WO1994015950A1 - Purification d'immunoglobuline - Google Patents

Purification d'immunoglobuline Download PDF

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WO1994015950A1
WO1994015950A1 PCT/GB1994/000033 GB9400033W WO9415950A1 WO 1994015950 A1 WO1994015950 A1 WO 1994015950A1 GB 9400033 W GB9400033 W GB 9400033W WO 9415950 A1 WO9415950 A1 WO 9415950A1
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antibody
process according
column
medium
cells
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David Robert Young
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Wellcome Foundation Ltd
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Wellcome Foundation Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2812—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a process for the purification of antibodies.
  • Antibodies or immunoglobulins are proteinaceous bi-functional molecules. One region which is highly variable between the different antibodies, is responsible for binding to an antigen, for example many different infectious agents that the body may encounter, whilst the second, constant region is responsible for binding to the Fc receptors of cells and also activates complement. In this way antibodies represent a vital component of the immune response of mammals in destroying foreign microorganisms and viruses. Immunisation of an animal with an antigen results in the production of polyclonal antibodies, in other words, different antibodies with different specificities and affinities. For therapeutic applications it is advantageous to be able to produce antibodies from a single lymphocyte clone - such .antibodies are called monoclonal antibodies and are specific to a particular determinant of the original antigen. They can be obtained by the method of Kohler and Milstein (Nature, 1975, 22 ⁇ , 495-497).
  • a single antibody molecule of the IgG class is composed of two light chains and two heavy chains that are held together by interchain disulphide bonds. Each light chain is linked to a heavy chain by a disulphide bond and the two heavy chains are linked to each other by disulphide bonds. Each heavy chain has at one end a variable domain followed by a number of constant domains, each light chain has a variable domain at one end and a constant domain at the other end. The light chain variable domain is aligned with the variable domain of the heavy chain. The light chain constant domain is aligned with the first constant domain of the heavy chain. The remaining constant domains of the heavy chains are aligned with each other and form the Fc fragment, after limited cleavage of the polypeptide chain.
  • variable domains of each pair of light and heavy chains form the antigen binding site. Together with the first constant domain of the heavy chain and the constant domain of the light chain they form, after limited cleavage of the polypeptide chain, the Fab fragment.
  • the variable domains of each pair of heavy and light chains have the same general structure with each domain comprising a framework of four regions, whose sequences are relatively conserved, connected by three complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • SUBSTITUTE SHEET conformation and the CDRs form loops connecting, and in some cases comprising part of. the b-sheet structure.
  • the CDRs are held in close proximity by the framework regions and. with the CDRs from the other domain, contribute to the formation of the antigen binding site.
  • Antibodies which are intended for use in medical therapy may need to be administered repeatedly, so the need to remove foreign immunoglobulins is important as such administration may produce an immune response and induce nephrotoxicity.
  • Whole animal serum or serum albumin will contain other proteins, lipids and carbohydrates: these molecules may themselves raise an immune response but pose a greater danger of harbouring pathogens such as the agent which causes Bovine Spongiform Encephalopathy (BSE). Endotoxins may also be present which are undesirable as they produce potentially fatal pyrogenic responses.
  • BSE Bovine Spongiform Encephalopathy
  • Metal affinity chromatography (now termed IMAC. immobilised metal ion affinity chromatography) was first proposed as a useful method for the purification of proteins by Porath. J.. Carlsson. J.. Olsson. I. and Belfrage. G. ( 1975) Nature ⁇ 8- 598-599. It was found that metal ions chelated or bound to a matrix have an affinity for certain groups on protein surfaces.
  • the commonest chelator employed is iminodiacetic acid (Pharmacia supply a chelating Sepharose FF and Pierce supply iminodiacetic acid bound to agarose or TSK (Fractogel) HW-65F).
  • chelators including salicylic acid, aminosuccinic acid groups and biscarboxymethylamino groups can be used Mohr. P. and Pommerening. K.. ( 1985) eds. Affinity Cliromatograpny: Practical and Theoretical Aspects. Marcel Dekker. Copper, zinc and nickel ions are those most commonly employed in IMAC and in general the affinity of proteins for these ions is as follows: Cu > Ni > Zn. IMAC has advantages over biospecific affinity chromatography. Metal chelates are stable under a variety of solvent conditions and temperatures and can be recycled a large number of times with little drop in performance Arnold. F.H. ( 1991) Biotechnology 9, 151-156.
  • the gels can usually be loaded with high concentrations of metal ions and therefore high capacities are possible. Elution is relative! ⁇ ' easy and the ligand can be regnerated readily by replenishing the supply of cheiated metal. Moreover, the problems of ligand leaching are much reduced.
  • Antibodies have been purified from serum and ascites using IMAC. In serum and ascites antibody is present at concentrations of about 20mg ml and 5mg to lOmg/ml respectively. A number of workers have used both zinc and copper ions chelated to a number of matrices. Andersson. L. ( 1984) J. Chromatogr. 315. 167-174. fractionated serum on zinc iminodiacetate and zinc trisi carboxymethyi lethylenediamine chelates. Porath. J.. Carlsson. J.. Olsson. I. and Belfrage. G. ( 1975 > ⁇ Nature 258. 598-599). used chelated zinc and copper to adsorb im unoglobulins.
  • antibodies can be purified successfully from cell culture supernatant using metal chelate chromatographic techniques, even though antibody may be present at concentrations as low as 20 ⁇ g/ml. and typicaily between 30 and 80 ⁇ g. ml.
  • the invention therefore provides a process for the preparation of a purified antibody comprising culturing a cell-line capable of producing an antibody and applying an aqueous solution of antibody so cultured to a metal chelate chromatography column to absorb the antibody onto the column and eluting the antibody to obtain desired antibody in selected factions producd from the column.
  • This process is particularly suitable for large scale purification of antibody.
  • the metal ions are selected as appropriate and may include iron, cobalt, copper, zinc or nickel, divalent metal ions being particularly useful: the ions are normally chelated or bound to a matrix. Most preferred is the use of zinc.
  • Suitable chelators which may be used are referred to in the art (Porath et al Prot. Exp. & Purif. 2 263-281 ( 1992)). these are incorporated herein by reference and include iminodiacetic and salicvlic acid, aminosuccinic acid izrourjs and carboxvmethvlamino groups for example tris(carboxymethyl)ethylenediamine. delators are preferably bound to a matrix optionally through a space arm.
  • Proteins bind to metal ions mainly via amino acid residues which have electron donating side chains. Thus amino acid side chains most likely to be involved in metal ion binding are histidine residues. Free cysteine residues can also bind to chelated metal ions but in practice the cysteine residues of an antibody are rarely available in the reduced state and therefore are rarely involved in such interactions. Adsorption is usually performed at a pH at which the binding groups on the antibody surface are unprotonated. Thus antibodies are usually adsorbed to IMAC gels at non-acidic pH. Non-specific binding on IMAC gels can be suppressed by the addition of sodium chloride to all buffers at concentrations up to 1M. Phosphate and acetate buffers are recommended buffers.
  • citrate buffer at low pH and without the addition of sodium chloride can be successfully used, the low pH having a beneficial anti-viral effect and the absence of sodium chloride making it possible to immediately apply the purified aniibodj to an ion exchange chromatography column, should this be desired.
  • Elution of bound antibody can be effected in three ways.
  • a drop in pH will protonate the amino acid side chains involved in metal ion binding and so allow release of bound antibody.
  • a second method involves a ligand exchange and imidazole at neutral pH is common! ⁇ ' used. Thioi and indolyl groups are also effective.
  • the third possible method of elution involves use of EDTA to remove the chelated metal ions from the gel. This disrupts the metal ion-protein interaction. After elution regeneration and recharging of columns may be necessary to obtain reproducible elution profiles.
  • An antibody which may be purified in accordance with the invention includes any antibody that generally comprises equimolar proportions of light and hea ⁇ y chains.
  • the invention therefore includes purification of for example rat. mouse and human antibodies wherein the amino acid sequences of the heavy and light chains are homologous with those sequences of antibodies produced by lymphocytes in vivo or in vitro by hybridomas.
  • altered antibodies such as hybrid antibodies in which the heavy and light chains are homologous to a natural antibody but are combined in a way that would not occur naturally.
  • a bispecific antibody has antigen binding sites specific to more than one antigen.
  • the constant region of the antibody may relate to one or other of the antigen binding regions or ma ⁇ ' be from a further antibody.
  • chimaeric antibodies have variable regions from one antibody and constant regions from another.
  • chimaeric antibodies may be species/species chimaeras or class/class chimaeras.
  • Such chimaeric antibodies may have one or more further modifications to improve antigen binding ability or to alter effector functioning.
  • Another form of altered antibody is a humanised or CDR-grafted antibody including a composite antibody, wherein parts of the hypervariable regions in additon to the CDRs are transferred to the human framework. Additional amino acids in the framework or constant regions of such antibodies may be altered. Included in the definition of altered antibody are Fab fragments which are roughly equivalent to the Y branch portions of the heavy and light chains: these may be included incomplete fragments or fragments including part of the Fc region. Thus, within the scope of the invention is included, any altered antibody in which the amino acid sequence is not one which exists in nature.
  • An antibody to be purified according to the invention is preferentially an altered antibody most preferably a chimaeric antibody or CDR-grafted antibody
  • T cell markers such as CD2. CD3. CD4.
  • Further examples include CDR-grafted antibodies against various cancer cell marker antigens such as CD33 and CD38.
  • a cell-line which may be used is any cell line capable of producing an antibody as described above.
  • the cell line may be a hybridoma or a recombinant line such that antibodies are prepared using a recombinant expression system.
  • the preferred system is a mammalian expression system using Chinese hamster ovary (CHO) cells or myeloma cells.
  • the CHO cells may be dihydrofolate reductase (dhfr) deficient and so dependent on thymidine and hvpoxanthine for growth ( PNAS 77 1980. 4216-4220).
  • the parental dhfrCHO cell line is transfected with the antibody gene and dhfr gene which enables selection of CHO cell transformants of dhfr positive phenotype. Selection is carried out by culturing the colonies on media devoid of thymidine and hvpoxanthine. the absence of which prevents untransformed cells from growing and transformed cells from resalvaging the folate pathway and thereby bypassing the selection system. These transformants usually express low levels of the product gene by virtue of co-integration of both transfected genes.
  • the expression levels of the antibody gene may be increased by amplification using methotrexate (MTX).
  • This drug is a direct inhibitor of the dhfr enzyme and allows isolation of resistant colonies which amplify their dhfr gene copy number sufficiently to survive under these conditions. Since the dhfr and antibody genes are more closely linked in the original transformants. there is usually concommitant amplification, and therefore increased expression of the desired antibody gene.
  • GS glutamine synthetase
  • Msx methionine sulphoximine
  • Antibody is preferably grown in serum and/or protein free medium and is generally obtained in a form in which it is secreted in to the culture medium typically at antibody concentrations of 20ug ml to 80 ⁇ g/ml.
  • the harvested medium may then be filtered and/or concentrated by an ultrafiltration step to obtain an aqueous solution containing antibody at concentrations of typically 200 ⁇ g to 800 ⁇ g. mi. which is then subjected to a purification procedure according to the invention.
  • ion-exchange chromatography which exploits interactions between charged groups in a stationary phase and the sample which is in a mobile phase.
  • the stationary phase of an ion-exchange column may be a positively charged cation exchanger or a negatively charged anion exchanger.
  • the charged groups are neutralised by oppositely charged counter ions in the mobile phase, the counter ions being replaced during chromatography by more highly charged sample molecules.
  • cross- linked columns based for example on agarose for example S-Sepharose Fast Flow (Trademark) cation exchange column particularly S. Sepharose Fast Flow cation exchange (Trademark).
  • a membrane-based column could be employed.
  • the column is usually washed after application of the eluate from the metal ion chromatography column, with 20mM HEPES buffer pH " .5 and the antibody is eluted with the same buffer containing sodium chloride in the range 0.2M to 0.075M.
  • Size exclusion chromatography as its name suggests separates on the basis of the size of proteins. In general separation occurs when large molecules are excluded from entering the porous stationary phase and are carried straight through the column while progressively smaller molecules are increasingly able to enter the stationary phase and consequently have particularly longer elution times. It is the porosity of the stationary phase which therefore determines the separation achieved. Suitable materials are chemically bonded and provide resistance to compression for example an agarose and/or dextran composition such as Superdex ( Trademark). A preferred column is a Superdex 200 size exclusion medium. The eluate from the ion exchange column is preferably applied to the Superdex column and developed in buffer in the range pH5-8 preferably PBS pH " .2.
  • Each column is preferably protected by a filter which ma ⁇ ' be a 0.2m Gelman Aero sterilising filter (or in the case of the metal ion chromatography column a PALL posidvne SLK 7002 NFZP or a PALL DSLK2 filter (available from Pall Process Filtration Ltd. European House. Havant Street. Portsmouth 301 3PD) and for the other two columns a Millipak filter preferably Vlillipak 1 0 for the ion exchange column and Millipak 20 or 60 for the size exclusion column ( available from Millipore. The Boulevard. Blackmore Lane. Watford. Herts ).
  • a filter which ma ⁇ ' be a 0.2m Gelman Aero sterilising filter (or in the case of the metal ion chromatography column a PALL posidvne SLK 7002 NFZP or a PALL DSLK2 filter ( available from Pall Process Filtration Ltd. European House. Havant Street. Portsmouth 301 3PD) and for the other
  • the columns are preferably sanitised before use with an appropriate sanitant for example 0.5M NaOH for 16 hours for any of the columns or 1 N NaOH.
  • Sanitants were washed out with the appropriate sterile buffers before applying the protein solution. All solutions used in the process were preferably sterile and endotoxin free.
  • Purified antibodies are useful in medical therapy for treating numerous human disorders, generally as immun ⁇ suppressives more particularly for example T-cell mediated disorders including severe vasculitis. rheumatoid artliritis. systemic lupis. also autoimmune disorders such as multiple sclerosis, graft vs host disease, psoriarsis. juvenile onset diabetes. Sjogrens' disease, thyroid disease, myasthenia gravis. transplant rejection and asthma. These antibodies are also useful in treating cancers such as non- Hodgkin lymphoma and leukemias.
  • Figure 1. shows a typical zinc chelate column trace for the purification of Campath I H from cell culture supernatant.
  • EXAMPLE 1A Cloning of the Heaw and Light Chain cDNAs for Campath-I H
  • the myeloma cell line TF57 (Hale et al, ibid.) was used to generate size selected cDNA fractions of 0.9-1.2kb and 1.4-1.7kb for the light and heavy chain cDNAs respectively. These were used to make EcoRl linkered cDNA libraries in lgtlO. All procedures were as described by Huynh et al (DNA Cloning. Vol I: A Practical Approach. 1984. Glover.D( Editor). IRL Press.Oxford). The libraries were screened using [ J -P] nick translated probes specific for the variable regions to isolate full length cDNA clones. For the light chain cDNA.
  • the 5' untranslated leader was removed up to position -32 using Bal-31 exonuclease and a Hindlll linker added.
  • a unique Sad site 47bp upstream of the stop codon A Sacl-Hindlll synthetic oligonucleotide pair was used to regenerate this sequence and position the Hindlll site immediately after the stop codon.
  • the unique Ncol site overlapping the ATG start codon was used to re-build a 29bp untranslated leader, identical to that of the light chain, using a Hindlll-Ncol oligonucleotide pair.
  • the unique Nael site 12bp downstream of the stop codon was converted into a Hindlll site using linkers.
  • EXAMPLE IB Construction of Vectors: The human b-actin promoter was excised from pHbAPr-3-neo (which corresponds to pHbAPr-1-neo (Gunning et aL P.N.A.S.. 1987. 84, 483-35) except that the SV40 polyadenylation/termination signal has been replaced with the respective human b-actin signals) as a 2860 bp PvuII-Hindlll fragment, in which the PvuII site was subsequently converted to a Bglll site using linkers. To isolate the human b-actin polyadenylation and termination signals from pHbAPr-3-neo.
  • the basal dhfr vector called pi 04. was constructed as follows. The SphI site at position -128 in the SV40 promoter in pSV2dhfr (Subramani et al. Mol.Cell.Biol.. 1981. I. 854-864) was converted into a Sail site to remove all enhancer elements from the promoter. The weakened dhfr expression unit was then subcloned as a Sall-BamHI fragment into the homologous sites in pSVOd ( Mellon g al- Cell. 1981. 27. 2 " 9-288).
  • the pi 04 vector was digested with BamHI. phosphatased. and ligated with three other fragments consisting of the Bglll-Hindlll b-actin promoter, the Hindlll Campath- 1H light chain cDNA and the Hindlll-BamHI b-actin poly A/termination signals.
  • pNH316 the construct pdBPV-MMTneo (Law et al. Mol.Cell.Biol.. 1983. 3. 21 10-21 15 ) was digested with BamHI. phosphatased. and the fragment containing the neomycin gene isolated following separation on an agarose gel. This was ligated to the two b-actin fragments and the Campath- 1H heavy chain cDNA.
  • EXAMPLE 1C Expression o Campath-l H in CHO Cells:
  • the dhfr CHO cell line DUK-B11 (Urlaub et_al, P.N.A.S.. 1980. 77, 4216-4220) was grown in Iscove's MEM supplemented with 10% fetal bovine serum, and 4 ⁇ g ml each of hypoxanthine and thymidine. 1 O ⁇ g of pLD9 and pNH316 was co-precipitated onto ceils using the calcium phosphate method. (Gorman et al. DNA Cloning. 1985. Vol II. 143- 190.
  • Campath- IH antibod ⁇ ' in the culture medium.
  • the average yield was O.f ⁇ g'ml for non-amplified first round transformants.
  • Cell line 3D 1 .0-200 Cells were allowed to reach confluence in a T-175 tissue culture flask, then re-fed with fresh 50ml of tissue culture medium and left for a further 4 days.
  • the Campath- IH antibody that had accumulated in the medium during this period was measured by ELISA. Total cell counts on the day of assay were usually 2.5 X 10?.
  • the yield from the 3D1 1 cell line reflects a productivity of lOO ⁇ g/10 ⁇ cells/ day.
  • the co-transfection vectors pLD9 and pNH316 were further employed to evaluate an alternative amplification strategy to the one described above.
  • the dhfr CHO cells were co-transfected as usual, and two days later split directly into a series of flasks containing G418 (for neomycin selection) and increasing concentrations of MTX ranging from 3 X 10' ⁇ M to 10 _7 M. Following two weeks of this selection, the number of resistant colonies were counted and pooled for each flask. When the cell populations had stabilized, they were assayed for Campath- 1 H antibody titres and the results are shown in Table 2.
  • the contents of the flasks were pooled and added to an equal volume of medium - MTX without peptone or PEG. and were transferred to a 75cm- flask.
  • C IH 3D1 1 *44 cells from previous stage which had been growing serum-free for over 2 months were transferred to a SGi 1 litre fermenter with a stainless steel angled paddle turning at 70 ⁇ m. The temperature was set at 37°C. d ⁇ at 10% and pH control to 7- 7.2.
  • the fermenter was seeded on da ⁇ " 0 with 0.22 x 10" cells/ml in WCM4 medium with 0.1 % polyethylene glycol (PEG) 10.000 and 0.25% soy peptone, and was top gassed with O2.
  • the cells were routinely passaged using fresh medium and a split rate typically between 1 to 2 and 1 to 4.
  • Tissue culture medium was obtained from Example 2 and was purified on a zinc chelate column as follows:
  • the column was a zinc chelate column (Chelating Sepharose FF Pharmacia 26mm diameter. 10cm bed height. 50ml volume). It was run at 5cm min throughout.
  • the column was washed with 2-3 column volumes of Milli Q water and was then charged with 2-3 column volumes of Zinc Sulphate (50mM). It was then washed with a further 2-3 column volumes of Milli Q water before re-equilibrating with PBS pH7.2.
  • Figure 1 shows a typical trace from the column.
  • Example 4 The same protocol as Example 4 was employed but the column was eluted with 0.1M citric acid and 0.5M NaCl pl-13.0. Results shown in Table 3.
  • cDNA sequences encoding the light and heavy chains of the humanised antibody CAMPATH- IH were obtained as described in M. Page and M. Sydenham. Bio- Technology, Volume 9. page 64 (January 1991 ).
  • the light chain cDNA was cloned into the polyliner of pEE12 (a pBR322 - based vector containing the h-CMV-MEI promoter and the GS cDNA under control of the SV40 earl ⁇ ' region promoter).
  • the light chain cDNA was cloned into the HinDIII site of the polylinker so that expression of the light chain cDNA was under control of the h-CMV-MEI promoter.
  • the vector pEE12 corresponds to pEE6 (see EP-A-0338841 ) with the GS cDNA expression cassette driven by the SV40 promoter transcribing in the same direction as the h-CMV promoter.
  • the heavy chain cDNA was cloned into the vector pEE14 (see WO91 06657) which also includes the h-CMV-MEI promoter.
  • the complete heavy chain expresssion cassette (promoter/ heavy chain cDNApolyadenylation signal) was excised as a BamHlBglll fragment and cloned into the unique BamH I site of the pEE12 - light chain plasmid described above with an orientation such that both heavy and light chain genes are transcribed in the same direction.
  • Myeloma cells (cell line NSO - see J. Jarvis. Methods in Enzymology. " B. 3 ( 1981)) were grown exponentially in non-selective medium (DMEM/10% FCS).
  • the cells were dispersed into 30ml of the non-selective media referred to in (ii) above. 20ml of the suspension were distributed over four 96-well tissue culture trays (50 ⁇ 1 well). The remaining 10ml was diluted with a further 30ml of non-selective medium and distributed over five 96-well plates * 50 ⁇ l/well). 10ml of the latter dilution was diluted with a further 40ml of medium and distributed over five 96-well plates (50 ⁇ l/ml well). All of the plates were returned to a 37°C incubator overnight. lOOul of selective medium (DMEM without added glutamine and supplemented with 10% dialysed calf serum) was added to each well of the fourteen 96-well plates.
  • DMEM without added glutamine and supplemented with 10% dialysed calf serum
  • the plates were returned to the incubator and left until substantial c ll death, had occurred and discrete surviving colonies had appeared. Once colonies of giu .m ⁇ ne-independent transfectants could be seen, wells with single colonies were seiectged and spent tissue culture ⁇ upernatants were collected and assayed for human IgG secretion by an anti-human IgG ELISA. Wells with single colonies that were positive for IgG secretion were then expanded in culture using selective medium. The expansion was carried out via 24- and 6-well plates into flasks for amplification.
  • MSX L-methionine sulphoximine
  • the individual clones were expanded in selective medium to the T75 flask stage whilst monitoring antibody yields in case of loss of production of antibod ⁇ '.
  • Cells were removed from the flask with 0.05% versere solution, diluted in non-selective medium, and the viable cell density was determined by counting in a haemocytometer. The total number of cells of the clone could then be calculated and was generally about 1 to 4 x 10' .
  • the remainder of the cells (about I to 2 x 10 ') were pelleted at 200g for 5 minutes and resuspended in fresh selective medium at a cell density of 2 to 10 lO- ⁇ 'ml.
  • Cells were then distributed ove 96-well or 24-well plates, using 1 0 or 500 ⁇ l per we ' ll respectively (2 to 5 x 10. or 2 to 5 x 10 cells per well respectively).
  • cells were trsnferred to about 5 or 6 small flasks (T25) using about 5ml per flask (about x 10 cells ). In all cases plates were returned to the 37°C incubator overnight to allow the cells to recover.
  • MSX was added to selective medium at a range of final concentrations between 10 and 400 ⁇ M (typically using serial 2-fold dilutions). This medium was then added to the medium in the 96-well or 24-well plates, using either 100 ⁇ l or 500 ⁇ l per well respectively to yield final MSX concentrations of between 5 and 200 ⁇ M. Alternatively. MSX was added directly to the flasks containing the cells. Typicall ⁇ " 5 or 6 different concentrations of the MSX were used with about 2 to 5 x 10 cells at each MSX concentration.
  • cDNA sequences encoding the light and heavy chains of a humanised anti-CD4 ( ⁇ l ) antibod ⁇ ' were obtained as described in Gorman el al.. Proc. Natl. Acad. Sci. (USA). 8J£ 4181 ( 1991 ).
  • the light and heavy chain cDNA sequences were cloned into pEE12 using essentially the method of Example 6.
  • Myeloma cells ( cell line NSO) were transfected with plasmid DNA and transformants were selected and amplified using MSX also using essentially the method of Example 6.
  • a 50ml (2.6cm internal diameter x 10cm bed height) column of freshly poured Pharmacia Chelating Sepharose FF was poured and washed with water, charged with 50mM zince sulphate and washed once more with water in accordance with Example 3.
  • the column was equilibrated with 10 column volumes of PBS. pH. 7.2.
  • the column was then loaded to 6mg/ml of gel with tissue culture medium containing anti-CD4 monoclonal antibodv from NSO mveloma cells grown in culture.
  • the column was 94/15950

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Abstract

Procédé de préparation d'un anticorps purifié selon lequel on cultive une lignée cellulaire capable de produire un anticorps dans le milieu de culture, on dépose le milieu contenant l'anticorps dans une colonne de chromatographie à chelate metallique afin que l'anticorps se fixe sur la colonne, puis on élue l'anticorps fixé pour obtenir l'anticorps désiré situé dans des fractions sélectionnées de la colonne.
PCT/GB1994/000033 1993-01-09 1994-01-07 Purification d'immunoglobuline Ceased WO1994015950A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5759864A (en) * 1995-06-23 1998-06-02 Cedars Sinai Medical Center Methods for reducing background binding in antibody preparations
US11447547B1 (en) 2017-12-13 2022-09-20 Amgen Inc. Method of antigen-binding protein production

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US11447547B1 (en) 2017-12-13 2022-09-20 Amgen Inc. Method of antigen-binding protein production

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