WO1995000174A1

WO1995000174A1 - Storage of antibodies

Info

Publication number: WO1995000174A1
Application number: PCT/GB1994/001348
Authority: WO
Inventors: Dhanraj Samuel
Original assignee: Health Protection Agency; Public Health Laboratory Service Board
Current assignee: Public Health Laboratory Service Board; Public Health England
Priority date: 1993-06-25
Filing date: 1994-06-22
Publication date: 1995-01-05
Anticipated expiration: 1995-12-25
Also published as: AU6977894A; GB9313163D0

Abstract

A method of storing an antibody that binds to a target ligand with high affinity, by protecting the antibody during storage in a solution of a protective ligand to which the antibody binds with low affinity, the solution being free of the target ligand.

Description

STORAGE OF ANTIBODIES

This invention relates to methods of storing antibodies, including methods for freezing antibodies and methods of freeze-drying antibodies for storage, and methods of recovering antibody solutions from storage.

Antibodies, whether polyclonal or monoclonal, are used in a wide range of experimental and clinical applications, forming the basis of highly specific purification procedures and sensitive assays e.g. radioimmunoassays and having many therapeutic uses. The usefulness of antibodies is based on their ability to bind particular molecules (ligands) with high affinity, each antibody typically having a high binding strength for one specific ligand or a limited number of specific ligands.

It is a known problem with antibodies, typically used in aqueous solution, that the binding activity of antibody solutions decreases over time markedly at room temperatures and above. The decrease may be due to partial decomposition of the antibody, to hydrolysis of those parts responsible for ligand binding or degradation of the antibody by proteolytic enzymes.

One consequence of the loss in activity is that where antibodies in solution have to be kept at around room temperature, the antibody activity cannot be assured for ore than a short time. This means, for example, that diagnostic kits employing antibodies have short shelf lives. Also, once prepared, antibody solutions must be used whilst fresh, before the activity falls below a useful level.

It is known to store antibodies at reduced temperature to prolong their life. Even at reduced temperatures antibody activity may decrease with time, only more slowly than at room temperature. Antibody solutions can have glycerol added for storage down to about -20°C, or they can be frozen and stored around -20^βC or freeze-dried. In these latter two cases the antibody solution needs to be reconstituted before it is ready for use. A disadvantage of storing antibodies thus is that the antibodies are not available for use on demand but have to be recovered from storage first. Once at room temperature or higher the antibody solution loses its activity as described above.

It is an object of this invention to provide a method of storage of antibodies that prolongs their useful life at all temperatures, though particularly in solution at room temperature and above. It is also an object of this invention to provide improved methods of freezing antibodies, freeze-drying antibodies and reconstituting antibody from frozen and freeze-dried antibody solutions.

In a first aspect of the invention a method of protecting in storage an antibody having high affinity for one ligand comprises storing the antibody with a low affinity ligand. In an embodiment of the invention an antibody with high affinity for a first ligand is stored in a solution of a second ligand for which the antibody has lower affinity, and the solution is substantially free of the first ligand.

In a preferred embodiment, the antibody has affinity for the first ligand at least 10 times greater than for the second ligand, more preferably at least 100 times and very preferably at least 1000 times.

In the method of the invention, an antibody that binds with relatively high affinity to a target molecule such as a ligand is protected from deterioation (decrease in its specific binding activity) during storage by the use of a reagent that binds less well to the antibody that the target molecule binds to the antibody i.e. the target molecule exhibits relatively greater strength of binding for the antibody than does the protective reagent. Following storage of the antibody, the protective reagent is displaced in use by the target molecule that binds the antibody preferentially.

The method of the invention has the advantage that antibodies stored according thereto surprisingly lose their binding activity more slowly than antibodies stored using conventional methods.

In an embodiment of the first aspect of the invention, the antibody has

7 14 affinity for the first ligand of between 10 and 10 litres per o mole (1/mol) , preferably greater than 10 1/mol. In a further embodiment of the first aspect, the antibody has affinity for the second ligand of between 10 and 10 1/mol, preferably less than 10' 1/mol.

In a particular embodiment of the invention either (a) the antibody is dissolved in an aqueous solution of the second ligand or (b) the second ligand is dissolved in an aqueous solution of the antibody, and (c) the solution is then stored, preferably at 3°C-25⁰C.

The concentration of the second ligand is preferably chosen so that there is an excess of second ligand for all the available binding activity of the stored antibody, more preferably at least a 3~fold excess, most preferably at least a 10 fold excess.

In a second aspect the invention provides a method of freezing for storage a solution of an antibody having high affinity for a first ligand, comprising the steps:

(a) substantially removing from the solution the first ligand, if present,

(b) adding to the solution a second ligand to which the antibody binds with lower affinity than to the first ligand so as to protect the antibody as in the first aspect of the invention, and

(c) freezing the solution. In a third aspect the invention provides a method of freeze-drying an antibody solution comprising the steps of the second aspect and drying the frozen solution.

In a fourth aspect the invention provides a method of reconstituting a solution of an antibody having high affinity for a first ligand from a frozen or freeze-dried solution of the antibody, comprising the steps of

(a) dissolving the antibody in a solution of a second ligand to which the antibody binds with lower affinity than to the first ligand, or

(b) making a solution of the antibody and adding thereto the second ligand, the solution being substantially free of the first ligand. In a fifth aspect the invention provides a stored antibody, for example in a diagnostic, assay or other kit using antibodies, comprising an antibody having high affinity for a first ligand in association with a second ligand to which the antibody binds with lower affinity, and being substantially free of the first ligand.

The embodiments and preferred features of the second to fifth aspects of the invention correspond to those described in relation to the first aspect. The invention enables antibodies to be stored for long periods retaining more of their activity than previously possible. The invention can be used in diagnostic and assay kits comprising antibodies to give them a longer useful life and prolong their shelf life. The methods of the invention can be used in conjunction with conventional antibody storage methods and recovery from storage methods.

Most antibodies show their highest affinity for one ligand and lower affinity for a number of cross-reactive or partially cross-reactive ligands. In storage of any given antibody various potential protective storage ligands with lower relative affinity for the antibody may be available; often one of these will give particularly good results using the methods of the invention.

The invention is illustrated by the following examples:-

Example 1

Antibody - cross reacting ligand incubation

MATERIALS

Antibody: Anti-FITC monoclonal antibody - IgG from clone 2F3.1 Cross-reacting ligand: Sodium-fluorescein (Na-F)

Coating buffer: PBS-Azide

Microtitre wells: Nunc 12 x 8 well modules METHOD

Anti-FITC (29μg/ml) solutions were prepared in PBS-azide buffer. To each microtitre well was added 50μl of antibody and 50μl ligand (or buffer as control). The ligand for the anti-FITC antibody was fluorescein at 2μg/ml. The control buffer was PBS-azide. After two days incubation at 4°C, after which time the antibody binds to the microtitre wells, the wells were incubated at various temperatures (4 C, 20°C, 37°C or 56°C) for varying times (2h, 4h, 6h, 1 day, 4 days) before assessing antibody activity.

TESTING FOR ANTIBODY ACTIVITY

MATERIA1S

Antibody coated wells from above Anti-FITC with and without Na-F FITC-HRP conjugate

Diluent buffer - BSA-PBS-Tween-Bronidox Substrate (TMB)

METHOD

After the appropriate incubation time the antibody coated wells were removed and tested at room temperature. This involved washing the solution by aspiration of the contents and adding a washing buffer (PBS containing Tween 20) and aspirating three times. The antibody remaining immobilised onto the wells was assessed for activity as follows.

To the anti-FITC wells was added lOOμl FITC labelled enzyme (FITC-HRP) and incubated for one hour at 37"C. Active antibody binds this FITC-HRP conjugate. After washing as above, the enzyme content is monitored by adding substrate (Tetra ethylbenzidine, TMB) for between 5-20 minutes before stopping the reaction by adding 50μl of 2M H_Sθ4. The colour developed was recorded using a microtitre plate reader. The optical density value obtained is related to the amount of FITC-HRP conjugate bound, which in turn is related to the activity of the antibody on the plate.

RESULTS

The effect of temperature with time on anti-FITC antibody activity

The activity at 4°C at varying times without ligand was taken as 100# and activity of antibody at the varying temperatures with and without ligand were compared to this value.

In the absence of ligand anti-FITC retains activity at 20^βC for four days. However after four days at 37°C more than 3~% of the activity is lost. In comparison, in the presence of protecting ligand there is no loss in activity at 37°C after 4 days. At 56°C, 50# of activity is lost after one day and virtually all of the activity is lost after 4 days in the absence of protecting ligand. There was very little loss in anti-FITC activity in the presence of protecting ligand even after 4 days incubation at 56°C. At 4^βC there was an apparent loss of activity when protecting ligand was used. However, when the temperature of the solution was raised to room temperature virtually all antibody activity returned. CONCLUSION

There was significant protection of anti-FITC antibody activity in the presence of protecting Na-F ligand. This demonstrates that the principle of antibody protection and retention of antibody activity on storage with a cross-reacting ligand having lower affinity (in this case the affinity of the anti-FITC antibody is around 10 ^~-106 M-1 for

Na-F and around 10 -10⁹ M^-1 for FITC-HRP) .

Example 2

In a similar experiment to that described above, the storage of anti-biotin monoclonal antibody in the presence of varying concentrations of cross reacting ligand, namely biotin, was investigated. After incubation and washing as described above the anti-biotin wells were incubated with a biotin labelled HRP conjugate. The same standard assay steps were used as before. 5 6 -1 The affinity of the antibody for biotin was around 10 -10 M and

8 9 -1 for the biotin-HRP conjugate around 10 -10 M

RESULTS

[BIOTIN] % Antibody activity after 3 days at

4^βc 20^βC 37°C

1 mg/ml 100 104 99

100 μG/ml 100 95 95

10 μG/ml 100 97 86

1 μG/ml 100 94 82 no biotin 100 95 76

CONCLUSION

There was significant (p <0.01) protection of anti-biotin activity even with a level of cross reacting ligand as low as 1 μg/ml.

These Examples demonstrate that storage of an antibody that binds a ligand with high activity in the presence of a cross-reacting ligand that binds that antibody with lower activity increases the storage life of the antibody.

Claims

1. A method of protecting an antibody having high affinity for a first ligand against deterioration in storage comprising forming a solution of the antibody in the presence of a second ligand for which the antibody has lower affinity, the solution being substantially free of the first ligand.

2. A method according to Claim 1 in which the antibody has affinity for the first ligand at least 10 times greater than its affinity for the second ligand.

3. A method according to Claim 1 in which the antibody has affinity for the first ligand at least 100 times it affinity for the second ligand-.

4. A method according to Claim 1 in which the antibody has affinity for the first ligand at least 1000 times its affinity for the second ligand.

^~ . A method according to any of Claims 1-4 in which the

7 affinity of the antibody for the first ligand is between 10 and

10 141/mol, preferably greater than 1081/mol.

6. A method according to any previous claim in which the

3 affinity of the antibody for the second ligand is between 10^J and

10 7

10 1/mol preferably less than 10' 1/mol.

7. A method according to any previous claim comprising

(a) dissolving the antibody in an aqueous solution of the second ligand, or (b) dissolving the second ligand in an aqueous solution of the antibody, and

(c) maintaining the solution at a storage temperature, preferably between 3°C-25⁰C.

8. A method of freezing for storage a solution of an antibody having high affinity for a first ligand comprising the steps of:- a) substantially removing from the solution the first ligand, if present, b) protecting the antibody according to the method of any preceding claim, and c) freezing the solution.

9. A method of freeze-drying a solution of an antibody having high affinity for a first ligand comprising a method of freezing the antibody solution according to Claim 8 and subsequently drying the frozen solution.

10. A method of reconstituting a solution of an antibody having high affinity for a first ligand from a frozen or freeze-dried solution of the antibody, comprising:- a) dissolving the antibody in a solution of a second ligand to which the antibody binds with lower affinity than to the first ligand, or b) making a solution of the antibody and adding thereto the second ligand, the solution being substantially free of the first ligand. 11. A stored antibody, e.g. in a diagnostic, assay or other kit using antibodies, comprising an antibody having high affinity for a first ligand in association with a second ligand to which the antibody binds with lower affinity, and being substantially free of the first ligand.