NZ551594A - Novel carbamylated epo and method for its production - Google Patents

Abstract

Disclosed is a method for producing a carbamylated erythropoietin protein having less than about 40% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein as measured by ESI-mass spectrometry, which method comprises contacting an amount of erythropoietin with an amount of cyanate at a temperature, pH, and for a time period sufficient for the amine groups on the lysines and the N-terminal amino acids of the erythropoietin to become at least about 90% carbamylated.

Description

RECEIVED at IPONZ on 22 December 2009 551594 Novel carbamvlated EPO and method for its production Introduction The present invention is directed to a method of producing a carbamylated EPO compound. The compound, carbamylated erythropoietin (CEPO), which is characterised by being carbamylated on all or most of the primary amines of lysines and on the N-terminal amino acid of the molecule, and in addition this compound has a low level of carbamylation of the primaiy amines of other amino acids in the molecule. Furthermore, this 10 compound is free of aggregated proteins and polymers, and is suited for use in pharmaceutical compositions for treatment of diseases in for example the central or peripheral nervous system, and other tissues that express the central EPO receptor. One other surprising advantage of the present method of production is the fact that the method provides a product that contains less aggregated protein and less polymers, than the products achieved from other 15 known carbamylation methods described for erythropoietin.

Background of the invention The impairment of biological hematopoietic activity of carbamylated EPO has been shown by Satake, R. et al. (1990) Biochimica et Biophysica Acta; 1038: 125-129 and Mun, K-C. and 20 Golper, T.A. (2000) Blood Purif.; 18: 13-17. Brines et al. 2003, US patent application 20030072737 showed that the loss of the hematopoietic activity did not interfere with the tissue protective properties of EPO.

Carbamylation of proteins is widely known as a side effect of using urea in purification of 25 proteins and as a result of high urea serum levels. This is caused by spontaneously decomposition of urea to cyanate. Cyanate is responsible for the carbamylation of the primary amines of the protein hence the N-terminal end and lysines of a protein are susceptible to carbamylation (Figure 1). Additionally other potential amino acid residues susceptible to carbamylation are arginine, cysteine, tyrosine, aspartic acid, glutamic acid and RECEIVED at IPONZ on 22 December 2009 551594 2 histidine the reaction is however pH dependent and does not proceed as readily as with the N-terminal and lysine residue, Investigations to reveal if carbamylation of proteins was able to improve or impair the 5 biological activity of proteins have been conducted by H6rkk6, S. et al. (1992) Kidney International.; 41:1175-1181, Plapp, B.V. et al. (1971) Jour. Biol. Chem.; 246(4): 939-945, Satake, R. et al. (1990) Biochimica et Biophysica Acta; 1038:125-129 and Mun, K-C. and Golper, T.A. (2000) Blood Purif.; 18: 13-17. They investigated the biological effect of carbamylation of proteins by employing KCNO as the source of cyanate. They all observed a 10 decline or change in the biological activity as a result of increased carbamylation. The assessment of degree of carbamylation was based on two analytical methods: 1. Measurement of the decline in free amino groups using a trinitrobenzenesulfonic acid (TNBS) assay and 2. Amino acid analysis determining the lysines converted to homocitrulline residues.

Horkko, S. et al. (1992), carbamylated a low-density lipoprotein for the maximum of 6 hours at 37° with 2 M KCNO but did not obtain a fully carbamylated protein as measured with the TNBS assay.

Plapp, B.V. et al (1971), investigated the effect of time and obtained almost a fully 20 carbamylated bovine pancreatic deoxyribonuclease A after a 24 hours treatment with 1 M KCNO at37°C.

Mun, K-C, and Golper, T.A. (2000) investigated the effect of time with the maximum of 6 hours reaction time using 2 M KCNO. They also investigated the effect of increasing KCNO 25 concentration at 6 hours all reactions were at 37°C. Mun, K-C. and Golper, T.A, (2000) could not, from the experimental design, verify the exact degree of carbamylation (please refer to page 16 line 33-35).

We have now found that the carbamylation of EPO yielded polymers and aggregates hence making it unsuitable as a biopharmacutical. In addition we found that RECEIVED at IPONZ on 22 December 2009 551594 3 the formation of these polymers and aggregates was dependent on the process conditions for the carbamylation. Hence the development of a process with optimal parameters regarding pH, time, cyanate concentration, temperature, protein concentration and roost importantly the degree of protein polymerisation was needed. The present invention comprises an optimal 5 process for carbamylation yielding a product with a low degree of polymerisation and aggregation, and furthermore, surprisingly, we have found that a fully carbamylated EPO aiming at the N-terminal and all lysine residues (the latter occurs in a specified pH-range) was obtained. A subsequent step of the method of the invention was made in order to remove the formed aggregates and polymers. The resulting carbamylated pure EPO is 10 described herein, along with pharmaceutical compositions comprising the compound.

It has previously been illustrated that the degree of carbamylation depends on cyanate concentration and time. However it has not been described how to obtain a scaleable 15 carbamylation process for production of a biopharmaceutical.

The presence of aggregates by sub-optimal production has been associated with the induction of antibodies. And the presence of aggregates therefore results in a biopharmaceutical product unsuitable for use in humans.

The carbamylation and purification process described herein leads to a protein that is characterized as fully carbamylated with the lowest formation of polymers or aggregates as possible and with the minimum loss of end product. Hence making it an economical viable step.

Further processing of the carbamylated protein renders a product useful as a biopharmaceutical with only a minimal risk for the generation of an immunological response to the protein due to aggregates and polymers.

RECEIVED at IPONZ on 22 December 2009 (followed by page 4a) The analytical methods for assessment of full carbamylation are in addition to amino acid analysis; TNBS for free primary amino groups and finally a characterization of the product and digested product by MALDI-TOF.

The compound described herein which is erythropoietin that is fully carbamylated on free amino groups at the N-terminal and lysines of the molecule and further is not aggregated and not polymerised to a content of above 2.5 %, and contains a minimum of over- or under-carbamylated erythropoietin, may be used for the production of pharmaceutical compositions for the treatment of diseases responsive to the neuroprotective effects of native erythropoietin.

Summary of the invention In one aspect, the present invention provides a method for producing a carbamylated erythropoietin protein having less than about 40% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein as measured by ESI-mass spectrometry, which method comprises contacting an amount of erythropoietin with an amount of cyanate at a temperature, pH, and for a time period sufficient for the amine groups on the lysines and the N-terminal amino acids of the erythropoietin to become at least about 90% carbamylated.

In another aspect the present invention provides a method for producing a carbamylated erythropoietin protein having less than about 3% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein as measured by ESI-mass spectrometry, comprising purifying the carbamylated erythropoietin using anion exchange, cation exchange, hydrophobic interaction chromatography, reverse phase chromatography, affinity chromatography or size exclusion chromatography.

In another aspect the present invention provides a method for producing a carbamylated erythropoietin protein having less than about 3% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein as measured by ESI mass spectrometry, which method comprises: (a) contacting an amount of an erythropoietin protein with an amount of cyanate at a temperature, pH, and for a time period sufficient for at least about 90% of the amine residues on the lysine and the N-terminal amino acids of the erythropoietin to become RECEIVED at IPONZ on 22 December 2009 5515% (followed by page 4b) carbamylated; and (b) purifying the carbamylated erythropoietin protein using anion exchange, cation exchange, hydrophobic interaction chromatography, reverse phase chromatography, affinity chromatography or size exclusion chromatography.

In another aspect the present invention provides a method for producing a carbamylated erythropoietin protein having less than about 40% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein, which method comprises contacting an amount of erythropoietin with an amount of cyanate at a temperature, pH, and for a time period sufficient for the amine groups on the lysines and the N-terminal amino acids of the erythropoietin to become at least about 90% carbamylated and wherein the erythropoietin is contacted with the cyanate in the presence of potassium borate.

In another aspect the present invention provides a method for producing a carbamylated erythropoietin protein having less than about 40% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein, which method comprises contacting an amount of erythropoietin with an amount of cyanate at a temperature of 30°C to 34°C, pH, and for a time period sufficient for the amine groups on the lysines and the N-terminal amino acids of the erythropoietin to become at least about 90% carbamylated.

In another aspect the present invention provides a method for producing a carbamylated erythropoietin protein having less than about 40% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein, which method comprises contacting an amount of erythropoietin with an amount of cyanate at a temperature, pH, and for a time period sufficient for the amine groups on the lysines and the N-terminal amino acids of the erythropoietin to become at least about 90% carbamylated, and wherein the carbamylated erythropoietin protein is purified using anion exchange chromatography while maintaining the pH of both the running buffer and the elution buffer at 8.5 ± 0.2.

In another aspect the present invention provides a carbamylated erythropoietin protein prepared by a method according to the invention. 2331503_l.doc RECEIVED at IPONZ on 22 December 2009 551594^ (followed by page 5) The present invention relates to a scaleable protein carbamylation procedure for the production of biopharmaceuticals. Furthermore, described are the product of the process, pharmaceutical compositions comprising the compound, and the use of those compositions.

The carbamylation and purification process described in the present application leads to a protein that is characterized as fully carbamylated with the lowest formation of polymers or aggregates as possible and with the minimum loss of end product.

The carbamylation process has been optimised to yield a carbamylated protein with the lowest amount of polymer and aggregates making it an economical viable process. The final product additionally contains limited amounts of isoforms of over- and/or under-carbamylated erythropoietin (below or above 9 carbamylations per molecule). Under-carbamylated EPO would contain less than 9 carbamyl residues, i.e., not all of the eight lysines andN-terminus are carbamylated. Under-carbamylated EPO can have as a little 5 carbamyl residues and still not have classic erythropoietic activity, making it suitable for use in the present invention. Over-carbamylated EPO has more than 9 carbamyl residues and 551594 would have carbamylation at amino acids other than the eight lysine residues and the N-terminus. CEPO can have as many as 15 carbamyl residues and still have the desired effect, 1.e., no classic erythropoietic activity. At least about 90%. and most likely 95%, of the CEPO isoforms are carbamylated at the 8 lysine residues and N-terminus only.

Further processing of the carbamylated protein removes aggregated and polymerised product to a level of maximum 3% or 2.5 %, and thereby renders a product useful as a biopharmaceutical with only minimal risk of generation of an immunological response to the protein due to aggregates and polymers.

The analytical methods for assessment of carbamylation are in addition to amino acid analysis; TNBS for free primary amino groups and a characterization of the product and digested product by MALDI-TOF and LC-MS/MS.

Detailed description of the invention METHOD OF MAKING Six steps constitute the method for carbamylation of the proteins: 1. Concentration by ultrafiltration 2. Modification by carbamylation. 3. Desalting by gelfiltration 4. Purification by anion-exchange.

. Concentration and buffer exchange by ultra- and diqfiltration. 6. 0.22 /urn filtration The starting material of the present carbamylation process is advantageously purified human EPO, but can be any EPO form of animal or human type, in non-limiting example being it synthetic, recombinant human EPO or biologically or chemically modified human EPO, such 551594 as asialo-EPO, mutants of human EPO, i.e., a molecule where changes in the amino acid sequence axe introduced, EPO fragments, peptides of EPO, other proteins, or a mixture of proteins if several proteins are desired carbamylated.

The first step of the process involves a protein concentration adjustment by ultrafiltration wherein the protein concentration is adjusted for the purpose of keeping a low process volume. The protein concentration of 0.05-10 mg/ml or 0.05 -8 mg/ml is a preferred embodiment. A more preferred embodiment is 0.05-7 mg/ml and most preferred is 2-5 mg/ml. If the concentration is increased aggregates are increasingly formed. The 10 ultrafiltration is performed by means of a BioMax (Millipore) with a MWCO of 5 kDa.

Other filters may be applied. In addition the solubility of the protein may be adjusted by adding stabilizers.

After completion of the concentration step, the protein solution is mixed with K-borate tetra 15 hydrate, K-cyanate, with a pH 7-11 or pH 7-10, In a preferred embodiment, the pH is 8-10, and most preferred 9.0. The temperature ranges from 0°-60°C or 0°-50°C or 0°-40°C or 0°-< 37°C but a preferred embodiment is a temperature interval of 30-34°C preferably 32°C, for a time window of 10 minutes-30 days or 30 minutes-30 days or 1 hour-30 days or 1 hour-20 days or 1 hour-10 days or 1 hour-5 days or 1 hour-2 days or 1 hour-26 hours or 18-26 hours 20 or preferred 22 hours-26 hours, most preferably 24 hours. However these preferred intervals could be changed if other process parameters are changed, i.e., temperature, cyanate concentration and protein concentration.

If the temperature is below the limits the yield will be low as carbamylation will be slow and 25 inefficient. If the temperature limits are exceeded, the yield will be low due to increased aggregation. Another crucial parameter is time as the carbamylation will not be complete if the time is decreased or if time is increased the formation of aggregates are observed hence resulting in lower yield. 551594 7 Therefore a process with coherent parameters are presented, i.e., if the temperature is lowered the decreased carbamylation reaction can be compensated for by increasing the cyanate concentration and/or reaction time. Additionally, if reaction time is reduced the decreased carbamylation reaction can be compensated for by increasing temperature and/or cyanate 5 concentration. Finally in a process with reduced cyanate concentration the decreased carbamylation reaction can be compensated for by increasing the reaction time and/or temperature.

Therefore in conclusion one significant change of one crucial parameter (time, temperature, 10 cyanate concentration and protein concentration) would imply a change in one or more of the other crucial parameters in order to obtain a fully carbamylated molecule with low formation of aggregates and polymers.

The concentration of borate buffer may be 0.05-2 M but in a preferred embodiment 0.1-1 M 15 and most preferably 0.5 M as cyanate inherently hydrolyses and polymerizes under uptake of protons and lack of buffer capacity results in a drift of the pH of the solution.

In addition the cyanate concentration is preferred in the range of 0.05-10 M or 0.05-8 M or 0.05-6 M or 0.05-4 M or 0.05-2 M, a preferred embodiment being 0.05-1 M and most 20 preferably 0.5 M.

A concentration of 0.5 M borate buffer is required to control the pH drift caused by proton uptake of the 0.5 M cyanate concentration in use. A process using other salts of cyanate and borate may be employed. Additionally other reaction buffers than borate may be employed, 25 e.g., a carbonate buffer or phosphate buffer.

The desalting of the reaction mixture of protein and cyanate is performed by means of a chromatographic gelfiltration. The G-25 Fine (Amersham Biosciences) matrice is employed. The hold up time before sample application to the column is controlled and should not exceed 30 2 hours, as this would cause further carbamylation and polymer formation. The desalting and RECEIVED at IPONZ on 22 December 2009 551594 8 buffer change of proteins can be performed by dialysis, dia-ultrafiltration or by means of a chromatographic gelfiltration. Other gelfiltration matrices may be applied such as for example matrices of crosslinked polysaccharides or crosslinked mixed polysaccharides, polyacrylamide, polystyrene or matrices of ceramic nature. Furthermore the column height 5 may be varied in this step.

The carbamylation step may be adjusted to obtain a product with less than 40% aggregates and polymers or less than 30% or less than 25% or less than 20 % or less than 15 % or less than 12.5 % or less than 10% or less than 8% or less than 7%.

The removal of aggregates and polymers is perfomed by a purification step using anion exchange. It is observed that it can separate carbamylated EPO from remains of the starting material and from aggregates/polymers. The running buffer A is: 0.3% Tris (25 mM), 0.3% (50 mM) NaCl. pH 8.5 ± 0.2, and elution buffer B: 0.3% Tris (25 mM), 5.8% (1 M) NaCl. pH 15 8.5 ± 0.2. The gradient is performed with 0-30 % over 20 column volumes yielding the desired separation. The purification step may result in a product with less than 3% aggregates and polymers or less than 2.5% or less than 2% or less than 1.5% or less than 1 % or less than about 0.5%.

The elution and collection and pooling of the carbamylated EPO peak influence the distribution of the heterogeneity, i. e„ isoforms, of the eluted protein. In other words, the amounts of over- and under-carbamylated CEPO will vary depending on the collection and pooling procedure. A narrow pooling will lead to a lowering of the content of over- and/or under-carbamylated erythropoietin. Increasing the length of the gradient will allow for 25 selection of a more defined product by leaving out some species.

A composition of carbamylated EPO with less than about 40% over- and under-carbamylated isoforms by weight, as measured by ESI-mass spectrometry is described.

A more preferred embodiment is a CEPO with less than about 35% of over- and 30 under- carbamylated isoforms by weight, as measured by ESI-mass spectrometry. An even 551594 9 more preferred embodiment is a CEPO with less than about 30% over- and under-carbamylated isoforms by weight, as measured by ESI-mass spectrometry. An even more preferred embodiment is a CEPO with less than about 25% over- and under-carbamylated isoforms by weight, as measured by ESI-mass spectrometry. An even more preferred 5 embodiment is a CEPO with less than about 20% over- and under-carbamylated isoforms by weight, as measured by ESI-mass spectrometry. An even more preferred embodiment is a CEPO with less than about 15% over- and under-carbamylated isoforms by weight, as measured by ESI-mass spectrometry. An even more preferred embodiment is a CEPO with less than about 10% over- and under-carbamylated isoforms by weight, as measured by ESI-10 mass spectrometry. An even more preferred embodiment is a CEPO with less than about 5% over- and under-carbamylated isoforms by weight, as measured by ESI-mass spectrometry. An even more preferred embodiment is a CEPO with less than about 2% over- and under-carbamylated isoforms by weight, as measured by ESI-mass spectrometry. The most preferred embodiment is a CEPO with less than about 1 % over- and under-carbamylated 15 isoforms by weight, as measured by ESI-mass spectrometry.

In addition to influencing the overall content of the isoforms, the collection and pooling of the carbamylated EPO peak influences the distribution of over-carbamylated CEPO. It is preferred that the amount of over-carbamylated CEPO isoforms be less than about 35% by 20 weight as measured by ESI-mass spectrometry. It is even more preferred that the amount of over-carbamylated CEPO be less than about 30% by weight as measured by ESI-mass spectrometry, and even more preferred that the amount of over-carbamylated CEPO be less than about 25% by weight, and even more preferred that it be less than about 20%, and even more preferred that it be less than about 15%. Most preferably the amount of over-25 carbamylated EPO should be no more than about 10%, about 5,% or about 1% by weight of the total CEPO.

Other running and elution buffers may be employed as other anion exchange matrices and charged filters may be employed. The matrices in unlimiting example being of crosslinked RECEIVED at IPONZ on 22 December 2009 551594 polysaccharides or crosslinked mixed polysaccharides, polyacrylamide, polystyrene or matrices of ceramic nature.

In addition even cationexchange, hydrophobic interaction chromatography, reversed phase 5 chromatography, affinity chromatography and size exclusion chromatography may be used for the purification.

In the next step for the adjustment of concentration and buffer a dia/ultrafiltration tangential flow filtration unit is used. The carbamylated EPO is adjusted to a concentration >0.5 mg/ml 10 and the buffer changed to a 20 mM citrate, 100 mM NaCl buffer. The concentration and buffer change is performed by means of a BioMax (Millipore) with a MWCO of 5 kDa. Other filters may be applied.

Finally the purified biopharmacutical drug substance is 0.22 jam filtrated using a Millipak 15 (Millipore) to reduce germs. Any 0.22 (im filter may be used.

Using the method a fully carbamylated EPO is obtained with less than 3% or preferably less than 2.5% of aggregates as measured by SEC-HPLC. The full carbamylation of the 8 lysine residues is verified using amin oacid analysis determining the converted lysines to 20 homocitrulline. Furthermore the carbamylation was followed using the TNBS assay for determination of primary amines hence showing the complete carbamylation of lysines and the N-terminal.

In addition a thorough characterization using MALDI-TOF determined the change in the 25 intact mass both of the PNGase treated protein and for the protein with the N-glycans. In addition MALDI-TOF peptide mass fingerprint analysis/LC-MS/MS analysis, showed that all 8 lysines and the N-terminal are carbamylated. No other carbamylated amino acids were detected and no modifications of the glycans were detected. Furthermore, a reduced content of over- and under- carbamylated forms of EPO) is obtained in the final product. This 30 product is described herein.

RECEIVED at IPONZ on 22 December 2009 551594 11 Described is the composition obtained after the carbamylation step, but before the anion exchange purification, comprising a carbamylated EPO with less than about 40% by weight of aggregates and polymers, or less than about 30%, or less than about 5 25%, or less than about 20 %, or less than about 15 %, or less than about 12.5 %, or less than about 10%, or less than about 8% or less than about 7%, and an amount of cyanate.

Also described is the composition obtained after the anion exchange purification comprising a carbamylated EPO with less than about 3% by weight of 10 aggregates and polymers, or less than about 2.5%, or less than about 2%, or less than about 1.5%, or less than about 1% or less than about 0.5%. Further, this composition comprises isoforms consisting of over- or under-carbamylated EPO in amounts less than about 40 % by weight of the total carbamylated EPO, or more preferably less than about 35%, or less than about 30%, or less than about 25%, or less than about 20%, or less than about 15%, or less 15 than about 10%, or less than about 7.5%, or less than about 5%, or less than about 2%, and most prefereably less than about 1%. Further, the amount of over-carbamylated EPO in the composition may be less than about 35% by weight of the total carbamylated EPO, or more preferably less than about 30%, or less than about 25%, or less than about 20%, or less than about 15%„or less than about 10%, or less than about 7.5%, or less than about 5%, or less 20 than about 2%, and most preferably less than about 1%.

PHARMACEUTICAL COMPOSITIONS OF THE INVENTION Also described is the use of the described compounds for the production of pharmaceutical compositions to be used in humans or mammals for treatment of the 25 conditions described below.

Also described is a pharmaceutical composition comprising a therapeutically effective amount of carbamylated EPO, with less than about 3% by weight of aggregates and polymers, or more preferably less than about 2.5%, or less than about 2%, or less than about 1.5%, or less than about 1%, and most preferably, less than about 0.5% and RECEIVED at IPONZ on 22 December 2009 551594 12 further, this composition comprises isoforms consisting of over- or under- carbamylated EPO in amounts less than about 40 % by weight of total carbamylated EPO, or more preferably less than about 35%, or less than about 30%, or less than about 25%, or less than about 20 %, or less than about 15%, or less than about 10%, or less than about 5 %, or less than about 5 3%, or less than about 2%, and most preferably less than about 1%. Further, the amount of over-carbamylated EPO in the composition may be less than about 35% by weight of the total carbamylated EPO, or more preferably less than about 30%, or less than about 25%, or less than about 20%, or less than about 15%, or less than about 10%, or less than about 5%, or less than about 3%, or less than about 2% and most preferably less than about 1 % .

Also described herein is a pharmaceutical composition as described above containing the described compound which may be administerable to a mammal by any route that provides a sufficient level of the described compound in the vasculature to permit translocation across an endothelial cell barrier and beneficial effects on responsive cells. When used for the purpose of perfusing a tissue or organ, similar results are 15 desired. In the instance where the cells or tissue is non-vascularized and/or the administration is by bathing the cells or tissue with the described composition, the pharmaceutical composition provides an effective responsive cell-beneficial amount of a described compound.

The endothelial cell barriers across which the described compound may translocate include tight junctions, perforated junctions, fenestrated junctions, and any other 20 types of endothelial barriers present in a mammal. A preferred barrier is an endothelial cell tight junction.

The aforementioned compound is useful generally for the therapeutic or prophylactic treatment of human diseases of the central nervous system or peripheral nervous system which have primarily neurological or psychiatric symptoms, ophthalmic diseases, 25 cardiovascular diseases, cardiopulmonary diseases, respiratory diseases, kidney, urinary and reproductive diseases, gastrointestinal diseases and endocrine and metabolic abnormalities. In particular, such conditions and diseases include hypoxic conditions, which adversely affect excitable tissues, such as excitable tissues in the central nervous system tissue, peripheral nervous system tissue, or cardiac or retinal tissue such as, for example, brain, heart, or RECEIVED at IPONZ on 22 December 2009 551594 13 retina/eye. Therefore, the described compound can be used to treat or prevent damage to excitable tissue resulting from hypoxic conditions in a variety of conditions and circumstances. Non-limiting examples of such conditions and circumstances are provided in the table hereinbelow.

In the example of the protection of neuronal tissue pathologies treatable as described herein, such pathologies include those resulting from reduced oxygenation of neuronal tissues. Any condition which reduces the availability of oxygen to neuronal tissue, resulting in stress, damage, and finally, neuronal cell death, can be treated by the methods described herein. Generally referred to as hypoxia and/or ischemia, these conditions 10 arise from or include, but are not limited to, stroke, vascular occlusion, prenatal or postnatal oxygen deprivation, suffocation, choking, near drowning, carbon monoxide poisoning, smoke inhalation, trauma, including surgery and radiotherapy, asphyxia, epilepsy, hypoglycemia, chronic obstructive pulmonary disease, emphysema, adult respiratory distress syndrome, hypotensive shock, septic shock, anaphylactic shock, insulin shock, sickle cell crisis, cardiac 15 arrest, dysrhythmia, nitrogen narcosis, and neurological deficits caused by heart-lung bypass procedures.

In one embodiment, for example, the specific pharmaceutical compositions comprising the described composition can be administered to prevent injury or tissue damage resulting from risk of injury or tissue damage during surgical procedures, such as, for example, tumor 20 resection or aneurysm repair. Other pathologies caused by or resulting from hypoglycemia which are treatable by the methods described herein include insulin overdose, also referred to as iatrogenic hyperinsulinemia, insulinoma, growth hormone deficiency, hypocortisolism, drug overdose, and certain tumors.

Other pathologies resulting from excitable neuronal tissue damage include seizure disorders, 25 such as epilepsy, convulsions, or chronic seizure disorders. Other treatable conditions and diseases include, but are not limited to, diseases such as stroke (ischemic stroke, subarachnoid haemorrhage, Intracerebral haemorrhage), multiple sclerosis, hypotension, cardiac arrest, Alzheimer's disease, Parkinson's disease, cerebral palsy, brain or spinal cord RECEIVED at IPONZ on 22 December 2009 551594 14 trauma, AIDS dementia, age-related loss of cognitive function, memory loss, amyotrophic lateral sclerosis, seizure disorders, alcoholism, retinal ischemia, optic nerve damage resulting from glaucoma, and neuronal loss.

The specific composition and the methods described herein may be used to treat 5 inflammation resulting from disease conditions or various traumas, such as physically or chemically induced inflammation. Such traumas could include angitis, chronic bronchitis, pancreatitis, osteomyelitis, rheumatoid arthritis, glomerulonephritis, optic neuritis, temporal arteritis, encephalitis, meningitis, transverse myelitis, dermatomyositis, polymyositis, necrotizing fascilitis, hepatitis, and necrotizing enterocolitis.

Evidence has demonstrated that activated astrocytes can exert a cytotoxic role towards neurons by producing neurotoxins. Nitric oxide, reactive oxygen species, and cytokines are released from glial cells in response to cerebral ischemia (see Becker, KJ. 2001. Targeting the central nervous system inflammatory response in ischemic stroke. Curr Opinion Neurol 14:349-353 and Mattson, M.P., Culmsee, C., and Yu, Z.F. 2000. Apoptotic and Antiapoptotic 15 mechanisms in stroke. Cell TissueRes 301:173-187.). Studies have further demonstrated that in models of neurodegeneration, glial activation and subsequent production of inflammatory cytokines depends upon primary neuronal damage (see Viviani, B., Corsini, E., Galli, C.L., Padovani, A., Ciusani, E., and Marinovich, M. 2000. Dying neural cells activate glia through the release of a protease product. Glia 32:84-90 and Rabuffetti, M., Scioratti, C., Tarozzo, G., 20 Clementi, E., Manfredi, A.A., and Beltramo, M. 2000. Inhibition of caspase-l-like activity by Ac-Tyr-Val-Ala-Asp-chloromethyl ketone includes long lasting neuroprotection in cerebral ischemia through apoptosis reduction and decrease of proinflammatory cytokines. J Neurosci 20:4398-4404). Inflammation and glial activation is common to different forms of neuro degenerative disorders, including cerebral ischemia, brain trauma and experimental allergic 25 encephalomyelitis, disorders in which erythropoietin exerts a neuroprotective effect. Inhibition of cytokine production by erythropoietin could, at least in part, mediate its protective effect. However, unlike "classical" anti-inflammatory cytokines such as 11-10 and IL-13, which inhibit tumor necrosis factor production directly, erythropoietin appears to be active only in the presence of neuronal death.

RECEIVED at IPONZ on 22 December 2009 551594 While not wishing to be bound by any particular theory, it appears that this anti-inflammatory activity may be hypothetically explained by several non-limiting theories. First, since erythropoietin prevents apoptosis, inflammatory events triggered by apoptosis would be prevented. Additionally, erythropoietin may prevent the release of molecular signals from 5 dying neurons which stimulate the glia cells or could act directly on the glial cells reducing their reaction to these products. Another possibility is that erythropoietin targets more proximal members of the inflammatory cascade (e.g., caspase 1, reactive oxygen or nitrogen intermediates) that trigger both apoptosis and inflammation.

Furthermore, erythropoietin appears to provide anti-inflammatory protection without the 10 rebound affect typically associated with other anti-inflammatory compounds such as dexamethasone. Once again, not wishing to be bound by any particular theory, it appears as though this may be due to erythropoietin's affect on multipurpose neuro toxins such as nitric oxide (NO). Although activated astrocytes and microglia produce neurotoxic quantities of NO in response to various traumas, NO serves many purposes within the body including the 15 modulation of essential physiological functions. Thus, although the use of an antiinflammatory may alleviate inflammation by suppressing NO or other neuro toxins, if the anti-inflammatory has too long a half-life it may also interfere with these chemicals' roles in repairing the damage resulting from the trauma that led to the inflammation. It is hypothesized that the described compound is able to alleviate the inflammation 20 without interfering with the restorative capabilities of neurotoxins such as NO.

The specific compositions and methods described herein may be used to treat conditions of, and damage to, retinal tissue. Such disorders include, but are not limited to retinal ischemia, macular degeneration, retinal detachment, retinitis pigmentosa, arteriosclerotic retinopathy, hypertensive retinopathy, retinal artery blockage, retinal vein blockage, hypotension, and 25 diabetic retinopathy.

The methods and principles described herein may be used to protect or treat injury resulting from radiation damage or chemotherapy induced damage to excitable tissue. In nonlimiting example, to protect from damages caused by compounds like taxanes, RECEIVED at IPONZ on 22 December 2009 551594 16 cisplatin and other chemotherapeutics with the potential to induce peripheral neuropathies. A further utility of the methods described herein is in the treatment of neurotoxin poisoning, such as domoic acid shellfish poisoning, neurolathyrism, and Guam disease, amyotrophic lateral sclerosis, and Parkinson's disease, Also described herein is a method for enhancing excitable tissue function in a mammal by peripheral administration of a compound of the invention as described above. Various diseases and conditions are amenable to treatment using this method, and further, this method is useful for enhancing cognitive function in the absence of any condition or disease. These uses are described in further detail below and include enhancement of learning and training in both human and non-human mammals.

Conditions and diseases treatable by the methods described herein directed to the central nervous system include, but are not limited to, mood disorders, anxiety disorders, depression, autism, attention deficit hyperactivity disorder, and cognitive 15 dysfunction. These conditions benefit from enhancement of neuronal function. Other disorders treatable in accordance with these teachings include for example, sleep disruption, sleep apnea, and travel-related disorders; subarachnoid and aneurismal bleeds, hypotensive shock, concussive injury, septic shock, anaphylactic shock, and sequelae of various encephalitides and meningitides, for example, connective tissue 20 disease-related cerebritides such as lupus. Other uses include prevention of or protection from poisoning by neurotoxins, such as domoic acid shellfish poisoning, neurolathyrism, and Guam disease, amyotrophic lateral sclerosis, Parkinson's disease; postoperative treatment for embolic or ischemic injury; whole brain irradiation; sickle cell crisis; and eclampsia.

A further group of conditions treatable by the methods described herein include 25 mitochondrial dysfunction, of either a hereditary or an acquired nature, which are the cause of a variety of neurological diseases typified by neuronal injury and death. For example, Leigh disease (subacute necrotizing encephalopathy) is characterized by progressive visual loss and encephalopathy, due to neuronal drop out, and myopathy. In these cases, defective RECEIVED at IPONZ on 22 December 2009 551594 17 mitochondrial metabolism fails to supply enough high energy substrates to fuel the metabolism of excitable cells. An erythropoietin receptor activity modulator optimizes failing function in a variety of mitochondrial diseases. As mentioned above, hypoxic conditions adversely affect excitable tissues. The excitable tissues include, but are not 5 limited to, central nervous system tissue, peripheral nervous system tissue, and heart tissue. In addition to the conditions described above, the described methods are useful in the treatment of inhalation poisoning, such as carbon monoxide and smoke inhalation, severe asthma, adult respiratory distress syndrome, choking, and near drowning. Further conditions which create hypoxic conditions or by other means induce excitable tissue damage 10 include hypoglycemia that may occur in inappropriate dosing of insulin, or with insulin-producing neoplasms (insulinoma).

Various neuropsychologic disorders which are believed to originate from excitable tissue damage are treatable by the instant methods. Chronic disorders in which neuronal damage is involved and for which treatment is described herein include disorders 15 relating to the central nervous system and/or peripheral nervous system including age-related loss of cognitive function and senile dementia, chronic seizure disorders, Alzheimer's disease, Parkinson's disease, dementia, memory loss, amyotrophic lateral sclerosis, multiple sclerosis, tuberous sclerosis, Wilson's Disease cerebral and progressive supranuclear palsy, Guam disease, Lewy body dementia, prion diseases, such as spongiform encephalopathies, 20 e.g., Creutzfeldt-Jakob disease, Huntington's disease, myotonic dystrophy, Charcot-Marie-Tooth Disease, Freidrich's ataxia and other ataxias, as well as Gilles de la Tourette's syndrome, seizure disorders such as epilepsy and chronic seizure disorder, stroke, brain or spinal cord trauma, AIDS dementia, alcoholism, autism, retinal ischemia, glaucoma, autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric 25 disorders that include, but are not limited to, schizophrenia, schizoaffective disorder, attention deficit disorder hyperactivity, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, panic disorder, as well as unipolar and bipolar affective disorders. Additional neuropsychiatric and neurodegenerative disorders include, for example, those listed in the American Psychiatric RECEIVED at IPONZ on 22 December 2009 551594 18 Association's Diagnostic and Statistical Manual of Mental Disorders (DSM), the most current version, IV, of which in incorporated herein by reference in its entirety.

In another embodiment, recombinant chimeric toxin molecules comprising a compound described herein can be used for therapeutic delivery of toxins to treat a proliferative disorder, such 5 as cancer, or viral disorder, such as subacute sclerosing panencephalitis.

Table 1 lists additional exemplary, non-limiting indications as to the various conditions and diseases amenable to treatment by the aforementioned compounds.

Table 1 Cell, tissue or organ Dysfunction or patholoev Condition or disease Type Heart Ischemia Coronary artery disease Acute, chronic Stable, unstable Myocardial infarction Dressier's syndrome Angina Congenital heart disease Valvular Cardiomyopathy Prinzmetal angina Cardiac rupture Aneuiysmatic Septal perforation Angiitis Arrhythmia Tachy-, bradyarrhythmia Supraventricular, ventricular Conduction abnormalities Stable, unstable Hypersensitive carotid sinus node Congestive heart failure Left, right, bi-ventricular, systolic, diastolic Cardiomyopathies, such as idiopathic familial, infective, metabolic, storage disease, deficiencies, connective tissue disorder, infiltration and granulomas, neurovascular Myocarditis Autoimmune, infective, idiopathic Cor pulmonale Blunt and penetrating trauma Toxins Cocaine toxicity Vascular Hypertension Primary, secondary Decompression sickness Fibromuscular hyperplasia Aneurysm Dissecting, ruptured, enlarging 551594 19 Cell, tissue or organ Dysfunction or pathology Condition or disease Type Lungs Obstructive Asthma Chronic bronchitis, Emphysema and airway obstruction Ischemic lung disease Pulmonary embolism, Pulmonary thrombosis, Fat embolism Environmental lung diseases Ischemic lung disease Pulmonary embolism Pulmonary thrombosis Interstitial lung disease Idiopathic pulmonary fibrosis Congenital Cystic fibrosis Cor pulmonale Trauma Pneumonia and pneumonitides Infectious, parasitic, toxic, traumatic, burn, aspiration Sarcoidosis Pancreas Endocrine Diabetes mellitus, type I and II Beta cell failure, dysfunction Diabetic neuropathy Other endocrine cell failure of the pancreas Exocrine Exocrine pancreas failure pancreatitis Bone Osteopenia Primary Secondary Hypogonadism immobilisation Postmenopausal Age-related Hyperparathyroidism Hyperthyroidism Calcium, magnesium, phosphorus and/or vitamin D deficiency Osteomyelitis Avascular necrosis Trauma Paget's disease Skin Alopecia Areata Totalis Primary Secondary Male pattern baldness 551594 Cell, tissue or organ Dysfunction or pathology Condition or disease Type Vitiligo Localized generalized Primary secondary Diabetic ulceration Peripheral vascular disease Burn injuries Autoimmune disorders Lupus erythematodes, Sjiogren, Rheumatoid arthritis, Glomerulonephritis, Angiitis Langerhan's histiocytosis Eye Optic neuritis Blunt and penetrating injuries, Infections, Sarcoid, Sickle C disease, Retinal detachment, Temporal arteritis Retinal ischemia, Macular degeneration, Retinitis pigmentosa, Arteriosclerotic retinopathy, Hypertensive retinopathy, Retinal artery blockage, Retinal vein blockage, Hypotension, Diabetic retinopathy, and Macular edema Embryonic and fetal disorders Asphyxia Ischemia 551594 21 Cell, tissue or organ Dysfunction or pathology Condition or disease Type CNS Chronic fatigue syndrome, acute and chronic hypoosmolar and hyperosmolar syndromes, AIDS Dementia, Electrocution Encephalitis Rabies, Herpes Meningitis Subdural hematoma Nicotine addiction Drug abuse and withdrawal Cocaine, heroin, crack, marijuana, LSD, PCP, poly-drug abuse, ecstasy, opioids, sedative hypnotics, amphetamines, caffeine Obsessive-compulsive disorders Spinal stenosis, Transverse myelitis, Guillian Barre, Trauma, Nerve root compression, Tumoral compression, Heat stroke ENT Tinnitus Meuniere's syndrome Hearing loss Traumatic injury, barotraumas 551594 22 Cell, tissue or organ Dysfunction or pathology Condition or disease Type Kidney Renal failure Acute, chronic Vascular/ischemic, interstitial disease, diabetic kidney disease, nephrotic syndromes, infections, injury, contrast-induced, chemotherapy-induced, CPB-induced, or preventive Henoch S. Purpura Striated muscle Autoimmune disorders Myasthenia gravis Dermatomyositis Polymyositis Myopathies Inherited metabolic, endocrine and toxic Heat stroke Crush injury Rhabdomylosis Mitochondrial disease Infection Necrotizing fasciitis Sexual dysfunction Central and peripheral (e.g. erectile dysfunction) Impotence secondary to medication, (diabetes) Liver Hepatitis Viral, bacterial, parasitic Ischemic disease Cirrhosis, fatty liver Infiltrative/metabolic diseases Gastrointestinal Ischemic bowel disease Inflammatory bowel disease Necrotizing enterocolitis Organ transplantation Treatment of donor and recipient RECEIVED at IPONZ on 22 December 2009 551594 23 Cell, tissue or organ Dysfunction or patholosv Condition or disease Type Reproductive tract Infertility Vascular Autoimmune Uterine abnormalities Implantation disorders Endocrine Glandular hyper- and hypofunction As mentioned above, these diseases, disorders or conditions are merely illustrative of the range of benefits provided by the described compound. Accordingly, generally described is the therapeutic or prophylactic treatment of the consequences of mechanical 5 travuna or of human diseases. Therapeutic or prophylactic treatment for diseases, disorders or conditions of the CNS and/or peripheral nervous system are preferred. Therapeutic or prophylactic treatment for diseases, disorders or conditions which have a psychiatric component are described. Therapeutic or prophylactic treatment for diseases, disorders or conditions including, but not limited to, those having an ophthalmic, cardiovascular, 10 cardiopulmonary, respiratory, kidney, urinary, reproductive, gastrointestinal, endocrine, or metabolic component are described.

Such a pharmaceutical composition comprising the described compound may be administered systemically to protect or enhance the target ceils, tissue, or organ. Such administration may be parenterally, via inhalation, or transmucosally, e.g., 15 orally, nasally, rectally, intravaginally, sublingually, submucosally or transdermally.

Preferably, administration is parenteral, e.g., via intravenous or intraperitoneal injection, and also including, but is not limited to, intra-arterial, intramuscular, intradermal and subcutaneous administration.

For other routes of administration, such as by use of a perfusate, injection into an organ, or 20 other local administration, a pharmaceutical composition is described which results in similar levels of the compound as described above. A level of about 0.0 lpM -30 nM is preferred.

RECEIVED at IPONZ on 22 December 2009 551594 24 The pharmaceutical compositions described herein may comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally 5 recognized foreign pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. A saline solution is a preferred 10 carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, 15 ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. The described compounds can be 20 formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. Examples of suitable pharmaceutical carriers are 25 described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

Pharmaceutical compositions adapted for oral administration may be provided as capsules or 30 tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non- RECEIVED at IPONZ on 22 December 2009 551594 aqueous liquids); as edible foams or whips; or as emulsions. Tablets or hard gelatine capsules may comprise lactose, starch or derivatives thereof, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, stearic acid or salts thereof, Soft gelatine capsules may comprise vegetable oils, waxes, fats, semi-solid, or liquid polyols etc. Solutions and syrups may comprise water, polyols, and sugars.

An active agent intended for oral administration may be coated with or admixed with a material that delays disintegration and/or absorption of the active agent in the gastrointestinal tract (e.g., glyceryl monostearate or glyceryl distearate may be used). Thus, the sustained release of an active agent may be achieved over many hours and, if necessary, the active agent can be protected from being degraded within the stomach. Pharmaceutical compositions for oral administration may be formulated to facilitate release of an active agent at a particular gastrointestinal location due to specific pH or enzymatic conditions.

Pharmaceutical compositions adapted for transdermal administration are described as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Pharmaceutical compositions adapted for topical administration may be ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For topical administration to the skin, mouth, eye or other external tissues a topical ointment or cream is preferably used. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops. In these compositions, the active ingredient can be dissolved or suspended in a suitable carrier, e.g., in an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles, and mouthwashes.

Pharmaceutical compositions adapted for nasal and pulmonary administration may comprise solid carriers such as powders (preferably having a particle size in the range of 20 to 500 microns). Powders can be administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nose from a container of powder held close to the nose. Alternatively, RECEIVED at IPONZ on 22 December 2009 551594 26 compositions adopted for nasal administration may comprise liquid carriers, e.g., nasal sprays or nasal drops. Alternatively, inhalation directly into the lungs may be accomplished by inhalation deeply or installation through a mouthpiece into the oropharynx. These compositions may comprise aqueous or oil solutions of the active ingredient. Compositions 5 for administration by inhalation may be supplied in specially adapted devices including, but not limited to, pressurized aerosols, nebulizers or insufflators, which can be constructed so as to provide predetermined dosages of the active ingredient. In a preferred embodiment, pharmaceutical compositions described herein are administered into the nasal cavity directly or into the lungs via the nasal cavity or oropharynx.

Pharmaceutical compositions can be adapted for rectal administration such as suppositories or enemas. Pharmaceutical compositions can be adapted for vaginal administration such a pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions adapted for parenteral administration include aqueous and nonaqueous sterile injectable solutions or suspensions, which may contain antioxidants, buffers, 15 bacteriostats, and solutes that render the compositions substantially isotonic with the blood of an intended recipient. Other components that may be present in such compositions include water, alcohols, polyols, glycerine and vegetable oils, for example. Compositions adapted for parenteral administration may be presented in unit-dose or multi-dose containers, for example sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition 20 requiring only the addition of a sterile liquid carrier, e.g., sterile saline solution for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. In one embodiment, an autoinjector comprising an injectable solution of a described compound may be useful for emergency use by ambulances, emergency rooms, and battlefield situations, and even for 25 self-administration in a domestic setting, particularly where the possibility of traumatic amputation may occur, such as by imprudent use of a lawn mower. The likelihood that cells and tissues in a severed foot or toe will survive after reattachment may be increased by administering a described compound to multiple sites in the severed part as soon as RECEIVED at IPONZ on 22 December 2009 551594 27 practicable, even before the arrival of medical personnel on site, or arrival of the afflicted individual with severed toe at the emergency room.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human 5 beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a 10 hermetically-sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile saline can be provided so that the ingredients may be mixed prior to administration.

Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.

A perfusate composition is described for use in transplanted organ baths, for in situ perfusion, or for administration to the vasculature of an organ donor prior to organ harvesting. 20 Such pharmaceutical compositions may comprise levels of the described compound not suitable for acute or chronic, local or systemic administration to an individual, but will serve the functions intended herein in a cadaver, organ bath, organ perfusate, or in situ perfusate prior to removing or reducing the levels of the described compound contained therein before exposing or returning the treated organ or tissue to regular circulation.

Also described is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or RECEIVED at IPONZ on 22 December 2009 551594 28 biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration.

In another embodiment, for example, the described compound can be delivered in a controlled-release system. For example, the polypeptide may be administered using 5 intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit, Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, the compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 10 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); WO 91/04014; U.S. Patent No. 4,704,355; Lopez-Berestein, ibid, pp. 317-327; see generally ibid.). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, Florida, 1974; Controlled Drug 15 Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley: New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61,1953; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et ah, 1989, J. Neurosurg. 71:105).

In yet another embodiment, a controlled release system can be placed in proximity of the 20 therapeutic target, i. e., the target cells, tissue or organ, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, pp. 115-138 in Medical Applications of Controlled Release, vol. 2, supra, 1984). Other controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533).

In another embodiment, the described compound, as properly formulated, can be 25 administered by nasal, oral, rectal, vaginal, or sublingual administration.

In a specific embodiment, it may be desirable to administer the described compositions locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a RECEIVED at IPONZ on 22 December 2009 551594 29 wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as silastic membranes, or fibers.

Selection of the preferred effective dose will be determined by a skilled artisan based upon 5 considering several factors which will be known to one of ordinary skill in the art. Such factors include the particular form of described compound, and its pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc., which will have been established during the usual development procedures typically employed in obtaining regulatory approval for a pharmaceutical compound. Further factors in considering the dose 10 include the condition or disease to be treated or the benefit to be achieved in a normal individual, the body mass of the patient, the route of administration, whether administration is acute or chronic, concomitant medications, and other factors well known to affect the efficacy of administered pharmaceutical agents. Thus the precise dosage should be decided according to the judgment of the practitioner and each patient's circumstances, e.g., depending upon the 15 condition and the immune status of the individual patient, and according to standard clinical techniques.

A perfusate or perfusion solution is provided for perfusion and storage of organs for transplant, the perfusion solution including an amount of the described compound, effective to protect responsive cells and associated cells, tissues, 20 or organs. Transplant includes, but is not limited to, xenotransplantation, where a organ (including cells, tissue or other bodily part) is harvested from one donor and transplanted into a different recipient; and autotransplant, where the organ is taken from one part of a body and replaced at another, including bench surgical procedures, in which an organ may be removed, and while ex vivo, resected, repaired, or otherwise manipulated, such as for tumor removal, 25 and then returned to the original location. In one embodiment, the perfusion solution is the University of Wisconsin (UW) solution (U.S. Patent No. 4,798,824) which contains from about 1 to about 25 U/ml erythropoietin, 5% hydroxyethyl starch (having a molecular weight of from about 200,000 to about 300,000 and substantially free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone); 25mM KH2PO4; 3mM glutathione; 5mM RECEIVED at IPONZ on 22 December 2009 551594 adenosine; lOmM glucose; lOmM HEPES buffer; 5mM magnesium gluconate; l.SmM CaC12; 105mM sodium gluconate; 200,000 units penicillin; 40 units insulin; 16mg Dexamethasone; 12mg Phenol Red; and has apH of 7.4-7,5 and an osmolality of about 320 mOSm/1. The solution is used to maintain cadaveric kidneys and pancreases prior to transplant. Using the solution, preservation can be extended beyond the 30-hour limit recommended for cadaveric kidney preservation. This particular perfusate is merely illustrative of a number of such solutions that can be adapted for the present use by inclusion of an effective amount of the compound as described herein. In a further embodiment, the perfusate solution contains from about O.Olpg/ml to about 400 ng/ml of the described compound, or from about 40 to about 300 ng/ml of the described compound.

While the preferred recipient of a compound as described herein for the purposes herein throughout is a human, the methods herein apply equally to other mammals, particularly domesticated animals, livestock, companion and zoo animals. However, the invention is not so limiting and the benefits can be applied to any mammal.

THERAPEUTIC AND PREVENTATIVE USES OF THE COMPOUNDS OF THE INVENTION As noted in Example 1 below, the presence of erythropoietin receptors in the brain capillary human endothelium indicates that the targets of the described compounds are present in the human brain, and that the animal studies on these compounds of the invention are directly translatable to the treatment or prophylaxis of human beings.

Methods and compositions for enhancing the viability of cells, tissues, or organs which are not isolated from the vasculature by an endothelial cell barrier are provided by exposing the cells, tissue or organs directly to a pharmaceutical composition comprising a compound as described herein, or administering or contacting a described compound containing pharmaceutical composition to the vasculature of the tissue or organ. Enhanced activity of responsive cells in the treated tissue or organ is responsible for the positive effects exerted.

RECEIVED at IPONZ on 22 December 2009 551594 31 As described above, the invention is based, in part, on the discovery that erythropoietin molecules can be transported from the luminal surface to the basement membrane surface of endothelial cells of the capillaries of organs with endothelial cell tight junctions, including, for example, the brain, retina, and testis. Thus, responsive cells across the barrier are 5 susceptible targets for the beneficial effects of a compound as described herein, and other cell types or tissues or organs that contain and depend in whole or in part on responsive cells therein are targets for the methods described herein. While not wishing to be bound by any particular theory, after transcytosis of a described compound, the described compound can interact with an erythropoietin receptor on an responsive cell, for example, 10 neuronal, retinal, muscle, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelial, testes, ovary, pancreas, bone, skin, or endometrial cell, and receptor binding can initiate a signal transduction cascade resulting in the activation of a gene expression program within the responsive cell or tissue, resulting in the protection of the cell or tissue, or organ, from damage, such as by toxins, chemotherapeutic agents, radiation 15 therapy, hypoxia, etc. Thus, methods for protecting responsive cell-containing tissue from injury or hypoxic stress, and enhancing the function of such tissue are described in detail herein below. As noted above, the methods described herein are equally applicable to humans as well as to other animals.

In the practice of one described embodiment a mammalian patient may be undergoing 20 systemic chemotherapy for cancer treatment, including radiation therapy, which commonly has adverse effects such as nerve, lung, heart, ovarian, or testicular damage. Administration of a pharmaceutical composition comprising a compound as described above is performed prior to and during chemotherapy and/or radiation therapy, to protect various tissues and organs from damage by the chemotherapeutic agent, such as to protect the testes. 25 Treatment may be continued until circulating levels of the chemotherapeutic agent have fallen below a level of potential danger to the mammalian body.

In the practice of another described embodiment, various organs were planned to be harvested from a victim of an automobile accident for transplant into a number of recipients, some of which required transport for an extended distance and period of time. Prior to organ RECEIVED at IPONZ on 22 December 2009 551594 32 harvesting, the victim was infused with a pharmaceutical composition comprising a compound as described herein. Harvested organs for shipment were perfused with a perfusate containing a compound as described herein, and stored in a bath comprising a compound as described herein. Certain organs were continuously perfused 5 with a pulsatile perfusion device, utilizing a perfusate containing a compound as described herein.

Minimal deterioration of organ function occurred during the transport and upon implant and reperfusion of the organs in situ.

In another described embodiment, & surgical procedure to repair a heart valve required temporary cardioplegia and arterial occlusion. Prior to surgery, the patient was infused with 4 10 ng of a described compound per kg body weight. Such treatment prevented hypoxic ischemic cellular damage, particularly after reperfusion.

In any surgical procedure, such as in cardiopulmonary bypass surgery, a compound as described herein can be used. In one embodiment, administration of a pharmaceutical composition comprising a compound 15 as described above is performed prior to, during, and/or following the bypass procedure, to protect the function of brain, heart, and other organs.

In the foregoing examples in which a compound as described herein is used for ex-vivo applications, or to treat responsive cells such as neuronal tissue, retinal tissue, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary endothelial, testes, 20 ovary, or endometrial cells or tissue, also described is a pharmaceutical composition in dosage unit form adapted for protection or enhancement of responsive cells, tissues, or organs distal to the vasculature which comprises, per dosage unit, an effective non-toxic amount within the range from about 0.01 pg to 5 mg, 1 pg to 5 mg, 500pg to 5 mg, 1 ng to 5 mg, 500 ng to 5 mg, 1 |ig to 5 mg, 500 fig to 5mg, or 1 mg to 5 mg of described compound and 25 a pharmaceutically acceptable carrier. In a preferred embodiment, the amount of a compound described herein is within the range from about 1 ng to 5mg.

EPO administration was found to restore cognitive function in animals having undergone brain trauma. The compounds described herein would RECEIVED at IPONZ on 22 December 2009 551594 33 be expected to have the same cellular protective effects as EPO. After a delay of either 5 days or 30 days, EPO was still able to restore function as compared to sham-treated animals, indicating the ability of a EPO to regenerate or restore brain activity. Thus, also described herein is the use of a compound for the preparation of a pharmaceutical composition for the treatment of brain trauma and other cognitive dysfunctions, including treatment well after the injury (e.g., three days, five days, a week, a month, or longer). Also described is a method for the treatment of cognitive dysfunction following injury by administering an effective amount of a compound of the invention. Any compound as described herein may be used for this described aspect.

Furthermore, this restorative aspect described herein is directed to the use of any of the compounds described herein for the preparation of a pharmaceutical composition for the restoration of cellular, tissue, or organ dysfunction, wherein treatment is initiated after, and well after, the initial insult responsible for the dysfunction. Moreover, treatment using a described compound can span the course of the disease or condition during the acute phase as well as a chronic phase.

In the instance wherein a compound has erythropoietic activity, the compound may be administered systemically at a dosage between about 1 fig and about 100 jxg /kg body weight, preferably about 5-50 jjg/kg-body weight, most preferably about 10-30 jig/kg-body weight, 20 per administration. This effective dose should be sufficient to achieve serum levels of the compound greater than about 10,000,15,000, or 20,000 mU/ml of serum after compound administration. Such serum levels may be achieved at about 1,2, 3, 4,5, 6, 7, 8, 9, or 10 hours post-administration. Such dosages may be repeated as necessary. For example, administration may be repeated daily, as long as clinically necessary, or after an appropriate 25 interval, e.g., every 1 to 12 weeks, but preferably, every 1 to 3 weeks. The effective amount of compound and a pharmaceutically acceptable carrier may be packaged in a single dose vial or other container. The compound described herein, however is nonerythropoietic, i.e., it is capable of exerting the activities described herein without causing an increase in hemoglobin concentration or hematocrit. Such a non-erythropoietic compound is especially preferred in RECEIVED at IPONZ on 22 December 2009 551594 34 (followed by 34a) instances wherein the methods described herein are intended to be provided chronically. In another embodiment, a described compound is given at a dose greater than that of a corresponding dose (W/W) of natural erythropoietin which would be necessary to maximally stimulate eiythropoiesis. As noted above, a compound as described herein does not have erythropoietic activity, and therefore the above dosages expressed in units are merely exemplary for corresponding amounts of natural erythropoietin; herein above molar equivalents for dosages are provided which are applicable to any compound described herein.

The term "comprising" as used in this specification means "consisting at least in part of'.

When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

In the description in this specification reference may be made to subject matter which is not within the scope of the claims of the current application. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the claims of this application.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

EXAMPLES The present invention may be better understood by reference to the following non-limiting examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

RECEIVED at IPONZ on 22 December 2009 34a by 35) Example 1 Production of Carbamylated Erythropoietin The starting material of the process in this example was purified recombinant human EPO.

First the protein concentration was adjusted by ultrafiltration for the purpose of keeping a low process volume, The protein concentration was adjusted to 3 mg/ml. The ultrafiltration was performed by means of a BioMax (Millipore) with a MWCO of 5 kDa.

After completion of the concentration step, the EPO solution was mixed with 0.5 M K-borate tetra hydrate 0.5 M K-cyanate, at pH 9.0 the solution was incubated at 32°C for 24 hours.

The desalting of the reaction mixture of EPO and cyanate was performed by gelfiltration. The protein was desalted to a 25 mM Tris, 50 mM NaCl pH 8.5 buffer. A G-25 Fine (Amersham Biosciences) resin was employed. 551594 Using a flow of 90 cm/h on an approximately 15 cm high column a sample load of approximately 20% of the column volume was applied.

The desalted carbamylated EPO was collected for further processing.

• At this point, the polymer/aggregate content was 7.3 %.

The next step was the removal of aggregates and polymers performed by a purification step using anion exchange. A SOURCE 30Q (Amersham Biosciences) resin was employed for the purification. Approximately 4.5 mg/ml of carbamylated EPO was applied to the column. 10 The running buffer A was: 25 mM Tris, 50 mM NaCl pH 8.5, and elution buffer B: 25 mM Tris, 1 M NaCl pH 8.5. The gradient was performed with 0-30 % over 20 column volumes the main peak of carbamylated EPO was collected and pooled.

The pool from the purification step was adjusted to a concentration > 0.5 mg/ml and buffer 15 changed to a 20 mM citrate, 100 mM NaCl buffer using a dia/ultrafiltration tangential flow filtration unit. The concentration and buffer change was performed on a 0. lm2 BioMax (Millipore) with a MWCO of 5 lcDa.

Finally the purified biopharmacutical drug substance was 0.22 um filtrated using a Millipak 20 filter (Millipore) to reduce germs.

The process resulted in carbamylated EPO with properties making it useful as a biopharmaceutical; • The polymer/aggregate content was 0.5 % as determined by SEC-HPLC • The carbamylated lysines was 100% as determined by aminoacid analysis • The concentration was > 0.5 mg/ml Characterization using MALDI-TOF for the determination of the change in the intact mass 30 both of PNGase treated protein and for the protein with the N-glycans was performed. In 551594 36 addition MALDI-TOF peptide mass fingerprint analysis/LC-MS/MS analysis was perfomed as follows: 1. CEPO and EPO were purified on a POROS R1 column (POROS R1 reverse phase column 5 material, PerSeptive Biosystems (1-1259-06)). The column material was stored in 50% HiPerSolv for HPLC VWR 152525R before use. The R1 column was equilibrated and washed in 5% formic acid (33015, Riedl de Haen). The samples were eluted from the column with Agilent MALDI HCCA quality matrix solution (G2037A). The intact mass was determined by analysis on a Bruker Reflex IV MALDI-TOF instrument. 2. 0.3 pmol CEPO and/or EPO were treated over night with 1 unit of PNGase F and PNGase F/O-glycosidase. Total mass was determined using MALDI-TOF 3. CEPO and EPO (1.5pmol) were reduced in solution with 50ul lOmM DTT, 50mM NH4CO3 and subsequently alkylated in 50ul 55 mM iodoacetamide, 50mM NH4CO3. The samples were purified on POROS R1 column before trypsin digestion. A fraction of the digested sample was purified on POROS R2 columns before MALDI-TOF analysis (POROS 50 R2 PerSeptive Biosystems (1-1159-05)). The R2 column was equilibrated and washed in 0.1% trifluoroacetic acid (99+% spectrometric grade, Aldrich 302031-100ml). The samples 20 were eluted from the column with Agilent MALDI HCCA quality matrix solution (G2037A). The pool of peptides obtained by the tryptic digestion were treated with PNGase F and purified over POROS R2 columns, and characterized by MALDI-TOF. 4. CEPO and EPO were reduced in solution with DTT and alkylated with iodoacetamide. 25 The samples were purified on POROS R1 column before Glu-C digestion. A fraction of the digested sample was purified on POROS R2 columns before MALDI-TOF analysis. The pool of peptides obtained by the Glu-C digestion were treated with PNGase F and purified over POROS R2 columns. 551594 37 . To rule out the possibility of having partly carbamylated CEPO, intact EPO and CEPO were digested with Lys-C. The samples were analysed using MALDI-TOF.

The conclusion was that the 8 lysines and the N-terminal were carbamylated. No other 5 carbamylated amino acids were detected and no modifications of the glycans were detected.

References: General protein identification by MS: Mann M, Hojrup P, Roepstorff P. (1993) Use of mass spectrometric molecular weight 10 information to identify proteins in sequence databases, Biol Mass Spectrom 22, 338-345 Yates, J R. Speicher S, Griffin P R, Hunkapiller T. (1993) Peptide mass maps: a highly informative approach to protein identification, Anal Biochem 214, 397-408 Intact mass: Laugesen S, Roepstorff P. (2003) Combination of two matrices results in improved performance of MALDI MS for peptide mass mapping and protein analysis. J Am Soc Mass Spectrom. 14(9), 992-1002.

Digest/grafit: Larsen MR, Hojrup P, Roepstorff P. (2005) Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry. Mol Cell Proteomics. 4(2), 107-19 Additional studies were done to determine the degree and homogeneity of carbamylation of EPO, the specificity of carbamylation and identity of carbamylation sites, and the presence of potential unspecific modifications due to side reactions of the carbamylation and purification 551594 38 process. Samples were analyzed by total mass analysis of deglycosylated protein samples and by peptide mapping using endoproteases LysC and trypsin for digestion and LC/MS analysis for peptide evaluation.

Example 2 Total Mass Analysis Analysis was performed on three EPO samples carbamylated using the method of Example 1. All three samples were purified following the carbamylation reaction using anion exchange, as described in example 1. One of the CEPO samples (designated CEPO-CMC) was 10 prepared by CMC Biotech, from a 1 gram production scale (concentration: 0.82 mg/ml; buffer: 20 mM Na-citrate, 0.3 mM citric acid, 0.1 M NaCl, pH 6.9-7.3), The remaining two samples, designated CEPO-1 and CEPO-2, were prepared from a 70 mg laboratory production scale (concentration: 1.1 mg/ml; buffer: 25 mM Tris, 0.2.M NaCl, pH 8.3-8.7). These CEPO samples were compared to unmodified or starting EPO (concentration: 0.82 15 mg/ml; buffer: 2 mM Na-citrate, 0.3mM citric acid, 0.1 NaCl, pH6.9-7.3) and mock CEPO (EPO which has gone through the carbamylation process without the addition of K-cyanate) (concentration: 0.38 mg/ml; buffer: 20mM Na-citrate, 0.3 mM citric acid, 0.1 MNaCl, pH 6.9-7.3).

ESI-mass spectrometry Prior to the total mass analysis, the samples were enzymatically deglycosylated. Each sample was incubated overnight with 50 |ig of N-glycosidase F (Prozyme from Glyko), recombinant neuraminidase from A. ureafaciens and O-glycosidase at a protein concentration of 0.5 mg/ml. Completeness of the deglycosylation reaction was checked by SDS-PAGE by 25 loading 3 jag of each sample onto 12% Tris-glycine gels. The remaining material from each sample was used for mass analysis.

The deglycosylated samples were brought to a concentration of 4-5 M guanidinium hydrochloride by adding the appropriate volume of guanidinium hydrochloride stock solution 551594 39 and were subsequently desalted into a buffer containing 2% formic acid and 40% acenitril. The guanidinium hydrochloride was added in order to ensure high recovery of deglycosylated EPO and CEPO for mass spectrometric analysis. Mass measurement was performed with a Waters ESI-Q-Tof- or a Waters ESI-LCT-mass spectrometer provided with an ESI-nanospray 5 source of ionization. Evaluation of data was conducted by automatic deconvolation and by manual evaluation of specific peaks of interest with Mass Lynx 4.0 software. Quantitation of the relative ratios of the differently carbamylated CEPO species was done by calculation of relative ratios based on the signal intensities recorded in the m/z spectrum.

The deglycosylation of the samples resulted in the N-linked sugars being removed completely. However, the release of the O-linked sugars was incomplete, especially for the CEPO samples.

As expected, due to the carbamylation, the CEPO samples showed different mass spectra as 15 compared to the EPO and mock-CEPO samples. As shown in Table 2, deconvolution of the spectra resulted in masses for the major peaks as theoretically expected for the various samples. In all of the CEPO samples, only highly carbamylated CEPO molecules were found, with the major isoform being the fully carbamylated isoform with the complete carbamylation of the 8 lysines and the N-terminus, i. e, 9 carbamyl residues. The CEPO 20 samples showed heterogeneity in containing additional isoforms corresponding to 8, 10 and 11 carbamyl residues attached to the CEPO. The species containing 8 carbamyl residues would be designated as under-carbamylated, missing at least one carbamyl residue. The species with 10 and 11 carbamyl residues would be designated as over-carbamylated, where the extra carbamyl residues attached would be bound in a non-specific manner to an amino 25 acid other than a lysine. There were some minor signals in the spectra of all of the samples but none were considered to be non-specifically modified species. Some of the minor signals were found in both the EPO and CEPO samples and were considered to be contaminants already present in the starting EPO. 551594 40 Table 2 Deconvolved Masses of Deglycosylated CEPO- and EPO- samples obtained in Total Mass Analysis SAMPLE Mass Theoretically Mass Obtained Interpretation Expected Starting EPO 18239 18237 Corresponds Mock-CEPO 18239 18239 Corresponds CEPO-CMC 18626 (9x carb) 18583 8x carb 18626 9x carb 18669 lOx carb 18711 llx carb 18991 9x carb + O-sugar CEPO-1 18626 (9x carb) 18583 8x carb 18626 9x carb 18669 lOx carb 18711 llx carb 18989 9x carb + O-sugar CEPO-2 18626 (9x carb) 18584 8x carb 18626 9x carb 18669 lOx carb 18712 llx carb 18992 9x carb + O-sugar Table 3 shows the relative ratios of the various isoforms. Under-carbamylated CEPO ranges from about 1.5 to 5.5% of the total CEPO depending on the sample and over-carbamylated CEPO ranges from about 11 to 22% depending on the sample. CEPO-1 and CEPO-2 have 10 similar distribution of the isoforms while the CEPO-CMC has less under-carbamylated 551594 41 species as compared to the other two samples. Different production scales give rise to products with a different distribution, but as Table 3 shows for the laboratory production scale, the production may be repeated with similar outcome. As discussed earlier, at any given production scale, the distribution may be adjusted by adjusting the pooling from the 5 anion exchange column.

The numbers in Table 3 represent the minimum ratio of under- and over-carbamylated CEPO. The reason for this is the degree of carbamylation (8-fold to 11- fold) may not specify the exact degree of under, full and over-carbamylated CEPO. For example, a CEPO molecule 10 containing 8 carbamyl residues as determined by mass analysis may have only 7 carbamyl groups specifically linked to lysines and the remaining carbamyl residue would be non-speciflcally attached another amino acid. This situation would be considered only as under-carbamylated, even though there is non-specifically bound carbamyl groups. Conversely, a CEPO molecule containing 10 carbamyl residues may have attached to only 8 residues 15 specifically and two are bound non-specifically. In this situation, the CEPO isoform is considered only over-carbamylated, even though all of the lysines are not carbamylated.

Table 3 Degree and Heterogeneity of Carbamylation: Relative Ratios of Differently Carbamylated 20 CEPO Species No.

RELATIVE CONTENT Degree of Carbamylatio n CEPO-CMC* CEPO-1* CEPO-2** 1 8x carb 1.5% .1% .5% 2 9x carb 77% 83.3% 83.6% 3 lOx carb 19.8% .9% .2% 551594 42 4 1 lx carb 1.7% 0.7% 0.7% 2 No. 2-4 (>9x) 98.5% 94.9% 94.5% 6 E No. 3+4 (over carb.) 21.5% 11.6% .9% * n=2 **11=1 Example 3 5 LysC Peptide Mapping Peptide map analysis of the EPO and CEPO samples was performed using endoproteases LysC and trypsin for fragmentation. All peptide map analyses were conducted with degycosylated peptides. The enzymatic deglycosylation of the peptides was performed 10 simultaneously with the digestion with the endoprotease.

EPO and CEPO samples (about 150 \ig each) were denatured and reduced by incubation with guanidinium hydrochloride and DTT. Free sulfhydryl groups were alkylated with iodoacetic acid. The alkylated samples were desalted and the buffer was exchanged to the appropriate 15 buffer by using single use gel filtration columns.

The endoprotease, N-glycosidase and neuraminidase were added simultaneously to the alkylated EPO and CEPO samples. The samples were incubated overnight at 37°C. After incubation, about 5(Xg of each digest was applied to RP-HPLC/MS analysis using a Jupiter, 20 CI 8 RP-column from Phenomenix coupled to an ESI-LCT from Waters. The UV signal at 220 nM and the total ion counts (TIC) in the mass spectrometer were recorded. For identification and quantification of the peptides obtained, the TIC was evaluated. 551594 43 Because LysC would not be able to cleave carbamylated lysines, it would be expected that no fragments would be formed by digestion with LysC if all of the lysines are carbamylated, indicating specificity of carbamylation. In the case of under-carbamylation, specific fragments of CEPO should form. Table 4 lists the fragments theoretically formed from LysC 5 digestion for EPO, fully and over-carbamylated CEPO and under-carbamylated CEPO.

Table 4 LysC Peptide Mapping: Lists of Peptide/Fragments Theoretically Formed from Digestion of LysC of EPO, fully or over-carbamylated CEPO and under-carbamylated CEPO 10 EPO Peptide Name Amino acids from - to Mass Amino acid Sequence K1 1-20 2399.3 APPR LIDSR VLER YLLEAK K2 21-45 2804.2 EANITTGCAEHCS LNENEITVPDDTK K3 46-52 926.5 VNFYAWK K4 53-97 5022.7 RMEVGQQAVEV WQGLALLSEAVL RGQALLVNSSQP WEPLQLHVDK K5 98-116 1954.2 AVSGLR SLTTLLR ALGAQK K6 (+ O-sugar) 117-140 2863.3 E AISPPD AAS AAP LR TITADTFR K K7 141-152 1498.8 LFR VYSNFLR GK K8 153-154 259.2 LK 551594 44 K9 155-165 1242.5 LYTGEACRTGD CEPO (fully carbamylated and over-carbamylated) Fragment Name Amino acids from - to Mass CEPO (9x carb) 1-165 19227 CEPO (lOx carb) 1-165 19270 CEPO (1 Ix carb) 1-165 19313 Under-carbamylated CEPO (8x carbamylation) Non-carbamylated amino group Mass expected for N-terminal fragment Mass expected for C-terminal fragment None (9x carbamylated) 19227 — N-terminus 19184 -- Lys20 2443.9 16755.9 Lys45 5274.9 13924.8 Lys52 6227.0 12972.7 Lys97 11276.8 7923.0 Lys 116 13257.1 5942.6 Lysl40 16147.2 3052.4 Lys152 17672.0 1527.5 Lys154 17956.4 1244.3 As expected the LysC peptide pattern obtained for EPO and CEPO samples digested with LysC were completely different. Digestion of starting EPO and mock-CEPO with LysC resulted in the peptide pattern as expected. In both samples, all of the peptides K1 to K9 could be identified. Peptide K5 and K1 were partially cleaved in an unspecific manner, in 10 both the EPO and mock-CEPO. No significant additional peaks could be identified nor were peaks missing in the LysC map of mock-CEPO as compared to starting EPO. From these data, it can be concluded that no significant non-specific covalent modifications of the EPO protein occurred during the carbamylation and purification process. 551594 45 The peptide maps of the CEPO samples had a different peptide pattern from the starting EPO. There was a single major peak and some minor peaks. As shown in Table 5, the masses obtained from the peaks correlated well with masses expected either for uncleaved CEPO or 5 for fragments from LysC cleavage of under-carbamylated CEPO. The major peak (A) contained intact, deglycosylated, folly carbamylated CEPO (9x carbamyls) and over-carbamylated CEPO (lOx carbamyls) (Table 5). The four minor peaks (B-E) contained under-carbamylated CEPO (8x carbamyls), with different peaks containing under-carbamylated CEPO not carbamylated at certain lysines. Peaks B and C contained under-10 carbamylated CEPO specifically lacking carbamylation at Lys45, while peaks D and E contained under-carbamylated CEPO specifically lacking carbamylation at Lys97 (Table 5).

Table 5 LysC Peptide Mapping: Assignment of Masses Experimentally Obtained to CEPO Fragments 15 theoretically deriving from Under-carbamylated CEPO Peak No.

Mass obtained Interpretation Mass expected A 19228 CEPO (9x carb + 0-sugar) 19227 19271 CEPO (lOx carb + O-sugar) 19270 B 18867 CEPO (9x carb w/o O-sugar) 18862 13926 CEPO-fragment (aa46-165) 13924.3 C 5276 CEPO-fragment (aal-45) 5274.8 D 7924 CEPO-fragment 7922.4 551594 46 (aa98-165) E 11279 CEPO-fragment (aal-97) 11276.6 There were some differences in the relative ratios of the peaks for the different CEPO samples. CEPO samples 1 and 2 had more prominent peaks D and E than CEPO-CMC, indicating that they contained more under-carbamylated CEPO species.

It was difficult to quantitate the minor peaks as related to the major peak due to technical limitations. However using the peak areas from UV-detection as a crude indication for the relative ratios of the various species, it could be estimated that under-carbamylated CEPO may account for less than 10% of the CEPO isoforms in the samples and that CEPO-1 and CEPO-2 have double the amount of under-carbamylated isoforms than the CEPO-CMC.

Using the same procedure as was used for the total mass analysis, quantitation of the over-carbamylated species located in peak A was about 21% for CEPO-CMC and 12% for CEPO-1 and CEPO-2.

Overall, the data from the LysC peptide mapping is in good agreement with the total mass 15 analysis described in Example 2. CEPO-CMC contained less under-carbamylated CEPO isoforms while CEPO-1 and CEPO-2 contained more over-carbamylated CEPO isoforms. This peptide mapping also indicated that lysine 45 and lysine 97 may represent sites of under-carbamylation.

Example 4 Trypsin Peptide Mapping The digestion of the sample using trypsin is described in Example 3.

Digestion of EPO and mock CEPO with trypsin resulted in a peptide pattern as expected. In both samples, most of the peptides (T1 to T21) as expected for trypsin digestion could be 551594 PCTYDK2005/000477 47 identified, except for some small di- and tri-peptides (such as T21). See Table 6. As was the case with the LysC digestion, there were no significant additional peaks nor were peaks missing from the mock-CEPO, as compared to starting EPO. From these data, it was concluded that there were no non-specifically covalent modifications of the EPO protein 5 during carbamylation and purification process.

The peptide patterns of the CEPO samples were different from the unmodified EPO. Trypsin normally cleaves at lysine and arginine. thus it would be expected that in the case of fully carbamylated EPO only fragmentation would occur by cleavage of the arginines. Thus, after 10 the peptide pattern is obtained with trypsin, the specific carbamylation sites and unspecifically carbamylated peptides could be identified. The peptides expected from a tryptic digestion of CEPO molecules assuming cleavage at the arginines (R) only is set forth in Table 6.

Table 6 List of Peptides Expected for EPO and CEPO digested with Trypsin EPO Peptide name Amino acid from -to Mass (MH) Amino acid sequence T1 1-4 439.3 APPR T2 -10 763.4 LICDSR T3 11-14 513.3 VLER T4 -20 735.4 YLLEAK T5 21-45 2806.2 EANITTGCAEHCS LNENITVPDDTK T6 46-52 926.5 VNFYAWK T7 53-53 174.1 R T8 54-76 2525.3 MEVGQQAVEVW QGLALLSEAVLR 551594 48 T 9 77-97 2359.2 GQALLVNSSQPW EPLQLHVDK T10 98-103 601.4 AVSGLR Til 104-110 802.5 SLTTLLR Til 111-116 586.3 ALGAQK T13 (+ O-sugar) 117-131 1829.8 EAISPPDAAS AAP LR T14 132-139 923.5 TITADTFR T15 140-140 146.1 K T16 141-143 434.3 LFR T17 144-150 897.5 VYSNFLR T18 151-152 203.1 GK T19 153-154 259.2 LK T20 155-162 969.4 LYTGEACR T21 163-165 291.1 TGD CEPO (assumed cleavage only at Arg (R) due to complete carbamylation) Peptide Name Amino acids from -to Mass (MH) Amino acid sequence R1 (lx carb) 1-4 482.3 APPR R2 -10 763.4 LICDR R3 11-14 515.3 VLER R4 (3x carb) -53 4717.2 YLLEAKEANITT GCAEHCSLNENIT VPDDTKVNF Y A WKR R5 54-76 2525.3 MEVGQAVEVWQ GLALLSEAYLR R6 (lx carb) 77-103 2985.3 GQALLVNSSQPW 551594 49 EPLQLHVDICAVS GLR R7 104-110 802.5 SLTTLLR R8 (lx carb) (4- O-sugar) 111-131 2441.1 ALGAQKEAISPPD AASAAPLR R9 132-139 923.5 TITADTFR RIO (lx carb) 140-143 605.4 KLFR Rll 144-150 897.5 VYSNFLR R12 (2x carb) 151-162 1481.7 GKLKLYTGEACR R13 163-165 291.1 TGD All of the peptides expected from trypsin cleavage were identified as major peaks in all of the CEPO samples, with the exception of the C-terminal peptide R13. This peptide was also not 5 found in the starting EPO or mock-CEPO. The peaks of R1 to R12 resulting from specific cleavage of only arginines accounted for the vast majority of peptides detected in the CEPO samples. Furthermore, all lysine-containing peptides were detected almost exclusively in the fully carbamylated form. These data indicate a high degree of specific carbamylation at the lysines and N-terminal.

In addition to the major peptide, six minor peptides were also detected in all the CEPO samples. Three of the minor peptides were identified by mass analysis as a result of tryptic cleavage of over-carbamylated CEPO and the remaining three resulted from under-carbamylated CEPO.

Table 7 lists all the peptides identified in the tryptic maps of the three CEPO samples analyzed. The high abundant peptides, R1 to R12, formed from completely and specifically carbamylated EPO are in regular letters, the correctly carbamylated EPO is additionally denoted in bold type. Peptides most likely formed by cleavage of under-carbamylated CEPO 551594 are denoted in italic type and peptides formed by cleavage of over-carbamylated CEPO are denoted in underlined type.

Table 7 Tryptic Peptide Map: List of Peptides Identified in Tryptic Peptide Map of CEPO Peptide Mass Relative Ion Counts (%)* CEPO-1 Relative Ion Counts (%)* CEPO-2 Relative Ion Counts (%)* CEPO-CMC Rl_lxcarb 482.25 1.28 1.09 1.15 R2 763.35 7.87 8.49 8.15 R3 515.31 2.78 2.58 2.66 R4_bl_lxcarb (T6+T7_ lxcarb 1125.6 0.17 0.2 0.15 R4_3xcarb 4717.2 1.5 0.80 1.32 R5 2525.34 11.44 9.56 9,54 R5_ox 2541.34 0.15 0.15 0.21 R5_Na 2547.32 0.15 0.13 0.14 R5_al 1869.97 0.17 0.17 0.16 R5_bl 673.38 0.29 0.27 0.25 T9=R6al 2359.24 0.62 0.54 0.26 T10=R6_bl 602.35 0.95 0.8 0.65 R6_lxcarb 2985.60 .36 14.48 .78 R7 802.49 7.16 7.28 7.05 R7 lxcarb 845.49 + 1 V V R8_lxcarb 2076.10 0.16 0.15 0.16 R8_lxcarb 2076.10 0.5 0.44 0,63 R8_SA0_lxcar b 2441.1 9.19 9.34 9.20 551594 51 R9 923.47 9.85 11.11 .58 RIOlxcarb 605.37 .17 .40 .43 RIO 2xcarb 648.37 0.02 0.03 0.03 Rll 897.47 9.82 .70 .30 R12_al_2xcar b 806.46 0.20 0.22 0.17 R12_2xcarb 1481.73 7.91 7.90 7.84 R12 3xcarb 1524.74 0.28 0.26 0.59 R13 291.11 - — — *- intensity relative to the total number of counts in TIC 1 - found by manual evaluation; signal confirmed as specific Referring to Table 7, the minor peaks T9 and T10 containing peptides R6_al and R6_bl, respectively, most likely derive from the cleavage of under-carbamylated CEPO molecules, which are not carbamylated at Lys97. Peptide R4_bl i lxcarb is formed if the Lys45 amino acid is not carbamylated. Peptide R6_al (peak T9) is more abundant in the CEPO-1 and 10 CEPO-2 samples than the CEPO-CMC indicating that the former may have double the amount of under-carbamylated CEPO. Peptides R6_bl and R4_bl+lxcarb were present in equal amounts in all CEPO samples.

Calculation of the relative content of under-carbamylated CEPO from these data was difficult 15 due to technical limitations. However, talcing all the observations together, it was estimated that the under-carbamylation species was about 10% as it was deduced from the total mass analysis and LysC peptide mapping.

Three other minor peptides which co-eluted with other peptides were interpreted by mass as 20 containing one extra carbamyl residue, i.e., over-carbamylated CEPO. Peptides R10_2xcarb 551594 52 and R7_lxcarb were detected in trace amounts, where R12_3xcarb was found to have significant signal intensities. CEPO-CMC had a two-fold higher content of over-carbamylated CEPO species as CEPO-1 and CEPO-2. This is in agreement with the results from the total mass analysis. It could also be concluded from these data that the amino acid 5 sequence of 151 -62 of EPO is a site for unspecific carbamylation.

Again, for the same reasons as with the quantitation of under-carbamylation, the quantitation of over-carbamylated species was difficult. However, assuming that the ionization efficiency of the peptides only differing in degree of carbamylation is similar, it was calculated by the 10 relative ion counts of the R12-derivatives that about 3-7% of the CEPO is over-carbamylated species. This is lower than the amount of over-carbamylated isoforms calculated by total mass analysis.

In general, the following can be concluded from the total mass analysis and peptide mapping 15 data of the EPO and CEPO.

From the total mass analysis, the CEPO samples appeared to carbamylated to a rather high degree. About 95-98% of all molecules are folly carbamylated and contain at least 9 carbamyl residues (see Table 3). The LysC and tryptic mapping confirm the high degree of 20 carbamylation at specific sites and that most likely over 95% of the CEPO molecules are fully carbamylated at the 8 lysines and the N-terminus.

The data also showed found four isoforms of CEPO. Species with 8, 9,10 and 11 carbamyl residues were detected in the CEPO samples analyzed, with the 9 carbamyl isoform being the 25 dominant species. A minor portion of the CEPO molecules contained 8 carbamyl residues instead of 9 and were considered under-carbamylated. For CEPO-1 and CEPO-2 these isoforms were about 5% of the total and for the CEPO-CMC, this isoform made up about 1.5% of the total CEPO molecules. 551594 53 A more significant portion of the CEPO molecules were over-carbamylated, i.e., contain 10 or 11 carbamyl residues. Over -carbamylated CEPO ranges from about 11% for CEPO-1 and CEPO-2 to about 22% in CEPO-CMC.

The data from the peptide mapping showed a high degree of specificity of carbamylation at the 8 lysines and N-terminus. Moreover, the peptide mapping generally confirmed the results of the total mass analysis. At least about 90-95% of the CEPO molecules appeared to be specifically modified by carbamyl residues at all of the lysines and the N-terminus. However, this data also showed under- and over-carbamylation CEPO species. Due to some technical 10 limitations, the exact ratio of under-carbamylated species was hard to determine, but it is estimated to be in the range of up to about 10% which is in agreement with the numbers found in the total mass analysis. Moreover, two distinct positions on the EPO, Lys45 and Lys97, were identified as the ones where lack of carbamylation was most likely to occur.

In both sets of peptide mapping, over-carbamylated species were also found. Again, due to technical limitations, the exact amount of over-carbamylated species was hard to quantitate. From the data obtained, it could be speculated that too little over-carbamylated species were detected in the peptide mapping. Only one-third to one-half of the amount of over-carbamylated species was identified by the peptide mapping as compared to the total mass 20 analysis. A reasonable explanation for this discrepancy is that not all of the over- carbamylated peptides were identified at all or in the correct quantitiy. In the LysC map, an uncleaved CEPO species containing 10 carbamyl residues was detected in significant amounts and in the tryptic mapping three peptides were detected that contained one extra carbamyl residue. Two of these fragments were detected in only trace amounts. The third 25 peptide, CEPO peptide amino acids 152-162, close to the C-terminus, appears to account for one-third of the over-carbamylation and may be a site for non-specific carbamylation in EPO.

No significant amounts of other non-specific (not carbamyl related) modifications were detected in the CEPO samples or in the mock CEPO samples by any analysis performed. 551594 54

Claims (78)

1. Claims 5 1. A method for producing a carbamylated erythropoietin protein having less than about 40% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein as measured by ESI-mass spectrometry, which method comprises contacting an amount of erythropoietin with an amount of cyanate at a temperature, pH, and for a time period sufficient for the amine groups on the lysines and the N-terminal amino acids of the 10 erythropoietin to become at least about 90% carbamylated.
2. 2. The method of claim 1, wherein the carbamylated erythropoietin protein is human erythropoietin. 15
3. 3. The method of claim 1, wherein the carbamylated erythropoietin protein has less than about 30% aggregated protein.
4. 4. The method of claim 1, wherein the carbamylated erythropoietin protein has less than about 20% aggregated protein. 20
5. 5. The method of claim 1, wherein the carbamylated erythropoeitin protein has less than about 10% aggregated protein.
6. 6. The method of claim 1, wherein the carbamylated erythropoietin protein has less than 25 about 30% by weight of over- and under-carbamylated protein.
7. 7. The method of claim 1, wherein the carbamylated erythropoietin protein has less than about 20% by weight of over- and under-carbamylated protein. WO 2006/002646 551594 PCT/DK2005/000477 55
8. 8. The method of claim 1, wherein the carbamylated erythropoietin protein has less than about 10% by weight of over- and under-carbamylated protein,
9. 9. The method of claim 1, wherein the carbamylated erythropoietin protein has less than 5 about 30% by weight of over-carbamylated protein.
10. 10. The method of claim 1, wherein the carbamylated erythropoietin protein has less than about 20% by weight of over-carbamylated protein. 10
11. 11. The method of claim 1, wherein the carbamylated erythropoietin protein has less than about 10% by weight of over-carbamylated protein.
12. 12. The method of claim 1, wherein the concentration of erythropoietin protein contacted with the cyanate is from about 0.05 mg/ml to about 10 mg /ml. 15
13. 13. The method of claim 1, wherein the concentration of erythropoietin protein contacted with the cyanate is about 2 mg/ml to about 5 mg/ml.
14. 14. The method of claim 1, wherein the concentration of the cyanate is from about 0.05 M 20 to about 10 M.
15. 15. The method of claim 1, wherein the concentration of the cyanate is from about 0.05 M to about 2 M. 25
16. 16. The method of claim 1, wherein the temperature ranges from about 0°C to about 60° C.
17. 17. The method of claim 1, wherein the temperature ranges from about 30°C to about 34°C. 30 WO 2006/002646 551594 PCT/DK2005/000477 56
18. 18 The method of claim 1, wherein the pH is from about 7 to about 11.
19. 19. The method of claim 1, wherein the pH is from about 8 to about 10. 5
20. 20. The method of claim 1, wherein the time period is from about 10 minutes to about 30 days.
21. 21. The method of claim 1, wherein the time period is from about 1 hour to about 5 days, 10
22. 22. The method of claim 1, wherein the erythropoietin protein is contacted with the cyanate in the presence of a buffer.
23. 23. The method of claim 22, wherein the buffer is borate, 15
24. 24. The method of claim 22, wherein the concentration of the buffer is from about 0.05 M to about 2 M.
25. 25. The method of claim 22, wherein the concentration of the buffer is from about 0.1 M to about 1 M. 20
26. 26. The method of claim 22, wherein the concentration of the buffer is about 0.5M.
27. 27. The method of claim 1, wherein the concentration of the erythropoietin protein contacted with the cyanate is from about 0.05 mg/ml to about 10 mg/ml, the concentration of 25 the cyanate is from about 0.05 M to about 10 M, the temperature ranges from about 0°C to about 60°C, the pH is from about 7 to about 11, and the time is from about 10 minutes to thirty days.
28. 28. The method of claim 1, wherein the concentration of the erythropoietin protein 30 contacted with the cyanate is from about 2 mg/ml to about 5 mg/ml, the concentration of the WO 2006/002646 551594 PCT/DK2005/000477 57 cyanate is from about 0.05 M to about 2 M, the temperature ranges from about 30°C to about 34°C, the pH is from about 8 to about 10, and the time is from about 1 hour to 5 days.
29. 29. The method of claim 1, wherein the concentration of the erythropoietin protein 5 contacted with the cyanate is about 3 mg/ml, the concentration of the cyanate is about 0.5 M, the temperature is about 32°C, the pH is about 9.0, and the time period is about 24 hours.
30. 30. A method for producing a carbamylated erythropoietin protein having less than about 3% aggregated protein and less than about 40% by weight of over- and under-carbamylated 10 protein as measured by ESI-mass spectrometry, comprising purifying the carbamylated erythropoietin using anion exchange, cation exchange, hydrophobic interaction chromatography, reverse phase chromatography, affinity chromatography or size exclusion chromatography. 15
31. 31. The method of claim 30, wherein the carbamylated erythropoietin protein is human erythropoietin.
32. 32. The method of claim 30, wherein at least about 90% of the amine residues on the lysines and the N-terminal amino acid of the erythropoietin are carbamylated. 20
33. 33. The method of claim 30, wherein the carbamylated erythropoietin protein has less than about 2.5% aggregated protein.
34. 34. The method of claim 30, wherein the carbamylated erythropoietin protein has about 25 0.5% or less aggregated protein.
35. 35. The method of claim 30, wherein the carbamylated erythropoietin protein has less than about 30% by weight of over- and under-carbamylated protein. WO 2006/002646 551594 PCT/DK2005/000477 58
36. 36. The method of claim 30, wherein the carbamylated erythropoietin protein has less than about 20% by weight of over- and under-carbamylated protein.
37. 37. The method of claim 30 wherein the carbamylated erythropoietin protein has less than 5 about 10% by weight of over- and under-carbamylated protein.
38. 38. The method of claim 30, wherein the carbamylated erythropoietin protein has less than about 30% by weight of over-carbamylated protein. 10
39. 39. The method of claim 30, wherein the carbamylated erythropoietin protein has less than about 20% by weight of over-carbamylated protein.
40. 40. The method of claim 30, wherein the carbamylated erythropoietin protein has less than about 10% by weight of over-carbamylated protein. 15
41. 41. A method for producing a carbamylated erythropoietin protein having less than about 3% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein as measured by ESI mass spectrometry, which method comprises: (a) contacting an amount of an erythropoietin protein with an amount of cyanate 20 at a temperature, pH, and for a time period sufficient for at least about 90% of the amine residues on the lysine and the N-terminal amino acids of the erythropoietin to become carbamylated; and (b) purifying the carbamylated erythropoietin protein using anion exchange, cation exchange, hydrophobic interaction chromatography, reverse phase chromatography, 25 affinity chromatography or size exclusion chromatography.
42. 42. The method of claim 41, wherein the carbamylated erythropoietin protein is human erythropoietin. WO 2006/002646 551594 PCT/DK2005/000477 59
43. 43. The method of claim 41, wherein the carbamylated erythropoietin protein has less than about 2.5% aggregated protein.
44. 44. The method of claim 41, wherein the carbamylated erythropoietin protein has about 5 0.5% or less aggregated protein.
45. 45. The method of claim 41, wherein the carbamylated erythropoietin protein has less than about 30% by weight of over- and under-carbamylated protein. 10
46. 46. The method of claim 41, wherein the carbamylated erythropoietin protein has less than about 20% by weight of over- and under-carbamylated protein.
47. 47. The method of claim 41, wherein the carbamylated erythropoietin protein has less than about 10% by weight of over- and under-carbamylated protein. 15
48. 48. The method of claim 41, wherein the carbamylated erythropoietin protein has less than about 30% by weight of over-carbamylated protein.
49. 49. The method of claim 41, wherein the carbamylated erythropoietin protein has less 20 than about 20% by weight of over-carbamylated protein.
50. 50. The method of claim 41, wherein the carbamylated erythropoietin protein has less than about 10% by weight of over-carbamylated protein. 25
51. 51. The method of claim 41, wherein the concentration of erythropoietin protein contacted with the cyanate is from about 0.05 mg/ml to about 10 mg/ml.
52. 52. The method of claim 41, wherein the concentration of erythropoietin protein contacted with the cyanate is from about 2 mg/ml to about 5 mg/ml. 30 WO 2006/002646 PCT/DK2005/000477 60
53. 53. The method of claim 41, wherein the concentration of erythropoietin protein contacted with the cyanate is about 3 mg/ml.
54. 54. The method of claim 41, wherein the concentration of the cyanate is from about 0.05 5 M to about 10 M.
55. 55. The method of claim 41, wherein the concentration of the cyanate is from about 0.05 M to about 2 M. 10
56. 56 The method of claim 41, wherein the concentration of the cyanate is about 0.5 M.
57. 57. The method of claim 41, wherein the temperature ranges from about 0°C to about 60° C. 15
58. 58. The method of claim 41, wherein the temperature ranges from about 30°C to about 34° C.
59. 59. The method of claim 41, wherein the temperature is about 32°C. 20
60. 60. The method of claim 41, wherein the pH is from about 7 to about 11.
61. 61. The method of claim 41, wherein the pH is from about 8 to about 10.
62. 62. The method of claim 41, wherein the pH is about 9. 25
63. 63. The method of claim 41, wherein the time period is from about 10 minutes to about 30 days.
64. 64. The method of claim 41, wherein the time period is from about 1 hour to about 5 days. 30 WO 2006/002646 551594 PCT/DK2005/000477 61
65. 65. The method of claim 41, wherein the time period is about 24 hours.
66. 66. The method of claim 41, wherein the erythropoietin protein is contacted with the cyanate in the presence of a buffer. 5
67. 67. The method of claim 66, wherein the buffer is borate.
68. 68. The method of claim 66, wherein the concentration of the buffer is from about 0.05 M to about 2 M. 10
69. 69. The method of claim 66, wherein the concentration of the buffer is from about 0.1 M to about 1 M.
70. 70. The method of claim 66, wherein the concentration of the buffer is about 0.5M. 15
71. 71. The method of claim 41, wherein the concentration of erythropoietin protein contacted with the cyanate is from about 0.05 mg/ml to about 10 mg/ml, the concentration of the cyanate is from about 0.05 M to about 10 M, the temperature ranges from about 0°C to about 60°C, the pH is from about 7 to about 11, and the time is from about 10 minutes to thirty 20 days.
72. 72. The method of claim 41, wherein the concentration of erythropoietin protein contacted with the cyanate is from about 2 mg/ml to about 5 mg/ml, the concentration of the cyanate is from about 0.05 M to about 2 M, the temperature ranges from about 30°C to about 34°C, the 25 pH is from about 8 to about 10, and the time is from about 1 hour to 5 days.
73. 73. The method of claim 34, wherein the concentration of the erythropoietin protein contacted with the cyanate is about 3 mg/ml, the concentration of the cyanate is about 0.5 M, the temperature is about 32°C, the pH is about 9.0, and the time period is about 24 hours. 30 RECEIVED at IPONZ on 22 December 2009 55159462
74. 74. A method for producing a carbamylated erythropoietin protein having less than about 40% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein, which method comprises contacting an amount of erythropoietin with an amount of cyanate at a temperature, pH, and for a time period sufficient for the amine groups on the lysines and the N-terminal amino acids of the erythropoietin to become at least about 90% carbamylated and wherein the erythropoietin is contacted with the cyanate in the presence of potassium borate.
75. 75. A method for producing a carbamylated erythropoietin protein having less than about 40% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein, which method comprises contacting an amount of erythropoietin with an amount of cyanate at a temperature of 30°C to 34°C, pH, and for a time period sufficient for the amine groups on the lysines and the N-terminal amino acids of the erythropoietin to become at least about 90% carbamylated.
76. 76. A method for producing a carbamylated erythropoietin protein having less than about 40% aggregated protein and less than about 40% by weight of over- and under-carbamylated protein, which method comprises contacting an amount of erythropoietin with an amount of cyanate at a temperature, pH, and for a time period sufficient for the amine groups on the lysines and the N-terminal amino acids of the erythropoietin to become at least about 90% carbamylated, and wherein the carbamylated erythropoietin protein is purified using anion exchange chromatography while maintaining the pH of both the running buffer and the elution buffer at 8.5 ± 0.2.
77. 77. A method according to any one of claims 1, 30, 41, 74, 75 and 76, substantially as herein described with reference to any example thereof.
78. 78. A carbamylated erythropoietin protein prepared by a method according to any one of claims 1 to 77.