DESC:FIELD OF INVENTION
The present invention relates to protein purification methods. In particular, disclosed is a method for purifying fusion proteins using anion exchange chromatography.
BACKGROUND OF INVENTION
Fc-fusion proteins are bioengineered polypeptides that join the crystallizable fragment (Fc) domain of an antibody with another biologically active protein domain to generate a molecule with unique structure–function properties and significant therapeutic potential. The gamma immunoglobulin (IgG) isotype is often used as the basis for generating Fc-fusion proteins because of favorable characteristics such as recruitment of effector function and increased plasma half-life. Given the range of proteins that can be used as fusion partners, Fc-fusion proteins have numerous biological and pharmaceutical applications, which has launched Fc-fusion proteins into the forefront of drug development.
Fc-fusion proteins can be commercially manufactured using platform upstream and downstream methods based for monoclonal antibodies (mAb). However, in Fc-fusion proteins receptor domains generally contain one or more glycosylation sites (both N- and O-linked) in contrast to a single glycosylation site for mAbs. Also, the oligosaccharide structures are more varied and complex (complex and high mannose; bi-, tri- and tetra-antennary) in their receptor domains than IgG Fc (complex, bi-antennary) and can contain more sialic acid residues. Presence of large number of sialic acid residues can shift the isolectric point (pI) of Fc-fusion proteins into an acidic pH range and impart significantly more charge heterogeneity on them than that of the conventional mAbs. Hence, there are unique attributes of Fc-Fusion proteins that would require optimization or redesigning of the commercial process used for manufacture of mAbs. Glycosylation, including sialylation, plays a vital role in protein solubility, stability, serum half-life, activity, and immunogenicity. Specifically, sialylation is one of the critical attributes affecting the pharmacokinetics of a therapeutic protein and the level of sialic acid can have a significant impact in the phamaco-kinetics (PK) of the Fc-fusion protein molecules.
Cytotoxic T-lymphocyte associated protein 4–immunoglobulin (CTLA4-Ig) fusion protein is a highly glycosylated therapeutic fusion protein that contains multiple N- and O-glycosylation sites. In CTLA4-Ig fusion protein, 3 N-linked sites and one O-linked site were reported rendering the protein to be glycosylated variedly with different glycosylation patterns. While reduced sialylation being an issue, higher sialylation with di-, tri- or tetra-sialylated antennary structured glycosylated pattern are more common in Fc-fusion protein molecules. It becomes essential to obtain optimal sialylation content with a control on the di-, tri- or higher sialylated isoforms, as the level of sialic acid content is known to impact the pharmacokinetics of the protein significantly.
The primary objective of the invention is to find a suitable chromatography method to control the di- and tri-sialic acid isoforms in the Fc-protein composition.
SUMMARY OF THE INVENTION
The present invention discloses a method of controlling the percentage of sialylated isoforms in a composition comprising Fc-fusion proteins, by employing ion exchange chromatography. Particularly, the method, effectively controls di- and tri-sialic acid isoforms of the fusion protein in the fusion protein composition. More specifically, the invention discloses a weak anion-exchange chromatographic method, wherein the elution mode in the chromatographic step is chosen/altered between linear and step gradient to control or obtain the desired amount of di- and tri-sialic acid isoforms in the fusion protein composition. Additionally, the method also provides an increased recovery of the Fc-fusion protein.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
"Fc fusion protein" is a protein that contains an Fc region of an immunoglobulin fused or linked to a polypeptide. The heterologous polypeptide fused to the Fc region may be a polypeptide from a protein other than an immunoglobulin protein. For instance, the heterologous polypeptide may be a ligand polypeptide, a receptor polypeptide, a hormone, cytokine, growth factor, an enzyme, or other polypeptide that is not a component of an immunoglobulin. Such Fc fusion proteins may comprise an Fc region fused to a receptor or fragment thereof or a ligand from a receptor including, but not limited to, any one of the following receptors: both forms of TNFR (referred to as p55 and p75), Interleukin-1 receptors types I and II (as described in EP Patent No. 0460846, US Patent No. 4,968,607, and US Patent No. 5,767,064, which are incorporated by reference herein in their entirety), Interleukin-2 receptor, Interleukin-4 receptor (as described in EP Patent No. 0 367 566 and US Patent No. 5,856,296, which are incorporated by reference herein in their entirety), Interleukin-15 receptor, Interleukin-17 receptor, Interleukin-18 receptor, granulocyte- macrophage colony stimulating factor receptor, granulocyte colony stimulating factor receptor, receptors for oncostatin-M and leukemia inhibitory factor, receptor activator of NF-kappa B (RANK, as described in US Patent No. 6,271,349, which is incorporated by reference herein in its entirety), VEGF receptors, EGF receptor, FGF receptors, receptors for TRAIL (including TRAIL receptors 1,2,3, and 4), and receptors that comprise death domains, such as Fas or Apoptosis-Inducing Receptor (AIR). Fc fusion proteins also include peptibodies, such as those described in WO 2000/24782, which is hereby incorporated by reference in its entirety.
The "composition" to be purified herein comprises the protein of interest and one or more contaminants. The composition may be "partially purified" (i.e., having been subjected to one or more purification steps) or may be obtained directly from a host cell or organism producing the antibody (e.g., the composition may comprise harvested cell culture fluid).
As used herein the term "molar ratio of sialic acids to CTLA4-Ig fusion protein" is calculated and given as number of moles of sialic acid molecules per mole of protein (CTLA4-Ig molecules) or dimer. Further, it is referred as mol/mol ratio in the present invention.
As used herein, the term "glycoprotein" refers to a protein that is modified by the addition of one or more carbohydrates, including the addition of one or more sugar residues.
As used herein, the term "sialylation" refers to the addition of a sialic acid residue to a protein, including a glycoprotein. Sialic acid is a common name for a family of unique nine-carbon monosaccharides, which can be linked to other oligosaccharides. Two family members are N-acetyl neuraminic acid, abbreviated as Neu5Ac or NANA, and N-glycolyl neuraminic acid, abbreviated as Neu5Gc or NGNA. The most common form of sialic acid in humans is NANA. N-acetylneuraminic acid (NANA) is the primary sialic acid species present in CTLA4-Ig molecules. However, it should be noted that minor but detectable levels of N glycolylneuraminic acid (NGNA) are also present in CTLA4-Ig molecules. Furthermore, the method described herein can be used to determine the number of moles of sialic acids for both NANA and NGNA, and therefore levels of both NANA and NGNA are determined and reported for CTLA4-Ig molecules. , sialic acid is the terminal residue of both N-linked and O-linked oligosaccharides.
As used herein, the term “di- and tri-sialic acid isoforms” refers to the number of sialic acid residues on the N-linked oligosachharaides, wherein di-sialic acid isoforms refers to two and tri-sialic acid isoforms refers to three sialic acid residues terminally attached to N-linked oligosachharides. Further, higher sialic acid isoforms, such as four sialic acid residues terminally attached to N-linked oligosaccharides are referred as “tetra-sialic acid isoforms.” Higher sialylated isoforms includes four or more sialic acid residues terminally attached to N-linked oligosachharides.
“High molecular weight aggregates” as referred herein encompasses association of at least two molecules of a product of interest, e.g., Fc-Fusion protein. The association of at least two molecules of a product of interest may arise by any means including, but not limited to, non-covalent interactions such as, e.g., charge-charge, hydrophobic and van der Waals interactions; and covalent interactions such as, e.g., disulfide interaction or no reducible crosslinking. An aggregate can be a dimer, trimer, tetramer, or a multimer greater than a tetramer, etc.
Aggregate concentration can be measured in a protein sample using Size Exclusion Chromatography (SEC), a well-known and widely accepted method in the art. Size exclusion chromatography uses a molecular sieving retention mechanism, based on differences in the hydrodynamic radii or differences in size of proteins. Large molecular weight aggregates cannot penetrate or only partially penetrate the pores of the stationary phase. Hence, the larger aggregates elute first and smaller molecules elute later, the order of elution being a function of the size.
The term “Mixed Mode Chromatography” refers to a form of chromatography that uses a chromatographic support with at least two unique types of functional groups, each interacting with the molecule or protein of interest. Mixed mode chromatography generally uses ligands that have more than one type of interaction with target proteins and/or impurities. For example, a charge-charge type of interaction and/or a hydrophobic or hydrophilic type of interaction, or an electroreceptor-donor type interaction. In general, based on the difference in the total interaction, the target protein and one or more impurities can be separated under various conditions.
The term "Anion Exchange Chromatography'" refers to a form of ion-exchange chromatography that uses a support with functional groups that exchanges anions.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The present invention discloses a method of controlling the percentage of sialylated isoforms in a composition comprising Fc-Fusion proteins, wherein the said method comprises use of ion exchange chromatography.
In an embodiment, the method is used to control the percentage of di- and tri-sialylated isoforms in a composition comprising Fc-fusion proteins, wherein the said method comprises use of weak anion exchange chromatography and the mode of operation is bind and elute.
In another embodiment, the method is used to control the percentage of di- and tri-sialylated isoforms of an Fc-fusion protein in a composition comprising Fc-fusion proteins, comprising the steps of:
1. loading the composition comprising the Fc-fusion protein and di- and tri-sialylated isoforms on a weak anion exchange chromatography support,
2. optionally washing the anion exchange chromatography support with a wash buffer solution, and
3. eluting the composition comprising the Fc-fusion protein and di and tri-sialylated isoforms using a linear salt gradient or a step salt gradient,
wherein the linear salt gradient is employed when the desired percentage of di- and tri-sialylated isoforms in the eluate is between 15% and 25% and the step salt gradient is employed when the desired percentage of di- and tri-sialylated isoforms in the eluate is between 25% and 35%, and
wherein the reduction in percentage of di- and tri-sialylated isoforms and recovery achieved by employing the linear salt gradient is 7% and 75%, respectively, and that achieved by employing the step salt gradient is 4% and 95%, respectively.
In any of the above embodiments, the conductivity range of the elution buffer solutions used for eluting the protein of interest is between 3 and 22 mS/cm when a linear salt gradient elution is employed and between 12 and 16 mS/cm when a step salt gradient elution is employed.
In yet another embodiment, the said method comprises use of one or more chromatographic steps before weak anion chromatography, wherein the preceding chromatography does not comprise an ion exchange chromatography step. In particular, the polishing steps may be selected from a group comprising hydrophobic interaction chromatography, ion exchange chromatography, hydrophobic charge induction chromatography and Mixed Mode chromatography.
In another embodiment, the support used for weak anion exchange chromatography is selected from, Diethylaminoethyl (DEAE), diethyl- (2-hydroxy-propyl) aminoethyl (QAE) or DEAE Sepharose Fast Flow.
In any of the above mentioned embodiments, anion exchange chromatography is the last chromatographic step for the purification of the said Fc-fusion protein.
In any of the above mentioned embodiments, the method employs use of one or more steps such as viral inactivation, filtration and diafiltration. These steps may be interspersed between the chromatographic steps or after all the chromatographic steps.

In any of the above mentioned embodiments, the Fc-fusion protein is CTLA4-Ig fusion protein.

In any of the above mentioned embodiments, the Fc-fusion protein is abatacept.
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.
EXAMPLE
Example 1: Purification of CTLA4-Ig fusion protein using Weak Anion Exchange Chromatography
A CTLA4-Ig fusion protein, more specifically abatacept, was cloned and expressed in a Chinese Hamster Ovary cell line and the cell culture broth containing the expressed fusion protein was harvested, clarified and subjected to protein-A affinity chromatography. The eluate from protein-A affinity chromatography was subjected to low-pH incubation and depth filtration, and the obtained eluate was subjected to Hydrophobic Interaction Chromatography that was operated in flow-through mode. Tangential flow filtration (TFF) process was carried out after HIC chromatography to concentrate HIC process output and to exchange the buffer for the next chromatography step. The output of the TFF step comprising the protein of interest was subjected to Mixed Mode Chromatography by loading onto a Ceramic Hydroxyapatite (CHT) resin. The flow-through fraction obtained from CHT resin comprising the protein of interest was used as a starting material for the Weak AEX chromatography. Two chromatographic columns were packed separately with DEAE Sepharose FF resin and the AEX chromatography was carried out in two columns parallelly. The output of mixed mode chromatography was split into two fractions and loaded onto the two AEX columns independently, wherein the protein was interest was bound to the resin of the AEX column. Both columns were equilibrated with a buffer solution containing 60 mM Tris-Acetate (pH 7.5, conductivity 3 mS/cm). The resins were then washed with a buffer solution having the same composition as the one used for equilibration in the previous step. The bound CTLA4-Ig fusion protein was eluted using a salt gradient. Two different elution criteria were employed based on the percentage of di- and tri-sialic acid isoforms desired in the AEX eluate. The protein of interest in one column was eluted by employing a linear salt gradient elution using an elution buffer of the composition 60 mM tris acetate, 0.2 M NaCl (0 to 100%, 15 column volumes; pH 7.5, conductivity 22 mS/cm). The protein of interest in the other column was eluted by employing a step salt gradient elution using an elution buffer of the composition 60 mM tris acetate, 0.12 M NaCl (pH 7.5, conductivity 14 mS/cm). The outputs of both the columns were collected as fractions based on absorption of ultraviolet light at a wavelength of 280 nm (UV-280 signal) separately and analysed for the percentage of di- and tri-sialic acid isoforms using high performance hydrophilic interaction chromatography (HILIC-UPLC). Protein recovery using the linear gradient elution was found to be about 70% and using step gradient elution was found to be about 90%. The percentage of di- and tri-sialic acid isoforms in the load and eluate of AEX chromatography employing step gradient elution is shown in Table 1 and the one employing linear gradient elution is shown in Table 2.

Sample Batch 1 Batch 2 Batch 3
Di+Tri Sialic acid isoforms (%)
AEX load 35.3 33.8 28.4
AEX eluate 31 30.7 25.7
Table 1: Percentage of di-+tri-sialic acid isoforms in AEX load and AEX eluate in step gradient elution mode

Sample Batch 1 Batch 2 Batch 3
Di+Tri Sialic acid isoforms (%)
AEX load 22.6 30.3 22.6
AEX eluate 16.1 25.2 20.4
Table 2: Percentage of di-+tri-sialic acid isoforms in AEX load and AEX eluate in linear gradient elution mode

The eluate from AEX chromatography may then be subjected to one or more ultra/dia filtration steps and buffer exchange steps and/or sterile filtration to obtain a therapeutic composition to be administered for human use.
,CLAIMS:CLAIMS
I/We claim:
1. A method for controlling the percentage of di- and tri-sialylated isoforms of an Fc-fusion protein in a composition comprising Fc-fusion proteins, comprising the steps of:
a. loading the composition comprising the Fc-fusion protein and di- and tri-sialylated isoforms on a weak anion exchange chromatography support,
b. optionally washing the anion exchange chromatography support with a wash buffer solution, and
c. eluting the composition comprising the Fc-fusion protein and di and tri-sialylated isoforms using a linear salt gradient or a step salt gradient,
wherein the linear salt gradient is employed when the desired percentage of di- and tri-sialylated isoforms in the eluate is between 15% and 25% and the step salt gradient is employed when the desired percentage of di- and tri-sialylated isoforms in the eluate is between 25% and 35%, and
wherein the reduction in percentage of di- and tri-sialylated isoforms and recovery achieved by employing the linear salt gradient is 7% and 75%, respectively, and that achieved by employing the step salt gradient is 4% and 95%, respectively.
2. The method as claimed in claim 1, wherein elution is achieved with an elution buffer in a conductivity range of 3 – 22 mS/cm when a linear salt gradient is employed.
3. The method as claimed in claim 1, wherein elution is achieved with an elution buffer in a conductivity range of 12 – 16 mS/cm when a step salt gradient is employed.
4. The method as claimed in claim 1, wherein anion exchange chromatography is the final chromatographic step for the purification of the said Fc-fusion protein.
5. The method as claimed in claim 1, wherein the Fc-fusion protein is CTLA4-Ig fusion protein.
6. The method as claimed in claim1, wherein the Fc-fusion protein is abatacept.