DESC:FORM 2

THE PATENTS ACT, 1970

(39 OF 1970)

&

THE PATENTS RULES, 2003

COMPLETE SPECIFICATION
(See section 10; rule 13)

Title: A topical Insulin Eye drop formulation and method thereof

APPLICANT DETAILS:
(A) NAME: Entod Pharmaceuticals Ltd.

(B) NATIONALITY: Indian
(C) ADDRESS: Ashirwad Building, Swami Vivekananda Rd, Opp. Badi Masjid,
Santosh Nagar, Bandra West, Mumbai, Maharashtra 400050

PREAMBLE TO THE DESCRIPTION:
The following specification (particularly) describes the nature of the invention (and the manner in which it is to be performed):

A topical Insulin Eye drop formulation and method thereof

FIELD OF THE INVENTION:
The invention relates to the field of topical insulin eye drop formulation. Particularly, the present invention provides a topical insulin eye drop formulation and its methods of preparation. The formulation of the present invention treats neurotrophic keratitis (NK) caused by damage to the trigeminal nerve and abolishes both tearing and blinking reflexes, causing severe dry eye disease. The formulation is also useful for treating refractory neurotrophic corneal ulcers, which arise as a result of a rupture or a defect in the corneal epithelium, further causing an underlying inflammation and often giving rise to necrosis of the corneal stroma.

BACKGROUND OF THE INVENTION:
The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Neurotrophic keratopathy is characterized by impaired corneal innervation. A pathognomonic symptom of neurotrophic keratopathy is corneal hypoesthesia, which denotes diminished corneal sensation. This impairment can arise following viral corneal infections (e.g., HSV, VZV) in the context of systemic diseases (such as diabetes mellitus and multiple sclerosis) or can be due to iatrogenic causes such as corneal surgeries and posterior vitrectomies. Regardless of the cause, however, the clinical severity of neurotrophic keratopathy remains poorly elucidated, especially in terms of restoring corneal sensation.
Associated symptoms include irritation, foreign body sensation, conjunctival edema, hyperemia and blurred vision. Early diagnosis and early initiation of appropriate treatment are essential as lack of treatment may lead to vision loss due to corneal opacity, as well as persistent epithelial defects.
Insulin is a biologically active peptide closely related to insulin-like growth factor (IGF) that can stimulate the hepatotactic migration of human epidermal keratinocytes and is involved in cell growth, proliferation, metabolism and wound healing. The mechanism by which TI improves corneal wound healing is not yet fully understood. Insulin is found in the tear film of the eye. Insulin receptors are found in the corneal epithelium and ocular surface tissue. The presence of insulin and insulin receptors on the cornea and lacrimal glands suggests that insulin may contribute to corneal wound healing also detected insulin in tears and the expression of the insulin receptor and IGF-1 receptor (IGF-1R) on the human ocular surface. IGF-1 promotes corneal epithelial healing by increasing cell proliferation. The topical application of insulin can stimulate IGF-1R and treat DK.
Developing insulin eye drops involves solving several technical challenges to ensure their safety, efficacy. Following are some key technical problems that need to be addressed in the development of insulin eye drops:
• Stability of Insulin: Insulin is a protein molecule that is susceptible to degradation, especially in liquid formulations. Maintaining the stability of insulin in the eye drop formulation is critical to ensure its effectiveness over time. This involves selecting appropriate Excipients, Stabilizers, Buffers, Preservatives and storage conditions to prevent degradation during manufacturing, storage, and administration.
• Ocular Penetration: The eye has multiple barriers, including the cornea and conjunctiva, which limit the penetration of drugs into the ocular tissues. Overcoming these barriers to ensure sufficient penetration of insulin into the target tissues of the eye, such as the retina, is essential for therapeutic efficacy. This may involve optimizing the size, charge, and lipophilicity of insulin molecules or using permeation enhancers to improve ocular penetration.
• Bioavailability: Achieving adequate bioavailability of insulin from the eye drops is crucial for achieving therapeutic effects while minimizing systemic exposure. Factors such as drug concentration, formulation viscosity, and tear turnover rate can influence the bioavailability of insulin in the eye. Formulation optimization and pharmacokinetic studies are necessary to maximize ocular bioavailability while minimizing systemic absorption.
• Patient Comfort and Tolerability: Insulin eye drops should be well-tolerated by patients and should not cause irritation, stinging, or other adverse effects that could affect compliance with treatment. Formulation optimization to minimize ocular irritation, as well as conducting clinical trials to assess patient comfort and tolerability are important technical considerations in the development process.
• Regulatory Compliance: Developing insulin eye drops that meet regulatory requirements for safety, efficacy and quality is a complex technical challenge. This involves conducting preclinical studies to assess toxicity, pharmacokinetics, and pharmacodynamics, as well as designing and conducting clinical trials to demonstrate therapeutic efficacy and safety in humans.
Hence, there is a need of an efficient topical insulin eye drop formulation which can address following factors:
• Targeted Delivery to the Eye: The eye is a complex organ with various tissues and structures, including the cornea, conjunctiva, and retina. Thus, there is a need of topical Insulin eye drop formulation which target specific tissues within the eye.
• Enhanced Bioavailability: There is a need of Insulin eye drop formulation which improves the bioavailability and promotes rapid absorption compared to other delivery methods.
• There is also a need of a stable and effective formulation of insulin suitable for ocular administration.
To address the above-mentioned technical problems, the present invention provides an efficient topical insulin eye drop formulation, which can provide the above mentioned technical advantages for the treatment of neurotrophic keratitis (NK) and associated symptoms.
More Specifically, the invention is directed to Neurotrophic keratitis (NK) and improved re-epithelialization of ulcers refractory to standard treatment. The formulation of the present invention is considered as a second-line treatment after the lack of improvement with autologous serum eye drops.

OBJECTIVE OF THE INVENTION:
The primary object of the present invention is to overcome the drawbacks associated with prior art.
Another object of the present invention provides an insulin eye drop formulation for the treatment of neurotrophic keratitis and corneal ulcers.
Another object of the present invention is to provide an insulin eye drop formulation which has the potential to reduce certain side effects such as Inflammation, pain, swelling, infection at site of application compared with traditional insulin delivery methods, such as injection site reactions or hypoglycemia.
Another object of the present invention is to provide an insulin eye drop formulation which has rapid absorption of drugs into systemic circulation of eye and could potentially lead to faster onset of action.
Another object of the present invention is to provide an insulin drop formulation which can be administered through eye, particularly in cases where traditional treatments may not be effective.
Another object of the present invention is to provide an insulin drop formulation which promotes healing and reduce inflammation efficiently.
Another object of the present invention is to provide an insulin drop formulation with efficient targeted delivery of drugs to specific tissues.
Another object of the present invention is to provide an insulin drop formulatio which can target the tissues of the eye that are affected by conditions such as diabetic retinopathy or macular edema, providing localized treatment.
Another object of the present invention is to provide an insulin drop formulation which causes rapid absorption of drugs into systemic circulation. This could potentially lead to faster onset of action compared to other insulin delivery methods.
Another object of the present invention is to provide a method of preparing insulin drop formulation, with the technical advantages mentioned above.

SUMMARY OF THE INVENTION:
The Invention provides a topical insulin eye drop formulation comprising:
a) Human Insulin;
b) Phosphate buffer;
c) Propylene Glycol, present in an amount of 0.3%(w/v);
d) Polyethylene Glycol 400, present in an amount of 0.4%(w/v);
e) Disodium EDTA, present in an amount of 0.05 %(w/v);
f) Sodium perborate, present in an amount of 0.005 %(w/v);
g) Sodium chloride, present in an amount of 0.8% (w/v);
h) Sodium Hydroxide present in an amount to obtain the pH of 7.0;
i) Hydrochloric acid in an amount to obtain the pH of the formulation as 7.0.
The Invention provides also provides a method of preparing the topical insulin eye drop formulation, comprising the steps of:
a) Collecting 80% batch size of Water for injection purged with nitrogen where the temperature is maintained at 2°C to 8°C and pH of approximately 5.96;
b) Dispensing and adding the Di sodium EDTA and Sodium Perborate under continuous stirring for 10 minutes;
c) adding Propylene glycol, Polyethylene glycol 400, Sodium Dihydrogen phosphate (anhydrous), Sodium dihydrogen Phosphate (Dihydrate) under continuous stirring for 20 min and adjusting the pH to 3.5 - 4.0 by adding HCl or Sodium Hydroxide solution;
d) allowing the resulting solution to cool at temperature at 2°C - 8°C, followed by adding insulin and stirring for 10 minutes, to obtain the formulation of pH 7.

DETAILED DESCRIPTION OF THE DRAWINGS:
To further clarify the advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which are illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting in their scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings in which:
Fig. 1: illustrates the working mechanism of formulation of the present invention.

DETAILED DESCRIPTION:
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.
Before defining the formulation of the present invention, it is important to highlight (mentioned below) the key parameters required to prepare a formulation:
Formulation Development:
1. Selection of Active Pharmaceutical Ingredient (API): Selection of insulin in ophthalmic preparation several criteria need consideration to ensure safety and efficacy.
• Purity and Quality: Insulin API should meet strict purity standards to ensure safety and efficacy in ocular applications. High-quality insulin API minimizes the risk of impurities that could cause adverse effects or decrease efficacy.
• Biocompatibility: The selected insulin API should be biocompatible with ocular tissues to prevent irritation or inflammation upon administration. Compatibility studies should be conducted to assess the API's interaction with ocular tissues.
• Solubility: Insulin API should be sufficiently soluble in the chosen formulation to ensure uniform distribution and adequate dosage delivery to the eye. Poor solubility can lead to formulation challenges and inconsistent drug delivery.
• Bioavailability: The selected insulin API should have optimal bioavailability when administered to the eye, ensuring effective therapeutic concentrations are achieved at the target site. Factors such as formulation design and delivery method can influence bioavailability.
• Safety Profile: Comprehensive safety data, including potential adverse effects and toxicity, should be evaluated for the insulin API. Studies assessing ocular tolerance and systemic effects should be conducted to ensure patient safety.
• Regulatory Compliance: Insulin API used in eye preparations must comply with regulatory guidelines and pharmacopeial standards to ensure product quality, safety, and efficacy. Compliance with Good Manufacturing Practices (GMP) is essential for regulatory approval.
• Manufacturability: Consideration should be given to the manufacturability of the insulin API, including scalability, reproducibility, and cost-effectiveness of production processes.
• Compatibility with Excipients: Insulin API should be compatible with excipients used in the formulation to ensure stability and efficacy. Compatibility studies should be conducted to assess potential interactions between the API and excipients
In an embodiment, the invention comprises regular, Insulin aspart and recombinant form of human insulin. The Invention may comprise other forms of Insulin alone or in combination with other agent as well.
2. Insulin Stability: Insulin, as a peptide hormone drug, is susceptible to changes in stability when exposed to environmental factors under storage, light, Humidity, Temperature.
Insulin API should exhibit stability in the formulation and upon storage to maintain its potency and efficacy throughout the product's shelf life. Stability testing under relevant storage conditions is essential to ensure product quality.
3. Excipients Selection: Excipients like stabilizers, buffers, preservatives are chosen to maintain insulin stability and ensure compatibility with ocular tissues. In an embodiment, the present invention also comprises excipient generally used / known for a topical eye drop formulation.
In another embodiment, the present invention also comprises excipient generally used / known for a topical Insulin eye drop formulation.
4. Buffering Agents: Insulin formulations require a pH within a specific range to maintain stability and activity.
In an embodiment, the present invention comprises buffering agents such as phosphate buffers, borate buffers, citrate buffers that can help maintain the desired pH and prevent degradation of insulin. However, the Invention may comprise other type of Buffering agents alone or in combination with other agent as well.
5. Preservatives: Eye drop formulations require preservatives to prevent microbial contamination during storage and use. Sodium perborate preservatives used in ophthalmic preparations. However, preservatives should be carefully selected to ensure compatibility with insulin and minimize potential adverse effects on ocular tissues.
In an embodiment, the present invention comprises Sodium perborate preservative. However, the Invention may comprise other type of antioxidant/s or in combination with other agent as well.
6. Stabilizers/ Chelating agent: Stabilizers are used to protect insulin from degradation caused by factors such as temperature, light, or oxidation.
Figure 1 illustrates mechanism of the working of present invention. The insulin-like growth factor family of ligands (IGF-I, IGF-II, and insulin), receptors, and IGF-binding proteins are crucial participants in both human physiology and the process of tissue repair. So far, six IGFBP proteins have been recognized. They are responsible for preventing IGF-1-induced IGF-1R activation by binding to IGF-1 to prolong its half-life in the circulation. The insulin-like growth factor family plays a pivotal role in maintaining corneal integrity, as well as cell proliferation, migration, and metabolic regulation.
Receptors for IGF type 1 (IGF-1R), IGF type 2 (IGF-2R), insulin receptor (INSR), and the hybrid of IGF-1R and INSR (Hybrid-R) are present in the cornea. These receptors have different structures. For example, IGF-1R and INSR are similar to each other. They are transmembrane glycoproteins consisting of two beta subunits, each incorporating an intracellular tyrosine kinase domain, and two alpha subunits each with an extracellular ligand-binding domain. IGF-2R, on the other hand, is a monomeric protein with 15 different extracellular domains.
These receptors also occur intracellularly, performing specific roles. IGF-1R, INSR, and their hybrid molecule influence mitochondrial stability and also play a role in the expression of nuclear genes. IGF-1 and IGF-2 are among the most important polypeptide growth factors that act in all cellular layers of the cornea.
Receptors for IGF type 1 (IGF-1R), IGF type 2 (IGF-2R), insulin receptor (INSR), and the hybrid of IGF-1R and INSR (Hybrid-R) are present in the cornea. These receptors play a pivotal role in maintaining corneal integrity, as well as cell proliferation, migration, and metabolic regulation. Glucose uptake occurs via an alternative route through the GLUT1 transporter. IGF-1R, INSR, and their hybrid molecule influence mitochondrial stability and also play a role in the expression of nuclear genes. External modulation is performed by insulin-like growth factor-binding proteins (IGFBP), which bind to IGF-1, extending its half-life and inhibiting the activation of the IGF-1R receptor.
In an embodiment, the present invention comprises stabilizers comprising antioxidants like ascorbic acid or sodium metabisulfite and or Di sodium Edetate as a chelating agent. The Invention may comprise other type of antioxidant/s alone or in combination with other agent as well.
In an embodiment, the present invention provides an insulin eye drop formulation which has the potential to reduce certain side effects such as Inflammation, pain, swelling, infection at site of application.
In an embodiment, the present invention provides an insulin eye drop formulation which has rapid absorption of drugs into systemic circulation of eye and could potentially lead to faster onset of action.
In an embodiment, the present invention provides a topical Insulin Eye drop formulation of preferably formulated as Sterile, isotonic Solutions.
In an embodiment, the drug formulation comprises the strength in the range of 1IU to 100IU, particularly 1IU/ml, 5 IU/ml 12.5IU/ml and 25IU/ml
In an embodiment, the drug product contains a Sodium perborate as an antimicrobial agent in an amount sufficient to meet or exceed the PET requirements of the USP, glycerin and propylene glycol.
In an embodiment of the present invention, the concentration of Sodium perborate is in the range of 0.01 to 0.05 w/v %, the concentration of glycerin is in the range of 0.5 to 2.0 w/v %, and the concentration of propylene glycol 0.5 -2.0% w/v.
In an embodiment of the present invention, the invention has packaging for Insulin Eye Drops is LDPE bottles with Nozzles and HDPE cap.
In an embodiment, the composition provided by the present invention is mentioned below:
Sr. No Ingredients Qty mg/ml Qty % w/v Batch size: (250ml)
Qty (gm)
1 Human Insulin (r-DNA origin) 0.0347 0.00347 0.008675
2 Propylene Glycol 3 0.3 0.75
3 Polyethylene Glycol 400 4 0.4 1.00
4 Disodium Hydrogen Phosphate (Anhydrous) 0.1 0.01 0.025
5 Sodium Dihydrogen Phosphate
(Dihydtate) 0.06 0.006 0.015
6 Disodium EDTA 0.5 0.05 0.125
7 Sodium perborate 0.05 0.005 0.0125
8 Sodium chloride 8 0.8 2.00
9 Sodium Hydroxide q.s to pH 3.5-4.0 q.s to pH 3.5-4.0 q.s to pH 3.5-4.0
10 Hydrochloric acid q.s to pH 3.5-4.0 q.s to pH 3.5-4.0 q.s to pH 3.5-4.0
11 Water for injection q.s to ml q.s to 100ml q.s to 250ml

In an embodiment, the present invention provides manufacturing process based on the Cold process at 2-8°C of active pharmaceutical ingredient with other excipients. Manufacturing process include use of Glycerin and Propylene glycol and Sodium perborate in formulation.
In an embodiment, the present invention has manufacturing process as described below.
Preparation of solution:
Step- 1. Collect the 90% Batch size of Water for injection (Temperature 2-8°C) in manufacturing vessel, which is further purged with nitrogen throughout manufacturing process.
Step-2. Add and dissolve Glycerin and propylene glycol in step (1) and stir for 15 min
Step-3. Add and dissolve Sodium perborate in step 2 solution with under stirring for 15 min to get clear solution.
Step-4. Maintain the temperature of bulk solution at 2-8°C. Add and dissolve Human Insulin under stirring for 5 min to get clear solution.
Step-5. Check pH of solution and pH limit is 3.0 -8.0, Preferably 3.8 - 4.0 by using 0.1N Hydrochloric acid/ 0.1N Sodium hydroxide solution.
Step- 6. Make up the final volume of solution by using previously nitrogen purged water for injection and stir for 15 min.
Step-7. Filter the solution by using 0.2 µ Nylon 66 Filter.
Step-8. Filtered solution is filled in 5mL & 10mL LDPE bottles with nozzles and HDPE cap.
In an embodiment, the present invention provides insulin eye drops formulated to target specific tissues within the eye with enhanced bioavailability and rapid absorption compared to other delivery methods.
In an embodiment, the present invention provides insulin eye drops that minimize certain systemic side effects associated with traditional insulin administration.
In an embodiment, the present invention composition uses insulin is a protein molecule that is susceptible to degradation, especially in liquid formulations. Maintaining the stability of insulin in the eye drop formulation is critical to ensure its effectiveness over time. This involves selecting appropriate excipients, stabilizers, buffers, preservatives and storage conditions to prevent degradation during manufacturing, storage, and administration. The present invention composition is stable.
The drug formulation is further tested for pH Optimization. The In an embodiment, the pH of the formulation is optimized to ensure insulin stability and ocular site of installation. Drug product stability over this pH range is studied to mitigate the risk associated with change in pH between 3.0 to 8.0 this ranges, preferably pH 3.0 to 5.0
In an embodiment, following pH were evaluated for evaluating product (drug formulation) stability.
- pH at Lower side at pH 3.00
- pH at middle side at pH 4.00
- pH at Higher side at pH 5.00
In an embodiment, effect of Osmolarity Adjustment is evaluated. Formulation osmolarity is adjusted to match the tear film osmolarity for improved ocular tolerance. In an embodiment, Osmolarity range is studied in between 320 to 500 mOsm/kg, preferably 280 to 400 mOsm/kg.
The effect of inert gas is evaluated. Batches were compounded with and without nitrogen, to evaluate the effect of nitrogen on product CQA’s.
The effect of Temperature (Autoclaving) is also evaluated. Batches were compounded with and without autoclaving to study the impact of autoclaving on drug product attributes.
The Compatibility study is also performed. The preformulation study was performed to evaluate the compatibility of product with materials which come in contact with the product during manufacturing. Compatibility study was carried out with S.S vessel, Silicon tubing’s, Nylon, PVDF and PES filters. Compatibility study results indicate that drug product was compatible with the product contact materials like. S.S vessel, Silicon tubings, Nylon, PVDF and PES filters.
In the photostability, Batches are compounded and subjected to ICH Photostability conditions. The drug product samples were placed in primary pack, secondary pack and as dark control samples (wrapped in aluminium foil).
The drug formulation will also be tested as per below studies:
• Preservative efficacy studies (Sodium perborate)
• Extractable and Leachable Study (Stability time point 3M, 6M)
• Sterility
• Pharmacokinetic Studies: Following studies were conducted to study the pharmacokinetic properties:
Absorption Studies: Conducting in vitro and in vivo studies to evaluate the absorption kinetics of insulin from the eye drop formulation.
Tissue Distribution: Investigating the distribution of insulin within ocular tissues to assess its bioavailability and targeting efficacy.
Ocular Irritation Testing: Conducting ocular irritation studies to evaluate the safety and tolerability of the eye drop formulation.
In vitro and In vivo Toxicity Testing: Assessing the cytotoxicity and acute/chronic toxicity of the formulation using cell culture and animal models.
Clinical Trials: Conducting phase I, II, and III clinical trials to evaluate the safety, efficacy, and tolerability of insulin eye drops in patients.
Topical administration of insulin eye drops is an effective and safe for the treatment of neurotrophic keratitis, demonstrating potential for promoting epithelial regeneration and improving corneal barrier function. The concentration and frequency of administration should be tailored to the severity of the local condition and adjusted based on the initial therapeutic response. Improvement is usually rapid. Although insulin has an important role to play in the treatment of corneal epitheliopathies, therapies (anti-glaucoma, antibiotic, anti-inflammatory) that do not adversely affect corneal re-epithelialization should not be overlooked. The invention is essential to elucidate the precise mechanism of action of topical insulin in healing ulcers and epithelial defects and to determine the optimal concentration of insulin in eye drops for the treatment of epithelial damage.
The Invention is further described with the help of non-limiting examples:
Example 1:
The following excipients have been selected for the formulation of insulin eye drops (1 IU/mL to 25 IU/mL) based on compatibility with recombinant human insulin, safety in ophthalmic use, and regulatory acceptability. Each excipient serves a functional role in maintaining stability, tonicity, pH, and microbial control.
Excipient Function Concentration Roles
Propylene Glycol Co-solvent, Humectant 0.1-10% Improves solubility of insulin, stabilizes protein structure, and reduces surface adsorption; widely used in ophthalmic solutions.
Polyethylene Glycol 400 (PEG 400) Viscosity enhancer, Humectant 0.1–1% Enhances ocular retention time and provides comfort; compatible with insulin and safe for ophthalmic use.

Disodium Hydrogen Phosphate (Anhydrous) Buffer component q.s. for pH 7.2–7.4 Maintains pH in physiological range, essential for insulin stability. Used widely in ophthalmic preparations.
Sodium Dihydrogen Phosphate (Dihydrate) Buffer component q.s. for buffer system Used in combination with disodium phosphate to create a phosphate buffer; supports pH control without interfering with insulin.
Sodium Perborate Preservative 0.015–0.05% On contact with tear film, converts to hydrogen peroxide and then water + oxygen; used in oxidative preservative systems like GenAqua®. Less irritating than BAK.
Sodium Chloride Tonicity adjuster q.s. to 280–350 mOsm/kg Ensures isotonicity for ocular comfort; physiologically compatible.
Di sodium EDTA Chelating agent; Stabilizer 0.02 to 0.1% Disodium EDTA acts as a chelating agent, primarily to remove calcium deposits from the cornea, It also enhances the stability and effectiveness of solutions by binding to metal ions that could interfere with the formulation or its preservatives

Example 2:
Selection of Buffer System:
To identify a suitable buffer system that maintains insulin stability, ensures ocular compatibility, and provides pH control during the product’s shelf-life, various experiments were performed. Buffers were evaluated for their compatibility with insulin and excipients, stability under various storage conditions, and ability to maintain the formulation pH within the target range.
The impact of buffer systems i.e. Borate, Citrate, and Phosphate was determined on the initial stability and quality of insulin eye drop formulations at 25 IU/ml. It was observed that Phosphate buffer offers the best balance of low impurity levels and adequate assay performance, making it most suitable for long-term stabilization of insulin at pH 7.0, as shown in the below table. The citrate and Borate buffer suffered with the drawback of higher osmolality and relatively elevated A2 impurity, as compared to the Phosphate Buffer.
Test parameter Strength: 25IU/ml
Borate Buffer Citrate Buffer Phosphate Buffer
Initial Initial Initial
Description Clear colourless solution Clear colourless solution Clear colourless solution
pH 4.00 6.46 6.66
Osmolality (mOsm/kg) 366 340 329
Assay of Insulin (%)
With A21 97.90 89.39 100.58
Related substances (%)
Single maximum unknown impurity ND ND ND
A2 related impurity (NMT-5.0%) 0.875 0.446 0.449
Total impurity (%) 0.385 0.290 0.367

Thus, phosphate buffer was found to be the most suitable buffer system for further development of the insulin eye drop formulation. It demonstrated the highest assay value (100.58%), maintained a clear and stable solution, and showed acceptable levels of related impurities, including A2-related impurities well below the acceptable threshold (NMT 5.0%). The phosphate buffer also helps in maintaining initial stability profile and compatibility with insulin which improved product performance. Therefore, phosphate buffer is selected as the buffering agent for formulation optimization and stability studies.
Optimization of Phosphate Buffer concentration:
Three combinations of Disodium Hydrogen Phosphate (Anhydrous) and Sodium Dihydrogen Phosphate (Dihydrate) were evaluated at varying concentrations to establish a robust and reproducible pH range (3.0–7.0) suitable for insulin stability and patient comfort.
Name of buffering agent Concentrations (%w/v)
Trial-1 Trial-2 Trial-2
Disodium Hydrogen Phosphate (Anhydrous) 0.05 0.01 0.1
Sodium Dihydrogen Phosphate (Dihydrate) 0.03 0.006 0.06

Formulation composition given as below,
Insulin eye drops, 25IU/ml
Sr. No Ingredients Trial-1 Trial-2/ present method Trial-3
Qty % w/v Qty % w/v Qty % w/v
1 Human Insulin (r-DNA origin) 0.00347 0.00347 0.00347
2 Propylene Glycol 0.3 0.3 0.3
3 Polyethylene Glycol 400 0.4 0.4 0.4
4 Disodium Hydrogen Phosphate (Anhydrous) 0.05 0.01 0.1
5 Sodium Dihydrogen Phosphate
(Dihydtate) 0.03 0.006 0.06
6 Disodium EDTA 0.05 0.05 0.05
7 Sodium perborate 0.005 0.005 0.005
8 Sodium chloride 0.8 0.8 0.8
9 Sodium Hydroxide q.s to adjust pH 7.0 q.s to adjust pH
7.0 q.s to adjust pH
7.0
10 Hydrochloric acid q.s to adjust pH
7.0 q.s to adjust pH
7.0 q.s to adjust pH
7.0
11 Water for injection q.s to 100ml q.s to 100ml q.s to 100ml

Observations from the above trials:
1. Stability of Insulin: Insulin can degrade in high pH or when exposed to stress (like heat or light). A phosphate buffer at the right concentration helps maintain the stability of insulin over time.
2. pH and Absorption: The optimal buffer concentration ensures that the pH of the eye drop formulation is compatible with both the insulin molecule and the ocular surface. This increases the absorption of insulin when administered as an eye drop.
3. Ionic Strength and Osmolarity: The buffer concentration should mimic physiological conditions (around 300 mOsm/L) to prevent irritation or adverse effects.
The chosen concentration provides the right balance between these factors, enhancing insulin efficacy while minimizing side effects.
Among the three phosphate buffer concentrations evaluated for insulin eye drops, one concentration demonstrated superior results in terms of stability and compatibility. Therefore, it was selected for further development.
Effect of chelating agent or stabilizer:
Three different concentrations of EDTA were evaluated to assess their impact on the formation of related substances in insulin eye drops. The objective was to identify the concentration that provides optimal chelation activity without compromising insulin stability. The study showed that higher concentrations led to increased degradation, likely due to pH or ionic strength variations. Conversely, the lowest concentration offered insufficient protection against trace metal-catalyzed degradation.
Among the concentrations tested, one intermediate concentration was found to effectively minimize the formation of related substances while maintaining formulation stability. This optimized concentration was therefore selected for further development.
Name of buffering agent Concentrations (%w/v)
Trial-1 Trial-2/ present method Trial-2
Di sodium EDTA 0.01 0.05 0.1

Insulin eye drops, 25IU/ml
Sr. No Ingredients Trial-1 Trial-2/ present method Trial-3
Qty % w/v Qty % w/v Qty % w/v
1 Human Insulin (r-DNA origin) 0.00347 0.00347 0.00347
2 Propylene Glycol 0.3 0.3 0.3
3 Polyethylene Glycol 400 0.4 0.4 0.4
4 Disodium Hydrogen Phosphate (Anhydrous) 0.01 0.01 0.01
5 Sodium Dihydrogen Phosphate
(Dihydtate) 0.006 0.006 0.006
6 Disodium EDTA 0.01 0.05 0.10
7 Sodium perborate 0.005 0.005 0.005
8 Sodium chloride 0.8 0.8 0.8
9 Sodium Hydroxide q.s to adjust pH 7.0 q.s to adjust pH
7.0 q.s to adjust pH
7.0
10 Hydrochloric acid q.s to adjust pH
7.0 q.s to adjust pH
7.0 q.s to adjust pH
7.0
11 Water for injection q.s to 100ml q.s to 100ml q.s to 100ml

It was observed from the results of the 3 trials that optimum amount of EDTA is very important for the claimed composition.
Example 3:
pH Optimization studies:
pH of the formulation is optimized to ensure insulin stability and ocular site of installation. Drug product stability over this pH range is studied to mitigate the risk associated with change in pH between 3.0 to 8.0 this ranges, preferably pH 3.0 to 7.0 With the proposed following pH were evaluated for product stability.
- pH at Lower side at pH 3.00
- pH at middle side at pH 4.00
- pH at Higher side at pH 7.00

Example 3(a):
pH Optimization trial batch (pH-3.00)
Insulin eye drops, pH 3.00
Sr. No Ingredients Qty mg/ml Qty % w/v Batch Q. ty (gm)
1IU 5IU 25IU
1 Human Insulin (r-DNA origin) 0.0347 0.00347 0.008675 0.04337 0.2168
2 Propylene Glycol 3 0.3 0.75 0.75 0.75
3 Polyethylene Glycol 400 4 0.4 1.00 1.00 1.00
4 Disodium Hydrogen Phosphate (Anhydrous) 0.1 0.01 0.025 0.025 0.025
5 Sodium Dihydrogen Phosphate
(Dihydtate) 0.06 0.006 0.015 0.015 0.015
6 Disodium EDTA 0.5 0.05 0.125 0.125 0.125
7 Sodium perborate 0.05 0.005 0.0125 0.0125 0.0125
8 Sodium chloride 8 0.8 2.00 2.00 2.00
9 Sodium Hydroxide q.s to pH 3.5-4.0 q.s to pH
3.5-4.0 q.s to pH 3.5-4.0 q.s to pH 3.5-4.0 q.s to pH 3.5-4.0
10 Hydrochloric acid q.s to pH 3.5-4.0 q.s to pH
3.5-4.0 q.s to pH 3.5-4.0 q.s to pH 3.5-4.0 q.s to pH 3.5-4.0
11 Water for injection q.s to ml q.s to 100ml q.s to 250ml q.s to 250ml q.s to 250ml

In an embodiment, the specific process for the preparation of the composition is mentioned below:
Manufacturing process steps Critical checks
pH Clarity of solution
Collect 80% batch size of Water for injection purged with nitrogen (Temperature at 2°C to 8°C) 5.97 Clear
Dispensed and add the quantity of Di sodium EDTA and Sodium Perborate under continuous stirring for 10 min. - -
In above solution add Propylene glycol, Polyethylene glycol 400, Sodium Dihydrogen phosphate (anhydrous), Sodium dihydrogen Phosphate (Dihydrate) in continues stirring for 20 min and adjust pH to (3.5 - 4.0) with HCl 8.49 Clear
Check pH of solution and adjust the at pH 3.0 by using 0.1N Hydrochloric acid or 0.1N Sodium Hydroxide solution 3.01 Clear
Solution allows to cool at temperature at 2°C to 8°C, dispensed and add the quantity of insulin in above solution, stirred for 10 min. 3.00 Clear
Make up the final volume of solution by using purged WFI and stirred for 5 min. 3.01 Clear
Filter the solution by using 0.2µ Nylon 66 filter and filter solution was filled in 5ml LDPE bottles. 3.00 -

Analytical results:
Test parameter Strength: 1IU/mL Strength: 5IU/mL Strength: 25IU/mL
pH- 3.00
Initial Initial Initial
Description Clear colourless solution Clear colourless solution Clear colourless solution
pH 2.99 3.00 2.98
Osmolality (mOsm/kg) 507 508 510
Assay of Insulin (%)
Without A21 93.68 93.97 93.66
Assay of Insulin (%)
With A21 NA NA NA
Related substances (%)
Single maximum unknown impurity (%) ND ND ND
A2 related impurity ND ND ND
Total impurity (%) ND ND ND

Based on above results, it is observed that all three concentrations remained clear and colorless at pH 3.00, with no visual signs of aggregation or precipitation. The pH was well-maintained and consistent across all samples. All formulations showed hypertonicity (>500 mOsm/kg). All formulations exhibited good assay recovery (~93–94%), indicating good chemical stability of insulin at acidic pH, even at low concentrations. No degradation products or related substances were detected (ND).pH 3.00 supports chemical stability and purity of insulin across all strengths, even at low concentrations (1 IU/ml). However, the high osmolality is a major limitation/ drawback for ophthalmic use and must be addressed through formulation refinement.

Example 3(b):
pH Optimization trial batch (pH-4.00):
Insulin eye drops, pH 4.00
Sr. No Ingredients Qty mg/ml Qty % w/v Batch size: (250ml) Q. ty (gm)
1IU 5IU 25IU
1 Human Insulin (r-DNA origin) 0.0347 0.00347 0.008675 0.04337 0.2168
2 Propylene Glycol 3 0.3 0.75 0.75 0.75
3 Polyethylene Glycol 400 4 0.4 1.00 1.00 1.00
4 Disodium Hydrogen Phosphate (Anhydrous) 0.1 0.01 0.025 0.025 0.025
5 Sodium Dihydrogen Phosphate
(Dihydrate) 0.06 0.006 0.015 0.015 0.015
6 Disodium EDTA 0.5 0.05 0.125 0.125 0.125
7 Sodium perborate 0.05 0.005 0.0125 0.0125 0.0125
8 Sodium chloride 8 0.8 2.00 2.00 2.00
9 Sodium Hydroxide q.s to pH 4.0 q.s to pH
4.0 q.s to pH 4.0 q.s to pH 4.0 q.s to pH 4.0
10 Hydrochloric acid q.s to pH 4.0 q.s to pH
4.0 q.s to pH 4.0 q.s to pH 4.0 q.s to pH 4.0
11 Water for injection q.s to ml q.s to 100ml q.s to 250ml q.s to 250ml q.s to 250ml

Manufacturing procedure:
Manufacturing procedure Critical checks
pH Clarity of solution
Collect 80% batch size of Water for injection purged with nitrogen (Temperature at 2°C - 8°C) 6.20 Clear
Dispensed and add the quantity of Di sodium EDTA and Sodium Perborate under continuous stirring for 10 min. - -
In above solution add Propylene glycol, Polyethylene glycol 400, Sodium Dihydrogen phosphate (anhydrous), Sodium dihydrogen Phosphate (Dihydrate) in continues stirring for 20 min 8.51 Clear
Check pH of solution and adjust the pH of solution at pH- 4.00 by using 0.1N Hydrochloric acid. 3.98 Clear
Solution allows to cool at temperature at 2°C - 8°C, dispensed and add the quantity of insulin in above solution, stirred for 10 min. 4.02 Clear
Check the pH of solution. 4.01 Clear
Make up the final volume of solution by using purged WFI and stirred for 5 min. 4.01 Clear
Filter the solution by using 0.2µ Nylon 66 filter and filter solution was filled in 5ml LDPE bottles. 4.00 -

Analytical results:
Test parameter Strength: 1IU/mL Strength: 5IU/mL Strength: 25IU/mL
pH- 4.00
Initial Initial Initial
Description Clear colourless solution Clear colourless solution Clear colourless solution
pH 3.93 4.63 4.10
Osmolality (mOsm/kg) 293 502 282
Assay of Insulin (%)
With A21 52.53 79.86 98.24
Related substances (%)
Single maximum unknown impurity (%) ND ND ND
A2 related impurity ND ND ND
Total impurity (%) ND ND ND
Observations: Based on above results, all strengths appeared clear and colorless, meeting visual inspection standards. The pH of 5 IU/mL was slightly higher (4.63), likely due to buffer dilution ratios or insulin concentration effects. All values remained within an acceptable range for stability and ocular application (3.8–4.7). The osmolality of 1 IU/mL and 25 IU/mL are within the acceptable isotonic range (270–350 mOsm/kg). 5 IU/mL showed hypertonicity (502 mOsm/kg), which may lead to ocular irritation or discomfort if not corrected. This requires reformulation with reduced tonicity-adjusting agents. The assay of Insulin of strength 25 IU/mL showed excellent recovery (98.24%) which indicates formulation accuracy and stability at higher concentration.
5 IU/mL showed moderate assay (79.86%)-slightly lower than expected, may indicate adsorption, dilution error. 1 IU/mL had significantly lower assay (52.53%)- suggests insulin instability, adsorption to container, or detection limits at low concentrations.
All impurity levels, including A21, were Not Detected, indicating chemical purity and early-phase stability at pH 4.00.
Conclusion:
a) Thus, 25 IU/mL formulation demonstrates good performance and stability, with proper pH, assay, and impurity profile.
b) 5 IU/mL formulation requires osmolality adjustment and may need process optimization to enhance assay recovery.
c) 1 IU/mL formulation faces challenges in achieving consistent assay due to instability at low concentrations or analytical sensitivity.
Example 3( c):
pH Optimization trial at pH- 7.00
Insulin eye drops, pH 7.00
Sr. No Ingredients Qty mg/ml Qty % w/v Batch size: (250ml) Q. ty (gm)
1IU 5IU 25IU
1 Human Insulin (r-DNA origin) 0.0347 0.00347 0.008675 0.04337 0.2168
2 Propylene Glycol 3 0.3 0.75 0.75 0.75
3 Polyethylene Glycol 400 4 0.4 1.00 1.00 1.00
4 Disodium Hydrogen Phosphate (Anhydrous) 0.1 0.01 0.025 0.025 0.025
5 Sodium Dihydrogen Phosphate
(Dihydtate) 0.06 0.006 0.015 0.015 0.015
6 Disodium EDTA 0.5 0.05 0.125 0.125 0.125
7 Sodium perborate 0.05 0.005 0.0125 0.0125 0.0125
8 Sodium chloride 8 0.8 2.00 2.00 2.00
9 Sodium Hydroxide q.s to
pH 7.0 q.s to
pH 7.0 q.s to
pH 7.0 q.s to
pH 7.0 q.s to
pH 7.0
10 Hydrochloric acid q.s to
pH 7.0 q.s to
pH 7.0 q.s to
pH 7.0 q.s to
pH 7.0 q.s to
pH 7.0
11 Water for injection q.s to ml q.s to 100ml q.s to 250ml q.s to 250ml q.s to 250ml

Manufacturing procedure:
Manufacturing procedure Critical checks
pH Clarity of solution
Collect 80% batch size of Water for injection purged with nitrogen (Temperature at 2°C - 8°C) 5.97 Clear
Dispensed and add the quantity of Di sodium EDTA and Sodium Perborate under continuous stirring for 10 min. - -
In above solution add Propylene glycol, Polyethylene glycol 400, Sodium Dihydrogen phosphate (anhydrous), Sodium dihydrogen Phosphate (Dihydrate) in continues stirring for 20 min and adjust pH to (3.5 - 4.0) with HCl 8.41 Clear
Check pH of solution and adjust the at pH 7.00 by using 0.1N Hydrochloric acid. 6.98 Clear
Solution allows to cool at temperature at 2°C - 8°C, dispensed and add the quantity of insulin in above solution, stirred for 10 min. 7.01 Clear
Check the pH of solution. 7.01 Clear
Make up the final volume of solution by using purged WFI and stirred for 5 min. 7.02 Clear
Filter the solution by using 0.2µ Nylon 66 filter and filter solution was filled in 5ml LDPE bottles. 7.01 -

Analytical results:
Test parameter Strength: 1IU/mL Strength: 25IU/mL
pH- 7.00 pH- 7.00
Initial 15 Day
2°C-8°C 1M-2°C-8°C Initial 15 Day
2°C-8°C 1M-2°C-8°C
Description Clear colourless solution Clear colourless solution Clear colourless solution Clear colourless solution Clear colourless solution Clear colourless solution
pH 6.70 6.78 6.89 6.81 6.85 6.84
Osmolality (mOsm/kg) 291 288 294 283 280 281
Assay of Insulin (%)
With A21 96.75 95.04 97.10 104.23 101.88 104.47
Related substances (%)
Single maximum unknown impurity (%) ND ND 0.088 ND ND 0.20
A2 related impurity ND ND ND ND ND 0.282
Total impurity (%) ND ND 0.16 ND ND 0.512

Observation: Based on above results the drug product CQA’s like description, pH, osmolality is found satisfactory results. The assay of insulin both strengths-maintained potency across 15 days and 1 month. Notably, 25 IU/mL good response of concentration and assay. Related substance of impurities began to appear at 1 month at 1 IU/mL, total impurities were low (0.16%), within acceptable ICH limits. Strength At 25 IU/mL, total impurity increased to 0.512%, primarily due to A2-related degradation (0.282%). Although still within safe limits.
Thus, it can be concluded that pH 7.00 formulations demonstrate excellent physical appearance, assay stability, and ocular compatibility. 25IU/ml formulation shows good stability and minimal degradation at pH 7.00. Further development Trials are monitored at pH- 7.00.

Example 4:
Effect of temperature:
To assess the impact of temperature and humidity on the stability of insulin formulations, recognizing that insulin is a temperature-sensitive protein prone to degradation outside refrigerated conditions. Initial trials were conducted under recommended cold storage (2°C to 8°C). Further stability studies are planned at accelerated and intermediate conditions to understand formulation robustness.
Initial Conditions are studied at 2°C–8°C feasibility batches were stored under refrigerated conditions to minimize risk of degradation. Insulin retained potency and clarity. Minimal or no impurity formation observed up to 30 days.
Insulin eye drops, pH 7.00
Sr. No Ingredients Qty mg/ml Qty % w/v Batch size Q. ty (gm)
1IU 5IU 25IU
1 Human Insulin (r-DNA origin) 0.0347 0.00347 0.008675 0.04337 0.2168
2 Propylene Glycol 3 0.3 0.75 0.75 0.75
3 Polyethylene Glycol 400 4 0.4 1.00 1.00 1.00
4 Disodium Hydrogen Phosphate (Anhydrous) 0.1 0.01 0.025 0.025 0.025
5 Sodium Dihydrogen Phosphate
(Dihydrate) 0.06 0.006 0.015 0.015 0.015
6 Disodium EDTA 0.5 0.05 0.125 0.125 0.125
7 Sodium perborate 0.05 0.005 0.0125 0.0125 0.0125
8 Sodium chloride 8 0.8 2.00 2.00 2.00
9 Sodium Hydroxide q.s to
pH 7.0 q.s to
pH 7.0 q.s to
pH 7.0 q.s to
pH 7.0 q.s to
pH 7.0
10 Hydrochloric acid q.s to
pH 7.0 q.s to
pH 7.0 q.s to
pH 7.0 q.s to
pH 7.0 q.s to
pH 7.0
11 Water for injection q.s to ml q.s to 100ml q.s to 250ml q.s to 250ml q.s to 250ml

Manufacturing procedure:
Manufacturing procedure Critical checks
pH Clarity of solution
Collect 80% batch size of Water for injection purged with nitrogen (Temperature at 2°C - 8°C) 5.96 Clear
Dispensed and add the quantity of Di sodium EDTA and Sodium Perborate under continuous stirring for 10 min. - -
In above solution add Propylene glycol, Polyethylene glycol 400, Sodium Dihydrogen phosphate (anhydrous), Sodium dihydrogen Phosphate (Dihydrate) in continues stirring for 20 min and adjust pH to (3.5 - 4.0) with HCl 8.45 Clear
Check pH of solution and adjust the at pH 3.5 to 4.0 by using 0.1N Hydrochloric acid or 0.1N Sodium Hydroxide solution 6.99 Clear
Solution allows to cool at temperature at 2°C - 8°C, dispensed and add the quantity of insulin in above solution, stirred for 10 min. 7.02 Clear
Check the pH of solution. 7.03 Clear
Make up the final volume of solution by using purged WFI and stirred for 5 min. 7.02 Clear
Filter the solution by using 0.2µ Nylon 66 filter and filter solution was filled in 5ml LDPE bottles. 7.02 -

Test parameter Strength: 1IU/mL Strength: 25IU/mL
pH- 7.00 pH- 7.00
Initial 1Month-
2°C-8°C 1Month-25°C/60% RH 1Month-40°C/75% RH Initial 1Month-
2°C-8°C 1Month-25°C/60% RH 1Month-40°C/75% RH
Description Clear colourless solution Clear colourless solution Clear colourless solution Clear colourless solution Clear colourless solution Clear colourless solution Clear colourless solution Clear colourless solution
pH 6.70 6.89 6.78 6.75 6.81 6.85 6.82 6.79
Osmolality (mOsm/kg) 291 294 288 285 283 280 282 280
Assay of Insulin (%)
With A21 96.75 97.10 95.10 86.12 104.23 101.88 102.57 85.26
Related substances (%)
Single maximum unknown impurity (%) ND ND ND 14.122 ND ND 2.62 7.54
A2 related impurity ND ND ND 0.253 ND ND 0.253 0.211
Total impurity (%) ND ND ND 38.545 ND ND 3.478 17.72

Observations: Based on above results, 1IU/ml and 25IU/ml at 2°C–8°C and 25°C/60% RH conditions both strengths remained chemically and physically stable. Assay values remained well within acceptable limits (95–110%). Impurities were not detected or minimal, indicating good formulation integrity for at least 1 month at these conditions.
At 40°C/75% RH condition significant degradation observed at both strengths: Assay dropped to ~85%, nearing or falling below the ICH limit. Impurity levels rose dramatically, especially at 1 IU/mL, where total impurity reached 38.545%. Max unknown impurity >14.22% suggests major degradation products possibly analytical interference or protein aggregation breakdown. 25 IU/mL formulation also showed elevated impurities, though less extreme than 1 IU/ml. Total impurity: is 17.72%, primarily due to unknown and A2-related impurities.
Conclusion: Insulin is extremely unstable at 40°C, confirming the need for cold chain storage. Stability at 25°C/60% RH is acceptable.
Recommendations:
1. Strict storage at 2°C–8°C should be maintained for all commercial and clinical batches.
2. Room temperature (25°C) stability supports label claim of short-term exposure, subject to further long-term validation.
,CLAIMS:We Claim:

1. A topical insulin eye drop formulation comprising:
a) Human Insulin;
b) Phosphate buffer;
c) Propylene Glycol, present in an amount of 0.3%(w/v);
d) Polyethylene Glycol 400, present in an amount of 0.4%(w/v);
e) Disodium EDTA, present in an amount of 0.05 %(w/v);
f) Sodium perborate, present in an amount of 0.005 %(w/v);
g) Sodium chloride, present in an amount of 0.8% (w/v);
h) Sodium Hydroxide present in an amount to obtain the pH of 7.0;
i) Hydrochloric acid in an amount to obtain the pH of the formulation as 7.0.
2. The formulation as claimed in claim 1, wherein Human Insulin is present in the concentration of 0.00347% w/v.
3. The formulation as claimed in claim 1, wherein Human Insulin is present in the concentration of 25IU.
4. The formulation as claimed in claim 1, wherein Phosphate buffer comprises Disodium Hydrogen Phosphate (Anhydrous) and Sodium Dihydrogen Phosphate (Dihydrate).
5. The formulation as claimed in claim 4, wherein Disodium Hydrogen Phosphate (Anhydrous) is present in an amount of 0.01 %w/v.
6. The formulation as claimed in claim 4, wherein Sodium Dihydrogen Phosphate
(Dihydrate) is present in an amount of 0.006 %w/v.
7. The formulation as claimed in claim 1, is to be maintained at temperature of 25°C and 60% relative humidity.
8. A method of preparing the topical insulin eye drop formulation as claimed in claim 1, comprising the steps of:
a) Collecting 80% batch size of Water for injection purged with nitrogen where the temperature is maintained at 2°C to 8°C and pH of approximately 5.96;
b) Dispensing and adding the Di sodium EDTA and Sodium Perborate under continuous stirring for 10 minutes;
c) adding Propylene glycol, Polyethylene glycol 400, Sodium Dihydrogen phosphate (anhydrous), Sodium dihydrogen Phosphate (Dihydrate) under continuous stirring for 20 min and adjusting the pH to 3.5 - 4.0 by adding HCl or Sodium Hydroxide solution;
d) allowing the resulting solution to cool at temperature at 2°C - 8°C, followed by adding insulin and stirring for 10 minutes, to obtain the formulation of pH 7.
9. The method as claimed in claim 8, wherein the solution is filtered by 0.2µ Nylon 66 filter.