FIELD OF THE INVENTION

The present invention relates to a solid support conjugated to darbepoetin alpha and an antibody or a repertoire thereof obtainable from the said solid support.

BACK GROUND OF INVENTION

Proteins such as antibodies cytokines growth factors are important analytical diagnostic tools. In particular antibodies capable of binding a proteins or an antigen of interest are used in detection and quantitation of the protein or the antigens of interest.
Antibodies have also been used for affinity purification of proteins and antigens of interest.

Antibodies may be obtained from multiple sources; such an immune sera a host immunized with an antigen from hybridomas or transfectomas. Antibodies derived from trasnfectomas or hybridomas are monoclonal in nature with high degree of specificity to towards a particular antigen/ epitope.

Immune serum obtained from a host is usually polyclonal in nature and comprises of a repertoire antibodies of different specificities and affinities. Choice of monoclonal or polyclonal antibody or sera is dictated by the intent of use. Polyclonal sera may be preferred if the intended is affinity purification detection or quantitation of a protein of interest as the ability to bind multiple epitopes on the protein or antigen of interest can be exploited to increase the yield and purity of the target protein or antigen.

Cytokines such as erythropoietin and darbepoetin are described as major erythropoiesis stimulating agents. Erythropoiesis stimulating agents (ESA) are known to stimulate production of red blood cells in a mammal. Darbepoetin alpha is a second generation ESA derived from erythropoietin alfa (EPO). It is a recombinant protein and as opposed to erythropoietin has a longer serum half –life due to the presence of three additional glycosylation sites as compared to erythropoietin (US7217689 Macdougall et al J Am Soc Nephrol 1999; 10:2392-2395).

Currently antibodies prepared against erythropoietin are mainly used for the detection of darbepoetin due to their cross reactivity. However a serious limitation in the use of these antibodies is that they bind darbepoetin less efficiently. Hence there is a need for antibodies exhibiting enhanced binding to darbepoetin than what is currently available.

In order to obtain antibodies exhibiting enhanced binding to darbepoetin a host may be immunized with darbepoetin and the antibody obtained from immune sera purified on a darbepoetin conjugated solid support. However unavailability of such a solid support has impeded the availability of better reactive antibodies to darbepoetin. Therefore in addition to antibodies exhibiting improved binding to darbepoetin there is a need for a darbepoetin conjugated solid support that could be used for obtaining antibodies having higher specificity to darbepoetin.

The principal object of the present invention is to disclose a solid support conjugated to darbepoetin alpha. A further object of the invention is to provide an antibody repertoire obtainable from the said support wherein the repertoire exhibits improved binding to darbepoetin in comparison to the commercially available ones.

A solid support conjugated to darbepoetin may have many additional non limiting uses; such as for purification of other proteins or agents different analytical assay such as immunoprecipitation etc.

SUMMARY OF THE INVENTION

The present invention relates to a solid support conjugated to darbepoetin alpha and an antibody or a repertoire thereof obtainable from the said support wherein the improvement being the antibody or the repertoire thereof exhibit improved binding to darbepoetin in comparison to currently available polyclonal antibody.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1: Illustration of purified anti darbepoetin antibody derived from darbepoetin conjugated column as described in Example 2

Figure 2: Illustration of binding as determined by ELISA for an antibody repertoire obtained by using the darbepoetin conjugated support described in Example 3

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments of the disclosed invention encompass an insoluble immobilizable matrix conjugated to darbepoetin alpha wherein the antibody or the repertoire thereof obtainable from the said support exhibit significantly improved binding to darbepoetin as compared to commercially available polyclonal antibody.

Proteins can be conjugated to a chromatography support or matrix by various means. The primary or secondary amine groups present in a protein are often utilized for such conjugations. In some instances the sugar side chain present in glycoproteins is also utilized (http://www.piercenet.com/browse.cfm?fldID=CE4D6C5C-5946-4814-9904-C46E01232683). The choice of conjugation system depends on the nature of the protein. Cynogen Bromide (CNBr) and N-Hydroxy Succiniamide (NHS) are two of the most commonly used protein conjugation agents which utilize primary and secondary amine groups present in a protein (Richard R et al Guide to protein purification 430-431).
One embodiment the invention provides an insoluble immobilizable matrix conjugated to darbepoetin alpha.

In another embodiment the invention provides an insoluble immobilizable matrix conjugated to darbepoetin alpha wherein the conjugation is mediated through the primary and/or the secondary amine groups present in darbepoetin alpha.

In another embodiment of the invention the conjugation is mediated by means of N-Hydroxy Succiniamide (NHS).

In a further embodiment the invention provides a method of preparation of an antibody capable of binding darbepoetin comprising;

a. loading an antibody solution on to an insoluble immobilizable matrix conjugated to darbepoetin and

b. eluting the bound antibody under suitable conditions.

In another embodiment the invention provides an antibody obtainable from the insoluble immobilizable matrix conjugated to darbepoetin that exhibit about 2.0 fold or more improved binding to darbepoetin as compared to commercially available polyclonal antibody wherein the binding is determined by ELISA.

The antibody thus obtained may be used in various non limiting exemplary ways such as affinity purification detection and quantitation of proteins or moieties bearing reactive epitopes.

Definitions

The term ‘protein’ and ‘glycoprotein’ has been used interchangeably in context of the present invention. A protein or a glycoprotein may bear different functional groups on them such as amine carboxyl aldehyde thiol or methyl or ethyl.

The term ‘immune sera’ as used herein refer to a serum obtained from a host capable of invoking an immune response when immunized with a suitable antigen. In context of present invention ‘darbepoetin immune sera’ refers to a sera obtained after immunization of a capable host with darbepoetin.

The term ‘darbepoetin’ and ‘darbepoetin alpha’ has been used interchangeably in context of the present invention.

EXAMPLES

Example 1

Preparation & Conjugation of Darbepoetin

NHS sepharose beads were activated by incubating in 1mM hydrochloric acid (HCl) for sufficient time for swelling the beads. The swollen beads were packed in an empty PD10 column and washed repeatedly with 1mM HCl.

Darbepoetin alpha was buffer exchanged into coupling buffer as described in Table 1 using a 30kDa membrane concentrator. 1.5ml and 1ml of darbepoetin preparation at a final concentration of 7.3mg/ml was incubated for about 4hrs with the NHS activated sepharose beads respectively.

Post incubation the sepharose beads were first washed with 25 ml of blocking buffer followed by incubation with 5 ml of blocking buffer (Table 1) for about another 4 hrs.

The reaction was conducted at room temperature of about 25?C ? 2?C. Darbepoetin coupled matrices was washed with PBS and stored in 20% ethanol.

Buffer’s Description of buffer

Wash buffer 1 mM HCl

PBS buffer Phosphate buffer saline composition of (1X PBS for 1L; 8g NaCl; 0.2g KCL; 1.44 g Na2HPO4; 0.24 g KH2PO4; distilled water q.s;

Coupling buffer 0.1 M NaHCO3 0.5 M NaCl: pH 9.0 in coupling buffer

Blocking buffer 1 M ethanolamine in coupling buffer or 1 M tris-base in coupling buffer

Low pH buffer 0.1 M acetic acid in 0.5M NaCl

Storage solution 20% ethanol

Table 1: Buffers used in coupling experiment

Example 2

a. Antibody Enrichment from Darbepoetin Immune Sera

Immunoglobulin fraction was enriched on a protein A column by diluting darbepoetin immune sera equally with equilibration buffer (50mM Tris 150mM NaCl pH7.5) followed by loading onto a protein A affinity column. Post loading the column was washed with wash buffer (50mM Tris 150mM NaCl pH7.5) for about 5 column volumes. Elution was conducted at low pH using 50mM citrate buffer pH 3.5. The eluate was neutralized to pH 6.0 by adding 1M Tris buffer pH 9.0.

b. Antibody Preparation using Darbepoetin Conjugated Column

Either of darbepoetin immune sera derived from a host or enriched immunoglobulin preparation from step ‘a’ was used for purification of antibodies.

About 0.5ml of antibody preparation was used per 1ml of resin. The antibody preparation was diluted equally in phosphate buffered saline (‘PBS’) before loading onto the darbepoetin conjugated column. The column was incubated for about 30 minutes at room temperature accompanied with gentle agitation. The load was allowed to flow though the column followed washing with PBS for about five column volumes. Samples were eluted using 0.02M orthophosphoric acid at low pH. Fractions were directly collected into the neutralization buffer 1M Tris buffer pH 9.0 (10% of the eluate volume). Purified antibody preparation was characterized by ELISA and SDS-PAGE.

Figure 1 (Lane 7 8) is a representative SDS-PAGE profiles of purified antibody obtained from the darbepoetin conjugated column. Antibodies could be purified to homogeneity by using the darbepoetin conjugated column.

Lane M represents protein molecular weight markers with respective molecular weights. Lane 1 and 2 are control purified antibodies which are not specific to darbepoetin or erythropoietin. Lane 3 represents purified darbepoetin lane 4 is 1:20 dilution of immunoglobulin enriched fraction obtained after protein A purification. Lane 5 and lane 6 are 1:20 dilution of flowthrough from the darbepoetin conjugated column and 1:10 dilution of post load wash from the darbepoetin column respectively.

Example 3

Binding Study by ELISA

Antibody repertoire as obtained following the procedure disclosed in example 2 was used for determination of binding efficiency. Darbepoetin was coated in ELISA plates followed by incubation with different concentrations of antibodies derived either from the disclosed darbepoetin conjugated support or were commercially available. Anti rabbit IgG-HRP was used as the secondary antibody. The reaction was revealed by using 3 3’ 5 5’ – tetramethylbenzidine. Reaction was quenched with sulfuric acid. 0.05% Tween 20 prepared in PBS was used as wash buffer.

Binding efficiency was compared with commercially available anti erythropoietin rabbit polyclonal antibodies reactive to darbepoetin (Table 2 Figure 2).

The commercially available anti erythropoietin rabbit polyclonal antibodies cross reacted to darbepoetin. Antibody purified by using darbepoetin column in which NHS was used for conjugation exhibited maximal reactivity to darbepoetin in the group. The fold improvement in binding ranged from about 2.2 to about 19 fold of what was commercially available (Table 2).

Antibody Source Absorbance (ng/ml)

Fold Binding (50ng/ml)

NHS Column

100ng/ml 50ng/ml

Rabbit polyclonal Antibody to EPO Santa Cruz

(Cat No: SC-7956) 0.160 0.073 19

Antibody repertoire Darbepoetin Column-NHS 2.131 1.402 1

Rabbit polyclonal Antibody to EPO Abcam

(Cat No: AB30545/ Lot No:954648) 0.409 0.181 7.7

Rabbit polyclonal Antibody to EPO Abcam

(Cat No: AB30545/ Lot No:836899) 1.216 0.623 2.2

Rabbit polyclonal Antibody to EPO R&D systems

(Cat No: AB-286-NA) 1.201 0.674 2.08

Table 2: Binding of purified anti-darbepoetin alpha antibody to darbepoetin on darbepoetin coated ELISA plates and fold change in binding in comparison to commercially available polyclonal antibodies to erythropoietin.

WE CLAIM:

1. An insoluble immobilizable matrix conjugated to darbepoetin alpha, and an antibody or a repertoire obtainable thereof obtainable from the said matrix, wherein the obtained antibody or the repertoire thereof exhibits improved binding to darbepoetin alpha in comparison to binding of an anti erythropoietin antibody to darbepoetin alpha, wherein the binding is determined by ELISA.

2. The antibody or the repertoire thereof of claim 1, wherein the anti darbepoetin antibody or a repertoire thereof exhibits at least greater than 2 fold improvement in binding as compared to binding of an anti erythropoietin antibody to darbepoetin alpha, wherein the binding is determined by ELISA.

3. The antibody or the repertoire thereof of claim 1, wherein the anti darbepoetin antibody or a repertoire thereof exhibits about 15 fold or greater improvement in binding as compared to binding of an anti erythropoietin antibody to darbepoetin alpha, wherein the binding is determined by ELISA.

4. An insoluble immobilizable matrix of claim 1 wherein darbepoetin alpha, is conjugated to the matrix through the primary and/or the secondary amine groups and by means of N-Hydroxy Succiniamide (NHS).

5. A method of obtaining an antibody binding to darbepoetin alpha in comparison to binding of anti erythropoietin antibody to darbepoetin alpha, wherein the method comprises

a. loading an antibody solution on to an insoluble immobilizable matrix conjugated
to darbepoetin, and

b. eluting the bound antibody under suitable conditions.

6. A method according to claim 5, wherein the antibody solution is prepared in phosphate buffer at near neutral pH and the bound antibody is eluted at acidic pH.