FIELD OF THE INVENTION

The present invention relates to a method of separation of high mannose-type glycoforms using anion exchange chromatography

BACKGROUND OF THE INVENTION

N-glycans covalently attached to a protein/immunoglobulin molecule are essential for the structure and function of glycoprotein.

N-linked glycans typically have a core bi-antennary pentasaccharide, 2N-Acetylglucosamine and 3Mannose (2GlcNAc-3Man) residues. Nevertheless, glycoprotein composition is highly heterogeneous due to considerable variation in the outer-arm sugar residues of the core complex, resulting in diverse assortments of N-glycans each with differently structured glycoforms. The two major types of heterogeneity reported in N-glycans are high mannose-type and tri/tetra antennary complex (or hybrid) type oligosaccharides.

High mannose-type oligosaccharides include the core structure (2GlcNAc-3Man) with additional mannose residues (typically greater than 3). High mannose-type glycans are not usually present in IgG and rapid clearance of IgGs bearing high mannose-type glycans has been demonstrated in mice. Studies also indicate that the Man5 glycoform greatly affects the physiological properties of antibodies/protein bearing them, in particular, their potency, immunogenicity and clearance rate. (Wright, A. and Morrison, SL. J Immunol 160(7): 3393-3402, 1998 and Kanda et.al., Glycobiology 17: 104-118, 2007). A study by Chen et. al., (Glycobiology, 19: 240-249, 2009) conducted on a recombinant human antibody in human serum, recovered circulating mannosidases in serum that were shown to trim high mannose species, specifically Man6-Man9. In addition, high mannose glycoproteins are expected to be more immunogenic and can significantly affect the safety and efficacy of therapeutic proteins. Accordingly, it is desirable to separate high mannose-type glycoforms from a composition comprising a heterogeneous mixture of glycoforms.

The prior art discloses several methods for the separation of glycans or glycan mixtures, using affinity chromatography.

U.S. Patent Application No. 20020164328 discloses a method of purifying an antibody with desired glycan property using lectin affinity chromatography. Specifically, concanavalin A (ConA) affinity chromatography is used to recognize D-mannose and purification of mannose type N-glycans thereof.

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 high mannose-type sugar chains using a Ricinus communis lectin based affinity column. (The protein protocols handbook, 2nd edition. Edited by J.M. Walker, pages 917-931).

U.S. Patent Application No. 2003077806 discloses a method of separating alpha galactosidase A glycoforms using an immune-affinity or hydrophobic interaction chromatography. 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 describe the use affinity resins for the separation or purification of N-glycans 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 N-glycans. 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 separation of a glycoform from a glycoprotein composition. The objective of the current invention is to provide a method of separation of a glycoform, in particular, high mannose-type glycoforms 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 separation of high mannose-type glycoforms from a mixture of glycoform composition by anion exchange chromatography operated in bind-elute mode. Specifically, the invention provides a method of separation of high mannose-type glycoforms from an Fc containing protein using anion exchange chromatography.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is an illustration of a chromatogram from the procedure as described in example 2 using load and wash buffer solutions at pH 6.3, conductivity at 4.5 mS/cm. "Cond" represents the increase in conductivity in mS/cm. Peaks marked "FT" represents the flow-through.

Figure 2 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. "Cond" represents the increase in conductivity in mS/cm. Peaks marked "FT" represents the flow-through.

Figure 3 is a comparison of chromatographic 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 DESCRD?TION OF THE INVENTION

The present invention provides a method of separation of high mannose-type glycoforms using anion exchange chromatography.

In an embodiment, the invention provides a method of separation of high mannose-type glycoforms from a mixture of glycoform composition using anion-exchange chromatography, comprising steps of;

a) loading the said glycoform composition onto an anion exchange chromatographic resin in a buffer solution having a conductivity of about 4 mS/cm to 5.5 mS/cm.
b) separating the high mannose-type glycoform in the flow-through and
c) eluting the bound glycoforms from the said resin
wherein, the eluted glycoform composition is substantially free of high mannose-type glycoforms.

In an embodiment of the invention, the glycoform 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. Therefore, the term "high-mannose type glycoform" as used herein refers to a glycoform having more than 3 mannose residues. Without limitation, the term also includes glycoforms having 5,6,7,8 or 9 or greater number of mannose residues.

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 term "substantially free" as used herein refers to fractions or solution, which is free or has a reduced amount (relative to the input or load) of certain components.
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: Weak Anion Exchange Chromatography

The eluate from example 1 was loaded onto a weak anion exchange chromatographic resin (DEAE-Sepharose, Tricorn 5 x 20, 4 ml) that was pre-equilibrated with 5 column volume (CV) of 50 mM 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 high mannose-type glycoform in the flow-through. The bound TNFR:Fc protein was then eluted using a step-gradient elution in a buffer containing phosphate, sodium chloride, pH 6.3.

Alternatively, the protein can be eluted with a linear gradient of elution buffer using mixture of buffer A (50 mM phosphate buffer, pH 6.3) and buffer B (50 mM phosphate, 0.5 N sodium chloride, pH 6.3).

Example 3: Strong Anion Exchange Chromatography
Alternatively, the eluate from example 1 was loaded onto a strong anion exchange chromatographic resin (Q-Sepharose FF, VL 44 x 200, 300 mL) that was pre-equilibrated with 5 CV of 50 mM phosphate buffer, pH 6.3 at a conductivity of 4.5 mS/cm. The column was then washed with -15 CV of 50 mM phosphate buffer, pH 6.3 at a conductivity of 4.5 mS/cm to obtain the high mannose-type glycoform in the flow-through. The bound TNFR:Fc protein was then eluted using a step-gradient elution buffer containing phosphate, sodium chloride, pH 6.3

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 leakage of bound glycoforms along with high-mannose type glycoform in the flow-through, leading to inefficient separation. Hence, specific conductivity range of less than 6.5 mS/cm is preferred for the separation process.

Analytical HPLC data of eluate of example 3 revealed that eluate was enriched with other glycoforms and was substantially free (-2 %) of high mannose-type glycoform. About 95 % of high mannose-type glycoforms was obtained in FT1 and FT2 (flow-through fractions). Thus the anion exchange chromatographic method according to the invention resulted in a significant, 22-fold reduction of high mannose-type glycoforms from the final glycoprotein composition.

We claim:
1. A method of separation of high mannose glycoforms from a mixture of glycoform composition using ion-exchange chromatography, comprising the following steps:
(a) applying the mixture of glycoform composition onto an ion-exchange chromatographic resin using a buffer solution,
(b) separating the high mannose-type glycoform in the flow-through, and
(c) eluting the bound glycoforms from the said ion-exchange chromatographic resin, wherein, the eluted glycoform composition has reduced amount of high mannose-type glycoforms.

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

3. The glycoform of according to claim 1 is a glycoprotein, wherein the said glycoprotein is an Fc-containing protein or a fusion protein or an antibody molecule.

4. The glycoform of claim 3 is a glycoprotein, wherein the said glycoprotein is TNFR: Fc fusion protein and wherein the said protein is applied onto the anion-exchange chromatographic resin at a pH at least 1 unit greater than the pi of the 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 the conductivity of buffer solution is about 4-7.5 mS/cm and pH is about 6-7.

7. The process according to claim 1, 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.

8. 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.

9. 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.

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