BRPI9813391B1 - A process for producing an EPO composition and a process for enhancing the specific activity of an EPO composition - Google Patents

Description

PROCESS TO PRODUCE ONE. EPO COMPOSITION AND ftRA PROCESS TO INCREASE THE SPECIFIC ACTIVITY OF A. COMPOSITION OF

EPO The invention relates to novel compositions having high specific activity which are: characterized by a high proportion of N-stilactosamine units and / or tetraanthenary branches in carbohydrate disruption. More specifically, the invention relates to a process for producing such EPO products and for increasing their specific activity.

Erythropoietin (EPO) is a human glycoprotein; stimulates the production of red blood cells. A 3 only occurs in the blood plasma of healthy people very low concentrations, so it is not possible) to portion large amounts in this way. EP-B1-0) 605 and EP-B1-0 205 564 describe the production of recombinant aana EPO in CHO cells. The EPO described in EP-0 148 605 has a higher molecular weight of; urinary EPO and no O-glycosylation. EPO is described in EP-B1-0 205 564 from CHO cells and is now available in large quantities and in pure form.

In addition, the isolation of human urine martyr EPO from patients with plastic anemia is known (Miyake al., J. Biol. Chem. 252 (1977), 5558-5564).

Recombinant and urinary EPOs are isolated in a mixture of various isoforms known to differ in their sialylation degree. These EPO isoforms are endowed with different isoelectric points and can be separated by isoelectric focusing or capillary electrophoresis (see Tsao et al., Biotech. Bi-oeng. 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). Isoforms with the highest sialic acid number have the highest specific activity, while those with the lowest sialic acid have the lowest activity (see, for example, Imai et al., Eur. J. Biochem. 194 (1990), 457-462; EP-A-0 428 267).

Takeuchi et al., (Proc. Natl. Acad. Sci. USA 86 (1989), 7819-7822) describe the relationship between biological activity and sialic acid content and the relationship of biantenary and tetrantenary carbohydrate structures Takeuchi et al additionally conclude that the N-acetyl lactosamine units present in the EPO carbohydrate structure are unrelated to biological activity.

Fukuda et al. (Blood 73 (1989), 84-89) deals with the rate of elimination of EPO from blood circulation which is an important contribution to biological activity and concludes that EPO with a number of units of N Relatively large acetyl lactosamine is more rapidly removed from the circulation than EPO without lactosamine units. Mori-tomo et al. (Glycoconjugate J. 13 (1996), 1093-1120) describe the separation of EPO isoforms by mono-Q chromatography so that the individual fractions are then only composed of a few isoforms. Research on these fractions shows an equidistribution of all structures in all fractions. No correlation was found between the content of biantenary or tetraanthenary structures or the content of N-acetyl lactosamine units and the specific activity.

Thus, said prior art shows that there is a general relationship between biological activity and sugar structure, especially with respect to sialic acid content. However, there is no indication that the tetraantenary structure content and / or the N-acetyl lactosamine content are directly related to biological activity.

When EPO preparations were purified, it was surprisingly found that an increase in the content of tetraantenary carbohydrate structures and / or N-acetyl lactosamine units in the carbohydrate structure led to a significant improvement in specific biological activity. This is particularly applicable when EPO is produced in a human cell line according to European application 97 112 640.4.

Investigations of individual EPO preparations or comparative activity EPO isoforms whose carbohydrate structure essentially differs only in the content of N-acetyl lactosamine units (LE units) exhibit significantly higher activity for the preparations or isoforms with the content. higher units of N-acetyl lactosamine for the same sialic acid content and for the same degree of antenarity. In this respect, antenna is understood as the relative (in%) mean content of the N-chained biantenary, troutanary and tetraantenary carbohydrate chains of EPO preparations or isolated EPO isoforms relative to the total number of N-chained carbohydrate chains. In addition, it has been found that especially in preparations or isoforms with a high content of tetratenary structures, the content of total lactosamine units is extremely important for in vivo activity. An increase in the content of total N-acetyl lactosamine units, for example, in the form of further extensions of the LE unit core structure (called repeats), can greatly increase biological activity. In addition, it has been found that an increase in the content of tetraanthenary structures may improve biological activity.

Therefore, if an EPO preparation is to be produced with the highest specific activity possible and in a high yield, then the purification steps, production cells and / or their culture should be selected and optimized to the highest possible content of tetraantenary carbohydrate structures and / or the highest possible content of N-acetyl lactosamine units is achieved.

A first aspect of the present invention relates to an EPO composition which is composed essentially of glycosylated EPO molecules containing a ratio of at least 75%, preferably at least 80%, particularly preferably at least 85%. and even more preferably at least 90% of tetraantenary structures over the total number of carbohydrate chains, i.e. the sum of biantenary, triantenary and tetraantenary structures.

Another aspect of the invention relates to an EPO composition which is essentially composed of glycosylated EPO molecules containing an average number of at least 3.7, preferably at least 4.0, particularly preferably at least at least 4.3 and most preferably at least 4.5 units of N-acetyl lactosamine with reference to the average N-linked carbohydrate chain composition of the EPO molecule or a number of at least 11.1, preferably of at least 12.0, particularly preferably of at least 13.0 and most preferably of at least 13.5 units of N-acetyl lactosamine, with reference to all three N-carbohydrate structures. -chain (total N-glycosylation) of the EPO molecule.

Another aspect of the invention relates to an EPO composition which is composed essentially of glycosylated EPO molecules which have a product value of the number of total average N-acetyl lactosamine units per EPO molecule multiplied by the acid content. average sialic acid per EPO molecule is at least 130, preferably at least 135, particularly preferably at least 140 and even more preferably at least 160.

In this regard, the term "essentially" means that the desired EPO molecules are present in a proportion preferably of at least 80%, particularly preferably of at least 90%, and even more preferably of at least 80%. 95% relative to the number of total EPO molecules in the composition.

In yet another aspect, the present invention relates to an EPO composition which is composed of glycosylated EPO molecules which have an average proportion of at least 75%, preferably at least 80%. and particularly preferably at least 85% of tetraantenary structures relative to the total number of carbohydrate chains.

Additionally, the invention relates to an EPO composition which is composed of glycosylated EPO molecules which have an average number of at least 3.7, preferably at least 4.0 and particularly preferably at least at least 4.3 and most preferably at least 4.5 units of N-acetyl lactosamine with reference to the average N-chain carbohydrate chain composition of the EPO molecule or contain a number of at least 11.1, preferably at least 12.0, particularly preferably at least 13.0 and most preferably at least 13.5 N-acetyl lactosamine units with reference to all 3 N-linked carbohydrate structures of the EPO molecule. The maximum proportion of tetraanthene structures can reach up to 100% of the total carbohydrate chains, where each tetraantenary structure contains 4 units of N-acetyl lactosamine in the N-linked sugar core structure. Additional N-acetyl lactosamine units that occur as extensions of the core structure as so-called repeats may increase the number of N-acetyl lactosamine units per carbohydrate structure as well as total glycosylation. The number of N-acetyl lactosamine units per glycosylation site (i.e., by N-linked carbohydrate structure) can thus be up to 6 (tetraantenary structure and 2 additional N-acetyl lactosamine units as repeats) ( Figure 1) or may - in the case of structures with more than 2 additional N-acetyl lactosamine units - be even larger- The number of N-acetyl lactosamine units may reach up to 18 or higher with reference to glycosylation. total (three N-linked carbohydrate structures).

Yet another aspect of the invention relates to an EPO composition which is composed of glycosylated molecules that have an average value for the product of the number of average total N-acetyl lactosamine units of the EPO molecule multiplied by the sialic acid content. average per EPO molecule of at least 130, preferably of at least 135, particularly preferably of at least 140 and even more preferably of at least 160.

Another subject matter of the invention is an EPO composition which has the features of at least two or more of the aforementioned features. The composition according to the invention may be composed of one or more isoforms, that is, EPO molecules with different isoelectric points in isoelectric focusing. The composition according to the invention preferably comprises a mixture of at least 2, for example from 2 to 5 isoforms, in particular 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, in particular 200,000 IU / mg in vivo (normocythemic rat). The specific activity is preferably # in the range of about 200.G0v to 400,000 IU / mg or 450,000 IU / mg protein # more preferably # in the range of 250,000 to 400,000 IU / mg or 450,000 IU / mg protein .

In the composition according to coa. the present invention - the average sialic acid content or the number of average sialic acid residues # per EPG molecule is preferably particularly preferably at least 11 # 5 and most preferably at least minus 12.5. The EPO composition according to the invention may, on the one hand, be obtained from EPO molecules which are the product of exogenous DIA expression in mammalian cells, for example, in rodent cells such as CHQ or BHK cells as described in EP-B-0 205 564. Alternatively, the composition may also be composed of molecules. ... ds .EPO ..... which are the product of an exogenous DMA expression after gene activation in human cells, for example in immortalized cell lines such as Namalwa (Nadkarni et: al., Cancer 23 (1969), 64-79), HT108 (Rasheed et al., Cancer 33 (1973), 1027-1033) or HeLa S3 (Puck et al., J. Exp. Meth, 103 (1956), 273-284 ). Such processes are described in European patent application 97 112 640.4, the disclosure of which forms part of the present application.

Other important parameters for EPO biological activity are the proportion of repeating carbohydrate chains, that is, additional N-acetyl lactose units in relation to the total number of N-linked carbohydrate chains, as well as the value of the product of this ratio of repetitions and the ratio of tetraanthenary carbohydrate chains to the total number of carbohydrate chains. In the case of EPO from CEO cells, the proportion of repeats is preferably at least 30%, particularly preferably at least 35%, and more preferably at least 40%. In the case of EPO from human cells, such as HeLa cells, the proportion of repeats is preferably at least 10%, particularly preferably at least 12% and most preferably pelvis. Therefore, in the case of EPO from CHO cells, the value for the product of the proportion of carbohydrate chains with repeats of N-acetyl lactosamin in relation to the total number of carbohydrate chains and The ratio of tetraanthenary structures to the total number of carbohydrate chains is preferably at least 2400, particularly preferably at least 2800, and more preferably at least 3400. In the case of EPO from human cells, the value is preferably at least 800, particularly preferably at least 960 and more preferably at least 1100.

An EPO composition is preferably used which has been produced by culturing EPO production cells in a culture medium eating a low serum content, for example, a maximum of 1 I (v / v), or especially in a serum-free culture medium (cf. m. 96/35718). Examples of culture media are RPMI 1640 or DMEM. The EPO composition according to the invention may be formulated as a pharmaceutical preparation optionally together with standard pharmaceutical diluents, auxiliaries and carriers. The EPO composition according to the invention which may be used to produce a pharmaceutical preparation is preferably preferably at least 99% pure and particularly preferably at least 99.9% as determined by reverse phase HPLC ( for example on a Vydac C4 column) or / and size exclusion chromatography (e.g. on a TSK 2000SW Ultra-pac column).

In addition, the composition according to the invention has a DMA content of preferably <10 pg, particularly preferably <5 pg and even more preferably <1 pg DMA per 10,000 XU protein. In addition, the composition according to the invention is substantially free of bacterial impurities (<1 CFU / ml and endotoxins (<1 EU / 10,000 IU protein). DNA content can be determined by a hybridization test using whether radiolabelled or fluorescently labeled DNA Commercially available purified human DMA is, for example, used as the probe DNA Human DNA may additionally be used as a standard for the test. such hybridization test is on the order of about 0.3 pg / 10,000 IU EPO The genus and endotoxin content of the EPO preparation can be determined by standard procedures as described in Pharm.

An EPO composition which preferably has the desired aspects of the invention may be obtained by at least one of the following: (a) selecting a suitable production cell line that is capable of producing carbohydrate chains with a high proportion of structures. tetraantenary or / e. N-acehi-1 - lacto.s.amine units, (b) selection of suitable culture conditions for cell culture to produce carbohydrate chains with a high proportion of tetratenary structure units N-acetyl lactosamine, and (c) separating undesirable components from a known EPO molecule composition while enriching the carbohydrate chain-containing EPO molecules with a high proportion of tetraantenary or / and N structure units. -acetyl lactosamine. Providence (a) comprises selecting a suitable production cell. In this case, one may use cells which are known to have a tendency to produce the desired carbohydrate chain structures in high yield. Examples of such cell lines are hamster-derived cells such as CHO or BHK and human cell lines such as HeLa, Namalwa, HT1080, or cell lines derived therefrom. HeLa S3 cells or modified CHO cells are particularly preferred.

On the other hand, it is also possible to specifically produce suitable production cells by overexpressing certain glycosylation enzymes in the cell, for example by recombinant expression or / and by endogenous gene activation. Examples of such glycosylation enzymes are sialyl transferases, N-acetyl glycosaminyl transferases and galactosyl transferases. Providence (b) comprises the selection of suitable culture conditions in the cell culture. According to a first embodiment of the invention, the provision (b) comprises adding a mixture of at least two and preferably at least three carbohydrates to the culture medium. Carbohydrates are preferably selected from monosaccharides and disaccharides such as glucose, glycosamine, ribose, fructose, galactose, mannose, saccharin, lactose, mannose-1-phosphate, mannose-1-sulfate and mannose-6-sulfate. Suitable nutrient media, for example, contain glucose or / and mannose or / and galactose. Particularly good results were obtained with nutrient media containing a mixture of glucose, galactose and mannose, for example, in a mass ratio of 1: (0.5 - 3) :( 1-5) and in particular 1: (0 , 7 - 1,4) :( 1,8 - 4,0) where each carbohydrate is particularly preferably used in the form D (+). The total concentration of all sugars during fermentation is preferably in the range 0.1 to 10 g / l, particularly preferably in the range 2 to 6 g / l in the culture medium. The carbohydrate mixture is preferably added depending on the respective cell requirement, as explained in more detail below.

According to another preferred embodiment, the provision (b) comprises controlled nutrient addition which is preferably according to requirements comprising at least one essential amino acid for the cultured cell line or / and at least one carbohydrate, depending on the respective cell requirements. In this way considerably improved glycosylation is achieved in large fermenters (volume> 11, eg 50 - 10,000 1) even with high cell density fermentation (harvest cell density> 10 x 105 cells / ml and preferably> 20 x 105 cells / ml). For this purpose, the concentration of parameters that relate to the nutrient requirements of the cells is determined by confinement or at appropriate time intervals, for example at least one vet per day and their consumption rates are calculated. This enables the nutrient requirements of cells to be quantitatively and / or quantitatively determined. Such parameters may be nutrients or metatolic products of the cells, such as glutamine, ammonium, glucose, and / or lactate concentration and especially glutamine concentration.

Nutrients added according to this aspect of the invention comprise essential amino acids, for example glutamine and / or tryptophan and / or carbohydrates and preferably, preferably, non-essential amino acids, vitamins, residuals, salts and / or growth factors. for example, insulin. Nutrients particularly preferably include at least one essential amino acid and at least one eardrate. These nutrients are preferably measured in the culture medium in one. dissolved state. The nutrient solutions preferably contain glutamine and carbohydrates, especially a mixture of at least one carbohydrate as previously mentioned. A mixture of glucose, galactose and mannose is particularly preferably used. In addition, it is preferred that nutrients are added as needed throughout the cell growth phase, that is, depending on the concentration of the selected parameters that are measured in the culture medium. The quantitative ratio of glutamine to carbohydrate in the nutrient solution is preferably selected such that it essentially corresponds to the consumption ratio in the fermenter. This enables a substantially constant concentration of the individual substrates to be maintained in the fermenter. The glutamine concentration is preferably maintained at a value of <150 mg / 1 in the culture medium and prevents the development of an ammonia concentration> 2.3 mol / 1 in the culture medium. During fermentation, the total concentration of sugars is preferably in the range 0.1 to 10 g / l, particularly preferably in the range 2 to 6 g / l of culture medium as explained above. The nutrient solution that is used contains a glutamine to sugar ratio by weight which is preferably in the range of 1: 3 to 20 and particularly preferably of 1: 5 to 15 with reference to total sugars. When using a nutrient solution containing glutamine as well as the three sugars glucose, galactose and mannose, the ratio of glutamine to sugars by weight preferably comprises 1: (1 to 3) :( 1 to 5 ) :( 2 to 8) and particularly preferably from 1: (1.5 to 2.2) :( 1.5 to 3.6): (4 to 6). Preferably, the culture is performed as a repeated batch process, in which part of the culture broth is harvested after the growth phase and the remainder of the culture broth remains in the fermenter which is subsequently replenished with fresh medium for growth. operation volume. The process according to the invention enables the glycosylated EPO to be harvested in very high yields. Thus, the concentration at the time of harvest is, for example, at least 30 mg and in particular at least 40 mg EPO per 1 culture medium.

Yet another aspect of the invention is a process for isolating EPO from eukaryotic cells, wherein eukaryotic cells are grown in a suitable medium and EPO is isolated from the culture supernatant, the process being characterized by the fact that that the culture is carried out at a temperature of ≤ 36 ° C, preferably between 30 and 35.5 ° C and particularly preferably between 33 and 350 ° C. It has surprisingly been found that the ratio of EPO to the desired glycosylation can be considerably increased by lowering the temperature during the culture. Provision (c) comprises the separation of the unwanted components from the known EPO composition whose carbohydrate structure does not meet the specifications of the present invention. This may be accomplished, for example, by chromatographic purification of the EPO preparations, for example by affinity chromatography on triazine dye gels, preferably Cibacron Blue dye gels. The unwanted components can also be further separated using hydrophobic interaction chromatography and reverse phase HPLC. Suitable binders for this are butyl, pentyl, octyl, octadecyl and phenyl residues. The reverse phase chromatography step is preferably performed at a pH value in the range of 6.0 to 8.5 and particularly preferably from 7.0 to 8.0. Suitable eluents are, for example, acetonitrile, ethanol or isopropanol, preferably acetonitrile.

Suitable fractions can be determined, accumulated and finally further processed using capillary zone electrophoresis (CZE) analysis. In addition, EPO molecules with a high content of N-acetyl lactosamine units can be directly enriched by using tomato or potato lectins (Merkle, Cummings, J. Biol. Chem. 262 (1987), 8179-8189). Such lectins are preferably used, for example, in an immobilized form.

Another subject matter of the invention is a process for increasing the specific activity of an EPO composition whereby EPO molecules are enriched in the composition having (a) a high proportion of tetraanthenary carbohydrate structures, (b) a large number of N-acetyl lactosamine units, (c) a high value for the product of N-acetyl lactosamine units and sialic acid content, (d) a high proportion of N-acetyl repeats -lactosamine and / or (e) a high value for the product of the proportion of N-acetyl lactosamine repeats and the proportion of tetraantenary carbohydrate structures.

Such enrichment may be accomplished by one or more of the provisions (a), (b) and (c) mentioned above. The present invention is further elucidated by the following figures and examples. Figure 1: shows a figure of a tetraanthenary carbohydrate structure with additional N-acetyl lactosamine units (repeats) and sialic acids. Figure 2: shows the dependence of the relative proportion of individual EPO isoforms on carbohydrates added to the culture medium. Figure 3 shows the dependence of the biological activity of EPO preparations on carbohydrates added to the culture medium. Figure 4: shows the dependence of EPO isophone biological activity on the number of repeat N-acetyl lactosaroin repeat units.

EXAMPLE I: EPO Purification from Overcurrent and Culture Cell Lines Two processes were essentially used to purify EPO from human cell supernatants or CHO cells from cell cultures that differed in number and principle. chromatographic steps and were used depending on the medium composition and EPO concentration: Process 1: Step 1: blue Sepharose column 2nd step: Butyl Sepharose column 3rd step: hydroxyapatite column 4th step: concentration Process 2 ; 1st step: blue Sepharose 2S column step: hydroxyapatite column 3 * step: concentration (3rd step alternative: RP-HPLC) Example of a purification of a He La S3 cell culture supernatant containing 2% (v / v) fetal calf serum (FCS) by the 1: 1 procedure, Blue Sepharose Column A 5 ml Hi-Trap Blue column (Pharmacia ready blue-Sepharose column) was equilibrated with at least 5 volumes. buffer column (CV) column (20 mM Tris-HCl, pH 7.0; 5 mM CaCl2; 100 mM NaCl) * Subsequently 70 ml HeLa cell supernatant (containing about 245 pg EPO and 70-100 mg total were absorbed overnight at a flow rate of 0.5 ml / min in a recycling procedure.The column was washed with at least 5 CV of buffer B (20 mM Tris-HCl, pH 7.0; 5 mM CaCl 2; 250 mM NaCl) and at least 5 HP of buffer C (20 mM Tris-HCl, pH 7.0; 0.2 mM CaCl 2; 250 mM NaCl) at 0.5 ml / min. was monitored by measuring protein content in OD 280. EPO was eluted with D buffer (100 mM Tris-HCl, pH 7.0; 0.2 mM CaCl 2; 2 mM NaCl) at a flow rate of 0.5 ml / min. The elution solution was collected in fractions of 1 - 2 ml. The EPO content of fractions, wash solutions and eluate was determined by reverse phase (RP) -HPLC by applying an aliquot to a POROS R2 / H column (Boehringer Mannheim). Alternatively, an immunoblot was performed for the quantitative identification of fractions containing EPO.

Elution fractions containing EPO (8-12 ml) were pooled and applied to a butyl Sepharose column. The yield after the blue Sepharose column was about 175 μg EPO (which is about 70%). In general, the yield after blue-Sepharose was between 50 and 75%. 2, Butyl Sepharose Column (hydrophobic interaction chromatography) A self-made 2-3 ml butyl Sepharose column (material: Toyopearl butyl S650) was equilibrated with at least 5 CV of D-damper (100 mM Tris- HCl, pH 7.0; 0.2 mM CaCl2; 2 mM NaCl) and subsequently the EPO blue-Sepharose combination containing 1 (about 150 μς EPO) was absorbed at a flow rate of 0.5 ml. / min The column was washed with at least 5 CV of E buffer (20 mM Tris-HCl, pH 7.0; 2 mM NaCl and 10% isopropanol) at 0.5 ml / min. Successful washing was monitored by measuring protein content under OD 280. EPO was eluted with F buffer (20 mM Tris-HCl, pH 7.0; 2 mM NaCl and 20% isopropanol) at room temperature and at room temperature. a flow rate of 0.5 ml / min. The elution solution was collected in fractions of 1 - 2 ml. The EPO content of the fractions, wash solutions and eluate was determined by RP-HPLC by applying an aliquot to a POROS R2H column. Alternatively, an immunological blur point of the EPO containing fractions was performed.

Elution fractions containing EPO (10-15 ml) were combined and applied to a hydroxy apatite column. The yield of the butyl sepharose column was about 130 μg EPO (which is about 85%). Overall, the yield of butyl Sepharose was between 60 and 85% of the applied blue Sepharose combination. 3. Hydroxyapatite Column A 5 ml hydroxyapatite column (ECRA self-absorbent central absorbent CHT II from BioRAD) was equilibrated with at least 5 HP of buffer F (20 mM Tris-HCl, pH 7.0 ; 2 mM NaCl; 20% isopropanol) and subsequently the EPO-containing butyl Sepharose combination containing 2 (about 125 μg EPO) was absorbed at a flow rate of 0.5 ml / min. The column was washed with at least 5 CV of buffer G (20 mM Tris-HCl, pH 7.0; 2 M NaCl) at 0.5 ml / min. Washing success was monitored by measuring protein content under OD 280. EPO was eluted with H-buffer (10 mM Tris-HCl, pH 7.0; 80 mM NaCl) at a flow rate of 0.5 ml / ml. min The elution solution was collected in fractions of 1 - 2 ml. The EPO content of fractions, wash solutions and eluate was determined by RP-HPLC by applying an aliquot to a POROS R2 / H column.

The elution fractions containing EPO (3-6 ml) were combined. The hydroxyapatite column yield was about 80 μg EPO (which corresponds to about 60%). In general, the yield of the hydroxyapatite column was between 50 and 65% of the applied butyl sepharose combination. 4. Concentration The combined EPO fractions from the hydroxyapatite step were concentrated in centrifugation units with an exclusion size of 10 kD (eg Microsep from Filtron) to a concentration of 0.1-0.5 mg / ml. 0.01% Tween 20 was added and stored in aliquots at -20 ° C.

Yield scheme: The purity of the isolated EPO was about> 90%, usually even> 95%.

Process 2 wherein the butyl sepharose step was omitted was also used to increase EPO yield. This process can be applied especially to cell culture supernatants without or with the addition of 1% (v / v) CELL FIBER supplement and isolated EPO yields of approximately the same purity (90-95%). The presence of 5 mM CaCl 2 in the equilibrium damper (damper F) for the hydroxyapatite column led this process to improved agglutination and thus also to improved reproducible EPO elution behavior in the hydroxyapatite step. Consequently, process 2 was performed with the following buffers, using in principle the same procedure as for process 1: 1. Blue-Sepharose column: equilibrium buffer (buffer A): 20 mM Tris-HCl, pH 7.0 ; 5 mM

CaCl2; 100 mM NaCl Wash Buffer 1 (Buffer B): 20 mM Tris-HCl, pH 7.0; 5 mM

CaCl2; 250 mM NaCl Wash Buffer 2 (Buffer C): 20 ιαμ Tris-HCl, pH 7.0; 5 mM

CaCl2; 250 mM NaCl Elution Buffer (Buffer D): 100 mM Tris-HCl, pH 7.0; 5 mM

CaCl2; 2 M NaCl 2. Balance damper hydroxyapatite column (F damper): 50 mM Tris-HCl, pH 7.0; 5 mM

CaCl2; 1 M NaCl Wash Buffer (G Buffer): 10 mM Tris-HCl, pH 7.0; 5 mM

CaCl2; 80 mM NaCl Elution Buffer (H Buffer): 10 ιημ Na-phosphate, pH 7.0; 5 mM CaCl 2; 80 mM NaCl Yield scheme: The addition of 5 mM CaCl2 to the C to G buffers in process 1 also led to improved agglutination and more defined elution from the hydroxyapatite column.

Alternatively or additionally, the following steps may also be used to purify EPO: RP-HPLC, for example, with Vydac C4 material DEAE-Sepharose chromatography diafiltration liner Example 2: Purification of EPO from culture supernatants as Isoforms 1-8 (comparison) 1. EPO starting material from mammalian cells, e.g. CHO or human cells, was fermented by repeated batching. A 1000 l fermenter was inoculated with a preculture and the fermenter content was harvested after about 3 to 5 days. After harvesting, the cells were removed from the fermentation broth by centrifugation. The cell-free culture supernatant is adjusted to pH 5.0 - 5.2 with 1 mol / 1 acetic acid and filtered at 1-9 ° C. 2. Blue Sepharose Chromatography A chromatography column (Amicon P440 x 500, Amicon, GB) was filled with 60 - 80 l of blue Sepharose and regenerated with 0.5 N NaOH. Subsequently, the column was equilibrated with about 3 column volumes (CV) of acetate buffer. The cell-free culture supernatant adjusted to pH 5 was absorbed into the column at a temperature of 10 ± 5 ° C and a flow rate of 800 - 1400 ml / min. The column was washed again at the same flow rate and 5 ± 4 ° C with about 1 CV of wash buffer 1. This was followed by about 2 CV of wash buffer 2. Subsequently, the column was eluted with about 3 HP elution damper. The total protein peak was collected (about 30 - 6 ° 1) adjusted to pH 6.9 with HCl and stored at 5 ± 4 ° C until further processing. The product solution was concentrated at this chromatography step and a purity of about 40 - 50% was achieved.

Balance buffer: 20 mM In acetate, 5 mM

CaCl 2, 0.1 M NaCl, pH 5.0 ± 0.2 wash buffer 1: 20 mM In acetate, 5 mM

CaCl2, 0.25 M NaCl, pH 5.0 ± 0.2 Wash Buffer 2: 20 mM Tris-HCl / 5 mM CaCl2 / pH 6.5 ± 0.3 Elution Buffer: 100 mM Tris-HCl, 5 mM CaCl 2, 1 M NaCl, pH 9.0 ± 0.2 3. Butyl-Toyopearl Chromatography (hydrophobic chromatography) A chromatography column (Pharmacia BGP 300/500) was filled with 30 - 40 1 of butyl-Toyopearl and regenerated. with 4 M guanidine-HCl and 0.5 N NaOH. Subsequently the column was balanced with at least 3 CV of balance damper. The eluate from the blue Sepharose column was adjusted to 10% isopropanol and absorbed into the column at a temperature of 27 ± 2 ° C and a flow rate of 800 - 1200 ml / min. The column was washed again at the same temperature and flow rate of about 1 CV of equilibration damper and then with about 2 CV of wash damper. Subsequently, it was eluted with about 3 CV of elution damper. The total protein peak is collected (about 10 - 18 1), immediately diluted three times with dilution buffer and stored at 15 ° C until further processing. A purity of about 90% was achieved on this chromatography. Equilibrium Damper: 20 mM Tris-HCl, 5 mM CaCl2, 0.75 M NaCl, 10% isopropanol, pH 6.9 ± 0.2 Wash Buffer: 20 mM Tris-HCl, 5 mM CaCl2, 0.75 M NaCl, 19% Isopropanol, pH 6.9 ± 0.2 Elution Buffer: 20 mM Tris-HCl, 5 mM CaCl 2, 0.75 M NaCl, 27% Isopropanol, pH 6.9 ± 0.2 dilution buffer: 20 mM Tris-HCl, 5 mM CaCl2, pH 6.9 ± 0.2 4. Ultrogel Hydroxyapatite Chromatography A chromatography column (Amicon P440 x 500 or equivalent) was packed with 30 40 1 of Ultrogel hydroxyapatite. and regenerated with 0.5 N NaOH. Subsequently, the column was balanced with at least 4 CV of balance damper. The eluate from the butyl-Toyopearl column was absorbed into the column at a temperature of about 15 ° C and a flow rate of 500-1200 ml / min. The column was washed again at the same temperature and flow rate with about 1 CV of equilibration damper and then with about 2 CV of wash damper. Subsequently, it was eluted with about 3 CV of elution damper. The total protein peak was collected (about 10 - 18 1) and stored at 15 ° C until further processing. A purity greater than 95% was obtained on this chromatography.

Equilibrium Damper: 20 mM Tris-HCl, 5 mM CaCl2, 0.25 M NaCl, 9% Isopropanol, pH 6.9 ± 0.2 Wash Buffer: 10 mM Tris-HCl, 5 mM CaCl2, pH 6.9 ± 0.2 elution buffer: 10 mM Tris-HCl, 10 phosphate, 0.5 mM CaCl2, pH 6.8 ± 0.2 5. HPLC (RP-HPLC) reverse phase A preparative HPLC was performed using a Merck Prepbar 100 (or equivalent) separating apparatus at a temperature of 22 ± 4 ° C. The separation column (100 mm x 400 mm, 3.21) was packed with Vydac C4 material. Prior to use, the column was regenerated by repeatedly applying a damper gradient A to 100% solvent and balancing subsequently with damper A. The eluate from the hydroxyapatite column was acidulated with trifluoroacetic acid to about pH 2.5 and sterile filtrate. Subsequently, it was applied to the column at a temperature of 22 + 4 ° C and a flow rate of 250 - 31 ml / min. The column was eluted at the same temperature and flow rate with a gradient from damper A to damper B linear. The elution peak was collected in fractions. The eluate was immediately neutralized by first adding 4 volumes of HPLC dilution buffer.

Fractions that were at least 99% pure on the analytical HPLC were combined (combined volume about 4 - 6 1). Residual impurities were separated on this chromatography and a purity of greater than 99% was obtained. buffer A: 0.1% trifluoroacetic acid in water buffer B: 80% acetonitrile, 0.1% trifluoroacetic acid in water HPLC dilution buffer: 10 mM

Na / K phosphate, pH 7.5 ± 0.2 6. DEAE-Sepharose ff chromatography A chromatography column (Amicon P90 x 250 or equivalent) was filled with 100 - 200 ml gel per g of EPO applied and regenerated with 0.5 N NaOH. Subsequently, the column was first equilibrated with 100 mM Na / phosphate K buffer, pH 7.5 and then with at least 12 HP of equilibrium buffer. Subsequently, it was washed again with about 10 CV of equilibrium damper and then eluted with about 7 CV of elution damper. The total protein peak was collected (about 2 - 5 1), sterile filtered and distributed.

In this chromatography, the solvent from the HPLC step was separated and the residual impurities removed. Purity is greater than 99%. equilibrium buffer: 10 mM Na / phosphate K, pH 7.5 ± 0.2 wash buffer: 30 mM Na / phosphate K, pH 4.5 ± 0.1 elution buffer: 10 mM Na / phosphate K, 80 mM

NaCl, pH 7.5 ± 0.2.

Example 3: Purification of EPO from Culture Supernatants While Maintaining Isoforms 1-4 (Invention) 1. EPO starting material from mammalian cells, eg CHO or human cells, was fermented by a process of repeated batching. A 10 1 fermenter was inoculated with a preculture and the fermenter content was harvested after about 5 days. After harvesting, the cells were removed from the fermentation broth by centrifugation. The cell-free culture supernatant was adjusted to pH 5.0 - 5.2 with 1 mol / l acetic acid and filtered at 1 - 9 ° C. 2. Blue Sepharose Chromatography A suitable chromatography column was filled with 150 - 250 ml blue Sepharose and regenerated with 0.5 N NaOH. Subsequently, the column was equilibrated with about 3 column volumes (CV) of acetate buffer. The cell-free culture supernatant adjusted to pH 5 was absorbed into the column at a temperature of 10 ± 5 ° C and a flow rate of 1-2 CV / h. The column was washed again at the same flow rate and 5 ± 4 ° C with about 1 CV of scrubber damper 1. This was followed by about 2 CV of scrubber damper 2. Subsequently the column was eluted with about 3-6 CV of elution damper. The protein peak was collected in fractions. After EC analysis, appropriate fractions were combined, adjusted to pH 6.9 with HCl and stored at 5 ± 4 ° C until further processing. The product solution was concentrated at this chromatography step and the basic impurities and isoforms were separated.

Balance buffer: 20 mM In acetate, 5 mM

CaCl 2, 0.1 M NaCl, pH 5.0 ± 0.2 wash buffer 1: 20 mM In acetate, 5 mM

CaCl2, 0.25 M NaCl, pH 5.0 ± 0.2 Wash Buffer 2: 20 mM Tris-HCl, 5 mM CaCl2, pH 6.5 ± 0.3 Elution Buffer: 50 mM Tris-HCl, 5 mM CaCl2, 0.25 M NaCl, pH 8.0 ± 0.2 3. Butyl-Toyopearl Chromatography (hydrophobic chromatography) A suitable chromatography column was filled with 200 - 300 ml of Butyl-Toyopearl and regenerated with 4 M guanidine. -HCl and 0.5 N NaOH. Subsequently the column was balanced with at least 3 HP of balance damper. The eluate from the blue-Sepharose column was adjusted to 10% isopropanol and absorbed into the column at a temperature of 27 ± 2 ° C and a flow rate of 1-2 CV / h. The column was washed again at the same temperature and flow rate of about 1 CV of damper damper and then with about 2 HP of damper damper. Subsequently, it was eluted with about 5-10 CV of elution damper. Fractions were collected from the protein peak and immediately diluted three times with dilution buffer. After EC analysis, appropriate fractions were combined and stored at 15 ° C until further processing. In this chromatography, other basic isoforms were removed and a purity of about> 80% was achieved.

Equilibrium Damper: 20 mM Tris-HCl, 5 mM CaCl2, 0.2 M NaCl, 10% Isopropanol, pH 6.9 ± 0.2 Wash Buffer: 20 mM Tris-HCl, 5 mM CaCl2, 0.2 M NaCl, 17% Isopropanol, pH 6.9 ± 0.2 Elution Buffer: 20 mM Tris-HCl, 5 mM CaCl2, 0.2 M NaCl, 23% Isopropanol, pH 6.9 ± 0.2 Dilution Buffer: 20 mM Tris-HCl, 5 mM CaCl 2, pH 6.9 ± 0.2 4. Chromatography of Ultrogel Hydroxyapatite An appropriate chromatography column was filled with 150 - 200 ml Ultrogel hydroxyapatite and regenerated with 0.5 N NaOH . Subsequently, the column was balanced with at least 4 HP of balance damper. The eluate from the Butyl-Toyopearl column was absorbed into the column at a temperature of about 15 ° C and a flow rate of 1-2 CV / h. The column was washed again at the same temperature and flow rate with about 1 CV of equilibration damper and then with about 2 CV of wash damper. Subsequently, it was eluted with about 3 CV of elution damper. The total protein peak was collected and stored at 15 ° C until further processing. A purity greater than 95% was obtained on this chromatography.

Equilibrium Damper: 20mM Tris-HCl, 5mM CaCl2, 0.06M NaCl, 7.5% Isopropanol, pH 6.9 + 0.2 Wash Damper: 10mM Tris-HCl, 5mM CaCl2, pH 6.8 ± 0.2 elution buffer: 10 mM Tris-HCl, 10 mM K-phosphate, 0.5 mM CaCl2, pH 6.8 ± 0.2 5. Reverse phase HPLC (RP-HPLC) a semi-preparation HPLC using a Vydac C4 separation column (20 mm x 250 mm, about 80 ml) at a temperature of 22 ± 4 ° C. Prior to use, the column was regenerated by repeatedly applying a damper gradient A to 100% solvent and subsequently equilibrating with damper A. The eluate from the hydroxyapatite column was applied to the column at a temperature of 22 ° C. ± 4 ° C and at a flow rate of 8 - 15 ml / min. The column was then eluted at the same temperature and flow rate with a damper gradient Ά to linear damper B according to the HPLC protocol which is shown below. The elution peak was collected in fractions. The eluate was immediately neutralized by first adding 4 volumes of HPLC dilution buffer.

HPLC protocol: <*) wash conditions 3 and elution; 50% damper gradient Ba 100% damper B in 50 to 200 minutes, preferably 140 minutes.

Fractions that were at least 99% pure on the analytical HPLC and suitable according to the EC analysis were combined. Residual impurities and isoform residues were removed on this chromatography and a purity of greater than 99% was obtained. buffer A: 10 mM Na / phosphate K, pH 7.0 ± 0.2 buffer B: 10 mM Na / phosphate K, acetonitrile 80%, pH 7.0 HPLC dilution buffer: 10 mM

Na / Phosphate K, 100 mM NaCl, ph 7.5 ± 0.2 6. Diafiltration A diafiltrage apparatus of a suitable size was. equipped with a 10 JtD cassette and regenerated with 1 N NaOH. Subsequently, the apparatus was rinsed for bleach free using mass dampener. The HPLC column eluate was concentrated at a temperature of 5 ± 4 ° C and diafiltered against a 10 volume mass damper. The final concentration should be between 1 and 3 mg / ml.

This step serves to remove solvent residue from the HPLC step, to adjust the required mass dampening conditions and mass concentration of the active substance product.

Mass damper; 10 mM. Na / phosphate K, 100 mM NaCl, pH 7.5 ± 0.2 Example 4: Determination of specific EPO activity in vivo (bioassay in a normocythemic rat) Dose-dependent EPO activity in the multiplication and differentiation of precursor cells from Red blood cells were determined in vivo in rats by increasing blood reticulocytes following EPO administration.

For these various dosages of the EPO sample to be analyzed and a standard EPO (standardized to the EPO WHO standard), each was parenthetically administered to 8 rats. The rats were subsequently kept under constant defined conditions. Blood was collected from the rats 4 days after EPO administration and the reticulocytes were stained with orange acridine. Reticulocyte count per 30,000 erythrocytes was determined by microfluorimetry on a flow cytometer by analysis of the red fluorescence histogram. Biological activity was calculated from the values for sample and standard reticulocyte counts at different doses according to the procedure described by Linder for determining parallel straight-line pair concentration (Linder, "Planen und Auswerten von Versuchen"). ", 3rd edition, 1969, Birkehãuser Verlag, Basel).

The EPO CH01, CHO 2 and CHO 3 preparations were obtained by purifying EPO from serum-free CHO cell culture supernatants having biological activities of 248,000 (CHO 1), 225,000 (CHO 2). ) and 186,000 IU / mg (CHO 3), respectively. In four preparations in which EPO was purified from human cell culture supernatants, products with specific activities of 220,000 (HeLa 1), 198,000 (HeLa 2), 204,000 (HeLa 3), 176,000 (HeLa 4) were obtained. and 100,000 IU / mg (HeLa 5). The relationship of values for biological activity with parameters for sugar structure is shown in example 11.

Example 5: Determination of Sialic Acid Residue Content Sialic acid content was determined chromatographically by means of HPAEC-PAD (pulsed amperometric detection high pH anion exchange chromatography) in a Dionex system after enzymatic acid cell division neuraminidase sialics from Arthrobacter ureafaciens (A. ureaf., Boehringer Mannheim).

Preparations, each containing 22 μg EPO from various CHO and human cell line preparations (eg HeLa S3) were adjusted to an EPO concentration of 0.2 mg / ml at 5 mM. In phosphate buffer, pH 7.2. Half of each preparation was used to determine the exact amount of EPO by RP-HPLC. 5 mM U of neuraminidase from A. ureaf. Were added to the second half of the preparations and incubated overnight (about 18 hours) at 37 ° C. Subsequently, the digestion mixtures were equally distributed, diluted 20 times to 50 μΐ with H20 and 50 μΐ (corresponding to about 27 pmol EPO) were applied to the Dionex system. For this, the following chromatographic parameters were used: column: CarboPac PA 100 flow: 1.0 ml / min detector sensitivity: 300 nA gradient: t (min)% damper B 0 17 7 17 9 100 12 100 13 0 20 0 buffer A: 0.1 M NaOH buffer B: 0.1 M NaOH; 0.5 M In acetate The amount of sialic acids in the sample applied was determined with the aid of a calibration line that was obtained from values of a sialic acid standard that was also analyzed (Boehringer Mannheim). The sialic acid content (mol of sialic acid / mol of EPO) was calculated from the result of the sialic acid determination (Dionex system) and the determination of the amount of EPO used by RP-HPLC. The EPO from CHO cells had an average content of 12.9 moles (CHO 1), 11.8 moles (CHO 2) and 11.7 moles (CHO 3) of sialic acid per mol of EPO. The human cell-derived EPO preparations had an amount of 13.1 moles (HeLa 1), 13.2 moles (HeLa 2), 13.3 moles (HeLa 3), 11.6 moles (HeLa 4) and 10, 8 moles (HeLa 5) of sialic acid per mol of EPO (see also example 11).

Example 6: Determination of the proportions of biantenary, triantenary and tetraantenary carbohydrate structures N-linked carbohydrate structures were chromatographically analyzed by HPAEC-PAD in a Dionex system. Asialo oligosaccharides from EPO preparations from CHO and human (e.g., HeLa S3) cell lines were isolated by enzymatic cell division with N-glycosidase F (Boehringer Mannheim) and neuraminidase from A. ureaf. (Boehringer Mannheim).

10 or 30 μg EPO was desalted by mixing using Micro-Con ultracentrifugation units (Amicon, 10 kD exclusion size) and adjusted with 10 mM Na phosphate buffer, pH 7.2 to a concentration of 0 ° C. , 2 or 0.3 mg / ml. Subsequently, 1 U N-glycosidase F and 10 mU neuraminidase were added to each mixture and incubated overnight (about 18 hours) at 37 ° C. In order to separate the half EPO polypeptide from the cell-divided oligosaccharides, the mixtures were centrifuged by Ultrafree Centrifugation Units (Millipore, 10 kD exclusion size) after incubation and the Ultrafree waswashed twice again with 20 µg. of H2 O. The oligosaccharides contained in the filtrate were prepared to 150 μΐ with H20 and 100 μΐ of them were analyzed in the Dionex system. For this, the following chromatographic parameters were used: column: CarboPac PA 100 flow: 1.0 ml / min detector sensitivity: 300 nA gradient: t (min)% damper B 0 0 2 0 60 10 62 100 67 100 t (min)% buffer B 69 0 80 0 buffer A: 0.1 M NaOH buffer B: 0.1 M NaOH; 0.5 M In acetate The peaks were identified on a standard oligosaccharide complex type N-sugars chromatogram (Oxford Glyco Systems) and verified by enzymatic digestion of the EPO oligosaccharides with the enzyme endo-p-galactosidase and fucosidase and subsequent analysis in the Dionex system. The percentages of the biantennary, triantennary and tetraantenary structures were calculated by means of the peak areas representing the corresponding N-sugar structure relative to the total peak area (sum of the peak areas of the biantenary, triantennary and tetraantenary structures). CHO cell-derived EPO had a content of 4.2% biannaryary carbohydrate structures, 22.3% triannary carbohydrate structures, and 73.5% tetrahanaryary carbohydrate structures (CH03). and a content of 8.6% tetraantenary carbohydrate structures in the CHO 1 preparation and 78.6% in CHO 2. The contents of biantenary / triantenary / tetraantenary structures found in EPO preparations from human cell lines were 5.8 / 8.8 / 85., ... 4.% for HeLa 1, 5.1 / 12.7 / 82.2% for HeLa 2, 4.1 / 17.7 / 78.2% for HeLa 3, 10.1 / 19.2 / 70.6% for HeLa 4 and 12.6 / 25.4 / 62% for HeLa 5 (see also example 11).

Example 7: Determination of Average N-Acetyl Lactosamine Units and Average N-Acetyl Lactosamine Units (Repeats) The Total Number of N-Acetyl Lactosamine Units in the N-Chained Carb Structures of EPO (i.e., in the core carbohydrate structures plus repeats) was calculated from the peak areas of the chromatograms of the Example 6 experiments. The number of the mean content (n) of N-acetyl lactosamine units per chain of Carbohydrate was calculated as follows: η = Σ% (bi) x2 +% (tri) x 3 +% (tetra) x 4 +% (tri + lr) x 4+% (tetra + lr) x 5 +% (tri + 2r) x 5 +% (tetra + 2r) χβ where% (bi) = percentage of biantenary structures in relation to the total amount (100%) of N-linked carbohydrate structures% (tri) = percentage of structures tetraantennials without additional N-acetyl lactosamine units% (tetra) = percentage of tetraantenary structures without Additional N-acetyl lactosamine% (tri + 1r) = percentage of triantennary structures with 1 additional unit of N-acetyl lactosamine% (tetra + 1r) = percentage of tetraantennial structures with 1 additional unit of N-acetyl lactosamine% (tri + 2r) = percentage of triantennary structures with 2 additional units of N-acetyl lactosamine% (tetra + 2r) = percentage of tetraantennial structures with 2 additional units of N-acetyl lactosamine In the case of CHO cell-derived EPO, an average number of 4.3 (CHO 1), 4.4 (CHO 2) and 4.2 (CHO 3) N-acetyl lactosamine units per carbohydrate chain were found. In EPO preparations from human cells, a number of N-acetyl lactosamine units of 4.0 (HeLa 1), 4.0 (HeLa 2), 3.9 (HeLa 3), 3.75 (HeLa 4) and 3.6 (HeLa 5) (see also example 11).

Due to the fact that EPO contains 3 N-linked sugar structures, the total number of N-acetyl lactosamine units is three times higher. With reference to total EPO glycosylation, the number of N-acetyl lactosamine units in EPO from CHO cells is therefore 12.9 (CHO 1), 13.2 (CHO 2) and 12.6 (CHO). 3). In human cell EPO preparations, 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 N-acetyl lactosamine units per carbohydrate structure multiplied by the respective sialic acid content produced 55.5 (CHO 1), 52 (CHO 2) and 49.3 (CHO 3) values for EPO. from CHO cells. In the case of EPO preparations from 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).

With reference to total EPO glycosylation (3 N-linked carbohydrate structures) the product of the number of N-acetyl lactosamine units multiplied by the respective sialic acid content is for EPO from CHO 166.5 (CHO 1) cells. , 156 (CHO 2), and 147.9 (CHO 3), respectively. In EPO preparations from 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.

Another important parameter is the amount of N-acetyl lactosamine units that can be attached to the core carbohydrate structures, as. called repetitions (see, for example, Figure 1). The repetition content is specified as the percentage of carbohydrate structures that contain repetitions relative to the sum of all N-linked carbohydrate structures (bi + tri + tetra = 100%).

This repeat ratio may differ in EPO preparations from CHO cells and from human cells. Thus, repetition percentages of 39.6% (CHO 1), 51% (CHO 2) and 36.8% (CHO 3) were determined for preparations from CHO cells. Repetition percentages of 18% (HeLa 1), 16.5% (HeLa 2) 14.0% (HeLa 3), 12.2% (HeLa 4) and 9.8% (HeLa 5) were determined for the preparations from human cells (see example 11).

Example 8: Influence of EPO biological activity by control and feeding according to requirements Cultures were performed as repeated feeding batch processes as required (repeated feeding batch) at a temperature of 36.5 ° C. For this, whey-free protein-poor culture medium was placed in a stirring fermenter (total operating volume: 10 L) and inoculated once with an inoculum culture. Cell density after inoculation was in the range of 3 ± 1 x 10 5 withdrawing cells / ml.

After a growth phase of 144 ± 24 hours, part of the culture broth was harvested. The remaining broth remained in the fermenter and represented the inoculum for the next growth phase; For this purpose, the fermenter was refilled with fresh medium for the operating volume. The culture supernatant containing the EPO was obtained by centrifuging the fermentation culture.

Nutrient solution was fed continuously to the culture during the growth phase. For this purpose, a storage vessel containing nutrient solution was attached to the fermenter. The nutrient solution contained amino acids, vitamins, insulin, waste elements, salts, glutamine and carbohydrates. Two fermentations were performed as follows: In fermentation A the nutrient solution contained D - (+) - glucose as sugar and in fermentation B the sugars were D - (+) - glucose, D - (+) - galactose. and D - (+) - mannose. The mass ratio of glutamine to sugars was 1: 2.2: 3.6: 6 in fermentation B. The concentration of individual sugars in the nutrient solution was between 7.2 and 18 g / 1. Glutamine concentration in the culture was analyzed periodically in fermentation B and consumption was calculated. The momentary volume flow of the nutrient solution has been harmonized with the cells requirement for nutrients. In fermentation A, glutamine concentration was not used as a controlled variable. The nutrient solution in fermentation B contained a mixture of D - (+) - glucose, D - (+) - galactose and D - (+) - mannose sugars in a 2: 3: 5 mass ratio. The concentration of all sugars in the fermenter was maintained in the range of 2 to 6 g / 1 during culture by corresponding feeding. Cell density changed during growth to more than 20x10 5 cells / ml, typically 30 ± 10x10 5 cells / ml by the time of harvest. At the time of collection, the EPO concentration was typically 40 + 10 mg / 1. The concentration of human erythropoietin was determined, for example, by ELISA in the culture broths collected. A percentage distribution of the isoforms of this protein that occurred was / for example determined by separation with capillary zone electrophoresis (CZE). The table 1 shows a comparison of the distribution of EPO isoforms between a fermentation feed A with a glucose-containing nutrient solution and a fermentation feed B with a glucose-mannose-galactose-containing nutrient solution in a controlled and guided manner. requirements. The EPO isoform contents in fermentation B were calculated as percentages of the corresponding fermentation A isoforms. The latter were each standardized to 100%. <The data demonstrate that the desired higher glycosylated EPO isoforms 2-4 are present at a substantially higher proportion during fermentation compared to fermentation A.

Table 1 Name of isofor- 1 2 3 4 5 6 7 δ I

[ma at CZE f l l l l m \ r% i m i m ■ m e% i t% ij Fermentation A: n.d. 100 100 100 100 100 100 100 I

feeding with I

, Glycosis j: j _____ I

Fermentation B: n.d. 136 140 115 102 91 76 55 I

power with I

glucose, mannose I and galactose as 1 I quartet II II 1 n.d. = not determined as the value is below the detection limit. The isoform pattern obtained with the feed was reproducible in four successive harvests from a controlled feed and demand-driven fermentation of the nutrient solution.

Example 9: Influence of EPO biological activity by changing culture temperature The procedure was as described in Example 8 (fermentation B) in a demand-oriented controlled batch division feeding process, except that the fermentation temperature The titer was 35.0 ° C instead of 36.5 ° C and the fermentation was performed on a 1000 scale. Table 2 shows a comparison of the distribution of EPO isoforms between a fermentation C at 36.5 ° C and a fermentation D at 35.0 ° C, each with controlled feeding of a nutrient solution. The contents of EPO isoforms in fermentation D were calculated as percentages of the corresponding fermentation C isoforms. Each of the latter has been standardized to 100%. The data show that acid EPO isoforms 2 to 4 can be considerably increased by decreasing temperature.

Table 2 ______________________________________________ n.d. = not determined as the value is below the detection limit.

Example 10: Influence of EPO biological activity by changing carbohydrate composition in medium The following process shows that it is possible to change the quality of human erythropoietin by changing carbohydrate feed in the medium.

Two variants of the process described above (called fermentation E and fermentation F below) which differ in the composition of the medium used are set forth.

In both preparations the culture medium formulation is based on the modified eRDF medium. No serum has been used but instead recombinant insulin (protein additive only) and other supplements (eg selenite, putrescine, hydrocortisone, iron sulphate) which are usually used in serum-free or protein-free medium. The nutrient feed solution is also based on modified eRDF medium but does not contain KCl, Na2HP04 and NaCl salts. The main difference between fermentation E and F is the addition of various monosaccharides to the feed medium.

Fermentation E: The usual D- (+) -glucose sugar was used for E fermentation. The initial concentration was 3 g / 1. By properly feeding the glucose-containing nutrient solution, the glucose concentration in the culture broth was maintained at 3 ± 0.5 g / 1 throughout the culture. The culture period was typically 100 ± 20 h. EPO concentration was typically 40 ± 10 mg / 1 at the time of collection.

Fermentation F: In addition to D - (+) - glucose, D- (+) -galactose and D- (+) -manose sugars were added in a mass ratio of about 1: 2: 3 to the feed medium for Fermentation F. During cultivation, the concentration of all sugars was kept within a range of 0.25 g / l to 3.5 g / l by appropriate feeding. The culture period for this growth was typically 100 ± 20 hours. The EPO concentration at harvest period was typically 40 ± 10 mg / l. Erythropoietin was purified from culture supernatants. The purification procedure was designed (cf. example 2) such that the distribution of the relevant glycoprotein isoforms was not influenced. The isoform distribution of purified erythropoietin was determined as described above.

The carbohydrate structures of human erythropoietin isoforms and their distribution in harvested culture supernatants differed in fermentation E and fermentation F. Fermentation E had a considerably higher proportion of isoforms 2, 3 and 4 compared to fermentation. F. These differences are caused by the feeding of the mannose and galactose monosaccharides (cf. Figure 2). The biological activity determined by the normal rat test (example 4) relates to the distribution and carbohydrate structures of the EPO isoforms (Figure 3). The carbohydrate structures of EPO preparations that were obtained from culture supernatants E and F were examined with CZE and HPAEC analyzes. The content of the biostructures, biostructures and tetrastructures (antennas), the content of N-acetyl lactosamine (LE) units, the sialic acid (SA) content and the LE and SA product of the two EPO preparations are exposed. in Table 3.

Table 11 Example 11: Correlation of specific activity and carbohydrate structures In this example, investigations of examples on the biological activity dependence of individual EPO isoforms on carbohydrate structures are summarized. For this, isoforms (IF) from various sources of EPO were isolated and compared (different batches of EPO from CHO cells and human cells). 11.1 Isolation of individual EPO isoforms by isoelectric focusing (IEF) and Western blot A) Procedure for IEF gel electrophoresis and electroblot In order to isolate individual isoforms into pure form, an EPO solution composed of a mixture of several isoforms was desalted in ultra-free centrifuge units and concentrated (5-10 mg / ml). 350-1000μg of this solution were applied to an IEF polyacrylamide gel (Servalyt Precotes, pH 3-5, 300 μιη, 125 x 125 mm) (in 5-10 bands containing 70-100 μg EPO). per track). IEF was performed at 2500 V for 3.5 h at 5 ° C; subsequently, the gel was smudged with nitrocellulose (wet smear in tris / glycine buffer containing methanol but without SDS for 3 hours at 200 mA). After the smudging process, the gel was removed and the nitrocellulose membrane was stained with Ponceu S. The stained isoforms were cut and completely disassembled again with H2 O or TBS buffer (100 mM Tris, pH 7.4; 150 mM NacL). B) Extraction of isoforms from the membrane The disassembled nitrocellulose strips containing the respective isoforms were placed in 2 ml Eppendorf vessels (corresponding to 3-4 strips of the IEF gel), 1.5 ml acetone was added and the nitrocellulose was added. was dissolved by vortexing. It was incubated overnight at -20 ° C to optimally precipitate the EPO. Subsequently, the EPO-containing precipitate was isolated for 10 min in a benchtop 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. The EPO precipitate was subsequently dissolved in 20 mM Na-phosphate buffer, pH 7.2 which contained 0.01% Tween 20 and stored at -20 ° C until further analysis. C) Isolation of Isoforms from Pre-Fractionated EPO Solutions Individual isoforms were isolated as described in A) and B) with the qualification that the initial EPO solutions contained 7-8 instead of only 3-4 isoforms. . The starting material were EPO fractions that had been isolated by DE (anion exchanger) chromatography. These fractions contained only 3-4 isoforms (e.g., isoform 6-8 or isoform 1-4). In order to isolate the isoform packages, a suitable chromatography column was filled with 1-2 ml DEAE-Sepharose ff per 10 mg of applied EPO and regenerated with 0.5 M NaOH. Subsequently, the column was first balanced with 2 CV of neutralization damper and then with at least 5 CV of balance damper.

A purified EPO preparation comprising 8 isoforms was absorbed at a temperature of 5 ± 4 ° C and a flow rate of up to 15 CV / h. The column was then washed with 2 to 3 CV of equilibration buffer and subsequently rinsed with wash buffer until the pH value was 5.0 (about 5 CV). Several isoform packages were eluted by increasing the NaCl concentration in the elution buffer in 10 mM steps starting at 20 mM NaCl. The basic isoforms weakly agglutinated to the ion exchanger and correspondingly eluted at low ionic intensities, the acidic forms were eluted at higher NaCl concentrations up to 70 mM NaCl. The amount of isoforms eluted under a given concentration of NaCl strongly depends on the starting material and elution volume. As a rule, elution continued in the individual steps until OD 280 decreased to about 50% of the maximum value at this NaCl concentration. This corresponds to between 15 and 40 CV. Additional fractionation of eluted isoform packages within a NaCl concentration resulted in further separation of the isoforms. The column travel speed was up to 15 CV / hour.

Neutralization Damper: 100 mM Na / Phosphate K, pH 7.5 ± 0.2 Equilibrium Damper: 10 mM Na / phosphate K, pH 7.5 ± 0.2 Washing Damper: 30 mM NaAc / HAc, pH 5 0.0 ± 0.2 Elution Buffer: 10 mM NaAc / HAc, pH 5.0 ± 0.2, 20 mM NaCl, or stepwise increased concentration from 10 mM to 70 mM NaCl Individual pure isoforms were isolated from the isoform packages obtained in this manner by purification as described in A) and B).

Pure isoforms (IF) obtained from A-C were numbered according to their isoelectric point (pi) from acid to basic. Isoform 2 (IF2) is the most strongly acidic isolated isoform with the lowest pi. Isoform 8 is the most strongly acidic with the highest pi. Isoform 2 was the lowest pi isoform that could be isolated in adequate amounts relative to the starting mixture. Only 1-2% of isoform 1 was present in the starting mixture so that adequate amounts could not be obtained for complete analysis.

The following analyzes were performed to characterize the pure isoforms: determination of quantity and yield by RP-HPLC determination of purity and identity by capillary electrophoresis and isoelectric focusing. The yield of the individual isoforms was generally between 20% and 30% of the isoform used in the starting mixture. The purity of the isoforms was usually> 90%, most of the flow rates> 94%. 11.2 Results The following results were obtained for the purified isoforms (IF): - Relative distribution of N-linked carbohydrate structures (ratio of biantenary, triantenary and tetraantenary structures to total glycosylation) and repetition content - biological activity in normal rat test - sialic acid content.

These determinations were performed essentially by the procedures described above. The sialic acid content of the isolated isoforms was not determined separately for each individual isoform preparation / but was performed on 1-3 preparations as an example for each of EPO isoforms 2-8 from CHO cells or isoforms 2-6. EPO from human cells.

The complete / round number sialic acid values of each isoform were used to calculate the product of the N-acetyl lactosamine unit content (LE value) and the sialic acid (SA) content.

These rounded SA values were as follows for EPO from CHO cells and human cells: 14 (IF2), 13 (IF3), 12 (IF4), 1.1 .. (IF5), 10 (IF6), 9 (IF7 ) and 8 (IF8). Table 4 contains data on the correlation between specific activity and carbohydrate structures of the various EPO preparations from CHO cells (CHO 1, CHO 2, and CHO 3) as well as from human cells (HeLa 1 through 5). . The table shows the correlation between biological activity and the average total number of N-acetyl lactosamine (LE) units in the EPO molecule, the average sialic acid (SA) content, as well as the LE x SA product. Table 5 contains data on the correlation between biological activity and the average total number of N-acetyl lactosamine (LE) units in the EPO molecule, the average sialic acid (SA) content and the isoform LE x SA product. isolated from a batch of unfractionated EPO from CHO cells. Table 6 contains a comparison of various preparations (A and B) of an isoform (IF2 or IF5) which was isolated from various fractions of a pre-fractionated EPO batch from CHQ cells, i.e. 2 prepared ( A and B) of isoforms 2 and 5 were analyzed in each case. Prefractionation was performed by means of a DE anion exchanger as described in example 11.1C. The two preparations A and B of the isoforms IF2 and IF5 were isolated for different fractions of the DE column (IF2 from fractions 5 and 6 and IF5 from fractions 2 and 3). Fraction 5 or 2 from which IF2 or IF5 was isolated, respectively, was eluted earlier (under a lower salt concentration) from the DE-Sepharose column than fractions 6 or 3 from which respectively isolate IF2 / B or IF5 / B. However, preparations A and B of isoforms 2 or 5, respectively, did not differ in their properties on subsequent isoelectric focusing or capillary electrophoresis, that is, both IF2 or IF5 preparations had the same sialic acid content. Nevertheless, it was surprisingly found that the isoforms from preparation A had significantly higher biological activity than the corresponding isoforms from preparation B due to their higher LE value and higher repeating structure content. The dependence described in Table 6 for isoform biological activity on the total number of N-acetyl lactosamine units contained in the EPO molecule in the same sialic acid content was not only observed for isoforms 2 and 5, but also for other isoforms. Table 7 compares the corresponding isoforms from various sources of EPO (CHO cells or human HeLa S3 cells). Likewise, in this case a correlation between biological activity and the LE x SA value was observed.

Therefore, in all tables a correlation can be observed between the product of the number of units of N-acetyl lactosamine (LE) and the sialic acid (SA) content and biological activity. The high value of LE x SA product is always associated with a high biological activity.

Table 4 LE: N-acetyl lactosamine SA units: sialic acid content of EPO preparation 1: Percentage of all sugar structures with additional LE extensions in relation to the number of total sugar structures (bi + tri + tetra = 100%) Table 5 LE: N-acetyl lactosamine units (calculated as in example 7) SA: sialic acid content of the respective isoform 1: Percentage of all sugar structures with additional LE extensions relative to the number of total sugar structures (bi + tri + tetra = 100%) Table 6 LE: N-acetyl lactosamine units (calculated as in example 7) SA: sialic acid content of respective isoform 1: Percentage of all sugar structures with additional LE extensions in relation to the number of total sugar structures (bi + tri + tetra = 100%) Table 7 LE: N-acetyl lactosamine units (calculated as in example 7) SA: isoform sialic acid content respective 1: percentage of all sugar structures with additional LE extensions in relation to the number of total sugar structures (bi + tri + tetra = 100%) The dependence of EPO biological activity on the proportion of N-chain linked N-acetyl-linked carbohydrate structures Additional lactosamine (repeats are shown in Figure 4 using individual isoforms as an example. The isoforms were isolated from EPO preparations containing different proportions of repeating carbohydrate structures (about 50% EPO 1, about 40% EPO 2 and about 15% repeating structures of EPO 3). ). The biological activity of the corresponding isoforms (same sialic acid content and approximately the same antenarity) decreases as the proportion of carbohydrate structures containing repeats decreases in the isoforms. This characteristic can be observed for isoform 2 to at least isoform 7.

Claims (10)

Process for producing an EPO composition characterized in that the EPO composition is obtained by at least one of the following steps: (a) selecting a suitable production cell which is capable of producing high proportion carbohydrate chains from N-acetyl lactosamine units, in particular CH01, CH02, CH03, HeLa 1, HeLa 2, HeLa 3, HeLa 4 and HeLa 5; (b) selecting suitable culture conditions for cell culture to produce carbohydrate chains with a high proportion of N-acetyl lactosamine units, in particular, adding a carbohydrate mixture containing glucose and mannose and galactose and cultivating the a temperature between 33 and 35.0 ° C; and (c) separating undesirable components from a known EPO molecule composition while enriching the carbohydrate chain-containing EPO molecules with a high proportion of N-acetyl lactosamine units, in particular by reverse phase chromatography and affinity chromatography; wherein the EPO composition is enriched with EPO isoforms IF2, IF3 and IF4 as compared to a composition produced by said cells grown at 36.5 ° C and is composed essentially of glycosylated EPO molecules containing a number of at least 4.3 N-acetyl lactosamine units with reference to an N-linked carbohydrate chain of an EPO molecule or at least 13.0 N-acetyl lactosamine units with reference to the total N-glycosylation of an EPO molecule wherein EPO molecules are the product of endogenous DNA expression in human cells and wherein the proportion of carbohydrate chains with N-acetyl lactosamine repeats is at least 10% of the total number of carbohydrate chains. N-linked, and wherein the EPO composition has a specific in vivo activity of at least 175,000 IU / mg protein. Process according to Claim 1, characterized in that step (b) comprises controlled addition according to nutrient requirements comprising at least one essential amino acid. Process according to Claim 2, characterized in that the nutrient requirements of cells are determined depending on the concentration of glutamine measured in the culture medium. Process according to either of claims 2 or 3, characterized in that the nutrients are added as needed throughout the growth phase of the cells. Process according to Claim 1, characterized in that step (c) comprises a reverse phase chromatography step under a pH value in the range of 6 to 8. Process according to Claim 5, characterized in that acetonitrile, ethanol or isopropanol are used as eluent. Process according to Claim 1, characterized in that step (c) comprises an affinity chromatography step using triazine dyes. Process according to Claim 1, characterized in that step (c) comprises an affinity chromatography step using lecithins. A process for increasing the specific activity of an EPO composition characterized in that the EPO molecules are enriched in a composition such as that obtained by the process of claim 1 having a high value for the product in the proportion of N-repeats. acetyl lactosamine and in proportion to tetraanthenary carbohydrate structures, and are enriched such that the composition is composed essentially of glycosylated EPO molecules containing a number of at least 4.3 N-acetyl lactosamine units with reference to one chain. N-linked carbohydrate content of an EPO molecule or at least 13.0 N-acetyl lactosamine units with reference to the total N-glycosylation of an EPO molecule, where EPO molecules are the product of a DNA expression in human cells and wherein the proportion of carbohydrate chains with N-acetyl lactosamine repeats is at least 10% of the total number of c N-linked carbohydrate diseases, and wherein the EPO composition has a specific in vivo activity of at least 175,000 IU / mg protein. Process according to claim 9, characterized in that it is enriched in the case of human cell EPO with a product value in the average ratio of N-acetyl lactosamine repeats to the total number of carbohydrate chains. multiplied by the average proportion of tetraanthenary carbohydrate structures of at least 800.