FIELD OF THE INVENTION

The present invention relates to purification process of pharmacologically active IgG1 containing protein comprising at least affinity chromatography followed by mixed-mode chromatography. The present invention provides cytotoxic T-lymphocyte-associated 4-immunoglobulin (CTLA4-Ig) fusion protein by using at least affinity chromatography, mixed-mode chromatography and optionally one or more suitable purification steps that provides purified composition of the fusion protein, substantially free of impurities selected from Pre-Peak, product and process related impurities. Further, the present invention provides highly purified CTLA4-Ig fusion protein with reduced heterogeneity.

BACKGROUND OF THE INVENTION

Purification at large scale of proteins, e.g., therapeutic proteins including antibodies or fusion protein, is an increasingly important consideration for biopharmaceutical industries. With the advancement of biotechnology, fusion proteins are considered a promising therapeutic area for the treatment of diseases. However, fusion proteins are complex in nature as made of fusion of receptor (natural or modified) and immunoglobulin constant region (Fc including with or without hinge region or modified Fc). As per the complexity of the fusion proteins and its challenging purification process gives a motivation to minimize the impurities from fusion protein that proportionally affect the stability and functional efficacy. The recent advances in mammalian cell culture processes have significantly increased product titers as well as process and product-related impurities. Aggregation, charge variants, high molecular weight (HMW), low molecular weight (LMW) like impurities with the fusion protein has been a major problem that has been associated with a change in protein structure and being a hurdle in various upstream and downstream purification processes. The Fc-fusion proteins have elevated levels of aggregates, high molecular weight species (HMWs; up to 20%) and low molecular weight species (LMWs; up to 20%) within the product species. Glycosylation of proteins and the subsequent processing of the added carbohydrates can affect protein folding and structure, protein stability, including protein half-life, and functional properties of a protein. Desired glycosylation can be obtained through clone and upstream process. These impurities generated during the manufacturing process leads to decreased product yield, peak broadening, and reduce or loss of activity of molecule. The improvement or a step forward in Fc-fusion protein processing is crucially needed as conventional chromatography column requires various conditioning parameters of protein before loading onto chromatography column, lacking effective purification process to remove product and process related impurities, that directly impacts process time, cost, and labor effort in protein handling. The present invention provides a purification process by selecting and arranging the affinity chromatography, mixed-mode chromatography and optionally one or more suitable purification steps to remove impurities associated with the fusion protein. The process reduces or removes at least one impurity selected from Pre-Peak, HMW, LMW, HCP, and undesired glycan.

There is a present need for methods of producing and purifying a fusion protein of interest in sufficiently pure form to be suitable for pharmaceutical use.

SUMMARY OF THE INVENTION

In an embodiment, the invention provides a chromatography process to produce a composition enriched with fusion protein of interest and substantially reduced impurities selected from acidic variants, basic variants, high molecular weight (HMW), low molecular weight (LMW), pre-peak or aggregates.

In an embodiment, the invention provides a chromatography process to produce a composition enriched with fusion protein of interest and substantially reduced amount of acidic variant impurities.

In an embodiment, the invention provides a chromatography process to produce a composition enriched with fusion protein of interest and substantially reduced basic variant impurities.

In an embodiment, the invention provides a chromatography process to produce a composition enriched with fusion protein of interest and substantially reduced high molecular weight (HMW) impurities.

In an embodiment, the invention provides a chromatography process to produce a composition enriched with fusion protein of interest and substantially reduced low molecular weight (LMW) impurities.

In an embodiment, the invention provides a chromatography process to produce a composition enriched with fusion protein of interest and substantially reduced level of undesired glycans.

In an embodiment, the invention provides a chromatography process to produce a composition enriched with fusion protein of interest and substantially reduced pre-peak impurities or aggregates. In an embodiment the invention provides a mixed-mode (MMC) chromatography to produce a composition enriched with fusion protein of interest and substantially reduced pre-peak impurities or aggregates.

In an embodiment, a method for purifying a Fc-fusion protein from a protein sample comprising Fc-fusion protein and Pre-peak impurity:

- a) contacting the protein sample onto mixed-mode chromatography column;
- b) optionally perform washing the mixed-mode chromatography column;
- c) eluting the fusion protein from a mixed-mode chromatography column; wherein the eluted fusion protein comprises reduce amount of Pre-peak impurity.

In one aspect of such embodiment, the pre-peak impurity reduced by at least more than about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%. In an embodiment, the pre-peak impurity is below quantifiable limit.

In an embodiment, a method for purifying a Fc-fusion protein from a protein sample comprising:

- a) contacting the protein sample onto mixed-mode chromatography column;
- b) washing the mixed-mode chromatography column with buffer comprising a suitable additive;
- c) eluting the fusion protein from a mixed-mode chromatography column with reduced amount of impurities;
- wherein, the suitable additive is arginine or arginine derivatives.

In an embodiment, the concentration of arginine or arginine derivatives is selected from about 20 mM to about 100 mM.

In another embodiment, the concentration of arginine or arginine derivative is selected from about 60 mM to about 80 mM.

In an embodiment, a method for purifying a Fc-fusion protein from a protein sample comprising:

- a) contacting the protein sample onto mixed-mode chromatography column;
- b) washing the mixed-mode chromatography column with a suitable buffer comprising suitable additive;
- c) eluting the fusion protein from a mixed-mode chromatography column;
- wherein the elution buffer comprises arginine or arginine derivatives concentration selected from below 600 mM, preferably 300 mM.

In an embodiment, the eluted fusion protein from mixed-mode chromatography column comprises reduced amount impurity compared to elution without using arginine or arginine derivatives in wash and elution buffers.

In an embodiment, a process for purification of IgG1 containing protein comprising:

- a) collecting the IgG1 containing protein from the suitable mammalian expression system and the impurities;
- b) contacting the IgG1 containing protein on to a affinity chromatography;
- c) eluting the IgG1 containing protein;
- d) contacting the eluted IgG1 containing protein onto mixed-mode chromatography, optionally followed by other suitable purification steps, wherein mixed-mode chromatography is performed in bind-elute mode.

In an embodiment, the process bind-elute mode comprises: (i) loading the eluted IgG1 protein from Affinity chromatography at suitable pH and/or conductivity for binding (ii) performing washing at suitable pH and/or conductivity (iii) eluting the IgG1 protein at suitable pH and/or conductivity.

In an embodiment, the loading is performed at suitable pH selected from about 6.5 to about 7.5.

In an embodiment, the loading is performed at suitable conductivity selected from about 3 mS/cm to about 5 mS/cm.

In an embodiment, the invention provides an affinity chromatography and mixed mode chromatography to produce a composition comprising enriched CTLA-4 IgG1 fusion protein and significantly reduced high molecular weight (HMW) impurities selected from about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5% or less HMW.

In one aspect of this embodiment, the low HMW composition comprises about 0.9% or less HMW, about 0.8% or less HMW, about 0.7% or less HMW, about 0.6% or less HMW, about 0.5% or less HMW, about 0.4% or less HMW, about 0.3% or less HMW, about 0.2% or less HMW, about 0.1% or less HMW.

In an embodiment, the invention provides an affinity chromatography and mixed mode chromatography to produce a composition comprising enriched CTLA-4 IgG1 fusion protein and reduced LMW impurities selected from about 50%, about 40%, about 30%, about 20%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or less LMW or below quantifiable limit.

In an embodiment, a method for purifying a Fc-fusion protein from a protein sample comprising Fc-fusion protein and at least Pre-peak impurity:

- a) collecting first protein sample/mixture from the suitable mammalian expression system comprising Fc-fusion protein and the impurities;
- b) contacting the first protein sample/mixture to affinity chromatography column;
- c) eluting the Fc-fusion protein from affinity chromatography column to form second protein mixture;
- d) contacting the second protein mixture to mixed-mode chromatography column;
- e) eluting the fusion protein from a mixed-mode chromatography column to form third protein mixture;
- f) contacting the third protein mixture to anion exchange chromatography column;
- g) eluting the fusion protein from anion exchange chromatography column to form fourth protein mixture;
- h) contacting the fourth protein mixture to hydrophobic interaction chromatography column;
- i) eluting the fusion protein from hydrophobic interaction chromatography column to form fifth protein mixture;

wherein, the eluted Fc-fusion protein is substantially free of Pre-peak impurities.

In one aspect of such embodiment, the pre-peak impurity reduced by at least more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. In an embodiment the pre-peak impurity is below quantifiable limit.

In an embodiment, a method for purifying a Fc-fusion protein from a protein sample comprising Fc-fusion protein and at least Low molecular weight (LMW):

- a) collecting first protein sample/mixture from the suitable mammalian expression system comprising Fc-fusion protein and the impurities;
- b) contacting the first protein sample/mixture to affinity chromatography column;
- c) eluting the Fc-fusion protein from affinity chromatography column to form second protein mixture;
- d) contacting the second protein mixture to mixed-mode chromatography column;
- e) eluting the fusion protein from a mixed-mode chromatography column to form third protein mixture;
- f) contacting the third protein mixture to anion exchange chromatography column;
- g) eluting the fusion protein from anion exchange chromatography column to form fourth protein mixture;
- h) contacting the fourth protein mixture to hydrophobic interaction chromatography column;
- i) eluting the fusion protein from hydrophobic interaction chromatography column to form fifth protein mixture;

wherein, the eluted fusion protein is substantially free of low molecular weight (LMW) impurities.

In an embodiment, the invention utilizes chromatography column to produce a Fc-fusion protein composition comprising about 0.5% or less LMW, about 0.4% or less LMW, about 0.3% or less LMW, about 0.2% or less LMW, about 0.1% or less LMW.

In an embodiment, a method for purifying a Fc-fusion protein from a protein sample comprising Fc-fusion protein and at least High molecular weight (HMW):

- a) collecting first protein sample/mixture from the suitable mammalian expression system comprising Fc-fusion protein and the impurities;
- b) contacting the first protein sample/mixture to affinity chromatography column;
- c) eluting the Fc-fusion protein from affinity chromatography column to form second protein mixture;
- d) contacting the second protein mixture to mixed-mode chromatography column;
- e) eluting the fusion protein from a mixed-mode chromatography column to form third protein mixture;
- f) contacting the third protein mixture to anion exchange chromatography column;
- g) eluting the fusion protein from anion exchange chromatography column to form fourth protein mixture;
- h) contacting the fourth protein mixture to hydrophobic interaction chromatography column;
- i) eluting the fusion protein from hydrophobic interaction chromatography column to form fifth protein mixture;

wherein, the eluted fusion protein is substantially free of high molecular weight (HMW) impurities.

In an embodiment, the Fc-fusion protein composition comprising about 0.5% or less HMW, about 0.4% or less HMW, about 0.3% or less HMW, about 0.2% or less HMW, about 0.1% or less HMW.

In an embodiment, a method for purifying a Fc-fusion protein from a protein sample comprising Fc-fusion protein and undesired glycans:

- a) collecting first protein sample/mixture from the suitable mammalian expression system comprising Fc-fusion protein and the impurities;
- b) contacting the first protein sample/mixture to affinity chromatography column;
- c) eluting the Fc-fusion protein from affinity chromatography column to form second protein mixture;
- d) contacting the second protein mixture to mixed-mode chromatography column;
- e) eluting the fusion protein from a mixed-mode chromatography column to form third protein mixture;
- f) contacting the third protein mixture to anion exchange chromatography column;
- g) eluting the fusion protein from anion exchange chromatography column to form fourth protein mixture;
- h) contacting the fourth protein mixture to hydrophobic interaction chromatography column;
- i) eluting the fusion protein from hydrophobic interaction chromatography column to form fifth protein mixture;

wherein, the eluted fusion protein is substantially free of undesired glycans.

In such embodiment, undesired glycans selected from high mannose and afucosylated variant are reduced by 76.54% and about 24.35% respectively.

In such embodiment, high mannose reduced by less than 80% preferably less than 77%.

In such embodiment, afucosylated variants are reduced by less than 30% preferably less than 25%.

In one aspect of such embodiment, the all-column chromatography is performed in bind elute mode.

In one aspect of such embodiment, all column chromatography uses same buffer.

In one aspect of such embodiment, viral inactivation is performed with a suitable detergent for about 60 minutes.

In one aspect of such embodiment, pH-based virus inactivation is not performed in eluate of affinity chromatography.

In an embodiment, the invention is related to the process for the purification of the fusion protein which is free from at least one impurity selected from Host cell proteins (HCP) comprising:

- a) collecting the first protein sample/mixture from the suitable mammalian expression system comprising fusion protein and impurities;
- b) contacting the protein sample/mixture to affinity chromatography column;
- c) eluting the Fc-fusion protein from affinity chromatography column to form second protein mixture;
- d) contacting the second protein mixture to mixed-mode chromatography column;
- e) eluting the fusion protein from a mixed-mode chromatography column to form third protein mixture;
- f) contacting the third protein mixture to anion exchange chromatography column;
- g) eluting the fusion protein from anion exchange chromatography column to form fourth protein mixture;
- h) contacting the fourth protein mixture to hydrophobic interaction chromatography column;
- i) eluting the fusion protein from hydrophobic interaction chromatography column to form fifth protein mixture;

wherein the eluted fusion protein is substantially free of host cell proteins (HCP) impurities.

In an embodiment, the fusion protein comprises reduced HCP impurities at drug substance level after purification selected from about 10 ng/mg or less, 9 ng/mg or less, 8 ng/mg or less, 7 ng/mg or less, 6 ng/mg or less, 5 ng/mg or less, 4 ng/mg or less, 3 ng/mg or less, 2 ng/mg or less, 1 ng/mg or less.

In an embodiment, the invention provides an affinity chromatography and mixed mode chromatography to produce a composition comprising enriched fusion protein and significantly reduced high molecular weight (HMW) impurities about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5% or less.

In one aspect of this embodiment, the low HMW composition comprises about 0.5% or less HMW, about 0.4% or less HMW, about 0.3% or less HMW, about 0.2% or less HMW, about 0.1% or less.

In an embodiment, the invention provides an affinity chromatography and mixed mode chromatography to produce a composition comprising enriched fusion protein and substantially reduced LMW or fragments.

In such embodiment, LMW reduced by about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, or about 0.3% less.

In an embodiment, the low LMW composition comprises about 0.5% less LMW, about 0.4% or less LMW, about 0.3% or less LMW, about 0.2% or less LMW, about 0.1% or less LMW or below quantifiable limit.

In an embodiment, the invention is related to the purification of CTLA-4 IgG1 fusion protein from a protein mixture comprising:

- a) collecting the first protein sample/mixture from the suitable mammalian expression system comprising fusion protein and impurities;
- b) contacting the protein sample/mixture to affinity chromatography column;
- c) eluting the CTLA-4 IgG1 fusion protein from affinity chromatography column to form second protein mixture;
- d) contacting the second protein mixture to mixed-mode chromatography column;
- e) eluting the CTLA-4 IgG1 fusion protein from a mixed-mode chromatography column to form third protein mixture;
- f) contacting the third protein mixture to anion exchange chromatography column;
- g) eluting the fusion protein from anion exchange chromatography column to form fourth protein mixture;
- h) contacting the fourth protein mixture to hydrophobic interaction chromatography column;
- i) eluting the fusion protein from hydrophobic interaction chromatography column to form fifth protein mixture;

wherein the eluted CTLA-4 IgG1 fusion protein is substantially free of LMW and HMW impurities.

In an embodiment, the invention is related to the purification of CTLA-4 IgG1 fusion protein from a protein mixture comprising:

- a) collecting the first protein sample/mixture from the suitable mammalian expression system comprising fusion protein and impurities;
- b) contacting the protein sample/mixture to affinity chromatography column;
- c) eluting the CTLA-4 IgG1 fusion protein from affinity chromatography column to form second protein mixture;
- d) contacting the second protein mixture to mixed-mode chromatography column;
- e) eluting the CTLA-4 IgG1 fusion protein from a mixed-mode chromatography column to form third protein mixture;
- f) contacting the third protein mixture to anion exchange chromatography column;
- g) eluting the CTLA-4 IgG1 fusion protein from anion exchange chromatography column to form fourth protein mixture;
- h) contacting the fourth protein mixture to hydrophobic interaction chromatography column;
- i) eluting the CTLA-4 IgG1 fusion protein from hydrophobic interaction chromatography column to form fifth protein mixture;

wherein the eluted fusion protein is substantially free of Pre-peak, LMW and HMW impurities.

In an embodiment, the invention is related to the purification of CTLA-4 IgG1 fusion protein from a protein mixture comprising:

- a) collecting the first protein sample/mixture from the suitable mammalian expression system comprising fusion protein and impurities;
- b) contacting the protein sample/mixture to affinity chromatography column;
- c) eluting the CTLA-4 IgG1 fusion protein from affinity chromatography column to form second protein mixture;
- d) contacting the second protein mixture to mixed-mode chromatography column;
- e) eluting the CTLA-4 IgG1 fusion protein from a mixed-mode chromatography column to form third protein mixture;
- f) contacting the third protein mixture to anion exchange chromatography column;
- g) eluting the CTLA-4 IgG1 fusion protein from anion exchange chromatography column to form fourth protein mixture;

wherein the eluted fusion protein is substantially free of Pre-peak, LMW and HMW impurities.

In an embodiment, the invention provides an affinity chromatography, mixed mode chromatography and anion exchange chromatography to produce a composition comprising enriched CTLA-4 IgG1 fusion protein and significantly reduced high molecular weight (HMW) impurities selected from about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5% or less HMW.

In an embodiment, the invention provides an affinity chromatography, mixed mode chromatography and anion exchange chromatography to produce a composition comprising enriched CTLA-4 IgG1 fusion protein and reduced LMW impurities selected from about 50%, about 40%, about 30%, about 20%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or less LMW or below quantifiable limit.

In an embodiment, the invention provides an affinity chromatography, mixed mode chromatography and anion exchange chromatography to produce a composition comprising enriched CTLA-4 IgG1 fusion protein and reduced undesired glycans. In such embodiment, undesired glycans are selected from high mannose are reduced by less than 80%, preferably 77%.

In such embodiment, undesired glycans selected from high mannose and afucosylated variants.

In such embodiment, high mannose reduced by less than 80%, preferably less than 77%.

In such embodiment, afucosylated variants are reduced by less than 30%, preferably less than 25%.

In such embodiment, the high mannose is reduced by about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 79%, about 78%, about 77%, about 76%, and about 75%.

In an embodiment the present invention provides a large-scale purification process of CTLA4-Fc fusion protein to maintain the quality and/or quantity of purified protein composition.

DETAIL DESCRIPTION OF FIGURES

FIG. 1—Process Chromatogram of Affinity chromatography (Protein A).

FIG. 2—Process chromatogram of Mixed-mode chromatography (MMC).

FIG. 3—Process chromatogram of Anion exchange chromatography (AEX).

FIG. 4—Process Chromatogram of Hydrophobic interaction chromatography (HIC).

FIG. 5—CE-SDS Chromatogram of Final DS.

FIG. 6—SEC-HPLC Chromatogram of Final DS.

DETAIL DESCRIPTION OF INVENTION

As used herein the term “column” or “resin” or “chromatographic resin or chromatographic column” are interchangeable.

The term “comprises” or “comprising” is used in the present description, it does not exclude other elements or steps. For purpose of the present invention, the term “consisting of” is considered to be an optional embodiment, of the term “comprising of”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which optionally consists only of these embodiments. As used throughout the specification and in the appended claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise.

The term “about”, as used herein, is intended to refer to ranges of approximately 10-20% greater than or less than the referenced value. In certain circumstances, one of skill in the art will recognize that, due to the nature of the referenced value, the term “about” can mean more or less than a 10-20% deviation from that value.

The term “clarified harvest cell culture fluid” or “HCCF” or “harvest cell culture fluid” used herein are interchangeable refers to the protein mixture obtained from mammalian cell culture containing protein of interest along with other impurities.

The term “affinity chromatography” refers to chromatography processes of separating biochemical mixtures based on a highly specific interaction between e.g., antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid. Examples of such chromatographic resins include, but are not limited to Protein A resin, Protein G resin, Protein L resin, immobilized metal ion affinity chromatography etc.

The term “Protein A affinity chromatography” use for the separation or purification of substances and/or particles using protein A, where the protein A is generally immobilized on a solid phase.

Protein A is a 40-60 kD cell wall protein originally found in Staphylococcus aureus. The binding of fusion protein to protein A resin is highly specific. Protein A affinity chromatography columns for use in protein A affinity chromatography herein include, but are not limited to, Protein A immobilized on a polyvinylether solid phase, e.g., the Eshmuno® columns (Merck, Darmstadt, Germany), Protein A immobilized on a pore glass matrix, e.g., the ProSep® columns (Merck, Darmstadt, Germany) Protein A immobilized on an agarose solid phase, for instance the MABSELECT™ SuRe™ columns (GE Healthcare, Uppsala, Sweden).

The term “MabSelect SuRe LX” used herein is a protein A affinity resin with very high dynamic binding capacity at extended residence times. The ligand is alkali-stabilized protein A-derived (E. coli) shows alkali tolerance, high capacity and low ligand leakage in combination with the rigid base matrix.

The term “mixed mode chromatography” or “MMC” refers to chromatography a combination of two or more chemical interactions within in a solid support. The examples of chemical interactions that can be combined in mixed mode supports include but are not limited to cation exchange, anion exchange, hydrophobic interaction, hydrophilic interaction, hydrogen bonding, pi-pi bonding, and metal affinity.

The term “Nuvia aPrime 4A” used herein is a hydrophobic anion exchange resin designed with a distinct balance of modes optimized for biomolecule interactions. The resin's ligand density and hydrophobicity are engineered to facilitate selective and reversible binding of target molecules for high purity and yield. Nuvia aPrime offers a wide design space and straightforward method development to purify even the most complex molecules The resin has aromatic hydrophobic anion exchanger chemistry.

The term “anion exchange chromatography” or “anion exchange column” or “AEX” used herein is a form of ion exchange chromatography (IEX), which is used to separate molecules based on their net surface charge. Anion exchange chromatography, more specifically, uses a positively charged ion exchange resin with an affinity for molecules having net negative surface charges. Anion exchange chromatography is used both for preparative and analytical purposes and can separate a large range of molecules, from amino acids and nucleotides to large proteins. Here, we focus on the preparative anion exchange chromatography of proteins.

As used herein the term “bind and elute mode” or “B/E” refers to purification process wherein the fusion protein of interest binds to chromatography resin. At least about 90% fusion protein of interest bind to chromatographic resin. At least about 60% or about 70% or about 80% fusion protein of interest binds to chromatographic resin. However, process and product related impurities does not bind the chromatographic resin. At least about 50% process and product related impurities does not bind to chromatographic resin. At least about 60% or about 70% or about 80% process and product related impurities does not bind to chromatographic resin.

In an embodiment, the anion exchange is strong anion exchange. The term “POROS XQ” used herein is a Thermo Scientific POROS XQ Strong Anion Exchange Resin are designed for charge based chromatographic separations of biomolecules including recombinant proteins, monoclonal antibodies. POROS XQ has high and consistent protein capacity across a broad range of salt concentrations. The resin has quaternary amine groups.

The term “hydrophobic interaction chromatography” or “HIC” refers to a column containing a stationary phase or resin and a mobile or solution phase in which the hydrophobic interaction between a protein and hydrophobic groups on the matrix serves as the basis for separating a protein from impurities including fragments and aggregates of the subject protein of interest, other proteins or other protein fragments and other contaminants such as cell debris, or residual impurities from other purification steps. The stationary phase comprises a base matrix or support such as a cross-linked agarose, silica or synthetic copolymer material to which hydrophobic ligands are attached. HIC relies on separation of proteins on the basis of hydrophobic interactions between non-polar regions on the surface of proteins and insoluble, immobilized hydrophobic groups on the matrix. The absorption increases with high salt concentration in the mobile phase and the elution is achieved by decreasing the salt concentration of the eluant.

The term “Poros Benzyl” used herein is Hydrophobic Interaction Chromatography (HIC) resin engineered for improved impurity removal during large-scale downstream purification of biomolecules and its capacity maintains excellent pressure flow characteristics, helps increase in productivity. The resin has POROS base bead with aromatic hydrophobic benzyl ligand, suited for purification.

The term “viral reduction/inactivation”, as used herein, is intended to refer to a decrease in the number of viral particles in a particular sample (“reduction”), as well as a decrease in the activity, for example, but not limited to, the infectivity or ability to replicate, of viral particles in a particular sample (“inactivation”).

The term used “pre-peak” demonstrates the analysis of the product related variant. The peak with the shortest retention time arising just before the main peak or monomer which contains product related impurities. Pre-peak may include but not limited to misfolded and aggregates of CTLA4-Ig.

The term “buffer” or “suitable buffer” refers to a solution that can resist pH change upon the addition of an acidic or basic components. It is able to neutralize small amounts of added acid or base, thus maintaining the pH of the solution relatively stable. This is important for processes and/or reactions which require specific and stable pH ranges. Buffer solutions have a working pH range and capacity which dictate how much acid/base can be neutralized before pH changes, and the amount by which it will change.

The term used herein “Buffer B” or “Elution buffer B” in mixed mode chromatography are interchangeable. For a non-limiting example, it refers to the buffer solution of 20 mM sodium phosphate, 300 mM L-Arginine HCl at pH 7.2±0.2, conductivity 21.0±3.0 mS/cm.

The term used herein “Buffer B” or “Elution buffer B” in anion exchange chromatography are interchangeable. For a non-limiting example, it refers to the buffer solution of 20 mM sodium phosphate, 300 mM sodium chloride at pH 7.2±0.2, conductivity 30.0±3.0 mS/cm.

The term used herein “Buffer B” or “Elution buffer B” in hydrophobic interaction chromatography are interchangeable. For a non-limiting example, it refers to the buffer solution of 20 mM sodium phosphate at pH 7.2±0.2, conductivity 2.7±0.3 mS/cm. The term “neutralization” used herein refers to bringing back the acidic pH of the protein A eluate to a neutral pH by using basic solution.

The term used “aggregates” are classified based on types of interactions and solubility. Soluble aggregates are invisible particles and cannot be removed with a filter. Insoluble aggregates can be removed by filtration and are often visible to the human eye. Both types of aggregates cause problems in biopharma development. Covalent aggregates arise from the formation of a covalent bond between multiple monomers of a given peptide. Disulfide bond formation of free thiols is a common mechanism for covalent aggregation. Oxidation of tyrosine residues can lead to formation of bityrosine which often results in aggregation. Reversible protein aggregation typically results from weaker protein interactions they include dimers, trimers, multimers among others.

The term “Mannose” used herein refers to the high mannose.

The term “Afucosylation” used herein refers to the protein molecule do not have any fucose sugar units.

The term “undesired glycan” used herein refers to the N-glycan of the fusion protein that is beyond the acceptable range. In an embodiment, the acceptable N-glycan ranges includes high mannose, afucosylation, total sialylation which should be in desired range. The N-glycan profile of fusion protein should be in the acceptable range as mentioned above to comply the regulatory requirements.

The term “Host cell protein” or “HCP” refers to process-related protein impurities produced by the host organism during biotherapeutic manufacturing and production. HCPs may cause immunogenicity in individuals or reduce the potency, stability or overall effectiveness of a drug.

The term “Host cell DNA” or “HCD” used herein are interchangeable and refers to residual DNAs (rDNAs) are trace/low quantity of DNA originating from the organisms used in the production process of biopharmaceutical products.

The term used “high molecular weight” or “HMW” used herein are interchangeable and refers to product-related impurities that contribute to the size heterogeneity of fusion protein drug product. The formation of HMW species within a therapeutic fusion protein drug product as a result of protein aggregation can potentially compromise both drug efficacy and safety (e.g., eliciting unwanted immunogenic response). HMW is considered critical quality attribute that are routinely monitored during drug development and as part of release testing of purified drug product during manufacturing. In certain embodiment, the HMW relates to aggregates dimer, trimer or tetramer of CTLA4-Ig or monoclonal antibodies.

The term used “low molecular weight” or “LMW” used herein are interchangeable and refers to species which is a protein backbone-truncated fragments & considered as product-related impurities that contribute to the size heterogeneity of fusion protein. LMW species often have low or substantially reduced activity relative to the monomeric form of the fusion protein and can lead to immunogenicity or potentially impact pharmacokinetic properties in vivo. As a result, LMW species are considered critical quality attributes that are routinely monitored during drug development and as part of release testing of purified drug product during manufacturing. The LMW has molecular weight selected from less than about 80 kDa or less, 75 kDa or less, 70 kDa or less, 65 kDa or less, 60 kDa or less, 55 kDa or less, 50 kDa or less, 45 kDa or less, 40 kDa or less, 35 kDa or less, 30 kDa or less, 25 kDa or less, 20 kDa or less, 15 kDa or less, 10 kDa or less.

The term “substantially pure fusion protein” used herein includes a fusion protein that is substantially free of impurity selected from product or process related impurities. The fusion protein is free of acidic variants, basic variants, low molecular weights and high molecular weights, substantially pure fusion protein has purity less than about 99% or less than about 98% or less than about 97% or less than about 95% or less than about 92% or less than about 90% or less than about 88% or less than about 85% or less than about 82% less than about 80% or less than about 75% or less than about 70% or less than about 65% or less than about 60% or less than about 50%.

The term “protein mixture” or “protein sample” used herein are interchangeable and refers to the solutions containing protein of interest along with other impurities.

The term “first protein mixture” or “first protein sample” or “HCCF” or “Harvest cell culture fluid” used herein are interchangeable which refers to the protein solution or mixture or sample obtained from mammalian cell culture before performing any chromatography column.

The term “second protein mixture” or “second protein solution” or “second protein sample” used herein are interchangeable which refers to the eluate of affinity chromatography.

The term “Neutralized Protein A Eluate” or “NPEL” used herein are interchangeable and it refers to the protein A eluate which has been further neutralized to a neutral pH. The term “Neutralized Protein A Elute” or “NPEL” also considering herein as “second protein mixture” or “second protein solution” or “second protein sample”.

The term “third protein mixture” or “third protein solution” or “third protein sample” used herein are interchangeable which refers to the eluate of mixed mode chromatography (MMC).

The term “fourth protein mixture” or “fourth protein solution” or “fourth protein sample” used herein are interchangeable which refers to the eluate of anion exchange chromatography (AEX).

The term “fifth protein mixture” or “fifth protein solution” or “fifth protein sample” used herein are interchangeable which refers to the eluate of Hydrophobic Interaction chromatography (HIC).

The term “Residence time” refers to the amount of time a compound spends on the column after it has been injected. If a sample containing several compounds, each compound in the sample will spend a different amount of time on the column according to its chemical composition i.e., each will have a different retention time. Retention times are usually quoted in units of seconds or minutes.

The term “IgG1 containing protein” used herein refers to antibody or fusion protein. Fusion proteins or Fc-fusion protein are receptor fused with IgG (‘ligand traps’) have also been generated. TNF receptor 2-Fc (etanercept), rilonacept (Arcalyst—an IL-1 Trap), vascular endothelial growth factor trap (aflibercept), CTLA4-Fc fusion proteins (Abatacept and belatacept).

In another embodiment, antibodies are selected from IgG1, IgG2, IgG3, IgG4 and fusion proteins. In certain embodiments the antibodies are selected from Etanercept, Rituximab, Palivizumab, Infliximab, Trastuzumab, Alemtuzumab, Adalimumab, Ibritumomab tiuxetan, Omalizumab, Cetuximab, Bevacizumab, Natalizumab, Eculizumab, Certolizumab pegol, Ustekinumab, Canakinumab, Golimumab, Ofatumumab, Tocilizumab, Denosumab, Belimumab, Ipilimumab, Brentuximab vedotin, Pertuzumab, Trastuzumab emtansine, Raxibacumab, Obinutuzumab, Siltuximab, Ramucimmab, Vedolizumab, Blinatumomab, Nivolumab, Pembrolizumab, Darucizumab, Necitumumab, Dinutuximab, Secukinumab, Mepolizumab, Alirocumab, Evolocumab, Daratumumab, Elotuzumab, Ixekizumab, Reslizumab, Olaratumab, Bezlotoxumab, Atezolizumab, Obiltoxaximab, Sarilumab, Ocrelizumab, Tildrakizumab, Romosozumab, Brolucizumab, and Crizanlizumab.

In an embodiment, the CTLA4-IgG1 or CTLA4-Fc is Abatacept or Belatacept.

The terms “CTLA4-Ig” or “CTLA4-Ig molecule” or “CTLA4-Fc molecule” used herein are interchangeable and refers to a protein molecule that comprises at least a polypeptide having a CTLA4 extracellular domain or portion thereof and an immunoglobulin constant region or portion thereof. The extracellular domain and the immunoglobulin constant region can be wild-type, or mutant or modified, and mammalian, including human or mouse. The polypeptide can further comprise additional protein domains. A CTLA4-Ig molecule can also refer to multimer forms of the polypeptide, such as dimers, tetramers, and hexamers. A CTLA4-Ig molecule is also capable of binding to CD80 and/or CD86. In an embodiment, the CTLA4-Ig is Abatacept.

The term “contacting” used herein refers to the loading of the protein mixture into the chromatography column.

The term “Drug Substance” or “DS” used herein are interchangeable and refers to an active ingredient intended to furnish pharmacological activity. The DS also refers to final protein mixture after all purification steps and substantially free from impurities.

In an embodiment, the present invention provides a combination of chromatographic methods selected from protein A chromatography, mixed mode chromatography, anion exchange chromatography and hydrophobic interaction chromatography to purify at least one impurity present in the mixture of CTLA-4 IgG1 fusion protein.

In an embodiment, the invention is related to a process for the purification of fusion protein by using Protein A (ProA) chromatography works in the bind and elute mode.

In another embodiment, the invention is related to a process for the purification of fusion protein followed by Mixed mode chromatography (MMC) works in the bind and elute mode.

In another embodiment, the invention is related to a process for the purification of fusion protein followed by Anion exchange chromatography (AEX) works in bind and elute mode.

In another embodiment, the invention is related to a process for the purification of fusion protein followed by Hydrophobic interaction chromatography (HIC) works in the bind and elute mode.

In yet another embodiment, the invention is related to the purification of fusion protein mixture comprising:

- a) loading the fusion protein mixture onto first chromatography column;
- b) optionally performing the washing;
- c) eluting the fusion protein mixture from said first chromatography column;
- d) optionally performing the filtration;
- e) loading the fusion protein mixture obtained from step (C) or (D) onto mixed-mode chromatography;
- f) optionally performing the washing;
- g) eluting the fusion protein mixture from said mixed-mode chromatography;

wherein the first chromatography column is selected from Protein-A column, Hydrophobic interaction chromatography, anion exchange chromatography and cation exchange chromatography.

In an embodiment, a method for purifying a Fc-fusion protein from a protein sample comprising Fc-fusion protein and Pre-peak impurity:

- a) contacting the protein sample onto mixed-mode chromatography column;
- b) optionally perform washing the mixed-mode chromatography column;
- c) eluting the fusion protein from a mixed-mode chromatography column;

wherein the eluted fusion protein comprises reduce amount of Pre-peak impurity.

In an embodiment, a process for purification of IgG1 containing protein comprising:

- a) collecting the IgG1 containing protein from the suitable mammalian expression system and the impurities;
- b) contacting the collecting the IgG1 containing protein Affinity chromatography;
- c) eluting the IgG1 containing protein;
- d) contacting the eluted IgG1 containing protein onto mixed-mode chromatography, optionally followed by other suitable purification steps, wherein mixed-mode chromatography is performed in bind-elute mode.

In one aspect of such embodiment, the pre-peak impurity reduced by at least more than about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%. In an embodiment the pre-peak impurity is below quantifiable limit.

In one aspect of this embodiment, the low HMW composition comprises about 0.5% or less HMW, about 0.4% or less HMW, about 0.3% or less HMW, about 0.2% or less HMW, about 0.1% or less HMW.

In an embodiment, the hydrophobic interaction chromatography reduces HMW and/or LMW by less than 50% preferably 70%.

In yet another embodiment, the invention is related to the purification of fusion protein by performing protein A chromatography, which is followed by mixed mode chromatography (MMC), which is followed by anion exchange chromatography (AEX), which is followed by hydrophobic interaction chromatography (HIC).

In an embodiment, the invention is related to the purification of fusion protein by performing protein A chromatography, which is followed by mixed mode chromatography (MMC), optionally further comprises one or more chromatography step.

In an embodiment, the invention is related to the purification of fusion protein by performing protein A chromatography, which is followed by hydrophobic interaction chromatography (HIC), optionally further comprises one chromatography step.

In an embodiment, the invention is related to the purification of fusion protein by performing protein A chromatography, followed by optional viral inactivation or removal, which is followed by mixed mode chromatography (MMC), optionally further comprises one or more chromatography step.

In an embodiment, the invention is related to the purification of fusion protein by performing protein A chromatography, followed by mixed mode chromatography (MMC), optionally further followed by viral inactivation or removal, optionally further comprises one or more chromatography steps.

In one aspect of such embodiment, the third protein mixture is further incubated with detergent or surfactant for viral inactivation selected from about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 80 minutes.

In one aspect of such embodiment, the detergent or surfactant is selected from Triton X100 or Octo X100, sugar-based detergent n-dodecyl-β-D-maltoside, preferably Triton X100.

In an embodiment, the invention is related to the purification of fusion protein by performing protein A chromatography, followed by optional viral inactivation or removal, which is followed by hydrophobic interaction chromatography (HIC), optionally further comprises one chromatography step.

In an embodiment, the viral inactivation or removal performed using a surfactant or detergent which eliminates the use of harsh acid based viral inactivation or removal.

In an embodiment, the invention provides a chromatography process to produce a composition enriched with fusion protein of interest and substantially reduced amount of acidic variant impurities.

In an embodiment, the invention provides a chromatography process to produce a composition enriched with fusion protein of interest and substantially reduced basic variant impurities.

In an embodiment, the invention provides a chromatography process to produce a composition enriched with fusion protein of interest and substantially reduced high molecular weight (HMW) impurities.

In an embodiment, the invention provides a chromatography process to produce a composition enriched with fusion protein of interest and substantially reduced low molecular weight (LMW) impurities.

In an embodiment, the invention provides a chromatography process to produce a composition enriched with fusion protein of interest and substantially reduced level of undesired glycans.

In an embodiment, a method for purifying a Fc-fusion protein from a protein sample comprising Fc-fusion protein and Pre-peak impurity:

- a) contacting the protein sample onto mixed-mode chromatography column;
- b) optionally perform washing the mixed-mode chromatography column;
- c) eluting the fusion protein from a mixed-mode chromatography column wherein the eluted fusion protein comprises reduce amount of Pre-peak impurity.

In one aspect of such embodiment, the pre-peak impurity reduced by at least more than about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%. In an embodiment the pre-peak impurity is below quantifiable limit.

In an embodiment, a method for purifying a Fc-fusion protein from a protein sample comprising:

- a) contacting the protein sample onto mixed-mode chromatography column;
- b) washing the mixed-mode chromatography column with buffer comprising a suitable additive;
- c) eluting the fusion protein from a mixed-mode chromatography column with reduced amount of impurities;
- wherein, the suitable additive is arginine or arginine derivatives.

In an embodiment, the concentration of arginine or arginine derivatives is selected from about 20 mM to about 100 mM.

In another embodiment, the concentration of arginine or arginine derivatives is selected from about 60 mM to about 800 mM.

In another embodiment, the concentration of arginine or arginine derivatives is selected from about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM and about 300 mM.

In an embodiment, a method for purifying a Fc-fusion protein from a protein sample comprising:

- a) contacting the protein sample onto mixed-mode chromatography column;
- b) washing the mixed-mode chromatography column with a suitable buffer comprising suitable additive;
- c) eluting the fusion protein from a mixed-mode chromatography column;
- wherein the elution buffer comprises arginine or arginine derivatives concentration selected from about below 600 mM, preferably 300 mM.

In an embodiment, a method for purifying a Fc-fusion protein from a protein sample comprising Fc-fusion protein and impurity:

- a) contacting the protein sample onto mixed-mode chromatography column;
- b) optionally perform washing the mixed-mode chromatography column;
- c) eluting the fusion protein from a mixed-mode chromatography column;
- wherein the elution is performed suitable buffer and arginine & the eluted fusion protein comprises reduce amount of impurity.

In an embodiment, the arginine concentration is selected from 0.1M to 0.5M preferably 0.3M.

In such embodiment, the impurities are selected from pre-peak, HMW and LMW.

In an embodiment, a process for purification of IgG1 containing protein comprising:

- a) collecting the IgG1 containing protein from the suitable mammalian expression system and the impurities;
- b) contacting the IgG1 containing protein affinity chromatography;
- c) eluting the IgG1 containing protein;
- d) contacting the eluted IgG1 containing protein onto mixed-mode chromatography, optionally followed by other suitable purification steps; wherein mixed-mode chromatography is performed in bind-elute mode.

In an embodiment, the invention provides an affinity chromatography and mixed mode chromatography to produce a composition comprising enriched CTLA-4 IgG1 fusion protein and reduced LMW impurities selected from about 50%, about 40%, about 30%, about 20%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or less LMW or below quantifiable limit.

In an embodiment, a method for purifying a Fc-fusion protein from a protein sample comprising Fc-fusion protein and at least Pre-peak impurity:

- a) collecting first protein sample/mixture from the suitable mammalian expression system comprising Fc-fusion protein and the impurities;
- b) contacting the first protein sample/mixture to affinity chromatography column;
- c) eluting the Fc-fusion protein from affinity chromatography column to form second protein mixture;
- d) contacting the second protein mixture to mixed-mode chromatography column; e) eluting the fusion protein from a mixed-mode chromatography column to form third protein mixture;
- f) contacting the third protein mixture to anion exchange chromatography column;
- g) eluting the fusion protein from anion exchange chromatography column to form fourth protein mixture;
- h) contacting the fourth protein mixture to hydrophobic interaction chromatography column;
- i) eluting the fusion protein from hydrophobic interaction chromatography column to form fifth protein mixture;

wherein, the eluted Fc-fusion protein is substantially free of Pre-peak impurities.

In an embodiment, a method for purifying a Fc-fusion protein from a protein sample comprising Fc-fusion protein and at least Low molecular weight (LMW):

- a) collecting first protein sample/mixture from the suitable mammalian expression system comprising Fc-fusion protein and the impurities;
- b) contacting the first protein sample/mixture to affinity chromatography column;
- c) eluting the Fc-fusion protein from affinity chromatography column to form second protein mixture;
- d) contacting the second protein mixture to mixed-mode chromatography column;
- e) eluting the fusion protein from a mixed-mode chromatography column to form third protein mixture;
- f) contacting the third protein mixture to anion exchange chromatography column;
- g) eluting the fusion protein from anion exchange chromatography column to form fourth protein mixture;
- h) contacting the fourth protein mixture to hydrophobic interaction chromatography column;
- i) eluting the fusion protein from hydrophobic interaction chromatography column to form fifth protein mixture;

wherein, the eluted fusion protein is substantially free of low molecular weight (LMW) impurities.

In an embodiment, a method for purifying a Fc-fusion protein from a protein sample comprising Fc-fusion protein and at least High molecular weight (HMW):

- a) collecting first protein sample/mixture from the suitable mammalian expression system comprising Fc-fusion protein and the impurities;
- b) contacting the first protein sample/mixture to affinity chromatography column;
- c) eluting the Fc-fusion protein from affinity chromatography column to form second protein mixture;
- d) contacting the second protein mixture to mixed-mode chromatography column;
- e) eluting the fusion protein from a mixed-mode chromatography column to form third protein mixture;
- f) contacting the third protein mixture to anion exchange chromatography column;
- g) eluting the fusion protein from anion exchange chromatography column to form fourth protein mixture;
- h) contacting the fourth protein mixture to hydrophobic interaction chromatography column;
- i) eluting the fusion protein from hydrophobic interaction chromatography column to form fifth protein mixture;

wherein, the eluted fusion protein is substantially free of high molecular weight (HMW) impurities.

In an embodiment, the Fc-fusion protein composition comprising about 0.5% or less HMW, about 0.4% or less HMW, about 0.3% or less HMW, about 0.2% or less HMW, about 0.1% or less HMW.

In an embodiment, a method for purifying a Fc-fusion protein from a protein sample comprising Fc-fusion protein and undesired glycans:

- a) collecting first protein sample/mixture from the suitable mammalian expression system comprising Fc-fusion protein and the impurities;
- b) contacting the first protein sample/mixture to affinity chromatography column;
- c) eluting the Fc-fusion protein from affinity chromatography column to form second protein mixture;
- d) contacting the second protein mixture to mixed-mode chromatography column;
- e) eluting the fusion protein from a mixed-mode chromatography column to form third protein mixture;
- f) contacting the third protein mixture to anion exchange chromatography column;
- g) eluting the fusion protein from anion exchange chromatography column to form fourth protein mixture;
- h) contacting the fourth protein mixture to hydrophobic interaction chromatography column;
- i) eluting the fusion protein from hydrophobic interaction chromatography column to form fifth protein mixture;