Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Reexamination Certificate
1994-06-14
2003-04-22
Lilling, Herbert J. (Department: 1651)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
C435S177000, C435S150000, C435S181000, C514S002600, C514S012200, C514S013800, C514S014800, C514S054000, C525S050000, C525S054100, C525S054110, C525S535000, C424S094300, C530S350000, C530S357000, C530S388200, C530S323000, C530S388230, C530S388850
Reexamination Certificate
active
06552170
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to active derivatives of polyethylene glycol and related hydrophilic polymers and to methods for their synthesis for use in modifying the characteristics of surfaces and molecules. The invention also relates to polypeptides that have been covalently bonded to such active derivatives and methods for making the same.
BACKGROUND OF THE INVENTION
Polyethylene glycol (“PEG”) has been studied for use in pharmaceuticals, on artificial implants, and other applications where biocompatibility is of importance. Various derivatives of PEG have been proposed that have an active moiety for permitting PEG to be attached to pharmaceuticals and implants and to molecules and surfaces generally. For example, PEG derivatives have been proposed for coupling PEG to surfaces to control wetting, static buildup, and attachment of other types of molecules to the surface, including proteins or protein residues.
PEG derivatives have also been proposed for affinity partitioning, for example, of enzymes from a cellular mass. In affinity partitioning, the PEG derivative includes a functional group for reversible coupling to an enzyme that is contained within a cellular mass. The PEG and enzyme conjugate is separated from the cellular mass and then the enzyme is separated from the PEG derivative, if desired.
In still further examples, coupling of PEG derivatives (“PEGylation”) is desirable to overcome obstacles encountered in the clinical use of biologically active molecules. Published PCT Publication No. WO 92/16221 states, for example, that many potentially therapeutic proteins have been found to have a short half life in the blood serum. For the most part, proteins are cleared from the serum through the kidneys. The systematic introduction of relatively large quantities of proteins, particularly those foreign to the human system, can give rise to immunogenic reactions that, among other problems, may lead to rapid removal of the protein from the body through formation of immune complexes. For other proteins, solubility and aggregation problems have also hindered the optimal formulation of the protein.
PEGylation decreases the rate of clearance from the bloodstream by increasing the apparent molecular weight of the molecule. Up to a certain size, the rate of glomerular filtration of proteins is inversely proportional to the size of the protein. The ability of PEGylation to decrease clearance, therefore, is generally not a function of how many PEG groups are attached to the protein, but the overall molecular weight of the altered protein. Decreased clearance can lead to increased efficiency over the non-PEGylated material. See, for example, Conforti et al.,
Pharm. Research Commun
. vol. 19, pg. 287 (1987) and Katre et al.,
Proc. Natl. Acad. Sci. U.S.A
. vol. 84, pg. 1487 (1987).
In addition, PEGylation can decrease protein aggregation (Suzuki et al.,
Biochem. Biophys. Acta
vol. 788, pg. 248 (1984)), alter protein immunogenicity (Abuchowski et al.,
J. Biol. Chem
. vol. 252 pg. 3582 (1977)), and increase protein solubility as described, for example, in PCT Publication No. WO 92/16221.
PEGylation of proteins illustrates some of the problems that have been encountered in attaching PEG to surfaces and molecules. The vast majority of PEGylating reagents react with free primary amino groups of the polypeptide. Most of these free amines are the epsilon amino group of lysine amino acid residues. Typical proteins possess a large number of lysines. Consequently, random attachment of multiple PEG molecules often occurs leading to loss of protein activity.
In addition, if the PEGylated protein is intended for therapeutic use, the multiple species mixture that results from the use of non-specific PEGylation leads to difficulties in the preparation of a product with reproducible and characterizable properties. This non-specific PEGylation makes it difficult to evaluate therapeutics and to establish efficacy and dosing information. The site selective PEGylation of such proteins could lead to reproducibly-modified materials that gain the desirable attributes of PEGylation without the loss of activity.
The need to reproducibly create complexes of two or more linked bioactive molecules or compounds also exists. In certain cases, the administration of multimeric complexes that contain more than one biologically active polypeptide or drug leads to synergistic benefits. For example, a complex containing two or more identical binding polypeptides may have substantially increased affinity for the ligand or active site to which it binds relative to the monomeric polypeptide. Alternatively, a complex comprised of (1) a bioactive protein that exerts its effect at a particular site in the body and (2) a molecule that can direct the complex to that specific site may be particularly beneficial.
A need also exists for hydrolytically-stable activated polymers which form linkages which are also hydrolytically stable. Otherwise, in certain cases, the reactive group can be rendered inactive before the desired reaction takes place or the conjugate formed after reaction has a short half life in aqueous media, such as blood or plasma.
For example, Zalipsky U.S. Pat. No. 5,122,614 describes that PEG molecules activated with an oxycarbonyl-N-dicarboximide functional group that can be attached under aqueous, basic conditions by a urethane linkage to the amine group of a polypeptide. Activated PEG-N-succinimide carbonate is said to form stable, hydrolysis-resistant urethane linkages with amine groups. The amine group is shown to more reactive at basic pHs of about 8.0 to 9.5, and reactivity falls off sharply at lower pHs. Hydrolysis of the uncoupled PEG derivative, however, also increases sharply at pHs of 8.0 to 9.5. Zalipsky avoids the problem of an increase in the rate of reaction of the uncoupled PEG derivative with water by using an excess of PEG derivative to bind to the protein. By using an excess of PEG derivative, sufficient reactive amino sites are bound to PEG to modify the protein before the PEG derivative becomes hydrolyzed and unreactive.
Zalipsky's method is adequate for nonspecific attachment of the lysine fraction of a protein to a PEG derivative at one active site on the PEG. If the rate of hydrolysis of the PEG derivative is substantial, however, then it can be problematic to provide attachment at more than one active site on the PEG molecule, since a simple excess does not slow the rate of hydrolysis.
For example, a linear PEG with active sites at each end will attach to protein at one end but the reactive site at the other end can react with water to form a relatively nonreactive hydroxyl moiety instead of a PEG linking two protein groups. A similar problem arises if it is desired to couple a molecule to a surface by a PEG linking agent because the PEG is first attached to the surface or couples to the molecule, and the opposite end of the PEG derivative must remain active for a subsequent reaction. If hydrolysis is a problem, then the opposite end typically becomes inactivated.
Zalipsky U.S. Pat. No. 5,122,614 also describes several other PEG derivatives from prior patents. PEG-succinoyl-N-hydroxysuccinimide ester is said to form ester linkages that have limited stability in aqueous media. PEG-cyanuric chloride is said to be toxic and is non-specific for reaction with particular functional groups on a protein which can lead to protein inactivation. PEG-phenylcarbonate is said to produce toxic hydrophobic phenol residues that have an affinity for proteins. PEG activated with carbonyldiimidizole is said to be too slow in reacting with protein functional groups, requiring long reaction times to obtain sufficient modification of the protein.
Still other PEG derivatives have been proposed for attachment to functional groups other than the epsilon amino group of lysine. Maleimide, for example, is specific for cysteine sulfhydryl but the maleimide functionality is subject to hydrolysis.
Accordingly, a need exists for reagents and methods for reproducibly creating complexes whose
Brewer Michael T.
Kohno Tadahiko
Thompson Robert C.
Amgen Inc.
Gaul Timothy J.
Levy Ron K.
Lilling Herbert J.
Odre Steven M.
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