FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLITE SPECIFICATION
[See section 10 and rule]
TITLE: PROCESS FOR PREPARATION OF STERICALLY STABILIZED LIPOSOMES CONTAINING RECOMBINANT PROTEIN AND USE THEREOF
Applicant: Claris Lifesciences Limited,
Claris Corporate Headquarters, Nr. Parimal Crossing, Ellisbridge, Ahmedabad - 380 006, India.
The following specification particularly describes the invention and the manner in which it is to be performed.

DESCRIPTION
FIELD OF INVENTION
The present invention relates to modified compositions of sterically stabilized vesicles for encapsulating biopharmaceuticals, more specifically recombinant proteins. More particularly, the present invention relates to sterically stabilized unilamellar vesicles (SSUV) for encapsulating modified pharmaceutical compositions of recombinant human or animal or plant or microbial derived proteins, glycoproteins, enzymes, peptides and their pharmaceutically acceptable derivatives, having long circulating half life with an effective therapeutic dose to create biological response to treat the human disease like cancer for example, Leukemia. The protein delivery system to date has some limitations particularly repeated dosing for being effective. The repeated use of unrelated proteins for example asparaginase, streptokinase etc. leads to toxicity by generating immune responses and thereby the efficacy and availability of the same is drastically reduced. Liposomal drug delivery system has ability to overcome these problems by reducing dosing frequency. However, the drawback of the liposomal encapsulation of L - Asparaginase is that they have been cleared from the circulation by reticuloendothelial system. In present invention, we would like to address these issues and develop efficacious system for protein delivery to increase its systemic circulation half life as well as therapeutic index. The objective of the present invention is to develop a pegylated liposomal drug delivery system of biopharmaceuticals. more particularly recombinant proteins in which protein is captured or associated in active state, while polyethylene glycol (PEG) moiety, which is attached to the lipid molecule, and not to the protein molecule as in case of Oncaspar, protects the liposomes clearance by reticuloendothelial system in human body and proteolytic degradation of the L - Asparaginase in serum. In present invention, different grades of PEG moiety according to their molecular weight was used to develop pegylated liposomes. Therefore, the proposed drug delivery system, that is pegylated liposomes of L - Asparaginase increases its therapeutic efficacy and systemic circulation time with reduced toxicity.

BACKGROUND AND PRIOR ART
[001] Leukemia is a cancer of the bone marrow and blood. European Physicians in 19 century were the earliest observers of patients who had markedly increased white cell counts and term Weisses Blut or white blood emerged as a designation for the disorder, Later, the term Leukemia was used to indicate the disease. The term myelogenous or lymphocytic denote the cell type involved. The major forms of leukemia are divided into four categories, which are Acute or Chronic Myelogenous and Lymphocytic Leukemia. Acute Lymphocytic Leukemia (ALL) results from an acquired genetic injury to the DNA of a single cell in the marrow. The patients with ALL need L -Asparaginase therapy.
[002] Bacterial L- Asparaginase (L - Asparaginase Amidohydrolase E. C. 3.5.1.1) are enzymes of high therapeutic value due to their use in treatment of leukemia. L - Asparaginase was used as therapeutics in Acute Lymphocytic Leukemia, because malignant cells are dependent on a exogenous source of L -Asparagine for survival. The normal cells, however, are able to synthesize L -Asparagine. and thus are less affected by its rapid depletion produced by treatment with the enzyme L - Asparaginase. The most common therapeutic indication of L - Asparaginase are; Treatment of Hodgkin disease, Acute Lymphocytic Leukemia, Acute Myelocytic Leukemia, Acute Myleomonocytic Leukemia, Chronic Lymphocytic Leukemia, Reticulosarcoma and Melanosarcoma. Commercially used enzymes are obtained from Escherichia coli (Elspar, Oncospar) Erwinia carotovora (Erwinase), Erwinia chrysanthemi, Wolinelaa succinogens and other genera.
[003] Liposomes are spherical vesicle with a membrane composed of a phopholipid and cholesterol bilayer. Phospholipid molecules composed of hydrophobic tail group and hydrophilic head group. Such molecules are termed as amphipathic molecules and have tendency to form bilayer when comes in contact with aqueous fluid and can be used to encapsulate biologically active materials. Some limitation of biologically active compounds, such as lower water solubility can be effectively minimized by encapsulating this compounds in liposomes. Generally, they act as carriers for therapeutic agents, and can carry therapeutic agents to target cells. It is desirable to have an increased circulation time for liposomes. However, the rapid clearance of liposomes from

systemic circulation by reticuloendothelial system has been an important barrier blocking the use of liposomes for systemic therapeutic applications. Opsonin protein as well as lipoproteins and phospholipases present in body fluid destabilize liposomes which is the major drawback associated with liposomes. Uptake by Mononuclear Phagocyte System (MPS) cells generally leads to irreversible sequestering of encapsulated drug, thereby eliminating any beneficial effects as well as posing potential risk of toxicity to these cells. Phagocytic and endocytic cellular uptake of liposomes and its contents, carried out by macrophage cells of MPS.can also occur in all other tissues. These cells originate from bone marrow, but forms a resident population through out the body. Following the intravenous administration, liposomes primarily comes in contact with macrophages in liver, spleen and bone marrow where they are removed from circulation. The various interactions of the liposomes are; (1) Exchange of materials, primarily lipids and proteins with cell membrane, (2) Adsorption or binding of liposome to cells, (3) Cellular internalization of liposomes by endocytosis or phagocytosis once they are bound to cells and (4) Fusion of bound liposomes to cell membrane. All these interactions depends upon the lipid composition, type of cell, presence of specific receptors and various other features. Most of the biotechnology based therapeutic agents are not expected to survive macrophage uptake. Results of studies of small peptide hormone in various liposomes showed complete loss of activity as liposomes are cleared from blood stream.
[004] In the case of present invention offered, lipid vesicles may be prepared either by ultra sonication and high pressure homogenization followed by extrusion and to encapsulate protein molecules, principles of passive loading were utilized. To determine encapsulation efficiency, free L - Asparaginase may be separated by using chromatographic method, more specifically, either by G-25 gel filtration chromatography or by ion exchange chromatography. While for determination of encapsulated proteins various methods especially, Micro Bradford Method, Enzyme Activity Assay and /or RP-HPLC may be utilized. Moreover, RP-HPLC method may also used to identify the ratio of protein to phospholipid content which enables to determine encapsulation efficiency of the process. Present invention utilized xenograft model of mammel cancer to determine potency and efficacy of final formulations.

SUMMARY OF THE PRESENT INVENTION
[005] Aim of the present invention was to improve pharmacokinetic profile of L - Asparaginase by novel drug delivery system. In order to achieve our goal, long circulating liposomes, more preferably long circulating pegylated liposomes were utilized. The concept behind the use of pegylated liposomes is to provide stability to liposomes against reticuloendothelial system in serum, as serum proteins identifies liposomes as foreign body and cleared it from the serum. Thus, present invention claims that, L - Asparaginase will remain active when formulated as pegylated liposomes, and is available to exert pharmacological effect on administration. The present invention holds promise to treat cancer, especially Leukemia.
[006] In present invention, sterically stabilized unilamellar vesicle that is stealth liposomes of nanometric size more preferably 100 -150 nm can be prepared by using different ratios of phospholipids to PEG coated lipids. Advantageously, these formulations has shown stability of more than 80 to 90% in serum. The method of preparation of liposomes, more specifically pegylated liposomes, and analytical methods for determination of encapsulation efficiency, determination of enzyme activity, flow cytometry analysis of cells, and other methods were developed and validated previously. Liposomes prepared using this method has been shown more than 85 to 90% L -Asparaginase association with pegylated liposomes which in terms of entrapped L - Asparaginase or entrapped as well as surface adsorbed L -Asparaginase, wherein the surface adhere protein must be within defined limit. In present invention, protein may also have a static interactions with PEG moiety which might increase the encapsulation and may create the effects of supper-loaded liposomes. The in vivo performance of the pegylated liposomal L - Asparaginase has been compared with parent L - Asparaginase and Oncaspar, and it was observed that, oncaspar has the advantage of low dosage and minimal immune responses.
[007| To overcome this problem with liposomes, various modifications in the processes/methods for liposome preparation have been disclosed in the literature. The liposomes containing high amount of distearylphosphatidyl choline (DSPC) mixed with cholesterol have shown prolonged systemic circulation. In another example preparation of liposome using PEG -

phosphatidylethanolamine has proven to offer better protection from MPS uptake and increased blood circulation time.
[008] The EU patent EP0485143 entitled "Liposomal composition and process for their production" assigned to Instituto Nacional De Engenharia E Technologia Industrial, has described encapsulation of hydrophobic derivative of L- Asparagenase that is palmitoyl - L - Asparaginase in Jiposomes prepared usingsphingomyelin, dioleoylphosphatidylcholine, dioleoylphospatidylglycerol dioleoylphosphatidylglycerol, phosphatidylglycerol, dimyristoylphosphatidy-lcholine dimyristoylphosphatidylcholine, phosphatidylglycerol dimyristoylphosphatidylcholine, dimyristoylphosphatidylcholine, dicetylphosphate, phosphatide acid, distearoylphosphatidylcholine and cholesterol. While. present invention offers sustained release and long biological half life of L -Asparaginase by using pegylated liposomes as carriers which has advantages of reduced clearance of liposomes from blood and hence provide effective therapy. Moreover, in present invention, L - Asparaginase is used in its parent form and not in its any derivative(s).
[009] The US patent application 6043094 has described methods of liposome based therapy, wherein the outer surface of liposomes that contains an affinity moiety, can specifically affect binding to a biological surface at which therapy is aimed.
[010] The US patent application 2005/0202091 has described a pharmaceutical composition of erythropoietin, which is stabilized with a combination of a poloxamer - polyol and polyhydric alcohol.
[011] The US patent application 7179484 has described liposomes containing lipophilic chemical agent, wherein liposomes has been coated with proteins like albumin to enhance stability. The coating of liposomes with emulsifying protein may complicate the analysis of the biopharmaceutical products like Asparaginase. Application of such protein emulsification for delivering the protein based product has great therapeutic concern, because along with the therapeutically active protein, other denatured protein used in emulsification may also be injected. Therefore, there is a need of vesicles with long circulation time without using the protein emulsifier agent.
[012] As required, detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are

merely exemplary of the present invention, which can be embodied in various forms. Therefore, specific structural, functional, formulation or process details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art. Further, the terms and phrases used herein are not intended to be limiting but rather to provide an understandable description of the present invention.
[013] The terms "a" or "an", as used herein, are defined as one or more than one. The term plurality is defined as two or more than two. The term another, is defined as at least a second or more. The terms including and/or having, as used herein, are defined as comprising (that is open language).
[014] An embodiment of the present invention provides pharmaceutical compositions comprising effective amounts of sterically stabilized unilamellar vesicles containing pharmaceutical compositions of human or animal or plant or microbial derived proteins, glycoproteins, enzymes, peptides or its pharmaceutically acceptable derivatives, having long circulating half life with therapeutic effective dose to create biological responses in order to treat the Acute Lymphocytic Leukemia. Formulation comprises of one or more than one pharmaceutically acceptable cryopreservative, diluents, emulsifiers, stabilizers, carriers, adjuvants, solubilizers and antioxidants.
[015] An embodiment of the present invention provides sterically stabilized unilamellar vesicles with prolonged circulation time without using the protein emulsifier agent. In an embodiment, encapsulation is achieved by pH gradient and/or salt gradient technique at temperature ranges from 4°C to 50°C which leads to better encapsulation efficiency of biopharmaceuticals, more specifically proteins. In an embodiment, mPEG - DSPE is used along with other phospholipids to form the sterically stabilized unilamellar vesicles of nanometric size, more specifically of 80-120 nm.
[016] In an embodiment of the present invention, the surface coating to form stealth liposomes has been provided by the hydrophilic polymer chains which gives colloidal stability and serves to protect the liposomes loaded with an effective amount of recombinant proteins or glycoproteins, phosphoproteins from human, bacteria, fungi, virus, animal and plant preferably, an enzyme like L - Asparaginase type II, and increases the sustained release effect of such liposomes by decreasing or protecting them from uptake by reticuloendothelial

batch size, (on anhydrous basis). Weigh HSPC, mPEG-DSPE 2000 and cholesterol and dissolve it in organic mixture followed by filtration from sterile 0.2 micron filter- - Close the bottle with rubber stopper and store it at 10°C to 35°C. Start spray drying of this lipid solution using selected parameters. After completion of spray drying, keep the assembly aside for cooling. After cooling collect spray dried powder and add ammonium sulphate solution to it and start hydration at 60 °C with sonication for 1 hour. Shake the bottle at regulator time interval during hydration. As the Hydration step completed, starts homogenization. Keep Homogenizer at S0°c in water bath and start homogenization of hydrated liposome solution. Homogenize it for 4 passes of 10000 to 15000psi After homogenization analyze for particle size, pH and volume of liposome solution. Take sodium chloride in volumetric flask and dissolve it with WFI and make up the volume 1 liter with WFI and filter it from sterile 0.2-micron filter. Take Sephadex G 50 and mix it with 0.9% sodium chloride solution, keep it at room temperature for 5 hours. After then, sterile it 121°C for 20 min and store it 2 to 8°C. Take L- Histidine in dry flask, dissolve it with WFI, adjust pH 6.50 with 5 M sodium hydroxide solution and make up the volume 3 lit with WFI, check the pH and filter it from sterile 0.2 micron filter. Take sephadex slurry in glass syringe and make a slurry column. Prepare the sephadex column. Sepadex column first saturate with 10 ml of 0.2 M Histidine buffer solution, then add 22 ml of lipid solution in column and keep it for 3 minute at room temperature. After then add 20 ml of histidine buffer in middle of the sephadex column and start to collect the liposome solution. Collection should start when slightly hazzy solution comes out from column. When start. the clear solution, stop the collection of the solution { Approx 22 ml Histidine buffer is require for 10 ml lipid solution ). After then regenerate the sepahdex column with 0.9 % sodium chloride solution"- Used this column three times for external ammonium sulphate removal. Check the ammonium sulphate content form liposome solution and pH and volume of total liposome solution. Take the liposomal solution and filter it from 1 micron and 0.45 micron polycarbonate filter paper. By membrane extruder. now take this sloution in glass bottle and keep this at 30 to 37°C temperature in water bath. When temperature of the solution reaches, add the L Asparagenase solution to it. Slightly shake the bottle, and analyze for pH of the solution. Then keep the solution for 12 hr, 24 hr and 48 hr. After completion of drug loading, filter the

daltons. In an embodiment, the DSPE may also contain the multiarm PEG of 1000 dalton or 2000 dalton molecular weight. The mPEG may also be branched with same molecular weight arm or with different molecular weight arms.
[019] An embodiment of the present invention provides a composition comprising stabilizing agent and or pharmaceutically acceptable excipients selected from, for example, amino acid like L- methionine, L - Arginine, Glycine. Histidine, Alanine and salt thereof, Sugars like, sucrose, mannose, trehalose, mannitol, glycerol and benzyl alcohol, or any combination thereof.
[020] An embodiment of the present invention provides a method for formulating a biomolecule with enhanced systemic circulation time and reduced clearance of liposome by reticuloendothelial system. In an embodiment of present invention, the biomolecules may be recombinant proteins, glycoproteins, peptides, or pharmaceutical ly accepted derivative from human, plant, fungi, bacteria preferably a protein belongs to a class of EC 3.5.1.1 enzyme, in sterically stabilized unilamellar vesicles, which is surface engineered with hydrophilic polymer like polyethylene glycol chains having molecular weight in the 350 to 12,000 daltons, more preferably 750, 2000 and/or 5000 daltons molecular weight, covalently attached with distearoylphosphatidylethanolamine (DSPE). In an embodiment, DSPE may bear the multiarm PEG of 2000 dalton molecular weight. In an embodiment of present invention, hydrophilic polymer for surface engineering, bound covalently to head group of vesicle forming phospholipid. In an embodiment of the present invention, the method comprises of several steps amongst which, initial step is selection of predetermined combination of lipids followed by dissolution of these lipids in suitable organic solvent(s). In an embodiment, process of preparation of liposomes more specifically pegylated liposomes involves hydration of lipid film by using aqueous solution more preferably using aqueous buffer like citrate buffer, phosphate buffer, preferably kosmotropes especially ammonium sulfate containing buffer with or with out biopharmaceutical stabilizing agent and or pharmaceulically acceptable excipients like amino acid such as L- Methionine, L - Arginine, Glycine, Histidine, Alanine and salt thereof, Sugars like, sucrose, mannose, trehalose, mannitol, glycerol and benzyl alcohol, or any combination thereof. In an embodiment, the process comprises homogenizing and extruding to achieve

required particle size of stealth liposomes. In an embodiment the required particle size is in the range of 50-300 nm, more preferably 80 to 120 nm. An embodiment of the present invention provides a process of encapsulation of biopharmaceutical, which can be driven by pH gradient using 4.5-11 pH, preferably 3.5-7.5 pH, where in the liposome has internal pH of 4.5-6.5 pH, more preferably 4.5-7.0 pH and immediate environment has a pH of 5.6-10.5, preferably, 7.0-8.0 pH and/or salt gradient with or with out pharmaceutical stabilizing agent and/or pharmaceutically acceptable excipients like amino acid like L- Methionine, L- Arginine, Glycine, Histidine, Alanine and salt thereof, Sugar like, sucrose, mannose, trehalose, mannitol, glycerol and benzyl alcohol, in any combination thereof.
[021] An embodiment of the present invention provides a process of encapsulation of biopharmaceutical like L - Asparaginase enzyme, which can be driven by a salt gradient using 10 mM-1000 raM, preferably 10-300 mM with or without biopharmaceutical stabilizing agent and/or pharmaceutically acceptable excipients like amino acid(s) more specifically L-methionine, L-Arginine, Glycine, Histidine, Alanine and salt thereof, sugar(s) like, sucrose, mannose. trehalose, mannitol, glycerol and benzyl alcohol or in any combination thereof
[022] In another embodiment of the present invention, the process further comprises of rehydration process of pegylated liposomes carried out using suitable aqueous buffer, like citrate buffer, phosphate buffer, preferably kosmotropes especially ammonium sulfate containing buffer.
[023] An embodiment of the present invention provides a novel formulation comprised of biopharmaceuticals in therapeutically effective amount to treat blood disorders such as Acute lymphocytic leukemia. Therapeutically effective amount is defined as the amount necessary for the in vivo performance to treat Acute lymphocytic leukemia in mammals especially in humans. This amount may vary depending upon factors such as the type and extent of disease, the condition of the patient and the primary cause of the anemia. The frequency of administration may be reduced by using the therapeutically effective amount of the composition of the present invention. The dosing frequency may also vary due to difference in response to the dose by different patients.

[024] The various embodiments of the present invention provide Pegylated Liposomal compositions with increased circulation time and stability as well as the desirable biological activity. These pegylated liposomes of biopharmaceuticals such as therapeutic proteins, glycoproteins, enzymes more preferably L - Asparaginase either originated frorn E.coli or recombinantly expressed, enables to overcome problems of short half life. Moreover, it enhances efficacy of the L - Asparaginase and increases its plasma residence time. In addition, pegylated liposomes of L - Asparaginase prevents self aggregation of liposome due to its stealth nature, resulting in increased systemic circulation time, and also the dysopsonization phenomenon, where PEG actually promotes binding of certain proteins that, then, masks the vesicle and prevents their clearance by reticuloendothelial system.
[025] In another embodiment of the present invention, the activity of modified composition can be determined by known methods for example, in vitro bioassay or in vivo bioassay using SCID mice. The Composition of the present invention can be administered in a therapeutically effective dose to patients at a relatively lower frequency and/or dosage. The compositions/formulations of the present invention may be presented in unit dosage form and may be prepared by any of the method well known in art. The suitable formulations include the aqueous sterile injection solution, which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood. In an embodiment of the present invention, the formulation may be prepared in unit-dose or multiple doses in pre-filled injection, ampoules or vials and may be in lyophilized form.
[026] In the foregoing disclosure, exemplary embodiments of the invention have been described. However, one of ordinary skill in the art appreciates that various modifications and changes can be made without departing from the scope of the invention. Accordingly, the provisional specification is to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of the invention. The benefits, advantages. solutions to problems, and any element(s)/feature(s)/step(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as critical, required, or essential features or elements with reference to the disclosure.

DETAILED DESCRIPTION OF THE INVENTION
[027] The present invention provides a modified process for the preparation of
pegylated liposomal drug delivery system of L Asparagenase
[028] A process of preparation of pegylated liposomal L Asparagenase
injection can be describe as following steps:
[029] The first step is preparation of organic mixture by mixing chloroform and
methanol in a ratio of 1:1.
[030] According to [029], chloroform and methanol are mixed in 500 to 1000
ml volume of bottle.
[031] According to [030], the bottle containing solution is sonicated for 10
minutes in ultrasonicator.
[032] The second step is the preparation of lipid solution by taking weighed
quantity of Hydrogenated phosphatidylcholine (HSPQ, N-(Carbonyl-methoxy-
polyehtyleneglycol 2000)-1, 2-disteroyl-sn-glycero-2-phosphoethanolamine
sodium salt (MPEG -DSPE 2000) and cholesterol, which is dissolve in organic
mixture and passed through 0.2 micron filter paper.
[033] According to [032], the concentration of lipicl solution should be in the
range of 10 to 30 percentages.
[034] According to [033], the solution is taken into bottle and then closed
with rubber stopper.
[035] According to [034], the bottle is stored at 10 to 35 °C temperature.
[036] According to [035], the solution is taken for spray drying.
[037] The third step is spray drying.
[038] As per the [037], the spray drying is carried out at selected inlet and
outlet temperature, air pressure, airflow and pump flow.
(039} As per the [038], the inlet temperature is 60 °C.
[040] As per the [039], the inlet temperature is ranged from 50 °C to 90 °C.
[041] As per the [038], the outlet temperature is 30 oC.
[042] As per the [041]. the range of outlet temperature is 30 °C to 60 °C.
[043] As per the [038], the selected compressed air pressure is ranging from
0.6 to 0.7 bars.
[044] As per the [038], the pump flow is 0.2 ml/minute.
[045] As per the [044], the pump flow is 0.1 to 0.6 ml/minute.

[046] As per the [038], allow the assembly along with the spray-dried powder
to cool down considerably in an air-conditioned environment.
[047] As per the [046], the spray-dried powder is collected in dry glass bottle
and check the moisture content of this spray dried powder.
[048] The fourth step is preparation of ammonium sulphate solution.
[049] As per the [048], Ammonium sulphate is taken in flask in a quantity of 1
to 10 mg/ml and it is dissolved in water for injection and the pH is adjusted by
using selected solution.
[050] As per the [049], the selected solution is Hydrochloric acid (HC1) and
Sodium Hydroxide (NaOH) to adjust the pH at 5.40.
[051] As per the [050], the pH is in the range of 4.0 to 6.5.
[052] As per the [051], after adjusting pH the voiume is adjusted with Water
for injection, again the pH is adjusted and after that the solution is passed
through 0.2 micron filter.
[053] As per the [049], the concentration of ammonium sulphate solution is
ranged from 0.01 M to 0.4 M.
[054] The fifth step is hydration of spray-dried lipid.
[055] As per the [054], weigh required quantity of spray dried lipid powder
and add 0.15M-ammonium sulphate solution to it for hydration. Hydration is
started at 60 °C.
[056] As per the [055], the temperature is ranged from 35 °C to 70 °C.
[057] As per the [056], the solution is taken for sonication for 1 hour with
frequent shaking.
[058] As per the [057], the time is ranging from 30 minute to 120 minute.
[059] The sixth step .is Homogenization
[060] As per the [059], the clean homogenizer is kept in water bath which is
previously heated at 80 °C.
|061] As per the [060], the temperature is maintained between 50 °C & 90 °C
[062] As per the [060], start Homogenization of hydrated solution of
liposome at 10000 PS! lo 1 50000 PS1 for 4 passes.
[063] As per the [062], the number of passes of homogenization is ranging
from 2 to 8.
[064] As per the [060], after homogenization the liposomal solution is
analysed for particle size, which should be in the range of 80 nm to 150 nm,
and the pH is ranged from 4.0 to 6.5.

[065] The seventh step is preparation of 0.9% sodium chloride.
[066] As per the [065], take sodium chloride in volumetric flask and dissolved
it with water for injection to get 1 liter final volume and then passed it through
0.2-micron filter.
[067] As per the [066], the concentration of sodium chloride is ranging from
0.5 to 1.5%.
(068] The eighth step is preparation of Sephadex slurry.
[069] As per the [068], sephadex G 50 is added in 0.9% sodium chloride
solution and the solution is put at room temperature for 5 hours.
[070] As per the [069], the time is ranged from 2 to 10 hours.
[071] As per the [069], the solution is sterile at 121 °C for 20 minutes and
stores it between the 2 to 8 °C temperatures.
[072] As per the [071 ], the slurry is used for the sephadex column preparation.
Before using it for the column preparation, the temperature of the slurry should
equal to room temperature.
[073] The eighth step is preparation of 0.2M Histidine buffer solution.
[074] As per the [073], L-Histidine is taken in dried flask and dissolved with
water for injection and the pH is adjusted at 6.50 with 5 M sodium hydroxide
solution and the final volume is made with water for injection and then passed
it from 0.2 micron filter.
[075] As per the [074], the pH is ranged from 5.0 to 7.50 and the
concentration of preparation is ranging from 0.05 to 0.5 M.
[076] The tenth step is removal of external ammonium sulphate.
[077] As per the [076], the sephadex slurry is taken in glass syringe and by
that way column is prepared.
[078] As per the [077], the sephadex column is first saturated with 0.2 M
Histidine buffer solution and after that is saturated with lipid solution and keep
a side for 3 minutes.
[079] As per the [078], the time should be ranging from 1 to 15 minutes.
[080] As per the [079], the histidine solution is again added in the sephadex
column and then the liposome solution is started to collect.
[081] As per the [080], the liposome solution is started to collect when the
slightly hazy solution is came out.
[082] As per the [077] to [081], repeat both steps to get more liposome
solution till the hazy solution came out.

[083] As per the [082], after completion of collection of liposome solution,
the sephadex column is regenerated with 0.9% sodium chloride solution.
[084] As per the [083], all the steps are followed thrice for the removal of
external ammonium sulphate.
[085] As per the [084], the solution is checked for the ammonium sulphate
content, for pH and the final volume is measured.
[086] The eleventh step is preparation of sucrose solution.
[087] As per the [086]. take sucrose and dissolved it in water for injection and
filter the solution from 0.2 micron sterile filter and autoclave it.
[088] As per the [087], the sucrose solution is added in liposome solution and
then measure the total volume of liposome solution.
[089] As per the [088], the solution is passed through 1.2 micron and 0.45
micron sterile filter by membrane extrusion.
[090] The twelfth step is preparation of L - Asparagenase solution.
[091] As per the [090], take required quantity of L - Asparagenase and
dissolved with required quantity of water for injection and filter it from sterile
0.2 micron filter.
[092] The thirteenth step is the drug loading.
[093] As per the [092], the liposome solution is taken in flask and kept it at 52
°C in water bath.
[094] As per the [093], the temperature is ranging between 35 °C & 60 °C.
[095] As per the [094], when the temperature is reached, add the drug solution
in the flask and slightly shake it followed by analyzing the pH of the solution.
[096] As per the [095], keep the solution for 4 hours at 52 °C.
[097] As per the [096], the time is ranged from 30 minutes to 24 hours and the
temperature is ranged from 35 °C to 60 °C.
[098] As per the [097], when the temperature of the solution reaches to room
temperature, starts the filtration from 0.2-micron filter
[099] As per the [098], take final drug-loaded solution in 5-liter flask, and
start the filtration from 0.2-micron sterile filter.
Example 1:
Prepare the organic mixture of chloroform and methanol in the ration of 1:1. Take 175 ml chloroform and 175 ml methanol in 500 to 1000 ml HPLC grade bottle. Sonicate them for 10 minutes in ultrasonicator . First weigh lipids as per

batch size, (on anhydrous basis). Weigh HSPC, mPEG-DSPE 2000 and cholesterol and dissolve it in organic mixture followed by filtration from sterile 0.2 micron filter. . Close the bottle with rubber stopper and store it at 10°C to 35°C. Start spray drying of this lipid solution using selected parameters. After completion of spray drying, keep the assembly aside for cooling. After cooling collect spray dried powder and add ammonium sulphate solution to it and start hydration at 60 °C with sonication for 1 hour. Shake the bottle at regulator time interval during hydration. As the Hydration step completed, starts homogenization. Keep Homogenizer at 80°c in water bath and start homogenization of hydrated liposome solution. Homogenize it for 4 passes of 10000 to 15000psi. After homogenization analyze for particle size, pH and volume of liposome solution. Take sodium chloride in volumetric flask and dissolve it with WFI and make up the volume 1 liter with WFI and filter it from sterile 0.2-micron filter. Take Sephadex G 50 and mix it with 0.9% sodium chloride solution, keep it at room temperature for 5 hours. After then, sterile it 12TC for 20 min and store it 2 to 8°C. Take L- Histidine in dry flask, dissolve it with WFI, adjust pH 6.50 with 5 M sodium hydroxide solution and make up the volume 3 lit with WFI, check the pH and filter it from sterile 0.2 micron filter. Take sephadex slurry in glass syringe and make a slurry column. Prepare the sephadex column. Sepadex column first saturate with 10 ml of 0.2 M Histidine buffer solution, then add 22 ml of lipid solution in column and keep it for 3 minute at room temperature. After then add 20 ml of histidine buffer in middle of the sephadex column and start to collect the liposome solution. Collection should start when slightly hazzy solution comes out from column. When start the clear solution, stop the collection of the solution (Approx 22 ml Histidine buffer is require for 10 ml lipid solution ). After then regenerate the sepahdex column with 0.9 % sodium chloride solution. Used this column three times for external ammonium sulphate removal. Check the ammonium sulphate content form liposome solution and pH and volume of total liposome solution. Take the liposomal solution and filter it from 1 micron and 0.45 micron polycarbonate filter paper. By membrane extruder. Now take this sloution in glass bottle and keep this at 30 to 37°C temperature in water bath. When temperature of the solution reaches, add the L Asparagenase solution to it. Slightly shake the bottle, and analyze for pH of the solution. Then keep the solution for 12 hr, 24 hr and 48 hr. After completion of drug loading, filter the

solution from 0.2 micron polycarbonate filter by membrane extruder. The process of preparation of pegylated Liposomal injection of L Asparaginase is novel in terms of type and amount of lipid used, concentration of all ingredient used, as well as buffer system. HSPC, Cholesterol and mPEG-DSPE 2000 was used as fipidic agent and the optimized concentration of these lipids provide better entrapment efficiency and Good stability. In this process of preparation of pegylated Liposomal injection of L Asparaginase mPEG - DSPE 2000 is used to provide stealth nature to liposomes .The optimized amount of HSPC was used as phospholipid to form bilayer. Organic mixture of methanol and chloroform in a ratio of 1:1 was used as solvent system in the invention. The recombinant protein used is L - Asparagenase in the invention. The process for preparation of liposomes is unique in terms of pegylation as well as process parameters of sonication, spray drying, homogenization, extrusion, and Iyophilization as these parameters are derived and optimized from lab trials. In the invention the pegylation was achieved using mPEq - DSPE 2000.All lipids were Dissolved in organic mixture and pass through 0.2 Micron filter paper.The lipids dissolved in organic solvent was spray dried using selected parameters. The selected parameters for spray drying are; inlet temperature of 50 °C to 90 °C; outlet temperature is 30 °C to 6rj °C; air flow of 0.1 to 0.6.spray-dried lipids was hydrated using Ammonium sulphate solution at temperature range from 35 °C to 70. The hydration has perform with sonication to form large vesicles.After hydration, homogenization is started at 6000 to 20000 psi and at 50 °C to 90 °C temperatnre.After homogenization, external ammonium sulphate solution was removed nsing sephadex treatment. After removal of external ammonium sulphate, drug loading is started using passive loading technique.Drug is loaded by pH gradient technique. The drug loaded liposomes is sterilized by aseptic filtration using 0.2 micron filter.

We claim:
1. A process of preparing Pegylated Liposomal Drug Delivery System of recombinant L Asparagenase comprising the following steps:
i) Preparing the organic mixture of chloroform & methanol,
ii)preparation of lipid solution by taking weighed
quantity of Hydrogenated phosphatidylcholine (HSPC), N-(Carbonyf-methoxypolyehtylene glycol 2000)-l,2-disteroyl-sn-glycero-2- phosphoethanol-amine phosphoethanolamine sodium salr (MPEG 2000-DSPE) and cholesterol , which is dissolve in organic mixture ,
iii) spray drying the selected lipid solution :
iv) preparing the ammonium sulphate solution separately,
v) carrying out the hydration of spray-dried lipid wherein spray dried lipid powder mix ammonium sulphate solution .
vi) carrying out the homogenization of the solution ,
vii) preparing the sodium hydrochloride solution ,
viii) preparing Sephadex slurry.
ix) preparing Histidine buffer solution ,
x) removing the external ammonium sulphate .
xi) preparing sucrose solution ,
xii) preparing of L -Asparagenase solution,
xiii),final]y , drug loading the solution.
2. Process as claimed in claim 1 wherein organic mixture of chloroform & methanol is prepared by mixing chloroform and methanol in a ratio of 1:1.
3. Process as claimed in claim 1 wherein preparation lipid solution comprises y lakin gweighed quantity of Hydrogenated phosphatidylcholine (HSPC), N-(Carbonyl- methoxypolyehtyleneglycol 2000)-l, 2-disteroyI-sn-gIycero-2-phosphoethanolamine sodium salt (MPEG 2000-DSPE) and cholesterol, which

is dissolve in organic mixture and passing through 0.2
micron filter for the filtration process,concentrating lipid solution between 10 and 30 percentages, stored at 10 to 35 °C temperature,selected solution is taken for spray dry.
4. Process as claimed in claim 1 wherein step of spray drying comprises
selecting parameters like inlet temperature,outlet temperature, compressed air
pressure, air flow and pump flow etc. ,
1. the selected inlet temperature is ranged from 50 °C to 90 T;
2. the selected outlet temperature is ranged from 30 °C to 60 T;
3. the selected compressed air pressure is is ranged from 0.6 to 0.7;
5. Process as claimed in claim 1 wherein preparation of the ammonium
sulphate solution comprises taking ammonium sulphate in a quantity of 1 to
10 mg/ml and it is dissolved with water for injection adjusting pH 4-6.5 by
Hydrochloric acid (HC1) and Sodium Hydroxide (NaOH) concentrating
ammonium sulphate solution from 0.01 M to 0.4 M.
6. Process as claimed in claim 1 wherein hydration of spray dried mixture are
carried by mixing spray dried lipid powder with 0.15M-ammonium sulphate
solution at selected at 35 °C to 70 °C, the solution is taken for sonication for
30 minute to 120 minute with frequent shaking.
7. Process as claimed in claim 1 wherein step of preparation of sodium hydrochloride solution comprises sodium chloride dissolved in water and then it is passed through 0.2-micron filter, the concentration of sodium chloride is ranging from 0.5 to 1.5 %.
8. Process as claimed in claim 1 wherein preparation Sephadex slurry comprises sephadex G 50 is added in 0.9% sodium chloride solution and the solution is put at room temperature for 2 to 10 hours , solution is sterile at 121 °C for 20 minutes and stores it between the 2 to 8 °C temperatures.
9. Process as claimed in claim 1 wherein preparation of Histidine buffer
solution comprises dissolving L-Histidine with water and the pH is adjusted at
5,0 to 7.50 with 5 M sodium hydroxide solution.

10. Process as claimed in claim 1 wherein required quantity of L -Asparagenase dissolved with required quantity of water for injection and filter it from sterile 0.2 micron filter.