DESC:A PHARMACEUTICAL FORMULATION OF A THERAPEUTIC ANTIBODY AND PREPARATIONS THEREOF
INTRODUCTION
The present invention relates to pharmaceutical formulations of anti-CD38 antibodies, in particular the invention discloses suitable compositions for stabilizing the antibody from lower to higher concentration.
BACKGROUND
Over the past two decades, recombinant DNA technology has led to the commercialization of many proteins, particularly antibody therapeutics. The effectiveness of these therapeutic antibodies is majorly dependent on the stability, route of administration and their dosage forms and concentrations. This in turn, necessitates therapeutic antibodies to be formulated appropriately to retain the stability and activity of a therapeutic antibody.
Formulations for each route of administration and dosage forms may be unique and, therefore, have specific requirements. Further, route of administration of a therapeutic antibody depends on various factors such as specific treatment schedule, mode of delivery, periodical follow up with physician, dosage and concentration to be administered, etc.,. Though bioavailability of a therapeutic antibody is to its maximum when administered via intravenous route of administration but, the amount of antibody that can be administered via the intravenous route is limited due to the physico-chemical properties and stability of the antibody in solution. Similarly, for high concentration antibody formulation subcutaneous route of administration is preferable but, volume of injection that can be administered may be a limitation unless a specific class of excipients are incorporated in the formulation. Hence, to inject large volume of injection through subcutaneous route of administration, most of the antibody formulations are co-formulated with hyaluronidase enzyme, which degrades hyaluronic acid present in extracellular matrix by hydrolytic degradation. In addition, most of high concentration antibody formulations are prone for oxidation upon storage.
Therapeutic antibodies are generally approved as intravenous and/or subcutaneous administrations that preferably aids administration of specific concentration and dose of antibody drugs. Existing antibodies for therapeutic use is generally approved as both low and high concentrations enabling intravenous and subcutaneous respectively. Majority of the approved therapeutic antibodies have been formulated in different buffer compositions/excipients for varying concentrations of antibody considering the permeability, solubility and stability of the antibody in such concentrations. Except for a few, the state of the art in the domain of antibody formulation does not substantiate use of a single, same buffer or excipients composition to stabilize a wide concentration range of an antibody. Although the latter render numerous advantages in terms of recovery and stability of an antibody in addition to saving cost, time etc., the challenge in stabilizing an antibody at its wide concentration range is multi-fold and still exists. The objective of the invention is to address the above, in addition to addressing rest other challenges, such as formation of aggregates, fragments and charge variants.
SUMMARY OF THE INVENTION
The present invention discloses a pharmaceutical formulation of a monoclonal antibody (Mab), comprising, Mab1 or Mab2 and succinate buffer having pH of 5.0 to 6.0, wherein the buffer stabilizes the antibody at concentrations ranging from 20 mg/ml to 200 mg/ml. The formulation may additionally comprise one or more pharmaceutically acceptable excipients.
The disclosed formulation composition (buffer and/or excipients composition) of the invention stabilizes Mab1 or Mab2 antibody from lower to higher concentrations, and maintains stability under accelerated conditions. The stable formulation composition of Mab1 or Mab2 antibody, obtained in the specific buffer, can be used to prepare low and high concentrations of antibody without varying the formulation composition. In other words, a single antibody drug substance composition can be used to prepare different drug product concentrations, without a change in the buffer or excipient components (i.e., formulation composition). The disclosed Mab1 or Mab2 antibody formulations of the invention are anti-CD38 antibodies, which binds to CD38 antigen and Mab1 antibody is daratumumab and Mab2 antibody is isatuximab.
Additionally, the invention discloses a method of controlling fragmentation in anti-CD38 antibody in a high concentration formulation comprising, preparing the antibody composition in succinate buffer at a pH of 5.0 to 6.0, wherein the method controls the rate of fragmentation of the antibody. The method may additionally comprise a step of addition of excipients along with the buffer.
In addition, the invention discloses a method of controlling charge variants formation, in particular, basic variants formation in an anti-CD38 antibody composition, by formulating the antibody in succinate buffer, wherein the method controls the formation of basic variants.
The invention further discloses a method of controlling pH drift in a high concentration anti-CD38 antibody composition, the method comprises preparing the antibody composition in succinate buffer at a pH 5.0 – 6.0, wherein the prepared composition controls a drift in pH, during storage.
In another aspect, the invention discloses a method of obtaining a high concentration anti-CD 38 antibody formulation comprising a step of preparation of the antibody in succinate buffer composition, having pH of 5.0 to 6.0, and comprising trehalose and surfactant. The antibody concentration in the said composition is 100 mg/ml to 200 mg/ml. The invention also discloses a method to control fragmentation in the antibody composition.
The disclosed formulations of the invention are not co-formulated with any enzyme. In particular, the disclosed high concentration formulations do not require a hyaluronidase enzyme. And also, the disclosed formulations of the invention may not require an anti-oxidant or methionine to stabilize anti-CD38 antibody at higher concentrations.
DETAILED DESCRIPTION
In antibody therapeutics, different concentrations of antibody are desired for different dosage and routes of administrations. Invariably, in the state of the art, formulation composition has been varied for varying concentrations of the antibody to accommodate the solubility and stability of the antibody at its low and higher concentrations. Use of a single buffer and excipients composition suiting/stabilizing both low and high concentrations (viz., a wide range of concentration) of antibody are minimal in the state of the art due to the multiple challenges associated with it, including fragmentation/degradation, oxidation, aggregation, etc., that is in particular, common with high concentration formulations.. The present invention addresses the above.
The present invention discloses pharmaceutical formulations of an antibody in succinate buffer, wherein the antibody is Mab1 or Mab2. The disclosed Mab1 or Mab2 antibody formulations of the invention are anti-CD38 antibodies, which binds to CD38 antigen.
The present invention discloses pharmaceutical formulations of an antibody in histidine buffer, wherein the antibody is anti-CD38 antibody.
In an embodiment, the invention discloses a pharmaceutical formulation of antibody comprising, anti-CD38 antibody, buffer, one or more stabilizers/pharmaceutically acceptable excipients and surfactant.
In the above said embodiment, the buffer is an organic buffer or an inorganic buffer, and/or its salts or combinations thereof. Organic buffers include succinate buffer, histidine buffer, lactate buffer or citrate buffer and inorganic buffer includes phosphate buffer.
In any of the above mentioned embodiments, the stabilizers or pharmaceutically acceptable excipients are sugar or amino acid or salt, or combinations thereof.
In an embodiment, the invention discloses a pharmaceutical formulation of antibody comprising, anti-CD38 antibody, succinate buffer having pH of 5.0 to 6.0, wherein the antibody is at a concentration ranging from 20 mg/ml to 200 mg/ml.
In the above mentioned embodiment, the formulation further comprises one or more following excipients: sugar, sodium chloride or surfactant.
In the above mentioned embodiments, the formulation does not require anti-oxidants or methionine.
In any of the above mentioned embodiments, the anti-CD38 antibody, at high concentrations, is stabilized without hyaluronidase.
In the above mentioned embodiment, the high concentration antibody ranges from about 50 mg/ml to 200 mg/ml.
In an embodiment, the invention discloses a method of stabilizing low to high concentrations of anti-CD38 antibody, in it’s aqueous formulation composition, wherein the method comprises formulating an anti-CD38 antibody in succinate buffer composition, having a pH of 5.0 to 6.0, and wherein the antibody is at a concentration ranging from 20 mg/ml to 200 mg/ml. The stable composition obtained enables administration of the antibody as intravenous or subcutaneous without a change in the buffer or excipient components.
In another embodiment, the invention discloses a method of preparing a stable anti-CD38 antibody in it’s liquid/aqueous composition, wherein the method comprises addition of succinate buffer to the antibody composition during pre-formulation and/or during the formulation steps of the antibody production.
In the above mentioned embodiment, the method additionally comprises a step of addition of one or more pharmaceutically acceptable excipients sugar, salt, amino acid or surfactant, following addition of the buffer to the antibody composition. Alternatively, these excipients can be added along with the buffer in pre-formulation steps.
In an embodiment, the invention discloses a method of controlling pH drift in a high concentration anti-CD38 antibody composition, the method comprises preparing the antibody composition in about 5 mM to 50 mM succinate buffer, at a pH ranging from pH 5.0 – pH 6.0, wherein the method controls a drift in pH in the said antibody composition. The anti-CD38 antibody concentration in the said composition is 50 mg/ml to 200 mg/ml.
In an embodiment, the invention discloses a method of controlling pH drift in a high concentration anti-CD38 antibody composition, the method comprises preparing the antibody composition in about 25 mM succinate buffer, pH 5.0 – 6.0, wherein the method controls a drift in pH in the said composition. The anti-CD38 antibody concentration in the said composition is 50 mg/ml to 200 mg/ml.
In yet another embodiment, the invention discloses a method of controlling fragmentation in an anti-CD38 antibody formulation, the method comprises preparation of an anti-CD38 antibody composition in succinate buffer, at a pH ranging from pH 5.0 to pH 6.0.
In another embodiment, the invention discloses a method of controlling fragmentation of anti-CD38 antibody, in it’s composition, the method comprises preparing the antibody in succinate buffer having pH of 5.0 to 6.0, wherein the method controls fragmentation of the antibody in the composition. The anti-CD38 antibody concentration in the said composition is 50 mg/ml to 200 mg/ml.
In yet another embodiment, the invention discloses a method of controlling fragmentation of anti-CD38 antibody, in it’s composition, the method comprises preparing the antibody composition in about 5 mM to 50 mM succinate buffer, having a pH of about 5.0 to about 6.0, wherein the method controls fragmentation of the antibody. The anti-CD38 antibody concentration in the said composition is 50 mg/ml to 200 mg/ml.
In an embodiment, the invention discloses a method of maintaining monomer content of anti-CD38 antibody in it’s composition, the method comprises addition of succinate buffer to the antibody composition during pre-formulation and/or at the formulation steps of the antibody production, followed by addition of sugar and surfactant.
In the above mentioned embodiment, 95% or more of anti-CD38 antibody in the antibody composition is maintained in monomeric form, when stored at 40 ? for four weeks or at 25 ? for four weeks.
In an embodiment, the invention discloses a method of controlling aggregation of anti-CD38 antibody in it’s composition, the method comprises addition of succinate buffer to the antibody composition during pre-formulation and/or at the formulation steps of the antibody production, followed by addition of sugar and surfactant. The method controls aggregation of the anti-CD38 antibody upon storage under accelerated or stress conditions. The accelerated condition is storage of antibody composition at 25 ? and stress condition is storage of sample at 40 ?.
In the above mentioned embodiment, the method additionally comprises addition of sodium chloride along with the addition of other excipients.
In an embodiment, the invention discloses a method of controlling visible or sub-visible particle formation in anti-CD38 antibody in it’s composition, wherein the method comprises formulating an anti-CD38 antibody in succinate buffer composition, having pH of 5.0 to 6.0, wherein the method controls formation of visible or sub-visible particles of anti-CD38 antibody.
In an embodiment, the invention discloses a method of controlling formation of charge variants of anti-CD38 antibody in it’s composition, the method comprises formulating an anti-CD38 antibody in succinate buffer composition having pH of 5.0 to 6.0, wherein the method controls formation of charge variants of anti-CD38 antibody. The buffer herein, is added during pre-formulation stage and/or at the formulation stage of the antibody production.
In an embodiment, the invention discloses a method of controlling deamidation of anti-CD38 antibody in it’s composition, the method comprises formulating an anti-CD38 antibody in succinate buffer composition having pH of 5.0 to 6.0, wherein the method controls deamidation thereby formation of acidic variants of anti-CD38 antibody. The buffer herein, is added during pre-formulation stage and/or at the formulation stage of the antibody production.
In yet another embodiment, the invention discloses a method of controlling basic variants formation in an anti-CD38 antibody in it’s composition, the method comprises preparation of anti-CD38 antibody in succinate buffer, wherein the method controls formation of basic variants of anti-CD38 antibody in the formulation. The buffer herein, is added during pre-formulation and/or at the formulation steps of the antibody production.
In an embodiment, the invention discloses a pharmaceutical formulation of anti-CD38 antibody comprising, an anti-CD38 antibody, succinate buffer, trehalose and surfactant, wherein the antibody in the pharmaceutical formulation is stable at a concentration range from about 20 mg/ml to 200 mg/ml. The formulation may additionally comprise a sodium chloride.
In the above mentioned embodiment, the pharmaceutical formulation may not require an anti-oxidant, such as methionine, to stabilize anti-CD38 antibody.
In any of the above mentioned embodiments, the disclosed formulations of anti-CD38 antibody are not co-formulated with any enzyme or hyaluronidase.
In an embodiment, the invention discloses a high concentration anti-CD38 antibody formulation, consisting essentially of, 100 mg/ml to 200 mg/ml anti-CD38 antibody, succinate buffer, sugar and surfactant.
In the above mentioned embodiment, the formulation further comprises sodium chloride.
In the above mentioned embodiment of aforesaid embodiment, wherein the sugar is sucrose or trehalose or mannitol.
In another embodiment, the invention discloses a pharmaceutical formulation of anti-CD38 antibody comprising, an anti-CD38 antibody, succinate buffer, trehalose, sodium chloride and surfactant, wherein the antibody in the pharmaceutical formulation is stable at a concentration range from about 20 mg/ml to 200 mg/ml.
In an embodiment, the invention discloses a pharmaceutical formulation of high concentration anti-CD38 antibody comprising, 120 mg/ml of an anti-CD38 antibody, succinate buffer, trehalose and surfactant.
In the above mentioned embodiment, the formulation further comprises sodium chloride.
In yet another embodiment, the invention discloses a pharmaceutical formulation of anti-CD38 antibody comprising, an anti-CD38 antibody, succinate buffer, sucrose, sodium chloride and surfactant, wherein the antibody in the pharmaceutical formulation is stable at a concentration range from about 20 mg/ml to 200 mg/ml.
In an embodiment, the invention discloses a pharmaceutical formulation of anti-CD38 antibody comprising, an anti-CD38 antibody, succinate buffer, mannitol, sodium chloride and surfactant, wherein the antibody in the pharmaceutical formulation is stable at a concentration range from about 20 mg/ml to 200 mg/ml.

In an embodiment, the invention discloses a high concentration anti-CD38 antibody formulation comprising, 100 mg/ml to 200 mg/ml anti-CD38 antibody, succinate buffer, sugar and surfactant, optionally containing sodium chloride, and wherein the formulation does not contain methionine.
In an embodiment, the invention discloses a high concentration anti-CD38 antibody formulation comprising, 100 mg/ml to 200 mg/ml anti-CD38 antibody, succinate buffer, sugar and surfactant, optionally containing sodium chloride, and wherein the formulation does not contain hyaluronidase enzyme.
In an embodiment, the invention discloses a high concentration anti-CD38 antibody formulation comprising, 100 mg/ml to 200 mg/ml anti-CD38 antibody, succinate buffer, sugar and surfactant, optionally containing sodium chloride, and wherein the formulation does not contain methionine and/or hyaluronidase enzyme.
In an embodiment, the invention discloses pharmaceutical formulation of anti-CD38 antibody comprising an anti-CD38 antibody, histidine buffer having pH of 5.0 to 6.0, sugar and surfactant.
In the above mentioned embodiment, wherein the sugar is trehalose or sucrose.
In the above mentioned embodiment of aforesaid embodiment, the anti-CD38 antibody formulation further comprises sodium chloride.
In another embodiment, the invention discloses pharmaceutical formulation of anti-CD38 antibody comprising, an anti-CD38 antibody, histidine buffer, trehalose and sodium chloride. The disclosed formulation of anti-CD38 antibody does not contain hyaluronidase enzyme and further may not require an anti-oxidant/methionine to stabilize the antibody at higher concentrations.
In the above mentioned embodiment, the formulation further comprises surfactant.
In another embodiment, the invention discloses a method of preparing an anti-CD38 antibody composition, the method comprises addition of histidine buffer to the antibody composition during pre-formulation and/or at the formulation steps of the antibody production.
In the above mentioned embodiment, the method further comprises a step of addition of one or more pharmaceutically acceptable excipients sugar, salt, amino acid or surfactant, following addition of histidine buffer to the antibody composition.
In an embodiment, the invention discloses a method of stabilizing an anti-CD38 antibody in it’s liquid/aqueous composition, the method comprises formulating the Mab1 antibody in histidine buffer composition having pH of 5.0 to 6.0, wherein the method stabilizes the antibody at a concentration ranging from 20 mg/ml to 200 mg/ml.
In the above mentioned embodiment, the formulation further comprises one or more following excipients: sugar, sodium chloride or surfactant.
In an embodiment, the invention discloses a method of stabilizing low to high concentrations of anti-CD38 antibody, in it’s aqueous formulation composition, the method comprises formulating the anti-CD38 antibody in histidine buffer composition having pH of 5.0 to 6.0, wherein the antibody is at a concentration ranging from 20 mg/ml to 200 mg/ml. The stable composition obtained enables administration of the antibody as intravenous or subcutaneous without a change in the buffer or excipient components.
In an embodiment, the invention discloses a method of maintaining monomer content of anti-CD38 antibody, in it’s composition, the method comprises addition of histidine buffer to the antibody composition during pre-formulation and/or at the formulation steps of the antibody production, followed by addition of sugar and surfactant.
In the above mentioned embodiment, 95% or more of anti-CD38 antibody in the antibody composition is maintained in monomeric form when stored at 40 ? for four weeks or at 25 ? for four weeks.
In an embodiment, the invention discloses a method of controlling aggregation of anti-CD38 antibody, in it’s composition, the method comprises addition of histidine buffer to the antibody composition during pre-formulation and/or at the formulation steps of the antibody production, followed by addition of sugar and sodium chloride.
In an embodiment, the invention discloses a method of controlling formation of charge variants of anti-CD38 antibody in it’s composition, the method comprises formulation of anti-CD38 antibody in histidine buffer composition having pH of 5.0 to 6.0, wherein the method controls formation of charge variants of anti-CD38 antibody in it’s composition. The buffer herein, is added during pre-formulation stage and/or at the formulation stage of the antibody production.
In an embodiment, the invention discloses a pharmaceutical formulation of anti-CD38 antibody comprising, anti-CD38 antibody, histidine buffer, trehalose and surfactant, wherein the antibody in the pharmaceutical formulation is stable at a concentration range from about 20 mg/ml to 200 mg/ml.
In the above mentioned embodiment, the formulation optionally comprises sodium chloride.
In an embodiment, the invention discloses a pharmaceutical formulation of anti-CD38 antibody comprising, anti-CD38 antibody, histidine buffer, mannitol and surfactant, wherein the antibody in the pharmaceutical formulation is stable at a concentration range from about 20 mg/ml to 200 mg/ml.
In an embodiment, the invention discloses a pharmaceutical formulation of anti-CD38 antibody comprising, an anti-CD38 antibody, histidine buffer, sucrose, sodium chloride and surfactant, wherein the antibody in the pharmaceutical formulation is stable at a concentration range from about 20 mg/ml to 200 mg/ml.
In an embodiment, the invention discloses an aqueous or liquid formulations of anti-CD38 antibody as disclosed in Table 1.
Mab1 antibody concentration (mg/mL) Buffer
(5 -50 mM)
pH 5.0 to 6.0 Sugar
(50 -200 mM)
Salt
(25 mM- 100 mM) Surfactant

(0.01-0.05%)
20 mg/ml succinate trehalose Sodium chloride polysorbate
20 mg/ml succinate Sucrose Sodium chloride polysorbate
20 mg/ml succinate mannitol Sodium chloride polysorbate
20 mg/ml succinate trehalose -- polysorbate
20 mg/ml histidine trehalose -- polysorbate
20 mg/ml histidine mannitol Sodium chloride polysorbate
20 mg/ml histidine sucrose Sodium chloride polysorbate
20 mg/ml histidine trehalose Sodium chloride
20 mg/ml histidine trehalose Sodium chloride polysorbate
120 mg/ml succinate trehalose -- polysorbate
120 mg/ml succinate trehalose Sodium chloride polysorbate
Table 1: aqueous antibody formulations of anti-CD38 antibody.
In the above mentioned embodiment, the disclosed formulations of Table 1, wherein the surfactant is polysorbate. Alternatively, the surfactant can be poloxamer.
In all of the above mentioned embodiments of the invention, the concentration of the antibody in the antibody composition or formulation is about 20 mg /ml to about 200 mg/ml. The concentration of the antibody in the formulation is 20 mg/ml or 30 mg/ml or 40 mg/ml or 50 mg/ml or 60 mg/ml or 80 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 195 mg/ml or 200 mg/ml.
In any of the above mentioned embodiments, the claimed compositions of anti-CD38 antibody is suitable to formulate daratumumab or isatuximab antibody.
In any of the above mentioned embodiments, the Mab1 and Mab2 antibodies are anti-CD38 antibodies, which binds to CD38 antigen.
In any of the above mentioned embodiments, the Mab1 antibody is daratumumab and the Mab2 antibody is isatuximab.
In any of the above mentioned embodiments, the disclosed formulations may be free of anti-oxidant. Preferably, the anti-oxidant is methionine.
In any of the above mentioned embodiments, viscosity of anti-CD38 antibody formulation is less than 20 cP.
In any of the above mentioned embodiments, viscosity of high concentration anti-CD38 antibody is less than 10 cP. More specifically, viscosity of anti-CD38 antibody formulation is less than 6 cP, wherein the concentration of antibody is 120 mg/ml.
In any of the above mentioned embodiments, pH of anti-CD38 antibody formulation is from 5.0 to 6.0.
In any of the above mentioned embodiments, the formulations are free of visible particles.
In any of the above mentioned embodiments, the formulation of anti-CD38 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-CD38 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-CD38 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 comprises anti-CD38 antibody, succinate buffer or it’s derivatives or combination thereof, sugar, and surfactant.
In any of the above mentioned embodiment, the claimed formulations of anti-CD38 antibody are suitable for treatment of multiple myeloma.
DEFINITIONS
The term “about” as used herein, would mean and include a variation of up to 20 % from the particular value.
The term “antibody” includes whole antibodies or any antigen binding fragment (i.e., “antigen-binding portion”) or single chains or fusions thereof.
The term “Mab1 antibody” herein refers to an antibody referenced and mentioned in IMGT as “Mab/DB ID 301” with IMGT/2D structure-DB card number ID 9128. Link to the referenced Mab1 sequence is https://www.imgt.org/3Dstructure-DB/cgi/details.cgi?pdbcode=9128. Mab1 antibody is an anti-CD38 antibody, corresponds to the sequence of daratumumab.
The tem “Mab2 antibody” herein refers to an antibody referenced and mentioned in IMGT as “Mab/DB ID 539” with IMGT/2D structure-DB card number ID 10068. Link to the referenced Mab2 sequence is https://www.imgt.org/3Dstructure-DB/cgi/details.cgi?pdbcode=10068. Mab2 antibody is an anti-CD38 antibody, corresponds to the sequence of isatuximab.
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 provide 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 or minimal 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 it 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 may include dimers, multimers, etc., and these elute first, followed by monomer, and the clipped antibody variants or degradants which may be eluted last. The clipped antibody variants are known as fragments or low molecular weight species of the antibody, which can be a result of deamidation, oxidation, isomerization and/or hydrolysis. 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. In order to maintain the appropriate activity of an antibody, in particular of a therapeutic antibody, it is desirable to reduce the formation of aggregate or fragmentation of products and hence control the monomer content to a target value.
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. 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. 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 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/stabilizers refer to the additives or carriers, which 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, sugars, amino acids, salts, surfactants, polymers, or it’s derivatives and/or it’s combination thereof.
The term “sugar/s” as used refers to one or more monosaccharides, disaccharides, or polysaccharides or oligosaccharides. Examples of sugars include, but are not limited to, sucrose, trehalose, glucose, dextrose, raffinose and others.
The term “polyol” refers to multiple hydroxyl group containing organic compounds. Examples of polyols include, but are not limited to, mannitol, sorbitol, and others.
The term “surfactant” refers to a substance that absorbs to surfaces or interfaces to reduce surface or interfacial tension. Surfactants are 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 “salt” refers to a substance produced by the reaction of an acid with a base. A salt consists of the positive ion (cation) of a base and the negative ion (anion) of an acid. 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 “antioxidant” mentioned herein refers to an agent that inhibits the oxidation of other molecules and is not part of buffer component. Example of antioxidant is methionine.
The term “visible particles” mentioned herein refers to insoluble particulates in a liquid composition, of size measuring greater than or equal to 100 µm (=100 µm). Formation of these insoluble particulates formation may be caused by degradation of excipients present in the formulation and/or due to protein aggregation or degradation or from any leachates from the container holding the composition.
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
Mab1 antibody, suitable for storage in the present pharmaceutical composition is produced by standard methods known in the art. For example, Mab1 antibody, which is an anti-CD38 antibody (daratumumab) 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 Mab1 antibody 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 Mab1 antibody may be purified using standard chromatography techniques such as affinity chromatography, ion-exchange chromatography and combinations thereof. The purified Mab1 antibody solution can additionally be subjected to one or more filtration steps, and the solution obtained is subjected to further formulation studies.
Example 1: Formulation of antibody in specific buffers and stabilizers
Purified Mab1 antibody approximately, 20 mg/ml in acetate buffer background was obtained from downstream chromatographic steps. Post which, Mab1 antibody in acetate buffer was buffer exchanged with either histidine or succinate buffer. Excipients, sucrose or trehalose or mannitol, sodium chloride and polysorbate-20 were added to the buffer or buffer exchanged formulation based on the requirement of final composition of a formulation. The final composition of all Mab1 antibody formulations are given in Table 2.
All the formulations were subjected to accelerated stability studies at 40 ºC for four weeks, and at room temperature (i.e., at 25 ºC) for four weeks and at 2-8 ºC for four weeks. The samples were then analyzed for high molecular weight (HMW) content, monomer content and low molecular weight (LMW) content using size exclusion chromatography (SEC) [results are given in Table 3 (a) to 3 (c)] and charge variants using ion-exchange chromatography [results are given in Table 4(a) to 4 (c)]. Further, these formulations were also checked for change in pH using pH meter, data is given in Table 5.
Table 2: Compositions of formulations prepared as per example-1
Sample Name Composition
F1 20 mg/ml Mab1 antibody,25 mM histidine buffer, 140 mM mannitol, 60 mM NaCl, 0.04 % polysorbate-20, pH 5.5
F2 20 mg/ml Mab1 antibody, 25 mM histidine buffer, 140 mM sucrose, 60 mM NaCl, 0.04 % polysorbate-20, pH 5.5
F3 20 mg/ml Mab1 antibody, 25 mM histidine buffer, 140 mM trehalose, 60 mM NaCl, 0.04 % polysorbate-20, pH 5.5
F4 20 mg/ml Mab1 antibody, 25 mM succinate buffer, 140 mM mannitol, 60 mM NaCl, 0.04 % polysorbate-20, pH 5.5
F5 20 mg/ml Mab1 antibody, 25 mM succinate buffer, 140 mM sucrose, 60 mM NaCl, 0.04 % polysorbate-20, pH 5.5
F6 20 mg/ml Mab1 antibody, 25 mM succinate buffer, 140 mM trehalose, 60 mM NaCl, 0.04 % polysorbate-20, pH 5.5
F7 20 mg/ml Mab1 antibody, 25 mM histidine buffer, 60 mM NaCl, 0.04 % polysorbate-20, pH 5.5
F8 20 mg/ml Mab1 antibody, 25 mM histidine buffer, 140 mM trehalose, 0.04 % polysorbate-20, pH 5.5
F9 20 mg/ml Mab1 antibody, 25 mM histidine buffer, 140 mM trehalose, 60 mM NaCl, pH 5.5

Table 3(a): High molecular weight content (i.e., aggregate content) of formulations as measured by SEC.
Sample name % of Aggregate content
at 40 ? at 25 ? at 5 ?
T0 T4W T0 T4W T0 T4W
F1 2.82 5.21 2.82 2.14 2.82 2.4
F2 2.92 2.36 2.92 2 2.92 2.33
F3 2.86 2.33 2.86 2.01 2.86 2.31
F4 3.05 2.79 3.05 2.18 3.05 2.51
F5 3.03 2.97 3.03 2.35 3.03 2.56
F6 3.02 2.69 3.02 2.33 3.02 2.5
F7 2.99 5.49 2.99 2.49 2.99 2.4
F8 2.78 3.44 2.78 2.27 2.78 2.37
F9 2.81 2.12 2.81 2.04 2.81 2.34
W-indicates weeks, T0-represents data at zero time point

Table 3(b): Percentage monomer content of formulations as measured by SEC.
Sample name % of monomer content
at 40 ? at 25 ? at 5 ?
T0 T4W T0 T4W T0 T4W
F1 96.81 93.46 96.81 97.39 96.81 97.32
F2 96.69 96.31 96.69 97.51 96.69 97.39
F3 96.78 96.37 96.78 97.54 96.78 97.41
F4 96.61 95.78 96.61 97.34 96.61 97.16
F5 96.64 95.55 96.64 97.15 96.64 97.08
F6 96.6 95.91 96.6 97.12 96.6 97.14
F7 96.6 93.02 96.6 97.03 96.6 97.25
F8 96.86 95.08 96.86 97.22 96.86 97.28
F9 96.83 96.41 96.83 97.46 96.83 97.31
W-indicates weeks, T0-represents data at zero time point

Table 3(c): Low molecular weight content of formulations as measured by SEC.
Sample name % of Low molecular weight content
at 40 ? at 25 ? at 5 ?
T0 T4W T0 T4W T0 T4W
F1 0.36 1.32 0.36 0.47 0.36 0.29
F2 0.39 1.32 0.39 0.49 0.39 0.28
F3 0.36 1.31 0.36 0.46 0.36 0.28
F4 0.33 1.44 0.33 0.47 0.33 0.33
F5 0.32 1.49 0.32 0.5 0.32 0.36
F6 0.38 1.4 0.38 0.54 0.38 0.36
F7 0.41 1.5 0.41 0.48 0.41 0.35
F8 0.37 1.47 0.37 0.51 0.37 0.34
F9 0.37 1.47 0.37 0.5 0.37 0.35
W-indicates weeks, T0-represents data at zero time point

Table 4(a): Acidic variants content of formulations as measured by IEX.
Sample name % of Acidic variants content
at 40 ? at 25 ? at 5 ?
T0 T4W T0 T4W T0 T4W
F1 14.01 30.72 14.01 17.91 14.01 14.5
F2 14.04 29.23 14.04 17.68 14.04 14.42
F3 14.14 31.2 14.14 14.05 14.14 14.16
F4 14.23 33.18 14.23 16.72 14.23 15.76
F5 13.86 33.43 13.86 19.34 13.86 17.09
F6 13.43 33.63 13.43 19.01 13.43 16.56
F7 13.53 30.97 13.53 17.14 13.53 14.34
F8 14.33 32.19 14.33 18.26 14.33 14.95
F9 13.84 27.76 13.84 17.9 13.84 15.55
W-indicates weeks, T0-represents data at zero time point

Table 4(b): Main peak content of formulations as measured by IEX.
Sample name % of main peak content
at 40 ? at 25 ? at 5 ?
T0 T4W T0 T4W T0 T4W
F1 76.68 61.42 76.68 72.47 76.68 76.81
F2 76.7 60.87 76.7 73.08 76.7 76.57
F3 77.39 58.08 77.39 76.7 77.39 75.79
F4 76.82 57.58 76.82 74.13 76.82 74.31
F5 77.42 56.9 77.42 71.07 77.42 73.17
F6 76.73 57.63 76.73 71.57 76.73 73.12
F7 76.48 58.83 76.48 73.14 76.48 75.6
F8 76.37 57.38 76.37 73.7 76.37 75.35
F9 77.19 61.47 77.19 72.7 77.19 74.38
W-indicates weeks, T0-represents data at zero time point

Table 4(c): Basic variants content of formulations as measured by IEX.
Sample name % of Basic variants content
at 40 ? at 25 ? at 5 ?
T0 T4W T0 T4W T0 T4W
F1 9.31 7.87 9.31 9.63 9.31 8.69
F2 9.25 9.91 9.25 9.24 9.25 9.01
F3 8.47 10.72 8.47 9.25 8.47 10.05
F4 8.95 9.24 8.95 9.15 8.95 9.94
F5 8.72 9.66 8.72 9.59 8.72 9.74
F6 9.85 8.74 9.85 9.42 9.85 10.33
F7 9.98 10.2 9.98 9.73 9.98 10.06
F8 9.31 10.43 9.31 8.04 9.31 9.71
F9 8.96 10.77 8.96 9.4 8.96 10.07
W-indicates weeks, T0-represents data at zero time point
Table 5: pH measurements of formulations as measured by pH meter.
Sample name pH
at 40 ? at 25 ? at 5 ?
T0 T4W T0 T4W T0 T4W
F1 5.61 5.83 5.61 5.69 5.61 5.46
F2 5.61 5.43 5.61 5.58 5.61 5.44
F3 5.61 5.66 5.61 5.80 5.61 5.93
F4 5.46 5.65 5.46 5.54 5.46 4.96
F5 5.47 5.33 5.47 5.31 5.47 5.15
F6 5.47 5.63 5.47 5.60 5.47 5.60
F7 5.62 5.72 5.62 5.73 5.62 5.45
F8 5.57 5.73 5.57 5.33 5.57 5.80
F9 5.61 5.73 5.61 5.41 5.61 5.65
Example 2: High concentration Mab1 antibody formulations
Purified Mab1 antibody approximately 20 mg/ml in acetate buffer background was obtained from downstream chromatographic steps. And, Mab1 antibody in acetate buffer was buffer exchanged with succinate buffer or alternatively with a histidine buffer in a separate experiment. The buffer exchanged antibody (in it’s respective buffer composition) was concentrated upto 190 mg/ml to obtain high concentration Mab1 antibody formulation. The concentration of the obtained high concentration Mab1 antibody was further adjusted to 120 mg/ml and one or more excipients such as sorbitol, trehalose, methionine, polysorbate-20 were added.
Details of the final formulation compositions of high concentration Mab1 antibody prepared as per Example-2 is given in Table 6. Viscosity of a few formulations (i.e., HF1 and HF4) are measured using m-VROC® viscometer at room temperature and viscosity of these two formulations found to be in the range of 5.3 mPa.s to 5.8 mPa.s.
All high concentration Mab1 antibody formulations of Table 6 were then subjected for accelerated stability studies at 40 ºC for one week. Post which, the samples were analyzed for LMW species and monomer content using SEC [results are given in Table 7] and also checked for main peak content, and, basic variants using ion-exchange chromatography [Table 8]. Further, these formulations were also checked for change in pH using pH meter, data is given in Table 9(a).
Table 6: High concentration Mab1 antibody formulations prepared as per example-2.
Sample name Composition
HF1 120 mg/ml Mab1 antibody 25 mM succinate buffer, 140 mM trehalose, methionine, 0.04 % polysorbate-20, pH 5.5
HF2 120 mg/ml Mab1 antibody, 25 mM succinate buffer, 140 mM trehalose, 0.04 % polysorbate-20, pH 5.5
HF3 120 mg/ml Mab1 antibody, 8 mM histidine buffer, 140 mM trehalose, methionine, 0.04 % polysorbate-20, pH 5.5
HF4 120 mg/ml Mab1 antibody, 8 mM histidine buffer, 140 mM sorbitol, methionine, 0.04 % polysorbate-20, pH 5.5

Table 7: HMW, monomer, and LMW content of formulations prepared as per example-2, measured by SEC.

Sample name SEC Data at 40 ?
% HMW % Monomer % LMW
T0 T1W T0 T1W T0 T1W
HF1 3.24 3.70 96.73 96.10 0.04 0.30
HF2 3.37 3.50 96.57 96.30 0.06 0.20
HF3 2.90 2.30 96.80 91.30 0.30 6.50
HF4 3.10 3.50 96.60 86.60 0.30 9.90
W-indicates weeks, T0-represents data at zero time point

Table 8: Acidic variants, main peak, and basic variants content of formulations prepared as per example-2, measured by IEX.

Sample name IEX Data at 40 ?
% Acidic variants % Main peak content % Basic variants
T0 T1D T0 T1D T0 T1D
HF1 18.3 18.5 71.5 70.7 10.2 10.8
HF2 18.1 18.4 71.5 70.5 10.4 11.1
HF3 16.7 17.2 73.9 73.7 9.4 9.2
HF4 17.7 17.7 73.0 73.5 9.3 8.8
D-indicates days, T0-represents data at zero time point

Table 9: pH measurements of formulations as measured by pH meter.

Sample name pH
at 40 ?
T0 T1W
HF1 5.56 5.57
HF2 5.56 5.63
HF3 5.89 4.32
HF4 5.88 6.88
Further, pH values of neutralized eluate (NTEL) of Mab1 used for formulating high concentration Mab1 formulation was also measured using pH meter.
Sample name pH
at 2-8 ?
T0 T2M
NTEL 5.61 6.38

26th July, 2023 Signature:
V.R. Srinivas, Ph.D., LL.B
(Head–IPM, Biologics)
For, Dr. Reddy’s Laboratories Limited
,CLAIMS:CLAIMS
We claim:
1.A pharmaceutical formulation of anti-CD38 antibody comprising an anti-CD38 antibody, succinate buffer and one or more stabilizers, wherein the antibody is at a concentration ranging from 20 mg/ml to 200 mg/ml.

2.The pharmaceutical formulation as claimed in claim 1, further comprises surfactant.
3. The pharmaceutical formulation as claimed in claim 1, wherein the one or more stabilizers include sugar, salts, or amino acid.

4. The pharmaceutical formulation as claimed in claim 3, wherein the sugar is sucrose or trehalose or mannitol

5. The pharmaceutical formulation as claimed in claim 3, wherein the salt is sodium chloride.

6. The pharmaceutical formulation as claimed in claim 3, wherein the amino acid does not include methionine.

7. The pharmaceutical formulation as claimed in claim 1 or claim 3, does not contain hyaluronidase enzyme.

8. A method of controlling fragmentation of anti-CD38 antibody in it’s composition, wherein the method comprises preparing the antibody composition in succinate buffer, at a pH value ranging from pH 5.0 – pH 6.0, wherein the method controls fragmentation of the antibody in the said composition.

9. The method as claimed in claim 8, wherein the concentration of antibody is 100 mg/ml to 200 mg/ml.
10. The method as claimed in claim 8, further comprises sugar and/or amino acid or surfactant.

11. A method of controlling deamidation of anti-CD-38 antibody in it’s composition, wherein the method comprises preparing the antibody composition in succinate buffer, pH 5.0 to 6.0, wherein the method controls deamidation of the antibody in the said composition.

26th July, 2023 Signature:
V.R. Srinivas, Ph.D., LL.B
(Head–IPM, Biologics)
For, Dr. Reddy’s Laboratories Limited