| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | August 10, 1999 |
| PATENT TITLE |
Use of hydrophobic interaction chromatography to purify polyethylene glycols |
| PATENT ABSTRACT |
The present invention relates to a process for purifying polyethylene glycols (PEGs) which utilizes hydrophobic interaction chromatography (HIC) to separate the PEGs based on their size and on their end-group functionality. The purified PEGs can be used to modify biologically active molecules and improve overall production of such molecules. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | September 18, 1997 |
| PATENT REFERENCES CITED |
Abstract (Biot.--068.) Seely et al Manufacturing of Recombinant Tumor Necrosis Factor Binding Protein "Dumbbell" Using a 20K Peg Bis-Vinylsulfone Linker. Waters Bulletin (1995) Columns for Gel Permeation and Size Exclusion Chromatography. Murphy et al J. of Chrom. (211) 160-165. Chen, et al., Biochem. Bviophys. Acta, 660: 293-298 (1988). Sada, et al., Journal Fermentation Bioenginerring 71: 137-139 (1991). Davis, et al., "Biomedical Polymers. Polymeric Materials and Pharmaceuticals for Biomedical Use", Eds. Goldberg & Nakajima, pp. 441-452, Academic Press, N.Y. (1980). Murphy, et al., Journal Chromat., 211: 160-165 (1981). Lai et al., Journal High Res. Chrom. & Chrom. Comm., 7: 494-496 (1984). Barka & Hoffman, Journal Chromat., 389: 273-278 (1987). Engelhardt & Mathes, Journal Chromat.., 185: 305 (1979). Seely, et al. BIOT--068 (1997) "Manufacturing of Recombinant Tumor Necrosis Factor Binding Protein "Dumbbell " Using a 20K PEG Bis-Vinylsulfone Linker". Waters Bulletin (1995) "Columns for Gel Permeation and Size Exclusion Chromatography". |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A process for the preparation of a pegylated tumor necrosis factor (TNF) inhibitor, said process comprising: (a) purifying a polyethylene glycol (PEG) using hydrophobic interaction chromatography (HIC); wherein said PEG has a molecular weight greater than 5000 daltons and is selected from the group consisting of PEG-bis-chlorosulfone and PEG-bis-vinyl sulfone, and wherein said HIC utilizes polymeric resins wherein a hydrophobic pendant group selected from the group consisting of octyl, butyl, phenyl, and ether is located on the surface of a hydrophilic vinyl copolymer backbone resin; (b) reacting the HIC-purified PEG with said TNF inhibitor; and (c) isolating the reaction product using chromatographic techniques. 2. A process according to claim 1, wherein said TNF inhibitor is selected from the group consisting of a 30 kDa TNF inhibitor, a 40 kDa TNF inhibitor, or closely related analogues thereof. 3. A process according to claim 1, wherein said TNF inhibitor is a TNF inhibitor mutein. 4. A process according to claim 3, wherein said TNF inhibitor mutein is c105 30 kDa TNF inhibitor. 5. A process according to claim 1, wherein said reaction product has the formula R.sub.1 -X-R.sub.2, wherein: X comprises an HIC-purified PEG having a first reactive group and a second reactive group, and wherein said first reactive group is a Michael acceptor; and wherein said PEG has a molecular weight greater than 5000 daltons is selected from the group consisting of PEG-bis-chlorosulfone and PEG-bis-vinyl sulfone; R.sub.1 comprises a TNF inhibitor having a reactive thiol moiety, said TNF inhibitor covalently bonded to said HIC-purified PEG by reaction of said thiol moiety with said Michael acceptor; and R.sub.2 comprises a TNF inhibitor or a nonbiologically-active group bonded to said PEG by reaction with said second reactive group. 6. A process according to claim 5, wherein X is polyethylene glycol-bis-vinyl sulfone and wherein R.sub.1 and R.sub.2 is c105 30 kDa TNF inhibitor. 7. A substantially homogenous pegylated TNF inhibitor produced by the process of claim 1. 8. A pharmaceutical composition comprising a substantially homogenous pegylated TNF inhibitor produced by the process of claim 1, and a pharmaceutically acceptable diluent, adjuvant or carrier. |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The invention relates to an efficient process for purifying polyethylene glycols (PEGS) which utilizes hydrophobic interaction chromatography (HIC) to separate the PEGs based on size and end-group functionality. More particularly, the invention is directed to a process for purifying 20K polyethylene glycol bis-vinylsulfone ("20K PEGbv") which utilizes a Butyl or Phenyl HIC resin to separate high molecular weight PEG impurities and PEG mono-vinylsulfone from the 20K PEGbv. The process does not require the addition of organic solvents and the purified 20K PEGbv can be used to modify certain biologically active proteins. BACKGROUND OF THE INVENTION Polyethylene glycols ("PEGs") are long chain, linear synthetic polymers composed of ethylene oxide units. The ethylene oxide units can vary such that PEG compounds can be obtained with molecular weights ranging from approximately 200-100,000. PEG typically is colorless, odorless, soluble in water, stable to heat, does not hydrolyze or deteriorate, and is non-toxic. As such, PEGs have been extensively studied for use in pharmaceuticals, on artificial implants, and other applications where biocompatibility is of importance. The attachment of PEG to proteins ("PEGylation") has been shown to: (1) increase the plasma half-life of the proteins, see e.g., U.S. Pat. No. 5,349,052, Delgado et al., issued Sep. 20, 1994; (2) alter the biochemical and physical properties of the protein, including increasing solubility, Chen et al., Biocem. Biophys. Acta, 660: 293-298 (1988), and improving protection against proteolysis, Sada, et al., J. Fermentation Bioengineering 71: 137-139 (1991); and (3) reduce antigenicity, Davis et al., "Biomedical Polymers. Polymeric Materials and Pharmaceuticals for Biomedical Use", (Eds. Goldberg & Nakajima) pp 441-452, Academic Press, N.Y. (1980). Various derivatives of PEG have been proposed that have an active moiety for permitting PEG to be attached to proteins. The vast majority of PEGylating reagents react with free primary amino groups of the polypeptide. Most of these free amines are the .epsilon.-NH.sub.2 group of lysine amino acid residues; and, because typical proteins possess a large number of lysine residues, random attachment of multiple PEG molecules often occurs, which can lead to loss of protein activity. Published PCT Publication No. WO 95/13312 describes, inter alia, water soluble sulfone-activiated PEGs which are highly selective for coupling with thiol moieties instead of amino moieties on molecules and on surfaces. These PEG derivatives are stable against hydrolysis for extended periods in aqueous environments at pHs of about 11 or less, and can form linkages with molecules to form conjugates which are also hydrolytically stable. The linkage by which the PEGs and the biologically active molecule are coupled includes a sulfone moiety coupled to a thiol moiety and has the structure PEG-SO.sub.2 -CH.sub.2 -CH.sub.2 -S-W, where W represents the biologically active molecule, and wherein the sulfone moiety is vinyl sulfone or an active ethyl sulfone. One example of a biologically active molecule which can be modified by such sulfone-activated polymers is TNF binding protein ("TNFbp")(also referred to as TNF inhibitor). TNFbp is of therapeutic interest in the treatment of disease states such as rheumatoid arthritis and septic shock. Several forms of native TNFbp, e.g., 30 kDa and 40 kDa TNFbp have been described in detail; see e.g., published European Patent Application No. 90 113 673.9, incorporated herein by reference. Furthermore, various TNFbp muteins, e.g., c105 30 kDa TNFbp mutein (the asparagine residue at position 105 of the native human protein is changed to cysteine) have been prepared and described; see published PCT Publication No. WO 92/16221, incorporated herein by reference. PCT Publication No. WO 92/16221 further describes TNFbp forms represented by the formula R.sub.1 -X-R.sub.2 (referred to as a "dumbbell", compound), wherein R.sub.1 and R.sub.2 are biologically active TNFbp groups and X is a non-peptidic polymeric spacer with at least one Michael acceptor group, e.g. a sulfone-activated PEG. One particular compound, referred to as c105 TNFbp mutein dumbbell, is a homodumbbell compound wherein R.sub.1 and R.sub.2 is a c105 30 kDa TNFbp mutein, X is 20 K polyethylene glycol bis-vinylsulfone (20 K PEGbv), and wherein R.sub.1 and R.sub.2 are attached site-specifically at the cysteine 105 residue to the PEGbv. This c105 TNFbp mutein dumbbell was reported to be many times more active than the unPEGylated native 30 kDa TNFbp, unPEGylated c105 30 kDa TNFbp mutein or the PEGylated 30 kDa TNFbp "monobell" (R.sub.1 -X). A problem commonly encountered by those skilled in the art when producing compounds such as the c105 TNFbp mutein dumbbell is that the procedures used to synthesize the 20 K PEGbv, or other polymer, have several drawbacks, depending on the synthesis procedure used. One particularly problematic drawback is that the resultant 20 K PEGbv will often contain high molecular weight PEG impurities and PEG mono-vinylsulfone. These impurities can then adversely affect the overall yield and purity of the desired dumbbell compound. The need exists, therefore, for methods which would further purify the PEGbv, or other polymers, and thereby enhance the overall purity and yield of the desired dumbbell compound, and make utilization of such compounds as therapeutic agents more commercially practicable. A number of groups have reported on the use of reversed-phase high performance liquid chromatography (rpHPLC) to separate low- and medium-molecular weight (<5K) PEGs on the basis of size; see e.g., Murphy et al., J. Chromat., 211: 160-165 (1981); Lai et al., J. High Res. Chrom. & Chrom. Comm., 7: 494-496 (1984); Barka & Hoffman, J. Chromat., 389: 273-278 (1987). These techniques, while relatively effective, suffer from their use of expensive resins and organic solvents. Also, these methods are analytical rather than preparative procedures. There have been no reports of successful separation of larger molecular weight PEGs using HPLC. Medium- and high-molecular-weight (1K-40K) PEGs have been analyzed by size exclusion chromatography on a variety of derivatized silica supports; Engelhardt & Mathes, J. Chromat., 185: 305 (1979). Hydrophobic interaction chromatography (HIC) has achieved acceptance as an analytical and preparative purification method for biomolecules. The chemical principles underlying HIC are similar to those involved in salt precipitation, and related to those associated with reversed phase liquid chromatography (RPLC), in that all operate on the basis of hydrophobicity. HIC is characterized by adsorption of molecules to a weakly hydrophobic surface at high salt concentrations, followed by elution with a decreasing salt gradient. Thus, HIC combines the non-denaturing characteristics of salt precipitation and the precision of chromatography to yield excellent activity recoveries. Moreover, because HIC operates at lower binding energy, it does not require the use of expensive organic solvents in the mobile phase. To date, there have been no reports concerning the use of HIC resins to separate PEGs, especially high molecular weight PEGs. SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a process for purifying non-peptidic polymers, in particular, sulfone-activated polymers, which utilizes HIC to separate the polymers based on size and end-group functionality. Importantly, purifications carried out in this manner do not require the addition of organic solvents. More importantly, the purified polymers can be used to modify various proteins and provide biologically-active conjugates with dramatically improved purity and overall yield as compared to conjugates formed with non-purified polymers. Polymers contemplated for use include water soluble polymers such as polyethylene glycol ("PEG") and related hydrophilic polymers. The biologically-active molecule can be a synthetic, a naturally occurring, or a modified naturally occurring molecule. In one aspect of the invention, a PEG or other non-peptidic polymer linker, having a molecular weight ranging from 5 kDa to 100 kDa, is applied to, and eluted from, an HIC resin using conventional chromatographic methods. In a preferred embodiment, the linker is eluted from the resin using a reverse linear salt gradient, such that the various linker forms are separated based on size and/or end-group functionality. Preferably, the linker is a sulfone-activated PEG linker and the HIC comprises a Butyl or Phenyl HIC resin. In a particularly preferred embodiment, the invention is directed to a process for purifying 20K polyethylene glycol bis-vinylsulfone ("20K PEGbv") utilizing a Toyopearl.RTM. Butyl-650M HIC resin to separate high molecular weight PEG impurities and PEG mono-vinylsulfone from the 20K PEGbv. In yet another aspect of the invention, an HIC-purified, non-peptidic polymer linker is reacted with a biologically-active molecule in order to provide a dumbbell compound having improved overall purity and yield as compared to previously described compounds containing the same molecule. Preferably, the HIC-purified polymer linker is a sulfone-activated PEG, and the biologically-active molecule is selected from the group consisting of TNF inhibitors, interleukin-1 receptor antagonists ("IL-1ra's"), exon six peptide of platelet-derived growth factor ("PDGF"), and interleukin-2 ("IL-2") inhibitors and receptors ("IL-2r"). In a particularly preferred embodiment, the HIC-purified polymer linker is 20K PEGbv, the biologically-active molecule is TNFbp. Pharmaceutical compositions containing the compounds of the present invention are also included within the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts the elution profile of various PEG diols from a ToyoPearl.RTM. Butyl 650M HIC column. PEG-containing fractions were detected with Nessler's reagent and the molecular weights confirmed by SEC HPLC as described in Example 3. FIG. 2 depicts the elution profile of 20K PEGbv and high molecular weight PEGbv from a ToyoPearl.RTM. Phenyl 650S HIC column (2 mg/mL bed volume loading). PEGbv concentration was determined by Nessler's reagent and the % molecular weight impurity was determined by SEC HPLC as described in Example 3. FIG. 3 depicts the elution profile of 20K PEGbv (2 mg/mL bed volume loading) from a ToyoPearl.RTM. Butyl 650M HIC column. PEG-containing fractions were detected with Nessler's reagent and turbidity measured at 600 nm. FIG. 4 depicts the elution profile of on-HIC-purified 20K PEGbv from a SEC-HPLC column. FIG. 5 depicts the elution profile of HIC-purified 20K PEGbv from a SEC-HPLC column. A ToyoPearl.RTM. Butyl 650M HIC column (8 mg/mL bed volume loading) was used for the HIC purification. FIG. 6 depicts a typical POROS HS cation-exchange elution profile for a TNFbp PECylation mixture (the mixture utilized non-HIC-purified 20K PEGbv). The various TNFbp peaks are shown as indicated. FIG. 7 depicts the POROS HS cation-exchange elution profile for a TNFbp PEGylation mixture. The PEGylation mixture was prepared as described in Example 5 and utilized ToyoPearl.RTM. Butyl 650M HIC-purified 20K PEGbv. FIG. 8 depicts the POROS HS cation-exchange elution profile for a TNFbp PEGylation mixture. The PEGylation mixture was prepared as described in Example 5 and utilized non-HIC-purified 20K PEGbv. FIG. 9 depicts the elution profile of TNFbp Dimer from a SP-Sepharose HP column. A.sub.280 absorbance is plotted vs. time. The TNFbp Dimer was prepared using ToyoPearl.RTM. Butyl 650M HIC-purified 20K PEGbv. FIG. 10 depicts the elution profile of TNFbp Dimer from a SP-Sepharose HP column. A.sub.280 absorbance is plotted vs. time. The TNFbp Dimer was prepared using non-HIC-purified 20K PEGbv. FIG. 11 depicts the POROS HS cation-exchange elution profile for an "early eluting" fraction from a 20K PEGbv ToyoPearl.RTM. Butyl 650M HIC purification. The PEGylation mixture was prepared as described in Example 6. DETAILED DESCRIPTION The processes by which hydrophobic interaction chromatography can be used to purify polyethylene glycols, and other polymers, are described in more detail in the discussion below and are illustrated by the examples provided below. The examples show various aspects of the invention and include results of the use of butyl and/or phenyl HIC resins to separate high molecular weight PEG impurities from a sulfone-activated PEG, 20K PEGbv. The results were surprising in that HIC also separated PEG mono-vinylsulfone from the 20K PEGbv, and the resultant purified 20K PEGbv, when used to PEGylate TNPbp, provided a biologically active conjugate having dramatically improved overall yield and purity as compared to conjugates prepared using non-HIC-purified 20K PEGbv. HIC contemplated for use in the present invention utilizes polymeric resins wherein a hydrophobic pendant group (e.g. octyl, butyl, phenyl or ether) is located on the surface of a hydrophilic vinyl copolymer backbone resin, including, but not limited to, a methacrylic, agarose, or vinylic backbone. A silica based backbone could also be used. Preferred HIC columns will have either a butyl or phenyl ligand on a methacrylic backbone. The term "PEG" as used herein means any of several condensation polymers of ethylene glycol. PEG is also known as polyoxyethylene, polyethylene oxide, polyglycol, and polyether glycol. PEG can also be prepared as copolymers of ethylene oxide and many other monomers. The end groups of the PEG can be derivatized in a variety of ways to include vinylsulfone moieties, aldehyde moieties, methoxy groups, or other types of reactive or nonreactive end-groups. The polymeric derivatives of the present invention have active sulfone moieties. "Active sulfone" means a sulfone group to which a two carbon group is bonded having a reactive site for thiol-specific coupling on the second carbon from the sulfone group at about pH or less. Examples of active sulfones include, but are not limited to, vinyl sulfone and activated ethyl sulfone. The sulfone-activated polymer can be further substituted as long as the thiol-specific reactivity at the second carbon is maintained at about pH 9 or less. The sulfone-activated polymers can be synthesized as described in co-pending U.S. patent application Ser. No. 08/259,413, specifically incorporated herein by reference, or prepared as described in Example 1 below. The activated polymeric derivatives of the present invention can have more than one reactive group. The derivatives can be monofunctional, bifunctional, or multifunctional. The reactive groups may be the same (homofunctional) or different (heterofunctional) as long as there is at least one active sulfone moiety. Two particularly useful homobifunctional derivatives are PEG-bis-chlorosulfone and PEG-bis-vinyl sulfone. general method for purifying the non-peptidic polymers includes the following steps: (1) synthesize the non-peptidic polymer linker; (2) apply the non-peptidic polymer linker to an HIC resin; (3) isolate the desired polymer linker form by eluting the polymer from the HIC resin using conditions, e.g., reverse linear salt gradient, which allow for separation of the various linker forms based on size and/or end-group functionality. Alternatively, the HIC purification step could be incorporated into the polymer linker synthesis procedure. Biologically-active molecules include, but are not limited to, pharmaceuticals, vitamins, nutrients, nucleic acids, amino acids, polypeptides, enzyme co-factors, steroids, carbohydrates, organic species such as heparin, metal containing agents, receptor agonists, receptor antagonists, binding proteins, receptors or portions of receptors, extracellular matrix proteins, cell surface molecules, antigens, haptens, and chelating agents. "Polypeptides" and "proteins" are used herein synonymously and mean any compound that is substantially proteinaceous in nature. Also included within the scope of the invention are truncated forms of the native polypeptide or polypeptides which are modified to contain an amino acid residue in a position where one was not present in the native protein before such modification ("mutein"). Many polypeptides useful in the present invention are set forth in published PCT Publication No. WO 92/16221, specifically incorporated herein by reference. In general, TNFbp useful in the present invention has the sequence of human TNFbp, or closely related analogues thereof. One TNFbp, referred to as 30 kDa TNFbp, or TNFbp I, is the extracellular portion of the p55 TNF receptor. In vivo, the extracellular portion of the receptor is shed and circulates in the bloodstream as a 30 kDa glycosylated protein which binds TNF. The purification and amino acid and nucleic acid sequences of this TNFbp are set forth in published European Patent Application No. 90 113 673.9, which is incorporated herein by reference. This reference also teaches the recombinant production of glycosylated and deglycosylated forms of 30 kDa TNFbp. Although the actual molecular weight of the deglycosylated form of this inhibitor is approximately 18 kDa, the term "30 kDa TNF inhibitor" includes the glycosylated and deglycosylated forms. The purification and amino acid and nucleic acid sequences of another TNF inhibitor, called 40 kDa TNFbp or TNFbp-II, is also set forth in European Patent Application No. 90 113 673.9. This inhibitor, in its naturally-occurring form, is the glycosylated extracellular portion of the p75 or p85 TNF receptor. Although the molecular weight of the deglycosylated form is not 40 kDa, both the glycosylated and deglycosylated forms of this TNFbp are referred to as "40 kDa TNF inhibitor. Furthermore, PCT Publication No. WO 92/16221, incorporated herein by reference, describes the preparation of various TNFbp muteins, e.g., c105 30 kDa TNFbp mutein (the asparagine residue at position 105 of the native human protein is changed to cysteine). While the procedures described in the published references above are the preferred methods for producing recombinant TNFbp forms, modifications and changes could be made to that process as known in the art. As stated above, the HIC-purified, sulfone-activated polymers can be reacted with a thiol-containing biologically active molecule to provide biologically active conjugates. The linkage between the thiol reactive group and the sulfone-activated polymer is a covalent bond, and the general method for preparing such conjugates includes the following steps: (1) select the desired biologically-active molecule; (2) synthesize the desired non-peptidic polymer linker; (3) purify the polymer using HIC chromatography; (4) react the HIC-purified, activated polymer with the molecule; (5) isolate the reaction product using chromatographic techniques well known in the art. (6) determine the biological activity of the conjugate formed using the relevant bioassay. The conjugates of the present invention can be used for a variety of purposes including, but not limited to, in-vitro diagnostic assays and the preparation of pharmaceutical compositions. Conjugates containing TNFbp can be used to treat TNF-mediated diseases such as adult respiratory distress syndrome, pulmonmary fibrosis, arthritis, septic shock, inflammatory bowel disease, multiple sclerosis, graft rejection and hemorrhagic trauma. Pharmaceutical compositions containing many of the conjugates or compounds (collectively, the "conjugates") of the present invention can be prepared. These conjugates can be in a pharmaceutically-acceptable carrier to form the pharmaceutical compositions of the present invention. The term "pharmaceutically acceptable carrier" as used herein means a non-toxic, generally inert vehicle for the active ingredient, which does not adversely affect the ingredient or the patient to whom the composition is administered. Suitable vehicles or carriers can be found in standard pharmaceutical texts, for example, in "Remington's Pharmaceutical Sciences", 16th Ed., Mack Publishing Co., Easton, Pa. (1980), incorporated herein by reference. Such carriers include, for example, aqueous solutions such as bicarbonate buffers, phosphate buffers, Ringer's solution and physiological saline. In addition, the carrier can contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation. Although the invention has been described and illustrated with respect to certain purification processes which utilize hydrophobic interaction chromatography for purifying non-peptidic polymers based on size and end-group functionality, it will be apparent to one of ordinary skill that additional embodiments may exist without departing from the scope of the invention. |
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