DESC:
Field of the Invention:
The present invention relates to an ion exchange chromatography method for the separation of highly glycosylated protein and its charge variants using a non-linear gradient approach, in which the ionic strength of the elution buffer is alternately increased and decreased for fixed time intervals for better resolution of glycosylated charge variants of the protein with closely associated and different isoelectric point.

Background of the Invention:
Proteins are important in biopharmaceuticals as they are widely used for the treatment of several diseases including cancers (e.g., Interferon, monoclonal antibodies), heart attacks, strokes, cystic fibrosis (e.g. Enzymes, Blood factors), inflammation diseases (e.g. Tumor Necrosis Factors), anemia (e.g., Erythropoietin), hemophilia (e.g. Blood clotting factors), etc. One of the important challenges in the development of these proteins is to identify an efficient method to analyze the charge variants present in glycosylated proteins.
Monoclonal antibodies (mAb) have heterogeneous variants due to a series of post-translational modifications that arise during cell culture, purification, and storage. Such modifications may include oxidation, deamidation, amino acid substitution or deletion, differential glycosylation, glycation, isomerization, succinimide formation, N-terminal pyroglutamic acid formation, and C-terminal lysine clipping. Some of these modifications can alter the charge distribution on the surface of the mAb and result in charge variants.
Fusion proteins are prepared by recombinant gene expression, which is created by joining two or more genes that originally coded for separate proteins. Fc-fusion proteins are the proteins, wherein Fc region of human Immunoglobulin G1 (IgG1) is fused with another protein of interest. The active form of the Fc fusion proteins are dimers with a certain degree of glycosylation.
Conventional methods employed in the industry for separation, purification, identification, and characterization of the charge variants include ion-exchange chromatography, isoelectric focusing gel electrophoresis, and capillary isoelectric focusing.
Ion exchange chromatography (IEX) is an efficient technique for charge variant separation in biomolecules like proteins. This technique takes advantage of the differences in the charged properties of the protein molecules to separate them based on their charge distribution. IEX involves use of a stationary phase that includes charged functional groups. By adjusting the pH and salt content of the buffer, these functional groups interact with the oppositely charged molecules in the samples and enable their preferential elution from the column. Positively charged molecules are separated using cation exchange chromatography, whereas negatively charged molecules are separated using anion exchange chromatography. In the biopharmaceutical sector, the purification and analysis of protein charge variations, such as monoclonal antibodies and Fc fusion proteins, is frequently done using IEX. To get the best separation and purity of the separated charged variants, thorough procedure, and parameter optimization is necessary. Ion exchange chromatography work efficiently for most glycosylated proteins. However, in the case of highly glycosylated protein molecules or monoclonal antibodies or fusion proteins ion exchange HPLC conventional approach of the Ion-exchange method does not work, and it is difficult to get desired level of separation to quantify charge variants in the product.
Image capillary isoelectric focusing (iCEF) is an effective method for separating and quantifying charge variants in highly glycosylated charge variants. Image capillary isoelectric focusing is advantageous over ion exchange chromatography due to its high resolution, small sample volume, and quick run times. However, the iCIEF technique necessitated high-end sensitive equipment and a labor-intensive approach to sample preparation, as well as a large amount of protein for each analysis. Furthermore, the iCIEF equipment is limited in its ability to overlay multiple chromatograms in a single window.
Prior arts disclose various methods for the separation and analysis of glycosylated proteins such as antibodies and fusion proteins. US10712322 patent describes a method for analyzing polypeptide compositions like antibodies using pH gradient ion exchange chromatography. It combines ionic strength gradients and pH gradients to distinguish the polypeptide from charge variations of the polypeptide.
US 20190056350 describes the use of image capillary isoelectric focusing (iCIEF) to analyze glycosylated fusion proteins such as Aflibercept.

Summary of the Invention
The invention discloses a method for separation and analysis of composition comprising charge variants or isoforms of proteins using ion exchange chromatography comprising, loading the protein on an ion exchange matrix using a loading buffer with an initial ionic strength and eluting the charge variant using elution buffer with higher ionic strength, wherein the ionic strength of elution buffer is alternatively increased and decreased after every two-minute interval for better separation of charge variants.
In some embodiments first, the ion exchange matrix is saturated up to 10 % of elution buffer, then the elution buffer ionic strength first increases by 0.5% to 2% in every 2 min interval, then elution buffer ionic strength decreases to 2% to 5% in for 2 min interval, this alternative change in elution buffer ionic strength is carried out up to 100% of elution buffer ionic strength. In some embodiment, the alternative change is elution buffer ionic strength is up to 30 % of elution buffer ionic strength, during this period all charge variants are separated in 15 minutes to 80 minutes of the window.
As described here the invention provides a simple and cost-effective method for better separation and analysis of protein charge variants using ion exchange chromatography, in particular, this method is useful for proteins with high glycosylation. For example, this method can be used for charge variants of monoclonal antibodies or Fc fusion proteins like VEGF-Trap/aflibercept or abatacept.
In another aspect, the invention relates to a method for the separation and analysis of charge variants in glycosylated protein.
In another aspect, the invention relates to a method for the separation and analysis of antibody comprising charge variants, the method comprising;
a) loading the sample comprising antibody onto an ion exchange matrix, with loading buffer,
b) eluting the antibody and one or more charge variants from the ion exchange matrix using elution buffer,
wherein the ionic strength of elution buffer is first increased at about 0.5% to about 2% about every 2 min interval, then the ionic strength of the elution buffer is decreased to about 2% to about 5% in for about every 2 min interval and alternative change in elution buffer ionic strength is carried out up to 30 % of elution buffer ionic strength;
c) collecting the antibody and charge variants.
In another embodiment, the invention relates to a method for separation and analysis of Fc fusion protein comprising charge variants, the method comprising:
a) loading the sample comprising Fc fusion protein onto an ion exchange matrix, with loading buffer,
b) eluting the Fc fusion protein and one or more charge variants from ion exchange matrix using elution buffer wherein the elution buffer ionic strength is first increase to about 0.5% to about 2% in about every 2 min interval, then elution buffer ionic strength is decreased to about 2% to about 5% in for 2 min interval and alternative change in elution buffer ionic strength is carried out up to 30 % of elution buffer ionic strength.
c) collecting the Fc fusion protein and charge variants.
In some embodiment the collected Fc fusion protein and charge variants can be further analyzed by the methods well known in prior arts such as IEF analysis and iCIEF analysis.
In another embodiment the Fc fusion protein is aflibercept or abatacept.

Description of the Drawings
Figure 1: Chromatographic profile of aflibercept analyzed on strong cation exchange column using step and linear gradient.
Figure 2: Chromatographic profile of aflibercept analyzed on strong cation exchange column using non-linear gradient.
Figure 3: Chromatographic profile of abatacept analyzed on strong anion exchange column using non-linear gradient.

Detailed Description of Invention
The present invention describes a cost effective and high-resolution method for separation and analysis of charge variants of a polypeptide using ion exchange chromatography. The charge heterogeneity can be reported in terms of percentages of charge variants or isoforms using the ion exchange method that is explained here. The instant method is simple and cost effective compared to image capillary isoelectric focusing (iCEF).
In an embodiment, the invention provides a method for separation and analysis of charge variants or isoforms of protein comprising; loading the protein onto an ion exchange matrix using a loading buffer with at initial ionic strength and eluting the charge variant using elution buffer with higher ionic strength, wherein the ionic strength of elution buffer is alternatively increased and decrease after every fixed time interval for better separation of protein charge variants, such that the protein charge variants separate as independent entities as they elute from the chromatographic material.
The terms "polypeptide" and "protein," as they are used here, are interchangeable and refer to polymers of amino acids that can be linear or branched and can be any length. The protein may undergo modifications, such as glycosylation or the formation of disulfide bonds, among others. Antibodies are also considered proteins.
Thus, in an embodiment, the invention relates to a method for the separation of charge variants from a composition comprising protein and charge variants, the method comprising;
a) binding the composition comprising protein and charge variants onto an ion exchange material;
b) eluting the protein and charge variants from the ion exchange material using an elution buffer
wherein the ionic strength of the elution buffer slowly rises along with the continuous rise in gradient pulse and bring back to low at every fixed interval of time; and
c) collecting the protein and charge variants.

The term “antibody” used herein in is the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. Antibody may be selected from Fc fusion protein for example but not limited to VEGF-Trap/aflibercept and abatacept, Fab the heavy chain and light chains of antibodies or other antibody fragments such as scFv, Diabodies, Tribodies, Tetrabodies, Bis - scFv, Minibodies Fab2 (bispecific), Fab3 (tri-specific).
In another embodiment, the invention relates to a method for the separation of charge variants from a composition comprising antibody and charge variants, the method comprising;
a) binding the composition comprising antibody and charge variants to an ion exchange material;
b) eluting the antibody and charge variants from the ion exchange material using and elution buffer wherein the ionic strength of elution buffer is slowly rise along with continuous rise in gradient pulse and bring back to low at every fixed interval of time; and
c) collecting the antibody and charge variants.

The term “charge variant” refers to a modified protein whose charge has been altered. These modifications include but not limited to oxidation, deamidation, and glycosylation. In some embodiments, the protein is a glycoprotein, meaning that the glycan is added to the protein, such as by adding sialic acid or its derivatives, but is not limited to this.
The term used here “loading buffer” is a buffer or solution used for the loading of the protein to the chromatographic material. The loading buffer may have fixed pH and ionic strength or selected as per the properties of the protein or method known in the art such that the loading buffer helps protein and charge variants to bind with ion exchange material.
The term “elution buffer” as used here refers to the buffer or solution used for the elution of the protein and charge variants of protein form the ion exchange chromatographic material. In an embodiment, the elution buffer has higher pH and/or ionic strength than the loading buffer. In another embodiment, the pH and/or ionic strength of loading buffer is measured as 0% and of the elution buffer is measured as 100%. The require percentage of elution buffer can be prepared by mixing loading buffer with 100% elution buffer, for example, 100 ml of 5% elution buffer means can be prepared by mixing 95 ml of loading buffer and 5 ml of elution buffer. In an embodiment, the elution of charge variants is accomplished through the nonlinear gradient formation. For example, if the gradient of the elution buffer is increased for a fixed time interval, then it is decrease for a fixed time interval and then again increases for a fixed time interval. In some embodiment, the ion exchange chromatography material is first saturated up to about 10% of pH and/or ionic strength of elution buffer then the elution buffer ionic strength is first to increase about 0.5% to about 2% in every 2 min interval, then elution buffer ionic strength decreases to about 2% to about 5% in for 2 min interval and alternative change in elution buffer ionic strength continued up to 100% elution buffer pH and/or ionic strength or till the elution of all the charge variants of the protein. In a non-limiting example, the instant method describes here results in higher resolution of glycosylated charge variants of VEGF-trap/ Aflibercept or Abatacept was observed to achieve up to about 70% or elution buffer strength.
In another embodiment the elution buffer ionic strength is first increase about 0.5% to about 2% in every about 1 to about 2 min intervals, then elution buffer ionic strength is decrease to about 2% to about 5% in for 2 min interval and alternative change in elution buffer ionic strength is carried out with same gradient method for separation of charge variant species
In another embodiment, the ionic strength of elution buffer is raised about <0.5% for 1 min, about <1% for 2 min, about <1% for 3 min, about <1% for 4 min, about <1% for 5 min and then the ionic strength is decreased about <0.5% for 1 min, about <1% for 2 min, about <1% for 3 min, about <1% for 4 min, about <1% for 5 min.
In another embodiment the ion exchange chromatography material is first saturated about 8% to about 10% of elution buffer for about 12 min, during this period charge variants are not expected to separate out from the column. This is considered as saturation, equilibration, and binding of the protein to the column, then increasing about 0.5% to about 2% of elution buffer percentage in every 2 min intervals and sudden decrease of elution buffer percentage to about 2% to about 5% in every 2 min intervals up to 30 % of elution buffer concentration.
Thus, in another embodiment, the invention relates to a method for separation of charge variants from a composition comprising protein and charge variants, the method comprising;
a) binding the composition comprising protein and charge variants to an ion exchange material,
b) eluting the protein and charge variants from the ion exchange material elution buffer wherein the elution buffer ionic strength is first increase about 0.5% to about 2% in every 2 min intervals, then elution buffer ionic strength is decrease to about 2% to about 5% in for 2 min interval and alternative change in elution buffer ionic strength is carried out with same gradient method for separation of charge variant species
c) collecting the protein and charge variants.

In another embodiment, the invention relates to a method for separation of charge variants from a composition comprising antibody and charge variants, the method comprising;
a) binding the composition comprising antibody and charge variants to an ion exchange material,
b) eluting the antibody and charge variants from the ion exchange material elution buffer wherein the elution buffer ionic strength is first increase about 0.5% to 2% in every 2 min intervals, then elution buffer ionic strength is decrease to about 2% to about 5% in for 2 min interval and alternative change in elution buffer ionic strength is carried out with same gradient method for separation of charge variant species and
c) collecting the antibody and charge variants.

In an embodiment, the ion exchange chromatography material is a cation exchange material. The cation exchange material may be a membrane, a monolith, or resin. In some embodiments, a cation exchange chromatography material is used for a separation of charge variants of polypeptide, e.g., an antibody or fragment thereof or fusion protein, with isoelectric point (pI) greater than about7. For example, the antibody or fragment thereof or fusion protein may have a pI of 7-12. In some embodiment, the cation exchange chromatography material is strong cation exchanger material for example, but not limited to, sulfonic acid functional groups, or a weak cation exchanger material, for example, but not limited to carboxylic, phosphonic, and phosphoric acid functional group.
In an embodiment, the ion exchange chromatography material is an anion exchange material. The anion exchange material may be a membrane, a monolith, or resin. In some embodiments, the anion exchange chromatography material is used for a separation of charge variants of a polypeptide, e.g., an antibody or fragment thereof or fusion protein, with a pI less than about 6. For example, the antibody or fragment thereof or fusion protein may have a pI of 4-6. In some embodiment, the anion exchange chromatography material may strong anion exchanger material, for example but not limited to, diethyl-2-hydroxypropylaminoethyl (QAE), triethylaminoethyl (TEAE), or trimethyl amino ethyl groups or weak anion exchanger material, for example but not limited to, diethylaminoethyl (DEAE) or amino ethyl groups.
In another embodiment, the invention relates to a method for separation of charge variants from a composition comprising Fc fusion protein and charge variants, the method comprising;
a) binding the composition comprising Fc fusion protein and charge variants to an ion exchange material,
b) eluting the Fc fusion protein and charge variants from the ion exchange material elution buffers,
wherein the elution buffer ionic strength is first increase about 0.5% to about 2% in every 2 min intervals, then elution buffer ionic strength is decrease to about 2% to about 5% in for 2 min interval and alternative change in elution buffer ionic strength is carried out with same gradient method for separation of charge variant species,
c) collecting the Fc fusion protein and charge variants.

In another embodiment the Fc fusion protein is VEGF-Trap or aflibercept.
In another embodiment the Fc fusion protein is abatacept.
In an embodiment, the ion exchange chromatography material is a cation exchange material. The cation exchange material may be a membrane, a monolith, or resin. In some embodiments, a cation exchange chromatography material is used for a separation of charge variants of polypeptide, e.g., an antibody or fragment thereof or fusion protein, with isoelectric point (pI) greater than about 8. For example, the antibody or fragment thereof or fusion protein may have a pI of 6-9. In some embodiment, the cation exchange chromatography material is strong cation exchanger material for example, but not limited to, sulfonic acid functional groups or a weak cation exchanger material, for example, but not limited to carboxylic, phosphonic, and phosphoric acid functional group.
In an embodiment, the ion exchange chromatography material is an anion exchange material. The anion exchange material may be a membrane, a monolith, or resin. In some embodiments, the anion exchange chromatography material is used for a separation of charge variants of a polypeptide, e.g., an antibody or fragment thereof or fusion protein, with a pI less than about 6. For example, the antibody or fragment thereof or fusion protein may have a pI of 4-6. In some embodiment, the anion exchange chromatography material may be strong anion exchanger material, for example but not limited to, diethyl-2-hydroxypropylaminoethyl (QAE), triethylaminoethyl (TEAE), or trimethyl amino ethyl groups or weak anion exchanger material, for example but not limited to, diethylaminoethyl (DEAE) or amino ethyl groups.
In another embodiment, the invention relates to a method for separation of charge variants from a composition comprising aflibercept and related molecules charge variants, the method comprising;
a) binding the composition comprising aflibercept and related molecules to a cation exchange material,
b) eluting the aflibercept and related molecules from the cation exchange material elution buffer wherein the cation exchange column is first equilibrated about 8 to about 10% of elution buffer for about 12 min, increasing about 0.5% to about 2% of elution buffer percentage in every 2 min intervals and sudden decrease of elution buffer percentage to about 2% to about 5% in every 2 min intervals up to 70% of elution buffer concentration; and
c) collecting the aflibercept and related molecules.

In another embodiment, the invention relates to a method for separation of charge variants from a composition comprising abatacept and related molecules, the method comprising;
a) binding the composition comprising abatacept and charge variants to an anion exchange material,
b) eluting the abatacept and charge variants from the anion exchange material elution buffer wherein the cation exchange column is first equilibrated about 8% to about 10% of elution buffer for about 12 min, increasing about 0.5% to about 2% of elution buffer percentage in every 2 min intervals and sudden decrease of elution buffer percentage to about 2% to about 5% in every 2 min intervals up to 70% % of elution buffer concentration; and
c) collecting the abatacept and related molecules.

The term “about” is intended to encompass a range of values 10% of the specified value(s). For example, the phrase “about 5% is intended to encompass +10% of 5%, i.e., from 4.5% to 5.5%, inclusive.
In another embodiment the chromatography material is chromatography column, used for liquid chromatography. In some embodiments, the chromatography column is high performance liquid chromatography (HPLC); for example, a cation exchange HPLC column or an anion exchange HPLC column.
In an embodiment, the ionic strength of the elution buffer is from about 0 mM to about 1000 mM. In further embodiments, the elution buffer comprises about 0 mM NaCl to about 1000 mM NaCl. In another embodiment the elution buffer comprises about 1000mM NaCl.
In some embodiments, the separation and analysis of charge variant of protein in performed by high performance liquid chromatography.
In an embodiment, the anion exchange chromatography is used for the protein having pI about 4.0, about 4.5, about 5.0, about 5.5, about 6.0
In an embodiment, the cation exchange chromatography is used for the protein having pI about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0
The examples which follow are illustrative of the invention and are not intended to be limiting.

Example 1: Buffer and sample preparation
Buffer/Mobile phase preparation Loading buffer or Buffer A;10 mM MES, pH 6.25): 1.95 g of MES was weighed and dissolved in 800 mL of purified water. pH was adjusted to 6.25 with 1M NaOH, and the volume was increased to 1000 mL with purified water. Sonicated for 10 minutes after filtering with a 0.2 filter.

Elution buffer or Buffer B: (10 mM MES + 1 M Sodium chloride, pH 6.25): 1.95 g MES and 58.4 g sodium chloride were weighed and dissolved in 800 mL of purified water. pH was adjusted to 6.25 with 1M sodium hydroxide, and the volume was increased to 1000 mL with purified water. Sonicated for 10 minutes after filtering with a 0.2 filter.
1M Sodium hydroxide: 4.01 g of sodium hydroxide was weighed and dissolved in 80 mL of purified water. Purified water was used to make up the volume to 100 ml.

Sample Preparation
Purified protein of interest is diluted with loading buffer as described above to get concentration of 1.0 mg/ml.

Example 2: Separations and Analysis of Aflibercept charge variant using linear gradient method
Sample of aflibercept was prepared as described in example 2. The analysis of aflibercept was carried out by Agilent 1260/1290 infinity series HPLC with UV detector using YMC BioPro SP-F, 100 X 4.6 mm I. D, 3µ column. The elution was carried out as per table 1 (Loading Buffer A: 10 mM MES, pH 6.25 and Elution Buffer B: 10 mM MES + 1 M Sodium chloride, pH 6.25). Further extracted ion chromatogram of protein was generated (Fig. 1)

Table 1.
Time (min) Mobile Phase A (%) Mobile Phase B (%) Flow Rate
0 100 0 0.8 mL/min
8 100 0
8.01 94 6
12 94 6
12.01 93 7
16 93 7
16.01 to 46 min 92.5 to 89 7.5 till 11% (0.5% increase per 4 minutes
46 to 64 89 to 85 11 to 15% (1% increase per 4 minutes)
64.01 to 75 0 100
75 to 80 100 0
Column temperature 30 °C
Injection Volume 50 µL
Run Time 80 min
Sampler 2 – 8 °C
Detection wavelength 280 nm

In Fig.1 peak elution spanning from RT ~ 15 min to ~ 50 min was observed. Upon analysis of chromatogram, acidic and basic charge variants were not distinctly separated, or poor resolution observed.

Example 3: Separation and analysis of Aflibercept charge variants using non-linear gradient method
The analysis of aflibercept (1mg/ml) was carried out by Agilent 1260/1290 infinity series HPLC with UV detector using YMC BioPro SP-F, 100 X 4.6 mm I. D, 3µ column. The gradient was carried out as per table 2 (loading Buffer A: 10 mM MES, pH 6.25 and elution Buffer B: 10 mM MES + 1 M Sodium chloride, pH 6.25). Further extracted ion chromatogram of protein was generated (Fig. 2)

Table 2.
Time (min) Mobile Phase A (%) Mobile Phase B (%) Flow Rate
0 100 0 0.8 mL/min
2 100 0
4 98 2
6 96 4
8 94 6
10 98 2
12 92 8
14 98 2
16 91.5 8.5
18 98 2
20 to 80 91 to 80 Within 2 mins increase 0.5% and decrease up to 2%
82 78 22
84 to 87 0 100
87.01 to 90 100 0
Column temperature 30 °C
Injection Volume 50 µL
Run Time 90 min
Sampler 2 – 8 °C
Detection wavelength 280 nm

In Fig.2, the entire elution window was spread from RT ~ 18 min to ~ 95 min. Peaks shape found to be sharp and well separated charge variants were observed.
When comparing Figs. 1 and 2, it was clear that the non-linear gradient method separates aflibercept charge variants better than the gradient method.

Example 4: Separation and analysis of Abatacept charge variants using non-linear gradient method
Sample for analysis was prepared by diluting abatacept with loading Buffer A to achieve the concentration of 1mg/ml. The analysis of was carried out by Agilent 1260/1290 infinity series HPLC with UV detector using Shim Pack Bio IEX Q-NP, 4.6 X 100 mm, 5 µ column. The gradient was carried out as per table 3 (loading buffer A: 50 Tris-base, pH 7.2 and loading buffer B: 50 Tris-base + 1 M Sodium chloride, pH 7.2). Further extracted ion chromatogram of protein was generated (Fig. 3)

Table 3:
Time (min) Buffer A% Buffer B% Flow rate
0 100 0 0.5 mL/min
6 95 5
8 90 10
10 98 2
14 89.5 10.5
16 98 2
18 89 11
20 to 86 87.5 to 80 Within 2 mins increase 0.5% and decrease up to 2%
88 78 22
90 76 24
92 to 96 0 100
96.01 to 100 100 0
Column temp. 30 °C
Inj. Volume 20 µL
Run Time 100 min
Wavelength 280 nm

In Fig.3, the entire elution window was spread from RT ~ 11 min to ~ 95 min. Peaks shape found to be sharp and well separated charge variants were observed.
,CLAIMS:
1. A method for separation of charge variants from a composition comprising protein and charge variants, the method comprising:
d) binding the composition comprising protein and charge variants to an ion exchange material;
e) eluting the protein and charge variants from the ion exchange material elution buffer wherein the elution buffer ionic strength is first increased from about 0.5% to about 2% in about every 2 min intervals, then elution buffer ionic strength is decreased to about 2% to about 5% for about 2 min interval and alternative change in elution buffer ionic strength is carried out with same gradient method for separation of charge variant species; and
f) collecting the protein and charge variants.

2. A method for separation of charge variants from a composition comprising antibody and charge variants, the method comprising;
d) binding the composition comprising antibody and charge variants to an ion exchange material;
e) eluting the antibody and charge variants from the ion exchange material elution buffer wherein the elution buffer ionic strength is first increased about 0.5% to about 2% in about every 2 min intervals, then elution buffer ionic strength is decreased to about 2% to about 5% for about 2 min interval and alternative change in elution buffer ionic strength is carried out with same gradient method for separation of charge variant species; and
f) collecting the antibody and charge variants.

3. A method for separation of charge variants from a composition comprising Fc fusion protein and charge variants, the method comprising;
d) binding the composition comprising Fc fusion protein and charge variants to an ion exchange material;
e) eluting the Fc fusion protein and charge variants from the ion exchange material elution buffers,
wherein the elution buffer ionic strength is first increased about 0.5% to about 2% in about every 2 min intervals, then elution buffer ionic strength is decrease to about 2% to about 5% for about 2 min interval and alternative change in elution buffer ionic strength is carried out with same gradient method for separation of charge variant species; and
f) collecting the Fc fusion protein and charge variants.

4. The method as claimed in claim 1, 2 or 3, wherein the alternative change in elution buffer ionic strength is carried out for about 15 minutes to about 80 minutes.

5. The method as claimed in claim 1or 2 or 3, wherein about 5% to about 10% of elution buffer is passed though ion-exchange material prior to alternative change in elution buffer gradient.

6. The method as claimed in claim 1 or 2 or 3, wherein the charge variants are oxidized or deamidated or glycosylated charge variants.

7. The method as claimed in claim 1 or 2 or 3, wherein the ion exchange material is a cation exchange material.

8. The method as claimed in claim 1 or 2 or 3, wherein the ion exchange material is an anion exchange material.

9. The method as claimed in claim 3, wherein the Fc fusion protein is VEGF-Trap or aflibercept or abatacept.

10. The method as claimed in claim 1, 2 or 3, wherein the elution buffer strength is 1mM to 1000mM.