DESC:FIELD OF THE INVENTION
The present invention relates to protein purification methods. In particular, disclosed is a method for purifying a fusion proteins using affinity chromatography.
BACKGROUND OF THE 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 as used monoclonal antibody (mAb) production. However, Fc-fusion proteins, receptor domains generally contain one or more glycosylation sites (both N- and O-linked) in contrast to single glycosylation site in mAb’s. Further, the oligosaccharide structures are more varied and complex (complex and high mannose; bi-, tri-, and tetraantennary) in the receptor domains than the ones in IgG Fc domains (complex, bianntennary), and former can contain more sialic acid residues. The latter quality can shift the pI of Fc-fusion proteins into an acidic pH range and impart significantly more charge heterogeneity on them than mAbs have. Hence, there are unique attributes of Fc-Fusion proteins that would require optimization or redesigning of the commercial process used for manufacture of mAbs.
Protein-A chromatography is one of the most widely used capture steps for therapeutic proteins such as antibodies and Fc-fusion proteins. Protein-A is a surface protein produced by the cell wall of gram positive bacterium Staphylococcus aureus. It is composed of five homologous immunoglobulin-binding domains – A, B, C, D and E. Initially, wild-type Protein-A has been used extensively as a ligand to capture immunoglobulins. Currently, Protein-A variants recombinantly produced by protein engineering are used as ligands in many techniques for improving the column performance. However, low pH is used for elution from Protein-A resins, which leads to aggregation in therapeutic proteins. The object of the current invention is to provide a method to purify an Fc-fusion protein using a modified Protein-A resin.
SUMMARY OF THE INVENTION
The present invention discloses a method for purifying the Fc-Fusion proteins from the contaminants, the method comprises use of an affinity chromatography (Protein-A chromatography) with steps of load and elution, to purify the Fc-fusion protein. Specifically, the pH of the elution step results in purified Fc protein. In particular, the pH of the elution buffer in Protein-A chromatography is about 4.0 and further, the ‘Protein-A’ ligand bound to the chromatography matrix is modified in domain C. More specifically, the Protein-A ligand used in the current invention is a mutated multimer of domain C.
The mildly acidic pH, i.e., pH of about 4.0 of the elution buffer, results in improved removal of the aggregates from the fusion protein composition. Furthermore, the mildly acidic pH, i.e., pH of about 4.0 to 4.2, of the elution buffer, inhibits the additional formation of aggregates in fusion protein composition which is prone to aggregation to low acidic pH.

DETAILED DESCRIPTION OF THE INVENTION
Definitions
"Fc fusion protein" is a protein that contains an Fc region fused or linked to a heterologous polypeptide. For instance, the heterologous polypeptide may be a ligand polypeptide, a receptor polypeptide, a hormone, cytokine, growth factor, an enzyme. A fusion protein is expressed from a fusion gene in which a nucleotide sequence encoding a polypeptide sequence from one protein (e.g. an Fc region) is appended in frame with, and optionally separated by a linker from, a nucleotide sequence encoding a polypeptide sequence from a different protein. The fusion gene can then be expressed by a recombinant host cell to produce the single fusion protein.
As used herein, "purifying a protein" refers to a process that reduces the amounts of substances that are different than the target protein and are desirably excluded from the final protein composition. The term “contaminants” as used herein can include proteins (e.g., soluble or insoluble proteins, or fragments of proteins, including undesired fragments of the protein of interest, such as half antibodies), lipids (e.g., cell wall material), endotoxins, viruses, nucleic acids (e.g., chromosomal or extrachromosomal DNA, t-RNA, rRNA, or mRNA), or combinations thereof, or any other substance that is different from the target Fc region-containing protein of interest. The contaminant can originate from the host cell that produced the Fc region-containing protein of interest. For example, in some embodiments, the impurity or contaminant is a host cell protein, host cell nucleic acid (DNA or RNA), or other cellular component of a prokaryotic or eukaryotic host cell that expressed the Fc region-containing protein of interest. The contaminants can also be the impurity or contaminant is not derived from the host cell, e.g., the impurity or contaminant could be a protein or other substance from the cell culture media or growth media, a buffer, or a media additive.
The term “chromatography material” as used herein denotes a solid material comprising a bulk core material to which chromatographic functional groups are attached, preferably by covalent bonds. The bulk core material is understood to be not involved in the chromatography process, i.e. the interaction between the polypeptide to be separated and the chromatographic functional groups of the chromatography material. It is merely providing a three dimensional framework to which the chromatographic functional groups are attached and which ensures that the solution containing the substance to be separated can access the chromatographic functional group. Preferably said bulk core material is a solid phase. Thus, preferably said “chromatography material” is a solid phase to which chromatographic functional groups are attached, preferably by covalent bonds.
When used herein, the term “Protein A” encompasses Protein A recovered from a native source thereof, Protein A produced synthetically (e.g. by peptide synthesis or by recombinant techniques), and variants thereof which retain the ability to bind proteins which have a CH2/CH3 region. Protein-A has been used traditionally as a ligand for antibody purification. Wild type Protein-A is composed of five immunoglobulin-binding domains – namely E, D, A, B and C. The term ‘Protein-A ligand modified in domain C’ as used here in, represents a recombinant Protein-A ligand with a multimer of domain C, wherein domain C is modified to have mutations in few amino acids as compared to the wild-type domain C of Protein-A. An example of a commercially available Protein-A resin having modified domain C is KanCapA™ 3G.
“Protein A chromatography” refers to the separation or purification of substances and/or particles using protein A, where the protein A is generally immobilized on a solid phase such as cellulose. A protein comprising a CH2/CH3 region may be reversibly bound to or adsorbed by the protein A.
The term “High molecular weight aggregates” as referred herein encompasses association of more than 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 nonreducible crosslinking. An aggregate can be a trimer, tetramer, or a multimer greater than a tetramer, etc. The term “protein aggregates,” includes any higher order species of the Fc-containing protein.
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.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The present invention discloses a method for purifying the Fc-Fusion proteins from the contaminants, the method comprises use of a Protein-A affinity chromatography, comprising steps of load and elution performed with appropriate buffers, and wherein the pH of elution buffer is about 4.0.
The present invention discloses a method for purifying the Fc-Fusion proteins from the contaminants, the method comprises Protein-A chromatography wherein the Protein-A is modified in domain C and the pH of the elution buffer in Protein-A chromatography is about 4.0.
In any of the above embodiments, the pH of the elution buffer in Protein-A chromatography is 4.2.
In any of the above mentioned embodiments, the fusion protein is CTLA4-IgG1 fusion protein.
In any of the above mentioned embodiments, the fusion protein is abatacept.
In any of the above mentioned embodiments, the contaminants are high molecular weight aggregates.
In any of the above mentioned embodiments, the percentage of high molecular weight aggregates is about or less than 4.5%.
In any of the above mentioned embodiments, the purity of the fusion protein composition is more than 95%.
In any of the above mentioned embodiments, the percentage reduction in percentage of high molecular weight aggregates in the fusion protein composition is about 80% as compared to percentage of high molecular weight aggregates in the fusion protein composition obtained otherwise i.e. elution buffer has pH about 3.5.
In any of the above mentioned embodiments, the percentage reduction in percentage of high molecular weight aggregates in the fusion protein composition is about 80% as compared to percentage of high molecular weight aggregates in the fusion protein composition obtained otherwise i.e. when Protein-A ligand is native or may be modified except at domain C.
One embodiment of the invention discloses a method for purifying the CTLA4-IgG1 fusion protein from the high molecular weight aggregates, the method comprising protein-A chromatography wherein the Protein-A is modified in domain C and the pH of the elution buffer in Protein-A chromatography is about 4.0, wherein the purified CTLA4-IgG1 fusion comprises less than 4.5% of the high molecular weight aggregates.
One embodiment of the invention discloses a method for purifying the abatacept from the high molecular weight aggregates, the method comprising Protein-A chromatography wherein the Protein-A is modified in domain C and the pH of the elution buffer in Protein-A chromatography is about 4.0, wherein the purified abatacept fusion comprises less than 4.5% of the high molecular weight aggregates.
In one embodiment of the invention, the % high molecular weight aggregates in the Protein-A affinity chromatography eluate is reduced in direct proportionality to the increasing pH of the elution buffer, when the elution is carried out independently with buffer of different pH values. For example, the % high molecular weight aggregate in Protein-A chromatography eluate is 22.5% at pH 3.4 of the elution buffer (120 mM acetate), the % high molecular weight aggregate in Protein-A chromatography eluate is 18.0% at pH 3.8 of the elution buffer (100 to 120 mM acetate), the % high molecular weight aggregate in Protein-A chromatography eluate is 13.0% at pH 4.0 of the elution buffer (100 to 120 mM acetate), and the % high molecular weight aggregate in Protein-A chromatography eluate is 4.5% at the pH 4.2 of the elution buffer (90 to 100 mM acetate).
In any of the above mentioned embodiments, the method comprises use of one or more polishing chromatography steps after Protein-A chromatography. In particular, the polishing steps may be selected from ion exchange chromatography, hydrophobic ion exchange chromatography, hydrophobic charge induction chromatography, mixed-mode chromatography.
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.
The invention is better understood with the help of the following examples. These examples, however, should not be construed to limit the scope of the invention.
EXAMPLES
Abatacept 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 as described below.
Example 1: Protein-A (Resin A) chromatography
The clarified cell culture broth was loaded onto a Protein-A chromatography column (Resin A) that was equilibrated with equilibration buffer (30 mM sodium acetate, 0.15 M sodium chloride pH 7.0). The column was then washed with a high-salt buffer. The bound abatacept (Sample 1A) was eluted using elution buffer 1 (120 mM sodium acetate) having pH of 3.5. In an alternate, the bound abatacept (Sample 1B) was eluted using elution buffer 2 (90-100 mM acetate+150 mM sodium chloride) having pH of 4.2.
The % of high molecular weight aggregates in Sample 1A and Sample 1B are represented in Table 1.
Example 2: Protein-A (KanCapA™ 3G) chromatography
The resin employed for Protein-A chromatography in this example (KanCapA™ 3G) is modified at C domain (C’’) of the native Protein-A molecule.
The clarified cell culture broth was loaded onto a Protein-A chromatography column (Resin B) that was equilibrated with equilibration buffer (30 mM sodium acetate, 0.15 M sodium chloride pH 7.0). The column was then washed with a high-salt buffer. The bound abatacept was eluted using elution buffer 1 (120 mM sodium acetate) having pH of 3.5 (Sample 2A). In an alternate, the bound abatacept was eluted using elution buffer 2 (90-100 mM acetate+150 mM sodium chloride) having pH of 4.2 (Sample 2B). When abatacept was eluted using a buffer devoid of sodium chloride (i.e., the elution buffer 2 with 90 – 100 mM acetate, but devoid of sodium chloride), the % high molecular weight aggregates was found not to be significantly different (Sample 2C) when compared to the values obtained in Sample 2B.
The percentage of high-molecular weight aggregates in samples (as per examples 1 and 2) are represented in Table 1.

Sample Name % High molecular weight aggregates
Sample 1A 25%
Sample 1B 25%
Sample 2A 25%
Sample 2B 6.3%
Sample 2C 4.5%
Table 1
,CLAIMS:We Claim:
1. A method of purifying an Fc-fusion protein from high-molecular weight aggregates, the method comprising steps of:
(a) contacting a solution comprising the Fc-fusion protein and high molecular weight aggregates with a chromatography material comprising a cellulose-based Protein-A ligand, wherein the Fc-fusion protein binds to the chromatography material, and wherein the Protein-A ligand is modified in domain C,
(b) optionally washing the chromatography material with a wash buffer solution and
(c) eluting the bound Fc-fusion protein from the chromatography material using a buffer solution at pH between 4.0 and 4.2, thereby separating the Fc-fusion protein from high molecular weight aggregates.
2. The method of purification as claimed in claim1, wherein the solution comprising the Fc-fusion protein in step (a) comprises between 20% and 30% high molecular weight aggregates, as measured by size exclusion chromatography.
3. The method of purification as claimed claim 1, wherein the eluate from chromatography in step (c) comprises about 4.5% high molecular weight aggregates, as measured by size exclusion chromatography.
4. The method of purification as claimed in claim 1, wherein the purity of the Fc-fusion protein obtained in the eluate of chromatography is greater than 95%.
5. The method of purification as claimed in claim 1, wherein the method may be followed by one or more polishing steps selected from ion exchange chromatography, hydrophobic interaction chromatography, and mixed mode chromatography.
6. The method of purification as claimed in claim 1, wherein the method may be followed by one or more purification steps selected from viral inactivation, filtration and diafiltration.
7. The method of purification as claimed in claim 1, wherein the Fc-fusion protein is a CTLA4-Ig fusion protein.
8. The method of purification as claimed in claim 1, wherein the Fc-fusion protein is abatacept.