DESC:Field of the invention
The present invention provides a pharmaceutical formulation of daratumumab antibody with suitable excipients.
Background of the invention
CD38 is a Type II glycosylated 45 kilo Dalton (kDa) membrane protein that was identified as a lymphocyte marker. CD38 has a role in leukocyte homeostasis through modulation of hematopoietic cell survival and differentiation (Richards JO, et al.)1 CD38 functions as a receptor binding to CD31 and is involved in cell adhesion and signal transduction. The function of CD38 in signal transduction appears to be versatile depending on the cell lineage, the differentiation stage, and, possibly, the association with different co-receptors (Richards JO, et al., 2008)2. CD38 is also an ectoenzyme catalyzing the synthesis and hydrolysis of cyclic adenosine-diphosphate-ribose (cADPR) from nicotinamide adenine dinucleotide (NAD+) to ADP-ribose 3. These reaction products are implicated in calcium mobilization4 and intracellular signaling (Derer S, et al, MAbs. 2014; 6(2):409-21)5. The antibodies are either injected or infused via subcutaneous, intravenous, intradermal, transdermal, intraperitoneal, intranasal or intramuscular administration. The amount of antibody that can be administered via this all route is limited by the physico-chemical properties of the antibody, in particularly by its solubility and stability in a suitable liquid formulation and by the volume of the infusion fluid 6. Present invention discloses novel compositions of daratumumab antibody with suitable excipients.
Summary of the invention
The present invention provides a pharmaceutical formulation of daratumumab antibody. In one aspect, the pharmaceutical formulation of the present invention comprises therapeutically effective amount of daratumumab antibody and suitable chelating agents selected from ethylene diamine tetraacetic acid (EDTA) or diethylenetriamine pentaacetate (DTPA) along with other suitable excipient(s). In one more aspect, the pharmaceutical formulation of the present invention comprises therapeutically effective amount of daratumumab antibody, chelating agent selected from ethylene diamine tetraacetic acid (EDTA) or diethylenetriamine pentaacetate (DTPA) and one or more buffer(s), wherein buffer(s) is selected from succinate buffer, arginine buffer, glycine buffer, citrate buffer, monosodium glutamate buffer, tris buffer, aspartate buffer, arginine-arginine buffer, arginine-aspartate buffer, arginine-citrate buffer, arginine-phosphate buffer, arginine-histidine buffer, arginine-succinate buffer, arginine-acetate buffer, arginine-glutamate buffer, aspartate-glutamate buffer, sodium adipic buffer, citrate-phosphate buffer, and suitable combination thereof optionally with other suitable excipient(s). The present invention provides method of preparing compostions of daratumumab antibody with suitable pharmaceutical excipient(s) as described herein.
Brief description of drawings
Figure 1 illustrates the rate of aggregation of daratumumab protein formulations, F-1, F-2, F-3, F-4, F-5, F-6, during storage under thermal stress condition at 50 °C ± 2 °C, RH 75 % ± 5 %, as assessed by HP-SEC. Rate of aggregation observed with the control formulation has been exhibited for comparison purpose.
Figure 2 illustrates the rate of aggregation of daratumumab protein formulations, F-7, F-8, F-9, F-10, F-11, during storage under thermal stress condition at 50 °C ± 2 °C, RH 75 % ± 5%, as assessed by HP-SEC. Rate of aggregation observed with the control formulation has been exhibited for comparison purpose.
Figure 3 illustrates the rate of aggregation of daratumumab protein formulations, F-12, F-13, F-14, F-15, F-16, F-17, during storage under thermal stress condition at 50 °C ± 2 °C, RH 75 % ± 5 % as assessed by HP-SEC. Rate of aggregation observed with the control formulation has been exhibited for comparison purpose.
Figure 4 illustrates the rate of aggregation of daratumumab protein formulations, F-18, F-19, F-20, F-21, F-22, F-23, during storage under thermal stress condition at 50 °C ± 2 °C, RH 75 % ± 5 % as assessed by HP-SEC. Rate of aggregation observed with the control formulation has been exhibited for comparison purpose.
Figure 5 illustrates the rate of aggregation of daratumumab protein formulations, F-24, F-25, F-26, F-27, F-28, F-29, during storage under thermal stress condition at 50 °C ± 2 °C, RH 75 % ± 5 % as assessed by HP-SEC. Rate of aggregation observed with the control formulation has been exhibited for comparison purpose.
Figure 6 illustrates the rate of aggregation of daratumumab protein formulations, F-30, F-31, F-32, F-33, F-34, F-35, during storage under thermal stress condition at 50 °C ± 2 °C, RH 75 % ± 5 % as assessed by HP-SEC. Rate of aggregation observed with the control formulation has been exhibited for comparison purpose.
Figure 7 illustrates the formation of total impurities (HMW+LMW) of daratumumab protein formulations, F-1, F-2, F-3, F-4, F-5, F-6, during storage under thermal stress condition at 50 °C ± 2 °C, RH 75 % ± 5 % as assessed by HP-SEC. Total impurities observed with the control formulation has been exhibited for comparison purpose.
Figure 8 illustrates the formation of total impurities of daratumumab protein formulations, F-7, F-8, F-9, F-10, F-11, during storage under thermal stress condition at 50 °C ± 2 °C, RH 75 % ± 5 % as assessed by HP-SEC. Total impurities observed with the control formulation has been exhibited for comparison purpose.
Figure 9 illustrates the formation of total impurities of daratumumab protein formulations, F-12, F-13, F-14, F-15, F-16, F-17, during storage under thermal stress condition at 50 °C ± 2 °C, RH 75 % ± 5 % as assessed by HP-SEC. Total impurities observed with the control formulation has been exhibited for comparison purpose.
Figure 10 illustrates the formation of total impurities of daratumumab protein formulations, F-18, F-19, F-20, F-21, F-22, F-23, during storage under thermal stress condition at 50 °C ± 2 °C, RH 75 % ± 5 % as assessed by HP-SEC. Total impurities observed with the control formulation has been exhibited for comparison purpose.
Figure 11 illustrates the formation of total impurities of daratumumab protein formulations, F-24, F-25, F-26, F-27, F-28, F-29, during storage under thermal stress condition at 50 °C ± 2 °C, RH 75 % ± 5 % as assessed by HP-SEC. Total impurities observed with the control formulation has been exhibited for comparison purpose.
Figure 12 illustrates the formation of total impurities of daratumumab protein formulations, F-30, F-31, F-32, F-33, F-34, F-35, during storage under thermal stress condition at 50 °C ± 2 °C, RH 75 % ± 5 % as assessed by HP-SEC. Total impurities observed with the control formulation has been exhibited for comparison purpose.
Figure 13 illustrates thermal transition of daratumumab in formulation comprising succinate buffer (F-1) by DSC.
Figure 14 illustrates thermal transition of daratumumab in formulation comprising acetate buffer (F-24) by DSC.
Figure-15 illustrates the rate of aggregation of daratumumab protein formulations, F-1, F-3, F-24, F-26, during storage under accelerated condition at 30 °C ± 2 °C; 75 % RH ± 5 % as assessed by HP-SEC. Rate of aggregation observed with the control formulation has been exhibited for comparison purpose.
Figure 16 illustrates the formation of HMW species of daratumumab protein formulations, F-1, F-3, F-24, F-26, upon photo exposed condition as assessed by HP-SEC. HMW species observed with the control formulation has been exhibited for comparison purpose.
Figure 17 illustrates the formation of LMW species of daratumumab protein formulations F-1, F-3, F-24, F-26, upon photo exposed condition as assessed by HP-SEC. LMW species observed with the control formulation has been exhibited for comparison purpose.
Figure 18 illustrates thermal transition of daratumumab in formulation comprising succinate buffer (F-3) by DSC.
Figure 19 illustrates thermal transition of daratumumab in formulation comprising acetate buffer (F-26) by DSC.
List of abbreviations used herein in the specification
CD-38: Cluster of differentiation 38
DSC: Differential scanning calorimetry
DTPA: Diethylenetriaminepentaacetic acid (Pentetic acid)
EDTA: Ethylenediaminetetraacetic acid
FT: Freeze-thaw
HMW: High molecular weight
HP-ß-CD: Hydroxypropyl beta cyclodextrin
LMW: Low molecular weight
mM: milimolar
NA: Not available
PEG: Polyethylene glycol
P20: Polysorbate 20
Definitions
The term “pharmaceutical formulation” refers to preparations which are in such form as to permit the biological activity of the active ingredients to be unequivocally effective, and which contain no additional components which are significantly toxic to the subjects to which the formulation would be administered. The term “pharmaceutical formulation”, “formulation”, “pharmaceutical composition”, or “composition” can be used here interchangeably.
The term “Control formulation” referred herein within the current specification comprises of 20 mg / mL of daratumumab in 25 mM sodium-acetate of pH 5.5 containing 60 mM sodium chloride, 25.5 mg / mL mannitol, and 0.04 % polysorbate 20, which can be referred herein after as “control daratumumab formulation” or “control formulation”.
In a pharmacological sense, in the context of the present invention, a “therapeutically effective amount” or “effective amount” of a daratumumab antibody refers to an amount effective in the prevention or treatment of a disorder for the treatment of which the daratumumab antibody is effective. A “disorder” is any condition that would benefit from treatment with the antibody. This includes chronic and acute disorders or diseases including those pathological conditions, which predisposes the subject to the disorder in question.
The term “buffer” or “buffer solution” or “buffer system” refers to generally aqueous solution comprising a mixture of an acid (usually a weak acid) and conjugate base. A buffered solution prevents change of pH of the solution due to the “buffering capacity” imparted by the “buffering agent(s)”. The pH of a “buffer solution” will change very little upon addition of a small quantity of strong acid or base due to the “buffering effect” imparted by the “buffering agent”.
The term “buffer system” comprises one or more buffering agent(s) and / or an acid/base conjugate(s) thereof, and more suitably comprises one buffering agent only and an acid/base conjugate thereof. The overall pH of the composition comprising the relevant buffer system is generally a reflection of the equilibrium concentration of each of the relevant buffering species (i.e. the balance of buffering agent(s) to acid/base conjugate(s) thereof). The buffer system may comprise dual buffers where two separate buffers are mixed to obtain target pH.
The term “buffering agent” refers to an acid or base component (usually a weak acid or weak base) of a buffer or buffer solution. A buffering agent maintain the pH of a given solution at or near to a pre-determined value, and the buffering agents are generally chosen to complement the pre-determined value. The term “buffering agent” and “buffers” can be used here interchangeably.
The term “pharmaceutical excipient” or “suitable excipient” refers to an agent that may be added to a formulation to stabilize the active drug substance in the formulated form to adjust and maintain osmolality and pH of the pharmaceutical preparations. Examples of used excipient(s) include, but are not limited to, suitable buffer(s), suitable carbohydrate(s), suitable surfactant(s), suitable amino acid(s), suitable anti-oxidant(s), suitable chelating agent(s) and combination thereof.
The “lyophilized formulation” or “freeze dried” formulation is a dosage form, which is prepared by lyophilization or freeze drying process. The lyophilization was performed with conventional lyophilization technique known in the literatures involving steps such as freezing, primary drying, secondary drying and optionally annealing.
“Liquid formulation” refers to a composition of matter that is found as a liquid, characterized by free movement of the constituent molecules among themselves but without the tendency to separate at room temperature. Liquid formulations include aqueous and non-aqueous liquid, with aqueous formulations being preferred. An aqueous formulation is a formulation in which the solvent or main solvent is water, preferably water for injection (WFI) 7
A “stable” formulation is one in which the active substance therein i.e. protein or antibody essentially retains its physical stability and/or chemical stability and/or biological activity upon storage. Active substance according to the present invention is a daratumumab antibody. Preferably, the formulation essentially retains its physical and chemical stability, as well as its biological activity upon storage. The storage period is generally selected based on the intended shelf-life of the formulation. Various analytical techniques for measuring protein stability are available in the art. Stability can be measured at a selected temperature for a selected time period. The formulation of the current invention is stable between +2 °C and +8 °C for at least one month. Furthermore, the formulation of the current invention has been observed to remain stable under frozen condition and can withstand multiple freeze-thaw stress, at very low temperature conditions, for example at – 70 ?C, the said compositions have been found to remain stable even after 10 freeze-thaw cycles. Stability can be evaluated qualitatively and/or quantitatively in a variety of different ways, including evaluation of aggregate formation (for example using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometric analysis; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS-C) analysis; evaluating biological activity or antigen binding function of the antibody; etc. Instability may involve any one or more of: aggregation, deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation), isomerization (e.g. Asp isomerization), clipping/hydrolysis/fragmentation (e.g. hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc.
The term “tonicity agent” or “tonicifier” or “tonicity modifier” is a compound which renders the formulation isotonic. Carbohydrate(s) such as sugar(s) or sugar alcohol (polyols),amino acid(s), glycerin may act as a tonicity agent. The term “isotonic” is known in the art and can mean, for example, that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonicity can be measured using a vapor pressure or ice-freezing type osmometer. Cryoprotectant also contribute to the tonicity of the formulations.
The term carbohydrate is used interchangeably with sugar and the word amino acid is used interchangeably with antioxidant.
The term “stabilizer” as used herein in the present invention refers to a compound which stabilizes an active ingredient(s) (daratumumab antibody) under various temperature storage conditions such as 25°C or 40 °C, or 50 °C or – 15 ?C or – 20 ?C or – 40 ?C or – 70 ?C or – 80 ?C, Buffer(s), carbohydrate(s) such as sugar(s) or sugar alcohol (polyols), surfactant(s), amino acid(s) may act as a stabilizers either alone or in suitable combination.
The term “anti-oxidant” as used herein in the present invention refers to a compound which prevents oxidation of active ingredient(s) (daratumumab antibody) under storage condition. Amino acid such as methionine etc and chelating agent such as pentetic acid (DTPA), EDTA, act as anti-oxidant.
Chelating agents, or chelators, constitute a well-known type of multifunctional organic compounds that are capable of forming complexes of multivalent metal ions such as, for example, calcium, magnesium, zinc, iron, chromium, and lead 8
The term “about” as used herein in the present invention refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods, and other similar considerations. The term “about” also encompasses amounts that differ due to aging of a formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a formulation with a particular initial concentration or mixture.
The term “patient” or “subject” is used in its conventional sense to refer to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a complex or a composition of the present invention, and includes animals. The term “animal” refers to a human or non-human animal, including, but not limited to, farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese and non-human primates, including, but not limited to, monkeys, chimpanzees and other apes and monkey species. The term does not denote a particular age. Thus, adult, juvenile and newborn individuals are of interest.
The term “comprising” or “comprises” as used herein in the present invention refers to the compositions and methods include the listed elements, but do not exclude other unlisted elements.
Unless otherwise defined, all technical and scientific terms used herein in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Embodiments of the invention
In one embodiment, the present invention provides a pharmaceutical formulation of therapeutically effective amount of daratumumab comprising chelating agent, wherein chelating agent is selected from ethylene diamine tetraacetic acid (EDTA) and diethylenetriamine pentaacetate (DTPA) along with other suitable excipient(s), wherein the suitable excipient(s) is selected from buffer, carbohydrate and suitable combination thereof.
In further embodiment, the present invention provides the pharmaceutical formulation of therapeutically effective amount of daratumumab wherein the formulation is further comprising buffer selected from acetate buffer, histidine buffer, succinate buffer, citrate buffer, arginine buffer, glycine buffer, monosodium glutamate buffer, tris buffer, aspartate buffer, arginine-arginine buffer, arginine-aspartate buffer, arginine-citrate buffer, arginine-phosphate buffer, arginine-histidine buffer, arginine-succinate buffer, arginine-acetate buffer, arginine-glutamate buffer, aspartate-glutamate buffer, sodium adipic buffer, citrate-phosphate buffer wherein buffer is present in the range of about 1 mM to about 120 mM.
In furthermore embodiment, the present invention provides the pharmaceutical formulation of therapeutically effective amount of daratumumab wherein the formulation is further comprising carbohydrate selected from sucrose or Trehalose or HP-ß-CD wherein carbohydrate is present in the range of about 1 mM to about 300 mM.
In one of the further embodiment, the present invention provides the pharmaceutical formulation of therapeutically effective amount of daratumumab further comprising surfactant and / or amino acid wherein surfactant is polysorbate 20 present in the range of about 0.001 mg / mL to 5 mg / mL and amino acid is methionine in the range of about 1 mM to 250 mM.
In one embodiment, the formulation of daratumumab according to the present invention comprising chelating agent selected from EDTA or DTPA in the range of about 0.001 mM to about 50 mM.
In one embodiment, the formulation of daratumumab according to the present invention wherein daratumumab is present in the range of 1.0 % w / v (10 mg / mL) to 25 % w / v (250 mg / mL) .
In a preferred embodiment, the pharmaceutical formulation of therapeutically effective amount of daratumumab of the present invention comprises daratumumab 20 mg / ml, 10 mM sodium succinate buffer, 257 mM Sucrose or 230 mM Trehalose or 235 mM HP-ß-CD, 0.04 mM DTPA, and / or 0.04 % Polysorbate 20 and / or 6.7 mM L-methionine In a more preferred embodiment, daratumumab 20 mg / ml, 10 mM sodium succinate buffer, 257 mM Sucrose, 6.7 mM L-methionine, 0.04 mM DTPA, 0.04 % polysorbate 20
In a one of more preferred embodiment, the pharmaceutical formulation of therapeutically effective amount of daratumumab of the present invention comprises daratumumab 20 mg / ml, 10 mM Sodium succinate buffer, 230 mM Trehalose, 6.7 mM L-methionine, 0.04 mM DTPA, 0.04% polysorbate 20.
In one of the other embodiment, the pharmaceutical formulation of therapeutically effective amount of daratumumab of the present invention comprises daratumumab 20 mg / ml , 25 mM sodium acetate buffer, 250 mM Sucrose or 257 mM Trehalose or 2.8 % mM HP-ß-CD, 0.04 mM DTPA, and / or 0.04 % Polysorbate 20 and / or 6.7 mM L-methionine.
In of the preferred embodiment, the pharmaceutical formulation of therapeutically effective amount of daratumumab of the present invention comprises daratumumab 20 mg / ml, 25 mM sodium acetate buffer, 250 mM sucrose, 6.7 mM L-methionine, 0.04 mM DTPA, 0.04 % polysorbate 20 .
In of the more preferred embodiment, the pharmaceutical formulation of therapeutically effective amount of daratumumab of the present invention comprises daratumumab 20 mg / ml, 25 mM sodium acetate buffer, 257 mM trehalose, 6.7 mM L-methionine, 0.04 mM DTPA, 0.04 % polysorbate 20 .
In one of the other embodiment, the pharmaceutical formulation of therapeutically effective amount of daratumumab of the present invention comprises daratumumab 20 mg / ml, 10 mM histidine buffer, 290 mM sucrose or 290 mM trehalose or 118 mM HP-ß-CD, 0.04 mM DTPA, and / or 0.04 % Polysorbate 20 and / or 6.7 mM L-methionine or
In one of the further embodiment, the pharmaceutical formulation of therapeutically effective amount of daratumumab of the present invention comprises daratumumab 20 mg / ml, 10 mM sodium citrate buffer, 250 mM sucrose or 272 mM trehalose or 235 mM HP-ß-CD, 0.04 mM DTPA, and / or 0.04 % Polysorbate 20 and / or 6.7 mM L-methionine
In one of the further embodiment, the pharmaceutical formulation of therapeutically effective amount of daratumumab of the present invention comprises daratumumab 20 mg / ml, 29 mM arginine citrate, 290 mM sucrose or 290 mM trehalose or 235 mM HP-ß-CD, 0.04 mM DTPA, and / or 0.04 % Polysorbate 20 and / or 6.7 mM L-methionine or
In one of the still further embodiment, the pharmaceutical formulation of therapeutically effective amount of daratumumab of the present invention comprises daratumumab 20 mg / ml, 10 mM sodium glutamate, 245 mM sucrose or 257 mM trehalose or 235 mM HP-ß-CD, 0.04 mM DTPA, and / or 0.04 % Polysorbate 20 and / or 6.7 mM L-methionine.
In further embodiment, the pharmaceutical formulation of the present invention is a liquid dosage form, a freeze-dried dosage form, or a frozen dosage form.
In one of the embodiments, the pharmaceutical formulation as embodied herein the present invention further comprises a hyaluronidase preferably, rHuPH20 in the range of 50 U / mL to about 5,000 U / mL.
In a further embodiment, the present invention provides route of administration to a subject by subcutaneous, intravenous, intradermal, transdermal, intraperitoneal, intranasal or intramuscular administration.
Detailed description of the invention
The present invention provides a pharmaceutical formulation of daratumumab. The terms daratumumab antibody or daratumumab or daratumumab monoclonal antibody are used interchangeably in the present invention. Daratumumab sequence has been disclosed in International Nonproprietary Names for Pharmaceutical Substances (INN, Vol. 24, No. 1, 2010) 9 for daratumumab product. The amount of daratumumab according to the present invention is in the range of 1.0 % w / v to 25 % w / v or its equivalent amount in mg / mL i.e. the amount of daratumumab according to the present invention is in the range of 10 mg / mL to 250 mg / mL. The amount of daratumumab according to present invention include each integer and non-integer number between the particular ranges. For example, 1.0 % w / v, 2.0 % w / v, 3.0 % w / v, 5.0 % w / v, 7.5 % w / v, 9.0 % w / v, 10.8 % w / v, 10.7 % w / v, 12.5 % w / v, 15 % w / v etc.
In one embodiment, a pharmaceutical formulation of therapeutically effective amount of daratumumab comprises chelating agent, wherein chelating agent is selected from ethylene diamine tetra acetic acid (EDTA) and diethylenetriamine pent acetate (DTPA) with other suitable excipient(s). The other suitable excipient(s) is selected from buffer(s), surfactant(s), carbohydrate(s), amino acid(s), acid(s), base(s) and suitable combination thereof. The chelating agent diethylenetriaminepentaacetic acid (DTPA) is also known as pentetic acid. In one embodiment, the amount of chelating agent(s) used herein in the present invention is in the range of about 0.001 mM to about 50 mM. The amount of chelating agent(s) in the range of about 0.001 mM to about 50 mM according to present disclosure include each integer and non-integer number between a particular range. For example, the amount of chelating agent(s) is in the range of about 0.001 mM to about 50 mM includes about 0.001 mM, about 0.5 mM, about 1.0 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM and any non-integer values like 10.5 or 5.8 etc.
In a further embodiment, the pharmaceutical formulation of the present invention comprises therapeutically effective amount of daratumumab and one or more buffer(s) wherein buffer(s) is selected from succinate buffer, arginine buffer, glycine buffer, citrate buffer, monosodium glutamate buffer, tris buffer, arginine-citrate buffer, arginine-phosphate buffer, arginine-succinate buffer, arginine-acetate buffer, arginine-glutamate buffer, citrate-phosphate buffer, and suitable combination thereof optionally with other suitable excipient(s). A skilled person can prepare buffers by methods known in the art. The buffer(s) may also act as stabilizer in the pharmaceutical formulation of the present invention. The amount of buffer used herein in the present invention is in the range of 1 mM to about 120 mM. The amount of buffer in the range of about 1 mM to about 120 mM according to present invention include each integer and non-integer number between a particular range. For Example, the amount of buffer in the range of about 1 mM to about 120 mM includes about 1 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 100 mM, about 120 mM and any non-integer values in between such as 10.5 mM, 12.8 mM and so on. In one embodiment, the buffers included in the pharmaceutical formulation according to the present invention is a buffer that maintains the pH of the composition at a pH, ranging from about pH 4.0 to about pH 8.0, preferably about pH 5.0 to about pH 7.5. The pH range 4.0 to 8.0 according to the present invention includes pH 4.0, pH 5.0, pH 5.5, pH 6.0, pH 7.0 or pH 8.0 or any non-integer number in between them (e.g., pH 6.3 or pH 6.5 or pH 6.7 or pH 6.8 or pH 7.4 or pH 7.5 and the like). The other suitable excipient(s) of the formulation is selected from one or more surfactant(s), carbohydrate(s), amino acid(s), acid(s), base(s), chelating agent(s) and suitable combination thereof.
In one of the embodiments, the present invention provides a pharmaceutical composition comprising daratumumab, a hyaluronidase and suitable excipients as embodied herein in the present invention. The invention also provides a pharmaceutical composition comprising daratumumab and a hyaluronidase rHuPH20. The concentration of hyaluronidase in the present invention is in the range of 50 U/mL to about 5,000 U/mL. In some embodiments of the present invention, the pharmaceutical composition of daratumumab comprises from about 750 U to about 75,000 U of the hyaluronidase.
In a further embodiment, the present invention provides method of preparing daratumumab composition with suitable pharmaceutical excipient(s). In one more embodiment, the pharmaceutical formulation of the present invention is a liquid dosage form or a freeze-dried dosage form or a frozen dosage form, preferably liquid dosage form. In one of the embodiments, the present invention provides the pharmaceutical formulation of daratumumab, which is stable under 2 °C to 8 °C for at least one month. The pharmaceutical formulation of daratumumab according to the present invention is stable under 2 °C to 8 °C for at least 3 months, for at least 6 months, or for at least 12 months. In one of the embodiments, the present invention provides pharmaceutical composition of daratumumab that has been observed to show reduced rate of formation of HMW species compared to the control formulation. The daratumumab formulations of the present invention were observed to show reduced rate and extent of the formation of HMW species when compared to the control formulation in temperature-dependent stress study. Rate of formation of aggregates species formed was observed under temperature stressed condition at 50 °C ± 2 °C, RH 75 % ± 5 % and 30 °C ± 2 °C, 75 % RH ± 5 % for at least 30 days. To estimate the level of impurities formation, analytical HP-size exclusion chromatography (HP-SEC) was performed. The said analytical method used in the present invention is well known to a skilled person and a brief description of the same is provided herein in below for the sake of reference.
In one of the embodiments, the pharmaceutical formulation of each embodiment of the present invention maintains stability of daratumumab at least after one freeze-thaw cycle, preferably, maintains stability of daratumumab at least after 10 freeze-thaw cycles, more preferably, maintains stability of daratumumab at least after 30 freeze-thaw cycles.
In preferred embodiment, the pharmaceutical formulation of each embodiment of the present invention maintains structural integrity of daratumumab upon multiple freeze-thaw cycles, preferably at least after one freeze-thaw cycle, preferably, maintains structural integrity of daratumumab at least after ten freeze-thaw cycles, more preferably, maintains structural integrity of daratumumab at least after thirty freeze-thaw cycles. In one of the embodiments, the present invention provides pharmaceutical composition of daratumumab antibody that has been observed to show reduced extent of formation of HMW species compared to the control formulation. In one of the embodiments, the present invention provides pharmaceutical composition of daratumumab that has been observed to show reduced extent of formation of LMW species compared to the control formulation. The daratumumab formulations of the present invention were observed to show reduced extent of the formation of HMW species and LMW species when compared to the control formulation upon photo-exposure stress study. Extent of formation of aggregates species i.e. HMW species and LMW species formed was observed upon 1x photo exposed condition wherein daratumumab compositions of the present invention were exposed to light providing an overall illumination of not less than 1.2 million lux hours and an integrated near ultraviolet energy of not less than 200 Watt hours / m2 for 1 complete photo exposure cycle. In one of the embodiments, the extent of formation of aggregates species i.e. HMW species and LMW species formed was observed upon 2x photo exposed condition wherein daratumumab compositions of the present invention were exposed to light providing an overall illumination of 2.4 million lux hours and an integrated near ultraviolet energy of 400 Watt hours / m2 for 1 complete photo exposure cycle. To estimate the level of HMW species and LMW species formation, analytical HP-size exclusion chromatography (HP-SEC) was performed.
Analytical methods used in the present invention:
Freeze-thaw study protocol:
Prior to analysis, samples were subjected to freezing and thawing cycles. Samples are the daratumumab formulations as mentioned herein in the present invention.
Before initiating the freezing and thawing, all samples were stored at – 70 °C ± 10 °C. After completion of each FT cycle, samples were analysed for purity by HP-SEC and compared with the FT-control sample.
For each FT cycle, freezing was performed at – 70 °C ± 10 °C for at least about 2 hours and thawing was performed at room temperature. The FT-control (not subjected to FT) sample was stored between + 2 °C and + 8 °C in liquid form.
To perform multiple repeated freezing and thawing cycles, each aliquots were filled with approximately 100 µL of the formulated daratumumab in a 0.5 mL cryo-tube as container closure system and subjected to FT study. After completion of respective FT cycles, each sample was analysed for purity by HP-SEC.
HP-SEC analysis:
To estimate the level of aggregates or fragments analytical HP-size exclusion chromatography (HP-SEC) was performed. The said analytical method used in the present invention is well known to a skilled person and a brief description of the same is provided below for the sake of reference.
Samples were analyzed to estimate the aggregates by HP-size exclusion chromatography (HP-SEC) using TSK gel G3000 SWXL column (7.8 mm I.D × 30 cm L). Samples were loaded and eluted isocratically in the presence of sodium phosphate buffer of pH 6.8, at a flow rate of 0.5 mL / min. Elution was monitored with UV214 nm detection.
Photo-exposure method for evaluation of degradation of antibody
Photo-exposure stress study includes two types of daratumumab compositions unexposed compositions and photo exposed compositions. Unexposed compositions were stored in refrigeration condition i.e. + 2 °C and + 8 °C. Photo exposed compositions were filled in vials without primary label. The vials filled with daratumumab compositions were exposed to light providing an overall illumination of not less than 1.2 million lux hours and an integrated near ultraviolet energy of not less than 200 Watt hours / m2 for 1 complete photo exposure cycle (1x photo exposed). Further, to check degradation / stability under more worst condition, daratumumab compositions, which were filled in vials, were photo exposed till 2.4 million lux hours and UV energy not less than 400 Watt hours / m2 (2x photo exposed).
Detailed description of excipients which applies to each formulation of each embodiment described herein above of the present invention
Buffers
Buffers suitable for use in each of the formulations of the present invention include buffers that are compatible with the daratumumab antibody, preferably daratumumab and suitable for administration. Buffers of each of the formulations of the present invention are selected from acetate buffer, histidine buffer, succinate buffer, arginine buffer, glycine buffer, citrate buffer, monosodium glutamate buffer, tris buffer, aspartate buffer, arginine-arginine buffer, arginine-aspartate buffer, arginine-citrate buffer, arginine-phosphate buffer, arginine-histidine buffer, arginine-succinate buffer, arginine-acetate buffer, arginine-glutamate buffer, aspartate-glutamate buffer, sodium adipic buffer, citrate-phosphate buffer and suitable combination thereof optionally with other suitable excipient(s). The buffer(s) may act as stabilizer in the pharmaceutical formulation of the present invention. Buffers of each of the formulations of the present invention can be prepared by a skilled person by method known in the art. The amount of buffer used herein in the present invention is in the range of 1 mM to about 120 mM. The amount of buffer in the range of about 1 mM to about 120 mM according to present invention include each integer and non-integer number between a particular range. For Example, the amount of buffer in the range of about 1 mM to about 120 mM includes about 1 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 100 mM, about 120 mM. In one embodiment, the buffers included in the pharmaceutical formulation according to the present invention is a buffer that maintains the pH of the composition at a pH, ranging from about pH 4.0 to about pH 8.0, preferably about pH 5.0 to about pH 7.5. The pH range 4.0 to 8.0 according to the present invention includes pH 4.0, pH 5.0, pH 5.5, pH 6.0, pH 7.0 or pH 8.0 or any non-integer number in between them (e.g., pH 6.3 or pH 6.5 or pH 6.7 or pH 6.8 or pH 7.4 or pH 7.5 and the like.
Amino acids
Amino acids suitable for use in each of the formulations of the present invention include amino acids that are compatible with the daratumumab antibody, preferably daratumumab and suitable for administration. Examples of suitable amino acids include, but are not limited to, arginine, glycine, asparagine, glutamine, lysine, threonine, histidine, glutamic acid, aspartic acid, isoleucine, valine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, cysteine or combination of any of the above. The amino acid(s) used herein in the present invention may have role as an aggregation inhibitor or as a stabilizer or as an anti-oxidant. In one embodiment, the amount of amino acid(s) used herein in the present invention is in the range of 1 mM to 200 mM .In one of the embodiment the amount of amino acid in the range of about 1 mM to about 250 mM according to present disclosure include each integer and non-integer number between a particular range. For Example, the amount of amino acid in the range of about 1 mM to about 250 mM includes about 1 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 100 mM, about 150 mM, about 170 mM, about 200 mM, about 250 mM.
Chelating agents
Chelating agents suitable for use in each of the formulations of the present invention include chelating agents that are compatible with the daratumumab antibody and suitable for administration. The chelating agent is selected from diamine tetraacetic acid (EDTA) and diethylenetriamine pentaacetate (DTPA). The diethylenetriaminepentaacetic acid (DTPA) is also known as pentetic acid. In one embodiment, the amount of chelating agent(s) used herein in the present invention is in the range of about 0.001 mM to about 50 mM. The amount of chelating agent(s) in the range of about 0.001 mM to about 50 mM according to present disclosure include each integer and non-integer number between a particular range. For Example, the amount of chelating agent(s) in the range of about 0.001 mM to about 50 mM includes about 0.001 mM, about 0.5 mM, about 1.0 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM.
Carbohydrates
The carbohydrate according to each of the formulations of the present invention may include sugar, sugar alcohol (polyol), a derivatized sugar, an esterified sugar and sugar polymer. Carbohydrate may use as a bulking agent or tonicity modifier or stabilizer herein in the present invention. Some carbohydrate of interest include, but are not limited to, for example, monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; sugar alcohol such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glycerol), pyranosyl sorbitol, myoinositol, and the like. Preferred sugars suitable for use in each of the formulations of the present invention include sugars that are compatible with the pharmaceutical formulation of daratumumab antibody of the present invention suitable for administration to a subject. Examples of suitable sugars include, but not limited to, sucrose, mannitol, sorbitol, maltose, trehalose, hydroxypropyl-beta-cyclodextrin (ß-HPCD), hydroxypropyl-gamma-cyclodextrin (?-HPCD) lactose, erythritol, isomalt, glucose, fructose, galactose, glucosamine, and the like, and suitable combinations thereof. In one embodiment, the amount of sugar(s) used herein in the present invention is in the range of 1 mM to 300 mM. The amount of sugar in the range of about 1 mM to about 300 mM according to present disclosure include each integer and non-integer number between a particular range. For Example, the amount of sugar in the range of about 1 mM to about 300 mM includes about 1 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 260 mM, about 300 mM.
Surfactants or detergents
Surfactants suitable for use in each of the formulations of the present invention include surfactants that are compatible with the pharmaceutical formulation of daratumumab antibody. Surfactant(s) may use as a stabilizer or an aggregation inhibitor in the pharmaceutical formulation of the present invention. Examples of surfactants according to each of the formulations of the present invention include, but not limited to polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (e.g. Brij), alkylphenylpolyoxyethylene ethers (e.g. Triton-X), polyoxyethylene-polyoxypropylene copolymer (e.g. Poloxamer, Platonic), polyethylene glycol (PEG), polyethyleneimine, sodium dodecyl sulphate (SDS) and the like. In one embodiment of the present invention, the pharmaceutical formulation of daratumumab antibody according to each of the formulations of the present invention comprises surfactant, wherein the surfactant is polyoxyethylensorbitan-fatty acid esters (Tweens). In another embodiment of the present invention, the pharmaceutical formulation of daratumumab antibody according to each of the formulations of the present invention comprises polysorbate 20 or polysorbate 80. The polysorbate 20 is sold under the trademark Tween 20™. In another embodiment of the present invention, the amount of surfactant according to each of the formulations of the present invention is in the range of about 0.001 mg / mL to 5 mg / mL. The amount of surfactant according to each of the formulations of the present invention include each integer and non-integer number between particular concentration range. For Example, the amount of surfactant about 0.001 mg / mL to about 5 mg / mL include 0.001 mg / mL, 0.005 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 5.0 mg / mL, or any integer or non-integer number in between them.
Acids and Bases
Acids and bases according to the present invention can be a part of buffering agent or acid/base conjugate or as a pharmaceutical excipient. Examples of acid according to the present invention may include, but are not limited to hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, succinic acid, sulfuric acid, fumaric acid, and any combinations thereof. Examples of bases according to the present invention include, but are not limited to sodium hydroxide, ammonium hydroxide and potassium hydroxide. In further embodiment of the present invention, the buffering agent(s) or buffering species or buffering component(s) used in each of the formulations according to the present invention is present in amount about 0.01 mg / mL to about 100 mg / mL. The concentration range of the buffering agent(s) or buffering species or buffering component(s) used in the formulations of present invention include each integer and non-integer number between particular ranges. For Example, the concentration range about 0.01 mg / mL to about 100 mg / mL includes 0.01 mg / mL, 0.1 mg / mL, 1.0 mg / mL, 1.08 mg / mL, 1.28 mg / mL, 2 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 50 mg / mL, 100 mg / mL or any integer and non-integer number between said concentration range. The formulation of the current invention is stable between +2 °C and +8 °C for at least one month. The amount of active substance i.e. daratumumab antibody and the amount of pharmaceutical excipient(s) used herein in the present invention, in % w / v or its equivalent mg / mL or its equivalent mM can be used interchangeably here in the present invention.
The composition according to the present invention finds therapeutic use in single-dose form or in multi-dose form. In one of the embodiments, the pharmaceutical formulation according to the present invention may further comprises suitable preservatives as known in the art. The present invention includes stable frozen, liquid or freeze dried daratumumab composition as embodied according to the present invention. The lyophilization process to prepare formulation of the present invention can be performed by a skilled person using the techniques available in the art, which includes various steps such as freezing, primary drying, secondary drying and optionally annealing.
The formulations of the present invention may be suitable for any use, including both in vitro and in vivo uses. In one embodiment, the formulation of the present invention is suitable for administration to a subject via a mode of administration, including, but not limited to, subcutaneous, intravenous, intradermal, transdermal, intraperitoneal, intranasal and intramuscular administration. The formulations of the invention may be used in the treatment of a disorder in a subject. Also included in the invention are devices that may be used to deliver the formulation of the invention. Examples of such devices include, but are not limited to, a glass vial, a syringe, a pen, an implant, an ampoule, a needle-free injection device and a patch.

Examples
The following non-limiting examples describe different formulations of daratumumab antibody, which can be prepared as per the present invention. It will be appreciated that other excipients can also be added as necessary to these formulations and such addition of excipients are considered to be within the scope of a person skilled in the art and are to be included within the scope of the present invention. The following examples describe experiments relating to present invention.
Example 1: Daratumumab formulations with succinate buffer in combination with sugar, antioxidant, chelating agent and / or surfactant
Table 1: Details of Formulations
Formulation Buffer Stabilizer Antioxidant chelating agent Surfactant
F-1 10 mM Sodium succinate pH 5.57 Sucrose
(257 mM) L-methionine
(6.7 mM) DTPA
(0.04 mM) P20
(0.04 %)
F-2 10 mM Sodium succinate pH 5.53 Sucrose
(257 mM) NA DTPA
(0.04 mM) P20
(0.04 %)
F-3 10 mM Sodium succinate pH 5.56 Trehalose
(230 mM) L-methionine
(6.7 mM) DTPA
(0.04 mM) P20
(0.04 %)
F-4 10 mM Sodium succinate pH 5.57 Trehalose
(230 mM) NA DTPA
(0.04 mM) P20
(0.04 %)
F-5 10 mM Sodium succinate pH 5.89 HP-ß-CD
(235 mM) L-methionine
(6.7 mM) DTPA
(0.04 mM) NA
F-6 10 mM Sodium succinate pH 5.81 HP-ß-CD
(235 mM) NA DTPA
(0.04 mM) NA
Daratumumab: 20 mg / ml

Daratumumab was formulated in sodium succinate buffer of pH 5.5 ± 0.5 comprising sucrose, trehalose and HP-ß-CD at desired concentration(s), as described above. Other excipient(s), like antioxidant, chelating agent and polysorbate 20 can be added as shown in Table 1. A person skilled in the art can prepare daratumumab formulation in accordance with conventional techniques such as those disclosed in the prior art10 and can fill the formulated solution in suitable container-closure system (like vials, cartridges, syringes etc.) for storage at suitable temperature for further use.
In one approach, stability of daratumumab in the said formulations was evaluated through freeze-thaw stress study (cold stress), for 30 cycles, at – 70 ?C. In another approach, stability of daratumumab in the said formulations were also assessed, under thermal stress condition at 50 °C ± 2 °C; RH 75 % ± 5 % for one month. Further, the Formulation 1 (F-1) and Formulation 3 (F-3) were subjected to 30 °C ± 2 °C, 75 % RH ± 5 % RH for one month, to assess the stability of daratumumab in the said formulations. Additionally, photo-exposure study was conducted with F-1 and F-3 to assess the photo-stability or photo-degradation of daratumumab in the said formulations. The treated or exposed samples were analyzed for purity and size variants profile (HMW and LMW) of daratumumab by HP-SEC, under native condition, and compared with respect to the control formulation. Results are summarized in Table 7 and Table 9 and illustrated in Figure 1, Figure 7, Figure 15, Figure 16 and Figure 17. The said formulations were observed to show reduced rate and extent of the formation of HMW species and LMW species of daratumumab as compared to that of the control formulation.
Differential scanning calorimetry (DSC):
Conformational integrity through thermal denaturation of daratumumab samples was assessed by DSC in F-1 and F-3 and compared to that of the control formulation. DSC analysis was performed on MicroCal VP capillary equipment (Malvern, USA). All samples were diluted to 1 mg / mL with the respective formulation media and subjected to heat denaturation experiments by DSC, wherein temperature of the samples was increased from 10 °C to 95 °C, at an up-scanning rate of 1 °C / min. Samples were analyzed against the respective formulation media, which was considered as the blank for each sample. Thermal transitions obtained with the samples were recorded in the form of Tm values including the Tonset. Total enthalpy of thermal transitions for each sample was calculated by deconvoluting the thermograms using Origin 7 SR4 v7.0552 software via Non-2-State model for multiple transition temperatures.
Daratumumab samples were observed to show three clear thermal transitions, Tm1, Tm2 and Tm3, as illustrated in Figure 13, Figure 18 and Table 8. The first transition (Tm1) represents unfolding of CH2 domain, the second transition (Tm2) represents Fab domain unfolding and the third transition (Tm3) represents CH3 domain unfolding.
Daratumumab samples when formulated in F-1 and F-3 were observed to show better stability than the control formulation, as illustrated in Figure 13 and Figure 18. Results are presented in Table 8.
Thermal transition and total enthalpy values obtained with daratumumab in F-1 and F-3 were found to be higher than that of the control formulation, indicating a better thermodynamic stability of daratumumab in the said formulations than the control formulation. Also, the Tonset values of daratumumab in F-1 and F-3 formulations were found to be higher than that of the control formulation, indicating higher thermodynamic stability of the daratumumab in F-1 and F-3 than the control formulation.
Example 2: Daratumumab formulations with histidine buffer in combination with sugar, antioxidant, chelating agent and surfactant
Table 2: Details of Formulations
Formulation Buffer Stabilizer Antioxidant chelating agent Surfactant
F-7 10 mM Histidine pH 5.64 Sucrose
(290 mM) L-methionine
(6.7 mM ) DTPA
(0.04 mM) P20
(0.04 %)
F-8 10 mM Histidine pH 5.59 Sucrose
(290 mM) NA DTPA
(0.04 mM) P20
(0.04 %)
F-9 10 mM Histidine pH 5.64 Trehalose
(290 mM) L-methionine
(6.7 mM ) DTPA
(0.04 mM) P20
(0.04 %)
F-10 10 mM Histidine pH 5.61 Trehalose
(290 mM) NA DTPA
(0.04 mM) P20
(0.04 %)
F-11 10 mM Histidine pH 5.609 HP-ß-CD
(118 mM) NA DTPA
(0.04 mM) NA
Daratumumab: 20 mg / ml

Daratumumab was formulated in histidine buffer of pH 5.5 ± 0.5 comprising sucrose, trehalose and HP-ß-CD at desired concentration(s), as described above. Other excipient(s), can be added as shown in Table 2. A person skilled in the art can prepare daratumumab formulation in accordance with conventional techniques such as those disclosed in the prior art10 and can fill the formulated solution in suitable container-closure system (like vials, cartridges, syringes etc.) for storage at suitable temperature for further use.
In one approach, stability of daratumumab in the said formulations was evaluated through freeze-thaw stress study (cold stress), for 10 cycles, at – 70 ?C. In another approach, stability of daratumumab in the said formulations were also assessed, under thermal stress condition at 50 °C ± 2 °C; RH 75 % ± 5 % for one month. The treated or exposed samples were analyzed for purity and size variants profile (HMW and LMW) of daratumumab by HP-SEC, under native condition, and compared with respect to the control formulation. Results are summarized in Table 7 illustrated in Figure 2 and Figure 8. The said formulations were observed to show reduced rate and extent of the formation of HMW species and LMW species of daratumumab as compared to that of the control formulation.
Example 3: Daratumumab formulations with sodium citrate buffer in combination with sugar, antioxidant, chelating agent and surfactant
Table 3: Details of Formulations
Formulation Buffer Stabilizer Antioxidant chelating agent Surfactant
F-12 10 mM Sodium Citrate pH 5.5 Sucrose
(250mM) L-methionine
(6.7 mM) DTPA
(0.04 mM) P20
(0.04 %)
F-13 10 mM Sodium Citrate pH 5.5 Sucrose
(250 mM) NA DTPA
(0.04 mM) P20
(0.04 %)
F-14 10 mM Sodium Citrate pH 5.5 Trehalose
(272 mM) L-methionine
(6.7 mM) DTPA
(0.04 mM) P20
(0.04 %)
F-15 10 mM Sodium Citrate pH 5.5 Trehalose
(272 mM) NA DTPA
(0.04 mM) P20
(0.04 %)
F-16 10 mM Sodium Citrate pH 5.5 HP-ß-CD
(235 mM) L-methionine
(6.7 mM) DTPA
(0.04 mM) NA
F-17 10 mM Sodium Citrate pH 5.5 HP-ß-CD
(235 mM) NA DTPA
(0.04 mM) NA
Daratumumab: 20 mg / ml

Daratumumab was formulated in sodium citrate buffer of pH 5.5 ± 0.5 comprising sucrose, trehalose and HP-ß-CD at desired concentration(s), as described above. Other excipient(s), can be added as shown in Table 3. A person skilled in the art can prepare daratumumab formulation in accordance with conventional techniques such as those disclosed in the prior art10 and can fill the formulated solution in suitable container-closure system (like vials, cartridges, syringes etc.) for storage at suitable temperature for further use.
In one approach, stability of daratumumab in the said formulations was evaluated through freeze-thaw stress study (cold stress), for 10 cycles, at – 70 ?C. In another approach, stability of daratumumab in the said formulations were also assessed, under thermal stress condition at 50 °C ± 2 °C; RH 75 % ± 5 % for one month. The treated or exposed samples were analyzed for purity and size variants profile (HMW and LMW) of daratumumab by HP-SEC, under native condition, and compared with respect to the control formulation. Results are summarized in Table 7 illustrated in Figure 3 and Figure 9.The said formulations were observed to show reduced rate and extent of the formation of HMW species and LMW species of daratumumab as compared to that of the control formulation.
Example 4: Daratumumab formulations with arginine citrate buffer in combination with sugar, antioxidant, chelating agent and surfactant
Table 4: Details of Formulations
Formulation Buffer Stabilizer Antioxidant chelating agent Surfactant
F-18 29 mM Arginine Citrate pH 5.55 Sucrose
(290 mM) L-methionine
(6.7 mM) DTPA
(0.04 mM) P20
(0.04 %)
F-19 29 mM Arginine Citrate pH 5.57 Sucrose
(290 mM) NA DTPA
(0.04 mM) P20
(0.04 %)
F-20 29 mM Arginine Citrate pH 5.55 Trehalose
(290 mM) L-methionine
(6.7 mM) DTPA
(0.04 mM) P20
(0.04 %)
F-21 29 mM Arginine Citrate pH 5.55 Trehalose
(290 mM) NA DTPA
(0.04 mM) P20
(0.04 %)
F-22 29 mM Arginine Citrate pH 5.80 HP-ß-CD
(235 mM) L-methionine
(6.7 mM) DTPA
(0.04 mM) NA
F-23 29 mM Arginine Citrate pH 5.83 HP-ß-CD
(235 mM) NA DTPA
(0.04 mM) NA
Daratumumab: 20 mg / ml

Daratumumab was formulated in arginine-citrate buffer of pH 5.5 ± 0.5 comprising sucrose, trehalose and HP-ß-CD at desired concentration(s), as described above. Other excipient(s), can be added as shown in Table 4. A person skilled in the art can prepare daratumumab formulation in accordance with conventional techniques such as those disclosed in the prior art10 and can fill the formulated solution in suitable container-closure system (like vials, cartridges, syringes etc.) for storage at suitable temperature for further use.
In one approach, stability of daratumumab in the said formulations was evaluated through freeze-thaw stress study (cold stress), for 10 cycles, at – 70 ?C. In another approach, stability of daratumumab in the said formulations were also assessed, under thermal stress condition at 50 °C ± 2 °C; RH 75 % ± 5 % for one month. The treated or exposed samples were analyzed for purity and size variants profile (HMW and LMW) of daratumumab by HP-SEC, under native condition, and compared with respect to the control formulation. Results are summarized in Table 7 illustrated in Figure 4 and Figure 10. The said formulations were observed to show reduced rate and extent of the formation of HMW species and LMW species of daratumumab as compared to that of the control formulation.
Example 5: Daratumumab formulations with sodium acetate buffer in combination with sugar, antioxidant, chelating agent and surfactant
Table 5: Details of Formulations
Formulation Buffer Stabilizer Antioxidant chelating agent Surfactant
F-24 25 mM Sodium acetate pH 5.55 Sucrose
(250 mM) L-methionine
(6.7 mM) DTPA
(0.04 mM) P20
(0.04 %)
F-25 25 mM Sodium acetate pH 5.59 Sucrose
(230 mM) L-methionine
(6.7 mM) NA P20
(0.04 %)
F-26 25 mM Sodium acetate pH 5.59 Trehalose
(257 mM) L-methionine
(6.7 mM) DTPA
(0.04 mM) P20
(0.04 %)
F-27 25 mM Sodium acetate pH 5.59 Trehalose
(257 mM) NA DTPA
(0.04 mM) P20
(0.04 %)
F-28 25 mM Sodium acetate pH 5.78 HP-ß-CD
(2.8 %) L-methionine
(6.7 mM) DTPA
(0.04 mM) NA
F-29 25 mM Sodium acetate pH 5.78 HP-ß-CD
(235 mM) NA DTPA
(0.04 mM) NA
Daratumumab: 20 mg / ml

Daratumumab was formulated in sodium acetate buffer of pH 5.5 ± 0.5 comprising sucrose, trehalose and HP-ß-CD at desired concentration(s), as described above. Other excipient(s), can be added as shown in Table 5. A person skilled in the art can prepare daratumumab formulation in accordance with conventional techniques such as those disclosed in the prior art10 and can fill the formulated solution in suitable container-closure system (like vials, cartridges, syringes etc.) for storage at suitable temperature for further use.
In one approach, stability of daratumumab in the said formulations was evaluated through freeze-thaw stress study (cold stress), for 30 cycles, at – 70 ?C. In another approach, stability of daratumumab in the said formulations were also assessed, under thermal stress condition at 50 °C ± 2 °C; RH 75 % ± 5 % for one month. Further, the Formulation 24 (F-24) and Formulation 3 (F-26) were subjected to 30 °C ± 2 °C, 75 % RH ± 5 % RH for one month, to assess the stability of daratumumab in the said formulations. Additionally, photo-exposure study was conducted with F-24 and F-26 to assess the photo-stability or photo-degradation of daratumumab in the said formulations. The treated or exposed samples were analyzed for purity and size variants profile (HMW and LMW) of daratumumab by HP-SEC, under native condition, and compared with respect to the control formulation. Results are summarized in Table 7 and Table 9 and illustrated in Figure 5, Figure 11, Figure 15, Figure 16 and Figure 17. The said formulations were observed to show reduced rate and extent of the formation of HMW species and LMW species of daratumumab as compared to that of the control formulation.
Differential scanning calorimetry (DSC):
Conformational integrity through thermal denaturation of daratumumab samples was assessed by DSC in F-24 and F-26 and compared to that of the control formulation. DSC analysis was performed on MicroCal VP capillary equipment (Malvern, USA). All samples were diluted to 1 mg / mL with the respective formulation media and subjected to heat denaturation experiments by DSC, wherein temperature of the samples was increased from 10 °C to 95 °C, at an up-scanning rate of 1 °C / min. Samples were analyzed against the respective formulation media, which was considered as the blank for each sample. Thermal transitions obtained with the samples were recorded in the form of Tm values including the Tonset. Total enthalpy of thermal transitions for each sample was calculated by deconvoluting the thermograms using Origin 7 SR4 v7.0552 software via Non-2-State model for multiple transition temperatures.
Daratumumab samples were observed to show three clear thermal transitions, Tm1, Tm2 and Tm3, as illustrated in Figure 14, Figure 19 and Table 8. The first transition (Tm1) represents unfolding of CH2 domain, the second transition (Tm2) represents Fab domain unfolding and the third transition (Tm3) represents CH3 domain unfolding.
Daratumumab samples when formulated in F-24 and F-26 were observed to show better stability than the control formulation, as illustrated in Figure 14 and Figure 19. Results are presented in Table 8.
Thermal transition and total enthalpy values obtained with daratumumab in F-24 and F-26 were found to be higher than that of the control formulation, indicating a better thermodynamic stability of daratumumab in the said formulations than the control formulation. Also, the Tonset values of daratumumab in F-24 and F-26 formulations were found to be higher than that of the control formulation, indicating higher thermodynamic stability of the daratumumab in F-24 and F-26 than the control formulation.
Example 6: Daratumumab formulations with sodium glutamate buffer in combination with sugar, antioxidant, chelating agent and surfactant
Table 6: Details of Formulations
Formulation Buffer Stabilizer Antioxidant chelating agent Surfactant
F-30 10 mM Sodium glutamate pH 5.62 Sucrose
(245 mM) L-methionine
(6.7 mM) DTPA
(0.04 mM) P20
(0.04 %)
F-31 10 mM Sodium glutamate pH 5.62 Sucrose
(245 mM) NA DTPA
(0.04 mM) P20
(0.04 %)
F-32 10 mM Sodium glutamate pH 5.63 Trehalose
(257 mM) L-methionine
(6.7 mM) DTPA
(0.04 mM) P20
(0.04 %)
F-33 10 mM Sodium glutamate pH 5.63 Trehalose
(257 mM) NA DTPA
(0.04 mM) P20
(0.04 %)
F-34 10 mM Sodium glutamate pH 5.79 HP-ß-CD
(235 mM) L-methionine
(6.7 mM) DTPA
(0.04 mM) NA
F-35 10 mM Sodium glutamate pH 5.81 HP-ß-CD
(235 mM) NA DTPA
(0.04 mM) NA
Daratumumab: 20 mg / ml

Daratumumab was formulated in sodium glutamate buffer of pH 5.5 ± 0.5 comprising sucrose, trehalose and HP-ß-CD at desired concentration(s), as described above. Other excipient(s), can be added as shown in Table 6. A person skilled in the art can prepare daratumumab formulation in accordance with conventional techniques such as those disclosed in the prior art10 and can fill the formulated solution in suitable container-closure system (like vials, cartridges, syringes etc.) for storage at suitable temperature for further use.
In one approach, stability of daratumumab in the said formulations was evaluated through freeze-thaw stress study (cold stress), for 10 cycles, at – 70 ?C. In another approach, stability of daratumumab in the said formulations were also assessed, under thermal stress condition at 50 °C ± 2 °C; RH 75 % ± 5 % for one month. The treated or exposed samples were analyzed for purity and size variants profile (HMW and LMW) of daratumumab by HP-SEC, under native condition, and compared with respect to the control formulation. Results are summarized in Table 7 illustrated in Figure 6 and Figure 12. The said formulations were observed to show reduced rate and extent of the formation of HMW species and LMW species of daratumumab as compared to that of the control formulation. Freeze-thaw study experimental results:
Table: 7 Purity of daratumumab in various formulations upon repeated freeze-thaw stress

Formulations Initial 10th FT
% HMW % Principal % LMW % HMW % Principal % LMW
Control formulation 0.22 99.64 0.13 1.31 98.77 0.09
F-1 0.20 99.72 0.07 0.24 99.66 0.10
F-2 0.23 99.67 0.11 0.27 99.65 0.09
F-3 0.23 99.67 0.10 0.25 99.71 0.04
F-4 0.23 99.66 0.11 0.27 99.69 0.03
F-5 0.20 99.70 0.10 0.21 99.75 0.04
F-6 0.20 99.69 0.11 0.26 99.64 0.11
F-7 0.12 99.80 0.09 0.15 99.78 0.08
F-8 0.12 99.79 0.09 0.14 99.78 0.08
F-9 0.12 99.75 0.11 0.10 99.79 0.08
F-10 0.12 99.73 0.14 0.13 99.80 0.05
F-11 0.10 99.77 0.13 0.12 99.82 0.06
F-12 0.27 99.68 0.05 0.28 99.62 0.09
F-13 0.31 99.63 0.05 0.34 99.56 0.10
F-14 0.28 99.68 0.04 0.21 99.71 0.08
F-15 0.28 99.67 0.05 0.34 99.58 0.09
F-16 0.28 99.67 0.05 0.27 99.61 0.12
F-17 0.31 99.64 0.05 0.33 99.55 0.12
F-18 0.26 99.60 0.14 0.25 99.57 0.18
F-19 0.27 99.59 0.14 0.25 99.59 0.16
F-20 0.26 99.61 0.13 0.27 99.58 0.15
F-21 0.25 99.61 0.14 0.26 99.59 0.16
F-22 0.25 99.61 0.14 0.23 99.60 0.17
F-23 0.26 99.59 0.15 0.24 99.60 0.16
F-24 0.24 99.63 0.13 0.23 99.62 0.14
F-25 0.24 99.64 0.12 0.24 99.63 0.13
F-26 0.24 99.63 0.12 0.23 99.65 0.13
F-27 0.26 99.62 0.13 0.24 99.63 0.13
F-28 0.24 99.64 0.12 0.22 99.65 0.13
F-29 0.24 99.64 0.12 0.25 99.61 0.15
F-30 0.24 99.60 0.16 0.26 99.56 0.18
F-31 0.25 99.59 0.16 0.23 99.59 0.17
F-32 0.25 99.58 0.16 0.23 99.61 0.17
F-33 0.23 99.6 0.16 0.23 99.60 0.18
F-34 0.24 99.59 0.18 0.22 99.61 0.17
F-35 0.24 99.59 0.18 0.22 99.59 0.18

Differential scanning calorimetry (DSC) experimental results:
Table 8: Experimental data of thermal transition of daratumumab in different formulations
Sr. No Samples Thermal transitions (?C) ? H
(Cal / mol)
Ton Tm1 Tm2 Tm3 Tm4
1 Control formulation 60.16 68.89 74.32 78.22 82.79 9.15 ? 105
2 F-1 62.38 71.31 75.71 80.43 83.95 9.72 ? 105
3 F-3 62.27 71.83 75.93 80.35 84.25 10.22 ? 105
4 F-24 62.62 69.90 75.84 - 82.86 9.62 ? 105
5 F-26 56.65 64.38 74.00 75.77 81.26 10.03 ? 105

Table: 9 Purity of daratumumab in various formulations upon repeated freeze-thaw stress
Formulations Initial FT-10 FT-20 FT-30
%
HMW %
Principal %
LMW %
HMW % Principal % LMW % HMW % Principal % LMW %
HMW %
Principal %
LMW
Control
formulation 0.74 99.21 0.04 0.82 99.13 0.06 0.76 99.16 0.05 0.84 99.11 0.04
F-1 0.57 99.35 0.07 0.57 99.37 0.07 0.56 99.37 0.08 0.56 99.36 0.07
F-3 0.57 90.36 0.07 0.56 99.37 0.07 0.57 99.36 0.07 0.57 99.36 0.07
F-24 0.59 99.39 0.02 0.56 99.4 0.02 0.56 99.41 0.07 0.6 99.39 0.02
F-26 0.59 99.39 0.02 0.53 99.41 0.05 0.57 99.4 0.03 0.59 99.39 0.02

Incorporation by reference
The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.
Equivalents
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.
References
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3. Differential Fc-Receptor Engagement Drives an Anti-tumor Vaccinal Effect Author links open overlay panel David J.DiLillo1Jeffrey V.Ravetch1.
4. Patent publication: WO2020219681A1
5. Increasing Fc?RIIa affinity of an Fc?RIII-optimized anti-EGFR antibody restores neutrophil-mediated cytotoxicity Stefanie Derer,Pia Glorius,Martin Schlaeth,Stefan Lohse,Katja Klausz,Umesh Muchhal,John R Desjarlais,Andreas Humpe,Thomas Valerius &Matthias Peipp.
6. Patent application: US 2021/0393775.
7. Patent publication: US10758621.
8. de Weers et al, J. Immunol. 186:1840-1848, 2011; U.S. Pat. No. 7,829,673
9. WHO Drug Information, Vol. 24, No. 1, 2010 Recommended INN: List 63 41 International Non-proprietary Names for Pharmaceutical Substances (INN).
10. Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA1 1995.

,CLAIMS:
We claim:
1. A pharmaceutical formulation of therapeutically effective amount of daratumumab comprising chelating agent, wherein chelating agent is selected from ethylene diamine tetraacetic acid (EDTA) and diethylenetriamine pentaacetate (DTPA) along with other suitable excipient(s), wherein the suitable excipient(s) is selected from buffer, carbohydrate and suitable combination thereof.

2. The pharmaceutical formulation of therapeutically effective amount of daratumumab as claimed in claim 1 wherein buffer is selected from acetate buffer, histidine buffer, succinate buffer, citrate buffer, arginine buffer, glycine buffer, monosodium glutamate buffer, tris buffer, aspartate buffer, arginine-arginine buffer, arginine-aspartate buffer, arginine-citrate buffer, arginine-phosphate buffer, arginine-histidine buffer, arginine-succinate buffer, arginine-acetate buffer, arginine-glutamate buffer, aspartate-glutamate buffer, sodium adipic buffer, citrate-phosphate buffer wherein buffer is present in the range of about 1 mM to about 120 mM.

3. The pharmaceutical formulation of therapeutically effective amount of daratumumab as claimed in claim 1 wherein carbohydrate is selected from sucrose or Trehalose or HP-ß-CD wherein carbohydrate is present in the range of about 1 mM to about 300 mM.

4. The pharmaceutical formulation of therapeutically effective amount of daratumumab as claimed in claim 1 to claim 3 further comprising surfactant and / or amino acid wherein surfactant is polysorbate 20 present in the range of about 0.001 mg / mL to 5 mg / mL and amino acid is methionine in the range of about 1 mM to 250 mM.

5. The pharmaceutical formulation as claimed in claim 1 wherein chelating agent in the range of about 0.001 mM to about 50 mM.

6. The pharmaceutical formulation as claimed in as claimed in claim 1 to claim 5 wherein the therapeutically effective amount of daratumumab is present in the range of 1.0 % w / v (10 mg / mL) to 25 % w / v (250 mg / mL).

7. The pharmaceutical formulation as claimed in any of preceding claims selected from

a) daratumumab 20 mg / ml, 10 mM sodium succinate buffer, 257 mM Sucrose or 230 mM Trehalose or 235 mM HP-ß-CD, 0.04 mM DTPA, and / or 0.04 % Polysorbate 20 and / or 6.7 mM L-methionine;
b) daratumumab 20 mg / ml, 10 mM sodium succinate buffer, 257 mM Sucrose, 6.7 mM L-methionine, 0.04 mM DTPA, 0.04 % polysorbate 20;
c) daratumumab 20 mg / ml, 10 mM Sodium succinate buffer, 230 mM Trehalose, 6.7 mM L-methionine, 0.04 mM DTPA, 0.04% polysorbate 20;
d) daratumumab 20 mg / ml , 25 mM sodium acetate buffer, 250 mM Sucrose or 257 mM Trehalose or 2.8 % mM HP-ß-CD, 0.04 mM DTPA, and / or 0.04 % Polysorbate 20 and / or 6.7 mM L-methionine;
e) daratumumab 20 mg / ml, 25 mM sodium acetate buffer, 250 mM sucrose, 6.7 mM L-methionine, 0.04 mM DTPA, 0.04 % polysorbate 20;
f) daratumumab 20 mg / ml, 25 mM sodium acetate buffer, 257 mM trehalose, 6.7 mM L-methionine, 0.04 mM DTPA, 0.04 % polysorbate 20;
g) daratumumab 20 mg / ml, 10 mM histidine buffer, 290 mM sucrose or 290 mM trehalose or 118 mM HP-ß-CD, 0.04 mM DTPA, and / or 0.04 % Polysorbate 20 and / or 6.7 mM L-methionine;
h) daratumumab 20 mg / ml, 10 mM sodium citrate buffer, 250 mM sucrose or 272 mM trehalose or 235 mM HP-ß-CD, 0.04 mM DTPA, and / or 0.04 % Polysorbate 20 and / or 6.7 mM L-methionine;
i) daratumumab 20 mg / ml, 29 mM arginine citrate, 290 mM sucrose or 290 mM trehalose or 235 mM HP-ß-CD, 0.04 mM DTPA, and / or 0.04 % Polysorbate 20 and / or 6.7 mM L-methionine and
j) daratumumab 20 mg / ml, 10 mM sodium glutamate, 245 mM sucrose or 257 mM trehalose or 235 mM HP-ß-CD, 0.04 mM DTPA, and / or 0.04 % Polysorbate 20 and / or 6.7 mM L-methionine.

8. The pharmaceutical formulation as claimed in claims 1 to 7, wherein the formulation further comprises a hyaluronidase preferably, rHuPH20 in the range of 50 U / mL to about 5,000 U / mL.

9. The pharmaceutical formulation as claimed in claims 1 to 8 wherein the formulation is administered to a subject by subcutaneous, intravenous, intradermal, transdermal, intraperitoneal, intranasal or intramuscular route of administration.

Dated this the 4th day of January 2024.

(HARIHARAN SUBRAMANIAM)
IN/PA-93
of SUBRAMANIAM & ASSOCIATES
Attorneys for the applicants