IE84931B1 - Purified IgG Antibodies - Google Patents

Abstract

ABSTRACT The present invention relates to a process for obtaining a purified preparation of an anti—CD,.,52 lgG antibody produced using a recombinant mammalian expression system, for example, a monoclonal antibody against the antigen CD,.,52.

Description

Introduction The present invention relates to a process for obtaining a purified preparation of an anti—CD,.,52 lgG antibody produced using a recombinant mammalian expression system, for example, a monoclonal antibody against the antigen CD,.,52.

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, E, 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.

The 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). The four framework regions largely adopt a fl-sheet conformation and the CDRs form loops and in some cases comprising part of, the fl-sheet structure. The CDRs are held in close proximity by the regions and. with the CDRs from the other domain, contribute to the connecting. framework formation of the antigen binding site.

The antigen CDwS2 (G.Hale g; 31. Tissue Antigens 1990 lfi, pp 118-127) is an abundant molecule widely distributed on most, if not all, human lymphocytes. It is also present on the surface of the majority of malignant lymphocytes, but not haemopoietic cells, nor is it expressed on granulocytes, platelets, erythroid or myeloid bone marrow cells. A number of monoclonal antibodies of different isotypee have been raised against this antigen and reported in the literature, (G.Hale g; 31 Tissue Antigens, 1990, 11, pp 118-127). One of these antibodies. an Igcl antibody, has been humanised (Nature. 1988, 322, 323-327 and EPD328404). This antibody is known as Campath 1H (Campeth is a trademark of The Wellcome Foundation Ltd). A preparation of this antibody has been used to treat patients suffering from gen Hodgkins' lymphoma. (G.Ha1e g; 31, Lancet. 1988, pp 139$-1399).

Campath 1B was originally purified in a one-step process on a Protein A Sepharose column (EP032840A). Protein A is a group specific ligand which binds to the Fc region of immunoglobulin: other therefore immunoglobulins contained in serum present in the culture medium with the interest thereby Maths. in will co-purify immunoglobulin of contaminating the end product (P.A. Underwood g; 31.

Enzymol. 1.21 pp. 301-306 (1986). and as. Underwood at 51. J.Immunol.

Meths 6_0, 33, 1983). 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, serum sickness and in severe cases anaphytactic shock. 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.

Other contaminants in the culture medium containing the expressed antibody, include host cell and viral nucleic acid. Also aggregates of antibodies as they too may act as immunogens and cause an undesirable immune response.

The present invention therefore provides a process for obtaining a purified preparation of an anti-CDw52 IgG antibody prepared using a recombinant expression system, which process comprises: (a) applying an aqueous solution of the antibody to a Protein A or Protein G column so as to absorb the antibody onto the column and then eluting the antibody with an acidic solution of a pH of from 3.0 to 3.5. (b) applying the acidic eluate to an ion-exchange column of charged particles so as to absorb the antibody onto the column and then eluting the antibody with an aqueous solution of counter-charged ions; and (c) applying the aqueous eluate to a size exclusion column of porous particles so as to retain non—antibody molecules in the porous particles and to obtain the desired antibody in selected fractions eluted from the column which contain less than 2% of antibody aggregate as measured on size-exclusion chromatography.

We also describe a purified preparation of an anti-CDw52 lgG antibody prepared using a recombinant expression system, which preparation is obtainable by a process according to the invention. Such preparations exhibit: (i) on size exclusion chromatography: a single peak under non-reducing conditions and two major peaks under reducing conditions; (ii) on conventional SDS polyacrylamide gel electrophoresis: one main band using a non—reduced sample and two main bands using a reduced sample; and (iii) on reversed phase HPLC: a single peak under non-reducing conditions and two major peaks under reducing conditions.

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. This analytical technique is particularly good for determining levels of aggregate in the purified preparation. The stationary phase is a wide pore silica gel which may be modified with diol groups preferably a gel such as Zorbax GF4SO-GP250 (Trademark of Dupont) or TSK gel C3000 SWXL or C4000 SWXL. The mobile phase is generally in the -pH range h-8 more preferably 6-7.5 advantageously around pH6.8. This phase is advantageously a mixture of a phosphate such as disodium hydrogen orthophosphate and a sulphate such as sodium or potassium sulphate and water. The molarity of such a mixture is _generally 2SmM to 1M most preferably around S0mM.

SDS polyacrylamide gel electrophoresis (SDS PAGE) gives information about the number and type of proteins present in a mixture. their relative abundance and a measure of their molecular weights. SDS is an anionic detergent, it is reacted with the proteins before electrophoresis. Most protein SDS complexes are soluble and will migrate through a polyacrylamide gel towards the anode, under the influence of an electrical charge. Rate of migration is generally inversely related to the logarithm of the molecular weight of the protein. It is convenient to carry out the SDS analysis on a gradient gel which may be flat bed or vertical slabs or rods. The gradient is advantageously 10-22% or more preferably B-18%. The gel is preferably Pharmacia Excel (Trademark) gel.

Reversed phase HPLC separates on the basis of hydrophobitity. As with other HPLC techniques there is a polymeric stationary phase, of for example polystyrene/divinylbenzene. The mobile phase is usually a combination of a weak aqueous buffer or a dilute acid and a water miscible organic solvent. For effective separation of proteins the mobile phase is generally a gradient system, required to achieve separation and is preferably linear for convenience.

The stationary phase for analysis of immunoglobulin may be an organic polymeric matrix such as Polymer Labs PLRP-S generally of particle size around 8pM; the pore size is preferably 30nm or 100nm (BOOA or 1000A). advantageously a mixture of an acid such as formic, acetic or trifluoroacetic acid, The mobile phase is water and acetonitrile. The acid and the water are preferably present in the ratio 5:3.

An antibody can be reduced to its component heavy and light chains by reduction of the disulphide bonds under denaturing conditions with for example Guanidinium chloride and dithiothreitol. Subsequent alkylation of the free thiol groups, for example with iodoacetamide iodoacetic acid, assists in preventing the bonds from reforming.

A measure of purity is provided by the specific activity of the antibody preparation.

Specific activity may be determined by the method set out in the Examples. A preparation purified according to the process of the invention preferably has a specific activity of greater than 0.8 Kilo Units/mg, ideally greater than 0.9 Kilo Units/rng, most preferably around 1.0 Kilo Units/mg.

A purified preparation of an anti-CDw52 antibody purified according to the process of the invention ideally, is substantially free from host cell contaminants such as host cell proteins, nucleic acids and endotoxins. Specific activity provides information about the levels of host cell protein in the preparation. Endotoxin levels may be measured by the LAL (Limulus Amoebocyte Lysate) method described in Parenteral Quality Control, M.J. Alles Lat; Marcel Dekker lnc., New York.

A preparation purified according to the process of the invention is also essentially free from aggregate, as measured on size exclusion chromatography. It is desirable for these levels to be less than 2%, ideally less than 0.5%.

The antibody is prepared using a recombinant expression system. The preferred system is a mammalian expression system using Chinese hamster ovary (CHO) cells. These may be dihydrofolate reductase (dhfr) deficient and so dependent on thymidine and hypoxanthine 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 hypoxanthine, 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 concomitant amplification, and therefore increased expression of the desired antibody gene.

Another expression system for use with CHO or myeloma cells is the glutamine synthetase (GS) amplification system described in W087/04462. This system involves the transfection of a cell with a gene encoding the GS enzyme and the desired antibody gene. medium. These selected clones are then subjected to inhibition of the GS enzyme Cells are then selected which grow in glutamine free using methionine sulphoximine (Msx). The cells, in order to survive, will amplify the GS gene with concomitant amplification of the gene encoding the antibody.

Anti CDw52 antibody is preferably obtained in a form in which it is secreted in to the culture medium. The harvested medium may then be filtered and/or concentrated by an ultrafiltration step to obtain an aqueous solution which is subjected to a purification procedure comprising: (a) applying an aqueous solution of the antibody thus produced to a Protein A or Protein G column so as to absorb the antibody onto the column and then eluting the antibody with an acidic solution; (b) applying the acidic eluate to an ion—exchange column of charged particles so as to absorb the antibody onto the column and then eluting the antibody with an aqueous solution of counter-charged ions; and (c) applying the aqueous eluate to a size exclusion column of porous particles so as to retain non-antibody molecules in the porous particles and to obtain the desired antibody in selected fractions eluted from the column.

Protein A is a group specific ligand which binds to the Fc region of most lgG. It is synthesised by some strains of Staphylococcus aureus and can be isolated from culture supematants then insolubilised by coupling to agarose beads or silica. An alternative method is to use whole bacteria of a strain which carries large amounts of protein A on the bacterial cell surface. Both types of gel preparation are available commercially. (Protein A — Pharmacia. Whole bacteria Calbiochem, |gG sorb).

(Alan Johnstone and Robin Thorpe lmmunochemistry in practice, Blackwell Scientific Publn. Chpt. 10). An alternative to Protein A is Protein G (Analytical Chem. Vol. 61 (13) 19891317).

The column which is most preferably used is a Protein A Sepharose (Trademark) column particularly Protein A Sepharose Fast Flow (Trademark). Ideally the column is washed with tris or phosphate buffered saline around pH7.0 and the antibody is eluted at acid pH 3.0 — 3.5 advantageously pH 3.0 using an acid such as citric acid for example in a concentration of about 0.1M.

Ion-exchange chromatography 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. It is preferable to use cross-linked columns based for example on agarose for example S-Sepharose Fast Flow (Trademark) cation exchange (Trademark).

The column column cation exchange particularly S.Sepharose Fast Flow Alternatively a membrane-based colum could be employed. is usually washed after application of the eluate from the Protein A column, with 20mM HEPES buffer pH 7.5 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. large molecules are excluded from entering the porous stationary phase In general separation occurs when and are carried straight through the colum while progressively smaller molecules are increasingly able to enter the stationary phase and consequently have particularly longer elution times. It is the stationary phase which therefore determines the chemically bonded and porosity of the separation achieved. Suitable materials are provide resistance to compression for example an agarose and/or dextran composition such as Superdex (Trademark). A preferred column The eluate from the ion is a Superdex 200 size exclusion medium. exchange column is preferably applied to the Superdex colum and developed in buffer in the range pH5-8 preferably PBS pH 7.2.

Each colum is preferably protected by a filter which may be a 0.2p Gelman Acro sterilising filter or in the case of the Protein A column a PALL posidyne SLK 7002 NFZP or a FALL DSLK2 filter (available from Pall Process Filtration Ltd. European House, Havant Street, Portsmouth 301 3PD) and for the other two columns e Hillipak filter preferably Millipak 100 for the ion exchange column and Millipak 20 or 60 for the size exclusion column (available from Millipore, The Boulevard, Blackmore Lane, Watford, Herts.) The columns are preferably sanitised before use with an appropriate sanitant for example 0.5M Na0H for 16 hours for any of the columns, or 2% hibitane gluconate in % ethanol for the Protein A column or 1M NaOH for the other two columns.

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.

Additional steps may be added to the purification procedure set out above.

Ultrafiltration may be used to further reduce viral and host cell nucleic acid contamination. This may be carried out using commercially available ultrafiltration units such as Viresolve/70’ or Viresolve/180’ membranes additionally, PLMK regenerated cellulose 300k cut off membrane all available from Millipore, The Boulevard, Blackmore Lane, Watford. Herts. An alternative method to reduce virus contamination is microflltration using a Nylon membrane in cartridge form for example Nylon 66.0.04um membrane from PALL.

A purification step to remove contaminating DNA may be introduced, for example, a wash of the Protein A column using NaCl in the range 1M-3M in buffer at neutral pH preferably PBS at pH7.2. Glycine may be added to the NaCl preferably at about 1.5M in the pH range 8.8-9.0.

Recombinant DNA technology has provided the ability to develop altered antibodies of two basic types. The first type, referred to as chimeric antibodies, is where the rodent constant domains only are replaced by equivalent domains of human origin (Morrison 91 gl, P.N.A.S., 1984, Q, 6851-6855; Boulianne gt al, Nature, 1985, gfl, 268-270; and Neuberger gt gl, Nature, 1985, Q1. 268-270). The second type is where the murine constant domains and the murine framework regions are all human patient and so do not elicit an anti-antibody response to the same degree.

A human antibody could also be used.

Accordingly the purified anti-CDw52 antibody may be a rat, mouse or human antibody wherein the amino acid sequences of the heavy and light chains are homologous with those sequences of antibody produced by the species lymphocytes 13 mg or ig _\_'_it£ by hybridomas. Preferably a anti-CDw52 antibody is an altered antibody such as a hybrid antibody in which the heavy and light chains are homologous to a natural antibody but are combined in a way that would not occur naturally. The antibody may be chimaeric antibody which has variable regions from one antibody and constant regions from another. Thus, chimaeric Such chimaeric antibodies may have one or more further modifications to improve antibodies may be species/species chimaeras or class/class chimaeras. 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 addition to the CDRs are transferred to the human framework. Additional amino acids in the framework or constant regions of such antibodies may be altered. Thus, we describe any anti-CDw52 altered antibody in which the amino acid sequence is not one which exists in nature. However, CDR-grafted antibodies are most preferred of which Campath 1H (Trademark of the Wellcome Foundation Ltd.) is an example. The antibody chain DNA sequences including the CDRs of Campath 1H are set out in EP0328404. We also describe a purified preparation of an anti-CDw52 antibody wherein the antibody comprises one or more of the CDR sequences set out in EP0328404.

Purified anti-CDw52 antibodies are useful in medical therapy for treating numerous human disorders, generally as immunosuppressives more particularly for example T-cell mediated disorders including severe vasculitis, rheumatoid arthritis, systemic lupis, also autoimmune disorders such as multiple sclerosis, graft vs host disease, psoriasis, juvenile onset diabetes, Sjogrens’ disease, thyroid disease, myasthenia gravis, transplant rejection and asthma. These antibodies are also useful in treating cancers such as non-Hodgkins lymphoma and leukaemias.

We also describe the use of a purified preparation of an anti-CDw52 antibody in the manufacture of a medicament for the treatment of any of the aforementioned disorders. A human being having any such disorder can be treated by administering to said individual a therapeutically effective amount of a purified preparation of an anti-CDw52 antibody.

The dosages of such antibodies will vary with the condition being treated and the recipient of the treatment, but will be in the range 1 to about 100mg for an adult patient, preferably 1-10 mg, usually administered daily for a period between 1 and days. administered for 5-10 days followed by 6-15mg for a further 5-10 days.

A two part dosing regime may be preferable wherein 1-5mg are Also described are formulations containing a purified preparation of an anti CDw52 antibody. Such formulations preferably include, in addition to antibody, a physiologically acceptable diluent or carrier possibly in admixture with other agents such as other antibodies and antibiotics. Suitable carriers include but are not limited to physiological saline, phosphate buffered saline, phosphate buffered saline glucose and buffered saline. Alternatively, the antibody may be lyophilised (freeze—dried) and reconstituted for use when needed, by the addition of an aqueous buffered solution as described above. Routes of administration are routinely parenteral including intravenous, intramuscular, subcutaneous and intraperitoneal injection or delivery.

The accompanying drawings show: (Figures 2 to 6 relate to a purified preparation of Campath 1H).

Figure 1 (a) the pLD9 construct containing expression cassettes for the ‘crippled’ dhfr selection/amplification marker and the Campath-1H light chain cDNA. The small box with the dashed arrow is the weakened SV40 promoter; the larger dotted box with an arrow is the [3-actin promoter; po|yA refers to respectively sourced polyadenylation and termination signals; the small box with ori contains the SV40 origin of replication; (b) the pNH316 construct containing expression cassettes for the neomycin selection marker and the Campath-1H heavy chain cDNA. The box with an arrow and MT refers to the mouse metallothionein promoter. Restriction sites indicated are:- H, Hindlll; Bg, Bgtll; B, BamHl; R1, EcoR1.

Figure 2 SDS polyacrylamide gel of non-reduced and reduced Campath 1H showing a single main band.

Figure 3 Reversed phase high performance chromatograph of non-reduced Campath 1H showing a single peak. .13.

Eizurs_é.

Reversed phase high performance chromatograph of reduced and carboxymethylaced Campath 1H showing two resolved peaks corresponding to the heavy and light chains of the antibody.

Eizure_i.

High performance size exclusion chromatograph of non-reduced Campath lh showing a single peak. £iznre_i.

High performance size exclusion chromatograph of reduced Campath 1H —showing two major peaks.

P t o o Ca o c ggguzgg LA; Cloning gfi gag Heagz and Ligng Chain ggflgs fig; gagpgtn-lfi The complementarity determining regions from the rat Campath-lG monoclonal were originally grafted directly into genomic human heavy and light chain frameworks (Winter e5_gl, flggggg, 1988, 312, 323-327).

These constructs were engineered for expression in the myeloma cell line Y0 and resulted in yields of Campath-1H of up to Spg/ml following and l.A-l.7kb for the light and heavy chain CDNA were screened Presa,0xford). The -‘-‘Q. for the variable regions to isolate full light chain cDNA, the 5' leader was removed up to position -32 using Bal-31 exonuclease Hindlll linker added. For the 3' end, use was made of a unique SacI site 47bp upstream of the A SacI-HindIII oligonucleotide pair was used to regenerate this sequence and position the HindIII site imediately after the stop codon» For the 5' end of the heavy chain cDNA, the unique Ncol site overlapping the ATG start re-build a 29bp untranslated leader, identical to translated probes specific length cDNA clones. For the untranslated and a stop codon. synthetic codon was used to that of the light chain. using a HindIII-Ncol oligonucleotide pair.

At the 3' end, the unique Nael site 12bp downstream of the stop codon was converted into a HindIII site using linkers; mmmm converted to a Bg1II site using linkers. site was subsequently To isolate the human fl-actin polyadenylation and termination signals from pHflAPr~3-neo. an Sphl site l.4kb downstream of the unique HindIII site was converted to a The basal dhfr vector called ploh, was Sphl site at position -128 in the SVAO 1981, 1, enhancer promoter in pSV2dhfr remove all expression unit was To construct pLD9, the p104 vector was digested with BamHI, phosphatased. and ligated with three other fragments consisting of the BglII-HindIII 5-actin promoter, the HindIII Campatholfl light chain cDNA and the HindIII-Bamhl fl—actin polyA/termination signals. To The dhfr- cao cell line oux-311 {Urlaub g_c_a_l, E.u.a.s., 1950, 11, 4216-4220) was grown in Iscove's HEM supplemented with 10: fetal bovine serum. and Aug/ml each of hypoxanthine and thymidine. 10p; of pLD9 and pNH3l6 was co-precipitated onto cel phosphate method. (German 5.5.31. I2.‘lA_9.12ninz. 1 -Academic Press,N.Y.) and selected for the ls using the calcium 985, Vol II. 143-190, double phenotype of dhfr+/neo resistance by using the medium above except that 10% dialysed serum was used, the hypoxanthine/thymid G418 (Gibco) was included at SO0ug/ml. the In som included directly in first round s transformants. Several hundred resistant assayed for the production of Campath-lH ant colonies ine were omitted, and e experiments MIX was election for dhfr+ were pooled and ibody in the culture medium. The average yield was 0.5;;/ml for non-amplified first round transformants.

Each pooled cell population was then cultured in MTX. and after two weeks, resistant colonies w titred for Campath-1H production. in yield of up to 80-fold (Table 1). These cloned. screened for Campath-1H yield, and two isolated, called A37 and 3D9 (Table 1). These further in the presence of l0’6H MTX, then The increase in expression screened as above. was not so dramatic final, amplification stage nevertheless, when re-fed at confluence and left There was a considerable the presence of l0'7M ere again pooled and increase cells were dilution high producer lines were both amplified dilution cloned and at this second. and as seen previously; for a further 4 days, the cell lines A39 and 3Dll were capable of producing up to 200pg/ml of Campath-1H.

‘ Accuulated Construct Selection stage ' Campath-1H (pg/ml) pLD9 + pNH3l6 dhfr+/neo basal pool 0.5 1o'7n MIX amplified pool 13.40 Cell lines A37 and 3D9 40 1o‘6M nrx amplified pool so-9o Cell line A39 100 Cell line 3Dll A 150-200 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-1H 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 107. The yield from the 3D1l cell line reflects a productivity of l00ug/106 cells/day. . ;7 .

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 l0.9M to lO'7H. 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-1H antibody titres and the results are shown in Table 2. As the MTX level was increased, there was a marked decrease in the number of surviving dhfr+_ colonies. but they express proportionately more Campath-lH. Thus. in la one selection at high concentrations of MTX, it is possible to isolate cell populations which produce up to 60-fold increase in antibody yield compared to step. direct _ cell populations selected for basal dhfr levels.

IALE_2 Accumulated Selection (M HTX) dhfr+ colonies Campath-1H (pg/ml) No MTX S00 0.5 3 x 1o’9 40 2 o'3 5 7 3 x 1o’8 5 3o ‘above, were considerably _ Colonies at each MTX selection stage were pooled and assayed as described in the legend of Table l. following another was repeated This selection co-transfection of cells, and in this instance. the entire population was selected in medium containing G418 and 3 X l0-8M MTX. This generated a larger pool of resistant colonies which were subsequently procedure pooled and re-amplified twice more using MIX concentrations of 5 X lO'7M, then 3 X l0 EM. At this stage, the cells were dilution cloned The two‘ highest producer cell of producing antibody levels up to and screened for Canpath-1H levels. lines isolated were capable 100-lS0pg/ml and were designated as lines hF1l and 5E10.

The growth rates of these cell lines, and the A39/3Dll lines described slower than the parental non-transformed dhfr' CHO cells. This is usually a cannon feature of these cells once they have been engineered to express high quantities of a product gene. The yields from the SE10 and 4F1l cell lines proved to be quite variable over time, and the latter appeared to have only a limited passage life lasting about 3 weeks before entering crisis and death.

This instability was not evident at all in the other cell lines, although in general, the lines isolated from the second amplification procedure, including SE10, were usually more fickle to culture. of all the lines. the 3Dll coupled good growth and stability with high the Campath-lH yields. To ensure the propagation of these features, Dll cell line was dilution cloned once more to generate the 3Dll* line and this similarly produced Campath-1H yields up to 200ug/ml. ;x.amp.ls_2.

..* C ow u C Ah ClH 3Dll* cells growing as a monolayer in Iscoves + 10! FBS Flow non-essential amino acids. l0'6h Methotrexate and antibiotics were approximately 90! confluent. These cells were removed from the .19. plastic with trypsin/versene. washed in Iscoves medium without supplements centrifuged and resuspended at S x 104/ml in WCHA medium set out in Table below + 0.25% peptone + 0.1% polyethylene glycol (PEG) 10,000 +0.51 fetal bovine serum (FBS) without methotrexate (MIX). Three 25cm2 flasks were set up with 10ml of cell suspension + hypoxanthine (H), thymidine (T) or HT. These flasks were incubated at .s°c in st coz incubator.

After six days, 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 7Scm2 flask. " ' ‘" These cells were used to seed a 500ml Techno spinner. incubated at .5°C spinning at 40 rpm. Cells continued growing serum free for a -period of over five months and although it was found that the cells needed a period or adaptation. the growth rate and viability steadily to be 73.1 this decreased to 47.4 hours over improved. The population doubling time was calculated hours over approximately 7 weeks: the subsequent 20 days then stabilised. Antibody secretion remained high at levels in excess of 60 ug/ml. It was determined that the gene copy number in these cells did not decrease according to band intensity using Northern blot analysis.

In fermenters, these cells produced antibody in excess of 70pg/ml and regularly achieved levels of l0Oug/ml or more. These cells are donated c1a 3911* 44. . 20 .

Iscoves DMEM (Iscoves N and Melcher (1978), J.Exp.Med. 1, 47, ) modified to exclude BSA, transferrin and lecithin.

S ml/litre + 200mM L glutamine + 50 mg/litre L proline + 50 mg/litre L threonine + 50 mg/Litre L methionine + 50 mg/litre L cysteine + 50 mg/litre L tyrosine + 25 mg/litre ascorbic acid + 0.062 mg/litre vitamin B6 + 1.36 mg/litre vitamin B12 + 0.2 mg/litre Iipoic acid + 0.088 mg/litre methyl linoleete + luu methotrexate + 1 mg/litre Peso" + 1 mg/litre znsoa + 0.0025 mg/litre CuS04 + 5 mg/litre recombinant insulin (Nucellin) + 50,000 Iu/litre polymyxin + 20,000 Iu/litre neomycin + 0.16 mg/litre putrescine-2 HCL.

Clfl 3D1l*44 cells from previous serum-free for over 2 months were stage which had transferred to a Sci 1 litre been growing fermenter with a stainless steel angled paddle turning at 70rpm. The temperature was set at 37°C, doz at 10% and pH control to 7-7.2. The fermenter was seeded on day 0 with 0.22 x 106 cells/ml in VCM4 medium with 0.1% polyethylene glycol (PEG) 10,000 and 0.25% soy peptone, and . was top gassed with 02. The cells were routinely passaged using fresh medium and a split rate typically between 1 to 2 and 1 to 4.

On day 33 the top gassing was replaced with deep sparging which is expected to cause more physical damage to the cells.

On day 50 onwards VCM5 (see Table below) was used together with peptone and PEG instead of WCM4.

On day 53 the PEG was replaced with 0.1% pluronic F68. The resulting growth and antihbdy levels achieved were in excess of lOOug/ml in fermenters.

EMLHSJJM Iscoves DMEM modified to exclude BSA. transferrin and lecithin. + 5 ml/litre mm L glutamine + 50 mg/litre L proline + 50 mg/litre L threonine + 50 mg/litre L methionine + 50 mg/litre L cysteine + 50 mg/litre L tyrosine + 25 mg/litre L ascorbic acid + 0.062 mg/litre Vitamin 36 + 1.36 mg/litre Vitamine 312 + 2 mg/litre Ferric citrate + 1 mg/litre Zinc sulphate + 0.0025 mg/lit Copper sulphate + 50,000 IU/litre Polymyxin + 20,000 IU/litre Neomycin + 3 pl/litre Ethanolamine + 0.16 mg/litre Putrescine + 5 mg/litre Recombinant Insulin (Nucellin) . 22 .

All components in WCM4 and WCH5 are commercially available. £zsanls_l The purification method used was based on chromatggraphy through three coluns. The gels used were 7.8Sml Protein A Sepharose 4 Fast Flow, Pharmacia code No. 17Oh (10cm x 1cm); 7.85 ml 5 Sepharose Fast Flow cation exchanger, Pharmacia code No. l701 (l0cm x lcm); and 120ml Superdex 200 size exclusion medium, Phsrmscia code No.17O1 (60cm x l.6cm). sterilising filter.

The hardware of the protein A column system was washed through with IN The Each column was protected by a 0.2 pm Gelman Acro NaOH and left in this solution for 2h hours to remove endotoxin. _gel was then packed into the Pharmacia C10/20 column and sanitized with 2% hibitane gluconate in 20% ethanol. Since, according to the manufacturer, 8 Sepharose and Superdex 200 gels are both stable in IN Naofl for prolonged periods these gels were packed into their columns (a Pharmacia C10/20 and a C16/100 column respectively) washed through with 1N NaOH and then left to stand in this solution for 24 hours to remove endotoxin and sanitize the column systems.

The solutions for column operation and sanitization were manufactured using pyrogen free distilled water and sterile filtered to 0.2 pm through Millipore Millipack 100 filters. Samples of all solutions were assayed for endotoxin by LAL test and only those with low values subsequently used. .material was washed from. the _at room .23.

Qolmnmnautian Eggggig 5 figphggggg 4 E33; Elgw ggl, Tissue culture medium from Example 1 containing Campath 1H antibody was supplied and filtered to 0.2pm through a sterile PALL posidyne SLK7002 NFZP filter in a Sealkleen housing. The 2% hibitane gluconate in 203 removed with distilled water and the system equilibrated with the tris buffered saline pH 7.5 (T.B.S.). with 1.75 litres of crude Campath 1H (7l.4mg) at a flow rate of 300 cm/hour (235 ml/hour) at a temperature of 20°C 3 5°C. Unbound column system. with 5 bed volumes (39.25ml.) of T.B.S. pH 7.5 at the same flow rate. The Protein A gel was eluted at 300 cm/hour with 0.1M citric acid pH 3.0 for <24 hours monitored at A280nm sanitize the protein A column th81'tO1 used to system was The protein A column was then loaded temperature. The elution profile was using a Pharmacia UVI single path monitor and the protein peak isolated. The elution peak volume was l8.9ml and lml removed and assayed for Campath 1H by ELISA as described below. of this was Se Fa w The 1N Na0H was washed from the S.Sepharose column system with 20mM Hepes pH 7.5 until the column washings were at pH 7.5. the colum at a The remaining l7.9ml. of Protein A column eluate was loaded onto flow rate of ’300 cm/hour (235ml/hour). Unbound material was washed from the column system with 7 bed volumes (5Sml) of 20mH Hepes pH 7.5 at the same flow rate. The S.Sepharose gel was eluted by a step elution, using 0.2M NaCl in 20mM Hepes pH 7.5 cm/hour. The elution peak was collected by trace using a Pharmacia Eluate collection at a flow rate of 300 UV1 monitor at A280nm (Zmm path length 0.5 AUFS). was started at approximately 20% deflection and continued until the .race had declined to 70% deflection. The elution volume was 10ml and lml of this was sampled for assay for Campath 1H by ELISA as described below. _ . 24 . iu2erdsx_ZQ9_zal The 1N NaOH was washed from the Superdex colum system with PBS pH 7.2 until the column washings were at pH 7.2. The remaining 9ml of S Sepharose eluate was loaded onto the Superdex column with a syringe via a Hillipore Hillex GV filter and the filter washed through with 2ml of PBS pH .7.2. The column was developed with PBS pH 7.2 at 30 cm/hour (60ml/hour). The size exclusion peeks were monitored using a Pharmacia UVI monitor at A280nm. the aggregate peak from the monomer peak.

As the peaks eluted fractions were taken in order to separate the monomer peak'fraction had a volume of 17.6ml. £nzxma_Linkad_Immn2a2:hant_esaax.L£LI£el This is a standard Sandwich Enzyme Immunoassay (lnihxznnna in which anti-human Igc, made from imune-purified goat antiserum, is attached to the solid phase as a capture layer. Detection of captured antigen (Campath lfl Ig) is achieved with a peroxidase - labelled goat anti-human Igc. The assay is sequential with samples of Campath 1H being diluted in a buffer containing casein and hydrolysed gelatin.

Incubation periods of 1 hour and 30 minutes, are used at temperature of 37°C. 3',3',5,s' Tetramethylbenzidine (run) hydrogen peroxide substrate are added to reveal any bound peroxidase. be determined and Campath 1H chromsgen plus Optical densities at 450nm can concentrations read from a standard curve of known concentrations The protein content of the monomer peak was estimated at Azflonm using an extinction coefficent (E 131:") of 1.35 (optionally 1.32) and a sample examined for aggregate content by HPLC size exclusion colum.

The remaining material was sterile filtered through a Hillipore Millex CV filter (0.2um pore size) and filled into 29 0.5ml aliquots in sterile Sarstedt tubes. The majority of the semis tubes were stored at 4°C however 6 tubes were stored at -7006. Samples from é°C storage were sent for assays as detailed in the following examples.

The Campath ELISA results are shown in the table below. £amnssh_1H Sample Titre Vol.(ml) Total mg Wgt.C1H 8 by ELISA of bulk C13 in applied recovery/ * * " bulk ' next col. column Crude 60.8ug/ml 1750 71.4 71.4 ---- -- ‘Protein A é.2mg/ml 18.9 79.4 75.2 111.2 eluate S sepharose 6.4mg/ml 10.0 64.0 57.6 85.0 eluate Superdex 2.Smg/ml 17.6 44.0 ---- 76.4 IIIOIIOIIIB 1’ The overall recovery across the three column system, based on the recovery across each colum is 61.6%.

Eamnle Enzml Crude 1.25 <0.625 Superdex monomer peak Example 4 Conventional SDS polyacrylamide gel electrophoresis was carried out on a flatbed 8-18% gradient Pharmacia Excelgel. The results are shown in Figure 2.

Example 5 Characterisation of Campath 1H by reversed phase high performance liquid chromatography.

A 50u| sample of the product of Example 3 (designated G-Dev-95) in phosphate buffered saline (PBS) at a concentration of 2.4mg/ml was subjected to reversed phase high performance liquid chromatography (RP~HPLC) under the following conditions: Column: PLRP-S O.1um (1000A) (pore size); 8pM (particle size) 15 x 0.46 cm from Polymer Laboratories Ltd UK.

Mobile phase utilised a formic acid/water/acetonitrile system: Formic acid: water (5:3) CH3CN Component A in the following gradient: % A 80 80 65 0 O 80 80 % B 20 20 35 100 100 20 20 Time (mins) 0 5 38 41 50 51 65 The column was run at ambient temperature at a flow rate of 1ml/min" and UV detection on LDC Spectro Monitor D variable wavelength was carried out at a wavelength of 280nm with a sensitivity of 0.1 a.u.f.s..

Result As can be seen in Figure 3 chromatography of Campath 1H utilising the above system gave a single sharp peak. The peaks eluting after 30 minutes are due to the mobile phase.

Example 6 Characterisation of reduced and carboxymethylated Campath 1 by reverse phase high performance liquid chromatography (RPHPLC). a. Reduction and carbo>_ Campath 1H may be reduced into its component heavy and light chains by utilising standard reduction and carboxymethylation procedures, which firstly reduce the disulphide bonds and prevents them reforming by alkylating the free thiol groups.

To 1ml Campath 1H G Dev-95 from Example 3, in phosphate buffered saline at a concentration of 2.4 mglml was added 1m| of 8M Guanidinium chloride in 0.5M Tris/HCI pH 9.0 buffer and 120pl of 7% Dithiothreitol in the same buffer. The mixture was incubated for two hours at 37°C.

After incubation 120ul of 9% iodoacetic acid in 0.5M Tris/HCI buffer was added and the mixture was left in the dark for 1 hour. The resulting reduced carboxymethylated material was designated Campath 1H RCM. b RP HPLC characterisation of Campath 1H RCM of the material from Example 6a was subjected to reverse phase high performance liquid chromatography under the following conditions.

Column PLRP-S 0.1um (1000A) (pore size); 8pm (particle size) 15 x 0.46 cm from Polymer laboratories Ltd. UK.

Mobile phase used a water, formic acid and acetonitrile gradient as depicted in the table below: A - Formic acid B - water C - Acetonitrile z A '50 so 29.4 o 0 so 0 a 3 35 35 20.6 o o 35 35 s c_ 15 15 so 1oo 1oo 1s 15 Time (mins) 0 , 5 7o 72 32 32 . 1 95 The colum was run at a flow rate of lml/min at ambient temperatures and followed by UV absorbance at a wavelength of 280nm a.u.f.s.

B:-.9211; As can be seen from Figure 4, the resulting material resolves into two peaks, corresponding to the heavy and light chains.

Essmzl..¢_.Z Characterisation of Campath lH by high performance size exclusion chromatography for the determination of high molecular weight components in Campath 1H.

A 50ul sample of the product of Example 3 (G-Dev -95) in phosphate buffered saline (at a concentration of 2.hmg/ml) was subjected to high performance size exclusion chromatography under the following conditions. colun - TSK gel C3000 Swxl 30cm x O.78cm i.d.

Hobile phase - 0.05M Na2H P04 + 0.1M N42 S04 adjusted with H3PO4 to pH 6.8 . to heavy and light. chains in expected ratio.

Flow rate - 0.75 ml/min.

The column was run for twenty four minutes at ambient temperature and followed by UV absorbance at a wavelength of 280 nm.

A second 50p1 sample using Campath 1H reduced in accordance with Example 6a) was analysed by the same method.

Results: The results in Figure 5 show a clean single peak indicating low levels of aggregate. Levels of between 0.5 and 2.0% are generally The results in Figure 6 show two main peaks The peaks at total achieved. corresponding permeation volumm (ca.l5-l8 minutes) are due to reagents. co a The complement lysis assay is a measure of antibody function expressed as specific activity, determined by the ability of a purified preparation of an anti-CDW52 antibody of known concentration to bind to a pre-determined number of cells and effect cell lysis.

The assay is carried out on Campath lfi using Karpas 422 cells (established from B-cell non-Hodgkin lymphoma cell line - Dyer at al (1990) Blood, 1} 70h-714) expressing Campath the cell surface. 1.2 x 107 cells were loaded with radiolabel by incubating antigen on for 2 hours at 37°C in a C02 incubator in the presence of 600uCi of 51 Cr (sodium chromate). .3 ml of the loaded cells in medium (total volume 23.5ml), were added to l2.5ml of normal human serum and 150u1 of the mixture were pipetted into the wells of a microtitre plate. .30. pl samples of the final eluate from three purification runs were mixed with the cells and incubated for 30 minutes at 4°C followed by 90 minutes at 37°C. was centrifuged at 2000 rpm for 5 minutes and the radioactivity in l00p1 of cell supernatant was counted Complement lysis activity in Kilo Units/ml was The culture on a game counter. calculated from a standard curve of a reference preparation (1000 Units/ml)._ The results are set out in Table 3.

The concentration.of Campath 1H in the Sopl samples of final eluate was estimated using.samples in PBS pH 7.2 read on a spectrophotometer at 280nm. The results are expressed in Table 3 as optical density in mg/ml.

From this data the specific activity in Kilo Units/mg is determined by using the equation: Kflzml The results indicate that purified preparations of Campath 1H are functional.

Claims (1)

1.CLAIMS A process for obtaining a purified preparation of an anti-CDw52 lgG antibody prepared using a recombinant expression system, which process comprises: a) applying an aqueous solution of the antibody to a Protein A or Protein G column so as to absorb the antibody onto the column and then eluting the antibody with an acidic solution of a pH of from 3.0 to 3.5; b) applying the acetic eluate to an ion-exchange column of charged particles so as to absorb the antibody onto the column and then eluting the antibody with an aqueous solution of counter-charged ions; and c) applying the aqueous eluate to a size exclusion column of porous particles so as to retain non-antibody molecules in the porous particles and to obtain the desired antibody in selected fractions eluted from the column which contain less than 2% of antibody aggregate as measured on size exclusion chromatography. A process as claimed in claim 1, wherein the said aqueous solution of antibody has been obtained by harvesting the antibody from a culture medium and subjecting the harvested medium to filtration and/or concentration by an ultrafiltration step. A process as claimed in claim 1 or 2, wherein the column in step (a) is washed with tris- or phosphate-buffered saline at a pH around 7.0 and then the antibody is eluted at a pH of from 3.0 to 3.5. A process as claimed in any one of the preceding claims, wherein the aqueous solution of the antibody is applied to a Protein A column in step (a). A process as claimed in claim 4, wherein the column is eluted with citric acid. A process as claimed in any one of the preceding claims, wherein the ion- exchange column is a cation-exchange column in step (b). A process as claimed in any one of the preceding claims, wherein the antibody obtained in step (c) is subjected to ultrafiltration. A process as claimed in any one of the preceding claims. wherein the antibody is prepared using CHO cells. A process as claimed in any one of claims 1 to 7, wherein the antibody is prepared from CHO or myeloma cells using the glutamine synthetase amplification system. A process as claimed in any one of the preceding claim, wherein the antibody is a chimeric antibody. A process as claimed in any one of claims 1 to 9, wherein the antibody is a CDR-grafted antibody. A process as claimed in any one of claims 1 to 9, wherein the antibody is a human antibody. A process as claimed in any one of the preceding claims, wherein step (0) is carried out to obtain the desired antibody in selected fractions eluted from the column which contain less than 0.5% of antibody aggregate as measured on size exclusion chromatography. A process as claimed in any one of the preceding claims further comprising formulating the purified preparation with a physiologically acceptable diluent or carrier. A process according to claim 14, wherein the formulation is Iyophilized. A process substantially as hereinbefore described with reference to the