FORM 2
THE PATENT ACT, 1970
(39 of 1970)

COMPLETE SPECIFICATION
1. METHOD OF MANUFACTURE OF PYROGEN FREE HYDROXYETHYL STARCH
2. (a) CLARIS LIFESCIENCES LIMITED, a Company incorporated in India under
the Companies Act, 1956 having its registered office at 6th Floor, A-Wing, Sangeeta Complex, Nr.Parimal Crossing, Eliisbridge, Ahmedabad-380 006 at state India Nationality an Indian Company
The following complete specification described the nature of the invention and
the manner in which it is to be performed is attached herewith as mark 'A'
IVIC-THOT1 op- rr)A-wup*vc-ru*L<= op PVR0fe,e^> ^Ke-& HYPKoXYeTi-{7L ±-c fr^-Oh
3
For CLARIS Lifesciences Ltd.
Signature
AUTHORISED SIGNATORY
4 We claim: As per mark'B'

5. Abstract of the invention is attached as mark 'A'
To
The Controller of Patents
The Pattaent Office,
At.Mumbai

- 8 FEB 2001

Title: Method of manufacture of pyrogen free Hydroxyethyl starch
Inventor: Kalpesh M. Bhalja
Applicant: CLARIS Lifesciences LTD, Ahmedabad, India
Abstract:
This invention relates to a novel and superior process of manufacturing Hydroxyethyl starch which is essentially free from pyrogen. It involves few unit operations, some of which are capable of performing in parallel, such as pretreatment of waxy starch, molecular size reduction by acid hydrolysis, attaching hydroxyethyl group, precipitation of HES, clarification, bioburden reduction, pyrogen removal by ultra filtration and spray drying. In another aspect this invention relates to manufacturing pyrogen free hydroxyethyl starch using a method which is very simple, repeatable, very economic and capable of using for low cost and commercial scale production of Hydroxyethyl starch.

Page 1 of 17

Complete Specification Background of the invention
Hydroxyethyl starch having average molecular wt of between 190000 and 600000 and having molar substitution value of from 0.45 to 0.8 is used as a plasma volume expander. Presently Dextran-40, Dextran-70 and Dextran-110 and to some extent PVP is used as plasma volume expander along with HES. However due to certain side reactions such as anaphylactic shock etc. with Dextrans and PVP, HES is preferred.
Various methods have been published in various patent as, mentioned in the reference
list, for the manufacture of HES. None of the method is however able to produce low cost
HES. All the processes are silent on the pyrogen aspect of HES. HES when used as plasma volume expander must be pyrogen free. Unless the starting material is free from
pyrogen it is very difficult and very expensive to produce pyrogen free end product.
Patent as mentioned in ref-1-4 also describe the method for HES manufacture. None of the method mentions any steps to reduce the load of Microorganism and pyrogens. Furthermore none of the methods are viable on commercial scale due to complexity, length of time and very high cost involved.
following are given critical drawbacks of these method;
Method as mentioned in ref- 5
t Calls for use of expensive equipments such as continuous flow centrifuge in the
pretreatment stage
The process nowhere mention the desired molecular wt range in the acid modification stage thus, lacking specificity
The hydroxyethylation stage mention reaction at very high temp of say between 190°F and 222°F which on a large scale results in development of dark yellow to brown colour which is very difficult to remove and makes the produtcion on large scale very expensive. Moreover the reaction requires use of water jacketed SS autoclave necessiating heavy investement for large scale production.
The process requires use of very large quantity of solvent and two drying processes, increasing time and cost of production
The process lack various qualitative aspects required in the manufacturing of products for parenteral application such as microfiltration to reduce bioburden and ultrafiltration to reduce level of pyrogen.
The product produced by this method leaves very high amount of sulphated ash and sodium chloride content and pH of the product is also high.


If we refer to the method as mentioned in patent as given in ref-4, it describes the method of manufacture in brief as follows
1 acid hydrolysis
1.1 differential centrifugation to remove dark colored impurities
1.2 Normal centrifugation and removal of brown colored impurities using spatula and repetition of centrifugation
2 stabilization of hydrolyzed starch
2.1 removal of flocculent gray impurities by filtration using loosely packed glass fibre
2.2 washing with copious quantity of distilled water ,2.3 drying
3 Hydroxyethylation in nonaqueous medium at 37°C for 24 hours with stirring for at
least first 2 hours and then neutralization and washing with isopropanol, acetone
and ethanol.
It will be seen that the process is very lengthy and when operated on large or commercial scale, requires considerable amount of sophisticated equipments such as,
* Differential centrifuge,
* Scrapper to remove brown sediments
* Special equipment for fast addition of glacial acetic acid and measurement of neutralization condition, since addition of glacial acetic acid and finding out whether sufficient acid has been added would take longer time in normal large scale production conditions.
* Special column containing loosely packed glass fibers.
* Special tightly closed vessel of large capacity with magnetic stirrer and cooling facility to cool to a temperature as low as 2°C and as high as 37°C.
More over the post hydroxyethylation stage requires use of very large quantities of three solvents such as isopropanol, acetone and ethanol which makes the production of low-cost HES on large scale almost impossible and unviable. Moreover about seven solvent washes are required to get a product free from glycol and acetates which incerase the complexity, cost and time of the process substantially. Moreover these solvents can create environmental related problems if not handleded carefully.
The method as mentioned in patent of ref- 2 uses a single stage reaction both for "hydroxyethylation and hydrolysis to produce HES of average molecular wt of from 40,000 to 80,000. The hydrolysis is so fast that at Ohours about 25% of the starch with m.wt of less than 80000 is produced. This method is, therefore, not capable of producing HES with average m.wt of between 190000 to 500000.
Further it calls for use of very high quantity of more than one solvent for precipitation and extraction of HES, increasing process complexity in terms of time, handling inventory and cost in large scale/commercial set up. E.g as mentioned in example given
Page 3 of 17

about 500ml of pyridine, 1000ml of ethanol, and some quantity of acetone are used for just lOg of the starch. This makes the process unviable for commercial production of HES.
The method as mentioned in patent of ref-1 lacks specificity with respect to desired molecular wt and no process for achieving the desired m.wt is described. Further more the method uses very high quantity of ethylene oxide which not only increases the cost of the product but also generates very high quantity of glycol, subsequent removal of which calls for special process with substantial increase in the cost of final product.
Keeping all the difficulties in mind, the present process has been designed to produce on a commercial scale, pyrogen free and low cost HES with definite average molecular wt and molar substitution value.

Page 4 of 1.7

Summary of the invention
The present method provides very simple and economic process to produce on commercial scale the parenteral grade or pyrogen free Hydroxyethyl starch.
The waxy starch is prewashed using suitable alkali to reduce the protein content to a level of less than 0.2%, preferably less than 0.1%. The starch with low protein content so obtained is then hydrolyzed by Hydrochloric acid at a concentration of 0.6M to 0.8M HC1, preferably 0.685 to 0.7M HC1, in slurry and at a temperature of between 45 °C and 55 °C , preferably 48.5°C and 50°C for a period of 8 to 9hours to achieve average m.wt. of from 185000 to 255000 with constant, slow speed agitation. Hydrolysis, under the same condition as mentioned above but for 6hrs to 7hrs yield starch having average m.wt of from 440000 to 560000. The hydrolyzed starch so obtained is neutralized with alkali to a pH of 4.5 to 7.5 preferably 5.0 to 7.0 and washed using suitable mariner, preferably centrifuged to reduce chloride content in the washing to less than 50ppm.
The hydrolyzed and washed starch is then suspended in water for injection to achieve concentration of about 20% to 27% solids and pH of this slurry is adjusted using suitable alkali to 12.3 to 12.6. The slurry is then heated to about 40°C to 44°C in SS jacketed tank with mechanical stirrer. Once the desired temperature is reached, liquid Ethylene Oxide, about 13% to 15% of starch weight on dry basis, to achieve molar substitution of 0.45 to 0.55 and 20% to 22% to achieve molar substitution of from 0.7 to 0.8,as the case is, is injected into it. Heating is then stopped and reaction is allowed to proceed for about 2 to 2.5 hours at a temp of about 48°C to 52°C. The heat of reaction is normally enough to maintain the above temperature throughout the process without need for any external heating. It is necessary to keep the pH between 12.3 to 12.5 during the course of reaction adding alkali, if required.
At the end of reaction period the almost clear and pale yellow liquid obtained is neutralized using 6N hydrochloric acid to a pH of from 5.0 to 6.5.
The neutralized liquid is then extracted by precipitating using about 1.2times volume of isopropyl alcohol. The precipitate of HES obtained is dissolved in minimum qt. Of water for injection and precipitation step is repeated using quantity of isopropyl alcohol, which is about 1.1 to 1.2times of solution quantity. Chloride content is measured and if more than 0.6% the precipitation step is repeated once more.
The final precipitate, thus obtained, are then dissolved in water for injection sufficient to produce a concentration of about 10-12% w/v of HES and filtered through filter pads via filter press and then passed through 1.0 micron and 0.2 micron cartridge filter to reduce bioburden.
The filtered solution is then passed through ultrafilter cassette having m.wt cut off limit of 30KD. The product solution is then subjected to spray drying. Final product so obtained is HES free from pyrogen, white in colour satisfying the quality criteria as mentioned above.
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Detailed description of the invention
The entire manufacturing process can be divided into following seven major steps:
1 Pretreatment of waxy starch
2 Molecular wt. Adjustment of waxy starch by acid hydrolysis
3 Attachment of hydroxyethyl groups by reacting with ethylene oxide.
4 Solvent precipitation of HES to reduce chloride and glycol content
5 Coarse and micro filtration to produce clear and low bioburden solution
6 Ultrafiltration to reduce pyrogen and other impurities
7 Spray drying
Each step are described in details as follows:
1 Pretreatment of waxy starch
Waxy starch (maize, potato, rice or wheat) is suspended in purified or equivalent grade water and stirred for about lOmin, so as to get final solid concentration in the slurry to be about 15-25%. The temperature of the water taken is kept between 25°C and 37°C preferably 30°C to 35°C, by heating if required.
The suspension is made in a vessel, inert plastic is preferred, with such a surface area, so that about lcm2 area is available for each 2g to 4g of waxy starch ( on dry basis).
The starch slurry (or suspension) is then made alkaline using appropriate quantity of 3M to 4.5M of Sodium hydroxide or Potassium Hydroxide solution, preferably Sodium Hydroxide (cheaper than Potassium hydroxide) to obtain a final pH of between 11.0 to 12.0 preferably 11.6 to 11.8.
The alkali as mentioned above is added slowly and with continuous stirring to prevent gelation of waxy starch.
Once the desired pH is achieved, stirring is stopped and the slurry is allowed to stand for about 3 to 6 hours, preferably 4-5 hours for reaction of protein with alkali.
At the end of standing period, the supernatant liquid that is yellow to dark yellow in colour due to protein extraction is removed by decantation.


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The solid remained in the vessel is again suspended (second extraction)in purified water as mentioned above, with stirring, to get a solid concentration of about 15-25% and pH is adjusted between 11.0 to 12.0 preferably 11.6 to 11.8, if required, using alkali as mentioned above.
The slurry is allowed to stand for about 3 to 5 hours, preferably 4 hours for reaction of remaining protein with alkali.
The process of alkali extraction is repeated third time in the same manner as of second extraction.
The solids, of starch, remained is suspended in purified water to get final solid concentration of 15% to 25%. The slurry is then neutralized using 4M Hydrochloric acid so that final pH is between 5.0 & 7.0, preferably 6.0. Slurry is stirred for about lOmin.
The slurry is then centrifuged and washed with water such that final chloride content of the washing is not more than 50ppm.
The washed starch with specified low chloride content is then removed from the centrifuged and dried, using suitable method such as tray drying or spray drying at 70°C to 80°C preferably 75°C until the moisture content is reduced to 12%. Drying at high temperature may produce off white to cream colored product.
The washed starch can be used for hydrolysis without drying if used within 2-3 hours. In such case solid content of sample is measured and volume is made accordingly.
2 Molecular wt. Adjustment of waxy starch by acid hydrolysis
The waxy starch obtained after treatment as mentioned above ( step-1) is suspended in water for injection such that the solid concentration in the suspension is from 37%w/v to 42%w/v.
The suspension can be made into plastic tank of suitable size made from virgin high density polyethylene or polypropylene or jacketed glass lined stainless steel vessel with the facility of mechanical stirrer having 60 to l00rpm speed or such other suitable tank. In the case of plastic tank suitable portable stirrer with about 60-100rpm speed can be used for stii ring the suspension. For heating, the plastic tank can be pul into another plastic tank with double the diameter of reaction tank in which hot water can be added to heat the suspension in the reaction tank. This process thus uses very simple low cost equipment for this step without compromising quality.
The suspension is then heated using hot water in the jacket or external tank such that temperature of the suspension is maintained between 48.5°C & 50°C.
In the starch suspension thus made and heated, the 6M to 8M Hydrochloric acid is added in quantity sufficient to make the final Hydrochloric acid concentration in the slurry
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between 0.6M to 0.9M, preferably 0.68M to 0.7M, when a portion is titrated against standard alkali using suitable indicator.
The Hydrochloric acid at the temperature mentioned above, hydrolyzes the starch and reduces the molecular weight to the desired level of from 185000 to 255000 and 440000 to 560000 depending on the reaction time allowed. The reaction time is, time period allowed between the completion of Hydrochloric acid addition at the start of hydrolysis to the start of neutralization reaction. The reaction time of from 8hours to l0hours, preferably 9hours to 9hours 30min is kept for obtaining size reduction to a level of from 185000 to 255000. The reaction time of from 5hours to 7hours, preferably 6hours to 6hours 15min is kept for obtaining size reduction to a level of from 440000 to 560000. During the entire course of reaction the starch suspension is kept stirred using suitable stirrer as mentioned above.
About 15 min before the end of specified reaction period, heating is stopped and cooling is started by circulating cold water in the jacket or filling cold water in the external plastic tank such that the temperature reaches to less than 40°C in 15 minutes. At the end of specified reaction time the hydrolyzed starch suspension, having' temperature below 40°C, is neutralized by adding alkali such as Sodium hydroxide or Potassium hydroxide at a concentration of 4.0M to 6.0M so as to bring the pH of suspension to from 5.0 to 6.5. The neutralization is completed in about 10 to 15 minutes.
At the end of neutralization as mentioned above, the hydrolyzed starch suspension is diluted with water for injection such that solid concentration in the diluted suspension is about 10%w/v to 20% w/v. The diluted starch suspension is stirred for about lOmin and then allowed to stay undisturbed for about 4 to 6hours. During this time most of the solids will settle down. The supernatant liquid containing light floating impurities is removed by decantation.
The solids settled is again suspended in water for injection, stirred and centrifuged to wash off chlorides to a level of less than 50ppm as measured by titrating the washing water collected from centrifuge by standard Silver Nitrate titration method.
3 Attachment of hydroxyethyl groups by reacting with ethylene oxide.
The hydrolysed and washed starch, as obtained in step-2 is then suspended in water for injection in jacketed SS tank of suitable capacity to achieve concentration of about 20%w/v to 27%w/v of solids.
pH of this suspension is adjusted using about 4M Sodium Hydroxide or Potassium Hydroxide solution to a pH value of 11.5 to 13.0 preferably 12.0 to 12.3.
The suspension is then stirred at about 100 to 200rpm and heated to about 40°C to 44°C. Once the desired temperature is reached, liquid Ethylene Oxide, about 13% to 16% of
Page 8 of 17

starch weight on dry basis, to achieve molar substitution of 0.45 to 0.55 and 20% to 25% to achieve molar substitution of from 0.7 to 0.8, as the case is, is injected into it. Heating is then stopped and reaction is allowed to proceed for about 2 to 3.0 hours at a temp of about 45°C to 55°C. The heat of reaction is normally enough to maintain the above temperature throughout the process without need for any external heating.
PH is measured every 30min interval during the course of reaction and maintained between 12.3 and 12.5 adding alkali, if required.
At the end of reaction period the almost clear and pale yellow liquid obtained is neutralized using 6N hydrochloric acid to a pH of from 5.0 to 6.5.
4 Solvent precipitation of HES to reduce chloride and glycol content
The neutralized liquid is then precipitated and extracted using suitable solvent such as acetone, methanol, or isopropyl alcohol. This step can be performed in the same SS tank or in a suitable plastic tank with arrangement of slow speed (60 to 100rpm) stirrer.
For the initial precipitation solvent quantity of about 1.2time to 1.3times the total volume of liquid is required to effect complete precipitation of Hydroxyethyl starch. The solvent is added slowly and with constant stirring. The precipitate is then allowed to settle for about 15 to 30 min. The upper liquid layer is then removed by decantation.
The precipitates so obtained are dissolved in minimum quantity of water for injection. The precipitation is repeated using solvent quantity of about 1.2times to 1.3times the total volume of liquid present as mentioned in procedure for initial precipitation as above.
The precipitation stage is repeated third time as mentioned above after dissolving precipitates as obtained after second precipitation.
At the end of third precipitation the solids are dissolved in minimum quantity of water for injection. Solid content is found out by evaporating the liquid at about 100°C in an oven. Chloride is determined by dissolving known quantity of solid in water for injection. If the chloride content is less than 0.6%w/v further precipitation is not carried out. If chloride is more than 0.6%w/v the fourth precipitation is carried out as mentioned in second precipitation.
5 Coarse and micro filtration to produce clear and low bioburden
HES solution
The final precipitate, thus obtained, are then dissolved in water for injection sufficient to produce a concentration of about 10%-20%preferably 1.0-12% w/v of HES. To this solution activated charcoal quantity from 0.3% to 1.2% is added and mixed by stirring for about 30min. The solution is then clarified by filtering through filter pads ( Seitz-

EKSl)using filter press. The clear filtrate is collected in SS tank. The solution is then filtered through 1.0 micron and 0.2/0.45 micron filter cartridge to reduce bioburden in the solution and filtrate is collected in sanitized tank.
6 Ultrafiltration to reduce pyrogen and other impurities
The filtered solution is then passed through ultrafilter cassette having m.wt cut off limit of 30KD. The 30KD cartridge can remove all compounds which are below 30000 M.Wt which includes pyrogens, residual salts, glycols, low molecular wt fragments of HES etc. The ultra filtration is carried out until the solid content is increase to about 18% to 22%. Ultrafiltration reduces the chloride content to less than 0.5%. The solution obtained at the product outlet is collected in SS tank for subsequent spray drying.
7 Spray drying
The ultra filtered solution is then spray dried at a temperature of about 80°C in a spray dryer of suitable capacity. The powder of HES is collected in virgin polyethylene bag and sealed suitably.
The powder so obtained is then analyzed for the criteria as mentioned in table-1
Example-l
1 250 ml of purified water, heated to about 37°C, is filled into a 500ml capacity
plastic beaker with a surface area of about 48cm2 (diameter about 7.8cm). lOOg waxy maize starch is then slowly added into the water with continuous stirring using magnetic stirrer. Once all the starch is added and dispersed fully, further purified water is added into beaker to make volume to about 400ml. 3.0ml of 4M sodium Hydroxide is then added to slurry prepared above with stirring to get pH of 11.7. Finally the volume was made with purified water to 500ml. Slurry was then stirred for about lOmin and allowed to settle. First decantation was carried out after 5.5hrs when yellow supernatant liquid was removed. Again the volume was made with purified water (temp.30°C) to 500ml and slurry stirred. PH was found to be 11.6. No alkali was therefore added. Second decantation was carried out after 3.5hours and the process repeated as mentioned for second decantation.
2.0ml of 4M Hydrochloric acid was added to the slurry made after third decantation and pH was found to be 6.1.
The slurry was then centrifuged at about 1500rpm and washed twice with water for injection to achieve chloride content of less than 50ppm in the supernatant liquid. The yield obtained was 94% when calculated on dry basis.


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2 The wet mass with dry wt of about 95g of starch on dry weight basis is then
suspended in water for injection, in 500ml conical flask, to get total volume of 250ml.
The suspension is then heated on hot plate stirring continuously using magnetic stirrer,
till temperature of 49°C is achieved.
About 21ml of 8M Hydrochloric acid is added to get molarity of suspension to 0.688M HC1. The suspension was heated at the above mentioned temperature for 9hours. Cooled to 35°C by putting the flask in cold water shaking for 5min. The slurry was then neutralized using 26.5ml of 6M Sodium Hydroxide solution to a pH of 5.6.
Water for injection was added to make total volume to 500ml and mixed for 5min. Suspension was then allowedto stand for about 5hours. The supemetant was decanted and solids were suspended in about 300ml of water for injection. The suspension was then washed with about 300ml of water for injection using laboratory centrifuge. The chloride content in final washing was found to be 45ppm.
3 The solids were then suspended in water for injection to a final volume of 400ml in 1000ml fkisk. The solid concentration was 20.1%w/v. About 8ml of 6m Sodium Hydroxide added to get pH of 12.4. The suspension was heated on hotplate to a temp, of 44°C. ETO 11.9g (14% of 85g) was then added into the suspension. Temperature was raised to 48°C and solution kept stirring for about 2hours and 20min. The liquid became almost transparent and slight pale yellow and was cooled to 35°C using cold water. 6ml of 8N HC1 was added to get pH of 5.2.
4 The liquid was transferred to 2000ml beaker and 480ml of Isopropyl alcohol(IPA) was added with constant stirring using portable mechanical stirrer. The mixture was then allowed to settle for lOmin. The ppt were dissolved in 100ml of water for injection and 130ml of IP A was added. Again the ppt were dissolved in 100ml water for injection and 125ml IP A added.
The ppt were now dissolved in 100ml water, solids were determined by evaporating at 90°C and chloride was measured. Chloride was 0.5%w/w. Yield was 72g.
5 20% solution was made and 600mg of activated charcoal( 0.83%w/w) was added
and stirred for about 30min. Solution was filtered through filter pad and vacuum filtered
to get clear almost colourless liquid. The filtrate was then passed through 1 micron and
0.2 micron membrane filter and tray dried at 80°C.
The yield was 71g i.e 71%.
Example-2
1 250 lit of purified water, heated to about 36°C, was filled into a 4501it capacity
plastic trough with a surface area of about 15000cm2 (l=w=r125cm). 70 kg waxy maize starch ( on dry wt basis) is then slowly added to into the water with continuous stirring


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using mechanical stirrer. Once all the starch is added and dispersed fully, further purified water was added into trough to make volume to about 4001it. About 1.51it of 4M sodium • Hydroxide is then added to slurry prepared above with stirring to get pH of 11.7. Finally the volume was made with purified water to 4501it. Slurry was then stirred for about lOmin and allowed to settle.
First decantation was carried out after 5hrs when yellow supernatant liquid was removed. Again the volume was made with purified water (temp.30°C) to 4501it and slurry stirred. PH was found to be 11.63. No alkali was therefore added.
Second decantation was carried out. after 3.5hours and the process repeated for third decantation.
500ml of 4M Hydrochloric acid was added to the slurry made after third decantation to adjust pH between 5.0 and 7.0, and pH was found to be 6.1.
The slurry was then centrifuged at about 1200rpm and washed with purified to achieve chloride content of less than 50ppm in the supernatant liquid.
The yield was 95% on dry wt basis measured by drying a known quantity centrifuged solid at 80°C for 5hours. Thus the yield was 66.5kg.
2 The wet mass with dry wt of about 66.5kg of starch was then suspended in water
for injection, in 5001it plastic( HDPE) tank , to get total volume of 170 lit. The
suspension is then heated with hot water in external plastic tank, stirring continuously
using mechanical stirrer at 100RPM. Temperature of 49°C was achieved in 35min.
About 15 lit of 8M Hydrochloric acid is added to get molarity of suspension to 0.694M HC1. The suspension was heated at the above mentioned temperature for 9hours. Cooled to 38°C by using cold water(12°C) and stirring for 15min. The slurry was then neutralized using 19.21it of 6M Sodium Hydroxide solution to a pH of 5.5.
Water for injection was added to make total volume to 4501it and mixed for 5min. Suspension was then allowed to stand for about 5hours. The supernatant was decanted and solids were suspended in about 4001it of water for injection. The suspension was then washed with about 3001it of water for injection using, 1500RPM speed, centrifuge. The chloride content in final washing was found to be 45ppm. The yield was found to be 90% i.e. 59.8kg.
3 The solids were then suspended in water for injection to a final volume of 3001it
in 5001it SS, jacketed tank with stirrer. The solid concentration was about 20%w/v. About 6. Hit of 6m Sodium Hydroxide added to get pH of 12.4. The suspension was heated to a temp, of 43°C by circulating hot water at 75°C. 8.4kg of ETO (14% of 59.8kg) was then injected into the suspension. Temperature was raised to 50°C due to heat of reaction and solution kept stirring for about 2hours and 20min. PH was adjusted once after l hour, from 12.20 to 12.4 using 300ml, 6M NaOH. The liquid became almost transparent and

slight pale yellow at the end of 2hrs and 20min. and was cooled to 39°C using cold water circulation in the jacket. 4.5lit of 8N HC1 was added jo get pH of 5.3.
4 • The liquid was transferred, in equal quantity( 2 x 1551its) into two plastic tanks
each of about 450 lit capacity. 1801it of Isopropyl alcohol(IPA) was added with constant
stirring using portable mechanical stirrer into each of the two tanks. The mixture was
then allowed to settle for 30min. The ppt of each tank were dissolved in 401it of water
for injection and 601it of IP A was added into each of the two tanks. Again the ppt were
dissolved in 401it water for injection and 601it IP A added as above.
The ppt were now dissolved in 501it of water for injection, solids were determined by evaporating a sample at 90°C and chloride was measured. Chloride was 0.48%w/w. Yield was 50kg ( 84%).
5 Volume was made to 2501it (20%w/v)with water for injection and 400g of
activated charcoal( 0.8%w/w) was added and stirred for about 30min. Solution was
filtered through filter pad using filter press to get clear and almost colourless liquid. The
filtrate was then passed through lmicron and 0.2 micron cartridge filter and spray dried
at 80°C.
The result of analysis of HES produced in examples 1 & 2 are mentioned in iable-2
Table-2

SR. Quality criteria Results Limit
Example-1 Example-2
01 Description White White White powder
02 Solubility Complies Complies Freely soluble in water, insoluble in organic solvents
03 Moisture 0.98% 1.07% NMT 3.0%
04 Residue on ignition 0.67% 0.75% NMT 0.8%
05 Total chloride 0.41% 0.22% NMT 0.5%
06 PH of 10% solution 5.82 5.63 5.0 to 6.5
07 Heavy metal Complies Complies NMT lOppm
08 Arsenic Complies Complies NMT lppm
09 Kjeldahl Nitrogen, dry basis 0.005% 0.01% NMT 0.05%
10 Glycol NMT 0.1%
11 Residual Iso propyl alcohol NMT 0.1%
12. Light transmission, 10% aqueous solution, at 450nm, water blank 98.2% 98.3% NLT 97%
13 Av molecular weight 218436 244843 180000 to 260000
14 Molar sustitution, morgan method 0.495 0.52 0.45- 0.55 arid 0.7- 0.8
15 Bioburden 12cfu 23cfu NMT lOOcfu/g
16 Pyrogen, 10% aqueous solution Passes Passes Passes rabit pyrogen test as mentioned in IP/BP/USP


Pagel3ol"17

The advantage of the method is that few operations such as pre-treatment and acid hydrolysis can be performed independently and parallel reducing the total time considerably. Thus the total time, with these operation performed parallel is well below 24hours for commercial scale production with typical batch size of say 50kg.
References;
1 Japanese patent Office, patent no Sho 49-5193 by Takeda
2 Japan patent agency, Public Patent Application No. 45-6556
3 The patent Office, London, INT CL C08B31/12, application no56060/72 by ISAOSATODA
4 The patent Office, London, INT CL C08B 31/12, application No. 43391/74 by Charles Trevor Greenwood
5 Preparation and characterization of HES by T.J.Schoch

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Claims
We claim
1 A superior process for the manufacture of Pyrogen free Hydroxyethyl starch in which at all the stages of manufacture including washing, water for injection IP/BP grade is used
2 A superior process for the manufacture of Pyrogen free Hydroxyethyl starch as mentioned in claim-1 in which, the waxy starch ( e.g. maize, potato, rice wheat, sorahum etc) is washed with alkali to reduce protein content to a level of less than 0.1% w/w and such other impurities removable by alkali..
3 A process according to claim 1 & 2 in which waxy starch of low protein content (less than 0.1%) is subjected to acid hydrolysis using Hydrochloric acid at a temperature of 48°C to 50°C.
4 A process according to claim 2 & 3 in which acid hydrolysed starch is neutralized to pH of 5.0 to 7.0 using suitable alkali and centrifuged to wash off excess salt generated. The water used for washing is Water for Injection grade.
5 A process according to claim 2 to 4 in which hydrolysed starch in aqueous suspension is made alkaline to a pH of about 11.5 to 13.0 preferably 12.3 to 12.6 and is reacted with ethylene oxide (100% purity) so as to achieve final molar substitution of from 0.45 to 0.55 and 0.7 to 0.8 depending on the final product required.
6 A process according to claim 5 in which hydroxyethylation is carried out at a temperature of 45°C to 55°C for about 2.0 hours to 3.0 hours.
7 A process according to claim 5 and 6 in which hydroxyethylation is carried out using ETO concentration of between 13% and 25%to achieve a final molar substitution as mentioned in claim 5.
8 A process according to claim 5 to 7 in which the solution or liquid after hydroxyethylation is neutralized using Hydrochloric acid to get pH of from 5.0 to 6.5.
9 A process according to claim 8 in which neutral hydroxyethyl starch solution is extracted by precipitating using suitable organic solvent as mentioned in the example.
10 A process according to claim 9 in which solvent extraction is repeated till chloride content is less than 0.5%.


Page 15 of 17

11 A process according to claim 9 and 10 in which ppt of hydroxyethyl starch(HES) is dissolved in water for injection to get final concentration of about 10% to 20% preferably 10-12% w/v and in which about 0.3 %w/v to 1.2%w/v of activated charcoal is added.
12 A process according to claim 11 in which HES solution is coarse filtered using suitable filterpad through suitable filterpress to get clear and almost colorless liquid.
13 A process according to claim 12 in which the filtered HES solution is then microfiltered using l.Omicron and 0.2 micron Nylon filter cartridge of suitable size to reduce microorganism( bioburden) load.
14 A process according to claim 1 to 13 in which the filtered HES solution is passed through Ultrafiltration module with 30KD molecular wt cut off limit to reduce any pyrogen, and any salt and glycol present.
15 A process of claim 14 in which solution from retentate or product side is collected in a sanitized tank and spray dried in suitable spray drier.
16 The final dried product as obtained is pyrogen free HES of desired m.wt and molar sustitution and low level of bioburden.
17 The HES thus prepared as mentioned from claiml to claim 16, meets the following quality criteria as mentioned in table-1 below.

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Table-l

SR. Quality criteria Results Limit

01 Description White powder
02 Solubility Freely soluble in water, insoluble in organic solvents
03 Moisture NMT 3.0%
04 Residue on ignition NMT 0.8%
05 Total chloride NMT 0.5%
06 PH of 10% solution 5.0 to 6.5
07 Heavy metal NMT lOppm
08 Arsenic NMT lppm
09 Kjeldahl Nitrogen, dry basis NMT 0.05%
10 Glycol NMT 0.1%
11 Residual Iso propyl alcohol NMT 0.1%
12 Light transmission, 10% aqueous solution, at 450nm, water blank NLT 97%
13 Av molecular weight 180000 to 260000 and 425000 to 600000
14 Molar sustitution , morgan method 0.45-0.55 and 0.7-0.8
15 Bioburden NMT lOOcfu/g
16 Pyrogen, 10% aqueous solution Passes rabit pyrogen test as mentioned in IP/BP/USP