DESC:Process of purifying anti-CD38 antibody

Field of the Invention
The invention provides process of purifying anti-CD38 antibody. Preferred anti-CD38 antibody according to present invention is daratumumab.

Background of the Invention
With the continuous development of biomedicine, antibody have shown an increasingly important position. Separation and purification of the culture containing the target antibody is an indispensable step in the production process. It has always been the continuous endeavor of industrial scientists to optimize the purification process steps of the antibody to increase the efficiency and also removing contaminants and thereby improve the purity and yield of the antibody.
Present invention provides efficient purification process of anti-CD38 antibody, preferably daratumumab which reduces product related impurities like high molecular weight (HMW) and Low molecular weight (LMW).

Summary of the invention
The present invention provides process of purification of anti-CD38 antibody, preferably daratumumab which comprises affinity chromatography followed by cation exchange chromatography followed by mixed-mode chromatography. Aspects of the present invention further comprises non-chromatography steps such as viral inactivation at low pH, pH neutralization, ultrafiltration-diafiltration, nano filtration, which are well known to a person skilled in the art.
Abbreviations
CEX: cation exchange chromatography
DF: Diafiltration
DNA: Deoxyribonucleic acid
HP-SEC: High performance size exclusion chromatography
MMC: Mixed mode 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

Brief description of figures
Figure 1- Figure 1 demonstrate elution profile of daratumumab by protein A chromatography as described in example 1.
Figure 2- Figure 2 demonstrate elution profile of daratumumab by cation exchange chromatography as described in example 1.
Figure 3- Figure 3 demonstrate elution profile of daratumumab by mixed mode chromatography as described in example 1.
Figure 4- Figure 4 demonstrate elution profile of daratumumab by protein A chromatography as described in example 2.
Figure 5- Figure 5 demonstrate elution profile of daratumumab by cation exchange chromatography as described in example 2.
Figure 6- Figure 6 demonstrate elution profile of daratumumab by mixed mode chromatography as described in example 2.
Figure 7- Figure 7 demonstrate purity of daratumumab by HP-SEC chromatography.

Definitions
Unless otherwise specified, a or an means "one or more."
The term about is used herein to mean plus or minus ten percent (10%) of a value. For example, "about 100" refers to any number between 90 and 110.
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 “flow-through-and-wash mode” refers to an operational approach to chromatography in which the buffer conditions are established so that the desired protein flows through the column upon application while undesired substances remain bound to matrix or retained by the matrix, thus achieving their removal in the subsequent steps.
The term “protein A chromatography” or “protein A column chromatography” or “r-PA chromatography” refers to capture of anti-CD38 antibody on resin containing protein A as a ligand, based on affinity of the anti-CD38 antibody, preferably, daratumumab to certain epitopes of protein A.
The term “mixed mode column chromatography” or “mixed mode chromatography” refers to chromatographic method that utilize at least two different forms of interaction between the stationary phase and analytes in order to achieve their separation. Preferably, mixed mode column or membrane comprises two different types of resins. Preferably, the mixed mode column comprising hydrophobic interaction resin and anion exchange resin are used in the present invention. The term “analytes” used herein is known to a skilled person and it is referred as the substances to be separated during chromatography.
The term “sequential” or “sequentially” according to present invention refers to a specific order of chromatography steps with or without intermediates step(s) of purification process of the current invention. Intermediate steps include non-chromatography steps such as viral inactivation at low pH, pH neutralization, ultrafiltration-diafiltration (UF/DF), nano filtration etc., which are referred herein to tune up purified sample solution obtained from the previous step with the subsequent purification step. The intermediate steps referred herein does not include any chromatography step between the purification steps describe herein the present invention.
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 "viral inactivation" refers to any treatment that makes a virus unable to infect biological samples or to replicate. Viral inactivation can be achieved by different techniques such as the incubation of the biological sample with solvents and /or detergents, which causes viral inactivation through the solubilization of the viral envelope; the incubation in low or high pH, which leads to the denaturation of the viral proteins; the pasteurization treatment, in which viral protein denaturation is achieved by high temperatures. Viral inactivation according to the present invention is performed at low pH ranges from pH 3.5 to pH 3.8.
The term "incubation" refers to the operation of keeping a material in certain conditions, comprising conditions for which the material undergoes modifications. In the process of viral inactivation, the incubation of a material with a solution having chemical characteristics that cause viral inactivation, leads to the generation of a material free of active viral contaminants.

Embodiments of the invention
In one embodiment, the present invention provides process of purification of anti-CD38 antibody, preferably, daratumumab comprises affinity chromatography followed by cation exchange chromatography followed by mixed-mode chromatography.
In a further embodiment of this invention, the affinity chromatography according to the present invention comprises protein A resin or protein G resin or protein L resin, preferably protein A (r-PA) resin.
In further embodiment of this invention, the r-PA chromatography according to the present invention is performed in bind-elute mode.
In further embodiment of this 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-CD38 antibody, preferably, daratumumab 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-CD38 antibody, preferably, daratumumab in protein A chromatography is performed at a pH lower than 7.0, preferably pH range from about pH 3.0 to pH 4.0.
In one embodiment of r-PA purification step according to the present invention, elution of the anti-CD38 antibody, preferably, daratumumab in protein A chromatography is performed at conductivity higher than 1 mS / cm, more preferably 1 mS / cm to 100 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 10 mS / cm to 25 mS / cm (ii) second wash at about pH 7.0 to 7.6 and / or conductivity at about 25 mS / cm to 90 mS / cm (iii) third wash at about pH 4.0 to pH 6.5 and / or conductivity at about 1 mS / cm to 10 mS / cm (iv) ) elution of the anti-CD38 antibody, preferably, daratumumab in the range of pH 3.0 – 4.5 and / or conductivity at about 1 mS / cm to 100 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 7.40 and / or conductivity at about 17.084 mS / cm (ii) second wash at about pH 7.33 and / or conductivity at about 83.879 mS / cm (iii) third wash at about pH 5.53 and / or conductivity at about 1.946 mS / cm (iv) elution of the anti-CD38 antibody, preferably, daratumumab at about pH 3.52, and / or conductivity at about 12.984 mS / cm.
In further embodiment of this invention, the cation exchange chromatography according to the present invention is performed in bind-elute mode.
In further embodiment of this invention, the cation exchange column (CEX) purification step according to the present invention includes one column wash steps wherein (i) first wash is with equilibration buffer at suitable pH and / or conductivity, (ii) elution of the anti-CD38 antibody, preferably, daratumumab at same pH and / or higher conductivity than first 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) elution of the anti-CD38 antibody, preferably, daratumumab in the range of pH 5.0 – 6.0 and / or conductivity at about 10 mS / cm to 40 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 5.53 and / or conductivity 2.039 mS / cm and (ii) elution of the anti-CD38 antibody, preferably, daratumumab at about pH 5.53 and / or conductivity at about 26.181mS / cm.
In further embodiment of this invention, the mixed mode chromatography (MMC) according to the present invention is performed in flow-through-wash mode.
In one embodiment of present invention, the mixed mode chromatography (MMC) purification step according to the present invention includes column wash steps wherein (i) first wash is with equilibration buffer at suitable pH and / or conductivity and (ii) elution of the anti-CD38 antibody, preferably, daratumumab is performed at about lower pH and / or conductivity than first wash, wherein the MMC is performed in flow-through-wash mode.
In one embodiment of the mixed mode column (MMC) purification step according to the present invention includes column wash steps wherein (i) first wash is with equilibration buffer at about pH 6.0 to pH 8.0 and / or conductivity 5 mS / cm to 20 mS / cm and (ii) elution of the anti-CD38 antibody, preferably, daratumumab is performed at about pH 1.0 to pH 5.0 and / or conductivity 1 mS / cm to 4 mS / cm.
In a specific embodiment of the mixed mode column (MMC) purification step according to the present invention includes column wash steps wherein (i) first wash is with equilibration buffer at about pH 7.43 and / or conductivity at about 15.316 mS / cm and (ii) elution of the anti-CD38 antibody, preferably, daratumumab is performed at about pH 2.31 and / or conductivity at about at about 1.719 mS / cm.
In a specific embodiment of the mixed mode column (MMC) purification step according to the present invention includes column wash steps wherein (i) first wash is with equilibration buffer at about pH 7.43 and / or conductivity at about 15.316 mS / cm and (ii) elution of the anti-CD38 antibody, preferably, daratumumab is performed at about pH 2.31 and / or conductivity at about at about 1.719 mS / cm wherein elution of the anti-CD38 antibody is performed in flow through and wash mode.
In further embodiment of this invention, the present invention provides the process of purification of anti-CD38 antibody, preferably, daratumumab, from a crude mixture comprising a series of chromatography as embodied herein above and ultrafiltration-diafiltration steps.
In further embodiment of this invention, the present invention provides the process of purification of anti-CD38 antibody, preferably, daratumumab, from a crude mixture comprising a series of chromatography as embodied herein above, clarification, low pH viral inactivation, pH neutralization, nano-filtration, ultrafiltration-diafiltration steps.
In further embodiment of this invention, the present invention provides process of purification of anti-CD38 antibody, preferably, daratumumab wherein the said process comprises following steps sequentially:
1. Clarification;
2. r-PA chromatography;
3. Viral inactivation at low pH;
4. Reconditioning by UF/DF;
5. Cation exchange chromatography;
6. Mixed-mode chromatography;
7. Virus clearance by nano-filtration;
8. Ultrafiltration/Diafiltration (UF/DF).

Detailed description of the present invention
The present invention provides process of purifying anti-CD38 antibody. Preferred anti-CD38 antibody according to present invention is daratumumab. The purification process of present invention comprises series of chromatography steps in specific order. The specific order is the order of chromatography steps as mentioned here in present invention. The chromatography steps of purification process of the present invention comprises membrane chromatography or column chromatography, preferably column chromatography. The process according to the present invention provides at least 99 % pure anti-CD38 antibody, preferably daratumumab. In one embodiment, the present invention provides process of purification of anti-CD38 antibody, preferably, daratumumab comprises affinity chromatography followed by cation exchange chromatography followed by mixed mode chromatography. The said chromatography steps are performed in specific order as mentioned herein above i.e. in any circumstances, first chromatography step according to the present invention is affinity chromatography, which is followed by cation exchange chromatography, which is second chromatography step according to the present invention. The cation exchange chromatography is followed by mixed mode chromatography, which is the third chromatography step according to the present invention. There may be non-chromatography steps such as clarification, low pH viral inactivation, pH neutralization, ultrafiltration-diafiltration steps, virus clearance, nano filtration performed between and / or before each chromatography steps mentioned herein present invention. In a further embodiment of this invention, the affinity chromatography according to the present invention comprises protein A resin or protein G resin or protein L resin, preferably protein A (r-PA) resin. Protein A chromatography step 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. Protein A 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-CD38 antibody, preferably daratumumab from a crude mixture by using a protein A 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 embodiment of this 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-CD38 antibody, preferably, daratumumab 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-CD38 antibody, preferably, daratumumab in protein A chromatography is performed at a pH lower than 7.0, preferably pH range from about pH 3.0 to pH 4.0. In one embodiment of r-PA purification step according to the present invention, elution of the anti-CD38 antibody, preferably, daratumumab in protein A chromatography is performed at conductivity higher than 1 mS / cm, more preferably 1 mS / cm to 100 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 10 mS / cm to 25 mS / cm (ii) second wash at about pH 7.0 to 7.6 and / or conductivity at about 25 mS / cm to 90 mS / cm (iii) third wash at about pH 4.0 to pH 6.5 and / or conductivity at about 1 mS / cm to 10 mS / cm (iv) elution of the anti-CD38 antibody, preferably, daratumumab in the range of pH 3.0 – 4.5 and / or conductivity at about 1 mS / cm to 100 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 7.40 and / or conductivity at about 17.084 mS / cm (ii) second wash at about pH 7.33 and / or conductivity at about 83.879 mS / cm (iii) third wash at about pH 5.53 and / or conductivity at about 1.946 mS / cm (iv) elution of the anti-CD38 antibody, preferably, daratumumab at about pH 3.52, and / or conductivity at about 12.984 mS / cm. r-PA column elute is further treated through various non-chromatography steps such as viral inactivation at low pH, pH neutralization, ultrafiltration/diafiltration before load on next column step. The said non-chromatography steps are performed according to procedure known in the art. Reconditioned protein A column purified material is then loaded on cation exchange column chromatography. Cation exchange chromatography is performed in bind-elute mode to further purify anti-CD38 antibody, preferably daratumumab. Product-related and process related impurities are removed during this step. The present invention provides atleast 97 % purity of daratumumab after cation exchange column chromatography. The column matrix for cation exchange chromatography according to the present invention is selected from SP Sepharose, SP-5PW, Capto™ 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. Capto™ SP ImpRes. Capto™ 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. SP Sepharose is an agarose based cation exchanger with a sulfopropyl group.
In further embodiment of this invention, the cation exchange column (CEX) purification step according to the present invention includes one column wash steps wherein (i) first wash is with equilibration buffer at suitable pH and / or conductivity, (ii) elution of the anti-CD38 antibody, preferably, daratumumab at same pH and / or higher conductivity than first 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) elution of the anti-CD38 antibody, preferably, daratumumab in the range of pH 5.0 – 6.0 and / or conductivity at about 10 mS / cm to 40 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 5.53 and / or conductivity 2.039 mS / cm and (ii) elution of the anti-CD38 antibody, preferably, daratumumab at about pH 5.53 and / or conductivity at about 26.181mS / cm.
CEX column purified daratumumab was followed by mixed mode column chromatography (MMC). After the second column step i.e. CEX as mentioned above, solution containing the daratumumab is reconditioned substantially to match up to the pH and conductivity of the mixed mode column equilibration conditions. Column is equilibrated with a buffer of pH about 7.4. The desired protein i.e. daratumumab is recovered from the column in the flow-through-and-wash fraction. For carrying out mixed mode chromatography according to the present invention, other mixed mode which also can be used are selected from Capto adhere impRes, Diamond MIX –A Mustang, Capto MMC, Poly Hi-Propyl, Toyopearl MX-Trp-650M, Eshmuno HCX media, etc. Capto Adhere ImpRes (CAI) has been used in the present invention.
In one embodiment of this invention, the mixed mode chromatography (MMC) according to the present invention is performed in flow-through-wash mode. In one embodiment of present invention, the mixed mode chromatography (MMC) purification step according to the present invention includes column wash steps wherein (i) first wash is with equilibration buffer at suitable pH and / or conductivity and (ii) elution of the anti-CD38 antibody, preferably, daratumumab is performed at about lower pH and / or conductivity than first wash, wherein the MMC is performed in flow-through-wash mode. In one embodiment of the mixed mode column (MMC) purification step according to the present invention includes column wash steps wherein (i) first wash is with equilibration buffer at about pH 6.0 to pH 8.0 and / or conductivity 5 mS / cm to 20 mS / cm and (ii) elution of the anti-CD38 antibody, preferably, daratumumab is performed at about pH 1.0 to pH 5.0 and / or conductivity 1 mS / cm to 4 mS / cm. In a specific embodiment of the mixed mode column (MMC) purification step according to the present invention includes column wash steps wherein (i) first wash is with equilibration buffer at about pH 7.43 and / or conductivity at about 15.316 mS / cm and (ii) elution of the anti-CD38 antibody, preferably, daratumumab is performed at about pH 2.31 and / or conductivity at about at about 1.719 mS / cm. In a specific embodiment of the mixed mode column (MMC) purification step according to the present invention includes column wash steps wherein (i) first wash is with equilibration buffer at about pH 7.43 and / or conductivity at about 15.316 mS / cm and (ii) elution of the anti-CD38 antibody, preferably, daratumumab is performed at about pH 2.31 and / or conductivity at about at about 1.719 mS / cm wherein elution of the anti-CD38 antibody is performed in flow through and wash mode.
In one of the embodiments of the present invention, the buffer used in the r-PA chromatography, cation exchange chromatography and mixed mode 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 70 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 55 mM, 60 mM, 65mM, 70 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 sodium chloride, arginine, glycine, preferably sodium chloride. The concentration of the said salts is in the range of 25 mM to 1000 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, 500 mM, 550 mM, 600 mM, 700 mM, 800 mM, 900 mM and 1000 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, 21.3 mS / cm, 24 mS / cm, 26 mS / cm, 28 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, 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, 70 mS / cm, 80 mS / cm, 85 mS / cm, 90 mS / cm etc. 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 2.0, pH 2.5, pH 3, pH 3.2, pH 3.5, pH 3.6, pH 3.7, pH 4.0, pH 4.1, pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.5, pH 5.7, 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, and pH 8.7 for each relevant step of various chromatography steps as described herein.
The purification process of the present invention may comprise other non-chromatography steps such as clarification, viral inactivation at low pH, pH neutralization, ultrafiltration-diafiltration (UF/DF), nano 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. The chromatography steps can be column chromatography or membrane chromatography.
In further embodiment of this invention, the present invention provides the process of purification of anti-CD38 antibody, preferably, daratumumab, from a crude mixture comprising a series of chromatography according to the present invention and ultrafiltration-diafiltration steps.
In further embodiment of this invention, the present invention provides the process of purification of anti-CD38 antibody, preferably, daratumumab, 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 nano filtration.
In further embodiment of this invention, the present invention provides process of purification of anti-CD38 antibody, preferably, daratumumab wherein the said process comprises following steps sequentially:
1. Clarification;
2. r-PA chromatography;
3. Viral inactivation at low pH;
4. Reconditioning by UF/DF;
5. Cation exchange chromatography;
6. Mixed-mode chromatography;
7. Virus clearance by nano-filtration;
8. Ultrafiltration/Diafiltration (UF/DF).
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. daratumumab 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, ultrafiltration / diafiltration before load on next column step. The said non-chromatography steps are performed according to procedure known in the art. Reconditioned protein A column purified material is then loaded on cation exchange column chromatography. CEX column purified daratumumab was loaded on mixed mode chromatography (MMC) in flow-through-and-wash mode. MMC flow through and wash fraction elute is further treated through various non-chromatography steps such as viral clearance by nano-filtration, ultrafiltration/diafiltration.
Examples
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 daratumumab
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 (r-PA) column chromatography
The clarified supernatant after reconditioning was passed through a protein A affinity column to capture anti-CD38 antibody, daratumumab 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 about 17.084 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 25 mM sodium acetate buffer of pH 5.53 and conductance 1.946 mS /cm (second wash). After the second wash step, elution of the daratumumab was conducted with a 50 mM sodium acetate buffer containing 100 mM sodium chloride of pH 3.52 at a conductance 12.984 mS / cm. Figure 1 illustrates the rPA column chromatography profiles obtained with 20L scale batch . Daratumumab eluted after this step shows at least 90 % purity when analyzed by analytical HP-SEC shown in Table 1.
Step 3: Viral inactivation at low pH
r-PA column-eluted daratumumab antibody was incubated at low pH condition, pH 3.5 to 3.6 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 by UF/DF
Following low-pH treatment, pH-neutralization of the antibody solution was performed with the addition of 2M tris base in a controlled manner, and adjusted to pH about 5.5 to 6.0. Neutralized antibody solution was further reconditioned by UF/DF to tune up the next column condition. The protein A column-eluted antibody was reconditioned by UF / DF using 30 kDa MWCO membrane filter against sodium acetate 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 5: Cation exchange column chromatography
Cation exchange (CEX) column chromatography was performed to separate the product-related and process-related impurities containing daratumumab antibody solution obtained from step 4.
The diafiltered antibody solution was loaded onto the CEX column at about 30 to 50 gm protein / L of matrix, equilibrated with 25 mM sodium acetate 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. Strong cation exchange matrix (cross-linked agarose with sulphopropyl functional group) was used as a CEX column matrix in this example. However, other CEX column matrices can also be used at this step. Loading was conducted at a linear flow rate of about 250 cm / h. Subsequently, elution of daratumumab protein was carried out at a conductivity about 26.181 mS / cm with 25 mM sodium acetate buffer of pH 5.5 ± 0.2 containing 250 mM NaCl at the same linear flow rate of 250 cm / h. At the end, column cleaning was performed with 0.5 M sodium hydroxide followed by WFI. Figure 2 illustrates the cation exchange column chromatography profiles obtained with 20L scale batch. Daratumumab eluted after this step shows at least 97 % purity when analyzed by analytical HP-SEC shown in Table 1.
Step 6: Mixed mode column chromatography
Diafiltered protein solution containing the desired monoclonal antibody was passed through a mixed mode column equilibrated with 25 mM Tris-Cl; pH 7.43; conductivity 15.316 mS / cm. The desired protein was recovered in flow-through-and-wash fraction. After the mixed mode purification step, purity of daratumumab was observed to be atleast 99 %, as assessed by HP-SEC.
The purified preparation may be suitably formulated for use as a pharmaceutical substance for human use. Figure 3 illustrates the mixed mode column chromatography profiles obtained with 20L scale batch.
Step 7: Nano-filtration
After the mixed mode column step, the solution containing the daratumumab antibody undergoes a nano-filtration step for virus clearance. No significant loss of protein or aggregation is observed during and after the nano-filtration step, as assessed by HP-SEC. After nano-filtration, purity of daratumumab was observed to remain more than 99 % .
Step 8: Ultrafiltration-diafiltration (UF/DF)
After nano-filtration, daratumumab protein solution was subjected to UF / DF for buffer exchange to prepare the purified bulk of daratumumab by using 30 kDa MWCO membrane filter. After buffer-exchange, daratumumab protein solution was further concentrated by ultrafiltration and collected at about 12 – 15 mg / mL. Polysorbate 80 at 0.2 mg / mL was added to the concentrated protein solution, under stirring condition and protein concentration was adjusted to 10 mg / mL by dilution to prepare the purified bulk of daratumumab.
Table 1: Purity of daratumumab after each column chromatography by HP-SEC
Column chromatography Purity analysis by HP-SEC
% HMW species % Principal peak % LMW species
Protein A 1.32 91.38 7.29
Cation exchange chromatography (CEX) 0.45 98.15 1.40
Mixed mode chromatography (MMC) 0.10 99.83 0.07

Example 2: Purification of daratumumab
Clarification of harvested cell culture fluid (HCCF) by centrifugation and filtration
At the end of cell culture process, cell culture broth was harvested and the HCCF was subjected to clarification by centrifugation at 10,500 ×g for 30 min; 23 °C (set-point). The clarified supernatant containing the soluble daratumumab protein was collected in a 50 L pressure vessel and passed through a single-use polyethersulfone (PES; 0.45 + 0.2 µm combined; 0.6 m2 surface area) filter, at 1.0 – 2.0 bar(s) pressure, to obtain the clarified cell culture fluid (CCCF). CCCF was collected in another vessel for reconditioning.
Reconditioning of the clarified cell culture fluid (CCCF) to prepare rPA load
The CCCF was reconditioned to match the equilibration buffer conditions of the first column step, rPA chromatography, in terms of pH and conductivity. pH was adjusted to 7.4 ± 0.2 with the addition of 2 M Tris-base solution. Conductivity was adjusted to 18 ± 2 mS / cm with the addition of NaCl solution from a 3 M stock, under stirring conditions at room temperature. The CCCF was passed through a 0.2 ?m filter before loading on to the rPA column.
rPA (recombinant Protein A) affinity column chromatography
rPA affinity column chromatography was performed to selectively capture and isolate the daratumumab protein in bind-elute mode from the reconditioned CCCF. Daratumumab protein present in the reconditioned crude CCCF was loaded (below the breakthrough point: 54 g protein / L of matrix) onto the agarose-based rPA resin, equilibrated with 25 mM Tris-Cl buffer of pH 7.4 ± 0.2 containing NaCl (conductivity 18.0 ± 2.0 mS / cm), under room temperature conditions. Loading was conducted at a linear flow rate of about 200 cm / h, while maintaining a contact time of at least 6 minutes for binding of protein to matrix. Majority of the undesired host cell contaminating proteins (HCPs) was observed to wash out of the column in flow-through-and-wash fraction, as expected. Post loading and equilibration buffer wash, the following steps were performed for the removal of loosely and / or non-specifically bound undesired residual impurities from the rPA column.
a) Wash I with 25 mM Tris-Cl buffer of pH 7.4 ± 0.2 containing 1.0 M NaCl, conductivity 85.0 ± 5.0 mS / cm; with about 3 column bed volumes
b) re-equilibration buffer wash with 25 mM Tris-Cl buffer of pH 7.4 ± 0.2 containing 150 mM NaCl (conductivity 18.0 ± 2.0 mS / cm); with about 2 column bed volumes and
c) Wash II with 25 mM Na-acetate buffer of pH 5.5 ± 0.1, conductivity 2.0 ± 0.5 mS / cm; with about 3 column bed volumes.
Elution of daratumumab protein from the column was carried out at pH 3.5 in 50 mM Na-acetate buffer containing 100 mM NaCl (conductivity 13.0 ± 2.0 mS / cm) at the same flow rate, mentioned above. Daratumumab protein was observed to elute out of the rPA column as a single peak in about 3 column bed volumes and collected in a clean de-pyrogenated glass bottle.
Peak collection criteria: Ascending portion, from about 20 mAU ? Descending portion, minimum up to 20 mAU.
Post-elution, the column was washed with an acidic solution of pH 2.5 – 2.6 to strip-out the residual bound proteins and subjected to CIP with 0.5 N NaOH. rPA. Figure 4 illustrates the rPA column chromatography profiles of daratumumab purified from crude CCCF at 20 L bioreactor-scale.
Low-pH hold for viral inactivation
pH of the rPA column eluted daratumumab protein solution was adjusted between pH 3.5 and 3.6 with the addition of 10 % acetic acid and was incubated for 60 ± 10 min, under static condition at room temperature (about 23 ?C) conditions. The low-pH incubation step is carried out for the inactivation of potential enveloped viruses. No visible precipitation or considerable protein aggregation was observed during or after low-pH hold.
pH neutralization
At the end of incubation, pH of the daratumumab protein solution was neutralized to pH 6.0 ± 0.2 with the addition of 2 M Tris-base solution, under stirring condition and passed through a 0.2 µm filter. No visible precipitation or protein aggregation was observed during or after pH neutralization.
Buffer exchange by UF / DF-I to prepare sCEX column load
Upon pH-neutralization, daratumumab protein solution was subjected to UF / DF-I to tune up with the equilibration buffer conditions of the next column step, in terms of pH and conductivity. UF / DF-I was performed at ambient temperature conditions, using 30 kDa MWCO membrane filters (cellulose based membrane; 0.3 m2 surface area), while maintaining a maximum transmembrane pressure (TMP) of 0.50 bar, with an average flux (max.) rate of about 30 L / m2 / h. Constant volume diafiltration was carried out for at least 8 dia-volumes, in the presence of sCEX column equilibration buffer – 25 mM Na-acetate buffer of pH 6.0 ± 0.2; conductivity 2.0 ± 0.5 mS /cm to achieve the desired pH and conductivity. Post-UF / DF-I, daratumumab protein solution was passed through a 0.2 µm filter and loaded onto the strong CEX column for further purification.
sCEX column chromatography
sCEX chromatography was performed in bind-elute mode to further purify the daratumumab protein, mainly for the removal of product-related and process-related impurities.
The diafiltered daratumumab protein solution was loaded onto the strong cation exchange column at about 40 g protein / L of matrix, which was equilibrated with 25 mM Na-acetate buffer of pH 6.0 ± 0.2 (conductivity 2.0 ± 0.5 mS / cm), under room temperature condition. Protein loading was conducted at a linear flow rate of 300 cm / h, while maintaining a contact time of at least 5 minutes for binding of protein to matrix. After loading, column was further washed with the equilibration buffer to remove loosely bound proteins, if any. Subsequently, elution of daratumumab protein was carried out with 250 mM Na-acetate buffer of pH 5.5 ± 0.2; conductivity 25 ± 5.0 mS / cm, at the same linear flow rate specified above, in linear gradient mode. Majority of daratumumab protein (loaded) was observed to elute out of the sCEX column with a minor peak 1 and major peak 2. The minor peak was collected separately. The major Peak 2 was collected in three sub-fractions (P2a, P2b and P2c) with pre-defined criteria Peak 2 fraction 1(P2a): Collected from about 30 mAU on the ascending portion to about 1500 mAU on the ascending portion of the peak, as indicated in Figure 5.
Peak 2 fraction 2 (P2b): Collected from about 1500 mAU on the ascending portion to about 1500 mAU on the descending portion of the peak
Peak 2 fraction 3 (P2c): Collected from about 1500 mAU on the descending portion to end of the peak.
At the end of protein elution, column cleaning was performed with 0.5 M NaOH followed by WFI wash. Figure 5 illustrates the sCEX column chromatography profile.
The major fraction P2b was selected for further purification of daratumumab.
Buffer exchange by UF / DF-II to prepare mixed mode column load sCEX purified daratumumab solution was subjected to UF / DF-II to tune up with the equilibration buffer conditions of the next column step, in terms of pH and conductivity. UF / DF-II was performed at ambient temperature conditions, using 30 kDa MWCO membrane filters (cellulose based membrane; 0.3 m2 surface area), while maintaining a maximum transmembrane pressure (TMP) of 0.50 bar, with an average flux (max.) rate of about 36 L / m2 / h. Prior to diafiltration, the protein solution was concentrated by about 3-fold (20 mg / mL). Post-concentration, constant volume diafiltration was carried out for at least 8 dia-volumes with 25 mM Tris-Cl buffer of pH 7.4 ± 0.2 containing 125 mM NaCl, conductivity 14.0 ± 2.0 mS / cm to achieve the desired pH and conductivity, as specified for the mixed mode column equilibration buffer. Post-UF / DF-II, daratumumab protein solution was passed through a 0.2 µm filter and loaded onto a mixed mode column for further purification.

Mixed mode column chromatography
Mixed mode column chromatography was performed in flow-through-and-wash mode for further purification of daratumumab protein for the removal of residual product-related and process-related impurities.
The reconditioned daratumumab protein solution was loaded on to the column at about 40 g protein / L of matrix, which was equilibrated with 25 mM Tris-Cl buffer of pH 7.4 ± 0.2 containing 125 mM NaCl (conductivity 14 ± 2 mS / cm). A contact time of about 4.5 minutes was maintained between protein and column matrix, during loading. In flow-through-and-wash daratumumab protein was collected in three fractions with pre-defined peak cutting criteria –
Fraction collection criteria –
Fraction 1 – Collected from about 30 mAU on the ascending portion to about 500 mAU of the ascending portion of the peak.
Fraction 2 – Collected from about 500 mAU on the ascending portion to about 500 mAU on the descending portion of the peak
Fraction 3 – Collected from about 500 mAU on the descending portion to about 150 mAU.
Finally, the drug substance preparation process was advanced with the fraction 2, since it was observed to exhibit the desired level of purity > 99%.
Post-elution, the column was washed with 25 mM citric acid (pH 2.5 ± 0.5; conductivity 1.5 ± 0.5 mS / cm) solution to remove the residual tightly bound proteins, predominantly the HMW species. Column sanitization was performed with 0.5 M NaOH solution and NaOH was washed out with WFI.
Figure 6 illustrates the mixed mode column chromatography profiles obtained with 20L scale bioreactor batch.
Virus clearance by nano-filtration
Post-mixed-mode column purification, the daratumumab protein recovered (about 40 g protein / batch) in the pooled fraction was passed through a 0.2 µm pre-filter followed by a nano-filter (204 cm2 total surface area; PES membrane; average cut-off 20 nm) at 1.7 bar pressure (set-point). Nano-filtration was performed as one of the orthogonal virus removal steps in the purification process of daratumumab.
Buffer exchange by UF / DF-III and preparation of formulated bulk
Nano-filtered daratumumab protein solution was subjected to UF / DF-III for drug substance buffer exchange using 30 kDa MWCO membrane filter (cellulose based membrane; 0.2 m2 surface area). UF / DF-III was carried out at a maximum TMP of 0.50 bar. In the first step of UF / DF, about 10 – 25-fold concentration of daratumumab protein was performed, after which constant volume diafiltration was carried out with the desired buffer solution of pH 5.5 ± 0.20 (conductivity 1.5 ± 0.5 mS / cm), for at least 8 dia-volumes. Diafiltration was monitored and controlled for pH and conductivity of the retentate protein solution to achieve the target values. After achieving the target pH and conductivity, the daratumumab protein solution was recovered at about 55 mg / mL. To the protein concentrate, a chelator, an anti-oxidant, a stabilizer and a surfactant that were dissolved in the same buffered solution, were added and mixed. The final volume was adjusted to obtain daratumumab at 20 mg / mL.
Terminal filtration (0.2 µm)
The formulated bulk containing purified daratumumab was passed through a 0.2 µm filter (150 cm2 surface area), under aseptic conditions, and collected in a sterile, non-pyrogenic container to obtain the bulk drug substance of daratumumab.
Storage of the bulk drug substance of daratumumab
The bulk drug substance of daratumumab was aliquoted in non-pyrogenic, single-use, sterile cryo-bags and stored under frozen condition at – 25 ± 5 °C.
Example 3- Size exclusion chromatography (HP-SEC) of the drug substance
Independent batch produced daratumumab drug substance sample were analyzed by analytical HP-SEC to assess the level of purity and presence of product-related size variants (HMW and LMW species), under native conditions. Samples were injected onto a TSK-Gel G3000SWXL (7.8 mm x 30 cm; 5 µm) column for HPLC analysis. HP-SEC analysis was performed in isocratic mode with 50 mM sodium phosphate buffer of pH 6.8, containing 300 mM NaCl, as mobile phase. Separation of protein size variants was conducted at a flow rate of 0.5 mL / min, 25 ?C column temperature and recorded with UV detection at 214 nm.
Purity of daratumumab drug substance preparation was found to be > 99.5 %. Less than 0.5 % HMW aggregates and barely detectable amount of LMW species of daratumumab were observed in the drug substance materials, as illustrated in Figure 7. Daratumumab eluted shows more than 99% purity as shown in table 2. The presence of impurities, including low molecular weight impurities, can significantly impact the quality, safety, and efficacy of the product. HMW and LMW impurities can contribute to the instability of daratumumab, leading to aggregation, precipitation, or degradation of the protein. This can compromise product shelf life, potency, and overall quality of the product.

Table 2: Purity of daratumumab by HP-SEC
Sample % relative distribution by HP-SEC
% HMW species % Principal peak % LMW species
Daratumumab after purification 0.02 99.94 0.03
Daratumumab drug substance 0.04 99.93 0.04

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 there.

,CLAIMS: We claim:
1. A process of purification of anti-CD38 antibody, preferably, daratumumab, which comprises the steps of affinity chromatography, followed by cation exchange chromatography, followed by mixed mode chromatography.

2. The process as claimed in claim 1, wherein affinity chromatography comprises protein A resin (r-PA) or protein G resin or protein L resin, preferably protein A resin (r-PA).

3. The process as claimed in claim 2, wherein said r-PA chromatography is performed in bind-elute mode.

4. The process as claimed in claim 3, which comprises 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; and elution of the anti-CD38 antibody, preferably, daratumumab is performed at lower pH and / or higher conductivity than the third wash.

5. The process as claimed in claim 4, comprising purification step wherein elution of the anti-CD38 antibody, preferably, daratumumab is performed at a pH lower than 7.0, preferably at a pH range from about pH 3.0 to pH 4.0, more preferably about pH 3.6 and / or elution of the anti-CD38 antibody, preferably, daratumumab is performed at conductivity higher than 1 mS / cm, more preferably about 1 mS / cm to 100 mS / cm.

6. The process as claimed in claim 4, comprising: (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 25 mS / cm to 90 mS / cm (iii) third wash at about pH 4.0 to pH 6.5 and / or conductivity at about 1 mS / cm to 10 mS / cm (iv) elution of the anti-CD38 antibody, preferably, daratumumab in the range of pH 3.0 -¬¬¬ 4.5 and / or conductivity at about 1 mS / cm to 100 mS / cm.

7. The process as claimed in claim 6, comprising: i) first wash with equilibration buffer at about pH 7.40 and /or conductivity at about 17.084 mS / cm (ii) second wash at about pH 7.33 and / or conductivity at about 83.879 mS / cm (iii) third wash at about pH 5.53 and / or conductivity at about 1.946 mS / cm (iv) elution of the anti-CD38 antibody, preferably, daratumumab at about pH 3.52, and / or conductivity at about 12.984 mS / cm.

8. The process as claimed in claim 1 wherein cation exchange chromatography is performed in bind elute mode.

9. The process as claimed in claim 8 wherein cation exchange column (CEX) comprises of at least one column wash step wherein the wash step is performed with equilibration buffer at suitable pH and / or conductivity and elution of the anti-CD38 antibody, preferably, daratumumab is carried out at the same pH and / or higher conductivity, than the first wash.

10. The process as claimed in claim 9 which 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) elution of the anti-CD38 antibody, preferably, daratumumab in the range of pH 5.0 to 6.0 and / or conductivity at about 10 mS / cm to 40 mS / cm.

11. The process as claimed in claim 10 which comprises: (i) first wash is with equilibration buffer at about pH 5.53 and / or conductivity 2.039 mS / cm and (ii) elution of the anti-CD38 antibody, preferably, daratumumab at about pH 5.53 and / or conductivity at about 26.18mS / cm.

12. The process as claimed in claim 1 wherein mixed mode chromatography (MMC) is performed in flow-through-wash mode.

13. The process as claimed in claim 12 which comprises column wash steps wherein (i) first wash is with equilibration buffer at suitable pH and / or conductivity and (ii) elution of the anti-CD38 antibody, preferably, daratumumab is performed at about lower pH and / or conductivity than first wash, wherein the MMC is performed in flow-through-wash mode.

14. The process as claimed in claim 13 which comprises column wash steps wherein (i) first wash is with equilibration buffer at about pH 6.0 to pH 8.0 and / or conductivity 5 mS / cm to 20 mS / cm and (ii) elution of the anti-CD38 antibody, preferably, daratumumab is performed at about pH 1.0 to pH 5.0 and / or conductivity 1 mS / cm to 4 mS / cm.

15. The process as claimed in claim 14 which comprises column wash steps wherein (i) first wash is with equilibration buffer at about pH 7.43 and / or conductivity at about 15.316 mS / cm and (ii) elution of the anti-CD38 antibody, preferably, daratumumab is performed at about pH 2.31 and / or conductivity at about at about 1.719 mS / cm.

16. The process as claimed in any preceding claim wherein the amount of low molecular weight species (LMW) is less than 0.1 %.

17. The process of purification of anti-CD-38 antibody, preferably, daratumumab as claimed in any preceding claim, which comprises of following steps sequentially:
a) Clarification;
b) r-PA chromatography;
c) Viral inactivation at low pH;
d) Reconditioning by UF/DF (optional);
e) Cation exchange chromatography;
f) Mixed-mode chromatography;
g) Virus clearance by nano-filtration;
h) Ultrafiltration / Diafiltration (UF / DF).

Dated this 19th day of February 2024.

(HARIHARAN SUBRAMANIAM)
IN/PA-93
Of SUBRAMANIAM & ASSOCIATES
ATTORNEYS FOR THE APPLICANTS