Title:  Method for the separation and purification of immunoglobulins

United States Patent:  6,207,807

Assignee:  Tecnogen S.C.p.A, (Piana Di Monte Verna, IT)

Filed:  December 3, 1998

Foreign Application Priority Data:   Jun 21, 1995[IT] (MI95A1328); Apr 29, 1996[IT] (MI96A0831)

A peptide of formula (I) (H₂ N--X₁ --Thr--X₂ --CO)_n --R (I) where X₁ and X₂, different one another, are an amino acid residue of arginine or tyrosine in configuration L or D, wherein the hydroxy group of threonine and tyrosine and the guanidine moiety of arginine may be protected by a compound conventionally used in peptide chemistry for protecting the hydroxy group and the guanidine moiety, respectively, n is 1,2, 3 or 4, and R, when n is 2,3 or 4, is a group suitable for forming a dimer, trimer or tetramer, while, when n is 1, R is OH, a single amino acid residue, or a peptide chain containing up to 7 amino acid residues.

SUMMARY OF THE INVENTION

Thr is an amino acid residue of threonine having configuration L or D, provided, however, that X₁ is arginine when X₂ is tyrosine, and X₁ is tyrosine when X₂ is arginine.

Preferably, at least one amino acid residue of the sequence (S) has D configuration.

Even more preferably, two or all the three amino acid residues of the sequence (S) have D configuration.

It is therefore a first object of this invention to provide a peptide of formula (I)

(H₂ N--X₁ --Thr--X₂ --CO)_n --R (I)

where

X₁ and X₂, different one another, are an amino acid residue of arginine or tyrosine in configuration L or D, wherein the hydroxy group of threonine and tyrosine and the guanidine moiety of arginine may be protected by a compound conventionally used in peptide chemistry for protecting the hydroxy group and the guanidine moiety, respectively, n is 1,2, 3 or 4, and

R, when n is 2,3 or 4, is a group suitable for forming a dimer, trimer or tetramer, while, when n is 1, R is OH, a single amino acid residue, or a peptide chain comprising up to 7 amino acid residues.

As used herein the terms "dimer", "trimer" and "tetramer" are intended to mean a peptide comprising 2, 3, or 4 sequences (S).

A typical example of a group suitable for forming a dimer (n=2) is a lysine residue. A typical example of a group suitable for forming a trimer (n=3) is a dipeptide lysil-lysine of formula Lys-Lys. A typical example of a group suitable for forming a tetramer (n=4) is a branched tripeptide of formula Lys-Lys(.epsilon.-Lys).

A typical example of a tetramer of formula (I) has the following formula

(H₂ N--X₁ --Thr--X₂ --CO)₄ --Lys₂ --Lys--Gly--OH (IA)

where

X₁ and X₂ have the above mentioned meanings and wherein the hydroxy group of threonine and tyrosine and the guanidine moiety of arginine may be protected by a compound conventionally used in peptide chemistry for protecting the hydroxy group and the guanidine moiety, respectively.

Many protecting groups for protecting the hydroxy group in peptide synthesis are reported in the literature (G. A. Grant, Synthetic peptides: a user's guide, Freeman, N.Y., 1992).

Typical examples of said protecting groups are: ter-butyl (tBu) (La Joie, G. Crivici, A., Adamson, J. G. "Synthesis" 571-572 (1990)) and the benzyl group (Yojima "Tetrahedron" 44:805-819 (1988)).

Many groups useful for protecting the guanidine moiety of arginine are also known from the literature (Grant, G. A. Synthetic peptides: A user's guide, Freeman, N.Y., 1992).

Typical examples of said protecting groups are: 2,2,5,7,8-pentamethylcroman-6-sulphonyl (Pmc) and 4-methoxy-2,3,6-trimethylbenzene (Mtr) (Ramage & Green, "Tetrahedron Letters, 28,2287 (1987); Fujino et al." Chem. Pharm. Bull., 29,2825 (1981).

Typical examples of thus protected compounds of formula (I) are the compounds Boc-D-Arg(Pbf)-D-Thr(tBu)-D-Tyr(tBu)-OMe of Example 1(d), and (H₂ N-Arg(Pmc)-Thr(OtBu)-Tyr(OtBu)-CO)₄ -Lys₂ -Lys-Gly-OH.

When n is 1 and R is a peptide comprising from one to seven amino acid residues, all the amino acids comprised in the sequence may be different or equal to each other and have L or D configuration. The D configuration is the preferred one. Furthermore, simple and cheap amino acids will be preferred.

Specific examples of R for n equal to 1 are, Gly or Ala, Gly-Gly, Gly-Ala, Ala-Gly, Ala-Ala, Gly-Gly-Gly, Ala-Ala-Ala, Gly-Gly-Gly-Gly (SEQ ID NO: 1), Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 2), Gly-Ala-Gly-Ala-Gly (SEQ ID NO: 3), Ala-Gly-Ala-Gly-Ala-Gly-Ala (SEQ ID NO: 4).

The peptides of formula (I) may be readily prepared according to both the conventional liquid phase peptide preparation and solid-phase peptide preparation techniques.

The preparation according to the solid-phase technique is preferably carried out by means of an automatic synthesizer. A typical example of a suitable automatic synthesizer is the model 431 A from Applied Biosystems (Foster City, Calif., USA). Preferably, the preparation is performed according to the synthesis procedures recommended by the manufacturer, said procedures being usually based on known methods well described in the literature (Atherton & Sheppard, 1989, Solid Phase Peptide Synthesis: A practical approach, IRL Press, Oxford).

It is a third object of this invention to provide the use of a compound of formula (I) to form complexes with at least one immunoglobulin in a separation process of said immunoglobulin or mixture of immunoglobulins.

Examples of immunoglobulins capable of forming complexes owing to non covalent binding to compounds of formula (I), are: mouse IgG, rat IgG, chicken IgY, goat IgG, bovine IgG, human IgG, human IgA, and of other species, human IgM and of other species.

A typical example of a method for the separation and purification of an immunoglobulin comprises:

(i) immobilizing on an affinity chromatography support a compound capable of binding non covalently itself to at least one immunoglobulin,

(ii) packing said affinity chromatography support in a chromatographic column,

(iii) equilibrating said column with a buffer capable of promoting an interaction between immunoglobulin and the immobilized compound,

(iv) loading said column with a fluid comprising at least one immunoglobulin,

(v) washing said column with at least one liquid capable of eluting the impurities without interfering with the interaction between immunoglobulin and the immobilized compound,

(vi) eluting said immunoglobulin previously adsorbed on the column with a dissociating eluent,

and is characterized in that:

the compound capable of binding itself non covalently to at least one immunoglobulin is a compound of formula (I), where X₁, X₂, n and R have the meanings shown above.

Steps from (i) to (vi) are carried out according to conventional techniques.

Preferably, the support for affinity chromatography is preactivated with epoxyde groups for direct coupling to peptides and proteins. Typical examples of suitable supports are the resin activated-CH SEPHAROSE.TM. 4B (N-hydroxysuccinimide containing agarose) from Pharmacia (Sweden), the resin PROTEIN PAK.TM. epoxy-activated affinity resin) (Waters, USA) the resin EUPERGIT.TM. C30 N (Rohm & Haas, Germany), or AFFI-GEL.TM. from BioRad (USA).

Step (i) is preferably carried out in the presence of a weakly basic buffer solution having a pH value of from 8.5 to 9.0.

Step (iii) is preferably performed with a neutral buffer such as, for example, a 25 mM Bis-Tris solution having pH 6.5, or a 50 mM phosphate buffer solution having pH 7.0.

Step (v) is preferably carried out by using a neutral buffer having a low ionic strength such as, for example, a 25 mM Bis-Tris solution having pH 6.5.

Examples of dissociating eluents useful in step (vi) comprise acid or basic aqueous solutions. Typical examples comprise aqueous solutions of acetic acid at pH 2.5 or of sodium bicarbonate at pH 9.0.

This separation and purification technique is widely described in the literature [Narayanan, S. R., "Preparative affinity chromatography of proteins" J. Chromatogr., 658:237-258 (1994), as well as references quoted therein; Lowe, C. R., "Laboratory technique in Biochemistry and Molecular Biology", Work and Burdon, vol. 7, part 2, Elsevier, N. Holland, Amsterdam; Ey et al. Immunochemistry, 15:429 (1978)].

The compounds of this invention may also be used in the qualitative or quantitative determination of immunoglobulins according to the well known ELISA technique.

A typical example of a method for quantitative determination of an immunoglobulin or a mixture of immunoglobulins according to the ELISA technique comprises:

(1) immobilizing a compound capable of binding itself non covalently to at least one immunoglobulin on a microtiter plate for ELISA determination,

(2) incubating a sample containing the immunoglobulin or the immunoglobulins to be determined on said microtiter plate,

(3) washing said microtiter plate,

(4) detecting the thus formed immobilized complex compound/ immunoglobulin,

and is characterized in that

the compound capable of binding itself non covalently to at least one immunoglobulin is a compound of formula (I) where X₁, X₂, n and R have the above mentioned meanings.

The analytical determination of immunoglobulins according to the ELISA technique is widely described in the literature ("Immunochemistry in practice", Johnstone & Thorpe, (1987), Blackwell, Oxford, UK).

Preferably, step 1 is carried out using a plastic microtiter plate such as, for example, of PVC, with 96 well filled with 0.1 M sodium bicarbonate solutions having pH 9.0 and containing variable amounts of a ligand (0-50 .mu.g/well). After 24 h incubation, excess solution is removed, and the microtiter plates are washed with phosphate buffer and the wells are filled with a 3% bovine albumin solution to eliminate a specific interaction sites.

In step 2, microtiter plates are washed with phosphate buffer and the wells are filled with solutions containing an immunoglobulin, preferably derivatized with biotin. Microtiter plates are then incubated for 4-18 h at 20-37^oC.

Washing in step 3 is preferably carried out with phosphate buffer.

Step 4 is performed by adding to each well a solution of avidin conjugated to peroxidase. After 2 h incubation, microtiter plates are washed, preferably again with a phosphate buffer. Then a solution of o-phenylenediamine is added and color formation is detected with a suitable ELISA reader.

Claim 1 of 16 Claims

What is claimed is:

1. A method for the separation and purification of immunoglobulins which comprises:

(i) immobilizing on an affinity chromatography support a compound which non covalently binds to at least one immunoglobulin,

(ii) packing said affinity chromatography support in a chromatographic column,

(iii) equilibrating said column with a buffer capable of promoting an interaction between immunoglobulin and the immobilized compound,

(iv) loading said column with a fluid comprising at least one immunoglobulin,

(v) washing said column with at least one liquid capable of eluting the impurities without interfering with the interaction between immunoglobulin and the immobilized compound,

(vi) eluting said immunoglobulin previously adsorbed on the column with a dissociating eluent,

wherein

said compound which non covalently binds to at least one immunoglobulin is a compound of formula (I)

(H₂ N--X₁ --Thr--X₂ --CO)_n --R (1)

where

X₁ and X₂, different from one another, are an amino acid residue of arginine or tyrosine in configuration L or D, wherein the hydroxy group of threonine and tyrosine and the guanidine moiety of arginine is optionally protected by a compound conventionally used in peptide chemistry for protecting the hydroxy group and the guanidine moiety, respectively, n is 1,2, 3 or 4, and

R, when n is 2,3 or 4, is a group suitable for forming a dimer, trimer or tetramer, and when n is 1,

R is a single amino acid residue, or a peptide chain comprising 2 to 7 amino acid residues.

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