DESC:
Field of the Invention
The present invention relates to a process for the purification of Fc-fusion proteins. More specifically, the invention relates to a process for the purification of Fc-fusion proteins through chromatographic steps that are carried out in bind elute mode to get purified product, which is free from the process-related impurities and product-related impurities. The present invention also relates to a process for reducing the high mannose species in the purified glycoprotein.

Background of the Invention
Proteins are important in biopharmaceuticals as they are widely used to cure several diseases including diabetes (e.g. Insulin), cancers (e.g. Interferon, monoclonal antibodies), heart attacks, strokes, cystic fibrosis (e.g. Enzymes, Blood factors), inflammatory diseases (e.g. Tumor Necrosis Factors), anemia (e.g. Erythropoietin), hemophilia (e.g. Blood clotting factors), etc. One of the major challenges in the development of these proteins is to identify efficient and competent process for the large-scale purification of these proteins. Numerous processes are available for the large-scale purification of the proteins of interest from the harvest cell culture broth, but still few impurities remain in the purified protein of interest which can prove to be detrimental to the long term stability as well as quality of the protein of interest. Another problem in the manufacturing of proteins is to identify efficient method to obtain a desired level of glycosylation.

Although several processes have been reported for the purification of glycoproteins and Fc fusion proteins from the culture broth, due to variability in the cell expression system it has been observed that general purification processes often fail to adequately purify the protein of interest from the process related impurities and product related impurities. The protein of interest produced by the host cells during cell culture or fermentation has to be purified from certain process related impurities such as host cell-derived proteins (HCP), host-cell DNA (HCDNA), process additives; and certain product-related impurities such as degradation products, monomers, low molecular weight impurities (LMWs), and high molecular weight impurities (HMWs), oxidized species, clipped products, unfolded products, and inappropriately glycosylated protein. These impurities are undesirable in the purified protein of interest and their levels need to be kept within the acceptable levels to render the product safe for human therapeutic use.

Fusion proteins are prepared by recombinantly expressing the genes which are created by joining two or more genes that originally code 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. Aflibercept is a recombinant fusion protein consisting of Vascular Endothelial Growth Factor (VEGF)-binding portions from the extracellular domains of human VEGF Receptors 1 and 2 (VEGFR-1 and VEGFR-2) fused to the Fc portion of the human immunoglobulin G1 (IgG1). Aflibercept is a dimeric glycoprotein with a protein molecular weight of 97 kilo Daltons (kDa) and contains glycosylation, constituting an additional 15% of the total molecular mass, resulting in a total molecular weight of 115 kDa. Aflibercept is produced by recombinant DNA technology in a Chinese hamster ovary (CHO) K-1 mammalian expression system. Aflibercept is marketed as Eylea® by Regeneron for the treatment of various ocular conditions, including wet type age related macular degeneration (AMD), and it is formulated for intravitreal administration. Aflibercept is also marketed as Zaltrap® by Sanofi-Aventis for the treatment of certain types of cancer and formulated for intravenous administration.

The US patent US7,070,959 describes expression and several aspects of the purification of Fc-fusion proteins including Aflibercept. The US ‘759 describes process for the purification of Aflibercept, which involves Protein A chromatography using Sepharose column followed by tangential flow filtration and size exclusion chromatography.

Various processes for the purification of glycosylated proteins and fusion proteins are reported in the prior art references such as US20200002373, US20200017544, US20200055894, WO2020252260, US20210171570, US20210284684, US20140323698, US20160083454, etc.

Despite significant advances in the understanding of various chromatography techniques and availability of various buffer systems, protein purification platforms, therapeutic proteins chromatography field remains significantly challenging and unpredictable.

Several chromatography methods have been explored in the state of the art for purification of proteins at commercial scale for removal of impurities from the therapeutic proteins. Protein chromatography methods are primarily carried out in two modes i.e. flow-through mode and bind-elute mode. Flow through chromatography methods rely on the property that protein of interest does not bind or binds minimally to the chromatography column and purified protein is recovered in flow through, while impurities are allowed to bind to the column. In bind-elute mode the protein of interest is first allowed to bind to the chromatography column under suitable conditions and then the conditions are so altered by means of suitable elution solvent or buffer system such that the bonding of the protein to the column could be reversed. Washing the column with suitable wash solvents of buffer systems allow for impurities to be separated from the protein of interest.

The selection of appropriate approach between flow through mode and bind elute mode in each chromatography steps such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography or mixed mode chromatography, is a crucial step. Especially, the choice between bind elute and flow through mode in multimodal chromatography is more complex than when using a single method, because multiple types of interactions are occurring in the multimodal chromatography, and the strength of these individual interactions often depends on the overall process conditions.

The state of art has described various approaches for purification of Fc-fusion proteins. However, these processes involve multiple chromatography steps involving various combinations of flow through and bind elute mode. The Fc-protein purification processes described in the prior art involve at least one chromatography step using flow through mode. The polishing chromatography steps, wherein chromatography is carried out in flow through mode, the eluted protein contains impurities with similar properties. To remove such impurities in the flow through, additional multiple polishing chromatography steps are required.

Further, in state of the art, bioreactor conditions are generally optimized to get desired level of glycosylated species in the molecule. The glycosylated species do not change during typical downstream purification for any antibodies or fusion proteins. Antibodies and Fc-fusion proteins bearing high levels of N-linked mannose-5 glycan (Man5) have been reported to exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and rapid clearance rate. Hence, there is need in the art to identify a downstream step which reduces the high mannose species such as Man-5 and Man-6 from the product to control their level even if the cell culture produces the same in higher amount. Further, the processes described in the state of art require additional steps for obtaining desired level of glycosylation in the glycoprotein of interest. This makes these methods cumbersome, time consuming and expensive. Therefore, present inventors have developed an efficient and economically feasible process for the purification of protein which involves fewer chromatography steps, which produces protein of interest with desired glycosylation.

Summary of the Invention
In an aspect, the invention relates to a process for the purification of Fc-Fusion protein comprising affinity chromatography, mixed mode chromatography and ion exchange chromatography, wherein all the chromatography steps are carried out in bind elute mode.

In another aspect, the invention relates to a process for the purification of Fc-fusion protein comprising:
a) passing a solution comprising Fc-fusion protein and impurities through first chromatography column whereby Fc-fusion protein binds to the column,
b) eluting Fc-fusion protein from first chromatography column to obtain eluate,
c) passing the eluate obtained from step (b) through second chromatography column whereby Fc-fusion protein binds to the second chromatography column,
d) eluting Fc-fusion protein from second chromatography column to obtain eluate
e) passing eluate obtained from step (d) through third chromatography column whereby Fc-fusion protein binds to the third chromatography column,
f) eluting Fc-fusion protein from third chromatography column
wherein, the first chromatography is affinity chromatography.
wherein, the second chromatography column and the third chromatography column are selected from mixed-mode chromatography column and ion exchange chromatography column.

In one more aspect, the present invention relates to a process for reducing the high mannose species in the purified glycoprotein, comprising:
a) contacting glycoprotein solution containing high level of mannose species with cation exchange resin, whereby protein binds to the cation exchange resin; and
b) eluting the glycoprotein from cation exchange resin to obtain glycoprotein containing lower level of high mannose species.

In an embodiment, the invention relates to a process for the purification of aflibercept comprising affinity chromatography, mixed mode chromatography and ion exchange chromatography, wherein all the chromatography steps are carried out in bind elute mode.

Description of the Drawings
Figure 1: Chromatogram of Protein A chromatography in bind elute mode performed according to present invention.
Figure 2: Chromatogram of Mixed mode chromatography in bind elute mode performed according to present invention.
Figure 3: Chromatogram of Cation exchange chromatography of in bind elute mode performed according to present invention.

Detailed Description of Invention
In one embodiment, the present invention provides a process for the purification of Fc-fusion protein comprising:
a) passing a solution comprising Fc-fusion protein and impurities through first chromatography column whereby Fc-fusion protein binds to the column,
b) eluting Fc-fusion protein from first chromatography column to obtain eluate,
c) passing eluate obtained from step (b) through second chromatography column whereby Fc-fusion protein binds to the second chromatography column,
d) eluting Fc-fusion protein from second chromatography column to obtain eluate,
e) passing eluate obtained from step (d) through third chromatography column whereby Fc-fusion protein binds to the third chromatography column,
f) eluting Fc-fusion protein from third chromatography column to obtain purified protein,
wherein, the first chromatography column is affinity chromatography column,
wherein, the second chromatography column and the third chromatography column are selected from mixed-mode chromatography column and ion exchange chromatography column.

The Fc-Fusion protein is selected from a group comprising of but not limited to aflibercept, belatacept, rilonacept, romiplostim, abatacept, alefacept, etanercept or conbercept. In one embodiment the Fc-fusion protein is aflibercept.

In first aspect of the invention, the affinity chromatography is carried out using Protein A or Protein G affinity chromatography. The Protein A affinity chromatography is carried out using a resin selected from the group comprising of MabSelect SuReTM LX (Cytiva), MabSelect™ (GE Cytiva), and ProSep® Ultra Plus (Millipore) resin, in a bind elute mode. The Fc-fusion protein is eluted by pH gradient. Affinity chromatography column is equilibrated with buffers selected from the group comprising of but not limited to Tris, Bis, and phosphate buffers. In one of the aspect, affinity chromatography column is equilibrated with a Tris buffer containing sodium chloride and EDTA at a pH about 8.0 ± 0.5. After equilibration, solution containing Fc-fusion protein and impurities is loaded onto the column. After passing the solution containing Fc-fusion protein onto the column, it is washed with wash buffers in order to remove impurities from the protein. Washing of the column is carried with same or different buffers selected from Tris, Bis, acetate or phosphate buffers, preferably buffers is selected from Tris and acetate buffer. The pH of the wash buffers is selected from the pH range from about 5 to about 8. After washing the column, protein of interest is eluted from the affinity column with elution buffer by using pH gradient from pH about 5 to about 3. The elution buffer is selected from acetate buffer, glycinate buffer and citrate buffer, preferably with the acetate buffer.

Protein A chromatography step according to present invention removes both process related impurities such as HCP, HCDNA and product related impurities such as oxidized species, HMW and LMWs.

The obtained protein A chromatography eluate is taken further for two polishing chromatography steps viz. mixed mode chromatography and ion exchange chromatography. In one aspect of the invention, these two chromatography steps are carried in either order i.e. carrying out first mixed mode chromatography and then ion exchange chromatography; or first carrying out ion exchange chromatography and then mixed mode chromatography. In a preferred aspect of the invention, mixed mode chromatography is carried out first and then ion exchange chromatography step.

The pH of the eluate containing protein obtained from protein A chromatography is optionally reduced for viral inactivation and subsequently neutralized to pH about 5 before loading the eluate onto the first polishing chromatography column selected from the mixed mode chromatography and an ion exchange chromatography, preferably a mixed mode chromatography.

In second aspect of the invention, mixed mode chromatography is carried out in bind elute mode using pH gradient between pH about 9.0 to about 5.0, preferably between pH 8.0 to 6.0. According to the invention, mixed mode chromatography is carried out using resin selected from the group comprising of PPA-HyperCelTM (propylphenyl amine) (Pall), HEA-HyperCelTM (hexylamine) (Pall), or CaptoAdhere® (Cytiva). The mixed mode chromatography column is equilibrated with equilibration buffers selected from Tris buffer, Bis buffer and phosphate buffer. Thereafter, the eluate obtained from the protein A chromatography step is loaded onto mixed mode chromatography column. After loading protein solution, column is optionally, washed with a wash buffer. The wash buffer is selected from Tris buffer, Bis buffer and phosphate buffer at pH about 8 ± 1, for efficient removal of HCP, HCDNA, HMWs and residual protein A. Thereafter, protein of interests is eluted with elution buffer selected from acetate buffer, glycinate buffer, citrate buffer, phosphate buffer and preferably phosphate buffer. The elution is carried out at pH about 6.0.

In one of the embodiments, the mixed mode chromatography column is equilibrated with 50 mM Tris having pH about 8.0 ± 1 and then protein solution is loaded onto the mixed mode chromatography column. The mixed column is then washed with a Tris buffer of pH about 8.0 ± 1 to remove impurities. Thereafter, protein of interest is eluted with phosphate buffer at pH about 6.0 ± 1.

In third aspect of the invention, the eluate obtained from the mixed mode chromatography step is further purified by using ion exchange chromatography in a bind elute mode and pH gradient from about pH 5.0 (± 1) to about pH 6.5 (± 1). The ion exchange chromatography according to present invention is an anion exchange chromatography or a cation exchange chromatography; preferably cation exchange chromatography.

In one embodiment, cation exchange chromatography is carried out using weak cation exchange resins is selected from the group comprising of Fractogel® COO-(M) (Millipore), carboxymethyl (CM), sulfoethyl(SE), sulfopropyl(SP), phosphate(P), sulfonate(S), NuviaTM S (Bio-Rad), CaptoTM S (Cytiva) and Gigacap S (Tosoh) resins. The column is equilibrated with buffers selected from Tris buffer, Bis buffer, phosphate buffer and acetate buffer. Thereafter, the eluate obtained from the mixed mode chromatography is loaded onto the cation exchange chromatography column. Thereafter, column is optionally washed with the buffer selected from Tris buffer, Bis buffer, phosphate buffer and acetate buffer, preferably sodium phosphate buffer at pH about 6.5 (± 0.5). In one aspect of the invention, the cation exchange chromatography carried out according to present invention, brings down the level of Man5 and Man6 species by about 50 % to about 60 % in the purified product.

In one more embodiment, the present invention provides to a process for the purification of aflibercept comprising:
a) passing a solution comprising aflibercept and impurities through Protein A affinity chromatography column whereby aflibercept binds to protein A column,
b) eluting aflibercept from protein A chromatography column to obtain first eluate,
c) passing eluate obtained from step (b) through mixed mode chromatography column whereby aflibercept binds to the mixed mode chromatography column,
d) eluting aflibercept from the mixed mode chromatography column to obtain second eluate
e) passing the eluate obtained from step (d) through cation exchange column whereby aflibercept binds to the cation exchange column, and
f) eluting aflibercept from cation exchange column to obtain purified aflibercept.

In one more embodiment, present invention relates to a method of treating ophthalmic diseases by administering a suitable amount of Aflibercept to a patient in need thereof wherein aflibercept is prepared according to the present invention.

The examples which follow are illustrative of the invention and are not intended to be limiting.

Example 1 Purification of fusion protein using Affinity Chromatography
3 CV of equilibration buffer containing 50 mM Tris, 150 mM sodium chloride and 5 mM EDTA with pH 8.2 were passed through MabSelect SuRe LX® column. After equilibration, protein solution obtained after clarification of culture broth was loaded onto the column. After loading the protein solution, column was washed with 3 CV of first wash buffer containing 50 mM Tris, 150 mM NaCl and 5 mM EDTA at pH 8.2; and then second wash buffer containing 50 mM Sodium Acetate at pH about 5.0. Thereafter 5 CV of elution buffer containing 100 mm Sodium acetate with pH 3.40 was loaded onto the column and eluate was collected.

Table 1: Impurities removal details in Protein A chromatography
Impurities Protein A Chromatography Load Protein A chromatography Eluate
HMW (%) 12 5
LMW (%) 10 1
Oxidized species (%) 9 5
HCP (ppm) 700000 4000
HCDNA (pg/ml) 1000000 BQL (<750 )

Example 2 Purification using mixed mode chromatography
3 CV of equilibration buffer containing 50 mM Tris having pH 8.0 were passed through Capto Adhere® column to equilibrate the column. After equilibration, protein eluate solution obtained from the protein A chromatography was loaded onto the column. After loading the protein solution, column was washed with 3 CV of wash buffer containing 50 mM Tris at pH 8.0. Thereafter 10 CV of elution buffer containing 50 mm Sodium acetate with pH 6.0 was loaded onto the column and eluate was collected.

Table 2: Impurities removal details in Capto Adhere® chromatography

Impurities Mixed Mode chromatography load Mixed Mode chromatography eluate
HMW (%) 5 1
Oxidized species (%) 5 3
HCP (ppm) 4000 50

Example 3 Purification using Cation Exchange Chromatography

5 CV of equilibration buffer containing 50 mM Tris having pH 8.0 were passed through Fractogel® COO- (M) column to equilibrate the column. After equilibration, protein eluate solution obtained from the mixed mode chromatography was loaded onto the column. After loading the protein solution, column was washed with 3 CV of wash buffer containing 100 mM sodium phosphate at pH 5.5 to remove impurities. Thereafter, 6.5 CV of elution buffer containing 50 mm Sodium phosphate of pH 6.5 was loaded onto the column and eluate was collected.

Table 3: Impurities removal details in Fractogel® COO Chromatography

Impurities Cation exchange chromatography load Cation exchange chromatography eluate
HMW (%) 1 <0.2
LMW (%) 1 <0.2
HCP (ppm) 100 <20

Table 4: Detail of Man5 and Man6 species present in the Cation Exchange column load and eluate collected:

Parameters Chromatography load Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Fraction 6 Fraction 7
Actual amount of Man5 and Man6 in sample ( %) 2.5 0.68 0.91 0.73 1.03 1.31 1.64 2.05
% of Man5 and Man6 present in sample wrt load NA 27 36 29 41 52 66 82
% of Man5 and Man6 removed in fractions wrt load NA 73 64 71 59 48 34 18
,CLAIMS:
1. A process for the purification of Fc-fusion protein comprising:
a) passing a solution comprising Fc-fusion protein and at least one impurity through first chromatography column whereby Fc-fusion protein binds to the column,
b) eluting the Fc-fusion protein from first chromatography column to obtain eluate,
c) passing the eluate obtained from step (b) through second chromatography column whereby Fc-fusion protein binds to the second chromatography column,
d) eluting Fc-fusion protein from second chromatography column to obtain eluate,
e) passing the eluate obtained from step (d) through third chromatography column whereby Fc-fusion protein binds to the third chromatography column, and
f) eluting Fc-fusion protein from third chromatography column
wherein, the first chromatography is affinity chromatography
wherein, the second chromatography column and the third chromatography column are selected from mixed-mode chromatography column and ion exchange chromatography column.

2. The process according to claim 1, wherein Fc-fusion protein is selected from a group comprising of aflibercept, belatacept, rilonacept, romiplostim, abatacept, alefacept, etanercept and conbercept.

3. The process according to claim 1, wherein Fc-fusion protein is aflibercept.

4. The process according to claim 1, wherein ion exchange chromatography is cation-exchange chromatography.

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

6. The process according to claim 1, wherein second chromatography is mixed mode chromatography.

7. The process according to claim 1, wherein third chromatography is ion-exchange chromatography.
8. The process according to claim 1, wherein affinity chromatography is Protein-A chromatography.

9. A process for reducing the high mannose species in the glycoprotein, comprising:
a) contacting glycoprotein solution containing high level of mannose species with cation exchange resin, whereby protein binds to the cation exchange resin; and
b) eluting the glycoprotein from cation exchange resin to obtain glycoprotein containing lower level of high mannose species.

10. The process according to claim 9, wherein the level of high mannose species is lowered by about 50 % to 60 %.

11. The process according to claim 9, wherein cation exchange resin is resin selected from the group comprising of Fractogel® COO-(M) (Millipore), carboxymethyl (CM), sulfoethyl(SE), sulfopropyl(SP), phosphate(P), sulfonate(S), NuviaTM S (Bio-Rad), CaptoTM S (Cytiva) and Gigacap S (Tosoh) resins.

12. The process according to claim 9, wherein glycoprotein is eluted with the buffer selected from the group comprising of Tris buffer, Bis buffer, phosphate buffer and acetate buffer.