WO2011063195A2

WO2011063195A2 - Purification of modified cytokines

Info

Publication number: WO2011063195A2
Application number: PCT/US2010/057364
Authority: WO
Inventors: Darshan Koticha; Aniket B. Chaudhari; Kishore Jahagirdar
Original assignee: Dr. Reddy's Laboratories Ltd.; Dr. Reddy's Laboratories, Inc.
Priority date: 2009-11-20
Filing date: 2010-11-19
Publication date: 2011-05-26
Also published as: WO2011063195A3

Abstract

Methods for purifying modified cytokines. A process includes the use of a cation exchange chromatographic technique for the purification of a desired cytokine. The purified cytokines can be used in therapeutic compositions.

Description

PURIFICATION OF MODIFIED CYTOKINES

INTRODUCTION

Aspects of the present invention relate to processes for separating low pi isoforms of modified cytokines from mixtures of isoforms.

Erythropoiesis stimulating proteins (ESPs), such as erythropoietin and analogs of erythropoietin, are glycoprotein hormones that are the principal homeostatic regulators of red blood cell production. Though natural erythropoietin is produced by the kidney, its large scale production for therapeutic purposes is achieved by recombinant DNA methods. Purified recombinant human

erythropoietin (rHuEPO) and its analog darbepoetin alpha are used in the treatment of medical indications associated with inadequate red blood cell supply, such as anemia, chronic renal failure perisurgery, as well in the treatment of side effects associated with human immunodeficiency virus and hepatitis C virus infections, and cancer chemotherapy.

Variability in the sugar moieties that make up the glycan chains of human erythropoietin and its analogue darbepoetin alpha contribute to the

microheterogenity in the isoform compositions of these glycoproteins. This, in turn, has profound effects on the physico-chemical properties and biological activities of erythropoietin and darbepoetin alpha compositions.

Negatively charged sialic acid residues typically cap the ends of a glycan chain of most glycoproteins. Human erythropoietin and darbepoetin alpha that is expressed in Chinese hamster ovary (CHO) cells exhibit variable degrees of glycosylation and sialylation. Variability in the glycan chains, in particular in the sialic acid content, results in erythropoietin and darbepoetin alpha isoforms that differ in their overall charge and isoelectric point (Rush RS, et al., Analytical Chemistry 1995, 67:1442-52). Thus erythropoietin and darbepoetin alpha isoforms with higher sialic content have lower isoelectric point (pi), than those with lower sialic acid content. Compositions enriched in low pi isoforms exhibit higher bioactivity then those with higher content of high pi isoforms (M. Takeuchi et al., Proceedings of the National Academy of Sciences, Vol. 86(20), pp. 7819-22, 1989; Zanette et al., 2003 Journal of Biotechnology, Vol. 101 (3), pp.275-287, 2003). Variability in the extent of sialylation, in turn, affects the in vivo activity of erythropoietin and darbepoetin compositions. J. C. Egrie et al., British Journal of Cancer, Vol. 84, pp. 3-10, 2001 and J. C. Egrie et al., Glycoconugate Journal, Vol. 10, pp. 263-269, 1993 report a direct correlation between sialic acid content of human erythropoietin and its serum half-life and physiological activity. Isoforms with more sialic acid residues exhibit slower clearance in vivo and higher physiological activity. Also Imai et al. European Journal of Biochemistry, Vol. 194, pp. 457-462, 1990 report that erythropoietin isoforms with high sialic acid content exhibit higher specific activity than those with lower sialic acid content. Likewise, darbepoetin alpha, the erythropoietin analogue that has 5 N-linked carbohydrate chains with up to 22 sialic acid residues as compared to 3 N-linked carbohydrate chains and 14 sialic acid residues in human erythropoietin, exhibits three-fold longer serum half-life and higher in vivo activity than human erythropoietin (J. C. Egrie et al., British Journal of Cancer, Vol. 84, pp. 3-10, 2001 ; and S. Elliot et.al. Blood, Vol. 96, 8 2a, 2000). Also, as in the case of human erythropoietin, low pi isoforms of darbepoetin alpha exhibit higher specific activity than isoforms with higher pi. Hence darbepoetin alpha isoforms with lower pi are of greater therapeutic value. For example, the product sold as Aranesp® by Amgen, the approved and marketed form of darbepoetin alpha, comprises essentially low pi isoforms of the protein, having a pi range of 3-3.9 (Francoise Lasne et al.,

Analytical Biochemistry, Vol. 31 1 , pp. 119-126, 2002).

However, darbepoetin alpha is expressed in cell culture as a

heterogeneous mixture of isoforms in the pi range of about 3 to 8. Hence, there is a need for efficient and effective methods for the separation of low pi isoforms from higher pi isoforms of darbepoetin alpha.

The literature discloses various methods for the purification of

erythropoietin and analogues of erythropoietin.

International Application Publication No. WO 00/27869 discloses a process for purifying erythropoietin, consisting of a sequence of hydrophobic interaction, anion exchange, cation exchange, and size exclusion chromatographic steps.

International Application Publication No. WO 03/045996 discloses chromatographic purification of recombinant human erythropoietin by reverse phase chromatography, anion exchange, and size exclusion chromatography. N. Inoue et al., Biol, and Pharm. Bulletin, Vol. 17(2), pp. 180-184, 1994 describe a method for the purification of human erythropoietin from urine that involves ion exchange, gel permeation, affinity chromatography, and reverse phase chromatography.

International Application Publication No. WO 86/07594 discloses a method of purifying erythropoietin from a fluid, comprising the steps of subjecting the fluid to reverse phase liquid chromatographic separation involving an immobilized C4 or C6 resin, followed by a selective elution of bound erythropoietin from the resin with an aqueous 50 to 80 percent ethanol solution, at a pH of from about 4.5 to 8.0, and isolating erythropoietin containing fractions of the eluate.

International Application Publication No. WO 96/35718 describes a preparation of protein having erythropoietin activity that is obtained from serum- free cell culture medium, using a purification process involving dye

chromatography, hydrophobic chromatography, chromatography on

hydroxyapatite, hydrophobic chromatography, and anion exchange

chromatography.

However the purification methods described above involve complex series of chromatographic steps. Given the enormous therapeutic importance of erythropoiesis stimulating proteins in general, and darbepoetin alpha in particular, simple and efficient methods for the separation and purification of low pi isoforms of erythropoiesis stimulating proteins are desired.

SUMMARY

Aspects of the present disclosure provide processes for the separation and purification of low pi isoforms of darbepoetin alpha from higher pi isoforms, embodiments comprising at least one cation exchange chromatographic step in the bind-elute mode and one cation exchange chromatographic step in the flow- through mode.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is an illustration of a chromatogram from the procedure of Example 3 Fig. 2 is an isoelectric focusing gel from cation exchange chromatography performed as described in Example 3 Fig. 3 is an illustration of a chromatogram from the procedure of Example 4 Fig. 4 is an isoelectric focusing gel of samples of the load and eluate fractions from cation exchange chromatography performed as described in

Example 4

Fig. 5 is an illustration of a chromatogram from the procedure of Example 5

Fig. 6 is an isoelectric focusing gel from cation exchange chromatography performed as described in Example 5

DETAILED DESCRIPTION

Recombinant glycoproteins expressed in CHO cells exhibit variable glycosylation and sialylation. Erythropoiesis-stimulating proteins, such as human erythropoietin and analogs of erythropoietin are glycoprotein hormones that are the principle homeostatic regulators of red blood cell production and used in the treatment of anemia caused by chronic renal failure.

The present invention provides novel and efficient methods for the isolation of low pi isoforms of darbepoetin alpha from a mixture comprising a

heterogeneous mixture of low and high pi isoforms.

In embodiments, the invention provides a method for the separation of low pi isoforms of darbepoetin alpha from solutions comprising low and high pi isoforms, the solutions having pH values that are equal to or greater than the pi of the low pi isoforms, using cation exchange chromatography including:

a) loading a solution onto a cation exchange resin to bind low pi isoforms to the cation exchange resin; and

b) eluting the low pi isoforms with an elution buffer at pH values less than the pH of the loaded solution, and increasing the conductivity of the elution buffer during the elution.

The loading solution may, for example, have pH values about 3.7 to about 4.2, or about 3.8 to about 4. The conductivities of the loading solution may, for example, be less than or equal to 4 mS/cm, or about 0.5 mS/cm to about 3 mS/cm, or about 1.5 mS/cm. The elution buffer may have pH values less than about 3.7, or between about 3 and about 3.5, or about 3.3. The conductivities of the elution buffer may be increased, in a gradient or in a step-wise manner, from about 0.4 mS /cm to about 20 mS/cm. ln embodiments, the cation exchange chromatography step is either preceded by or followed by a second cation exchange chromatography step, performed in the flow-through mode.

In embodiments, the above two cation exchange chromatographic steps may be preceded by or followed by an anion exchange or mixed mode

chromatographic step.

In embodiments, the above two cation exchange chromatographic steps may be preceded by and followed by anion exchange or mixed mode

chromatographic steps.

In embodiments, the invention provides methods for separating low pi isoforms of darbepoetin alpha from a solution comprising low and high pi isoforms, the solution having pH values equal to or greater than the pi of the low pi isoforms, using cation exchange chromatography comprising:

a) loading the solution onto a cation exchange resin to bind the low pi isoforms to the cation exchange resin; and

b) eluting the low pi isoforms with an elution buffer having pH values about the same as the pH of the load solution and increasing the conductivity of the elution buffer during the elution.

The loading solution may have pH values about 3.7 to about 4.2, or about pH 4. The conductivities of the loading solution may be less than or equal to 4 mS/cm, or about 0.5 mS/cm to about 3 mS/cm, or about 1.5 mS/cm. The elution buffer have pH values about 3.7 to about 4.2, or about pH 4. The conductivities of the elution buffer may be increased, in a gradient or in a step-wise manner, from about 1.5 mS /cm to about 20 mS/cm.

In embodiments, the cation exchange chromatography is either preceded or followed by a second cation exchange chromatography, performed in the flow- through mode.

In embodiments, the above two cation exchange chromatographic steps may be followed by an anion exchange or a mixed mode chromatographic step.

In embodiments, the above two cation exchange chromatographic steps may be preceded and followed by anion exchange or mixed mode

chromatographic steps. ln embodiments, the low pi isoforms are isolated using a process comprising at least two cation exchange chromatography steps, one of which is performed in the flow-through mode, and the other is performed in the bind-elute mode. The flow-through and bind-elute modes of chromatography may be performed in either order, with the cation exchange in the bind-elute mode either preceding or following the cation exchange in the flow-through mode.

In embodiments, the two cation exchange chromatographic steps are carried out in succession, that is, without any intervening chromatographic steps.

In embodiments, the two cation exchange chromatographic steps may be preceded by an anion exchange or a mixed mode chromatographic step.

In embodiments, the two cation exchange chromatographic steps may be followed by an anion exchange or a mixed mode chromatographic step.

In embodiments, the two cation exchange chromatographic steps may be preceded by and followed by anion exchange or mixed mode chromatographic steps.

The embodiments mentioned herein may optionally comprise tangential flow filtration, concentration, diafiltration, buffer exchange, or ultrafiltration steps, between the chromatographic steps.

The embodiments mentioned here may include one or more viral inactivation, sterile filtration, or viral filtration steps.

Anion exchange chromatography mentioned in the embodiments may be carried out using any weak or strong anion exchange chromatographic resin, or a membrane that can function as a weak or a strong anion exchanger. In

embodiments, a Q Sepharose chromatographic resin (e.g., Q Sepharose® Fast Flow, a strong anion exchanger with a quaternary amine group attached to a highly cross-linked agarose base matrix, from GE Healthcare Life Sciences) or membrane may be used.

Cation exchange chromatographic steps mentioned in the embodiments may be carried out using any weak or strong cation exchange chromatographic resin or a membrane that can function as a weak or a strong cation exchanger. In embodiments, a weak cation exchange column, such as CM Sepharose (CM Sepharose® Fast Flow, a weak cation exchanger with a carboxymethyl group attached to cross-linked agarose, 6% base matrix, from GE Healthcare Life Sciences) or a resin or a membrane with a similar function may be used.

Mixed mode chromatography as mentioned in the embodiments refers to chromatographic systems, in which more than one chromatographic separation principle, for example ionic and hydrophobic, are at work. Thus a mixed mode resin refers to a solid phase with cationic or anionic and hydrophobic moieties or ligands. The term "solid phase" is used to mean any non-aqueous matrix. Mixed- mode chromatographic ligands show either hydrophobic or charged interactions or both. For example, a mixed-mode chromatographic column used in embodiments is a Capto™ adhere column (Capto™ adhere is a multimodal strong anion exchanger anion exchanger with a N-benzyl-N-methylethanolamine group attached to highly cross-linked agarose base matrix, from GE Healthcare Life Sciences) or any mixed mode resin which functions in a similar manner.

The buffering agents used for making a buffer solution may comprise sodium acetate, sodium citrate, or a phosphate buffer, and/or other salts or derivatives.

The term "isoform," as used herein, refers to proteins with similar amino acid sequences that differ with respect to charge and therefore isoelectric point, as a result of differences in glycosylation, acylation, deamidation, or sulphation.

The "isoelectric point" or "pi" is the pH value at which a particular molecule or surface carries no net electrical charge. The pi of a polypeptide refers to the pH at which the polypeptide's positive charge balances its negative charge. The pi can be estimated by various methods known to those skilled in the art, e.g., from the net charge of the amino acid and/or sialic acid residues on the polypeptide or by using isoelectric focusing, chromatofocusing, etc.

Low pi isoforms refer to isoforms having pi of about 4 or less.

"Flow-through mode" as used herein refers to processes wherein the desired protein is not bound to a chromatographic resin but is instead obtained in the unbound or "flow-through" fraction during loading or post loading washes of a chromatography resin. The desired protein in the flow-through fluid may be collected in various fractions and pooled together, or may be collected as a single fraction. Certain specific aspects and embodiments are more fully described in the following examples. These examples should not, however, be construed to limit the scope of the invention as defined by the appended claims. EXAMPLE 1

Protein Expression and Harvest.

Chinese hamster ovary (CHO) production cell lines are made by

transduction of the CHO-S parental cell line with a retrovector from the

darbepoetin alpha expression vector. The pooled population of cells is diluted to very low cell density (e.g., 1-3 viable cells/200 μΙ_ of medium) and plated in 96 well microtiter plates to establish clonal cell lines that originate from single cells.

Clones are screened for darbepoetin alpha production and clones with high productivity are selected for expression.

The cells expressing darbepoetin alpha are expanded from the master cell bank in four stages of spinners and one stage of seed reactor before, being inoculated into a production reactor.

PF CHO medium is used for culturing the cells in spinners in order to obtain good cell growth and high viability. The PF-CHO medium contains, per liter of medium: PF-CHO main powder 6.0 g, PF-CHO base powder 10.4 g, L-glutamine 0.58 g, Pluronic F-68 (block copolymer nonionic surfactant, mol. wt. 8400) 1.0 g, and sodium bicarbonate 2.0 g. The pH of the medium is adjusted to 7 before inoculation. Cells from the master cell bank are inoculated in a spinner bottle containing PF-CHO medium at an initial cell count of 0.2 million cells/mL. The spinner bottles are incubated in a 5% CO₂ incubator maintained at 37°C. After 72 hours of incubation, cells are inoculated in a 6 L seed reactor containing 4 L of SFM-6(1) medium. The SFM-6(1) medium contains, "DMEM/F-12" (GIBCO®, media about 15.6 g/L, comprising the following inorganic salts: Calcium Chloride, Cupric sulfate, Potassium Chloride, Magnesium Sulfate or Chloride, Sodium Chloride, Sodium Dihydrogen Phosphate, Sodium Bicarbonate, Zinc sulfate; the following amino acids L-Alanine, L-Arginine, L-Asparagine, L-Aspartic acid, L- Cysteine, L-Glutamic acid, L-Glutamine, Glycine, L-Histidine, L-lsoleucine, L- Leucine, L-Lysine, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L- Threonine, L-Tryptophan, L-Tyrosine, L-Valine; the following lipids and vitamins: Biotin, D-Calcium-Pantothenate, Choline Chloride, Folic Acid, myo-lnositol, Niacinamide, Nicotinamide, Pyridoxine, Riboflavin, Thiamine, Vitamin B12

(cobalamin), Thymidine, Linoleic Acid, Lipoic Acid; and other components including D-Glucose, Phenol Red, Hypoxanthine, Sodium Pyruvate, Putrescine (1 ,4-diamino butane), and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)), amino acids, insulin, vitamins, trace elements, ferric ammonium citrate - 2.5 mg, plant peptone, bicarbonate, and fructose. In the seed reactor, the pH is maintained at 7 and temperature of the culture is controlled at 37°C. Dissolved oxygen is maintained at 40% by controlling agitation and aeration. After 72 hours, the culture is aseptically harvested and cells are transferred to a 10 L production reactor containing 9 L of SFM-6(2) medium (SFM-6(1), ferric ammonium citrate - 10 mg), at an initial cell density of 0.2 million cells/mL. The culture is harvested after 12 days to collect the supernatant containing the desired product. EXAMPLE 2

Anion Exchange Chromatography or Capto™ adhere Chromatography

After clarification of the crude extract from Example 1 , the clarified cell culture broth is concentrated and the conductivity is reduced by diafiltration (using a tangential flow filtration (TFF) with a molecular weight cut off of 30 kDa) using a 25 mM Tris, 60 mM NaCI buffer having pH 7.1. The concentrated cell culture broth is then loaded into a Q Sepharose column (400 ml_, XK 50/20) that has been pre- equilibrated with 5 column volumes (CV) of 25 mM Tris + 60 mM NaCI, pH 7.1 buffer. The column is then washed with 5 CV of the equilibration buffer (25 mM Tris, 60 mM NaCI, pH 7.1). This is followed by a low pH wash with 80 mM sodium acetate, 50 mM NaCI buffer, pH 4. Another wash with the equilibration buffer is performed. The desired protein that has been loaded onto the column is eluted with 25 mM Tris, 300 mM NaCI buffer having pH 7.1.

Alternatively, a Capto adhere N-benzyl-N-methylethanolamine ligand (mixed mode chromatographic column) can be used in place of a Q-Sepharose column. The Capto adhere column (400 ml_, XK 50/20) is pre-equilibrated with 5 CV of 20 mM phosphate, 60 mM NaCI, pH 7.1±0.2 buffer. The column is then washed with 5 CV of the equilibration buffer (20 mM phosphate, 60mM NaCI, pH 7.1 ±0.2). This is followed by a low pH wash with 80 mM sodium acetate, 120 mM NaCI buffer, pH 4. Another wash with the equilibration buffer is performed. The desired protein that is loaded onto the column is eluted with 20mM phosphate, 140-300 mM NaCI buffered at pH 7.1 ±0.2. EXAMPLE 3

Cation Exchange Chromatography

Eluate from Example 2 is concentrated and the conductivity and pH reduced by diafiltration by a TFF step using 100 mM sodium acetate buffer, pH 3.84, conductivity 1.5 mS/cm. This step acts as a buffer exchanging step wherein the pooled eluate of a Q Sepharose column is brought into the 100 mM sodium acetate buffer. The buffer exchanged sample is then loaded onto a CM- Sepharose column (20 ml, VL 11/21) that has been pre-equilibrated with 5 CV of 100 mM sodium acetate buffer, pH 3.84, conductivity 1.5 mS/cm. The column is washed with approximately 5 CV of equilibration buffer (100 mM sodium acetate buffer, pH 3.84) until the absorbance returns to baseline. The protein is eluted with 10 CV of 83.4 mM sodium acetate buffer with pH 3.3 and a conductivity of about 0.4 mS/cm. Fractions of 10 mL (0.5 CV) are collected in tubes containing 1 mL of 1 M Tris-HCI, pH 9. This adjusts the pH of the eluate to approximately 7. The elution is continued with 83.4 mM sodium acetate buffer, pH 3.3 at different conductivities (5 CV each) 1 mS/cm, 2mS/cm, 4 mS/cm, 8 mS/cm and 20 mS/cm.

Figure 1 is an illustration of a chromatogram from the procedure as described in this example. The line marked "COND" represents the step-wise increase in conductivity in mS/cm. Peaks A, B, C, D, E, and F represents the eluate obtained at conductivities 0.39 mS/cm, 1 mS/cm, 2 mS/cm, 4 mS/cm, 8 mS/cm, and 20.5 mS/cm, respectively.

Figure 2 is an isoelectric focusing gel of the cation exchange

chromatography performed as described in this example. Lane 1 is the loaded sample. Lane 2 corresponds to a fraction obtained by acid elution at pH 3.3 and conductivity 0.4mS/cm. Lanes 3-7 correspond to fractions obtained by acid elution at pH 3.3 and conductivities 1 mS/cm, 2 mS/cm, 4 mS/cm, 8 mS/cm, and 20 mS/cm, respectively. Lane 9 is an internal reference standard. EXAMPLE 4

Cation Exchange Chromatography

As an alternative to Example 3, the eluate from Example 2 may be subjected to cation exchange chromatography.

Eluate from Example 2 is concentrated and the conductivity and pH reduced by diafiltration by a TFF step using 100 mM sodium acetate buffer, pH 3.84, conductivity 1.5 mS/cm. This step acts as a buffer exchanging step wherein the pooled eluate of Q Sepharose column is brought into 100mM sodium acetate buffer of pH 3.84 conductivity 1.5 mS/cm. The buffer exchanged sample is then loaded onto a CM Sepharose column (20 ml_, VL 11/21) that has been pre- equilibrated with 5 CV of 100 mM sodium acetate buffer, pH 3.84, conductivity 1.5 mS/cm. The column is washed with approximately 5 column volumes of equilibration buffer (100 mM sodium acetate buffer, pH 3.84) until the absorbance returns to baseline. The protein is eluted with 10 CV of 100 mM sodium acetate buffer, pH 3.84 with the conductivity increasing from 1.5 mS/cm to 20 mS/cm. The rate of increase in conductivity is 1.85 mS/cm per CV wash by elution buffer.

Figure 3 is an illustration of a chromatogram from the procedure as described in this example. The line marked "Cond" represents the increase in conductivity in mS/cm. The peak marked "FT" represents the flow-through obtained on washing the column with equilibration buffer. Peak A represents the eluate obtained with a linear increase in conductivity of 1.5 mS/cm to 20 mS/cm.

Figure 4 is an isoelectric focusing gel of samples of the loaded sample and eluate fractions of the cation exchange chromatography performed as described in this example. Lane 1 is an internal reference standard. Lanes 2-5 show the fractions obtained at pH 3.8 and increasing conductivities from 9 mS/cm to 14.5 mS/cm.

EXAMPLE 5

Cation Exchange Chromatography

As an alternative to Examples 3 or 4, the eluate from Example 2 may be subjected to cation exchange chromatography. Eluate from Example 2 is concentrated and the conductivity and pH reduced by diafiltration by a TFF step using 100 mM sodium acetate buffer, pH 3.84, conductivity 1.5 mS/cm. This step acts as a buffer exchanging step wherein the pooled eluate of Q-Sepharose column is brought into 100 mM sodium acetate buffer of pH 3.84 conductivity 1.5 mS/cm. The buffer exchanged sample is then loaded onto a CM Sepharose column (20 ml_, VL 11/21) that is pre-equilibrated with 5 CV of 100 mM sodium acetate buffer, pH 3.84, conductivity 1.5 mS/cm. The column is washed with approximately 5 column volumes of equilibration buffer (100 mM sodium acetate buffer, pH 3.84) until the absorbance returns to baseline. The protein is eluted with 10 CV of 83.4 mM Acetate buffer pH 3.3, conductivity 0.3 mS/cm. Fractions of 10 ml_ (0.5 CV) are collected in tubes containing 1 ml_ of 1 M Tris-HCI, pH 9. This adjusts the pH of the eluate to approximately 7. The column is washed with equilibration buffer. The elution is then continued with 10 CV of 100 mM sodium acetate buffer, 140 mM NaCI, pH 3.84 with the conductivity increasing from 1.5 mS/cm to 20 mS/cm. The rate of increase in conductivity is 1.85 mS/cm per CV wash by elution buffer.

Figure 5 is an illustration of a chromatogram from the procedure as described in this example. The line marked "Cond" represents the increase in conductivity, in mS/cm. Peak A represents the flow-through obtained on washing the column with equilibration buffer. Peak B represents the eluate obtained with elution buffer of pH 3.3, conductivity 0.3 mS/cm. Peak C represents the eluate obtained with elution buffer of pH 3.8 and a linear increase in conductivity of 1.5 mS/cm to 20 mS/cm.

Figure 6 is an isoelectric focusing gel from cation exchange

chromatography performed as described in this example. Lanes 1 and 8 are an internal reference standard. Lane 2 contains the loaded sample. Lane 3

corresponds to the flow-through obtained in a wash step. Lanes 4 and 5

correspond to fractions obtained by acid elution at pH 3.3, conductivity 0.3 mS/cm. Lanes 6 and 7 correspond to fractions obtained by elution at pH 3.8, conductivity 1.5 mS/cm to 20 mS/cm. EXAMPLE 6

Cation Exchange Chromatography

The eluate fractions from any of Examples 3-5 are pooled, concentrated, and exchanged with buffer containing 83.4 mM sodium acetate, pH 3.3, using TFF and loaded onto a CM Sepharose column (4 ml_, Tricon 5/20) pre-equilibrated with 5 CV of 83.4 mM sodium acetate buffer with pH 3.3. The desired product is obtained in the flow-through. After loading, the column is washed with 25 CV of 83.4 mM sodium acetate buffer, pH 3.3. The desired protein is also obtained in the flow-through of the wash step. Impurities bound to the column are subsequently eluted with 25 mM Tris, 500 mM NaCI buffer, pH 7.1.

EXAMPLE 7

Anion Exchange or Capto adhere Chromato raphy

The flow-through fractions from Example 6 are loaded onto a Q Sepharose column (1 ml_, Hitrap 7/2.5) that has been pre-equilibrated with buffer containing 25 mM Tris, 60 mM NaCI, pH 7.1. The column is washed with the same buffer. Desired product is bound to the column and is eluted with 40 mM phosphate, 280 mM NaCI buffer at pH 6. Alternatively, Capto adhere (1 ml_, Hitrap 7/2.5) can be used in place of a Q Sepharose column. In this case, the column is pre- equilibrated with 5 CV of 20 mM phosphate buffer of pH 6 and washed again with the same buffer (5 CV). Desired protein bound to the column is then eluted with 4 CV of buffer containing 40 mM phosphate and 280 mM NaCI, pH 6. This step acts as a concentration and buffer exchanging step, thus eliminating the need for another TFF operation.

EXAMPLE 8

Isoelectric Focusing

Eluate fractions of Examples 3 and 4 are analyzed by isoelectric focusing (IEF). The IEF gel is prepared using water, urea, 30% acrylamide, and

ampholytes, pH range 2-4 and 3-10 (Bio-Lyte^®3/10, from Bio-Rad Laboratories). The above components are mixed gently and 10% w/v ammonium persulfate and TEMED (tetramethylethylenediamine) are added to the mixture and the mixture is cast in a gel sandwich apparatus (BIORAD Mini Protean Cell) and fitted with a comb. The gel is allowed to polymerize for 45 minutes at room temperature. A small amount of protein solution (sample) is mixed with an equal volume of sample buffer (glycerol, ampholyte and water) and protein samples are loaded into the gel. The gel is then placed in a BIORAD Mini Protean Cell assembly and filled with a cathode buffer (25 mM sodium hydroxide) and anode buffer (25 mM orthophosphoric acid) in separate compartments. The flow-through fractions are run at 200 V constant voltage for 1.5 hours, for pre-focusing of ampholytes at room temperature, and then the voltage is increased to 400 V and run for the next 1.5 hours at room temperature. After the run, the gel is carefully removed and stained by silver staining.

Claims

CLAIMS:

1. A process for the isolation of low pi isoforms of darbepoetin from a mixture of darbepoetin isoforms, comprising:

loading the mixture onto a cation exchange resin to bind the low pi isoforms the cation exchange resin; and

eluting the low pi isoforms with an elution buffer having a pH value less than the pH value of the loaded mixture, while increasing the conductivity of the elution buffer.

2. A process according to claim 1 , wherein the conductivity is increased by a linear gradient.

3. A process according to claim 1 , wherein the conductivity is increased by a step gradient.

4. A process according to claim 1 , wherein a cation exchange procedure is preceded by an anion exchange or a mixed mode chromatography procedure.

5. A process according to claim 1 , wherein a cation exchange procedure is preceded by another cation exchange procedure, performed in flow- through mode.

6. A process according to claim 1 , wherein a cation exchange procedure is followed by another cation exchange chromatography, performed in flow-through mode.

7. A process according to either of claims 5 or 6, wherein two cation exchange procedures are either preceded by or followed by an anion exchange or a mixed mode chromatography step.

8. A process according to either of claims 5 or 6, wherein two cation exchange procedures are preceded and followed by an anion exchange or a mixed mode chromatography step.

9. A process for the isolation of low pi isoforms of darbepoetin from a mixture of darbepoetin isoforms, comprising:

loading the mixture onto a cation exchange resin to bind the low pi isoforms to the cation exchange resin; and eluting the low pi isoforms with an elution buffer having a pH that is similar to the pH of the loaded mixture, while increasing the conductivity of the elution buffer.

10. A process according to claim 9, wherein the conductivity is increased by a linear gradient.

11. A process according to claim 9, wherein the conductivity is increased by a step gradient.

12. A process according to claim 9, wherein a cation exchange procedure is preceded by an anion exchange or a mixed mode chromatography step.

13. A process according to claim 9, wherein a cation exchange procedure is preceded by another cation exchange procedure performed in flow- through mode.

14. A process according to claim 9, wherein a cation exchange procedure is followed by another cation exchange procedure performed in flow- through mode.

15. A process according to either of claims 13 or 14, wherein two cation exchange procedures are either preceded by or followed by an anion exchange or a mixed mode chromatography step.

16. A process according to either of claims 13 or 14, wherein two cation exchange chromatography steps are preceded by and followed by an anion exchange or a mixed mode chromatography step.

17. A process for the isolation of low pi isoforms of darbepoetin from a mixture of darbepoetin isoforms, comprising:

loading the mixture on a cation exchange resin to bind the low pi isoforms to the cation exchange resin;

eluting with a first elution buffer having a pH value less than the pH value of the loaded mixture; and

eluting with a second elution buffer having a pH value similar to the pH value of the loaded mixture, while increasing the conductivity of the second elution buffer.

18. A process according to claim 17, wherein the conductivity is increased by a linear gradient.

19. A process according to claim 17, wherein the conductivity is increased by a step gradient.

20. A process according to claim 17, wherein a cation exchange procedure is preceded by an anion exchange or a mixed mode chromatography step.

21. A process according to claim 17, wherein a cation exchange procedure is preceded by another cation exchange procedure performed in flow- through mode.

22. A process according to claim 17, wherein a cation exchange procedurey is followed by another cation exchange procedure performed in flow- through mode.

23. A process according to either of claims 21 or 22, wherein two cation exchange procedures are either preceded by or followed by an anion exchange or a mixed mode chromatography step.

24. A process according to either of claims 21 or 22, wherein two cation exchange procedures are preceded by and followed by an anion exchange or a mixed mode chromatography step.

25. A process for separating low pi isoforms of darbepoetin from a mixture of darbepoetin isoforms, comprising at least two cation exchange chromatography steps, wherein one cation exchange chromatography step is performed in flow-through mode and another cation exchange chromatography step is performed in bind-elute mode.

26. A process according to claim 25, wherein two cation exchange chromatography steps are carried out in succession.

27. A process according to claim 25, wherein cation exchange

chromatography is performed in bind-elute mode, followed by cation exchange chromatography performed in flow-through mode.

28. A process according to claim 25, wherein cation exchange chromatography is performed in flow-through mode, followed by cation exchange chromatography performed in bind-elute mode.

29. A process according to claim 25, wherein two cation exchange chromatography steps are preceded by an anion exchange or a mixed mode chromatography step.

30. A process according to claim 25, wherein two cation exchange chromatography steps are followed by an anion exchange or a mixed mode chromatography step.

31. A process according to claim 25, wherein two cation exchange chromatography steps are preceded and followed by an anion exchange or a mixed mode chromatography step.

32. A process according to any of claims 1 , 9, 17, or 25, further comprising a tangential flow filtration, concentration, diafiltration, ultrafiltration, or buffer exchange step between chromatographic steps.

33. A process according to any of claims 1 , 9, 17, or 25, further comprising viral inactivation, sterile filtration, or viral filtration steps.