FIELD OF THE INVENTION
The present invention relate to processes for separating low pI isoforms of modified cytokines from mixtures of isoforms.

BACKGROUND OF THE INVENTION
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. 311  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 OF THE INVENTION
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 OF THE INVETION
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.
In 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.
In 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 µL 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-Isoleucine  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-Inositol  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 NaCl 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 NaCl  pH 7.1 buffer. The column is then washed with 5 CV of the equilibration buffer (25 mM Tris  60 mM NaCl  pH 7.1). This is followed by a low pH wash with 80 mM sodium acetate  50 mM NaCl 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 NaCl 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 NaCl  pH 7.1±0.2 buffer. The column is then washed with 5 CV of the equilibration buffer (20 mM phosphate  60mM NaCl  pH 7.1±0.2). This is followed by a low pH wash with 80 mM sodium acetate  120 mM NaCl 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 NaCl 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 1M Tris-HCl  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 1M Tris-HCl  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 NaCl  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 NaCl buffer  pH 7.1.

EXAMPLE 7
Anion Exchange or Capto adhere Chromatography
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 NaCl  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 NaCl 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 NaCl  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.

WE CLAIM:
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 allow binding of the low pI isoforms to the resin; and
eluting the low pI isoforms with a buffer at a pH value less than the pH value of the load  while increasing the conductivity of the buffer.
2. 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 allow binding of the low pI isoforms to the resin; and
eluting the low pI isoforms with an elution buffer at a pH similar to the pH of the load  while increasing the conductivity of the buffer.
3. 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 allow binding of the low pI isoforms to the resin;
eluting with a first elution buffer at a pH less than the pH of the load; and
eluting with a second elution buffer at a pH similar to the pH of the load  while increasing the conductivity of the second elution buffer.
4. A process according to any of claims 1  2 or 3  wherein the cation exchange chromatography is preceded by an anion exchange or a mixed mode chromatography.
5. A process according to any of claims 1  2 or 3  wherein the cation exchange chromatography is preceded or followed by another cation exchange chromatography  performed in flow-through mode.
6. 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 of the chromatography step is performed in flow-through mode and another is performed in bind-elute mode.
7. A process according to claim 6  wherein the said two cation exchange chromatography steps are carried out in succession.
8. A process according to claim 6  wherein cation exchange chromatography is performed in bind-elute mode  followed by cation exchange chromatography performed in flow-through mode.
9. A process according to claim 6  wherein cation exchange chromatography is performed in flow-through mode  followed by cation exchange chromatography performed in bind-elute mode.
10. A process according to any of claims 6  7  8 or 9  wherein the two cation exchange step are either preceded by or followed by an anion exchange or a mixed mode chromatography.