WO2012020080A2 - LIGANDS FOR ANTIBODY AND Fc-FUSION PROTEIN PURIFICATION BY AFFINITY CHROMATOGRAPHY II - Google Patents

LIGANDS FOR ANTIBODY AND Fc-FUSION PROTEIN PURIFICATION BY AFFINITY CHROMATOGRAPHY II Download PDF

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WO2012020080A2
WO2012020080A2 PCT/EP2011/063831 EP2011063831W WO2012020080A2 WO 2012020080 A2 WO2012020080 A2 WO 2012020080A2 EP 2011063831 W EP2011063831 W EP 2011063831W WO 2012020080 A2 WO2012020080 A2 WO 2012020080A2
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Prior art keywords
ligand
antibody
protein
aromatic ring
substituted
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PCT/EP2011/063831
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French (fr)
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WO2012020080A3 (en
Inventor
Holger Bittermann
Klaus Burkert
Marc Arnold
Oliver Keil
Thomas Neumann
Inge Ott
Kristina Schmidt
Daniel Schwizer
Renate Sekul
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Graffinity Pharmaceuticals Gmbh
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Publication of WO2012020080A2 publication Critical patent/WO2012020080A2/en
Publication of WO2012020080A3 publication Critical patent/WO2012020080A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
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    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
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    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
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    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3253—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure not containing any of the heteroatoms nitrogen, oxygen or sulfur, e.g. aromatic structures
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    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
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    • B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
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    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
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    • C07D213/72—Nitrogen atoms
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    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/38—Nitrogen atoms
    • C07D277/44—Acylated amino or imino radicals
    • C07D277/48—Acylated amino or imino radicals by radicals derived from carbonic acid, or sulfur or nitrogen analogues thereof, e.g. carbonylguanidines
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    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
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    • C07D277/68—Benzothiazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
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    • C07D285/01—Five-membered rings
    • C07D285/02—Thiadiazoles; Hydrogenated thiadiazoles
    • C07D285/04—Thiadiazoles; Hydrogenated thiadiazoles not condensed with other rings
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    • C07D285/02—Thiadiazoles; Hydrogenated thiadiazoles
    • C07D285/04—Thiadiazoles; Hydrogenated thiadiazoles not condensed with other rings
    • C07D285/12—1,3,4-Thiadiazoles; Hydrogenated 1,3,4-thiadiazoles
    • C07D285/125—1,3,4-Thiadiazoles; Hydrogenated 1,3,4-thiadiazoles with oxygen, sulfur or nitrogen atoms, directly attached to ring carbon atoms, the nitrogen atoms not forming part of a nitro radical
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    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/62—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
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Definitions

  • the present invention relates to the field of protein separation, preferably the purification of monoclonal and polyclonal antibodies and fusion proteins containing an immunoglobulin Fc segment, by affinity separation techniques, in particular chromatography using small molecule ligands.
  • Immunoglobulins are a class of soluble proteins found in body fluids of humans and other vertebrates. They are also termed “antibodies” and play a key role in the processes of recognition, binding and adhesion of cells. Antibodies are oligomeric glycoproteins which have a paramount role in the immune system by the recognition and elimination of antigens, in general bacteriae and viruses.
  • the polymeric chain of antibodies is constructed such that they comprise so-called heavy and light chains.
  • the basic immunoglobulin unit consists of two identical heavy and two identical light chains connected by disulfide bridges. There are five types of heavy chains ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ), which determine the immunoglobulin classes (IgA, IgG, IgD, IgE, IgM).
  • the light chain group comprises two subtypes, ⁇ and ⁇ .
  • IgGs are soluble antibodies, that can be found in blood and other body fluids. They are built by B-cell derived plasma cells as response to and to neutralize bacterial or other pathogens.
  • An IgG is an Y-shaped glycoprotein with an approximate molecular weight of 150 kDa, consisting of two heavy and two light chains. Each chain is distinguished in a constant and in a variable region. The two carboxy terminal domains of the heavy chains are forming the Fc fragment ("constant fragment"), the amino terminal domains of the heavy and light chains are recognizing the antigen and are named Fab fragment ("antigen-binding fragment").
  • Fc fusion proteins are created through a combination of an antibody Fc fragment and a protein or protein domain that provides the specificity for a given drug target. Examples are domain antibody-Fc fusion proteins, where the two heavy chains of the Fc fragments are linked either to the variable domains of the heavy (V R ) or light chains (V L ) of specific antibodies. Other Fc fusion proteins are combinations of the Fc fragment with any type of therapeutic proteins or protein domains. The Fc part is considered to add stability and deliverability to the protein drug.
  • Therapeutic antibodies and Fc fusion proteins are used to treat various diseases, prominent examples include rheumatoid arthritis, psoriasis, multiple sclerosis and many forms of cancer.
  • Therapeutic antibodies can be monoclonal or polyclonal antibodies. Monoclonal antibodies are derived from a single antibody producing cell line, showing identical specificity towards a single antigen. Possible treatments for cancer involve antibodies that are neutralizing tumour cell specific antigens. Bevacizumab (Avastin, Genentech) is a monoclonal antibody which neutralizes the vascular endothelial growth factor (VEGF), thereby preventing the growth of new blood vessels into the tumour tissue.
  • VEGF vascular endothelial growth factor
  • Therapeutic fusion proteins such as Etanercept (Enbrel, Amgen, TNF-Receptor domain linked to Fc fragment) or Alefacept (Amevive, Biogen poute, Biogen poute, Biogen poute, Biogen poute, Biogen poute, Biogen poute, Biogen poute, Biogen poute, Biogen poute, Biogen poute, Biogen poute, Biogen pout, Biogen pout, LFA-3 linked to Fc portion of human IgGl) are used or developed as drugs against autoimmune diseases.
  • Etanercept Enbrel, Amgen, TNF-Receptor domain linked to Fc fragment
  • Alefacept Amevive, Biogen poute, Biogen pout, LFA-3 linked to Fc portion of human IgGl
  • Protein bioseparation which refers to the recovery and purification of protein products from various biological feed streams is an important unit operation in the food, pharmaceutical and bio technological industry. More and more therapeutic monoclonal antibodies (Mabs) and fusion proteins are entering the market or are currently in clinical development. Such proteins require an exceptionally high purity which is achieved by elaborate multi-step purification protocols. Downstream processing and purification constitute about 50 to 80 % of the manufacturing cost, hence considerable efforts are under way to develop new or improve existing purification strategies (1).
  • Affinity chromatography is one of the most effective chromatographic methods for protein purification. It is based on highly specific protein-ligand interactions. The ligand is immobilized covalently on the stationary phase which is used to capture the target protein from the feed stock solution. Affinity ligands can bind their target with high specificity and selectivity, enabling up to thousand fold higher enrichment at high yields even from complex mixtures.
  • affinity chromatography on protein A is the first step in most Mab and Fc fusion protein purification schemes. Protein A is a cell wall associated protein exposed on the surface of the bacterium Staphylococcus aureus.
  • Protein A binds with nanomolar affinity to the constant part (Fc domain) of immunoglobulins from various species, in particular to human subtypes IgGl, IgG2 and IgG4 (2).
  • the use of Protein A is limited by leaching into the product and poor stability under harsh conditions applied for sanitization and cleaning in place procedures.
  • the chemical stability of Protein A can be improved by using genetically engineered Protein A variants for Mab purification.
  • the high costs of Protein A resins have resulted in the search for suitable alternatives, in particular selected from small molecules.
  • Mabsorbent A2P (Prometic Biosciences) is a small molecule ligand for the purification of immunoglobulins. However, the ligand does not show an appropriate selectivity (3) and is not applied in process chromatography.
  • Another approach uses mixed mode chromatography as primary capture step in antibody purification.
  • the most common material is based on immobilized 2-mercaptoethylpyridine (MEP HyperCel, Pall Corporation), which effectively captures IgG from fermentation broth but shows lower clearance of host cell proteins (HCP) in comparison to protein A. (4).
  • Synthetic affinity ligands that are more readily available, preferably cheaper than protein- based ligands, are more robust under stringent conditions and have a selectivity comparable to or even higher than Protein A would provide a suitable solution for antibody and Fc fusion protein purification.
  • Synthetic small molecule affinity ligands are of particular interest for the purification of therapeutic proteins due to their generally higher chemical stability and their lower production costs. Synthetic affinity ligands that are more readily available, preferably cheaper than protein-based ligands, are more robust under stringent conditions and have a selectivity comparable to or even higher than Protein A would provide a suitable solution for antibody and Fc fusion protein purification. Depending on the target protein, such affinity ligands should preferably offer the same broad applicability as Protein A, recognizing the constant Fc region of IgG type immunoglobulins and Fc fusion proteins. The problem underlying the present invention is the provision of a matrix comprising a small affinity ligand binding to the Fc region of antibodies and Fc fusion proteins.
  • the present invention is directed to the use, for affinity purification of a protein, preferably an antibody or fragment of an antibody, of a ligand-substituted matrix comprising a support material and at least one ligand covalently bonded to the support material, the ligand being represented by formula (I)
  • L is the linking point on the support material to which the ligand is attached
  • Sp is a spacer group
  • v is 0 or 1 ;
  • R 1 is hydrogen or Ci to C 4 alkyl, preferably hydrogen or methyl; and more preferably hydrogen;
  • R is hydrogen or Ci to C 4 alkyl, preferably hydrogen or methyl; and more preferably hydrogen;
  • Ar 1 is a divalent 5- or 6-membered substituted or unsubstituted aromatic ring
  • Ar is 5- or 6-membered aromatic ring which may be unsubstituted or which is
  • Ar 1 may also be denoted AR 1
  • Ar2 may also be denoted AR 2.
  • Sp may also be denoted V. This is the nomenclature as used in the patent application EP 10172684.2 of which the present application claims priority. Still, R 1 and R 2 as defined in the context with formula (I) correspond to R 4 and R 5 , respectively, as defined and shown in the said priority document.
  • Ligands according to the present invention bind to polyclonal and monoclonal IgG of human origin, in particular human IgGi, IgG 2 and IgG 4 and polyclonal and monoclonal IgGs from different animal species such as rabbit and mouse immunoglobulins.
  • the present invention is also directed to the ligand-substituted matrix, and the ligands (which may also be termed "compounds") attached to the matrix at the linking point L, optionally via a spacer group Sp.
  • L in the above formula (I) is the linking point, also referred to as "point of attachment".
  • point of attachment The person skilled in the art is aware of appropriate linking points/points of attachment.
  • linking point L is either directly connected with the support material or via a spacer.
  • L is part of a moiety resulting from the reaction of an appropriate functional group on the support material with a corresponding functional group on the precursor compound to form the ligand.
  • Examples of functional groups on the support material include carboxylic acid, epoxide, amine, hydroxyl, maleimide, aldehyde, ketone, sulfonic acid, hydrazine, oxime ether, thiol, azide, and alkyne groups.
  • L is a bond, preferably a single bond, which is directly attached to the support material, in general via an appropriate functional group on the material.
  • support material refers to polymers known to the person skilled in the art and available on the market which lend themselves for the purposes of the present invention. A definition of "support material” is provided below.
  • the support material comprises functional groups for the attachment of molecules, in general those of the present invention.
  • the functional groups on the support material are regarded as a part of the support material; this also applies in cases where the ligands of the present invention are connected to the support material via a bond (i. e. L is a bond and does not comprise a spacer group Sp, see below), and wherein the bond is directly formed between the respective ligand according to the invention and the functional group on the support material.
  • "Precursor groups” or “functional groups” in the present context are groups which are capable of a chemical reaction with complementary “precursor groups” or “functional groups”, under formation of a chemical bond. If necessary, additional reagents may be used for the formation of said chemical bond.
  • L is connected to a spacer group -Sp-.
  • L is a bond or a chemical unit resulting from the reaction of an appropriate functional group on the support material with an appropriate functional group (or a "precursor group”) on the spacer group Sp. Accordingly, the chemical reaction between the functional group on the support material and the functional group (or “complementary group") on the spacer group Sp connects the ligand according to the invention with the support material via the linking point L.
  • v in formula (I) is 0, then the ligand is directly bonded to L. In other embodiments the ligand is bonded to the support material via a spacer group Sp. This applies when v in formula (I) is different from 0.
  • the spacer group Sp preferably is a hydrocarbon group which may contain, in addition to C and H atoms, further atoms. Appropriate further atoms are known to the person skilled in the art. Preferred atoms include O, S, N, P, Si.
  • the spacer group Sp preferably contains a connecting unit/final functionality which covalently connects the ligand with the support material via linking point L.
  • connecting unit/final functionalities are known to the person skilled in the art.
  • the connecting unit is attached to a carbon chain that may be interrupted by one or more heteroatoms such as oxygen atoms or carboxamide groups.
  • the connecting unit is attached to an alkylene or an alkylene - polyoxyalkylene group such as -CH 2 -CH 2 -(0-CH 2 -CH 2 )x- with x ranging from 1 to 10, preferably from 1 to 6.
  • the functional groups on the spacer group are attached to a hydrocarbon group which may contain one or more heteroatoms such as N, O, P, S, Si.
  • the spacer group therefore is a hydrocarbon group containing one or more heteroatoms preferably selected from. N, O, P, S, Si.
  • Appropriate chemical entities/chemical units contained in Sp are known to the person skilled in the art.
  • Sp in general contains at least one unit selected from alkylene, alcohol, amine, amide, carbonyl, alkyleneoxy and phosphate.
  • the alkylene unit preferably has 1-30, preferably 1-12, in particular 1-6 carbon atoms.
  • the alcohol can be a primary, secondary or tertiary alcohol.
  • the amine can be a non organic or an organic amine.
  • the amine furthermore, can be a diamine or a triamine, preferably a diamine.
  • the amide can be a non organic or an organic amide, and can be derived from a diamine or a triamine, preferably a diamine.
  • the alkyleneoxy unit can comprise a linear or a branched alkylene unit.
  • the alkyleneoxy unit is preferably an ethylenoxy or a propyleneoxy unit, preferably an ethylenoxy unit.
  • the unit can also comprise two or more of the above cited entities/units, e.g. an amino acid.
  • the amino acid can be a natural or a non natural amino acid.
  • the amino acid can be linear or branched. Branched amino acids may serve as a branching point, enabling the connection of more than one ligand moiety to one point of attachment ("L").
  • Examples for preferred amino acids include 6-aminohexanoic acid, 8-amino-3,6- dioxaoctanoic acid, 5-amino-3-oxapentanoic acid, glycine, beta-alanine, lysine, ornithine, glutamic acid, 2,3-diaminopropionic acid. 2,4-diaminobutyric acid and serine.
  • the spacer group Sp may consist of one unit as described before, or of more than one unit. If the spacer group Sp consists of more than one unit as described before, said units are interconnected by connecting groups. Such connecting groups include but are not restricted to amide, urea, hydrazide, oxalic diamide, oxime, hydrazone, triazole, thiourea, isourea, ether, with amide and urea being preferred.
  • a phosphate group may as well serve as a connecting group.
  • the phosphate group may be linked to one, two or three units as described before. If the phosphate group is linked to three units as described before, it may serve as a branching point, enabling the connection of more than one ligand moiety to one point of attachment C'L").
  • n is an integer from 1-6, i.e. 1, 2, 3, 4, 5 or 6.
  • Further preferred units of the spacer group Sp include but are not restricted to amino acids, which may be natural or unnatural amino acids, in particular 6-aminohexanoic acid, 8-amino-3,6-dioxaoctanoic acid, 5-amino-3-oxapentanoic acid, lysine and glutamic acid.
  • the named units and functional groups are thus part of the spacer group Sp and of the linking point L.
  • spacer groups Sp are -NH-(CH 2 ) n NH-, -NH-((CH 2 ) 2 0) n - (CH 2 ) 2 -NH-, wherein n is integer from 1-30, preferably 1-12. In one embodiment, n is an integer from 1-6, i.e. 1, 2, 3, 4, 5 or 6.
  • Typical examples for preferred spacer groups Sp include the following:
  • the total length of the spacer group Sp is less than 100 atoms, preferably less than 60 atoms, in particular less than 30 atoms.
  • Sp may also be branched.
  • the term "branched" in the present context means that more than 1 ligand of the present invention according to formula (II) is present on the spacer group Sp. This embodiment allows the linkage of more than one ligand to one matrix attachment point (linking point) L.
  • “Final functionality” or “connecting unit” in the present context designates a unit formed in the reaction between the precursor groups (functional group) on the support material and on the ligand of the invention and/or on the spacer group Sp.
  • the connecting unit incorporates the bond between the support material and the ligands of the present invention (which may include the spacer Sp), resulting in the ligand-substituted matrix.
  • Connecting units incorporated in L and/or Sp are known to the person skilled in the art and may include the following groups: ether, thioether, C-C single bond, alkenyl, alkynyl, ester, amine, amide, carboxylic amide, sulfonic ester, sulfonic amide, phosphonic ester, phosphonic amide, phosphoric ester, phosphoric amide, phosphoric thioester, thiophosphoric ester, secondary amine, secondary alcohol, tertiary alcohol, 2-hydroxyamine, urea, isourea, imidocarbonate, alkylthiosuccinic imide, dialkylsuccinic imide, triazole, ketone, sulfoxide, sulfone, oxime ether, hydrazone, silyl ether, diacyl hydrazide, acyl sulphonyl hydrazide, N- acyl sulphonamide, hydra
  • the ligand is directly bonded to L. In other embodiments the ligand is bonded to the support material via a spacer group Sp.
  • R 1 and R 2 are each independently selected from: hydrogen; and Ci to C 4 alkyl, typically linear and branched Ci to C 4 alkyl which may comprise a cycloalkyl unit such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec- butyl, tert-butyl, cyclopropyl, methylcyclopropyl.
  • R 1 and R 2 are each independently selected from hydrogen and methyl. More preferably, R 1 and R 2 are all hydrogen.
  • Ar 1 can be an alicyclic or heterocyclic aromatic ring.
  • Ar 1 is an alicyclic aromatic ring, i.e. Ar 1 is phenylene. If Ar 1 is a heterocyclic aromatic ring it comprise one, two or more heteroatoms which are preferably selected from N, S, O and combinations thereof.
  • Examples of appropriate 5- or 6-membered heterocylic aromatic rings include but are not restricted to pyrrole, furan, thiophene, imidazole, oxazole, isoxazole, thiazole, isothiazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole (4H-l,2,4-triazole and lH-l,2,4-triazole), lH-tetrazole, 2H-tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, 1,2,3-triazine, 1,2,4-triazine, and 1,3,5-triazine.
  • Preferred 5- or 6-membered heterocylic aromatic rings are imidazole, thiazole, and pyridine with pyridine being a preferred embodiment.
  • the divalent 5- or 6-membered aromatic ring including the preferred embodiments described above may be substituted or unsubstituted. Typically the 5- or 6-membered aromatic ring is substituted, preferably monosubstituted.
  • appropriate substituents are Ci to C 4 alkoxy, typically linear and branched Ci to C 4 alkoxy which may comprise a cycloalkyl unit such as methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy, tert-butoxy, cyclopropoxy, methylcyclopropoxy, and cyclobutoxy; Ci to C 4 alkyl, typically linear and branched Ci to C 4 alkyl which may comprise a cycloalkyl unit such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, tert-butyl, cyclopropy
  • Ar is 5- or 6-membered aromatic ring which may be unsubstituted or may be
  • the 5- or 6-membered aromatic ring of Ar is alicyclic, i.e. it is a benzene ring, or it is heterocyclic and comprises at least one heteroatom which is preferably selected from N, S, O and combinations thereof. More preferably, the 5- or 6-membered heterocyclic aromatic ring contains two or more nitrogen atoms or a nitrogen atom and an oxygen atom or a nitrogen and a sulfur atom.
  • Examples of appropriate first 5- or 6-membered heterocyclic aromatic rings of Ar directly bonded to the NR group group in formula (I) include but are not restricted to pyrrole, furan, thiophene, imidazole, oxazole, isoxazole, thiazole, isothiazole, pyrazole, 1,2,3-triazole, 1,2,4- triazole (4H-l,2,4-triazole and lH-l,2,4-triazole), lH-tetrazole, 2H-tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, 1,3,4-thiadiazole, 1,2,3-triazine, 1,2,4-triazine, and 1,3,5- triazine,.
  • Preferred 5- or 6-membered heterocylic aromatic rings are thiazole, oxazole, 1,3,4- thiadiazole, pyrazole, imidazole, isoxazole, pyrrole, furan, 1,2,4-triazole, pyridine, 1,2,4- triazine, pyridazine, and pyrimidine, with thiazole, oxazole, 1,3,4-thiadiazole, and pyrazole being most preferred.
  • a thiazole ring the NR group is typically bonded to the thiazole ring in 2-position.
  • an oxazole ring the NR group is typically bonded to the oxazole ring in 2-position.
  • the NR group is typically bonded to the oxazole ring in 2-position.
  • the NR group is typically bonded to the pyrazole ring in 3-position, 4-position or 5-position, preferably in 3-position.
  • the NR group is typically bonded to the pyridine ring in 3 position (5 position).
  • the first 5- or 6-membered aromatic ring is attached to a further 5- or 6-membered aromatic ring ("second aromatic ring of Ar " in the following) via a single bond.
  • second aromatic ring of Ar in the following
  • first aromatic ring of Ar in the following
  • first aromatic ring of Ar is benzene, thiazole, pyrazole or pyridine.
  • first aromatic ring of Ar in the following
  • the NR group and the second aromatic ring are typically positioned meta to each other.
  • the NR group is typically bonded to the thiazole ring in 2-position.
  • the second aromatic ring is preferably bonded to the thiazole ring in 4-position.
  • the NR group is typically bonded to the pyrazole ring in 3-position.
  • the second aromatic ring is preferably bonded to the pyrazole ring in 5-position.
  • the NR group is bonded to the pyrazole ring in 5-position and the second aromatic ring is bonded to the pyrazole ring in 3-position.
  • the NR group is bonded to the pyrazole ring in 3-position and the second aromatic ring is bonded to the pyrazole ring in 1 -position.
  • the first aromatic ring is fused to a second 5- or 6-membered ring, preferably aromatic ring as part of a multicyclic, preferably bicyclic ring system.
  • a benzene ring as first aromatic ring and being part of a fused ring system
  • it is preferably fused to the second ring via the ortho and meta position or meta and para position
  • the NR group is typically bonded to the oxazole ring in 2-position.
  • the oxazole ring is preferably fused to the second ring via the 4- and 5-position.
  • a thiazole ring as first aromatic ring and being part of 2
  • the NR group is typically bonded to the thiazole ring in 2-position.
  • the thiazole ring is preferably fused to the second ring via the 4- and 5-position.
  • the NR group is typically bonded to the pyrazole ring in 3-position, 4-position or 5-position, preferably in 3- position.
  • the pyrazole ring is preferably fused to the second ring via the 4- and 5-position.
  • the second aromatic ring of Ar in alternative (a) including the preferred embodiments described above is typically a 5- or 6-membered alicyclic or heterocyclic ring.
  • appropriate 5- or 6-membered aromatic rings include but are not restricted to benzene, pyrrole, furan, thiophene, imidazole, oxazole, isoxazole, thiazole, isothiazole, pyrazole, 1,2,3- triazole, 1,2,4-triazole (4H-l,2,4-triazole and lH-l,2,4-triazole), lH-tetrazole, 2H-tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, 1,2,3-triazine, 1,2,4-triazine, and 1,3,5-triazine.
  • Preferred second aromatic rings are benzene, furan, thiophene, pyrrole, pyridine, thiazole, pyrimidine, and pyrazole, with benzene, thiophene, pyridine, pyrimidine, thiazole, furan and thiophene being more preferred, and benzene, thiophene, pyridine, pyrimidine being most preferred.
  • a furan, thiophene or pyrrole ring as second aromatic ring in alternative (a) those rings are preferably bonded to the first aromatic ring via their 2- or 3-position, with the 2-position being most preferred. In case of pyridine, the 2-position is a preferred position.
  • Ar is l-methyl-3-(2-thienyl)pyrazol-5-yl, 4-(2- pyridyl)thiazol-2-yl or 5-methyl-l-phenylpyrazol-3-yl. These compounds are also preferred.
  • the second ring of Ar in alternative (b) including the preferred embodiments described above is typically an aromatic or non aromatic 5- or 6-membered alicyclic or heterocyclic ring.
  • appropriate 5- or 6-membered rings include but are not restricted to benzene, pyrrole, furan, thiophene, imidazole, oxazole, isoxazole, thiazole, isothiazole, 1,2,5- thiadiazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole (4H- 1,2,4-triazole and 1H- 1,2,4-triazole), lH-tetrazole, 2H-tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, 1,2,3-triazine, 1,2,4- triazine, 1,3,5-triazine, 1,2,5-thiadiazole, cyclopentene, cyclohexene, cyclohep
  • Preferred second rings are cyclopentene, benzene, furan, thiophene, pyrrole, pyridine, thiazole, pyrimidine, pyrazole, with benzene, thiazole, furan, pyridine, 1,2,5- thiadiazole, 1,3-dioxalane and thiophene being more preferred, benzene, pyridine, 1,2,5- thiadiazole being most preferred. 2
  • Ar is benzothiazol-6-yl, benzothiazol-2-yl, benzoxazol-2-yl, 5,6-dihydro-4H-cyclopentathiazol-2-yl, benzo-[l,2,5]-thiadiazol-4-yl, 5- methoxyquinolin-8-yl, 4-methoxybenzothiazol-2-yl and quinolin-8-yl.
  • Preferred embodiments include 5-methoxyquinolin-8-yl, quinolin-8-yl, 4-methoxybenzothiazol-2-yl and benzo- [l,2,5]thiadiazol-4-yl.
  • the 5- or 6-membered aromatic ring is attached to at least one (preferably one or two) substituent selected from Ci to C 4 alkyl, typically linear and branched Ci to C 4 alkyl which may comprise a cycloalkyl unit such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, tert-butyl, cyclopropyl, methylcyclopropyl, and cyclobutyl; C 2 to C 4 alkenyl; C 2 to C 4 alkynyl such as ethynyl; a halogen such as fluoro, chloro, bromo, and iodo; Ci to C 4 haloalkyl such as trifluoromethyl; hydroxyl- substituted Ci to C 4 alkyl, typically mono- or dihydroxyalkyl such as mono- or dihydroxyethy
  • the substituent(s) may be bonded to the ring via ring carbon and/or ring heteroatoms, preferably via carbon and/or nitrogen ring atoms.
  • Preferred substituents of the 5- or 6-membered heterocylic aromatic ring in alternative (c) are cyclopropyl, ethynyl, methyl, ethyl, and trifluoromethyl.
  • the at least one substituent is preferably positioned meta to the NR group.
  • Ar is methylthiazol-2-yl and 5-ethyl-[l,2,4]thiadiazol- 2-yl.
  • the aromatic rings of Ar i.e. the first aromatic ring or the second aromatic ring or both, in alternative (a) and (b) may optionally carry one or more further substituents which are typically selected from the substituents mentioned above for alternative (c).
  • the substituent(s) may be bonded to the ring(s) via ring carbon or ring heteroatoms, preferably via carbon or nitrogen atoms.
  • Preferred substituents of the first and/or second aromatic ring of Ar are Q to C 4 alkyl such as methyl; halo such as bromo and chloro; and haloalkyl such as trifluoromethyl. If the first aromatic ring is a pyrazole ring it is typically methyl-substituted, preferably N-methyl-substituted, more preferably in 1 -position. 2
  • Typical examples of a substituted Ar are 5-methoxyquinolin-8-yl, 4-and methoxybenzothiazol-2-yl.
  • a typical example for a non substituted Ar is quinolin-8-yl.
  • the first aromatic ring is a pyridine ring it typically does not carry further substituents.
  • the group in alpha position to the carbonyl function is an amino group NR 3 , wherein R 3 can have various meanings as defined below.
  • the ligand-substituted matrix of the invention in these cases, can be depicted as in the following figure (II), which is covered by the above figure (I).
  • the NR group is not shown as a part of the linking point L or the spacer group Sp, but as a part of the ligand as such, for clarity reasons. It has to be kept in mind, however, that NR together with V forms the spacer group Sp as shown in figure (I), or is a part of the linking point L.
  • R is selected from: hydrogen; and C ⁇ to C 4 alkyl, typically linear and branched Ci to C 4 alkyl which may comprise a cycloalkyl unit such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, tert-butyl, cyclopropyl, methylcyclopropyl.
  • R 3 is selected from hydrogen and methyl. More preferably, R 3 is hydrogen.
  • R 3 as defined in the context with and shown in formula (II) corresponds to R 3 as defined and shown in the patent application EP 10172684.2 of which the present application claims priority.
  • the present invention also includes the ligands of the present invention (which, after being attached to the support material) form together with the support material the ligand- substituted matrix which is used in the method of the present invention.
  • the ligands are in accordance with the following formulae wherein the symbols Sp, Ar 1 and Ar 2 have the meanings defined above, including the preferred meanings:
  • the ligands according to formula (III) comprise the spacer group Sp attached to the ligand.
  • the ligands according to formulae (III) can serve as precursors for the synthesis of further compounds not having the spacer group attached.
  • the resulting compounds are also included in the present invention.
  • Preferred ligands of the matrix according to the present invention are depicted below wherein in each formula the very left N atom is connected to the linking point L which is not shown. All formulae below show the spacer group attached to the ligands:
  • N- ⁇ 3 (3-Aminopropyl)carbamoyl]pyridin-5-yl ⁇ -N'-(lH-indazol-5-yl)urea (L21) N- ⁇ 3 - [(3 - Aminopropyl)carbamoyl] -4-methoxyphenyl ⁇ -N '-( 1 -methylindazol-3 -yl)urea (L22)
  • the synthesis of the ligands (I) can, for example, be carried out on insoluble supports, also known as resins, e.g. polystyrene resins, preferably pre-loaded with the desired linker bearing a reactive group, e.g. an amino group, to which a building block, e.g. an Fmoc protected or unprotected amino acid, may be attached by amide formation.
  • a building block e.g. an Fmoc protected or unprotected amino acid
  • the remaining parts of the molecule are attached by any reaction leading to an appropriate bond formation and, where necessary, additional synthetic steps.
  • the compounds including the linker moieties are released from the insoluble support/resin by a suitable cleavage protocol known to the person skilled in the art and purified by chromatographic methods also known to the person skilled in the art.
  • antibody means an immunoglobulin, including both natural or wholly or partially synthetically produced and furthermore comprising all fragments and derivatives thereof which maintain specific binding ability. Typical fragments are Fc, Fab, heavy chain, and light chain.
  • the term also comprises any polypeptide having a binding domain which is homologous or largely homologous, such as at least 95% identical when comparing the amino acid sequence, to an immunoglobulin binding domain. These polypeptides may be derived from natural sources, or partly or wholly synthetically produced.
  • An antibody may be monoclonal or polyclonal and may be of human or non-human origin or a chimeric protein where the human Fc part is fused to murine Fab fragments (chimeric therapeutic antibodies with ending ...ximab, e.g. Rituximab) or to Fab fragments comprising human and murine sequences (humanized therapeutic antibodies with the ending ...zumab, e.g. Bevacizumab) or linked to different Fab fragments (bispecific antibodies).
  • the antibody may have been produced by recombinant expression systems, e.g. CHO cells or NSO cells.
  • the antibody may be a member of any immunoglobulin class, preferentially IgG, in case of human and humanized antibodies more preferentially IgGi, IgG 2 , IgG 3 and IgG 4 .
  • the antibody comprises an Fc fragment or domain, as it is supposed that the ligands according to the present invention bind to an antibody's Fc part.
  • an antibody of the present invention most preferably is an antibody containing an Fc fragment or domain of an immunoglobulin class, preferably IgG, more preferably of human IgG or of polyclonal or monoclonal IgG of human origin, in particular of IgGi, IgG 2 and IgG 4 .
  • Fc fusion protein refers to any combination of a Fc fragment with one or more proteins or protein domains.
  • Fc fusion proteins include, but are not limited to, chimers of the human IgGi Fc domain with soluble receptor domains (therapeutic proteins with the ending....cept) such as Etanercept (human IgGi Fc combined with two tumor necrosis factor receptor domains) or Rilonacept (human IgGi fused with interleukin-1- receptor domain).
  • Etanercept human IgGi Fc combined with two tumor necrosis factor receptor domains
  • Rilonacept human IgGi fused with interleukin-1- receptor domain
  • the Fc fusion protein may be enzymatically or chemically produced by coupling the Fc fragment to the appropriate protein or protein domain or it may be recombinantly produced from a gene encoding the Fc and the protein/protein domain sequence.
  • the Fc fusion protein may be wholly or partially synthetically produced.
  • the Fc fusion protein may also optionally be a multimolecular complex.
  • a functional Fc fusion protein will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.
  • the present invention is drawn to the affinity purification, preferably by affinity chromatography, of antibodies or Fc fusion proteins from complex mixtures e.g. fermentation supematants or plasma of human or animal origin, making use of the affinity ligands of formula (I) and preferred embodiments thereof, as disclosed elsewhere in the specification.
  • the present invention comprises a method for purifying a protein, preferably an immunoglobulin or Fc fusion protein, by affinity purification, preferably affinity chromatography.
  • the affinity ligands according to the present invention bind to the Fc region of an antibody.
  • Bevacizumab (Avastin®, Genentech/Roche) is a humanized monoclonal antibody. It stops tumor growth by preventing the formation of new blood vessels by targeting and inhibiting the function of the protein vascular endothelial growth factor-A (VEGF-A) that stimulates new blood vessel formation (angiogenesis).
  • VEGF-A protein vascular endothelial growth factor-A
  • Tocilizumab (RoActemra®, Genentech/Roche) is a humanized monoclonal antibody. It is directed against the Interleukin-6 receptor and blocks the action of the proinflammatory cytokine Interleukin-6. It is approved for the treatment of rheumatoid arthritis.
  • Palivizumab (Synagis®, Abbott) is a humanized monoclonal antibody directed against the A- epitope of the respiratory syncytial virus (RSV) F-protein. It is used in the prevention of RSV infections.
  • RSV respiratory syncytial virus
  • Polyclonal human and rabbit IgGs are investigated to demonstrate the universal applicability of the described ligands for the affinity purification of immunoglobulins via the Fc part.
  • the ligands according to the general formula (I) are attached to a support matrix of an appropriate support material, resulting in a ligand-substituted matrix, typically a matrix for affinity purification, preferably affinity chromatography (also referred to as affinity matrix in the context of the present invention) for protein separation.
  • a ligand-substituted matrix typically a matrix for affinity purification, preferably affinity chromatography (also referred to as affinity matrix in the context of the present invention) for protein separation.
  • affinity matrix also referred to as affinity matrix in the context of the present invention
  • the ligands of the general formula are attached to the support matrix via L, optionally including a spacer Sp.
  • the present invention includes an affinity matrix for protein separation, comprising a support matrix comprising a support material and at least one ligand as specified in the specification beforehand, wherein the ligand is attached thereto via L.
  • the support matrix may comprise any appropriate support material which is known to the person skilled in the art.
  • the material may be soluble or insoluble, particulate or non- particulate, or of a monolithic structure, including fibers and membranes, porous or non- porous. It provides a convenient means of separating ligands of the invention from solutes in a contacting solution.
  • support matrix examples include carbohydrate and crosslinked carbohydrate matrices such as agarose, Sepharose, Sephadex, cellulose, dextran, starch, alginate or carrageenan; synthetic polymer matrices such as polystyrene, styrene- divinylbenzene copolymers, polyacrylates, PEG polyacrylate copolymers, polymethacrylates, (e.g. poly(hydroxyethylmethacrylate), polyvinyl alcohol, polyamides or perfluorocarbons; inorganic matrices such as glass, silica or metal oxides; and composite materials.
  • the affinity matrix is prepared by providing a support matrix of an appropriate support material and attaching a ligand of formula (I) thereto. Methods for attaching the ligand (I) to the support material are known to the person skilled in the art.
  • the present invention also relates to a method for affinity purification, preferably affinity chromatography, of a protein wherein a protein to be purified is contacted with the ligand- substituted matrix as described before.
  • affinity purification refers to any separation technique involving molecular recognition of a protein by a ligand of formula (I).
  • the ligand may be immobilized on a solid support facilitating separation of the ligand-antibody complex later on. Separation techniques may include, but are not limited to, affinity chromatography on packed columns, monolithic structures or membranes. The term further includes adsorption in batch-mode or affinity precipitation.
  • purification techniques are composed by an initial recognition phase where ligand is contacted with antibody in crude.
  • impurities are separated from the ligand-antibody complex or (e.g. column chromatography) or ligand- antibody complex is separated from impurities (e.g. affinity precipitation).
  • the antibody is released from the ligand-antibody complex by alteration of chemical and/or physical conditions like change in pH, ionic strength and/or addition of modifiers like organic solvents, detergents or chaotropes.
  • 96 and 384-well filter plates having hydrophilic membrane filters with 0.45 ⁇ average pore size were purchased from Pall GmbH (Dreieich/Germany). Top frits made from polyethylene with 10 ⁇ average pore size were provided by Porex (Bautzen/Germany).
  • General purpose microtiterplates for collection of fractions and analytical assays were ordered from Greiner Bio One GmbH (Frickenhau sen/Germany). Analytical assays were read out using a Fluostar Galaxy plate reader from BMG Labtech GmbH (Offenburg/Germany).
  • Antibodies used in the present invention were Bevacizumab (Avastin, F. Hoffmann-La Roche, Switzerland), Tocilizumab (RoActemra, F. Hoffmann-La Roche, Switzerland), Palivizumab (Synagis, Abbott, USA), poly-IgG from human serum (Sigma-Aldrich, USA) and poly-IgG from rabbit serum (Sigma-Aldrich, USA). Immobilized papain for preparation of antibody fragments was purchased from Thermo Scientific (Bonn/Germany).
  • Flowthrough from protein A chromatography (referred to as host cell proteins) was derived from the supernatant of antibody-producing CHO cell culture in serum-free medium.
  • Graffinity has developed high density chemical microarrays comprising thousands of small molecules immobilized onto gold chips.
  • the arrays are constructed using maleimide -thiol coupling chemistry in combination with high density pintool spotting.
  • glass plates are microstructured by photolithographical methods in order to define up to 9,216 sensor fields per array.
  • a subsequently applied gold coating provides the basis for the SPR effect and enables the formation of a binary, mixed self assembled monolayer (SAM) of two different thiols.
  • SAM binary, mixed self assembled monolayer
  • One of the thiols exhibits a reactive maleimide moiety to which thiol-tagged array compounds can couple to during pintool spotting.
  • the resulting microarrays comprise 9,216 sensor fields, each containing multiply copies of a defined and quality controlled array compound.
  • SPR minimum The angle and wavelength position of this reflectance minimum strongly depends on the refractive index of the medium adjacent to the metal surface. Processes which alter the local refractive index in close proximity to the metal surface (such as antibody binding to the immobilized ligands) can therefore be monitored sensitively.
  • the chemical microarray is incubated with the target protein under optimized screening conditions.
  • SPR shift a shift in the wavelength dependent SPR minimum (referred to as the SPR shift) occurs with respect to the buffer control and thus indicates protein-ligand interaction.
  • the SPR imaging approach utilizes an expanded beam of parallel light to illuminate the entire microarray sensor area at a fixed angle. The reflected light is then captured by means of a CCD camera. While scanning over a range of wavelengths covering the SPR resonance conditions, reflection images are recorded stepwise. Automated spot finding routines and subsequent grey-scale analysis of the obtained pictures lead to SPR resonance curves of all 9,216 sensor fields per chip in parallel. Screening of Antibodies and Fragments thereof by SPR
  • the platform allowed screening of a proteins, antibodies and fragments thereof against Graffinity's immobilized compound library of 110,000 small molecules in a high-throughout fashion.
  • the SPR screening conditions were optimized individually before screening of the targets against the entire library.
  • optimized screening parameters were a) the composition of the screening buffer including appropriate detergents, b) the concentration of the antibody or protein target in solution and c) the surface density of immobilized ligands on the chip surface.
  • Neumann et al. (5) summarizes the screened targets and the corresponding screening conditions applied. In total four different full-length antibodies were screened and compared. Additionally, the Fc and Fab fragments of two antibodies were obtained by proteolytic digestion and were subjected to array screening as well.
  • the screening campaign included also antibody free host cell protein (HCP) of CHO cell lines as controls.
  • HCP antibody free host cell protein
  • N-Fmoc-aminoaryl carboxylic acid (0.25 mmol) and HATU (0.25 mmol) were dissolved in NMP (approx. 1.5 mL) and treated with DIPEA (0.5 mmol). After 2 min, the mixture was added to 0.15 mmol of pre-swollen 1,3-diaminopropane-trityl polystyrene resin. After shaking for 4 h, the resin was washed with DMF and dichloromethane (each solvent several times). Removal of the Fmoc group was effected by treatment with 25% piperidine in DMF for approx.
  • N- ⁇ 3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl ⁇ -N'-(5-methoxyquinolin-8-yl)urea (L6) Prepared from N-Fmoc-5-amino-2-methoxybenzoic acid and 8-amino-5-methoxyquinoline following General Procedure A.
  • ESI-MS 424 (M+1).
  • 1,3-Diaminopropane-trityl -resin (0.1-0.15 mmol) was pre-swollen in NMP.
  • aminoarylcarboxylic acid to be used, two different coupling protocols were applied.
  • 5-Aminonicotinic acid The carboxylic acid (0.3 mmol) and HOBt (0.3 mmol) were suspended in NMP (1.5 mL) and treated with DIC (0.3 mmol). After vigorous shaking for 2 min, the suspension was added to the resin. The reaction mixture was agitated for 1 h, then the resin was washed with DMF, methanol and dichloromethane (each solvent several times) and dried in a stream of dry nitrogen prior to the next step.
  • N-Fmoc-5-Amino-2-methoxybenzoic acid N-Fmoc-4-aminobenzoic acid, N-Fmoc-3-amino-
  • arylisocyanates are also suitable for larger batches.
  • a batch of 2.5 mmol of quinoline-8-ylisocyanate was prepared by multiplying all quantities listed before by 5. After the reaction time the resulting thick slurry was gently heated until it was completely liquified. Subsequently, aliquots equivalent to approx. 0.5 mmol of the isocyanate were added to different pre-loaded resins and treated as described before.
  • N- ⁇ 5-[(3-Aminopropyl)carbamoyl]-2-methylphenyl ⁇ -N'-(quinolin-8-yl)urea (L20) Prepared from N-Fmoc-3-amino-4-methylbenzoic acid and 8-aminoquinoline following General Procedure B. ESI-MS: 378 (M+l).
  • 1,3-Diaminopropane-trityl -resin (1.5 mmol) was pre-swollen in NMP.
  • N-Fmoc-5-Amino-2- methoxybenzoic acid (2 mmol) and HATU (2 mmol) were dissolved in NMP (15 mL) and treated with DIPEA (4 mmol). After 2 min, the solution was added to the resin. The mixture was agitated for 1 h, then the resin was washed with DMF (3 times) and treated with 25% piperidine in DMF for 30 min. After this time, the resin was washed with DMF, methanol and dichloromethane (each solvent several times) and dried over night in a stream of dry nitrogen.
  • Dried ligands were redissolved in DMSO at an approximate concentration of 100 mM based on gravimetric analyses. Precise concentrations of stock solutions were determined by the o-phthaldialdehyde assay (6). Dye-ligand molecules were quantitated by measuring absorbance at 340 nm. Measurements were calibrated with l-(3-aminopropyl)imidazole.
  • Ligands from example 2 were immobilized on NHS-activated Sepharose 4 FF for subsequent chromatography and batch adsorption experiments. Coupling was achieved by formation of an amide bond between the amino group of the aminopropyl linker on ligands and the NHS- activated carboxylic group of the pre-activated resin.
  • Resins were assessed in packed mode by microtiter plate chromatography. For each column, approximately 30 ⁇ of resin were transferred to a 384-well filter plate and sealed with appropriate top frits. Columns with rProtein A Sepharose FF and Mabsorbent A2P HF were included as controls.
  • Unbound protein was washed from columns with 5 cv of PBS prior to elntion with 5 cv of glycine buffer at pH 2,5. Volumes transferred were spun through columns at 50 g for 1 - 2 min. During injection of samples, acceleration was reduced to 10 g and centrifugation time increased to 5 - 10 min. Effluent fractions were collected in 384- well plates and concentrations of protein analyzed by Bradford assay. Protein masses m, ⁇ 3 ⁇ 4 and m. (see below) were calculated as the product of fraction volume and measured protein concentration.
  • Binding and e!ution of several antibodies was demonstrated. Among them were the three humanized therapeutic antibodies Bevacizumab, Tocilizumab and Palivlzumab and a human poly-IgG (h-pol -IgG) mixture Isolated from human serum. In some cases, purified Bevacizumab Fab and Fc fragments or a mixture of rabbit IgGs (r-poly-IgG, isolated from serum) were used as additional feed. Selectivity of ligands toward antibodies was determined by investigation of binding behavior of host ceil proteins. Results for commercial resins rProtein A Sepharose FF irProtein A), Mabsorbent A2P (A2P) and MEP Hypercel (MEP) were included for comparison.
  • the fraction of 'bound' protein was caicnlated as the difference between the total mass of protein Injected n3 ⁇ 4 and the mass of protein detected in the flowthrough n3 ⁇ 4. divided by the total mass of protein injected . m su ,.
  • the 'yield' of protein was calculated as the mass of protein eiuted m i; , divided by the mass of protein injected n3 ⁇ 4 . .
  • ligands from example 2 showed either reduced binding of at least one of the tested antibodies, lower yield of at least one of the testes antibodies or higher binding of HCP (resulting selectivity indices were 1.5 or lower).

Abstract

The present invention relates to the use of a ligand-substituted matrix comprising a support material and at least one ligand covalently bonded to the support material, the being represented by formula (I) wherein L is the linking point on the support material to which the ligand is attached; V is a spacer group; v is 0 or 1; R1 is hydrogen or C1 to C4 alkyl, preferably hydrogen or methyl; and more preferably hydrogen; R2 is hydrogen or C1 to C4 alkyl, preferably hydrogen; Ar1 is a divalent 5- or 6-membered substituted or unsubstituted aromatic ring; and Ar2 is 5- or 6-membered aromatic ring which may be unsubstituted or which is (a) attached to a further 5- or 6-membered aromatic ring via a single bond; or (b) fused to a further 5- or 6-membered aromatic or non aromatic ring, as part of a multicyclic ring system; or (c) attached to at least one substituent selected from C1 to C4 alkyl; C2 to C4 alkenyl; C2 to C4 alkynyl; a halogen; C1 to C4 haloalkyl; hydroxyl-substituted C1 to C4 alkyl; C1 to C4 alkoxy; hydroxyl-substituted C1 to C4 alkoxy; C1 to C4 alkylamino; C1 to C4 alkylthio; and combinations thereof, for affinity purification of a protein.

Description

Ligands for Antibody and Fc-Fusion Protein Purification by Affinity Chromatography
II
The present invention relates to the field of protein separation, preferably the purification of monoclonal and polyclonal antibodies and fusion proteins containing an immunoglobulin Fc segment, by affinity separation techniques, in particular chromatography using small molecule ligands.
Immunoglobulins are a class of soluble proteins found in body fluids of humans and other vertebrates. They are also termed "antibodies" and play a key role in the processes of recognition, binding and adhesion of cells. Antibodies are oligomeric glycoproteins which have a paramount role in the immune system by the recognition and elimination of antigens, in general bacteriae and viruses.
The polymeric chain of antibodies is constructed such that they comprise so-called heavy and light chains. The basic immunoglobulin unit consists of two identical heavy and two identical light chains connected by disulfide bridges. There are five types of heavy chains (α, γ, δ, ε, μ), which determine the immunoglobulin classes (IgA, IgG, IgD, IgE, IgM). The light chain group comprises two subtypes, λ and κ.
IgGs are soluble antibodies, that can be found in blood and other body fluids. They are built by B-cell derived plasma cells as response to and to neutralize bacterial or other pathogens. An IgG is an Y-shaped glycoprotein with an approximate molecular weight of 150 kDa, consisting of two heavy and two light chains. Each chain is distinguished in a constant and in a variable region. The two carboxy terminal domains of the heavy chains are forming the Fc fragment ("constant fragment"), the amino terminal domains of the heavy and light chains are recognizing the antigen and are named Fab fragment ("antigen-binding fragment").
Fc fusion proteins are created through a combination of an antibody Fc fragment and a protein or protein domain that provides the specificity for a given drug target. Examples are domain antibody-Fc fusion proteins, where the two heavy chains of the Fc fragments are linked either to the variable domains of the heavy (VR) or light chains (VL) of specific antibodies. Other Fc fusion proteins are combinations of the Fc fragment with any type of therapeutic proteins or protein domains. The Fc part is considered to add stability and deliverability to the protein drug.
Therapeutic antibodies and Fc fusion proteins are used to treat various diseases, prominent examples include rheumatoid arthritis, psoriasis, multiple sclerosis and many forms of cancer. Therapeutic antibodies can be monoclonal or polyclonal antibodies. Monoclonal antibodies are derived from a single antibody producing cell line, showing identical specificity towards a single antigen. Possible treatments for cancer involve antibodies that are neutralizing tumour cell specific antigens. Bevacizumab (Avastin, Genentech) is a monoclonal antibody which neutralizes the vascular endothelial growth factor (VEGF), thereby preventing the growth of new blood vessels into the tumour tissue.
Therapeutic fusion proteins such as Etanercept (Enbrel, Amgen, TNF-Receptor domain linked to Fc fragment) or Alefacept (Amevive, Biogen Idee, LFA-3 linked to Fc portion of human IgGl) are used or developed as drugs against autoimmune diseases.
Protein bioseparation which refers to the recovery and purification of protein products from various biological feed streams is an important unit operation in the food, pharmaceutical and bio technological industry. More and more therapeutic monoclonal antibodies (Mabs) and fusion proteins are entering the market or are currently in clinical development. Such proteins require an exceptionally high purity which is achieved by elaborate multi-step purification protocols. Downstream processing and purification constitute about 50 to 80 % of the manufacturing cost, hence considerable efforts are under way to develop new or improve existing purification strategies (1).
Affinity chromatography is one of the most effective chromatographic methods for protein purification. It is based on highly specific protein-ligand interactions. The ligand is immobilized covalently on the stationary phase which is used to capture the target protein from the feed stock solution. Affinity ligands can bind their target with high specificity and selectivity, enabling up to thousand fold higher enrichment at high yields even from complex mixtures. Typically, affinity chromatography on protein A is the first step in most Mab and Fc fusion protein purification schemes. Protein A is a cell wall associated protein exposed on the surface of the bacterium Staphylococcus aureus. It binds with nanomolar affinity to the constant part (Fc domain) of immunoglobulins from various species, in particular to human subtypes IgGl, IgG2 and IgG4 (2). However, the use of Protein A is limited by leaching into the product and poor stability under harsh conditions applied for sanitization and cleaning in place procedures. The chemical stability of Protein A can be improved by using genetically engineered Protein A variants for Mab purification. Yet, the high costs of Protein A resins have resulted in the search for suitable alternatives, in particular selected from small molecules.
Mabsorbent A2P (Prometic Biosciences) is a small molecule ligand for the purification of immunoglobulins. However, the ligand does not show an appropriate selectivity (3) and is not applied in process chromatography.
Another approach uses mixed mode chromatography as primary capture step in antibody purification. The most common material is based on immobilized 2-mercaptoethylpyridine (MEP HyperCel, Pall Corporation), which effectively captures IgG from fermentation broth but shows lower clearance of host cell proteins (HCP) in comparison to protein A. (4).
Synthetic affinity ligands that are more readily available, preferably cheaper than protein- based ligands, are more robust under stringent conditions and have a selectivity comparable to or even higher than Protein A would provide a suitable solution for antibody and Fc fusion protein purification.
Synthetic small molecule affinity ligands are of particular interest for the purification of therapeutic proteins due to their generally higher chemical stability and their lower production costs. Synthetic affinity ligands that are more readily available, preferably cheaper than protein-based ligands, are more robust under stringent conditions and have a selectivity comparable to or even higher than Protein A would provide a suitable solution for antibody and Fc fusion protein purification. Depending on the target protein, such affinity ligands should preferably offer the same broad applicability as Protein A, recognizing the constant Fc region of IgG type immunoglobulins and Fc fusion proteins. The problem underlying the present invention is the provision of a matrix comprising a small affinity ligand binding to the Fc region of antibodies and Fc fusion proteins.
The problem is solved by the embodiments of the present invention as laid out hereinafter.
The present invention is directed to the use, for affinity purification of a protein, preferably an antibody or fragment of an antibody, of a ligand-substituted matrix comprising a support material and at least one ligand covalently bonded to the support material, the ligand being represented by formula (I)
O R1 O R2
II I II I
L— (Sp)-C— Ar— — C— — Ar2 (I) wherein
L is the linking point on the support material to which the ligand is attached;
Sp is a spacer group;
v is 0 or 1 ;
R1 is hydrogen or Ci to C4 alkyl, preferably hydrogen or methyl; and more preferably hydrogen;
R is hydrogen or Ci to C4 alkyl, preferably hydrogen or methyl; and more preferably hydrogen;
Ar1 is a divalent 5- or 6-membered substituted or unsubstituted aromatic ring; and
Ar is 5- or 6-membered aromatic ring which may be unsubstituted or which is
(a) attached to a further 5- or 6-membered aromatic ring via a single bond; or
(b) fused to a further 5- or 6-membered aromatic or non aromatic ring, as part of a multicyclic ring system; or
(c) attached to at least one substituent selected from Ci to C4 alkyl; C2 to C4 alkenyl; C2 to C4 alkynyl; a halogen; Ci to C4 haloalkyl; hydroxyl-substituted Q to C4 alkyl; Ci to C4 alkoxy; hydroxyl-substituted Ci to C4 alkoxy; Q to C4 alkylamino; Ci to C4 alkylthio; Ci to C4 alkylnitrile, and combinations thereof.
Ar 1 may also be denoted AR 1 , and Ar2 may also be denoted AR 2. Furthermore, Sp may also be denoted V. This is the nomenclature as used in the patent application EP 10172684.2 of which the present application claims priority. Still, R 1 and R 2 as defined in the context with formula (I) correspond to R4 and R5, respectively, as defined and shown in the said priority document.
Ligands according to the present invention bind to polyclonal and monoclonal IgG of human origin, in particular human IgGi, IgG2 and IgG4 and polyclonal and monoclonal IgGs from different animal species such as rabbit and mouse immunoglobulins.
The present invention is also directed to the ligand-substituted matrix, and the ligands (which may also be termed "compounds") attached to the matrix at the linking point L, optionally via a spacer group Sp.
L in the above formula (I) is the linking point, also referred to as "point of attachment". The person skilled in the art is aware of appropriate linking points/points of attachment.
It is understood that the linking point L is either directly connected with the support material or via a spacer. Typically L is part of a moiety resulting from the reaction of an appropriate functional group on the support material with a corresponding functional group on the precursor compound to form the ligand.
Examples of functional groups on the support material include carboxylic acid, epoxide, amine, hydroxyl, maleimide, aldehyde, ketone, sulfonic acid, hydrazine, oxime ether, thiol, azide, and alkyne groups.
In one embodiment, L is a bond, preferably a single bond, which is directly attached to the support material, in general via an appropriate functional group on the material. In this context, the term "support material" refers to polymers known to the person skilled in the art and available on the market which lend themselves for the purposes of the present invention. A definition of "support material" is provided below. In general, the support material comprises functional groups for the attachment of molecules, in general those of the present invention. In the context of the present invention, the functional groups on the support material are regarded as a part of the support material; this also applies in cases where the ligands of the present invention are connected to the support material via a bond (i. e. L is a bond and does not comprise a spacer group Sp, see below), and wherein the bond is directly formed between the respective ligand according to the invention and the functional group on the support material.
"Functional group" in the present context refers, on the one hand, to appropriate groups ("precursor groups") on the support material and, on the other hand, to appropriate groups ("precursor groups") present in the ligands according to the invention (e.g. present on the C=0 group, or present on the spacer Sp). "Functional group" in the present context also refers to appropriate groups ("precursor groups") on the spacer group Sp. "Precursor groups" or "functional groups" in the present context are groups which are capable of a chemical reaction with complementary "precursor groups" or "functional groups", under formation of a chemical bond. If necessary, additional reagents may be used for the formation of said chemical bond. As is clear from the foregoing, during the reaction the functional groups/precursor groups are transformed into a chemical bond under a transformation of the original functionality into a "final functionality" or "connecting unit". From the foregoing, it is clear that a functional group/precursor group also is or can be a "complementary group".
Examples of functional groups on the support material are known to the person skilled in the art and include, but are not limited to -OH, -SH, -NH2, >NH -COOH, -S03H, -CHO, - NHNH2, -P(=0)(OH)2, -0-PH(=0)(OH), -P(OH)2, -0-P(=0)(OH)2, ester, ether, thioether, epoxide, natural or non natural amino acids, carboxylic amides, maleimide, ketone, sulphoxide, sulfone, urea, isourea, imidocarbonate, sulphonamide, sulphonylhydrazide, sulphonic ester, phosphate, phosphonate, phosphoric amide, phosphonic amide, alkyne, oxime ether, oxime, azide, alkoxyamine, semicarbazide, sulphonic halide, phosphonic halide, phosphoric halide, carboxylic active esters, sulphonic active esters, phosphonic active esters, phosphoric active esters, phosphoramidite, cyanate, thiocyanate, isocyanate, isothiocyanate, maleimide, vinyl sulphonyl, CI, Br, I, alkene, alkyl phosphonium.
Examples of functional groups attached to the -C=0 group on the ligands of the invention are known to the person skilled in the art and include, but are not limited to hydroxy, thiol, F, CI, Br, ester, ether, thioether, carboxylic acid, epoxide, amine, amide, amino acids, carboxylic amides, maleimide, aldehyde, ketone, sulphonic acid, sulphoxide, sulphone, urea, isourea, imidocarbonate, sulphonamide, sulphonic ester, phosphate, phosphonate, phosphoric amide, phosphonic amide, hydrazine, oxime, azide, alkene, alkyne,, hydroxylamine, thiourea, isocyanate, isothiocyanate, N-hydroxysuccinimidyl, 1-hydroxybenzotriazolyl, 7-aza-l- hydroxybenzotriazolyl, 6-chloro- 1 -hydroxybenzotriazolyl.
In an embodiment of the invention, the bond or the chemical unit resulting from the reaction of an appropriate functional group on the support material with an appropriate functional group (or a "precursor group") on the -C=0 group on the ligands of the invention constitutes the linking point L by which the ligands of the invention are attached to the support material/the matrix. Accordingly, the chemical reaction between the functional group on the support material and the functional group (or "complementary group") on the -C=0 group on the ligands of the invention connects the ligand according to the invention with the support material via the linking point L.
In a further embodiment of the invention, L is connected to a spacer group -Sp-. In this embodiment, L is a bond or a chemical unit resulting from the reaction of an appropriate functional group on the support material with an appropriate functional group (or a "precursor group") on the spacer group Sp. Accordingly, the chemical reaction between the functional group on the support material and the functional group (or "complementary group") on the spacer group Sp connects the ligand according to the invention with the support material via the linking point L.
If v in formula (I) is 0, then the ligand is directly bonded to L. In other embodiments the ligand is bonded to the support material via a spacer group Sp. This applies when v in formula (I) is different from 0.
The spacer group Sp preferably is a hydrocarbon group which may contain, in addition to C and H atoms, further atoms. Appropriate further atoms are known to the person skilled in the art. Preferred atoms include O, S, N, P, Si. The hydrocarbon group may be linear or branched or cyclic. Sp is linked to the -C=0 group of the ligands according to the invention, in general Sp is found in alpha-position to the -C=0 group. Furthermore, Sp contains functional groups (precursor groups) by which the ligands of the invention can be covalently linked with the matrix in a chemical reaction under formation of a final functionality (which may also be termed connecting unit).
The spacer group Sp preferably contains a connecting unit/final functionality which covalently connects the ligand with the support material via linking point L. Appropriate connecting unit/final functionalities are known to the person skilled in the art. Preferably the connecting unit is derived from suitable precursor groups including -NH2, >NH, -SH, - COOH, -SO3H, -CHO, -OH, -NHNH2, -P(=0)(OH)2, -0-PH(=0)(OH), -P(OH)2, -O- P(=0)(OH)-NHNH2, natural or non natural amino acids, carboxylic amides, ethers, ester, thioether, epoxide, maleimide, ketone, sulphoxide, sulphone, urea, isourea, imidocarbonate, sulphonamide, sulphonylhydrazide, sulphonic ester, phosphate, phosphonate, phosphoric amide, phosphonic amide, hydrazine, oxime, azide, alkoxyamine, semicarbazide, sulphonic halide, phosphonic halide, phosphoric halide, carboxylic active esters, sulphonic active esters, phosphonic active esters, phosphoric active esters, phosphoramidite, cyanate, thiocyanate, alkyl halide, alkyl sulphonate, isocyanate, isothiocyanate, vinyl sulphonyl, carboxylic halide, alkene and alkyne groups. Typically the connecting unit is attached to a carbon chain that may be interrupted by one or more heteroatoms such as oxygen atoms or carboxamide groups. In one embodiment, the connecting unit is attached to an alkylene or an alkylene - polyoxyalkylene group such as -CH2-CH2-(0-CH2-CH2)x- with x ranging from 1 to 10, preferably from 1 to 6.
Typically the functional groups on the spacer group are attached to a hydrocarbon group which may contain one or more heteroatoms such as N, O, P, S, Si. The spacer group therefore is a hydrocarbon group containing one or more heteroatoms preferably selected from. N, O, P, S, Si. Preferably the atom which is linked to the C=0 group of the ligands of the invention is nitrogen, oxygen, carbon or sulfur. Appropriate chemical entities/chemical units contained in Sp are known to the person skilled in the art. Sp in general contains at least one unit selected from alkylene, alcohol, amine, amide, carbonyl, alkyleneoxy and phosphate. The alkylene unit preferably has 1-30, preferably 1-12, in particular 1-6 carbon atoms. The alcohol can be a primary, secondary or tertiary alcohol. The amine can be a non organic or an organic amine. The amine, furthermore, can be a diamine or a triamine, preferably a diamine. The amide can be a non organic or an organic amide, and can be derived from a diamine or a triamine, preferably a diamine. The alkyleneoxy unit can comprise a linear or a branched alkylene unit. The alkyleneoxy unit is preferably an ethylenoxy or a propyleneoxy unit, preferably an ethylenoxy unit. The unit can also comprise two or more of the above cited entities/units, e.g. an amino acid. The amino acid can be a natural or a non natural amino acid. The amino acid can be linear or branched. Branched amino acids may serve as a branching point, enabling the connection of more than one ligand moiety to one point of attachment ("L"). Examples for preferred amino acids include 6-aminohexanoic acid, 8-amino-3,6- dioxaoctanoic acid, 5-amino-3-oxapentanoic acid, glycine, beta-alanine, lysine, ornithine, glutamic acid, 2,3-diaminopropionic acid. 2,4-diaminobutyric acid and serine.
The spacer group Sp may consist of one unit as described before, or of more than one unit. If the spacer group Sp consists of more than one unit as described before, said units are interconnected by connecting groups. Such connecting groups include but are not restricted to amide, urea, hydrazide, oxalic diamide, oxime, hydrazone, triazole, thiourea, isourea, ether, with amide and urea being preferred. A phosphate group may as well serve as a connecting group. The phosphate group may be linked to one, two or three units as described before. If the phosphate group is linked to three units as described before, it may serve as a branching point, enabling the connection of more than one ligand moiety to one point of attachment C'L").
Preferred units of the spacer group Sp include but are not restricted to amines, preferably organic amines, diamines, like ethylenediamine, piperazine, homopiperazine, -(CH2)n-C(=0), -NH-((CH2)20)n-(CH2)„-NH-; -NH-(CH2)n-NH-, -NH-(CH2)n-, -NH-CH-(C(=0)NH-, -NH- (0-C2H4)n-CH2-C(=0)- wherein n is an integer from 1-30, preferably 1-12. In one embodiment, n is an integer from 1-6, i.e. 1, 2, 3, 4, 5 or 6. Further preferred units of the spacer group Sp include but are not restricted to amino acids, which may be natural or unnatural amino acids, in particular 6-aminohexanoic acid, 8-amino-3,6-dioxaoctanoic acid, 5-amino-3-oxapentanoic acid, lysine and glutamic acid. The named units and functional groups are thus part of the spacer group Sp and of the linking point L.
Particularly preferred examples of spacer groups Sp are -NH-(CH2)nNH-, -NH-((CH2)20)n- (CH2)2-NH-, wherein n is integer from 1-30, preferably 1-12. In one embodiment, n is an integer from 1-6, i.e. 1, 2, 3, 4, 5 or 6. Typical examples for preferred spacer groups Sp include the following:
-N'H-CH(C(=0)NH-(CH2)3-N"H-)((CH2)2C(=0)NH-(CH2)3-N'"H-) with N' being attached to the linking point L and both N" and N'" being attached to the C(=0) of two individual ligands;
-NH-(CH2)3-N'(CH3)- with N being attached to the linking point L and N' being attached to the C(=0) of the ligand;
-N'H-(CH2)3-NH-C(=0)-CH(-N"H-)(-(CH2)4-N' "H-) with N' being attached to the linking point L and both N" and N" ' being attached to the C(=0) of two individual ligands;
-N'H-(CH2)3-NH-C(=0)-CH(-NH-C(=0)-CH2-(0-C2H4)2-N"H-)(-(CH2)4-NH-C(=0)-CH2- (0-C2H4)2-N'"H-) with N' being attached to the linking point L and both N" and N' " being attached to the C(=0) of two individual ligands;
-N'H-(CH2)3-NH-C(=0)-CH(-NH-C(=0)-CH2-0-C2H4-N"H-)(-(CH2)4-NH-C(=0)-CH2-0- C2H4-N' "H-) with N' being attached to the linking point L and both N" and N' " being attached to the C(=0) of two individual ligands;
-N'H-(CH2)3-NH-C(=0)-CH2-(0-C2H4)2-N"H- with N' being attached to the linking point L and N" being attached to the C(=0) of the ligand;
-N'H-(CH2)3-NH-C(=0)-CH2-(0-C2H4)2-NH-C(=0)-CH2-(0-C2H4)2-N"H- with N' being attached to the linking point L and N' ' being attached to the C(=0) of the ligand;
-N'H-(C2H4-0)2-C2H4-NH-C(=0)-NH-C2H4-(0-C2H4)2-N'H- with N' being attached to the linking point L and the other N' being attached to the C(=0) of the ligand;
-N'H-(CH2)5-C(=0)-NH-(CH2)3-N"H- with N' being attached to the linking point L and N" being attached to the C(=0) of the ligand;
-N'H-C2H4-(0-C2H4)2-NH-C(=0)-(CH2)5-N"H- with N' being attached to the linking point L and N" being attached to the C(=0) of the ligand;
-N'H-(CH2)6-NH-C(=0)-CH2-(0-C2H4)2-N"H- with N' being attached to the linking point L and N" being attached to the C(=0) of the ligand;
-NH-CH2-CH2-CH2-NH with -NH- being attached to the linking point L (particularly preferred);
-NH-(CH2)3-NH- with N being attached to the linking point L and the other N being attached to the C(=0) of the ligand (particularly preferred).
Typically, the total length of the spacer group Sp is less than 100 atoms, preferably less than 60 atoms, in particular less than 30 atoms. In one embodiment of the invention, Sp may also be branched. The term "branched" in the present context means that more than 1 ligand of the present invention according to formula (II) is present on the spacer group Sp. This embodiment allows the linkage of more than one ligand to one matrix attachment point (linking point) L.
"Final functionality" or "connecting unit" in the present context designates a unit formed in the reaction between the precursor groups (functional group) on the support material and on the ligand of the invention and/or on the spacer group Sp. The connecting unit incorporates the bond between the support material and the ligands of the present invention (which may include the spacer Sp), resulting in the ligand-substituted matrix.
Connecting units incorporated in L and/or Sp are known to the person skilled in the art and may include the following groups: ether, thioether, C-C single bond, alkenyl, alkynyl, ester, amine, amide, carboxylic amide, sulfonic ester, sulfonic amide, phosphonic ester, phosphonic amide, phosphoric ester, phosphoric amide, phosphoric thioester, thiophosphoric ester, secondary amine, secondary alcohol, tertiary alcohol, 2-hydroxyamine, urea, isourea, imidocarbonate, alkylthiosuccinic imide, dialkylsuccinic imide, triazole, ketone, sulfoxide, sulfone, oxime ether, hydrazone, silyl ether, diacyl hydrazide, acyl sulphonyl hydrazide, N- acyl sulphonamide, hydrazide, N-alkoxy amide, acyl semicarbazide, acyloxy amide, sulphonyloxy amide, phosphoryloxy amide, phosphonyloxy amide, acyl hydrazone, N- carboxamidoisourea, N-carboxamidoimidocarbonate, N-carboxamidoisothiourea, N- carboxamidoimidothiocarbonate, acyl urea, acyl thiourea, N-carboxamidourea, N- carboxamidoisourea, alkylthioethylsulphonyl, hydrazinylsuccinimide, acylhydrazinylsuccinimide, alkoxyaminosuccinimide, aminosuccinimide, hydrazinylethylsulphonyl, acylhydrazinylethylsulphonyl, alkoxyaminoethylsulphonyl, aminoethylsulphonyl, tetrazole, alkoxyamidine.
If v in formula (I) is 0, then the ligand is directly bonded to L. In other embodiments the ligand is bonded to the support material via a spacer group Sp.
In an embodiment of the present invention, R 1 and R 2 are each independently selected from: hydrogen; and Ci to C4 alkyl, typically linear and branched Ci to C4 alkyl which may comprise a cycloalkyl unit such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec- butyl, tert-butyl, cyclopropyl, methylcyclopropyl. Preferably R 1 and R 2 are each independently selected from hydrogen and methyl. More preferably, R 1 and R 2 are all hydrogen.
Ar1 can be an alicyclic or heterocyclic aromatic ring. In one embodiment Ar1 is an alicyclic aromatic ring, i.e. Ar1 is phenylene. If Ar1 is a heterocyclic aromatic ring it comprise one, two or more heteroatoms which are preferably selected from N, S, O and combinations thereof. Examples of appropriate 5- or 6-membered heterocylic aromatic rings include but are not restricted to pyrrole, furan, thiophene, imidazole, oxazole, isoxazole, thiazole, isothiazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole (4H-l,2,4-triazole and lH-l,2,4-triazole), lH-tetrazole, 2H-tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, 1,2,3-triazine, 1,2,4-triazine, and 1,3,5-triazine. Preferred 5- or 6-membered heterocylic aromatic rings are imidazole, thiazole, and pyridine with pyridine being a preferred embodiment.
In formula (I) Ar1 is bonded to a C=0 group and an NR1 group. In some embodiments the C=0 and NR1 group are in meta position to each other. If Ar1 is phenylene the C=0 group and the NR1 group are positioned ortho, meta or para, preferably meta or para, most preferably meta, to each other. If the aromatic ring is imidazole, the C=0 group can in some embodiments be bonded in 2-position and the NR1 group in 4-position. If the aromatic ring is thiazole, the C=0 group can in some embodiments be bonded in 4-position and the NR1 group in 2-position. If the aromatic ring is pyridine the C=0 group and the NR1 group are preferably positioned meta to each other whereas the nitrogen ring atom may have different positions. In some embodiments the C=0 group is typically bonded in 3 -position of the pyridine ring and the NR1 group in 5-position.
The divalent 5- or 6-membered aromatic ring including the preferred embodiments described above may be substituted or unsubstituted. Typically the 5- or 6-membered aromatic ring is substituted, preferably monosubstituted. Examples of appropriate substituents are Ci to C4 alkoxy, typically linear and branched Ci to C4 alkoxy which may comprise a cycloalkyl unit such as methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy, tert-butoxy, cyclopropoxy, methylcyclopropoxy, and cyclobutoxy; Ci to C4 alkyl, typically linear and branched Ci to C4 alkyl which may comprise a cycloalkyl unit such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, tert-butyl, cyclopropyl, methylcyclopropyl, and cyclobutyl; hydroxyl; and combinations thereof. Preferred substituents include methoxy, ethoxy, and methyl, with methoxy being most preferred.
In some preferred embodiments Ar1 is methoxy-substituted phenylene wherein the methoxy radical is positioned ortho to the C=0 group and para to the NR^group (the C=0 group and the NR1 group are positioned meta to each other). In some embodiments the Ar1 is methyl- substituted phenylene wherein the methyl radical is positioned para to the C=0 group and otho to the NR^roup (the C=0 group and the NR1 group are positioned meta to each other). In yet other preferred embodiments Ar1 is a pyridine ring wherein the C=0 group is bonded in 3-position and the NR^roup in 5-position. In still other preferred embodiments Ar1 is unsubstituted phenylene wherein the C=0 and the NRlgroup are positioned meta or para to each other.
Ar is 5- or 6-membered aromatic ring which may be unsubstituted or may be
(a) attached to a further 5- or 6-membered aromatic ring via a single bond; or
(b) fused to a further 5- or 6-membered aromatic or non aromatic ring, as part of a multicyclic ring system; or
(c) attached to at least one substituent selected from Ci to C4 alkyl; C2 to C4 alkenyl; C2 to C4 alkynyl; a halogen; Ci to C4 haloalkyl; hydroxyl-substituted Q to C4 alkyl; Q to C4 alkoxy; hydroxyl-substituted Q to C4 alkoxy; Ci to C4 alkylamino; Q to C4 alkylthio; Ci to C4 alkylnitrile, and combinations thereof.
The 5- or 6-membered aromatic ring of Ar is alicyclic, i.e. it is a benzene ring, or it is heterocyclic and comprises at least one heteroatom which is preferably selected from N, S, O and combinations thereof. More preferably, the 5- or 6-membered heterocyclic aromatic ring contains two or more nitrogen atoms or a nitrogen atom and an oxygen atom or a nitrogen and a sulfur atom.
Examples of appropriate first 5- or 6-membered heterocyclic aromatic rings of Ar directly bonded to the NR group group in formula (I) include but are not restricted to pyrrole, furan, thiophene, imidazole, oxazole, isoxazole, thiazole, isothiazole, pyrazole, 1,2,3-triazole, 1,2,4- triazole (4H-l,2,4-triazole and lH-l,2,4-triazole), lH-tetrazole, 2H-tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, 1,3,4-thiadiazole, 1,2,3-triazine, 1,2,4-triazine, and 1,3,5- triazine,. Preferred 5- or 6-membered heterocylic aromatic rings are thiazole, oxazole, 1,3,4- thiadiazole, pyrazole, imidazole, isoxazole, pyrrole, furan, 1,2,4-triazole, pyridine, 1,2,4- triazine, pyridazine, and pyrimidine, with thiazole, oxazole, 1,3,4-thiadiazole, and pyrazole being most preferred. In case of a thiazole ring the NR group is typically bonded to the thiazole ring in 2-position. In case of an oxazole ring the NR group is typically bonded to the oxazole ring in 2-position. In case of a 1,3,4-thiadiazole ring the NR group is typically bonded to the oxazole ring in 2-position. In case of a pyrazole ring the NR group is typically bonded to the pyrazole ring in 3-position, 4-position or 5-position, preferably in 3-position. In case of pyridine ring the NR group is typically bonded to the pyridine ring in 3 position (5 position).
In alternative (a) of Ar described above for formula (I) the first 5- or 6-membered aromatic ring is attached to a further 5- or 6-membered aromatic ring ("second aromatic ring of Ar " in the following) via a single bond. In these embodiments, it is especially preferred that the 5- or
2 2
6-membered aromatic ring directly bonded to the NR group ("first aromatic ring of Ar " in the following) is benzene, thiazole, pyrazole or pyridine. In case of a benzene ring as first aromatic ring the NR group and the second aromatic ring are typically positioned meta to each other. In case of a thiazole ring as first aromatic ring, the NR group is typically bonded to the thiazole ring in 2-position. The second aromatic ring is preferably bonded to the thiazole ring in 4-position. In case of a pyrazole ring as first aromatic ring, the NR group is typically bonded to the pyrazole ring in 3-position. The second aromatic ring is preferably bonded to the pyrazole ring in 5-position. In other embodiments the NR group is bonded to the pyrazole ring in 5-position and the second aromatic ring is bonded to the pyrazole ring in 3-position. In other embodiments the NR group is bonded to the pyrazole ring in 3-position and the second aromatic ring is bonded to the pyrazole ring in 1 -position.
In alternative (b) the first aromatic ring is fused to a second 5- or 6-membered ring, preferably aromatic ring as part of a multicyclic, preferably bicyclic ring system. In case of a benzene ring as first aromatic ring and being part of a fused ring system, it is preferably fused to the second ring via the ortho and meta position or meta and para position In case of a oxazole ring as first aromatic ring and being part of a fused ring system, the NR group is typically bonded to the oxazole ring in 2-position. The oxazole ring is preferably fused to the second ring via the 4- and 5-position. In case of a thiazole ring as first aromatic ring and being part of 2
a fused ring system, the NR group is typically bonded to the thiazole ring in 2-position. The thiazole ring is preferably fused to the second ring via the 4- and 5-position. In case of a pyrazole ring as first aromatic ring and being part of a fused ring system, the NR group is typically bonded to the pyrazole ring in 3-position, 4-position or 5-position, preferably in 3- position. The pyrazole ring is preferably fused to the second ring via the 4- and 5-position.
The second aromatic ring of Ar in alternative (a) including the preferred embodiments described above is typically a 5- or 6-membered alicyclic or heterocyclic ring. Examples of appropriate 5- or 6-membered aromatic rings include but are not restricted to benzene, pyrrole, furan, thiophene, imidazole, oxazole, isoxazole, thiazole, isothiazole, pyrazole, 1,2,3- triazole, 1,2,4-triazole (4H-l,2,4-triazole and lH-l,2,4-triazole), lH-tetrazole, 2H-tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, 1,2,3-triazine, 1,2,4-triazine, and 1,3,5-triazine. Preferred second aromatic rings are benzene, furan, thiophene, pyrrole, pyridine, thiazole, pyrimidine, and pyrazole, with benzene, thiophene, pyridine, pyrimidine, thiazole, furan and thiophene being more preferred, and benzene, thiophene, pyridine, pyrimidine being most preferred. In case of a furan, thiophene or pyrrole ring as second aromatic ring in alternative (a) those rings are preferably bonded to the first aromatic ring via their 2- or 3-position, with the 2-position being most preferred. In case of pyridine, the 2-position is a preferred position.
In typical embodiments of alternative (a) Ar is l-methyl-3-(2-thienyl)pyrazol-5-yl, 4-(2- pyridyl)thiazol-2-yl or 5-methyl-l-phenylpyrazol-3-yl. These compounds are also preferred.
The second ring of Ar in alternative (b) including the preferred embodiments described above is typically an aromatic or non aromatic 5- or 6-membered alicyclic or heterocyclic ring. Examples of appropriate 5- or 6-membered rings include but are not restricted to benzene, pyrrole, furan, thiophene, imidazole, oxazole, isoxazole, thiazole, isothiazole, 1,2,5- thiadiazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole (4H- 1,2,4-triazole and 1H- 1,2,4-triazole), lH-tetrazole, 2H-tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, 1,2,3-triazine, 1,2,4- triazine, 1,3,5-triazine, 1,2,5-thiadiazole, cyclopentene, cyclohexene, cycloheptene and 1,3- dioxalane. Preferred second rings are cyclopentene, benzene, furan, thiophene, pyrrole, pyridine, thiazole, pyrimidine, pyrazole, with benzene, thiazole, furan, pyridine, 1,2,5- thiadiazole, 1,3-dioxalane and thiophene being more preferred, benzene, pyridine, 1,2,5- thiadiazole being most preferred. 2
In typical embodiments of alternative (b) Ar is benzothiazol-6-yl, benzothiazol-2-yl, benzoxazol-2-yl, 5,6-dihydro-4H-cyclopentathiazol-2-yl, benzo-[l,2,5]-thiadiazol-4-yl, 5- methoxyquinolin-8-yl, 4-methoxybenzothiazol-2-yl and quinolin-8-yl. Preferred embodiments include 5-methoxyquinolin-8-yl, quinolin-8-yl, 4-methoxybenzothiazol-2-yl and benzo- [l,2,5]thiadiazol-4-yl.
In alternative (c) of Ar described above for formula (I) the 5- or 6-membered aromatic ring is attached to at least one (preferably one or two) substituent selected from Ci to C4 alkyl, typically linear and branched Ci to C4 alkyl which may comprise a cycloalkyl unit such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, tert-butyl, cyclopropyl, methylcyclopropyl, and cyclobutyl; C2 to C4 alkenyl; C2 to C4 alkynyl such as ethynyl; a halogen such as fluoro, chloro, bromo, and iodo; Ci to C4 haloalkyl such as trifluoromethyl; hydroxyl- substituted Ci to C4 alkyl, typically mono- or dihydroxyalkyl such as mono- or dihydroxyethyl; Ci to C4 alkoxy, typically linear and branched Ci to C4 alkoxy which may comprise a cycloalkyl unit such as methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i- butoxy, sec-butoxy, tert-butoxy, cyclopropoxy, methylcyclopropoxy, and cyclobutoxy; hydroxyl- substituted Ci to C4 alkoxy, typically monohydroxyl-substituted Ci to C4 alkoxy; Ci to C4 alkylamino; Ci to C4 alkylthio, Ci to C4 alkylnitrile, and combinations thereof. The substituent(s) may be bonded to the ring via ring carbon and/or ring heteroatoms, preferably via carbon and/or nitrogen ring atoms. Preferred substituents of the 5- or 6-membered heterocylic aromatic ring in alternative (c) are cyclopropyl, ethynyl, methyl, ethyl, and trifluoromethyl. The at least one substituent is preferably positioned meta to the NR group. In typical embodiments of alternative (c) Ar is methylthiazol-2-yl and 5-ethyl-[l,2,4]thiadiazol- 2-yl.
It is understood that the aromatic rings of Ar , i.e. the first aromatic ring or the second aromatic ring or both, in alternative (a) and (b) may optionally carry one or more further substituents which are typically selected from the substituents mentioned above for alternative (c). The substituent(s) may be bonded to the ring(s) via ring carbon or ring heteroatoms, preferably via carbon or nitrogen atoms. Preferred substituents of the first and/or second aromatic ring of Ar are Q to C4 alkyl such as methyl; halo such as bromo and chloro; and haloalkyl such as trifluoromethyl. If the first aromatic ring is a pyrazole ring it is typically methyl-substituted, preferably N-methyl-substituted, more preferably in 1 -position. 2
Typical examples of a substituted Ar are 5-methoxyquinolin-8-yl, 4-and methoxybenzothiazol-2-yl. A typical example for a non substituted Ar is quinolin-8-yl.
If the first aromatic ring is a pyridine ring it typically does not carry further substituents.
In a preferred embodiment of the present invention, the group in alpha position to the carbonyl function is an amino group NR 3 , wherein R 3 can have various meanings as defined below. The ligand-substituted matrix of the invention, in these cases, can be depicted as in the following figure (II), which is covered by the above figure (I).
R3 O R1 O R2
I II I II I
L— ( V)v— N— C— Ar1— N— C— N— Ar2 (n)
In the above figure (II), the NR group is not shown as a part of the linking point L or the spacer group Sp, but as a part of the ligand as such, for clarity reasons. It has to be kept in mind, however, that NR together with V forms the spacer group Sp as shown in figure (I), or is a part of the linking point L.
In an embodiment of the present invention, R is selected from: hydrogen; and C\ to C4 alkyl, typically linear and branched Ci to C4 alkyl which may comprise a cycloalkyl unit such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, tert-butyl, cyclopropyl, methylcyclopropyl.
Preferably R 3 is selected from hydrogen and methyl. More preferably, R 3 is hydrogen.
The other symbols Ar 1 and Ar 2 have the meanings and preferred meanings as defined above in the context of figure (I).
R 3 as defined in the context with and shown in formula (II) corresponds to R 3 as defined and shown in the patent application EP 10172684.2 of which the present application claims priority. The present invention also includes the ligands of the present invention (which, after being attached to the support material) form together with the support material the ligand- substituted matrix which is used in the method of the present invention. The ligands are in accordance with the following formulae wherein the symbols Sp, Ar 1 and Ar 2 have the meanings defined above, including the preferred meanings:
O R1 O R2
Sp— C— Ar1— N— C— N— Af (III)
The ligands according to formula (III) comprise the spacer group Sp attached to the ligand.
The ligands according to formulae (III) can serve as precursors for the synthesis of further compounds not having the spacer group attached. The named compounds are generated after cleavage of the spacer group and, as the case may be, conversion of the -C=0 containing functionality present in the part of the molecule which binds to antibodies, to any other appropriate functionality known to the person skilled in the art. Appropriate reactions to that end are known to person skilled in the art. The resulting compounds are also included in the present invention.
Preferred ligands of the matrix according to the present invention are depicted below wherein in each formula the very left N atom is connected to the linking point L which is not shown. All formulae below show the spacer group attached to the ligands:
N- 5-[(3-Aminopropyl)carbamoyl]-3-pyridyl}-N'-[3-(2-methylpyrimidin-4-yl)phenyl]urea
Figure imgf000019_0001
N-{5-[(3-Aminopropyl)carbamoyl]-3-pyridyl}-N'-(5-methylthiazol-2-yl)urea (L2)
Figure imgf000019_0002
N- { 5- [(3 - Aminopropyl)carbamoyl] -3 -pyridyl } -N '-(2-methylbenzothiazol-6-yl)urea (L3 )
Figure imgf000020_0001
N-{5-[(3-Aminopropyl)carbamoyl]-3-pyridyl}-N'-(benzoxazol-2-yl)urea (L4)
Figure imgf000020_0002
N-{5-[(3-Aminopropyl)carbamoyl]-3-pyridyl}-N'-(5,6-dihydro-4H-cyclopentathiazol-2- yl)urea (L5)
Figure imgf000020_0003
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(5-methoxyquinolin-8-yl)urea (L6)
Figure imgf000020_0004
N-{3-[(3-Aminopropyl)carbamoyl]phenyl}-N'-(5-ethyl-[l,3,4]thiadiazol-2-yl)urea (L7)
Figure imgf000020_0005
N-{5-[(3-Aminopropyl)carbamoyl]-3-pyridyl}-N'-(8-quinolinyl)urea (L8)
Figure imgf000020_0006
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(quinolin-8-yl)urea (L9)
Figure imgf000021_0001
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(4-methoxybenzothiazol-2-yl)urea (L10)
Figure imgf000021_0002
N-{5-[(3-Aminopropyl)carbamoyl]-3-pyridyl}-N'-(4-methoxybenzothiazol-2-yl)urea (Ll l)
Figure imgf000021_0003
N-{5-[(3-Aminopropyl)carbamoyl]-3-pyridyl}-N'-[l-methyl-3-(2-thienyl)pyrazol-5-yl]urea (L12)
Figure imgf000021_0004
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-[4-(2-pyridyl)thiazol-2-yl]urea (L13)
Figure imgf000021_0005
N- { 3 - [(3 - Aminopropyl)carbamoyl]phenyl } -N '- [4-(2-pyridyl)thiazol-2-yl] urea (L 14)
Figure imgf000021_0006
N-{3 (3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(2-methylbenzothiazol-6-yl)urea (L15)
Figure imgf000022_0001
N-{ 3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(benzothiazol-2-yl)urea (L16)
Figure imgf000022_0002
N-{3 (3-Aminopropyl)carbamoyl]-4-metho^
(L17)
Figure imgf000022_0003
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(4-trifluoromethylphenyl)urea (L18)
Figure imgf000022_0004
N-{3-[(3-Aminopropyl)carbamoyl]phenyl}-N'-(quinolin-8-yl)urea (L19)
Figure imgf000022_0005
N-{5-[(3-Aminopropyl)carbamoyl]-2-methylphenyl}-N '-(quinolin-8-yl)urea (L20)
Figure imgf000022_0006
N-{3 (3-Aminopropyl)carbamoyl]pyridin-5-yl}-N'-(lH-indazol-5-yl)urea (L21)
Figure imgf000022_0007
N- { 3 - [(3 - Aminopropyl)carbamoyl] -4-methoxyphenyl } -N '-( 1 -methylindazol-3 -yl)urea (L22)
Figure imgf000023_0001
N-{3-[(3-Aminop1 pyl)carbamoyl]-4-methoxyphenyl}-N '-(thiazol-2-yl)urea (L23)
Figure imgf000023_0002
N- { 3 - [(3 - Aminopropyl)carbamoyl] -4-methoxyphenyl } -N '-(5 -methylthiazol-2-yl)urea (L24)
Figure imgf000023_0003
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N '-(3-cyanophenyl)urea (L25)
Figure imgf000023_0004
N- { 3 - [(3 - Aminopropyl)carbamoyl] -4-methoxyphenyl } -N '-(3 ,4-methylenedioxyphi
(L26)
Figure imgf000023_0005
N-{ 3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N '-(2-methoxypyridin-5-yl)urea (L27)
Figure imgf000023_0006
N- { 3 - [(3 - Aminopropyl)carbamoyl] -4-methoxyphenyl } -N '-(5 -methyl- 1 -phenylpyrazol-3 - yl)urea (L28)
Figure imgf000023_0007
N-{4-[(3-Aminopropyl)carbamoyl]phenyl}-N'-(quinolin-8-yl)urea (L29)
Figure imgf000024_0001
The synthesis of the ligands (I) can, for example, be carried out on insoluble supports, also known as resins, e.g. polystyrene resins, preferably pre-loaded with the desired linker bearing a reactive group, e.g. an amino group, to which a building block, e.g. an Fmoc protected or unprotected amino acid, may be attached by amide formation. After deprotection of the building block (if applicable), the remaining parts of the molecule are attached by any reaction leading to an appropriate bond formation and, where necessary, additional synthetic steps. Finally, the compounds including the linker moieties are released from the insoluble support/resin by a suitable cleavage protocol known to the person skilled in the art and purified by chromatographic methods also known to the person skilled in the art.
The term "antibody" means an immunoglobulin, including both natural or wholly or partially synthetically produced and furthermore comprising all fragments and derivatives thereof which maintain specific binding ability. Typical fragments are Fc, Fab, heavy chain, and light chain. The term also comprises any polypeptide having a binding domain which is homologous or largely homologous, such as at least 95% identical when comparing the amino acid sequence, to an immunoglobulin binding domain. These polypeptides may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal and may be of human or non-human origin or a chimeric protein where the human Fc part is fused to murine Fab fragments (chimeric therapeutic antibodies with ending ...ximab, e.g. Rituximab) or to Fab fragments comprising human and murine sequences (humanized therapeutic antibodies with the ending ...zumab, e.g. Bevacizumab) or linked to different Fab fragments (bispecific antibodies). The antibody may have been produced by recombinant expression systems, e.g. CHO cells or NSO cells. The antibody may be a member of any immunoglobulin class, preferentially IgG, in case of human and humanized antibodies more preferentially IgGi, IgG2, IgG3 and IgG4. In the most preferred embodiment of the present invention, the antibody comprises an Fc fragment or domain, as it is supposed that the ligands according to the present invention bind to an antibody's Fc part. Accordingly, an antibody of the present invention most preferably is an antibody containing an Fc fragment or domain of an immunoglobulin class, preferably IgG, more preferably of human IgG or of polyclonal or monoclonal IgG of human origin, in particular of IgGi, IgG2 and IgG4.
The term "Fc fusion protein" refers to any combination of a Fc fragment with one or more proteins or protein domains. Examples of Fc fusion proteins include, but are not limited to, chimers of the human IgGi Fc domain with soluble receptor domains (therapeutic proteins with the ending....cept) such as Etanercept (human IgGi Fc combined with two tumor necrosis factor receptor domains) or Rilonacept (human IgGi fused with interleukin-1- receptor domain). The Fc fusion protein may be produced by any means. For instance, the Fc fusion protein may be enzymatically or chemically produced by coupling the Fc fragment to the appropriate protein or protein domain or it may be recombinantly produced from a gene encoding the Fc and the protein/protein domain sequence. Alternatively, the Fc fusion protein may be wholly or partially synthetically produced. The Fc fusion protein may also optionally be a multimolecular complex. A functional Fc fusion protein will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids.
The present invention is drawn to the affinity purification, preferably by affinity chromatography, of antibodies or Fc fusion proteins from complex mixtures e.g. fermentation supematants or plasma of human or animal origin, making use of the affinity ligands of formula (I) and preferred embodiments thereof, as disclosed elsewhere in the specification.
Accordingly, in a preferred embodiment, the present invention comprises a method for purifying a protein, preferably an immunoglobulin or Fc fusion protein, by affinity purification, preferably affinity chromatography. The affinity ligands according to the present invention bind to the Fc region of an antibody.
Bevacizumab (Avastin®, Genentech/Roche) is a humanized monoclonal antibody. It stops tumor growth by preventing the formation of new blood vessels by targeting and inhibiting the function of the protein vascular endothelial growth factor-A (VEGF-A) that stimulates new blood vessel formation (angiogenesis).
Tocilizumab (RoActemra®, Genentech/Roche) is a humanized monoclonal antibody. It is directed against the Interleukin-6 receptor and blocks the action of the proinflammatory cytokine Interleukin-6. It is approved for the treatment of rheumatoid arthritis.
Palivizumab (Synagis®, Abbott) is a humanized monoclonal antibody directed against the A- epitope of the respiratory syncytial virus (RSV) F-protein. It is used in the prevention of RSV infections.
Polyclonal human and rabbit IgGs are investigated to demonstrate the universal applicability of the described ligands for the affinity purification of immunoglobulins via the Fc part.
When practicing the invention, the ligands according to the general formula (I) are attached to a support matrix of an appropriate support material, resulting in a ligand-substituted matrix, typically a matrix for affinity purification, preferably affinity chromatography (also referred to as affinity matrix in the context of the present invention) for protein separation. The ligands of the general formula are attached to the support matrix via L, optionally including a spacer Sp.
Accordingly, the present invention includes an affinity matrix for protein separation, comprising a support matrix comprising a support material and at least one ligand as specified in the specification beforehand, wherein the ligand is attached thereto via L.
The support matrix may comprise any appropriate support material which is known to the person skilled in the art. The material may be soluble or insoluble, particulate or non- particulate, or of a monolithic structure, including fibers and membranes, porous or non- porous. It provides a convenient means of separating ligands of the invention from solutes in a contacting solution. Examples of support matrix include carbohydrate and crosslinked carbohydrate matrices such as agarose, Sepharose, Sephadex, cellulose, dextran, starch, alginate or carrageenan; synthetic polymer matrices such as polystyrene, styrene- divinylbenzene copolymers, polyacrylates, PEG polyacrylate copolymers, polymethacrylates, (e.g. poly(hydroxyethylmethacrylate), polyvinyl alcohol, polyamides or perfluorocarbons; inorganic matrices such as glass, silica or metal oxides; and composite materials. The affinity matrix is prepared by providing a support matrix of an appropriate support material and attaching a ligand of formula (I) thereto. Methods for attaching the ligand (I) to the support material are known to the person skilled in the art.
The present invention also relates to a method for affinity purification, preferably affinity chromatography, of a protein wherein a protein to be purified is contacted with the ligand- substituted matrix as described before.
The term "affinity purification" (which is used interchangeably with the term "affinity separation") refers to any separation technique involving molecular recognition of a protein by a ligand of formula (I). The ligand may be immobilized on a solid support facilitating separation of the ligand-antibody complex later on. Separation techniques may include, but are not limited to, affinity chromatography on packed columns, monolithic structures or membranes. The term further includes adsorption in batch-mode or affinity precipitation.
Independently of the flavour, purification techniques are composed by an initial recognition phase where ligand is contacted with antibody in crude. In a second phase, either impurities are separated from the ligand-antibody complex or (e.g. column chromatography) or ligand- antibody complex is separated from impurities (e.g. affinity precipitation). In a third step, the antibody is released from the ligand-antibody complex by alteration of chemical and/or physical conditions like change in pH, ionic strength and/or addition of modifiers like organic solvents, detergents or chaotropes.
The invention will now be illustrated by the following examples, which shall not be construed to limit the invention. Documents cited in the specification:
1. A.Cecilia, A.Roque, C.R. Lowe, M.A. Taipa: Antibodies and Genetically Engineered Related Molecules: Production and Purification, Biotechnol. Prog. 2004, 20, 639-654
2. K.L.Carson: Flexibility-the guiding principle for antibody manufacturing, Nature
Biotechnology, 2005, 23, 1054-1058; S.Hober, K.Nord, M. Linhult: Protein A chromatography for antibody purification, J. Chromatogr. B. 2007, 848, 40-47
3. T.Arakawa, Y.Kita, H.Sato, D. Ejima, Protein Expression and Purification. 2009, 63, 158-163
4. S. Ghose, B. Hubbard, S.M. Cramer, Journal of Chromatography A, 2006, 1122, 144- 152
Examples
Materials and Methods
If not otherwise stated, all chemicals and solvents were of analytical grade, with the exception of example 2. Reagents used in example 2 were of preparative to analytical grade depending on particulate requirements and availability.
96 and 384-well filter plates having hydrophilic membrane filters with 0.45 μιη average pore size were purchased from Pall GmbH (Dreieich/Germany). Top frits made from polyethylene with 10 μηι average pore size were provided by Porex (Bautzen/Germany). General purpose microtiterplates for collection of fractions and analytical assays were ordered from Greiner Bio One GmbH (Frickenhau sen/Germany). Analytical assays were read out using a Fluostar Galaxy plate reader from BMG Labtech GmbH (Offenburg/Germany).
Column chromatography with antibody and purification of antibody fragments was conducted on a Waters HPLC system (Waters GmbH, Eschborn/Germany). Omnifit column housings (Diba Industries Ltd, Cambridge/United Kingdom) were used for packing of columns. NHS- activated Sepharose 4 FF, rProtein A Sepharose FF and Superdex 70 chromatography media were bought from GE Healthcare (Uppsala/Sweden). Mabsorbent A2P HF was purchased from Prometic Life Sciences (Cambridge/United Kingdom) and MEP Hypercel from Pall Corporation (Port Washington NY, USA).
Analytical chromatography of ligands was conducted on a Shimadzu HPLC system (Shimadzu Deutschland GmbH, Duisburg/Germany) including a diode array detector and single-quad mass spectrometer. The monolithic C18 reversed phase column was purchased from Merck KGaA (Darmstadt/Germany). Solvents used in analyses were of mass spectrometry grade.
Antibodies used in the present invention were Bevacizumab (Avastin, F. Hoffmann-La Roche, Switzerland), Tocilizumab (RoActemra, F. Hoffmann-La Roche, Switzerland), Palivizumab (Synagis, Abbott, USA), poly-IgG from human serum (Sigma-Aldrich, USA) and poly-IgG from rabbit serum (Sigma-Aldrich, USA). Immobilized papain for preparation of antibody fragments was purchased from Thermo Scientific (Bonn/Germany).
Flowthrough from protein A chromatography (referred to as host cell proteins) was derived from the supernatant of antibody-producing CHO cell culture in serum-free medium.
Coomassie brilliant blue dye reagent for Bradford assay, was purchased from Thermo S cientif ic (B onn/Germany ) . Example 1
SPR Screening of chemical microarrays
Graffinity has developed high density chemical microarrays comprising thousands of small molecules immobilized onto gold chips. The arrays are constructed using maleimide -thiol coupling chemistry in combination with high density pintool spotting. For the construction, glass plates are microstructured by photolithographical methods in order to define up to 9,216 sensor fields per array. A subsequently applied gold coating provides the basis for the SPR effect and enables the formation of a binary, mixed self assembled monolayer (SAM) of two different thiols. One of the thiols exhibits a reactive maleimide moiety to which thiol-tagged array compounds can couple to during pintool spotting. The resulting microarrays comprise 9,216 sensor fields, each containing multiply copies of a defined and quality controlled array compound.
Surface plasmons are collective oscillations of electrons at a metal surface which can be resonantly excited by polarized light of appropriate wavelength and incidence angle. At surface plasmon resonance conditions, the light intensity reflected from the gold chip exhibits a sharp attenuation (SPR minimum). The angle and wavelength position of this reflectance minimum strongly depends on the refractive index of the medium adjacent to the metal surface. Processes which alter the local refractive index in close proximity to the metal surface (such as antibody binding to the immobilized ligands) can therefore be monitored sensitively. For SPR detection, the chemical microarray is incubated with the target protein under optimized screening conditions. If targets bind to the immobilized fragments a shift in the wavelength dependent SPR minimum (referred to as the SPR shift) occurs with respect to the buffer control and thus indicates protein-ligand interaction. The SPR imaging approach utilizes an expanded beam of parallel light to illuminate the entire microarray sensor area at a fixed angle. The reflected light is then captured by means of a CCD camera. While scanning over a range of wavelengths covering the SPR resonance conditions, reflection images are recorded stepwise. Automated spot finding routines and subsequent grey-scale analysis of the obtained pictures lead to SPR resonance curves of all 9,216 sensor fields per chip in parallel. Screening of Antibodies and Fragments thereof by SPR
The platform allowed screening of a proteins, antibodies and fragments thereof against Graffinity's immobilized compound library of 110,000 small molecules in a high-throughout fashion. For each of the targets, the SPR screening conditions were optimized individually before screening of the targets against the entire library. Among such optimized screening parameters were a) the composition of the screening buffer including appropriate detergents, b) the concentration of the antibody or protein target in solution and c) the surface density of immobilized ligands on the chip surface. Neumann et al. (5) summarizes the screened targets and the corresponding screening conditions applied. In total four different full-length antibodies were screened and compared. Additionally, the Fc and Fab fragments of two antibodies were obtained by proteolytic digestion and were subjected to array screening as well. In order to identify antibody specific ligands, the screening campaign included also antibody free host cell protein (HCP) of CHO cell lines as controls.
The experimental results of the microarray screening campaigns were analyzed by manual hit selection using in-house developed software routines as well as by an in-depth data analysis by commercially available data mining tools. The analysis resulted in a number of hit series which showed clear binding to antibodies and their Fc fragments but only weak to negligible binding to the HCP controls. Such primary hit series were used for hit confirmation in secondary assays and could be used as starting point for the synthesized of focused libraries around the primary hits. For example, the Fc specific ligand LI showed distinct binding to the full length Bevacizumab and its Fc fragment but only minor binding to the Bevacizumab Fab fragment. Furthermore it clearly bound the other full length antibodies Anti RhD IgG, Tocilicumab and Palivizumab. Example 2
Synthesis of Ligands
General Procedure A: Synthesis of Ν,Ν'-diarylureas starting from electron-rich support-bound arylamines (L6, L7, L9, L10)
The required N-Fmoc-aminoaryl carboxylic acid (0.25 mmol) and HATU (0.25 mmol) were dissolved in NMP (approx. 1.5 mL) and treated with DIPEA (0.5 mmol). After 2 min, the mixture was added to 0.15 mmol of pre-swollen 1,3-diaminopropane-trityl polystyrene resin. After shaking for 4 h, the resin was washed with DMF and dichloromethane (each solvent several times). Removal of the Fmoc group was effected by treatment with 25% piperidine in DMF for approx. 30 min, whereupon the resin was washed thoroughly as described before, followed by drying in a stream of nitrogen or in high vacuum. Next, a freshly prepared solution of chloroformic acid 4-nitrophenyl ester (0.5 mmol) and DIPEA (1 mmol) in dry dioxane (1.5 mL) was added. The resin was agitated for 30 min, then the solution was removed and the resin was quickly washed with dioxane. Subsequently, the moist resin was dried cautiously in high vacuum for several minutes. After transferring the dried resin to a glass vial, the required aniline (0.3 mmol), DIPEA (0.3 mmol) and dry dioxane (1.5 mL) were added, the vial was tightly closed and shaken overnight at 40 °C. Finally, the resin was washed with DMF and dichloromethane (each solvent several times) and the target compound was cleaved from the resin by treatment with dichloromethane-TFA-triethylsilane 85: 10:5 (twofold cleavage), followed by rinsing with dichloromethane. After evaporation to dryness, the residue was purified by preparative reversed-phase HPLC. This procedure may result in the formation of varying amounts of symmetric urea as by-product which is usually easily separable from the desired product.
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(5-methoxyquinolin-8-yl)urea (L6)
Figure imgf000032_0001
Prepared from N-Fmoc-5-amino-2-methoxybenzoic acid and 8-amino-5-methoxyquinoline following General Procedure A. ESI-MS: 424 (M+1).
N-{3-[(3-Aminopropyl)carbamoyl]phenyl}-N'-(5-ethyl-[l,3,4]thiadiazol-2-yl)urea (L7)
Figure imgf000033_0001
Prepared from N-Fmoc-3-aminobenzoic acid and 2-amino-5-ethyl-[l,3,4]thiadiazole following General Procedure A. ESI-MS: 349 (M+1).
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(quinolin-8-yl)urea (L9)
Figure imgf000033_0002
Prepared from N-Fmoc-5-amino-2-methoxybenzoic acid and 8-aminoquinoline following General Procedure A. ESI-MS: 394 (M+1).
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(4-methoxybenzothiazol-2- yl)urea (L10)
Figure imgf000033_0003
Prepared from N-Fmoc-5-amino-2-methoxybenzoic acid and 2-amino-4- methoxybenzothiazole following General Procedure A. ESI-MS: 430 (M+1). General Procedure B: Synthesis of Ν,Ν'-diarylureas by pre-formation of isocyanates in solution (LI, L2, L3, L4, L5, L8, Ll l, L12, L13, L14, L15, L16, L17, L19, L20, L21, L22, L23, L24, L27, L28, L29)
1,3-Diaminopropane-trityl -resin (0.1-0.15 mmol) was pre-swollen in NMP. Depending on the aminoarylcarboxylic acid to be used, two different coupling protocols were applied.
5-Aminonicotinic acid: The carboxylic acid (0.3 mmol) and HOBt (0.3 mmol) were suspended in NMP (1.5 mL) and treated with DIC (0.3 mmol). After vigorous shaking for 2 min, the suspension was added to the resin. The reaction mixture was agitated for 1 h, then the resin was washed with DMF, methanol and dichloromethane (each solvent several times) and dried in a stream of dry nitrogen prior to the next step.
N-Fmoc-5-Amino-2-methoxybenzoic acid, N-Fmoc-4-aminobenzoic acid, N-Fmoc-3-amino-
4- methylbenzoic acid and N-Fmoc-3-aminobenzoic acid: The carboxylic acid (0.2 mmol) and HATU (0.2 mmol) were dissolved in NMP (1.5 mL) and treated with DIPEA (0.4 mmol). After 2 min, the solution was added to the resin. The mixture was agitated for 45-90 min, then the resin was washed with DMF (1-3 times) and treated with 25% piperidine in DMF for SO- SO min. After this time, the resin was washed with DMF, methanol and dichloromethane (each solvent several times) and dried in a stream of dry nitrogen prior to the next step. This preparation is also suitable for larger batches. For example, a batch of 1,3-diaminopropane trityl resin preloaded with 5-amino-2-methoxybenzoic acid equivalent to a total loading of 1.5 mmol was prepared by multiplying all quantities listed before by 10. Of this batch, aliquots equivalent to approx. 125 μιηοΐ were used for the next synthetic step (see below).
Pre-formation of the isocyanate: Chloroformic acid 4-nitrophenyl ester (0.2-0.5 mmol, typically 0.5 mmol) was dissolved in dry dioxane in a glass vial. To this solution, a solution or suspension of the required arylamine (0.2-0.5 mmol, typically 0.5 mmol) and DIPEA (0.4-1 mmol, typically 1 mmol) in dry dioxane (1 mL) was added dropwise. The mixture was shaken for 1-1.5 h at room temperature, then the dry resin prepared before was added, the vial was closed thoroughly and heated to 80 °C (employed only for 5-amino-2-methoxybenzoic amide and 3-aminobenzoic amide) or to 100 °C (suitable for all resins, and particularly required for
5- aminonicotinic amide) for 1-1.25 h. After cooling to room temperature, the resin was washed with DMF, methanol and dichloromethane (each solvent several times), and the target compound was cleaved from the resin by treatment with dichloromethane-TFA-triethylsilane 85: 10:5 (twofold cleavage), followed by rinsing with dichloromethane. After evaporation to dryness, the residue was purified by preparative reversed-phase HPLC.
The pre-formation of arylisocyanates is also suitable for larger batches. For example, a batch of 2.5 mmol of quinoline-8-ylisocyanate was prepared by multiplying all quantities listed before by 5. After the reaction time the resulting thick slurry was gently heated until it was completely liquified. Subsequently, aliquots equivalent to approx. 0.5 mmol of the isocyanate were added to different pre-loaded resins and treated as described before.
N-{5-[(3-Aminopropyl)carbamoyl]-3-pyridyl}-N'-[3-(2-methylpyrimidin-4- yl)phenyl]urea (LI)
Figure imgf000035_0001
Prepared from 5-aminonicotinic acid and 3-(2-methylpyriminin-4-yl)aniline following General Procedure B. ESI-MS: 406 (M+l).
N-{5-[(3-Aminopropyl)carbamoyl]-3-pyridyl}-N'-(5-methylthiazol-2-yl)urea (L2)
Figure imgf000035_0002
Prepared from 5-aminonicotinic acid and 2-amino-5-methylthiazole following General Procedure AE. ESI-MS: 335 (M+l).
N-{5-[(3-Aminopropyl)carbamoyl]-3-pyridyl}-N'-(2-methylbenzothiazol-6-yl)urea (L3)
Figure imgf000035_0003
Prepared from 5-aminonicotinic acid and 6-amino-2-methylbenzothiazole following General Procedure B. ESI-MS: 385 (M+l). N-{5-[(3-Aminopropyl)carbamoyl]-3-pyridyl}-N'-(benzoxazol-2-yl)urea (L4)
Figure imgf000036_0001
Prepared from 5-aminonicotinic acid and 2-aminobenzoxazole following General Procedure B. ESI-MS: 355 (M+l).
N-{5-[(3-Aminopropyl)carbamoyl]-3-pyridyl}-N'-(5,6-dihydro-4H-cyclopentathiazol-2- yl)urea (L5)
Figure imgf000036_0002
Prepared from 5-aminonicotinic acid and 2-amino-5,6-dihydro-4H-cyclopentathiazole following General Procedure B. ESI-MS: 361 (M+l).
N-{5-[(3-Aminopropyl)carbamoyl]-3-pyridyl}-N'-(8-quinolinyl)urea (L8)
Figure imgf000036_0003
Prepared from 5-aminonicotinic acid and 8-aminoquinoline following General Procedure B. ESI-MS: 365 (M+l).
N-{5-[(3-Aminopropyl)carbamoyl]-3-pyridyl}-N'-(4-methoxybenzothiazol-2-yl)urea (Lll)
Figure imgf000036_0004
Prepared from 5-aminonicotinic acid and 2-amino-4-methoxybenzothiazole following General Procedure B. ESI-MS: 401 (M+l). N-{5-[(3-Aminopropyl)carbamoyl]-3-pyridyl}-N'-[l-methyl-3-(2-thienyl)pyrazol-5- yl]urea (L12)
Figure imgf000037_0001
Prepared from 5-aminonicotinic acid and 5-amino-l-methyl-3-(2-thienyl)pyrazole following General Procedure B. ESI-MS: 400 (M+l).
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-[4-(2-pyridyl)thiazol-2-yl]urea (L13)
Figure imgf000037_0002
Prepared from N-Fmoc-5-amino-2-methoxybenzoic acid and 2-amino-4-(2-pyridyl)thiazole following General Procedure B. ESI-MS: 427 (M+l).
N-{3-[(3-Aminopropyl)carbamoyl]phenyl}-N'-[4-(2-pyridyl)thiazol-2-yl]urea (L14)
Figure imgf000037_0003
Prepared from N-Fmoc-3-aminobenzoic acid and 2-amino-4-(2-pyridyl)thiazole following General Procedure B. ESI-MS: 397 (M+l).
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(2-methylbenzothiazol-6- yl)urea (L15)
Figure imgf000037_0004
Prepared from N-Fmoc-5-amino-2-methoxybenzoic acid and 6-amino-2-methylthiazole following General Procedure B. ESI-MS: 414 (M+l). N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(benzothiazol-2-yl)urea (L16)
Figure imgf000038_0001
Prepared from N-Fmoc-5-amino-2-methoxybenzoic acid and 2-aminobenzothiazole following General Procedure B. After urea formation, the highly insoluble symmetric urea derivative that had formed as a by-product was removed by careful flotation in methanol. ESI-MS: 400 (M+l).
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(benzo-[l,2,5]thiadiazol-4- yl)urea (L17)
Figure imgf000038_0002
Prepared from N-Fmoc-5-amino-2-methoxybenzoic acid and 4-aminobenzo-[l,2,5]thiadiazole following General Procedure B. ESI-MS: 401 (M+l).
N-{3-[(3-Aminopropyl)carbamoyl]phenyl}-N'-(quinolin-8-yl)urea (L19)
Figure imgf000038_0003
Prepared from N-Fmoc-3-aminobenzoic acid and 8-aminoquinoline following General Procedure B. ESI-MS: 364 (M+l).
N-{5-[(3-Aminopropyl)carbamoyl]-2-methylphenyl}-N'-(quinolin-8-yl)urea (L20)
Figure imgf000038_0004
Prepared from N-Fmoc-3-amino-4-methylbenzoic acid and 8-aminoquinoline following General Procedure B. ESI-MS: 378 (M+l).
N-{3-[(3-Aminopropyl)carbamoyl]pyridin-5-yl}-N'-(lH-indazol-5-yl)urea (L21)
Figure imgf000039_0001
Prepared from 5-aminonicotinic acid and l-Boc-5-aminoindazole following General Procedure B. Complete Boc deprotection was ensured by prolonged treatment of the target compound with trifluoroacetic acid solution. ESI-MS: 354 (M+l).
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(l-methylindazol-3-yl)urea (L22)
Figure imgf000039_0002
Prepared from N-Fmoc-5-amino-2-methoxybenzoic acid and 3-amino-l-methylindazole following General Procedure B. ESI-MS: 397 (M+l).
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(thiazol-2-yl)urea (L23)
Figure imgf000039_0003
Prepared from N-Fmoc-5-amino-2-methoxybenzoic acid and 2-aminothiazole following General Procedure B. After urea formation, the highly insoluble symmetric urea derivative that had formed as a by-product was removed by thorough washing of the resin with DMSO. ESI-MS: 350 (M+l). N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(5-methylthiazol-2-yl)urea (L24)
Figure imgf000040_0001
Prepared from N-Fmoc-5-amino-2-methoxybenzoic acid and 2-amino-5-methylthiazole following General Procedure B. ESI-MS: 364 (M+l).
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(2-methoxypyridin-5-yl)urea (L27)
Figure imgf000040_0002
Prepared from N-Fmoc-5-amino-2-methoxybenzoic acid and 5-amino-2-methoxypyridine following General Procedure B. ESI-MS: 374 (M+l).
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(5-methyl-l-phenylpyrazol-3- yl)urea (L28)
Figure imgf000040_0003
Prepared from N-Fmoc-5-amino-2-methoxybenzoic acid and 3-amino-5-methyl-l- phenylpyrazole following General Procedure B. ESI-MS: 423 (M+l).
N-{4-[(3-Aminopropyl)carbamoyl]phenyl}-N'-(quinolin-8-yl)urea (L29)
Figure imgf000040_0004
Prepared from N-Fmoc-4-aminobenzoic acid and 8-aminoquinoline following General Procedure B. ESI-MS: 364 (M+l).
General Procedure C: Synthesis of Ν,Ν'-diarylureas by reaction of anilines with aryl isocyanates (LI 8, L25, L26)
1,3-Diaminopropane-trityl -resin (1.5 mmol) was pre-swollen in NMP. N-Fmoc-5-Amino-2- methoxybenzoic acid (2 mmol) and HATU (2 mmol) were dissolved in NMP (15 mL) and treated with DIPEA (4 mmol). After 2 min, the solution was added to the resin. The mixture was agitated for 1 h, then the resin was washed with DMF (3 times) and treated with 25% piperidine in DMF for 30 min. After this time, the resin was washed with DMF, methanol and dichloromethane (each solvent several times) and dried over night in a stream of dry nitrogen. Of this resin, aliquots of approx. 125 μιηοΐ were placed in glass vials and treated with the respective aryl isocyanates (0.5 mmol) in dry dioxane (1.5 mL) at 100 °C for 1 h. After washing the resins with DMF, methanol and dichloromethane (each solvent several times), the target compounds were cleaved from the support by twofold treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by rinsing of the resins with dichloromethane and evaporation of the solvents. The residues were purified by preparative reverse-phase HPLC.
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(4-trifluoromethylphenyl)urea (L18)
Figure imgf000041_0001
Prepared from 4-trifluoromethylphenylisocyanate following General Procedure C. ESI-MS: 411 (M+l). N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(3-cyanophenyl)urea (L25)
Figure imgf000042_0001
Prepared from 3-cyanophenylisocyanate following General Procedure C. ESI-MS: 368 (M+l).
N-{3-[(3-Aminopropyl)carbamoyl]-4-methoxyphenyl}-N'-(3,4- methylenedioxyphenyl)urea (L26)
Figure imgf000042_0002
Prepared from 3,4-methylenedioxyphenylisocyanate following General Procedure C. ESI-MS: 387 (M+l).
Example 3: Immobilization of Ligands Experimental
Dried ligands were redissolved in DMSO at an approximate concentration of 100 mM based on gravimetric analyses. Precise concentrations of stock solutions were determined by the o-phthaldialdehyde assay (6). Dye-ligand molecules were quantitated by measuring absorbance at 340 nm. Measurements were calibrated with l-(3-aminopropyl)imidazole. In coupling reactions, one volume of ligand dissolved at an approximate concentration of 15 to 20 mM in 90% DMSO and 10% N-Methyl-2-pyrrolidone containing 1 M N,N- Diisopropylethylamine was added to one volume of settled NHS-activated Sepharose 4 FF (GE Healthcare) equilibrated with isopropanol. Reactions were conducted for 2 h at 25 °C while shaking vigorously. The supernatant of reactions was withdrawn and resins were washed twice with appropriate solvent. Remaining active groups on resins were blocked with 1 M Ethanolamine for 1 h at 25 °C. Final resins were washed and stored in 20% ethanol at 4 °C until used in subsequent experiments. Reaction yields were determined based on mass balances after analysis of ligand concentrations in all fractions.
Summary and Results
Ligands from example 2 were immobilized on NHS-activated Sepharose 4 FF for subsequent chromatography and batch adsorption experiments. Coupling was achieved by formation of an amide bond between the amino group of the aminopropyl linker on ligands and the NHS- activated carboxylic group of the pre-activated resin.
The table below gives the results for coupling reactions with ligands LI to LI 5 from example 2. On average, an immobilized ligand density of 16.5 μιηοΐ per ml of resin was obtained. Reaction yields averaged at 78.5 %.
Initial Supernatant Immobilized Yield
Ligand (μπιοΐ per ml (μπιοΐ per ml (μπιοΐ per ml
(%) gel) gel) gel)
LI 22.4 2.3 20.1 89.7
L2 22.2 4.1 18.1 81.6
L3 23.5 3.3 20.2 85.9
L4 22.2 3.2 19.0 85.7
L5 20.3 3.1 17.2 84.7
L6 23.8 6.1 17.7 74.5
L7 22.8 4.2 18.6 81.7
L8 10.8 2.2 8.6 79.9
L9 20.1 4.7 15.4 76.7
L10 19.6 3.6 16.0 81.5
L13 24.9 9.8 15.1 60.7
L14 16.4 7.0 9.4 57.3
L15 23.2 4.5 18.7 80.8
Averages 16.5 78.5 The table below gives the results for coupling reactions with ligands LI 6 to L29 from example 2. On average, an immobilized ligand density of 15.9 μιηοΐ per ml of resin was obtained. Reaction yields averaged at 65.2 %. ligand Initial Supernatant Immobilized Yield
(μηιοΐ per ml (μηιοΐ per ml (μπιοΐ per ml (%)
gel) gel) gel)
L16 23.9 12.4 11.5 48.2
L17 26.8 10.4 16.3 61.0
L18 22.1 4.9 17.2 77.9
L19 25.6 7.5 18.1 70.6
L20 - - - -
L21 31.0 7.4 23.5 76.0
L22 18.4 4.8 13.6 74.1
L23 21.8 10.4 11.4 52.3
L24 24.3 10.3 14.0 57.7
L25 25.2 8.6 16.6 65.9
L26 25.5 12.8 12.6 49.6
L27 24.4 9.6 14.8 60.7
L28 23.7 5.2 18.5 78.1
L29 24.6 6.1 18.5 75.1
Averages 15.9 65.2
Example 4: Chromatographic Evaluation of Ligands (from Example 2) Experimental
Resins were assessed in packed mode by microtiter plate chromatography. For each column, approximately 30 μΐ of resin were transferred to a 384-well filter plate and sealed with appropriate top frits. Columns with rProtein A Sepharose FF and Mabsorbent A2P HF were included as controls.
Columns were equilibrated with 6.7 column volumes (cv) of phosphate buffered saline (0.15 M NaCl, 20 mM sodium phosphate, pH 7.3; PBS) prior to injection. Either 3.3 cv of whole IgGs dissolved at 0.75 rng mi"'1. Fab or Fc fragments dissolved at 0.25 mg ; (all three in PBS) or host ceil proteins (HCP) were injected onto columns. Antibody fragments of Bevacizumab (Fab and Fc) were prepared by cleavage of whole igG with papain protease (7) according to the protocol of the manufacturer. Unbound protein was washed from columns with 5 cv of PBS prior to elntion with 5 cv of glycine buffer at pH 2,5. Volumes transferred were spun through columns at 50 g for 1 - 2 min. During injection of samples, acceleration was reduced to 10 g and centrifugation time increased to 5 - 10 min. Effluent fractions were collected in 384- well plates and concentrations of protein analyzed by Bradford assay. Protein masses m, π¾ and m. (see below) were calculated as the product of fraction volume and measured protein concentration.
Summary and Results
Binding and e!ution of several antibodies was demonstrated. Among them were the three humanized therapeutic antibodies Bevacizumab, Tocilizumab and Palivlzumab and a human poly-IgG (h-pol -IgG) mixture Isolated from human serum. In some cases, purified Bevacizumab Fab and Fc fragments or a mixture of rabbit IgGs (r-poly-IgG, isolated from serum) were used as additional feed. Selectivity of ligands toward antibodies was determined by investigation of binding behavior of host ceil proteins. Results for commercial resins rProtein A Sepharose FF irProtein A), Mabsorbent A2P (A2P) and MEP Hypercel (MEP) were included for comparison.
The fraction of 'bound' protein was caicnlated as the difference between the total mass of protein Injected n¾ and the mass of protein detected in the flowthrough n¾. divided by the total mass of protein injected . m su ,.
bound =— : -
The 'yield' of protein was calculated as the mass of protein eiuted mi;, divided by the mass of protein injected n¾..
yield = ^
m The 'selectivity' of resins was calculated as the fraction of bonnd antibody Β,¾5 divided by the fraction of bound host cell proteins BRCP-
B ,
selectivity =
BHcr
The fraction of 'bound' protein and the 'yield' of protein were calculated for each resin based on data for whole !gG-aniibodies. In the case of Bevacizumab fragments and host cell proteins, only the fraction of 'bonnd' protein is given. Selectivity of resins was calculated from data for Bevaeizumab whole IgG and host cell proteins. Due to limited experimental precision, selectivity was truncated beyond values of 10.
Chromatography results for resins from example 3 and reference resins axe given in the tables below.
Bevacizumab Tocilizumab Palivizumab h-poly-IgG r-poly-IgG HCP
IgG Fab Fc
Ligand Bound Yield Bound Bound Bound Yield Bound Yield Bound Yield Bound Yield Bound Selectivil
(%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%)
L1 100 85 - - 9 9 3 0 21 15 - 58 1 .7
L2 100 80 - - 16 19 18 14 31 24 - 52 1 .9
L3 100 89 - - 20 25 9 5 28 22 - 54 1 .9
L4 100 94 - - 26 33 9 9 38 30 - 54 1 .9
L5 100 96 - - 53 56 35 40 54 45 - 68 1 .5
L6 100 93 - - 100 93 100 97 95 75 - 55 1 .8
L7 100 84 - - 36 38 24 25 45 36 - 56 1 .8
L8 100 89 - - 84 82 67 66 75 61 - 40 2.5
L9 100 93 - - 100 96 100 96 95 77 - 53 1 .9
L10 100 99 - - 100 92 100 97 95 79 - 62 1 .6
L1 1 100 100 - - 56 58 20 25 45 40 - 61 1 .6
L12 100 98 - - 62 65 25 27 39 33 - 61 1 .6
L13 100 96 - - 70 66 61 67 68 62 - 47 2.1
L14 100 93 - - 32 38 23 23 39 33 - 52 1 .9
L15 100 82 - - 76 75 62 62 65 57 - 53 1 .9
Bevacizumab Tocilizumab Palivizumab h-poly-IgG r-poly-IgG HCP
Fab Fc
Ligand Bound Yield Bound Bound Bound Yield Bound Yield Bound Yield Bound Yield Bound Selectivit
(%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) rProtA 100 94 - - 100 95 100 100 95 79 - - 0 10.0
A2P 100 84 - - 97 77 100 89 100 82 - - 100 1 .0
MEP 100 83 - - 100 71 100 59 95 57 - - 77 1.3
Bevacizumab Tocilizumab Palivizumab h-poly-lgG HCP
Bound Yield Bound Yield Bound Yield Bound Yield Bound Selectivity
Ligand
(%) (%) (%) (%) (%) (%) (%) (%) (%)
L16 100 98 98 98 98 79 100 99 60 1 .7
L17 100 98 100 100 97 95 97 87 48 2.1
L18 100 94 98 98 93 78 100 87 68 1 .5
L19 100 100 98 98 82 71 100 100 56 1 .8
L20 100 100 93 93 78 78 80 80 57 1 .8
L21 100 93 9 9 7 0 35 33 48 2.1
L22 100 96 62 62 25 19 60 60 42 2.4
L23 100 92 31 31 7 7 9 0 50 2.0
L24 100 93 55 55 29 29 48 48 49 2.0
L25 100 89 55 55 27 27 40 30 58 1 .7
L26 100 96 52 52 32 32 51 47 54 1 .9
L27 100 98 22 22 7 7 34 29 38 2.6
L28 100 96 64 64 65 51 86 81 59 1 .7
L29 100 100 82 82 61 47 95 95 55 1 .8
Controls bound all antibodies to nearly 100%. Whereas Protein A bound no host cell proteins (HCP) at all, A2P and MEP Hypercel showed high binding properties towards them. Resulting selectivity indices were 10.0 for Protein A, 1.0 for A2P and 1.3 for MEP Hypercel. Among the tested ligands from example 2, L6, L9, L17 and L10 showed best results in respect of antibody binding, yield and selectivity. All of them bound all tested human whole IgG antibodies to more than 90% and thus have binding characteristics of generic Fc -binders. Yields of Bevacizumab, Tocilizumab and Palivizumab after chromatography varied between 92% and 99% (100% Tocilizumab yield for L17). Yield of poly-IgG was slightly lower and ranged from 75% to 79% (87% for L17). Selectivity indices were 1.8 for L6, 1.9 for L9, 2.1 for L17 and 1.6 for L10.
Other ligands from example 2 showed either reduced binding of at least one of the tested antibodies, lower yield of at least one of the testes antibodies or higher binding of HCP (resulting selectivity indices were 1.5 or lower).
Documents cited in the examples:
5. T. Neumann, HD Junker, K. Schmidt, R. Sekul, SPR Based Fragment Screening:
Advantages and Applications, Current Topics in Medicinal Chemistry 2007; 7: 1630-1642
6. Roth M. Fluorescence reaction for amino acids. Anal Chem 197; 43(7):880-2.
7. Rousseaux J, Rousseaux-Prevost R, Bazin H. Optimal conditions for the preparation of Fab and F(ab')2 fragments from monoclonal IgG of different rat IgG subclasses. J. Immunol. Methods 1983; 64(1-2): 141-146.

Claims

Claims
Use, for affinity purification of a protein, preferably an antibody or fragment of an antibody, of a ligand-substituted matrix comprising a support material and at least one ligand covalently bonded to the support material, the ligand being represented by formula (I)
O R1 O R2
II I II I
L— (Sp)-C— Ar— N— C— N— Ar2 m wherein
L is the linking point on the support material to which the ligand is attached;
Sp is a spacer group;
v is 0 or 1 ;
R1 is hydrogen or Ci to C4 alkyl;
R is hydrogen or Q to C4 alkyl;
Ar1 is a divalent 5- or 6-membered substituted or unsubstituted aromatic ring; and Ar is 5- or 6-membered aromatic ring which may be unsubstituted or which is
(a) attached to a further 5- or 6-membered aromatic ring via a single bond; or
(b) fused to a further 5- or 6-membered aromatic or non aromatic ring as part of a multicyclic ring system; or
(c) attached to at least one substituent selected from Ci to C4 alkyl; C2 to C4 alkenyl; C2 to C4 alkynyl; a halogen; Ci to C4 haloalkyl; hydroxyl-substituted Ci to C4 alkyl; Ci to C4 alkoxy; hydroxyl-substituted Ci to C4 alkoxy; Ci to C4 alkylamino; Ci to C4 alkylthio; Q to C4 alkylnitrile, and combinations thereof.
The use of claim 1 wherein Ar1 is phenylene, preferably methoxy-substituted phenylene, or a pyridine ring.
The use of claim 1 or 2 wherein the C=0 and the NR1 group are bonded to Ar1 in meta position to each other.
The use of any of claims 1 to 3 wherein the 5- or 6-membered aromatic ring of Ar is heterocyclic. The use of any of claims 1 to 4 wherein the 5- or 6-membered aromatic ring of Ar contains two or more nitrogen atoms or a nitrogen atom and an oxygen atom or a nitrogen atom and a sulfur atom.
2
6. The use of any of claim 1 wherein the 5- or 6-membered aromatic ring of Ar is benzene.
7. The use of claim 1 wherein Ar are 5-methoxyquinolin-8-yl, quinolin-8-yl, 4- methoxybenzothiazol-2-yl, benzo-[l,2,5]thiadiazol-4-yl.
8. The use according to any of claims 1 to 7 wherein the support material comprises a material selected from carbohydrates or crosslinked carbohydrates, preferably agarose, cellulose, dextran, starch, alginate and carrageenan, Sepharose, Sephadex; synthetic polymers, preferably polystyrene, styrene-divinylbenzene copolymers, polyacrylates, PEG-Polycacrylate copolymers polymethacrylates, polyvinyl alcohol, polyamides and perfluorocarbons; inorganic materials, preferably glass, silica and metal oxides; and composite materials.
9. The use according to any of claims 1 to 8 wherein the protein is an antibody, preferably an IgG type antibody, or an Fc fusion protein.
10. The use of claim 9 wherein the purification is attained by binding of the ligand of the ligand- substituted matrix to an Fc fragment or domain of the antibody or the fusion protein.
11. The use according to claims 9 or 10 wherein the Fc fragment or domain or the antibody belongs to the IgG antibody class, more preferably to human, humanized and chimeric antibodies, IgG or to polyclonal or monoclonal IgG of human origin, in particular to IgGi, IgG2, IgG3 and IgG4.
12. A ligand-substituted matrix as defined in any of claims 1 to 8.
13. A method of synthesis of a ligand-substituted matrix according to claim 12 wherein a ligand according to formula (I) as specified in any of claims 1 to 7 is attached to the support material.
A method for affinity purification, preferably affinity chromatography, of a protein, wherein a protein to be purified is contacted with a ligand-substituted matrix as defined in claims any of claims 1 to 8.
15. The method of claim 14 wherein the protein is an antibody or an Fc fusion protein and the ligand binds to the Fc region of the antibody or the Fc fusion protein.
A ligand as shown in formula (II), wherein Sp, Ar 1 , Ar 2 and Am have the meaning defined in claims 1-7.
O R1 O R2
- i I 11 I 2
Sp— C— Ar— — C— — Ar2 (m)
PCT/EP2011/063831 2010-08-12 2011-08-11 LIGANDS FOR ANTIBODY AND Fc-FUSION PROTEIN PURIFICATION BY AFFINITY CHROMATOGRAPHY II WO2012020080A2 (en)

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