Chemically modified novel erythropoietin stimulating protein compositions and methods

Abstract

The present invention broadly relates to the field of protein modification, and, more specifically, the attachment of water soluble polymers to novel erythropoietin stimulating protein (NESP).

Claims

What is claimed is: 1. A substantially homogenous preparation of a chemically modified hyperglycosylated protein, Novel Erythropoietin Stimulating Protein (NESP), wherein said NESP is chemically modified with polyethylene glycol (PEGylated); and wherein said preparation is comprised of a mixed population of mono-PEGylated NESP and poly-PEGylated NESP and at least 95% PEGylated NESP molecules. 2. A preparation of claim 1 wherein said NESP has the sequence identified in SEQ ID NO:1. 3. A preparation of claim 1 wherein said polyethylene glycol has a molecular weight of between 2 kD and 100 kD. 4. A preparation of claim 3 wherein said polyethylene glycol has a molecular weight of between 5 kD and 30 kD. 5. A pharmaceutical composition comprising: (a) a substantially homogenous preparation of a chemically modified hyperglycosylated protein, PEGylated NESP, said PEGylated NESP comprising a mixed population of mono-PEGylated NESP and poly-PEGylated NESP; (b) fewer than 5% non-PEGylated NESP molecules; and (c) a pharmaceutically acceptable diluent, adjuvant or carrier.
BACKGROUND OF THE INVENTION Novel erythropoietin stimulating protein (NESP) is a hyperglycosylated erythropoietin analog having five changes in the amino acid sequence of rHuEPO which provide for two additional carbohydrate chains. More specifically, NESP contains two additional N-linked carbohydrate chains at amino acid residues 30 and 88 (numbering corresponding to the sequence of human EPO)(see PCT Application No. US94/02957, herein incorporated by reference in its entirety). NESP is biochemically distinct from EPO, having a longer serum half-life and higher in vivo biological activity; Egrie et al., ASH 97 , Blood , 90:56a (1997). NESP has been shown to have ˜3 fold increase in serum half-life in mice, rats, dogs and man; Id. In mice, the longer serum half-life and higher in vivo activity allow for less frequent dosing (once weekly or once every other week) compared to rHuEPO to obtain the same biological response; Id. A pharmacokinetic study demonstrated that, consistent with the animal studies, NESP has a significantly longer serum half-life than rHuEPO in chronic renal failure patients, suggesting that a less-frequent dosing schedule may also be employed in humans; MacDougall, et al., J American Society of Nephrology , 8:268A (1997). A less frequent dosing schedule would be more convenient to both physicians and patients, and would be particularly helpful to those patients involved in self-administration. Other advantages to less frequent dosing may include less drug being introduced into patients, a reduction in the nature or severity of the few side-effects seen with rHuEPO administration, and increased compliance. Although the extended half-life of NESP offers the advantage of less frequent dosing relative to EPO, there are still potential indications, such as chemotherapy, which may require an even longer therapeutic half-life than NESP currently demonstrates. A common approach often used to extend the half-lives of proteins in vivo is the chemical conjugation of a water soluble polymer, such as polyethylene glycol (PEG), to the protein of interest. Generally, polyethylene glycol molecules-are connected to the protein via a reactive group found on the protein. Amino groups, such as those on lysine residues or at the N-terminus, are convenient for such attachment. A variety of approaches have been used to attach the polyethylene glycol molecules to the protein (PEGylation). For example, Royer (U.S. Pat. No. 4,002,531) states that reductive alkylation was used for attachment of polyethylene glycol molecules to an enzyme. Davis et al. (U.S. Pat. No. 4,179,337) disclose PEG:protein conjugates involving, for example, enzymes and insulin. Shaw (U.S. Pat. No. 4,904,584) disclose the modification of the number of lysine residues in proteins for the attachment of polyethylene glycol molecules via reactive amine groups. Hakimi et al. (U.S. Pat. No. 5,834,594) disclose substantially non-immunogenic water soluble PEG:protein conjugates, involving for example, the proteins IL-2, interferon alpha, and IL-1ra. The methods of Hakimi et al. involve the utilization of unique linkers to connect the various free amino groups in the protein to PEG. Kinstler et al. (U.S. Pat. Nos. 5,824,784 and 5,985,265) teach methods allowing for selectively N-terminally chemically modified proteins and analogs thereof, including G-CSF and consensus interferon. Importantly, these modified proteins have advantages as relates to protein stability, as well as providing for processing advantages. PEGylation approaches such as those described above are traditionally applied to non-glycosylated proteins derived from bacterial expression systems in order to render improvements in solubility and in vivo circulating half-lives (such properties are typically conferred to glycosylated proteins (glycoproteins) through the carbohydrate moieties added in the course of eukaryotic expression). The effects of PEGylation on the in vivo half-lives of non-glycosylated proteins is generally thought to derive from the physicochemical and dynamic properties of PEG conferring a larger hydrodynamic volume and total mass to the conjugate, thus reducing the rate of renal clearance. Additional benefits typically include increased solubility and decreased immunogenicity for the conjugate. However, not all proteins respond equally to PEGylation and there is no guarantee of improved performance. The present invention is based upon the surprising finding that a highly glycosylated protein, e.g., NESP, can be PEGylated to provide a pharmaceutical composition with an even more dramatic sustained duration profile than NESP, allowing for a once every 4-6 week dosing for raising hematocrit and treating anemia, and thus providing tremendous therapeutic advantage. SUMMARY OF THE INVENTION The present invention relates to a substantially homogenous preparation of chemically modified NESP (or analog thereof) and related methods. The present invention further relates to a substantially homogenous preparation of N-terminally chemically modified NESP (or analog thereof). The present invention further relates to a preparation of chemically modified NESP represented as a mixed population of either monosubstituted positional isoforms or polysubstituted forms. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 depicts the design strategy for NESP PEGylation: (A) PEG polymer size is varied from 5 kD, 20 kD and 30 kD; (B) PEG polymer conformation can be either linear or branched with total molecular weights of 10 kD, 20 kD or 40 kD PEG; and (C) preparations of PEG:NESP with different degrees of substitution can be isolated to include: mono-PEG, di-PEG or, in some cases, tri-PEG NESP. FIG. 2 depicts the various reaction chemistries for PEGylation of NESP: (A) reductive alkylation of NESP with PEG-aldehyde; (B) acylation of NESP with N-succinimidyl ester of PEG; and (C) PEGylation of the NESP polysaccharide side chains by limited periodate oxidation of the carbohydrate with the resultant aldehyde reacted with PEG-hydrazide to form a hydrazone linkage followed by subsequent reduction with sodium cyanoborohydride to stabilize the linkage. FIG. 3 is a graph depicting in vivo activity data of various 5 kD poly-PEG:NESP conjugates vs. unmodified NESP (▪). Samples -▴-, -▾-, --, and -♦- are mixtures of 5 kD poly-PEG:NESP with progressively lower degrees of substitution. % iron uptake is plotted vs. ng/mL administered. FIG. 4 is a graph depicting prolongation of elevated hemoglobin (HGB) levels in response to treatment with various PEG:NESP conjugates relative to unmodified NESP. Single bolus injection of 100 μg/kg NESP (♦), 20 kD linear mono-PEG:NESP conjugate derived from NHS-ester activated methoxy-PEG (▪), 20 kD linear (˜80% mono-PEG:NESP and 20% di-PEG:NESP) conjugate derived by reductive alkylation from aldehyde activated PEG (▾), and a saline control (). HGB (g/dL) is plotted vs. # days post-treatment. FIG. 5 is a graph depicting prolongation of elevated reticulocyte levels in response to treatment with various PEG:NESP conjugates relative to unmodified NESP. Single bolus injections of 100 μg/kg*NESP (O), 20 kD linear mono-PEG:NESP (), 5 kD linear mono-PEG:NESP (▾) and 5 kD linear di-PEG:NESP conjugates (♦) derived by reductive alkylation from aldehyde activated methoxy-PEG, a 20 kD branched mono-PEG:NESP (▪) conjugate from NHS-ester activated PEG, and a saline control (▴). Absolute reticulocyte count is plotted vs. # days post-treatment. FIG. 6 is a graph depicting prolongation of elevated hemoglobin levels in response to treatment with various PEG:NESP conjugates relative to unmodified NESP. Single bolus injections of 100 μg/kg NESP (O), 20 kD linear mono-PEG:NESP (), 5 kD linear mono-PEG:NESP (▾) and 5 kD linear di-PEG:NESP conjugates (♦) derived by reductive alkylation from aldehyde activated methoxy-PEG and a 20 kD branched mono-PEG:NESP conjugate (▪) from NHS-ester activated PEG. HGB (g/dL) is plotted vs. # days post-treatment. FIG. 7 depicts a Q Sepharose HP column chromatogram of the 5 kD poly-PEG:NESP conjugate. The column was a HiTrap Q Sepharose HP column which utilized a 50 mM NaCl to 200 mM NaCl linear gradient to elute the product. FIG. 8 depicts a Q Sepharose HP column chromatogram of the 20 kD mono-PEG:NESP conjugate. The column was a HiTrap Q Sepharose HP column which utilized a 50 mM NaCl to 200 mM NaCl linear gradient to elute the product. FIG. 9 depicts a Q Sepharose HP column chromatogram of the 30 kD mono-PEG:NESP conjugate. The column was a HiTrap Q Sepharose HP column which utilized a 50 mM NaCl to 200 mM NaCl linear gradient to elute the product. FIG. 10 is a graph depicting reticulocyte response of anemic mice after single bolus injections of 3 μg/kg 30 kD mono-PEG:NESP conjugate (▾), 3 μg/kg 20 kD mono-PEG:NESP conjugate (▪), and 3 μg/kg 5 kD poly-PEG:NESP conjugate mixture (). Absolute reticulocyte count is plotted vs. # days post-treatment. FIG. 11 is a graph depicting reticulocyte response of anemic mice after single bolus injections of 10 μg/kg 30 kD mono-PEG:NESP conjugate (▾), 10 μg/kg 20 kD mono-PEG:NESP conjugate (▪), and 10 μg/kg 5 kD poly-PEG:NESP conjugate mixture (). Absolute reticulocyte count is plotted vs. # days post-treatment. FIG. 12 is a graph depicting reticulocyte response of anemic mice after single bolus injections of 30 μg/kg 30 kD mono-PEG:NESP conjugate (▾), 30 μg/kg 20 kD mono-PEG:NESP conjugate (▪), and 30 μg/kg 5 kD poly-PEG:NESP conjugate mixture () vs. 30 μg/kg unmodified NESP (O). Absolute reticulocyte count is plotted vs. # days post-treatment. FIG. 13 is a graph depicting hemoglobin response of-anemic mice after single bolus injections of 3 μg/kg 30 kD mono-PEG:NESP conjugate (▾), 3 μg/kg 20 kD mono-PEG:NESP conjugate (▪), and 3 μg/kg 5 kD poly-PEG:NESP conjugate mixture (). HGB (g/dL) is plotted vs. # days post-treatment. FIG. 14 is a graph depicting hemoglobin response of anemic mice after single bolus injections of 10 μg/kg 30 kD mono-PEG:NESP conjugate (▾), 10 μg/kg 20 kD mono-PEG:NESP conjugate (▪), and 10 μg/kg 5 kD poly-PEG:NESP conjugate mixture (). HGB (g/dL) is plotted vs. # days post-treatment. FIG. 15 is a graph depicting hemoglobin response of anemic mice after single bolus injections of 30 μg/kg 30 kD mono-PEG:NESP conjugate (▾), 30 μg/kg 20 kD mono-PEG:NESP conjugate (▪), and 30 μg/kg 5 kD poly-PEG:NESP conjugate mixture () vs. 30 μg/kg unmodified NESP (O). HGB (g/dL) is plotted vs. # days post-treatment. FIG. 16 is a graph depicting reticulocyte response of normal mice after single bolus injections of 3 μg/kg 30 kD mono-PEG:NESP conjugate (▾), 3 μg/kg 20 kD mono-PEG:NESP conjugate (▪), and 3 μg/kg 5 kD poly-PEG:NESP conjugate mixture (). Absolute reticulocyte count is plotted vs. # days post-treatment. FIG. 17 is a graph depicting reticulocyte response of normal mice after single bolus injections of 10 μg/kg 30 kD mono-PEG:NESP conjugate (▾), 10 μg/kg 20 kD mono-PEG:NESP conjugate (▪), and 10 μg/kg 5 kD poly-PEG:NESP conjugate mixture (). Absolute reticulocyte count is plotted vs. # days post-treatment. FIG. 18 is a graph depicting reticulocyte response of normal mice after single bolus injections of 30 μg/kg 30 kD mono-PEG:NESP conjugate (▾), 30 μg/kg 20 kD mono-PEG:NESP conjugate (▪), and 30 μg/kg 5 kD poly-PEG:NESP conjugate mixture () vs. 30 μg/kg unmodified NESP (O). Absolute reticulocyte count is plotted vs. # days post-treatment. FIG. 19 is a graph depicting hemoglobin response of normal mice after single bolus injections of 3 μg/kg 30 kD mono-PEG:NESP conjugate (▾), 3 μg/kg 20 kD mono-PEG:NESP conjugate (▪), and 3 μg/kg 5 kD poly-PEG:NESP conjugate mixture (). HGB (g/dL) is plotted vs. # days post-treatment. FIG. 20 is a graph depicting hemoglobin response of normal mice after single bolus injections of 10 μg/kg 30 kD mono-PEG:NESP conjugate (▾), 10 μg/kg 20 kD mono-PEG:NESP conjugate (▪), and 10 μg/kg 5 kD poly-PEG:NESP conjugate mixture (). HGB (g/dL) is plotted vs. # days post-treatment. FIG. 21 is a graph depicting hemoglobin response of normal mice after single bolus injections of 30 μg/kg 30 kD mono-PEG:NESP conjugate (▾), 30 μg/kg 20 kD mono-PEG:NESP conjugate (▪), and 30 μg/kg 5 kD poly-PEG:NESP conjugate mixture () vs. 30 μg/kg unmodified NESP (O), HGB,(g/dL) is plotted vs. # days post-treatment. FIG. 22 depicts size exclusion HPLC chromatograms of the 5 kD poly-PEG:NESP (—), the 20 kD mono-PEG:NESP ( - - - ) and 30 kD mono-PEG:NESP ( - - - ). The SEC column was a Tosohaas TSK 3000 SW×1 (5 micron-7.8 mm×30 cm) which utilized 100 mM NaHPO 4 , 10% ethanol, 150 mM NaCl, pH 6.9, to elute the products. DETAILED DESCRIPTION OF THE INVENTION To discover if the in vivo therapeutic half-life of a glycoprotein such as NESP would benefit from PEGylation, a variety of different PEG:NESP conjugates were synthesized and tested in vivo for prolonged erythropoiesis. In order to both optimize the potential effects of PEGylation and to identify the preferred sites and chemistries of PEG attachment, a design strategy was employed wherein polymer length, conformation, and both the degree and sites of attachment were varied (see FIG. 1 ). Methods for preparing the PEGylated NESP of the present invention generally comprise the steps of (a) reacting NESP with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby NESP becomes attached to one or more PEG groups, and (b) obtaining the reaction product(s). Because the specific sites of NESP modification might significantly alter the intrinsic activity of the conjugate, three different PEGylation chemistries were explored (see FIG. 2 ). The first approach utilizes reductive alkylation to conjugate a PEG-aldehyde (O-(3-Oxopropyl)-O′-methylpolyethylene glycol) to a primary amine of NESP. Under appropriate conditions, this approach has been demonstrated to yield PEG conjugates predominately modified through the α-amine at the protein N-terminus. Because the PEG is linked through a secondary amine by reductive alkylation there is the potential to preserve the charge at the protein N-terminus. The second chemistry applied to PEGylation of NESP was the acylation of the primary amines of NESP using the NHS-ester of methoxy-PEG (O-[(N-Succinimidyloxycarbonyl)-methyl]-O′-methylpolyethylene glycol). In contrast to the previous chemistry, acylation with methoxy-PEG-NHS results in an amide linkage which will eliminate the charge from the original primary amine. The final attachment chemistry evaluated utilized a mild oxidation of NESP under conditions selected to target the pendant diol of the penultimate glycosyl unit sialic acid for oxidation to an aldehyde. The resultant glycoaldehyde was then reacted with a methoxy-PEG-hydrazide (O-(Hydrazinocarbonylmethyl)-O′-methylpolyethylene glycol) to form a semi-stable hydrazone between the PEG and NESP. The hydrazone was subsequently reduced by sodium cyanoborohydride to produce a stable PEG:NESP conjugate. The present methods each provide for a substantially homogenous mixture of polymer:protein conjugate. “Substantially homogenous” as used herein means that only polymer:protein conjugate molecules are observed. As ascertained by peptide mapping and N-terminal sequencing, one example below provides for a preparation which-is at least 90% polymer:protein conjugate, and at most 10% unreacted protein. Preferably, the PEGylated material is at least 95% of the preparation (as in the working example below) and most preferably, the PEGylated material is 99% of the preparation or more. The polymer:protein conjugate has biological activity and the present “substantially homogenous” PEGylated NESP preparations provided herein are those which are homogenous enough to display the advantages of a homogenous preparation, e.g., ease in clinical application in predictability of lot to lot pharmacokinetics. One may also choose to prepare a mixture of polymer:protein conjugate molecules, and the advantage provided herein is that one may select the proportion of mono-polymer:protein conjugate to include in the mixture. Thus, if desired, one may prepare a mixture of various protein with various numbers of polymer moieties attached (i.e., di-, tri-, tetra-, etc.) and combine said conjugates with the mono-polymer:protein conjugate prepared using the present methods, and have a mixture with a predetermined proportion of mono-polymer:protein conjugate. Initial experiments designed to evaluate and optimize PEG:protein reaction stoichiometries revealed that PEGylation by reductive alkylation using PEG-aldehyde was surprisingly somewhat inefficient, requiring substantially higher molar ratios of PEG to protein than typically observed with non-glycosylated proteins. Similarly, acylation with PEG-NHS esters was also slower and less efficient than expected. It was thus evident that the PEGylation of non-glycosylated proteins was not necessarily predictive of the PEGylation of glycosylated proteins and that further optimization of reaction conditions was necessary. The polymer molecules contemplated for use in the PEGylation approaches described herein may be selected from among water soluble polymers or a mixture thereof. The water soluble polymer may be selected from the group consisting of, for example, polyethylene glycol, monomethoxy-polyethylene glycol, dextran, poly-(N-vinyl pyrrolidone), propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol), dextran, HPMA, Fleximer™, and polyvinyl alcohol. The polymer selected should be water soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. For the acylation reactions, the polymer(s) selected should have a single reactive ester group. For the present reductive alkylation, the polymer(s) selected should have a single reactive aldehyde group. A preferred reactive PEG-aldehyde is polyethylene glycol propionaldehyde, which is water stable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (see, U.S. Pat. No. 5,252,714). The polymer may be branched or unbranched. Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable. A particularly preferred water-soluble polymer for use herein is polyethylene glycol, abbreviated PEG. As used herein, polyethylene glycol is meant to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol. The proportion of polyethylene glycol molecules to protein molecules will vary, as will their concentrations in the reaction mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is no excess unreacted protein or polymer) will be determined by the molecular weight of the polyethylene glycol selected and on the number of available reactive groups (typically ∝ or ∃ amino groups) available. As relates to molecular weight, the higher the molecular weight of the polymer, the fewer number of polymer molecules which may be attached to the protein. Similarly, branching of the polymer should be taken into account when optimizing these parameters. Generally, the higher the molecular weight (or the more branches) the higher the polymer:protein ratio. In the present invention, several different linear PEG polymer lengths were evaluated (5 kD, 20 kD and 30 kD). Similarly, conjugates of two-armed branched PEG polymers (10 kD, 20 kD and 40 kD) were also tested. From each preparation, samples of mono-substituted and di-substituted PEG:NESP were isolated to investigate the effects of secondary sites of PEGylation. In general, for the PEGylation reactions contemplated herein, the preferred average molecular weight is about 2 kDa to about 100 kDa (the term “about” indicating ±1 kDa). More preferably, the average molecular weight is about 5 kDa to about 40 kDa. The ratio of water-soluble polymer to NESP will generally range from 1:1 for monoPEG-, 2:1 for diPEG etc, and the mass ratios for PEG:protein would run ˜1:7 for 5 kD mono-PEG to ˜1:1.3 for 30 kD monoPEG. The method of obtaining the PEGylated NESP preparation may be by purification of the PEGylated material from a population of non-PEGylated NESP molecules. For example, presented below is an example where mono- and/or di-PEGylated NESP is separated using ion exchange size chromatography. Size exclusion chromatography is used as an analytical tool to characterize the purified products. The present invention also provides a method for selectively obtaining N-terminally chemically modified NESP. The method comprises reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved. The reaction is performed at pH which allows one to take advantage of the pK a differences between the ε-amino groups of the lysine residues and that of the α-amino group of the N-terminal residue of the protein. By such selective derivatization attachment of a water soluble polymer to a protein is controlled: the conjugation with the polymer takes place predominantly at the N-terminus of the protein and no significant modification of other reactive groups, such as the lysine side chain amino groups, occurs. The preparation will preferably be greater than 80% mono-polymer:protein conjugate, and more preferably greater than 95% mono-polymer:protein conjugate. NESP of the present invention is a hyperglycosylated EPO analog comprising two additional glycosylation sites with an additional carbohydrate chain attached to each site. NESP was constructed using site-directed mutagenesis and expressed in mammalian host cells. Details of the production of NESP are provided in co-owned PCT Application No. US94/02957. New N-linked glycosylation sites for rHuEPO were introduced by alterations in the DNA sequence to encode the amino acids Asn-X-Ser/Thr in the polypeptide chain. DNA encoding NESP was transfected into Chinese Hamster Ovary (CHO) host cells and the expressed polypeptide was analyzed for the presence of additional carbohydrate chains. In a preferred embodiment, NESP will have two additional N-linked carbohydrate chains at residues 30 and 88. The numbering of the amino acid sequence is that of human erythropoietin (EPO). The amino acid sequence of NESP is that depicted in SEQ ID NO: 1. It is understood that NESP will have the normal complement of N-linked and O-linked glycosylation sites in addition to the new sites. The NESP of the present invention may also, include conservative amino acid changes at one or more residues in SEQ ID NO: 1. These changes do not result in addition of a carbohydrate chain and will have little effect on the biological activity of the analog. In general, comprehended by the present invention are pharmaceutical compositions comprising effective amounts of protein or derivative products of the invention together with pharmaceutically acceptable diluents, stabilizers, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Polysorbate 20, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435:1712 which are herein incorporated by reference. An effective amount of active ingredient is a therapeutically, prophylactically, or diagnostically effective amount, which can be readily determined by a person skilled in the art by taking into consideration such factors as body weight, age, and therapeutic or prophylactic goal. The PEG:NESP compositions of the present invention may also include a buffering agent to maintain the pH of the solution within a desired range. Preferred agents include sodium acetate, sodium phosphate, and sodium citrate. Mixtures of these buffering agents may also be used. The amount of buffering agent useful in the composition depends largely on the particular buffer used and the pH of the solution. For example, acetate is a more efficient buffer at pH 5 than pH 6 so less acetate may be used in a solution at pH 5 than at pH 6The preferred pH range for the compositions of the present invention is pH 3.0-7.5. The compositions of the present invention may further include an isotonicity adjusting agent to render the solution isotonic and more compatible for injection. The most preferred agent is sodium chloride within a concentration range of 0-150 mM. As used herein, and when contemplating PEG:NESP conjugates, the term “therapeutically effective amount” refers to an amount which gives an increase in hematocrit that provides benefit to a patient. The amount will vary from one individual to another and will depend upon a number of factors, including the overall, physical condition of the patient and the underlying cause of anemia. For example, a therapeutically effective amount of rHuEPO for a patient suffering from chronic renal failure is 50 to 150 units/kg three times per week. The amount of rHuEPO used for therapy gives an acceptable rate of hematocrit increase and maintains the hematocrit at a beneficial level (usually at least about 30% and typically in a range of 30% to 36%). A therapeutically effective amount of the present compositions may be readily ascertained by one skilled in the art using publicly available materials and procedures. The invention provides for administering PEG:NESP conjugates less frequently than NESP and/or EPO. The dosing frequency will vary depending upon the condition being treated, but in general will be about one time per 4-6 weeks. It is understood that the dosing frequencies actually used may vary somewhat from the frequencies disclosed herein due to variations in responses by different individuals to the PEG:NESP conjugates; the term “about” is intended to reflect such variations. The present invention may thus be used to stimulate red blood cell production and correct depressed red cell levels. Most commonly, red cell levels are decreased due to anemia. Among the conditions treatable by the present invention include anemia associated with a decline or loss of kidney function (chronic renal failure), anemia associated with myelosuppressive therapy, such as chemotherapeutic or anti-viral drugs (such as AZT), anemia associated with the progression of non-myeloid cancers, and anemia associated with viral infections (such as HIV). Also treatable are conditions which may lead to anemia in an otherwise healthy individual, such as an anticipated loss of blood during surgery. In general, any condition treatable with rHuEPO and/or NESP may also be treated with the PEG:NESP conjugates of the invention. The invention also provides for administration of a therapeutically effective amount of iron in order to maintain increased erythropoiesis during therapy. The amount to be given may be readily determined by one skilled in the art based upon therapy with rHuEPO. PEG:NESP conjugates prepared in accordance with the present invention is preferably administered by injection intraperitoneally, subcutaneously, or intramuscularly. However, it would be clear to one skilled in the art that other routes of delivery could also be effectively utilized using the compositions of the present invention. The following examples are offered to more fully illustrate the invention, but are not to be construed as limiting the scope thereof. Example 1 describes the preparation and testing of PEG:NESP conjugates prepared by coupling either 5 kD or 20 kD methoxy-PEG-hydrazides to NESP through aldehydes generated in the NESP carbohydrate chains by sodium periodate oxidation. Example 2 describes the preparation and testing of PEG:NESP conjugates prepared utilizing 20 kD PEG polymers as NHS-PEG esters and PEG-aldehydes to produce PEG-NESP conjugates by acylation and reductive alkylation respectively. Example 3 demonstrates the effects on activity of the degree of substitution and variations of the polymer size and conformation for various PEG:NESP conjugates. Example 4 describes the efficacy of three PEG:NESP conjugates: 20 kD mono-PEG:NESP; the 5 kD poly-PEG:NESP mixture; and 30 kD mono-PEG:NESP, as examined at three different doses relative to a NESP control, in an anemic mouse model. In Example 5, three different PEG-NESP conjugates were evaluated in a normal mouse bioassay to compare and contrast their erythropoietic potential and duration. EXAMPLE 1 PEG:NESP conjugates were produced by coupling either 5 kD or 20 kD methoxy-PEG-hydrazides to NESP through aldehydes generated in the NESP carbohydrate chains by sodium periodate oxidation. The degree of modification was controlled by varying the sodium periodate concentration during oxidation. The conjugates were prepared by first oxidizing NESP (2-4 mg/ml in 50 mM sodium acetate) with either 1 mM or 10 mM sodium meta-periodate (Sigma) for thirty minutes at room temperature in 100 mM sodium acetate, pH 5.6. The periodate is then removed by buffer exchange into 100 mM sodium acetate, pH 5.4. Methoxy-PEG-hydrazide (Shearwater Polymers) is then added at 5-100 fold molar excess polymer:protein, with 100-fold excess preferred. The intermediate hydrazone linkage was further reduced by addition of 15 mM sodium cyanoborohydride (Sigma) and allowed to react overnight at 4° C. The resultant conjugates were then fractionated by size exclusion FPLC using a Superdex 75, 26 mm×60 cm column (Pharmacia) eluted with 20 mM sodium phosphate, 150 mM NaCl, pH 7.2. The resultant preparations ranged in size from ˜40 kD to ˜200 kD, as estimated by SDS-PAGE. Samples of PEG:NESP were tested for receptor binding in an in vitro EIA format. The in vitro assay is a displacement assay wherein the PEG:NESP conjugates compete for binding of the EPO receptor with an EPO-HRP conjugate used as a reporter. The in vitro assay results suggest that the PEG:NESP conjugates had a lower apparent affinity for the NESP receptor. Bioactivity of various PEG:NESP conjugates was then evaluated in vivo by monitoring iron uptake in rodents after a single subcutaneous dose of conjugate. In the assay, mice are preconditioned in a hyperbaric chamber to suppress expression of endogenous erythropoietin, then dosed with a single, subcutaneous bolus injection of NESP or a PEG:NESP conjugate. After five days, the mice receive an intravenous injection of Fe 59 isotope as a tracer to monitor iron uptake in the red blood cells. Two days after the administration of Fe 59 , the animals are sacrificed and analyzed for iron uptake as a function of dose. Initially, several pools of 5 kD poly-PEG:NESP conjugates with varying degrees of PEGylation were tested for iron uptake as a function of conjugate dose. The in vivo assay results are depicted in FIG. 3, and demonstrated that the PEG:NESP conjugates prepared by coupling PEG-hydrazide to oxidized NESP perform comparably to NESP alone in the iron uptake bioassay. EXAMPLE 2 This example describes the preparation and testing of PEG:NESP conjugates prepared utilizing NHS-PEG esters and PEG-aldehydes produced from 20 kD PEG polymers. Reaction stoichiometries and buffer conditions were optimized for each chemistry to produce 20 kD mono-PEG:NESP conjugates in good yield. A 20 kD mono-PEG:NESP derived by acylation of NESP with the 20 kD methoxy-PEG-NHS ester was prepared, as well as a mixture (˜80%/20%) of 20 kD mono/di-PEG:NESP derived by reductive alkylation of NESP with 20 kD methoxy-PEG-aldehyde. The reaction with methoxy-PEG-aldehyde (Shearwater Polymers) can be carried out from pH 4-6 with the optimum being at pH 5.2. The concentration of NESP in the reaction mixture was 4 mg/ml in 50 mM sodium acetate. The molar excess of PEG aldehyde used was 5-20 fold, and sodium cyanoborohydride was added to a final 15 mM concentration. The reaction was stirred for 1 hour at ambient temperature and then for 18 hours at 5° C. Upon completion of the reaction, the mixture was diluted to a conductivity of less than 5 mS/cm, the pH raised to 7.0, and the mixture loaded onto a Q Sepharose HP column (Pharmacia). The products were eluted from the column utilizing a linear gradient from 50 mM NaCl to 200 mM NaCl buffered in 10 mM Bis-Tris-Propane, pH 7.0. This purification allows for separation of species based on the number of PEG molecules attached to NESP. The reaction with PEG activated NHS ester, methoxy-SPA-PEG (Shearwater Polymers), was carried out at pH 8.0 at a NESP concentration from 2-4 mg/ml in 50 mM Bicine buffer. A buffered solution of NESP was added to 10-20 molar equivalents of PEG. The reaction was stirred for 1 hour at ambient temperature. Upon completion of the reaction, the mixture was diluted to a conductivity of less than 5 mS/cm, the pH raised to 7.0, and the sample loaded onto a QHP column (Pharmacia). The products were eluted with a linear gradient from 50 mM NaCl to 200 mM NaCl buffered in 10 mM Bis-Tris-Propane, pH 7.0. The two isolated PEG:NESP conjugates, a 20 kD mono-PEG:NESP (NHS) and a mixture (˜80%/20%) of 20 kD mono/di-PEG:NESP (aldehyde) were then tested in a murine in vivo bioassay. The murine bioassay measures reticulocytes, a red blood cell-precursor, and hemoglobin as monitors of erythropoiesis in response to a single dose of NESP or PEG:NESP in normal mice. Specifically, the bioassay measures the intensity and duration of an increased hemoglobin and reticulocyte response resulting from subcutaneous bolus injections of 100 μg/kg in female BDF 1 mice. The assay results are depicted in FIG. 4, and the results of the study indicated a significant increase and prolongation of the hemoglobin response from the PEG:NESP conjugates relative to an equivalent dose of NESP alone. EXAMPLE 3 This example demonstrates the effects on activity of the degree of substitution and variations of the polymer size and conformation for PEG:NESP conjugates. Using both methoxy-PEG-aldehyde and methoxy-PEG-NHS based chemistries, a variety of PEG:NESP conjugates were synthesized from 5 kD, 20 kD and 30 kD linear polymers as well as 10 kD, 20 kD and 40 kD branched polymers. From these reactions, preparations of mono-substituted and di-substituted PEG:NESP were isolated chromatographically and tested for prolonged erythropoiesis in the mouse bioassay. The reaction with methoxy-PEG-aldehyde (Shearwater Polymers) was run with a NESP concentration of 4 mg/ml and a 25-fold molar excess of PEG in 20 mM NaOAc, pH 5.0, with sodium cyanoborohydride added to a final concentration of 20 mM. The reaction was stirred overnight at 4° C., diluted 4-fold with 20 mM Tris, pH 7.2, and the pH adjusted to pH 7.4 with NaOH. The diluted reaction mixture was then loaded onto a 5 ml HiTrap Q Sepharose HP column (Pharmacia). The PEGylated NESP isoforms were resolved by elution with a 0-150 mM NaCl gradient in 20 mM Tris, pH 7.2. The reaction with methoxy-PEG-NHS ester (Shearwater Polymers) was run with a NESP concentration of 4 mg/ml and a 5-7 fold molar excess of PEG in 50 mM Bicine buffer, pH 8. The reaction was stirred overnight at 4° C., then diluted 4-fold with 20 mM Tris, pH 7.2 and the pH adjusted to pH 7.4 with NaOH. The diluted reaction mixture was then loaded onto a 5 ml HiTrap Q Sepharose HP column (Pharmacia). The PEGylated NESP isoforms were resolved by elution with a 0-150 mM NaCl gradient in 20 mM Tris, pH 7.2 (see FIGS. 5 - 7 ). These process schemes were employed for each of the 5 kD, 20 kD and 30 kD linear polymers as well as the 10 kD, 20 kD and 40 kD branched PEG-NHS esters. The various conjugates are listed in Table 1 below: TABLE 1 Conjugation Degree of PEG polymer Chemistry Substitution 5 kD linear mPEG-NHS ester mono/di-PEG 20 kD linear mPEG-NHS ester mono-PEG 20 kD linear mPEG-NHS ester di-PEG 30 kD linear mPEG-NHS ester mono-PEG 30 kD linear mPEG-NHS ester di-PEG 5 kD linear mPEG-aldehyde mono-PEG 5 kD linear mPEG-aldehyde di-PEG 20 kD linear mPEG-aldehyde mono-PEG 30 kD linear mPEG-aldehyde mono-PEG 30 kD linear mPEG-aldehyde di-PEG 10 kD branched branched mPEG-NHS mono/di-PEG ester 20 kD branched branched mPEG-NHS mono-PEG ester 40 kD branched branched mPEG-NHS mono-PEG ester 20 kD branched branched mPEG-aldehyde mono-PEG 40 kD branched branched mPEG- mono-PEG aldehyde 5 kD linear mPEG-hydrazide high (>7 PEGs) 5 kD linear mPEG-hydrazide low (1-5 PEGs) 20 kD linear mPEG-hydrazide high (>7 PEGs) 20 kD linear mPEG-hydrazide medium (˜4-7 PEGs) 20 kD linear mPEG-hydrazide low (1-5 PEGs) Each purified isoform was then tested in a murine in vivo bioassay for prolonged erythropoietic activity as measured by changes in reticulocyte and hemoglobin determinations after single, subcutaneous bolus injections of 100 μg/kg in normal, female BDF 1 mice. Each mono-substituted PEG:NESP conjugate from the linear and branched polymer series showed significant and comparable prolongation of the erythropoietic effect (see FIGS. 8 and 9 ). The di-substituted PEG:NESP conjugates from the 20 kD and 30 kD PEG polymers were considerably less active, but unexpectedly, the 5 kD di-substituted PEG:NESP conjugate demonstrated an equivalent activity to the mono-substituted counterpart. All of the mono-substituted, branched PEG:NESP conjugates demonstrated prolonged activity comparable to the analogous mono-substituted linear PEG:NESP conjugates. These examples thus demonstrate the enhanced duration of erythropoietic stimulation by a variety of PEG:NESP conjugates using single-dose, bolus injections in normal mouse models. EXAMPLE 4 This example describes the efficacy of three PEG:NESP conjugates: 20 kD mono-PEG:NESP; the 5 kD poly-PEG:NESP mixture; and 30 kD mono-PEG:NESP, as examined at three different doses relative to a NESP control, in an anemic mouse model. To induce an anemic condition, mice were pretreated with cis-platinin at 1 mg/kg/day for 3 days, followed by a 7 day rest period. After 3 ten day cycles, the mice were dosed with single, bolus injections of 30 μg/kg, 10 μg/kg or 3 μg/kg of the 20 kD mono-PEG:NESP, 30 kD mono-PEG:NESP or the 5 kD poly-PEG:NESP conjugates and compared to a NESP alone control at 30 μg/kg. Reticulocyte and hemoglobin levels were monitored as a function of time and in response to the single dose of each drug (see FIGS. 10 - 15 ). These data demonstrate the unexpected advantages of an ˜3 fold dose reduction and significant increases in erythropoietic half-life for the PEG:NESP conjugates relative to NESP alone, in that the results demonstrate a clear dose dependence for both the magnitude and duration of either the reticulocyte or hemoglobin response to the PEG:NESP conjugates. In some cases the 30 kD mono-PEG:NESP conjugate appears to modestly outperform the 5 kD poly-PEG:NESP conjugate, which modestly outperforms the 20 kD mono-PEG:NESP conjugate, suggesting that the 30 kD mono-PEG:NESP conjugate might be a preferred configuration. EXAMPLE 5 In this example, three different PEG-NESP conjugates were evaluated in a normal mouse bioassay to compare and contrast their erythropoietic potential and duration. The three compounds tested were: 30 kD mono-PEG:NESP derived by acylation with the 30 kD PEG-NHS ester, the 20 kD mono-PEG:NESP derived by reductive alkylation with the 20 kD PEG-aldehyde and the 5 kD poly-PEG:NESP mixture derived by reductive alkylation with the 5 kD PEG-aldehyde. Each PEG:NESP conjugate was tested as a single bolus, subcutaneous dose at 30 μg/kg, 10 μg/kg or 3 μg/kg. Unmodified NESP was used as a control at 30 μg/kg in a single, bolus injection. The erythropoietic response and duration were monitored as a function of reticulocyte counts or hemoglobin concentration (see FIGS. 16-21) as a function of time. These data show that all three PEG:NESP forms are capable of inducing a strong erythropoietic response with significant dose reduction. Moreover, these PEG:NESP conjugates demonstrate a prolonged efficacy relative to the unmodified NESP. Materials and Methods The present NESP may be prepared according to the above incorporated-by-reference PCT Application No. US94/02957. The conjugates prepared-herein were also characterized using size exclusion chromatography (SEC) as an analytical tool. The SEC column was a Tosohaas TSK 3000 SW×1 (5 micron-7.8 mm×30 cm) which utilized 100 mM NaHPO 4 , 10% ethanol, 150 mM NaCl, pH 6.9, to elute the products. A representative chromatograph is depicted in FIG. 22 . While the present invention has been described in terms of certain preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations which come within the scope of the invention as claimed. 1 1 165 PRT Homo sapiens 1 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Asn Glu Thr 20 25 30 Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp 50 55 60 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80 Leu Val Asn Ser Ser Gln Val Asn Glu Thr Leu Gln Leu His Val Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110 Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly Asp 165

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Patent Citations (9)

    Publication numberPublication dateAssigneeTitle
    US-4002531-AJanuary 11, 1977Pierce Chemical CompanyModifying enzymes with polyethylene glycol and product produced thereby
    US-4179337-ADecember 18, 1979Davis Frank F, Placzuk Nicholas C, Theodorus Van EsNon-immunogenic polypeptides
    US-4904584-AFebruary 27, 1990Genetics Institute, Inc.Site-specific homogeneous modification of polypeptides
    US-5252714-AOctober 12, 1993The University Of Alabama In HuntsvillePreparation and use of polyethylene glycol propionaldehyde
    US-5641663-AJune 24, 1997Cangene CorporationExpression system for the secretion of bioactive human granulocyte macrophage colony stimulating factor (GM-CSF) and other heterologous proteins from steptomyces
    US-5824784-AOctober 20, 1998Amgen Inc.N-terminally chemically modified protein compositions and methods
    US-5834594-ANovember 10, 1998Hoffman-La Roche Inc.Polyethylene-protein conjugates
    US-5985265-ANovember 16, 1999Amgen Inc.N-terminally chemically modified protein compositions and methods
    WO-9505465-A1February 23, 1995Amgen Inc.Erythropoietin analogs

NO-Patent Citations (3)

    Title
    Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems, vol. 9, No. 3,4, pp. 249-304, 1992.*
    Egrie, et al., "Novel Erythropoiesis Stimulating Protein (NESP) has a longer Serum Half-life and greater in vivo Biological Activity Than Recombinant Human Erythropoietin (rHuEPO)", Blood, vol. 90, pp. 56a, (1997).
    MacDougall, et al., "Comparison of the Pharmacokinetics of Novel Erythropoiesis Stimulating Protein (NESP) and Epoetin Alfa (rhEPO) in Dialysis Patients", Journal of the American Society of Nephrology, vol. 8, pp. 268A, (1997).

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    US-2003012775-A1January 16, 2003Michael Brandt, Apollon PapadimitriouPEG conjugates of NK4
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    US-9187546-B2November 17, 2015Novo Nordisk A/SCompositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants
    US-7985838-B2July 26, 2011Baxter International Inc., Baxter Healthcare S.A.Factor VIII polymer conjugates
    US-2007087961-A1April 19, 2007Wolfram Eichner, Katharina Lutterbeck, Norbert Zander, Ronald Frank, Harald Conradt, Helmut KnollerConjugates of hydroxyalkyl starch and erythropoietin
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    EP-3045187-A1July 20, 2016Amgen, IncInjector and method of assembly
    US-2010016562-A1January 21, 2010Merck Patent GmbhReducing the immunogenicity of fusion proteins
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    US-8003760-B2August 23, 2011Baxter International Inc., Baxter Healthcare S.A.Factor VIII polymer conjugates
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    US-9731024-B2August 15, 2017Baxalta Incorporated, Baxalta GmbHNucleophilic catalysts for oxime linkage
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    US-7879319-B2February 01, 2011Merk Patent GmbhHeterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
    WO-2015187797-A1December 10, 2015Amgen Inc.Système d'administration de médicament pouvant être commandé et son procédé d'utilisation
    US-7148321-B2December 12, 2006Emd Lexigen Research Center Corp.Expression technology for proteins containing a hybrid isotype antibody moiety
    WO-2016100055-A1June 23, 2016Amgen Inc.Drug delivery device with live button or user interface field
    US-7842661-B2November 30, 2010Novo Nordisk A/SGlycopegylated erythropoietin formulations
    US-8076292-B2December 13, 2011Novo Nordisk A/SFactor VIII: remodeling and glycoconjugation of factor VIII
    US-2009186822-A1July 23, 2009Amgen Inc.Thrombopoietic Compounds
    US-2010311670-A1December 09, 2010Nobert Zander, Ronald FrankConjugates of hydroxyalkyl starch and a protein, prepared by native chemical ligation
    US-8748571-B2June 10, 2014Amgen Inc.Thrombopoietic compounds
    US-7537923-B2May 26, 2009Biomarin Pharmaceutical Inc.Compositions of prokaryotic phenylalanine ammonia-lyase and methods of treating cancer using compositions thereof
    US-9789196-B2October 17, 2017Antriabio, Inc.Site-specific insulin-polymer conjugates
    US-8475765-B2July 02, 2013Fresenius Kabi Deutschland GmbhHydroxyalkyl starch derivatives
    WO-2017039786-A1March 09, 2017Amgen Inc.Adaptateur d'ensemble de seringue pour une seringue
    US-8633157-B2January 21, 2014Novo Nordisk A/SGlycopegylated erythropoietin
    US-2008213277-A1September 04, 2008Amgen Inc.Hepcidin, hepcidin antagonists and methods of use
    US-2010316702-A1December 16, 2010The Regents Of The University Of CaliforniaCompositions and methods for regulating erythropoeitin expression and ameliorating anemia and stimulating erythropoiesis
    WO-2011156373-A1December 15, 2011Amgen Inc.Drug delivery device
    US-8716239-B2May 06, 2014Novo Nordisk A/SGranulocyte colony stimulating factor: remodeling and glycoconjugation G-CSF
    US-7534595-B2May 19, 2009Biomarin Pharmaceutical Inc.Compositions of prokaryotic phenylalanine ammonia-lyase and methods of using compositions thereof
    US-8143380-B2March 27, 2012Amgen Inc.Therapeutic peptides
    WO-2016100781-A1June 23, 2016Amgen Inc.Dispositif d'administration de médicament doté d'un capteur de proximité
    US-7973150-B2July 05, 2011Merck Patent GmbhReducing the immunogenicity of fusion proteins
    WO-2015119906-A1August 13, 2015Amgen Inc.Drug delivery system with electromagnetic field generator
    US-2008008695-A1January 10, 2008Vellard Michel C, Fitzpatrick Paul A, Kakkis Emil D, Wendt Daniel JCompositions of prokaryotic phenylalanine ammonia-lyase and methods of using compositions thereof
    US-2008020978-A1January 24, 2008Gegg Colin V Jr, Johnson Eileen J, Miranda Leslie P, Walker Kenneth W, Holder Jerry R, Wright Marie E, D Amico Derin CCGRP peptide antagonists and conjugates
    US-9187532-B2November 17, 2015Novo Nordisk A/SGlycosylation of peptides via O-linked glycosylation sequences
    US-7932364-B2April 26, 2011Novo Nordisk A/SCompositions and methods for the preparation of human growth hormone glycosylation mutants
    WO-2014081780-A1May 30, 2014Amgen Inc.Drug delivery device
    US-7186804-B2March 06, 2007Emd Lexigen Research Center Corp.IL-2 fusion proteins with modulated selectivity
    US-9145450-B2September 29, 2015Amgen Inc.Thrombopoietic compounds
    US-8044174-B2October 25, 2011Amgen Inc.Thrombopoietic compounds
    US-8916518-B2December 23, 2014Fresenius Kabi Deutschland GmbhCoupling proteins to a modified polysaccharide
    US-8926973-B2January 06, 2015Merck Patent GmbhReducing the immunogenicity of fusion proteins
    US-7553653-B2June 30, 2009Biomarin Pharmaceutical Inc., The Scripps Research InstituteVariants and chemically-modified variants of phenylalanine ammonia-lyase
    US-2009263369-A1October 22, 2009Biomarin Pharmaceutical Inc.Compositions of Prokaryotic Phenylalanine Ammonia-Lyase and Methods of Treating Cancer Using Compositions Thereof
    US-8841439-B2September 23, 2014Novo Nordisk A/SNucleotide sugar purification using membranes
    US-2011112025-A1May 12, 2011Baxter Healthcare S.A., Baxter International Inc.Factor viii polymer conjugates
    EP-3103880-A1December 14, 2016Ambrx, Inc.Polypeptides d'insuline modifiés et utilisations de ceux-ci
    US-2010278802-A1November 04, 2010Biomarin Pharmaceutical Inc.Compositions of prokaryotic phenylalanine ammonia-lyase and methods of treating cancer using compositions thereof
    US-2011206651-A1August 25, 2011Baxter Healthcare S.A., Baxter International Inc.Factor viii polymer conjugates
    US-8618044-B2December 31, 2013Amgen Inc.Thrombopoietic compounds
    US-2005080014-A1April 14, 2005Chuan-Fa Liu, Ulrich Feige, Cheetham Janet C.Thrombopoietic compounds
    EP-3045190-A1July 20, 2016Amgen, IncInjector and method of assembly
    US-8809501-B2August 19, 2014Baxter International Inc., Baxter Healthcare SaNucleophilic catalysts for oxime linkage
    US-8247381-B2August 21, 2012Biogenerix AgBranched water-soluble polymers and their conjugates
    US-7226998-B2June 05, 2007Emd Lexigen Research Center Corp.Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
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    US-7790433-B2September 07, 2010Biomarin Pharmaceutical Inc.Compositions of prokaryotic phenylalanine ammonia-lyase and methods of treating cancer using compositions thereof
    US-2003166163-A1September 04, 2003Emd Lexigen Research Center Corp.Immunocytokines with modulated selectivity
    US-2010062973-A1March 11, 2010Fresenius Kabi Deutschland GmbhProduction of bioactive glycoproteins from inactive starting material
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    US-2010173830-A1July 08, 2010Baxter Healthcare S.A., Baxter International Inc.Factor viii polymer conjugates
    US-7714114-B2May 11, 2010Nektar TherapeuticsConjugates of an EPO moiety and a polymer
    US-7795210-B2September 14, 2010Novo Nordisk A/SProtein remodeling methods and proteins/peptides produced by the methods
    US-7645860-B2January 12, 2010Baxter Healthcare S.A., Baxter International Inc.Factor VIII polymer conjugates
    US-7625564-B2December 01, 2009Novagen Holding CorporationRecombinant human EPO-Fc fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo
    US-8278418-B2October 02, 2012Ambrx, Inc.Modified animal erythropoietin polypeptides and their uses
    US-2009324478-A1December 31, 2009Hinman Norman D, Wyman Charles SMethod for Making Silicon-Containing Products
    US-9493499-B2November 15, 2016Novo Nordisk A/SProcess for the production of purified cytidinemonophosphate-sialic acid-polyalkylene oxide (CMP-SA-PEG) as modified nucleotide sugars via anion exchange chromatography
    US-8071728-B2December 06, 2011Baxter International Inc., Baxter Healthcare S.A.Factor VIII polymer conjugates
    US-2010210831-A1August 19, 2010Merck Patent GmbhImmunocytokine Sequences and Uses Thereof
    US-8168592-B2May 01, 2012Amgen Inc.CGRP peptide antagonists and conjugates
    EP-2594284-A1May 22, 2013Amgen Inc.Lyophilized therapeutic peptibody formulations
    US-8466277-B2June 18, 2013Fresenius Kabi Deutschland GmbhCoupling low-molecular substances to a modified polysaccharide
    US-8404809-B2March 26, 2013Novo Nordisk A/SGlycopegylated factor IX
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    WO-2017189089-A1November 02, 2017Amgen Inc.Drug delivery device with messaging label
    US-7576193-B2August 18, 2009Merck Patent GmbhHeterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
    US-2009297522-A1December 03, 2009Novagen Holding CorporationRecombinant human epo-fc fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo
    US-2007036752-A1February 15, 2007Emd Lexigen Research Center Corp.IL-2 fusion proteins with modulated selectivity
    WO-2015061389-A1April 30, 2015Amgen Inc.Drug delivery system with temperature-sensitive control
    US-8791070-B2July 29, 2014Novo Nordisk A/SGlycopegylated factor IX
    EP-3216800-A1September 13, 2017Ambrx, Inc., Eli Lilly & CompanyPolypeptides d'érythropoïétine animale modifiés et leurs utilisations
    US-9150848-B2October 06, 2015Novo Nordisk A/SConjugated factor VIII molecules
    US-8637007-B2January 28, 2014Baxter International Inc., Baxter Healthcare SaFactor VIIa-polysialic acid conjugate having prolonged in vivo half-life
    US-6838260-B2January 04, 2005Emd Lexigen Research Center Corp.Heterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
    US-7538092-B2May 26, 2009Fresenius Kabi Deutschland GmbhPharmaceutically active oligosaccharide conjugates
    US-9320797-B2April 26, 2016Amgen Inc.Pharmaceutical formulations
    US-2006204473-A1September 14, 2006Blatt Lawrence M, Seiwert Scott D, Jin HongSynthetic hyperglycosylated, and hyperglycosylated protease-resistant polypeptide variants, oral formulations and methods of using the same
    US-2005192211-A1September 01, 2005Emd Lexigen Research Center Corp.Fc-erythropoietin fusion protein with improved pharmacokinetics
    US-7465447-B2December 16, 2008Merck Patent GmbhFc-erythropoietin fusion protein with improved pharmacokinetics
    US-2011112026-A1May 12, 2011Baxter Healthcare S.A., Baxter International Inc.Factor viii polymer conjugates
    US-2004180035-A1September 16, 2004Emd Lexigen Research Center Corp.IL-15 immunoconjugates and uses thereof
    US-2011200555-A1August 18, 2011Fresenius Kabi Deutschland GmbhConjugates of hydroxyalkyl starch and a protein
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    US-2005238723-A1October 27, 2005Norbert Zander, Harald Conradt, Wolfram EichnerMethod of producing hydroxyalkyl starch derivatives
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    US-2010113365-A1May 06, 2010Baxter Healthcare S.A., Baxter International Inc.Factor viii polymer conjugates
    US-7169754-B2January 30, 2007Hoffmann-La Roche Inc.Erythropoietin composition
    US-8840879-B2September 23, 2014Fresenius Kabi Deutschland GmbhConjugates of hydroxyalkyl starch and a protein
    US-8066994-B2November 29, 2011Merck Patent GmbhProteins comprising an IgG2 domain
    US-7994117-B2August 09, 2011Amgen Inc.Thrombopoietic compounds
    US-2010173831-A1July 08, 2010Baxter Healthcare S.A., Baxter International Inc.Factor viii polymer conjugates
    US-2011028693-A1February 03, 2011Baxter International Inc., Baxter Healthcare S.A.Blood coagulation protein conjugates
    US-2009092607-A1April 09, 2009Merck Patent GmbhFc-erythropoietin fusion protein with improved pharmacokinetics
    EP-2574628-A1April 03, 2013Amgen Inc.Anticorps de ferroportine et procédés d'utilisation
    US-2008207562-A1August 28, 2008Fresenius Kabi Deutschland GmbhConjugates of Hydroxyalkyl Starch and Active Substance, Prepared by Chemical Ligation Via Thiazolidine
    US-8404834-B2March 26, 2013Fresenius Kabi Deutschland GmbhHydroxyalkyl starch derivatives and process for their preparation
    US-2009081218-A1March 26, 2009Novagen Holding CorporationFusion proteins
    US-7767405-B2August 03, 2010Merck Patent GmbhImmunocytokine sequences and uses thereof
    US-2010099851-A1April 22, 2010Alios Biopharma, Inc.Synthetic hyperglycosylated, protease-resistant polypeptide variants, oral formulations and methods of using the same
    US-2011054152-A1March 03, 2011Fresenius Kabi Deutschland GmbhHydroxyalkyl Starch Derivatives
    US-9557340-B2January 31, 2017Biomarin Pharmaceutical Inc.Assays for detection of phenylalanine ammonia-lyase and antibodies to phenylalanine ammonia-lyase
    US-2008206182-A1August 28, 2008Fresenius Kabi Deutschland GmbhConjugates of a Polymer and a Protein Linked by an Oxime Group
    US-2006188472-A1August 24, 2006Fresenius Kabi Deutschland Gmbh, A Germany CorporationHAS-active ingredient conjugates
    US-2011112028-A1May 12, 2011Baxter Healthcare S.A., Baxter International Inc.Factor viii polymer conjugates
    US-2007258944-A1November 08, 2007Emd Lexigen Research Center Corp.Multiple cytokine protein complexes
    EP-2803675-A2November 19, 2014Amgen, IncFerroportin-Antikörper und Anwendungsverfahren
    US-2006034836-A1February 16, 2006Emd Lexigen Research Center Corp.Enhancing the circulating half-life of antibody-based fusion proteins
    US-9657098-B2May 23, 2017Intrinsic Lifesciences, LlcAnti-hepcidin antibodies and uses thereof
    US-7803618-B2September 28, 2010Merck Patent GmbhRecombinant tumor specific antibody and use thereof
    US-9175078-B2November 03, 2015Amgen Inc.Ferroportin antibodies and methods of use
    US-2011112027-A1May 12, 2011Baxter Healthcare S.A., Baxter International Inc.Factor viii polymer conjugates
    US-7541328-B2June 02, 2009Fresenius Kabi Deutschland GmbhCoupling proteins to a modified polysaccharide
    US-2007092486-A1April 26, 2007Avigenics, Inc.Glycolated and glycosylated poultry derived therapeutic proteins
    US-7141651-B2November 28, 2006Emd Lexigen Research Center Corp.Multiple cytokine protein complexes
    WO-2015187799-A1December 10, 2015Amgen Inc.Systems and methods for remotely processing data collected by a drug delivery device
    WO-2012135315-A1October 04, 2012Amgen Inc.Adaptateur de flacon et système
    WO-2014149357-A1September 25, 2014Amgen Inc.Injector and method of assembly
    WO-2015171777-A1November 12, 2015Amgen Inc.Autoinjector with shock reducing elements
    US-8067543-B2November 29, 2011Baxter International Inc., Baxter Healthcare S.A.Factor VIII polymer conjugates
    EP-2093235-A1August 26, 2009Alios Biopharma Inc.Hyperglycosylierte Varianten des Interferons alfacon-1
    US-7956032-B2June 07, 2011Novo Nordisk A/SGlycopegylated granulocyte colony stimulating factor
    US-7790415-B2September 07, 2010Merck Patent GmbhEnhancing the circulating half-life of antibody-based fusion proteins
    US-2010098716-A1April 22, 2010Novagen Holding CorporationRecombinant human epo-fc fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo
    US-2007048855-A1March 01, 2007Alejandra Gamez, Lin Wang, Woomi Kim, Mary Straub, Patch Marianne G, Emil Kakkis, Dan Oppenheimer, Paul Fitzpatrick, Robert Heft, Stevens Raymond CVariants and chemically-modified variants of phenylalanine ammonia-lyase
    WO-2015187793-A1December 10, 2015Amgen Inc.Drug delivery system and method of use
    US-7531341-B1May 12, 2009Biomarin Pharmaceutical Inc.Compositions of prokaryotic phenylalanine ammonia-lyase and methods of using compositions thereof
    US-2008254020-A1October 16, 2008Amgen Inc.Therapeutic Peptides
    US-2005063943-A1March 24, 2005Klaus Sommermeyer, Wolfram Eichner, Sven Frie, Cornelius Jungheinrich, Roland Scharpf, Katharina LutterbeckConjugated of hydroxyalkyl starch and an active agent
    US-2004147431-A1July 29, 2004Apollon PapadimitriouErythropoietin composition
    US-9029331-B2May 12, 2015Novo Nordisk A/SGlycopegylated granulocyte colony stimulating factor
    US-8470991-B2June 25, 2013Merck Patent GmbhImmunocytokine sequences and uses thereof
    US-2003044423-A1March 06, 2003Lexigen Pharmaceuticals Corp.Expression technology for proteins containing a hybrid isotype antibody moiety
    US-8071727-B2December 06, 2011Baxter International Inc., Baxter Healthcare S.A.Factor VIII polymer conjugates
    US-2005202538-A1September 15, 2005Merck Patent GmbhFc-erythropoietin fusion protein with improved pharmacokinetics
    US-8053410-B2November 08, 2011Novo Nordisk Health Care A/GPegylated factor VII glycoforms
    US-8361961-B2January 29, 2013Biogenerix AgO-linked glycosylation of peptides
    US-8637640-B2January 28, 2014Baxter International Inc., Baxter Healthcare SaBlood coagulation protein conjugates
    US-9050304-B2June 09, 2015Ratiopharm GmbhMethods of treatment using glycopegylated G-CSF
    US-8916360-B2December 23, 2014Novo Nordisk A/SGlycopegylated erythropoietin
    US-7262166-B2August 28, 2007Amgen Inc.Chemically modified novel erythropoietin stimulating protein compositions and methods
    US-7067110-B1June 27, 2006Emd Lexigen Research Center Corp.Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
    WO-2015061386-A1April 30, 2015Amgen Inc.Injecteur et procédé d'assemblage
    US-9005625-B2April 14, 2015Novo Nordisk A/SAntibody toxin conjugates
    EP-3045188-A1July 20, 2016Amgen, IncInjektor und verfahren zur montage
    US-2007059282-A1March 15, 2007Emd Lexigen Research Center Corp.Immunocytokine sequences and uses thereof
    US-2003166877-A1September 04, 2003Lexigen Pharmaceuticals Corp.Reducing the immunogenicity of fusion proteins
    US-2006182711-A1August 17, 2006Bossard Mary J, Gayle StephensonConjugates of an EPO moiety and a polymer
    US-8911967-B2December 16, 2014Novo Nordisk A/SOne pot desialylation and glycopegylation of therapeutic peptides
    US-8642737-B2February 04, 2014Baxter International Inc., Baxter Healthcare SaNucleophilic catalysts for oxime linkage
    US-9040664-B2May 26, 2015Antriabio, Inc.Materials and methods for preparing protein-polymer conjugates
    US-2006121062-A1June 08, 2006Wolfram Eichner, Dirk DormannConjugated hydroxyalkyl starch allergen compounds
    US-2007083006-A1April 12, 2007Pr Pharmaceuticals, Inc.Method for preparation of site-specific protein conjugates
    WO-2016061220-A2April 21, 2016Amgen Inc.Dispositif d'injection de médicament comportant des témoins visuels et sonores
    US-7517526-B2April 14, 2009Merck Patent GmbhEnhancement of antibody-cytokine fusion protein mediated immune responses by combined treatment with immunocytokine uptake enhancing agents
    US-8008252-B2August 30, 2011Novo Nordisk A/SFactor VII: remodeling and glycoconjugation of Factor VII
    US-8632770-B2January 21, 2014Novo Nordisk A/SGlycopegylated factor IX
    US-2009292110-A1November 26, 2009Defrees ShawnEnzymatic modification of glycopeptides
    US-8063015-B2November 22, 2011Novo Nordisk A/SGlycopegylation methods and proteins/peptides produced by the methods
    US-2010113364-A1May 06, 2010Baxter Healthcare S.A., Baxter International Inc.Factor viii polymer conjugates
    WO-2017120178-A1July 13, 2017Amgen Inc.Auto-injector with signaling electronics
    EP-2594285-A1May 22, 2013Amgen Inc.Lyophilized therapeutic peptibody formulations
    US-2002037841-A1March 28, 2002Apollon PapadimitriouErythropoietin composition
    US-2003105294-A1June 05, 2003Stephen Gillies, Kin-Ming Lo, Yan Lan, John WesolowskiEnhancing the circulating half life of antibody-based fusion proteins
    US-9200049-B2December 01, 2015Novo Nordisk A/SRemodeling and glycopegylation of fibroblast growth factor (FGF)
    US-8071724-B2December 06, 2011Baxter International Inc., Baxter Healthcare S.A.Factor VIII polymer conjugates
    EP-2620448-A1July 31, 2013Amgen Inc.Anti-hepcidin antibodies and methods of use
    US-8017739-B2September 13, 2011Fresenius Kabi Deutschland GmbhConjugates of hydroxyalkyl starch and a protein
    US-8791066-B2July 29, 2014Novo Nordisk A/SBranched PEG remodeling and glycosylation of glucagon-like peptide-1 [GLP-1]
    US-8969532-B2March 03, 2015Novo Nordisk A/SMethods for the purification of polypeptide conjugates comprising polyalkylene oxide using hydrophobic interaction chromatography
    US-9283260-B2March 15, 2016Amgen Inc.Lyophilized therapeutic peptibody formulations
    US-2002147311-A1October 10, 2002Gillies Stephen D., Christa Burger, Kin-Ming LoEnhancing the circulating half-life of antibody-based fusion proteins
    US-2005234230-A1October 20, 2005Norbert Zander, Harald Conradt, Wolfram EichnerHydroxyalkyl starch derivatives
    WO-2009094551-A1July 30, 2009Amgen Inc.Anticorps anti-ferroportine et procédés d'utilisation
    US-8716240-B2May 06, 2014Novo Nordisk A/SErythropoietin: remodeling and glycoconjugation of erythropoietin
    US-7985839-B2July 26, 2011Baxter International Inc., Baxter Healthcare S.A.Factor VIII polymer conjugates
    US-7462350-B2December 09, 2008Emd Serono Research Center, Inc.Cancer treatments including administering IL-2 fusion proteins with modulated selectivity
    WO-2013055873-A1April 18, 2013Amgen Inc.Injecteur et procédé d'assemblage
    US-2010297106-A1November 25, 2010Christopher James Sloey, Camille Vergara, Jason Ko, Tiansheng LiPharmaceutical Formulations
    US-2010297078-A1November 25, 2010Fresenius Kabi Deutschland GmbhMethod for producing a hydroxyalkyl starch derivative with two linkers
    US-8053561-B2November 08, 2011Baxter International Inc., Baxter Healthcare S.A.Pegylated factor VIII
    US-2006100163-A1May 11, 2006Michele Orlando, Jurgen Hemberger, Jeanne Delbos-KrampePharmaceutically active oligosaccharide conjugates
    US-2006025573-A1February 02, 2006Merck Patent GmbhReducing the immunogenicity of fusion proteins
    US-2009047251-A1February 19, 2009Wolfram Eichner, Martin Schimmel, Frank Hacket, Elmar Kraus, Norbert Zander, Ronald Frank, Harald Conradt, Klaus Langer, Michele Orlando, Klaus SommermeyerConjugates of hydroxyalkyl starch and a protein
    US-2010305033-A1December 02, 2010Fresenius Kabi Deutschland GmbhHydroxyalkyl starch derivatives and process for their preparation
    EP-2594286-A1May 22, 2013Amgen Inc.Lyophilized therapeutic peptibody formulations
    EP-2816059-A1December 24, 2014Amgen, IncAnticorps anti-hepcidine et procédés d'utilisation
    US-2006019877-A1January 26, 2006Conradt Harald S, Eckart Grabenhorst, Manfred Nimtz, Norbert Zander, Ronald Frank, Wolfram EichnerHasylated polypeptides
    US-7202208-B2April 10, 2007Hoffman-La Roche Inc.Erythropoietin composition
    US-8071726-B2December 06, 2011Baxter International Inc., Baxter Healthcare S.A.Factor VIII polymer conjugates
    US-8207112-B2June 26, 2012Biogenerix AgLiquid formulation of G-CSF conjugate
    US-7560263-B2July 14, 2009Biomarin Pharmaceutical Inc.Compositions of prokaryotic phenylalanine ammonia-lyase and methods of treating cancer using compositions thereof
    US-2007178112-A1August 02, 2007Novagen Holding CorporationRecombinant human EPO-Fc fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo
    EP-3045189-A1July 20, 2016Amgen, IncInjecteur et procédé d'assemblage
    US-9156899-B2October 13, 2015Eli Lilly And Company, Ambrx, Inc.Modified animal erythropoietin polypeptides and their uses
    US-8067548-B2November 29, 2011Novagen Holding CorporationFusion proteins having mutated immunoglobulin hinge region
    US-2005186174-A1August 25, 2005Bossard Mary J.Compositions comprising two different populations of polymer-active agent conjugates
    US-2006264377-A1November 23, 2006Amgen Inc.Chemically modified novel erythropoietin stimulating protein compositions and methods
    US-2006217293-A1September 28, 2006Michele Orlando, Jurgen HembergerCoupling low-molecular substances to a modified polysaccharide
    US-9688759-B2June 27, 2017Amgen, Inc.Ferroportin antibodies and methods of use
    US-2009047268-A1February 19, 2009Biomarin Pharmaceutical Inc.Compositions of prokaryotic phenylalanine ammonia-lyase and methods of treating cancer using compositions thereof
    EP-3072548-A1September 28, 2016Amgen, IncDispositif d'administration de médicaments
    US-2008102083-A1May 01, 2008Neose Technologies, Inc.Compositions and Methods for the Preparation of Human Growth Hormone Glycosylation Mutants
    US-9534032-B2January 03, 2017Amgen Inc.Thrombopoietic compounds
    US-2009258017-A1October 15, 2009Callahan William J, Remmele Jr Richard L, Gayathri Ratnaswamy, Latypov Ramil F, Dingjiang LiuLyophilized therapeutic peptibody Formulations
    US-2009088561-A1April 02, 2009Merck Patent GmbhEnhancing the circulating half-life of antibody-based fusion proteins
    US-2008274948-A1November 06, 2008Fresenius Kabi Deutschland GmbhConjugates of Hydroxyalkyl Starch and G-Csf
    US-2011201022-A1August 18, 2011Biomarin Pharmaceutical Inc.Assays for detection of phenylalanine ammonia-lyase and antibodies to phenylalanine ammonia-lyase
    US-2004072299-A1April 15, 2004Gillies Stephen D., Kin-Ming LoMultiple cytokine protein complexes
    US-7815893-B2October 19, 2010Fresenius Kabi Deutschland GmbhHydroxyalkyl starch derivatives
    US-8287850-B2October 16, 2012Fresenius Kabi Deutschland GmbhConjugates of hydroxyalkyl starch and a protein, prepared by reductive amination
    EP-2594287-A1May 22, 2013Amgen Inc.Lyophilisierte therapeutische Peptidkörperformulierungen
    US-9644014-B2May 09, 2017Ambrx, Inc., Eli Lilly And CompanyModified animal erythropoietin polypeptides and their uses
    US-7582288-B2September 01, 2009Merck Patent GmbhMethods of targeting multiple cytokines
    US-2006263856-A1November 23, 2006Emd Lexigen Research Center Corp.Expression technology for proteins containing a hybrid isotype antibody moiety
    US-2010297060-A1November 25, 2010Merck Patent GmbhEnhancement of antibody-cytokine fusion protein mediated immune responses by combined treatment with immunocytokine uptake enhancing agents
    US-2005181985-A1August 18, 2005Jurgen Hemberger, Michele OrlandoCoupling proteins to a modified polysaccharide
    WO-2017160799-A1September 21, 2017Amgen Inc.Réduction de la probabilité de casse du verre dans des dispositifs d'administration de médicament
    US-2006194952-A1August 31, 2006Emd Lexigen Research Center Corp.Enhancing the circulating half-life of antibody-based fusion proteins
    US-8071725-B2December 06, 2011Baxter International Inc., Baxter Healthcare S.A.Factor VIII polymer conjugates
    WO-2017100501-A1June 15, 2017Amgen Inc.Auto-injecteur avec capuchon de signalisation
    EP-2594288-A1May 22, 2013Amgen Inc.Lyophilized therapeutic peptibody formulations
    US-2009076237-A1March 19, 2009Baxter Healthcare S.A., Baxter International Inc.Factor VIII Polymer Conjugates
    US-8569233-B2October 29, 2013Eli Lilly And Company, Ambrx, Inc.Modified animal erythropoietin polypeptides and their uses
    US-7982010-B2July 19, 2011Baxter International Inc., Baxter Healthcare S.A.Factor VIII polymer conjugates
    US-2010160610-A1June 24, 2010Nektar TherapeuticsConjugates of an EPO Moiety and a Polymer
    US-9795683-B2October 24, 2017Lipoxen Technologies Limited, Baxalta Incorporated, Baxalta GmbHGlycopolysialylation of non-blood coagulation proteins
    US-2003166566-A1September 04, 2003Amgen Inc.Chemically modified novel erythropoietin stimulating protein compositions and methods
    US-2002193570-A1December 19, 2002Gillies Stephen D., Kin-Ming Lo, Yan LanHeterodimeric fusion proteins useful for targeted immune therapy and general immune stimulation
    US-2003049227-A1March 13, 2003Gillies Stephen D., Yan Lan, Sylvia HoldenEnhancement of antibody-cytokine fusion protein mediated immune responses by combined treatment with immunocytokine uptake enhancing agents