WO2006011839A1

WO2006011839A1 - Chromatography method

Info

Publication number: WO2006011839A1
Application number: PCT/SE2005/001134
Authority: WO
Inventors: James Van Alstine; Jan Bergström; Dag LINDSTRÖM; Joachim Stadler; Tobias SÖDERMAN
Original assignee: Ge Healthcare Bio-Sciences Ab
Priority date: 2004-07-29
Filing date: 2005-07-08
Publication date: 2006-02-02
Also published as: SE0401951D0

Abstract

The present invention relates to a method for purification of least one pegylated compound, which comprises to provide a separation matrix comprised of a porous support, which presents an average pore radius of at least 40 nm and to the surfaces of which functional groups have been immobilised; and to contact said matrix with a liquid that comprises pegylated compound(s) to allow interaction of one or more such compounds with the functional groups; and to recovering pegylated compound(s) as one or more fractions. The interaction is provided at functional groups located at the outer surface as well as located on the pore surfaces of the support. Thus, the porosity of the separation matrix allows for the pegylated compounds to enter the pores.

Description

CHROMATOGRAPHY METHOD

Technical field

The present invention relates to the field of biotechnology, and more specifically to the purification of biological compounds, such as proteins, antibodies and the like. More specifically, the present invention relates to a method of liquid chromatography, which is especially useful for separation and/or isolation of polymer-modified compounds, such as pegylated proteins.

Background

In the pharmaceutical and biopharmaceutical industry, new therapeutic proteins and ex¬ isting FDA-approved proteins are often modified with compounds that enhance their physical properties, such as solubility, hydrolytic stability and aggregation, as well as their biomedical properties, such as antigenicity, proteolytic stability, serum circulation time, and ease of delivery. At present, modification with poly(ethylene glycol) (PEG), commonly known as pegylation, is the most widely used for therapeutic applications. However, other compounds, such as PEG derivatives and neutral hydrophilic polymers, e.g. dextran, are alternatively used for such modification. The same kind of modification is also applied to other molecules than proteins, such as low molecular weight organic drugs and drug candidates.

Thus, pegylated proteins and low molecular weight drugs constitute an important class of biopharmaceuticals, which is commonly produced by pegylation of pre-purified mole¬ cules. The reaction mixture will then contain unreacted PEGs, unmodified molecules and pegylated molecules. The pegylated molecules can comprise one PEG polymer, known as monopegylated molecules, or a number of PEG polymers, often denoted oligopegy- lated molecules. Since unreacted PEG exhibits both colloidal and detergent properties, and under some solution conditions may precipitate or cause precipitation of proteins there is a well-known risk of interference in the subsequent purification. For example, if chromatography is used to purify the target substances, the unreacted PEGs could pro¬ mote precipitation in the separation matrix. Accordingly, in this field, it is important to be able to efficiently remove unreacted PEG from a process as early as possible. Further, in some cases it may be desired to separate monopegylated molecules from oligopegy- lated molecules, and consequently another need is a process that allows such separation.

Ultrafiltration has been suggested to remove unreacted PEG. However, this requires a significant size difference between the PEG and the pegylated molecules, which is not always the case. In addition, ultrafiltration is difficult and costly to scale up, and hence not suitable for large-scale processing.

Due to its versatility and sensitivity to the compounds, chromatography is often the pre¬ ferred purification method in the biotechnological field. The term chromatography em¬ braces a family of closely related separation methods, which are all based on the princi¬ ple that two mutually immiscible phases are brought into contact. More specifically, the target compound is introduced into a mobile phase, which is contacted with a stationary phase. The target compound will then undergo a series of interactions between the sta¬ tionary and mobile phases as it is being carried through the system by the mobile phase. The interactions exploit differences in the physical or chemical properties of the compo¬ nents in the sample.

Ion exchange is a well-known method frequently used for isolation of biological com¬ pounds, such as proteins. The basis for the ion exchange process is the competitive bind¬ ing of ions of one kind, such as proteins, for ions of another kind, such as salt ions, to an oppositely charged matrix known as the ion exchanger. The interaction between the pro¬ teins and the ion exchanger depends on several factors, such as net charge and surface charge distribution of the protein, the ionic strength and the nature of the particular ions in the solvent, the proton activity (pH) etc.

Kato et al. discloses a study of chromatographic conditions to obtain high resolution in protein separation by ion-exchange on a macroporous anion-exchange matrix (Yoshio Kato; Koji Nakamura; Takashi Kitamura; Teruhiko Tsuda; Masazumi Hasegawa; Hiroo Sasaki: "Effect of chromatographic conditions on resolution in high-performance ion- exchange chromatography of proteins on macroporous anion-exchange resin", Journal of Chromatography A, 1031 (2004) 101-105). The macroporous matrix was TSKgel Bio- Assist Q (Tosoh), which presents a particle diameter of 10μm and a pore diameter of 400 nm. The conclusion of the study was that the resolution continuously became higher as the gradient time and the column length became longer. However, it was also noted that the gradient times of up to several hours have not been popular in high-performance ion- exchange.

Ion-exchange chromatography has been suggested for viral separation. WO 98/39467 (Calydon Inc.) relates to adenovirus vectors specific for cells expressing carcinogenic antigen and describes the purification of pegylated adenovirus using the anion exchanger Q Sepharose™ XL (Amersham Biosciences AB, Uppsala, Sweden). Q Sepharose™ XL is a strong anion exchanger comprised of highly cross-linked agarose to which dextran is attached, and its functional groups are quaternary ammonium groups. However, in order to obtain a high capacity, the purification of large compounds will require a separation matrix that presents a relatively large surface area available for interaction. It has been noticed with some commercial ion-exchange matrices that the capacity is not satisfactory for efficient large scale preparation of pegylated compounds.

WO 96/40731 (Mount Sinai School of Medicine of the City University of New York) relates to pegylated proteins, and discloses how IgG modified with PEG is isolated using ultraconcentration followed by size exclusion chromatography and anion exchange chromatography. Size exclusion chromatography, also known as gel filtration is a sepa¬ ration principle that has been in use for more than forty years for desalting and purifica¬ tion of various macromolecules. The separation is obtained by retention of components based on the size in solution, so that the largest molecules are excluded from those sta¬ tionary phase pores that are not big enough and elute earlier in the chromatogram. The smaller molecular weight components enter the stationary phase pores to a larger extent and, as a result, elute later in the chromatogram. The series of steps described will in ad¬ dition to the ultraconcentration equipment require two chromatography columns, and is hence a relatively costly process. As the skilled person will appreciate, the retentate re¬ sulting from the size exclusion chromatography may require conditioning such as pH ad- justment before it can be applied to the next column. Furthermore, it is well known that size exclusion chromatography can only achieve efficient separations at relatively low loadings, resulting in low capacity.

US 6,025,324 (Hoffmann-La Roche Inc.) relates to specific pegylated recombinant obese (ob) protein. More specifically, the ob protein is expressed in a biologically active and soluble state, and thereafter purified to homogeneity and pegylated. The purification is obtained by a combination of anion exchange column chromatography, hydrophobic in¬ teraction column chromatography and gel filtration. Anion exchange chromatography and hydrophobic interaction chromatography can be carried out in any order; however the use of either must precede gel filtration. Thus, like the above discussed WO 96/40731, this purification is both costly and time-consuming.

Finally, EP 1 124 633 (Centre Nationale de Ia Research Scientific) disclose a device for removing biomolecules, which device is comprised of an ultrafiltration module; a dialy¬ sis module; and a column comprising as adsorbent gel. The gel is a polysaccharide ma¬ trix, onto which is grafted a polymer grafted to a ligand that presents affinity to a metal, such as iminodiacetic acid (IDA), which is frequently used in immobilised metal affinity chromatography (IMAC). The grafted polymer may be polyethyleneglycol (PEG) or polypropylene glycol (PPG). The gel presents a cut-off molecular weight of 2-60 kDa, such as 20 kDa, and the biomolecules are exemplified with beta2 -microglobulin.

A similar approach is disclosed in US 5,147,537 (Chisso Corp.) which discloses affinity chromatography using an insoluble carrier to which antibodies modified with activated polyethyleneglycol (PEG) have been immobilised. The activated PEG is e.g. a con¬ densed material of methoxy polyethylene glycol and cyanuric acid. Since the antibodies are modified at a location other than their binding sites, they are useful for purification of antigens directed to the antibodies. The advantage of the described carrier is that the an¬ tibody ligands are protected by the activated PEG against degradation such as by prote- ases present in a protein solution. US 2003/0113798 (Burmer et al) relates to antigenic peptides for G-protein cell recep¬ tors and antibodies relating thereto. Methods are disclosed, wherein antigenic peptides are selected. The antigenic peptides so produced, and antibodies against them, are also disclosed, as well as derivatives of both antigenic peptides and antibodies, wherein the term "derivative" embraces pegylated forms. To purify antibodies or their derivatives, a liquid mixture is loaded onto a conventional HIC column, such as Phenyl Sepharose™ which is comprised of crosslinked 90 um agarose beads functionalised with phenyl groups via ethers. As is well known, the agarose supports used for conventional HIC, ion-exchangers and other functionalised supports are as a general rule less porous and of smaller particle size than gel filtration media.

However, there is still a need in this field of improved purification schemes enabling fast isolation of large target compounds, such as pegylated compounds, at high separation efficiencies. To improve process economy, such purification schemes should preferably allow efficient isolation of pegylated compounds in a single step. There is also a need of novel separation matrices that allow such purification.

Summary of the invention

Thus, one object of the present invention is to provide a method of liquid chromatogra- phy, which enables purification of one or more pegylated compounds with an improved capacity compared to conventional chromatography matrices. This can be achieved by a method as defined in claim 1, wherein a highly porous separation matrix provided with functional groups is utilised.

Another object of the present invention is to provide a method of liquid chromatography, which comprises, purification of pegylated compounds directly from the reaction mix¬ ture. This can be achieved by a method as disclosed above, wherein the liquid applied to the separation matrix is a reaction mixture. However, the reaction mixture in which compounds are pegylated is commonly of a relatively high salt concentration. Thus, one object of the invention is to provide such a method, which avoids the need of diluting the reaction mixture before it is contacted with the separation matrix. This can be achieved by providing a separation matrix that exhibits salt tolerant functional groups.

A specific object of the present invention is to provide a method of separating mono- pegylated compounds from other components of a liquid. Such other components are e.g. oligopegylated compounds; unreacted i.e. non-pegylated proteins and/or PEG.

Another object of the present invention is to provide a method as described above, which allows purification of large pegylated compounds, preferably pegylated proteins, such as pegylated antibodies.

Another object of the present invention is to provide a novel use of conventional size ex¬ clusion media, which has been provided with functional groups such as ion exchange groups, hydrophobic groups, affinity groups or the like.

Yet another object of the present invention is to provide a kit for purification of pegy¬ lated compounds, such as proteins.

A further object of the invention is to provide a separation matrix that enables one or more of the objects above.

The objects above can be achieved by a method as defined in one or more of the ap¬ pended claims. Further objects and advantages of the present invention will appear from the detailed description that follows.

Brief description of drawings

Figure 1 is a reference chromatogram that shows the purification obtained of pegylated protein on a commercially available separation matrix (SP Sepharose™ XL 6FF (Amer- sham Biosciences, Uppsala, Sweden). Figure 2 is a chromatogram that shows the purification in a method according to the pre¬ sent inventiom of pegylated protein. Definitions

The term "separation matrix" means a material that is useful as the stationary phase in chromatography. Commonly used chromatographic separation matrices are comprised of a support to which functional groups are immobilised. The support may be porous or non-porous. In the field of chromatography, a separation matrix is sometimes denoted a resin, or a chromatography media. The term "functional groups" means in the context of chromatography groups capable of sufficient interaction, such as adsorption, to impart separation of different compounds. In the field of chromatography, functional groups, or molecules that comprise functional groups, are often denoted ligands. The "surface" of a separation matrix as used herein includes both the external surface of the matrix and the pore surfaces.

The term "purification" means herein isolation of one component from other compo¬ nents. The term "capture" refers to the initial step of a separation procedure from crude or clari- fied feed. Most commonly, a capture step includes clarification, concentration, stabilisa¬ tion and a significant purification from soluble contaminants.

The term "eluent" is used in its conventional meaning in this field, i.e. a buffer of suit¬ able pH and/or ionic strength to release one or more compounds from a separation ma¬ trix. The terms "size exclusion" and "gel filtration" are used herein interchangeably and means separation of compounds based on their molecular size in a sieving effect, as dis¬ cussed above. In gel filtration, the medium is in the form of spherical particles that have been chosen for their chemical and physical stability and inertness (lack of reactivity and adsorptive properties). For a review of this technique, see e.g. "Gel Filtration - Princi- pies and Methods", Amersham Biosciences handbook no. 18-1022- 18

The term "gel filtration media" is used herein for a media i.e. a separation matrix used for either group separation, wherein the components of a sample are separated into two major groups according to size, or high resolution fractionation of biomolecules accord¬ ing to differences in their molecular size. Such high resolution fractionation is commonly used to isolate one or more components, to separate monomers from aggregates, to de- termine molecular weight or to perform a molecular weight distribution analysis. Thus, the selectivity of a gel filtration medium depends solely on its pore size distribution. The term "K_a" is used in its conventional meaning for the dissociation constant of an acid, and consequently "pK_a value" denotes the -log K_a value.

Detailed description of the invention

In a first aspect the present invention relates to liquid chromatography method for purifi¬ cation of at least one pegylated compound, which method comprises the steps of (a) providing a separation matrix comprised of a porous support, to the surfaces of which functional groups have been immobilised, wherein the support presents an average pore radius of at least about 40 nm;

(a) contacting said matrix with a liquid that comprises pegylated compound(s) to allow interaction of one or more compounds with the functional groups; and

(b) recovering pegylated compound(s) as one or more fractions.

The separation matrix provided in step (a) is comprised of a porous support, which was originally a gel filtration media, to which functional groups have been immobilised. Thus, the support as such has not been optimised for presenting functional groups, but is instead of the kind developed for a separation technique wherein the matrix is chosen for its inertness, i.e. lack of reactivity and adsorptive properties. Thus, the present invention shows that a combination of a porous support, the properties of which corresponds to a media the selectivity of which depends solely on its pore size distribution, and conven¬ tional chromatographic functional groups results in unexpectedly good purification of pegylated compounds.

In one embodiment, the interaction of step (b) is an adsorption and the pegylated com¬ pounds are recovered by contacting the separation matrix with an eluent that releases pe¬ gylated compound(s). In step (c), the interaction is provided at functional groups located at the outer surface as well as located on the pore surfaces of the support. Thus, the po- rosity of the separation matrix is selected to allow for pegylated compounds to enter the pores. The functional groups of the separation matrix provided in step (a) of the present method may be immobilised directly or indirectly to the support. The kind of the functional groups selected will depend on the nature of the pegylated product. Thus, in one em¬ bodiment, said functional groups are selected from the group that consists of charged groups, such as cationic groups or anionic groups; hydrophobic groups; affinity groups; and metal chelating groups. Such groups are well known see e.g. Protein Purification — Principles, High Resolution Methods and Applications, Janson and Rydenl989 VCH Publishers, Inc.

In one embodiment, the functional groups are positively charged and consequently the separation matrix is comprised of a support to which anion-exchange ligands have been immobilised. In an alternative embodiment, the functional groups are negatively charged and consequently the separation matrix is comprised of a support to which cation- exchange ligands have been immobilised. In these embodiments, the selectivity of the matrix will be essentially ion exchange-based. In a specific embodiment, the functional groups are salt tolerant ion-exchanging or multimodal groups; see e.g. WO 01/38227; WO 02/05959; and WO 03/024588 (all in the name of Amersham Biosciences AB).

In yet another embodiment, the functional groups are hydrophobic, and the separation matrix is comprised of a support to which hydrophobic interaction chromatography

(HIC) ligands have been immobilised. In this embodiment, the selectivity of the matrix will be essentially HIC-based.

In an alternative embodiment, the functional groups are groups capable of forming hy- drogen bonds with a target molecule. Such groups have been disclosed in patent applica¬ tion SE 0302509-5 (Bergstrόm et al), which was secret at the time of filing of the present application. In brief, groups capable of forming hydrogen bonds with a target compound are in a first embodiment proton donating groups, which are capable of forming hydro¬ gen bonds with proton accepting groups on the target compound. Such groups may be present on polymer chains in a repetitive manner, to provide a satisfactorily bond for ad¬ sorbing a large target compound, such as a pegylated protein. The hydrogen donating groups may exhibit pK_a values of below 8.0, such as below 7 and preferably below, such as below 5. In an advantageous embodiment, the functional groups of the separation ma¬ trix are predominantly carboxyl groups, which are easily deprotonated into carboxylate ions, which allows breaking the hydrogen bond simply by increasing the pH to a value above the pK_a value of carboxylic acid. The functional groups capable of forming hy¬ drogen bonds can be present on a polymer chain immobilised to the matrix, such as poly(acrylic) acid, poly(meth)acrylic acid, poly(aryl) acid, polyphenol-containing poly¬ mers etc. In an advantageous embodiment of the present separation matrix, the polymer chains are predominantly poly(acrylic acid) chains.

The porous support used in the present method may be in any suitable form, such as es¬ sentially spherical particles; a monolith; a filter or membrane; a chip, a surface, capillar¬ ies or the like. In the most advantageous embodiment, the support is in the form of es¬ sentially spherical particles having an average particle diameter (dry state) of 10-200 μm, such as 10-100 μm. In a specific embodiment, the average particle diameter is 30-60 μm, such as about 50 μm. In an alternative embodiment, the average particle diameter is be¬ low about 55 μm, such as 10-55 μm.

As mentioned above, the support presents an average pore radius of at least about 40 nm. In one embodiment, the support presents an average pore radius of at least about 50 nm; such as at least about 60 nm. In a specific embodiment, the average pore radius is at least about 70 nm or even at least about 80 nm. All the values given herein relating to radii are understood to be hydrodynamic radii, and they preferably exhibit a standard deviation of ±40 (for a reference to the relationship between hydrodynamic radius and molecular weight, see P.L. Dubin (Ed), Aqueous size-exclusion Chromatography: Pore size distri¬ butions, 1988 Elsevier science Publishers B.V. Amsterdam, Chapter 5, pages 119-155). The pore distribution may be narrow or broad, and as the skilled person in this field will appreciate, a certain extent of connectivity between the pores is advantageous.

The nature of the support may alternatively be defined by its cut-off molecular weight for dextran. Thus, in one embodiment, its cut-off molecular weight for dextran is at least about 10 kDa, such as at least about 10 kDa. In other words, pegylated compounds of a hydrodynamic radius equal to a dextran with a molecular weight below 10⁷ kDa, or even below 10⁸ kDa can be purified by the present method at higher loads than matrices of lower exclusion limits will allow.

Alternatively, the porosity of the separation matrix used in the present method is defined in terms of its K_AV value. By measuring the K_AV value of a porous support, the extent of penetration of a target compound into the pores can be described. A higher value means that a substance can penetrate and reach into the porous support to a high degree, while a lower value indicates that a substance in principle can only reach the outer surfaces of the support. Thus, the available surface for interaction and/or adsorption to a separation matrix will be larger at a high K_AV value than at a lower value. In the present method, the K_AV value of dextran of molecular weights of 1000,000; 100,000; 20,000 and 10,000 for the separation matrix provided in step (a) will be about 0.44; 0.62; 0.75 and 0.8, re- spectively. (For a definition of K_Av see L. Hagel in "Protein Purification, Principles, High Resolution, and Applications", J-C Janson and L Ryden (Eds), VCH Publishers Inc. New York, 1989, p. 99.)

The porous support may be of any suitable organic or inorganic material. In one em- bodiment, the support is made from a synthetic polymer, preferably cross-linked syn¬ thetic polymers, such as styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters, vinyl amides etc. Such polymers are easily produced according to standard methods, see e.g. "Styrene based polymer sup¬ ports developed by suspension polymerization" (R Arshady: Chimica e L'Industria 70(9), 70-75 (1988)).

In an alternative embodiment, the support is comprised of a cross-linked carbohydrate material, such as agarose, agar, starch, pectin, cellulose, dextran, chitosan, konjac, carra- geenan, gellan, and alginate. In the most preferred embodiment, the support is porous cross-linked agarose. The carbohydrate support of the invention is easily prepared by the skilled person in this field in accordance with standard methods, such as inverse suspen- sion gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964). To obtain an im¬ proved rigidity of the support, the support may be prepared as described in US patent no 6,602,990 (Berg et al).

Alternatively, the support is a commercially available product for size exclusion separa¬ tion, such as Sephacryl™, e.g. Sephacryl™ S 500 HR or Sephacryl™ S 1000 HR (Am- ersham Biosciences, Uppsala, Sweden). Such a commercial product can easily be modi¬ fied by the skilled person in this field with ligands that comprises functional groups (for an overview, see e.g. Immoblised Affinity Ligand Techniques, Hermanson, MaIHa and Smith, 1992 by Academic Press, Inc).

The pegylated compound may be any pegylated organic substance or pegylated biologi¬ cal compound such as a protein, e.g. an antibody, such as a monoclonal antibody, or an antibody fragment; a nucleic acid; such as DNA, RNA, plasmids; a virus; such as retro- virus, adenovirus, etc; a cell; such as a human eukaryotic cell e.g. a stem cell or progeni¬ tor cell; etc. As discussed above, pegylated compounds are commonly used in the phar¬ maceutical industry. Hence, illustrative examples of pegylated compounds are biophar- maceuticals, such as hormones, insecticides, and industrial catalysts, such as enzymes and ribozymes. It has been observed that PEG molecules of the same molecular weight as proteins behave in size exclusion chromatography as if they were proteins of a much higher molecular weight. For example, a PEG molecule that presents a molecular weight of 20,000 kDa has approximately the same molecular weight as an immunoglobulin that presents a molecular weight of 150,000 kDa.

The liquid that comprises at least one pegylated compound may be a mixture comprising unmodified compound, pegylated compound and PEG. In one embodiment, the liquid is the reaction mixture wherein the compound(s) were pegylated. PEG is available from commercial sources, and is normally polydisperse in relation to the stated molecular weight. Illustrative nominal weights of PEG are 2000-50,000; such as 5000 to 20,000. It is also understood that a mixture of differently sized PEG molecules may be used to pe- gylate the compound. Methods for pegylating compounds such as proteins are well known in this field and easily performed by the skilled person. In the present context, it is to be understood that the term "pegylated" as used in the present application is in¬ tended to embrace modification with polyethylene glycol (PEG) as well as any similar polyether substances that modify a compound to achieve equivalent functions or proper- ties. Examples of such similar polyethers are polypropylene glycol (PPG), PEG-PPG block copolymers, PEG-PPG copolymers, Pluronic™ (BASF) and other PEG-PPG-PEG triblock polymers, ethylhydroxyethylcellulose (EHEC) and similar polymers, polymer¬ ised allylglycidyl ether, polymerised phenyl glycidyl ether, plus various surfactants. Al¬ ternatively, a polysaccharide polymer such as dextran is used to modify the protein for the herein disclosed purposes.

In an advantageous embodiment, the present method is performed in a chromatography column that comprises the separation matrix, and the liquid is passed across said matrix by any conventional means such as by pumping or by gravity. The suitable flow rate and contact time for step (b) depends e.g. on the kind of functional groups; the nature of the compound to be separated and the nature of the support. In a specific embodiment, the load of liquid such as reaction mixture on the column is up to about 2 mg/ml, such as about 4 mg/ml separation matrix. The skilled person in this field can easily select suit¬ able conditions and buffers for each case using common general knowledge or standard textbooks in the field. In case of adsorbed pegylated compounds, the elution of step (c) may comprise a stepwise or linear gradient, and the skilled person in this field can easily select suitable conditions and buffers using common general knowledge or standard textbooks in the field. The separation matrix may be washed with a suitable buffer or wash liquid.

As appears from the above, the product recovered from step (c) will be substantially pure. In one embodiment, the product is a monopegylated compound that presents a pu¬ rity of at least about 90%, preferably at least about 95% such as 96%, and most prefera¬ bly at least about 98% such as about 99%. The present method also encompasses a case where it is desired to purify a desired liquid from a pegylated contaminant to obtain a clean liquid.

In a second aspect, the present invention relates to the use of a separation matrix com- prised of a porous support, to the surfaces of which functional groups have been immobi¬ lised, for the purification of pegylated compounds, wherein the support presents an aver¬ age pore radius of at least about 40 nm, e.g. 46 nm, and preferably at least about 50 nm. In one embodiment, said separation matrix is used for capture in liquid chromatography. The separation matrix used according to this aspect may be any one of the above de- scribed. Thus, in one embodiment, the separation matrix has a cut-off molecular weight for dextrans of at least about 10 kDa, such as at least about 10 kDa (for a correlation to molecular weight, see e.g. P.L. Dublin (Ed.) Aqueous Size-Exclusion Chromatography. 1988 Elsevier Science Publishers B.V. Amsterdam, Chapter 5). Some matrices are pref¬ erably defined by the use a cut-off molecular weight, especially if they are gels, i.e. in a wet state. The standard method in this field for determination of pore sizes is by mercury porosimetry, which however requires a dry sample. As a porous gel dries, its pore struc¬ ture will change and eventually collapse. Accordingly, when pore sizes are defined for porous gels, they have commonly been estimated indirectly by packing a column with a size exclusion gel, running an experiment and noting retention data for model com- pounds of known molecular weight.

The porous support and its functional groups may be as described above.

The present invention also relates to a kit for the purification of pegylated proteins, which kit is comprised of a separation matrix comprised of a porous support, to which functional groups have been immobilised; a suitable buffer; and written instructions that describes the use of the kit for purification of pegylated compounds, such as pegylated proteins. The support presents an average pore radius of at least about at least about 40 nm, e.g. 46 nm, and preferably at least about 50 nm. In a specific embodiment, the in- structions describe purification of monopegylated compounds. The components of the kit are preferably present in separate compartments of the kit. The support may be in the form of essentially spherical particles; a monolith; a membrane or the like, and may be further defined as described above. In an advantageous embodiment, the kit comprises spherical particles packed in a chromatography column. In a specific embodiment, the present kit also comprises luer adaptors, tubing connectors, and domed nuts. The present kit may be for laboratory scale purification or large scale purification of pegylated com¬ pounds, such as proteins e.g. antibodies.

Finally, the present invention relates to a process of preparing a porous separation ma¬ trix, which process comprises to provide a porous support, which presents an average pore radius within the range of 40-100 nm; and to immobilise functional groups to the surfaces of said support. The invention also comprises a process of preparing a separa¬ tion matrix for purification of pegylated compounds, which method comprises to provide a porous support which presents an average pore radius of at least about 40 nm; and to immobilise functional groups to the surfaces of said support. The immobilisation may comprise ether coupling, thioether coupling, amine coupling or the like, which are all well known methods in this field (for standard methods for immobilisation, see e.g. Im¬ mobilized Affinity Ligand Techniques, Hermanson et al, Greg T. Hermanson, A. Krishna Mallia and Paul K. Smith, Academic Press, INC, 1992). Further, the size exclu¬ sion media may be allylated and/or epoxy activated in accordance with well known method before the coupling of the ligands that comprise functional groups thereon. In an advantageous embodiment, the size exclusion media is defined by a cut-off molecular weight for dextrans of at least about 10⁷ kDa, such as at least about 10⁸ kDa. Further de¬ tails regarding the size exclusion media and the functional groups may be as described above.

Accordingly, the present invention also relates to a separation matrix comprised of a po¬ rous support, to the surfaces of which functional groups have been immobilised, wherein the support presents an average pore radius of at least about 40 nm. The functional groups may be any of the ones described above in the context of the first aspect of the invention. In one embodiment, the functional groups are hydrophobic interaction chro¬ matography (HIC) ligands. HIC- ligands are well known and readily available in this field. In an advantageous embodiment of the present, the porous support is allyl dex- tran/bisacrylamide, such as Sephacryl™ S 500.

A further aspect of the invention is a chromatography column packed with a separation matrix as defined above. In an advantageous embodiment, the column is made from a conventional material, such as a biocompatible plastic, e.g. polypropylene, or glass. The column may be of a size suitable for laboratory scale or large-scale purification, prefera¬ bly of large-scale. In a specific embodiment, the column according to the invention is provided with luer adaptors, tubing connectors, and domed nuts. Thus, the present inven- tion also encompasses a kit comprised of a chromatography column packed with a sepa¬ ration matrix as described above; at least one buffer; and written instructions for purifi¬ cation of pegylated compounds in separate compartments.

Detailed description of the drawings Figure 1 is a chromatogram that shows the purification obtained of pegylated protein on a commercially available separation matrix (SP Sepharose™ XL 6FF (Amersham Bio- sciences, Uppsala, Sweden), obtained as described in the experimental part below. In Figure 1, the load of pegylated protein is 1.5 mg/ml mobile phase. The blocks provided underneath the curve show the results of analysis of the material obtained in each peak, as obtained by size exclusion chromatography. The first peak represents mainly oligo- pegylated protein; the second peak represents the target pegylated protein; and the third peak represents non-modified protein. As appears from the figure, the purity of the target pegylated protein in the second peak is merely about 80%, while a substantial part thereof is also to be found in the first and third peaks. More specifically, peaks (frac- tions) B6-B8 comprises 89% oligomer; 3.5% target-PEG; and 7.5% low molecular spe¬ cies. Peaks (fractions) B12-C3 comprises 19% oligomer; and 80% target-PEG. Finally, peaks C6 and C7 comprises 94% protein and 2% target pegylated protein, respectively. Thus, Figure 1 illustrates the problems of using a conventional kind ion exchanger in the purification of pegylated proteins. Figure 2 is a chromatogram that shows the purification obtained of pegylated protein on a separation matrix provided according to the present invention, showing the results of analysis as explained above and obtained as described in the experimental part below. In Figure 2, conditions corresponded to 4.6/150, 4 mg/ml load, flow 150 ch/h starting at 25%B.

As appears from Figure 2, the method according to the invention results in a second peak from which substantially all of the target pegylated protein can be obtained as an essen¬ tially pure product.

EXPERIMENTAL PART

The following examples are provided to illustrate the present invention and should not be construed as limiting the scope of the invention as defined by the appended claims. All references given below and elsewhere in the present specification are hereby included herein by reference.

Example 1

Synthesis of a sulfopropyl (SP) cation exchanger by allylation and bisulphite coupling Allylation of Sephacryl™ S-500 HR (Amersham Biosciences, Uppsala, Sweden), which is a porous size exclusion chromatography matrix without functional groups, was per¬ formed in the following way.

200 g beads drained by suction on a glass filter were charged to a 500 ml reaction vessel. 40 ml distilled water, 20 ml 50% sodium hydroxide, 0.8 g sodium borohydride, 24g so¬ dium sulphate were added during stirring. The temperature was 5O⁰C and finally 200 ml allylglycidylether were added. The reaction continued overnight and the product was neutralised with acetic acid and washed with ethanol and distilled water. The allylation was determined to 154 μmol allyl groups per ml of gel.

To couple the SP groups to the support, 100 g gel allylated as described above allylated was drained by suction on a glass filter and mixed with 100 ml distilled water and 40 g sodium bisulphite. 50 % sodium hydroxide was added during stirring until pH was 6.7. The temperature was 25⁰C and air was bubbled into the reaction vessel through a capil¬ lary. The reaction continued during stirring overnight at 25⁰C. The product was washed with distilled water. The ion exchange capacity was determined to 76 μmol H⁺ per ml of gel.

Example 2 Protein purification

A comparative chromatogram was first obtained by contacting a reaction mixture com¬ prising mono- and oligo-pegylated 30 kDa protein and unreacted PEG with a conven- tional ion exchanger (SP Sepharose™ XL, Amersham Biosciences, Uppsala, Sweden), which presents functional sulfopropyl (SP) groups on a matrix of a relatively small pore size. The PEG used was PEG 20,000. The chromatography was performed in accordance with well known standard methods on an AKT A™ system (Amersham Biosciences, Uppsala, Sweden). The column size was as follows: ά=A.β mm, h=150 mm, and the flow rate was 150 cm/h. As appears from Figure 1, only a rough separation was obtained, and the mono-pegylated protein fraction was not pure.

A sample of the same reaction mixture was then applied to a separation matrix obtained as described in Example 1, under the corresponding conditions. As appears from Figure 2, a substantially pure fraction of mono-pegylated protein as obtained.

Example 3(a)

Functional quaternary ammonium (Q) groups An alternative separation matrix according to the present invention was prepared as fol- lows. Sephacryl™ S-500 HR (Amersham Biosciences, Uppsala, Sweden), which is a product marketed for size exclusion chromatography and consequently does not present any functional groups, was drained by suction on a glass filter, and 200 g of the beads were added to a reaction vessel. 8.0 g sodium hydroxide and 0.2 g sodium borohydride were stirred with 40 ml distilled water to a clear solution and charged to the vessel. 400 ml glycidyltrimetylammonium chloride (GMAC) was pumped into the reaction vessel in 2 hours. The temperature was kept at 25⁰C and the reaction continued during the night (18 hours). The product, herein denoted Q-Sephacryl™, was neutralised with 60 % ace¬ tic acid and washed with distilled water. The ion-exchange capacity was determined to 34 μmol Cl^" per ml of gel.

Example 3(b)

Functional amine groups Example 2a: Diethylamine coupling

Another alternative separation matrix according to the present invention was prepared as follows. 25 ml of the gel Sephacryl™ S500 HR (Amersham Biosciences, Uppsala, Swe- den), which had been allylated with allylglycidyl ether (AGE) and NaOH according to standard methods to an allyl content of 140 μmol/ml, was vacuum drained and placed in a 600 ml beaker together with in 400 ml water. Bromine was added drop wise until a lasting yellow colour appeared in the suspension. The brominated gel was then washed on a glass filter funnel with more than 500 ml distilled water.

25 g of the above described brominated and vacuum drained gel was charged in a 100 ml round flask together with 4 g distilled water and 7 g diethylamine. The reaction was run at room temperature over night. The reaction was discontinued by washing the gel with about 2 bed volumes of water and then resuspension thereof in about 50 ml of water. The pH was adjusted to 5-6 by addition of a water: concentrated hydrochloric acid solution of 1 : 1. An additional wash with more than 500 ml of water was performed.

The ion exchange capacity (amount of ion exchanger) was determined to 63 μmol using standard methods.

Claims

1. A liquid chromatography method for purification of at least one pegylated com¬ pound, which method comprises the steps of

(a) providing a separation matrix comprised of a porous support, to the surfaces of which functional groups have been immobilised, wherein the support presents an average pore radius of at least about 40 nm; and

(b) contacting said matrix with a liquid that comprises pegylated compound(s) to al¬ low interaction of one or more compounds with functional groups at the outer sur¬ face and the pore surfaces of the support; and (c) recovering pegylated compound(s) as one or more fractions.

2. A method according to claim 1, wherein the interaction of step (b) is an adsorption and the pegylated compounds are recovered by contacting the separation matrix with an eluent that releases pegylated compound(s).

3. A method according to claim 1 or 2, wherein the separation matrix is comprised of a gel filtration media, to which functional groups have been immobilised.

4. A method according to any one of the preceding claims, wherein in step (c), pegy¬ lated compounds enter the pores and are adsorbed at the pore surfaces.

5. A method according to any one of the preceding claims, wherein said functional groups are selected from the group that consists of charged groups, such as cationic groups or anionic groups; hydrophobic groups; affinity groups; and metal chelating groups.

6. A method according to claim 5, wherein the separation matrix is comprised of a po¬ rous support, to the surfaces of which ion-exchange ligands have been immobilised.

7. A method according to claim 5, wherein the separation matrix is comprised of a po- rous support, to the surfaces of which hydrophobic interaction chromatography (HIC) ligands have been immobilised.

8. A method according to any one of the preceding claims, wherein in step (b), a liquid is passed across the separation matrix at a load below 4 mg per ml separation matrix.

9. A method according to any one of the preceding claims, wherein the liquid from which the pegylated compounds are purified is the reaction mixture wherein the compounds were pegylated.

10. A method according to any one of the preceding claims, which is a capture step.

11. A method according to any one of the preceding claims, wherein in step (c), mo- nopegylated compound(s) are recovered in a separate fraction.

12. A method according to any one of the preceding claims, wherein the pegylated com- pounds are pegylated proteins.

13. A method according to any one of the preceding claims, wherein the support is com¬ prised of essentially spherical particles, the average diameter of which is 30-60 μm, such as 50 μm.

14. A method according to any one of the preceding claims, wherein the porous support is made from crosslinked copolymer of allyldextran and N₅N- methylenebisacrylamide.

15. A method according to claim 14, wherein the porous support is Sephacryl™ S 500.

16. A process of preparing a hydrophobic separation matrix for purification of pegylated compounds, which process comprises to provide a porous support, which presents an average pore radius of at least 40 nm; and to immobilise functional groups to the sur¬ faces of said support.

17. A process according to claim 16, wherein the porous support is a gel filtration media.

18. A process according to claim 16 or 17, wherein said functional groups are hydropho¬ bic interaction chromatography (HIC) ligands.

19. A separation matrix comprised of a porous support, to the surfaces of which func¬ tional groups have been immobilised, wherein the support presents an average pore radius of at least about 40-100 nm.

20. A matrix according to claim 19, wherein the support is comprised of essentially spherical particles, the average particle diameter of which is in the range of 10-55 μm.

21. A separation matrix according to claim 19 or 20, wherein the functional groups are ion-exchange ligands or hydrophobic interaction chromatography (HIC) ligands.

22. A matrix according to any one of claims 19-21, wherein the porous support is a gel filtration media.

23. A matrix according to any one of claims 19-22, wherein the porous support is made from crosslinked copolymer of allyldextran and N,N-methylenebisacrylamide.

24. A matrix according to claim 23, wherein the porous support is Sephacryl™ S 500.

25. A chromatography column packed with a separation matrix as defined in any one of claims 19-24.