DESC:INTRODUCTION
The present invention is related to an aqueous, buffer free formulation of an antibody molecule, stabilized at a particular pH, without any buffering agent. The disclosed formulation stabilizes the antibody from about 10 mg/ml to about 200 mg/ml which are suitable for intravenous or subcutaneous route of administration.
BACKGROUND
There are at least 130 monoclonal antibodies which have been approved by regulatory authorities to treat various diseases. And, these antibodies are available as either solid or liquid form and can be administered via various routes of administration which includes intravenous, subcutaneous, intramuscular, and intravitreal route of administration. Formulations for each route of administration and dosage forms may be unique and, therefore, have specific requirements. Solid dosage forms, such as lyophilized powders, are generally more stable than liquid (aqueous) formulations. However, reconstitution of the lyophilized formulation requires a significant vial overfill, care in handling and involves high production cost relative to a liquid formulation. While liquid formulations are advantageous in these and are usually preferred for injectable protein therapeutics (in terms of convenience for the end user and ease of preparation for the manufacturer), this form may not always be feasible given the susceptibility of proteins to denaturation, aggregation and oxidation under stresses such as temperature, pH changes, agitation etc.,. All of these stress factors could result in the loss of biological activity of a therapeutic protein / antibody.
In particular, high concentration liquid formulations are susceptible to degradation and/or aggregation. Nevertheless, high concentration formulations may be desirable for subcutaneous or intravenous route of administration, as the frequency of administration and injection volume is reduced. On the other hand, specific treatment schedule and dosing might require a low concentration formulation and prefer intravenous route of administration for more predictable delivery and complete bioavailability of the therapeutic drug.
A formulation combination with increased concentration of protein and /or stabilizers may increase the viscosity of the formulation, in turn increasing the injection time and pain at the site of injection and also pose difficulties during processing of the drug substance. Further, it has been reported that some of the buffers are not suitable for parenteral route of administration as it causes pain at the injection site in case of citrate buffer and irritation at injection in case of glutamate buffer. Hence, it is necessary to develop an improved formulation, which stabilizes a protein at a wide range of its concentration and suitable for in different route of administration (intravenous or subcutaneous), pose a significant developmental challenge. Further, every protein or antibody with its unique characteristics and properties of degradation, adds to the complexity in the development of a stable formulation and may demand a specific formulation.
SUMMARY OF THE INVENTION
The present invention discloses a buffer free formulation of an anti-CD20 antibody comprising, at least 10 mg/ml anti-CD20 antibody, water and surfactant. The antibody formulated in water maintains solubility and stability, during long-term liquid storage or other processing steps, such as freeze/thawing.
In particular, the invention discloses a buffer free formulation of an anti-CD20 antibody, comprising 10 mg/ml to 200 mg/ml an anti-CD20 antibody, sugar, water and surfactant, stabilized at a pH of about 5.0 to about 6.5. The disclosed anti-CD20 antibody formulation of the invention does not require any specific buffering agent to maintain/stabilize the pH of the formulation.
In another aspect, the invention discloses a method of stabilizing an anti-CD20 antibody in an aqueous solution, at a pH of about 5.0 to about 6.5, without a buffering agent comprising steps of; expressing and purifying anti-CD20 antibody to obtain anti-CD20 antibody composition, subjecting the said antibody composition to diafiltration with a diafiltration media comprising water, followed by addition of one or more pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients are sugar, salt, amino acid or surfactant.
The formulations and methods disclosed in the invention stabilizes anti-CD20 antibody, in concentrations ranging from about 10 mg/ml to about 200 mg/ml.
In yet another aspect, the disclosed buffer free anti-CD20 antibody formulation exhibits colloidal stability. And the viscosity of the disclosed anti-CD20 formulations is less than 20 cP.
The disclosed formulations of the invention specifically control fragmentations and/or aggregation and/or charge variants of anti-CD20 antibody in it’s composition. Further, these formulations are able to control formation of lysine variants, methionine oxidation, succinimide formation of anti-CD20 antibody in it’s composition when the composition is stored at real time and/or at accelerated or stress conditions.
The disclosed formulations of the antibody exhibits stability under accelerated conditions such as at 40 ºC for at least two weeks.
DETAILED DESCRIPTION
The present invention discloses a buffer free formulation of an anti-CD20 antibody, comprising anti-CD20 antibody, water and surfactant, stabilized at a pH of about 5.0 to about 6.5. Wherein the formulation is devoid of any buffering agent.
In one embodiment, the invention discloses a buffer free formulation of an anti-CD20 antibody, stabilized at a pH of about 5.0- about 6.5, comprising anti-CD20 antibody, water and surfactant, wherein the pH of the anti-CD20 antibody formulation is maintained without any buffering agent.
In the above mentioned embodiment, the anti-CD20 antibody formulation optionally contains one or more pharmaceutically acceptable excipients and the one or more pharmaceutically acceptable excipients comprise sugar or polyol or salt or surfactant.
In an embodiment, the invention discloses a buffer free formulation of an anti-CD20 antibody, stabilized at a pH of about 5.0- about 6.5, comprising at least 10 mg/ml anti-CD20 antibody, water, sugar, and surfactant, wherein the pH of the formulation is maintained without any buffering agent.
In an embodiment, the invention discloses a buffer free formulation of an anti-CD20 antibody, stabilized at a pH of about 5.0- about 6.5, comprising at least 10 mg/ml – 200 mg/ml anti-CD20 antibody, water, and surfactant, wherein the pH of the formulation is maintained without any buffering agent.
In an embodiment, the invention discloses a buffer free formulation of an anti-CD20 antibody, stabilized at a pH of about 5.0- about 6.5, comprising at least 10 mg/ml – 200 mg/ml anti-CD20 antibody, water, sugar, and surfactant, wherein the pH of the formulation is maintained without any buffering agent.
In an embodiment, the invention discloses a buffer free formulation of an anti-CD20 antibody, stabilized at a pH of about 5.0- about 6.5, comprising at least 120 mg/ml anti-CD20 antibody, water, sugar, and surfactant, wherein the pH of the formulation is maintained without any buffering agent.
In the above mentioned embodiment, the anti-CD20 antibody optionally contains amino acid and/or salts.
In an embodiment the invention discloses buffer free formulations of Table 1.
Anti CD20 antibody concentration
10 mg/ml to 200 mg/ml Stabilizer (10 mM to 200 mM)
Sugar Surfactant
10 mg/ml to 200 mg/ml Sugar Polysorbate or poloxamer
10 mg/ml to 200 mg/ml Amino acid
10 mg/ml to 200 mg/ml Amino acid Polysorbate or poloxamer
10 mg/ml to 200 mg/ml Salt
10 mg/ml to 200 mg/ml Amino acid and salt Polysorbate or poloxamer
10 mg/ml to 200 mg/ml Sugar and salt Polysorbate or poloxamer
10 mg/ml to 200 mg/ml Amino acid, sugar and salt Polysorbate or poloxamer
10 mg/ml to 200 mg/ml Amino acid, sugar and salt
Table 1: Buffer free anti-CD20 antibody formulations
In an embodiment, the invention discloses a buffer free formulation of an anti-CD20 antibody, stabilized at a pH of about 5.0 to about 6.5, comprising at least 10 mg/ml of anti-CD20 antibody, water, sugar and surfactant, wherein the formulation is stable for at least two weeks when stored at 40 ºC.
In the above said embodiment, the anti-CD20 antibody formulation is stable and maintains at least 98% of monomeric content of the antibody, when stored at 40 ºC for two weeks.
In an embodiment, the invention discloses a buffer free formulation of an anti-CD20 antibody, stabilized at a pH of about 5.0 to about 6.5, comprising at least 10 mg/ml of anti-CD20 antibody, water, sugar and surfactant, wherein the formulation is stable for two weeks when stored at 40 ºC and aggregate content of the antibody is less than 1.5% after storage at 40 ºC for two weeks.
The anti-CD20 antibody formulations disclosed in the invention are biologically active.
In an embodiment, the invention discloses a buffer free formulation of an anti-CD20 antibody, stabilized at a pH of about 5.0 to about 6.5, comprising 30 mg/ml of anti-CD20 antibody, water, trehalose and surfactant, wherein the formulation is stable for at least two weeks when stored at 40 ºC
In another embodiment, the invention discloses a method of stabilizing an anti-CD20 antibody in a solution, at a pH of about 5.0 to about 6.5 without a buffering agent, the method comprising;
a) expressing and purifying an anti-CD20 antibody to obtain the antibody composition;
b) subjecting the purified antibody composition to a diafiltration step using water as a diafiltration medium,
c) obtaining the antibody composition in water.
In the above mentioned embodiment, wherein the antibody concentration is 10 mg/ml to 200 mg/ml
In the above mentioned embodiment, the antibody concentration is 120 mg/ml.
In the above mentioned embodiment, the method further comprises addition of one or more pharmaceutically acceptable excipients to the antibody composition obtained from step c) to obtain anti-CD20 antibody solution, wherein the pharmaceutically acceptable excipients are sugar or polyol, amino acid, salt or surfactant.
In the above mentioned embodiment of aforesaid embodiment, the antibody composition obtained in step c) may optionally subjected for ultrafiltration to concentrate up to 200 mg/ml.
In the above mentioned embodiment of aforesaid embodiment, wherein the antibody solution obtained by the said method exhibits stability at 2-8? or at 25? or at 40? for one day to 28 days.
In an embodiment, the invention discloses a method of controlling fragmentation of an anti-CD20 antibody in an aqueous buffer free formulation, stabilized at a pH of about 5.0 to about 6.5 wherein the method comprises preparation of the anti-CD20 antibody in a composition comprising water, sugar and surfactant, and wherein the pH of the formulation is maintained without any buffering agent. The obtained anti-CD20 antibody formulation from the said method is stable at 40 ºC for two weeks and content of fragmentation of the antibody is less than 2% when stored at 40 ? for two weeks.
In an embodiment, the invention discloses a method of controlling aggregation in an anti-CD20 antibody in an aqueous buffer free formulation, stabilized at a pH of about 5.0 to about 6.5, wherein the method comprises preparation the antiCD20 antibody in a composition comprising water, sugar and surfactant, and wherein the pH of the formulation is maintained without any buffering agent. The obtained anti-CD20 antibody formulation from the said method is stable at 40 ºC for two weeks and aggregate content is less than 2.5%.
In an embodiment, the invention discloses a method of controlling formation of charge variants in an anti-CD20 antibody in an aqueous buffer free formulation, stabilized at a pH of about 5.0 to about 6.5, wherein the method comprises preparation of the anti-CD20 antibody in a composition comprising water, sugar and surfactant, and wherein the pH of the formulation is maintained without any buffering agent. The obtained anti-CD20 antibody formulation from the said method is stable at 40 ºC for two weeks.
In an embodiment, the invention discloses a method of controlling formation of acidic variants in an anti-CD20 antibody in an aqueous buffer free formulation, stabilized at a pH of about 5.0 to about 6.5, wherein the method comprises preparation of the anti-CD20 antibody in a composition comprising water, sugar and surfactant, and wherein the pH of the formulation is maintained without any buffering agent. The obtained anti-CD20 antibody formulation from the said method is stable at 40 ºC for two weeks and change in acidic variants content of the antibody is less than 10% when stored at 40 ºC for two weeks.
In an embodiment, the invention discloses a method of controlling formation of lysine variants in an anti-CD20 antibody in an aqueous buffer free formulation, stabilized at a pH of about 5.0 to about 6.5, wherein the method comprises preparation of the anti-CD20 antibody in a composition comprising water, sugar and surfactant, and wherein the pH of the formulation is maintained without any buffering agent. The obtained anti-CD20 antibody formulation from the said method is stable at 40 ºC for two weeks and change in lysine variants content of the antibody is less than 1% when stored at 40 ºC for four weeks.
In an embodiment, the invention discloses a method of controlling methionine oxidation in an anti-CD20 antibody in an aqueous buffer free formulation, stabilized at a pH of about 5.0 to about 6.5, wherein the method comprises preparation of the anti-CD20 antibody in a composition comprising water, sugar and surfactant, and wherein the pH of the formulation is maintained without any buffering agent. The obtained anti-CD20 antibody formulation from the said method is stable at 40 ºC for two weeks and methionine oxidation content of the antibody is less than 1% when stored at 40 ºC for four weeks.
In an embodiment, the invention discloses a method of controlling succinimide formation in an anti-CD20 antibody in an aqueous buffer free formulation, stabilized at a pH of about 5.0 to about 6.5, wherein the method comprises preparation of the anti-CD20 antibody in a composition comprising water, mannitol, salt and surfactant, and wherein the pH of the formulation is maintained without any buffering agent. The obtained anti-CD20 antibody formulation from the said method is stable at 40 ºC for two weeks and succininide oxidation content of the antibody is less than 1% when stored at 40 ºC for four weeks.
In an embodiment, the invention discloses a method of controlling formation of sub-visible particle formation in an anti-CD20 antibody in an aqueous buffer free formulation, stabilized at a pH of about 5.0 to about 6.5, wherein the method comprises preparation of the anti-CD20 antibody in a composition comprising water, sugar and surfactant, and wherein the pH of the formulation is maintained without any buffering agent. The obtained anti-CD20 antibody formulation from the said method is stable at 40 ºC for two weeks and methionine oxidation content of the antibody is less than 1% when stored at 40 ºC for four weeks.
In the above said embodiments, the concentration of anti-CD20 antibody is 10 mg/ml or 20 mg/ml or 30 mg/ml or 40 mg/ml or 50 mg/ml, ‘or’ 60 mg/ml, ‘or’ 70 mg/ml, ‘or’ 80 mg/ml, ‘or’ 90 mg/ml, ‘or’ 100 mg/ml, ‘or’ 110 mg/ml, ‘or’ 120 mg/ml, ‘or’ 130 mg/ml, ‘or’ 140 mg/ml, ‘or’ 150 mg/ml, ‘or’ 160 mg/ml, ‘or’ 170 mg/ml, ‘or’ 180 mg/ml, ‘or’ 190 mg/ml, ‘or’ 200 mg/ml.
In any of the above mentioned embodiments, the claimed 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 said embodiment, the buffer free anti-CD20 antibody formulation exhibits stability at room temperature for at least 3 days, at least 7 days or at least 14 days or at least 28 days.
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 ± 0.5.
In any of the above mentioned embodiments, wherein the sugar is trehalose, sucrose, 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 amino acid is acidic amino acid or basic amino acid or hydrophobic amino acid.
In any of the above mentioned embodiments, wherein is surfactant is polyosrbate-20 or polysorbate-80 or poloxamer-188.
In any of the above mentioned embodiments, the disclosed anti-CD20 antibody formulations are stable and withstands thermal stress, shear stress, freeze thaw induced stress.
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 stable liquid anti-CD20 antibody are compatible with lyophilization process and the lyophilization process does not impact quality attributes of the antibody.
In any of the above mentioned embodiments, the stable anti-CD20 antibody formulation’s osmolality is less than 600 mOsm/kg, preferably less than 350 mOsm/kg.
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 autoinjector device contains anti-CD20 antibody, sugar, salt and surfactant.
In any of the above mentioned embodiments, wherein the anti-CD20 anibody is a chimeric, humanized or human anti-CD20 antibody or fusion proteins or conjugates thereof.
In any of the above mentioned embodiments, wherein the anti-CD20 antibody is either Type-I or Type-II anti-CD20 antibody.
In any of the above mentioned embodiments, the anti-CD20 antibody is rituximab, ofatumumab, obinutuzumab, ocrelizumab, 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.
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 “buffer or buffering agent” used interchangeably herein this document, refers to an agent which resists any change in pH of a solution, near a chosen value, up on addition of acid or base
The term "stable" formulation refers to the formulation wherein the antibody therein retains its physical stability and/or chemical stability and/or biological activity.
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 or minimal (or to the extent of acceptable standards) 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 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.
The term “fragments” herein refers to a part of large entity such as part of protein or antibody which consists of less than the entire amino acid sequence of the protein or the antibody which are formed due to terminal or internal deletion of a portion of the protein/antibody.
The term “charge variants” herein refers to an antibody variant which has net positive or negative charge and contains either lower or higher isoelectric point (pI) than the antibody of interest. Examples of charge variants include acidic variants and basic variants. The acidic variants of an antibody can be formed due to deamidation of glutamine and aspargine and sialylation which may impart net negative charge to the antibody and resulted in decrease in pI of the antibody. The basic variants of an antibody can be formed due to C-terminal lysine variation, oxidation, glycine amidation, succinamide formation, removal of sialic acids which may impart net positive charge to the antibody and resulted in increase in pI of the antibody.
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: Buffer free anti-CD20 antibody formulations
As part of experimental design, to prepare a buffer free `ocrelizumab formulation, approximately 8 mg/ml ocrelizumab in acetate buffer background was obtained from downstream chromatographic steps. The obtained ocrelizumab sample was buffer exchanged at least three times with a composition comprising water, post which, concentrated up to 80 mg/ml and then diluted to desired concentration of 30 mg/ml using a formulation buffer comprising 20-50 mg/ml trehalose and 0.2 mg/mL polysorbate-80 were added to the sample.
The sample (i.e; ocrelizumab formulation) was then subjected for accelerated stability conditions such as at 40 ºC for two weeks and various quality attributes were checked and measured. The quality attributes such as monomer content, low molecular weight species (LMW), high molecular weight species (HMW) using size exclusion chromatography (SEC) [results are given in Table 2], charge variants of the antibody using ion-exchange chromatography (IEX) [results are given in Table 3] Osmolality and pH of the buffer free formulation given in Table 4.
Table 2: SEC data of buffer free anti-CD20 antibody formulations when stored at 40 ºC for two weeks.

Formulation details %Monomer content at 40 ºC % HMW species at 40 ºC % LMW content at 40
T0 T1W T2W T0 T1W T2W T0 T1W T2W
S1- 30 mg/ml ocrelizumab, trehalose, polysorbate 80 98.1 97.6 97.5 1.57 1.99 1.14 0.29 0.42 1.33
T0-indicates a value at zero time point; ND-not detected
Table 3: IEX data of buffer free anti-CD20 antibody formulation

Sample ID % Main peak content at 40 ºC % Acidic species at 40 ºC % Basic species at 40 ºC
T0 T1W T2W T0 T1W T2W T0 T1W T2W
S1 60.9 56.1 47.1 28.2 38.1 39.4 11.4 9.0 13.5
T0-indicates a value at zero time point;
Table 4: Quality attributes of buffer free anti-CD20 formulation prepared as per Exampe-1

Sample ID Osmolality at 40 ºC pH at 40 ºC

T0 T1W T2W T0 T1W T2W
S1 186 189 237 5.79 6.73 5.05
T0-indicates a value at zero time point;
Further, the sample was maintained at 40 ? for four weeks and the sample is subjected for mass spectrometry to analyze various variants such as lysine variants, Results are given in Table 5. In addition, other variants such as oxidized variants and succinimide variants were also measured and Data is not given here.

Sample ID Lysine variants at 40 ºC
T0 T4W
S1 0.40 0.43
Table 5: Mass spectrometry data of buffer free anti-CD20 antibody formulation of Example-1.
,CLAIMS:We claim:

1. A buffer free formulation of an anti-CD20 antibody, stabilized at a pH of about 5.0- about 6.5, comprising anti-CD20 antibody, water and surfactant, wherein the pH of the anti-CD20 antibody formulation is maintained without any buffering agent.

2. The buffer free formulation as claimed in claim 1, optionally contains one or more pharmaceutically acceptable excipients selected from sugar or amino acid or polyol or salt or surfactant.

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

4. The buffer free formulation as claimed in claim 2, wherein the sugar is trehalose, sucrose, mannitol or sorbitol.

5. The buffer free formulation as claimed in claim 2, wherein the salt is sodium chloride.

6. The buffer free formulation as claimed in claim 2, wherein the amino acid is acidic amino acid or basic amino acid or hydrophobic amino acid.

7. The buffer free formulation as claimed in claim 2, wherein the surfactant is polysorbate-20 or polysorbate-80 or poloxamer-188.

8. The buffer free formulation as claimed in claim 1, wherein the anti-CD20 antibody is rituximab, ofatumumab, obinutuzumab, ocrelizumab, or ubilituximab or conjugates thereof.
9. The buffer free formulation as claimed in claim 1, is a stable liquid/aqueous formulation and can be lyophilized as lyophilized powders.