MXPA00005048A

MXPA00005048A - Erythropoietin with high specific activity

Info

Publication number: MXPA00005048A
Application number: MXPA/A/2000/005048A
Authority: MX
Inventors: Hans Koll; Josef Burg; Karlheinz Sellinger; Anton Haselbeck
Original assignee: Roche Diagnostics Gmbh
Priority date: 1997-12-03
Filing date: 2000-05-23
Publication date: 2001-07-03

Abstract

The invention relates to new EPO compositions with a high specific activity, characterised by a high proportion of N-acetyl-lactosamin units and/or tetra-antenna branches in the carbon hydrate structure. The invention also relates to a method for preparing such EPO products.

Description

ERYTHROPOYETINE WITH HIGH SPECIFIC ACTIVITY DESCRIPTION OF THE INVENTION The invention relates to novel compositions of EPO (erythropoietin) with high specific activity which are characterized by a high content of N-acetyl-lactosamine units and / or tetraantennial branches in the carbohydrate structure. The invention also relates to a process for obtaining these EPO products. Erythropoietin (EPO) is a human glucopropein that stimulates the production of red blood cells. EPO occurs only in the blood plasma of healthy people at very small concentrations, so it is not possible to provide higher amounts in this way. EP-B1-0 148 605 and EP-B1-0 205 564 describe the production of human, recombinant EPO in CHO cells. The EPO described in EP-B1-0 148 605 has a higher molecular weight than urinary EPO and does not have O-glycosylation. The EPO described in EP-B1-0 205 564 from CHO cells is now available in large quantities and in pure form. In addition, isolation of human EPO from the urine of patients with aplastic anemia is known (Miyake et al., J. Biol. Chem. 252 (1977) 5558-5564). Recombinant and urinary EPO are isolated as a mixture of several isoforms that are known to differ in their degree of sialization. These EPO isoforms have different isoelectric points and can be separated by isoelectric focusing or capillary electrophoresis (see Tsao et al., Biotech, Bioeng 40 (1992), 1190-1196, Nieto et al., Anal. Commun. 33 (1996 ), 425-427, Tran et al., J. Chromatogr. 542 (1991), 459-471; Bietot et al., J. Chromatogr. 759 (1997), 177-184; Watson et al., Anal. Biochem. 210 (1993), 389-393). The isoforms with the highest number of sialic acids have the highest specific activity, while those with the least number have the least activity (see, for example, Imai et al., Eur. J. Biochem. 194 (1990), 457 -462; EP-A-0 428-267). Takeuchi et al., (Proc. Nati. Acad. Sci.

USA 86 (1989), 7819-7822) describes a relationship between the biological activity with the sialic acid content and the ratio of ratantenary carbohydrate bi- and tet structures. Takeuchi: et al. Further concludes that the N-acetyllactosamine units present in the carbohydrate structures of EPO have no correlation with biological activity. Fukuda et al., (Blood 73 (1989), 84-89) deals with the rate of elimination of EPO from the blood circulation, which essentially cooperates with biological activity and concludes that an EPO with greater Number of units of N-acetyllactosamine is eliminated more rapidly from the circulation than an EPO without lactosamine units. Morimoto et al., (Glycoconjugate J. 13 (1996), 1093-1120) describes the separation of EPO isoforms by means of mono-Q chromatography, so that the individual fractions are each composed of only a few isoforms. The investigations carried out with these fractions show an equidistribution of all the structures in all the fractions. No relationship is found between the content of bi- or triantennary structures or the content of N-acetyllactosamine units and the specific activity. In this way, the prior art shows that there is a general correlation between the biological activity and the structure of sugars, especially in relation to the content of sialic acids. However, no indication is found that the content of tetraantenarian structures and / or the content of N-acetyllactosamine have a direct correlation with biological activity. It was surprisingly found that during purification of EPO preparations an increase in the content of tetraantenary carbohydrate structures and / or N-acetyllactosamine units in the carbohydrate structure leads to a significant improvement in specific biological activity. This is especially true for the manufacture of EPO in a human cell line according to European Application 97 112 640.4. The comparative activity investigations of EPO preparations or individual EPO isoforms, whose carbohydrate structure is essentially differentiated only by the content of N-acetyllactosamine units (LE units), show a significantly higher activity for the preparations or isoforms with higher content of N-acetyllactosamine units for the same content of sialic acid and for the same degree of antenaridad. In this respect, antennarity is understood in this context as the average, relative (in%.) Content of N-linked, bi-, tri- and tetraantennary carbohydrate chains of the EPO preparations or of the isolated EPO isoforms, in relation to the total number of N-linked carbohydrate chains Furthermore, it was found that especially in preparations or isoforms with an increased content of triantennary structures, the total content of lactosamine units has a considerable importance for in vivo activity. of an increase in the total content of N-acetyllactosamine units, for example in the form of additional extensions of the core structure with LE units (so-called repeats), biological activity can be considerably increased. An increase in the content of tetraantenarian structures can be achieved by an improvement in biological activity. n EPO with the highest possible specific activity and with a high yield, it is necessary to optimize and select the purification steps, the production cells and / or the culture thereof to achieve a content as high as possible of carbohydrate structures tetraantennary and / or the highest possible content of N-acetyl-lactosamine units. A first aspect of the present invention relates to an EPO composition comprising essentially glycosylated EPO molecules, containing a proportion of at least 75%, preferably at least 80%, particularly preferably at least 85%, and most preferably at least 90% of tetraantenarian structures, relative to the total number of carbohydrate chains, ie the sum of bi-, tri- and tetraantenarian structures. A further aspect of the invention relates to an EPO composition comprising essentially glycosylated EPO molecules, containing an average number of at least 3.7, preferably of at least 4.0, particularly preferably of at least 4.3, and most preferably preferably at least 4.5 units of N-acetyllactosamine, relative to the average composition per chain of N-linked carbohydrate of the EPO molecule, or a number of at least 11.1 preferably of at least 12.0, particularly preferably at least 13.0, and most preferably at least 13.5 units of N-acetyllactosamine, relative to all three N-linked carbohydrate structures (total N-glycosylation) of the EPO molecule. A further aspect of the invention relates to an EPO composition comprising essentially glycosylated EPO molecules, which have a value for the product of the average total number of units of N-acetyllactosamine per EPO molecule multiplied by the average content of sialic acid per EPO molecule of at least 130, preferably at least 135, particularly preferably at least 140, and most preferably at least 160. In this regard, the term "essentially" means that the EPO molecules desired are present in the composition in a proportion of preferably "at least 80%, particularly preferably at least 90%, and most preferably at least 95%, relative to such number of EPO molecules .. Yet another aspect of the invention relates to an EPO composition comprising glycosylated EPO molecules, containing an average ratio of at least 75%, preferably at least 80%, and especially preferably at least 85% of tetraantennial structures, in relation to the total number of carbohydrate chains. The invention further relates to an EPO composition comprising glycosylated EPO molecules, containing an average number of on average at least 3.7, preferably at least 4.0, particularly preferably at least 4.3, and most preferably at minus 4.5 units of N-acetyllactosamine, relative to the average composition per N-linked carbohydrate chain of the EPO molecule, respectively a number of at least 11.1, preferably of at least 12.0, particularly preferably of at least 13.0 , and most preferably at least 13.5 units of N-acetyllactosamine, relative to all three N-linked carbohydrate structures of the EPO molecule. The maximum proportion of tetraantenarian structures can reach up to 100% of all the carbohydrate chains, each ratantenary structure containing 4 units of N-acetyllactosamine in the core structure of the N-linked sugar. Additional units of N-acetyllactosamine, the so-called Repetitions, which exist as extensions in the core structure, can increase the number of N-acetyl-lactosamine units, both by carbohydrate structure and also in total glycosylation. The number of units of N-acetyllactosamine per glycosylation site (ie, by N-linked carbohydrate structure) can therefore amount to 6 (tetraantenary structure and two additional units of N-acetyl-lactosamine in the Repetition form) (see Figure 1), or, with structures with more than two additional units of N-acetyllactosamine, may be even larger. In relation to total glycosylation (three N-linked carbohydrate structures), the number of N-acetyllactosamine units may increase to 18 or more. • Even a further aspect of the invention refers to an EPO composition that is composed of glycosylated EPO molecules, which have an average value for the product of the average total number of N-acetyllactosamine units of the EPO molecule multiplied by the average sialic acid content per EPO molecule of at least 130, preferably at least 135, particularly preferably at least 140, and most preferably at least 160. An additional material of the invention is an EPO composition having the characteristics of at least two or more of the previously mentioned aspects. The composition according to the invention may comprise one or more isoforms, ie EPO molecules with different isoelectric points during isoelectric focusing. Preferably, the composition according to the invention comprises a mixture of at least 2, for example 2 to 5, isoforms, especially a mixture of 3 or 4 isoforms. The specific activity of the composition according to the invention is preferably at least 175,000 IU / mg, especially at least 200,000 IU / mg, in vivo (normocytemic mouse). Especially preferred is the specific activity in the range of about 200,000 to 400,000 ul / mg or 450,000 Ul / mg of protein, most preferred is the activity in the range of 250,000 to 400,000 or 450,000 Ul / mg of protein. The average sialic acid content or the average number of sialic acid residues per EPO molecule is, in the composition according to the invention, preferably 11 to 14, particularly preferably at least 11.5, and most preferably at least 12.5. The EPO composition according to the invention can be obtained, on the one hand, from EPO molecules, which are the product of an expression of exogenous DNA in mammalian cells, for example in rodent cells, such as for example cells CHO or BHK, as described in EP-B-0 205 564. Alternatively, the composition can also be composed of EPO molecules, which are the product of an endogenous DNA expression after gene activation in human cells, by example in immortalized cell lines, such as for example Namalwa (Nadkarni et al., Cancer 23 (1969), 64-79), HT 1080 (Rasheed et al., Cancer 33 (1973), 1027-1033) or HeLa S3 (Puck). et al, J. Exp. Meth. 103 (1956), 273-284). These methods are described in European patent application 97 112 640.4, the description of which forms part of the present application. Additional important parameters for the biological activity of EPO are the proportion of repeating carbohydrate chains, ie additional units of N-acetyllactosamine relative to the total number of N-linked carbohydrate chains, as well as the value of the product of this ratio of repeats and the proportion of tetraantenary carbohydrate chains in relation to the total number of carbohydrate chains. For CHO cell EPO, the ratio of repeats is preferably at least 30%, particularly preferably at least 35%, and most preferably at least 40%. For the EPO of human cells, such as for example HeLa cells, the proportion of repeats is preferably at least 10%, particularly preferably at least 12%, and most preferably at least 14%. Therefore, for CHO cell EPO, the value for the product of the ratio of carbohydrate chains with N-acetyllactosamine repeats relative to the total number of carbohydrate chains and the proportion of tetraantennial structures relative to the total number of carbohydrate chains is preferably at least 2400, particularly preferably at least 2800, and most preferably at least 3400. For the EPO of human cells, the value is preferably at least 800, particularly preferably at least 960, and most preferably at minus 1100. Preferably, an EPO composition that was manufactured by culturing EPO producing cells in a culture medium with low serum content, for example with a maximum content of 1% (v / v) serum, is used. , or especially in a serum-free culture medium (see WO 96/35718). Examples of suitable culture media are RPMI 1640 or DMEM. The EPO composition according to the invention can be formulated as a pharmaceutical preparation, optionally together with pharmaceutically customary diluents, auxiliaries and carriers. The EPO composition according to the invention, which must be used for the manufacture of a pharmaceutical preparation, has a purity of preferably at least 99%, and particularly preferably at least 99.9%, determined by reverse phase HPLC ( for example on a Vydac C4 column) and / or by size exclusion chromatography (for example on a TSK 2000S Ultrapac column). In addition, the composition of the present invention has a DNA content of preferably <; 10 μg, particularly preferably < 5 μg and most preferably from < 1 μg DNA per 10,000 IU of protein. In addition, the composition of the present invention is preferably free of bacterial impurities (< 1 CFU / ml) and endotoxins (< 1 EU / 10,000 ul of protein). The determination of the DNA content can be carried out by a hybridization assay with radioactive DNA or fluorescent label. As a DNA probe, for example, commercially available purified human DNA is used. Human DNA can also be used as a standard for the assay. The lower limit of detection of such a hybridization assay is approximately 0.3 pg / 10,000 IU of EPO. The content of germs and endotoxins in the preparation of EPO can be determined with standardized methods, such as those described in Pharm. Eu or USP. An EPO composition preferably having the desired characteristics according to the invention can be obtained by means of at least one of the following measures: (a) selection of an appropriate production cell line, which is capable of producing carbohydrate chains with a high proportion of tetraantenarian structures and / or N-acetyllactosamine units, (b) selection of appropriate culture conditions during cell culture to produce carbohydrate chains with a high proportion of tetraantenarian structures and / or N-acetal units -lact osamine, and (c) separation of unwanted components from a known composition of EPO molecules, by concentrating EPO molecules containing carbohydrate chains with a high proportion of tetraantenarian structures and / or Na cetyl-lactosamine units. Measure (a) comprises the selection of a cell from. appropriate production. On the one hand, cells having the known tendency to manufacture the desired carbohydrate structures in high yield can be used here. Examples for such cell lines are cells derived from the hamster, such as CHO or BHK, and human cell lines, such as HeLa, Namalwa, HT1080 or cell lines derived therefrom. Especially preferred are HeLaS3 cells or modified CHO cells. On the other hand, appropriate production cells can also be generated in a targeted manner, by over-expressing certain glycosylation enzymes, for example by means of recombinant expression and / or by endogenous gene activation. Examples of such glycosylation enzymes are sialyl transferases, N-acetyl-glucosaminyl transferases and galactosyl transferases. Measure (b) comprises selection of appropriate culture conditions during cell culture. In a first embodiment of the invention, measure (b) comprises the addition of a mixture of at least two, and preferably at least three, carbohydrates to the culture medium. Carbohydrates are preferably selected from mono- and disaccharides, such as glucose, glucosamine, ribose, fructose, galactose, mannose, sucrose, lactose, mannose-1-phosphate, mannose-1-sulfate and mannose-6-sulfate. For example, nutritive media containing glucose and / or mannose and / or galactose are suitable. Especially good results were achieved with nutritive media containing a mixture of glucose, galactose and mannose, for example in the weight ratio of 1: (0.5-3): (1-5), and especially of 1: (0.7-2.4) ): (1.8-4.0), the carbohydrates being used in each case, particularly preferably in the D (+) form. The total concentration of all sugars in the culture medium during the fermentation is preferably in the range of 0.1 to 10 g / 1, particularly preferably in the range of 2 to 6 g / 1 of culture medium. Preferably, the aggregate of the carbohydrate mixture is made depending on the demand of the cells in each case, as will be explained below. According to another preferred embodiment, the measure (b) comprises the aggregate controlled and preferably in accordance with the demand for nutrients, which comprise at least one amino acid essential for the cultured cell line and / or at least one carbohydrate, depending on the of the demand of the cells in each case. In this way, a clearly improved glycosylation is obtained, even with a fermentation with high cell density (cell density during harvest >; 10 x 105 cells / ml and preferably > 20 x 105 cells / ml in large fermentors (volume> 1 1, for example 50-10,000 1). For this purpose, it is determined continuously or at appropriate time intervals, for example at least once a day. The concentration of parameters that are related to the demand of nutritive substances in the cells, and the speed of consumption of such parameters is determined. In this way, the nutritional substances required for the demand of the cells can be determined quantitatively or qualitatively. Such parameters may be nutritive substances or metabolic products of the cells such as, for example, the concentration of glutamine, ammonium, glucose and / or lactate, especially the concentration of glutamine. The added nutrients according to this aspect of the invention comprise essential amino acids, for example glutamine and / or tryptophan, and / or carbohydrates, as well as preferably also non-essential amino acids, vitamins, trace elements, salts and / or growth factors by insulin example. Particularly preferably, the nutritional substances comprise at least one essential amino acid and at least one carbohydrate. These nutritive substances are added to the culture medium, preferably in the dissolved state. The nutrient solutions preferably comprise at least glutamine and carbohydrates, especially a mixture of at least 2 carbohydrates of those previously mentioned. A mixture of glucose, galactose and mannose is particularly preferably used. Furthermore, it is preferred that the aggregate of the nutrients be made during the entire growth phase of the cells according to the demand, that is, in dependence on the concentration measured in the culture medium of the selected parameters. The weight ratio between glutamine and carbohydrates in the nutrient solution is preferably selected so that it essentially corresponds to the consumption ratio in the fermenter. In this way, it is possible to achieve in the fermentor a broadly constant concentration of individual substrates. Preferably, the concentration of glutamine is maintained at a value which is < 150 mg / l in the culture medium and which prevents the formation of an ammonium concentration in the culture medium > 2.3 mmol / l. The total concentration of sugar during fermentation, as it is. previously exposed, is preferably in a range of 0.1 to 10 g / 1, particularly preferably in a range of 2 to 6 g / 1 of culture medium. The nutrient solution used contains a weight ratio between glutamine and sugars in the range of preferably 1: 3 to 20, and particularly preferably 1: 5 to 15, relative to the total sugar. When using a nutrient solution containing glutamine and the three sugars glucose, galactose and mannose, the weight ratio between glutamine and the sugars is preferably in the range of 1: (1 to 3): (1 to 5): (2 a) 8), and especially preferably from 1: (1.5 to 2.2): (1.5 to 3.6): (4 to 6). ' The cultivation is preferably carried out in the form of batches with repeated feeding, according to which after a growth phase a part of the culture broth is harvested leaving the rest of the culture broth in the fermenter, which is then again filled with fresh medium up to the work volume. With the process according to the invention, glycosylated EPO can be harvested with very high yields. Thus, for example, the concentration at the time of harvest is at least 30 mg, and especially at least 40 mg of EPO per 1 of culture medium. Yet another aspect of the invention is a process for obtaining EPO from eukaryotic cells, with which eukaryotic cells are cultured in an appropriate medium and EPO is obtained from the culture supernatant, the process is characterized in that the culture is performs at a temperature of <; of 36 ° C, preferably between 30 and 35.5 ° C, and especially preferably between 33 and 35.0 ° C. It was surprisingly determined that by decreasing the temperature during cultivation the proportion of EPO with the desired glycosylation clearly increases. Measurement (c) comprises the separation of unwanted components from a known composition of EPO, whose carbohydrate structure does not meet the specifications of the present application. This separation can be carried out, for example, by chromatographic purification of the EPO preparations, for example by means of affinity chromatography on gels with triazine dye, especially gels with Cibacron Blue dye. Another separation of undesired components can also be carried out by means of hydrophobic interaction chromatography and inverted phase chromatography. Suitable ligands for this are butyl, pentyl, octyl, octadecyl and phenyl residues. The inverted phase chromatography step is preferably carried out with a pH value in the range of 6.0 to 8.5, and especially preferably 7.0 to 8.0. Suitable eluents are, for example, acetonitrile, ethanol or isopropanol, especially acetonitrile. With the help of the capillary electrophoresis analysis (CZE), the appropriate fractions can be determined, collected and finally subjected to the final elaboration. In addition, direct concentration of EPO molecules with a high content of N-acetyl-Lactosamine units can be achieved by the use of tomato or potato lectins (Merkle, Cummings, J. Biol. Chem. 262 (1987)). , 8179-8189). Preferably, these lectins are used, for example, in an immobilized form. Another object of the invention is a process for increasing the specific activity of an EPO composition, in which the EPO molecules which have (a) a high proportion of tetraantennary structures, are concentrated in the composition, (b) a large number of units of N-acetyllactosamine, (c) a high value for the product of the number of units of N-acetyllactosamine multiplied by the content of sialic acid, (d) a high proportion of repeats of N-acetyllactosamine, and / or (e) a high value for the product of the number of repeats of N-acetyllactosamine and the proportion of tetraantenary carbohydrate structures. This concentration can be carried out by one or more of the measures (a), (b) and (c) previously mentioned. The present invention will be further explained by means of the following figures and examples. Figure 1 shows a figure of a tetraantenary carbohydrate structure with additional units of N-acetyllactosamine (repetitions) and sialic acids. Figure 2 shows the dependence of the relative proportion of individual EPO isoforms, with respect to the carbohydrates added to the culture medium, Figure 3 shows the dependence of the biological activity of EPO preparations, with respect to the carbohydrates added to the medium of culture, and Figure 4 shows the dependence of the biological activity of EPO ispformas, with respect to the proportion of the repeating units of N-acetyllactosamine.

Example 1. Purification of EPO from supernatants of cell line culture. For the purification of EPO from cell cultures of human cell lines or CHO cells, essentially two methods were used, which are differentiated by the number and beginning of the chromatographic steps and which were used depending on the composition of the medium and the EPO concentration: Method 1 Step 1 Column Blue-Sepharose Step 2 Column Butyl sepharose Step 3 Column hydroxyapatite Step 4 Concentration Method 2 Step 1: Column Blue-Sepharose Step 2 Column Butyl sepharose Step 3: Concentration Step 'Alternative 3 RP-HPLC Example of a purification of a culture supernatant of HeLa S3 cells with 2% (v / v) fetal calf serum (FKS) according to method 1: 1. Blue-Sepharose Column A 5 ml Hi-Trap-Blue column (prefabricated Blue-Sepharose column from Pharmacia) was equilibrated with at least 5 column volumes (SV) of buffer A (20 M Tris-HCl, pH 7.0; 25 mM CaCl; 100 mM NaCl). Then 70 ml of Hela cell supernatant (containing approximately 245 μg EPO and 70-100 mg of total protein) were absorbed overnight with a circulation process with a flow rate of 0.5 ml / min. The column was washed with at least 5 SV of buffer B (20 mM Tris-HCl, pH 7.0, 5 mM CaCl 2; 250 mM NaCl) and at least 5 SV of C buffer (20 mM Tris-HCl, pH 7.0, 0.2 mM CaCl2, 250 M NaCl) with a flow rate of 0.5 ml / min. The result of the washing was monitored by measuring the protein content at an optical density (OD) of 280. The EPO elution was carried out with buffer D (100 mM Tris-HCl, pH 7.0, 0.2 mM CaCl 2, 2 M NaCl ) with a flow of 0.5 ml / min. The eluted solution was collected in fractions of 1-2 ml. The EPO content of the fractions, the washing solutions and the eluent was determined with reverse phase HPLC (RP) by means of the application of an aliquot on a POROS R2 / H column (Boehringer Mannheim). Alternatively, for the qualitative identification of fractions containing EPO, a dot transfer was performed.

Fractions containing EPO (8-12 ml) were combined in a mixture and absorbed on a column of butyl sepharose. The yield after the Blue-Sepharose column amounted to approximately 175 μg of EPO (corresponding to approximately 70%). The yield after Blue-Sepharose was generally comprised between 50-75%. 2. Butyl-sepharose column (interactive hydrophobic chromatography) A non-commercial butyl-sepharose column prepared by the investigators (material: Toyopearl Butyl S650) was equilibrated with at least 5 SV of buffer D (100 M Tris-HCl, pH 7.0, 0.2 mM CaCl 2, 2 M NaCl) and then the Blue-Sepharose mixture from step 1 containing EPO (approximately 150 μg EPO) was added at a flow rate of 0.5 ml / min. The column was washed with at least 5 SV of buffer E (20 mM Tris-HCl, pH 7.0, 2 M NaCl and 10% isopropanol) at a flow rate of 0.5 ml / min. The result of the washing was monitored by measuring the OD protein content of 280. The EPO elution was carried out with F buffer (20 mM Tris-HCl, pH 7.0, 2 M NaCl and 20% isopropanol) with a flow of 0.5 ml / min. The eluted solution was collected in fractions of 1-2 ml. The EPO content of the fractions, washing solutions and eluent was determined with RP-HPLC by the application of an aliquot on a POROS R2 / H column. Alternatively, for the qualitative identification of fractions containing EPO, an immunological transfer was made. Fractions containing EPO (10-15 ml) were collected in a mixture and applied on a hydroxyapatite column. The yield of the butyl-sepharose column was about 130 μg (corresponding to about 85%). In general, the yield of the butyl-sepharose column was between 60-85% of the mixture applied to the Blue-Sepharose column. 3. Hydroxyapatite column A 5 ml hydroxyapatite column (BioRAD Econo-Pac CHT II prefabricated column) was equilibrated with at least 5 SV of F buffer (20 mM Tris-HCl, pH 7.0, 2M NaCl, 20% isopropanol) and then the butyl sepharose mixture from step 2 containing EPO (approximately 125 μg EPO) was added with a flow rate of 0.5 ml / min. The column was washed with at least 5 SV of buffer G (20 mM Tris-HCl, pH 7.0, 2M NaCl) at 0.5 ml / min. The result of washing was monitored by the measurement of the protein content at OD of 280. The elution of the EPO was carried out with H buffer (10 mM Na phosphate, pH 7.0, 80 mM NaCl) with a flow rate of 0.5 ml / min. . The eluted solution was collected in fractions of 1-2 ml. The EPO content of the fractions, the washing solutions and the eluent was determined by means of RP-HPLC, applying an aliquot on a POROS R2 / H column. Fractions containing EPO (3-6 ml) were pooled in a mixture. The yield of the hydroxyapatite column was about 80 μg of EPO (corresponding to about 60%). In general, the yield of the hydroxyapatite column was between 50-65% of the applied amount of the butyl sepharose mixture. 4. Concentration The mixed EPO fractions from the step with. Hydroxyapatite were concentrated at the concentration of 0.1-0.5 mg / ml in centrifugation units with an exclusion size of 10 kD (eg Filtron's Microsep.), 0.01% Tween 20 was added and stored in aliquots at -20. ° C.

Performance scheme The purity of the isolated EPO was about > 90%, including even > 95% To increase EPO yield, method 2 without the butyl-sepharose step was also used. This method is applicable especy with supernatants of cell cultures without. added to FKS or with 1%. { v / v) of FKS and provides isolated EPO of approximately equal purity (90-95%). The presence of 5 mM CaCl2 in the equilibrium buffer (buffer F) used for the hydroxyapatite column led to improved absorption with this method, and with this also to a reproducible behavior of the EPO elution during the hydroxyapatite step. Therefore, method 2, with a course in principle equal to that of method 1, was carried out with the following dampers: 1. Column of Blue-Sepharose: Balance buffer Tris-HCl 20 mM, pH 7.0; (Shock Absorber A): 5 mM CaCl2, 100 mM NaCl Wash buffer 1 Tris-HCl 20 mM, pH 7.0; (Shock absorber B.): 5 mM CaCl2, 250 mM NaCl-Wash buffer 1 Tris-HCl 20 mM, pH 7.0; (Shock absorber C.): 5 mM CaCl2, 250 mM NaCl Wash buffer 1 Tris-HCl 100 mM, pH 7.0; (Shock Absorber D.): CaCl2 5 M, NaCl 2M 2. Hydroxyapatite Column Shock absorber of 50 mM Tris-HCl, pH 7.0; equilibrium 5 mM CaCl 2, 1 M NaCl (Shock Absorber F): 10 mM Tris-HCl wash buffer, pH 7.0; 1 (Shock absorber G.): 5 M CaCl 2, 80 mM NaCl 10 M α-Phosphate Wash Buffer, pH 7.0; 1 (Shock absorber H.): 0.5 mM CaCl2, 80 mM NaCl Performance scheme The addition of 5 mM CaCl 2 to buffer B to G of method 1 also led to better absorption and better defined elution of the hydroxyapatite column. Alternatively or additionally, the following steps can still be used for the purification of EPO: RP-HPLC, for example with Vydac C4 mater chromatography with DEAE-sepharose ff, diafiltration.

Example 2: Purification of EPO from culture supernatants with retention of isoforms 1 - 8 (Comparison) 1. Starting materEPO was fermented from mammalian cells, for example CHO or human cells, according to a repeated batch process. A 1000 1 fermentor was inoculated with a preculture, and the contents of the fermenter were harvested after 3 to 5 days. After harvest, the cells were separated from the fermentation broth by centrifugation. The cell-free supernatant of the culture is adjusted with 1 mol / l of acetic acid at pH 5.0 - 5.2 and filtered at 1-9 ° C. 2. Chromatography with Blue-Sepharose. A chromatographic column (Amicon P440 x 500, Amicon, GB) was filled with 60-80 1 Blue-Sepharose and regenerated with 0.5 N NaOH. The column was then equilibrated with approximately three column volumes (SV) of acetate cushion. The supernatant of the cell-free culture, adjusted to pH 5, was absorbed through the column at a temperature of 10 ± 5 ° C and with a flow rate of 800 - 1400 ml / min. The column was washed with 1 SV of wash buffer 1 with the same flow rate and at 5 ± 4 ° C. Then, it was followed with approximately 2 SV of wash shock absorber 2. The column was then eluted with approximately 3 SV of elution buffer. The total protein peak was collected (approximately 30-60 1) adjusted with HCl to pH 6.9 and stored until further processing at 5 ± 4 ° C. With this chromatographic step, the product solution was concentrated and a purity of approx. 40 - 50%. mM Na Acetate buffer, • equilibrium: 5 mM CaCl2, 0.1 M NaCl, pH 5.0 + 0.2 'Wash buffer 1 Na 20 .mM acetate, 5 mM CaCl2, 0.25 M NaCl, pH 5.0 + 0.2 Wash damper 1: 20 mM Tris HCl, 5 mM CaCl 2, pH 6.5 ± 0.3 Elution buffer; 100 mM Tris HCl, 5 mM CaCl 2, 1 M NaCl, pH 9.0 ± 0.2 3. Chromatography with Butyl-Toyopearl (hydrophobic chromatography) A chromatographic column (Pharmacia BPG 300/500) was filled with 30-40 1 of butyl-toyopearl and regenerated with 4 M guanidine-HCl and 0.5 N NaOH. The column was then set in equilibrium with at least 3 SV of equilibrium damper. The product eluted from the Blue-Sepharose column was adjusted to 10% isopropanol and absorbed onto the column at a temperature of 27 + 2 ° C and at a flow rate of 800-1200 ml / min. The column was washed with the same temperature and flow rate with approx. 1 SV of balance buffer and then with approx. 2 SV of wash buffer. It was then eluted with approx. 3 SV elution buffer. The total protein peak is collected (approx 10 - 18 1), diluted immediately three times with dilution buffer and stored at 15 ° C until further processing. With this chromatography a purity of approx. 90% Shock absorber of 20 mM Tris-HCl, 5 mM CaCl2, equilibrium NaCl: 0.75 M, 10% isopropanol, pH 6.9 ± 0.2 Shock absorber of 20 mM Tris-HCl, 5 mM CaCl2, washed NaCl: 0.75 M, 19% isopropanol, pH 6.9 ± 0.2 Shock absorber of 20 mM Tris-HCl, 5 mM CaCl2, NaCl elution: 0.75 M, 27% isopropanol ', pH 6.9 ± 0.2 Shock absorber of 20 mM Tris-HCl, 5 mM CaCl2, pH dilution: 6.9 ± 0.2 4. Chromatography with hydroxyapatite Ultrogel A chromatographic column (Amicon P440 x 500 or equivalent) was packed with 30 - 40 1 of hydroxyapatite Ultrogel and regenerated with 0.5 N NaOH. The column was then equilibrated with at least 4 SV of buffer 'of balance. The product eluted from the Butyl Toyopearl column was absorbed onto the column at a temperature of approx. 15 ° C and with a flow rate of 500 - 1200 ml / min. The column was washed with the same temperature and flow rate with approx. 1 SV of balance buffer and then with approx. 2 SV of wash buffer. It was then eluted with approx. 3 SV elution buffer. The total protein peak was collected (ca 10 - 18 1) and stored at 15 ° C until further processing. With this chromatography a purity lower than 95% was achieved.

Shock absorber of 20 mM Tris-HCl, 5mM CaCl2, equilibrium: 0.25 M NaCl, 9% isopropanol, pH 6.9 ± 0.2 Wash buffer 10 mM Tris-HCl, 5mM CaCl2, pH 6.8 + 0.2 Shock absorber 10 mM Tris-NCl, Elution phosphate: 10mM K, 0.5mM CaCl2, pH 6.8 ± 0.2 . Reverse Phase HPLC (RP-HPLC) Preparative HPLC was performed using a Merck Prepbar 100 separation apparatus (or equivalent) at a temperature of 22 ± 4 ° C. The separation column (100 mm x 400 mm, 3.2 1) was packed with a Vydac C4 material. Prior to its use, the column was regenerated by repeated application of a gradient from buffer A to 100% solvent, and then equilibrated with buffer A. The eluate from the hydroxyapatite column was acidified with trifluoroacetic acid to ca. pH 2.5 and filtered sterilely. Subsequently, it was applied to the column at a temperature of 22 ± 4 ° C and a flow velocity of 250-310 ml / min. The column was eluted at the same temperature and flow rate with a linear gradient of buffer A to buffer B according to the following HPLC protocol. The elution peak was collected in fractions. The eluate was diluted immediately upon first adding 4 volumes of HPLC dilution buffer. The fractions that during the analytical HPLC showed a purity of at least 99% (volume of the mixture approximately 4 - 6 1) were combined. With this chromatography, trace impurities were separated and a better purity of 99% was achieved.

Shock absorber A: 0.1% trifluoroacetic acid in water Shock absorber B 80% acetonitrile, 0.1% trifluoroacetic acid in water, 10 mM Na / K phosphate dilution buffer, pH 7.5 HPLC: ± 0.2 6. Chromatography with DEAE-Sepharose ff A chromatographic column (Amicon P90 x 250 or equivalent) was filled with 100-200 ml of gel per g of EPO in the applied amount and then regenerated with 0.5 N NaOH. The column was then equilibrated, first with 100 mM Na / K phosphate buffer, pH 7.5, and then with at least 12 SV of equilibrium buffer. The product eluted from the HPLC column was absorbed on the column at a temperature of 5 + 4 ° C and with a flow rate of approx. 150 ml / min. The column was washed with equal temperature and flow rate with at least 5 SV of equilibrium buffer and then with at least 10 SV of wash buffer. Next, the column was again washed with approx. 10 SV of equilibrium buffer and then eluted with approx. 7 SV elution buffer. The total protein peak was collected (approx 2 - 5 1), sterile filtered and packed. With this chromatography the solvent was removed from the HPLC step and the trace impurities were removed. Purity is better than 99%. mM Na / K phosphate buffer, pH balance: 7.5 ± 0.2 Wash buffer: 30 mM Na acetate, pH 4.5 ± 0.1 10 mM Na / K phosphate elution buffer, 80 mM NaCl, pH 7.5 ± 0.2 Example 3: Purification of EPO from culture supernatants with retention of isoforms 1-4 (Invention! 1. Starting material EPO was fermented from mammalian cells, for example CHO or human cells, according to the repeated batch process. A 10 1 fermenter was inoculated with a preculture, and the contents of the fermenter were harvested after approx. 5 days. After harvest, the cells were separated from the fermentation broth by centrifugation. The cell-free supernatant of the culture is adjusted with 1 mol / l of acetic acid at pH 5.0 - 5.2 and filtered at 1-9 ° C. 2. Chromatography with Blue-Sepharose. An appropriate chromatographic column was filled with 150 to 250 ml of Blue-Sepharose and regenerated with 0.5 N NaOH. The column was then equilibrated with approximately three column volumes (SV) of acetate buffer. The supernatant of the cell-free culture, adjusted to pH 5, was absorbed through the column at a temperature of 10 ± 5 ° C and with a flow rate of 1-2 SV / h. The column was washed with 1 SV of wash buffer 1 with the same flow rate and at 5 ± 4 ° C. Then it was followed with approximately 2 SV of wash buffer 2. The column was then eluted with about 3-6 SV of elution buffer. The protein peak was collected in fractional form. After EC analysis, the appropriate fractions were pooled, adjusted with HCl to pH 6.9 and stored at 5 ± 4 ° C until further elaboration. With this chromatographic step, the product solution was concentrated and the impurities and the basic isoforms separated. mM Na acetate buffer, 5 M CaCl2, equilibrium: 0.1 M NaCl, pH 5.0 ± 0.2 20 mM Na acetate buffer, 5 mM CaCl2, 1 wash: 0.25 M NaCl, pH 5.0 ± 0.2 Tris HCl buffer 20 mM, 5 M CaCl2, washed pH 2: 6.5 ± 0.3 Shock absorber of 50 mM Tris-HCl, 5 mM CaCl2, NaCl elution: 0.25 M, pH 8.0 ± 0.2 3. Chromatography with Butil-Toyopearl (hydrophobic chromatography) An appropriate chromatographic column was filled with 200-300 ml of butyl-toyopearl and regenerated with 4 M guanidine-HCl and 0.5 N NaOH. The column was then equilibrated with at least 3 SV of balance cushion. The product eluted from the Blue-Sepharose column was adjusted to 10% isopropanol and absorbed on the column at a temperature of 27 -2 ° C and at a flow rate of 1-2 SV / h. The column was washed with the same temperature and flow rate with approx. 1 SV of balance buffer and then with approx. 2 SV of wash buffer. It was then eluted with approx. 5 - 10 SV elution buffer. From the protein peak, fractions were collected and immediately diluted three times with dilution buffer. After EC analysis, appropriate fractions were pooled and stored at 15 ° C until further processing. - With this chromatography additional basic isoforms were separated and a purity was achieved - > 80% Shock absorber of 20 mM Tris-HCl, 5 M CaCl2, equilibrium NaCl: 0.2 M, 10% isopropanol, pH 6.9 ± 0.2 Shock absorber of 20 mM Tris-HCl, 5 mM CaCl2, washed NaCl: 0.2 M, 17% isopropanol, pH 6.9 ± 0.2 Shock absorber of 20 mM Tris-HCl, 5 mM CaCl2, NaCl elution: 0.2 M, 23% isopropanol, pH 6.9 ± 0.2 Shock absorber of 20 mM Tris-HCl, 5 mM CaCl2, pH dilution: 6.9 ± 0.2. Chromatography with hydroxyapatite Ultrogel An appropriate chromatographic column was packed with 150-200 ml of Ultrogel hydroxyapatite and regenerated with 0.5 N NaOH. The column was then equilibrated with at least 4 SV of equilibration buffer. The product eluted from the Butyl Toyopearl column was absorbed onto the column at a temperature of approx. 15 ° C and with a flow rate of 1 - 2 SV / h. The column was washed with the same temperature and flow rate with approx. 1 SV of balance buffer and then with approx. 2 SV of wash buffer. It was then eluted with approx. 3 SV elution buffer. The total protein peak was collected and stored at 15 ° C until further processing. With this chromatography a purity lower than 95% was achieved. Shock absorber of 20 mM Tris-HCl, 5 mM CaCl2, equilibrium: 0.06 M NaCl, 7.5% isopropanol, pH 6.9 ± 0.2 Wash buffer: 10 mM Tris-HCl, 5 mM CaCl2,. pH 6.8 ± 0.2 Shock absorber 10 mM Tris-NCl, K phosphate elution: 10 mM, 0.5 mM CaCl2, pH 6.8 ± 0.2 . Reversed Phase HPLC (RP-HPLC) Semi-preparative HPLC was performed with a Vydac C4 separating column (20 mm by 250 mm, approximately SO ml) at a temperature of 22 ± 4 ° C. Before use, the column was regenerated by repeatedly applying a buffer A to 100% solvent, and subsequently equilibrated with buffer A. The eluate from the hydroxyapatite column was applied to the column at a temperature of 22. ± 4 ° C and a flow rate of 8-15 ml / min. The column was eluted at the same temperature and flow rate with a linear gradient of buffer A to buffer B according to the following HPLC protocol. The elution peak was collected in fractions. The eluate was diluted immediately upon first adding 4 volumes of HPLC dilution buffer.

HPLC protocol (*) wash 3 and elution conditions: gradient from 50% shock absorber B to 100% shock absorber B in 50 to 200 min. Preferably 140 min.

The fractions that show with analytical HPLC a purity of aL least 99% and that are appropriate according to the CE analysis were gathered. With this chromatography, trace impurities and residues of basic isoforms were separated and a purity of better than 99% was achieved.

Shock absorber 10 mM Na / K phosphate, pH 7.0 ± 0.2 Shock absorber B 10 mM Na / K phosphate, 80% acetonitrile, pH 7.0 ± 0.2 10 mM Na / K phosphate buffer, NaCl HPLC dilution: 100 mM, pH 7.5 ± 0.2 6. Diafiltration A diafiltration apparatus of appropriate size was equipped with a 10 kD cartridge and regenerated with 1 N NaOH. The apparatus was then rinsed to the absence of alkali with a volumetric buffer. The product eluted from the SPLC column was concentrated at a temperature of 5 ± 4 ° C and diafiltered against 10 volumes of volumetric buffer. The final concentration should be between 1 - 3 mg / ml.

This step is used for the removal of solvent residues from the HPLC pass / for the adjustment of the conditions required by the volumetric absorber and the concentration d of the active substance of the volumetric absorber.

Volumetric cushion: 10 mM Na / K phosphate, 100 mM NaCl, pH 7.5 ± 0.2 Example 4: Determination of the specific activity of EPO in vivo (Bioassay in normocytemic mouse) "EPO activity, dose dependent, for the multiplication and differentiation of erythrocyte precursor cells was determined in vivo in mice by increasing reticulocytes in the blood after EPO administration. To this end, eight mice were administered parenterally in one of the "" '' different doses of the sample from the US and an EPO standard (calibrated with the standard of EPO WHO). Next, the mice were maintained under defined constant conditions. Four days after the EPO administration, a blood sample was taken from the mice and the reticulocytes were colored with acriidine orange. The determination of the number of reticulocytes per 30,000 erythrocytes was performed microfluorimetrically in the flow cytometer, analyzing the red fluorescence histogram. The calculation of the biological activity was made from the values for the numbers of reticulocytes of the sample and the standard with different doses, according to the method of determining the content of pairs with parallel lines, described by Linder (Linder, Plañen und Auswerten von Versuchen, 3. Auflage, 1969, Birkenháuser Verlag, Basel). During the purification of EPO from culture supernatants of CHO cells grown in a serum-free medium, the preparations EPO CH01, CH02 and CH03, which had a biological activity of 248,000 (CHOl), 225,000 were obtained.

(CH02) and 186,000 (CH03) Ul / mg respectively. In four batches, gums which were purified EPO from supernatants of human cell culture, products were obtained with specific activities of 220,000 (HeLa), 198,000 (HeLa2), 204,000 (HeLa3), 176,000 (HeLa4) and 100,000 (HeLa5} Ul / mg, respectively The correlation of the values for the biological activity with the parameters ^, of the structure of the sugar is indicated in Example 11.

Example 5: Determination of the content of sialic acid residues. The determination of the sialic acid content was performed chromatographically by means of HPAEC-PAD (High pH Anion Exchange Chromatography with Pulsed Amperometric Detection = high-anion exchange chromatography with pulsating amperometric detection) in a Dionex system, after the enzymatic separation of sialic acids with neuraminidase from Arthrobacter ureafaciens (A. ureaf., Boehringer Mannheim). Preparations each containing 22 μg of EPO for various preparations of CHO and human cell lines (for example HeLa S3) were adjusted to an EPO concentration of 0.2 mg / ml in 5 mM Na phosphate buffer, pH 7.2. One half of each batch was used for the exact determination of the amount of EPO with RP-HPLC. To the second half of each batch, 5 mM U of neuraminidase from Aureas were added to each one and incubated overnight (approximately 18 h) at 37 ° C. The digested batches were then divided into two halves, diluted twenty times to 500 μl with H20, and 50 μl of this dilution (corresponding to approximately 27 pmol of EPO) were applied to the Dionex system. For this, the following chromatographic parameters were used: Column: Carbo Pac PA 100 Flow rate: 1.0 ml / min Detector sensitivity 300 nA Gradient T (min)% of shock absorber B 0 17 7 17 9 100 12 100 13 0 20 0 Shock absorber A: 0.1 M NaOH Shock absorber B 0.1 M NaOH; Na acetate 0.5 M The amount of sialic acid in the applied sample was determined with the help of a calibration line obtained from the sialic acid standard (Boehringer Mannheim) analyzed in parallel. The content of sialic acid (mol sialic acid / mol EPO) was calculated from the result of the determination of sialic acid (Dionex system) and the determination by RP-HPLC of the amount of EPO used The CHO cell EPO had an average content of 12.9 mol (CH01), 11.8 mol (CH02) or 11.7 mol (CH03) of sialic acid per mole of EPO, respectively EPO preparations from human cells had a ratio of 13.1 mole (HeLa), 13.2 mole (HeLa2) ", 13.3 mole (HeLa3), 1 .6 mole (HeLa4) or 108 mole ( HeLa5) of sialic acid per mole of EPO, respectively (see also Example 11).

Example 6: Determination of the proportions of bi-, tri- _-tetraantenarian carbohydrate structures "" "'The analysis of the N-linked carbohydrate structures was performed chromatographically with HPAEC-PAD with a Dionex system The asialo oligosaccharides of preparations EPO of CHO and human cell lines (for example HeLa S3) were obtained by enzymatic cleavage with N-glucosidase F (Boehringer Mannheim.) And A.ureaf neuraminidase (Boehringer Mannheim), 10 μg or 30 μg per mixture were desalified with MicroCon ultracentrifugation units (Amicon, exclusion size 10 kD} and were adjusted with 10 mM Na phosphate buffer, pH 7.2, at a concentration of 0.2 mg / ml, respectively 0.3 mg / ml. Then, 1 U of N-glucosidase F and 10 mU of neuraminidase were added to each batch and incubated overnight (approximately 18 h) at 37 ° C. To separate the proportion of the EPO polypeptide from the dissociated oligosaccharides, the batches were centrifuged after incubation through Ultrafree • centrifugation units (Milipore, exclusion size 10 kD), and the Ultrafree device was then washed twice with 20 μl of H20. The oligosaccharides obtained in the liquid that passed through the centrifuge were taken with H20 to 150 μl, and 100 ml of this liquid were analyzed with the "Dionex." System. For this purpose the following chromatographic parameters were used: Column: Carbo Pac PA 100 Flow rate: 1.0 ml / min Sensitivity of the detector. 300 nA Gradient: T (min)% of buffer B 0 0 2 0 60 10 62 100 67 100 69 0 80 0 Shock absorber A: 0.1 M NaOH Shock absorber B: 0.1 M NaOH; Na acetate 0.5 M The identification of the peaks in a chromatogram of N-linked sugars of the complete type was performed with normal oligosaccharides (Oxford Glyco Systems) and verified by the enzymatic digestion of the EPO oligosaccharides with the enzyme endo-β-galactosidase, respectively fucoside, followed of the analysis with the Dionex system. The calculation of the percentage proportions of 'bi-, tri- and tetraantenarian structures was made with the surface of the peaks that represent the corresponding structure of M-linked sugar, in relation to the total peak surface (sum of peak surfaces of structures bi-, tri- and tetraantenaries). EPO derived from CHO cells had a ratio of 4.2% biantennary carbohydrate structures, 22.3% triantennial carbohydrate structures and 73.5% (CHO 3), 86.7% (CHO 1) or 78.6% (CHO 2)., Respectively , tetraantenary carbohydrate structures With the EPO preparations of human cell lines proportions were obtained in biantennary / triantennial / tetraantennial structures for HeLa 1 of 5.8 / 8.8 / 85.4%, for HeLa 2 of 5.1 / 12.7 / 82.2%, for HeLa 3 of 4.1 / 17.7 / 78.2%, for HeLa 4 of 10.1 / 19.2 / 70.6% and for HeLa 5 of 12.6 / 25.4 / 62% (see also Example 11).

Example 7: Determination of the average content of N-acetyllactosamine units and of the average proportion of additional units N-acetyllactosamine (Repetitions) The total number of N-acetyllactosamine units in the N-linked carbohydrate structures of EPO (ie, in the core carbohydrate structures plus Repetitions) was calculated from the peak surfaces of the chromatograms of the experiments in Example 6. The calculation of the average content (n) of N-acetyllactosamine units by carbohydrate chain was the following.

N = S% (bi) x 2 +% (tri) x 3 +% (tetra) x 4 +% (tri + Ir) x 4 +% (tetra + Ir) x 5 +% (tri + 2r) x 5 +% (tetra + 2r) x 6 where% (bi) = percentage proportion of biantennary structures in relation to the total amount (100%) of N-linked carbohydrate structures (tri) = percentage proportion of triantennarian structures without additional unit of N-acetyllactosamine (tetra) = percentage proportion of tetraantennary structures without additional unit of N-acetyllactosamine tri + Ir) proportion- percentage of structures; triantenary with 1 additional unit of N-acetyllactosamine% (tetra + percentage ratio of lr structures) = tetraantennial with 1 additional unit of N-acetyllactosamine% (tri + 2r) = percentage ratio of triantennial structures with 2 additional units of N-acetyllactosamine% (tetra + percentage ratio of structures, 2r) = tetraantennial with 2 additional units of N-acetyllactosamine With CHO cell EPO, an average number of 4.3 (CH01), 4.4 (CH02) or 4.2 was found.

(CH03) of N-acetyllactosamine units per carbohydrate chain, respectively. In the EPO preparations of human cells a number of N-acetyllactosamine units of 4.0 (HeLa 1), 4.0 (HeLa 2), 3.9 (HeLa 3), 3.75 was found.

(HeLa 4), or 3.6 (HeLa 5), respectively (see also Example 11). Because EPO contains 3 N-linked structures, the total number of N-acetyl-lactosamine N-linked units is three times greater. In relation to total glycosylation of EPO, the number of units of N-acetyllactosamine is therefore, for CHO cell 12.9 (CHO 1), 13.2 (CHO 2) or 12.6 (CHO 3) cell, respectively. For EPO preparations of human cells, the corresponding values were 12.0 (HeLa 1), 11.9 (HeLa 2), 11.7 (HeLa 3), 11.25 (HeLa 4) and 10.8 (HeLa 5). The product of the number of units of N-acetyl-lactosamine per carbohydrate structure multiplied by the corresponding sialic acid content resulted for EPO of CHO- cells at values of 55.5 (CHO 1), 52 (CHO 2) or 49.3 (CHO 3), respectively. With the EPO preparations of human cells, the corresponding values were 52.4 (HeLa 1), 52.5 (HeLa 2), 51.3 (HeLa 3), 43.5 (HeLa 4) and 38.9 (HeLa 5). In relation to total glycosylation of EPO (3 N-linked carbohydrate structures), the product of the number of N-acetyllactosamine units multiplied by the sialic acid content in each case for EPO of CHO cells is 166.5 (CHO 1), 156 (CHO 2) or 147.9 (CHO 3), respectively. For EPO preparations of human cells, the corresponding values were 157. 2 (HeLa 1), 157.5 (HeLa 2), 153.9 (HeLa 3), 130. 5 (HeLa 4) and 116.7 (HeLa 5), see also Example 11. An important additional parameter is the ratio of; the so-called Repetitions, that is to say of the N-acetyllactosamine units that can be linked to the carbohydrate structures (see, for example, Figure 1). The content of Repetitions is indicated as a percentage proportion of the carbohydrate structures containing Repetitions in relation to the sum of all the N-linked carbohydrate structures (bi + tri + tetra = 100%). This ratio of repeats may be different for EPO preparations from CHO cells and from human cells. Thus, repeating ratios of 39.6% (CHO 1), 51% (CHO 2) or 36.8% (CHO 3)., Respectively, were determined for the CHO cell preparations. Repetitions of 18% (HeLa 1), 16.5% (HeLa 2), 14.0% (HeLa 31), 12.2% (HeLa 4) and 9.8% (HeLa 5) (see Example 11).

Example 8: Influence exerted on the biological activity of EPO by controlled feeding and according to the demand The crops were performed as an operation in batches with repeated feeding with a feed according to the demand (Repeated Fed Batch) at a temperature of 36.5 ° C . To this end, a culture medium poor in protein and free of serum was placed in a stirred fermentor (total work volume: 10 1) and inoculated once with an inoculation culture. The cell density after inoculation was in the range of 3 ± 1 × 10 5 live cells / ml. After a growth phase of 144 ± 24 hours, a part of the culture broth was harvested. The rest of the culture broth remained in the fermenter and was the inoculum for the next growth phase; for this purpose, the fermenter was again filled with fresh medium up to the work volume. The culture supernatant containing EPO was obtained by centrifugation of the fermentation culture. During the growth phase, the crop is continuously fed with nutrient solution. To this end, a container containing a nutritive solution was coupled to the fermenter. The nutrient solution contained amino acids, vitamins, insulin, trace elements, salts, glutamine and carbohydrates. Two fermentations were carried out as follows with fermentation A, the nutrient solution contained as sugar D- (+) -glucose and with fermentation B the sugars D- (+) - glucose, D - (+) - galactose and D - (+) - crafty. The weight ratio between glutamine and sugars was in fermentation B 1: 2, 2: 3, 6: 6. The concentration of the individual sugars in the nutrient solution was in the range between 7.2 and 18 g / 1. During fermentation B, the concentration of glutamine in the culture was analyzed periodically and the consumption was calculated. The momentary flow of the nutrient solution was adequate to the demand of the cells in nutritive substances. During fermentation A, the glutamine concentration was not used as a control variable. The solution of nutrients from fermentation B contained a mixture of the sugars D- (+) - glucose, D- (+) - galactose and D- (t) -manose in the weight ratio of 2: 3: 5. By means of the corresponding feed, the concentration of all the sugars in the fermenter in a range of 2 to 6 g / 1 was maintained during cultivation. The cell density at the time of harvest was modified by growth to more than 20 x: 105 cells / ml, typically at 30 ± 10 x 10 5 cells / ml. At the time of harvest, the EPO concentration was typically 40 ± 10 mg / l. In the harvested culture broths the concentration of human erythropoietin was determined, for example by means of Elisa. A percentage distribution of the present isoforms of this protein was determined for example by means of separation with capillary zone electrophoresis (CZE). Table 1 shows the comparison of the distribution of the EPO isoforms between a fermentation A with feeding by a solution of nutrients with glucose 'and a fermentation B with controlled feeding and according to the demand for a solution of nutrients containing glucose, mannose and galactose. The proportion of the EPO isoforms with fermentation B was calculated as a proportion percentage of the isoforms corresponding to the fermentation A. The latter were normalized in each case to 100%. The data show that the desired EPO isoforms 2-4 with higher glycosylation are present during fermentation B in a considerably higher proportion in relation to fermentation A.

Table 1 n.d. = not determinable, because the value is below the detection limit.

The model of iso forms obtained with the feeding could be reproduced with four successive harvests of a fermentation with controlled feeding and according to the demand of the nutritive solution Example 9: Influence exerted on the biological activity of EPO by the modification of the culture temperature. The embodiment was carried out as described in Example 8 (fermentation B) with the Fed-Spli tbatch process (batches with repeated feeding) with controlled feeding and according to the demand, except that the fermentation temperature was 35.0 ° C instead of 36.5 ° C and the fermentation was performed on the scale of 1,000 1. Table 2 shows the comparison of the distribution of the EPO isoforms between a fermentation C at 36.5 ° C and a fermentation D at 35.0 ° C, in each case with controlled feeding of a nutritive solution. The proportion of EPO isoforms with fermentation D was calculated as a percentage proportion of the isoforms corresponding to fermentation C. The latter were normalized in each case to 100%. The data show that decreasing the temperature considerably increases the acid isoforms of EPO 2 to 4.

Table 2 n.d. = not determinable, because the value is below the detection limit.

Example 10: Influence exerted on the biological activity of EPO by the modification of the carbohydrate composition The process described below demonstrates the possibility of modifying the quality of human erythropoietin by modifying the supply of carbohydrates in the feeding medium. Two variants of the above-described process are shown (called in the following fermentation E and fermentation F), which are differentiated by the composition of the media used. For both preparations, the formulation of the culture medium was based on the modified eRDF medium. Serum was not used, but recombinant insulin (only protein added) and other supplements (for example selenite, putrescine, hydrocortisone, iron sulfate), usually used with serum-free or protein-free media respectively. The feed nutrient solution is also based on a modified eRDF medium, but does not contain the KCl, Na2HPO and NaCl salts. The determining difference between fermentations E and F is the addition of different monosaccharides to the feeding medium.

Fermentation E: For the fermentation E the D- (+) -glucose sugar usually used was used. The initial concentration was 3 g / 1. By means of an appropriate feeding with the nutrient solution containing g-lucosa, the glucose concentration in the culture broth was kept constant at 3 ± 0.5 g / 1 throughout the culture. The duration of the culture was typically 100 ± 20 h. The EPO concentration at the time of harvest was typically 40 ± 10 mg / l.

Fermentation F: For the fermentation F, the sugars D- (+) -galactose and D- (+) -sugar were used in addition to D- (+) -glucose in the weight ratio of approximately 1: 2: 3 By means of corresponding feeding, during the culture the concentration of all the sugars in a gamma comprised between 0.25 g / 1 and 3.5 g / 1 was maintained. The culture duration for this growth was typically 100 ± 20 hours. The EPO concentration at the time of harvest was typically 40 ± -10 mg / l. The erythropoietin was purified from the culture supernatants. The purification process carried out was designed in such a way (see Example 2) that the distribution of the relevant isoforms of the glycoprotein was not influenced. The distribution of the isoforms of the purified erythropoietin was determined as mentioned above. The carbohydrate structures of the human erythropoietin isoforms and their distribution in the supernatants of the harvested cultures of fermentation E and fermentation F are different. Fermentation F shows a proportion of isoforms 2, 3 and 4 clearly greater in relation to fermentation E. These differences were caused by a feeding with the monosaccharides mannose and galactose (see Figure 2). 'There is a correlation of activity Biological determined by the test with the normocitémic mouse (Example 4) with the distribution and the carbohydrate structures of the EPO isoforms (Figure 3). The carbohydrate structures of the EPO preparations obtained from the supernatant of cultures E and F were determined by analysis CZE and HPAEC. Table 3 shows the antenarity (contained in bi-, tri- and tetra structures), the content in units of N-acetyllactosamine (LE), the content of sialic acid (SA) and the product LE x; SA of the two EPO preparations.

Table 3 Example 11: Correlation of the specific activity with the carbohydrate structures In this Example the investigations on the dependence of the activity of individual EPO isoforms of the carbohydrate structures have been summarized. To this end, isoforms (IF) were isolated and compared from different EPO sources (different EPO batches of CHO cells and human cells). 11. 1 Isolation of individual EPO isoforms by means of isoelectric focusing (IEF) and Western Transfer A) Performing electrophoresis on IEF gel and elect roblott ing on nitrocellulose For the isolation of individual isoforms in pure form, an EPO solution comprising a mixture of several isoforms was desalted and concentrated in Ultrafree centrifugation units (5-10 mg / ml). 350 - 1,000 μg of this solution were applied on a polyalkylamide gel prepared for IEF of Serva (Servalyt Precotes, pH 3-5 300 μm 125 x 125 mm.), In 5 - 10 tracks with 70 - 100 μg EPO per track.

IEF was performed at 2,500 V for 3.5 h at 5 ° C; the gel was then transferred by dotting on nitrocellulose (wet blot in Tris / glycine buffer with methanol, but in SDS, for 3 h at 200 mA). After the blotting process, the gel was separated, and the nitrocellulose membrane was stained with Ponceau S. The colored isoforms were trimmed and again fully decolorized with H20 or TBS buffer (100 mM Tris, pH 7.4, 250 mM NaCl).

B 'Extraction of the isoforms of the membrane The strips of nitrocellulose discolored with the corresponding isoforms were transferred to 2 ml Eppendorf containers (corresponding to 3-4 tracks of the IEF gel), 1.5 ml of acetone were added and the nitrocellulose was dissolved by agitation with vertex formation For optimal precipitation of EPO, it was incubated overnight at -20 ° C. The precipitate containing EPO was then isolated by centrifugation for 10 min in a tabletop centrifuge at 14,000 rpm. The precipitate was washed 2-3 times with 1 ml of acetone and then dried at room temperature or 37 ° C under a stream of nitrogen. Then, the EPO precipitate was dissolved in 20 M Na-phosphate buffer, pH 7.2, dissolved with 0.01% Tween 20 and stored at -20 ° C until further analysis.

C) Isolation of isoforms from pre-fractionated EPO solutions The ailing of individual isoforms was carried out as mentioned above in A) and B), with the limitation that the initial EPO solutions used did not contain 7-8, but only 3-4 isoforms The starting material were EPO fractions obtained by DE chromatography (anion exchanger). These fractions contained only 3-4 isoforms (eg isoforms 6-8 or isoforms 1-4). For the isolation of the "isoform packages" an appropriate chromatographic column was filled with, 1-2 ml of DEAS-sepharose ff per 10 mg EPO in the applied amount, and regenerated with 0.5 M NaOH. The column was then placed in equilibrium, first with 2 SV of neutralization buffer and then with at least 5 SV of equilibrium damper. A purified EPO preparation comprising 8 isoforms was absorbed at a temperature of 5 ± 4 ° C and with a flow rate of up to 15 SV / h. Then, the column was washed with 2 to 3 SV of equilibrium buffer and then rinsed with wash buffer until the pH value was 5.0. (approx 5 SV), Elution of different packets of isoforms was achieved by increasing the concentration of NaCl in the elution buffer in steps of 10 mM, starting with 20 mM. The basic isoforms bind weaker to the ion exchanger, and therefore elute with lower ionic strength; Acid isoforms elute with higher concentrations of NaCl up to 70 mM NaCl. The amount of the isoforms eluted with a certain concentration of NaCl depends strongly on the starting material and the volume of elution. In general it was eluted in the individual steps until the OD280 had decreased to approximately 50% of the maximum value at this NaCl concentration. This corresponded to a volume between 15 and 40 SV. Further separation of the isoforms was achieved by additional fractionation of isoform packages of products eluted within a concentration of NaCl. The flow of the column increased up to 15 SV / h. 100 mM Na / K phosphate buffer, neutralization: pH 7.5 ± 0.2 Na / K -100 - M phosphate balance buffer, pH 7.5 ± 0.2 Wash buffer: 30 mM NaAc / Hac, pH 5.0 ± 0.2 Elution buffer : NaAc / Hac 10 mM, pH 5.0 ± 0.2, NaCl 20 mM, respectively increase in concentration in steps of 10 mM to 70 mM NaCl.

From the packets of isoforms thus obtained, individual pure isoforms were obtained by means of a purification as mentioned above in A) and B). The numbering of the pure isoforms (IF) obtained from A-C was made according to their isoelectric point (pl) of acid to alkaline. Isoform 2 (IF2) is the isoform isolated most strongly acid with the lowest pl, isoform 8 is the most alkaline with the highest pl. Isoform 2 was the isoform with the lowest pl that could be isolated from the starting mixture in sufficient amounts. The starting mixture contained only 1-2% of isoform 1, so that it was not possible to obtain sufficient amounts for a complete analysis. The following analyzes were carried out for the characterization of the pure isoforms: - determination of the quantity and yield by means of RP-HPLC - determination of the purity and identity by means of capillary electrophoresis and soelectric approach. The yield in individual isoforms was generally comprised between 20% and 30% of the isoforms contained in the initial mixture. The purity of the isoforms was in general > 90%, in most cases even > 94% 11. 2 Results The following data were collected from the purified isoforms (IF): - relative distribution of the N-linked carbohydrate structures (proportion of bi-, tri- and tetraantenarian structures in total glycosylation) and the content of Repetitions. - biological activity in the normocitémic mouse assay - sialic acid content. These tests were carried out essentially according to the methods already described. The sialic acid content of the isolated isoforms was not determined separately for each individual isoform preparation, but was determined, for example, in 1-3 preparations for each of the 2-8 EPO isoforms of CHO cells and those for EPO 2-6 isoforms of human cells, respectively. To calculate the product content of units of N-acetyllactosamine (LE value) multiplied by the sialic acid content (SA), sialic acid values rounded to whole numbers of each isoform were used. These rounded SA values were for EPO of CHO cells and human cells the following: 14 (IF2), 13 (IF3), 12 (IF4), 11 (IF5), 10 (IF6), 9 (IF7) and (IF8) . Table 4 contains data on the correlation between the specific activity and the carbohydrate structures of different EPO preparations of CHO cells (CHO 1, CHO 2, and CHO 3), as well as human cells (HeLa 1 to 5). The Table shows the correlation between the biological activity and the total average number of units of N-acetyllactosamine (LE) in the EPO molecule, the average sialic acid content (SA) and the LE x SA product. Table 5 contains indications about the correlation between. the biological activity and the average total number of units of N-acetyllactosamine (LE) in the FPO molecule, the average sialic acid content (SA) and the LE x SA product of isoforms isolated from an FPO load of CHO cells not pre-fractioned Table 6 contains a comparison of various preparations (A and B) of an isoform (IF2, respectively IF5), which was isolated from different fractions of a pre-fractionated EPO charge of CHO cells, ie from each of the isoforms 2 and 5 two preparations (A and B) were analyzed. The prefraction was carried out with an ion exchanger DE, - as mentioned above in Example ll.l.C. The two preparations A and B of the IF2 isoform, respectively IF5, were isolated from different fractions of the DE column (A and B of IF 2 of fractions 5 and 6, respectively, and AYB of IF 5 of fractions 2. and 3, respectively). Fraction 5 or 2, from which IF2 / A or IF5 / A was isolated, - respectively, eluted earlier (with lower salt concentration) from the DE sepharose column than fraction 6 or 3, from which IF2 / B or IF5 / B was isolated, respectively. The preparations A and B of isoform 2 or 5 are not differentiated, however, by their behavior during isoelectric focusing or during capillary electrophoresis carried out below, that is, the two preparations of IF2 or IF5 have the same acid value sialic. Surprisingly it was determined, however, that the isoforms of preparation A, due to their higher LE value, respectively their higher proportion in repeat structures (Repeat), have a significantly higher biological activity than the corresponding isoforms of drug B. The dependence of the biological activity of an isoform of the total number of N-acetyllactosamine units contained in the EPO molecule with the same content of sialic acid, dependence described in Table 6, was not only observed for isoforms 2 and 5, but also for other isoforms. Table 7 compares the corresponding isoforms of various EPO sources (CHO cells or human HeLa S3 cells). Also here is a correlation of the biological activity with the LE x SA value. Therefore, in all the Tables a correlation of the product of the number of units of N-acetyllactosamine (LE) multiplied by the content of sialic acid with the biological activity is recognized. A high value of the product LE x SA is always associated with a high biological activity.

Table 4 LE: Units of N-acetyl-lactosamine SA: Sialic acid content of the EPO preparation 1: Proportion% of all sugar structures with additional LE extensions in relation to total sugar structures (bi + tri + tetra = 100%) LE: N-acetyllactosamine units (calculated as in example 7) SA: Sialic acid content of the corresponding isoform 1: Proportion% of all sugar structures with additional LE extensions in relation to the total sugar structures (bi + tri + tetra = 100%) Table 6: LE: N-acetyllactosamine units (calculated as in Example 7) SA: Sialic acid content of the corresponding isoform 1: Proportion% of all sugar structures with additional LE extensions in relation to the total sugar structures (bi + tri + tetra = 100%) Table 7: LE: N-acetyllactosamine units (calculated as in Example 7) SA: Sialic acid content of the corresponding isoform 1: Proportion% of all sugar structures with additional LE extensions in relation to the total sugar structures (bi + tri + tetra = 100%) Figure 4 shows with the example of individual isoforms the dependence of the biological activity of EPO on the proportion of N-linked carbohydrate structures with additional units of N-acetyllactosamine (Repetitions). The isoforms were isolated from EPO preparations with varying proportions of carbohydrate structures containing Repetitions (EPO 1 with about 50%, EPO 2 with about 40% and EPO 3 with about 15% of structures containing Repetitions). The biological activity of corresponding isoforms (equal sialic acid content and approximately equal antenarity) is less, the lower the proportion of carbohydrate structures that contain repetitions in the isoforms. This characteristic can be observed from the isoform 2 to at least the isoform 7.

Claims

1. Composition of EPO (erythropoietin), wherein, it is essentially composed of glycosylated EPO containing on average a number of at least 4.3 units of N-acetyllactosamine with reference to an N-linked carbohydrate chain of an EPO molecule or in average at least 13.0 units of N-acetyllactosamine with reference to the total N-glycosylation of an EPO molecule.

2. EPO composition wherein, it is composed of glycosylated EPOs containing on average an average number of at least 4.3 units N-acetyllactosamine with reference to an N-linked carbohydrate chain or on average at least 13.0 N units -acetyl-lactosamine with reference to the total N-glycosylation of an EPO molecule.

3. The EPO composition according to claim 1 or 2, wherein the number of N-acetyllactosamine units is at least 4.5 with reference to the N-linked carbohydrate chain or 13.5 with reference to the total glycosylation.

4. The EPO composition, wherein it is composed essentially of glycosylated EPO molecules having a value for the product of the average number of N-acetyllactosamine units with reference to the N-linked carbohydrate chain of an EPO molecule multiplied by the average content of sialic acid per molecule of EPO and at least 43.3 or at least 130 relative to the total N-glycosylation of an EPO molecule.

5. EPO composition, where it is composed of glycosylated EPO molecules having an average value for the product of the average number of N-acetyllactosamine units with reference to an N-linked carbohydrate chain of a multiplied EPO molecule for the average content of sialic acid per EPO molecule of at least 43.3 or at least 130 with reference to the total N-glycosylation of an EPO molecule.

The EPO composition according to claim 4 to 5, wherein the product value is at least 46.7 with reference to the N-linked carbohydrate chain of at least 140 with reference to total N-glycosylation.

The EPO composition wherein it has the characteristics of at least two of claims 1, 2, 4 and 5.

The EPO composition according to one of the preceding claims, wherein it comprises a mixture of 2 to 5 isoforms.

9. The EPO composition according to claim 11, wherein it comprises a mixture of 3 to 4 isoforms.

The EPO composition according to one of the preceding claims, wherein it has a specific in vivo activity of at least 175,000 IU / mg of protein.

The EPO composition according to one of the previous claims, wherein it has a specific in vivo activity of at least 200,000 IU / mg of protein.

The EPO composition according to one of the previous claims, wherein the average content of sialic acid per molecule is at least 11.

The EPO composition according to one of the previous claims, wherein the EPO molecules are the product of an exogenous DNA expression in mammalian cells.

The EPO composition according to claim 3, wherein it is composed of glycosylated EPO molecules of CHO cells in which the proportion of carbohydrate chains with N-acetyllactosamine extensions (repeats) relative to the total number of n-linked carbohydrate chains is at least 30

15. The EPO composition according to claim 14, wherein the value for the product of the ratio of carbohydrate chains with N-acetyllactosamine repeats relative to the total number of carbohydrate chains and the proportion of tetraantenarian structures relative to the number Total carbohydrate chains is at least 2400.

16. The EPO composition according to one of claims 1 to 12, wherein the EPO molecules are the product of an endogenous DNA expression in human cells.

17. The EPO composition according to claim 16, wherein the proportion of the carbohydrate chains with N-acetyl-lactosamine repeats relative to the total number of carbohydrate chains at least 10%.

18. The EPO composition according to claim 17, where the value for the product of the proportion of carbohydrate chains with N-acetyllactosamine repeats in relation to the total number of carbohydrate chains and the proportion of tetraantennial structures in relation to the total number of carbohydrate chains is at least 800.

19. The EPO composition according to claims 13 or 16, wherein the cells are cultured in a serum-free medium.

20. The pharmaceutical preparation, wherein it contains an EPO composition according to one of claims 1 to 19 as the active substance together optionally with the pharmaceutical, common diluents, excipients and carriers.

21. The process for producing an EPO composition, in particular according to one of claims 1 to 20, wherein the EPO composition with the desired characteristics is obtained by at least one of the following measurements: a) selection of a cell of adequate production that is capable of producing carbohydrate chains with a high proportion of N-acetyllactosamine units, b) selection of suitable culture conditions for the cellular structure in order to produce carbohydrate chains with a high proportion of N units. acetyl-lactosamine, and c) separation of undesired components from a known composition of EPO molecules as long as the EPO molecules containing carbohydrate chains are enriched with a high proportion of N-acetyllactosamine units.

22. The process according to claim 21, wherein the measure (b) comprises adding a mixture of at least two carbohydrates and preferably at least 3 carbohydrates to the culture medium.

23. The process according to claim 22, wherein a carbohydrate mixture containing glucose and / or mannose and / or galactose is used. -

24. The process according to claim 21, wherein the measure (b) comprises the controlled addition according to the needs of the nutrients comprising at least one amino acid- Essential and / or at least one carbohydrate depending on the requirements of the cells.

25. The process according to claim 24, wherein the nutrient requirements of the cells are determined depending on the concentration of glutamine measured in the culture medium.

26. The process according to claim 24 or -25, where the nutrients are added according to the needs on the phase of complete growth of the cells.

The process according to one of claims 24 to 26, wherein the nutrients comprise a mixture of at least 2 carbohydrates and preferably at least 3 carbohydrates.

28. The process according to one of claims 21 to 27, wherein the measure (b) comprises cultivating at a temperature between 30 and 35.5 ° C, preferably between 33 and 35.0 ° C.

29. The process according to claim 28, wherein the measure (c) comprises a step of - Inverted phase chromatography to a value. from. pH .in the range of 6-8.

30. The process according to claim 29, wherein acetonitrile, ethanol and isopropanol are used as the eluent.

31. The process according to claim 21, wherein the measure (c) comprises an affinity chromatography step using triazine dyes.

32. The process according to claim 21, wherein the measure (c) comprises an affinity chromatography step using lectins.

33. The process for increasing the specific activity of an EPO composition, wherein the EPO molecules are enriched in the composition having (a) a large number of N-acetyllactosamine units, (b) a high value for the product of the number of N-acetyllactosamine units and the sialic acid content, (c) a high proportion of repeats of N-acetyllactosamine and / or (d) a high value for the product of the repeating ratio of N-acetyllactosamine and the proportion of tetraantenary carbohydrate structures.

34. The process according to claim 33, wherein an average number of at least 4.3 units of N-acetyllactosamine is enriched with reference to an N-linked carbohydrate chain of an EPO molecule or on average at least 13.0 units. of N-acetyllactosamine in relation to the total N-glycosylation of an EPO molecule.

35. The process according to claim 33, wherein a value for the product of the average number of N-acetyllactosamine units is enriched with reference to an N-linked carbohydrate chain of an EPO molecule multiplied by the average content of sialic acid of at least 43.3 or at least 130 relative to the total N-glycosylation of an EPO molecule.

36. The process according to claim 33, wherein (a) in the case of EPO of CHO cells is enriched at an average ratio of at least 30% N-acetyllactosamine repeats relative to the total number of carbohydrate chains or (b) in the case of EPO of human cells at an average ratio of to. less 10% repeats of N-acetyllactosamine relative to the total number of carbohydrate chains.

37. The process according to claim 36, wherein (a) in the case of EPO of CHO cells is enriched in a product value of the average rate of N-acetyllactosamine repeats relative to the total number of chains of carbohydrate multiplied by the average ratio of tetraantenary carbohydrate structures of at least 2400 or (b) in the case of EPO from human cells to a product value of the average rate of N-acetyllactosamine repeats relative to the number total carbohydrate chains multiplied by the average proportion of tetraantenary carbohydrate structures and at least 800.

38. The process. according to one of claims 33-37, wherein the enrichment is achieved by one or more of the measures (a), (b) and (c) according to claim 21.