FIELD OF THE INVENTION

The present invention relates to a method of enrichment of sialylated glycoforms using anion exchange chromatography

BACKGROUND OF THE INVENTION

Recombinant proteins, in particular monoclonal antibodies and Fc-fusion proteins constitute an important therapeutic sector of the biopharmaceutical industry (Chirino and Mire-Sluis 2004). However, these molecules are prone to several types of glycosylation including sialylation that can decrease or increase their efficacy.

Variation of glycoprotein sialylation has significant clinical implications. Highly sialylated glycoforms of erythropoietin (eg., darbepoetin) are found to be long-acting and more effective in humans. Studies also report sialylated immunoglobulin (IgG) molecules to exhibit enhanced anti-inflammatory properties (Kaneko Y., etal, Science, 313 (5787); 2006: 670-673). Thus, in past several strategies were designed to improve sialylation of IgG or IgG-Fc fusion molecules including overexpression of sialic acid transferase enzymes or alteration of certain culture conditions. However, separation and enrichment of these sialylated glycoforms remains a challenge in the downstream process.
The prior art discloses several methods for the separation of glycoforms, using affinity chromatography.

U.S. Patent Application No. 20020164328 discloses a method of purifying an antibody with desired glycan property using lectin affinity chromatography. Tamao Endo in his review (Journal of chromatography A, 720; 1996: 251-261) discusses various methods of preparing lectin immobilized affinity chromatographic columns and its use in the fractionation and purification of N-linked oligosaccharides from a glycoprotein composition.

Yamamoto et.al teaches a method of separation of sialylated sugar chains using Maackia amurensis leukoagglutinin immobilized affinity column. (The protein protocols handbook, 2nd edition. Edited by J.M. Walker, pages 917-931).

U.S. Patent Application No. 20100151584 describes a multi-dimensional chromatographic method involving a combination of anion exchange and a reverse phase chromatographic technique for the separation of N-glycans.

The prior-art describes the use of affinity resins for the purification of sialylated glycoforms from a glycoprotein composition. However, affinity resins typically suffer the leaching of the affinity agent from the solid support, resulting in contamination of the purified product with the affinity agent, which in turn renders the glycoprotein unsuitable for use in pharmaceutical preparations. The prior-art also discloses use of a combination of chromatographic techniques for the separation of glycoforms. However use of multiple chromatographic resins add to the process complexity and may result in considerable reduction in the glycoprotein yield.

Thus, there is a clear need for an efficient and effective method of enrichment of sialylated glycoform composition. The objective of the current invention is to provide a method of enrichment of sialylated glycoform composition using anion exchange chromatography. The method described in the invention is suitable for large-scale separation of the glycoform and in turn alleviates the difficulties discussed in prior-art.

SUMMARY OF THE INVENTION

The present invention provides a method of enrichment of sialylated glycoforms in a protein composition by anion exchange chromatography operated in bind-elute mode. Specifically, the invention provides a method of enrichment of sialylated glycoforms in an Fc containing protein composition using an anion exchange chromatography.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an illustration of a chromatogram from the procedure as described in example 3 using load and wash buffer solutions at pH 6.3, conductivity at 4.5 mS/cm. The line marked "Cond" represents the increase in conductivity in mS/cm. Peaks marked "FT" represents the flow-through.
Figure 2 is a comparison of chromatographic elution profiles at varying conductivity values of 4.5, 5.5, 6.5 and 7.5 mS/cm, from the procedure as described in example 3. Peaks marked "FT" represents the flow-through. "Cond" represents the increase in conductivity in mS/cm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of enrichment of sialylated glycoforms using anion exchange chromatography.

In an embodiment, the invention provides a method of enrichment of sialylated glycoforms in a protein composition using anion-exchange chromatography, comprising steps of;

a) loading the protein composition onto an anion exchange chromatographic resin
b) washing the asialylated glycoforms in the flow-through and
c) eluting the bound protein from the said resin in a buffer having a conductivity of about 12 mS/cm to about 16 mS/cm

wherein, the eluted protein composition is enriched with sialylated glycoforms.
In an embodiment of the invention, the protein is an Fc-containing protein.
In another embodiment, the Fc-containing protein may be a fusion protein or an antibody molecule.
In a further embodiment, the Fc-containing protein may be a TNFR: Fc fusion protein.
In yet another embodiment, the TNFR: Fc protein may be loaded onto the anion exchange chromatographic resin at a pH at least 1 unit greater than the pi of the said protein.
The anion exchange chromatographic step in the invention may include one or more wash steps prior to the elution of the antibody.

The anion exchange chromatographic step may be preceded or followed by an affinity, hydrophobic interaction, ion-exchange, mixed mode or a size-exclusion chromatography and/or a membrane that could perform similar function. The additional chromatographic resins mentioned here may be used to remove impurities such as host cell proteins, nucleic acids, aggregates, endotoxins, etc.,
The embodiments mentioned herein may include one or more tangential flow filtration, depth filtration, diafiltration, ultrafiltration or concentration steps.

The embodiments mentioned herein may include one or more viral inactivation steps or sterile filtration or nano filtration steps.

The embodiments mentioned herein may include one or more neutralization steps.

"Anion exchange resin" mentioned in the embodiments refers to a solid phase which has a positively charged ligand such as quaternary amino groups, attached thereto. The anion exchange resin can be any weak or strong anion exchange chromatographic resin or a membrane, which could function as a weak or a strong anion exchanger. Commercially available anion exchange resins include, but are not limited to, DEAE cellulose, Poros PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX Sepharose Fast Flow, Capto DEAE, Q Sepharose high Performance, QAE SEPHADEX and FAST Q SEPHAROSE from GE Healthcare, Macro-Prep DEAE and Macro-Prep High Q from Biorad, Q-Ceramic Hyper D, DEAE-Ceramic Hyper D, from Pall Corporation. In embodiments of the invention, a weak or a strong anion exchange resin is used. DEAE Sepharose® (GE Healthcare Life Sciences) is used as a weak anion exchange resin and is made using a highly cross-linked agarose matrix attached to a diethylaminoethyl functional group. Q- Sepharose Fast Flow® (GE Healthcare Life Sciences) is used as a strong anion exchange resin and is made using a highly cross-linked, 6 % agarose matrix attached to -O-CH2CHOHCH20CH2CHOHCH2N+(CH3)3 functional group.

The term "glycan" refers to a monosaccharide or polysaccharide moiety.

The term "glycoform" as used herein denotes a glycoprotein containing a particular glycan structure or structures.

The term "sialylated glycoforms" as used herein refers to a glycoform having one or more sialic acid residues attached at the non-reducing end.

The term "Fc-containing protein", as used herein, refers to any protein having at least one immunoglobulin constant domain selected from the CHI, hinge, CH2, CH3, CH4 domain, or any combination thereof.

The term "composition", as used herein comprises the target protein and one or more impurities or contaminants including glycans/mixtures of glycans and/or glycoforms.

The term "flow-through" as used herein refers to species that are not bound or loosely bound to the chromatographic resin, and obtained in the "flow-through" fraction.

The term 'bind elute mode' as used herein refers to an operation mode of a purification method, wherein a glycoprotein is bound to the chromatographic resin when loaded, and subsequently eluted with an elution buffer.

The buffering agents used in the buffer solutions include, and are not limited to citrate, phosphate, hydrochloride, acetate, chloride, succinate, MES, MOPS, TRIS or ammonium and their salts or derivatives as well as combinations of these.

The invention is more fully understood by reference to the following examples. These examples should not, however, be construed as limiting the scope of the invention.

EXAMPLES

Example 1: Protein A Chromatography

The clarified cell culture broth containing TNFR:Fc fusion protein was subjected to a protein A affinity chromatography (Prosep ultra VA, VL 32 x 200, 160 ml) that was equilibrated with equilibration buffer, 50 mM Sodium acetate pH 7.0 and 0.15 M NaCl. The equilibration was then followed by a wash with a high salt buffer containing sodium acetate pH 7.0 with 0.75 M NaCl. The bound protein was eluted with 0.2 M Acetic acid and 50 mM Sodium acetate. The affinity step was majorly performed to remove impurities such as host cell proteins, host cell DNA etc.

Example 2: Anion Exchange Chromatography

The eluate from example 1 was loaded onto an anion exchange chromatographic resin (Q-Sepharose FF, VL 44 x 200, 300 mL) that was pre-equilibrated with 5 column volume (CV) of 50 raM phosphate buffer, pH 6.3 at a conductivity of 4.5 mS/cm. Post loading, the column was washed with ~ 15 CV of 50 mM phosphate buffer, pH 6.3 at a conductivity of 4.5 mS/cm to obtain the asialylated (non-sialylated) molecules in the flow-through. The bound TNFR:Fc protein enriched with sialylated glycoforms was then eluted using a step-gradient elution using a buffer containing 50 mM phosphate, 100 mM NaCl, pH 6.3 and at an increased conductivity of 15 - 16 mS/cm.

Alternatively, the protein was eluted with a linear gradient of elution buffer using buffer A (50 mM phosphate buffer, pH 6.3) and buffer B (50 mM phosphate, 0.5 N NaCl, pH 6.3) and at a linear increase in conductivity.

The example was repeated with load and wash buffer conductivity values of 5.5, 6.5 and 7.5 mS/cm. However, loading and washing with buffer at conductivity values 6.5 and 7.5 mS/cm resulted in elution of sialylated glycoform along with asialylated forms in the flow-through, leading to significant loss of sialylated forms in the eluate. Hence, specific conductivity range (i.e., less than 6.5 mS/cm) is preferred for the enrichment process.

We claim:

1. A method of enrichment of sialylated glycoforms from a mixture of protein composition using ion-exchange chromatography, comprising the following steps:

(a) applying the mixture of protein composition onto an ion-exchange chromatographic resin using a buffer solution,
(b) washing the said ion-exchange chromatographic resin to remove asialylated glycoforms in the flow-through, and
(c) eluting the bound protein from the said ion-exchange chromatographic resin, wherein, the eluted protein composition has increased amount of sialylated glycoforms.

2. The process according to claim 1, wherein the ion exchange chromatography is anion-exchange chromatography.

3. The sialylated glycoform according to claim 1 is a glycoprotein having at least one silaic acid residue attached to glycoprotein, wherein the said glycoprotein is an Fc-containing protein or a fusion protein or an antibody molecule.

4. The sialylated glycoform according to claim 3 is a glycoprotein, wherein the said glycoprotein is TNFR: Fc fusion protein and wherein the said protein present in a composition is applied onto the anion-exchange chromatographic resin at a pH at least 1 unit greater than the pi of the said protein.

5. The process according to claim 1, wherein the chromatographic resin used is a weak or strong anion exchange resin or a membrane used as a weak or strong anion exchanger.

6. The process according to claim 1, wherein one or more wash steps are included prior to the elution of the protein enriched in sialylated glycoform.

7. The process according to claim 1, wherein the elution of bound sialylated glycoform is performed using step gradient or linear gradient elution method.

8. The process according to claim 1, wherein the conductivity of elution buffer is about 12-16 mS/cm and pH is about 6-7 and wherein the buffer solution used is selected from the group consisting of citrate buffer, phosphate buffer, hydrochloride buffer, acetate buffer, chloride buffer, succinate buffer, MES buffer, MOPS buffer, TRIS buffer or ammonium buffer.

9. The process according to claim 1, wherein the said process is preceded or succeeded by an affinity or hydrophobic interaction or ion-exchange or mixed mode or size-exclusion or membrane chromatography.

10. The process according to claim 1, wherein the ion exchange is preceded or succeeded by one or more steps selected from tangential flow filtration, depth filtration, diafiltration, ultrafiltration, concentration, viral inactivation, sterile filtration, nano filtration, or neutralization step.