DESC:INTRODUCTION
The present invention relates to the field of therapeutic antibody formulations. More specifically, the present invention relates to the field of stable formulations of antibodies and antigen-binding fragments against human CD-20 antigen and method for preparing the same.
BACKGROUND
Recombinant therapeutic monoclonal antibodies (mAbs) require a mammalian expression system (for example CHO cells) to be produced. The molecular weight of a monomer of a therapeutic mAb ranges between 140-160 kDa, which is usually higher than other chemically synthesized oligopeptides. mAb therapeutics are either lyophilized or supplied as liquid-based formulations for parenteral transmission to achieve highest level of bioavailability in patients.
The hallmarks of any biologically active protein are the conformational integrity of the core amino acid sequence of the protein and intact multiple functional groups of the protein.
However, the protein instability and degradation as a result of the harsh pH and salt conditions in the downstream process is one of the challenges for the maximum recovery of the purified product. Furthermore, other factors such as protein’s intrinsic instability, aggregate formation due to self-association, insolubility and viscosity are the other major constraints for formulating the therapeutic mAbs or mAb based therapeutics. Chemical factors like oxidation, hydrolysis, disulfide exchange, deamidation accelerate the degradation of therapeutic mAbs.
The optimization of the mAb formulation is performed in such a way that the highest concentration is achieved at the minimum achievable volume for parenteral route of administration without affecting the overall quality of the mAb.
Therefore, it is crucial to develop a suitable formulation buffer including appropriate excipients that would stabilize a therapeutic monoclonal antibody against various physico-chemical factors that would reduce the shelf life of mAbs.
The selection of excipients in the formulation buffer depends on the compatibility with the protein, concentration of the protein, buffer, other excipients present and also the concentration of the excipients. Therefore, the correct screening and selection of the formulation buffers is a challenge to obtain the accurate formulation of the mAb in the buffer which is free from aggregate formation, free of colloidal particles, lesser acidic/basic variants.
Hence, there remains a need in the art for improved pharmaceutical formulations comprising anti-CD-20 antibodies that are sufficiently stable and suitable for administration to patients.
SUMMARY OF THE INVENTION
The present invention discloses a pharmaceutical formulation of an anti-CD-20 antibody formulation, wherein the formulation comprises anti-CD20 antibody, buffer, stabilizer and surfactant and pH of the formulation is about 5.0 to about 6.5.
The invention comprises obtaining a stable anti-CD20 antibody formulation in succinate containing buffer, in particular, sodium succinate buffer.
In another aspect the invention discloses a method of preparing/obtaining a stable anti-CD20 antibody composition wherein the method comprises, preparing sodium succinate buffer in a specific way and obtaining the anti-CD20 antibody composition in the prepared sodium succinate buffer.
In one aspect the invention particularly discloses a method of reducing light induced aggregation or fragmentation of anti-CD20 antibody in it’s composition, wherein the method comprises obtaining anti-CD20 antibody in a buffer composition comprising sodium succinate buffer or histidine buffer or it’s derivatives.
In one aspect the invention particularly discloses a method of reducing basic variants formation of anti-CD20 antibody in the anti-CD20 antibody composition, wherein the method comprises obtaining ant-CD20 antibody in a buffer composition comprising sodium succinate buffer or histidine buffer or it’s derivatives. The disclosed method specifically controls pyroglutamate formation and/or lysine variants formation and/or methionine oxidation or succinimide formation of an anti-CD20 antibody thereby reduces formation of basic variants in anti-CD20 antibody composition.
In another aspect the invention particularly discloses a method of reducing fragmentation of anti-CD20 antibody in the anti-CD20 antibody composition, wherein the method comprises obtaining ant-CD20 antibody in a buffer composition comprising sodium succinate buffer or histidine buffer or it’s derivatives. Additionally, the disclosed method controls aggregation of anti-CD20 antibody in it’s composition.
The disclosed formulations and the methods of the present invention stabilizes anti-CD20 antibody from lower to higher concentration (i.e; from 10 mg/ml to 200 mg/ml) and suitable for intravenous as well as subcutaneous formulations.
The pH of the formulation of the present invention can range from about 5.0 to about 6.5.
The disclosed formulations and the methods of the invention exhibit stability under at least one of the following accelerated conditions that includes a temperature ranging from 25 ? to 40 ? and for a period of time ranging from 1 day to 28 days/4 weeks. The antibody in the said formulation is stable and maintains 98% or more (= 98 %) of monomeric content of the antibody in the formulation even after storage for two to four weeks at 40 ºC.
DETAILED DESCRIPTION
The present invention discloses a pharmaceutical formulation of an anti-CD20 antibody.
In an embodiment, the invention discloses a pharmaceutical formulation of an anti CD-20 antibody comprising:
(i) an anti-CD20 antibody and
(ii) a buffer having pH of about 5.0 to about 6.5.
In another embodiment, the invention discloses a pharmaceutical formulation of an anti-CD20 antibody comprising:
(i) an anti-CD20 antibody,
(ii) a buffer having pH of about 5.0 to about 6.5 and
(iii) a surfactant.
In the above mentioned embodiment, the disclosed formulation further comprises one or more pharmaceutically acceptable excipients or stabilizers.
In the above mentioned embodiment, the formulation comprises one or more pharmaceutically acceptable excipients which includes sugars, amino acids, or polymers or salts.
In the above mentioned embodiment of the invention, the said buffer is a succinate buffer or an acetate buffer or a citrate buffer or a histidine buffer or a phosphate buffer or combination thereof.
In the above mentioned embodiment, the succinate buffer is a sodium succinate buffer.
In any of the above mentioned embodiments, the buffer is sodium succinate buffer, that is prepared in a specific way with the following steps;
a) dissolution of succinic acid in water to obtain succinic acid solution
b) preparation of sodium hydroxide solution by dissolving sodium hydroxide in water
c) mixing of sodium hydroxide solution to succinic acid solution in a particular molar ratio to obtain sodium succinate buffer.
In the above embodiment, the molar ratio of succinic acid to sodium hydroxide is 1:1 to 1:3.
In another embodiment, the invention discloses a pharmaceutical formulation of an anti-CD20 antibody comprising:
(i) 10 mg/ml to 200 mg/ml an anti-CD20 antibody,
(ii) succinate buffer or it’s derivatives or salts or combinations thereof
(iii) one or more stabilizers and;
(iv) a surfactant.
In an embodiment, the invention discloses a pharmaceutical formulation of an anti-CD20 antibody comprising:
i) 10 mg/ml to 200 mg/ml an anti-CD20 antibody,
ii) 10-50 mM sodium succinate buffer,
iii) one or more stabilizers comprise mannitol, or trehalose, or sucrose or sorbitol; or sodium chloride or derivatives thereof and;
iv) a surfactant.
In an embodiment, the invention discloses a pharmaceutical formulation of an anti-CD20 antibody comprising:
i) 120 mg/ml an anti-CD20 antibody,
ii) 10-50 mM sodium succinate buffer,
iii) one or more stabilizers comprise mannitol, or trehalose, or sucrose or sorbitol; or sodium chloride or derivatives thereof and;
iv) a surfactant.
In any of the above mentioned embodiments, the succinate buffer or it’s derivatives or salts or combination thereof, is a sodium succinate buffer. The said buffer is prepared in a specific way in a specific molar ratio of succinate and sodium hydroxide. The specific molar ratio is in between 1:1 to 1:3. The buffer composition may further contain at least one or more pharmaceutically acceptable excipients/stabilizer. The said excipients can be added during pre-formulation and/or formulation stage of the antibody production.
In an embodiment, the invention discloses a pharmaceutical formulation of an anti-CD20 antibody comprising:
(i) 10 mg/ml to 200 mg/ml an anti-CD20 antibody,
(ii) histidine buffer or it’s derivatives or salts or combinations thereof
(iii) one or more stabilizers and;
(iv) a surfactant.
In an embodiment, the invention discloses a pharmaceutical formulation of an anti-CD20 antibody comprising:
i) 10 mg/ml to 200 mg/ml an anti-CD20 antibody,
ii) 10-50 mM histidine buffer,
iii) one or more stabilizers comprise mannitol, or trehalose, or sucrose or sorbitol; or sodium chloride or derivatives thereof and;
iv) a surfactant.
In an embodiment, the invention discloses a pharmaceutical formulation of an anti-CD20 antibody comprising:
i) 120 mg/ml an anti-CD20 antibody,
ii) 10-50 mM histidine buffer,
iii) one or more stabilizers comprise mannitol, or trehalose, or sucrose or sorbitol; or sodium chloride or derivatives thereof and;
iv) a surfactant.

In an embodiment, the invention discloses a pharmaceutical formulation of an anti-CD20 antibody comprising:
i) 10 mg/ml to 200 mg/ml an anti-CD20 antibody,
ii) 10-50 mM histidine acetate buffer,
iii) one or more stabilizers comprise mannitol, or trehalose, or sucrose or sorbitol; or sodium chloride or derivatives thereof and;
iv) a surfactant.

In an embodiment, the invention discloses a pharmaceutical formulation of an anti-CD20 antibody comprising:
v) 10 mg/ml to 200 mg/ml an anti-CD20 antibody,
vi) 10-50 mM histidine-succinate buffer,
vii) one or more stabilizers comprise mannitol, or trehalose, or sucrose or sorbitol; or sodium chloride or derivatives thereof and;
viii) a surfactant.
In any of the above mentioned embodiments, the histidine buffer or it’s derivative or salts or combinations thereof, is a histidine buffer or a histidine-citrate buffer or a histidine-phosphate buffer. The histidine buffer composition may also further contain at least one pharmaceutically acceptable excipients/stabilizer. The said composition can be added during pre-formulation and formulation stage of the antibody production.
In an embodiment, the invention discloses a pharmaceutical formulation of an anti-CD20 antibody comprising:
i) 10 mg/ml to 200 mg/ml an anti-CD20 antibody,
ii) 10-50 mM citrate-phosphate buffer,
iii) one or more stabilizers comprise mannitol, or trehalose, or sucrose or sorbitol; or sodium chloride or derivatives thereof and;
iv) a surfactant.

In an embodiment, the invention discloses formulations of Table-1 as disclosed below.
Table 1: Anti-CD20 antibody formulation composition
Anti-CD20 Antibody concentration Buffer
(10-50 mM) Stabilizer
(50 mM to 200 mM) Surfactant
(0.02%)
10 mg/ml-200 mg/ml Histidine Trehalose Polysorbate/Poloxamer
10 mg/ml-200 mg/ml Histidine Mannitol Polysorbate/Poloxamer
10 mg/ml-200 mg/ml Histidine Sucrose Polysorbate/Poloxamer
10 mg/ml-200 mg/ml Histidine Sorbitol Polysorbate/Poloxamer
10 mg/ml-200 mg/ml Succinate Trehalose Polysorbate/Poloxamer
10 mg/ml-200 mg/ml Succinate Mannitol Polysorbate/Poloxamer
10 mg/ml-200 mg/ml Succinate Sucrose Polysorbate/Poloxamer
10 mg/ml-200 mg/ml Succinate Sorbitol Polysorbate/Poloxamer
10 mg/ml-200 mg/ml Histidine-succinate Trehalose Polysorbate/Poloxamer
10 mg/ml-200 mg/ml Citrate-phosphate Trehalose Polysorbate/Poloxamer
In an embodiment, the invention discloses a method of obtaining a stable formulation of anti-CD20 antibody, wherein the method comprises,
a. dissolution of succinic acid in water to obtain succinic acid solution
b. preparation of sodium hydroxide solution by dissolving sodium hydroxide in water
c. mixing of sodium hydroxide solution to succinic acid solution in a specific molar ratio to obtain sodium succinate buffer and;
d. preparation of anti-CD-20 antibody in a buffer composition comprising the said sodium succinate buffer; wherein the antibody solution obtained from step d) is stable.
In the above mentioned embodiment, the molar ratio between succinic acid to sodium hydroxide is 1:1 to 1:3 to obtain sodium succinate buffer composition.
In the above mentioned embodiment, the molar ratio between succinic acid to sodium hydroxide is 1:1, 1:1.1; 1:1.2; 1:1.3, 1:1.4; 1:1.5; 1:1.6; 1:1.7; 1:1.8; 1:1.9; 1:2; 1:2.1; 1:2.1; 1:2.2; 1:2.3; 1:2.4; 1:2.5; 1:2.6; 1:2.7; 1:2.8; 1:2.9 and 1:3. obtain sodium succinate buffer composition.
In the above mentioned embodiment of aforesaid embodiment, wherein the concentration of antibody is about 10 mg/ml to 200 mg/ml.
In above mentioned embodiment of aforesaid embodiment, the buffer composition may additionally comprise one or more pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients include sugar, polyol, polymers, amino acid or it’s derivatives, salts.
In an embodiment, the invention discloses a method of preparing a buffer composition to obtain a stable formulation of anti-CD20 antibody, wherein the method comprises,
a) dissolution of L-histidine and L-histidine hydrochloride in water in a specific molar ratio to obtain histidine buffer
b) preparation of anti-CD20 antibody in a buffer composition comprising the said histidine buffer.
In the above mentioned embodiment, the molar ratio between L-histidine to L-histidine hydrochloride is between 1:3 to 1:5.
In another embodiment the invention discloses a method to prepare a pharmaceutical formulation of anti-CD20 antibody comprising formulating an anti-CD20 antibody in a buffer composition comprising sodium succinate buffer, sugar and/or surfactant.
In an embodiment, the invention discloses a method of controlling aggregation of an anti-CD-20 antibody, in an anti-CD20 antibody composition, wherein the method involves preparation of anti-CD20 antibody in succinate buffer or histidine buffer or it’s derivatives or salts or combinations thereof.
In an embodiment, the invention discloses a method of controlling agitation induced aggregation of an anti-CD-20 antibody, in an anti-CD20 antibody composition, wherein the method involves preparation of anti-CD20 antibody in succinate buffer or histidine buffer or it’s derivatives or salts or combinations thereof.
In an embodiment, the invention discloses a method of controlling light induced aggregation of an anti-CD-20 antibody, in an anti-CD20 antibody composition, wherein the method involves preparation of anti-CD20 antibody in succinate buffer or histidine buffer or it’s derivatives or salts or combinations thereof.
In the above mentioned embodiment, the buffer composition may not require methionine or anti-oxidant or chelating agent to control light induced aggregation of anti-CD20 antibody.
In another embodiment, the invention discloses a method to obtain a stable anti-CD20 formulation against light induced aggregation of anti-CD20 antibody comprising; formulating/preparation of anti-CD20 antibody in a buffer composition comprising sodium succinate buffer, sugar and surfactant.
In another embodiment, the invention discloses a method to obtain a stable anti-CD20 formulation against light induced aggregation of anti-CD20 antibody comprising; formulating/preparation of anti-CD20 antibody in a buffer composition comprising histidine buffer, sugar and surfactant.
In another embodiment, the invention discloses a method to obtain a stable anti-CD20 antibody formulation against light induced aggregation/degradation of anti-CD20 antibody comprising; formulating/preparation of anti-CD20 antibody in a buffer composition comprising sodium succinate buffer and one or more stabilizers.
In any of the above mentioned embodiment, sodium succinate is prepared in a specific way in a specific molar ratio of succinate and sodium hydroxide. The specific molar ratio is in between 1:1 to 1:3.
In the above mentioned embodiment, the formulation additionally/optionally comprises surfactant.
In another embodiment, the invention discloses a method to obtain a stable anti-CD20 antibody formulation against light induced aggregation/degradation of anti-CD20 antibody comprising; formulating/preparation of anti-CD20 antibody in a buffer composition comprising histidine buffer and one or more stabilizers.
In the above mentioned embodiment, the formulation additionally/optionally comprises surfactant.
In an embodiment, the invention discloses a method of controlling fragmentation of an anti-CD-20 antibody, in an anti-CD20 antibody composition, wherein the method involves formulating/preparation of anti-CD20 antibody in a buffer composition comprising sodium succinate buffer. The buffer composition further comprises sugar and surfactant.
In another embodiment, the invention discloses a method to obtain a stable formulation against light induced fragmentation of anti-CD20 antibody comprising; formulating/preparation of anti-CD20 antibody in a buffer composition comprising sodium succinate buffer. The buffer composition further comprises sugar and surfactant.
In the above mentioned embodiment, sodium succinate is prepared in a specific way in a specific molar ratio of succinate and sodium hydroxide. The specific molar ratio is in between 1:1 to 1:3.
In another embodiment, the invention discloses a method to obtain a stable formulation against light induced fragmentation of anti-CD20 antibody comprising; formulating anti-CD20 antibody in a buffer composition comprising histidine buffer, sugar and surfactant.
In an embodiment, the invention discloses a method of controlling charge variants formation of an anti-CD-20 antibody, in an anti-CD20 antibody composition, wherein the method comprises preparation of anti-CD20 antibody in a buffer composition comprising succinate buffer or histidine buffer or it’s derivatives or salts or combinations.
In an embodiment, the invention discloses a method of controlling deamidation of an anti-CD-20 antibody, in an anti-CD20 antibody composition, wherein the method comprises preparation of anti-CD20 antibody in a buffer composition comprising succinate buffer or histidine buffer or it’s derivatives or salts or combinations.
In an embodiment, the invention discloses a method of controlling formation of basic variants in an anti-CD20 antibody composition wherein the method comprises preparation of anti-CD20 antibody in a buffer composition comprising succinate buffer or histidine buffer or it’s derivatives or salts or combinations. The method specifically controls formation of pyroglutamate variants, succinimide variants and lysine variants thereby reduces formation of basic variants.
In an embodiment, the invention discloses a method of controlling formation of pyroglutamate variants in an anti-CD20 antibody composition wherein the method comprises preparation of anti-CD20 antibody in a buffer composition comprising succinate buffer or histidine buffer or it’s derivatives or salts or combinations.
In an embodiment, the invention discloses a method of controlling formation of lysine variants in an anti-CD20 antibody composition,
wherein the method comprises preparation of anti-CD20 antibody in a buffer composition comprising succinate buffer or histidine buffer or it’s derivatives or salts or combinations.
In an embodiment, the invention discloses a method of controlling formation of succinimide variants in an anti-CD20 antibody composition, wherein the method comprises preparation of anti-CD20 antibody in a buffer composition comprising succinate buffer or histidine buffer or it’s derivatives or salts or combinations.
In an embodiment, the invention discloses a method of imparting colloidal stability to an anti-CD-20 antibody, in an anti-CD20 antibody composition, wherein the method comprises preparation of anti-CD20 antibody in a buffer composition comprising succinate buffer or histidine buffer or it’s derivatives or salts or combinations.
In another embodiment the invention discloses, a method of controlling oxidation of Met34 and Met83 of heavy chain of anti-CD20 antibody in a pharmaceutical composition of anti-CD20 antibody, wherein the method comprises preparation of the antibody composition in a buffer composition comprising succinate buffer or histidine buffer, sugar, and surfactant.
In another embodiment, the invention discloses a method of controlling visible particle formation in an anti-CD20 antibody composition, wherein the method comprises preparation of the antibody composition in a buffer composition comprising succinate buffer or histidine buffer, sugar, and surfactant.
In another embodiment, the invention discloses a method of controlling sub-visible particle formation in an anti-CD20 antibody composition wherein the method comprises preparation of the antibody composition in a buffer composition comprising succinate buffer or histidine buffer, sugar, and surfactant.
In the above mentioned embodiment, sodium succinate is prepared in a specific way in a specific molar ratio of succinate and sodium hydroxide. The specific molar ratio is in between 1:1 to 1:3.
In any of the above mentioned embodiments, the pH of the disclosed formulation of the present invention is in the range from about 5.0 to about 6.5.
In any of the above mentioned embodiments, the pH of the disclosed formulation of the present invention is 5.5 ± 0.3.
The disclosed formulations of the present invention stabilize an anti-CD20 antibody at various concentration levels i.e., from about 10 mg/ml to about 200 mg/ml and are suitable for intravenous as well as subcutaneous routes of administration. Further, viscosity of the anti-CD20 antibody formulations is less than 20 cP, specifically, less than 10 cP.
In any of the above mentioned embodiments, the one or more pharmaceutically acceptable excipients/stabilizers is a sugar, polyol, amino acid, or salt.
In any of the above mentioned embodiments, wherein the sugar is trehalose or sucrose.
In any of the above mentioned embodiments, wherein the polyol is mannitol or sorbitol.
In any of the above mentioned embodiments, wherein the salt is sodium chloride.
In any of the above mentioned embodiments, wherein the surfactant is polysorbate or poloxamer.
In any of the above mentioned embodiments, the sodium succinate buffer is prepared by following steps:
a. dissolution of succinic acid in water
b. preparation of sodium hydroxide solution by dissolving sodium hydroxide in water
c. mixing of sodium hydroxide to succinic acid solution in a particular molar ratio to obtain sodium succinate buffer.
In the above mentioned embodiment, the molar ratio between succinic acid to sodium hydroxide is 1:1 to 1:3.
In any of the above mentioned embodiments, the claimed anti-CD20 antibody formulations of the invention exhibit stability under at least one of the following conditions, wherein the temperature range from 25 ºC to 50 ºC for a period of time which includes from 1 day to 6 months.
In any of the above mentioned embodiments, wherein the formulation may not require an anti-oxidant or chelating agent to stabilize anti-CD20 antibody.
In the above mentioned embodiment, the chelator includes ethylenediamine tetraacetic acid (EDTA) or ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or diethylenetriamine pentaacetate (DTPA) or like.

In any of the above mentioned embodiments, the anti-CD20 antibody is a chimeric or humanized or human anti-CD20 antibody or conjugates or fusion proteins thereof.
In the above mentioned embodiment, the anti-CD20 antibody is either a type-I or type-II anti-CD20 antibody.
In the above mentioned embodiment, the anti-CD20 antibody is rituximab, ofatumumab, obinutuzumab, ocelizumab or ubilituximab or conjugates thereof.
In any of the above mentioned embodiments, wherein the conjugates is a drug or an antibody or any other therapeutic molecule.
In any of the above mentioned embodiments, wherein the fusion proteins include an anti-CD 20 antibody or antigen fragment thereof fused to another protein.
In all of the above mentioned embodiments of the invention, the concentration of the antibody in the formulation is about 10 mg /ml to about 200 mg/ml. Preferably, the concentration of the antibody in the formulation is 10 mg/ml, or 25 mg/ml, or 50 mg/ml, or 60 mg/ml, or 70 mg/ml, or 80 mg/ml, 90 mg/ml, or 100 mg/ml, or 110 mg/ml, or 120 mg/ml, or 150 mg/ml or 160 mg/ml, or 170 mg/ml or 175 mg/ml or 180 mg/ml or 190 mg/ml or 195 mg/ml or 200 mg/ml.
In any of the above said embodiments of the invention, the anti-CD20 antibody formulation is stable and contains less than 1.5 % of high molecular weight (HMW) species in the formulation, even after storage at 40 ºC for four weeks.
In any of the above said embodiments of the invention, the anti-CD20 antibody formulation is stable and contains less than 2% of fragments in the formulation, even after storage at 40 ºC for four weeks.
In any of the above mentioned embodiments, the anti-CD20 antibody maintains at least 90% of monomeric content of the antibody after storage at 40 ºC for four weeks
In any of the above mentioned embodiments, the anti-CD20 antibody formulation’s osmolality is less than 600 mOsm/kg, preferably less than 300 mOsm/kg.
The anti-CD20 antibody formulations disclosed in the invention are biologically active.
In any of the above mentioned embodiments, the formulation of anti-CD20 antibody is a stable liquid (aqueous) formulation, which can be used for parenteral administration. Parenteral administration includes intravenous, subcutaneous, intra peritoneal, intramuscular administration or any other route of delivery generally considered to be falling under the scope of parenteral administration and as is well known to a skilled person.
In any of the above embodiments of the invention, the stable liquid/aqueous formulation is suitable and can be lyophilized as lyophilized powders. Further, the lyophilized formulation of anti-CD20 antibody can be reconstituted with appropriate diluent to achieve the liquid formulation suitable for administration.
In any of the above mentioned embodiments, the liquid/aqueous anti-CD20 antibody are compatible with lyophilization process and the lyophilization process does not impact quality attributes of the antibody.
Another aspect of the invention provides a vial, pre-filled syringe or autoinjector device, or any other suitable device comprising any of the subject formulations described herein. In certain embodiments, the aqueous formulation, stored in the vial or pre-filled syringe or an auto injector device comprise anti-CD20 antibody, succinate buffer or acetate buffer or citrate buffer or histidine buffer or it’s derivatives or combination thereof, sugar and surfactant.
DEFINITIONS
The term "about" refers to a range of values that are similar to the stated reference value and includes a range of values that fall within 10 % or less, of the stated reference value.
The term “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. The “antibody” as used herein encompasses whole antibodies or any antigen binding fragment (i.e., “antigen-binding portion”) or fusion protein thereof.
The term "stable" formulation refers to the formulation wherein the antibody therein retains its physical stability and/or chemical stability and/or biological activity upon storage.
Stability studies provides evidence of the quality of an antibody under the influence of various environmental factors during the course of time. ICH’s “Q1A: Stability Testing of New Drug Substances and Products,” states that data from accelerated stability studies can be used to evaluate the effect of short-term excursions higher or lower than label storage conditions that may occur during the shipping of the antibodies.
Various analytical methods are available for measuring the physical and chemical degradation of the antibody in the pharmaceutical formulations. An antibody "retains its physical stability" in a pharmaceutical formulation if it shows substantially no signs of aggregation, precipitation and/or denaturation upon visual examination of color and/or clarity, or as measured by UV light scattering or by size exclusion chromatography. An antibody is said to “retain its chemical stability” in a pharmaceutical formulation when its shows no or minimal formation of product variants which may include variants as a result of chemical modification of antibody of interest such as deamination, oxidation etc. Analytical methods such as ion exchange chromatography and hydrophobic ion chromatography may be used to investigate the chemical product variants.
The term ‘monomer’ as used herein describes antibodies consisting of two light chains and two heavy chains. The monomer content of an antibody composition is typically analyzed by size exclusion chromatography (SEC). As per the separation principle of SEC the large molecules or molecules with high molecular weight (HMW) elute first followed by smaller or lower weight molecules. In a typical SEC profile for an antibody composition, aggregates that may include dimers, multimers, etc., elute first, followed by monomer, and the clipped antibody variants or degradants may be eluted last. In some circumstances the aggregate peak or the degradant peaks may not elute as a baseline separated peaks but instead as a shoulder or abnormal broad peaks.
The term ‘main peak’ as used herein refers to the peak that elutes in abundance (major peak) during a cation exchange chromatography. The peak that elutes earlier than the main peak, during a cation exchange chromatography, with a charge that is acidic relative to the main peak is termed acidic variant peak. The peak that elutes later than the main peak, during a cation exchange chromatography, with a charge that is relatively basic than the main peak is termed as basic variant peak. The main peak content can be determined by Ion exchange chromatography (IEC). There are two modes of IEC available viz., cation and anion exchange chromatography. Positively charged molecules bind to anion exchange resins while negatively charged molecules bind to cation exchange resins. In a typical cation exchange chromatographic profile of an antibody composition acidic variants elute first followed by the main peak and thereafter lastly the basic variants will be eluted. The acidic variants are a result of antibody modifications such as deamidation of asparagine residues. The basic variants are a result of incomplete removal of C-terminal lysine residue(s). In general, in an antibody a lysine residue is present at the C-terminal end of both heavy and light chain. An antibody molecule containing lysine at both heavy and light chain is referred to as K2 variant, the antibody molecule containing lysine residue at either one of heavy and light chain is referred to as K1 variant and antibody molecule having none is K0 molecule. Carboxypeptidase B (CP-B enzyme) enzyme acts on the C-terminal lysine residues present on K2 and K1 variants and thus converting them as K0 molecules. As per circumstances of the case, the IEC analysis can be carried out for samples digested with carboxypeptidase B (CP-B) enzyme. In a typical stability study it is expected that a stable formulation leads to reduction in formation of charge variants (acidic and basic variants), during the study, and hence minimize any reduction in main peak content.
Pharmaceutically acceptable excipients refer to the additives or carriers, which may contribute to stability of the antibody in formulation. The excipients may encompass stabilizers and tonicity modifiers. Examples of stabilizers and tonicity modifiers include, but not limited to, salts, surfactants, and derivatives and combination thereof. The at least one stabilizer in a pharmaceutical formulation of the present invention can be a polyethylene glycol (PEG), ß-cyclodextrin or equivalents thereof.
The term sugar/s as used herein includes organic compounds having general formula of all carbohydrates of the general formula Cn(H2O)n. Sugars can be referred to monosaccharides, disaccharides, and polysaccharides. Examples of sugars include, but are not limited to, sucrose, trehalose, glucose, dextrose, raffinose and others.
The term “polyol” or “sugar alcohol” as used herein includes an organic compound containing multiple hydroxyl groups. Examples of polyols include mannitol, sorbitol, xylitol etc.,
Surfactant refers to pharmaceutically acceptable excipients used to protect the protein formulations against various stress conditions, like agitation, shearing, exposure to high temperature etc. The suitable surfactants include but are not limited to polyoxyethylensorbitan fatty acid esters such as Tween 20™ or Tween 80™, polyoxyethylene-polyoxypropylene copolymer (e.g. Poloxamer, Pluronic), sodium dodecyl sulphate (SDS) and the like or combination thereof.
Examples of salts include, but not limited to, sodium chloride, potassium chloride, magnesium chloride, sodium thiocyanate, ammonium thiocyanate, ammonium sulfate, ammonium chloride, calcium chloride, zinc chloride and/or sodium acetate.
The term "opalescence" or "opalescent appearance" refers to the degree of turbidity detected in a solution, e.g., a protein preparation, as a function of the concentration of one or more of the components in the solution, e.g., protein and/or salt concentration. The degree of turbidity can be calculated by reference to a standard curve generated using suspensions of known turbidity. Reference standards for determining the degree of turbidity for pharmaceutical compositions can be based on the United States Pharmacopeia or European Pharmacopeia criteria. Here, in this invention to measure opalescence, first Formazine solution has been prepared by mixing equal volumes of a hydrazine sulfate solution and hexamethylenetetramine solution and then diluted to prepare various reference opalescence standards. The opalescence standards includes ROS-I, ROS-II, ROS-III and ROS-IV.
Nephelometry is a turbidometric method used to detect the presence of soluble aggregates or to indicate opalescence. The output is listed in terms of nephelometric turbidity units (NTUs).
“Pre-formulation steps” refers to any or multiple steps performed before formulating the protein into a therapeutic product. Examples of such steps include, chromatography, filtration, (ultrafiltration, sterile filtration, nano filtration, diafiltration, tangential flow filtration, depth filtration), or any other steps performed to concentrate the protein or to exchange the buffer to a different/suitable buffer. The filtration steps mentioned herein may be performed in a tangential flow filtration mode.
”Formulation steps” refers to steps which are followed after the downstream chromatographic and filtration steps to prepare drug product from drug substance, the latter obtained from the pre-formulation steps.
The term “chelators/chelating agents” refers to a compound which can form at least one bond with a metal atom. A chelating agent is typically a multidentate ligand that can be used in compositions as a stabilizer to complex with species, which might otherwise promote instability. Exemplary chelating agents include aminopolycarboxylic acids, hydroxyaminocarboxylic acids, N- substituted glycines, 2- (2-am ino-2-oxocthyl) aminoethane sulfonic acid (BES), deferoxamine (DEF), niacinamide, desoxycholates, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), nitrilotriacetic acid (NTA), N-2-acetamido-2- iminodiacetic acid (ADA), bis(am inoethyl)glycolether, N,N,N',N'-tetraacetic acid (EGTA), trans- diaminocyclohexane tetraacetic acid (DCTA), N- hydroxyethyliminodiacetic acid (HIMDA), N,N-bis-hydroxyethylglycine (bicine), N- (trishydroxymethylmethyl) glycine (tricine), glycylglycine, sodium desoxycholate, ethylenediamine; propylenediamine; diethylenetriamine; triethylenetetraamine (trien), ethylenediaminetetraaceto EDTA; disodium EDTA, EDTA, calcium EDTA oxalic acid and malate.
The term “antioxidant” refers to an agent that inhibits the oxidation of other molecules and is not part of buffer component. Examples of antioxidants herein include citrate, methionine, lipoic acid, uric acid, glutathione, tocopherol, carotene, lycopene, cysteine, phosphonate compounds, e.g., etidronic acid, desferoxamine and malate.
Certain specific aspects and embodiments of the invention are more fully described by reference to the following examples. However, these examples should not be construed as limiting the scope of the invention in any manner.

EXAMPLES
An anti-CD20 antibody, ocrelizumab, suitable for storage in the present pharmaceutical composition is produced by standard methods known in the art. For example, ocrelizumab is prepared by recombinant expression of immunoglobulin light and heavy chain genes in a mammalian host cell such as Chinese Hamster Ovary cells. Further, the expressed ocrelizumab is harvested and the crude harvest is subjected to standard downstream process steps that include purification, filtration and optionally dilution or concentration steps. For example, the crude harvest of ocrelizumab may be purified using standard chromatography techniques such as affinity chromatography, ion-exchange chromatography and combinations thereof. The purified ocrelizumab solution can additionally be subjected to one or more filtration steps, and the solution obtained is subjected to further formulation studies.
Example 1: Anti-CD20 antibody formulations.
Purified ocrelizumab antibody sample, approximately 8 mg/ml in acetate buffer back ground was obtained from downstream chromatographic step. The antibody containing sample was buffer exchanged with different buffer compositions comprising succinate buffer (i.e., succinic acid and di sodium succinate hexahydrate), histidine buffer (L histidine and L-histidine hydrochloride), histidine-succinate buffer (L histidine and disodium succinate hexahydrate) and citrate phosphate. Post which, the samples were concentrated to a concentration ranging between 40 mg/ml to 120 mg/ml and the concentration was further adjusted to 30 mg/ml with formulation buffer having pH 5.3 ± 0.3 comprising excipients such as sugar at a concentration range of 10 mg/ml to 60 mg/ml and surfactant at a concentration range of 0.1 to 0.5 mg/ml. Details of the formulation are given in Table 2.
All ocrelizumab formulations were subjected for accelerated stability studies at 40 ?C for four weeks. The samples were then analyzed for low molecular weight (LMW) species, high molecular weight species (HMW) and monomer content using size exclusion chromatography (SEC) [results are given in Table 3] and also checked for particle formation, and opalescence [Table 4] and charge variants using ion-exchange chromatography [Table 5], at zero time point (before subjecting the samples for accelerated stability condition at 40 ?) and also at T4W (sample stored at 40? for four weeks).
Table 2: Compositions of anti-CD20 antibody formulations prepared as per Example-1
Sample Name Composition
S1 30 mg/ml ocrelizumab, 25 mM histidine buffer, trehalose, polysorbate-80
S2 30 mg/ml ocrelizumab, 25 mM histidine buffer, mannitol, polysorbate-80
S3 30 mg/ml ocrelizumab, 25 mM histidine buffer,, sorbitol, polysorbate-80
S4 30 mg/ml ocrelizumab, 25 mM succinate buffer, trehalose, polysorbate-80
S5 30 mg/ml ocrelizumab, 25 mM succinate buffer, mannitol, polysorbate-80
S6 30 mg/ml ocrelizumab, 25 mM succinate buffer, sucrose, polysorbate-80
S7 30 mg/ml ocrelizumab, 25 mM succinate buffer,, sorbitol, polysorbate-80
S8 30 mg/ml ocrelizumab, 23 mM sodium succinate buffer, trehalose polysorbate-80
S9 30 mg/ml ocrelizumab, 25 mM histidine-acetate buffer,, trehalose, polysorbate-80
S10 30 mg/ml ocrelizumab, 25 mM histidine-succinate buffer, trehalose, polysorbate-80
S11 30 mg/ml ocrelizumab, 25 mM citrate-phosphate buffer, trehalose, polysorbate-80
Table 3: Visual inspection of anti-CD20 antibody formulations as per example 1
Sample name Visual inspection
T0 T4W at 40?
S1 Opalescent, Between ROS II to III, No visible particles Opalescent, Between ROS II to III, No visible particles
S2 Opalescent, Between ROS II to III, No visible particles Opalescent, Between ROS II to III, No visible particles
S3 Opalescent, Between ROS II to III, No visible particles Opalescent, Between ROS II to III, No visible particles
S4 Opalescent, Between ROS II to III, No visible particles Opalescent, Greater than ROS IV, No visible particles
S5 Opalescent, Between ROS II to III, No visible particles Opalescent, Between ROS II to III, No visible particles
S6 Opalescent, Between ROS II to III, No visible particles Opalescent, Greater than ROS IV, precipitates visible
S7 Opalescent, Between ROS II to III, No visible particles Opalescent, Between ROS II to III, No visible particles
S8 Opalescent, Between ROS II to III, No visible particles Opalescent, Between ROS II to III, No visible particles
S9 Opalescent, Between ROS II to III, No visible particles Opalescent, Between ROS II to III, No visible particles
S10 Opalescent, Between ROS II to III, No visible particles Opalescent, Between ROS II to III, No visible particles
S11 Opalescent, Between ROS II to III, No visible particles Opalescent, Between ROS II to III, No visible particles
Table 4: SEC data on anti-CD20 antibody formulations prepared as per example-1
Sample name SEC data at 40 ?
% Monomer at 40 ? % HMW at 40 ? % LMW at 40 ?
T0 T4W T0 T4W T0 T4W
S1 98.5 97.4 1.22 1.00 0.32 1.51
S2 98.5 97.4 1.21 0.97 0.31 1.58
S3 98.5 97.3 1.27 1.13 0.27 1.60
S4 98.6 56.6 0.97 1.13 0.45 42.24
S5 98.5 74.1 1.01 1.02 0.46 24.90
S6 98.6 NAV 1.01 NA 0.44 NA
S7 98.5 73.7 1.01 1.17 0.48 25.16
S8 98.6 97.8 1.04 0.95 0.33 1.21
S9 98.5 97.5 1.21 1.06 0.28 1.42
S10 98.5 97.5 1.22 1.10 0.28 1.41
S11 98.4 97.2 1.33 1.45 0.27 1.33
Table 5: IEX data on anti-CD20 antibody formulations prepared as per example-1
Sample name IEX data at 40 ?
% acidic variants at 40 ? % main peak content at 40 ? % basic variants at 40 ?
T0 (?/week) T0 (?/week) T0 (?/week
S1 27.0 6.88 61.4 -6.68 11.7 10.99
S2 27.0 6.89 60.9 -6.45 12.2 10.57
S3 26.7 6.88 61.1 -6.54 12.2 11.03
S4 13.5 7.08 51.3 -15.28 35.2 NAV
S5 14.8 4.23 49.6 -1.97 35.6 27.11
S6 15.7 3.71 52.8 -13.24 31.5 NAV
S7 14.9 4.62 49.5 -5.16 35.7 37.58
S8 26.7 5.35 60.1 5.89 13.2 15.09
S9 27.0 6.74 61.3 6.37 11.7 10.12
S10 26.4 6.89 61.6 -6.43 11.9 10.00
S11 26.5 6.48 60.9 -6.20 12.7 11.71

Table 6: Quality attributes of anti-CD 20 antibody formulations prepared as per Example-1.
Sample name pH at 40 ? Osmolality at 40 ?
T0 T4W T0 T4W
S1 5.39 5.77 236 244
S2 5.38 5.74 233 245
S3 5.44 5.89 257 299
S4 5.39 5.63 210 328
S5 5.38 5.52 203 218
S6 5.36 4.12 244 353
S7 5.40 5.53 204 228
S8 5.37 5.70 165 174
S9 5.39 5.85 215 228
S10 5.34 5.71 233 244
S11 5.39 5.65 251 269

Further, a few of the samples (S4 and S8) were subjected for mass spectrometry to analyze various variants such as lysine variants and pyroglutamate variants [Table 7].
Table 7: Mass spectrometry data of few of anti-CD20 antibody formulations of Example-1

Sample ID Pyroglutamate variants at 40 ºC Lysine variants at 40 ºC
T0 T4W T0 T4W
S4 0.38 1.19 0.75 0.63
S8 0.49 0.93 0.62 (T1W) 0.52

In addition, the samples were also measured for oxidation variants and succinimide variants and data is not given here.
Example 2: High concentration Anti-CD20 antibody formulations.
Purified rituximab antibody sample, approximately 8 mg/ml in acetate buffer back ground was obtained from downstream chromatographic step. The antibody containing sample was buffer exchanged with Histidine acetate. Post which, the samples were concentrated to a concentration ranging between 40 mg/ml to 190 mg/ml with formulation buffer having pH 6.5 ± 0.3 comprising excipients such as sugar at a concentration range of 80 mg/ml to 150 mg/ml, surfactant at a concentration range of 0.1 to 0.7 mg/ml, amino acid at a range of 5 mg/ml to 12 mg/ml, polyethylene glycol (PEG) and diethylenetriamine pentaacetate (DTPA). Details of the formulation are given in Table 8.
All rituximab formulations were subjected for accelerated stability studies at 40 ? for four weeks. The samples were then analyzed for low molecular weight (LMW) species, high molecular weight species (HMW) and monomer content using size exclusion chromatography (SEC) [results are given in Table 9].
Table 8: Compositions of high concentration anti-CD20 antibody (rituximab) formulations prepared as per Example-2

Sample name Buffer Sugar Excipients Protein Conc(mg/mL)


S12 Histidine acetate (25mM) Trehalose
(100mg/mL) Glycine, L-arginine, PS-80 170
S13 L-arginine, PS-80, Proline
S14 L-methionine, L-arginine, PS-80, DTPA
S15 PEG 3350, L-arginine, PS-80
S16 Sucrose (150mg/mL) Glycine, L-arginine, PS-80 166
S17 L-arginine, PS-80, Proline
S18 L-methionine, L-arginine, PS-80, DTPA
S19 PEG 3350, L-arginine, PS-80
S20 Sorbitol (80mg/mL) Glycine, L-arginine, PS-80 189
S21 L-arginine, PS-80, Proline
S22 L-methionine, L-arginine, PS-80, DTPA
S23 PEG 3350, L-arginine, PS-80
Table 9: Quality attributes and SEC data on anti-CD20 antibody formulations prepared as per Example-2

Sample name pH Protein concentration (mg/mL) SEC data at 40 ?
%HMWs
%LMW
T0 T2W ? T2W T1M ? T0 T2W T1M ? T0 T2W T1M ?
S12 6.7 6.7 0.0 182.5 177.7 -4.8 1.7 1.8 2.1 0.4 0.0 0.3 0.5 0.5
S13 6.7 6.7 0.0 182.2 173.7 -8.4 1.7 1.7 2.0 0.3 0.0 0.3 0.5 0.5
S14 6.7 6.7 0.0 189.6 178.2 -11.4 1.6 1.7 1.9 0.3 0.0 0.3 0.5 0.5
S15 6.7 6.7 0.0 189.8 179.1 -10.7 1.6 1.8 2.1 0.5 0.0 0.3 0.5 0.5
S16 6.7 6.7 0.0 183.9 178.0 -5.9 1.7 1.6 2.0 0.3 0.0 0.3 0.5 0.5
S17 6.7 6.7 0.0 187.5 176.0 -11.5 1.6 1.7 1.9 0.3 0.0 0.3 0.6 0.6
S18 6.7 6.7 0.0 188.2 178.2 -10.0 1.6 1.6 2.0 0.4 0.0 0.3 0.5 0.5
S19 6.7 6.7 0.0 197.3 181.1 -16.2 1.6 1.8 1.8 0.2 0.0 0.3 0.6 0.6
S20 6.8 6.8 0.0 170.6 156.6 -14.1 1.7 1.7 1.9 0.2 0.1 0.3 0.6 0.5
S21 6.8 6.8 0.0 169.6 157.1 -12.5 1.7 1.7 1.9 0.2 0.0 0.3 0.6 0.6
S22 6.8 6.8 0.0 176.3 157.9 -18.4 1.6 1.7 1.8 0.2 0.0 0.3 0.6 0.6
S23 6.8 6.8 0.0 175.5 161.1 -14.4 1.7 1.7 1.9 0.2 0.1 0.3 0.6 0.5
,CLAIMS:We claim:

1. A pharmaceutical formulation of an anti-CD20 antibody comprising:
(i) an anti-CD20 antibody,
(ii) a buffer having pH of about 5.0 to about 6.5,
(iii) one or more stabilizers selected from mannitol, or trehalose, or sucrose or sorbitol,
(iv) a surfactant.

2. The pharmaceutical formulation as claimed in claim 1, wherein the anti-CD20 antibody concentration ranges from 10 mg/ml to 200 mg/ml.

3. The pharmaceutical formulation as claimed in claim 1, wherein the surfactant is polysorbate or poloxamer.

4. The pharmaceutical formulation as claimed in claim 1, further comprises one or more pharmaceutically acceptable excipients selected from amino acids or polymers or chelating agents or salts or combination thereof.

5. A pharmaceutical formulation of an anti-CD20 antibody comprising:
i) 10 mg/ml to 200 mg/ml an anti-CD20 antibody,
ii) 10-50 mM histidine buffer,
iii) one or more stabilizers selected from mannitol, or trehalose, or sucrose or sorbitol,
iv) a surfactant.
6. A pharmaceutical formulation of an anti-CD20 antibody comprising:
i) 10 mg/ml to 200 mg/ml an anti-CD20 antibody,
ii) 10-50 mM histidine acetate buffer,
iii) one or more stabilizers selected from mannitol, or trehalose, or sucrose or sorbitol,
iv) a surfactant.

7. The pharmaceutical formulation as claimed in claim 5 or 6, wherein the surfactant is polysorbate or poloxamer.

8. The pharmaceutical formulation as claimed in claim 1, 5 or 6, further comprises one or more pharmaceutically acceptable excipients selected from amino acids or polymers or chelating agents or salts or combination thereof.

9. The pharmaceutical formulation as claimed in claim 1, 5 or 6, wherein the anti-CD20 antibody is rituximab, ofatumumab, obinutuzumab, ocrelizumab, or ubilituximab or conjugates thereof.

10. The pharmaceutical formulation as claimed in claim 1, 5 or 6, is a stable liquid (aqueous) formulation.