DESC:FORM 2

THE PATENTS ACT, 1970
(39 of 1970)
&
The Patent Rules, 2003

COMPLETE SPECIFICATION
(See section 10 and rule 13)

TITLE OF THE INVENTION

“PROCESS OF PURIFYING ANTI-RANKL ANTIBODY”

We, CADILA HEALTHCARE LIMITED, an Indian company incorporated under the Companies Act, 1956, of Zydus Corporate Park, Scheme No. 63, Survey No. 536, Khoraj (Gandhinagar), Nr. Vaishnodevi Circle, Sarkhej –Gandhinagar Highway, Ahmedabad, Gujarat 382481, India,

The following specification particularly describes the invention and the manner in which it is to be performed:

Field of the Invention
The invention provides process of purifying antibody, preferably anti-RANKL antibody. Preferred anti-RANKL antibody according to present invention is denosumab.
Background of the Invention
Denosumab (trade names Prolia® and Xgeva®) is a human monoclonal antibody (mAb) for the treatment of osteoporosis, treatment-induced bone loss, metastases to bone, and giant cell tumor of bone. Denosumab is a RANKL inhibitor, which acts by preventing the development of osteoclasts. Denosumab was developed by the biotechnology company Amgen Inc. and its biosimilars are being developed by many pharmaceutical companies. A biosimilar product cannot be considered an identical copy of its innovator counterpart. It is always being continuous endeavor of industrial scientists to optimize the purification process steps of the antibody to increase the efficiency of removing contaminants and improve the purity and yield of the antibody and thus meet regulatory requirement for its commercialization.
Prior art disclosures: WO2005042569 discloses purification of protein other than denosumab by hydrophobic interaction chromatography (HIC).
WO2018051348 discloses purification of denosumab by mixed mode chromatography (Capto™ Adhere column).
None of the prior art discloses purification of anti-RANKL antibody, preferably denosumab by chromatography steps according to the invention of present disclosure i.e. affinity chromatography followed by hydrophobic interaction chromatography (HIC) and cation exchange chromatography (CEX).

Summary of the invention
The present invention provides process of purification of anti-RANKL antibody, preferably denosumab which comprises affinity chromatography, hydrophobic interaction chromatography and cation exchange chromatography. In a preferred aspect, the present invention provides process of purification of denosumab comprises affinity chromatography followed by hydrophobic interaction chromatography. Aspects of the present invention further comprises certain standard non-chromatography steps such as viral inactivation at low pH, pH neutralization, ultrafiltration-diafiltration, nano filtration, terminal filtration which are well known to a person skilled in the art.
Abbreviations
Anti-RANKL: anti- receptor activator for nuclear factor ? B ligand
CEX: cation exchange chromatography
DF: Diafiltration
DNA: Deoxyribonucleic acid
HP-SEC: High performance size exclusion chromatography
HIC: Hydrophobic interaction chromatography
Protein A: Protein A cross-linked agarose column
Protein G: Protein G cross-linked agarose column
Protein L: Protein L cross-linked agarose column
r-PA: recombinant protein A
UF: Ultrafiltration
ND: Not detected

Definitions
The term “about” refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods, and other similar considerations. The term “about” also encompasses amounts that differ due to aging of a composition with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a formulation with a particular initial concentration or mixture.
The term “bind-elute mode” refers to an operational approach to chromatography in which the buffer conditions are established so that the desired protein and some product related substances bind to the column upon application, which are eluted differentially by with modified buffer conditions and collected in fractions. Fractionation is most commonly achieved by applying an elution gradient, in which the concentration of one or more buffer components, or conditions such as pH, are increased or decreased. The increase or decrease may be essentially continuous, as in the case of so-called linear gradients, or incremental, as in the case of so-called step gradients.
The term “cation exchange chromatography” or “cation exchange column chromatography” refers to a form of ion-exchange chromatography that uses resins or packings with functional groups that separates cations.
The term “diafiltration step” refers to a total volume exchange during the process of diafiltration. The term “diafiltration” or “DF” is used to mean a specialized class of filtration in which the retentate is diluted with solvent and re-filtered, to reduce the concentration of soluble permeates components. Diafiltration may or may not lead to an increase in the concentration of retained components when performed with constant-volume mode. For example, in continuous diafiltration, a solvent is continuously added to the retentate at the same rate as the filtrate is generated. In this case, the retentate volume and the concentration of retained components do not change during the process. On the other hand, in discontinuous or sequential dilution diafiltration, an ultrafiltration step is followed by the addition of solvent to the retentate side; if the volume of solvent added to the retentate side is not equal or greater to the volume of filtrate generated, then the retained components will have a high concentration. Diafiltration may be used to alter the pH, ionic strength, salt composition, buffer composition, or other properties of a solution or suspension of macromolecules.
The term “protein A chromatography” or “protein A column chromatography” or “r-PA chromatography” refers to capture of anti-RANKL antibody on resin containing protein A as a ligand, based on affinity of the anti-RANKL antibody to certain epitopes of protein A.
As used herein, the terms “ultrafiltration” or “UF” refers to any technique in which a solution or a suspension is subjected to a semi-permeable membrane that retains macromolecules while allowing solvent and small solute molecules to pass through. Ultrafiltration may be used to increase the concentration of macromolecules in a solution or suspension.
As used herein, the terms “ultrafiltration-diafiltration” or “UF/DF” refers to any process, technique or combination of techniques that accomplishes ultrafiltration and / or diafiltration, either sequentially or simultaneously.
The term “hydrophobic interaction chromatography” or “HIC” refers to separation of proteins according to differences in their surface hydrophobicity by utilizing a reversible interaction between these proteins and the hydrophobic surface of a HIC medium.

Embodiments of the invention
In one embodiment, the present invention provides a process of purification of anti-RANKL antibody, preferably, denosumab which comprises affinity chromatography followed by hydrophobic interaction chromatography.
In a further embodiment, the present invention provides process of purification of anti-RANKL antibody, preferably, denosumab comprises affinity chromatography, hydrophobic interaction chromatography and cation exchange chromatography.
In a further embodiment, the process of purification of anti-RANKL antibody, preferably, denosumab comprises affinity chromatography followed by hydrophobic interaction chromatography followed by cation exchange chromatography.
In an alternate embodiment of the present invention, the process of purification of anti-RANKL antibody, preferably, denosumab comprises affinity chromatography followed by cation exchange chromatography followed by hydrophobic interaction chromatography.
In a further embodiment of the present invention, the affinity chromatography according to the present invention comprises protein A resin or protein G resin or protein L resin, preferably protein A resin (r-PA).
In a further embodiment of the present invention, the r-PA chromatography according to the present invention is performed in bind-elute mode.
In a further embodiment of the present invention, the r-PA purification step according to the present invention includes three column wash steps wherein (i) first wash is with equilibration buffer at suitable pH and / or conductivity, (ii) second wash at the same pH and / or at a conductivity higher than the first wash buffer (iii) third wash at a pH and / or conductivity lower than the second wash (iv) elution of the anti-RANKL antibody, preferably, denosumab at lower pH and / or higher conductivity than third wash.
In one embodiment of r-PA purification step according to the present invention, elution of the anti-RANKL antibody, preferably, denosumab in protein A chromatography is performed at a pH lower than 7.0, preferably pH range from about pH 3.0 to pH 4.5, more preferably about pH 3.5.
In one embodiment of r-PA purification step according to the present invention, elution of the anti-RANKL antibody, preferably, denosumab in protein A chromatography is performed at conductivity higher than 10 mS / cm, preferably at about 10 mS / cm to 40 mS / cm, more preferably 10 mS / cm to 20 mS / cm.
In a further embodiment of r-PA purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 7.0 to 7.6 and / or conductivity at about 1 mS / cm to 30 mS / cm (ii) second wash at about pH 7.0 to 7.6 and / or conductivity more than 30 mS / cm (iii) third wash at a pH lower than pH 7.0 and / or conductivity lower than or equal to 10 mS / cm (iv) elution of the anti-RANKL antibody, preferably, denosumab in the range of pH 3.0 – 4.5 and / or conductivity higher than 10 mS / cm.
In a furthermore embodiment of r-PA purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 7.0 to 7.6 and / or conductivity at about 10 mS / cm to 25 mS / cm (ii) second wash at about pH 7.0 to 7.6 and / or conductivity at about 30 mS / cm to 60 mS / cm (iii) third wash at about pH 4.8 to pH 6.5, preferably at about pH 5.0 and / or conductivity about 1 mS / cm to 10 mS / cm (iv) elution of the anti-RANKL antibody, preferably, denosumab in the range of pH 3.0 – 4.5, preferably at about pH 3.5 and / or conductivity at about 10 mS / cm to 40 mS / cm, preferably 10 mS / cm to 20 mS / cm.
In a furthermore embodiment of r-PA purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 7.0 to 7.6 and / or conductivity at about 10 mS / cm to 25 mS / cm (ii) second wash at about pH 7.0 to 7.6 and / or conductivity at about 30 mS / cm to 60 mS / cm (iii) third wash at about pH 4.8 to pH 6.5, preferably at about pH 5.5 and / or conductivity at about 1 mS / cm to 10 mS / cm (iv) elution of the anti-RANKL antibody, preferably, denosumab in the range of pH 3.0 – 4.5, preferably at about pH 3.5 and / or conductivity at about 10 mS / cm to 40 mS / cm, preferably 10 mS / cm to 20 mS / cm.
In a specific embodiment of r-PA purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 7.4 and / or conductivity at about 18 mS / cm (ii) second wash at about pH 7.4 and / or conductivity at about 48.0 mS / cm (iii) third wash at about pH 5.0 and / or conductivity at about 2.0 mS / cm (iv) elution of the anti-RANKL antibody, preferably, denosumab at about pH 3.5, and / or conductivity at about 12.0 mS / cm.
In a specific embodiment of r-PA purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 7.4 and / or conductivity at about 18 mS / cm (ii) second wash at about pH 7.4 and / or conductivity at about 48.0 mS / cm (iii) third wash at about pH 5.5 and / or conductivity at about 2.0 mS / cm (iv) elution of the anti-RANKL antibody, preferably, denosumab at about pH 3.5, and / or conductivity at about 12.0 mS / cm.
In further embodiment of the present invention, the hydrophobic interaction chromatography according to the present invention is performed in bind-elute mode.
In further embodiment of the present invention, the hydrophobic interaction column chromatography (HIC) purification step according to the present invention includes two column wash steps wherein (i) first wash is with equilibration buffer at suitable pH and / or conductivity, (ii) second wash at the same pH and / or at a conductivity lower than the first wash buffer (iii) elution of the anti-RANKL antibody, preferably, denosumab at same pH and / or lower conductivity than second wash.
In a furthermore embodiment of the hydrophobic interaction column chromatography (HIC) purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 5.0 to 8.0 and / or conductivity at about 150.0 mS / cm to 200.0 mS / cm (ii) second wash at about pH 5.0 to 8.0 and / or conductivity at about 100.0 mS / cm to 145.0 mS / cm (iii) ) elution of the anti-RANKL antibody, preferably, denosumab in the range of pH 5.0 – 8.0, preferably at about pH 6.8 and / or conductivity at about 45 mS / cm to 95 mS / cm, preferably 50 mS / cm to 70 mS / cm.
In a specific embodiment of the hydrophobic interaction column chromatography (HIC) purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 6.8 and / or conductivity at about 160.0 mS / cm (ii) second wash at about pH 6.8 and / or conductivity at about 135.0 mS / cm (iii) elution of the anti-RANKL antibody, preferably, denosumab at about pH 6.8 and / or conductivity at about 55 mS / cm.
In further embodiment of the present invention, the cation exchange chromatography according to the present invention is performed in bind-elute mode.
In further embodiment of the present invention, the cation exchange column (CEX) purification step according to the present invention includes two column wash steps wherein (i) first wash is with equilibration buffer at suitable pH and / or conductivity, (ii) second wash at the same pH and / or at a conductivity higher than the first wash buffer (iii) elution of the anti-RANKL antibody, preferably, denosumab at same pH and / or higher conductivity than second wash.
In a furthermore embodiment of the cation exchange column (CEX) purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 5.0 to 6.0 and / or conductivity at about 1.0 mS / cm to 4.0 mS / cm (ii) second wash at about pH 5.0 to 6.0 and / or conductivity at about 5.0 mS / cm to 12.0 mS / cm (iii) ) elution of the anti-RANKL antibody, preferably, denosumab in the range of pH 5.0 – 6.0, preferably at about pH 5.5 and / or conductivity at about 14 mS / cm to 40 mS / cm, preferably 15 mS / cm to 20 mS / cm.
In a specific embodiment of the cation exchange column (CEX) purification step according to the present invention comprises: (i) first wash is with equilibration buffer at about pH 5.5 and / or conductivity 2.0 mS / cm, (ii) second wash at pH about pH 5.5 and / or conductivity at about 10 mS / cm and (iii) elution of the anti-RANKL antibody, preferably, denosumab at about pH 5.5 and / or conductivity at about 17 mS / cm.
In a specific embodiment of the cation exchange column (CEX) purification step according to the present invention comprises: (i) first wash is with equilibration buffer at about pH 5.5 and / or conductivity 2.0 mS / cm, (ii) second wash at pH about pH 5.5 and / or conductivity at about 6.5 mS / cm and (iii) elution of the anti-RANKL antibody, preferably, denosumab at about pH 5.5 and / or conductivity at about 17 mS / cm.
In further embodiment of the present invention, the process of purification of anti-RANKL antibody, preferably, denosumab, from a crude mixture comprising a series of chromatography as embodied herein above and ultrafiltration-diafiltration steps.
In further embodiment of the present invention, the process of purification of anti-RANKL antibody, preferably, denosumab, from a crude mixture comprising a series of chromatography as embodied herein above, clarification, low pH viral inactivation, pH neutralization, ultrafiltration-diafiltration steps, virus clearance and terminal filtration.
In one embodiment of the present invention, the process of purification of anti-RANKL antibody, preferably, denosumab comprises following steps:
1. Clarification;
2. r-PA chromatography;
3. Viral inactivation at low pH;
4. pH neutralization;
5. Hydrophobic interaction chromatography.
In one more embodiment of the present invention, the process of purification of anti-RANKL antibody, preferably, denosumab comprises following steps:
1. Clarification;
2. r-PA chromatography;
3. Viral inactivation at low pH;
4. pH neutralization;
5. Hydrophobic interaction chromatography;
6. UF/DF-I;
7. Cation exchange chromatography;
8. Virus clearance by nano-filtration;
9. UF/DF-II;
10. Terminal filtration.
In an alternate embodiment of the present invention, the process of purification of anti-RANKL antibody, preferably, denosumab comprises following steps:
1. Clarification;
2. r-PA chromatography;
3. Viral inactivation at low pH;
4. pH neutralization;
5. UF/DF-I
6. Cation exchange chromatography;
7. UF/DF-II;
8. Hydrophobic interaction chromatography;
9. Virus clearance by nano-filtration;
10. UF/DF-III;
11. Terminal filtration.

Detailed description of the present invention
The present invention provides process of purifying anti-RANKL antibody. Preferred anti-RANKL antibody according to present invention is denosumab. The purification process of said antibody comprises chromatography steps. The chromatography steps of purification process comprises membrane chromatography or column chromatography, preferably column chromatography. The purification process according to the present invention comprises two column chromatography steps or three column chromatography steps. The purification process according to the present invention may also comprises non-chromatography steps such as clarification, viral inactivation, pH neutralization, UF/DF, virus clearance by nano filtration, terminal filtration. The purification process according to the present invention provides at least 99 % pure anti-RANKL antibody, preferably, denosumab. In one embodiment, the present invention provides process of purification of anti-RANKL antibody, preferably, denosumab comprises affinity chromatography followed by hydrophobic interaction chromatography. In one more embodiment, the present invention provides process of purification of anti-RANKL antibody, preferably, denosumab comprises affinity chromatography, hydrophobic interaction chromatography and cation exchange chromatography. In a further embodiment of the present invention, the process of purification of anti-RANKL antibody, preferably, denosumab comprises affinity chromatography followed by hydrophobic interaction chromatography followed by cation exchange chromatography. In an alternate embodiment of the present invention, the process of purification of anti-RANKL antibody, preferably, denosumab comprises affinity chromatography followed by cation exchange chromatography followed by hydrophobic interaction chromatography. In a further embodiment of the present invention, the affinity chromatography according to the present invention comprises protein A resin or protein G resin or protein L resin, preferably protein A resin (r-PA). Protein A chromatography step (r-PA chromatography) is useful to capture the protein from crude mixture and to elute the desired protein from the column with high level of purity in bind-elute mode. In further embodiment of the present invention, the r-PA chromatography according to the present invention is performed in bind-elute mode. r-PA chromatography as mentioned herein is performed as per WO2014/207763 published on 31 December 2014 along with suitable modifications as necessary, which are within the scope of a person skilled in the art. The present invention provides a purification process of anti-RANKL antibody, preferably, denosumab from a crude mixture by using a r-PA column chromatography, first to capture, and then elute the said antibody from the column with desired purity at low pH elution buffer. The crude mixture may include host-cell derived contaminating proteins, DNA, product-related substances and other impurities in addition to that of the antibody of interest. Protein G or protein L can be used as column matrix in the affinity chromatography step. Protein G is an immunoglobulin binding protein expressed in group C and G Streptococcal bacteria. Protein L is also an immunoglobulin binding protein isolated from surface of bacterial species Peptostreptococcus magnus, which binds antibodies through light chain interactions. They are coupled with agarose base to form column matrix. For example, protein G column comprises protein G covalently coupled with agarose base and protein L column comprises protein L which is covalently coupled with agarose base.
In further embodiments of the present invention, the r-PA purification step according to the present invention includes three column wash steps wherein (i) first wash is with equilibration buffer at suitable pH and / or conductivity, (ii) second wash at the same pH and / or at a conductivity higher than the first wash buffer (iii) third wash at a pH and / or conductivity lower than the second wash (iv) elution of the anti-RANKL antibody, preferably, denosumab at lower pH and / or higher conductivity than third wash.
In one embodiment of r-PA purification step according to the present invention, elution of the anti-RANKL antibody, preferably, denosumab in protein A chromatography is performed at a pH lower than 7.0, preferably pH range from about pH 3.0 to pH 4.5, more preferably about pH 3.5.
In one embodiment of r-PA purification step according to the present invention, elution of the anti-RANKL antibody, preferably, denosumab in protein A chromatography is performed at conductivity higher than 10 mS / cm, preferably at about 10 mS / cm to 40 mS / cm, more preferably 10 mS / cm to 20 mS / cm.
In a further embodiment of r-PA purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 7.0 to 7.6 and / or conductivity at about 1 mS / cm to 30 mS / cm (ii) second wash at about pH 7.0 to 7.6 and / or conductivity more than 30 mS / cm (iii) third wash at a pH lower than pH 7.0 and / or conductivity lower than or equal to 10 mS / cm (iv) elution of the anti-RANKL antibody, preferably, denosumab in the range of pH 3.0 – 4.5 and / or conductivity higher than 10 mS / cm.
In a furthermore embodiment of r-PA purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 7.0 to 7.6 and / or conductivity at about 10 mS / cm to 25 mS / cm (ii) second wash at about pH 7.0 to 7.6 and / or conductivity at about 30 mS / cm to 60 mS / cm (iii) third wash at about pH 4.8 to pH 6.5, preferably at about pH 5.0 and / or conductivity at about 1 mS / cm to 10 mS / cm (iv) elution of the anti-RANKL antibody, preferably, denosumab in the range of pH 3.0 – 4.5, preferably at about pH 3.5 and / or conductivity at about 10 mS / cm to 40 mS / cm, preferably 10 mS / cm to 20 mS / cm.
In a furthermore embodiment of r-PA purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 7.0 to 7.6 and / or conductivity at about 10 mS / cm to 25 mS / cm (ii) second wash at about pH 7.0 to 7.6 and / or conductivity at about 30 mS / cm to 60 mS / cm (iii) third wash at about pH 4.8 to pH 6.5, preferably at about pH 5.5 and / or conductivity at about 1 mS / cm to 10 mS / cm (iv) elution of the anti-RANKL antibody, preferably, denosumab in the range of pH 3.0 – 4.5, preferably at about pH 3.5 and / or conductivity at about 10 mS / cm to 40 mS / cm, preferably 10 mS / cm to 20 mS / cm.
In a specific embodiment of r-PA purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 7.4 and / or conductivity at about 18 mS / cm (ii) second wash at about pH 7.4 and / or conductivity at about 48.0 mS / cm (iii) third wash at about pH 5.0 and / or conductivity at about 2.0 mS / cm (iv) elution of the anti-RANKL antibody, preferably, denosumab at about pH 3.5, and / or conductivity at about 12.0 mS / cm.
In a specific embodiment of r-PA purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 7.4 and / or conductivity at about 18 mS / cm (ii) second wash at about pH 7.4 and / or conductivity at about 48.0 mS / cm (iii) third wash at about pH 5.5 and / or conductivity at about 2.0 mS / cm (iv) elution of the anti-RANKL antibody, preferably, denosumab at about pH 3.5, and / or conductivity at about 12.0 mS / cm.
r-PA column elute is further treated through various non-chromatography steps such as viral inactivation at low pH, pH neutralization before load on next column step. The next column step is either hydrophobic interaction chromatography or cation exchange chromatography according to the present invention. The non-chromatography steps are performed according to procedure known in the art.
Hydrophobic interaction chromatography (HIC) is performed to further purify the anti-RANKL antibody, preferably, denosumab with the removal of mainly, product-related high molecular weight (HMW) species variants and process-related impurities (HCP). In one embodiment, the HIC is performed in bind-elute mode. The column matrix for hydrophobic interaction chromatography according to the present invention is selected from phenyl sepharose, butyl sepharose, octyl sepharose. Preferably, column matrix for hydrophobic interaction chromatography according to the present invention is phenyl sepharose cross-linked with agarose.
In further embodiment of the present invention, the hydrophobic interaction column chromatography (HIC) purification step according to the present invention includes two column wash steps wherein (i) first wash is with equilibration buffer at suitable pH and / or conductivity, (ii) second wash at the same pH and / or at a conductivity lower than the first wash buffer (iii) elution of the anti-RANKL antibody, preferably, denosumab at same pH and / or lower conductivity than second wash.
In a furthermore embodiment of the hydrophobic interaction column chromatography (HIC) purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 5.0 to 8.0 and / or conductivity at about 150.0 mS / cm to 200.0 mS / cm (ii) second wash at about pH 5.0 to 8.0 and / or conductivity at about 100.0 mS / cm to 145.0 mS / cm (iii) ) elution of the anti-RANKL antibody, preferably, denosumab in the range of pH 5.0 – 8.0, preferably at about pH 6.8 and / or conductivity at about 45 mS / cm to 95 mS / cm, preferably 50 mS / cm to 70 mS / cm.
In a specific embodiment of the hydrophobic interaction column chromatography (HIC) purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 6.8 and / or conductivity at about 160.0 mS / cm (ii) second wash at about pH 6.8 and / or conductivity at about 135.0 mS / cm (iii) elution of the anti-RANKL antibody, preferably, denosumab at about pH 6.8 and / or conductivity at about 55 mS / cm.
In a specific embodiment of the hydrophobic interaction column chromatography (HIC) purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 6.8 and / or conductivity at about 160.0 mS / cm (ii) second wash at about pH 6.8 and / or conductivity at about 135.0 mS / cm (iii) elution of the anti-RANKL antibody, preferably, denosumab at about pH 6.8 and / or conductivity at about 60 mS / cm.
Cation exchange chromatography is performed in bind-elute mode to further purify anti-RANKL antibody, preferably, denosumab. Product-related and process related impurities are removed during this step. The column matrix for cation exchange chromatography according to the present invention is selected from SP Sepharose, SP-5PW, CaptoTM SP impRes and PARESTO SP35 Jetted. Preferably, column matrix for cation exchange chromatography according to the present invention is cation exchange matrix containing sulphopropyl functional group cross-linked with agarose i.e., SP Sepharose. SP Sepharose is an agarose-based cation exchanger with a sulfopropyl group.
CaptoTM SP impRes is a cation exchanger for high-throughput intermediate purification and polishing steps of biomolecules. The chromatography media is based on the high-flow agarose Capto product line provides good pressure/flow properties and a small bead size (approx. 40µm) that gives high resolution. The ligand is a well-established SP ligand; a sulfopropyl group.
SP-5PW is a cation exchange resin for biomolecule purification. It is composed of highly crosslinked polymethacrylate beads that have been functionalized with sulfopropyl (SP) cation exchange groups.
PARESTO SP35 Jetted is an agarose-based, acid cation exchange resin designed for biomolecule purification, including proteins, peptides and oligonucleotides. It is manufactured using ‘Jetting’ method that produces agarose beads with a very narrow particle size distribution.
In further embodiment of the present invention, the cation exchange column (CEX) purification step according to the present invention includes two column wash steps wherein (i) first wash is with equilibration buffer at suitable pH and / or conductivity, (ii) second wash at the same pH and / or at a conductivity higher than the first wash buffer (iii) elution of the anti-RANKL antibody, preferably, denosumab at same pH and / or higher conductivity than second wash.
In a furthermore embodiment of the cation exchange column (CEX) purification step according to the present invention comprises: (i) first wash with equilibration buffer at about pH 5.0 to 6.0 and / or conductivity at about 1.0 mS / cm to 4.0 mS / cm (ii) second wash at about pH 5.0 to 6.0 and / or conductivity at about 5.0 mS / cm to 12.0 mS / cm (iii) ) elution of the anti-RANKL antibody, preferably, denosumab in the range of pH 5.0 – 6.0, preferably at about pH 5.5 and / or conductivity at about 14 mS / cm to 40 mS / cm, preferably 15 mS / cm to 20 mS / cm.
In a specific embodiment of the cation exchange column (CEX) purification step according to the present invention comprises: (i) first wash is with equilibration buffer at about pH 5.5 and / or conductivity 2.0 mS / cm, (ii) second wash at pH about pH 5.5 and / or conductivity at about 10 mS / cm and (iii) elution of the anti-RANKL antibody, preferably, denosumab at about pH 5.5 and / or conductivity at about 17 mS / cm.
In a specific embodiment of the cation exchange column (CEX) purification step according to the present invention comprises: (i) first wash is with equilibration buffer at about pH 5.5 and / or conductivity 2.0 mS / cm, (ii) second wash at pH about pH 5.5 and / or conductivity at about 6.5 mS / cm and (iii) elution of the anti-RANKL antibody, preferably, denosumab at about pH 5.5 and / or conductivity at about 17 mS / cm.
In one of the embodiments of the present invention, the buffer used in the r-PA chromatography, hydrophobic interaction chromatography and cation exchange chromatography purification step according to the present invention is selected from tris, phosphate, acetate and citrate buffers. Preparation and use of such buffers and their compositions are known and are within the scope of a skilled person. The concentration of said buffer is in the range of 5 mM to 50 mM, preferably 10 mM to 30 mM. The concentration of buffer according to the present invention include each integer and non-integer number in specified range for each step of various chromatography steps as described herein. For example, it includes 5 mM, 10 mM, 12 mM, 15,5 mM, 17 mM, 20 mM, 25 mM, 30 mM, 37 mM, 40 mM, 45 mM, 50 mM.
The purification process of the present invention includes use of additives/salts. In one of the embodiments, the purification process according to the present invention includes use of additives/salts selected from ammonium sulphate, sodium chloride, arginine, glycine. The concentration of the said salts is in the range of 25 mM to 1200 mM, preferably 40 mM to 500 mM. The concentration of salts according to the present invention include each integer and non-integer number in specified range for each step of various chromatography steps as described herein. For example, it includes 25 mM, 30 mM, 40 mM, 50 mM, 75 mM, 100 mM, 125 mM, 140 mM, 150 mM, 300 mM, 500 mM, 550 mM, 600 mM, 800 mM, 900 mM, 1000 mM, 1100 mM.
The conductivity of buffer according to the present invention include each integer and non-integer number in specified range for each step of various chromatography steps as described herein. For example, it includes 0.1 mS / cm, 0.5 mS / cm, 0.8 mS / cm, 1.3 mS / cm, 1.5 mS / cm, 1.6 mS / cm 2.0 mS / cm, 2.1 mS / cm, 2.5 mS / cm, 2.6 mS / cm, 2.8 mS / cm, 3.0 mS / cm, 5 mS / cm, 5.5 mS / cm, 6 mS / cm, 6.5 mS / cm, 7.5 mS / cm, 9 mS / cm, 10 mS / cm, 11 mS / cm, 12 mS / cm, 13 mS / cm, 14 mS / cm, 15 mS / cm, 16 mS / cm, 17 mS / cm, 18 mS / cm, 18.5 mS / cm, 19 mS / cm, 21.3 mS / cm, 24 mS / cm, 25 mS / cm, 26 mS / cm, 28 mS / cm, 30 mS / cm, 31 mS / cm, 33.7 mS / cm, 35 mS / cm, 37 mS / cm, 39 mS / cm, 40 mS / cm 42 mS / cm, 45 mS / cm, 46 mS / cm, 47 mS / cm, 46.2 mS / cm, 48 mS / cm, 49 mS / cm, 50 mS / cm, 51 mS / cm, 55 mS / cm, 60 mS / cm, 81 mS / cm, 86 mS / cm, 91 mS / cm, 125 mS / cm, 135 mS / cm, 145 mS /cm, 150 mS / cm, 160 mS / cm, 170 mS / cm, 200 mS / cm. in a specified range for each step of various chromatography steps as described herein.
The pH of buffer according to the present invention include each integer and non-integer number in specified range for each step of various chromatography steps as described herein. For example, it includes pH 3, pH 3.2, pH 3.3, pH 3.5, pH 3.6, pH 3.7, pH 4, pH 4.2, pH 4.8, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.5, pH 5.7, pH 6.6, pH 6.8, pH 7.0, pH 7.2, pH 7.4, pH 7.6, pH 7.8, pH 8.0, pH 8.2, pH 8.4, pH 8.5, pH 8.7 for each relevant step of various chromatography steps as described herein.
The said process may comprise other non-chromatography steps such as clarification, viral inactivation at low pH, pH neutralization, ultrafiltration-diafiltration (UF/DF), nano filtration, terminal filtration. The non-chromatography steps according to the present invention is used here in the present invention before chromatography steps and / or between chromatography steps and / or after chromatography steps.
In further embodiment of the present invention, the process of purification of anti-RANKL antibody, preferably, denosumab from a crude mixture comprising a series of chromatography according to the present invention and ultrafiltration-diafiltration steps.
In further embodiment of the present invention, the process of purification of anti-RANKL antibody, preferably, denosumab from a crude mixture comprising a series of chromatography according to the present invention, clarification, low pH viral inactivation, pH neutralization, ultrafiltration-diafiltration steps, virus clearance and terminal filtration.
In one embodiment of the present invention, the process of purification of anti-RANKL antibody, preferably, denosumab comprises following steps:
1. Clarification;
2. r-PA chromatography;
3. Viral inactivation at low pH;
4. pH neutralization;
5. Hydrophobic interaction chromatography.

In one embodiment of the present invention, the process of purification of anti-RANKL antibody, preferably, denosumab comprises following steps:
1. Clarification;
2. r-PA chromatography;
3. Viral inactivation at low pH;
4. pH neutralization;
5. Hydrophobic interaction chromatography;
6. UF/DF-I;
7. Cation exchange chromatography;
8. Virus clearance by nano-filtration;
9. UF/DF-II;
10. Terminal filtration.

In an alternate embodiment of the present invention, the process of purification of anti-RANKL antibody, preferably, denosumab comprises following steps:
1. Clarification;
2. r-PA chromatography;
3. Viral inactivation at low pH;
4. pH neutralization;
5. UF/DF-I
6. Cation exchange chromatography;
7. UF/DF-I;
8. Hydrophobic interaction chromatography;
9. Virus clearance by nano-filtration;
10. UF/DF-III;
11. Terminal filtration.

After harvesting the batch, cells are separated from the culture broth by centrifugation followed by depth filtration in order to obtain clear supernatant containing the antibody of interest i.e. denosumab along with other soluble contaminants. The cell-free clarified supernatant is recovered and reconditioned to tune up with the protein A column equilibration buffer conditions for pH and conductivity. Protein A elute is further treated through various non-chromatography steps such as viral inactivation at low pH, pH neutralization before load on next column step. The next column step is either hydrophobic interaction chromatography or cation exchange chromatography. The non-chromatography steps are performed according to procedure known in the art. Reconditioned protein A column purified material is then loaded on either hydrophobic interaction chromatography or cation exchange column chromatography. The said column purified denosumab is reconditioned by UF/DF followed by either hydrophobic interaction chromatography or cation exchange chromatography. The column elute is further treated through various non-chromatography steps such as viral clearance by nano-filtration, ultrafiltration/diafiltration and 0.2 µm terminal filtration.
Here, the present invention is illustrated with the following non-limiting examples, which should not be interpreted as limiting the scope of the invention in any way:

Example 1: Purification of denosumab
Step 1: Cell separation or clarification
After harvesting the batch, cells are separated from the culture broth, first by centrifugation followed by depth filtration in order to obtain clear supernatant containing the protein of interest along with other soluble contaminants. Centrifugation is carried out at 10,500 g × 25 minutes. Depth filtration is performed by using 0.45 ? 0.22 µm membrane for further clarification. The clarified supernatant is reconditioned to tune up with the protein A column (r-PA) equilibration buffer condition e.g., pH and conductance.

Step 2: Protein A column chromatography
The clarified supernatant after reconditioning was passed through a protein A affinity column to capture anti-RANKL antibody, preferably, denosumab by the affinity matrix followed by its elution from the column at low pH. Protein A affinity column was used here in present example. However, other affinity column matrices can also be used at this step. Prior to loading, the column was equilibrated with a 25 mM Tris-Cl buffer of pH 7.4 containing 150 mM sodium chloride at a conductance of 18 mS /cm. Subsequent to loading, the column was washed with the same buffer (first wash). Following the first wash step, the column was washed with the high ionic strength buffer 25 mM Tris-Cl buffer of pH 7.4 containing 500 mM sodium chloride conductance 48 mS /cm (second wash). A third wash step was performed with 25 mM sodium acetate trihydrate buffer pH 5.5 having a conductance of 2.1 mS / cm (third wash). After the third wash step, elution of the denosumab was conducted with a 50 mM sodium acetate buffer containing 100 mM sodium chloride of pH 3.5 at a conductance 12 mS / cm. Denosumab eluted after this step shows at least 97% purity when analyzed by analytical HP-SEC shown in Table 1.

Step 3: Viral inactivation at low pH
r-PA column-eluted denosumab antibody was incubated at low pH condition, pH 3.7 - 3.8 for at least 45 min under room temperature conditions for potential viral inactivation, after which the antibody solution was passed through a 0.2 µm filter.

Step 4: Neutralization and reconditioning
Following low-pH treatment, pH-neutralization of the denosumab antibody solution was performed with the addition of an alkaline solution, like tris base or Na-citrate or ammonium hydroxide, in a controlled manner, and adjusted to pH about 6.8. Neutralized denosumab antibody solution was further reconditioned to match to the equilibration buffer conditions of the next HIC column step, with the addition of 2.2 M (NH4)2SO4 solution, under stirring condition. The final concentration of (NH4)2SO4 in the protein solution was adjusted to 1.1 M and the reconditioned solution was passed through a 0.2 µm filter, prior to loading onto the HIC column.

Step 5: Hydrophobic interaction chromatography (HIC)
Hydrophobic interaction chromatography was performed to remove product-related high molecular weight (HMW) species variants and process-related impurities (HCP). The reconditioned denosumab antibody solution was loaded onto HIC column at about 18 to 23 gm protein / L of matrix, equilibrated with 25 mM Tris-Cl buffer of pH 6.8 ± 0.2 and conductivity about 160.0 ± 10.0 mS / cm. (Wash 1) The column was washed with the same equilibration buffer, under room temperature conditions. Phenyl sepharose matrix was used as a HIC column matrix in this example. However, other HIC matrices known in the art can also be used at this step. Loading was conducted at a linear flow rate of about 90 cm / h. Prior to elution, an intermediate buffer wash was performed at conductivity about 135.0 ± 10.0 mS / cm with 25 mM Tris-Cl buffer of pH 6.8 ± 0.2 containing 0.9 M (NH4)2SO4. (Wash II) Subsequently, elution of denosumab was carried out at a conductivity about 55.0 ± 5.0 mS / cm with 25 mM Tris-Cl buffer of pH 6.8 ± 0.2 containing 0.35 M (NH4)2SO4 at the same linear flow rate of 90 cm / h. At the end, column cleaning was performed with 0.5 M sodium hydroxide followed by WFI. Denosumab eluted after this step shows at least 99 % purity when analyzed by analytical HP-SEC shown in Table 1.

Step 6: Ultrafiltration-diafiltration (UF/DF I)
The HIC column-eluted denosumab was subjected to UF / DF using 30 kDa MWCO membrane filter against sodium acetate trihydrate buffer of pH 5.5 in order to match to the next column (cation exchange column) step equilibration buffer conditions e.g., pH and conductance are adjusted up to the desired level. Diafiltered antibody solution was passed through a 0.22 µm filter, prior to loading on to the cation exchange column.

Step 7: Cation exchange column chromatography
Cation exchange (CEX) column chromatography was performed to separate the product-related and process-related impurities containing denosumab antibody solution obtained from step 6.
The diafiltered denosumab antibody solution was loaded onto the CEX column at about 30 to 40 gm protein / L of matrix, equilibrated with 25 mM sodium acetate trihydrate buffer of pH 5.5 ± 0.2 and conductivity about 2.1 mS / cm. (Wash 1) The column was washed with the same equilibration buffer, under room temperature conditions. Sulfopropyl (SP) –cross-linked agarose matrix was used as a CEX column matrix in this example. However, other CEX matrices known in the art can also be used at this step. Loading was conducted at a linear flow rate of about 105 cm / h. Prior to elution, an intermediate buffer wash was performed at conductivity about 10.0 mS / cm with 25 mM sodium acetate trihydrate buffer of pH 5.5 ± 0.2 containing 40 mM NaCl. (Wash II) Subsequently, elution of denosumab was carried out at a conductivity about 17.0 mS / cm with 25 mM sodium acetate buffer of pH 5.5 ± 0.2 containing 140 mM NaCl at the same linear flow rate of 105 cm / h. At the end, column cleaning was performed with 0.5 M sodium hydroxide followed by WFI. Denosumab eluted after this step shows at least 99% purity when analyzed by analytical HP-SEC shown in Table 1.

Step 8: Virus clearance by nano-filtration
The CEX column eluted denosumab protein was passed through a buffer-equilibrated 0.1 µm pre-filter followed by a nano-filter. During and after nano-filtration, no aggregation of denosumab protein was observed.

Step 9: Ultrafiltration-diafiltration (UF/DF II)
After nano-filtration, denosumab antibody was subjected to UF / DF using 30 kDa MWCO membrane filter against 18 mM sodium acetate buffer of about pH 5.0 to pH 5.2

Step 10: Terminal filtration
Finally, the purified bulk of denosumab obtained from step 9 was passed through 0.22 µm membrane filter, aseptically, and is stored either in the liquid form under cold condition or under frozen condition for long-term storage. The final purified denosumab exhibits more than 99.5 % purity.

Table 1: Purity of denosumab after each column chromatography by HP-SEC
Column chromatography Purity analysis by HP-SEC
% HMW species % Principal peak % LMW species
Protein A 2.0 98.0 ND
Hydrophobic interaction chromatography (HIC) 0.6 99.4 ND
Cation exchange chromatography (CEX) 0.11 99.89 ND

Example 2: Purification of denosumab
Step 1: Cell separation or clarification
After harvesting the batch, cells are separated from the culture broth, first by centrifugation followed by depth filtration in order to obtain clear supernatant containing the protein of interest along with other soluble contaminants. Centrifugation was carried out at 10,500 g × 25 minutes. Depth filtration is performed by using 0.45 ? 0.22 µm membrane for further clarification. The clarified supernatant is reconditioned to tune up with the protein A column (r-PA) equilibration buffer condition e.g., pH and conductance.

Step 2: Protein A column chromatography
The clarified supernatant after reconditioning was passed through a protein A affinity column to capture anti-RANKL antibody, preferably, denosumab by the affinity matrix followed by its elution from the column at low pH. Protein A affinity column was used here in present example. However, other affinity column matrices can also be used at this step. Prior to loading, the column was equilibrated with a 25 mM Tris-Cl buffer of pH 7.4 containing 150 mM sodium chloride at a conductance of 18 mS /cm. Subsequent to loading, the column was washed with the same buffer (first wash). Following the first wash step, the column was washed with the high ionic strength buffer 25 mM Tris-Cl buffer of pH 7.4 containing 500 mM sodium chloride conductance 48 mS /cm (second wash). A third wash step was performed with 50 mM sodium acetate trihydrate buffer pH 5.5 having a conductance of 2.1 mS / cm (third wash). After the third wash step, elution of the denosumab was conducted with a 50 mM sodium acetate buffer containing 100 mM sodium chloride of pH 3.5 at a conductance 12 mS / cm. Denosumab eluted after this step shows at least 97% purity when analyzed by analytical HP-SEC shown in Table 2.

Step 3: Viral inactivation at low pH
r-PA column-eluted denosumab antibody was incubated at low pH condition, pH 3.7 - 3.8 for at least 45 min under room temperature conditions for potential viral inactivation, after which the antibody solution was passed through a 0.2 µm filter.

Step 4: Neutralization and reconditioning
Following low-pH treatment, pH-neutralization of the denosumab antibody solution was performed with the addition of an alkaline solution, like tris base or Na-citrate or ammonium hydroxide, in a controlled manner, and adjusted to pH about 6.8. Neutralized denosumab antibody solution was further reconditioned to match to the equilibration buffer conditions of the next HIC column step, with the addition of 2.2 M (NH4)2SO4 solution, under stirring condition. The final concentration of (NH4)2SO4 in the protein solution was adjusted to 1.1 M and the reconditioned solution was passed through a 0.2 µm filter, prior to loading onto the HIC column.

Step 5: Hydrophobic interaction chromatography (HIC)
Hydrophobic interaction chromatography was performed to remove product-related high molecular weight (HMW) species variants and process-related impurities (HCP). The reconditioned denosumab antibody solution was loaded onto HIC column at about 18 to 23 gm protein / L of matrix, equilibrated with 25 mM Tris-Cl buffer of pH 6.8 ± 0.2 and conductivity about 160.0 ± 10.0 mS / cm. (Wash 1) The column was washed with the same equilibration buffer, under room temperature conditions. Phenyl sepharose matrix was used as a HIC column matrix in this example. However, other HIC matrices known in the art can also be used at this step. Loading was conducted at a linear flow rate of about 90 cm / h. Prior to elution, an intermediate buffer wash was performed at conductivity about 135.0 ± 10.0 mS / cm with 25 mM Tris-Cl buffer of pH 6.8 ± 0.2 containing 0.9 M (NH4)2SO4. (Wash II) Subsequently, elution of denosumab was carried out at a conductivity about 55.0 ± 5.0 mS / cm with 25 mM Tris-Cl buffer of pH 6.8 ± 0.2 containing 0.35 M (NH4)2SO4 at the same linear flow rate of 90 cm / h. At the end, column cleaning was performed with 0.5 M sodium hydroxide followed by WFI. Denosumab eluted after this step shows at least 99 % purity when analyzed by analytical HP-SEC shown in Table 2.

Table 2: Purity of denosumab after each column chromatography by HP-SEC
Column chromatography Purity analysis by HP-SEC
% HMW species % Principal peak % LMW species
Protein A 1.84 98.16 ND
Hydrophobic interaction chromatography (HIC) 0.65 99.35 ND

Incorporation by reference
The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

Equivalents
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.
,CLAIMS:We claim:
1. A process of purification of anti-RANKL antibody, preferably, denosumab comprises affinity chromatography followed by hydrophobic interaction chromatography.

2. The process of purification of anti-RANKL antibody, preferably, denosumab as claimed in claim 1 comprises affinity chromatography followed by hydrophobic interaction chromatography followed by cation exchange chromatography.

3. The process of purification of anti-RANKL antibody, preferably, denosumab as claimed in claim 1 or claim 2, wherein the affinity chromatography comprises protein A resin or protein G resin or protein L resin, preferably protein A resin (r-PA).

4. The process of purification of anti-RANKL antibody, preferably, denosumab as claimed in claim 3, wherein the r-PA chromatography according to the present invention is performed in bind-elute mode.

5. The process of purification of anti-RANKL antibody, preferably, denosumab as claimed in claim 4, wherein the r-PA purification step according to the present invention includes three column wash steps wherein (i) first wash is with equilibration buffer at suitable pH and / or conductivity, (ii) second wash at the same pH and / or at a conductivity higher than the first wash buffer (iii) third wash at a pH and / or conductivity lower than the second wash (iv) elution of the anti-RANKL antibody, preferably, denosumab at lower pH and / or higher conductivity than third wash.

6. The process of purification of anti-RANKL antibody, preferably, denosumab as claimed in claim 5, wherein the r-PA purification step comprises: (i) first wash with equilibration buffer at about pH 7.0 to 7.6 and / or conductivity at about 10 mS / cm to 25 mS / cm (ii) second wash at about pH 7.0 to 7.6 and / or conductivity at about 30 mS / cm to 60 mS / cm (iii) third wash at about pH 4.8 to pH 6.5, preferably at about pH 5.0 or pH 5.5 and / or conductivity at about 1 mS / cm to 10 mS / cm (iv) elution of the anti-RANKL antibody, preferably, denosumab in the range of pH 3.0 – 4.5, preferably at about pH 3.5 and / or conductivity at about 10 mS / cm to 40 mS / cm, preferably 10 mS / cm to 20 mS / cm.

7. The process of purification of anti-RANKL antibody, preferably, denosumab as claimed in claim 1 or claim 2, wherein the hydrophobic interaction chromatography according to the present invention is performed in bind-elute mode.

8. The process of purification of anti-RANKL antibody, preferably, denosumab as claimed in claim 7, wherein the hydrophobic interaction column chromatography (HIC) purification step includes two column wash steps wherein (i) first wash is with equilibration buffer at suitable pH and / or conductivity, (ii) second wash at the same pH and / or at a conductivity lower than the first wash buffer (iii) elution of the anti-RANKL antibody, preferably, denosumab at same pH and / or lower conductivity than second wash.

9. The process of purification of anti-RANKL antibody, preferably, denosumab as claimed in claim 8, wherein the hydrophobic interaction column chromatography (HIC) purification step comprises: (i) first wash with equilibration buffer at about pH 5.0 to 8.0 and / or conductivity at about 150.0 mS / cm to 200.0 mS / cm (ii) second wash at about pH 5.0 to 8.0 and / or conductivity at about 100.0 mS / cm to 145.0 mS / cm (iii) ) elution of the anti-RANKL antibody, preferably, denosumab in the range of pH 5.0 – 8.0, preferably at about pH 6.8 and / or conductivity at about 45 mS / cm to 95 mS / cm, preferably 50 mS / cm to 70 mS / cm.

10. The process of purification of anti-RANKL antibody, preferably, denosumab as claimed in claim 2, wherein the cation exchange chromatography according to the present invention is performed in bind-elute mode.

11. The process of purification of anti-RANKL antibody, preferably, denosumab as claimed in claim 10, wherein the cation exchange column (CEX) purification step includes two column wash steps wherein (i) first wash is with equilibration buffer at suitable pH and / or conductivity, (ii) second wash at the same pH and / or at a conductivity higher than the first wash buffer (iii) elution of the anti-RANKL antibody, preferably, denosumab at same pH and / or higher conductivity than second wash.

12. The process of purification of anti-RANKL antibody, preferably, denosumab as claimed in claim 11, wherein the cation exchange column (CEX) purification step comprises: (i) first wash with equilibration buffer at about pH 5.0 to 6.0 and / or conductivity at about 1.0 mS / cm to 4.0 mS / cm (ii) second wash at about pH 5.0 to 6.0 and / or conductivity at about 5.0 mS / cm to 12.0 mS / cm (iii) ) elution of the anti-RANKL antibody, preferably, denosumab in the range of pH 5.0 – 6.0, preferably about pH 5.5 and / or conductivity at about 14 mS / cm to 40 mS / cm, preferably 15 mS / cm to 20 mS / cm.

13. The process of purification of anti-RANKL antibody, preferably, denosumab as claimed in any preceding claim comprises following steps:
1. Clarification;
2. r-PA chromatography;
3. Viral inactivation at low pH;
4. pH neutralization;
5. Hydrophobic interaction chromatography.

14. The process of purification of anti-RANKL antibody, preferably, denosumab as claimed in any preceding claim comprises following steps:
1. Clarification;
2. r-PA chromatography;
3. Viral inactivation at low pH;
4. pH neutralization;
5. Hydrophobic interaction chromatography;
6. UF/DF-I;
7. Cation exchange chromatography;
8. Virus clearance by nano-filtration;
9. UF/DF-II;
10. Terminal filtration.

Dated this 08th day of May 2021.

(ASHISH KUMAR SHARMA)
IN/PA-858
Of SUBRAMANIAM & ASSOCIATES
AGENTS FOR THE APPLICANTS