CN1966682A - Glutamine-auxothrophic human cells capable of producing proteins and capable of growing in a glutamine-free medium - Google Patents

Abstract

A glutamine-auxotrophic human cell transfected with an exogenous DNA sequence encoding a protein or an exogenous DNA sequence capable of altering the expression of an endogenous gene encoding a protein and an exogenous DNA sequence encoding a glutamine synthetase, wherein these exogenous DNA sequences are located on one or more than one DNA construct, said transfected cell capable of producing said protein and capable of growing in a glutamine-free medium.

Description

The glutamine auxotroph human cell that can produce protein and can on the substratum of no glutamine, grow

The application is that the application number that 2003.01.17 submits to is 03802413.6 (PCT/EP03/00454), the dividing an application of application for a patent for invention that can " the glutamine auxotroph human cell who produces protein and can grow on the substratum of no glutamine " by name.

The invention relates to and novel can produce protein and can be grown in glutamine auxotroph human cell in the substratum of no glutamine.In addition, its be about the generation method of protein of novelty and about in glutamine auxotroph human cell glutamine synthetase (GS) but be used as the purposes of selective marker.

Cultivating the protein that is produced by mammalian cell is the protein that is used to provide treatment and diagnostic use.Now, it is the preferable source that is used for many key proteins of human and animal's medicine that mammalian cell is cultivated, especially those sizable, complicated and glycosylated (N.B.Finter etc., in extensive mammalian cell culture technique, 1990, A.S.Lubiniecki compiles, Marcel Dekker, Inc., New York).

For example, the mat cell cultures produces human protein's erythropoietin (EPO) description in WO 93/09222.Use is with the human fibrous tissue parent cell of the human EPO foreign gene of codified transfection, and human EPO has obtained considerable specific production rate.In the method (WO 94/12650) that further produces human EPO, being to use the human fibrosarcoma cell to give birth to the dna sequence dna transfection of the EPO that encodes in can activating is HT1080.Similarly method is described in WO99/09268.(Immortalised) human cell of immortality such as Namalwa, Hela S3 and HT 1080 cells are with activating interior dna sequence dna transfection of giving birth to the EPO of coding.

Being described in the clone that WO 93/09222, WO 94/12650 and WO 99/09268 be used for producing human EPO all cultivates in containing the substratum of glutamine.This is disadvantageous, because when glutamine is used as energy matrix by cultured cells, can produce this meta-bolites of ammonia, it has cytotoxicity and meeting cell growth inhibiting.Moreover, owing to its influence in the cell golgi body to pH, and can the glycosylation of arrestin matter.

Produce and organize plasminogen activator (a kind of glycosylated protein) in WO 87/04462, to describe in a large number.It is system that GS has been used as amplification therein, is used for the gene that common amplification codified is organized plasminogen activator (tPA) at glutamine prototroph Chinese hamster ovary (CHO) cell, and it is by the gene transfecting cell with codified GS.Yet as what find at EP-A 148 605, it is disadvantageous producing glycosylated human protein with Chinese hamster ovary celI.Can be different by its average carbohydrate components of Chinese hamster ovary celI synthetic protein with the glycosylated human proteinoid that generates naturally, this is because the human cell is contained α 2.3 sialytransferases and α 2.6 sialytransferases.Chinese hamster ovary celI only has α 2.3 sialytransferases, so can not produce the bond of α 2.6 end sialic acids and oligosaccharides part, Chinese hamster ovary celI lacks the ferment of sulfuration carbohydrate structure, though Chinese hamster ovary celI has α 1-6 fucosyltransferase (fucosyl residues of attached center), also lack α 1-3 fucosyltransferase (adhering to terminal fucosyl residues).The human cell then has both fucosyltransferases (Cumming D.A., 1991, Glycobiology volume 1, No.2,115-130, Jenkins N. and Curling E.M.A., 1994, enzyme and Micorbial Technology, volume 16,354-364; Lee etc., 1989, Journal of Biological Chemistry, volume 264,13848-13855).So, the feature that the glycosylated protein that is synthesized by Chinese hamster ovary celI can not be hopeful to obtain, for example in vivo biological activity as producing in the human cell.

In WO 89/10404, reported the manufacturing myeloma cell for example mouse merge independently method of knurl, mouse plasmoma cell and rat hybridoma cell glutamine, it is by transforming them with GS.Its further proof GS can be used for the gene that common amplification codified immunoglobulin molecules gently reaches the gene of heavy chain and be used for being amplified in altogether codified scleroproein amylase in the myeloma cell line.Yet rodents clone has shortcoming, promptly adhering to of N-glycoloyl neuraminic acid residue to replace the N-n acetylneuraminic acid n, can't vulcanize and the existing of α 1.3 galactotransferases.Therefore the oligosaccharide structure of synthetic glycoprotein can produce immunity the mankind in the rodents cell.

The present invention's purpose provides a kind of improved method, and it does not have above-mentioned shortcoming when being used to produce protein (especially when producing glycoprotein), and can obtain high protein and tire.

Utilize the glutamine auxotroph human cell of the described novelty of claim 1 and the method for the described novelty of claim 7, can reach this purpose.

According to the present invention, can obtain glutamine auxotroph human cell, described cell maybe can change the exogenous DNA array transfection of giving birth to genetic expression within the codified protein with the exogenous DNA array of (first) codified protein, and further with the exogenous DNA array transfection of (second) codified glutamine synthetase (GS) (that preferable is Mammals GS), wherein these exogenous DNA arrays are positioned at one or above DNA construction, and this cells transfected can produce this protein and can be grown in the substratum of no glutamine.

Fig. 1 showed cell is the cell concn figure of R223 suspension culture in the adaptation of serum free medium, cultivates via repeating successive.

Fig. 2 shows the adaptation summary of HT1080 cell suspension culture in serum free medium.

Fig. 3 showed cell is the cell concn figure of HT1080 suspension culture in the adaptation of serum free medium, cultivates via repeating successive.

Fig. 4 shows the IEF analysis that reaches the EPO of immune purifying from the R223 clone of no transfection from GS transfectant 3E10, and it is to be grown in industry high-density growth substratum.Swimming lane 2: (collection) 3E10 of collection; Swimming lane 3:3E10 peak; Swimming lane 4: the R223 clone peak of no transfection; Swimming lane 5: the R223 clone of the no transfection of collection.

Fig. 5 shows the GS-R223 transfectant 3E10 of R223 clone and from the IEF analysis of the EPO of the R223 clone of no transfection immunity purifying, is grown in traditional Iscove substratum.Swimming lane 4: the R223 clone of adding the no transfection that the Iscove substratum of glutamine collects certainly; Swimming lane 5: the GS-223 clone 3E10 that does not have the Iscove substratum collection of glutamine certainly.

Fig. 6 shown gel swimming lane 4 shown in Figure 5 from the top to the color atlas of bottom densitometric scan.

Fig. 7 shown gel swimming lane 5 shown in Figure 5 from the top to the color atlas of bottom densitometric scan.

" the proteinic exogenous DNA array of codified " or " can change the exogenous DNA array of giving birth to genetic expression within the codified protein " reaches " exogenous DNA array of codified GS " and is usually located on the DNA construction, for example expression vector or infectious vector.Plastid can be used as expression vector.Then can use as retrovirus, herpes virus, adenovirus, adeno-associated virus, mumps virus and poliovirus carrier as infectious vector.Preferable is expression vector, especially uses plastid.

" the proteinic exogenous DNA array of codified " can comprise additional sequences, for example regulating and controlling sequence such as promotor and/or enhanser, polyadenylation site and montage joint can comprise that maybe extra one or more target sequence of separating reaches the DNA (described in WO 93/09222) of codified selective marker optionally but be generally used for expression alien gene.

" exogenous DNA array of giving birth to genetic expression within the codified protein can be changed " and exogenous DNA array can be comprised, it is the gene product of coded protein not, but codified part this gene product (as exon), and can comprise additional sequences, for example regulating and controlling sequence and montage joint are generally used for expressing exogenous DNA array.But they may further include the DNA (described in WO 93/09222) of target sequence and codified selective marker optionally.

Usually, " can change the exogenous DNA array of giving birth to genetic expression within the codified protein " is the chromosomal DNA that inserts cell after cell is advanced in transfection.Homologous recombination or target are to be used for replacing or to make and to contain the regulation and control zone of giving birth to gene-correlation within the regulating and controlling sequence ineffective at this.Can use as promotor and/or enhanser as regulating and controlling sequence, its cause gene expression amount be higher than corresponding no cells transfected (such as WO 93/09222 description), suitable promotor can be the promotor of regulatable or continuous expression.Suitable promotor can be a strong promoter, it is to depend on employed clone, reaches late promoter, interferon alpha 1 promotor, mouse metallothionein promoter, the long end of Rous sarcoma virus repeated priming, beta-globin promotor, conalbumin promotor, Protalbinic acid promotor, mouse beta-globin promotor and human beta-globin promotor in early days as the main immediate early promoter of human cytomegalovirus (hCMV-MIE), SV 40 early stage late promoter, other adenovirus promoters, any polyomavirus or the papovavirus of reaching.

" exogenous DNA array of codified GS " according to the present invention can be subjected to the control of strong promoter and weak promoter, if exogenous DNA array is the gene that only needs to express codified GS, then use strong promoter, when but exogenous DNA array is as selective marker, and, then use weak promoter if GS is when being used for amplification.Suitable promotor can be the promotor of regulatable or continuous expression.Promotor can be selected, and for example, GS expresses in enough making the concentration of cells transfected growth, but does not produce a large amount glutamine metabolism product ammonia during cell cultures, is not more than 4mM usually, the preferable 2mM that is not more than, the better 2mM ammonia that is less than.

" but selective marker " provides selectable phenotype, and making discriminating and separating recipient cell becomes possibility.But GS can be used for being used as selective marker in the present invention, and to filter out successfully the glutamine auxotroph human cell of transfection, it incorporates and express the exogenous DNA array of codified GS into.

According to used clone, strong promoter can be to reach late promoter, interferon alpha 1 promotor, mouse metallothionein promoter, the long end of Rous sarcoma virus repeated priming, beta-globin promotor, conalbumin promotor, Protalbinic acid promotor, mouse beta-globin promotor and human beta-globin promotor in early days as hCMV-MIE, SV 40 early stage late promoter, other adenovirus promoters, any polyomavirus or the papovavirus of reaching.

According to used clone, weak promoter can be as the long end repetition of murine leukemia virus, simple exanthema virus thymidine kinase and the long end repetition of mouse mammary tumor virus, preferable, the gene of codified GS is under strong promoter control, more preferably under the control of hCMV-MIE promotor.A possible specific examples, but a kind ofly known by this technology as the purposes of selective marker from the scalable Mammals GS sequence of hamster and in mammalian cell, and such as WO 87/04462, WO 91/06657 and WO 89/01036 description; The embodiment of the invention uses this hamster GS expression unit and discrete screening method such as reference mentioned.

" exogenous DNA array of codified protein " or " can change the exogenous DNA array of giving birth to genetic expression within the codified protein " reaches " exogenous DNA array of codified GS " and is positioned on one or more DNA constructions, preferable, these exogenous DNA arrays are positioned at more than one, and better is positioned on two DNA constructions.If these exogenous DNA arrays are positioned on the DNA construction, they are combined on may function, and for example their expression can be subjected to same regulating and controlling sequence, as promotor and/or enhanser (such as WO 89/10404 description) drive.

Glutamine auxotroph human cell means that all do not express GS or express the very poor human cell of GS, thus can be grown in the substratum that contains glutamine, but can not grow or grow very poor at the substratum of no glutamine.The glutamine auxotroph human cell who is used for the present invention is glutamine auxotroph human cell or the immortal glutamine auxotroph human cell who will certainly die.The glutamine auxotroph human cell who will certainly die ties up to the glutamine auxotroph human cell who has finite life in the substratum, (claim again permanent or set up) glutamine auxotroph human cell of immortality ties up in the substratum as practises in this operator know with suitably succeeding transfer culture and time cultivation, has the glutamine auxotrophy cell of obvious infinite life.

The glutamine auxotroph human cell's who will certainly die example can be human fibrous tissue parent cell and human tire lung tissue cell, the glutamine auxotroph human cell's of immortality example can be human fibrosarcoma cell, as HT1080 clone (as DSMZ No.ACC-315 or ATCC No.CCL 121) and B-lymphoblast human cell such as HL60 (DSMZ No.Acc-3) or Namalwa (DSMZ Acc-24) clone.Be used for the preferably glutamine auxotroph human cell of immortality of the present invention, better, this immortal glutamine auxotroph human cell is with B-lymphoblast or fibrosarcoma cell, the fibrid sarcoma cell of more preferably choosing, best is with HT1080 clone (as ATCC No.CCL 121).

Glutamine auxotroph human cell can be by known genetic engineering technique with the exogenous DNA array transfection.

Depend on the exogenous DNA array transfection whether sequence is positioned on one or more DNA constructions, if sequence is positioned at more than on the DNA construction, transfection can take place respectively or cotransfection with each sequence.When transfection takes place respectively with each sequence, sequence transfection order is normally random, the preferable elder generation of transfection that takes place respectively with each sequence is with " this proteinic exogenous DNA array of codified " or " can change the exogenous DNA array of giving birth to genetic expression within this protein of codified " transfection, and then with " exogenous DNA array of codified GS " transfection, the glutamine auxotrophy cell of transfection can cultivate after transfection out of the ordinary and evaluating protein matter generates.

In order successfully to screen cells transfected, described cell is tender to be grown in the substratum of no glutamine.Cell can be grown directly upon the substratum of no glutamine or contain the substratum of glutamine earlier, progressively is diluted to the substratum of no glutamine then,, progressively dilutes with 2mM to 0mM then with glutamine concentration 10mM as earlier.Suitable screening method can be selected according to used clone.Show as described above and know, the present invention can produce protein and can be grown in glutamine auxotroph human cell system in the substratum of no glutamine and derive from codified this proteinic exogenous DNA array and maybe can change the exogenous DNA array of life genetic expression within the codified protein and this cell of exogenous DNA array transfection of codified glutamine synthetase, and wherein these exogenous DNA arrays are positioned on one or more DNA constructions.

The proteinic exogenous DNA array of codified maybe can change within the codified protein exogenous DNA array of giving birth to genetic expression can be after transfection according to as WO 94/12650 the gene amplification currently known methods of description amplify.Scalable gene codified ferment can be used for this purpose as DHFR (Tetrahydrofolate dehydrogenase), GS, adenosine deaminase, asparagine synthetase, aspartic acid transcarbamylase, metallothionein(MT)-1, ornithine decarboxylase, P-sugar-protein, ribonucleotide reductase, thymidine kinase or xanthine-guanine phosphoribosyl transferase.Contain clone that the amplification of these genes duplicates and for example can or contain in the substratum of relevant selective agent and survive at the substratum that does not have the ferment meta-bolites, relevant selective agent be for example under the DHFR situation for Rheumatrex (MTX) and under the GS situation for methionine(Met) sulphonamide (methionine sulphoximine) (MSX).

The protein that is produced by the glutamine auxotroph human cell of transfection of the present invention is sugar basedization and glycosylated protein, and glycosylated protein is meant the protein with at least one oligonucleotide chain.

Proteinic example of sugar basedization such as sugar based hormone, as luteinizing hormone-releasing hormone (LRH), thyroxine releasing hormone, Regular Insulin, Somatostatin, prolactin, thyroliberin, melanophore melanotropin, vasopressin, with and derivative such as Desmopressin, pitocin, thyrocalcitonin, Parathormone (PTH) or its fragment (as PTH (I-43)), tert-Amyloxycarbonyltetragastrin, secretin, pancreozymin, swash gall-bladder element, Angiotensin, human placenta's metakentrin, human cilium film gonad-stimulating hormone (HCG), caerulin and motilin; The pain killer material of sugar basedization such as enkephalin and derivative thereof (referring to US-A 4 277 394 and EP-A 031567), endorphin, daynorphin and kyotorphin; Sugar based ferment such as sugar based nerve conduction material be Magainin, neurotensin, bradykinin and Substance P for example; Sugar based somatomedin and the sugar based hormone and the growth factor receptors of nerve growth factor (NGF) family, epidermal growth factor (EGF) and fibrous tissue mother cell growth factor (FGF) family.

The example of glycosylated protein is hormone and hormone releasing hormone, as HGH, comprise the human growth hormone, the ox HGH, the HGH releasing hormone, Parathormone, thyroid-stimulating hormone, EPO, lipoprotein, α-1-antitrypsin, follicular stimulating hormone, thyrocalcitonin, lutropin, glucagon, thrombin such as Factor IX C, factors IX, the tissue factor and the von Willebrand factor, anticoagulin is protein C for example, atrionatriuretic factor, pulmonary surfactant, the plasminogen activator is urokinase or human urine or tissue-type plasminogen activator (t-PA) for example, zymoplasm, hemopoieticgrowth factor, enkephalin, RANTES (the T-cell expressing and the secretion of regulation and control activation nature), human MIP-2 matter (MIP-1-α), the for example human serum protein element of serum protein element, the Miller inhibition, Relaxin A chain, Relaxin B chain, preceding Relaxin, mouse gonad-stimulating hormone related peptides, microprotein is β-lactanase for example, DNase, statin, activator, feritin, vascular endothelial growth factor (VEGF), hormone or growth factor receptors, integrate plain, a-protein or D, the rheumatoid factor, neurotrophic factor for example is derived from bone neurotrophic factor (BDNF), neurotrophin-3,-4,-5 or-6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factor NGF-β for example, be derived from PDGF (PDGF), the fibrous tissue mother cell growth factor is FGF and bFGF for example, Urogastron (EGF), transforming growth factor (TGF) is TGF-α and TGF-β for example, comprise TGF-β 1, TGF-β 2, TGF-β 3, TGF-β 4 or TGF-β 5, insulin-like growth factor-I reaches-II (IGF-I and IGF-II), des (1-3)-IGF-I (brain IGF-I), insulin-like growth factor is conjugated protein, CD albumen (a group differential protein matter) is CD-3 for example, CD-4, CD-8 and CD-19, bone-inducing factor, immunotoxin, bone morphogenetic protein (BMP), cytokine and acceptor thereof, also has chimeric protein, the cytokine that comprises its acceptor, comprise for example tumor necrosis factor alpha and β, their acceptor (TNFR-1, EP 417 563 and TNFR-2, EP 417 014) and derivative, a kind of Interferon, rabbit is interferon-' alpha ' for example,-β, and-γ, cell group stimulating factor (CSFs), as M-CSF, GM-CSF and G-CSF, interleukin (ILs) arrives IL-10 as IL-1, superoxide-dismutase, T-cell receptors, surface membrane protein, the degraded speedup factor, virus antigen is the some of AIDS folder film for example, transport protein, homing receptor, addressin, modulin, antibody, chimeric protein such as immunoadhesin, and the glycosylated protein of any above-mentioned listed fragment.Preferable, the present invention produces glycosylated protein, and better the present invention produces the N-glycosylated protein, and best glycosylation hormone is that N-glycosylation and biological activity thereof are to rely on it as EPO, or especially produces EPO.

Any in the known suitable cultivation program of this technology and culture device all can be used to the to grow human cell of transfection of the present invention.Adding basic medium of common no glutamine of about 0.1 to 20% (preferable 0.5 to 15%) serum and common basic medium that serum-free does not have glutamine all can be used as substratum and uses, in addition, the yet available basic medium that does not contain the common no glutamine of protein for animal matter, preferable use serum-free does not have the common basic medium of glutamine.

The serum that can use such as foetal calf serum or adult bovine serum are preferably the use foetal calf serum.The basic medium that common available does not have glutamine is, the Eagle limit basic medium (MEM) that does not for example have glutamine, the improved Eagle substratum of the Dulbecco of no glutamine (DMEM), IscoveShi DMEM substratum (N.Iscove and the F.Melchers of no glutamine, Journal of Experimental Methods, 1978,147,923), the HamShi F12 substratum (R.G.Ham of no glutamine, Proceedings of National Academy ofScience, 1965,53,288), L-15 substratum (the A.Leibovitz of no glutamine, American Journalof Hygiene, 1963,78,173), the RPMI 1640 substratum (G.E.Morre etc. of no glutamine, TheJournal of the American Medical Association, 1967,199,519), the mixture of the special culture media of no glutamine and its proper ratio.The common cell cultures growth medium of reinforcement that is used for the high-density cells cultivation is known in this technology, and has been described in as GB 2251249, and it also is suitable as the substratum of the present invention's no glutamine very much.

Common additive can be added to the basic medium of common no glutamine.The normal additive that adds comprise in the serum protein and optionally can cell growth and/or cells survival the further composition of favourable influence is arranged.Protein in the serum such as bovine serum albumin (BSA), transferrin and/or Regular Insulin; Can cell growth and/or cells survival further composition such as soybean fat, selenium and the thanomin of favourable influence are arranged.The Amino acid that replaces glutamine and/or nucleosides can be added in the substratum example of Amino acid such as Isoleucine, leucine, Xie Ansuan, Methionin, l-asparagine, aspartic acid, L-glutamic acid, Serine, L-Ala according to used clone.Optionally, glutamine can be added in the basic medium of common no glutamine by lower concentration, is less than 1mg/l usually, and the preferable 0.5mg/l that is less than is to support its biosynthesizing effect (as changeing the amine reaction).

If the proteinic exogenous DNA array of codified maybe can change the exogenous DNA array of giving birth to genetic expression within the codified protein and amplify with scalable (amplifiable) gene after transfection, relevant selective agent can be added in the basic medium of common no glutamine, the concentration range that selective agent is used is decided according to used clone, usually with 10 μ M and higher concentration.

Glutamine auxotroph human cell can be that set dependence or set are independent, it can be used as parent material and obtains glutamine auxotroph human cell according to transfection of the present invention, according to the present invention, this cells transfected can produce protein and can be grown in the substratum of no glutamine.If use set to rely on the human cell, as HT1080 clone (ATCC No.CCL 121), it can adapt to becomes set independence HT1080 clone, can suspension growth in serum free medium, in document, do not describe as yet.

Adaptation can occur in the proteinic exogenous DNA array of codified and maybe can change within the codified protein before or after the exogenous DNA array transfection of the exogenous DNA array of giving birth to genetic expression and codified GS.Preferable, cell maybe can change the exogenous DNA array transfection of giving birth to genetic expression within the codified protein with the proteinic exogenous DNA array of codified earlier, make it adapt to suspension growth again, then further with the exogenous DNA array transfection of codified GS in serum free medium.If need, cells transfected can make it adapt to suspension growth does not have glutamine in serum-free substratum again.

The glutamine auxotroph human cell of the present invention's transfection can be that set dependence or set are independent, and can suspension growth have the substratum of glutamine in serum-free.The glutamine auxotroph human cell of preferable transfection is that set is independent, and can suspension growth have the substratum of glutamine at serum-free.

Adaptation make its become can suspension growth in the set independent cell of serum free medium, can be by adapting to cell in the first step with the substratum that contains serum and reaching.This can be via as the trypsin treatment cell, then shakes then or mat shakes and discharges cell and reach.Cell then reduces serum content at the second step mat then and adapts to serum free medium, when adapting to, if use selective agent, can reduce its consumption, to avoid cell growth inhibiting.Yet, cell also can grow in serum free medium by then reducing the serum content adaptation in the first step, second step adapt to become can suspension growth the set independent cell, by then shaking then as the trypsin treatment cell or mat shakes and discharges cell and reach, two steps also can be implemented simultaneously.

Preferable, clone contains the blood serum medium adaptation in the first step becomes set independent cell usefulness, and second step was adapted to serum free medium via then reducing serum content then by shaking the release cell.

Substratum can be as containing the basic of blood serum medium as described above.Can be added in the substratum at the selective agent that this defined, the concentration range that selective agent is used depends on used clone, uses 10 μ M and greater concns usually.It is about 0.1 to 20% that the substratum that contains serum usually adds, preferable 0.2 to 10%, 0.5 to 5% best serum, and operable serum is as above mentioned.Then reducing serum content can be by gradually reducing serum content, and for example from 10% to 1% reaches to 0%.

The glutamine auxotroph human cell of transfection of the present invention system is used in and produces in the method for protein, and by being fit to be used for expressing this protein and reclaiming this cell of culture medium culturing under this proteinic condition, the protein of generation is to describe as above.Common as described above no glutamine basic medium and common additive can be used as substratum, suitable culture condition be such as WO 96/39488 description known be used for mammalian cell vitro culture person.

Protein can be from cell culture separates with known isolation technique, and for example immune affine or ion-exchange tubing string graduation, precipitation, anti-phase HPLC, chromatographic analysis, the burnt collection method of chromatograph, SDS-PAGE, colloid filter.Practise in this operator should be appreciated that, the purification process of the polypeptide that is suitable for being interested in may need to do modification according to the characteristic that polypeptide is expressed in reconstitution cell is cultivated.

Embodiment

It is HT1080-R223 that embodiment 1. prepares human fibrosarcoma cell

It is the cell that is used for producing EPO on the industry that the set that contains how human EPO gene copy relies on human HT1080-R223 clone, created (Cambridge by Transkaryotic Therapies company, MA 02139 (US)), it is to be derived from set to rely on human fibrosarcoma HT 1080 clones.Parent HT1080 clone (ATTC No.CCL121) has the ability that produces EPO, it is by being similar to the DNA construction pREPO22 transfection of DNA construction pREPO18 (being described in WO 95,/31 560), except the DHFR gene be in the opposite direction and pREPO22 contain than pREPO18 and lack about 600 base pair homologous sequences.Later on this clone is called R223 clone.

The adaptation of embodiment 2.R223 clone with can suspension growth in serum free medium

Be released in by trypsin treatment and leave standstill in the bottle to adhere to the cell of incubation growth, be suspended in the special-purpose serum free medium that contains glutamine (further mentioning) with " HM9 ", add the foetal calf serum (dFBS) and the 500nM MTX of 10% dialysis, then with and shake flask cultivation.Begin after being grown in six days, cell is cultivated (Fig. 1) in the same substratum.In case believable growth pattern has been set up, just reduce the serum content to 1% of substratum, again, believable growth can be set up before the serum that adds is used up fully again.

Make the culture hypertrophy of adding serum and serum-free with assessment output, the results are shown in table 1, and adhere to the data of cultivation.After enough adaptations, the specific growth rate of suspension culture (even without serum) is cultivated the same with adhering to of interpolation serum.

From adhering to the adaptation when cultivating suspension culture, the synthetic specific speed of EPO descends 50% in adding the substratum of serum, from 24 to 12EU/10 ⁶Cell/hour, yet this speed can return to 18EU/10 substantially after adapting to the serum-free growth ⁶Cell/hour.

Table 1

Before or after adapting to serum free medium, the R223 cell is in the growth and the output of adhering to cultivation and suspension culture.

	Maximum cell number/mL * 10 -6	Specific growth rate h -1	Accumulation cell h/ml * 10 -6	EPO EU/ml	q EP0 EU/10 6Cell/h
						Adhere to cultivation (DMEM+10%dFBS 500nM MTX)	Inapplicable	0.0157	Inapplicable	Inapplicable	24
Suspension culture (HM9+10%dFBS 500nM MTX)	1.4	0.0185	334	4199	12
						Suspension culture (HM9 (serum-free) 500nM MTX)	1.1	0.0166	210	3600	18

The adaptation of embodiment 3.HT1080 (ATCC CCL121) with can suspension growth in serum free medium

HT1080 clone ATCC CCL121 is that (Rockville, Maryland USA) obtain, and the cell attachment incubation growth is in the DMEM that contains 10% foetal calf serum (FBS) at the beginning from American type culture collection.

The adaptation of suspension and serum-free can carry out according to the program of Fig. 2.In order to begin suspension culture, cell discharges from adhere to cultivation by trypsin treatment, and the cell resuspending that discharges is in the HM9 that has added 2%FBS, these are then cultivated with concussion, in case cell has been set up at the suspension growth of the HM9 that has added 2%FBS, do filial generation with identical substratum dilution culture and cultivate, believable HT1080 clone suspension growth was set up (Fig. 3) after 30 days.The substratum serum content reduces after that, loses fully at last.Cell continues to continue time incubation growth in serum-free.

Embodiment 4. ammonia are to the influence of R223 cell line growth and output

When with glutamine during as energy matrix, ammonia is the meta-bolites that cultured cells produces, and has cytotoxicity and can cell growth inhibiting, and in addition, its can be by the influence of pair cell Golgi body pH, the glycosylation of arrestin matter.The R223 cell of the cultivation of unavailability in bottle produces 5mM ammonia usually, cultivates at the fermenter that nutrient supply is arranged and then produces 10mM.

When assessing the influencing of ammonia at the beginning, in the R223 cell line growth earthquake bottle, there are not ammonification or ammonification (2,5 or 10mM), each the ammonia concentration that repeats to cultivate is to obtain under different pH values, mat changes the CO that applies gas ₂Content.Major objective herein is to determine ammonia to growth inhibiting degree, and the pH that test is lowered can overcome this growth-inhibiting.

Ammonia is found can cell growth inhibiting (table 2), and the pH that lowers can not relax the restraining effect that ammonia causes, though be used for reducing the pCO of the rising of pH ₂Itself may cause some growth-inhibiting.

Cultivation is only finishing after 3 days, and it is invalid that lengthening is cultivated, because the acid metabolic product accumulation of non-volatility can cause all culture pH several pH units that descend.

Table 2

In different cultivation pH values, ammonia is to the influence of growth of R223 cell and output.The pH CO that applies gas ₂Content is adjusted.

pCO 2 ％	The ammonia mM that adds	Initial pH	Every milliliter maximum cell number * 10 -6	Specific growth rate h -1	EPO EU/ml	q -EPOEU/ 10 6Cell/h
							5	0	7.26	0.9	0.020	1719	35
10	0	6.93	0.7	0.017	1580	40
							15	0	6.75	0.4	0.012	1186	56

5	2	7.15	0.8	0.018	1796	38
							10	2	6.88	0.7	0.017	1928	42
15	2	6.80	0.3	0.009	1404	78

5	5	7.18	0.6	0.018	2639	40
							10	5	6.91	0.6	0.014	1663	51
15	5	6.75	0.3	0.016	1626	45

5	10	7.17	0.5	0.016	2222	43
							10	10	6.90	0.6	0.014	1966	50
15	10	6.75	0.2	0.008	1891	86

Pass through to adjust substratum NaHCO at ensuing experiment (table 3) pH ₃Content adjust, and keep the CO that applies gas ₂Content is in the concentration of unrestraint effect.The pH scope of test is from pH 7.0 to 7.5 (be stressed that the medium pH of cultivating in bottle can not Be Controlled, and can descend greatly, even two days cultivation at first, the pH value of indication is the initial pH of cultivation respectively).Specific growth rate is at pH 7.5 (NaHCO ₃In 3g/l) time maximum, but under this pH, because the existence of 10mM ammonia reduces by half growth velocity and maximal cell concn, the synthetic specific speed of EPO reduces by four times (table 3).

As pH 7.25 (NaHCO ₃In 1.5g/l) time, compared to the cultivation of pH 7.5, specific growth rate reduces, and the synthetic specific speed of EPO increases, however because there is 10mM ammonia to exist, growth velocity is more unaffected, the synthetic specific speed of EPO is unaffected.

At minimum research pH, pH 7.0 (NaHCO ₃In 0.75g/l), specific growth rate further reduces but still not influenced by ammonia during than pH 7.5.The synthetic specific speed of EPO increases (this may be because growth velocity reduces) because of the existence of the ammonia that adds.

Table 3

Under different cultivation pH values, ammonia is to the influence of growth of R223 cell and output.PH is by changing NaHCO in the substratum ₃Content is adjusted.

Initial pH

Maximum cell number/ml * 10 -6

Specific growth rate h -1

EPO EU/ml

q EPOEU/ 10 6Individual cell/h

NaHCO 3 3g/l	7.49	1.3	0.030	951	16
						NaHCO 33g/l 10mM ammonia	7.51	0.5	0.016	256	4

NaHCO 3 1.5g/l	7.26	1.1	0.027	1151	24
						NaHCO 31.5g/l 10mM ammonia	7.27	0.7	0.021	1191	26

NaHCO 3 0.75g/l

7.02

0.9

0.020

995

26

NaHCO 30.75g/l 10mM ammonia

7.01

0.4

0.016

1009

42

Embodiment 5

The output that R223 clone is cultivated at 5 liters of scale fermenters

For R223 clone, implemented three 5 liters fermentation, be controlled between 6.95 with 7.15 different pH values (referring to table 4), each cultivation obtains spissated nutrition supply, it contains Amino acid and glucose, and design is kept main consumption nutrient in enough concentration.Culture is fine pH 7.15 and pH 7.05 growths, but can't grow 6.95 of pH, may be because be used for controlling the high concentration CO of this pH ₂

Table 4

The output and the metabolism of the R223 clone of fermenter cultivation.

Fermentation condition	Maximum cell number/ml * 10 -6	Doubly grow time hour	Cumulative cell hour/ml * 10 -6	EPO EU/ml	q EPO EU/10 6Cell count/h	Final ammonia mg/l
							pH 7.15 NaHCO 3 2g/l	1.0	46	197	10795	58	163
pH 7.05 NaHCO 3 2g/l	1.0	51	195	11414	64	157
							pH 6.95 NaHCO 3 2g/l	0.2	105	62	2995	70	Do not do

Embodiment 6. is adhering to the output of cultivation with the R223 and the GS transfectant of GS transfection.

Be used for the serum-free stock solution that cells transfected system suspends and adapt to from the R223 clone of embodiment 2, long during when these cells to the big many cells gathering of picture, make it become substantial single-cell suspension with trypsin treatment them again.

Transfection is from about 10 of embodiment 2 acquisitions ⁷Single suspend that to adapt to R223 clone separatory (at the phosphate-buffered salt of calcic or magnesium not) be to mix to contain the linear DNA of GS gene with 20 μ g (dna sequence dna pCMGS Bam H1 is described in Bebbington etc., 1992, Biotechnology 10,169-175) and use Biorad Gene Pulser (450 volts, 250 μ F) to impose electroporation.As control group person, the cell of equivalent separatory is made electroporation not adding under the DNA.

Cell does not add MTX with HM9 substratum dilution, contains 0 or 10%dFBS, and is distributed in 96 hole culture plate or 25cm ²Bottle, 35.5 to 37 ℃ of cultivations.

The HM9 substratum contains the 2mM glutamine at the beginning, but is diluted to 0.5mM after 1 day, changes the HM9 substratum of no glutamine then after ten days into, and will add MTX (500nM) again in culture at the 1st day or the 10th day.

As for not adding the control group cell cultures of making electroporation under the DNA, not growth.

With the cell of DNA electroporation, there are 15 GS transfectants to differentiate out at 96 porose discs, no matter whether initial substratum has MTX, all can obtain the GS transfectant.In 15 GS transfectants, what 7 successes were arranged proceeds to flask culture (table 5), and 8 remaining GS transfectants present unusual cell kenel, or growth is bad, abandons it.

Isolated each seven initial GS transfectant are provided with one group and duplicate and leave standstill flask culture, use the 10% HM9 substratum that adds the no glutamine of serum, every 1 to 4 day, sacrifice culture with the counting cells number, and with EPO ELISA measurement EPO concentration.Can estimate the synthetic specific speed of EPO of each GS transfectant from these data integrations, data preparation also comprises the R223 clone of no transfection in table 5 and (as a comparison) data, all GS transfectants present the synthetic specific speed of EPO of raising compared to the R223 clone of no transfection, best GS transfectant, 3E10 has the synthesis rate that is higher than five to six times of no transfection R223 clones.

Table 5

R223 clone GS transfectant and do not have the synthetic specific speed of EPO of the R223 clone of transfection.

Data do not add Rheumatrex from being grown in the cultivation of adhering to that contains 10%dFBS in the substratum of making transfectant, but are added in the culture in first day or the tenth day after transfection again.

The GS transfectant	Add the date of MTX (500nM) to the transfection dish	q EPO(adhering to) EU/10 6Cell count/h
			3B3
	10	59
			3E10	10	139
3E11	10	45
			4D9	10	35
8F11	1	47
			8F12	1	79
8G3	1	52
			The R223 clone of no transfection in the cultivation is adhered in assessment	Inapplicable	24

Embodiment 7. adapts to the GS transfectant in suspension culture and serum free medium.

By embodiment 6 resulting seven GS transfectants, GS transfectant #3E10 and GS transfectant #8G3 carry out suspension culture.

The cell attachment incubation growth includes the HM9 substratum of no glutamine in leaving standstill bottle, adds 10%dFBS and 500nM MTX, in 35.5 to 37 ℃.Cell mat after five or six days shakes release, and resuspending, begins after being grown in two or three days with shake flask cultivation in uniform temp in same substratum, and cell time is cultivated once in same medium, and hypertrophy is with assessment output then.Tire and the product synthesis rate is higher than the no transfection R223 clone twice (table 6) that is grown in 10%dFBS and 500nM MTX at least.

Table 6

The growth and the output of GS transfectant 3E10 and 8G3 earthquake bottle suspension culture.The HM9 substratum of no glutamine contains 10%dFBS and 500nM MTX.

The GS transfectant	Maximum cell number/ml * 10 -6	The maximum EPO EU/ml that tires	q -EPO(suspension) EU/10 6Individual cell/h
				3E10	0.7	11113	57
8G3	1.2	10171	33
				Be evaluated at the R223 clone of the no transfection of suspension culture growth, in 10% dFBS and 500nM MTX	1.4	4199	12

The suspension culture of GS transfectant 3E10 and 8G3, dFBS content is to gradually reduce in the HM9 substratum of no glutamine, 3E10 from 10% to 2% to 1% to 0.2% to 0.1% to 0%, 8G3 from 10% to 2% to 1%, and before each dFBS concentration further reduces, set up believable cell growth, 8G3 further is not adapted to 1%dFBS.

Embodiment 8.GS transfectant 3E10 is in the output and the metabolism of serum-free suspension culture.

The GS transfectant 3E10 suspension culture that is adapted to the serum-free growth among the embodiment 7 is incubated at serum-free is not had in the HM9 substratum of glutamine 35.5 to 37 ℃.For GS transfectant 3E10 clone, in the HM9 substratum that contains glutamine, it has the synthetic specific speed (table 7) of the EPO faster 2 to 3 times than the R223 clone of no transfection at the same terms in cultivation.

Table 7

GS transfectant 3E10 cell is in the growth and the output of serum-free suspension culture.

	Maximum cell number/ml * 10 -6	EPO EU/ml	q -EPO EU/10 6/h
				3E10 (the HM9 substratum of no glutamine)	1.1-1.2	9731-14234	40-67
R223 (the HM9 substratum adds the 6mM glutamine)	1.0-1.6	3075-5818	13-24

The minimizing that the high EPO output of GS cells transfected follows metabolism ammonia to discharge, be different from the R223 clone of the no transfection of flask culture typical case meeting generation 5mM ammonia in containing 6mM glutamine HM9 substratum, GS transfectant 3E10 only produces 1.8mM ammonia (table 8) in the HM9 of no glutamine substratum.

Table 8

The ammonia synthesis of GS transfectant 3E10 reduces.

	Maximum cell number/ml * 10 -6	Ammonia mM	Q ammonia micromole/10 6Individual cell/h
				R223 (the HM9 substratum that contains the 6mM glutamine)	0.9 1.2 1.0	4.8 5.0 5.5	30 23 27
3E10 (the HM9 substratum of no glutamine)	0.8	1.8	11

Embodiment 9

The assay products quality.

The EPO that is produced at flask culture by GS transfectant 3E10 is by immune purifying and analyze its sugared type and distribute, Fig. 4 shows isoelectric focusing method (IEF) the colloid analysis of EPO, it is when deriving from high cell concentration and collection grows in the 3E10 cell culture of the HM9 substratum of no glutamine, as described in Example 8, comprise from the sample that can compare of the no transfection R223 clone that is grown in the HM9 substratum.The EPO (compared to the R223 clone of no transfection) that produces from GS transfectant 3E10 presents sourer isomer, and analysis IEF glue is the relative reactivity (IRA) of isomer on the distribution of isomer and the theory of computation quantitatively.

Table 9

Analysis comes from the R223 clone of no transfection and the EPO isomer relative reactivity of GS transfectant 3E10.

			The band numbering	Specific activity *
	Alkali more	1
			2
		3
			4
		5
			6	0.071
		7			0.194
			8	0.273
		9			0.373
			10	0.658
		11			0.989
			12	0.999
		13			1.000
			14	0.796
		15
			16
	Sourer	17
			Other minor band

The R223 of no transfection
		Each isomer per-cent	Active (per-cent * specific activity)
5.2
		5.2
6.3
		11.4
10.5
		9.2	0.7
8.8	1.7
		8.7	2.4
5.5	2.1
		4.4	2.9
4.3	4.3
		4.5	4.5
1.4	1.4
		0.2	0.2
		14.4
Sum total=100%	Sum total=IRA=20.2

GS transfectant 3E10
		Each isomer per-cent	Active (per-cent * specific activity
2.5
		3.2
3.6
		6.0
6.2
		7.5	0.5
10.5	2.0
		11.6	3.2
9.3	3.5
		9.5	6.3
7.8	7.7
		6.1	6.1
5.3	5.3
		1.7	1.4
0.5
		8.7
Sum total=100%	Sum total=IRA=36.0

* activity data is from EP-A 0 428 267

Data have pointed out that GS transfectant 3E10 has sizable product quality-improving (table 9 and 10), and the IRA almost twice is higher than the R223 clone cultivation that the control group does not have transfection.

Also determined hypothesis N-polysaccharide electric charge " Z " from the EPO of GS transfectant 3E10, table 10 does not determine the R223 clone it " Z " of no transfection, yet GS transfectant 3E10 (273 in maximal cell concn and 265 when collecting) it " Z " value surpasses the value of the no transfection R223 of flask culture (183-228) clone." Z " equals Glycobiology according to Hermentin, and 1996,6, the 217-230 decision; (sialyated) isomer that indivedual %-are sialylated partly multiply by this corresponding isomer negative charge, can get Z, on its whether lack saliva acidic group/neutrality, single saliva, three-, four or five saliva decide.The mathematics sum total term of this product is Z.The Z numeral is about sugar-protein in the treatment that in vivo gives (Hermentin, clearance rate ibd.).

Table 10

EPO product attributional analysis from no transfection R223 clone and GS transfectant 3E10.

	EU/mL tires	The isomer relative reactivity	’Z’
				R223 parent (the HM9 substratum that adds the 6mM glutamine) peak is collected	3518	25.2	Do not do
6150	20.2	Do not do
			3E10 (the HM9 substratum of no glutamine) peak is collected		3921	43.6	273
6862	36.0	265

Embodiment 10. is in output, metabolism and the product quality of the GS transfectant 3E10 of 6 liters of fermenter serum-free suspension culture.

GS transfectant 3E10 is grown in batch culture in gaslift formula (airlift) fermenter, substratum is the HM9 of no glutamine, does not contain serum, and each cultivation has spissated nutrient supply, it contains Amino acid and glucose, and design makes the nutrient of main consumption maintain enough concentration.The result is presented at table 11, also comprises the parent R223 fermentation data of no transfection, is used for comparison.

The GS transfectant presents the viability of prolongation, so increase between incubation period, product synthetic specific speed is same as the parental cell line of no transfection, but the long cultivation life-span causes maximum production concentration to increase, the ammonia accumulation is lower than four times of GS transfectants at least, and GS transfectant product quality (measuring with IRA) is improved.

Table 11

GS transfectant 3E10 cell is in the growth and the output of serum-free suspension culture, and the EPO product quality of generation is quantitative with IRA.

Fermentation condition

Maximum cell number/ml * 10 -6

Maximum CCH*/ml * 10 -6

Collect day

Ammonia mg/L

The collection EU/ml that tires

Whole q EPO EU/10 6Cell count/h

IRA during collection

No glutamine	1.03	306	21	41	20936	69	43.6
								No glutamine	1.13	360	24	20	20150	52	42.6
No glutamine	0.94	319	24	15	18680	49	48.5

With the parent R223 of standard method with glutamine

1.11

216

13

165

12225

58

28

Number when * accumulating cell, the time integral of cell concn [Renard etc., 1988, Biotechnology Letters10 (9): 1-96].

The output and the metabolism of embodiment 11. serum-free suspension culture GS transfectant in based on the substratum of IscoveShi

The GS transfection 3E10 that is adapted to serum-free growth among the embodiment 7 cultivates the earthquake bottle, does not have the Iscove substratum of glutamine at serum-free, and parental cell line R223 is the substratum that correspondence is grown in equal interpolation glutamine.

Used substratum is the Dulbecco substratum that Iscove modifies, and contains Iscove additive (bovine serum albumin 0.4g/L, human complete (holo) transferrin 30mg/L), recombinant human Regular Insulin 10mg/L, LutrolF68 1g/L and thanomin 60 μ L/L.The substratum that is used to cultivate R223 clone contains the 4mM glutamine, does not then add glutamine but be used for GS cells transfected system, changes with the 4mM Sodium Glutamate to add the 4mM l-asparagine.

The GS cells transfected is that synthetic about 50% parent who is higher than no transfection of specific speed of the product of 3E10 is R223 (table 12), and cells transfected is that the ammonia of 3E10 generates specific speed low seven times (table 13).

Product is from these indivedual culture purified, and analyzes on isoelectric focusing method (IEF) colloid, and this analytical results is shown in Fig. 5, and it shows the IEF glue after the dyeing.IEF glue runs under pH 2.5 to 6.5 sex change conditions, and with Coomassie blue stain.

Fig. 5 explains orally
		Swimming lane 1,3,6 and 7	Blank
Swimming lane
2 and 7		The pI mark
	Swimming lane
4		Product from the R223 immunoaffinity purification of during containing the Iscove substratum of glutamine, growing
	Swimming lane 5		The product of immunoaffinity purification, it is to grow 3E10 from the commentaries on classics from the R223 clone of the GS transfection of growing the Iscoves of no glutamine substratum

Product from R223 has 13 visible bands at least, is distributed on the whole piece colloid; From the GS cells transfected is the product that 3E10 generates, than band (colloid top) strength ratio of alkali a little less than clone R223 product band, but clone 3E10 the band of acid (colloid below) then intensity increase.Have at least one extra acid band can detect in GS transfectant 3E10 product, but R223 product detecting is less than the parent, this shows that the sialylated degree from the product of the 3E10 of GS transfection increases.

Table 12

Growth and output at the GS transfectant 3E10 cell of serum-free suspension culture in the Iscove substratum

	Maximum keep alive cell count/mL	EPO EU/mL	q epo EU/10 6Cell count/h
				Be grown in the 3E10 of the Iscove substratum of no glutamine	0.55	1340	11.5
Be grown in the R223 that contains glutamine Iscove substratum	0.72	993	7.3

Table 13

Ammonia at serum-free suspension culture GS transfectant 3E10 cell in the Iscove substratum generates minimizing

	Maximum keep alive cell count/mL	Ammonia mM	Q ammonia receives and rubs/and 10 6Cell count/h
				Be grown in the 3E10 of the Iscove substratum of no glutamine	0.55	0.61	2.6
Be grown in the R223 that contains glutamine Iscove substratum	0.72	2.39	17.4

Further quantize the gels data of Fig. 5 by densitometric scan IEF-gel images.Calculated the relative proportion of the product of each band representative.Data are summarised in the table 14, and the relative proportion of each band is expressed as the per-cent that accounts for gross product on each swimming lane of gel.

Acid stronger band (8-14) has the highest biologic activity, and acid more weak band is active relatively low or do not have an activity.Obvious from gel (Fig. 5) and densitometric scan collection of illustrative plates (seeing Fig. 6, gel swimming lane 4 and Fig. 7, gel swimming lane 5), the product of GS cells transfected system, GSR223 is rich in the stronger isotype of acidity.For the clone of untransfected, R223, band 8-14 has accounted for 44% of gross product, and GS cells transfected system, GSR223, this ratio has reached 73%.In Fig. 6 and 7, the peak is to distinguish according to the numbering of gel band among Fig. 5.

	R223		The GS cells transfected is GSR223
					The band numbering	Account for the ratio of sum		Account for the ratio of sum
1	20.626	The summation of band 1-7=55.735%	1.185	The summation of band 1-7=26.865%
					2	9.604	1.346
3	3.291		1.913
					4	1.934	2.54
5	4.593		3.362
					6	7.870	7.265
7	7.817		9.254
					8	15.905	The summation of band 8-14=44.263%	18.347	The summation of band 8-14=73.137%
9	13.384	18.87
			10	8.103	14.339
11	5.522	11.978
			12	1.349	7.609
13	0	1.482
			14	0	0.512
	Amount to=99.998%					Amount to=100.002%

Claims (3)

1. But one kind in glutamine auxotroph cell with GS as selective marker, improve the method for the sialylated and/or N-polysaccharide electric charge of glycosylated protein in the exogenous DNA array cells transfected of the glutamine synthetase that is encoded.
2. 2. the method for claim 1 is characterized in that, described transfected cell can produce described protein, reclaims described glycosylated protein from described transfected cell.
3. 3. but in glutamine auxotroph cell, use GS as selective marker for one kind, the N-polysaccharide electric charge that is defined as the Z numeral by raising to be strengthening the method for quality of glycosylated protein product, and the clearance rate of sugar-protein is relevant in described Z numeral and the treatment that in vivo gives.

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