EP0188589A1 - Immunopurification process - Google Patents

Abstract

An immunopurification method for separating an antigen from a sample containing this antigen, with the intervention of an affinity matrix comprising a solid phase, to which is attached an antibody which has a positive thermo-dependent affinity such as that the affinity of the antibody is significantly greater at a relatively higher first temperature than at a relatively lower second temperature. The method involves bringing the sample into contact with the affinity matrix at the first temperature, so that the sample antigen associates with the affinity matrix, that unbound species are removed from the affinity matrix and that the antigen is dissociated from the affinity matrix at the second temperature, in order to obtain the antigen. The method reduces the need for a chemical reagent to carry out the dissociation, which reduces the risk of denaturing the antibody and / or the antigen. Examples are presented where the antigens are blood factor VIII and thrombin-antithrombin.

Description

IMMUNOPORIFICATIQN PROCESS Field of the Invention

This invention relates to an immunopurification process for separating an antigen from a sample containing the antigen, and to reagents for use in the process. Background to the Invention

The process of immunopurification (or immunoaffinity purification) is well known and is widely used for the isolation of a particular desired antigen from a sample. In general, the process involves contacting a sample containing the desired antigen with an affinity matrix comprising antibody to the antigen covalently attached to a solid phase. Antigen in the sample becomes bound to the affinity matrix through an immunochemical bond. The affinity matrix is then washed to remove any unbound species. The antigen is removed from the affinity matrix by altering the chemical composition of a solution in contact with the affinity matrix. The immunopurification may be conducted on a column containing the affinity matrix, in which case the solution is an eluent or in a batch process, in which case the affinity matrix is maintained as a suspension in the solution. An important step in the process is the removal of antigen from the matrix. This is commonly achieved by increasing the ionic strength of the solution in contact with the affinity matrix, for example, by the addition of a chaotrope such as urea, guanidine or an inorganic salt. An alteration of pH may also be effective to dissociate the immunochemical bond between antigen and the affinity matrix. These modifications of the chemical composition of the solution in contact with the affinity matrix, in many cases, have a detrimental effect on the antigen and/or the immobilised antibody often partially denaturing the antigen or antibody and thus reducing the efficiency of the immunopurification process. This effect is particularly marked where high affinity antibodies, such as certain types of monoclonal antibodies, are used in the affinity matrix (see for example published International patent application WO

83/03678). In such systems major changes in the chemical composition of the solution (either by the addition of a chaotrope or by altering the pH) are necessary in order to dissociate the immunochemical bond between antigen and affinity matrix.

It is known that certain polyclonal antibodies exhibit a negative temperature-dependent affinity, that is, a binding equilibrium constant which decreases with increased temperature (see Chard, T. An introduction to radioimmunoassay and related techniques North-Holland (1981) p 456 and Keane et al Clin. Chem. (1976) 22, 70). Monoclonal antibodies have also been described which exhibit a negative temperature-dependent affinity (Trucco, M. et al Human Immunology (1980) 3 233-243). Such monoclonal antibodies have been used in an immunopurification process, in which purified product is eluted by an increase in temperature. It is however generally disadvantageous to release a potentially labile protein at an elevated temperature.

It has now been discovered that certain antibodies exhibit a positive temperature-dependent affinity with the antigen for which they are specific, that is, a binding equilibrium constant which increases with increased temperature. This property has not previously been recognised and is entirely unexpected. It was discovered in the course of work unconnected with immunopurification. Summary of the Invention

In a first aspect the present invention provides an immunopurification process for separating an antigen from a sample containing the antigen, the process involving an affinity matrix comprising antibody to the antigen attached to a solid phase, wherein the antibody has a positive temperature-dependent affinity such that the affinity of the antibody is substantially higher at a first relatively higher temperature than at a second relatively lower temperature, the process comprising the steps of: contacting the sample with the affinity matrix at the first temperature, thereby causing antigen in the sample to become associated with the affinity matrix, removing unbound species from the affinity matrix, and dissociating the antigen from the affinity matrix at the second temperature, to obtain the antigen.

The process of the invention has the advantage that dissociation of the antigen from the affinity matrix is caused primarily by the reduction in temperature from the first temperature to the second temperature. This obviates, or at least reduces, the need for a chemical reagent to produce the required dissociation, thereby reducing the possibility of denaturation of the antigen. A chemical dissociation agent such as a chaotrope or an acid or base may if desired be employed to assist dissociation, but at a level which does not cause appreciable denaturation of the antigen and/or the immobilised antibody. Furthermore, elution of the antigen at a relatively low temperature is beneficial in maintaining the stability of a potentially labile antigen or immobilised antibody.

The process of the first aspect of the invention may be used to separate any antigen from a sample containing the antigen for which an antibody exhibiting a positive temperature-dependent affinity may be isolated. Particular examples of antigens which we have found may advantageously be purified in this way are thrombin-antithrombin complex and blood factor VIII. The use of the process for purification of blood factor VIII is particularly advantageous since the mild conditions necessary to cause dissociation of the antigen from the affinity matrix may not simultaneously cause dissociation of the two complexed components which form factor VIII namely factor VIII related antigen (Von Hillibrands factor) and the clotting factor. Factor VIII is important for the therapeutic application in the treatment of the symptons of diseases affecting blood clotting, such as haemophilia.

In a second aspect the invention provides an antibody having a positive temperature-dependent affinity such that the affinity of the antibody is substantially higher at a first relatively higher temperature than at a second relatively lower temperature.

The antibody may have specificity for any antigen to which an antibody having a positive temperature-dependent affinity can be raised and isolated. Specific examples of such antibodies are antibodies to blood factor VIII {preferably blood factor VIII related antigen)and thrombin-antithrombin (preferably the neoantigen formed in the complex) as described herein. Preferably the antibody shows a marked affinity change in the range from 4° to 37°C and, most preferably, in the range from 4° to ambient temperature. The first temperature may be, for example, ambient temperature and the second temperature may be for example 4°.

The antibody may be a monoclonal antibody, or a polyclonal antiserum or a fraction thereof. Preferably however, the antibody is monoclonal antibody. The antibody is provided for use in a process of the first aspect of the invention.

In a third aspect the invention provides a process for preparing an antibody having a positive temperature-dependent affinity comprising screening a library of antibodies to an antigen for an antibody having an affinity which is substantially higher at a first relatively higher temperature than at a second relatively lower temperature. Where the antibody is a monoclonal antibody the screening step may result in the identification of a hybridoma cell line producing monoclonal antibody having the desired positive temperature-dependent affinity characteristic.

In a fourth aspect, the invention provides an affinity matrix comprising an antibody attached to a solid phase wherein the antibody is an antibody of the second aspect of the invention. The solid phase may be any solid phase material suitable for the production of an affinity matrix. The solid phase may be, for example, CNBr activated Sepharose - to which antibody may be readily attached. Brief Description of the Drawings

Figure 1 - shows the temperature-dependence of the affinity of monoclonal antibody from cell-line ESTl for thrombin-antithrombin complex, and Figure 2 - shows the temperature dependence of the affinity of various monoclonal antibodies for human factor VIII related antigen. Detailed Description of Embodiments

The invention is now illustrated by way of the following Examples. EXAMPLE 1

Monoclonal antibodies to human α-thrombin were prepared and one monoclonal antibody, designated ESTl, showed a marked temperature-dependent affinity to human α-thrombin-antithrombin. Materials and Methods α-Thrombin α-Thrombin was prepared by barium citrate adsorption and elution of reject clinical Factor IX concentrate (Prowse et al, 1976) where the barium eluate was pre-adosrbed to SP-Sephadex C-50 (Pharmacia) in 50 mM Tris-HCl pH 7.0. Following activation with tissue factor/Factor V (Fenton et al, 1977) or ecarin (Franza et al, 1975), the resultant thrombin was purified by chromatography on SP-Sephadex (Prowse et al, 1976) and in some cases, heparin-agarose (Miller-Anderson et al, 1980). The product (specific activity at least 2400 NIH unit/mg) was at least' 90% pure by SDS-PAGE analysis (Weber and Osborn, 1979). Radiolabelling

Proteins were radiolabelled with ¹²⁵I by a modification of the chloramine T method of Greenwood et al (1963). Protein ( 5μg in 10μl) and chloramine T (50μg in 10μl) in 0.25 M phosphate buffer, pH 7.5 were added to 10μl (37,000 KBg) of carrier-free Na ¹²⁵I (Amersham). After mixing, the reaction was immediately terminated by adding 860 μl of 0.05 M phosphate buffer containing 13.5 μl Na₂S₂O₅, and carrier KI (1 mg in 100μl). Labelled protein was separated from unreacted iodide on a 10 x 1cm

Sephadex G-50 (Pharmacia) column pre-equilibrated and eluted with assay buffer (0.05 M phosphate, pH 7.5, containing 0.5 M NaCl, 0.25% gelatine and 1% Tween 20). A specific activity of 1,500-2,200 KBg/μg was achieved for α-thrombin. α-Thrombin was also radiolabelled by the method of Bolton and Hunter (1972), which yielded a tracer of specific activity 900 KBg/μg. Thrombin-Antithrombin Thrombin-antithrombin complex was produced by incubating thrombin with 10-fold molar excess of antithrombin (AB Kabi) for 30 minutes at room temperature. Labelled thrombin-antithrombin was produced as described above. The clotting and amidase activities of the product were 1% of those of uncomplexed thrombin. Production of Hybridomas

Twelve-week old Balb/c female mice were immunised with an intial intraperitoneal injection of 50μg or 10μg human α-thrombin in Freund's complete adjuvant with Tween 80. Six weeks later they received the same dose intraperitoneally in alum. In some cases this was followed by further boosts of the same dose in saline at 4-weekly intervals; in others the second boost was postponed until week 20.

The mice were bled and the sera titred for anti-thrombin antibody after each boost. The mouse selected for fusion (see Results) received its second boost at week 20, and was given a final injection intravenously in saline at week 24.

Three days after this last injection the spleen was removed and as many cells as possible dispersed by injection with Dulbecco A saline

(Kennett et al, 1978). Half of the 4 x 10⁷ cells obtained were used for Fusion A. The rest were pooled with those obtained when the remaining spleen was crushed between ground glass slides, a total of 20 x 10⁷ cells. The pool was separated on a nylon wool column to enrich for B cells (Julius et al, 1973). The adherent cells were recovered by agitation in Dulbecco A saline at 4°C. The 8.2 x 10⁷ cells recovered (41%) were used for Fusion B. The plasmacytoma cell line P3/NS-1/1.Ag4.1

(NS-1) (Kohler et al, 1976) was mixed at a ratio of 1:1 with the spleen cells and fused after the method of Kennett et al 1978 in the presence of 50% polyethylene glycol 4000 (BDH) at pH 7.5-8.0 and 37°C. The cells were immediately resuspended in hypoxanthine-aminopterin-thymidine (HAT) medium and dispersed at 2.2 x 10⁵ cells/0.1 ml/well in 96-well Costar tissue culture plates containing mouse thymocytes at 3 x 10⁵/0.1 ml/well. Media were as described by Oi and Herzenburg, (1980).

Supernatants were assayed for antithrombin antibody within 2-3 weeks, and positive cultures were then cloned and subcloned by limiting dilution.

Antibody was produced by bulk culture of clones in 150 cm² T flasks, or as ascitic fluid following intraperitoneal injection of 1 x 10⁵ cells into mice injected 10 and 3 days previously with 0.5 ml Pristane. The immunoglobulin fraction was isolated using protein A-Sepharose (Pharmacia) as described by Ey et al, (1979), and the elution pattern used to define the isotype.

Hybridoma cells were stored in liquid nitrogen in 90% foetal calf serum/10% DMSO. Radioimmunoassay

Mouse sera and hybrid supernatant were screened using a liquid phase radioimmunoassay. Samples (50μl) were incubated with 50μl 1²⁵I-thrombin (10ng/ml) and 100μl of assay buffer for 16 hours at 20°C. The buffer used minimised the non-specific binding of labelled thrombin, which was measured in tubes containing only ¹²⁵I-thrombin and buffer. Thrombin-antibody complexes were separated from unbound thrombin using Sepharose 4B (Pharmacia) coupled sheep anti-mouse immunoglobulin antiserum (Scottish Antibody Production Unit) prepared as described by Hunter and Budd (1981); 50 μl of 1:1 dilution in assay buffer was added and the tubes were incubated with shaking for 45 minutes at 20°C. Sepharose-bound material was separated from that remaining in the liquid phase by sedimentation of the particles through assay buffer containing 10% sucrose and removal of the supernatant by aspiration (Hunter, 1977). Rabbit poiyclonal anti-thrombin antiserum was routinely used as a positive control throughout this work. Separation of these samples was achieved using donkey anti-rabbit immunoglobulin antiserum (Scottish Antibody Production Unit) coupled to Sepharose 4B.

This type of assay was also used for characterisation studies of the monoclonal antibodies produced by the hybrid cell lines for the determination of the maximum binding of antigen, antibody was used in excess where possible. Antibody titres were established by serial dilution; the titre was taken as that concentration of antibody which bound 50% of the difference between maximum binding and non-specific binding of the tracer. For the estimation of affinity constant, a concentration of antibody binding 50-80% of the maximum was added, and 50μl of serial dilutions of antigen replaced 50μl of buffer in the screening assay. Other variations in assay conditions are detailed in the Results section. Immobilisation of anti-thrombin antibodies

Ig solution (1 ml containing 0.2-0.1 mg/ml IgG) was attached to 150 mg CNBr-activated Sepharose 4B (Pharmacia) in accordance with the manufacturer's instructions. Antibodies inactivated by this procedure were attached to a solid phase by linking to protein A-Sepharose 4B (2 mg protein A/ml gel) in 0.05 M phosphate buffer pH 8.5 containing 0.5 M NaCl. After mixing overnight at room temperature the solid phase was washed twice and then incubated with an equal volume of 1% paraformaldehyde for 1 hour at 37°C (MacSween and Eastwood, 1978). Excess paraformaldehyde was removed by washing overnight. Results

Production of anti-thrombin monoclonal antibodies

Although an immunisation schedule was followed which usually induces antibody production in mice, 50 mice received human α-thrombin, and only one responded with a serum antibody titre of >1/10,000; this mouse was selected for fusion. Two others developed lower serum antibody titres, but there was no detectable response by the majority of animals. Fusion A yielded 67 hybrids from 180 seeded wells, but only one produced anti-thrombin antibody. The clone derived from this hybrid is termed EST 8. Of 300 wells seeded after fusion B, 232 produced hybrids and 7 of these secreted anti-thrombin antibody. The resulting clones are

EST 1-7. Clone EST 1 is of particular relevance to the present invention.

Early attempts at further characterisation of the antibodies were frustrated by the existence in both culture medium and mouse ascitic fluid of potent inhibitors of human α-thrombin, probably mouse and calf antithrombin. The antibodies were therefore produced in bulk and purified on protein A-Sepharose. At the same time their isotypes were determined.

Figure 1 shows the results of the radioimmunoassay described above applied to supernatant from clone EST 1 using ¹²⁵I labelled thrombin-antithrombin complex. The radioimmunoassay shows the marked temperature dependence of the monoclonal antibody produced by the clone. Monoclonal antibody was shown to exhibit minimal binding to α-thrombin (selection of the clone was due to an unforeseen binding of human ¹²⁵I-thrombin tracer with bovine anti-thrombin). Experiments not described in detail indicate that monoclonal antibody from EST 1 is directed against a neoantigen on the thrombin-antithrombin complex.

The monoclonal antibody produced by hybrid cell-line EST 1 was immobilised upon a solid phase to produce an affinity matrix as described above, for use in immunopurification. EXAMPLE 2

In a further experiment cell lines producing monoclonal antibodies to human Factor VIII (FVIIIR;Ag) related antigen were prepared. FVIIIR:Ag

Preparations of VIIIR:Ag were made utilising cryoprecipitate or plasma as starting material. A typical preparation is described below:

Six donations of blood were taken from routine collection sessions and the plasma separated by centrifugation for 90,000 g min. at 4°C. The plasma was pooled in polycarbonate bottles and precipitated with 4% Ficoll 70 at 0°C for 2 hours. The resulting precipitate was harvested by centrifugation for 30,000 g min. at 0°C, dissolved in 35 ml of 15 mM citrate, 150 mM NaCl pH 6.9. The dissolved precipitate was applied to a column (2.5 x 65 cms) of Sepharose CL-4B (Pharmacia) equilibrated with citrate-saline-3 mM sodium azide. Fractions were screened for VIII:C by coagulation assay, for VIIIR:Ag, fibrinogen and fibronectin by Laurell assay and for total protein by absorbance at 280 nm. Fractions containing VIIIR:Ag without detectable fibrinogen were pooled. The pool was concentrated by dialysis against 50% PEG 6000 in citrate-saline, and the concentrate was stored frozen at -40°C. Prior to immunisation, samples were dialysed against 150mM NaCl, 50 mM phosphate pH 7.5 without any azide. SDS-polyacrylamide gel electrophoresis in the presence of reducing agant confirmed that the VIIIR:Ag had a sub-unit molecular weight of 220,000. Radiolabelling

FVIIIR:Ag for use as tracer in the radioimmunoassay was labelled with ¹²⁵I using iodogen reagent (Salacinski, P.R.R., et al, 1981). Production of hybridomas

Hybridomas were produced by the methodology described in Example 1. Five mice were immunised with an initial intraperitoneal injection containing 50 μg FVIIIR:Ag and subsequently boosted with 25 μg doses. Radioimmunoassay

Mouse sera and hybrid supernatants were screened using the following liquid phase radioimmunoassay.

Samples (50μl) were incubated with 1²⁵I-labelled tracer (500 pg/tube) in a final volume of 200μl for 16 h at room temperature. The assay diluent was 0.05 M phosphate buffer, pH 7.4 containing 2% horse serum (Wellcome Reagents Ltd.) and 1% Tween 20 (BDH Chemicals). Tubes containing only ¹²⁵I-FVIIIR:Ag and buffer gave the level of non-specific binding, which was 5-7%. FVIIIR:Ag bound to antibody was separated from free protein by shaking for 45 minutes at 20° with a second antibody immobilised on Sephacryl S-1000 (Wright and Hunter, 1982; see below) and sedimentation through assay buffer containing 10% sucrose as described in Example 1. The working range of this assay was 1 100 ng/ml of sample.

Screening for temperature dependence

Temperature-dependent clones are easily selected by incubation in the radioimmunoassays described at the two practicable temperature extremes, probably 4°C and 37°C. The ratio of tracer bound at these two temperatures reflects the degree of temperature dependence of the antigen-antibody reaction. Results

All 5 mice responded to this antigen with high serum antibody titres. 537 hybrids were derived from the spleen of one of these mice and all bound ¹²⁵I-FVIIIR:Ag. 36 were initially selected for further study and 19 of these were cloned. Figure 2 shows the effect of temperature on the affinity of monoclonal antibodies produced by cell lines 14.6, 10.1, 12.1, 7.6 and 8.6. The monoclonal antibody produced by cell line 10.1, in particular, shows a marked temperature-dependent affinity. Immobilisation of anti-FVIIIR:Ag antibodies

The high molecular weight of FVIIIR:Ag multimer necessitates the use of a solid phase support with a large pore size for immobilisation of anti-FVIIIR:Ag antibodies. The effect of temperature on elution of antigen was then tested by treating with lithium diiodosalicylate (LIS) at 20°C and 0°C. Method i. Preparation of Matrices

A series of five anti-FVIIIR:Ag monoclonal antibodies was coupled to Sephacryl SI000 using the method of Cohn and Wilcheck. A polyclonal sheep antibody was used as a control, ii. Immunoaffinity Chromatography

The immobilised antibodies (50μl) were incubated for 30 mins. with cryoprecipitate (0.5ml) containing I¹²⁵ FVIIIR:Ag tracer. The gel was then recovered by sedimentation and washed before treatment with the eluting agent, 0.25M LIS at 20°C and 0°C. Results

Percentage of I¹²⁵ FVIIIR:Ag eluted at 20°C and 0°C with 0.25M LIS

Antibody Number

Elution 7.6 8.6 10.1 12.1 14.6 Sheep

Temp. °C Polyclonal Ab

0°C 69 60 60 42 81 18;

20°C 79 58 37 31 77 18

For two of the monoclonal antibodies, 10.1 and 12.1 there was a significant increase in the recovery of I¹²⁵ FVIIIR:Ag with 0.25M LIS as the temperature is reduced. There was no such effect for monoclonal antibodies 7.6, 8.6, 14.6 or the sheep polyclonal. It will of course be understood that the present invention has been described above purely by way of example, and modifications of detail can be made within the scope and spirit of the invention.

References

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Claims

Claims :

1. An immunopurification process for separating an antigen from a sample containing the antigen, the process involving an affinity matrix comprising antibody to the antigen attached to a solid phase, wherein the antibody has a positive temperature-dependent affinity such that the affinity of the antibody is substantially higher at a first relatively higher temperature than at a second relatively lower temperature, the process comprising the steps of: contacting the sample with the affinity matrix at the first temperature, thereby causing antigen in the sample to become associated with the affinity matrix, removing unbound species from the affinity matrix, and dissociating the antigen from the affinity matrix at the second temperature, to obtain the antigen.

2. An antibody having a positive temperature-dependent affinity such that the affinity of the antibody is substantially higher at a first relatively higher temperature than at a second relatively lower temperature.

3. An antibody according to claim 2 for use in an immunopurification process as defined in claim 1.

4. A process for preparing an antibody having a positive temperature-dependent affinity comprising screening a library of antibodies for an antibody having an affinity which is substantially higher at a first relatively higher temperature than at a second relatively lower temperature.

5. An antibody produced by the process of claim 4.

6. An affinity matrix comprising an antibody according to claim 2 attached to a solid phase.

7. An immunopurification process according to claim 1 wherein the antigen is blood factor VIII.

8. An antibody according to claim 2 wherein the antibody has specificity for blood factor VIII.

9. An affinity matrix comprising an antibody according to claim 8 attached to a solid phase.

10. An immunopurification process according to claim 1 wherein the antigen is thrombin-antithrombin.

11. An antibody according to claim 2 wherein the antibody has specificity for thrombin-antithrombin.

12. An affinity matrix comprising an antibody according to claim 11 attached to a solid phase.