HK1076115B - Polyol-ifn-beta conjugates - Google Patents

Description

Polyol interferon beta conjugates

This application is a divisional application entitled polyol interferon beta conjugates, filed on 28.4.1999, application number 99805496.8.

Cross Reference to Related Applications

Priority of U.S. provisional application No. 60/083,339 is claimed in 35u.s.c. § 119(e), the entire content of which is incorporated herein by reference.

Technical Field

The present invention relates to polyol-IFN- β conjugates in which the polyol unit and Cys are¹⁷And (4) covalently bonding. Another object of the invention is the process of site-specific generation thereof and its use in the treatment, prognosis or diagnosis of bacterial infections, viral infections, autoimmune diseases and inflammatory diseases. The invention also relates to methods for the stepwise conjugation of two or more PEG molecules to a polypeptide.

Background

Human fibroblast interferon (IFN- β) has antiviral activity and also stimulates natural killer cells against tumorigenic cells. It is a polypeptide of about 20,000Da induced by viruses and double-stranded RNA. Derynk et al (Nature of nature,285: 542-547, 1980) the complete amino acid sequence of the protein was deduced from the nucleotide sequence of the fibroblast gene cloned by recombinant DNA techniques. It is 166 amino acids long.

Shepard et al (Nature of nature,294: 536-565, 1981) describe that the base mutation at position 842 abolishes its antiviral activity (position 141Cys → Tyr), and that variant clones with a deletion of nucleotides 1119-1121.

Mark et al (Proc.Natl.Acad.Sci.U.S.A.,81(18): 5662-5666, 1984) an artificial mutation was inserted by replacing the 469(T) base with (A) resulting in an amino acid transition from Cys → Ser at position 17. The resulting IFN- β was reported to be as active as the "native" IFN- β and to remain stable during long term storage (-70 ℃).

Covalent attachment of the hydrophilic polymer polyethylene glycol (PEG), also known as polyethylene oxide, to molecules is an important application in biotechnology and medicine. PEG in its most common form is a linear polymer with hydroxyl groups at each end:

HO-CH₂-CH₂O(CH₂CH₂O)_nCH₂CH₂-OH

this molecular formula can be simply represented by HO-PEG-OH, where-PEG-represents a polymer backbone without terminal groups:

"-PEG-" means "-CH₂CH₂O(CH₂CH₂O)_nCH₂CH₂-

PEG is commonly used as methoxy-PEG-OH, where one terminus is a relatively inert methoxy group and the other terminus is a hydroxyl group to be chemically modified.

CH₃O-(CH₂CH₂O)_n-CH₂CH₂-OH

Branched PEG is also commonly used. Branched PEG may be represented by R (-PEG-OH)_mWhere R represents, for example, a central core molecule of neopentyl glycol or glycerol and m represents the number of branched arms. The number of branching arms (m) can vary from three to 100 or more. The hydroxyl groups are chemically modified.

Other branched forms, as described in PCT patent application WO 96/21469, have a single end that is chemically modified. This PEG type can be expressed as (CH)₃O-PEG-)_pR-X, wherein p is equal to 2 or 3, R represents a central core of, for example, lysine or glycerol, and X represents a functional group such as a carboxyl group capable of chemical activation. While another branched form, the "pendant PEG" has a reactive group such as a carboxyl group (which is predominantly on the PEG backbone rather than at the end of the PEG chain).

In addition to these forms of PEG, polymers can also be prepared with weak or degradable bonds in the backbone. For example, Harris has shown in us patent application 06/026,716 that PEG can be prepared using ester linkages in the backbone of a polymer that can undergo hydrolysis. This hydrolysis (reaction) results in the polymer being cut into smaller molecular weight fragments, according to the reaction scheme:

-PEG-CO₂-PEG-+H₂O→-PEG-CO₂H+HO-PEG-

according to the invention, the term polyethylene glycol or PEG is meant to include all the above derivatives.

Co-polymers of ethylene oxide and propylene oxide are closely related in their chemistry to PEG and they can be used to replace many applications of PEG. They have the following general formula:

HO-CH₂CHRO(CH₂CHRO)_nCH₂CHR-OH wherein R is H or CH₃。

PEG is a useful polymer with properties of high water solubility and high solubility in many organic solvents. PEG is neither toxic nor immunogenic. When PEG and water-insoluble compounds are chemically combined (PEGylated), the resulting conjugates are generally water-soluble and soluble in many organic solvents.

PEG-protein conjugates are now used in protein replacement therapy and other therapeutic applications. For example, PEGylated Adenosine Deaminase (ADAGEN)^®) Will be used for the treatment of Severe Combined Immunodeficiency Disease (SCIDS), PEGylated L-asparaginase (ONCAPSPAR)^®) Is being used to treat Acute Lymphoblastic Leukemia (ALL), while pegylated interferon-alpha (INTRON (R) A) is being tested in a third phase trial for the treatment of hepatitis C.

For a review of PEG protein conjugates with clinical efficacy, we can see n.l. burnham,Am.J.Hosp.Pharm.，15：210-218，1994。

different methods for obtaining pegylated proteins have been developed. Conjugation of PEG to reactive groups found on proteins is typically accomplished using electrophilically activated PEG derivatives. PEG was conjugated to lysine residues and alpha-and epsilon-amino groups found at the N-terminus to give conjugates that constitute a product mixture.

Typically, these conjugates include conjugates in which the number of PEG molecules bound per protein molecule varies from 0 to the number of amino groups in the protein. For protein molecules that have been modified individually, PEG units can be attached at a number of different amine sites.

This type of non-specific PE curing has produced many conjugates that become almost inactive. The reduced activity is usually due to the shielding of the active binding domain of the protein as in many cytokines and antibodies. For example, Katre et al, in U.S. Pat. No. 4,766,106 and U.S. Pat. No. 4,917,888, describe PEGylation of IFN- β and IL-2 with a large excess of methoxy-polyethylene glycol-based N-succinimidyl glutaric acid and methoxy-polyethylene glycol-based N-succinimidyl succinic acid. Both proteins are produced in microbial host cells, which allow site-specific mutation of the free cysteine position to serine. Mutations are necessary in the microbial expression of IFN- β to promote protein folding. In particular, the IFN- β used in these experiments was the commercial product Betaseron^®Wherein Cys is¹⁷Is replaced by serine. In addition, the lack of glycosylation reduces its solubility in aqueous solutions. Non-specific pegylation results in increased solubility, but the main problem is reduced levels of activity and yield.

European patent application EP 593868 (entitled PEG-interferon conjugates), describes the preparation of PEG-IFN-alpha. However, the PEGylation reaction is not site-specific, and thus a mixture of positional isomers of PEG-IFN alpha conjugates is obtained (see also Monkarsh et al,ACS.Symp.Ser.，680：207-216，1997)。

the selective modification of the N-terminal residue of Megakaryocyte Growth and Development Factor (MGDF) with mPEG-propionaldehyde is demonstrated by Kinstler et al in European patent application EP 675201. This allows for batch-to-batch reproducible pegylation and pharmacokinetics. Gilbert et al, in U.S. Pat. No. 5,711,944, demonstrated PEGylation that produced IFN- α with optimal levels of activity. In this case laborious purification steps are required to obtain the optimal conjugate.

Most cytokines, as well as other proteins, do not have specific PEG binding sites and, in addition to the examples mentioned above, it is likely that some of the isoforms produced by the pegylation reaction are partially or completely inactive, resulting in loss of activity of the final mixture.

Site-specific mono-pegylation is therefore an ideal target in the preparation of these protein conjugates.

Woghiren et al inBioconjugate Chem.,4(5): thiol-selective PEG derivatives were synthesized for such site-specific PEGylation in 314-318, 1993. The stable thiol-protected PEG derivative is shown to be specifically conjugated as a disulfide-p-pyridine reactive group to the free cysteine of the protein papain. The newly formed disulfide bond of papain and the PEG ester bond can be cleaved under mild degradation conditions to regenerate the native protein.

The citation of any document herein is not an admission that such document is prior art or is prior art with respect to any patentable material of the present application. Any statement as to the contents and timing of any document is based on the information available to the applicant at the time of filing and is not an admission as to the correctness of the statement.

Disclosure of Invention

Provided herein are polyol-IFN- β conjugates, particularly PEG-IFN- β conjugates, in which the polyol unit is conjugated to Cys¹⁷And (4) covalently bonding. By reacting a thiol-reactive polyol agent with Cys in IFN- β¹⁷Residue reaction to obtain specific conjugate. It is expected that such conjugates will exhibit enhanced action in vivo. The goal is to achieve increased solubility at moderate pH, increased stability (reduced aggregation), reduced immunogenicity, and no loss of activity compared to "native" IFN- β. The result of such polymerization is to reduce the dosage required to achieve the desired effect, simplify and stabilize the formulation of the pharmaceutical composition, and possibly improve long-term efficacy.

The invention also provides a method for continuous stepwise conjugation of PEG molecules to polypeptides.

Drawings

FIG. 1 shows a diagram of Capillary Electrophoresis (CE) before purification of PEG-IFN- β.

FIGS. 2A-2C show PEG-IFN- β conjugate purification by size exclusion chromatography (Superose 12): figure 2A-first pass; FIG. 2B-two passes; figure 2C-third pass.

FIG. 3 shows SDS-PAGE chromatography of purified PEG-IFN- β conjugate from a third pass. Lanes 1 and 4 are protein molecular weight standards, lane 2 is "native" IFN- β, and lane 3 is a PEG-IFN- β conjugate.

FIG. 4 reports purified PEG-IFN- β conjugates (where IFN was mPEG-OPSS)_5kPegylation) of the sample.

FIG. 5 reports MALDI MS chromatography of purified PEG-IFN- β conjugates.

Fig. 6 shows "natural: comparison of the antiviral Activity of IFN- β and PEG-IFN- β conjugates. Samples of IFN- β at the indicated concentrations were incubated with WISH cells for 24 hours prior to challenge with the cytopathic dose of vesicular stomatitis virus. After a further 48 hours the cytopathic effect was determined by the MTT method.

FIG. 7 shows the binding of IFN- β and PEG-IFN in Daudi cells.

FIG. 8 shows the pharmacokinetics of IFN- β and PEG-IFN in mice after intravenous administration. The dashed line indicates the LOQ measurement for each standard curve.

FIG. 9 shows the pharmacokinetics of IFN- β and PEG-IFN in mice following subcutaneous administration. The dashed line indicates the LOQ measurement for each standard curve.

Detailed Description

The invention is based on polyol molecules, in particular PEG molecules, with Cys of human IFN-beta¹⁷The binding of residues was unexpectedly improved over (or to) native human interferon-betaLess retained and did not result in diminished) IFN- β biological activity. Thus, having a polyol molecule with Cys¹⁷Not only does the residue-bound IFN- β exhibit the same or increased IFN- β activity, but the polyol-IFN- β conjugate also provides desirable properties conferred by the polyol molecule, such as increased solubility.

"IFN- β", as used herein, refers to human fibroblast interferon, as obtained by isolation from biological fluids, or from prokaryotic or eukaryotic host cells using recombinant DNA techniques, and salts, functional derivatives, precursors and active fragments thereof, defined as containing a cysteine residue at position 17 in naturally occurring form.

According to the present invention, the polyol molecule in the polyol-IFN- β conjugate may be any water-soluble mono-or bifunctional poly (alkylene oxide) group having linear or branched chains. Typically, the polyol is poly (ethylene glycol) (PEG). However, one skilled in the art will recognize that other polyols, such as poly (glycerol) and copolymers of polyethylene glycol and polypropylene glycol, can also be suitably used.

The term "PEG molecule" as used herein refers to molecules including, but not limited to, linear or branched PEG, methoxy PEG, hydrolyzable or enzymatically cleavable PEG, pendant PEG, dendrimer PEG, copolymers of PEG and one or more polyols, and PEG and PLGA (poly (lactic/glycolic acid)) copolymers.

The definition "salt" as used herein refers both to salts of carboxyl groups and to salts of amino functions of compounds obtainable by known methods. Salts of carboxyl groups include inorganic salts such as sodium, potassium, calcium salts and salts with organic bases formed, for example, with amines such as triethanolamine, arginine or lysine. Salts of amino groups include salts with inorganic acids, such as hydrochloric acid, and salts with organic acids, such as acetic acid.

The definition "functional derivative" as used herein refers to a derivative which can be prepared according to known methods from functional groups present in the side chains or in the terminal N-C-group of amino acid molecules and which is included in the present invention when it is pharmaceutically acceptable, i.e. when it does not destroy the activity of the protein or confer toxicity on the pharmaceutical compositions containing them. Such derivatives comprise esters or fatty acids of the carboxyl group and N-acyl derivatives of the free amino group or O-acyl derivatives of the free hydroxyl group and are formed by acyl groups, such as chain acyl-or aroyl-groups.

A "precursor" is a compound that is converted to IFN- β in the human or animal body.

By "active fragment" of a protein, the invention refers to any fragment or precursor of the compound itself polypeptide chain, alone or in combination with a related molecule or residue, such as a sucrose or phosphate residue, or a polymer of polypeptide molecules, associated therewith, when such fragment or precursor exhibits the same IFN- β activity as the drug.

The conjugates of the invention can be prepared by any method known in the art. According to one embodiment of the invention, IFN- β and the PEGylating agent are reacted in a suitable solvent to isolate and purify the desired conjugate, e.g., by performing one or more chromatographic methods.

"chromatographic process" refers to any process for separating components of a mixture by linking them to a support (stationary phase) through which a solvent (mobile phase) flows. The separation principle of chromatography is based on the different physical properties of the stationary and mobile phases.

Some specific types well known in the chromatography literature include: liquid phase, high performance liquid phase, ion exchange, absorption, affinity, separation, hydrophobic, reverse phase, gel filtration, ultrafiltration or thin layer chromatography.

As used herein, "thiol-reactive pegylating agent" refers to any PEG derivative that is capable of reacting with the thiol group of a cysteine residue. It may be PEG containing, for example, disulfide para-pyridine, vinyl sulfone, maleimide, iodoacetamide, and other functional groups. According to a preferred embodiment of the invention, the thiol-reactive PEGylating agent is a dithiopyridine (OPSS) derivative of PEG.

The PEGylating agent is used in its mono-methoxylated form, in which only one end can be coupled, or in a bifunctional form, in which both ends can be coupled, for example, in the form of two IFN- β s covalently linked to a PEG molecule to form a conjugate. The molecular weight is preferably between 500 and 100,000.

A typical reaction scheme for the preparation of the conjugates of the invention is as follows:

the second line of the above procedure reports a method for cleaving the PEG-protein bond. The mPEG-OPSS derivatives are highly selective for free sulfhydryl groups and react rapidly under IFN- β stable acidic pH conditions. High selectivity can be demonstrated by a reduction in coupling to IFN- β and the native form of PEG.

The disulfide bond created between the protein and the PEG molecule has been shown to be stable in circulation, but it is reduced upon entry into the cellular environment. It is therefore desirable that the conjugate does not enter the cell and remains stable in the circulation until cleared.

It is noted that the above reaction is site-specific, in that the other two Cys residues at positions 31 and 141 in the naturally occurring form of human IFN- β do not react with the thiol-reactive PEGylating agent due to the formation of disulfide bridges.

The invention also features a method for the sequential, stepwise conjugation of two or more PEG molecules to a polypeptide. This method is based on the recognition that low molecular weight activated PEG reacts more completely with steric hindrance reactive sites on proteins than high molecular weight activated PEG. PEG modification of expensive therapeutic proteins in order for PEG conjugates to be practical to produce, must be cost effective. In addition, in order to reduce glomerular filtration and optimize the pharmacological properties of the PEG-protein conjugate, the conjugate must have an effective size comparable to a protein with a molecular weight of 70 kDa. This means that for site-specific modification to which one PEG is to be attached, it is preferred to attach one PEG derivative having a molecular weight above 20 kDa. If the modification sites are sterically crowded, reactive groups on large PEG molecules may be difficult to reach the modification sites and thus will result in low yields. Preferred methods for PEGylating polypeptides according to the invention improve the yield of site-specific PEGylation by first incorporating small heterobifunctional or homobifunctional PEG molecules that can react with sterically crowded sites due to their relatively small size. Subsequent conjugation of large molecular weight PEG derivatives with the small PEG yields high yields of the desired PEGylated protein.

According to the present invention, a method for the sequential stepwise conjugation of two or more PEG molecules to a polypeptide comprises: first, a low molecular weight hetero-bifunctional or homo-bifunctional PEG molecule is conjugated to a polypeptide, and then a mono-functional or bifunctional PEG molecule is conjugated to the end of the low molecular weight PEG molecule conjugated to the polypeptide. Two or more PEG molecules are successively and stepwise conjugated to the polypeptide (wherein the polypeptide is preferably IFN- β and wherein the Cys is located at a sterically crowded site)¹⁷Is a preferred site for PEG attachment), the PEG-polypeptide conjugate can be purified using one or more purification techniques such as ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography, and reverse phase chromatography.

The low molecular weight PEG molecules have the molecular formula:

W-CH₂CH₂O(CH₂CH₂O)_nCH₂CH₂-X, wherein W and X are groups that react with amine, thiol, carboxyl or hydroxyl functional groups, respectively, to bind low molecular weight PEG molecules and polypeptides. W and X are preferably independently selected from the group consisting of dithio-p-pyridine, maleimide, vinyl sulfone, iodoacetamide, amine, thiol, carboxyl, active ester, benzotriazole carbonate, p-nitrophenyl carbonate, isocyanate, and biotin. The low molecular weight PEG molecules preferably have a molecular weight in the range of about 100 to 5,000 daltons.

The monofunctional or bifunctional PEG molecule used to bind to the free end of the low molecular weight PEG conjugated to the polypeptide preferably has a molecular weight ranging from about 100 daltons to 200kDa, and is preferably methoxy PEG, branched PEG, hydrolysable or enzymatically cleavable PEG, pendant PEG, or dendrimer PEG. However, functional or bifunctional PEG also has the formula:

Y-CH₂CH₂O(CH₂CH₂O)_MCH₂CH₂-Z, wherein Y reacts with the free end group of the low molecular weight PEG molecule bound to the polypeptide and Z is-OCH₃Or a group that reacts therewith to form a bifunctional conjugate.

PEG-polypeptide conjugates produced by a process in which two or more PEG molecules are successively conjugated stepwise to a polypeptide, wherein the polypeptide is effective as an active ingredient, can be used to produce a medical or pharmaceutical composition for the treatment of a disease or disorder.

It is another object of the invention to provide the conjugates in a sufficiently purified form to render them suitable for use as pharmaceutical compositions, as active ingredients in the treatment, diagnosis or prognosis of bacterial and viral infections and autoimmune, inflammatory diseases and tumours. Such a pharmaceutical composition represents a further object of the present invention.

Non-limiting examples of such diseases include: septic shock, AIDS, rheumatoid arthritis, lupus erythematosus and multiple sclerosis.

Other embodiments and advantages of the invention will be demonstrated in the following description.

One embodiment of the invention is to administer a pharmacologically active amount of a conjugate of the invention to a person threatened by the occurrence of one of the diseases reported above or to a person already showing such a pathological state.

Any route of administration compatible with the active principle can be used. Parenteral administration, such as subcutaneous, intramuscular or intravenous injection, is preferred. The dose of active ingredient to be administered is determined on the basis of the medical prescription with reference to the age, weight and individual response of the patient.

For an average body weight of 75 kg, the dose may be between 10 micrograms and 1mg per day, whereas a preferred daily dose is between 20 micrograms and 200 micrograms.

Pharmaceutical compositions for parenteral administration can be prepared in injectable form containing the active principle and a suitable carrier. Carriers for parenteral administration are well known in the art and include, for example, water, saline solution, Ringer's solution and/or glucose. The carrier can contain small amounts of excipients to maintain stability and isotonicity of the pharmaceutical preparation. Solution preparation can be carried out in accordance with conventional formats.

The present invention has been described in terms of specific embodiments, but the description includes all variations and alternatives that may be suggested by those skilled in the art, not exceeding the intent and intent of the claims.

The invention will now be illustrated by the following examples, which are not intended to limit the invention in any way.

Example 1: preparation of PEG-IFN-beta conjugates

mPEG for IFN-beta_5kModification of OPSS

Recombinant human IFN- β stable at a 50mM sodium acetate buffer concentration of 0.37mg/ml, pH3.6, was used for PEG-IFN- β preparation. Approximately 1.0ml of 6M urea was added at a concentration of 0.37mg/ml (0.74mg, 3.7X 1)^-8Mole) was added to 2ml of IFN- β. Addition of mPEG as the molar remainder of 50M to 1M IFN- β_5K-OPSS, both reacted in polypropylene bottles at 37 ℃ for 2 hours or 50 ℃ for 1 hour. Prior to any purification step, the reaction mixture was analyzed by Capillary Electrophoresis (CE) mapping to determine the extent of PEG-IFN- β conjugate formation by PEGylation (FIG. 1). The typical yield of this reaction is 50% PEG-IFN- β. The reaction product was filtered from the reaction mixture using a 0.22mM syringe filter, and the filtrate was applied to a size exclusion chromatography column (Superose12 or Suoerdex 75, Pharmacia) and eluted with 50mM sodium phosphate, 150mM NaCl, pH7.5 buffer. FIG. 2A shows the elution profile of the PEG-IFN- β conjugate purified on a Superose12 size exclusion chromatography column. Peaks were collected and analyzed by SDS-PAGE (FIG. 3). Because the "natural" IFN- β peaks are close together (FIG. 2B), the fractions containing the PEG-IFN- β conjugate are pooled together and the concentrate is then re-applied to the same size exclusion column to re-purify the PEG-IFN- β conjugate. The procedure was repeated again (third time)Pass) to ensure purity (fig. 2C). FIGS. 4 and 5 show the capillary electrophoresis and MALDI MS chromatograms of purified PEG-IFN- β conjugates, respectively.

mPEG for IFN-beta_30KModification of OPSS

It is provided at 0.36mg/ml in 50mM sodium acetate buffer, pH3.6StabilizedRecombinant human IFN- β. Will dissolve in 3ml of deionized H₂About 36mg mPEG of O_30KOPSS was added to a concentration of 0.36mg/ml (1.08mg, 4.9X 10)^-8Molal) of 3ml of IFN- β. The two were allowed to react in a polypropylene tube at 50 ℃ for 2 hours. The reaction mixture was analyzed for the degree of modification by capillary electrophoresis. The typical yield of the reaction is less than 30%. The solution was then applied to a size exclusion chromatography column (Superose12, Pharmacia) and eluted with 50mM sodium phosphate, 150mM NaCl, pH7.0 buffer. Peaks were collected and the contents analyzed by SDS-PAGE.

Example 2: biological Activity of PEG-IFN- β conjugates

To evaluate the effect of pegylation on the antiviral activity of human recombinant IFN- β, human WISH amniotic cells were pre-cultured with freshly prepared IFN- β (same batch used for pegylation) or PEG-IFN- β conjugates. According to the method based on Novick et al,journal of immunology,129: 2244 (1982) as tested using the WISH-VSV cytopathic assay to determine IFN- β mediated antiviral activity. The materials used in this WISH test were as follows:

WISH cells (ATCC CCL 25)

The vesicular stomatitis virus stock (ATCC V-520-

IFN-. beta.human recombinant, Interpharm Laboratories LTD (32, 075-species, Batch #205035), 82X 10⁶IU/ml, specific activity: 222X 10⁶IU/mg

PEG-IFN- β conjugate prepared as in example 1, stored in PBS, pH7.4

WISH growth medium (MEM high glucose and Earls salts + 10% FBS + 1.0% L-Glutamine + penicillin/streptomycin (100U/ml, 100. mu.g/ml))

WISH test medium (MEM high glucose and Earls salt + 5% FBS + 1.0% L-Glutamine + penicillin/streptomycin (100U/ml, 100. mu.g/ml))

5mg/ml MTT in PBS, stored at-70 ℃.

The protocol for the WISH test is as follows:

IFN- β samples were diluted to the starting concentration in the 2XWISH assay medium.

3-fold dilutions of IFN- β samples in WISH assay medium were placed in flat bottom 96-well plates so that each well contained 50 microliters of diluted IFN- β sample (some control wells contained only 50 microliters of WISH assay medium)

WISH cells in the logarithmic growth phase were collected with a trypsin/EDTA solution, washed with WISH assay medium, and brought to a final concentration of 0.8X 10⁶Cells/ml.

50 microliters of WISH cell suspension (4X 10) was added⁴Cells/well) were added to each well. The final concentration of IFN- β contacted with the cells was 1X.

At 5% CO₂After 24 hours incubation in a humidified incubator, 50 microliters of a 1: 10 diluted (in WISH test medium) VSV stock (at a dose intended to lyse 100% WISH cells over 48 hours) was added to all wells except the virus-free control wells (these were added only to an equal volume of test medium).

After an additional 48 hours, 25 microliters of MTT solution was added to all wells, and the plates were then incubated in the incubator for an additional 2 hours.

The contents of the wells were removed by inverting the plate and 200 microliters of 100% ethanol was added to the wells.

After 1 hour, plates were read at 595nm using the Soft max Pro software package and the Spectramax spectrophotometer System (molecular devices).

Table 1.PEGylation and mock-PEGylation of the antiviral Activity of IFN-beta samples

IFN-beta samples*	EC50 **
		PEG-IFN-beta conjugates	3.9+/-0.7pg/ml
IFN-β	16.4+/-1.0pg/ml

^*Stock concentration of IFN-beta in samples determined by amino acid analysis

^**EC determined with the software program Microcal Origin 4.1₅₀(+/-S.D.)

As shown in FIG. 6 and Table 1 above, the PEG-IFN- β conjugate maintained a level of antiviral activity that was higher than that of the freshly prepared IFN- β parent batch.

It was observed that the PEG-IFN- β conjugates had approximately 4-fold greater biological activity than the freshly prepared IFN- β, which may also be the result of greater stability of the PEG-IFN- β conjugates after addition to the WISH cell assay medium as compared to the "native" IFN- β.

Example 3: in vitro assay of the relative Activity of PEG-IFN samples

The relative biological activities of PEG [30kD ] -IFN- β and PEG [2X20kD ] -IFN- β were determined by the WISH assay using the standard protocol described in example 2 (Table 2). Three independent experiments were performed by three different persons at different times.

TABLE 2 relative antiviral Activity of PEG-IFN- β

	Relative interferon activity*(from three studies)
					Sample (I)	Test 1	Test 2	Test 3	Average (S.D.)
PEG[30kD]-IFN-β	3.2X higher	3.1X higher	1.8X higher	3.0X (0.78) higher
					PEG[2×20kD]-IFN-β	4.2X higher	1.3X higher	0.85X higher	2.1X (1.8) higher

^*EC50 dose was compared to the standard IFN- β contained in each assay

^**The comparison was based on 330 micrograms/ml IFN-. beta.concentration. Assay of PEG [30kD ] with AAA]IFN-. beta.s (5.41. mu.g/ml) and PEG [2X20kD]Stock concentration of IFN- β (6.86. mu.g/ml).

At a fixed amount¹²⁵Presence of I-IFN-alpha 2aThe binding of PEG-IFN- β to its receptor on cells was evaluated as follows. Using the chloramine T method¹²⁵I radiolabeling IFN-. alpha.2a. The reaction was passed through a Sephadex G25 column and the fractions-containing protein (Pharmacia) were combined¹²⁵I combined with IFN alpha 2a in removal of free iodine. Quantitation with IFN-. alpha.2a ELISA assay (Biosource, USA)¹²⁵I-IFN-. alpha.2a and the specific activity was determined. The logarithmic phase of Daudi cells were collected and 2X 10 cells were assayed in the presence of PEG-IFN-. beta.or IFN-. alpha.2a diluted in RPMI1640 assay buffer containing 2% fetal bovine serum and 0.1% sodium azide at various concentrations⁶Cells and 0.5nM¹²⁵I-IFN-. alpha.2a was incubated at room temperature for 3 hours. At the end of the culture, cells were centrifuged through a layer of phthalate oil and the cell-bound radioactivity was counted in a gamma counter. In addition, PEG [30kD ]]IFN-. beta.and PEG [2X20kD]The binding of IFN- β to the receptor is very similar or close to the binding activity shown in FIG. 7.

In addition, relative activity was determined in Daudi cell (human B-cell lymphoma) antiproliferative assays (table 3). All IFNs were prepared at a 2X concentration of 200 ng/ml. The sample was diluted 3 times the length of the plate to a final volume of 100 microliters. Will be 1 × 10⁵Cells/well (100. mu.l) were added to each well and incubated in CO₂The cells were co-cultured in a humidified incubator at 37 ℃ for 72 hours. After 48 hours, tritium (E) (1. mu. Ci/well in 20. mu.l³H) Thymidine. At the end of the 72 hour culture, plates were collected using a Tomtek Plate Harvester. The results shown in table 3 indicate that no detectable loss of IFN was observed from pegylation. In fact, the activity was found to be slightly higher than that of free IFN- β. This may be due to the formation of inactive aggregates in the free IFN or to differences between the quantitative methods (amino acid analysis for PEG-IFN samples and RP-HPLC for IFN- β).

TABLE 3 Daudi antiproliferation test

	IC50Dosage form*	Fold increase for IFN
			IFN-beta (plate 1)	1153.1	-
PEG[30kD]-IFN(71A)	695.6	1.6X
			IFN-beta (plate 2)	1005.8	-
PEG[40kD]-IFN(71B)	629.4	1.7X

^*pg/ml

Example 4: pharmacokinetic study of intravenous drug administration in mice

Mice were injected with 100ng of IFN- β, PEG [30kD ] -IFN- β or PEG [2X20kD ] -IFN- β, followed by blood sampling at the indicated times. IFN- β serum concentrations were determined using IFN- β specific ELIAS (Toray industries), and the results are shown in FIG. 8. 28 female B6D2F1 mice (6-8 weeks) (approximately 20 grams each) were divided into 4 groups as follows: group 1 contained 9 mice that were bolus injected with 200 microliters of 500ng/ml human IFN- β (final dose of 100 ng/mouse); group 2(9 mice) received 200 microliters of an equivalent amount of PEG30kD-IFN- β; while group 3 received 200 microliters of equivalent amounts of PEG (2x20kD) -IFN- β; while group 4 was 3 groups of non-injected mice as negative controls. Blood samples were collected at 9 designated times by using the capillary orbital venous plexus (approximately 200 microliters/sample). Blood samples were allowed to clot for 1 hour at room temperature and microcentrifuge. The sera were stored at-70 ℃ until all sera were collected. The presence of biologically active human IFN- β in serum was determined by the Toray assay. The results indicate that the area under the curve (AUC) is significantly increased in the PEG-IFN sample over the free IFN- β, while PEG [2X20kD ] -IFN- β is higher than PEG [30kD ] -IFN- β.

Subcutaneous administration of drugs

Mice were injected subcutaneously with IFN- β and PEG-IFN (100 ng/mouse). Figure 9 shows that the total area under the curve (AUC) of the PEG-IFN sample is significantly increased compared to free IFN- β. Pharmacokinetic studies were consistent with PEG-IFN samples with longer half-lives and increased area under the curve.

Example 5: conjugation of Low molecular weight PEG molecules to Polypeptides

IFN-beta and OPSS-PEG_2K-hydrazide linkage

Recombinant human IFN- β was dissolved at 0.33mg/ml in 50mM sodium acetate buffer, pH 3.8. Approximately 3.6mg (40 moles more than protein moles) of a heterobifunctional PEG reagent, OPSS-PEG_2KHydrazide (dissolved in 2ml of deionized water) was added to 3ml of 0.33mg/ml (0.99mg) IFN-. beta.and the two were allowed to react in a polypropylene tube at 45 ℃ for 1 hour. The reaction mixture was then analyzed by capillary electrophoresis to determine the degree of modification. Generally, yields vary from 90-97% depending on the purity of the IFN- β and PEG reagents. The solution was then applied to a size exclusion chromatography column (Superdex 75, Pharmacia) and eluted with 5mM sodium phosphate, 150mM NaCl, pH7.0 buffer. Peaks were collected and analyzed by SDS-PAGE. The monopegylated IFN- β components are pooled together and then used in a further modification step with high molecular weight PEG.

IFN- β and (OPSS)₂-PEG₃₄₀₀Connection of

Recombinant human IFN- β was dissolved at 0.33mg/ml in 50mM sodium acetate buffer, pH 3.8. Approximately 6.1mg (40 moles more than protein moles) of homobifunctional PEG reagent, (OPSS)₂-PEG₃₄₀₀(dissolved in 2ml of deionized water) was added to 3ml of 0.33mg/ml (0.99mg) IFN-. beta.and the two were allowed to react in a polypropylene tube at 50 ℃ for 2 hours. The extent of modification was determined by monitoring the reaction by non-degrading SDS-PAGE and analyzing the final reaction mixture by capillary electrophoresis. The typical modification of this reaction with IFN- β is greater than 95%. The solution was then applied to a size exclusion chromatography column (Superdex 75, Pharmacia) and eluted with 5mM sodium phosphate, 150mM NaCl, pH7.0 buffer. Peaks were collected and their contents were analyzed by SDS-PAGE. The monopegylated IFN- β components are combined together.

Example 6: conjugation of a second PEG molecule to a Low molecular weight PEGylated polypeptide

With mPEG_30kAcetaldehyde (ALD) vs. IFN-S-S-PEG_2kModification of hydrazides

Will be 20 moles more mPEG than protein_30kALD to IFN-S-S-PEG in example 5_2kHydrazide in combined components. The reaction was allowed to proceed at room temperature (25 ℃) for 4 hours and the amount of modification was determined by applying the sample to a size exclusion chromatography column (Superose 6, Pharmacia). The amount of modification in this reaction depends on the PEG reagent and the reaction conditions, and is typically greater than 80%.

Having now fully described this invention, it is to be understood that one of ordinary skill in the art can, without undue experimentation, practice the same within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention.

While the invention has been described in conjunction with specific embodiments thereof, it will be understood that other modifications may be made. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

All references cited herein, including journal articles or abstracts, published or unpublished U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are incorporated by reference herein in their entirety, including the data, tables, figures, and text in all cited references, and additionally, all references cited herein are also incorporated by reference in their entirety.

Reference to known method steps, conventional method steps, known methods, or conventional methods is not an admission in any aspect that any aspect, description, or embodiment of the present invention is disclosed, taught, or suggested in the relevant art.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such changes and modifications are to be considered within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is also to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

Claims (11)

1. A method for stepwise and continuous conjugation of polyethylene glycol to a polypeptide, comprising the steps of:

reacting a polypeptide with a low molecular weight hetero-or homo-bifunctional polyethylene glycol molecule, said low molecular weight polyethylene glycol molecule having a molecular weight in the range of 100 to 5,000 daltons, said polyethylene glycol molecule having the formula:

W-CH₂CH₂O(CH₂CH₂O)_nCH₂CH₂-X

wherein W and X are groups which react with amine, thiol, carboxyl or hydroxyl functional groups, respectively, to bind low molecular weight polyethylene glycol molecules to the polypeptide; and

reacting the low molecular weight polyethylene glycol molecule bound to the polypeptide with a monofunctional or bifunctional polyethylene glycol molecule having a molecular weight in the range of 100 daltons to 200kDa, such that the monofunctional or bifunctional polyethylene glycol molecule binds to the free end of the low molecular weight polyethylene glycol molecule, forming a polyethylene glycol-polypeptide conjugate.

2. The method of claim 1, wherein the monofunctional or bifunctional polyethylene glycol molecule has the following formula:

Y-CH₂CH₂O(CH₂CH₂O)_mCH₂CH₂-Z，

wherein Y is reactive with the free end group of a low molecular weight polyethylene glycol molecule bound to the polypeptide and Z is-OCH₃Or a group that reacts with X to form a bifunctional conjugate.

3. The method of claim 2, wherein the monofunctional or bifunctional polyethylene glycol molecule is methoxy polyethylene glycol, branched polyethylene glycol, hydrolyzable or enzymatically hydrolyzable polyethylene glycol, pendant polyethylene glycol, or dendrimer polyethylene glycol.

4. The method of claim 1, wherein W and X are each independently selected from the group consisting of paradisulfide, maleimide, vinyl sulfone, iodoacetamide, hydrazide, acetaldehyde, succinyl ester, epoxide, amine, thiol, carboxyl, active ester, benzotriazole carbonate, p-nitrophenyl carbonate, isocyanate, and biotin.

5. The method of claim 1, wherein the low molecular weight polyethylene glycol molecule is a copolymer of polyethylene glycol.

6. The method of claim 1, wherein the monofunctional or bifunctional polyethylene glycol molecule is a copolymer of polyethylene glycol.

7. The method of claim 5, wherein the low molecular weight polyethylene glycol molecule and the monofunctional or bifunctional polyethylene glycol molecule are both copolymers of polyethylene glycol.

8. The method of claim 5, wherein the copolymer of polyethylene glycol is selected from the group consisting of polyethylene glycol/polypropylene glycol copolymer and polyethylene glycol/poly (lactic acid/glycolic acid) copolymer.

9. The method of claim 1, further comprising the step of purifying the polyethylene glycol-polypeptide conjugate after two polyethylene glycol molecules are successively bound to the polypeptide in steps.

10. The method of claim 9, wherein the purifying step further comprises one or more purification techniques selected from the group consisting of: ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, affinity chromatography and reverse phase chromatography.

11. The method of any one of claims 1-10, wherein the polypeptide is interferon- β.