WO1990009237A1

WO1990009237A1 - Supports for immunoaffinity separations

Info

Publication number: WO1990009237A1
Application number: PCT/GB1990/000192
Authority: WO
Inventors: Frank Mitchell Roberts; Kamran Beyzavi
Original assignee: Bioprocessing Limited
Priority date: 1989-02-08
Filing date: 1990-02-08
Publication date: 1990-08-23
Also published as: GB8902770D0

Abstract

An adsorbent for use in immunoaffinity separations includes a particulate porous support having a pore size of not less than 500 Å and a surface area of more than 20 m2/g of dry support to which at least 4 mg/ml of the ligand Protein A is bound.

Description

SUPPORTS FOR IMMUNOAFFINITY SEPARATIONS

This invention relates to a method for the separation of a substance from a mixture by the

technique of affinity chromatography.

It is well known to use affinity

chromatography for the separation of, for example, biochemical substances from a mixture. Affinity chromatography relies on non-covalent reversible ligand-ligate interactions between an adsorbent

comprising a ligand which is bound to an insoluble porous support and a ligate which is the substance to be separated.

In general, separation by affinity

chromatography involves four stages

(1) Adsorption:- The mixture containing the substance to be separated is passed over the adsorbent (i.e. the solid support to which the ligand is

attached) in conditions such that the ligate binds to the ligand.

(2) Washing:- Contaminating molecules not bound to the adsorbent are washed away, using

conditions such that the ligand-ligate complex is not dissociated. Thus the substance to be separated remains bound.

(3) Elution:- The ligand-ligate complex is caused to dissociate, by treatment with an appropriate solution, so releasing the substance to be separated.

(4) Regeneration:- The adsorbent on the solid support is returned to its original state for the next adsorption cycle, by treatment with appropriate reagents.

In order to obtain highly pure product, the adsorbent must meet certain requirements:- (i) the support must be sufficiently durable to withstand a number of separation cycles. withstand a number of separation cycles,

(ii) the support must be such that the ligand can be securely bonded thereto, e.g. covalently, so that it is not removed by repeated separation cycles, and such that its ligate-binding properties are unaffected.

(iii) the support must be porous, to give a large surface area at which ligand-ligate binding can occur. Pore size is inversely related to surface area, but the ligand-ligate interaction can be hindered if the pores are too small. Pore size also affects intra-particle diffusion rates.

(iv) the ligand-ligate interaction must be highly specific, and non-specific binding of contaminating molecules to the support, which may be released at the elution stage, must be minimised.

The most commonly used support media are (on a laboratory scale) polymers of carbohydrates such as beaded agarose. However, whilst such materials exhibit low non-specific binding, they are soft and

compressible, and so are of limited use on a large scale because sufficiently high flow rates of eluent and washing solutions cannot be achieved.

Less commonly used support media are inorganic materials which are rigid and incompresible. Higher flow rates can be achieved with these materials.

Suitable inorganic support media include siliceous materials and non-sileceous metal oxides.

Representative siliceous support media include

particulate porous glass, colloidal silica,

wollastonite, dried silica gel and bentonite.

Representative non-siliceous metal oxides include aluminia, hydroxy apatite and nickel oxide. Porous glass is a particularly preferred support medium because of its uniform pore size - i.e. the pore size distribution falls within a narrow range (90% of all pores have a diameter ±10% from the mean) and also because of its low attrition when used in a stirred tank or fluidised bed.

It is known to use Protein A as the ligand on various organic and inorganic supports, for the

separation of immunoglobulins. However, this is not entirely satisfactory because although immunoglobulins have two binding sites for the ligand, it is believed that on such supports binding occurs at one only of these sites, hence ligand-ligate interaction is

insufficiently strong and the ligate is not adequately attached to the support and therefore is not retained on the support at the washing stage.

It has now surprisingly been found that this disadvantage can be overcome by using a support having a specific pore size and a specific coverage of Protein A.

Accordingly the present invention provides an adsorbent comprising a particulate porous support having a uniform pore size which is not less than 500A and a surface area which is greater than 20m²/g of dry support and a ligand comprising Protein A in an amount of at least 4mg/ml of the particulate support.

Since the support is particulate, it, is convenient to define the concentration of the Protein A on the support on the basis of the volume of the support.

The use of a pore size of 1100Å is

particularly preferred.

It is believed that the provision of pores having the specified size results in the pores having an internal surface, the curvature of which ensures that the Protein A bound thereto adopts a spacial relationship appropriate to the binding sites of the ligate. Glass supports having the desired pore size can be produced, for example, by the procedures set out in US Patent No.3 549 524.

As a first stage in the preparation of the adsorbent from a porous glass support, the surface silanol groups of the porous glass may be reacted with an organo-silane compound of the general formula

R' - Si - (R)3

where commonly R represents an alkoxy group, and preferably is methoxy or ethoxy, and R' represents a functional group (such as an amino group, an aldehyde group or a carboxylic acid group). The organo-silane is covalently bonded to the surface of the porous glass via one or more alkoxy groups.

In the second stage, the functional group of the organo silanes is employed to bond covalently to the ligand, so securing the ligand to the porous glass support. Where the functional group is an aldehyde group, this can be used to bond directly to ligands such as proteins.

In the case where the ligand is smaller than the ligate, it is desirable that the ligand be spaced from the surface of the support. Thus R' may include a spacer arm comprising a linear chain of carbon atoms between the functional group and the silicon atom of the silane. Thus a suitable example of a precursor for R' is glycidoxypropyltrimethoxysilane,

wherein the epoxy group may be converted to an aldehyde group which acts as the ligand. Alternatively the functional group of the organo-silane may, in the second stage, be reacted with a suitable first

functional group of a bi-functional compound. Thus, in the case where the functional group of the organosilane is an amino group, the first functional group of the bi-functional compound may be an aldehyde group. Similarly, where the functional group of the

organosilane is an aldehyde or carboxylic acid group, the first functional group of the bifunctional compound may be an amino group. The second functional group of the bifunctional compound is then employed to bond covalently to the ligand, so securing the ligand to the porous glass support. Thus, in the case where the ligand is a protein, the second functional group of the bifunctional compound may be an aldehyde group.

Suitable examples of bifunctional compounds are

dialdehydes and diamines.

The adsorbent of the present invention is particularly suitable for use with mouse IgG1. However it has utility with other antibodies and sub-types.

It may be desirable that there is provided an inorganic support for use in combination with a ligand in affinity chromatography, which support comprises a plurality of sites wherein (i) moieties including functional groups capable of forming covalent bonds with molecules of the ligand are attached to some of said sites and (ii) moieties consisting of nonfunctional, non-polar groups are attached to the remainder of said sites.

The following Examples illustrate the invention.

Example 1

A sample of porous glass (1100Å pore size, 100 um particle size) was incubated in 5% (v/v) nitric acid at 80°C for 2 hours. The glass was washed several times with distilled water on a glass sintered funnel. After removing all the water by using the suction pump, the glass was transferred to a glass beaker and placed in an oven at 170°C overnight.

The glass was mixed with dimethyl sulphoxide containing 5% (v/v) 3-glycidoxypropyltrimethoxysilane and 0.5% (v/v) triethylamine in a round bottom flask and placed in a water-bath at 90°C for 24 hours. The glass was then washed exhaustively on a glass sintered funnel with dimethyl sulphoxide, 1-propanol, water and 0.5M sulphuric acid.

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The glass was transfered in to a round bottom flask and mixed with 0.5M sulphuric acid. The flask was placed in a water-bath at 90°C for 4 hours. The glass was cooled down and washed several times with distilled water on a sintered glass funnel.

The glass was transferred into a screw top bottle and mixed for 2 hours with 1 mM acetate buffer at pH 4 containing 0.24M sodium periodate. The glass was washed several times with water followed by acetone on a sintered glass funnel and air-dried.

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Eighty grams of the activated glass was added to 400 ml of 0.1M bicarbonate/0.15M sodium chloride solution, pH 8.0 containing 1600mg protein A in a screw-top bottle and mixed for 6 hours using a rollermixer. 0.2M glycine and 0.1M sodium cyanoborohydride as solid were added to the glass - protein A mixture and the mixing continued overnight. The glass was separated from the supernatant on a sintered glass tunnel and was washed several times with 0.1M

bicarbonate/0.15M sodium chloride solution, pH 8.0.

Total protein A immobilised to the glass was estimated by measuring the optical density of the supernatant and washes at 257nm. The glass was incubated in 0.1M bicarbonate/0.15M sodium chloride, pH 8.0 containing 0.1M sodium borohydride for 2 hours at 4°C. The glass was then exhaustively washed with 0.1M

bicarbonate/0.15M sodium chloride solution, pH 8.0 containing 1% (w/v) polyethylene glycol (20,000), 0.1M glycine/HCl at pH 2 and phosphate buffered saline (PBS) and stored in PBS containing 0.02% (w/v) sodium azide.

2.7ml of the immobilised protein A was packed into a column (8mm diameter, 60mm height). 2ml of PBS containing 100mg human IgG was applied to the column under gravity. The column was washed with 10ml of PBS and the bound human IgG was eluted with 2 x 5ml of 0.1M glycine/HCl at pH 2.0. The human IgG eluted was estimated by optical density measurements at, 280nm.

5mL of cell culture supernatant containingabout 10mg/ml of monoclonal mouse IgG1 was dialysed against 2 litres of 0.1M borate/0.15M sodium chloride buffer, pH 8.5 overnight. The supernatant was applied to the column and the column was washed with 20ml of the borate/NaCl buffer. The bound antibody was eluted with 0.1M citrate buffer at pH 3.0. The amount of antibody eluted was estimated by optical density measurements at 280nm. The purity of the antibody was determined by gradient 10-15 gel electrophoresis.

Results are shown in Table 1.

Table 1: Capacity of protein A-porous glass (CPG) for human and mouse immunoglobulins

Immobilised Human IgG Mouse IgG1 Purity of Protein A Eluted Eluted Mouse IgG1 (mg/ml CPG) (mg/ml CPG) (mg/ml CPG)

5.8 47.4 13.4 98

Example 2

670mg protein A dissolved in 400ml of 0.1M bicarbonate/0.15M sodium chloride solution, pH 8 was reacted with 80grams of activated glass. The

activation of the glass and immobilisation of protein A was as in Example 1.

The binding capacity of the resultant protein A - porous glass for human IgG and mouse IgG1 was determined as in Example 1 using a similar size column. Results are shown in Table 2.

Table 2: Capacity of protein A - porous glass for human and mouse IgG1

Immobilised Human IgG Mouse IgG1 Purity of Protein A Eluted Eluted Mouse IgG1 (mg/ml CPG ) (mg/ml CPG ) ( mg/ml CPG) %

2.4 21.3 1.85 98

Example 3

21.5mg protein A dissolved in 10ml of 0.1M bicarbonate/0.15M sodium chloride solution, pH 8 was reacted with 1 gram of activated glass (715A porous size, 100-130um particle size). The activation of the glass and immobilisation of protein A was as in Example The binding capacity of the resultant protein A - porous glass for human IgG and mouse IgGl was determined as in Example 1 using a similar size column. Results are shown in Table 3.

Table 3: Capacity of protein A - porous glass for

human and mouse IgG1

Immobilised Human IgG Mouse IgG1 Purity of Protein A Eluted Eluted Mouse IgG1 (mg/ml CPG) (mg/ml CPG) (mg/ml CPG) % 5.41 31.8 4.2 98

Claims

1. An adsorbent comprising a particulate porous support hoaving a uniform pore size which is greater than 500Å and a surface area which is greater than 20m²/g of dry support, and a ligand comprising protein A in an amount of at least 4mg/ml of the particulate support.

2. An adsorbent according to claim 1 where the pore size is 1100Å.

3. An adsorbent according to claim 1 or 2 wherein the support comprises a glass, silica, alumina or ceramic material.

4. An adsorbent according to claim 1, 2 or 3 wherein the ligand is bound to the support by means of a moiety derived from an organosilane compound.

5. An adsorbent according to any preceding claim wherein the organosilane compound has the general formula

where R represents a C₁ to C₃ alkoxy group and R¹ includes a functional group.

6. An adsorbent according to claim 5 wherein R¹ includes an aldehyde group or a precursor therefor.

7. An adsorbent according to claim 5 or 6 wherein R¹ includes a linear chain of carbon atoms between the functional group and the silicon atom.

8. An adsorbent according to any of claims 4 to 7 wherein the organo-silane compound is

glycidoxypropyltrimethoxysilane.

9. An adsorbent according to any of claims 4 to 6 wherein the moiety and the ligand are spaced from the support by means of a further moiety derived from a compound including two functional groups separated by a linear chain of carbon atoms.

10. An adsorbent according to claim 9 wherein said compound is a dialdehyde or diamine.