
TM), influenza vaccines and deactivation hepatitis A vaccine (VAQTA
TM).
The pH of cell culture medium, temperature, dissolved oxygen concentration and osmolarity (osmolarity) are also non-key in principle, and they depend on the type of selected cell.Preferably, pH, temperature, dissolved oxygen concentration and osmolarity are selected as: feasible growth and productivity for cell is best.Those skilled in the art will know that best pH, temperature, dissolved oxygen concentration and the osmolarity (seeing that for example WO 2004/099396) that how to find culture.Preferably, for technology of the present invention, when using the HER cell of E1 immortalization, select the pH between 6.6 to 7.6 for use, and/or select the temperature between 30 to 39 ℃ for use and/or select osmolarity between 260 to 400mOsm/kg for use.For keeping optimum process condition, the automatization that processing condition are controlled is that people want.For processing condition are optimized, for example be used to increase cells produce power in order to obtain the growth obstruction, in the training period, can use the change of culture condition.This can for example change by temperature change (for example from 37 to 32 ℃), pH change or osmolarity and realize.
Can on the cell culture medium of any kind that is suitable for culturing cell, carry out on the technological principle of the present invention.It is known selecting the guidance of cell culture medium and cell culture condition, for example be provided in Freshney, R.I.Culture of animal cells (a manual of basic techniques), 4thedition 2000, Wiley-Liss and in Doyle, A., Griffiths, J.B., Newell, D.G.Cell﹠amp; Tissue culture:Laboratory Procedures 1993, John Wiley ﹠amp; In the 8th and 9 chapters of Sons.
For example, cell culture medium can for example comprise carbohydrate source, salt and/or amino acid and/or VITAMIN and/or lipid and/or washing agent and/or buffer reagent and/or somatomedin and/or hormone and/or cytokine and/or trace elements as the cell culture medium component.The example in carbohydrate source comprises glucose, fructose, semi-lactosi and pyruvate salt.The example of salt comprises: magnesium salts, for example MgCl
2.6H
2O, MgSO
4And MgSO
4.7H
2O, molysite, for example FeSO
4.7H
2O, sylvite, for example KH
2PO
4, KCl; Sodium salt, for example NaH
2PO
4, Na
2HPO
4, and calcium salt, for example CaCl
2.2H
2O.Amino acid whose example comprises the proteinic amino acid of all known formation, for example Histidine, glutamine, Serine, methionine(Met).The example of VITAMIN comprises: ascorbate salt, vitamin H, choline chlorine, inositol, D-pantothenate, riboflavin.The example of lipid comprises: lipid acid, for example, linoleic plus oleic acid; The example of washing agent comprises Tween
80 and Pluronic
F68.The example of buffer reagent comprises HEPES and Na
2CO
3The example of somatomedin/hormone/cytokine comprises IGF (rhIGF-1), corticoid and (reorganization) Regular Insulin.The example of trace elements is well known by persons skilled in the art, and it comprises Zn, Mg and Se.Cell culture medium also can for example comprise other cell culture medium component, for example soy peptone or thanomin.
For according to producing the thing material next life of the present invention, if particularly biological substance will be used as activeconstituents in the pharmaceutical preparation, the substratum that the substratum that does not contain serum contains the serum source relatively is preferred.Its reason is: the substratum in serum source may be by virus pollution, the risk that exists protein (prionic) to infect, and may cause big obstacle to the downstream processing (that is being further purified from cell culture) of biopharmaceutical product to biological substance.Therefore, optimal process of the present invention carries out in the cell culture medium that does not comprise the serum of originating from animal (comprising the people).In view of also there is infection risk in the compound from the Mammals source, therefore, preferably, cell culture medium is the nonmammalian source, that is, cell culture medium does not comprise serum or the component from the Mammals source.More preferably, cell culture medium is non-animal-origin, that is, cell culture medium does not comprise serum or the component from animal (comprising the people) source.The example that can be used for cultivating the substratum that does not contain serum of PER.C6 cell comprises can the commercial substratum that obtains, for example, and EX Cell
TMVPRO substratum (SAFC), HyQ
CDM4Retino
TM(HyClone), IS ProVec CD (Irvine scientific), 293-SFM II (invitrogen).
In some preferred implementations, be maintained at the reactor or the stream that preferably advanced reactor by loopback is gathered in the crops the biological substance that produces in the technology of the present invention from inclusion wherein, or from the cell culture that shifts out by reactor, gather in the crops, perhaps from both, gather in the crops.Also can in the processing of so-called downstream, use the method (described method is that the technician is known) that depends on biological substance, the biological substance that from cell culture, produces in the results technology of the present invention.Downstream processing comprises usually with various combinations and some in sequence purification steps.In the downstream processing example of purification step be separating step (for example, by affinity chromatography and/or ion-exchange chromatography and/or by the aqueous two phase system extraction and/or by for example ammonium sulfate precipitation), be used for the concentrated biological material step (for example by ultrafiltration or diafiltration), be used for the step of exchange buffering liquid and/or be used to shift out or the step (for example, changing or the processing of solvent washing agent) of inactivation of viruses by virus filtration, pH.
In one aspect, the present invention relates to comprise mammalian cell, the HER cell of preferred E1-immortalization, the more preferably cell culture of PER.C6 cell, it has at least 50 * 10
6Individual cell/mL, preferably at least 60 * 10
6Individual cell/mL, particularly at least 90 * 10
6The survivaling cell density of individual cell/mL and 5g/L at least, more preferably 10g/L at least, the particularly biomass density of 11g/L at least.The concentration of biological substance can be up to the degree that solubleness allowed to biological substance in principle.The concentration of survivaling cell typically is no more than 200 * 10
6Individual cell/mL is preferably in 80-150 * 10
6In the scope of individual cell/mL.
Survivaling cell density for example can be measured like this: use the trypan blue method for removing, for example by using cell counter (can obtain from for example Innovatis (Cedex cell counter) is commercial) to carry out.
Cell culture represents to comprise the liquid of cell culture medium, cell and biological substance, described liquid be in reactor in cell culture medium the result of the technology of culturing cell, wherein, described cell produces described biological substance.
To set forth the present invention by following embodiment now, but following embodiment is not limited to the present invention.
Accompanying drawing is described
Fig. 1 has shown the survivaling cell density Y (10 at technology A (in batches), B (fed-batch) and C1 (technology of the present invention)
6.ml
-1) to process time X (my god) mapping.
Fig. 2 shown at the IgG concentration (with respect to the % of the IgG concentration among the technology A) among the reactor Z of technology A (in batches), B (fed-batch) and C1 (technology of the present invention) to process time X (my god) mapping.
Fig. 3 has shown the survivaling cell density Y (10 at technology A (in batches), B (fed-batch) and C2 (technology of the present invention)
6.ml
-1) to process time X (my god) mapping.
Fig. 4 shown at the IgG concentration (with respect to the % of the IgG concentration among the technology A) among the reactor Z of technology A (in batches), B (fed-batch) and C2 (technology of the present invention) to process time X (my god) mapping.
Fig. 5 has shown the survivaling cell density Y (10 at technology A (in batches), B (fed-batch) and C3 (technology of the present invention)
6.ml
-1) to process time X (my god) mapping.
Fig. 6 shown at the IgG concentration among the reactor Z of technology A and C3 (with respect to the % of the IgG concentration among the technology A3) to process time X (my god) mapping.
Fig. 7 shown at technology A, B and C3 cumulative yield Q (with respect to the % of the productive rate among the technology A, every liter of reactor volume) to process time X (my god) mapping.
Fig. 8 has shown the cell quantity Y (10 at technology C4 (technology of the present invention)
6.ml
-1) to process time X (my god) mapping.
Fig. 9 shown at the IgG concentration among the reactor Z of technology C4 (a kind of embodiment of technology of the present invention) (with respect to the % of the maximum IgG concentration that reaches) to process time X (my god) mapping.
Embodiment
Embodiment 1: to batch process, fed-batch technology with according to the comparison between the technology of the present invention
In this embodiment, will according to the performance of technology of the present invention with compare with fed-batch technology in batches.
Fig. 1 has shown the survivaling cell density Y (10 at technology A (in batches), B (fed-batch) and C1 (technology of the present invention)
6.ml
-1) to process time X (my god) mapping.
Fig. 2 shown at the IgG concentration (with respect to the % of the IgG concentration among the technology A) among the reactor Z of technology A (in batches), B (fed-batch) and C1 (technology of the present invention) to process time X (my god) mapping.
Sartorius Biostat B controller is all adopted in all fermentations, and temperature is controlled to be 36.5 ℃, and pH is controlled to be between 7.2 to 6.8, is to carry out under 50% air saturation and the 200rpm condition at DO.In all experiments, all use the PER.C6 clone (seeing WO2004/099396) of identical generation IgG.
Batch process A
Batch process carries out in Sartorius B5 container with the working volume of 4L.Cell is inoculated in the VPRO substratum (SAFC) that is supplemented with the 6mM L-glutaminate with 3 * 10e5 cell/mL, cultivated subsequently 17 days.
Fed-batch technology B
Fed-batch technology is carried out in Sartorius B5 container with the working volume of 4L.Cell is inoculated in the VPRO substratum (SAFC) that is supplemented with the 6mM L-glutaminate with 3 * 10e5 cell/mL.In the training period, add glucose and glutamine, surpass 15mM and 1mM respectively to keep concentration.Added amino acid and peptide from the 5th day, with the amino acid of supplement consumed.
Technology C1 of the present invention
Technology of the present invention is carried out in 2L Applikon container.The hollow-fibre membrane of operating with the ATF stream mode with ATF-2 system (RefineTechnology) with 100kDa molecular weight separation point (MWCO) (obtaining from General Electric (GE)) is used to keep cell and IgG product.Cultivation begins with 3 * 10e5 cell/mL in the VPRO substratum (SAFC) that is supplemented with the 6mM L-glutaminate.Use the ratio flow velocity (SFR) between 0.05 to 0.2nL/ cell/sky, be supplemented with the VPRO substratum (SAFC) of 6mM L-glutaminate via the suspended cell culture perfusion.The highest production concentration that obtains is 1.4g/L.
The training mode that technology of the present invention is mentioned than preamble has produced the survivaling cell density that increases and the production concentration of increase with the less time, and this can see from Fig. 1 and Fig. 2.
Embodiment 2: to batch process, fed-batch technology with according to the comparison between the technology of the present invention
In this embodiment, once more will according to the performance of technology of the present invention with compare with fed-batch technology in batches; In technology C2, CO
2Pressure Be Controlled, and the separation system of use 50kDa.
Fig. 3 has shown the survivaling cell density Y (10 at technology A (in batches), B (fed-batch) and C2 (technology of the present invention)
6.ml
-1) to process time X (my god) mapping.
Fig. 4 shown at the IgG concentration (with respect to the % of the IgG concentration among the technology A) among the reactor Z of technology A (in batches), B (fed-batch) and C2 (technology of the present invention) to process time X (my god) mapping.
Sartorius Biostat B controller is all adopted in all fermentations, and temperature is controlled to be 36.5 ℃, and pH is controlled to be between 7.2 to 6.8, is to carry out under 50% air saturation and the 200rpm condition at DO.In all experiments, all use the PER.C6 clone (seeing WO 2004/099396) of identical generation IgG (approximately 150kDa).
Batch process A
Batch process carries out in Sartorius B5 container with the working volume of 4L.With cell with 3 * 10
5Individual cell .mL
-1Be inoculated in the VPRO substratum (SAFC) that is supplemented with the 6mM L-glutaminate, cultivated subsequently 17 days.
Fed-batch technology B
Fed-batch technology is carried out in Sartorius B5 container with the working volume of 4L.With cell with 3 * 10
5Individual cell .mL
-1Be inoculated in the VPRO substratum (SAFC) that is supplemented with the 6mM L-glutaminate.In the training period, add glucose and glutamine, surpass 15mM and 1mM respectively to keep concentration.Added amino acid and peptide from the 5th day, with the amino acid of supplement consumed.
Technology C2 of the present invention
Technology of the present invention is carried out in 2L Applikon container.The hollow-fibre membrane of operating with the ATF stream mode with ATF-2 system (RefineTechnology) (GE) with 50kDa molecular weight separation point (MWCO) is used to keep cell and IgG product.Cultivation begins with 3 * 10e5 cell/mL in the VPRO substratum (SAFC) that is supplemented with the 6mM L-glutaminate.Use 0.05 to 0.2nL. cell
-1. day
-1Between SPR, be supplemented with the VPRO substratum (SAFC) of 6mM L-glutaminate via suspended cell culture perfusion.CO
2Pressure is controlled as and is lower than 15%.
The result
As from Fig. 3 and Fig. 4 as seen, technology according to the present invention is equating or shorter time (in batches 100% of the time; The fed-batch time 81%) in produced the survivaling cell density of remarkable increase and the production concentration of increase (2415%x batch-wise productive rate; The productive rate of 690%x fed-batch).
The total productivity of technology of the present invention is (with g.L
-1. day
-1) increase be that batch production power is (with g.L
-1. day
-1) 23.9 times, be that the fed-batch productivity is (with g.L
-1. day
-1) 8.5 times.In technology C2 of the present invention, produced 11.1g product/L.The obstruction of resident equipment did not take place during 17 days, even very under the situation of high-cell density.
Embodiment 3: to batch process, fed-batch technology with according to the comparison between the technology of the present invention
The performance of the technology that in this embodiment, once more will employing cell culture according to the present invention shifts out with compare with fed-batch technology in batches; In technology C3, shifted out cell culture.
Fig. 5 has shown the survivaling cell density Y (10 at technology A (in batches), B (fed-batch) and C3 (technology of the present invention)
6.ml
-1) to process time X (my god) mapping.
Fig. 6 shown at the IgG concentration among the reactor Z of technology A and C3 (with respect to the % of the IgG concentration among the technology A3) to process time X (my god) mapping.
Fig. 7 shown at technology A, B and C3 cumulative yield Q (with respect to the % of the productive rate among the technology A, every liter of reactor volume) to process time X (my god) mapping.
Sartorius Biostat B controller is all adopted in all fermentations, and temperature is controlled to be 36.5 ℃, and pH is controlled to be between 7.2 to 6.8, is to carry out under 50% air saturation and the 200rpm condition at DO.In all experiments, all use the PER.C6 clone (seeing WO 2004/099396) of identical generation IgG (approximately 150kDa).
Batch process A
Batch process carries out in Sartorius B5 container with the working volume of 4L.With cell with 3 * 10
5Individual cell .mL
-1Be inoculated in the VPRO substratum (SAFC) that is supplemented with the 6mM L-glutaminate, cultivated subsequently 17 days.
Fed-batch technology B
Fed-batch technology is carried out in Sartorius B5 container with the working volume of 4L.With cell with 3 * 10
5Individual cell .mL
-1Be inoculated in the VPRO substratum (SAFC) that is supplemented with the 6mM L-glutaminate.In the training period, add glucose and glutamine, surpass 15mM and 1mM respectively to keep concentration.Added amino acid and peptide from the 5th day, with the amino acid of supplement consumed.
Technology C3 of the present invention
Technology of the present invention is carried out in 2L Applikon container.The hollow-fibre membrane of operating with the ATF stream mode with ATF-2 system (RefineTechnology) (GE) with 100kDa molecular weight separation point (MWCO) is used to keep cell and IgG product.Cultivation begins with 3 * 10e5 cell/mL in the VPRO substratum (SAFC) that is supplemented with the 6mM L-glutaminate.Use 0.05 to 0.2nL. cell
-1. day
-1Between SPR, be supplemented with the VPRO substratum (SAFC) of 6mM L-glutaminate via suspended cell culture perfusion.When survivaling cell density greater than 10 * 10
6Individual cell .mL
-1The time, every day is with 10% emigrated cells culture of working volume, when survivaling cell density surpasses 30 * 10
6Individual cell .mL
-1The time, every day is with 30% emigrated cells culture of working volume.
The result
As seeing from Fig. 5, adopt technology of the present invention, reached higher survivaling cell density fast.In addition, Fig. 5 also shows, adopts the viability of technology cell of the present invention can keep the longer time, and for example technology C3 keeps having moved near 40 days, even because obstruction to resident equipment can not take place under the situation of high-cell density yet.
Fig. 6 shows, the production concentration of technology of the present invention is more much higher than the production concentration in the technology in batches.Gathered in the crops the product stream that contains product from technology C3 with about 200% to 250% times of final concentration among the batch process A.
Fig. 7 shows that technology of the present invention has formed maximum products, and technology of the present invention can be than technology A and fed-batch technology B keep the longer time in batches.At the 17th day, the cumulative yield of technology C3 was 8.1 times of batch process (A3) cumulative yield, was 2.1 times of fed-batch technology (B) cumulative yield.In addition, at the 17th day, batch process stopped.At the 21st day, the cumulative yield of technology C3 was 3.0 times of cumulative yield of fed-batch technology B.At the 21st day, fed-batch technology stopped.After 39 days, total cumulative yield of technology C3 is 25 times of batch process A cumulative yield, is 6 times of fed-batch technology B productive rate.
Can infer from this experiment, in technology according to the present invention, after cell density surpasses certain high level, can further improve the overall yield of the biomaterial of wanting by the removing of application cell culture.
Embodiment 4: cultivate and produce Chinese hamster ovary celI
In this embodiment, adopting the Chinese hamster ovary celI of producing IgG is to have carried out according to technology of the present invention, reduces to reduce the cell growth comprising temperature.
Fig. 8 has shown the cell quantity Y (10 at technology C4 (technology of the present invention)
6.ml
-1) to process time X (my god) mapping.
Fig. 9 shown at the IgG concentration among the reactor Z of technology C4 (a kind of embodiment of technology of the present invention) (with respect to the % of the maximum IgG concentration that reaches) to process time X (my god) mapping.
Sartorius Biostat B controller is adopted in fermentation, and temperature is controlled to be 36.5 ℃, and pH is controlled to be between 7.1 to 6.9, is to carry out under 40% air saturation and the 100rpm condition at DO.Temperature was reduced to 32 ℃ at the 5th day.
Technology C4 of the present invention
Technology of the present invention is carried out in 2L Applikon container.The hollow-fibre membrane of operating with the ATF stream mode with ATF-2 system (RefineTechnology) with 50kD molecular weight separation point (MWCO) (General Electric) is used to keep cell and IgG product.Cultivation is with 5 * 10 in the MTCM-49 substratum (Hyclone)
6Individual cell/mL begins.Use 0.1 to 0.4nL. cell
-1. day
-1Between SPR, via suspended cell culture perfusion culture base.CO
2Pressure is controlled as and is lower than 15%.
The result
Data presentation, when using the proteic Chinese hamster ovary celI of generation to be, technology of the present invention can be worked equally.The cell density and the production concentration that obtain increase to some extent than batch culture.Data show that also in technology according to the present invention, the cell growth can be hindered (for example, reducing by temperature), and the accumulation of the product in the culture systems can continue.
Embodiment 5: the technology of the present invention that adopts myeloma cell line to carry out
Also may be used on the myeloma cell according to technology of the present invention fastens.For reaching this purpose, Sartorius Biostat B controller is adopted in fermentation, and temperature is controlled to be 36.5 ℃, and pH is controlled to be between 7.2 to 6.8, is to carry out under 40% air saturation and the 100rpm condition at DO.Cultivation is to begin with 3 * 10e5 cell/mL inoculation myeloma cell in the SFM4Mab substratum (Hyclone) in 5L Sartorius container.The resident equipment of cell and product is with the hollow-fibre membrane with 30kD molecular weight separation point (MWCO) (General Electric) of ATF-4 system (RefineTechnology) with the operation of ATF stream mode.Use 0.1 to 0.4nL. cell
-1. day
-1Between SPR, via suspended cell culture perfusion SFM4Mab substratum (Hyclone).CO
2Pressure is controlled as and is lower than 15%.
Embodiment 6: the technology of the present invention that adopts mdck cell system to carry out
Fasten according to the mdck cell that technology of the present invention also may be used in the suspension through transforming.For reaching this purpose, Sartorius Biostat B controller is adopted in fermentation, and temperature is controlled to be 36.5 ℃, and pH is controlled to be between 7.2 to 6.8, is to carry out under 40% air saturation and the 100rpm condition at DO.Cultivation is to begin with 3 * 10e5 the cell/mdck cell of mL inoculation through transforming in the VP-SFM substratum (Invitrogen) in 5L Sartorius container.The resident equipment of cell and product is with the hollow-fibre membrane (GeneralElectric) with 30kD molecular weight separation point (MWCO) of ATF-4 system (Refine Technology) with the operation of ATF stream mode.Use 0.1 to 0.4nL. cell
-1. day
-1Between SPR, via suspended cell culture perfusion VP-SFM substratum (Invitrogen).CO
2Pressure is controlled as and is lower than 15%.
Claims (14)
- In reactor in cell culture medium in suspension the technology of culturing cell,Wherein, described cell produces biological substance, wherein, and at least a cell culture medium component of cell culture feed supplement, and,Wherein, the described cell culture that comprises described cell, described biological substance and described cell culture medium is in the separation system cocycle, and, wherein, described separation system is separated described biological substance and the material lower than described biological substance molecular weight, and wherein, described biological substance is retained in the described reactor or by loopback and advances described reactor.
- 2. according to the technology of claim 1, wherein, the part of the described material that molecular weight is lower continues to shift out from described cell culture.
- 3. according to the technology of claim 1-2, wherein, described cell is a mammalian cell, and preferably the HER cell of E1-immortalization is more preferably the PER.C6 cell.
- 4. according to the technology of claim 1-3, wherein, described biological substance is a recombinant protein, preferably antibody.
- 5. according to any described technology among the claim 1-4, wherein, described separation system is a strainer, and preferably film filter is more preferably hollow fiber filter.
- 6. according to the technology of claim 5, wherein, described cell culture with the cross-stream circulation, preferably circulates with mutual tangential flow on described strainer.
- 7. according to any described technology among the claim 1-6, wherein, carry out once operation at least from described reactor emigrated cells culture, and, add liquid to described reactor, preferably add cell culture medium, to compensate shifting out of described cell culture.
- 8. according to any described technology among the claim 1-7, wherein, cell culture condition is selected as: make that the ratio productivity and/or the cell growth rate of described cell are unrestricted.
- 9. technology according to Claim 8, wherein, described cell culture condition is selected as: make at least a concentration in the described component of described cell culture medium keep constant.
- 10. according to any described technology among the claim 1-9, wherein, the nutrition of Xiao Haoing is replenished by part or is preferred all additional at least, and this is by realizing to described these nutrition of reactor feed supplement.
- 11., wherein, randomly, gather in the crops described biological substance from described cell and/or from described cell culture according to any described technology among the claim 1-10.
- 12. comprise the cell culture of mammalian cell, it has at least 50 * 10 6The survivaling cell density of individual cell/mL and the biomass density of 5g/L at least.
- 13. according to the cell culture of claim 12, wherein, described biomass density is 10g/L at least, preferably 11g/L at least.
- 14. according to the cell culture of claim 12-13, wherein, described mammalian cell is the HER cell of E1-immortalization, preferably PER.C6 cell.
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| EP06014671 | 2006-07-14 | ||
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| CN2012103216278A Division CN102876628A (en) | 2006-07-14 | 2007-07-04 | Improved process for the culturing of cells |
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| CN103154233A (en) * | 2010-10-05 | 2013-06-12 | 诺沃—诺迪斯克保健股份有限公司 | Process for protein production |
| CN103517919A (en) * | 2011-05-13 | 2014-01-15 | 欧克塔医药公司 | A method of increasing the productivity of eucaryotic cells in the production of recombinant fviii |
| CN105579572A (en) * | 2013-09-30 | 2016-05-11 | 克鲁塞尔荷兰公司 | Method for the clarification of high density crude cell culture harvest |
| CN107286235A (en) * | 2010-07-08 | 2017-10-24 | 百深有限责任公司 | The production restructuring HMW vWF method in cell culture |
| CN108138128A (en) * | 2015-05-29 | 2018-06-08 | 葛莱高托普有限公司 | For the small-scale cultural method of suspension cell |
| CN110268043A (en) * | 2017-03-03 | 2019-09-20 | 富士胶片株式会社 | Cell culture apparatus and cell culture processes |
| WO2021008571A1 (en) * | 2019-07-16 | 2021-01-21 | 信达生物制药(苏州)有限公司 | Cell culture method and application thereof based on high-density and continuous inoculation |
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| CN107286235A (en) * | 2010-07-08 | 2017-10-24 | 百深有限责任公司 | The production restructuring HMW vWF method in cell culture |
| CN103154233A (en) * | 2010-10-05 | 2013-06-12 | 诺沃—诺迪斯克保健股份有限公司 | Process for protein production |
| CN107190034A (en) * | 2010-10-05 | 2017-09-22 | 诺沃—诺迪斯克保健股份有限公司 | Produce method of protein |
| CN103517919A (en) * | 2011-05-13 | 2014-01-15 | 欧克塔医药公司 | A method of increasing the productivity of eucaryotic cells in the production of recombinant fviii |
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