ADSORBENTS FOR PROTEIN PURIFICATION Field of the Invention

This invention relates to compounds and their use as affinity ligands for protein purification. Background of the Invention

Antibodies are immunoglobulin glycoproteins having a basic unit of a monomer structure. The monomer is a Y-shaped protein that consists of four polypeptide chains, two of which are identical heavy chains and two are identical light chains connected by disulphide bridges. There are five different types o! heavy chain (y, μ, α ₃ ε and δ) that distinguish the immunoglobulin classes (IgG, IgM, IgA, IgD and IgE, respectively). There are also two different types of light chain (A and K) resulting from different gene products.

IgG (a monomeric immunoglobulin approximately of 150 kD in size) provides antibody-based immunity against invading pathogens and, due to the high specificity that IgG has towards specific antigens within the body, it is the most commonly used reagent in immunological research and clinical diagnostics.

Monoclonal antibodies (herein termed Mabs) are antibodies that have identical specificity towards a single antigen and are generated from a cell line that has been produced from a single cloned cell. Mabs constitute the fastest growing sector in the biopharmaceutical industry where is it estimated that sales will reach $30 billion (US) by 2010. Antibody litres from mammalian cell cultures have continued to improve over the last 20 years and aiternative downstream processes and chromatography adsorbents are required to resolve the process bottlenecks in the processing of Mabs.

Antibody fragments (parts of whole antibody molecules) offer several advantages over whole antibodies. They are easier and more cost effective to manufacture, they have fewer side-effects in patients, by reducing the risk of cytokine release and its associated toxicity, due to the absence of the Fc (heavy chain) region, and they can be modified to include therapeutic payloads. There are several types of antibody fragments that are either lgG ^! domains prepared by specific endopeptidase enzyme digestion or that have been genetically engineered in cell lines. These include monovalent fragments such as Fab', Fab and scFv; bivalent fragments such as F(ab ^! ) ₂ diabodies and msntbodies; and multivalent fragments such as triabodies and fetrabodies.

Many antibody fragment products are in devefopment for use as therapeutics or in diagnostics. Recombinant antibody fragments are expected to have a significant share of the $6 billion (US) per year diagnostic market, from in vitro immunoassays to in vivo tumour and clot imaging applications (Hoiiiger, P., & Hudson P.J., Nat. Biotech 23 (9; 2005) 1126-

Most antibody fragment products iack a protein A-bϊnding site and therefore, unlike full-chain antibodies, cannot be purified by protein A affinity chromatography. In most instances, conventional chromatography techniques are used to purify antibody fragments. Protein L ₅ a protein with a molecular weight of 35000 Daltons derived from a bacterial species of Peptostreptococcus magnυs, is known to bind to antibody light chains and has been investigated for the purification of some antibody fragments but is not considered to be cost- effective and is not available in commercia! quantities.

WO97/1G887 discloses triazine-based compounds, useful as affinity adsorbents, of formula I

wherein R ₁ is H, alky!, hydroxyalkyl, cyclohexyl, NH ₂ , phenyl, naphthyl, 1-ρhenylpyrazole, indazoie, benzthiazole, benzoxazole or benzimidazote, any of which aromatic groups can be substituted with one or more of aikyl, alkoxy, acytoxy, acylamino, amino, NH ₂ , OH, CO ₂ H, sulphonyl, carbamoyl, suiphamoyl, alkylsuIphonyS and halogen; one X is N and the other is N, C-CI or C-CN;

Y is O, S or NR ₂ ;

Z is O, S or NR ₃ ;

R ₂ and R ₃ are each H ₁ alkyi, hydroxyaikyl, benzyl or β-phenylethyl;

Q is benzene, naphthalene, benzthiazole, benzoxazoie, 1 -phenylpyrazoie, indazoie or benzimidazole;

R ₄ , R ₅ and R ₆ are each H, OH, alky!, alkoxy, amino, NH ₂ , acyloxy, acylamino, CO ₂ H, sulphonic acid, carbamoyl, suiphamoyl, alkylsulphonyl or halogen; n is O to 6; p is O to 20; and A is a support matrix optionally linked to the X-containing ring by a spacer.

Compounds of formula ! are disclosed as having affinity for proteins such as immunoglobulins, insulin. Factor VII or human growth hormone.

Compounds of related structure are disclosed in WO00/67900 and WO03/097112. They have affinity for endotoxins.

Certain trϊazine-based compounds disclosed in WG97/1Q887 have affinity for immunoglobulins. An example of a compound showing such affinity is a compound of structure !!

Compounds such as II are able to remove immunoglobulins specifically from complex mixtures or feedstocks such as human plasma.

Another type of commonly encountered feedstock is industrially produced cell culture supernatant, in which monoclonal antibodies are present at concentrations up to 5 g/i of supernatant. Compounds such as I! may also be useful for specific removal of monoclonal antibody from these mixtures, although their performance is known to be compromised by the presence of cell culture additives such as PIuronic F-68.

Pluronic F-68 is an anti-foaming agent commonly used used in mammalian ce)! culture, it is a biock copolymer of poiyoxyethylene and polyoxypropytene, and has a molecular weight of approximately 8000 Da. Pluronic F-68 is used to protect cells from shear and air bubble damage, and is typically used in an amount of 1 g/L in cell culture supernatants. Its presence may reduce or abolish the ability of compounds such as S! to remove immunoglobulins from such feedstocks, which represents a considerable obstacle to the use of such ligands for direct capture of monoclonal antibodies from mammalian eel! culture media.

Summary of the Invention

Surprisingly, it has been found that certain compounds, many of which are novel, are useful for affinity-based purification of immunoglobulins, including but not limited to monoclonal antibodies and antibody fragments, even in the presence of compounds such as Piuronic F-68. Compounds for use in the invention are of formula I!!:

/ ⁵

wherein R-. is H, alkyi, aryi, hydroxyaikyl, cyclohexyi amino or a heterocyclic group e.g naphthyS, 1-phenyipyrazole, indazole, benzthiazole, benzoxazole or benzimidazole, any of which aromatic groups may comprise a further fused ring And can be substituted with one or more of alkyi, aryl, alkoxy, aryloxy, acyloxy, acylamino, amino, OH, CO ₂ H, sulphonyl, carbamoyl, suiphamoyi, alky!suiphony! and halogen, one X is N and the other is N, C-CI or C-CN,

Y is O, S or NR ₂₅

Z is O, S or NR ₃ ,

R ₂ and R ₃ are each H, aikyi, hydroxyalkyi, benzyl or β-phenylethyi; Q is benzene, naphthalene, benzthiazole, benzoxazole, 1-phenyIpyrazoie, indazole or benzimidazole,

R ₄ , R ₅ and R _s are each H, OH, alkyi, aryi, heterocyclic alkoxy, aryloxy, amino, acyloxy, acySamsno, CO ₂ H, sulphonic acsd, carbamoyl, suiphamoyi, alkylsuSpnonyi or halogen, or two or more of R ₄ , R ₅ and R ₆ are linked to form a cyclic structure, U and V are the same or different C-i ₁₀ straight-chain aϊkyiene groups optionally substituted by one or more of hydroxy!, alky!, aryl, hydroxyaikyl, β-phenylethyl and halogen such as CHOH, and

A is a support matrix optionally linked to the X-containing ring by a spacer

Further compounds of the invention include the corresponding ligands, in whsch A is replaced by a functional group, linked dfrectSy or indirectly to the tπazsne ring, which can be immobilised on a support matπx The terms isgand" and "adsorbent" may be used interchangeably, below Description of the Invention

WO97/10887, WO00/67900 and WO003/097112 disclose how combinatorial libraries of iigands can be haul on a solid support. Thesr disclosures, including examples of embodiments and procedures common to the present invention, are incorporated herein by reference During the screening of a set of these combinatorial libraries with a feedstock containing albumin, immunoglobulins and PIuronic F-68, a number of ligands were identified as being capable of selectively binding and eluting immunoglobulins, Compounds of formula III, for use in the invention, can be prepared by procedures known to those skilled tn the art Such procedures are described in the 3 PCT publications identified above, they can be readily adapted to the preparation of new compounds

In compounds for use m the invention, rt is preferred that R and/or QR ₄ R ₅ R ₆ is or includes a cyclic structure, erther or each cyclic structure preferably has a OH or SO ₃ H substituenf Preferably, each X ss N Further it is preferred that U and/or V is substituted, e g ss CHOH Such substituted compounds are novel

Preferred immunoglobuling-binding ligands or adsorbents of the invention are of formulae IV-XISl:

T he immunoglobulin-binding ligands described herein are useful for the purification immunoglobulins from complex mixtures including, but not limited to, human plasma and

recombinant fermentation supematants. This utility is demonstrated below in Example 2, oy chromatography experiments using a number of feedstocks.

The term "immunoglobulin" ss used herein to describe intact immunoglobulins themselves, including IgG, IgA, IgM and IgE, and also analogues that have the functional or structural characteristics of immunoglobulins, e.g in terms of affinity to a given compound described herein Thus, the anaiyte may be a protein that is a functional fragment of an immunoglobulin, or a structural analogue having one, more or all of the same binding sites, or a fusion protein

The optional Nnker may comprise any means of attaching lsgands of the invention to support matrices and providing a means of spacing the ligand from the surface of the support matrix The support matrix may comprise any material, soluble or insoluble, particulate or non-particulate, including fibres and membranes, porous or non-porous. It provides a convenient means of separating lsgands of the invention from solutes in a contacting solution. Examples of support matrix and optional linker A include carbohydrate matrices such as agarose, cellulose, dextran, starch, alginate or carrageenan, synthetic polymer matrices such as polystyrene, styrene-dtvmySbenzene copolymers, polymethacrylates, (e g, poly(hydroxyethylmethacfylate), polyvinyl alcohol, poiyamides or perfluorocarbons, inorganic matrices such as glass, silica or metai oxsdes;and composite materials The following Examples illustrate the invention

Example 1 - Synthesis of Adsorbents

The synthesis of adsorbents of the type described is explained in WO97/10887, WO00/67900 and WG003/097112 The synthesis of Adsorbent Xf described and is typical 6% cross-linked PuraBead agarose gel (650 g settled in RO water) was slurried with

RO water (650 mL), 10 M sodium hydroxide (NaOH) {88 ml), and epichSorohydnn (124 ml), The slurry was stirred over 19 hours Further 10 M sodium hydroxide (NaOH) (22 mL), and epichlorohydrsn (37 mL) was then added and the slurry stirred over 1.5 hours. After a sample was taken for analysis, the slurry was filtered then washed with RO water (12 x 1 L) Analysis for epoxy groups showed that the gel was deπvatised with 21 6 μmoi epoxy groups per g of settled gel

The gel was drained before RO water (780 mL) and 0.88 specific gravity ammonia solution (200 mL) were added The mixture was stirred and heated to 40 ⁰ C, then stirred at this temperature over 18 hours After a sample was taken for analysis, the slurry was filtered and then washed with 12 x 1 L RO water (12 x 1 L) TNBS analysis for amine groups showed that the ge ^s was deπvatised with 20 8 μmol amine groups per g of settled gel

Settled aminated gel (475 g) was slurried in 1 M potassium phosphate (475 ml_) and allowed to settle. 1 M potassium phosphate (140 mL) was then added, the mixture stirred vigorously, and acetone (70 mL) added. The mixture was cooled to O ⁰ C in an ice sail bath, before cyanuric chloride (11.9 g) in cold acetone (120 mL) was added in one portion The siurry was stirred over 1 hour at 0-4 ⁰ C, before being drained, then washed wrth 50% v/v aqueous acetone (5 x 500 mL), RO water (5 x 500 mL) ₃ with 50% v/v aqueous acetone (5 x 500 ml), and RO water (1O x 500 mL). Analysis revealed the attachment of 25 μmol dichlorotriazine groups per g of settled gei

The dichlorotπazinyl agarose (50 g) was slurried in RO water (55 mL) NorphenySephπne hydrochloride (1.99 g) was dissolved in RO water (15 mL), 10 M NaOH (0.95 mL) was added, and the mixture was cooled on see, prior Io addition to the dichlorotπaztnyi agarose. The mixture was reacted at 6θ'C over 19 hours. The gel was washed with 50% DMF (5 x 100 mL), RO water (5 x 100 mL), 0 1 M HCt (5 x 100 mL), 30% IPA/0.2 M NaOH (5 x 100 mL), RO water (10 x 100 mL), and 20% aqueous ethanoi (3 x 100 mL) before storage in the coid room in 20% aqueous ethanoi Example 2 - Chromatography

Chromatography experiments were performed wrth each of the adsorbents tabulated in Table 1. For all experiments a 1 cm diameter column was used with a bed height of 5 5 cm and column volume (CV) of 4.3 mL with a linear flow rate of 300 cm/h. The adsorbent was initially equilibrated wrth 10 CV of phosphate buffered saline (PBS), pH 7 4, and then loaded wrth pure IgG, IgG feedstock 1 (1 g/L IgG, 1 g/L Plurømc F-68, and other proteins to mimic eel culture supernatant) or 2 (1 g/L !gG, 1 g/L Pluronsc F-8θ wrth 5% foetal calf serum), or murine IgGi feedstock up to a concentration of 30 g/L of adsorbent The adsorbent was then washed with 10 CV of PBS, pH 7 4, before the IgG was eiuted wrth 5 CV of 50 my citric acid, pH 3.5. The adsorbent then underwent a ciean in place (CiP) with 5 CV of 0 5 M sodsum hydroxide followed by re-equslιbratιon of the adsorbent with 7 CV of PBS, pH 7 4

Subsequent to the chromatography experiment, the IgG content of the ioad, post- load wash, elution and CIP fractions were assessed by nephelometry, A280, HPLC or GPC, to assess the binding and etuison capacities and SDS PAGE analysis to assess purity The biπdmg and elutson capacities are summarised in Table 1

Pure IgG feed contained 1 g/L of polyclonal IgG sn PBS, pH 7 4 m the presence or absence of 1 g/L Pluroπic F-68 Mock feedstock 1 contained 1 g/L polyclonal IgG, 1 g/L horse skeletal myoglobin, 5 g/L human serum albumin and 1 g/L Pluroπic F-68 in CHO cell culture medsum. Mock feedstock 2 contained 1 g/L polyclonal IgG, 5% foetal bovine serum and 1 g/L Pluronic F-68 in GHO ceil cuϊture medium

Table 1

ESution buffer 50 mM citric acid, pH 3.5 with 30% ethylene glycol and 2 M NaCi.

The chromatographic performance of adsorbent Xl was further investigated, to assess the purification capability of the materia!. Experiments were completed using a 1 cm diameter column with a bed height of 2 5 cm and column volume (CV) of 2 0 mL and a linear flow rate of 50 cm/h (3 minute residence time) The adsorbent was initially equilibrated with 10 CV of phosphate buffered saline (PBS), pH 74. 60 mL o! IgGi in a CHO (Chinese Hamster Ovary) cell culture supernatant was loaded onto the column to a concentration of 54 g/L of adsorbent The adsorbent was then washed with 10 CV of PBS, pH 74, before the IgG was eluted with 5 CV of 50 mM sodium crtrate at pH 3 0 The adsorbent then underwent a clean in place (CfP) with 5 CV of 0 5 M sodium hydroxide followed by re- equilibration of the adsorbent with 7 CV of PBS ₅ pH 7 4 Fractions (2 mL) were collected throughout the chromatography and analysed for IgG content (Protein A HPLC) DNA content (Picogreen analysis) and total protein (Bradford total protein assay) The breakthrough profile of IgG ₁ for adsorbent Xl shows the binding capacity to be 21 8 g/L and the elutϊon capacity to be 20 9 g/L Using gel permeation chromatography, the pυπty of the eluted IgG was determined to be 92.8% and adsorbent Xl has a 2 log clearance of DNA

Chromatography experiments were completed with adsorbent XS using antibody fragments prepared by enzyme (pepsin) digestion Pepsin is a non-specific βndopeptidase that is only active at acid pH and is irreversibly denatured at neutral or alkaline pH Pepsin digestion results in the generation of one F(ab') ₂ fragment and severai small peptides of the Fc fragment Fragments o! human, ovsne and bovine polyclonal antibodies (mixed population of antibodies) were prepared by contacting the IgG with pepsin for 1 hour at 37 ⁰ C at pH 40 The digestion was halted by adjusting the pH above 7 0, and the F(ab') ₂ fragments were separated by ge! filtration

The chromatographic performance of adsorbent Xl was investigated to assess the purification capability of the materia! for antibody fragments Experiments were completed using a 1 cm diameter column with a bed height of 2 5 cm and column volume (CV) of 2.0 mL with a linear flow rate of 50 cm/h (3 minute residence tsme) The adsorbent was initially equilibrated with 10 CV of phosphate buffered saline (PBS), pH 7 4. Approximately 20 mg of F(ab') ₂ fragments were loaded per mL of adsorbent. The adsorbent was then washed with 10 CV of PBS, pH 7 4, before the fragments were eiuted with 5 CV of 50 mM sodium citrate at pH 3 0 The adsorbent then underwent a clean in place (CiP) with 5 CV of 0 5 M sodtunn hydroxide followed by re-equilibration of the adsorbent with 7 CV of PBS, pH 7 4 The controS for all experiments was Protein L adsorbent fusing the same experimental conditions as for adsorbent Xl) Each fraction from the chromatography column was collected and analysed ussng Western blot techniques This technique indicated that adsorbent Xl bound both hunan kappa and lambda iight chasn and ovine and bovsne F(ab') ₂ fragments, protein L b^nds only human kappa light chain and does not tand ovine and bovine fragments