DESC:
Field of the Invention
The present invention relates to protein purification. In particular, the invention is related to the separation of ranibizumab from cysteinylated ranibizumab.

Background of the invention
Recombinant DNA technology methods, for production of therapeutic proteins, generally adopt mammalian or bacterial based expression systems. Although, mammalian based expression systems have experienced a major boost with the advent of antibody-based therapeutics, however bacteria based expression systems continue to be widely used for production of non-glycosylated peptides and proteins. The bacterial expression systems are easy to manipulate, less time consuming and higher in yield.

In bacterial expression system, recombinant proteins are mostly expressed as inclusion bodies. Recovery of the therapeutically active proteins from the inclusion bodies involves multiple steps such as unfolding and refolding of the protein using harsh conditions in presence of chaotropic agents and reducing thiols. During the refolding process cysteine residues in protein (for example antibody, IgG, Fab fragments) are involved in disulfide bond formation. However, some of the cysteine residues, which do not have any role in disulfide bond formation can, form a disulfide bond with free cysteine or glutathione present in refolding buffer or solution. This process is known as cysteinylation (cysteine) or glutathionylation (glutathione). Cysteinylated/glutathionylated proteins are the part of product related impurities.

Ranibizumab is a monoclonal antibody fragment (Fab) which lacks an Fc region. The molecular weight of ranibizumab is approximately 48 kDa (23 kDa and 25 kDa for the light and heavy chain, respectively). Ranibizumab contains 10 cysteine residues forming 4 intra-chain and 1 inter-chain disulfide bonds. Ranibizumab is commercially available under the brand name Lucentis®. Lucentis® is used to treat the "wet form" of age-related macular degeneration and swelling in the retina caused by diabetes or by a blockage in the blood vessels. Ranibizumab binds to human vascular endothelial growth factor A (VEGF-A) and inhibits the biologic activity of VEGF-A. Ranibizumab is produced by an E. coli expression system.

WO2012/013682 discloses a method for purification of Fab fragment using cation exchange chromatography followed by anion exchange chromatography. US8044017 and US8710196 describe the importance of changing conductivities for separation of polypeptides. WO2013/067301 discloses an overloading method for protein purification in which the protein load amount is more than the dynamic binding capacity of the column. WO1999/062936 discloses purification of monomeric protein up to 99.5% purity using ion exchange chromatography using salt gradient.

Despite significant advances in the understanding of various chromatography techniques and availability of various buffer systems, protein purification platforms, protein chromatography remains significantly challenging and unpredictable. At industrial scale, there is no universal purification method available for any therapeutic protein. Protein purification methods are mostly governed by the type of protein, method for production, quantity and quality of impurities. Sometimes, even after two or more chromatographic purification steps some impurities are still present in the protein. Purification of any specific protein requires a novel solutions or approaches and these tasks become more challenging and unpredictable, when the protein of interest and the impurities demonstrate similar or proximal physiochemical properties, for example when both, the impurity and the protein of interest, are acidic or are basic or share similar hydrophobicity etc.

As discussed above, in therapeutic protein purification there is a substantial need and interest for developing efficient and economical method. Prior art methods disclose general method for impurity removal. In particular, prior arts are silent about reducing the heterogeneity in protein by separation of cysteinylated or glutathionylated proteins.

Thus, the main aim of the instant invention is to develop a cost effective and efficient process for removal of product related impurities such as cysteinylated or glutathionylated from a protein mixture during production of therapeutic protein. In particular, the instant invention provides a separation method of ranibizumab from cysteinylated ranibizumab.

SUMMARY OF THE INVENTION
In an embodiment, the invention provides an efficient and economic process for the purification of antibody or antibody’s fragment with free or unpaired cysteine residues from product related impurities.

In an embodiment, the instant invention provides a method for purifying antibody or antibody’s fragment from a composition comprising the antibody or antibody’s fragment and contaminants wherein the contaminants are product related impurities.

In an embodiment, the product related impurities are cysteinylated or glutathionylated antibody or fragment thereof.

In an embodiment, the invention describes production of heavy chain and light chain of antibody or its fragment thereof in bacteria in the form of inclusion bodies, solubilizing the heavy chain and light chain, refolding the heavy chain and light chain under suitable conditions to obtain refolded antibody or its fragment thereof, wherein solubilization or refolding buffer comprises cysteine or glutathione and purifying the refolded protein using ion exchange chromatography to remove cysteinylated antibody or fragment thereof. In an embodiment the ion exchange chromatography is anion exchange chromatography.

In an embodiment, the invention provides a large-scale separation method of antibody or fragment thereof from antibody or fragment thereof, the method comprises;

(a) loading the composition comprising antibody or fragment thereof and cysteinylated antibody or fragment thereof onto anion exchange resin using load buffer wherein the load buffer is at first pH and first conductivity,
(b) washing the anion exchange resin with wash buffer wherein the wash buffer is on second pH and second conductivity;
(c) eluting the antibody or fragment thereof with elution buffer at third pH and third conductivity, wherein the pH of elution buffer is altered as linear pH gradient,
(d) collection of purified antibody or fragment thereof;

wherein the molar concentration of load buffer, wash buffer and elution buffer is essentially same and wherein the elution buffer linear gradient causes the separation of antibody or fragment thereof from cysteinylated antibody or fragment thereof.

In certain embodiment, the antibody or its fragment thereof expressed in bacteria is Fab fragment. In certain embodiment the Fab fragment is ranibizumab.

In an embodiment, the invention provides a separation method of ranibizumab from cysteinylated ranibizumab, the method comprises, loading the composition comprising ranibizumab and cysteinylated ranibizumab onto anion exchange chromatography material, and eluting the ranibizumab, wherein the eluted ranibizumab has reduced amount of cysteinylated ranibizumab.

In an embodiment, the invention provides a large-scale separation method of ranibizumab from cysteinylated ranibizumab, the method comprises;

(a) loading the composition comprising ranibizumab and cysteinylated ranibizumab onto anion exchange resin using load buffer wherein the load buffer is at first pH and first conductivity,
(b) washing the anion exchange resin with wash buffer wherein the wash buffer is on second pH and second conductivity,
(c) eluting the ranibizumab with eluting buffer at third pH and third conductivity wherein the pH of elution buffer is altered as linear pH gradient, and;
(d) collection of purified ranibizumab;

wherein the molar concentration of load buffer, wash buffer and elution buffer is essentially same and wherein the elution buffer linear gradient cause the separation of ranibizumab from cysteinylated ranibizumab.

In some embodiment of above aspect, the first pH is greater than the third pH and first conductivity is lower than the third conductivity.

In some embodiment, the first pH and first conductivity are essentially the same as second pH and second conductivity.

In some embodiments, the pH gradient comprises a decrease in pH from about pH 9 to about pH 8.

In some embodiments, the pH gradient is generated using one or more buffer solutions. In further embodiments, one or more buffers are Tris-Cl, phosphate, or CAPS.

In some embodiment the loading buffer, wash buffer and elution buffer concentration are in the range of about 10mm to about 100mM.

In some embodiment the loading buffer, wash buffer and elution buffer pH are in the range of about 8.0 to about 10.

In some embodiment, the loading buffer, wash buffer and elution buffer conductivities are in the range of about 0.1mS/cm to about 5 mS/cm.

In some embodiment, the invention provides separation method of ranibizumab from cysteinylated ranibizumab the method comprises:

(a) loading the composition comprising ranibizumab and cysteinylated ranibizumab onto anion exchange resin using loading buffer comprising, pH about 9 to about 10, and conductivity about 0.5 to about 5 mS/cm,
(b) washing the antibody resin with wash buffer which is essentially same as loading buffer,
(c) applying the elution buffer comprising pH of about 8 to about 9 and conductivity about 1 to about 10 mS/cm onto anion exchange resin,
(d) collection of purified ranibizumab,

wherein the elution buffer is applied in linear gradient mode and molarity of loading buffer and elution buffer is essentially same.

In some embodiment the protein load on anion exchnage chromatography is about 1 mg/ml of resin to about 10 mg/ml of resin.

In some embodiment, separation of ranibizumab from cysteinylated ranibizumab using anion exchange chromatography step may be applied after removal of other process and product related impurities such as host cell impurities or aggregates.

In some embodiment, the eluted ranibizumab may be further purified using one or more chromatographic step.

Brief description of Drawings
Figure 1: Illustrates the chromatogram of Anion exchange chromatography carried out in linear gradient mode.

Figure 2: Reversed-phase HPLC chromatograms of ranibizumab after anion exchange chromatography: Cysteinylated (Fab-Cys fragments) and non-cysteinylated (Fab) are separated.

Figure 3: Reversed-phase HPLC chromatograms of ranibizumab after anion exchange chromatography: AEX eluate devoid of Cysteinylated Fab/Fab fragments.

Detailed Description of the Invention
The instant invention relates to purification of ranibizumab in linear gradient mode for separation of cysteinylated ranibizumab from target ranibizumab protein. The anion exchange chromatography is carried out in bind-elute mode wherein the ranibizumab and cysteinylated ranibizumab binds with ion exchange chromatographic column and linear pH gradient is applied to elute ranibizumab before to cysteinylated ranibizumab.

The terms "purifying" or “purified” or "separating," or "isolating," or purification” or “separation” as used interchangeably herein, refer to increasing the degree of purity of a polypeptide or protein of interest or a target protein from a protein mixture comprising the polypeptide and one or more impurities or contaminants including at least one of the product related impurities.

As used herein the term “antibody or fragment thereof” is used in the broadest sense and covers monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, bispecific antibodies, diabodies, and single-chain molecules, as well as antibody fragments (e.g., Fab, F(ab')2, and Fv).

As used herein, "buffered solution" refers to a solution, which resists changes in pH by the action of its acid-base conjugate components.

As used herein, "solubilization buffer" refers to a solution used for solubilization of inclusion bodies.

As used herein, "refolding buffer” or “refolding solution" refers to a solution used for recovering a desire refolded protein confirmation from solubilized inclusion bodies.

As used herein, the term “bind-elute mode” refers to a mode of purification by chromatography, wherein the Protein-of-interest when loaded on the column is bound to the chromatographic resin and is subsequently eluted with an elution buffer.

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.

As used herein "properly folded" or "biologically active" refers to a molecule with a biologically active conformation and perform desire biological activities.

"Anion exchange resin" or “Anion exchange chromatography” mentioned in the embodiments refers to a solid chromatographic support, which has a positively charged ligand such as a quaternary amino group attached thereto, capable of ionic interaction with a negatively charged protein or a functional group under suitable conditions. The anion exchange resin can be any weak or strong anion exchange resin or a membrane, which could function as a weak or a strong anion exchanger. Various commercially available anion exchange resins are known in the art and include without any limitation DEAE cellulose, Poros PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, MonoQ, MiniQ, Source 15Q and 3OQ, Q, DEAE and ANX Sepharose Fast Flow, Q Sepharose high Performance, QAE SEPHADEX and FAST Q SEPHAROSE from GE Healthcare, Macro-Prep DEAE and Macro-Prep High Q from Biorad, Q-Ceramic Hyper D, DEAE-Ceramic Hyper D, from Pall Corporation.

The term "gradient elution" is used herein to refer generally to conditions in which pH and/or conductivity is either increased or decreased using at least two buffers wherein the buffers are different in terms of pH or conductivity or both.

The term "linear gradient" is used here to refer to conditions in which pH and/or conductivity is either increased or decreased gradually using at least two buffers wherein the buffers are different in terms of pH or conductivity or both.

The term “essentially same” indicates that a value or parameter has not been altered by a significant effect. For example, a molarity of a load buffer is essentially the same as wash buffer means the difference between molarity is not significant. Most preferably the essentially same is used when both molarity are same.

The term "impurity" or “impurities” as used herein refers to any proteinaceous or non-proteinaceous molecular entity distinct from the than the protein of interest. The impurity includes, without limitation: host cell DNA, fragment, aggregate, another polypeptide, endotoxin, bacterial cell culture media component etc. The protein of interest may be described as the fraction of protein, substantially free of impurities, as described herein, and can be obtained as part of the flow-through from the anion exchange chromatography.

The term “cysteinylation” or “glutathionylation” refers to undesirable posttranslational modification in pharmaceutical proteins, which may lead to a conformational isoform with undesirable properties, such as low binding, low biological activity and low stability. Free cysteine of “protein” is involved in free disulfide bond formation. “cysteinylation” or “glutathionylation” involve the formation of di-sulfide bond with free cysteine of glutatheon respectively. The term “Cysteinylated” or “glutohionylated” refers to protein with free disulfide bond formation. In some embodiments the protein is ranibizumab. The term cysteinylated ranibizumab refers to ranibizumab wherein either heavy or light chain or both chains of ranibizumab are cysteinylated.

The following examples are provided to further illustrate the present invention but are not provided to in any way limit the scope of the current invention.

Example 1:
Solubilization and refolding of ranibizumab inclusion bodies
Solubilization and refolding of ranibizumab has already been disclosed in WO2017/029620. WO2017/029620 describe a method for production of active ranibizumab, which is incorporated herein by reference.

36 gm Inclusion bodies (IBs) obtained after fermentation, harvesting and IBs isolation-were solubilized in around 720 mL of first buffer comprised of 8 M Urea, 50 mM Tris at pH 9 and reduction was done using 4 mM DTT for 45 minutes. The solubilized and reduced IBs were then filtered with 0.8+ 0.2 µp? filter. Around 360 ml of solubilized and reduced IBs solution was then added to around 8640 mL of refolding buffer comprised of 0.6 M Arginine, 5% sorbitol, 2-10 mM of cystine and 50 mM Tris at pH 10 and temperature of 5 -10 °C within 1 h at constant flow rate. Refolding was carried out for further 14-24 hrs. The refolded ranibizumab was passed through ultrafiltration step to remove refolding buffer component and diafiltration to change the buffer for anion exchange chromatography.

Example 2: Anion exchange chromatography
The anion exchange chromatography was carried out using Fractogel® (Millipore) TMAE resin. TMAE resin beads having positive charge will bind the ranibizumab target protein which is having net negative charge at operating pH of chromatography. This step is mainly used for removal of product related impurities. Anion exchange chromatography was carried out as follows.

1. The pH of antibody composition comprising ranibizumab and cysteinylated ranibizumab was adjusted to 9.10 ± 0.15 and conductivity was adjusted to < 1.0 mS/cm.
2. The column was equilibrated with 3-5 CVs of equilibration buffer (50 mM Tris, pH 9.1, conductivity 0.6 mS/cm).
3. After the column equilibration, the antibody composition comprising ranibizumab and cysteinylated ranibizumab was loaded on anion exchange resin. The antibody load was about 7mg/ml of resin.
4. After the loading was completed, the column was washed with 3 - 5 CVs of equilibration buffer.
5. After the wash was over, elution was carried out with elution buffer (50 mM Tris, pH- 8.0, conductivity 2.4 mS/cm in linear gradient mode of 0-100% (i.e. equilibration buffer and elution buffer) in 20 CV and elution peak was collected.

The details of the chromatogram of anion exchange chromatography is provide in Figure 1. Figure 1 showed the presence of protein of interest and post elutes peaks. Both peaks were collected and characterized using reverse phase HPLC.

Example 3: Reverse Phase HPLC
The post eluted peak obtained in anion exchange chromatography were characterized using reverse phase HPLC ( Agilent 1260 HPLC with MWD and FLD) and column Agilent Zorbax 300 SB – C8, 150 X 4.6 mm, 5µ, Catalogue No.:883995-906 ) using the protocol as suggested by the instrument provider. The results of RP-HPLC are furnished in Figure 2 & Figure 3. The post-eluted peak were identified as cysteinylated Heavy chain and light chain of ranibizumab by LC-MS.
,CLAIMS:
1. A method for separation of antibody’s Fab fragments from a composition comprising antibody’s Fab fragments and cysteinylated or glutathionylated Fab fragments the method comprises, loading the composition onto anion exchange chromatography and eluting the antibody’s Fab fragments using liner pH gradient, wherein the eluted antibody’s Fab fragments are essentially free from cysteinylated or glutathionylated Fab fragments.

2. The method of claim 1 wherein the Fab fragments are ranibizumab.

3. A method for separation of ranibizumab from composition comprising ranibizumab and cysteinylated ranibizumab, the method comprises the steps of:
a. loading the composition onto anion exchange resin using load buffer wherein the load buffer is at first pH and first conductivity;
b. washing the anion exchange resin with wash buffer wherein the wash buffer is on second pH and second conductivity;
c. eluting the ranibizumab with eluting buffer at third pH and third conductivity wherein the pH of elution buffer is altered as linear pH gradient; and
d. collection of purified ranibizumab;
wherein the molar concentration of load buffer, wash buffer and elution buffer is essentially same; and wherein the elution buffer linear pH gradient cause the separation of ranibizumab from cysteinylated ranibizumab.

4. The method of claim 3 wherein the said first pH is greater than the third pH and first conductivity is lower than the third conductivity.

5. The method of claim 3 wherein the said the pH gradient comprises a decrease in pH from about pH 9 to about pH 8.

6. The method of claim 3 wherein the said loading buffer, wash buffer and elution buffer concentration are in the range of about 10mm to about 100mM.

7. The method of claim 3 wherein the said loading buffer, wash buffer and elution buffer pH are in the range of about 8 to about 10.

8. The method of claim 3 wherein the said loading buffer, wash buffer and elution buffer conductivities are in the range of about 0.1mS/cm to about 5 mS/cm.

9. The method of claim 3 wherein the said pH linear gradient is generated using one or more buffer solutions of Tris-Cl, phosphate, or CAPS.

10. The method of claim 3 wherein the said eluted ranibizumab may be further purified using one or more chromatography steps.