GB2438088A

GB2438088A - Process for cross-linking moieties

Info

Publication number: GB2438088A
Application number: GB0709127A
Authority: GB
Inventors: Graham Mock
Original assignee: Individual
Current assignee: Individual
Priority date: 2006-05-11
Filing date: 2007-05-11
Publication date: 2007-11-14
Anticipated expiration: 2027-05-11
Also published as: GB0609370D0; WO2007132207A3; GB2438088B; GB0709127D0; WO2007132207A2

Abstract

A process for linking a first moiety, wherein the first moiety is not a cell membrane or cell membrane fraction and is equipped with one or more free amino groups, comprising: conjugating the first moiety with a linker either of formula I or formula II: <EMI ID=1.1 HE=77 WI=129 LX=410 LY=867 TI=CF> <PC>wherein Y is an organic linker with 2-10 carbon atoms in the backbone; m and n are 1-3; and R<1>-R<20> are hydrogen or substituents with preferred compounds featuring SO3Na substituents on the rings; the first moiety-linker conjugate may then be reacted with a second moiety to form the conjugated product. The process is particularly for linking a first moiety, such as a nucleic acid or protein, with a second moiety, such as an antibody or enzyme. Preferred linkers are 4,4'-diisothiocyanato-2.2'-stilbenedisulfonic acid (DIDS) and dimers thereof of general formula II formed with a central amine compound. The conjugates formed, their medicinal or diagnostic use and dimer compounds of general formula II are also claimed.

Description

--2438088

PROCESS FOR CROSS-LINKING MOIETIES

The invention relates to a linkers for linking moieties such as nucleic acids, proteins, antibodies and polypeptides with other moieties such as enzymes, antibodies and solid supports, and processes using such linkers.

Crosslinking reagents are often used in the diagnostic, pharmaceutical, chemical, electronic and biotechnology industries to produce conjugates such as DNA and antibodies with enzymes, and proteins with biotin. These conjugates find applications as nucleic acid probes and in immunoassays, amongst others. Crosslinking reactions are also used in the immobilisation of macromolecules on solid supports such as glass, microprocessors, membranes, dipsticks and magnetic beads.

A number of cros slinking reagents and methods are known in the prior art.

For example, Pierce market a kit for controlled protein-protein crosslinking which depends on the reaction of a thiol with a maleiimide group (Perbio Science UK 05/06, Pierce 23456) and disuccinimidyl suberate, a homobifunctional agent (Pierce 21655). Other reagents include the photoactivatable sulfo-SANPAH (sulfosuccinimidyl 6-(4'-azido-2'-nitro-phenylamino)hexanoate).

A key disadvantage of the methods mentioned above, is that the thiol/maleiimide approach is an all-day procedure requiring several complicated steps using unstable reagents. In particular, the thiols readily oxidise and dimerise, and the maleiimides can hydrolyse. Furthermore, the homobifunctional reagents suffer from self-conjugation and polymerisation.

Additionally, rapid hydrolysis leading to poor yields and the required use of high concentrations of such toxic reagents are also disadvantages.

Photoactivatable reagents also show poor discrimination and are often low yielding.

There exists a need therefore for processes for linking moieties such as macromolecules which do not suffer from the above disadvantages.

The Applicant has now developed an improved method for the conjugation of moieties such as biological macromolecules. This method takes less time than prior art methods; and all reactions steps can be carried out in a single reaction vessel using a single buffer without the need to purify intermediates.

High concentrations of toxic reagents are avoided using the method of the invention. Furthermore, the final conjugate may be easily purified using standard means.

The invention therefore provides linkers which are suitable for linking a first moiety with a second moiety and processes using such linkers. The invention also provides "activated" first moieties which comprise first moieties conjugated to linkers of the invention in a form in which they can be used for conjugation to a second moiety, and processes for the production of such "activated" moieties.

The invention therefore provides a process for linking a first moiety with a linker, wherein the first moiety comprises one or more free amino groups and wherein the first moiety is not a cell membrane or cell membrane fraction, the process comprising the steps: (i) conjugating the first moiety with a linker of formula I: I R1 NR2 R5 s_ R10 or a dimerised linker of formula II:

II R

C*N...Y,,LT,,R2R5 R7 R14 R1yLr'C....

S R3 R8 R'. _L,)JLL R18 -R4 R6 9flj* R17 R1 R11

I

wherein -R' to R4, R7 to R' R'1 to R'4 and R'7 to R2 are independently selected from H, halogen, Cjio-alkyl, C.s-cycloalkyl, OH, O-CI..3-alkyl, SH, NH2, NO2, CO2H, C02-CI..3-alkyl, SO3H and SO3X, wherein X denotes a counter ion, for example, NH4, Na or K; R5, R6, R'5 and R'6 are independently selected from H, halogen, C1-6-alkyl, OH, O-CI..3-alkyl, SH, NH2, CO2H, and CO2-CI-3-alkyl; m and n are independently 1, 2 or 3, and Y denotes an organic linker with between 2 and 10 carbon atoms in the backbone.

The invention further relates to a process for conjugating a first moiety with a second moiety, wherein the first and second moieties independently comprise one or more free amino groups, the process comprising the steps: (1) conjugating the first moiety with a linker of formula I: I R1 R R5 R7 s-R3 R8 R4 R6 R'0 or a dimerised linker of formula II: R1 R2 cyr2 R5 R7 R14 R1Y"LI'C...

S R8 R'&E 1.)J.(L 18 R4 R6 9fl _C%N_YC._A...(&..l2R R17 R10 R11 wherein R' to R4, R7 to R' , R" to R'4 and R'7 to R2 are independently selected from H, halogen, Ci..io-alkyl, C8-cycloaIkyl, OH, O-C13-alkyl, SH, NH2, NO2, CO2H, C02-CI..3-alkyl, SO3H and SO3X, wherein X denotes a counter ion, for example, NH, Na or K; R5, R6, R35 and R'6 are independently selected from H, halogen, Ci-6-alkyl, OH, O-C1-3-alkyl, SH, NH2, CO2H, and CO2-Cl..3-alkyl; m and n are independently 1, 2 or 3, and Y denotes an organic linker with between 2 and 10 carbon atoms in the backbone, and then (ii) reacting the first moiety-linker conjugate formed in (i) with the second moiety.

Preferably, step (i) is carried out under the conditions: molar ratio of first moiety:linker 1:45 to 1:140; and/or time 15 to 120 minutes.

Preferably, step (ii) is carried out under the conditions: molar ratio of first moiety used in step (i):second moiety 1:1 to 1:8; and/or time 15 to 120 minutes.

In the context of the present invention, the following definitions are used both hereinbefore and hereinafter: By the following (pf is meant one double bond or a plurality of up to n conjugated double bonds wherein each pair of R5 and R6 groups may, independently of any other R5 and R6 pairs, be either in the cis or in the trans geometrical isomeric configuration; and wherein each R5 may be the same or different, and wherein each R6 may be the same or different. The same applies, mutatis mutandis, to groups comprising R'5 and R'6 and m. -The term Ci..io-alkyl denotes a branched or unbranched alkyl group having between 1 and 10 carbon atoms. The group includes methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl etc. The term C3..8-cycloalkyl denotes a cyclic alkyl group having between 3 and 8 carbon atoms in the ring. The group includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

The term O-Ci..3-alkyl denotes an alkoxy group having between 1 and 3 carbon atoms in the alkyl portion. The group includes methoxy, ethoxy, n-propoxy and iso-propoxy.

The term C02-CI-3-alkyl denotes an ester group having between 1 and 3 carbon atoms in the alkyl portion. The group includes methyl carboxylate, ethyl carboxylate, n-propyl carboxylate and iso-propyl carboxylate.

The term halogen denotes an atom of a group VII element selected from among fluorine, chlorine, bromine and iodine.

The term "free amino group(s)" refers to amino groups which are capable of reacting with the isothiocyanate groups of the linker. Examples of such free amino groups include those present in the amino acids lysine, arginine, asparagine and glutamine of polypeptides or at the N terminus of polypeptides.

Preferably, R' to R4, R7 to R10, R" to R'4 and R'7 to R20 are each independently selected from H, methyl, ethyl and SO3X.

Preferably, R5, R6, R'5 and R16 are each independently selected from H and C1..3 alkyl.

In some embodiments of the invention, only one or two of R' to R4 and R7 to R' are SO3X. Particularly preferably, only one of R' to R4 and/or only one of R7 to R' are SO3X. Most preferably, only one of R' to R4 and/or only one of R7 to R' are SO3X wherein R4 is SO3X and/or R7 is SO3X.

In other embodiments of the invention, only one or two of R" to R'4 and R'7 to R20 are SO3X. Particularly preferably, only one of R' to R'4 and/or only one of R'7 to R2 are SO3X. Most preferably, only one of R" to R'4 and/or only one of R'7 to R2 are SO3X wherein R'4 is SO3X and/or R'7 is SO3X.

m and n are preferably 1. If n = 1, then R5 and R6 are preferably arranged in the trans configuration. If m= 1, then R'5 and R'6 are preferably arranged in the trans configuration.

X is preferably Na.

In some embodiments of the invention, the linker is a compound of formula III: III R1 R2 r R8 o3x R9,,LJL N R10 or a dimerised linker of formula IV Iv R1 R2 R19 N'N'N SO3X R16 ml

I H H H SO3X s03x

R1 R1' or a compound of formula V CH3 R8 H3C R6 R1 or a dimerised linker of formula VI, VII or VIII:

VI

CH3 CH3 19 1 N SO3X SO3X R S#C*NIJ1IR5 i R8 H3C R4 R6 R9 I H H H H R2 R'7 R' R11 R' R2 19 I N S..-R14 R1 3c SO3X R6 CNYR15 R18 s03x R9 N

I H H H H

CH3 CH3 CH3 R2 R5 SO3X R14 S H3C _c( R8 S R4 R6 SO3X R1 CH3 wherein the R groups, X, m and n are as defined above.

In the compounds of formulae Ill-VIlI, R' to R20, where present, are preferably independently selected from H, methyl and ethyl. Most preferably, R' to R20, when present, are all H. A particularly preferred linker is 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS, Aldrich 28,561-7). The corresponding dimerised linker (i.e. the compound of formula IV, wherein R' to R3, R5, R6, R8 to R' , R" to R'3, R'5, R'6 and R'8 to R2 are all H, and n and m are both 1) is also particularly preferred.

The organic linker Y is preferably a linear C2-6-alkyl, most preferably C2-4-alkyl group, optionally substituted at one or more of the backbone carbon atoms by one or more groups selected from halogen, CI3-alkyl, O-CI.3-alkyl, CO2H and C02-CI-3-alkyl.

In one particularly preferred embodiment, Y is -CH2CH2-.

In another particularly preferred embodiment, Y is -(CH2)4CH(COOH)-.

In particularly preferred embodiments, the dimerised linkers are selected from the compounds of general formulae IX and X: XO3S)N

U-

wherein X is as defined above.

In the present invention, the first and second moieties, which may be the same or different, are preferably a chemical entity and/or a solid support.

Preferably, the chemical entity is an isolated or purified chemical entity.

Examples of chemical entities include macromolecules, most preferably biological macromolecules. Examples of biological macromolecules include nucleic acid molecules, proteins, polypeptides and peptides. Preferably, the biological macromolecule is in isolated or purified form, e.g. an isolated nucleic acid molecule or a purified protein, polypeptide or peptide. Examples of nucleic acid molecules include DNA and RNA. The nucleic acid molecule may be single-stranded or double-stranded or part single-and part double-stranded. In some preferred embodiments of the invention, the macromolecule is a single-stranded oligomer, preferably a DNA oligomer.

Examples of proteins and polypeptides include enzymes and antibodies.

Preferably the enzyme is an enzyme which is capable of producing a detectable reaction, such as horseradish peroxidase, alkaline phosphatase, beta galactosidase, glucose oxidase or a restriction enzyme.

In some embodiments of the invention, the first and/or second moiety is not a cell membrane or a cell membrane fraction. By the term "cell membrane or cell membrane fraction" is meant that the first and/or second moiety is not a biological cell membrane (e.g. a membrane from a red blood cell) or a fraction therefore, for example a fraction that has been obtained by centrifugation of the cell membrane in a solvent. In other embodiments, the first and/or second moiety is not a cell membrane protein, in particular not an anion channel protein.

The first and/or second moieties may also be a structural binding peptide such as streptavidin or avidin, or a label, such as biotin.

-10 -In some embodiments of the invention, one or both of the moieties may be an antibody. The antibodies may be of any suitable source, e.g. monoclonal, polyclonal, chimeric, bispecific, single-chain or fragments thereof. Antibody fragments include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH 1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals, or derived from phage or ribosome display libraries. Preferably, the antibodies are human, murine (e.g. mouse or rat), donkey, rabbit, goat, guinea pig, camel, horse, or chicken. The antibodies of the invention may be monospeciflc, bispecific, trispeciflc or of greater multispecificity.

The antibodies may be labelled in any suitable manner, thus allowing for detection in a suitable assay.

In other embodiments of the invention, one or both of the moieties is a solid support, such as a particle or bead, or magnetic bead, or matrix such as a sheet, gel, filter, membrane, fibre, tube, microtitre plate or column. Other examples of solid supports according to the invention are dipsticks, polymers, fibre optics and glass and electronic devices (e.g. diodes and transistors).

Such solid supports may either already comprise one or more free amino groups or they may be amino-functionalised.

In some embodiments of the invention, one or both moieties is a medicament, for example an antibiotic, antifungal or anticancer agent.

In other embodiments of the invention, one or both moieties is a dye, for example, fluorescein, rhodamine or Texas Red. Preferably the dye is fluoresce in.

In yet other embodiments of the invention, one or both moieties is a moiety as -11 -defined above which incorporates a radiolabel. The radiolabel may, for example, be 1251 or Europium.

The molar ratio of the first moiety to the linker in step (i) is preferably in the range 1:45 to 1:140. The molar ratio is chosen to maximise the efficiency of the conjugation without unduly lengthening the overall process time.

Preferably, the molar ratio is 1:60 to 1:120, more preferably 1:80 to 1:100, and most preferably about 1:90.

The molar ratio of the second moiety to the first moiety in step (ii) is preferably in the range 1:1 to 8:1. The molar ratio is chosen to maximise the efficiency of the conjugation without unduly lengthening the overall process time. Preferably, the molar ratio is 2:1 to 6:1, more preferably 3:1 to 6:1, and most preferably about 4:1.

The buffer is chosen as one that is suitable for conjugating the chosen first and second moieties with the chosen linker. Suitable buffers are well known in the art. Preferably the buffer is an aqueous buffer. If the first and/or second moiety is a biological molecule, the buffer should ideally be a physiologically-acceptable buffer. Examples of suitable aqueous buffers include PBS, borate-based buffers, Tns-HC1, etc. Most preferably, the buffer is a sodium borate buffer.

One or more co-solvents may also be used, e.g. DMF, DMSO and/or an alcohol, for example ethanol, propanol or butanol.

The pH will be chosen to maximise the efficiency of the conjugation.

Examples of suitable pHs include pH 5.5 to 9.5. If the first and/or second moiety is a biological molecule, the pH should ideally be a physiologically-acceptable pH, for example pH 6 to 9, more preferably pH 6 to 8.5, most preferably about pH 8.0.

The reaction times for steps (i) and (ii) are chosen to maximise the efficiency of the conjugation without unduly lengthening the overall process time. The reaction times for steps (i) and (ii) may the same or different. If times of less than 15 minutes are used, an undesirable amount of linker and first/second moieties may be unreacted. If times of more than 120 minutes are used, -12 -polymer formation may occur. Examples of suitable reaction times are about 15, about 30, about 45, about 60, about 75, about 90, about 105 or about minutes. Preferred reaction times are 15 to 45 minutes, 30 to 90 minutes and 45 to 75 minutes. Most preferably, the reaction time is about 30 minutes or about 60 minutes.

The temperature of the reaction is chosen to maximise the conjugation efficiency. The reaction temperatures for steps (i) and (ii) may the same or different. Examples of suitable temperatures are 4 to 50 C.

If the first and/or second moiety is a biological molecule, reaction temperatures should preferably be kept within physiological ranges, for example between 4 and 37 C. Preferably the reaction temperature is 4-37 C, most preferably about room temperature, e.g. about 21 C.

In one preferred embodiment of the invention, step (ii) is carried out directly after step (1). By the term "directly after", it is intended to mean that step (ii) is carried out without a delay or intermediate step after step (i).

In other preferred embodiments of the invention, when carrying out step (ii), the second moiety is added to the reaction mixture obtained from step (i), i.e. the first moiety-linker conjugate obtained from step (i) is not purified or is not treated in any other way before the second moiety is added to the reaction mixture.

Preferably, both step (i) and step (ii) are carried out in a single reaction vessel using the same buffer.

At the end of the conjugation process, the conjugate may be purified by any suitable process. Examples of purification processes include chromatographic means, for example ion exchange, anion/cation exchange chromatography, FPLC or reverse phase, hydrophobic interaction, gel filtration, membrane separation, extraction, gels, capillary electrophoresis, affinity chromatography, precipitation, centrifugation and osmosis. Preferred purification processes are anion exchange, gel filtration and membrane separation (e.g. dialysis).

For example, the conjugate may be purified by FPLC using a Tris/salt -13 -gradient.

After purification, the conjugate may be stored, for example at 4C in 20mM Tris p1-I 7.5, 0.1-0.5 M sodium chloride etc. Under such conditions, the conjugate should remain stable for at least 5 months.

Alternatively, the conjugate may be freeze-dried or iyophilised. Stabilising agents (such as BSA, antibacteriocides, detergents, surfactants and sugars) may also be added to the conjugates (either in solution, freeze-dried or lyophilised).

Preferred conjugates from step (i) are antibody-or enzyme-linker conjugates, particularly those wherein the enzyme is horseradish peroxidase or glucose oxidase, and particularly those wherein the linker is DIDS. Further preferred conjugates from step (i) include solid support-linker, for example "activated" beads, dip-sticks, micro-titreplates, polymers, fibre optics, glass and electronic devices.

Preferred conjugates from step (ii) include enzyme-antibody, enzyme-enzyme, enzyme-oligonucleotide, enzyme-DNA conjugates, enzymesolid support and antibody-solid support. Particularly preferred conjugates include horse-radish peroxidase-antibody and horse-radish peroxidase-oligonucleotide conjugates; glucose oxidase-antibody and glucose oxidase-oligonucleotide conjugates.

Specific examples of particularly preferred conjugates are horse radish peroxidase-DIDS-antibody and horse-radish peroxidase-DIDS-oligonucleotide conjugates; glucose oxidase-DIDS-antibody and glucose oxidase-DIDS-oligonucleotide conjugates.

In one particularly preferred embodiment of the invention, an enzyme is reacted with about a 90 fold molar excess of crosslinker for about 30 minutes.

The linker-enzyme conjugate is then reacted in about 2.4 fold molar excess (enzyme:antibody) with an antibody for 30 minutes. Preferably, the enzyme is horseradish peroxidase, the linker is DIDS, and the antibody is an anti-HCG antibody.

The invention also relates to a process for conjugating a first moiety with a second moiety, as herein defined, which additionally comprises the step of reacting one or more compounds of formula I with a diamine of formula XI:

XI

H2N NH2 wherein Y is as defined above, in order to produce a dimerised linker of formula II.

The linkers of the invention can be used, for example, to attach DNA or polypeptides to solid supports such as polystyrene beads, plastic wells, magnetic particles, electronics or optical fibres.

The invention also provides a kit comprising: (a) a linker of any one of formulae I, III, and V as herein defined; and (b) a diamine of formula XI as herein defined.

The invention also provides a kit comprising: (1) a first moiety-linker conjugate, wherein the linker is of any one gf formulae I, II, III, IV, V, VI, VU, VIII, IX or X; and (ii) a coupling buffer.

The first-moiety-linker conjugate is preferably in lyophilised or freeze-dried form.

The coupling buffer is preferably one which is suitable for coupling the first-moiety-linker conjugate to a second moiety, wherein the second moiety comprises one or more free amino groups. Most preferably, the coupling buffer is 0. 1M sodium borate, pH 8. The coupling buffer may be provided in dry or aqueous form.

The kit optionally includes instructions for coupling the first moiety-linker conjugate to a second moiety.

The invention also extends to conjugates which are produced using the processes of the current invention.

-15 -The invention further provides a compound of formula II: R' R2 R7 R14 R1 i)( s_ R8 R4 R17

I H H H H

R' R1' wherein R' to R4, R7 to R1 R" to R'4 and R17 to R2 are independently selected from H, halogen, Ci..io-alkyl, C3-s-cycloalkyl, OH, O-Ci3-aIkyl, SH, NH2, NO2, CO2H, C02-CI.3-alkyl, SO3H and SO3X, wherein X denotes a counter ion, for example, NH, Na or K; R5, R , R'5 and Rio are independently selected from H, halogen, C1-6-alkyl, OH, O-C1-3-alkyl, SH, NH2, CO2H, and C02-Cl-3-alkyl; m and n are each independently 1, 2 or 3, and Y denotes an organic linker with between 2 and 10 carbon atoms in the backbone.

Preferably, R' to R4, R7 to R' , R" to R14 and R'7 to R20 are each independently selected from H, methyl, ethyl and SO3X.

The invention further provides a compound of formula IX or X: soax IX XO3S'NN'C

H H H

S

NC x03S

-16 -x

N

S COOH S

wherein X is NH44, Na or K. The invention *also provides a moiety-linker conjugate, wherein the linker is one of formulae I, II, III, IV, V, VI, VII, VIII, IX or X as defined herein.

Preferably, the moiety is as defined herein.

The invention also provides a first moiety-linker-second moiety conjugate wherein the linker is one of formulae I, II, III, IV, V, VI, VII, VIII, IX or X as defined herein; and wherein the first and/or second moiety is as defined herein.

The invention also provides a conjugate as defined herein for use as a medicament; and a conjugate as defined herein for use in a diagnostic method practised on the human or animal body.

The invention also provides the use of a conjugate as defined herein in an ex vivo diagnostic method; and the use of a conjugate as defined herein in the manufacture of a diagnostic tool for use in a diagnostic method practised on the human or animal body.

The invention further provides the use of a conjugate as defined herein in the manufacture of a medicament for use in a method of treatment of the human or animal body.

The invention also provides a method of treating a patient comprising administering to that patient a conjugate as defined herein; and a method of diagnosing a condition in a patient comprising administering to that patient a conjugate as defined herein.

-17 -The following abbreviations are used throughout this specification: ESA Bovine serum albumin DIDS 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid DMF Dimethylformamide DMSO Dimethyl sulfoxide ELISA Enzyme-linked immuno-sorbent assay ELOSA Enzyme-linked oligo-sorbent assay FPLC fast protein liquid chromatography HCG human chorionic gonadotropin HPLC High performance liquid chromatography HRP horseradish peroxidase PBS phosphate-buffered saline Tris tris(hydroxymethyl)aminomethane

BRIEF DESCRIPTION OF FIGURES

Figure 1 illustrates the purification of a HRP-DIDS-anti-HCG antibody conjugate on an ion exchange column on an Akta Prime liquid chromatographic system.

Figure 2 illustrates the purification of a HRP-DIDS-anti-Inhibin A antibody conjugate on an ion exchange column on an Akta Prime liquid chromatographic system.

Figure 3 illustrates an ELISA of a HRP-DIDS-anti-Inhibin A conjugate of Example 3 in comparison with a commercial standard (control) assay for Inhibin A. Figure 4 shows the purification of a HRP-DIDS-oligonucleotide by anion exchange chromatography.

Figure 5 illustrates an ELOSA of two HRP-DNA conjugates of the invention.

-18 -

EXAMPLES

Example 1

Preparation of HRP-DIDS-anti-HCG antibody conjugate Materials Monoclonal anti-HCG antibody, British Biocell International, batch 7416, 5.3 mgs/ml.

Crosslinking agent DIDS, Aldrich 285617.

Horse radish peroxidase, Biozyme Labs, code HRI' 4B, Batch 935 C Sodium Borate buffer pH 8, 0.1 M Reagent concentrations Reagent m.wt mass(mg) moles in reaction HRP 40000 0.27 6.8 nmol DIDS 498 0.3 0.6 gmol ANTIBODY 150000 0.4 2.7 nmol Protocol To the HR.? dissolved in borate buffer (451d) was added DIDS in borate buffer (5p1). The reaction mixture was shaken for 0.5 hours and then the antibody (75.6d of supplied solution + 124d borate buffer) was added and the shaking continued for a further 0.5 hours.

The product was purified by anion exchange chromatography using a Q-Sepharose column and AKTA prime chromatography unit. The product was eluted in a gradient of 0 to 1M sodium chloride in 20mM Tris pH 7.5. (Fig 1).

Example 2

Preparation of bead-DIDS-anti-HCG antibody conjugate Antibodies can be linked to amino functionalised beads (for example, polystyrene) using a similar procedure to that described in Example 1.

Materials Polyscience amino-functionalised polystyrene beads 2pm.

Lot GKOO2O5O1T, 50mg/mi, 4nmoi of amine/mg.

-19 -DIDS and antibody as Example 1.

Protocol 1 mg of beads contained in a lml Eppendorf tube(with the cap removed and placed in a FALCON tube) were washed with borate buffer (2x300il) and concentrated by centrifugation (2000rpm, 4 minutes per buffer wash). To the concentrated beads was added 200pg DIDS dissolved in 50jtl borate buffer. The mixture was shaken at 50 revolutions per minute for 1 hour and then washed with borate buffer (2x300tl) and concentrated. 20i1 (0.1mg) of antibody in 80tl borate buffer was added to the beads and the mixture shaken for a further 1 hour. The conjugated beads were washed with borate buffer (2x300p1) and then stored at 4 C in 20mM Tris pH 7.5.

Enzyme antibody conjugates are quality controlled by immunoassay. A capture antibody is immobilised on polystyrene beads and the beads dried onto a membrane (dipstick). The immunogen+conjugate dissolved in an elution buffer is then applied to the dipstick and the mixture allowed to run up the stick. Enzyme substrate is added and any signal observed at the application point of the beads is noted.

Example 3

Preparation of HRP-DIDS-anti-Inhibin A conjugate An HRP-DIDS-anti-Inhibin A (a fertility marker) conjugate was prepared usinga similar procedure to that described in Example 1. Figure 2 shows the chromatographic (AKTA prime) purification profile obtained.

This conjugate was quality controlled using the DSL Ltd Inhibin A (DSL-10- 28100) ELISA kit. Figure 3 demonstrates the superior performance of the prepared conjugate versus a commercial control in an ELISA for Inhibin A. The level of detection of target Inhibin A by the present conjugate was lopgIml. In comparison, the commercial product was at least 10 times less sensitive (detection level of 250pg/ml). a

Example 4

Preparation of HRP-DIDS-oligonucleotide Materials Amino-link oligonucleotide to DNA target: Amine-30mer oligonucleotide in H20, lOOp.M, HPLC purified (Sigma Genosys); Cross linking reagent: DIDS; HRP: HRP 4B -Batch 947C (50mgs); and Sodium Borate buffer pH8.0, 0.1M.

Reagent concentrations Reagent m.wt. mass(mgs) mols in PW Sodium borate buffer vol. HRP 40000 0.675 l7nmols 45il DIDS 498 0.75 1.5tmol 5tl Amino-linked oligonucleotide 9395 0.063 6.7nmols 67j.tl (or 134jtl for 2x) Protocol To the HRP dissolved in sodium borate (45d) was added the coupling agent DIDS in borate buffer (5.tl). The reaction mixture was shaken for 0.5 hours and the oligonucleotide (67d or 134il for 2x) was added and the shaking continued for a further 0.5 hours (total volume =1 17tl or l84il). 20mM Tris pH 7.5 (250.tI) was added to stop the reaction.

The product was purified by anion exchange chromatography using a Q-Sepharose column and AKTA Prime chromatography unit. The product was eluted in a gradient of 0 to 1M sodium chloride in 20mM Tris pH7.5 (Figure 4).

Figure 5 demonstrates the comparative performance in an ELOSA of two HRP-DNA conjugates differing only in the structure of the linker connecting the DNA strand to the enzyme (linker 1 is DIDS, linker 2 is DIDS-lysine-DIDS).

Application of linker 1 led to a level of detection of 400pg/ml (1 fmol of target).

-21 -

Example 5

Kit comprising a pre-activated moiety A kit is produced comprising (i) a lyophilised or freeze-dried HRP-DIDS conjugate as described in Example 1, and (ii) an aqueous coupling buffer (O.1M sodium borate, pH 8).

At the time of use, the coupling buffer is added to the lyophilised or freeze-dried HRP-DIDS conjugate. An antibody is then added and conjugation is allowed to proceed for 0.5 hours. The reaction is stopped by the addition of mM Tris/HCI, pH 7.5.

Claims

-22 -

CLAIMS

1. A process for linking a first moiety with a linker, wherein the first moiety comprises one or more free amino groups and wherein the first moiety is a not a cell membrane or cell membrane fraction, the process comprising the steps: (i) conjugating the first moiety with a linker of formula 1: I R1 NR2 R5 R7 R'0 or a dimerised linker of formula II: II R1 R2 R5 R7 R14 R1r"Lr"C...

s__ RVIrL(i!(L(R s R1(J,(Jç)JL.Li8 R4 R17 R1 R'1 wherein R' to R4, R7 to R' to R'4 and R'7 to R2 are independently selected from H, halogen, Ci.1o-alkyl, C3..8-cycloalkyl, OH, O-C1-3-alkyl, SH, NH2, N02, CO2H, C02-Cl-3-alkyl, SO3H and SO3X, wherein X denotes a counter ion, for example, NH, Na or K; R5, R6, R'5 and R'6 are independently selected from H, halogen, CI-6-alkyl, OH, O-C13-alkyI, SH, NH2, CO2H, and C02-C1-3-alkyl; -23 -m and n are independently 1, 2 or 3, and Y denotes an organic linker with between 2 and 10 carbon atoms in the backbone.

2. A process for conjugating a first moiety with a second moiety, wherein the first and second moieties independently comprise one or more free amino groups, the process comprising the steps: (i) conjugating the first moiety with a linker of formula I:

I R7 R3 s_c R4 R6 R9 R' 

or a dimerised linker of formula II:

H R2 

R S R18 s._ R8 R7 R14 R' . N R4 R17

I H H H H

R1 Ru wherein R' to R4, R7 to R' ' R" to R'4 and R'7 to R2 are independently selected from H, halogen, Ci..io-alkyl, C3-8-cycloalkyl, OH, O-C1-3-alkyl, SH, NH2, NO2, CO2H, C02-C1-3-alkyl, SO3H and SO3X, wherein X denotes a counter ion, for example, NH44, Na or 24 -R5, R6, R'5 and R'6 are independently selected from H, halogen, C1-6-alkyl, OH, O-Cl3-alkyl, SH, NH2, CO2H, and C02-Ci..3-alkyl; m and n are independently 1, 2 or 3, and Y denotes an organic linker with between 2 and 10 carbon atoms in the backbone, and then (ii) reacting the first moiety-linker conjugate formed in (i) with the second moiety.

3. A process as claimed in claim 1 or claim 2, wherein step (i) is carried out under the cànditions: molar ratio of first moiety:linker 1:45 to 1:140.

4. A process as claimed in claim 1 or claim 2 or claim 3, wherein step (i) is carried out under the conditions: time 15 to 120 minutes 5. A process as claimed in any one of claims 2 to 4, wherein step (ii) is carried out under the conditions: molar ratio of first moiety used in step (i):second moiety 1:1 to 1:8.

6. A process as claimed in any one of claims 2 to 5, wherein step (ii) is carried out under the conditions: time 15 to 120 minutes.

7.' A process as claimed in any one of claims 1 to 6, wherein R' to R4, R7 to R' , R1' to R'4 and R'7 to R20, when present, are each independently selected from H, methyl, ethyl and SO3X.

-25 - 8. A process as claimed in any one of claims 1 to 7, wherein R5, R6, R'5 and R'6, when present, are each independently selected from H and Ci..3 alkyl.

9. A process as claimed in any one of claims 1 to 8, wherein only one or two of R' to R4 and R7 to R' are SO3X.

10. A process as claimed in claim 9, wherein only one of R' to R4 and/or only one of R7 to R' are SO3X.

11. A process as claimed in claim 10, wherein R4 is SO3X and/or R7 is s03x.

12. A process as claimed in any one of claims 1 to 11, wherein only one or two of R" to R4 and R17 to R20, when present, are SO3X.

13. A process as claimed in claim 12, wherein only one of R" to R'4 and/or only one of R'7 to R20, when present, are SO3X.

14. A process as claimed in claim 13, wherein R'4 is SO3X and/or R'7 is SO3X.

15. A process as claimed in any one of claims 1 to 14, wherein n is 1.

16. A process as claimed in claim 15, wherein R5 and R6 are arranged in the trans configuration.

17. A process as claimed in any one of claims ito 16, wherein mis 1.

18. A process as claimed in claim 17, wherein R'5 and R'6 are arranged in the trans configuration.

19. A process as claimed in any one of claims 1 to 18, wherein X is Na.

20. A process as claimed in any one of claims ito 19, wherein the linker is a compound of formula III: -26 -III R1 CNR2 R5 SO3X R3frnJ1( R8 SO3X R

R I R1 

or a dimerised linker of formula IV Iv R1 R20 R'6 R2 s03x R1&%) N

R

R3 O3XI6 N IR12R' " R1 3x

I H H H H

R1 R11 or a linker of formula V V Cl-ta S_CNR 7 so R4 R6 R10 or a dimerised linker of formula VI, VII or VIII: -27 -CH3 CH3 c#srLr2 R5 SO3X SOX R1'LrC...

s.._ H3C/ s R11/lL(JJL(L R4 R17 R1 R11 R1 R20 R5 R7 R14 S R3(('(L(C RIB SO3X s03x CH3 CH3 CH3 R2 R R5 SO3X R14 R1 H3C *._-(L(R8 R18 R4 R6 R9(N_rr rRI2 SO3X R' CH3 21. A process as claimed in claim 20, wherein R' to R20, when present, are independently selected from H, methyl and ethyl.

22. A process as claimed in claim 20, wherein R' to R20, when present, are all H.

-

23. A process as claimed in any one of claims 1 to 22, wherein the organic linker Y is a linear C2.o-alkyl, most preferably a C2-ealkyl group, optionally substituted at one or more of the backbone carbon atoms by one or more groups selected from halogen, C1-3-alkyl, O-Ci3-alkyl, CO2H and CO2-C13-alkyl.

24. A process as claimed in claim 23, wherein Y is -CH2CI-12-.

-28 - 25. A process as claimed in claim 23, wherein Y is -(CH2)4CH(COOH)-.

26. A process as claimed in any one of claims 1 to 6 wherein the linker is 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid, disodium salt (DIDS): Na NO3S N 27. A process as claimed in any one of claims 1 to 25, wherein the dimerised linker is selected from compounds of formulae IX and X:

H H

IX XO3S

H H II

XO3S,,LN.C x

S COOH S

28. A process as claimed in any one of claims ito 27, wherein the first moiety and/or the second moiety are independently selected from a chemical entity, preferably an isolated or purified chemical entity, and a solid support.

29. A process as claimed in any one of claims 1 to 28, wherein the first 29 -moiety andlor the second moiety is a macromolecule, preferably a biological macromolecule and most preferably an isolated or purified biological macromolecule.

30. A process as claimed in claim 29, wherein the first moiety and/or the second moiety are independently selected from a nucleic acid molecule, a protein, a polypeptide or a peptide.

31. A process as claimed in claim 30, wherein at least one of the moieties is a DNA oligomer, an enzyme or an optionally-labelled antibody.

32. A process as claimed in claim 31, wherein at least one of the moieties is horseradish peroxidase, alkaline phosphatase, beta galactosidase, glucose oxidase or a restriction enzyme.

33. A process as claimed in any one of claims 1 to 32, wherein the moiety or at least one moiety is a solid support, such as a particle, a bead or a matrix such as a sheet, gel, filter, membrane, fibre, tube, microtitre plate or column.

34. A process as claimed in any one of claims 1 to 33, wherein at least one moiety is a medicament or a dye.

35. A process as claimed in any one of claims 1 to 34, wherein at least one moiety incorporates a radiolabel.

36. A process as claimed in any one of the preceding claims, wherein the molar ratio of the first moiety:linker is 1:60 to 1:120, more preferably 1:80 to 1:100, and most preferably about 1:90.

37. A process as claimed in any one of claims 2 to 36, wherein the molar ratio of the second moiety to the first moiety is 2:1 to 6:1, more preferably 3:1 to 6:1, and most preferably about 4:1.

38. A process as claimed in any one of the preceding claims, wherein the process is carried out at a pH between 5.5 to 9.5.

39. A process as claimed in any one of the preceding claims, wherein the -30 -reaction time for step (i) is 15 to 45 minutes, 30 to 90 minutes or 45 to 75 minutes.

40. A process as claimed in any one of claims 2 to 39, wherein the reaction time for step (ii) is 15 to 45 minutes, 30 to 90 minutes or 45 to 75 minutes.

41. A process as claimed in any one of the preceding claims, wherein the reaction(s) are carried out at 4 to 50 C.

42. A process as claimed in any one of claims 2 to 41, wherein step (ii) is carried out directly after step (i).

43. A process as claimed in any one of claims 2 to 42, wherein, when carrying out step (ii), the second moiety is added to the reaction mixture obtained from step (i).

44. A process as claimed in any one of claims 2 to 43, wherein the first moiety-linker conjugate obtained from step (i) is not purified or is not treated in any other way before the second moiety is added to the reaction mixture.

45. A process as claimed in any one of the preceding claims, wherein the process additionally comprises step (ia) purifying the obtained first moiety-linker conjugate from step (i), and optionally resuspending the conjugate in a

suitable buffer.

46. A process as claimed in any one claims 2 to 45, wherein the process additionally comprises step (iia) purifying the obtained conjugate, and optionally resuspending the conjugate in a suitable buffer.

47. A process as claimed in claim 45, wherein the conjugate is an enzyme-linker conjugate.

48. A process as claimed in any one of claims 2-44 and 46, wherein the conjugate is an enzyme-antibody, enzyme-enzyme, enzyme-oligonucleotide or enzyme-DNA conjugate.

49. A process as claimed in claim 47 or claim 48 wherein the enzyme is horseradish peroxidase.

50. A process as claimed in claim 47 or claim 48 wherein the enzyme is glucose oxidase.

51. A process as claimed in claim 48 or claim 49 or claim 50 wherein the antibody is an anti-HCG antibody.

52. A process as claimed in any one of claims 2-44 and 46, wherein an enzyme is reacted with about a 90 fold molar excess of linker for about 30 minutes; and then the linker-enzyme conjugate is reacted in about
2.4 fold molar excess (enzyme:antibody) with an antibody for about 30 minutes.

53. A process as claimed in any one of the preceding claims, which additionally comprises the step of reacting one or more compounds of formula I with a diamine of formula XI: xl H2N NH2 in order to produce a dimerised linker of formula II.

54. A compound of formula II: R' R2 R2 R5 R7 R14 R1 N R3 (L(R s R1.(JJ " R18 R4 R17 R1 R11 wherein R1 to R4, R7 to R10R" to R'4 and R'7 to R2 are independently selected from -32 -H, halogen, Ci..io-alkyl, C38-cycloalkyl, OH, O-Ci..3-alkyl, SH, NH2, NO2, CO2H, C02-Cj..3-alkyl, SO3H and SO3X, wherein X denotes a counter ion, for example, NH4, Na or K; R5, R6, R'5 and R'6 are independently selected from H, halogen, CI.6-alkyl, OH, O-CI-3-alkyl, SH, NH2, CO2H, and C02-CI-3-alkyl; m and n are each independently 1, 2 or 3, and Y denotes an organic linker with between 2 and 10 carbon atoms in the backbone.

55. A compound as claimed in claim 54, wherein R' to R4, R7 to R' , R" to R'4 and RI7 to R2 are each independently selected from H, methyl, ethyl and SO3X.

56. A compound of formula IX or X: ix so3x xo3s Nc

S COOH S

wherein X is NH, Na or K4.

57. A kit comprising: (a) a linker of formula I, III or V as defined in any one of claims 1 to 22 and 26; and -33 - (b) a diamine as defined in any one of claims 23 to 25.

58. A kit comprising: (i) a moiety-linker conjugate, wherein the linker is one of formulae I, II, III, IV, V, VI, VII, VIII, IX or X as defined in any one of claims 1, 2, 7-35, 47-51; and (ii) a coupling buffer.

59. A kit as claimed in claim 58, wherein the moiety-linker conjugate is present in lyophilised form.

60. A kit as claimed in claim 58 or 59, wherein the moiety is as defined in any one of claims 28-35 or 47-5 1.

61. A conjugate produced by or producable by a process of any one of claims 1 to 53.

62. A moiety-linker conjugate, wherein the linker is one of formulae I, II, III, IV, V, VI, VII, VIII, IX or X as defined in any one of claims 1, 2, 7-35, or 47-51.

63. A moiety-linker conjugate as claimed in claim 62, wherein the moiety is as defined in any one.of claims 28-3 5 or 47-51.

64. A first moiety-linker-second moiety conjugate wherein the linker is one of formulae I, II, III, N, V, VI, VII, VIII, IX or X as defined in any one of claims 1, 2, 7-3 5, or 47-51; and wherein the first and/or second moiety is as defined in any one of claims 28-35 or 47-51.

65. A conjugate as defined in any one of claims 1-35, 47-5 1, 58 and 6 1-64 for use as a medicament.

66. A conjugate as defined in any one of claims 1-35, 47-5 1, 58 and 6 1-64 for use in a diagnostic method practised on the human or animal body.

67. Use of a conjugate as defined in any one of claims 1-35, 47-51, 58 and 6 1-64 in an ex vivo diagnostic method.

-34 - 68. Use of a conjugate as defined in any one of claims 1-35, 47-5 1, 58 and 61-64 in the manufacture of a diagnostic tool for use in a diagnostic method practised on the human or animal body.

69. Use of a conjugate as defined in any one of claims 1-35, 47-5 1, 58 and 61-64 in the manufacture of a medicament for use in a method of treatment of the human or animal body.

70. A method of treating a patient comprising administering to that patient a conjugate as defined in any one of claims 1-35, 47-5 1, 58 and 6 1-64.

71. A method of diagnosing a condition in a patient comprising administering to that patient a conjugate as defined in any one of claims 1-35, 47-5 1, 58 and 6 1-64.