FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
&
The Patent Rules, 2003
COMPLETE SPECIFICATION
(See sections 10 & Rule 13)
TITLE OF THE INVENTION
PURIFICATION PROCESS AND FORMULATION OF RECOMBINANT ANTI RHD MONOCLONAL ANTIBODIES
2. APPLICANT: Bharat Serums and Vaccines Ltd.,
Address: R&D Centre, 3rd Floor, Liberty Tower, Behind Reliable Plaza, Plot no. K-10, Kalwa Industrial estate, Airoli, Navi-Mumbai - 400708.
NAME NATIONALITY ADDRESS
Mr. Mahesh Gavasane Indian A-601, Rite Skyluxe, Off. 13th road, Subhash nagar, Chembur, Mumbai - 400071
Mr. Pranav Gupte Indian A-01 Om Laxminarayan Soc, Charai, Thane (west)-400601.
Mr. Lilesh Bhise Indian C-702, Kailash Park CHS, Lake Road, Tulshetpada, Bhandup (west), Mumbai-400078
Mr. Tushar Bhabal Indian Daisy-105, Mohan Highlands CHS, Katrap valley, Katrap, Badlapur (East), Thane - 421503
Ms. Aditee Kambli Indian 2708, Lodha Casa Viva- A wing, Majiwada, Thane (West) -400608
Dr. Abir Banerjee Indian D204, Anjor Apartments, Baner, Pune - 411045
PREAMBLE TO THE DESCRIPTION
The present invention relates to an improved purification process for isolating anti-Rhesus D monoclonal
antibodies and their antigen-binding fragments, the present invention also encompasses a stable formulation for the purified antibodies or fragments with improved shelf life and performance of the final product.
COMPLETE SPECIFICATION
The following specification describes the invention.

PURIFICATION PROCESS AND FORMULATION OF RECOMBINANT ANTIRHD MONOCLONAL ANTIBODIES
FIELD OF INVENTION
The present invention relates to an improved purification process for isolating anti-Rhesus D monoclonal antibodies and their antigen-binding fragments. The improved process involves a series of purification techniques, including affinity chromatography, low pH inactivation, anion exchange and cation exchange chromatography, virus/nanofiltration, ultrafiltration and the like. Without limitation to the embodiments described hereinafter, the present invention also encompasses a stable formulation for the purified antibodies or fragments with improved shelf life and performance of the final product.
BACKGROUND AND PRIOR ART
Rhesus D antigen, also known as RhD antigen, Rhesus factor, or Rh factor, is an antigen that may be present on the surface of human red blood cells. Individuals whose red blood cells have this antigen are typically referred to as "RhD-positive," while those without it are called "RhD-negative."
A person who is RhD-negative and has never been exposed to the RhD antigen will not produce antibodies against it. However, if a RhD-negative individual receives RhD-positive blood, they may become sensitized and develop anti-RhD antibodies. This can lead to complications, especially during pregnancy. If a RhD-negative woman gives birth to a RhD-positive baby, there is a risk of the baby's blood entering the mother's circulation, causing her to produce anti-RhD antibodies. While this usually does not harm the first baby, subsequent pregnancies with RhD-positive infants may result in hemolytic disease of the newborn (HDN) as the maternal anti-RhD antibodies may attack the baby's blood cells.

To prevent RhD-negative patients from becoming immunized against RhD-positive blood, anti-RhD antibodies may be given when there is a risk of exposure to RhD-positive blood. For instance, a RhD-negative patient may be administered anti-RhD antibodies prior to and/or shortly after giving birth to or having an abortion of an RhD-positive baby, experiencing bleeding across the placenta during pregnancy, or prior to or soon after undergoing any transfusion of blood components containing RhD-positive red blood cells.
Traditionally, polyclonal antibodies obtained from the blood plasma of RhD-negative individuals repeatedly immunized against RhD-positive red blood cells have been used as anti-RhD antibodies. However, use of polyclonal antibodies has drawbacks, including the ongoing need for multiple volunteer donors to meet the demand for antibodies and the risk of potential contamination with viruses or pathogens from the donor's blood.
In contrast, monoclonal antibodies, produced from a single parent cell's clone and constitute a homogenous population of antibodies as known in art offer advantages over polyclonal antibodies. The cell lines from which monoclonal antibodies are produced, developed and cultured in-vitro, and this means that Monoclonal antibodies have potential to be produced as and when required in large amounts and with high purity as needed. This makes monoclonal anti-RhD antibodies a promising alternative to the traditional polyclonal preparations.
The Anti-RhD monoclonal antibodies of the present invention was developed by the same inventors and is disclosed in the previous patent publication (US 8,529,903), which pertains to Anti-RhD monoclonal antibodies and their production methods.
The said monoclonal antibody as referred in the draft is also named as Trinbelimab, rAnti-Rh-D, rAnti-D, rAnti-Rh(0)-D, Recombinant Anti Rho-D Immunoglobulin,

Anti-D (recombinant), Rh Immune Globulin (RhIG), Rho(D) Immune Globulin, Anti-D (Immunoglobulin) (Recombinant), Anti-Rh(D) (Recombinant) antibody.
The inventors produced Recombinant Anti Rho-D Immunoglobulin from genetically engineered Chinese Hamster Ovary (CHO) cells secreting the rAnti-Rh-D antibody. rAnti-Rh-D antibody as described in US '903 patent, is a 1352-amino acid containing antibody, has a molecular weight of around 150 kDa and consists of two identical light and two identical heavy chains. The light chain consists of 216 amino acids and heavy chain consists of 460 amino acids. Light and heavy chains are interlinked by 18 disulfide bonds. Each light chain contributes to ~ 25 kDa molecular weight and the heavy chain to ~ 50 kDa. The Isoelectric point (pi) of rAnti-Rh-D is ~ 8.7 and the glycosylation site is in the Fc region at N-310 of heavy chain.
rAnti-Rh-D is used for treatment of Haemolytic disease of newborn (HDN). HDN is caused by the action of maternal RhD antibody in Rh negative pregnant women against the foetal RhD +ve RBCs. A dose of 300mg is given intramuscularly as soon as possible during first three days after delivery. The same is incorporated with reference herein in its entirety.
Following the successful launch of the formulation of said antibody in the market, it was observed that the impurity profile of the marketed formulation can be further improved with respect to the high molecular (HMW) and low molecular weight (LMW) impurities. The inventors optimized the process by making significant modifications, the optimized downstream process was found to be robust and yielded a stable formulation.
The new optimized and improved process ensures thorough purification of the Recombinant Anti-RliD monoclonal antibodies and enhanced product quality and

safety, meeting the international regulatory standards. Additionally, it increased the product's stability and shelf-life.
OBJECTS AND SUMMARY OF THE INVENTION:
According to a first aspect of the present invention there is provided an improved process for purification of an isolated Recombinant Anti Rho-D Immunoglobulin (rAnti-Rh-D) monoclonal antibody from cell culture harvest with an objective to remove the product and process related impurities. The newly devised process aims to overcome the challenges faced in eliminating HMW impurities, thereby enhancing the overall purification efficiency and product quality.
According.to the second aspect of the present invention there is provided an improved formulation comprising Anti-RhD monoclonal antibodies with an aim to achieve improved purity and enhanced stability while minimizing process and product-related impurities.
Yet another aspect of the invention is to optimize the downstream process and formulation to obtain a shelf-stable drug substance and drug product.
The following embodiments further describe the objects of the present invention in accordance with the best mode of practice. However, the disclosed invention is not restricted to the embodiments hereinafter described.
DETAILED DESCRIPTION:
The present invention can be more readily understood by reading the following detailed description of the invention and studying the included examples and experimental results.

The process of purifying a protein often involves several steps. The present invention encompasses an improved downstream process and, in some embodiments, involves at least four steps. However, the entire process of purifying the protein may include other steps before and/or after each of these steps.
According to a first aspect of the present invention there is provided an improved process for purification of an isolated Recombinant Anti Rho-D Immunoglobulin (rAnti-Rh-D) monoclonal antibody comprising the following steps:
a) affinity chromatography (AF) on the cell culture harvest comprising the isolated rAnti-Rh-D monoclonal antibody and collecting eluate comprising the purified monoclonal antibody.
b) low pH inactivation on eluant obtained in (a) to inactivate the enveloped viruses
c) Ion Exchange Chromatography to remove aggregates, endotoxins and other impurities.
d) optionally followed by other suitable purification steps.
In an embodiment of the present invention, the yield of the final composition after purification is at least 50% and high molecular weight (HMW) impurity is less than 0.5% and low molecular weight (LMW) impurity is less than 1.5%.
In an embodiment of the present invention, the ion exchange chromatography comprises anion exchange & cation exchange chromatography steps.
In an embodiment of the present invention, the improved downstream process for (rAnti-Rh-D) monoclonal antibody comprises of the following steps:
a) affinity chromatography (AF) on the cell culture harvest comprising the isolated rAnti-Rh-D monoclonal antibody and collecting eluate comprising the purified monoclonal antibody.
b) low pH inactivation on eluant obtained in (a) to inactivate the enveloped viruses.

c) Anion exchange chromatography
d) Cation exchange chromatography
e) Virus filtration
f) optionally followed by other suitable purification steps.
In an embodiment of the present invention, the suitable purification steps are Ultrafiltration and Aseptic filtration.
In another embodiment of the present invention, the purification process comprises steps of
a) affinity chromatography
b) low pH inactivation
c) Anion Exchange Chromatography
d) Cation exchange chromatography
e) Nanofiltration/virus filtration,
f) Ultrafiltration and
g) Aseptic filtration
In an embodiment of the present invention, an improved process for purification of an isolated rAnti-Rh-D monoclonal antibody from cell culture harvest comprises of the following steps:
a) carrying out affinity chromatography (AF) on the cell culture harvest comprising the isolated anti-RhD monoclonal antibody and collecting eluate comprising the purified monoclonal antibody.
b) carrying out low pH inactivation on eluant obtained in (a) to inactivate the enveloped viruses, pH of the affinity elute was adjusted to 3.5±0.1 using acetic acid or Tris buffer; followed by
c) an intermediate purification step, wherein affinity elute was neutralized to pH 7.4 with the 2M Tris Buffer and subjected to anion exchange step using resins.

d) polishing step using cation exchange polymers to remove the process and product related impurities wherein chromatography is performed in bind-elute mode.
e) virus filtration/nanofiltration to remove broad size of viruses depending on pore size of filter.
f) optionally ultrafiltration wherein buffer exchange is achieved using a diafiltration mode.
g) optionally aseptic filtration
wherein the yield of the final composition after purification is greater than or equal to about 95% and LMV is less than 1.5% and HMV is less than 0.5%.
As used herein, the term "anti-RhD antibody" refers to both whole antibodies and to fragments thereof that have binding specificity for RhD antigen. The binding affinity/specificity of an antibody can be measured by various assays, as will be known to and can be routinely implemented by one of ordinary skill in the art. For example, antibodies recognising and specifically binding to RhD antigen can be determined using one or more standard techniques as known to one of ordinary skill in the art, such as but not limited to: EIA/ELISA techniques, such as competitive EIA (enzyme linked-immunoassay); flow cytometry; and/or ADCC (antibody-dependant cellular toxicity) assays.
Recombinant Anti Rho-D Immunoglobulin is produced from genetically engineered Chinese Hamster Ovary (CHO) cells secreting the rAnti-Rh-D antibody.
As used herein the term "an isolated monoclonal antibody" refers to an antibody which has been produced by monoclonal techniques and which has been isolated from antibodies of other types. In other words, the only other antibodies present will be antibodies produced by cells of the same cell line (i.e., cells all originating from the same single parent cell) as the cell which produced the monoclonal antibody. This is of

course in contrast to, for example, polyclonal antibodies where the antibodies constitute a mixture of different antibodies originating from different plasma cells.
As used herein the term "chromatography" refers to the process by which a solute of interest, e.g., a protein of interest, in a mixture is separated from other solutes in the mixture by percolation of the mixture through an adsorbent, which adsorbs or retains a solute strongly due to properties of the solute, such as hydrophobicity, size and structure, under buffering conditions of the process.
Many chromatographic methods can be employed in antibody purification. For example, Gel-permeation (molecular sieve) chromatography (this technique is used for purification of enzymes, hormones, antibodies, nucleic acids, specific proteins, etc.); Size-exclusion chromatography (less popular final step for removing residual proteins and contaminants); tagged affinity chromatography (a recombinant antibody is produced as a fusion protein containing a terminal affinity tag such as a poly-histidine tag. The chromatography column contains media functionalized with a molecule that binds the tag with high affinity); affinity chromatography, ion exchange chromatography (cation exchange and anion exchange chromatography), and the like.
As used herein, "Adsorbent" refers to at least one molecule affixed to a solid support or at least one molecule that is, itself, a solid, which is used to perform chromatography.
As used herein, "Affinity chromatography" is popular method and refers to chromatography that utilizes the specific, reversible interactions between biomolecules, for example, the ability of Protein A to bind to an FC portion of an IgG antibody, rather than the general properties of a molecule, such as isoelectric point (pi), hydrophobicity, or size, to effect chromatographic separation. In practice, affinity chromatography is only a part of a multistep approach - that is affinity chromatography involves using an adsorbent, such as Protein A affixed to a solid support, to chromatographically separate

molecules that bind tightly to the adsorbent (See Ostrove (1990) in Guide to Protein Purification, Methods in Enzymology 182: 357-379, which is incorporated with reference herein in its entirety).
By "purifying" an antibody from a composition comprising the antibody and one or more contaminants is meant increasing the degree of purity of the antibody in the composition by removing (completely or partially) at least one contaminant from the composition. A "purification step" may be part of an overall purification process resulting in a "homogeneous" composition. "Homogeneous" is used herein to refer to a composition comprising at least about 70% by weight of the antibody of interest, based on total weight of the composition, preferably at least about 80% by weight, more preferably at least about 90% by weight, even more preferably at least about 95% by weight.
The terms "ion-exchange" and "ion-exchange chromatography" refer to a chromatographic process in which an ionizable solute of interest (e.g., a protein of interest in a mixture) interacts with an oppositely charged ligand linked (e.g., by covalent attachment) to a solid phase ion exchange material under appropriate conditions of pH and conductivity, such that the solute of interest interacts non-specifically with the charged compound more or less than the solute impurities or contaminants in the mixture. The contaminating solutes in the mixture can be washed from a column of the ion exchange material or are bound to or excluded from the resin, faster or slower than the solute of interest. "Ion-exchange chromatography" specifically includes cation exchange (CEX), anion exchange, and mixed mode chromatography. Cation exchange chromatography is a popular method for cleaning up an isolated monoclonal antibody. In this step, the antibody binds the solid phase, and contaminants are allowed to flow through or wash off the protein.

The complementary method, anion exchange chromatography, may also be employed, in which case contaminants are captured on the anion exchange column, while the antibody flows through. Two ion-exchange chromatography steps are often needed for the complete removal of cell-related contaminants.
The "composition" to be purified herein comprises the antibody of interest and one or more contaminants. The composition may be "partially purified" (i.e., having been subjected to one or more purification steps) or may be obtained directly from a host cell or organism producing the antibody (e.g., the composition may comprise harvested cell culture fluid).
A "buffer" is a solution that resists changes in pH by the action of its acid-base conjugate components. Various buffers which can be employed depending, for example, on the desired pH of the buffer are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., Ed. Calbiochem Corporation (1975).
An "equilibration buffer" is a buffer that is used to equilibrate the cation exchange material, prior to loading the composition comprising the antibody of interest and one or more contaminants onto the cation exchange material. Preferably the pH of the equilibration buffer herein is in the range from about 5.0 to about 6.0, preferably about 5.5. Preferably, the conductivity of the equilibration buffer herein is in the range from about 1 to about 8mS/cm, preferably from about 4 to about 8mS/cm, and most preferably from about 5 to about 8mS/cm.
In yet another embodiment of the present invention, the equilibrium buffer comprises buffers with or without salt additives viz. suitable buffers include, but are not limited to, phosphate buffers, Tris buffers, acetate buffers, and/or citrate buffers, glycine, histidine and its pharmaceutical salts thereof, HEPES, MOPS, arginine and its pharmaceutical slats thereof, proline and its pharmaceutical salts thereof and the combinations thereof

and suitable salts comprise of sodium chloride, sodium carbonate, potassium chloride, ammonium chloride, sodium acetate, potassium acetate, ammonium acetate, calcium salts, and/or magnesium salts or combinations thereof.
The term "wash buffer" is used herein to refer to the buffer that is passed over the cation exchange material following loading of a composition and prior to elution of the protein of interest. The wash buffer may serve to remove one or more contaminants from the cation exchange material, without substantial elution of the desired antibody product. According to the preferred embodiment of the invention, multiple buffer washings are used.
Affinity separation is the most selective type of chromatography which makes for an ideal capture step in purification processes. Affinity chromatography matrix may be with natural or derived or modified form of Protein-A, protein G and protein L as a ligand. In the present invention protein, A is preferred due to its high affinity for the Fc region of IgG-type antibodies forms.
The procedure employed for Protein A chromatography involved passage of clarified cell culture supernatant over the column atpH 6-8, to bind the antibodies and unwanted components such as host cell proteins and cell culture harvest media components and putative viruses, flow through the column. An optional intermediate wash step may be carried out to remove non-specifically bound impurities from the column, followed by elution of the product at pH 2.5-4.
Because of its high selectivity, high flow rate and cost-effective binding capacity and its capacity for extensive removal of process-related impurities such as host cell proteins, DNA, cell culture harvest media components and endogenous and adventitious virus particles, Protein A chromatography is typically used as the first step in an antibody purification process. After this step, the antibody product is highly pure and

more stable due to the elimination of proteases and other media components that may cause degradation.
There are currently three major types of Protein A resins, classified based on their resin backbone composition: glass or silica-based, e.g., Prosep vA, Prosep vA Ultra (Millipore); agarose-based, e.g., Protein A Sepharose Fast Flow, MabSelect (GE Healthcare); and organic polymer based, e.g., polystyrene-divinylbenzene Poros A and MabCapture (Applied Biosystems).
In one of the embodiments of the present invention Protein A is preferred and selected from MabSelect SuRe™ LX and rProtein A Sepharose®, preferably MabSelect SuRe™ LX.
Loading of clarified cell culture harvest supernatant can occur in a variety of buffers or salts including sodium, potassium, ammonium, magnesium, calcium, chloride, fluoride, acetate, phosphate, and/or citrate salts and/or Tris buffer.
In yet another embodiment of the present invention, suitable buffers include, but are not limited to, phosphate buffers, Tris buffers, acetate buffers, and/or citrate buffers, glycine, histidine and its pharmaceutical salts thereof, arginine and its pharmaceutical salts, proline and its pharmaceutical salts and combinations thereof and suitable salts include, but are not limited to, sodium chloride, potassium chloride, ammonium chloride, sodium acetate, potassium acetate, ammonium acetate, calcium salts, and/or magnesium salts or combinations thereof.
In yet another embodiment of the present invention, wherein antibody is recovered from the column with salts/additive comprising of sodium chloride, arginine and salts, proline and salts, and glycine or combinations thereof.

In another embodiment of the present invention, the protein to resin ratio is about 40 -50mg/ml, preferably 46mg/ml.
In another embodiment of the present invention, the resin cleaning is done with either with acid or alkali or its buffer in acidic or alkaline range respectively; preferably with 0.1 M acetic acid at pH 3.0 and/or 0.1 to 0.5 M NaOH.
In another embodiment of the present invention, the affinity chromatography elution is done in the pH gradient mode between pH 2.0 to pH 5.0.
In another embodiment of the present invention, the equilibrium buffer may comprise of phosphate buffers, Tris buffers, acetate buffers, and/or citrate buffers, glycine, histidine or its pharmaceutical salts, HEPES, MOPS, arginine or its pharmaceutical salts, proline or its pharmaceutical salts and or combinations thereof, with or without salt additives wherein the additive can preferably comprise of sodium chloride, potassium chloride, ammonium chloride, sodium acetate, potassium acetate, ammonium acetate, calcium salts, and/or magnesium salts or combinations thereof; preferably at pH 6. 0 to 8.0., preferably 6.8 to 7.5, most preferably at 7.3±0.1.
In another embodiment of the present invention, the equilibrium buffer is 50 mM phosphate at pH between 7.0-7.5.
In other embodiment of the present invention, the equilibrium buffer is 50 mM sodium phosphate with I50mM sodium chloride at pH between 7.0-7.5.
In another embodiment of the present invention, the equilibrium buffer can be alternate buffer system comprising of either Tris hydrochloride (lOmM-lOOmM at pH 7.0-9.0 with sodium chloride Or HEPES i.e., N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (5mM-50mM) at pH 6.-8-8.2 with sodium chloride Or MOPS i.e.,3-(N-

morpholino) propane sulfonic acid (lOmM-lOOmM) at pH 6.5-7.9 with sodium chloride.
In yet another embodiment of the present invention, affinity chromatography comprises loading of a clarified cell culture harvest supernatant containing the desired antibody to the column at a pH between 3.0-5.0 for binding, followed by column wash.
In another embodiment of the present invention, the column ash after cell culture harvest loading comprises:
a) washing with equilibration buffer comprising of high salt buffer or combination of high and lower pH buffer to remove impurities, if any
b) elution of antibody with suitable acid or its salt buffer at lower pH.
In another embodiment, the column wash can be done with in at least two wash cycles which comprises:
a) first wash with equilibration buffer 50 mM phosphate at pH between 7.0-7.5 and with 150mM sodium chloride preferably at pH 7.3 ±0.1.
b) second wash with 50 mM phosphate, pH 5-7, preferably at 6.0
c) third wash at a pH 4-6, preferably at 5.0
d) elution of antibody with 20 mM sodium acetate pH 3-4, preferably at 3.6
In another embodiment, the column wash can be done with in at least two wash cycles which comprises:
a) first wash with equilibration buffer 50mM-sodium phosphate with 150 mM sodium chloride; preferably atpH 7.3±0.1.
b) second wash with 50 mM phosphate with 0.5M sodium chloride, pH 5-7, preferably at 7.3±0.1.
c) third wash with 50mM sodium phosphate at a pH 4-6, preferably at 6.0±0.1.
d) elution of antibody with 20mM sodium acetate.

In another embodiment, the column wash alternatively can also be done with lOmM-lOOmM Tris hydrochloride and sodium chloride at pH 7.0-9.0 or 5mM -50mM HEPES and sodium chloride at pH 6.8-8.2, or lOmM-100 mM MOPS and sodium chloride at pH 6.5-7.9 and the elution of antibody can alternatively be done with 0.1M-0.2M sodium citrate or 0.1M-0.2 M glycine at pH 3.0-4.0.
In another embodiment, the equilibrium buffer may comprise of phosphate buffers, Tris buffers, acetate buffers, and/or citrate buffers, glycine, histidine or its pharmaceutically acceptable salts, HEPES, MOPS, arginine or its pharmaceutically acceptable salts, proline or its pharmaceutically acceptable salts, and combinations thereof with or without salt additives wherein the additive can preferably comprise of sodium chloride, potassium chloride, ammonium chloride, sodium acetate, potassium acetate, ammonium acetate, calcium salts, and/or magnesium salts or combinations thereof; preferably at a pH 6. 0 to 8.0., preferably 6.8 to 7.5, most preferably at 7.3±0.1
In a preferred embodiment, equilibration buffer and wash buffer component comprise of sodium hydroxide, ethanol, phosphate, acetate, glycine, histidine or its salts, arginine or its salts, proline, or its salts, HEPES, MOPS and citrate buffer and combinations thereof.
In another embodiment, the column wash after cell culture harvest loading comprises:
a) washing with equilibration buffer comprising of high salt buffer or combination of high and lower pH buffer to remove impurities, if any.
b) elution of antibody with acid or its salt buffers selected from group comprising of acetate, citrate, glycine, phosphoric acid, tartaric acid or combination thereof, or its salts buffer with molarity range between 0.1M- 0.2M and at lower pH between 2.0-5.0, preferably between 3.0-4.0 and most preferably at pH 3.6 ± 0.1.

In another embodiment, elution of the antibody is performed at apH ranging from about pH2.0topH5.0.
In yet another embodiment of the present invention, elution may be affected by lowering the pH that the elution is performed with additive in the buffer, wherein the additive is selected from the group comprising of Sodium chloride, arginine or its salts, potassium chloride, magnesium chloride, glycine and /or combinations thereof.
In another embodiment, the process with step (b) comprises adjusting the pH of the affinity elute between 3.0 - 4.0, most preferably at pH 3.5 using buffers selected from group comprising of acetic acid, hydrochloric acid, citric acid, phosphoric acid, tartaric acid or Tris, sodium hydroxide, potassium hydroxide and sodium carbonate, incubating for minimum 60 min in static condition at Room Temperature (RT) and finally adjusting the pH to 7.5.
In another embodiment, the amount of aggregates in an antibody preparation after affinity chromatography is less than 0.5%.
Thus, the higher buffering strength and at least two wash cycles with gradually reducing pH typically from about 7.3 to about 6.0 before elution at lower pH prevents sudden pH shock and provides better control of the process.
In an embodiment of the present invention, the second step of purification is a low pH inactivation process which comprises adjusting the pH of the affinity elute to 3.5±0.1. The process uses buffers comprising of acetic acid or Tris buffers, incubating for not less than 60 minutes in static condition at RT and adjusting the pH to 7.5. This provides neutralized affinity elute, which is subjected to anion exchange chromatography.

In a preferred embodiment, the anion exchange chromatography is performed either in resin or in membrane form.
Anion exchange chromatography uses a positively charged group (weakly basic such as diethylamino ethyl, DEAE or dimethyl aminoethyl, DMAE; or strongly basic such as quaternary amino ethyl, Q or trimethylammonium ethyl, TMAE or quaternary aminoethyl, QAE immobilized to the resin. Alternatively, membrane can also be used. It is a powerful tool to remove process-related impurities such as host cell proteins, DNA, endotoxin and leached Protein A, product-related impurities such as dimer/aggregate, endogenous retrovirus and adventitious viruses etc. It can be used either in flow-through mode or in bind and elute mode, depending on the pH of the antibody and impurities to be removed. For antibodies having an isoelectric point (pi) in the acidic to neutral range, bind and elute mode can be used to remove process-related and product-related impurities from the product. Vice versa, for antibodies having an isoelectric point in basic range, flow through mode can be used to remove process-related and product-related impurities from the product.
The antibody product pool is first loaded onto an anion exchange column and the antibody is then eluted in flow through mode, leaving most impurities bound to the column. The impurities are eluted from the column during the cleaning or regeneration step.
In the development and optimization of an anion exchange chromatography in flow-through mode, the operating pH should be above or close to the isoelectric point of the product to obtain a net positive charge or higher positive charge number on the surface of the antibody molecules, and hence for the impurities to achieve a higher binding during the chromatography step. To reach a high binding capacity for the impurities to the strong anion exchange resin with flow through mode, the ionic strength of the load

should be in low range and pH of flow through buffer nearer to isoelectric point range of the protein product i.e., between 7.0 - 8.0.
The anion exchange chromatography resin can be selected from resins comprising of Q Sepharose FF, Fractogel EMD TMAE and Nuvia aPrime 4A, preferably Q Sepharose FF or any other suitable anion exchange membranes. The anion exchange chromatography resin preferably has a particle size of less than 100 micrometers.
In an embodiment of the present invention anion exchange chromatography membrane used is Q-Sepharose FF with preferable particle size of about 90 micrometers and Nuvia a Prime 4 A with a preferable particle size of about 50 micrometers.
In another embodiment of the present invention, anion exchange chromatography equilibration and flow through buffer are selected from buffers comprising 20 mM Tris, 0.10 M glycine + 0.1 M Sodium chloride Buffer, at pH 7.4.
In another embodiment of the present invention, anion exchange chromatography equilibration and flow through buffers alternatively are selected from suitable buffer system comprising Tris Hydrochloride with sodium chloride at 1 OmM to lOOmM at pH between 7.0-9.0; HEPES with sodium chloride at 5mM to 50mM at pH between 6.8-8.2; MOPS with sodium chloride at lOmM to lOOmM at pH between 6.5-7.9.
In another embodiment of the present invention, anion exchange chromatography elution buffer is selected from buffers comprising 1 M sodium chloride. 0.10 M glycine + 0.2 M sodium chloride at pH 7.4.
The anion exchange chromatography step is followed by Cation exchange chromatography which uses a resin modified with negatively charged functional groups. They can be strong acidic ligands such as sulfo-propyl, sulfo-ethyl and sulfo-isobutyl

groups or weak acidic ligands such as carboxyl group. In this step the antibody is bound onto the resin during the loading step and eluted through either increasing conductivity or increasing pH in the elution buffer. The most negatively charged process-related impurities such as DNA, some host cell protein, leached Protein A and endotoxin are removed in the load and wash fraction. Cation exchange chromatography can also provide separation power to reduce antibody variants from the target antibody product such as deamidated products, oxidized species and N-terminal truncated forms, as well as high molecular weight species.
The dynamic binding capacity of mAbs on cation exchange resins depends on pH and conductivity.
In cation exchange chromatography, the resins are selected from group consisting of a carboxylate, sulfonate, a sulfo-ethyl based group, a sulfo-isobutyl based group, a carboxymethyl based group, sulfonic and carboxylic acid-based groups and orthophosphate-based group or combinations thereof. In another embodiment of the present invention, the cation exchange chromatography resin consists of either NUVIA HRS and/or Capto SP ImpRes; preferably Capto SP ImpRes.
In another embodiment of the present invention, the cation exchange chromatography is performed preferably in bind & elute mode.
In another embodiment of the present invention, the equilibration and wash buffer used in the cation exchange chromatography is 20 mM sodium acetate or 50 mM MES at pH
-5.
In another embodiment of the present invention, the Equilibration and wash buffer selected from range of suitable buffers preferably comprising sodium acetate, sodium citrate or combinations thereof.

In an embodiment of the present invention, the elution buffer used in the cation exchange chromatography is 20 mM sodium acetate (either alone or with 0.5 M Sodium chloride) or 50 mM MES + 0.3 M Sodium chloride and step Gradient at 25%B for 5CV and linear Gradient from 25 to 65%B in 30CV.
In an embodiment of the present invention, the elution buffer used in the cation exchange chromatography is 50 mM -200mM sodium citrate (either alone or with 0.1M to 0.5 M Sodium chloride) atpH 5.0-6.0.
In another embodiment of the present invention, Protein to resin ratio is about 20-30mg/ml, preferably 26mg/ml.
The cation exchange chromatography step was followed by virus filtration/nanofiltration step.
Mammalian cells used in the manufacture of mAbs, and other therapeutic recombinant proteins produce endogenous retroviruses and are occasionally infected with adventitious viruses during processing. Virus filtration can provide a size-based viral clearance mechanism that complements other virus clearance steps. Current virus-retentive filters are ultrafilters or microfilters with very small pores. Virus filtration membranes are made from hydrophilic polyether sulfone (PES), hydrophilic polyvinylidene (PVDF) and regenerated cellulose. According to the size distribution of viruses that are removed, virus filters can be categorized into retrovirus filters and parvovirus filters.
During virus filtration, fouling is typically caused by the presence of protein aggregate, DNA, partially denatured product, and other debris. Fouling can be significantly reduced using appropriate prefilters, and prefiltration of the feed solution can have a dramatic impact on virus filtration performance. Larger impurities can be removed using

0.1-0.2 urn microfilters, but impurities that are only marginally larger than the protein product are not easily removed using size-based methods. Prefiltration through adsorptive depth filters and charged membranes have been observed to provide significant protection to the virus filters. Different lots of viral filters and feed solutions used in viral filtration processes can give different filtration throughputs, whereas manufacturing variability of filter membrane permeability can be controlled with an acceptable range. The impurity content in feed streams from different manufacturing lots is often variable and freshness and storage conditions for feed solutions can also significantly affect throughput. This can be more pronounced if an in-process product pool from an earlier purification step is used as feed solution for viral filtration.
In the present invention, virus filtration/nanofiltration is done with suitable virus retention filters preferably using Planova 20N.
In a preferred embodiment, the ultrafiltration/diafiltration medium preferably comprises of 20mM L-histidine at pH 5.8±0.2 or lOmM sodium phosphate, phosphate, HEPES, Sodium acetate, sodium citrate and 20mM -100 mM L-arginine or L-proline or combinations thereof at pH 5.0 -7.0.
In an embodiment of the present invention, the purification process comprises adjustment of the pH of the culture supernatants to pH 7.2 with IN NaOH. Each supernatant is filtered through a 0.2m filter and loaded on a protein A column pre-equilibrated in phosphate-buffered saline (PBS). The column is washed with PBS (phosphate buffer system) to remove all the unbound material from the culture supernatant. The antibody bound to the protein A column is eluted with 20 mM sodium acetate pH 3.6. The eluate is adjusted to pH 3.5 ±0.1 with buffers comprising acetic acid or 2M Tris buffers, incubating for not less than 60 minutes in static conditions at RT and then adjusted to pH 7.5 after incubation. This neutralized eluate containing monoclonal antibody is further purified by anion exchange chromatography using Q Sepharose FF column, eluted with 0.10 M glycine+0.1 M Sodium chloride Buffer, at

pH 7.4. The elute is further purified using cation exchange chromatography using Capto SP ImpRes column, eluted with 20 mM sodium acetate at pH 5.5 + 0.5 M Sodium chloride, followed by filtration steps.
In another embodiment of the present invention, the purification process comprises adjustment of the pH of the culture supernatants to pH 7.2 with IN NaOH. Each supernatant is filtered through a 0.2m filter and loaded on a protein A column pre-equilibrated in Tris buffered saline (TBS). The column is washed with TBS (Tris buffer system) to remove all the unbound material from the culture supernatant. The antibody bound to the protein A column is eluted with 0.1M-0.2M sodium citrate pH 3.0-4.0. The eluate is adjusted to pH 3.5 ± 0.1 with buffers comprising acetic acid or 2M Tris buffers, incubating for not less than 60 minutes in static conditions at RT and adjusted to pH 7.5 after incubation. This neutralized eluate containing monoclonal antibody is further purified by anion exchange chromatography using Q Sepharose FF column, eluted with 0.10 M glycine+0.1 M Sodium chloride Buffer, at pH 7.4. The elute is further purified using cation exchange chromatography using Capto SP ImpRes column, eluted with 20 mM sodium acetate atpH 5.5 + 0.5 M Sodium chloride, followed by filtration steps.
In another embodiment of the present invention, the purification process comprises adjustment of the pH of the culture supernatants to pH 7.2 with IN NaOH. Each supernatant is filtered through a 0.2m filter and loaded on a protein A column pre-equilibrated in phosphate-buffered saline (PBS). The column is washed with PBS (phosphate buffer system) or TBS (Tris buffer system) or HEPES buffer system or MOPS buffer system to remove all the unbound material from the culture supernatant. The antibody bound to the protein A column is eluted with 20 mM sodium acetate pH 3.6. The eluate is adjusted to pH 3.5 ±0.1 with buffers comprising acetic acid or 2M Tris buffers, incubating for not less than 60 minutes in static conditions at RT and adjusted to pH 7.5 after incubation. This neutralized eluate containing monoclonal antibody is further purified by anion exchange chromatography using Q Sepharose FF

column, eluted with 0.10 M glycine+0.1 M Sodium chloride Buffer, or 0.1M glycine with 25mM sodium chloride or 0.1M glycine with 1M sodium chloride at pH 7.4 or alternatively, with Tris hydrochloride (lOmM-lOOmM) and sodium chloride at pH 7.0-9.0 or HEPES hydrochloride (5mM-50mM) and sodium chloride at pH 6.8-8.2 or MOPS hydrochloride (lOmM-lOOmM) and sodium chloride at pH 6.5-7.9. The elute is further purified using cation exchange chromatography using Capto SP ImpRes column, eluted with 20 mM sodium acetate at pH 5.5 + 0.5 M Sodium chloride or 50mM -200mM sodium citrate with 0.1 M-0.5 M sodium chloride, followed by filtration steps.
After virus filtration, Ultrafiltration is carried out. Ultrafiltration is a size-based separation, where species larger than the membrane pores are retained and smaller species pass through freely. Separation in ultrafiltration is achieved through differences in the filtration rates of different components across the membrane under a given pressure driving force. Buffer exchange is achieved using a diafiltration mode in which buffer of the final desired composition is added to the retentate system at the same rate in which filtrate is removed, thus maintaining a constant retentate volume. In another embodiment of the present invention, suitable buffers used comprise sodium acetate, sodium citrate, histidine, sodium phosphate, HEPES, 1-arginine, or 1-proline, or combinations thereof,
In other embodiment of the present invention, the ultrafiltration/diafiltration uses suitable buffers preferably comprising 20mM L-histidine at pH 5.8 ± 0.2, lOmM-lOOmM sodium phosphate or HEPES or sodium acetate or sodium citrate or 20mM -100 mM L-arginine at pH 5.0-7.0.
Ultrafiltration with membrane pores ranging from 1 to 20 nm can provide separation of species ranging in molecular weight from 500 Daltons to 1,000 kilodaltons.

Ultrafiltration membranes can be cast from a wide variety of polymers, including poly-sulfone, polyether sulfone, polyvinylidene fluoride and regenerated cellulose.
Ultrafiltration is normally carried out in tangential flow filtration (TFF) mode, in which fluid passes across the filter (crossflow), tangential to the plane of the filter surface. The primary advantage of TFF is that the crossflow continuously sweeps the filter surface. reducing the extent to which materials accumulate on the filter surface, and increasing filtration throughput.
Ultrafiltration systems can be operated with different control strategies. Ultrafiltration and diafiltration processes are typically developed using constant retentate pressure, constant trans-membrane pressure or constant filtrate flux.
In one of the embodiments of the present invention, the ultrafiltration membrane is selected from a wide variety of polymers, comprising poly-sulfone, polyether sulfone, polyvinylidene fluoride and regenerated cellulose and the like.
An important process development aspect of a final formulation step includes the final sterile filtration or bulk filtration of the product. In general, sterile filtration is an important concern for all intermediate purification pools, but more so at the end of the process where the highest protein concentrations are present and greatest value has been imparted onto the product. Proper scale-up techniques using equipment representative to manufacturing is critical.
In one of the embodiments of the present invention, aseptic filtration is carried by passing through a 0.22 um membrane filter. The filter for aseptic filtration was chosen from a wide variety of capsule filters having material of construction (MOC) viz. Polyether sulphone (PES), Modified Polyether sulphone (mPES), Cellulose acetate (CA), Regenerated Cellulose acetate (RCA) and the likes.

In another embodiment of the present invention, the overall recovery of an antibody is not less than 50%
In yet another embodiment of the present invention, purified antibody preparation contains less than 0.5% aggregate.
In another aspect of the present invention the purified antibody is formulated into pharmaceutical composition.
Therefore, in another embodiment of the present invention, a pharmaceutical composition comprises an isolated anti-RhD monoclonal antibody and at least one pharmaceutically acceptable carrier.
In yet another embodiment of the present invention, a stable formulation comprises of recombinant Anti Rh-D Immunoglobulin (rAnti-Rh-D) as an active ingredient, histidine, sucrose, and Polysorbate-80 (Tween 80).
In an embodiment of the present invention, the stable formulation comprises:
a) 150 to 300 micrograms of recombinant Anti Rho-D Immunoglobulin,
b) 5mM - 30mM Histidine buffer
c) 1-10% Sucrose,
d) 0.0001% to 0.1% of Polysorbate-80 (Tween 80), and
e) water for injection (WFI) as a solvent,
wherein the pH of the formulation is adjusted using sodium hydroxide and hydrochloric acid between 5.0 - 7.0, preferably at pH 5.8± 0.2 (i.e., between 5.6 - 6.0).
In another embodiment of the present invention, a stable formulation comprises rAnti-Rh-D as an active ingredient, sucrose, histidine and Polysorbate-80 (Tween 80) and comprises of the following manufacturing steps:

a) preparation of drug substance: diafiltration of pure rAnti-Rh-D in 20mM Histidine buffer at pH 5.8± 0.2.
b) preparation of drug product: dilution of drug substance with '5mM-30mM Histidine + 1%-10% sucrose at pH 5.8± 0.2 buffer, followed by adjusting pH to 5.8± 0.2 and filtration through 0.2u.m.
In an embodiment of the present invention, to avoid chances of aggregation occurring due to surface interaction of proteins with container closure system, one or more surfactant is added in the formulation.
In another embodiment of the present invention, stabilizers/tonicity agents can be added to have an optimal osmolality of solution from about 270 to about 330 mOsmol/kg range. The stabilizers are preferably selected from sodium chloride and sucrose.
In yet another embodiment of the present invention, pH of the formulation is adjusted using various buffers to keep target formulation pH between 5.0 - 7.0, preferably between 5.6 - 6.0, more preferably at pH 5.8 to avoid chances of having increase in the acidic variants and protein aggregation during stability.
The monoclonal antibodies can be formulated as desired dependent on the intended route of administration. For example, monoclonal antibodies may be formulated as injection (for example intra-muscular injection) analogous to conventional polyclonal anti-D formulations. Exemplary dosages range from 150 to 300 micrograms (as measured by agglutination titer). Exemplary carriers include phosphate-buffered saline, histidine buffered saline; and glycine saline buffer and/or combinations thereof.
In another embodiment, a stable formulation comprising rAnti-Rh-D as an active ingredient, sucrose, histidine and Polysorbate-80 (Tween 80) is prepared using the following steps:

a) preparation of drug substance: diafiltration of pure rAnti-Rh-D in 5mM-30mM histidine buffer at pH 5.8± 0.2, followed by addition of sucrose about 1% w/w to about 10% w/w and 0.0001% to 0.1%w/w of Polysorbate-80 (Tween 80) followed by adjusting pH to 5.8± 0.2 followed by 0.2um filtration.
b) preparation of drug product: dilution of drug substance with '5mM-30mM Histidine + 1%-10% sucrose at pH 5.8± 0.2 buffer, followed by adjusting pH to 5.8± 0.2 to required protein concentration of 150 to 300 micrograms of recombinant Anti Rh-D Immunoglobulin, followed by 0.2urn filtration.
The composition may comprise monoclonal antibodies of a single type only (i.e., the only antibodies present in the composition are antibodies produced by cells of the same cell line). Alternatively, the composition may comprise a combination of more than one type of monoclonal antibody. For example, the composition could comprise two or more distinct types of monoclonal antibodies such as a combination of two or all three monoclonal antibodies RhDl, RhD2 and/or RhD3. Alternatively, or additionally, the composition could comprise, in addition to monoclonal antibodies other anti-RhD monoclonal antibodies known from art.
In another embodiment of the present invention, the method of inhibiting or preventing immunization of a RhD-negative human patient against RhD-positive blood comprises administering a prophylactically effective amount of a monoclonal antibody.
Specific indications and/or circumstances in which the monoclonal antibodies may be administered correspond to those for which the existing anti-RhD polyclonal antibodies are administered.
The invention herein provides detailed method for purifying an antibody from a composition comprising the antibody and one or more contaminants. The composition is one resulting from the recombinant production of the antibody but may be that

resulting from production of the antibody by peptide synthesis (or other synthetic means), or the antibody may be purified from a native source of the antibody.
Figure 1 illustrates the improved downstream process, which integrates a sequence of advanced purification techniques.
EXAMPLES
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 isolated Recombinant Anti Rho-D Immunoglobulin (rAnti-Rh-D) monoclonal antibody from cell culture harvest Step 1: Affinity chromatography
The experiment was performed to understand the formation and control of LMW due to process conditions. The study was planned with clarified harvest. Impact of compressed air and nitrogen purging was studied with the clarified harvest.
The study was conducted at room temperature. The N2 (nitrogen) and compressed air overlay was done continuously for 65 h. at 250 millibar pressure in two different 250 mL glass bottles with 130 mL of clarified harvest. After 65 h, the harvest was subjected to Protein A chromatography with r-Protein A Sepharose resin. The elution samples were analyzed by SE-HPLC and CE-SDS.
Step 2: Low pH inactivation
The affinity elute was subjected to pH adjustment (3.5+0.1) by using the 2M acetic acid or 2M Tris buffer. The pH adjusted Protein A elute was incubated for 60 minutes in static condition at RT. After 60 min of incubation, the sample pH was adjusted to pH 7.5 to prepare load for next step of purification i.e., anion exchange chromatography (AEX).

The additional study was performed to see the impact on aggregation after holding the samples at different temperature and time point.
Step 3: Anion Exchange Chromatography (AEX)
After the affinity elution, the eluate was neutralized to pH 7.4 using a 2M Tris buffer.
Following this pH adjustment, the sample underwent anion exchange chromatography
(AEX).
For AEX step, various anion exchange resins were evaluated, including Fractogel EMD
TMAE (strong anion exchange) from Merck, Nuvia aPrime 4A (Anion + HIC), and Q
Sepharose FF (strong anion exchange). The chromatography was performed in negative
mode, also known as flow-through mode, wherein the target protein was observed in
the flow-through fraction, while all impurities were bound to the resin during the loading
phase and eluted during regeneration and sanitization steps.
For the cleaning of the chromatography equipment, a 0.5 M NaOH solution was used,
while the storage of the equipment was done using 20% ethanol.
Step 4: Cation Exchange Chromatography (CEX)
The pooled Fractogel TMAE fractions were subjected to cation exchange
chromatography (CEX) using the resin Capto SP ImpRes.
The buffer system employed for the CEX step consisted of an acetate buffer. During the
chromatographic process, a gradient was applied, starting from 0% to 100% buffer over
the course of 30 column volumes (CV).
The CEX step is designed to selectively bind and elute components based on their
charge properties. In this case, the target was to further purify the sample by removing
any remaining aggregates and fragments that might have been present after the previous
purification steps.
Step 5: Nanofiltration

After the Q-column step, the protein solution containing the desired monoclonal antibody underwent a nano-filtration step. After nano-filtration, purity of monoclonal antibody is expected to remain more than 95%.
Step 6: Ultrafiltration-Diafiltration (UF/DF)
After nano-filtration, protein solution was Dia filtered with desired media for the preparation of bulk drug substance.
Step 7: Microfiltration/Aseptic Filtration
Finally, the purified preparation containing the desired monoclonal antibody was passed through 0.22 urn membrane filter, aseptically, and was stored either in the liquid form, under cold condition or under frozen condition for storage. The final purified monoclonal antibody exhibits more than 95% purity, as assessed by HP-SEC.

Example 2: Drug substance (DS) preparation of isolated recombinant Anti Rho-D Immunoglobulin (rAnti-Rh-D) monoclonal antibody Table 1

Example Formulation Composition pH Protein
cone.
Post
Filtration
(mg/ml)
DS01 0.1M Glycine + 0.1M NaCl + Polysorbate-80 (Tween 80) (0.01%) at pH 6.0 5.983 7.474
DS02 0.1M Glycine + 5% Sucrose + Polysorbate-80 (Tween 80) (0.01%) at pH 6.0 6.044 6.625
DS03 0.1M Glycine + 0.1M NaCl + Polysorbate-80 (Tween 80) (0.01%) at pH 6.5 6.495 7.273
DS04 0.1M Glycine + 5% Sucrose + Polysorbate-80 (Tween 80) (0.01%) at pH 6.5 6.520 6.582
DS05 20mM Histidine + 0.1M NaCl + Polysorbate-80 (Tween 80) (0.01%) at pH 6.0 6.026 6.884
DS06 20mM Histidine + 5% Sucrose + Polysorbate-80 (Tween 80) (0.01%) at pH 6.0 6.051 6.269
DS07 20mM Histidine + 0.1M NaCl + Polysorbate-80 (Tween 80) (0.01%) at pH 6.5 6.491 6.881
DS08 20mM Histidine + 5% Sucrose + Polysorbate-80 (Tween 80) (0.01%) at pH 6.5 6.520 6.204
DS09 5mM Na-Citrate + 0.1M NaCl + Polysorbate-80 (Tween 80) (0.01%) at pH 5.5 5.510 6.983
DS 10 5mM Na-Citrate + 5% Sucrose + Polysorbate-80 (Tween 80) (0.01%) at pH 5.5 5.478 6.292

DS11
5mM Na-Citrate + 0.1M NaCl + Polysorbate-80 (Tween 80) (0.01%) at pH 6.0 5.995 6.862
DS12 5mM Na-Citrate + 5% Sucrose + Polysorbate-80 (Tween 80) (0.01%) at pH 6.0 5.991 6.406
DS 13 0.4 M Glycine + 0.1M NaCl at pH 6.5 6.490 6.710

Table 2: Drug product (DP) formulation of isolated recombinant Anti Rho-D Immunoglobulin (rAnti-Rh-D) monoclonal antibody

Example Formulation Composition pH Protein cone. Post
Filtration (mcg/ml)
DP 01 0.1M Glycine + 0.1M NaCl + Polysorbate-80 (Tween 80) (0.01%) at pH 6.0 6.041 293
DP 02 0.1M Glycine + 5% Sucrose + Polysorbate-80 (Tween 80) (0.01%) at pH 6.0 6.049 296
DP 03 0.1M Glycine + 0.1M NaCl + Polysorbate-80 (Tween 80) (0.01%) at pH 6.5 6.469 288
DP 04 0.1M Glycine + 5% Sucrose + Polysorbate-80 (Tween 80) (0.01%) at pH 6.5 6.05 292
DP 05 20mM Histidine + 0.1M NaCl + Polysorbate-80 (Tween 80) (0.01%) at pH 6.0 5.985 323
DP 06 20mM Histidine + 5% Sucrose + Polysorbate-80 (Tween 80) (0.01%) at pH 6.0 6.019 310
DP 07 20mM Histidine + O.lMNaCl + Polysorbate-80 (Tween 80) (0.01%) at pH 6.5 6.474 310
DP 08 20mM Histidine + 5% Sucrose + Polysorbate-80 (Tween 80) (0.01%) at pH 6.5 6.536 303
DP 09 5mM Na-Citrate + 0.1M NaCl + Polysorbate-80 (Tween 80) (0.01%) at pH 5.5 5.487 302
DP 10 5mMNa-Citrate + 5% Sucrose + Polysorbate-80 (Tween 80) (0.01%) at pH 5.5 5.555 300
DP 11 5mM Na-Citrate + 0.1M NaCl + Polysorbate-80 (Tween 80) (0.01%) at pH 6.0 5,966 333

DP 12 5mMNa-Citrate + 5% Sucrose + Polysorbate-80 (Tween 80) (0.01%) at pH 6.0 6.029 294
DP 13 0.4 M Glycine + 0.1M NaCl at pH 6.5 6.491 276
Procedure:
Drug Substance (DS) Preparation
The drug substance was prepared using rAnti-Rh-D at 40 % viability, affinity purified material was Dia-filtered in below mentioned buffer for respective formulations. i. 0.4 M Glycine + 0.1M NaCl, pH 6.5 (for DS/DP-13: Control set) ii. 0.1 M Glycine+ 0.1M NaCl, pH 6.5 (for DS/DP-01 &DS/DP-03) iii. 0.1 M Glycine, pH 6.5 (for DS/DP-02 & DS/DP-04) iv. 20mM Histidine, pH 6.0 (for DS/DP-05 to DS/DP-08) v. 5mM Na- Citrate, pH 6.0 (for DS/DP-09 to DS/DP-12)
For formulations with 5% sucrose, 50% stock prepared with respective base buffers and added to achieve 5% concentration in final bulk. Polysorbate-80 (Tween 80) was added to all formulations using 1% stock prepared in respective base buffers to achieve 0.01% concentration in final formulation. pH of respective formulations adjusted using 1M NaOH and 6M HC1 solutions followed by 0.2um filtration under laminar flow. Post filtration, protein content checked for formulations and aliquoted under sterile conditions for stability and Freeze thaw studies.
Drug Product (DP) Preparation
Formulations were prepared by adding Sucrose (50% stock solution) and Polysorbate-80 (Tween 80) (1% stock solution) in respective base buffers at optimum batch size. Depending on Protein concentration of respective DS formulation, volume replaced for formulation buffer with DS material followed by adjustment of pH using 1M NaOH and 6M HC1 solutions. After preparation of complete formulation, material was filtered using 0.2 pm filter under sterile condition (laminar) followed by determination of protein content using Spectrophotometer method. Post filtration and protein check, DP material aliquoted as (1ml in 2ml glass vial) and subjected to stability studies.

All formulations were carried out using the above procedure. Table 2 and 3 enlists compiled data for DS and DP formulations.
Optimization of Formulation using stability data:
1. Freeze-Thaw Stability of DS liquid bulk: Freeze-Thaw stability for rAnti-D drug substance bulk liquid was carried out at two different conditions, namely at -20°C and -80°C. The number of freeze-thaw cycles was kept at 5 cycles for this study at each temperature. After each Freeze-Thaw cycle, the samples were subjected to LMW by CE-SDS-NR and HMW by SEC-HPLC. The results are given in Figure 2-5. All 12 formulations (DS-1 to 12) with control formulation (DS-13) did not show much increase in %LMW and %HMW from the initial time point. Hence, it is evident that based on the above data all the formulations were stable for 5 cycles of freeze-thaw at -20°C and -80°C.
2. Stress Stability of DS Bulk and DP: The stress conditions were chosen based on previous stability data for the stress study of DS bulk and DP DS liquid Bulk at 25°C for 1-, 14-, 21-, and 30-days DP vials at 40°C for 7, 14, 21, and 30 days. The results are given in Fig 6-9. For the DS bulk solution of r-Anti-D formulations DS-l to DS-12, stress stability at 25°C seemed to show little effect on the LMW content except a nearly 2% increase was found in our current control formulation (DS-13), but there is not much increase in HMW content for all DS formulations including control formulation.
In the case of DP formulations, there is a considerable increase in LMW content for all the formulations (DP-1 to DP-13), in which DP-8 formulation with 20mM Histidine + 5% Sucrose + Polysorbate-80 (Tween 80) (0.01%) at pH 6.5 was performing the best and DP-3 formulation with 0.1M Glycine + 0.1M NaCl + Polysorbate-80 (Tween 80) (0.01%) at pH 6.5 showed a nearly 4-fold increase in LMW content after 4 weeks of exposure. Also, DP formulation DP-2 with 0.1M Glycine + 5% Sucrose + Polysorbate-

80 (Tween 80) (0.01%) at pH 6.0 showed a considerable increase in HMW content under the 40°C stress condition.
Accelerated Stability of DS Bulk and DP: The stress conditions were chosen based
on previous stability data for the accelerated stability study of DS bulk and DP.
DS liquid Bulk at 2-8°C for 0, 1, 2, 3, and 6 months
DP vials at 25°C for 0, 1, 2, 3, and 6 months. The results are given in Figure 10-12.
It is evident from the stability data/graphs that out of all the 12 formulations number 08 of both DS and DP performed well in all the stability conditions. The interim storage of formulation DS08 is also achieved till 6 months at 2-8° C with no impact on HMW/LMW content.
Table 3: Stability results of optimized formulation.

PARAMETERS Results
Initial (Zero - 80 deg C - 20 deg C 2-8 deg C

IM 3M 6M IM 2M 3M 6M IM 2M 3M 6M
point)
Purity %HMW 1.24 1.33 1.15 1.16 1.37 1.25 1.13 1.14 1.37 1.18 1.14 1.15
by SEC
HPLC
CE SDS %LMW 4.12 4.13 3.92 4.08 4.34 3.89 3.24 4.15 4.61 4.09 4.07 4.14
(NR)
i Based on the data, the inventors have achieved a significant milestone in the development of the rAnti-D API with 'DS08' formulation. The formulation's storage at temperatures between 2-8 degrees Celsius for a period of 6 months demonstrates its stability and robustness. During this storage period, there is no significant impact on the content of High Molecular Weight (HMW) and Low Molecular Weight (LMW)

components of the formulation. This indicates that the product remains consistent in terms of its molecular composition and does not undergo degradation or impurity accumulation during short-term storage under these controlled conditions. Based upon results obtained during further stability studies, the final pH was adjusted to 5.8 ± 0.2 from the initial pH of 6.5, while varying the concentration of Polysorbate-80 (Polysorbate-80 (Tween 80)) in the drug product formulation DP08 on dilution of DS with Histidine-Sucrose formulation buffer. This adjustment was aimed to achieve additional protection against product impurity and degradation pattern with both DS08 and DP08 formulations.
While this invention has been described in detail with reference to certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments. Rather, in view of the present disclosure, which describes the current best mode for practicing the invention, modifications and variations would present themselves to those skilled in the art without departing from the scope and spirit of the invention.

Claims:
1. A process for purification of Recombinant Anti Rho-D Immunoglobulin (rAnti-
Rh-D) monoclonal antibody from cell culture harvest comprising the following steps:
a) affinity chromatography (AF) comprising the isolation of rAnti-Rh-D monoclonal antibody from cell culture harvest and collecting eluate comprising the purified monoclonal antibody.
b) low pH inactivation on eluant obtained in (a) to inactivate the enveloped & non-enveloped viruses.
c) Ion Exchange Chromatography to remove aggregates, endotoxins and other impurities.
d) optionally followed by other suitable purification steps.

2. The process as claimed in claim 1, wherein the yield of the final composition after purification is at least 50% and high molecular weight (HMW) impurity is less than 0.5% and low molecular weight (LMW) impurity is less than 1.5%.
3. The process as claimed in Claim 1, wherein the ion exchange chromatography comprises anion exchange & cation exchange chromatography steps.
4. The process as claimed in claim 1, wherein it further comprises purification steps like Nanofiltration, Ultrafiltration/Diafiltration and Aseptic filtration.
5. The process as claimed in claim 1, wherein affinity chromatography matrix is with natural or derived or modified form of Protein-A as a ligand.
6. The process as claimed in claim 5, wherein Protein A ligand is MabSelect SuRe LX™.

7. The process as claimed in claim 1, wherein step (a) comprises loading of cell culture harvest containing the rAnti-Rh-D antibody to the column at suitable pH and/or conductance for binding, followed by column wash prior to elution of the desired antibody.
8. The process as claimed in claim 7, wherein antibody is recovered from the column with buffer components comprising of phosphate buffers, Tris buffers, acetate buffers, and/or citrate buffers, glycine, histidine, or its pharmaceutically acceptable salts, HEPES, MOPS, arginine or its pharmaceutically acceptable salts, proline or its pharmaceutically acceptable salts, and combinations thereof.

9. The process as claimed in claim 7, wherein antibody is recovered from the column with elution buffer with or without additives to ensure complete elution and/or stabilize the eluted protein wherein the additive can preferably comprise of sodium chloride, potassium chloride, ammonium chloride, sodium acetate, potassium acetate, ammonium acetate, calcium salts, and/or magnesium salts or combinations thereof.
10. The process as claimed in claim 7, wherein Protein to resin ratio is preferably between 20-60, more preferably between 30 to 50 and most preferably 46 mg/ml.
11. The process as claimed in claim 7, wherein resin cleaning is done either with
acid or alkali or its buffer in acidic or alkaline range, respectively.
12. The process as claimed in claim 7, wherein affinity chromatography elution is done at low pH conditions between 2.0-5.0, preferably between 3.0-4.0 and most preferably at pH 3.6 ± 0.1.
13. The process as claimed in claim 7, wherein equilibrium buffer may comprise of phosphate buffers, Tris buffers, acetate buffers, and/or citrate buffers, glycine, histidine

or its pharmaceutically acceptable salts, HEPES, MOPS, arginine or its pharmaceutically acceptable salts, proline or its pharmaceutically acceptable salts, and combinations thereof, with or without salt additives wherein the additive can preferably comprise of sodium chloride, potassium chloride, ammonium chloride, sodium acetate, potassium acetate, ammonium acetate, calcium salts, and/or magnesium salts or combinations thereof; preferably at a pH 6. 0 to 8.0., preferably 6.8 to 7.5. most preferably at 7.3±0.1.
14. The process as claimed in claim 7, wherein column wash after cell culture
harvest loading comprises:
a) washing with equilibration buffer comprising of high salt buffer or combination
of high and lower pH buffer to remove impurities, if any
b) elution of antibody with acid or its salt buffers selected from group consisting of
acetate, citrate, glycine, phosphoric acid, tartaric acid or combination thereof,
comprising of or its salt buffer with molarity range between 0.1 M - 0.2M and at
lower pH between 2.0-5.0, preferably between 3.0-4.0 and most preferably at pH
3.6±0.1.
15. The process as claimed in claim 1, wherein amount of aggregates in an antibody preparation after step (a) is less than 0.5%.
16. The process as claimed in claim 1, wherein step (b) comprises adjusting the pH of the affinity elute between 3.0 - 4.0, most preferably at pH 3.5 using buffers selected from group consisting of acetic acid, hydrochloric acid, citric acid, phosphoric acid, tartaric acid or Tris, sodium hydroxide, potassium hydroxide and sodium carbonate, incubating for minimum 60 min in static condition at Room Temperature (RT) and finally adjusting the pH to 7.5.

17. The process as claimed in claim 23 wherein cation exchange chromatography is performed in bind & elute mode.
18. The process as claimed in claim 24, wherein cation exchange chromatography resin is selected from NUVIA HRS, Capto SP ImpRes.
19. The process as claimed in claim 23-25, wherein cation exchange chromatography resin is Capto SP ImpRes.
20. The process as claimed in claim 23-26, wherein the Equilibration and wash buffer selected from range of suitable buffers preferably comprising 'sodium acetate, sodium citrate or combinations thereof.
21. The process as claimed in claim 23-26, wherein the Elution buffer selected from range of suitable buffers alone or with additive preferably comprising: '20 mM sodium acetate pH 5.0' (alone) or with an additive 0.5 M Sodium chloride at pH 5.5, or sodium citrate 50mM to 200mM (alone) or with an additive 0.1- 0.5M Sodium chloride at pH 5.0 to 6.0 comprising step Gradient at 25% of'20 mM Sodium Acetate with 0.5M NaCl pH 5.5' Or sodium citrate 50mMto 200mM with 0.1M-0.5M sodium chloride atpH 5.0 to 6.0 for 5CV and Linear Gradient from 25 % to 65% of '20 mM Sodium Acetate with 0.5M NaCl pH 5.5' Or sodium citrate 50mM to 200mM with 0.1M-0.5M sodium chloride at pH 5.0 to 6.0 in 30CV.
29. The process as claimed in claim 27-28, wherein Protein to resin ratio is about
20 -30mg/ml, preferably 26mg/ml.
30. The process as claimed in claim 4, wherein nanofiltration is performed using
suitable virus retention filters, preferably Planova 20 N.

17. The process as claimed in claim 1, wherein neutralized affinity elute (after low pH treatment) is subjected to anion exchange chromatography, either in resin or in membrane form.
18. The process as claimed in claim 17, wherein the anion exchange chromatography resin is selected from resins comprising of Q Sepharose FF, Fractogel EMD TMAE and Nuvia aPrime 4A or any other suitable anion exchange membranes.
19. The process as claimed in claim 17-18, wherein the anion exchange chromatography membrane used preferably is Q Sepharose FF.
20. The process as claimed in claim 17-19, wherein the equilibration and wash buffer comprise of 20 mM Tris, 0.025 M Sodium chloride in 0.1 M glycine Buffers, at pH between 7-8, preferably at pH 7.5.
21. The process as claimed in claim 17-20, wherein the equilibration and wash buffers are selected from buffer system comprising Tris Hydrochloride with sodium chloride at lOmM to lOOmM at pH between 7.0-9.0; HEPES with sodium chloride at 5mM to 50mM atpH between 6.8-8.2; MOPS with sodium chloride at lOmM to lOOmM at pH between 6.5-7.9.
22. The process as claimed in claim 17-21, wherein the flow-through buffer is selected from 1 M sodium chloride to 0.025 M Sodium chloride in 0.1M glycine Buffers, at pH between 7-8, preferably at pH 7.5.
23. The process as claimed in claim 17-22, is followed by cation exchange chromatography.

31. The process as claimed in claim 1-30, comprising steps of
a) affinity chromatography
b) low pH inactivation
c) Anion Exchange Chromatography
d) Cation exchange chromatography
e) Nanofiltration,
f) Ultrafiltration/ Diafiltration and
g) Aseptic filtration.
32. The process as claimed in claim 31 wherein the pH of the culture supernatants
adjusted to pH 7.2. and loaded on a protein A column pre-equilibrated in phosphate-
buffered saline (PBS), column is washed with PBS to remove all the unbound material
from the culture supernatant, antibody bound to the protein A column is eluted with 20
mM sodium acetate pH 3.6, eluate is neutralized with 2M Tris buffer adjusted to pH 7.5,
eluate containing monoclonal antibody is further purified by anion exchange
chromatography using Q Sepharose FF. column, eluted with 0.025 M Sodium chloride
in 0.1M glycine Buffer, at pH 7.5, elute is further purified using cation exchange
chromatography using Capto SP ImpRes column, eluted with 20 mM sodium acetate at
pH 5.5 + 0.5 M Sodium chloride, followed by Ultrafiltration/Diafiltration & finally,
aseptic filtration step.
33. The process as claimed in claim 31-32, wherein ultrafiltration/diafiltration
medium is selected from phosphate, acetate, citrate, succinate and combinations thereof.
34. The process as claimed in claim 31-33, wherein ultrafiltration/diafiltration
medium preferably comprises of 20mM L-histidine at pH 5.8± 0.2 or lOmM sodium
phosphate, HEPES, Sodium acetate, sodium citrate and 20mM -100 mM L-arginine or
L-proline or combinations thereof at pH 5.0 -7.0.

35. The process as claimed in claim 1, wherein the overall recovery of an antibody is in the range of not less than 50%.
36. The process as claimed in claim 1, wherein purified antibody preparation contains less than 0.5% aggregate.
37. A stable formulation comprising recombinant Anti Rho-D Immunoglobulin (r-Anti-Rh-D) as an active ingredient, histidine, sucrose and Polysorbate-80 (Tween 80).
38. The stable formulation as claimed in claim 37, comprises:

a) 150 to 300 micrograms of recombinant Anti Rh-D Immunoglobulin
b) 5mM-30mM Histidine buffer
c) 1%-10% Sucrose
d) 0.0001%-0.1% of Polysorbate-80 (Tween 80)
e) water for injection (WFI) as a solvent,
wherein the pH of the formulation is adjusted using sodium hydroxide and hydrochloric acid between pH 5.0 - 7.0, more preferably at pH 5.8 ± 0.2.
39. A stable formulation comprising rAnti-Rh-D as an active ingredient, Sucrose,
Histidine and Polysorbate-80 (Tween 80) prepared using the following steps:
a) preparation of drug substance: diafiltration of pure rAnti-Rh-D in 5mM-30mM
Histidine buffer atpH 5.8± 0.2, followed by addition of sucrose about 1% w/w to about 10% w/w and 0.0001% to 0.1% w/w of Polysorbate-80 (Tween 80) followed by adjusting pH to 5.8 ± 0.2 followed by 0.2um filtration.
f) preparation of drug product: dilution of drug substance with 5mM-30mM
Histidine + 1%-10% Sucrose at pH 5.8 ± 0.2 buffer, to required protein concentration
of 150 to 300 micrograms of recombinant Anti Rh-D Immunoglobulin, followed by
0.2um filtration.

40. A process of purification of an isolated rAnti-Rh-D monoclonal antibody from cell culture according to steps of claim 1 and administering the same as stable pharmaceutical composition.