Patent Description:
Diabetes mellitus is a metabolic disorder in which the ability to utilize glucose is more or less completely lost. For decades, insulin has been used in the treatment of diabetes mellitus for which several insulin formulations have been developed. Currently available rapid acting insulin includes insulin lispro, insulin aspart and insulin glulisine. Fast acting insulin shows faster absorption and rapid onset of action i.e. within <NUM> of administration. Thus, they are used for effective control of postprandial increase in the blood sugar level. These analogous are used for the treatment of type <NUM> (insulin-dependent) and type II (non-insulin-dependent) diabetes. For immediate and long term control of the glycemic level in the body, long acting insulin is given in combination with rapid acting insulin. Basal-bolus insulin therapy given as either multiple daily injections or by an insulin pump is a mainstay of diabetes treatment for achieving optimal glycemic control in type I diabetes. Attempts have been made to mix the different types of insulin in one injection in order to reduce the number of injections to be administered to a patient in the day. Due to difference in isoelectric points, mixing of different types of insulin prior to administration is not recommended as it leads to uncontrolled precipitation after mixing due to which it is difficult to predict the exact dose that has been administered by the patient.

Insulin glargine cannot be readily mixed with other insulin, insulin analogues or derivative having isoelectric points <NUM> to <NUM>, because the mixture causes glargine to precipitate prior to injection and administration of precipitated insulin makes it virtually impossible to administer a known and reliable dose. Several warnings have been issued by the regulatory agencies against mixing long and rapid-acting insulin's together (insulin glargine, LANTUS; Sanofi-Aventis; available from http://www. com/hcp/closing. aspx; insulin detemir rDNA origin, brand name LEVEMIR drug insert; Novo Nordisk, Bagsvaerd, Denmark).

Kaplan W et al. (<NUM>) discloses the effects of mixing glargine and short-acting insulin analogs on glucose control. Evans et al. (<NUM>) discloses that neither insulin glargine nor insulin detemir are suitable for mixing with other insulin analogues as this mixing substantially alters their pharmacokinetic properties. Lucchesi Mb et. (<NUM>) discloses mixing Insulin Lispro with Insulin glargine immediately before the subcutaneous injection decreases Insulin Lispro serum peak concentration without affecting the glycemic profile after <NUM> wk in this group with type I diabetes mellitus. <CIT> discloses biphasic pharmaceutical composition comprising an insulin analogue, derivative or metabolite having isoelectric point between <NUM> to <NUM>, zinc or salts thereof, isotonic agent optionally along with one or more pharmaceutically acceptable excipients. <CIT> discloses stabilized pharmaceutical formulations of insulin analogues and/or insulin derivatives. <CIT> discloses a pharmaceutical composition comprising readily dissociable molecular aggregates formed by combining an insulin, insulin analogue, derivative or metabolite having isoelectric point between <NUM> to <NUM> in combination with one or more an insulin, insulin analogue, derivative or metabolite having isoelectric point between <NUM> to <NUM> optionally along with one or more excipients. <CIT> discloses a pharmaceutical composition comprising human insulin, analogues or derivatives thereof, at least one or more amino acids and a halogenide optionally along with one or more pharmaceutically acceptable excipient(s).

There is a need to develop a stable and compatible insulin glargine composition which can be readily mixed with another insulin, insulin analogue or derivative having isoelectric point between <NUM> and <NUM>, and which will lead to reduction in the number of injections that a patient needs to administer.

In a first aspect of the invention, there is provided a stable biphasic pharmaceutical composition comprising: (a) insulin glargine in a suspension form, (b) insulin aspart, (c) at least two amino acids in a weight ratio of about <NUM>:<NUM>, and optionally, (d) one or more pharmaceutically acceptable excipients, wherein the pharmaceutical composition has a pH between about <NUM> to about <NUM>. The two amino acids are arginine and isoleucine. In an embodiment, insulin glargine is present in about 40IU to about 500IU. In another embodiment, insulin aspart is present in about 40IU to about 500IU. In another embodiment, the one or more pharmaceutically acceptable excipients comprise buffer, solubilising agent, isotonic agent, preservative, antioxidant, pH modifying agent or a combination thereof. In another embodiment, the composition is stable for at least <NUM> months at <NUM> and relative humidity of <NUM>%.

In a second aspect of the invention, there is provided a method of preparing a stable biphasic pharmaceutical composition comprising: (a) insulin glargine in a suspension form, (b) insulin aspart, (c) arginine and isoleucine in a weight ratio of <NUM>:<NUM>, and optionally (d) one or more pharmaceutically acceptable excipients, wherein the pharmaceutical composition has a pH between about <NUM> to about <NUM>, wherein the said method comprises steps of:.

The term "insulin" herein includes mammalian insulin, insulin human.

The term "insulin analogue" herein includes insulin NPH, insulin lispro, insulin lispro protamine, insulin glulisine, insulin aspart, insulin aspart protamine, Gly(A21) human insulin, Gly(A21) Lys(B28) human insulin, Gly(A21) Lys(B28) Pro(B29) human insulin, Gly(A21) Asp(B28).

The term "insulin derivative" herein includes B29-Nεmyristoyl-des(B30) human insulin (insulin detemir), Lys(B29)-Nε-(N-palmitoyl-. -glutamyl)-des(B30) human insulin (insulin degludec), B29-Nε-palmitoyl-des(B30) human insulin, B29-Nε-myristoyl human insulin, B29-Nε-palmitoyl human insulin, B28-Nε-myristoyl LysB28ProB29 human insulin, B28-Nε-palmitoyl LysB28ProB29 human insulin, B30-Nε-myristoyl-ThrB29LysB30 human insulin, B30-Nε-palmitoylThrB29LysB30 human insulin, B29-Nε-(N-lithocholyl-. -glutamyl)-des(B30) human insulin, B29-Nε- b(ω-carboxyheptadecanoyl)des(B30) human insulin, B29-Nε-( ω-carboxyheptadecanoyl) human insulin.

The term "stable" herein relates to a physical and/or chemical stability of pharmaceutical composition of insulin.

The term "compatible" herein relates to a physical and/or chemical compatibility of insulin glargine composition when mixed with another insulin, insulin analogue or derivative having isoelectric point between <NUM> and <NUM>.

The term "biphasic" herein relates to a composition of insulin glargine and another insulin, insulin analogue or derivative having isoelectric point between <NUM> and <NUM>, wherein insulin glargine is in suspension form and another insulin, insulin analogue or derivative having isoelectric point between <NUM> and <NUM> is in solution form.

The term "pharmaceutically acceptable excipients" herein relates to non-active pharmaceutical ingredients which are within the scope of sound medical judgment suitable for use in pharmaceutical products.

The term "isoelectric point" herein relates is the pH at which the insulin molecule carries no net electrical charge in the statistical mean.

For clarity, insulin glargine can also be denoted as Gly(A21), Arg(B31), Arg(B32) insulin human and insulin aspart can also be denoted as Asp(B28) insulin human.

In a first aspect of the invention, there is provided a stable biphasic pharmaceutical composition comprising: (a) insulin glargine in a suspension form, (b) insulin aspart, (c) arginine and isoleucine in a weight ratio of about <NUM>:<NUM>, and optionally, (d) one or more pharmaceutically acceptable excipients, wherein the pharmaceutical composition has a pH between about <NUM> to about <NUM>.

In an embodiment, the concentration of arginine or isoleucine ranges from about <NUM> to <NUM>, or about <NUM> to <NUM>, or about <NUM> to <NUM>. In an embodiment, the concentration of arginine is about <NUM> to <NUM>, or about <NUM> to <NUM>. Alternatively, the concentration of arginine is about <NUM>, or about <NUM>, or about <NUM>, or about <NUM>, or about <NUM>, or about <NUM>, or about <NUM>. In an embodiment, the concentration of isoleucine is about <NUM> to <NUM>, or about <NUM> to <NUM>. Alternatively, the concentration of isoleucine is about <NUM>, or about <NUM>, or about <NUM>, or about <NUM>, or about <NUM>, or about <NUM>, or about <NUM>, or about <NUM>, or about <NUM>, or about <NUM>, or about <NUM>, or about <NUM>. Alternatively, the concentration of arginine is about <NUM> and the concentration of isoleucine is about <NUM>.

In still another embodiment, the pharmaceutical composition is suitable for parenteral administration such as intramuscular, subcutaneous, intradermal and intravenous administration.

In another embodiment, insulin glargine is present in about 40IU to about 500IU. Alternatively, insulin glargine is present in about 80IU to about 400IU, or about 100IU to about 300IU, or about 200IU to about 300IU. Alternatively, insulin glargine is present in about 50IU, or about 60IU, or about 70IU, or about 80IU, or about 90IU, or about 100IU, or about 110IU, or about 120IU, or about 130IU, or about 140IU, or about 150IU, or about 160IU, or about 170IU, or about 180IU, or about 190IU, or about 200IU, or about 250IU, or about 300IU, or about 350IU, or about 400IU, or about 450IU. Each of this concentration constitutes an alternate embodiment of the invention. In another embodiment, insulin aspart is present in about 40IU to about 500IU. Alternatively, insulin aspart is present in about 80IU to about 400IU, or about 100IU to about 300IU, or about 200IU to about 300IU. Alternatively, insulin aspart is present in about 50IU, or about 60IU, or about 70IU, or about 80IU, or about 90IU, or about 100IU, or about 110IU, or about 120IU, or about 130IU, or about 140IU, or about 150IU, or about 160IU, or about 170IU, or about 180IU, or about 190IU, or about 200IU, or about 250IU, or about 300IU, or about 350IU, or about 400IU, or about 450IU. Each of this concentration constitutes an alternate embodiment of the invention.

The compositions of insulin glargine and insulin aspart can be provided in the form of a stable and biphasic composition and in fixed dose proportion, as percentage, <NUM>:<NUM>, or <NUM>:<NUM>, or <NUM>:<NUM>, or <NUM>:<NUM>, or <NUM>:<NUM>, <NUM>:<NUM>, or <NUM>:<NUM>, or <NUM>: <NUM> or <NUM>:<NUM>. In another embodiment, such fixed dose composition is stable for at least <NUM> months at <NUM> and relative humidity of <NUM>%.

In another embodiment, the stable and biphasic composition comprises insulin glargine in suspension form and wherein insulin glargine possess a particle size (D90) ranging from <NUM> to <NUM>, or about <NUM> to <NUM>, or about <NUM> to <NUM>.

In another embodiment, the one or more pharmaceutically acceptable excipients comprise buffer, solubilising agent, isotonic agent, preservative, antioxidant, pH modifying agent or a combination thereof.

The pH modifying agents as used herein refers to a combination of acid and alkali. The pH modifying agents can be selected from the group comprising of hydrochloric acid, o-phosphoric acid, citric acid, acetic acid, succinic acid, lactic acid, gluconic acid, tartaric acid, <NUM>,<NUM>,<NUM>,<NUM>-butane tetracarboxylic acid, fumaric acid or malic acid. Alkali is selected from the group comprising of sodium hydroxide, potassium hydroxide, sodium hydroxide, ammonium hydroxide, magnesium oxide, calcium hydroxide, calcium carbonate, magnesium carbonate, magnesium aluminum silicates, diethanolamine, monoethanolamine, sodium carbonate, sodium bicarbonate or triethanolamine and combination thereof.

The solubilizing agent are selected from the group consisting of partial and fatty acid esters and ethers of polyhydric alcohols such as of glycerol, sorbitol, glycine and the like (Span®, Tween®, in particular Tween® <NUM> and Tween®<NUM>, Myrj®, Brij®, Cremophore® or poloxamers, Pluronics® and Tetronics®), polysorbates (Tween™), sodium dodecyl sulfate (sodium lauryl sulfate), lauryl dimethyl amine oxide, cetyltrimethylammonium bromide (CTAB), polyethoxylated alcohols polyoxyethylene sorbitan, Octoxynol (Triton X100™), N,N-dimethyldodecylamine-N-oxide, hexadecyltrimethylammonium bromide (HTAB), polyoxyl <NUM> lauryl ether, Brij <NUM>™, bile salts (sodium deoxycholate, sodium cholate), polyoxyl castor oil (Cremophor™), nonylphenol ethoxylate (Tergitol™), cyclodextrins, lecithin and methylbenzethonium chloride (Hyamine™). The concentration of solubilizing agent (preferred is glycine) is about <NUM> to <NUM> or about <NUM>, or about <NUM>.

The isotonic agent as used herein includes salts, e.g., sodium chloride, dextrose, lactose or combination thereof.

The preservative as used herein include, but are not limited to, benzoic acid, butylparaben, ethyl paraben, methyl paraben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetypyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, <NUM>-penoxyethanol, phenyl mercuric nitrate, thimerosal, metacresol and combinations thereof. The concentration of preservative is about <NUM>/mL to about <NUM>/mL, or about <NUM>/ml, or about <NUM>/ml.

The antioxidant as used herein can be selected from the group comprising of ascorbate (sodium/acid), bisulite sodium, butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), cysteine / cysteinate HCl, dithionite sodium, gentisic acid, gentisic acid ethanolamine, glutamate monosodium, glutathione, formaldehyde sulfoxylate sodium, metabisulite potassium, metabisulite sodium, methionine, monothioglycerol (thioglycerol), propyl gallate, sulfite sodium, tocopherol alpha, alpha tocopherol hydrogen succinate, thioglycolate sodium and combination thereof.

The buffer as used herein include, but are not limited to, phosphate, acetate, citrate or TRIS (i.e. <NUM>-amino-<NUM>-hydroxymethyl-<NUM>,<NUM>-propanediol) buffer and corresponding salts.

In another embodiment of the invention, there is provided a stable biphasic pharmaceutical composition comprising: (a) insulin glargine in a suspension form, (b) insulin aspart, (c) arginine and isoleucine in a weight ratio of about <NUM>:<NUM>, and optionally (d) one or more pharmaceutically acceptable excipients, wherein the pharmaceutical composition has a pH between about <NUM> to about <NUM>, and wherein insulin glargine is present in about 40IU to about 500IU, and wherein inulin aspart is present in about 40IU to about 500IU, and wherein the one or more pharmaceutically acceptable excipients comprise buffer, solubilising agent, isotonic agent, preservative, antioxidant, pH modifying agent or combination thereof, and wherein the composition is stable for at least <NUM> months at <NUM> and relative humidity of <NUM>%.

In another embodiment of the invention, there is provided a stable biphasic pharmaceutical composition comprising: (a) insulin glargine in a suspension form, (b) insulin aspart, (c) arginine and isoleucine in a weight ratio of about <NUM>:<NUM>, and optionally (d) one or more pharmaceutically acceptable excipients, wherein the pharmaceutical composition has a pH between about <NUM> to about <NUM>, , and wherein insulin glargine is present in about 50IU, or about 60IU, or about 70IU, or about 80IU, or about 90IU, or about 100IU, or about 110IU, or about 120IU, or about 130IU, or about 140IU, or about 150IU, or about 160IU, or about 170IU, or about 180IU, or about 190IU, or about 200IU, or about 250IU, or about 300IU, or about 350IU, or about 400IU, or about 450IU, and wherein insulin aspart is present in about 50IU, or about 60IU, or about 70IU, or about 80IU, or about 90IU, or about 100IU, or about 110IU, or about 120IU, or about 130IU, or about 140IU, or about 150IU, or about 160IU, or about 170IU, or about 180IU, or about 190IU, or about 200IU, or about 250IU, or about 300IU, or about 350IU, or about 400IU, or about 450IU, and wherein the one or more pharmaceutically acceptable excipients comprise buffer, solubilising agent, isotonic agent, preservative, antioxidant, pH modifying agent or combination thereof, and wherein the composition is stable for at least <NUM> months at <NUM> and relative humidity of <NUM>%.

In another embodiment of the invention, there is provided a stable biphasic pharmaceutical composition comprising: (a) insulin glargine in a suspension form, (b) insulin aspart, (c) arginine and isoleucine in a weight ratio of about <NUM>:<NUM>, and optionally (d) one or more pharmaceutically acceptable excipients, wherein the pharmaceutical composition has a pH between about <NUM> to about <NUM>, and wherein insulin glargine and insulin aspart are present in a fixed dose proportion, as percentage, <NUM>:<NUM>, or <NUM>:<NUM>, or <NUM>:<NUM>, or <NUM>:<NUM>, or <NUM>:<NUM>, <NUM>:<NUM>, or <NUM>:<NUM>, or <NUM>: <NUM> or <NUM>:<NUM>.

In another embodiment of the invention, there is provided a stable biphasic pharmaceutical composition comprising: (a) insulin glargine in a suspension form, (b) insulin aspart, (c) arginine and isoleucine in a weight ratio of about <NUM>:<NUM>, and optionally (d) one or more pharmaceutically acceptable excipients, wherein the pharmaceutical composition has a pH between about <NUM> to about <NUM>, and wherein insulin glargine and insulin aspart are present in a fixed dose proportion, as percentage, <NUM>:<NUM>, or <NUM>:<NUM>, or <NUM>:<NUM>, or <NUM>:<NUM>, or <NUM>:<NUM>, <NUM>:<NUM>, or <NUM>:<NUM>, or <NUM>: <NUM> or <NUM>:<NUM>, and wherein the composition is stable for at least <NUM> months at <NUM> and relative humidity of <NUM>%.

Example <NUM> elaborates composition and manufacturing process for composition of compatible insulin glargine having a pH of <NUM>±<NUM> that is ready to mix (before injection) with insulin aspart solution or insulin human solution having a pH of <NUM>±<NUM>, in the proportion ranging from, as percentage, <NUM>:<NUM> to <NUM>:<NUM> (insulin glargine : insulin aspart or insulin human).

Defined quantity of m-cresol and glycerol (<NUM>%) were dissolved in <NUM>% final batch size of water for injection to prepare a preservative solution. The insulin glargine and zinc chloride were suspended in <NUM>% final batch size of water for injection to obtain slurry. To prepare a solution of insulin glargine, hydrochloric acid (1N) was added in the insulin glargine slurry at <NUM>/g of insulin glargine concentration (v/w). The defined quantity of glycine was then added in the dissolved insulin glargine solution and followed by addition of the preservative solution. To this, while stirring, calculated quantities of L-arginine and L-isoleucine (in the weight ratio of <NUM>:<NUM>) were added. The solution volume was made up to <NUM>% of final formulation batch size with water for injection. The pH of solution was further adjusted to <NUM>±<NUM> using hydrochloric acid (1N) or sodium hydroxide (1N). The final volume was made up to <NUM>% with water for injection. The resultant insulin glargine solution was sterile filtered using <NUM>. 2µ PES membrane filter and collected in a filling tank.

In a separate container, a defined quantity of phenol, m-cresol, glycerol (<NUM>%) and di-Sodium hydrogen phosphate dihydrate were dissolved in <NUM>% of final batch size volume of water for injection to form a preservative solution. The insulin aspart and zinc chloride were suspended in <NUM>% final batch size of water for injection to obtain slurry. To prepare a solution of insulin aspart, hydrochloric acid (1N) was added in the insulin aspart slurry at <NUM>/g of insulin aspart concentration (v/w). The prepared preservative solution was further added to above insulin aspart solution. The volume of solution from above step was made up with sterile water for injection to <NUM>% of final batch size. The solution pH was adjusted to <NUM>±<NUM> by using sodium hydroxide (1N) solution to prepare the insulin aspart solution. Post pH adjustment, volume of the above solution is made up with water for injection to <NUM>% of final batch. Prepared insulin aspart solution was filtered using <NUM>. 2µ PES membrane filter and collected in another filling tank.

In a separate container, defined quantity of m-cresol, glycerol (<NUM>%), citric acid monohydrate and tri-sodium citrate were dissolved in <NUM>% of final batch size volume of water for injection to form a preservative solution. The insulin human and zinc chloride were suspended in <NUM>% final batch size of water for injection to obtain slurry. To prepare a solution of insulin human, hydrochloric acid (1N) was added in the insulin human slurry at <NUM>/g of insulin human concentration (v/w). The prepared preservative solution was further added to above insulin human solution. The volume of solution from above step was made up with water for injection to <NUM>% of final batch size. The solution pH was adjusted to <NUM>±<NUM> by using sodium hydroxide (1N) solution to prepare the insulin human solution. Post pH adjustment, volume of the above solution is made up with water for injection to <NUM>% of final batch. Prepared insulin human solution was filtered using <NUM>. 2µ PES membrane filter and collected in another filling tank.

The solution formulations of insulin glargine and insulin aspart or insulin human are ready to be mixed with each other in the proportion ranging from, as percentage, <NUM>:<NUM> to <NUM>:<NUM>. Since these solutions are mixed before injection, therefore solutions are filled separately in the suitable container closure systems and can be mixed just before the injection in a suitable sterile container system. For instance, the final solution containing insulin glargine and insulin aspart or insulin human (100IU/ml, <NUM>/<NUM>) was prepared by mixing <NUM> of insulin glargine solution (100IU/ml) and <NUM> of insulin aspart or insulin human solution (100IU/ml) and the final solution containing insulin glargine and insulin aspart or insulin human (200IU/ml, <NUM>/<NUM>) was prepared by mixing <NUM> of insulin glargine solution (200IU/ml) and <NUM> of insulin aspart or insulin human solution (200IU/ml) in a suitable container has following composition:.

Three comparative compositions were made: composition devoid of L-isoleucine (2A), and composition devoid of L-arginine (2B) and altering the weight ratio of L-arginine and L-isoleucine that is <NUM>:<NUM> (2C). These compositions were compared with the composition of Example <NUM> (solution composition of insulin glargine and insulin aspart, 200IU/ml, not according to the invention).

In Example <NUM> compositions and comparative compositions, where the contact time (before injection in a syringe) for two components (insulin glargine and insulin aspart) is expected to be less than <NUM> seconds to about <NUM> minutes, the stability of acid labile insulin aspart part was carried up to <NUM> hours at <NUM> and relative humidity of <NUM>%. Before analysis, to separate the two insulin's components from each other, samples were passed through cation-exchange column. This was followed by analysis of related impurities using-"<NPL>".

As evident from the stability data that, the formation of high molecular weight impurities and related impurities with respect to insulin aspart in a premix with insulin glargine are more in comparative composition 2A, 2B and 2C than in Example <NUM> composition (not according to the invention).

Three comparative compositions were made: composition devoid of L-isoleucine (3A), and composition devoid of L-arginine (3B) and altering the weight ratio of L-arginine and L-isoleucine that is <NUM>:<NUM> (3C). These compositions were compared with the composition of Example <NUM> (solution composition of insulin glargine and insulin human, 200IU/ml, not according to the invention).

In Example <NUM> compositions and comparative compositions, where the contact time (before injection in a syringe) for two components (insulin glargine and insulin human) is expected to be less than <NUM> seconds to about <NUM> minutes, the stability of acid labile insulin human part was carried up to <NUM> hours at <NUM> and relative humidity of <NUM>%. Before analysis, to separate the two insulin's components from each other, samples were passed through cation-exchange column. This was followed by analysis of related impurities using-"<NPL>".

As evident from the stability data that, the formation of high molecular weight impurities and related impurities with respect to insulin human in a premix with insulin glargine are more in comparative composition 3A, 3B and 3C than in Example <NUM> composition of the invention.

Example <NUM> of elaborates composition and manufacturing process for composition of compatible insulin glargine suspension having a pH of <NUM>±<NUM> formulated with insulin aspart or insulin human solution having a pH <NUM>±<NUM>. The insulin glargine and insulin aspart or insulin human can be mixed in the proportion ranging from, as percentage, <NUM>:<NUM> to <NUM>:<NUM> (insulin glargine : insulin aspart or insulin human).

In a separate container, a defined quantity of phenol, m-cresol, glycerol (<NUM>%) and di-Sodium hydrogen phosphate dihydrate were dissolved in <NUM>% of final batch size volume of water for injection to form a preservative solution. The insulin aspart and zinc chloride were suspended in <NUM>% final batch size of water for injection to obtain slurry. To prepare a solution of insulin aspart, hydrochloric acid (1N) was added in the insulin aspart slurry at <NUM>/g of insulin aspart concentration (v/w). The prepared preservative solution was further added to above insulin aspart solution. The volume of solution from above step was made up with sterile water for injection to <NUM>% of final batch size. The solution pH was adjusted to <NUM>±<NUM> by using sodium hydroxide (1N) solution to prepare the insulin aspart solution. Post pH adjustment, volume of the above solution is made up with water for injection to <NUM>% of final batch.

In a separate container, defined quantity of m-cresol, glycerol (<NUM>%), citric acid monohydrate and tri-sodium citrate were dissolved in <NUM>% of final batch size volume of water for injection to form a preservative solution. The insulin human and zinc chloride were suspended in <NUM>% final batch size of water for injection to obtain slurry. To prepare a solution of insulin human, hydrochloric acid (1N) was added in the insulin human slurry at <NUM>/g of insulin human concentration (v/w). The prepared preservative solution was further added to above insulin human solution. The volume of solution from above step was made up with water for injection to <NUM>% of final batch size. The solution pH was adjusted to <NUM>±<NUM> by using sodium hydroxide (1N) solution to prepare the insulin human solution. Post pH adjustment, volume of the above solution is made up with water for injection to <NUM>% of final batch.

The suspension of insulin glargine and solution of insulin aspart or insulin human can be mixed with each other in the proportion ranging from, as percentage, <NUM>:<NUM> to <NUM>:<NUM>. The final suspension containing insulin glargine and insulin aspart or insulin human (100IU/ml, <NUM>/<NUM>) was prepared by mixing <NUM> of insulin glargine suspension (100IU/ml) and <NUM> of insulin aspart or insulin human solution (100IU/ml) and the final suspension containing insulin glargine and insulin aspart or insulin human (200IU/ml, <NUM>/<NUM>) was prepared by mixing <NUM> of insulin glargine suspension (200IU/ml) and <NUM> of insulin aspart or insulin human solution (200IU/ml) in a suitable container and has following composition:.

Three comparative biphasic compositions were made: composition devoid of L-isoleucine (5A), and composition devoid of L-arginine (5B) and altering the weight ratio of L-arginine and L-isoleucine that is <NUM>:<NUM> (5C). These compositions were compared with invention composition of example <NUM> (Biphasic composition of insulin glargine and insulin aspart, 200IU/ml).

In Example <NUM> (according to the invention) compositions and comparative compositions, where the contact time for two components (insulin glargine and insulin aspart) is expected to be at least <NUM> months, the stability of insulin glargine part was carried up to <NUM> months at <NUM> and relative humidity of <NUM>% (accelerated stability condition; considered as indicator of product stability at <NUM>-<NUM> up to <NUM> months). Before analysis, to separate the two insulin's components from each other, samples were passed through cation-exchange column. This was followed by analysis of related impurities using-"<NPL>".

As evident from the stability data that, the formation of high molecular weight impurities and related impurities with respect to insulin glargine in a biphasic composition are more in comparative composition 5A, 5B and 5C than in Example <NUM> composition of the invention.

Three comparative biphasic compositions were made: composition devoid of L-isoleucine (6A), and composition devoid of L-arginine (6B) and altering the weight ratio of L-arginine and L-isoleucine that is <NUM>:<NUM> (6C). These compositions were compared with a composition of Example <NUM> (biphasic composition of insulin glargine and insulin human, 200IU/ml).

In Example <NUM> (not according to the invention) compositions and comparative compositions, where the contact time for two components (insulin glargine and insulin human) is expected to be at least <NUM> months, the stability of insulin glargine part was carried up to <NUM> months at <NUM> and relative humidity of <NUM>% (accelerated stability condition; considered as indicator of product stability at <NUM>-<NUM> up to <NUM> months). Before analysis, to separate the two insulin's components from each other, samples were passed through cation-exchange column. This was followed by analysis of related impurities using-"<NPL>".

Claim 1:
A stable biphasic pharmaceutical composition comprising: (a) insulin glargine in a suspension form, (b) insulin aspart, (c) arginine and isoleucine in a weight ratio of <NUM>:<NUM>, and optionally, (d) one or more pharmaceutically acceptable excipients, wherein the pharmaceutical composition has a pH between <NUM> to <NUM>.