FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See Section 10 and Rule 13)
Formulations and a process to stabilize therapeutic drug
Intas Biopharmaceuticals Limited
An Indian company having its registered office at:
Plot No: 423/P/A/GIDC
Sarkhej-Bavla Highway
Moraiya, TaL: Sanand
Ahmedabad -382 213
Gujarat, India
The following specification particularly describes the invention and the manner in which it is to be performed:

FIELD OF THE INVENTION
The present invention relates to a formulation of polymer - drug conjugates with dual buffer system to minimize the cleavage between polymer and a therapeutic drug caused by physical stresses associated with deviations from recommended storage and transportation temperature of these therapeutic drug products, in particular at elevated temperatures and repeated freeze-thaw cycles. It also discloses a process to develop such formulations comprising therapeutic drugs and applications of the same in treatment of diseases.
BACKGROUND OF THE INVENTION
Therapeutic drugs, especially having short body-residence time (and hence short half-lives) require frequent administrations in order to maintain therapeutic blood levels of the drug. There are multiple examples of such therapeutic drugs that require frequent administration like Granulocyte colony stimulating factor (GCSF), Erythropoietin (EPO), Interferon alpha- 2a (IFN - a-2a), Interferon alpha- 2b (IFN-a-2b), etc. Currently, one of the most successful methods for stabilizing therapeutic drugs and increasing their half-lives is to use polymer therapeutics, i.e., conjugating a therapeutic drug to a polymer such as polyethylene glycol (PEG). Conjugating a therapeutic drug to a polymer can have many advantages including increase in body-residence time, decrease in immunogenicity and antigenicity, successful transportation of the drug across a cell membrane; protection against proteolysis and hence stability. These superior properties can increase effective potency, reduce overall dosage, increase patient tolerance and lower the cost of treatment to the patient.
Various hydrophilic polymers exhibiting high hydration and flexibility may be utilized for conjugation to a therapeutic drug. These polymers when conjugated offer many structural advantages including (a) protection of amino acid sequences sensitive to chemical degradation and microenvironment around therapeutic drugs (b) the masking of critical sites sensitive to metabolic enzymes degradation or to antibody recognition. This is due to the fact that the drug surface is properly covered by polymer molecules and the native structure is well protected by the external environment in the solution.
The most successful strategy for enhancing the potential of therapeutic and therapeutic drugs employs the use of polyethylene glycol (PEG) as modifying polymer and the strategy is termed as PEGylation. Conjugation of a therapeutic drug with PEG not only retains the biological activity and receptor recognition but also masks the drug's surface and increases its molecular size thereby reducing its renal

ultra filtration and hence increasing circulation half-life of the drug. Other reported biodegradable polymers for drug conjugation are albumin, hydroxyethyl starch (HES), polyamino acids, hyaluronic acid, polysialic acid, etc. These polymers also provide similar advantages as presented by PEG.
Examples of commercially available PEGylated products are Adagen®, Oncaspar®' PEG-Intron®, Pegasys®' Neulasta® and Somavert® and a dozen of other PEG conjugated drugs which are now in advanced clinical trials. Usually PEG moieties are attached to a therapeutic drug by first activating the PEG moiety and then conjugating it with the side chain of lysine residue and/or the N-terminal amino group on the drug.
PEG may be conjugated to various reactive amino acids present in biologicals which may include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, etc. Polymers may be conjugated covalently with therapeutic drugs through amine PEGylation and N-terminal PEGylation, carboxyl PEGylation, thiol PEGylation, hydroxyl PEGylation etc. to form amide, urethane, thiourea, ester, disulfide, carbonate bonds, etc.
In conjugated form, the bonds or linkages between polymer and therapeutic drug is accessible to solvent and external environment and therefore is highly prone to disruption by slight changes in its microenvironment. It is important that polymer - drug conjugates should be stable for longer periods of time to withstand manufacturing, storage and transportation conditions. However, the linkages or bonds between a polymer and the drug when exposed to external environment are highly prone to cleavage which could have significant impact on pharmacokinetic properties of the therapeutic drugs (Figure - 1). Therefore it is highly desirable to keep the bonds or linkages between a drug and a polymer more stable.
Figure - 2 shows the detailed mechanism of hydrolysis of polymers - drug conjugates under mild acidic or basic conditions. These conjugates are highly prone to hydrolysis due to variations in buffer pH as a result of non-maintenance of cold-chain. Different conjugation bonds e.g. ethers, esters, urethanes, anhydrides, thiourea, and amides etc. follow similar mechanism of hydrolysis under similar set of conditions.
Polymer - drug conjugates are highly prone to cleavage as slight changes in their microenvironment, such as changes in temperature pH and freeze-thaw cycles may disrupt the polymer - drug linkages. Therefore, these products are usually lyophilized and require cold chain for transportation, storage and delivery. Lyophilization process involves the use of additional excipients which serve to protect the active therapeutic drug during lyophilization. Cryoprotectants and stabilizers have been particularly used in

lyophilized formulations to render protection and stability to the polymer - drug conjugates. Lyophilization process involves a freeze-drying step which could potentially cause freeze-injury to the therapeutic drug lyophilized. Further, cold chain maintenance of lyophilized product is also essential during storage, transportation and handling to maintain the quality of these lyophilized products.
However, it is difficult to maintain the cold chain especially in tropical countries such as India where temperature can reach as high as 48 °C in summer. Mean Kinetic Temperature (MKT) is a fixed temperature that simulates the effects of temperature variation over a period of time and is calculated from the average temperature during storage. In a study conducted by Intas Biopharmaceuticals Ltd (Mody et al, Understanding variations in biosimilars: Correlation with risk and regulatory implications; Int. J. Risk Safety Med.; 22 (2010), 27-40.), it was observed that the MKT at various clearing and forwarding (C&F) agents can vary from less than -5 °C to more than 25 °C. As a result, not only the product undergo repeated freeze-thaw cycles but also temperature excursions beyond the recommended 2 - 8 °C. As a result ,many therapeutic drug products lose substantial potency from the time of manufacture to the of administration. According to a recent study conducted by the UK-based Medicines and Healthcare Products Regulatory Agency (MHRA), a staggering 43 % of critical and major product deficiencies are related to ineffective temperature control and monitoring during storage and transportation (Taylor J, (MHRA), Regulatory Expectations, IQPC Cool Chain 2008, Brussels, Belgium). Similarly, the World Health Organization (WHO) has maintained that as much as 25% of all vaccine products reach their destination in a degraded state. The responsibility for the cold chain ultimately resides with the manufacturer but accountability is shared across the supply chain. Maintaining the cold chain at a specific temperature range, normally 2°C to 8°C is both costly and challenging for resource constrained countries such as India. Since a denatured therapeutic drug corresponding to disruption of tertiary structure is generally useless, the objective of this invention is to scrupulous maintenance of polymer - drug conjugate stability and hence its biological function,
In the biopharmaceutical industry, assurance of long term stability of polymer - drug conjugate in aqueous formulations is a challenge. For short-term storage of polymer - drug conjugates (hours to days), a standard laboratory refrigerator at 4°C is satisfactory provided the buffer used to solvate the polymer -drug conjugates has all the necessary components necessary to stabilize the conjugate of interest. Polymer - drug conjugates can be stored long term (days to weeks) by quick-freezing the sample in appropriate buffers followed by storage at -20°C. Additions of stabilizers such as sugars, glycerol, surfactants etc., help prevent cleavage of the linkages or bonds between polymer and drug during freezing and

thawing. The frozen samples are then thawed to bring the drug in solution form during development and production of formulations in the biopharmaceutical industry.
A frozen sample usually is an anisotropic one and is comprised of several concentrated solute phases distributed throughout a bulk ice phase. The freeze induced stresses include cold denaturation, generation of ice-solution or ice-drug interface and freezing-induced concentration of drug and solutes which may lead to crystallization and pH shift of the solution. Such a heterogeneous microstructure leads to extensive light scattering and may induce potential stresses which may damage the linkages or bonds between polymers and drug irreversibly. Primarily these damages are in the form of cleavage or high molecular weight species formation and subsequent denaturation and therapeutic activity loss of therapeutic drug product. In the subsequent thawing step, additional damage could be caused by ice crystallization, which introduces extra interfacial stresses. Therefore, control of the primary stresses responsible for therapeutic drug damage remains a challenging step during freeze-thaw process.
The nature of additives and stabilizers in commercial formulations of therapeutic drugs can vary. However, the common feature of these formulations, both in frozen and in aqueous form is the presence of a buffer. A buffer is required to maintain the pH of the formulation where a therapeutic drug and its conjugates achieve maximum stability. In some cases, different buffers or buffer combinations at different pH values can be used to enhance the stability of formulation for a prolonged period of time. However, increasing ionic strength of buffering excipients during freezing can reduce their solubility and potentially also harm the linkages or bonds between polymer and drug. Buffering excipients can crystallize if their solubility limit is reached till saturation point resulting in significant shift in the pH of the formulation. Among common buffers used for formulation of biologies, the sodium phosphate buffer is particularly susceptible to pH decrease as much as 3 pH units from 7 to - 4 on precipitation of the dibasic salt below 0 °C. Even if the salts do not reach their solubility limits, their pKa value is sensitive to temperature, so pH shifts will occur during freezing which may initiate the hydrolysis reactions of the conjugation bonds and hence may considerably affect polymer - drug conjugate stability.
Patent EP 1314437 Al invention relates to antibody-containing preparations, particularly stabilized antibody-containing preparations with low loss of active ingredients even after long-term storage. The present invention also relates to processes for preparing polymer - drug conjugates-containing stabilized preparations, comprising adjusting the pH with a basic amino acid or a basic amino acid derivative or a salt thereof. The invention states that heat-induced aggregation can be controlled with stabilized preparations of the present invention formulated in a glycine buffer and/or a histidine buffer and that

aggregation can be further reduced by adding glycine and/or sucrose. According to processes for preparing a stabilized preparation containing a physiologically active biological drug of the present invention, heat-induced aggregation can be controlled to provide a stable preparation by adjusting the pH with a basic amino acid or a basic amino acid derivative or a salt thereof. Effect of the type of buffer on aggregation was studied. Five buffers (all 19 mM) were used to examine their effect on aggregation. The pHs of samples prepared by dissolving an antibody hPM-1 preparation in these buffers (at a concentration of about 1 mg/ml) are as follows. 1) Sodium phosphate (pH 6.8) 2) histidine-HCl (pH 7.1) 3) sodium citrate (pH 6.7) 4) Tris-HCl (pH 7.2) 5) glycine (pH 7.6). Effects of the type of buffer on sedimentation distribution and the effect of glycine and sucrose were studied.
Patent application WO/2009/006097 has been published on liquid protein formulations comprising GDF-5 for use at elevated temperatures. Liquid formulations of bone morphogenetic proteins are provided for prolonged use at elevated temperatures. More specifically, the invention relates to liquid formulations comprising rhGDF-5, trehalose, and one or more biocompatible excipients that provide stability to the protein for at least 30 days at temperatures up to body temperature. The invention is a liquid protein formulation. The formulation comprises a Bone Morphogenetic Proteins, BMP, at least a 50% w/v solution of trehalose, and at least one additional excipient selected from the group consisting of an amino acid, a trialkylammonium salt, a heat shock protein, a betaine, taurine, raffinose, myo-inositol, and potassium aspartate in an amount sufficient to stabilize the BMP as evidenced by retention of at least 80% of the main chromatography peak for at least 30 days storage at temperatures up to 37°C.
Veronese, F.M. et al, in Biomaterials, 2001, Vol. 22, pp. 405 - 471 discussed about the general problems in using PEG for conjugation to high or low molecular weight like methods of binding PEG to different functional groups of macromolecules, problems encountered in conjugation such as the evaluation of the number of PEG chains bound, the localization of the site of conjugation in polypeptides and the procedure to direct PEGylation to the desired site in the molecules, and reported the specific methods regarding reversible PEGylation, cross-linking reagents with PEG arms, PEG for enzyme solubilization in organic solvent and new polymers as alternative to PEG.
As it can be seen from the above prior arts, till now, very little work has been done to improve the long -term stability of polymer - drug linkages in solution form at elevated temperature and at frozen conditions. Many aspects of biopharmaceutical production and formulation processes are pH sensitive. Fluctuations in pH of the formulation may initiate the hydrolysis reactions of the conjugation bonds and hence may considerably affect polymer - drug conjugate stability. This disruption of polymer - drug

linkages is generally associated with the release of native therapeutic drugs and increased blood clearance. Maintaining the correct pH of a finished therapeutic drug product is critical to its stability, effectiveness, and shelf life, and keeping the pH as constant is an important consideration in designing formulations for administration that will be acceptable, as well as safe and effective. It is therefore crucial to ensure that the product is stored under conditions where the intact structure of the polymer - drug conjugate is maintained. Consequently, there remains a need in the art to develop a suitable formulation system to stabilize the polymer - drug conjugate when exposed to stress conditions associated with deviations from recommended storage and transportation temperature of these therapeutic drug products, in particular at elevated temperatures and repeated freeze-thaw cycles.
SUMMARY OF THE INVENTION
The present invention relates to a process for the stabilization of bonds or linkages between polymer and a therapeutic drug when present in solution form at elevated temperatures and repeated freeze-thaw cycles. The present invention describes a method of preparation of stable pharmaceutically acceptable formulation of polymer - drug conjugates to minimize the cleavage between polymer and a therapeutic drug caused by physical stresses associated with deviations from recommended storage and transportation temperature of these therapeutic drug products, in particular at elevated temperatures and repeated freeze-thaw cycles. The present invention can also stabilize the lyophilized polymer - drug conjugates after reconstitution when stored in liquid form.
The present invention describes the use of two or more buffers which can stabilize the bonds or linkages between polymer and a therapeutic drug when present in solution form at elevated temperatures and repeated freeze-thaw cycles over a broad pH range from 2 - 10, preferably 4-9. The first buffering excipient is selected from the acidic range (Group - A in Table - 1) which can maintain the pH in range from 2-7. The second buffering excipient is selected from the alkaline range (Group - B in Table - 1) which can maintain the pH in range from 7-11. Two buffering excipients thus selected may be used to prepare the buffer system and the pH may be adjusted to a value where a polymer - drug conjugates shows the maximum stability. The said formulation further comprises an ampholyte, an anti-oxidant, a non-reducing sugar, a non-ionic surfactant and optionally with preservative either alone or in combination such that the bonds or linkages between polymer and therapeutic drug when present in solution form can be maintained at elevated temperatures falls in the range of 25°C and 50°C and upto five freeze-thaw cycles to ensure a reasonable shelf-life. The samples in the present invention may be frozen at a temperature below -5 DC or less and thawed above freezing temperature of the solution.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure -1 shows the conjugation reaction of polymers with a model protein. It also shows that the protein
surface is fully covered by polymer after conjugation.
In Figure 1 - represents the covalent bonding; — represents the non-covalent (non-specific) bonding,
Figure - 2a, 2b & 2c: Detailed mechanism of hydrolysis of polymers - drug conjugates under different conditions cleavage by acids (Figure 2a) cleavage by bases (Figure 2b) and cleavage in neutral environment at elevated temperature (Figure 2c).
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of stabilization of bonds or linkages between polymer and a therapeutic drug when present in solution form at elevated temperatures and repeated freeze-thaw cycles. The present invention describes a method of preparation of stable pharmaceutically acceptable formulation of polymer - drug conjugates to minimize the cleavage between polymer and a therapeutic drug caused by physical stresses associated with deviations from recommended storage and transportation temperature of these therapeutic drug products, in particular at elevated temperatures and repeated freeze-thaw cycles. The present invention can also stabilize the lyophilized polymer - drug conjugates after reconstitution when stored in liquid form.
The present invention describes the use of two or more buffers which can stabilize the bonds or linkages between polymer and a therapeutic drug when present in solution form at elevated temperatures and repeated freeze-thaw cycles over a broad pH range from 2-10, preferably 4-9. The first buffering excipient is selected from the acidic range (Group -A in Table - 1) which can maintain the pH in range from 2-7. The second buffering excipient is selected from the alkaline range (Group - B in Table - 1) which can maintain the pH in range from 7-11. Two buffering excipients thus selected may be used to prepare the buffer system and the pH may be adjusted to a value where a polymer - drug conjugates shows the maximum stability. The said formulation further comprises an ampholyte, an anti-oxidant, a non-reducing sugar, a non-ionic surfactant and optionally with preservative either alone or in combination such that the bonds or linkages between polymer and a therapeutic drug when present in solution form can be maintained at elevated temperatures upto 25 °C or more and upto five or more freeze-thaw cycles for a prolonged period of time to ensure a reasonable shelf-life. The samples in the present invention may be frozen at a temperature below -5 °C or less and thawed above freezing temperature of the solution. The

present invention can also stabilize the lyophilized polymer - drug conjugates after reconstitution when stored in liquid form.
Thermostability of a polymer - drug conjugate is the quality to protect bonds or linkages between polymer and a therapeutic drug in solution from cleavage or rupture at relatively high temperature by keeping the microenvironment around the polymer conjugates as protected as possible. Thermostability with respect to the present invention refers to the storage stability of polymer conjugates, i.e. stability at 25 °C for at least two weeks or more.
Freeze-thaw stability of a polymer - drug conjugate is the quality to protect bonds or linkages between polymer and a therapeutic drug in solution from cleavage during repeated freeze-thaw cycles say 5 or more by keeping the microenvironment around the polymer conjugates as protected as possible. Freeze-thaw stability with respect to the present invention refers to the storage stability of polymer conjugates at C&F agents where MKT is poorly maintained and the product may undergo repeated freeze-thaw cycles. The present invention can also stabilize the lyophilized polymer - drug conjugates after reconstitution when stored in liquid form.
Thermostability and freeze-thaw stability as disclosed in the present invention is the quality of a polymer - drug conjugate to protect bonds or linkages between polymer and therapeutic drug when present in solution form during elevated temperatures and repeated freeze-thaw cycles. Thermostability and freeze-thaw stability with respect to the present invention refers to the storage stability of polymer conjugates where MKT is outside the recommended range and the product may not only undergo repeated freeze-thaw cycles but also has to withstand higher temperatures. The present invention also discloses a process to stabilize the said conjugates at stress conditions.
The present invention involves the usage of dual buffer system by which the bonds or linkages between polymer and therapeutic drug when present in solution can be protected. The dual buffers system which is used in the present invention comprising one buffer from acidic and one buffer from basic pH range. The lists of buffers which can be used to stabilize the polymer - drug conjugates are given in table - 1. The pH of the formulation is then adjusted to a pH value where polymer - drug conjugates shows maximum stability and the linkages or bonds conjugating a therapeutic drug to polymers remain intact.

The formulations of the present invention further comprising amphoteric or ampholyte substances with two or more groups having acidic as well as basic pKa values. The examples of such compounds are amino acids such as glycine or lysine or arginine etc.
Table -1: List of buffering excipients from acidic and basic pH range

Acidic excipients (pH 2-7) Basic excipients (pH 7-12)
Acetic acid
Acidic Amino acids e.g, Glutamic acid, Aspartic acid, Histidine, etc. Basic Amino acids e.g, Arginine, Cysteine, Lysine, Tyrosine
Adipic acid Tris (hydroxymethyl) aminomethane
Ascorbic acid Asparagine
Benzoic acid Sodium carbonate
Carbonic acid Sodium bicarbonate
Citric acid Ethyleneimine
Citrulline Purine
Formic acid Tricine
Fumaric acid HEPES
Glutaric acid Boric acid
Glycolic acid
Lactic acid

Malic acid

Malonic acid

Oxalic acid

Phosphoric acid and salts

Pyruvic acid

Succinic acid

Tartaric acid

The term "therapeutic drugs" described herein refer to biological drugs either animal, plant or recombinant origin, chemical drugs either extracted from plants, animals or synthesized in the laboratory etc.
According to the present invention wherein polymer conjugates of biologicals are selected from a group consisting of proteins and peptides enzymes, antigens, hormones, insulin, growth factors, interferons, monoclonal antibodies (mAbs), albumin, nucleic acids, either naked or encapsulated in polymeric either natural or synthetic, lipids, carbohydrates or non-polymeric matrix, small chemical entities either naked or encapsulated in polymeric either natural or synthetic, lipids, carbohydrates or non-polymeric matrix.
According to the present invention wherein the polymer is selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol (PPG), albumin, hydroxy ethyl starch (HES), polyamino acids, hyaluronic acid, polysialic acid, medium chain carboxylic acids etc. The present invention may be utilized to stabilize the covalent bonds between a biological drug and polymer and may consist of amide,

urethane, thiourea, ester, ether, and disulfide, carbonate bonds, etc. The present invention may be utilized to stabilize the covalent, ionic, Van der Walls interactions, hydrogen bonds or any other non-specific bonds.
The following examples illustrate the invention in more detail:
Example 1: Determination of optimum pH of PEG-IFNα
Aqueous solutions of PEG-IFNa 2b was prepared in water for Injection (WFI) without buffers were prepared and different pHs were adjusted viz, 5,0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 by using HC1 or NaOH. Samples were incubated at 40°C and studied for aggregated, intact and depegylated PEG-IFNα 2b by size exclusion - high performance liquid chromatography (SE-HPLC) after 0, 2,4 and 7 Hours to determine the optimum pH.
Table 2: SE-HPLC results of PEG IFNa 2b at different pH when incubated at 40°C.

pH % values

2Hrs 4Hrs 7Hrs

Agg Monopeg Depeg Agg Monopeg Depeg Agg Monopeg Depeg
Initial 7.56 87.17 5.27 7.56 87.17 5.27 7.56 87.17 5.27
5.0 13.88 80.25 5.88 17.96 76.05 5.98 25.90 68.15 5.93
5.5 14.51 80.08 5.39 19.03 76.80 4.16 25.84 68.68 5.47
6.0 15.04 79.47 5.48 19.27 75.60 5.12 26.87 68.14 4.98
6.5 8.66 85.53 5.80 10.49 82.81 6.69 15.00 78.25 6.73
7.0 9.42 84.02 6.54 10.81 81.38 7.80 14.55 76.88 8.56
7.5 7.03 85.20 7.70 7.61 82.99 9.38 10.46 78.64 10.89
8.0 15.50 74.39 10.09 22.18 66.94 10.87 37.15 50.24 12.60
Table 2 shows the comparison of aggregated (Agg), intact and depegylated PEG-IFNa 2b at different pHs after 0, 2, 4 and 7 hours at 40 °C. From Table 2 it was observed that PEG-IFNa 2b is more stable in the pH range of 6.5 to 7.5. There was slight increase in aggregation from 7% to 10% between 0 to 4 hours at pH range of 6.5 to 7.5 as compared to other lower and higher pH values. There was no significant increase in depegylation from 0 (5%) to 7 hrs (8.5%) at pH range of 6.5 to 7.5. At pH 6.5, PEG IFN a 2b is more stable as approximately 90% conjugated protein was recovered in terms of purity after 7 hours incubation at 40°C.
Example 2: Optimization of molar concentration of Tris benzoate buffer
Tris-benzoate (TB) buffer was selected for protection of PEG-IFNa 2b urethane bond. Tris buffer was selected from basic buffer side and Benzoate buffer was selected from the acidic side to prepare the Tris-benzoate dual buffer system to be used in present invention. Aqueous solutions containing PEG-IFNa 2b in tris-benzoate buffer were prepared at pH 6.5. Tris-benzoate buffer was prepared at five different molar strengths i.e. 10,

12.5, 15, 20 and 30 mM. The formulations also contained other excipients like sucrose and polysorbate 80 as stabilizers. The formulation of PEG-IFNa 2b having similar composition as that of Viraferonpeg® was prepared in phosphate buffer (In-house Viraferonpeg® ) as control. Details of the formulations are given in Table 3. Samples were incubated at 40°C for and then studied for aggregated, intact and depegylated PEG-IFNa 2b by SE-HPLC after 24 hours to determine the optimum molar concentration of the buffer. The results are shown below in Table 4. The results shows that the Tris-benzoate buffer containing formulations are able to protect the PEG-IFN bond and there is not more than 15 % of depegylation and about 9 - 12 % aggregation has occurred after 24 hours incubation at 40°C. In contrast the innovators composition showed that there is more than 46% of aggregation and only 47% of intact conjugate could be recovered. This clearly shows that the presence of dual buffers protects the bond or linkage between the polymer and the drug in polymer - drug conjugates.
Table 3: Compositions of PEG-IFNα 2b aqueous formulations

Formulation code Molar strength of Tris -Benzoic acid (mM) Details of the formulation
F-1 10
F-2 12.5 PEG-IFNa 2b - 0.15 mg/ml
F-3 15 Sucrose -118.4 mg/ml
F-4 20 Polysorbate 80 - 0.148 mg/ml
F-5 30
Control (In-house Viraferonpeg® ) PEG-IFNa2b (0.15 mg/ml), Anhydrous dibasic sodium phosphate (2.22mg/ml), Monobasic sodium phosphate dihydrate (2.22g/ml), Sucrose (118.4mg/ml), Polysorbate 80 (0.148 mg/ml)
Table 4: SE-HPLC data of PEG-IFN at different molar strengths of buffer system after 24 hours at
40 °C

Formulation Code Agg Monopeg Depeg
F-1 9.4 77.7 12.9
F-2 9.97 76.32 13.7
F-3 10.2 76.05 13.75
F-4 12.28 73.66 14.06
F-5 11.05 78.75 10.2
Control(In-house Viraferonpeg4 46.5 47.9 5.7
Example 3: Effect of freeze thaw cycles
Freeze thaw studies were conducted on formulations F-3 , F-4 and In-house Viraferonpeg® (Table 3). In-house Viraferonpeg was used as control. Samples were frozen at -20°C for 1 hr and thawed in water bath at

25 °C for 30 minutes. These freeze-thaw cycles were repeated 5 times and samples were withdrawn after 3rd and 5th cycle for SEC-HPLC analysis.
The results are shown below in Table 5. From the results it was observed that PEG-IFNa 2b is more stable in tris-benzoate buffer as compared to the innovator's buffer i.e. phosphate buffer. In control sample 25.7% aggregate got generated after 5 freeze thaw cycles whereas in tris-benzoate buffer (F-3 and F-4) it was just 6.0 to 7.0 % aggregates after 5 cycles of freeze -thaw,
Table 5: SEC data of PEG TEN at different freeze thaw cycles.

Sample % Area of peak

3 FT - Cycles 5 FT - Cyclea

Agg Monopcg Depeg Agg Monopeg Depeg
Control(In-house Viraferonpeg®) 7.0 91.1 1.9 25.7 69.6 4.7
F-3 5.9 86.7 7.4 6.8 89.3 3.9
F-4 5.7 91.0 3.3 5.9 90.5 3.6
Novel formulations described in the present invention offers the following advantages:
1. Involve operational simplicity as it minimizes the use of stabilizers.
2. Involve use of buffers which protect the bonds or linkages between polymer and therapeutic drug when present in solution form during elevated temperatures and repeated freeze-thaw cycles.
3. Provide better stability to the aqueous formulation of drug conjugates to maintain its activity for a prolonged period of time to guarantee a reasonable shelf-life.
4. Provide better stability to the aqueous formulation to maintain its activity even at a higher temperature and repeated freeze-thaw cycles.
5. May be presented as liquid formulation and hence lyophilization step may be avoided.
6. Avoid cold-chain storage and transportation
7. Cost effective and enhanced patient convenience.

We Claim
1. A process for the preparation of stable pharmaceutically acceptable formulation of polymer - drug conjugates to minimize the cleavage between the polymer and a therapeutic drug when present in solution form at elevated temperatures and repeated freeze-thaw cycles by using two or more buffering excipients especially with two buffering excipients.
2. A process as claimed in claim 1 wherein the first buffer excipients is selected from the buffering excipients which maintains the pH in acidic range and the second buffering excipient is selected from the buffering excipients which maintains the pH in alkaline range.
3. A process as claimed in claim 1 wherein the first buffering excipient is selected from the group consisting of Acetic acid, Acidic Amino acids, Adipic acid, Ascorbic acid, Benzoic acid, Carbonic acid, Citric acid, Citrulline, Formic acid, Fumaric acid, Glutaric acid, Glycolic acid, Lactic acid, Malic acid, Malonic acid, Oxalic acid, Phosphoric acid and salts, Pyruvic acid, Succinic acid, Tartaric acid.
4. A process as claimed in claim 1 wherein the second buffering excipient is selected from the group consisting of Basic Amino acids, Tris (hydroxymethyl) aminomethane, Asparagine, Sodium carbonate, Sodium bicarbonate, Ethyleneimine, Purine, Tricine, HEPES, Boric acid.
5. A pharmaceutically acceptable formulation as claimed in claim 1 further comprises an ampholyte, an anti-oxidant, a non-reducing sugar, a non-ionic surfactant and optionally with preservative either alone or in combination such that the bonds or linkages between polymer and therapeutic drug when present in solution form can be maintained at elevated temperatures.

6. A process as claimed in claim 1 wherein elevated temperatures falls in the range of 25 °C to 50 °C and the number of freeze-thaw cycles upto 4 or 5.
7. A process as claimed in claim 1 stabilizes the polymer - drug conjugates even at a frozen temperature below -5 °C or less and thawed above freezing temperature of the solution.