WO 2006/066598 PCT/DK2005/000828
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ANTIBODY BINDING AFFINITY LIGANDS
FIELD OF'THE INVENTION
The present invention relates to affinity ligands covalently bound to a solid support material,
such as a polymer matrix, and uses thereof in the purification and/or isolation of
biomolecules, such as proteins, in particular antibodies, such as monoclonal antibodies. The
affinity ligands comprise two different domains or functional groups: (i) a hydrophobic
domain and (ii) a cationic domain.
BACKGROUND OF THE INVENTION
Affinity chromatography enables selectively and reversibly adsorbing biological substances,
such as monoclonal antibodies, to a complementary binding substance, such as an affinity
ligand immobilised on a solid support material packed in an affinity column.
Affinity columns often contain a solid support material, usually a porous, polymer matrix, to
which a suitable ligand is covalently attached directly or by means of a linker. A sample
containing biological substances having an affinity for the ligand can be brought into contact
with the affinity ligand covalently immobilised to the solid support material under suitable
binding conditions which promote a specific binding between the ligand and the biological
substances having an affinity for the ligand. The column can subsequently be washed with a
buffer to remove unbound material, and in a further step biological substances having an
affinity for the ligand can be eluted and obtained in a purified or isolated form. Accordingly,
the ligand should preferably exhibit specific and reversible binding characteristics to the
biological substance, such as an antibody, which is desired to purify or isolate.
Antibodies have one or more copies of a Y-shaped unit, composed of four polypeptide chains.
Each Y contains two identical copies of a "heavy" chain, and two identical copies of a "light
chain", named as such by their relative molecular weights.
Antibodies can be divided into five classes: IgG, IgM, IgA, IgD and IgE, based on the number
of Y units and the type of heavy chain. The heavy chain determines the subclass of each
antibody. Heavy chains of IgG, IgM, IgA, IgD, and IgE are known as gamma, mu, alpha,
delta, and epsilon, respectively. The light chains of any antibody can be classified as either a
kappa (k) or lambda (A) type (a description of molecular characteristics of the polypeptide).

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For pharmaceutical applications, the most commonly used antibody is IgG which can be
cleaved into three parts, two F(ab) regions and one Fc region, by the proteolytic enzyme
papain, or into two parts, one F(ab')2 and one Fc region by the proteoiytic enzyme pepsin.
The F(ab) regions comprise the "arms" of the antibody, which are critical for antigen binding.
The Fc region comprises the "tail" of the antibody and plays a role' in immune response, as
well as serving as a useful "handle" for manipulating the antibody during some
immunochemical procedures. The number of F(ab) regions on the antibody, corresponds with
its subclass, and determines the "valency" of the antibody (loosely stated, the number of
"arms" with which the antibody may bind its antigen).
The term "antibody" means an immunoglobuiin, whether natural or wholly or partially
synthetically produced. All fragments and derivatives thereof which maintain specific binding
ability are also included in the term. Typical fragments are FC, FAB, heavy chain, and light
chain. The term also covers 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 immunoglobuiin binding domain. These polypeptides may be derived from
natural sources, or partly or wholly synthetically produced. An antibody may be monocionai
or polyclonal. The antibody may be a member of any immunoglobuiin class, including any of
the human classes: IgG, IgM, IgA, IgD, and IgE. Derivatives of the IgG class, however, are
preferred in one embodiment of the present invention.
The term "antibody fragment" refers to any derivative of an antibody which is less than full-
length. Preferably, the antibody fragment retains at least a significant portion of the specific
binding ability of the full-length antibody. Examples of antibody fragments include, but are
not limited to, Fab, Fab1, F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments. The antibody
fragment may be produced by any means. For instance, the antibody fragment may be
enzymatically or chemically produced by fragmentation of an intact antibody or it may be
recombinantly produced from a gene encoding the partial antibody sequence. Alternatively,
the antibody fragment may be wholly or partially synthetically produced. The antibody
fragment may optionally be a single chain antibody fragment. Alternatively, the fragment
may comprise multiple chains which are linked together, for instance, by disulfide linkages.
The fragment may also optionally be a multimolecular complex. A functional antibody
fragment will typically comprise at least about 50 amino acids and more typicality will
comprise at least about 200 amino acids.
"Single-chain Fvs" (scFvs) are recombinant antibody fragments consisting of only the variable
light chain (VL) and variable heavy chain (VH) covalently connected to one another by a
polypeptide linker. Either VL or VH may be the amino-terminal domain. The polypeptide linker

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may be of variable length and composition so long as the two variable domains are bridged
without se-ious steric interference. Typically, the linkers are comprised primarily of stretches
of glycine and serine residues with some glutamic acid or lyslne residues interspersed for
solubility. "Diabodies" are dimerlc scFvs. The components of diabodies typically have shorter
peptide linkers than most scFvs and they show a preference for associating as dimers. An
"Fv" fragrrient is an antibody fragment which consists of one VH and one VL domain held
together by non-covalent interactions. The term "dsFv" is used herein to refer to an Fv with
an engineered intermolecular disulfide bond to stabilize the VH- VL pair. A "F(ab')2" fragment
Is an antibody fragment essentially equivalent to that obtained from immunoglobulins
(typically IgG) by digestion with an enzyme pepsin at pH 4.0-4.5. The fragment may be
recombinantly produced. A "Fab"' fragment is an antibody fragment essentially equivalent to
that obtained by reduction of the disulfide bridge or bridges joining the two heavy chain
pieces in the F(ab')2 fragment. The Fab' fragment may be recombinantly produced. A "Fab"
fragment is an antibody fragment essentially equivalent to that obtained by digestion of
immunoglobulins (typically IgG) with the enzyme papain. The Fab fragment may be
recombinantly produced. The heavy chain segment of the Fab fragment is the Fd piece. A
wFc" region is a constant region of a particular class of antibody.
The bonding between antigens and antibodies is dependent on hydrogen bonds, hydrophobic
bonds, electrostatic forces, and van der Waals forces. These are all bonds of a weak, non-
covalent nature, yet some associations between an antigen and an antibody can be quite
strong. Accordingly, the affinity constant for antibody-antigen binding can span a wide range,
extending from below 105 mol"1 to more than 1012 mol'1. Affinity constants are affected by
temperature, pH and solvent. Apart from an affinity of an antibody for a ligand, the overall
stability of an antibody-ligand complex is also determined by the valency of the antigen and
antibody and the structural arrangement of the interacting parts.
Accurate affinity constants can only be determined for monoclonal antibodies which are
genetically identical molecules recognising one single epitope on the antigen whereas for
polyclonai! antibodies a broad distribution of affinities may contribute to an apparent affinity
constant. The apparent affinity constant may also be caused by the fact that polyclonai
antibodies may recognise more than one single epitope on the same antigen. Since
antibodies normally harbour more than one binding domain per molecule multiple, co-
operative bondings take place between antibodies and their antigens; this effect is termed
avidity. As monoclonal antibodies react with only one single epitope on the antigen they are
more vulnerable to the loss of epitope through chemical treatment of the antigen than
polyclonai antibodies. This can be offset by pooling two or more monoclonal antibodies to the
same antigen.

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Monoclonal antibodies can be raised by fusion of B lymphocytes with immortal cell cultures to
produce hybridomas. Hybridomas will produce many copies of the exact same antibody - an
essential feature in the development of antibodies for therapeutic or diagnostic applications.
Currently, the most explored affinity ligand for the purification and isolation of biomolecules,
such as monoclonal antibodies is Protein A. Protein A is a widely used ligand, however, the
ligand may suffer from several shortcomings, such as problems relating to instability
concomitant with leaching from the column and the need to remove it from the final product
or insufficient sanitation of the chromatography resin and moreover, Protein A is fairly
expensive.
Hence, there exists a need for novel, stable, inexpensive iigands for isolating antibodies, in
particular monoclonal antibodies, or analogues, derivatives, fragments and precursors
thereof, whether derived from natural or recombinant sources.
SUMMARY OF THE INVENTION
In one aspect of the present invention, there is provided a solid support material having
covalently immobilized thereon an affinity ligand, said ligand comprising one or more
hydrophobic functional group(s) and one or more cationic functional group(s),
wherein at least one hydrophobic functional group is separated from at least one cationic
functional group by a through bond distance of from 5 A to 20 A,
wherein said ligand has a molecuiar weight of from 120 Da to 5,000 Da.
In another aspect of the present invention, there is provided a solid support material having
covalently immobilized thereon an affinity ligand, said ligand having one or more hydrophobic
functional group(s) and one or more heteroaromatic functional group(s),
wherein at least one hydrophobic functional group is separated from at least one
heteroaromatic functional group by a through bond distance of from 5 A to 20 A, and
wherein said ligand has a molecular weight of from 120 Da to 5,000 Da.
In a further aspect of the present invention, there is provided a method for the'isolation of
biomolecules, such as proteins, e.g. antibodies, in particular monoclonal antibodies, or
derivatives thereof, the method comprising the steps of (i) providing a solid support material

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having covalently immobilized thereon an affinity ligand as defined herein, (ii) providing a
sample putatively containing an antibody having an affinity for said ligand, (iii) contacting
said ligand with said sample putatively containing said antibody, (iv) binding selectively said
antibody when said antibody is contained in said sample and (v) isolating selectively said
antibody when said antibody is contained in said sample.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1: Resin B2 selectivity analysis. 1 = fermentation supernatant, 2 = flow through (cycle
1), 3 = elution (cycle 1), 4 = regeneration/sanitation (cycle 1), 5 = washing (cycle 2), 6 =
elution (cycle 2), 7 = no protein, 8 = mAb reference sample.
Figure 2: Resin B3 selectivity analysis. 1 = fermentation supernatant, 2 = flow through (cycle
1), 3 = elution (cycle 1), 4 = regeneration/sanitation (cycle 1), 5 = washing (cycle 2), 6 =
elution (cycle 2), 7 = regeneration/sanitation (cycle 2), 8 = mAb reference sample.
Figure 3; Resin Dl selectivity analysis. 1 = mAb reference sample, 2 = fermentation
supernatant, 3 = flow through (cycle 1), 4 = elution (cycle 1), 5 = regeneration/sanitation
(cycle 1), 6 = washing (cycle 2), 7 = elution (cycle 2), 8 = regeneration/sanitation (cyde 2).
Figure 4: Resin D2 selectivity analysis. 1 = mAb reference sample, 2 = fermentation
supernatant, 3 = flow through (cycle 1), 4 = elution (cycle 1), 5 = regeneration/sanitation
(cycle 1), 6 = washing (cycle 2), 7 = elution (cycle 2), 8 = regeneration/sanitation (cycle 2).
DETAILED DISCLOSURE OF THE INVENTION
As mentioned above, the present invention relates to novel solid support materials having
covalently immobilised thereon an affinity ligand, wherein the ligand comprises a particular
set of functional groups. Such materials are particularly useful for the purification and
isolation of biomolecules, such as proteins, e.g. antibodies, in particular monoclonal
antibodies, or derivatives thereof.
Ligands
When used herein, the term "ligand" means a molecule which can bind a target compound,
for example an antibody, in particular a monoclonal antibody. Ligands preferably bind their
binding partners at least In a substantially specific manner ("specific binding").

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"Specific binding" refers to the property of a ligand to: (1) bind to a binding partner, e.g. a
monoclonal antibody, (2) preferentially such that the relative mass of bound binding partner
e.g., a monoclonal antibody, is at least two-fold, such as 50-fold, for example 100-fold, such
as 1000-fold, or more greater than the relative mass of other bound species thajn the binding
partner, e.g. a monoclonal antibody. By relative mass of bound compound is me;ant the mass
of bound compound minus the mass of unbound compound divided by the total mass of
binding partner, i.e.
relative mass of bound compound = (mass of bound compound - mass of unbound
compound)/(mass of bound compound + mass of unbound compound),
compound being binding partner or other species.
The term "binding partner" means any biological molecule which is bound by a particular
ligand, preferably in a substantially specific manner. A binding partner may be shared by
more than one ligand. Preferred binding partners are antibodies including polyclonal
antibodies and monoclonal antibodies. Further preferred binding partners are antibody
fragments from monoclonal antibodies or polyclonal antibodies.
The ligands according to the invention include enriched or resolved optical isomers at any or
all asymmetric atoms as are apparent from the description or depiction herein. Both racemic
and diasteromeric mixtures, as well as the individual optical isomers can be isolated or
synthesized so as to be substantially free of their enantiomeric or diastereomeric partners,
and these are all within the scope of the invention.
Experiments have surprisingly shown that certain classes of affinity ligands, i.a. one where
the ligands comprise one or more hydrophobic functional group(s) and one or more cationic
functional group(s) bind selectively to mAbs. Another promising class of affinity ligands is the
one where the ligands comprise one or more hydrophobic functional group(s) and one or
more heteroaromatic functional group(s).
It was further found that the at least one hydrophobic functional group preferably should be
separated from the at least one cationic functional group by a through bond distance of from
5 A to 30 A, for example a through bond distance of from 5 A to 20 A, such as ja through
bond distance of from 5 to 19 A, for example a through bond distance of from 5 to 18 A, such
as a through bond distance of from 5 to 17 A, for example a through bond distance of from 5
to 16 A, such as a through bond distance of from 5 to 15 A, for example a through bond
distance of from 5 to 14 A, such as a through bond distance of from 5 to 13 A, for example a
through bond distance of from 5 to 12 A, such as a through bond distance of from 5 to 11 A,

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for example a through bond distance of from 5 to 10 A, such as a through bond distance of
from 6 to 14 A, or such as a through bond distance of from 7 to 20 A, for example a through
bond distance of from 7 to 19 A, such as a through bond distance of from 7 to 18 A, for
example a through bond distance of from 7 to 17 A, such as a through bond distance of from
7 to 16 A, for example a through bond distance of from 7 to 15 A, such as a through bond
distance of'from 7 to 14 A, for example a through bond distance of from 7 to 13 A, such as a
through bond distance of from 7 to 12 A, for example a through bond distance of from 7 to
11 A, such as a through bond distance of from 7 to 10 A, for example a through bond
distance of from 8 to 12 A, or for example a through bond distance of from 9 to 20 A, such as
a through bond distance of from 9 to 18 A, for example a through bond distance of from 9 to
16 A, such as a through bond distance of from 9 to 14 A, for example a through bond
distance of from 9 to 12 A, such as a through bond distance of from 9 to 11 A, for example a
through bond distance of about 10 A.
Through bond distance is the shortest intra molecular through bond distance between
covalently linked chemical entities along the chemical bonds. It is calculated by adding the
individual atom-atom bond distances along the shortest intramolecular path. Typical atom-
atom bond distances are i.2 A to 1.4 A.
The distance through space between the at least one hydrophobic functional group and the at
least one cationic functional group of the ligand is preferably less than 30 A, such as less
than 28 A, for example less than 26 A, such as less than 24 A, for example less than 22 A,
such as less than 20 A, for example less than 18 A, such as about or less than 16 A, for
example less than 15 A, such as about or less than 14 A, for example less than 13 A, such as
about or less than 12 A, for example less than 11 A, such as about or less than 10 A, for
example about or less than 8 A, such as about 6 A, for example in the range of from 5 to 20
A, such as a distance through space of from 5 to 19 A, for example a distance through space
of from 5 to 18 A, such as a distance through space of from 5 to 17 A, for example a distance
through space of from 5 to 16 A, such as a distance through space of from 5 to 15 A, for
example a1 distance through space of from 5 to 14 A, such as a distance through space of
from 5 to |13 A, for example a distance through space of from 5 to 12 A, such as a distance
through space of from 5 to 11 A, for example a distance through space of from 5 to 10 A,
such as a distance through space of from 7 to 20 A, for example a distance through space of
from 7 to Il9 A, such as a distance through space of from 7 to 18 A, for example a distance
through space of from 7 to 17 A, such as a distance through space of from 7 to 16 A, for
example a distance through space of from 7 to 15 A, such as a distance through space of
from 7 to 14 A, for example a distance through space of from 7 to 13 A, such as a distance
through space of from 7 to 12 A, for example a distance through space of from 7 to 11 A,
such as a distance through space of from 7 to 10 A, for example a distance through space of

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from 9 to 20 A, such as a distance through space of from 9 to 18 A, for example a distance
through space of from 9 to 16 A, such as a distance through space of from 9 to 14 A, for
example a distance through space of from 9 to 12 A, such as a distance through space of
from 9 to 11 A.
Through bond distances and distances through space can be calculated or determined by the
person skilled in the art according to state of the art techniques. Molecular modelling can also
be used for determining the minimum distance between atoms of different ligand functional
groupi. Molecular modelling can be performed e.g. with Sybyl/Mendyl 5.4 (Tripos Associates,
St. Louis, Mo.) using an Evans and Sutherland PS390 graphics computer equipped with a
stereographic viewer. Structures of suitable ligands can be provided via construction with the
Concord program or from libraries followed by energy minimization. Energy calculations can
be made with Sybyl/Mendyl force field and a 1.2 A van der Waals parameter for hydrogen.
Charges can be calculated using the Gasteigner-Huckel method which includes sigma-bonding
and pi-bonding.
The ligand preferably has a molecular weight of less than 5,000 Da, such as less than 4,000
Da, for example less than 3,500 Da, such as less than 3,000 Da, for exampie iess than 2,500
Da, such as less than 2,000 Da, such as less than 1,800 Da, for example less than 1,600 Da,
such as less than 1,500 Da, such as less than 1,400 Da, for example less than 1,300 Da,
such as less than 1,200 Da, such as less than 1,100 Da, for example less than 1,000 Da.
Additionally, the ligand preferably has a molecular weight of more than 120 Da, such as more
than 140 Da, for example more than 160 Da, such as more than 180 Da, for example more
than 200 Da, such as more than 220 Da, for example a molecular weight of more than 240
Da.
In order to reduce the degree of non-specific binding to the cationic groups, the ligand may
further comprise one or more anionic groups in order to compensate some of the positive
charge on the ligand. The anionic groups include, but are not limited to, carboxylate,
sulfonate, sulphate, phosphate and other negatively charged ionisable groupings, and can
e.g. be disposed upon groups pendant from the ligand.
Ligand functional groups
In one main group of suitable ligands, each ligand comprises one or more hydrophobic
functional group(s) and one or more cationic functional group(s).

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In another main group of suitable ligands, each ligand comprises one or more hydrophobic
functional c roup(s) and one or more heteroaromatic functional group(s).
As used herein, a cationic functional group is an organic group which has a positive charge in
the pH range 3-7. Primary, secondary, and tertiary amines are typical examples of cationic
groups. Guanidine is a further relevant example. Further examples of cationic functional
groups are given in the section "Cationic functional groups" below.
A hydrophobic functional group is an organic group capable of binding to the surface of a bio-
molecule mainly by hydrophobic interaction. Hydrophobic functional groups are characterised
by being essentially nonLpolar and uncharged at normal physiological conditions. Hydrophobic
residues are repelled by aqueous solution so as to seek the inner positions in the
conformation of a ligand when the ligand is in an aqueous medium. Also, hydrophobic
residues will seek towards hydrophobic pockets or grooves of ligand binding partners when
the ligand is associated with a binding partner under normal physiological conditions.
When used herein, the term "normal physiological condition" means conditions that are
typical inside a living organism or a ceii. While it is recognized that some organs or organisms
provide extreme conditions, the intra-organlsma! and intra-cellular environment normally
varies around pH 7 (i.e., from pH 6.5 to pH 7.5), contains water as the predominant solvent,
and exists at a temperature above 0°C and below 50°C. It will be recognized that the
concentration of various salts depends on the organ, organism, cell, or cellular compartment
used as a reference.
Organic hydrophobic groups generally have a high content of carbon atoms. Typical examples
of hydrophobic groups are linear and branched alkanes, cyclic hydrocarbons, aromatic
compounds, and combinations of linear and branched alkanes, cyclic hydrocarbons, and
aromatic compounds. Also substituted variants of such groups are considered as being
hydrophobic as long as the relative content of carbon is above a certain limit. However, the
percentage of carbon atoms is not the only parameter, which influences the hydrophoblcity.
Also the position and nature of other atoms play an important role. E.g. an ether is typically
more hydrophobic than an alcohol with the same number of carbon atoms and oxygen atoms,
and an ester is more hydrophobic than a di-ol with the same elemental composition. When
one or more possible npn-carbon and non-hydrogen atoms of an organic group are at
primary positions the relative number of carbon atoms must be higher for the group to be
hydrophqbic than when possible non-carbon and non-hydrogen atoms are at secondary,
tertiary, or quarternary positions. Keeping this in mind, we define a hydrophobic group as an
organic groups with 75% or more of its non-hydrogen atoms being carbon atoms, such as
80% or more, preferably 85% or more of its non-hydrogen atoms being carbon atoms. E.g.

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the lower value, 75%, applies to ethers and esters, the intermediate value, 80%, applies to
amides and secondary and tertiary amines, whereas the upper value, 85%, applies to
alcohols and primary amines. Examples of hydrophobic functional groups are given in the
section 'Ligand functional groups' below.
The at least one hydrophobic functional group can comprise or consist of one or more groups
selected from "alkyl", "cyclic alkyl", "substituted alkyl", "aryl", "substituted aryl", "alkenyl",
"substituted alkenyl", "alkynyl", "substituted alkynyl", "aralkyl", "substituted aralkyl",
"heterocyclyl", "substituted heterocydyl", "heterocyclylalkyl", "substituted heterocyclylalkyl",
"alkylaminoalkyi", "substituted alkylaminoalkyl", "dialkylaminoalkyl", "substituted
dialkylaminoalkyl", "heterocyclyloxyalkyl", "substituted heterocyclyloxyalkyl",
"arylaminoalkyl", "substituted aryiaminoalkyl", "heterocyclylaminoalkyl", "substituted
heterocyclylaminoalkyl", "alkylaminoalkoxy", "substituted alkylaminoalkoxy",
"dialkylaminoalkoxy", "substituted dialkylaminoalkoxy", "heterocyclyloxy", and "substituted
heterocyclyloxy", as defined herein below.
Hydrophoblc functional groups comprising aliphatic residues
The at least one hydrophobic functional group can comprise or consists of an optionally
substituted aliphatic residue and/or an optionally substituted aromatic residue. Aliphatic
residues generally refer to hydrocarbons such as e.g. alkyl, alkylene and alkynyl residues
which can be substituted or non-substituted.
"Alkyl" as used herein includes straight chain alkyl groups such as methyl, ethyl, propyl,
butyl, pentyl, hexyl, heptyl, octyl, nonyl, deo/l, undecyl, dodecyl and the like. "Alkyl" also
includes branched chain isomers of straight chain alkyl groups, including but not limited to,
the following which are provided by way of example: -CH(CH3)2, -CH(CH3)(CHZCH3),
-CH(CH2CH3)2, -C(CH3)3, -C(CH2CH3)3/ -CH2CH(CH3)2, -CH2CH(CH3)(CH2CH3),
-CH2CH(CH2CH3)2, -CH2C(CH3)3, -CH2C(CH2CH3) 3, -CH(CH3)CH(CH3)(CH2CH3),
-CHzCHiCHCCHs)^ -CH2CH2CH(CH3)(CH2CH3), -CH2CH2CH(CH2CH3)2, -CH2CH2C(CH3)3,
-CH2CH2C(CH2CH3)3, -CH(CH3)CH2CH(CH3)2, -CH(CH3)CH(CH3)CH(CH3)CH(CH3)2/
-CH(CH2CH3)CH(CH3)CH(CH3)(CH2CH3), and others.
The aliphatic residue can be an optionally substituted linear aliphatic residue or an optionally
substituted branched aliphatic residue. The aliphatic residue can also be an optionally
substituted cyclic alkyl. "Cyclic alkyl" includes groups such as cyclopropyl, cyclobutyl,
cydopentyl, cyclohexyl, cyclohepty!, and cyclooctyl and such rings substituted with straight
and branched chain alkyl groups as defined above, and also includes polycyclic alkyl groups

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such as, but not limited to, adamantyl norbornyl, and bicyclo[2.2.2]octyl and such rings
substituted with straight and branched chain alkyl groups as defined above. Thus,
unsubstitutsd alkyl groups include primary alkyl groups, secondary alkyl groups, and tertiary
alkyl groups. Unsubstituted alkyl groups may be bonded to one or more carbon atom(s),
oxygen atom(s), nitrogen atom(s), and/or sulfur atom(s) in a ligand.
The cyclic aliphatic residue can e.g. comprise or consist of a C5-C16 cycloalkyl group. Shorter
chain lengths can also occur, typically when the cycloalkyl is substituted with an aryl or
heteroaryl residue.
"Substituted alkyl" refers to an unsubstituted alkyl group as defined above in which one or
more bonds to a carbon(s) or hydrogen(s) are replaced by a bond to non-hydrogen and non-
carbon atoms such as, but not limited to, a halogen atom in halides such as F, Cl, Br, and I;
and oxygen atom in groups such as hydroxyl groups, alkoxy groups, aryloxy groups, and
ester groups; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups,
sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as
amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-
oxides, imides, and enamines; a silicon atom in groups such as in trialkylsilyl groups,
dialkylarylsilyl groups, alkyldlarylsilyl groups, and triarylsilyl groups; and other heteroatoms
in various other groups.
Substituted alkyl groups also include groups in which one or more bonds to a carbon(s) or
hydrogen(s) atom is replaced by a bond to a heteroatom such as oxygen in carbonyl,
carboxyl, and ester groups; nitrogen in groups such as imines, oximes, hydrazones, and
nitriles. Substituted alkyl groups also include, among others, alkyl groups in which one or
more bonds to a carbon or hydrogen atom is/are replaced by one or more bonds to a halogen
atom. Other substituted alkyl groups include those in which one or more bonds to a carbon or
hydrogen atom is replaced by a bond to an oxygen atom such that the substituted alkyl
group contains a hydroxyl, alkoxy, aryloxy group, or heterocyclyloxy group. Stili other alkyi
groups include alkyl groups that have an amine, alkylamine, dialkylamine, arylamine,
(alkyl)(aryl)amine, diarylamine, heterocyclylamine, (alkyl)(heterocyclyl)amine,
(aryl)(heterocyclyl)amine, or diheterocyclylamine group.
In one embodiment, an1 aliphatic functional group is preferably substituted with an aryl group
such as an (C6-Ci2) aryl group mentioned herein below, which may in turn also be
substituted, as also described herein. An example of a substituted aryl group includes an
"aralkyl group", which can be substituted or non-substituted.

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Accordingly, "aralkyl" refers to unsubstituted alkyl groups as defined above in which a
hydrogen or carbon bond of the unsubstituted alkyl group is replaced with a bond to an aryl
group as defined above. For example, methyl (-CH3) is an unsubstituted alkyl group. If a
hydrogen atom of the methyl group is replaced by a bond to a phenyl group, such as if the
carbon of the methyl was bonded to a carbon of benzene, then the compound is an
unsubstituted aralkyl group (i.e., a benzyl group). Thus includes, but is not limited to, groups
such as benzyl, diphenylmethyl, and 1-phenylethyl (-CH(C6H5)(CH3)), 2-phenylethyl group,
2-naphthylethyl group, and the like.
"Substituted aralkyl" has the same meaning with respect to unsubstituted aralkyl groups that
substituted aryl groups had with respect to unsubstituted aryl groups. However, a substituted
aralkyl group also includes groups in which a carbon or hydrogen bond of the alkyl part of the
group is replaced by a bond to a non-carbon or a non-hydrogen atom. Examples of
substituted aralkyl groups include, but are not limited to, -CH2C(=O)(C6H5), and -CH2(2-
methylphenyl) among others.
In one embodiment, the optionally substituted aliphatic residue comprises or consists of a C5-
C20 alkyl group. Shorter chain lengths can aiso occur, typically when the alkyl is substituted
with an aryl or heteroaryl residue. Further examples of alkyl groups substituted with aryl or
heteroaryi include, for example, a linear (C1-C10), branched (C4-Ca0) or cyclic (C5-C10) group,
such as a methyl group, ethyl group, propyl group, such as a n-propyl group and an isopropyl
group, butyl group, such as n-butyl group, isobutyl group, t-butyl group, n-amyl group,
pentyl group, such as neopentyl group, cyclopentyl group, hexyl group, such as n-hexyl
group, cyclohexyl group, heptyl group, octyl group, such as n-octyl group, nonyl group, such
as n-nonyl group, decyl group, such as n-decyl group, undecyl group, dodecyl group, mentyl
group, 2,3,4-trimethyl-3-penty! group, 2,4-dimethyl-3-penty! group, and the like.
In one embodiment, a C5-C2o alkyl group can also be substituted, for example with a halogen
atom, an alkoxy group, an aryloxy group, an alkylthio group, or an arylthio group. Examples
of the halogen atom are a fluorine atom, a chlorine atom, a bromine atom, and an iodine
atom. Examples of the alkoxyl group include, for example, a (C1-C4 alkoxy group such as
methoxy group, ethoxy group, n-propoxy group, t-butoxy group or the like. Examples of the
alkylthio group include, for example, those comprised of the (C1-C10) alkyl group, as
described above, and thio group, and specific examples thereof include, for example, n-
propylthio group, t-butylthio group or the like. Examples of the arylthio group include, for
example, those comprised of the (C6-C12) aryl group, as described above, and a thio group,
and specific examples thereof include, for example, a phenylthip group or the like. Examples
of the aryloxy group, which may be present on the aryl, heteroaryl, and saturated
hydrocarbon groups, for example, those comprised of the (CB-C12) aryl group, as described

WO 2006/066598 PCT/DK2005/000828
13
above, and an oxy group, and specific examples thereof include, for example, a phenoxy
group or the like.
The alkyl groups described herein above can contain one or more carbon-carbon double
bonds (alkeinyl groups) or one or more carbon-carbon triple bonds (alkynyl groups).
"Alkenyl" refers to straight and branched chain and cyclic groups such as those described
with respect to unsubstituted alkyl groups as defined above, except that at least one double
bond exists between two carbon atoms. Examples include, but are not limited to vinyl, -
CH=C(H)(CH3), -CH=C(CH3)2, -C(CH3)=C(H)2, -C(CH3)=C(H)(CH3), -C(CH2 CH3)=CH2,
cydohexenyl, cyclopentenyl; cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among
others.
"Substituted alkenyl" has the same meaning with respect to unsubstituted alkenyl groups
that substituted alkyl groups had with respect to unsubstituted aikyi groups. A substituted
alkenyl group includes alkenyl groups in which a non-carbon or non-hydrogen atom is bonded
to a carbon double bonded to another carbon and those in which one of the non-carbon or
non-hydrogen atoms is bonded to a carbon not involved in a double bond to another carbon.
"Alkynyl" refers to straight and branched chain groups such as those described with respect
to alkyl groups as defined above, except that at least one triple bond exists between two
carbon atoms. Examples include, but are not limited to -CC(H), -CC(CH3), -CC(CH2CH3), -
C(H2)CC(H), -C(H)2CC(CH3), and -C(H)2CC(CH2CH3) among others.
"Substituted alkynyl" has the same meaning with respect to unsubstituted alkynyl groups
that substituted alkyl groups had with respect to unsubstituted alkyi groups. A substituted
alkynyl group includes alkynyl groups in which a non-carbon or non-hydrogen atom Is bonded
to a carbon triple bonded to another carbon and those in which a non-carbon or non-
hydrogen atom is bonded to a carbon not involved in a triple bond to another carbon.
Further examples of substituted alkyl groups are described herein below.
"Alkylamirjioalkyl" refers to an unsubstituted alkyl group as defined above in which a carbon
or hydrogen bond is replaced by a bond to a nitrogen atom that is bonded to a hydrogen
atom and'an unsubstituted alkyl group as defined above. For example, methyl (-CH3) is an
unsubstituted alkyl group. If a hydrogen atom of the methyl group is replaced by a bond to a
nitrogen atom that is bonded to a hydrogen atom and an ethyl group, then the resulting
compound is -CH2-N(H)(CH2CH3) which is an unsubstituted alkylaminoalkyl group.

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"Substituted alkyiaminoalkyl" refers to an unsubstituted aikylaminoalkyl group as defined
above except where one or more bonds to a carbon or hydrogen atom in one or both of the
alkyl groups is replaced by a bond to a non-carbon or non-hydrogen atom as described above
with respect to substituted alkyl groups except that the bond to the nitrogen atdm in all
aikylaminoalkyl groups do not by itself qualify all aikylaminoalkyl groups as beinp substituted.
However, substituted alkyiaminoalkyl groups do include groups in which the hydrogen
bonded to the nitrogen atom of the group is replaced with a non-carbon and non-hydrogen
atom.
"Dialkylaminoalkyl" refers to an unsubstituted alkyl group as defined above in which a carbon
bond or hydrogen bond is replaced by a bond to a nitrogen atom which is bonded to two
other similar or different unsubstituted alkyl groups as defined above.
"Substituted dialkylaminoalkyl" refers to an unsubstituted dialkylaminoalkyl group as defined
above In which one or more bonds to a carbon or hydrogen atom in one or more of the alkyi
groups is replaced by a bond to a non-carbon and non-hydrogen atom as described with
respect to substituted alkyl groups. The bond to the nitrogen atom in all dialkylaminoalkyl
groups do not by itself qualify all dialkylaminoaikyi groups as being substituted.
"Heterocyclyloxyalkyl" refers to an unsubstituted alkyl group as defined above in which a
carbon bond or hydrogen bond is replaced by a bond to an oxygen atom which is bonded to
an unsubstituted heterocyclyl group as defined above.
"Substituted heterocyclyloxyalkyl" refers to an unsubstituted heterocyclyloxyalkyl group as
defined above in which a bond to a carbon or hydrogen group of the alkyl group of the
heterocyclyloxyalkyl group is bonded to a non-carbon and non-hydrogen atom as described
above with respect to substituted alkyl groups or in which the heterocyclyl group of the
heterocyclyloxyalky! group is a substituted heterocyclyl group as defined above.
"Arylaminoalkyl" refers to an unsubstituted alkyl group as defined above in which a carbon
bond or hydrogen bond is replaced by a bond to a nitrogen atom which is bonded to at least
one unsubstituted aryl group as defined above.
"Substituted arylaminoalkyl" refers to an unsubstituted arylaminoalkyl group as defined
above except where either the alkyl group of the arylaminoalkyl group is a substituted alkyl
group as defined above or the aryl group of the arylaminoalkyl group is a substituted aryl
group except that the bonds to the nitrogen atom in all arylaminoalkyl groups do not by itself
qualify all arylaminoalkyl groups as being substituted. However, substituted arylamirioalkyl
groups do include groups in which the hydrogen bonded to the nitrogen atom of the group is

WO 2006/066598 PCT/DK2005/000828
15
replaced with a non-carbon and non-hydrogen atom.
"Heterocyclylaminoalkyl" refers to an unsubstituted alkyl group as defined above in which a
carbon or hydrogen bond is replaced by a bond to a nitrogen atom which is bonded to at
least one umsubstituted heterocyclyl group as defined above.
"Substituted heterocyclylaminoalkyl" refers to unsubstituted heterocydytaminoalkyl groups as
defined above in which the heterocyclyl group is a substituted heterocyclyl group as defined
above and/or the alkyl group is a substituted alkyl group as defined above. The bonds to the
nitrogen atom in all heterocyclylaminoalkyl groups do not by itself qualify all
heterocyclylaminoalkyl groups as being substituted. However, substituted
heterocyclylaminoalkyl groups do include groups in which the hydrogen bonded to the
nitrogen atom of the group is replaced with a non-carbon and non-hydrogen atom.
"Alkylaminoalkoxy" refers to an unsubstituted alkyl group as defined above in which a carbon
or hydrogen bond is replaced by a bond to an oxygen atom which is bonded to the parent
compound and in which another carbon or hydrogen bond of the unsubstituted alkyl group is
bonded to a nitrogen atom which is bonded to a hydrogen atom and an unsubstituted alkyl
group as defined above.
"Substituted alkylaminoalkoxy" refers to unsubstituted alkylaminoalkoxy groups as defined
above in which a bond to a carbon or hydrogen atom of the alkyl group bonded to the oxygen
atom which is bonded to the parent compound is replaced by one or more bonds to a non-
carbon and non-hydrogen atoms as discussed above with respect to substituted alkyl groups
and/or if the hydrogen bonded to the amino group is bonded to a non-carbon and non-
hydrogen atom and/or if the alkyl group bonded to the nitrogen of the amine is bonded to a
non-carbon and non-hydrogen atom as described above with respect to substituted alkyl
groups. The presence of the amine and alkoxy functionality in all alkylaminoalkoxy groups do
not by itself qualify all such groups as substituted alkylaminoalkoxy groups.
"Unsubstituted dialkylarninoalkoxy" refers to an unsubstituted alkyl group as defined above in
which a carbon or hydrogen bond is replaced by a bond to an oxygen atom which is bonded
to the parent compound and in which another carbon or hydrogen bond of the unsubstituted
alkyl group is bonded to a nitrogen atom which is bonded to two other similar or different
unsubstituted alkyl groups as defined above.
"Substituted dialkylaminoalkoxy" refers to an unsubstituted dialkyiaminoalkoxy group as
defined above in which a bond to a carbon or hydrogen atom of the alkyl group bonded to the
oxygen atom which is bonded to the parent compound Is replaced by one or more bonds to a

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non-carbon and non-hydrogen atoms as discussed above with respect to substituted alkyl
groups and/or if one or more of the alkyl groups bonded to the nitrogen of the amine is
bonded to a non-carbon and non-hydrogen atom as described above with respect to
substituted alkyl groups. The presence of the amine and alkoxy functionality in all
dialkylaminoalkoxy groups do not by itself qualify all such groups as substituted!
dialkylaminoalkoxy groups.
"Heterocydyloxy" refers to a hydroxyl group (-OH) in which the bond to the hydrogen atom is
replaced by a bond to a ring atom of an otherwise unsubstituted heterocyclyl group as
defined above.
"Substituted heterocydyloxy" refers to a hydroxyl group (-OH) in which the bond to the
hydrogen atom is replaced by a bond to a ring atom of an otherwise substituted heterocyclyl
group as defined above.
Hydrophobic functional groups comprising aromatic residues
Aromatic residues can be optionally substituted aryl or heteroaryl residues. "Aryl" includes,
but is not limited to, groups such as phenyl, biphenyl, anthracenyl, naphthenyl by way of
example. Although "aryl" includes groups containing condensed rings such as naphthalene, it
does not include aryl groups that have other groups such as alkyl or halo groups bonded to
one of the ring members, as aryl groups such as tolyl are considered herein to be substituted
aryl groups as described herein below. Aryl groups may be bonded to one or more carbon
atom(s), oxygen atom(s), nitrogen atom(s), and/or sulfur atom(s) in the ligand.
"Substituted aryl group" has the same meaning with respect to unsubstituted aryl groups
that substituted alkyl groups had with respect to unsubstituted alkyl groups. However, a
substituted aryl group also includes aryl groups in which one of the aromatic carbons is
bonded to one of the non-carbon or non-hydrogen atoms described above and also includes
aryl groups in which one or more aromatic carbons of the aryl group is bonded to a
substituted and/or unsubstituted alkyl, alkenyl, or alkynyl group as defined herein. This
includes bonding arrangements in which two carbon atoms of an aryl group are bonded to
two atoms of an alkyl, alkenyl, or alkynyl group to define a fused ring system (e.g.
dihydronaphthyl or tetrahydronaphthyl).
Examples of aryl and heteroaryl include, for example, a (C5-C12) aryl group such; as a pheny!
group, tolyl group, naphthyl group, biphenyl group or the like, and a (C4-C5) heteroaryl
group, or pyridyl group, or the like.

WO 2006/066598 PCT/DK2005/000828
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Also, when the at least one hydrophobic functional group comprises or consists of an
optionally substituted aromatic residue, the aromatic residue can be selected from the group
consisting of aromatic residues comprising or consisting of fluorenyl, pyrroyl, furanyl, thienyl,
thiophenyl, thiazolyl, isoindolyl, quinolinyl, isoquinolinyl, oxazolyl, and purinyl. Further
examples include, but is not limited to tetrahydrothiophenyl, sulfur oxidized
tetrahydrothiophenyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolenyl, benzimidazolyl,
piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl,
tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,
azocinyl, triazinyl, 6H-l,2,5-thiadiazinyl, 2H,6H-l,5,2-dithiazinyl, thianthrenyi, pyranyl,
isobenzofuranyl, chromenyl, xanthenyl, phenoxathiinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl,
pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, lH-indazoly, 4H-quinolizinyl,
phthalazinyi, naphthyridinyi, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl,
carbazolyl, p-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl,
phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl,
imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyi, quinuclidinyl, morpholinyl,
oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, and isatinoyl.
By way of example and not limitation, carbon bonded heterocydes can be bonded at position
2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a
pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,
tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, position 2, 4, or 5 of an
oxazole, imidazole or thiazole, position 3,4, or 5 of an isoxazole, pyrazole, or isothlazole,
position 2 br 3 of an aziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6, 7, or 8
of a quinol'ine or position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline. Still more typically, carbon
bonded heterocydes include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-
pyridazinyl, 4-pyridazinyl, 5-pyridazinyi, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl,
5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-tniazolyl, 4-
thiazolyl, or 5-thiazolyl.
By way of example and not limitation, nitrogen bonded heterocydes are bonded at position 1
of an aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,
imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,
piperidine piperazine, indole, indoline, lH-indazole, position 2 of an isoindole, or isoindoline,
position 4of a morpholine, and position 9 of a carbazole, or p-carboline. Typically, nitrogen
bonded heterocydes include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and
1-piperidihyl.
As will be dear from the above, the heteroaromatic group can also be selected from the
group consisting of heteroaromatic groups comprising or consisting of optionally substituted,

WO 2006/066598 PCT/DK2005/000828
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fused heteroaromatic compounds. Examples include e.g. indole, benzothiophene,
benzotriazene and quinoline.
In one embodiment, the aromatic residue can be substituted with one or more optionally
substituted aliphatic groups, such as the optionally substituted aliphatic groups mentioned
herein immediately above, such as the linear, branched or cyclic (C1-C10) alkyl gfoup, for
example a methyl group, an ethyl group, an isopropyl group, a n-butyl group, a t-butyl
group, a n-amyl group, a n-hexyl group and the like.
The aromatic residue can be substituted with one or more heteroatoms, or substituted with
one or more aromatic groups, or substituted with one or more heteroaromatic groups.
Also, the aromatic residue can e.g. be substituted with a substituted alkyl or aryl or
heteroaryl, wherein the aromatic residue, or the alkyl or the aryl or the heteroaryl is
substituted with a heteroatom selected from O, N, S and halogen, or substituted with one or
more groups selected from hydroxyl, amino, thiol, halogen, carbonyl, carboxyiic acid, ether
and ester.
The aryl and heteroaryl groups can also be substituted, for example, with a halogen atom, an
alkoxy group, an aryloxy group, an alkylthio group, or an arylthio group.
Examples of the halogen atom are a fluorine atom, a chlorine atom, a bromine atom, and an
iodine atom. Examples of the alkoxyl group include, for example, a (C1-C4) alkoxy group such
as methoxy group, ethoxy group, n-propoxy group, t-butoxy group or the like. Examples of
the alkylthio group include, for example, those comprised of trie (C1-C10) alkyl group, as
described above, and thio group, and specific examples thereof include, for example, n-
propylthio group, t-butylthio group or the like. Examples of the1 arylthio group include, for
example, those comprised of the (C5-Ci2) aryl group, as described; above, and a thio group,
and specific examples thereof include, for example, a phenylth|io group or the like. Examples
of the aryloxy group, which may be present on the aryl, heteroaryl, and saturated
hydrocarbon groups, for example, those comprised of the (C6-C12) aryl group, as described
above, and an oxy group, and specific examples thereof include, for example, a phenoxy
group or the like.
The term "heterocyclyl" as used herein refers to both aromatic and non-aromatic ring
compounds including monocyclic, bicyclic, and polycyclic ring compounds such jas, but not
limited to, quinuclidyl, containing 3 or more ring members of which one or morje is a
heteroatom such as, but not limited to, N, O, and S. Although "heterocyclyl" includes
condensed heterocyclic rings such as benzimidazolyl, it does riot include heterocyclyl groups
that have other groups such as alkyl or halo groups bonded to one of the ring members as

WO 2006/066598 PCT/DK2005/000828
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compounds such as 2-methylbenzimidazolyl are substituted heterocyclyl groups. Examples of
heterocycly groups include, but are not limited to: unsaturated 3 to 8 membered rings
containing :. to 4 nitrogen atoms such as, but not limited to pyrrolyl, pyrrolinyl, imidazolyl,
pyrazolyl, pyridyi, dihydrbpyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (e.g. 4H-1,2,4-
triazolyl, lH-l,2,3-triazolyl, J2H-l,2,3-triazolyl etc.), tetrazolyl, (e.g. lH-tetrazolyl, 2H
tetrazolyl, etc.); saturated 3 to 8 membered rings containing 1 to 4 nitrogen atoms such as,
but not limited to, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl; condensed unsaturated
heterocyclic groups containing 1 to 4 nitrogen atoms such as, but not limited to, indolyl,
isoindolyl, indolinyl, indolizinyl, benzimidazolyl, quinoiyl, isoquinolyl, indazolyl, benzotriazolyl;
unsaturated 3 to 8 membered rings containing 1 to 2 oxygen atoms and 1 to 3 nitrogen
atoms such as, but not limited to, oxazolyl, isoxazolyl, oxadiazolyl (e.g. 1,2,4-oxadiazolyl,
1,3,4-oxadiazolyl, 1,2,5-oxa'diazolyl, etc.); saturated 3 to 8 membered rings containing 1 to
2 oxygen atoms and 1 to 3 hitrogen atoms such as, but not limited to, morpholinyl;
unsaturated condensed heterocyclic groups containing 1 to 2 oxygen atoms and 1 to 3
nitrogen atoms, for example, benzoxazolyl, benzoxadiazolyi, benzoxazinyt (e.g. 2H-1,4-
benzoxazinyl etc.); unsaturated 3 to 8 membered rings containing 1 to 3 sulfur atoms and 1
to 3 nitrogen atoms such as, but not limited to, thlazolyl, isothiazolyl, thiadiazolyl (e.g. 1,2,3-
thiadiazolyl, 1,2,4-thiadiazoiyi, l,3,4-thi3di3zo!y!, 1,2,-5-thiadiazolyl, etc.); saturated 3 to 8
membered rings containing! 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms such as, but not
limited to,'thiazolodinyl; saturated and unsaturated 3 to 8 membered rings containing 1 to 2
sulfur atoms such as, but nbt limited to, thienyl, dihydrodithiinyl, dihydrodithionyl,
tetrahydrothiophene, tetraljiydrothiopyran; unsaturated condensed heterocyclic rings
containing 1 to 2 sulfur atopis and 1 to 3 nitrogen atoms such as, but not limited to,
benzothiazolyl, benzothiadiazolyl, benzothiazinyl (e.g. 2H-l,4-benzothiazinyl, etc.),
dihydrobehzothiazinyl (e.g.[ 2H-3,4-dihydrobenzothiazinyl, etc.), unsaturated 3 to 8
membered rings containing oxygen atoms such as, but not limited to furyl; unsaturated
condensed heterocyclic rings containing 1 to 2 oxygen atoms such as benzodioxolyl (e.g. 1,3-
benzodioxoyl, etc.); unsaturated 3 to 8 membered rings containing an oxygen atom and 1 to
2 sulfur atoms such as, but not limited to, dihydrooxathiinyl; saturated 3 to 8 membered
rings containing 1 to 2 oxygen atoms and 1 to 2 sulfur atoms such as 1,4-oxathiane;
unsaturated condensed' rings containing 1 to 2 sulfur atoms such as benzothienyl,
benzodith inyl; and unsaturated condensed heterocyclic rings containing an oxygen atom and
1 to 2 oxygen atoms such as benzoxathlinyl. Heterocyclyl group also include those described
above in whlch one or more S atoms in the ring is double-bonded to one or two oxygen
atoms (siilfoxides and sulfdnes). For example, heterocyclyl groups include
tetrahydrothiophene, tetrahydrothiophene oxide, and tetrahydrothiophene 1,1-dioxide.
Preferred'heterocyclyl groups contain 5 or 6 ring members. More preferred heterocyclyl
groups include morpholine, piperazine, piperidine, pyrrolidine, imidazole, pyrazole, 1,2,3-
triazole, 1,2,4-triazole, tetrazole, thiomorpholine, thiomorpholine in which the S atom of the

WO 2006/066598 PCT/DK2005/000828
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thiomorpholine is bonded to one or more 0 atoms, pyrrole, homopiperazine, oxazolidin-2-
one, pyrrolidin-2-one, oxazole, quinuclidine, thiazole, isoxazole, furan, and tetrahydrofuran.
"Substituted heterocyclyl" refers to an unsubstituted heterocyclyl group as defined above in
which one of the ring members is bonded to a non-hydrogen atom such as described above
with respect to substituted alkyl groups and substituted aryl groups. Examples, include, but
are not limited to, 2-methylbenzimidazolyl, 5-methylbenzimidazolyl, 5-chloroberizthiazolyl, 1-
methyl piperazinyl, and 2-chloropyridyl among others.
"Heterocyclylalkyl" refers to unsubstituted alkyl groups as defined above in which a hydrogen
or carbon bond of the unsubstituted alkyl group is replaced with a bond to a heterocyclyl
group as defined above. For example, methyl (-CH3) is an unsubstituted alkyl group. If a
hydrogen atom of the methyl group is replaced by a bond to a heterocyclyl group, such as if
the carbon of the methyl was bonded to carbon 2 of pyridine (c ne of the carbons bonded to
the N of the pyridine) or carbons 3 or 4 of the pyridine, then the compound is an
unsubstituted heterocyclylalkyl group.
"Substituted heterocyclylalkyl" has the same meaning with respect to unsubstituted
heterocyclylalkyl groups that substituted araikyl groups had wil:h respect to unsubstituted
aralkyl groups. However, a substituted heterocyclylalkyl group also includes groups in which
a non-hydrogen atom is bonded to a heteroatom in the heteroeyclyl group of the
heterocyclylalkyl group such as, but not limited to, a nitrogen atom in the piperidine ring of a
piperidinylalkyl group.
In one particular embodiment, the optionally substituted aromatic residue may be selected
from the group consisting of aromatic and heteroaromatic residues comprising or consisting
of phenyl, naphthyl, fluorenyl, pyridine, furane, thiophene, indol, isoindole, quinoline,
isoquinoline, oxazole, pyramidine and purine.
The substituted aromatic residues may also be substituted with one or more aliphatic groups,
or substituted with one or more heteroatoms, or substituted with one or more aromatic
groups, or substituted with one or more heteroaromatic groups.
The at least one hydrophobic functional group may also comprise or consists of an optionally
substituted heteroaromatic residue, e.g. a heteroaromatic residue selected fron the group
consisting of heteroaromatic residues comprising or consisting of furane, pyrro e and
thiophene, or a heteroaromatic residue selected from the group consisting of heteroaromatic
residues comprising or consisting of fused heteroaromatic compounds, such as,a fused

WO 2006/066598 PCT/DK2005/000828
21
heteroaromjatic compound selected from the group consisting of indole, benzothiophene,
benzotriazene and quinoline.
In the above embodiments, the aromatic residue may be substituted with alkyl or aryl or
heteroaryl.
Also, the aromatic residue, or the alkyl or the aryl or the heteroaryl may be substituted with
a heteroatom selected from O, N, S and halogen, and/or the aromatic residue, or the alkyl or
the aryl or the heteroary! may be substituted with one or more groups selected from
hydroxyl, amino, thiol, halogen, carbonyl, carboxylic acid, ether and ester.
In one embodiment, the iigand comprises at least one amino acid residue comprising a
hydrophobic group, e.g. a hydrophobic group which comprises or consists of an aromatic
group, or a hydrophobic group which comprises or consists of an aliphatic group.
Cationic functional groups
Cationic functional groups are positively charged either due to a permanent positive charge
or due to an association with an H ion at under normal physiological conditions. Cationic
functional groups are attracted by aqueous solution so as to seek the surface positions in the
conformatipn of a Iigand when the Iigand is in an aqueous medium under normal
physiological conditions. Cationic functional groups will seek towards anionic groups of Iigand
binding partners when the Iigand is associated with a binding partner under normal
physiological conditions.
The cationic functional group is preferably selected from cationic groups comprising one or
more positively charged nitrogen(s), one or more phosphorous atom(s) and/or one or more
sulphur atom(s).
Preferred cationic groups comprise a permanent, positively charged nitrogen from groups
such as e.g. alkyl ammonium, such as trimethyl ammonium, or triethylammonium, or
dimethylammonium, or benzyldimethylammonium, or guanidinium, or a positively charged
nitrogen from positively charged heterocycles, such as imidazolinium, piperidinium and
pyrrolidini jm. Guanidinium is particularly preferred.
Other preferred catlonic groups are mono- and disubstituted amines, such as monoalkyl
amines, dialkyi amines, heterocyclic amines and aromatic amines which have a partial

WO 2006/066598 PCT/DK2005/000828
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positive charged in aqueous solutions with pH in the range of 3-8, in particular in ,the range
of 3-7.
The positively charged nitrogen can also be donated by an amino acid residue, sufch as lysine,
arginine, histidine, ornithine, citrulline, diaminobutyric acid, diaminopropionic acid,
diaminopentanoic acid, diaminohexanoic acid, diaminopimelic acid, homoarginine,
homocitrulline, p-aminophenylalanine and 3-aminotyrosine. Arginine is particularly preferred.
The side chain of an amino acid can provide both one or more hydrophobic functional
group(s) and one or more cationic functional group(s). Normally, when amino acids are
providing both of said functional groups, at least one hydrophobic functional group and at
least one cationic functional group are provided by different amino acids of the ligand. Apart
from amino acids, the ligand can comprise other ligand residues as described herein below in
more detail.
The term "amino acid" within the scope of the present invention is used in its broadest sense
and is meant to include naturally-occurring L-amino acids or residues thereof. The commonly
used one- and three-letter abbreviations for naturaiiy-occurring amino acids are used herein
(Lehninger, Biochemistry, 2d ed., pp. 71-92, (Worth Publishers: New York, 1975). The term
also includes D-amino acids (and residues thereof) as well as chemically-modified amino
acids, such as amino acid analogues, including naturally-occurring amino acids that are not
usually incorporated into proteins, such as norleucine, as well as chemically-synthesized
compounds having properties known in the art to be characteristic of an amino acid.
For example, analogues or mimetics of phenylalanine or proline, which allow the same
conformational restriction of a ligand as natural Phe or Pro, are included within the definition
of amino acid. Such analogues and mimetics are referred to herein as "functional
equivalents" of an amino acid. Other examples of amino acids are listed by Roberts and
Vellaccio, The Peptides: Analysis, Synthesis, Biology, Eds. Gross and Meiehofer, Vol. 5, p.
341 (Academic Press, Inc.: N.Y. 1983).
The amino acids are typically linked by amide bonds, but other bonds such as e.g. any one or
more bonds selected from -NHN(R)CO-; -NHC(R)CO; -NHC(RR')CO-; -NHC(=CH[R)CO-;
-NHC6H4CO-; -NHCH2 CHRCO-; -NHCHRCH2CO-; -COCH2-; -COS-; -CONR-; -COOj-; -CSNH-;
-CH2NH~; -CH2CH2-; -CH2S-; -CH2SO-; -CH2SO2-; -CH(CH3)S-; -CH^CH-; -NHCoi;
-NHCONH-; -CONHO-; -C(=CH2)CH2-; -PO2-NH-; -PO2"CH2-; -PO2"CH2N+-; -SO2Nrk- can also
link amino acid residues of the ligands according to the invention. R (and R') denotes a
functional group, such as e.g. a hydrophobic functional group or a cationic groupj, or another

WO 2006/066598 PCT/DK2005/000828
23
structural entity linking said aforementioned groups. The currently most preferred bond
between "amino acids" is the amide bond.
Examples of amino acids that are generally capable of being incorporated into ligands
according to the present invention are listed herein below:
Glycyl; aminopolycarboxylic acids, e.g., aspartic acid, p-hydroxyaspartic acid, glutamic acid,
p-hydroxyglutamic acid, p-methylaspartic acid, (3-methylglutamic acid, p,p-dimethylaspartic
add, Y-hydroxyglutamic acid, p,y-dihydroxyglutamic acid, p-phenylglutamic acid, y-
methyleneglutamic acid, 3-aminoadipic acid, 2-aminopimelic acid, 2-aminosuberic acid and 2-
aminosebacic acid residues; amino acid amides such as glutaminyl and asparaginyl;
polyamino- or polybasic-monocarboxylic acids such as arginine, lysine, p-aminoalanine, y
aminobutyrine, ornithine, citruline, homoarginlne, homocitmlline, 5-hydroxy-2,6-
diaminohexanoic acid (commonly, hydroxylysine, including allohydroxylysine) and
diaminobutyric acid residues; other basic amino acid residues such as histidinyl;
diaminodicarboxylic acids such as a,a'-diaminosuccinic acid, a.a'-diaminoglutaric acid, a,a'-
diaminoadipic acid, a,a'-diaminopimelic acid, a,a'~diamino-p~riydroxypimelic acid, a,a'-
diaminosuberic acid, a'-diaminoazelaic acid, and aja'-diaminosebacic acid residues; imino
acids such as proline, 4- or 3-hydroxy-2-pyrro!idine- carboxylic acid (commonly,
hydroxyproline, including allohydroxyproline), y-methylproline, pipecolic acid, 5-
hydroxypipecolic acid, -N([CH2]nCOORpR)2, wherein n is 1, 2, 3, 4, 5 or 6 and RPR is -H or a
protecting group, and azetidine-2-carboxylic acid residues; a mono- or di-alkyl (typically Ct-
C25 branched or normal) amino acid such as alanine, valine, leucine, allylglycine, butyrine,
norvaline, norleucine, heptyline, a-methylserine, a-amino-a-methyl-y-hydroxyvaleric add, a-
amino-a-methyl-6-hydroxyvaleric acid, a-amino-a-methyl-e-hydroxycaproic acid, isovaline,
a-methylglutamic acid, a-aminoisobutyric acid, a-aminodiethylacetic acid, o-
aminodiisopropylacetic acid, a-aminodi-n-propylacetic acid, a-aminod iisobutylacetic acid, a-
aminodi-n-butylacetic acid, a-aminoethylisopropylacetic acid, a-amino-n-propylacetic acid, a-
aminodiisoamyacetic acid, a-methylaspartic acid, a-methylglutarnic acid, I-
aminocydopropane-1-carboxylic acid; isoleucine, alloisoleucine, tert-leucine, p-
methyltryptophan and a-amino-a-ethyl-p-phenylpropionic acid residues; p-phenylserinyl;
aliphatic a-amino-p-hydroxy acids such as serine, p-hydroxyleucine, p-hydroxynorieucine, p-
hydroxync rvaline, and a-amino-a-hydroxystearic acid residues; a-Amino, a-, y-, 5- or E-
hydroxy acids such as homoserine, y-hydroxynorvaline, 5-hydroxynorvaline and e-
hydroxynorleuclne residues; canavinyl and canalinyl; y-hydroxyornithinyl; 2-Hexosaminic
acids such as D-glucosaminic acid or D-galactosaminic acid residues; a-amino-p-thiols, such
as penicillamine, p-thiolnorvaline or p-thiolbutyrine residues; other sulfur containing amino
acid residues including cysteine; homocystine; p-phenylmethionine; methionine; S-allyl-(L)-
cysteine sulfoxide; 2-thiolhistidine; cystathionine; and thiol ethers of cysteine or

WO 2006/066598 PCT/DK2005/000828
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homocysteine; phenylalanine, tryptophan and ring-substituted amino acids such as the
phenyl- or cyclohexylamino acids, a-aminophenylacetic acid, a-aminocyclohexylacetic acid
and a-amino-p-cyclohexylpropionic acid; phenylalanine analogues and derivatives comprising
aryl, lower alkyl (C1-C6), hydroxy, guanidino, oxyalkylether, nitro, sulfur or halo- substituted
phenyl (e.g., tyrosine, methyltyrosine and o-chloro-, p-chloro-, 3,4-dicloro, o-, in- or p-
methyl-, 2,4,6-trimethyl-, 2-ethoxy-5-nitro, 2-hydroxy-5-nitro and p-nitro-phenylalanine);
furyl-, thienyl-, pyridyl-, pyrimidinyl-, purine or naphthylalanines; and tryptophan analogues
and derivatives including kynurenine, 3-hydroxykynurenine, 2-hydroxytryptophan and 4-
carboxytryptophan residues; a-amino substituted amino acid residues including ,sarcosine (N-
methylglycine), N-benzylglycine, N-methylalanine, N-benzylalanine, N-methylphenylalanine,
N-benzylphenylalanine, N-methylvaline and N-benzylvalinej and a-Hydroxy and substituted
a-hydroxy amino acid residues including serine, threonine, allothreonine, phosphoserine and
phosphothreonine residues. Also of interest are hydrophobic amino acids such as mono-or di-
alkyl or aryl amino acids, cycloalkylamino acids, and the like.
Preferred ligands
Selected llgand residues as cited herein below are denoted:
PPC: 4-Phenyl piperidine-4-carboxy!ic acid
Dap: Diaminopropionic acid
L-Orn: L-Omithine
DPPAA: 2,4-Di-tert-pentylphenoxy acetic acid
DMBA: 3,5-Dimethoxybenzoic acid
TMPPA: 3-(3,4,5-Trimethoxyphenyl)propionic acid
DBHPA: 3-(3,5-Di-tert-butyl-4-hydroxyphenyl)propionic acid
1H2NA: l-Hydroxy-2-naphthoic acid
DPPA: 3,3-Diphenylpropionic add
SAA: Salicylic acid
DBBA: 3,5-Di-tert-butylbenzoic acid
DPPBA: 4-(2,4-Di-tert-pentylphenoxy)butyric acid
TEBA: 3,4,5-Triethoxybenzoic acid
PCAA: a-Phenyl cyclopentane acetic acid
MDCA: 3,4-(Methylenedioxy) cinnamic acid
Gly: Glydne
L-Phe: L-Phenylalanine
L-Arg: L-Arginine
L-His: L-Histidine

WO 2006/066598 PCT/DK2005/000828
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L-Trp: L-Tryptophan
L-Pro: L-Froline
L-Asn: L-Asparagine
L-Lys: L-Lysine
L-Asp: L-Aspartic acid
D-Phe: D-Phenylalanine
D-Arg: D-Arginine
D-Tyr: D-Tyrosine
D-Ser: D-Serine
D-Trp: D-Tryptophan
D-Pro: D-Proline
D-Leu: D-Leucine
Ahx: 6-Aminohexanoic acid
Aib: a-Aminoisobutyric acid
DBHBA: 3,5-Di-tert-butyl-4-hydroxybenzoic acid
INA: Isonipectolc acid
Nle: L-Norleucine
PPC: 4-Phenyi-piperidine 4-carboxy!ic acid
SAA: Salicylic acid
3HBA: 3-Hydroxybenzoic acid
4HBA: 4-Hydroxybenzoic acid
In the main class of preferred affinity ligands, each ligand comprises one or more
hydrophobic functional group(s) and one or more cationic functional group(s), wherein at
least one hydrophobic functional group is separated from at least one cationic functional
group by a through bond distance of from 5 A to 20 A, and wherein said ligand has a
molecular weight of from 120 Da to 1,500 Da.
In one preferred embodiment, the affinity ligand comprising or consisting of covalently linked
residues X1-X2-X3, wherein optionally Xlf X2 and/or X3, in particular Xi and/or X3, is
associated with a linker residue, L
Preferably X1, X2, X3, and the optional linker residue are preferably highly stable molecules,
which can withstand repeated exposure to harsh chemical conditions (e.g. 1 M strong acid or
1 M strong base) and biological conditions (e.g. high protease activity). Likewise, the
respective bonds between each of X1, X2, X3| and the optional linker residue are highly stable
and can withstand harsh chemical and biological conditions.

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The residue X1 is preferably selected from the group consisting of Arg, Phe, PPC, DBHBA,
SAA, DAP, DAB, (DBHBA)2-DAP, (MDCA)2-DAP, DPBBA, DBBA, PCAA, DPPAA, Trp, TMPPA,
DBHPA, wherein each member is optionally further selected from an isolated an'd optically
active residue and a racemic mixture comprising both of the optically active iso^ners of the
same residue. In one preferred variant, Xi is selected from the group consisting of L-Arg, D-
Lys, D-Phe, D-Pro, INA, PPC, DBHBA, 3HBA, 4HBA and SAA.
The residue X2 is preferably selected from the group consisting of Arg, Asn, Leu, Lys, Phe,
Pro, PPC, DAP, DAB, His, Trp, Tyr, Ser, wherein each member is optionally further selected
from an isolated and optically active residue and a racemic mixture comprising both of the
optically active isomers of the same residue. In one preferred variant, X2 Is selected from the
group consisting of L-Arg, L-Asn, D-Leu, D-Lys, D-Phe, D-Pro, L-Pro, AIB, AHX, INA, Nle and
PPC.
The residue X3 is preferably selected from the group consisting of Arg, Asn, Pro, PPC, Asp,
Om, (lH2NA)Dap wherein each member is optionally further selected from an isolated and
optically active residue and a racemic mixture comprising both of the optically active isomers
of the same residue. In particular, X3 is preferably selected from the group consisting of L-
Arg, L-Asn, D-Lys, D-Phe, D-Pro, L-Pro and PPC.
A currently promising set of ligands having affinity for an antibody, such as a monoclonal
antibody, are the ligands comprising or consisting of one or more of the sets (corresponding
to XrX2-X3):
D-Phe-(L)Arg-(L)Arg
L-Arg-D-Phe-(L)Arg
PPC-D-Pro-(L)Arg
PPC-D-Leu-PPC
INA-D-Phe-PPC
PPC-Aib-PPC
D-Phe-(L)Arg-(L)Arg
L-Arg-D-Phe-(L)Arg
L-Arg-(L)Arg-D-Phe
PPC-(L)Arg-D-Pro
D-Pro-PPC-(L)Arg
L-Arg-D-Pro-PPC
L-Arg-D-Lys-(L)Arg
L-Arg-(L)Arg-D-Lys
D-Lys-INA-(L)Arg

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INA-(L)Arg-D-Lys
PPC-Ahx-PPC
PPC-Nle-PFC
SAA-(L)Arg-(L)Pro
SAA-(L)Pro-(L)Arg
DBHBA-(L)Arg-(L)Asn
DBHBA-(L)Asn-(L)Arg
3HBA-(L)Arg-(L)Pro
3HBA-(L)Pro-(L)Arg
4HBA-(L)Arg-(L)Pro
4HBA-(L)Pro-(L)Arg
In another currently promising embodiment, the ligand comprises or consists of less than 5,
such as less than 4 residues, for example 3 residues selected from the group consisting of D-
Leu; D-Lys; D-Phe; D-Pro; L-Arg; L-Asn; L-Pro; Ahx; Aib; DBHBA; INA; Nle; PPC; SAA; 3HBA
and 4HBA. The ligand can thus consist of 3 residues, Xi-X2-X3, individually selected from the
group consisting of D-Leu; D-Lys; D-Phe; D-Pro; L-Arg; L-Asn; L-Pro; Ahx; Aib; DBHBA; INA;
Nle; PPC; SAA; 3HBA and 4HBA.
When the iligand comprises or consists of 3 residues, X1-X2-X3,
X1 is preferably selected from the group consisting of D-Lys; D-Phe; D-Pro; L-Arg; DBHBA;
INA; PPC; SAA; 3HBA and 4HBA,
X2 Is preferably selected from the group consisting of D-Leu; D-Lys; D-Phe; D-Pro; L-Arg; L-
Asn; L-Pro; Ahx; AIB; INA; Nle; PPC; and
X3 is preferably selected from the group consisting of D-Lys; D-Phe; D-Pro; L-Arg; L-Asn; L-
Pro; PPC.
In a further embodiment, the ligand comprises or consists of 3 covalentiy linked residues, X1-
X2-X3,
wherein said covalentiy linked residues are further covalentiy linked to the linker residue L of
the entity]' L-PM, wherein PM is the solid support material, preferably a polymer matrix
optionally in cross-linked and/or beaded form,
wherein X1 is a natural or non-natural amino acid in D- and/or L-configuration, or a carboxylic
acid residue comprising an optionally substituted aromatic group,

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wherein X2 is a natural or non-natural amino acid in either D- and/or L-configuration, or a
carboxylic acid residue comprising an optionally substituted aromatic group, witfi the proviso
that X2 is not a threonine residue, and
wherein X3 is a natural or non-natural amino acid in either D- and/or L-configuration, or a
carboxylic acid residue comprising an optionally substituted aromatic group,
wherein at least one of X1, X2 and X3 comprises a cationic functional group, and
wherein at least one of X1;X2 and X3 comprises a hydrophobic functional group.
wherein X1 is selected from L-Arg, D-Lys, D-Phe, D-Pro, INA, PPC, DBHBA, 3HBA, 4HBA and
SAA;
wherein X2 is selected from L-Arg, L-Asn, D-Leu, D-Lys, D-Phe, D-Pro, L-Pro, AIB, AHX, INA,
NLE and PPC; and
wherein X3 is selected from L-Arg, L-Asn, D-Lys, D-Phe, D-Pro, L-Pro and PPC.
In a still further promising embodiment, the ligand comprising or consisting of 3 covalently
linked residues, Xi-X2-X3,
wherein said covalently linked residues are covalently linked to a linker L of the entity L-PM,
wherein X1 is a natural or non-natural amino add in D- and/or L-configuration, or a carboxylic
acid residue comprising an optionally substituted aromatic group,
wherein X2 is a natural or non-natural amino acid in either D- and/or L-configuration, or a
carboxylic add residue comprising an optionally substituted aromatic group, with the proviso
that X2 is not a threonine residue, and
wherein X3 is a natural or non-natural amino acid in either D- and/or L-configuiation, or a
carboxylic acid residue comprising an optionally substituted aromatic group,
wherein at least one of X1; X2 and X3 comprises a cationic functional group, and
wherein at least one of X1, X2 and X3 comprises a hydrophobic functional group.

WO 2006/066598 PCT/DK2005/000828
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wherein X1 is selected from L-Arg, D-Lys, D-Phe, D-Pro, INA, PPC, DBHBA, 3HBA, 4HBA and
SAA;
wherein X2 is selected from L-Arg, L-Asn, D-Leu, D-Lys, D-Phe, D-Pro, L-Pro, AIB, AHX, INA,
NLE and PPC; and
wherein X3 is selected from L-Arg, L-Asn, D-Lys, D-Phe, D-Pro, L-Pro and PPC.
In one embodiment, the linker residue L attached to the polymer matrix is cleavable by acids,
bases, temperature, light, or by contact with a chemical reagent. As before, the linker
attached to the polymer matrix may be one selected from (3-formylindol-l-yl)acetic acid,
2,4-Dimethoxy-4'-hydroxy-benzophenone, HMPA, HMPB, HMPPA, Rink acid, Rink amide,
Knorr linker, PAL linker, DCHD linker, Wang linker and Trityl linker. LJgands bound to a
polymer matrix via cleavable linker can be used for analytical purposes, e.g. for diagnostic
applications.
In one embodiment of the present invention, X1, X2, and X3 are chosen from the group of
natural amino acids and their stereoisomers.
It has been found that the inclusion of at least one amino acid comprising a side chain
guanidino or amino group, in particular guanidino groups, provide typically provide
advantageous properties with respect to binding capacity. Hence, particularly relevant amino
acids to include in the ligands are arginine, homoarginine, lysine, homolysine and ornitine, in
particular arginine and homoarginine.
A particularly promising subclass of ligands is the one where the ligands comprise two
arginine moieties. Hence, particularly favoured ligands within this embodiment are,


WO 2006/066598 PCT/DK2005/000828
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H2N-(L)Arg-(D)Phe-(L)Arg-Gly-OH.
In a further embodiment Xt, X2/ and X3 are chosen from the group of natural and unnatural
amino acids.
Hence, for a still further promising subclass of ligands,
X1 is chosen from the group consisting of Arg, Phe, PPC, DBHBA, SAA, DAP, DAB, (DBHBA)2-
DAP, (MDCA)2-DAP, DPBBA, DBBA, PCAA, DPPAA, Trp, TMPPA, and DBHPA,
X2 is chosen from the group consisting of Arg, Asn, Leu, Lys, Phe, Pro, PPC, DAP, DAB, His,
Trp, Tyr, and Ser,
X3 is chosen from the group consisting of Arg, Asn, Pro, PPC, Asp, Orn, and (lH2NA)Dap.

Particularly favoured ligands within this subclass are:

WO 2006/066598 PCT/DK2005/000828
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In a still further subclass of ligands, each ligand comprises one or more substituted or un-
substituted phenyl or naphtyl groups and one or more primary amine or guanidine.
Particularly favoured ligands within this embodiment are,


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WO 2006/066598 PCT/DK2005/000828
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Alternative affinity ligands
Another currently promising class of affinity ligands is the one where the ligands contain one
or more hydrophobic groups and one or more heteroaromatic groups.
Hence, in another main class of preferred affinity ligands, each ligand comprises one or more
hydrophobic functional group(s) and one or more heteroaromatic functional group(s),
wherein at least one hydrophobic functional group is separated from at least ohe
heteroaromatic functional group by a through bond distance of from 5 A to 20|A, and wherein
said ligand has a molecular weight of from 120 Da to 5,000 Da.
Preferably, the affinity resin having such an affinity ligand immobilised thereoii has a binding
capacity larger than 5 mg monoclonal antibody per mL of affinity resin.
The term "heteroaromatic functional group(s)" is intended to encompass the heteroaromatic
species defined under "Hydrophobic functional groups comprising aromatic residues" and
"Cationic functional groups" hereinabove.
The specifications and preferences with respect to through bond distances, through space
distances, molecular weight, etc. given further above for the main class of affinity ligands

WO 2006/066598 PCT/DK2005/000828
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also apply for this class of affinity ligands, although it should be understood that "distances"
referred to are between the hydrophobic functional group(s) and heteroaromatic functional
group(s).
An example of a ligand within this class is

Solid support material
The ligandis according to the invention are covalently immobilised to a solid support material,
such as a polymer resin, a surface of a polymeric material, a glass surface, etc, and can be
used for purifying and/or isolating antibodies, such as monoclonal antibodies.
The solid support material typically comprises a polymer matrix, e.g. a cross-linked polymer
matrix. Most often, the polymer matrix is beaded, alternatively, the polymer matrix is
prepared as a monolithic unit. Preferably, at least the surface of the beaded polymer matrix
comprises! hydrophilic rnoieties.
Hence, the solid support material is typically in the form of a resin, a monolith, a filter, a
plate, such as a micro-array, a fibre, a sensor, such as a cantelever, a surface plasmon
resonance sensor, or a quartz crystal microbalance.
A resin is a porous solid material used for separation of compounds in a fluid. Resins can be
cross-linked polymers, ceramic materials, such as inorganic oxides, e.g. aluminium oxide,
silicium oxide. Resins are provided as particles, such as spherical particles or irregular
particles.
The ligands are characterised by a binding capacity which is preferably larger than 5 mg of
antibody per mL of wet resin, such as larger than 6 mg of antibody per mL of resin, for

WO 2006/066598 PCT/DK2005/000828
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example larger than 7 mg of antibody per mL of resin, such as larger than 8 mg of antibody
per mL of resin, for example larger than 9 mg of antibody per mL of resin, such as larger
than 10 mg of antibody per mL of resin, for example larger than 12 mg of antibody per mL of
resin, such as larger than 14 mg of antibody per mL of resin, for example larger than 16 mg
of antibody per mL of resin, such as larger than 18 mg of antibody, per mL of resin, for
example larger than 20 mg of antibody per mL of-resin, such as larger than 22 mg of
antibody per mL of resin, for example larger than 24 mg of antibody per mL of resin, such as
larger than 26 mg of antibody per mL of resin, for example larger than 28 mg of antibody per
mL of resin, such as larger than 30 mg of antibody per mL of resin, for example larger than
35 mg of antibody per mL of resin, such as larger than 40 tng of antibody per mL of resin, for
example larger than 45 mg of antibody per mL of resin, such as larger than 50 mg of
antibody per mL of resin, such as e.g. at the most 500 mg of antibody per mL of resin.
The expression "binding capacity" denotes the capacity of the resin to bind a target
compound, such as e.g. an antibody. The binding capacity is given in mg target (e.g.
antibody) per mL of resin (or other form of beaded material), and can be measured by
passing a solution of the pure target with a known concentration through the resin and
measuring the volume until breakthrough of the target compound. The binding capacity is
then calculated as the breakthrough volume multiplied by the concentration and divided by
the bed volume. As used herein, binding capacity denotes the capacity of the resin to bind an
antibody. The binding capacity is given in mg antibody per milliliter of resin, and can be
measured by passing an aqueous buffered solution (50 mM Na-Phosphate, pH=7.0) of the
pure antibody with a known concentration and recording the development of the UV
absorbance (280 nm) of the solution exiting the resin. The breakthrough volume is the
volume of solution that has exited from the resin at the point where the UV absorbance has
reached 5% of its terminal value. The binding capacity is then calculated as the breakthrough
volume multiplied by the concentration of antibody in the feed solution and divided by the
bed volume.
When the ligand is attached to the surface of an essentially non-porous material, such as e.g.
and array surface or a sensor surface, the binding capacity of the ligand is measured as mass
of protein bound per surface area. The ligands are characterised by a binding capacity which
is preferably larger than 1 ng of antibody per cm2, such as larger than 5 ng of antibody per
cm2, for example larger than 10 ng of antibody per cm2, such as larger than 50 ng of
antibody per cm2, for example larger than 100 ng of antibody per cm2.
Suitable examples of resins are disclosed herein below in more detail.

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Polymer matrices
The ligand can be associated covalently to a solid support such as a porous, inorganic matrix
or a polymer matrix, optionally in cross-linked and/or beaded form or in a monolithic porous
entity. Preferably, the pores of the polymer matrix are sufficiently wide for the target protein
to diffuse through said pores and interact with the ligand on the inner surface of the pores.
For a monoclonal antibody with molar mass approx. 150 kDa an average pore diameter of
50-200 nm is preferred, such as approx. 100 nm. A number of commercially available
suitable polymer resins are available, e.g. Sepharose™, Fractogel™, CIMGEL™, Toyopearl.
The beaded and optionally cross-linked polymer matrix in one embodiment comprises a
plurality of hydrophilic moieties. The hydrophilic moieties can be polymer chains which, when
cross-linked, form the cross-linked polymer matrix. Examples include e.g. polyethylene glycol
moieties, polyamine moieties, polyvinylamine moieties, and polyol moieties.
In some embodiments, the core and/or the surface of a beaded polymer matrix comprises a
polymeric material selected from the group consisting of polyvinyls, polyacrylates,
polyacrylamides, polystyrenes, polyesters and polyamides.
The beaded polymer matrix can also be selected from the group consisting of PS, POEPS,
POEPOP, SPOCC, PEGA, CLEAR, Expansin, Polyamide, 3andagel, PS-BDODMA, PS-HDODA,
PS-TTEGDA, PS-TEGDA, GDMA-PMMA, PS-TRPGDA, ArgoGel, Argopore resins, ULTRAMINE,
crosslinked LUPAMINE, high capacity PEGA, Silica, Fractogel, Sephadex, Sepharose, Glass
beads, crosslinked polyacrylates, and derivatives of the aforementioned; in particular, ttie
polymer matrix is selected from the group consisting of SPOCC, PEGA, HYDRA, POEPOP, PEG-
polyacrylate copolymers, polyether-polyamine copolymers, and cross-linked polyethylene di-
amines.
Apart from the above-mentioned examples, any material capable of forming a polymer
matrix can in principle be used in the production of beads of the invention. Preferably, the
core material of a bead is polymeric. In some embodiments, the core comprises or consists of
hydrophilic polymeric material. In other embodiments, the core comprises or consists of
hydrophobic polymeric material. In some embodiments, the surface of the beads comprises
or consists of the same material as the core.
Resins useful for large-scale applications may be one of the above mentioned or other
commercial resins such as Sephadex™, Sepharose™, Fractogel™, CIMGEL™, Toyopearl,
crosslinked agarose, and macroporous polystyrene or polyacrylate. The matrix may also be of

WO 2006/066598 PCT/DK2005/000828
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a mainly inorganic nature, such as macroporous glass or clay minerals, or combinations of
resins and and inorganics, such as Ceramic HyperD™.
Polymer beads according to the invention can be prepared from a variety of polymerisable
monomers, including styrenes, acrylates and unsaturated chlorides, esters, acetates, amides
and alcohols, including, but not limited to, polystyrene (including high density polystyrene
latexes such as brominated polystyrene), polymethylmethacrylate and other pojyacrylic
acids, polyacrylonitrile, polyacrylamide, polyacroleln, polydimethylsiloxane, polybutadiene,
polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine,
polyvinylbenzylchloride, polyvinyltoluene, polyvinylidenechloride and polydivinylbenzene. In
other embodiments, the beads are prepared from styrene monomers or PEG based macro-
monomers. The polymer is in preferred embodiments selected from the group consisting of
polyethers, polyvinyls, polyacrylates, polymethacrylates, polyacylamides, polyurethanes,
polyacrylamides, polystyrenes, polycarbonates, polyesters, polyamides, and combinations
thereof. Highly preferred surface and core moieties include cross-linked PEG moieties,
polyamine moieties, polyvinylamine moieties, and polyol moieties.
A preferred hydrophobic polymer to be used for production of beads of the composition of the
invention is PS-DVB (polystyrene divinylbenzene). PS-DVB has been widely used for solid-
phase peptide synthesis (SPPS), and has more recently demonstrated utility for the polymer-
supported preparation of particular organic molecules (Adams et al. (1998) J.Org.Chem.
63:3706-3716). When prepared properly (Gr0tli et al. (2000) J.Combi.Chem.2:108-119), PS-
DVB supports display excellent properties for chemical synthesis such as high loading,
reasonable swelling in organic solvents and physical stability.
In one embodiment of the present invention, the ligand is associated to the surface of a
sensor or an array plate and used to detect and/or quantify antibodies in a biological sample.
When used herein, the term "biological sample" includes natural samples or samples obtained
from industrial processes, e.g. recombinant processes, and include "body fluid?1, i.e. any
liquid substance extracted, excreted, or secreted from an organism or tissue cf an organism.
A body fluid need not necessarily contain cells. Body fluids of relevance to the present
invention include, but are not limited to, whole blood, serum, urine, plasma, cerebral spinal
fluid, tears, milk, sinovial fluid, and amniotic fluid.
In a further embodiment, a plurality of llgands are associated to the surface of an array plate
and arranged in a plurality of spots, with each spot representing one ligand. Such a
functions llzed array can be used to detect the presence of antibodies in a solution. Such an

WO 2006/066598 PCT/DK2005/000828
41
array can be used for diagnostic applications to detect the presence of certain antibodies in a
biological sample.
In a further embodiment, a plurality of ligands are associated to the binding surface of a
cantilever sensor for detection and optionally quantification of antibodies. A plurality of
affinity ligands can be associated to a plurality of cantilevers with each cantilever
representing one ligand. Such a functionalized array can be used to detect the presence of
various antibodies in a solution. Such a multi-sensor can be used for diagnostic applications
to detect the presence of certain antibodies in a biological sample.
Linkers
The above-mentioned ligand is covalently immobilized to a solid support material, possibly
through a linker. In preferred embodiments, the ligand is covalently attached to a linker
which is covalently attached to the polymer matrix. General techniques for linking of affinity
ligands to solid support materials can be found in Hermanson, Krishna Mallia and Smith,
Immobilized Affinity Ligand Techniques", Academic Press, 1992.
Linkers are used for linking the ligand to a solid support such as e.g. a polymer matrix or an
inorganic support. Preferably, the linker forms a strong and durable bond between the ligand
and the solid support. This is particularly important, when the solid support material of the
present invention is to be used for repeated purification of monoclonal antibodies or
fragments thereof.
However, in one embodiment of the present invention, linkers can be selectively cieavable.
This can be useful when the solid support is to be used for analytical purposes.
Amino adds and polypeptldes are examples of typical linkers. Other possible linkers include
carbohydrates and nucleic acids.
In one emaodiment, the linker residue L attached to the polymer matrix is cieavable by acids,
bases, temperature, light, or by contact with a chemical reagent. In particular, the linker
attached to the polymer matrix can be (3-formy!indol-l-yl)acetic add, 2,4-dimethoxy-4'-
hydroxy-bsnzophenone, HMPA, HMPB, HMPPA, Rink acid, Rink amide, Knorr linker, PAL
linker, DCHD linker, Wang linker and Trityl linker.

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The ligand can be associated with the solid support through a linker having a length of
preferably less than 50 A, such as a length of from 3 to 30 A, for example a lencth of from 3
to 20 A, such as a length of from 3 to 10 A.
The linker can be attached to a hydrophobic functional group or to a cationic fun:tional
group, or to a structural entity of the ligand joining a hydrophobic functional gro jp and a
cationic functional group. In one embodiment, the linker is attached to a cationic functional
group. Preferably, however, the linker is attached to the affinity ligand via a carboxylic acid
group, or an amino group, in particular via a carboxylic acid group.
The linker may also comprise a plurality of covalently linked subunits, e.g. such that the
subunits are selected from identical and non-identical linker subunits. In one variant, the
linker is flexible and comprises from 3 to preferably less than 50 identical or non-identical,
covalently linked subunits.
In a preferred embodiment of the present invention, the linker L is selected from the group
consisting of glycine, alanine, 3-aminopropionic acid, 4-aminobutanoic acid, and HMBA.
The linker can also be selected from the group consisting of polydispersed polyethylene
glycol; monodispersed polyethylene glycol, such as triethylene glycol, tetraethylene glycol,
pentaethylene glycol, hexaethylene glycol, heptaethylene glycol; an amino acid; a dipeptide;
a tripeptide; a tetrapeptide; a pentapeptide; a hexapeptide; a heptapeptide; octapeptide; a
nonapeptide; a decapeptide, a polyalanine; a polyglydne, a polylysines, a polyarginine,
including any combination thereof.
Preferred embodiments
One preferred embodiment relates to a solid support material having covalently immobilized
thereon an affinity ligand, said ligand comprising one or more hydrophobic functional
group(s) and one or more cationic functional group(s),
wherein at least one hydrophobic functional group is separated from at least one cationic
functional group by a through bond distance of from 5 A to 20 A,
wherein said ligand has a molecular weight of from 120 Da to 1,500 Da;
wherein the ligand comprises one or more amino acids having side chain guanidino groups
and one or more amino acids having side chain substituted phenyl or naphtyl groups.

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In view of the above, it has been found that particularly interesting ligands are those selected
from the group consisting of DBBA-(L)His-(L)Arg-Gly-OH and (DBHBA)2-DAP-(L)Arg-(L)Arg-
Gly-OH.
A composition
The preser t invention also provides a solid support material having covalently immobilized
thereon a plurality of different affinity ligands. Such a composition can be used to separate
various antibodies from a biological sample.
Method for. the isolation of antibodies
Another aspect of the present invention relates to a method for the Isolation of biomolecules,
such as proteins, e.g. antibodies, in particular monoclonal antibodies, the method comprising
the steps of (i) providing a solid support material having covalently immobilized thereon an
affinity ligand as defined herein, (ii) providing a sample putatively containing an antibody
having an affinity for said ligand, (iii) contacting said Hgand with said sample putatively
containing said antibody, (iv) binding selectively said antibody when said antibody is
contained in said sample and (v) isolating selectively said antibody when said antibody is
contained in said sample.
In one variant, the method comprises the steps of providing a ligand as defined herein,
attaching said ligand to a solid support, such as e.g. a beaded and/or cross-linked polymer
matrix, providing a sample putatively containing an antibody having an affinity for said
ligand, contacting said ligand with said sampie putatively containing said antibody, binding
selectively said antibody when said antibody is contained in said sample and isolating
selectively said antibody when said antibody is contained in said sample.
Preferably, the binding capacity of the ligand is larger than 5 mg of antibody per mL of resin,
or even more as described further above under "Solid Support Material".
It should be understood that the handling of the solid support material and the procedure
essentially follows that of conventional affinity chromatographic techniques.
EXAMPLES
The present invention is further illustrated by the following non-limiting examples.

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The examples describe how large libraries can be designed and tested for their affinity
towards monoclonal antibodies.
Materials and Methods
All reagents and solvents obtained from commercial suppliers and used without :urther
purification. All solvents used were of HPLC grade kept over molecular sieves. Fiinoc-
protected amino acids and HMBA linker were obtained from Bachem AG, Bubendorf,
Switzerland. PPC and ACC were purchased from Neosystem, Strasbourg, France. Sepharose™
was purchased from Sterogene and Fractogel™ was purchased from Merck. All the ligands
were synthesised on PEGA1900 (Versabeads™ A) resin. A 20 well multiple column peptide
synthesizer was used for the combinatorial library synthesis. 1H NMR spectra were recorded
in CDCI3 solutions with a Varian Unity Inova 500 MHz spectrometer. Chemical shifts are
reported in 5 values relative to tetramethylsilane. The ESI-mass spectra were recorded on a
Waters Global Ultima mass spectrometer and MALDI-TOF spectra were recorded on a Bruker
Reflex III mass spectrometer using a-cyano-4-hydroxycinnamic acid as matrix. Analytical and
preparative reverse-phase HPLC separations were performed on a Waters HPLC system using
analytical Zorbax 300SB-C18 (4.5 x 50 mm) and Delta PAK (25 x 300 mm) Ci8 columns with a
flow rate of 1 cm3min'1 and 10 cm3min'1, respectively. Detection was at 215 nm on a
multiwavelength detector (Waters 490E) for analytical purposes and a photodiode array
detector (Waters M991) was used for preparative separations. A solvent system consisting of
A: 0.1% TFA in water and B: 0.1% TFA in 90:10 acetonitrile/water was used.
Labelling of Fc fragment
The Fc fragment of an antibody was purified by dialysis in bicarbonate buffer (pH 8). The
purified Fc fragment was treated with Oregon Green dye (50 x) in DMF and kept for 1 hour at
room temperature. The reaction was stopped by adding hydroxylamine (1.5 M, pH 8.5). The
labelled polypeptide was purified by dialysis in bicarbonate buffer (pH 8).

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Example 1. Synthesis of ligands

The solid phase synthesis followed the scheme:
All compounds are synthesized on a polyethyleneglycol-acrylamide based amino functional
resin, PEGA1900. The ligands were attached to the glycine derivatised resin via base labile p-
hydroxymethylbenzoic acid HMBA linker.
The building block (acid/amino acid; 3 equiv) dissolved in DMF and /V-ethylmorpholine (4
equiv) and TBTU (2.88 equiv) were added. The reaction mixture was kept for 5 min and
added to the swollen resin in DMF for 2h. The resin was washed with DMF, EtOH-DMF and
DCM.
Incubation of synthesised compounds in labelled Fc fragment
The beads (10 mg) were washed with water (10 x), bicarbonate buffer (pH 8,10 x), and
were suspended in bicarbonate buffer for 1 h. The labelled polypeptide was then added to the
resin and kept overnight at room temperature. The resin was washed with bicarbonate buffer
(pH 8) and water.
The fluorescence on the beads was recorded by using a fluorescence microscope and a digital
camera. A mercury lamp equipped with an emission filter provided excitation in the visible
blue rangje. The images of the beads were recorded in water.

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The below formulas illustrate the building blocks which were used for the synthesis.
The below table lists the ligands and the corresponding apparent level of fluorescence
determined by simply looking at the samples and comparing the apparent colour intensities
between samples. On an arbitrary scale from 0 to 3, where 0 indicates no fluorescence and 3
indicates the strongest level of fluorescence observed


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Example 2. Chromatographic evaluation ofligands (from Example 1)
Preparing the chromatography column
The new rejsins were assessed in packed mode. 1 mL of each resin was packed into an HR5
chromatogr aphy column (GE Healthcare) applying standard conditions.
After packing, the column had a bed height of 3.9 cm and a total volume of 0.76 mL
Equilibration. Load. Elution
Before loading the matrix with the monoclonal antibody, the column was equilibrated with 6
column volumes equilibration buffer containing 50 mM Na-phosphate (pH 7.0; cond.
9 mS cm"1). After equilibration a monoclonal antibody solution in equilibration buffer (protein
cone. 1.14 mg/mL) was loaded to the column. Subsequently, the column was washed with 6
column volymes of equilibration buffer and eluted with 6 column volumes of elution buffer 1
(25 mM sodium acetate pH 5.5; cond. 1.8 mS cm"1) and 6 column volumes of elution buffer 2
(25 mM sodium acetate buffer pH 3.8; 0.5 mS cm"1). The column was stored in 20% ethanol.
Afterwards the matrix was regenerated with 40 mM phosphoric acid and 20 mM sodium
hydroxid. The flow rate for equilibration, load and wash was 60 cm/h, elution and
regeneration was performed at a flow rate of 30 cm/h.
Results for Ligand 55 coupled on Fractoqel

Process ste'p Volume mAb content
[mL] [mg/mL] [mg] Step yield [%]
Load 6.6 1.10 7.26 100
Flow trough 6.5 0.19 1.24 17.0
Wash 4.5 0.02 0.09 1.20
Elution 1 4.5 0.19 0.86 11.8
Elution 2 4.0 1.05 4.2 57.9
Regeneration 1.5 0.49 0.74 10.1
Recovery 98

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Results for Ligand 16 coupled on Fractogel

Summary and results:
Binding and elution of mAbs were shown with both ligands applying not optimizsd operation
conditions. For llgand 55, only limited material is in the flow through whereas nothing was
detected in the flow through with ligand 16.
Example 3
The ligands TMPPA-(L)Trp-(L)Arg-Gly-OH, DBHPA-(L)Arg-(L)Orn-Gly-OH, (DPPA)2-DAP-
(L)Arg-(L)Orn-Giy-OH and (DBHBA)2-DAP-(L)Arg-(L)Arg-Gly-OH w.ere synthesized on amino
activated Toyopearl resin (supplied by Tosoh). The binding capacity of each of the resulting
affinty resins (B2, B3, Dl, and D2 respectively) towards mAb was evaluated by( performing
the folowing sequence of steps two times (cycle 1 and cycle 2),
1) passing an aqueous solution of mAb (50 mM Na-Phosphate, pH=7.0) througt a column
packed with the resin while recording the development of the UV absorbance of: the permeate
and collecting the liquid sample exiting the column (flow through),
2) passing an aqueous elution buffer (25 mM Acetate pH=3.7) through the column and
collecting the liquid sample exiting the column (elution),
3) passing an aqueous sanitising buffer (1 M acetic acid) through the column and collecting
the liquid sample exiting the column (regeneration/sanitation).
The mass of mAb, mi, i=l,2,3, was then determined for each sample in each cycle.

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The 'binding capacity' is calculated as the total mass of mAb added to the column, m0,
multiplied by the volume of buffer added to the column at the point where the UV absorbance
has reached 5% of its terminal value, V5%, divided by the total volume of buffer used, Vo, and
divided by the volume of wet resin, Vresin-

It is clear from the table that all of the tested ligands have binding capacities higher than 5
mg/mL.
In order to determine the selectivity of resins B2 and B3 towards mAb, the experiment was
repeated, but this time the crude fermentation supernatant was used in step 1. After the
experiment, the purity of the samples was determined by SDS Page. The results are shown
for B2, B3, Dl, and D2 in figures 1, 2, 3, and 4, respectively.
It can be seen from Figures 1-4 that all four tested ligands show selectivity towards mAb with
B2 and Dl being especially selective at the given conditions.

50
CLAIMS - AMENDED UNDER ARTICLE 34 PCT
1. A solid support material having covaiently immobilized thereon an affinity ligand, said
llgand comprising one or more hydrophobic functional group(s) and one or more catlonlc
functional group(s),
wherein at least one hydrophobic functional group Is separated from at least one catlonic
functional group by a through bond distance of from 5 A to 20 A,
wherein said llgand consists of less than 5 residues and has a molecular weight of from 120
Da to 1,000 Da.
2. The solid support material according to claim 1, wherein said affinity resin has a binding
capacity fargerthan 5 mg monoclonal antibody per mL of affinity resin.
3. The solid support material according to any one of the preceding claims, wherein the
affinity ligand comprising or consisting of covaiently linked residues X1-X2-X3/ wherein
optionally X1, Xz and/or X3 is associated with a linker residue.
4. The solid support material according to claim 3, wherein residue Xx is selected from the
group consisting of Arg, Phe, PPC, DBHBA, SAA, DAP, DAB, (DBHBA)2-DAP, (MDCA)2-DAP,
DPBBA, DBBA, PCAA, DPPAA, Trp, TMPPA, and DBHPA.
5. The solid support material according to any one of the claims 3-4, wherein residue X2 is
selected from the group consisting of Arg, Asn, Leu, Lys, Phe, Pro, PPC, DAP, DAB, His, Trp,
Tyr, and Ser.
6. The solid support material according to any one of the claims 3-5, wherein residue X3 is
selected from the group consisting of Arg, Asn, Pro, PPC, Asp, Orn, and (lH2NA)Dap.
7. The solid support material according to any one of the claims 3-6, wherein the ligand
comprises or consists of 3 covaiently linked residues, X1-X2-X3,
wherein said covaiently linked residues are further covaiently linked to the linker L of the
entity L-PM, wherein L is a linker, and PM is the solid support material, preferably a polymer
matrix optionally in cross-linked and/or beaded form,

51
wherein X1 is a natural or non-natural amino add in D- and/or L-configuration, or a carboxylic
acid residue comprising an optionally substituted aromatic group,
wherein X2 is a natural or non-rnatural amino acid in either D- and/or.L-configuration, or a
carboxylic acid residue comprising an optionally substituted aromatic group, with the proviso
that X2 is not a threonine residue, and
wherein X3 Is a natural or non-natural amino add in either D- and/or L-configuration, or a
carboxylic add residue comprising an optionally substituted aromatic group,
wherein at least one of X1, X2 and X3 comprises a cationlc functional group, and
wherein at least one of X1, X2 and X3 comprises a hydrophobic functional group.
wherein X1 is selected from L-Arg, D-Lys, D-Phe, D-Pro, INA, PPC, DBHBA, 3HBA, 4HBA and
SAA;
wherein X2 is selected from L-Arg, L-Asn, D-Leu, D-Lys, D-Phe, D-Pro, L-Pro, AIB, AHX, INA,
NLE and PPC; and
wherein X3 is selected from L-Arg, L-Asn, D-Lys, D-Phe, D-Pro, L-Pro and PPC.
8. The solid support material according to any one of the preceding claims, wherein the
ligand is one selected from the group consisting of
H2N~(D)Phe-(L)Arg-(L)Arg-Gly-OH (SEQ ID NO: 1),
H2N~(L)Arg-(D)Pbe-(L)Arg-Gly-OH (SEQ ID NO; 2),
HN-PPC-(D)Pro-(L)Arg-Gly-OH (SEQ ID NO: 3),
DBBA-(L)His-(L)Arg-Gly-OH,
DBBA-Hts-Arg-Gly-OH,
DBBA-His-Arg-Arg-Gly-Orl (SEQ ID NO: 5),
DPPBA-(L)Phe-(L)Arg-Gly-OH,
PCAA-(L)Phe-(L)Arg-Gly-OH,
DPPBA-(L)Lys-(L)Arg-Gly-OH,
SAA-(L)Arg-(L)Pro-Gly-OH,
SAA-(L)Pro-(L)Arg-Gly-OH,
DBHBA-(L)Arg-(L)Asn-Gly-OH,
DBHBA-(L)Asn-(L)Arg-Gly-OH,

52
DPPBA-(L)Trp-(L)Arg-Gly-OH,
(MDCA)2-DAP-(L)Arg-(L)Orn-Gly-OH (SEQ ID NO: 6),
(MDCA)2-DAP-(L)Arg-(L)Asp-Gly-OH (SEQ ID NO: 7),
DPPAA-(L)Phe-(L)Arg-Gly-OH,
DPPAA-PPC-(L)Arg-Gly-OH,
DBHBA-(D)Phe-(D)Arg-Gly-OH,
DPPAA-(D)tyr-(D)Arg-Gly-OH,
(DPPA)rDAP-(L)Arg-(L)Orn-Gly-OH (SEQ ID NO: 10),
(DBHBA)z-DAP-(L)Arg-(L)Arg-Gly-OH (SEQ ID NO: 11),
DBHBA-DAP-Arg-Gly-OH,
DBHBA-DAP-Arg-Arg-Gly-OH {SEQ ID NO: 11),
(DBHBA)2-DAP-Arg-Gly-OH,
(DBHBA)3-DAP-Arg-Arg-Gly-OH (SEQ ID NO: 11),
TMPPA-(L)Trp-(L)Arg-Gly-OH, and
DBHPA-(L)Arg-(L)Orn-Gly-OH.
9. The solid support material according to any one of the preceding claims, wherein the
ligand is selected from the group consisting of DBBA-(L)His-(L)Arg-Gly-OH and (DBHBA)2-
DAP-(L)Arg-(L)Arg-G!y-OH (SEQ ID NO: 11).
10. A method for the isolation of antibodies or derivatives thereof, the method comprising the
steps of (i) providing a solid support material having covalently Immobilized thereon an
affinity ligand as defined in any one of claims 1-9, (ii) providing a sample containing an
antibody having an affinity for said ligand, (ill) contacting said ligand with said sample
containing said antibody, (Iv) binding selectively said antibody when said antibody is
contained in said sample and (v) isolating selectively said antibody when said antibody is
contained in said sample.

The present application discloses a
solid support material having covalently immobilized
thercon an affinity ligand, said ligand comprising
one of more hydrophobic functional group(s) and
one or more cationic functional group(s) or one or
more heteroaromatic functional group(s), wherein at
least one hydrophobic functional group is separated
from at least one cationic/heteroaromatic functional
group by a through bond distance of from 5 Å to 20
Å, wherein said ligand has a molecular weight of
from 120 Da to 5,000 Da. Typically, the affinity resin
has a binding capacity larger than 5 mg monoclonal
antibody per mL of affinity resin. A method for
the isolation of biomolecules, such as proteins, in
particular antibodies, such as monoclonal antibodies,
or derivatives thereof, is also disclosed.