FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, Rule 13]
A PROCESS FOR REDUCING ENDOTOXIN LEVELS IN CRUDE HUMAN CHORIONIC GONADOTROPIN.
SANZYME LIMITED, A COMPANY INCORPORATED UNDER THE COMPANIES ACT, 1956, WHOSE ADDRESS IS A-2, SILVER BELLE, SRINIVAS BAGADKAR MARG, J.B. NAGAR, ANDHERI (EAST), MUMBAI -400 059, MAHARASHTRA, 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 process for reducing endotoxin levels in crude human chorionic gonadotropin (hCG). More particularly the present invention relates to a process for reducing the endotoxin in crude human chorionic gonadotropin to pharmaceutically acceptable levels.
Background of the Invention
Human chorionic gonadotropin is a glycoprotein hormone found in the urine of the pregnant women. It is produced during pregnancy by the developing embryo after conception and later by the syncytidtrophoblast, part of the placenta. Human chorionic gonadotropin is well established as pregnancy hormone which plays an important role in reproductive physiology during gestation. It is extensively used parenterally as an ovulation inducer in lieu of luteinizing hormone, commonly in patients that undergo IVF treatment. Administration of hCG is also used in certain circumstances to enhance the production of progesterone as it supports the corpus luteum. In the male, hCG injections are used to stimulate the leydig cells to synthesize testosterone necessary for spermatogenesis from the Sertoli cells. Typical uses for hCG in men include hypogonadism and fertility treatment. Human chorionic gonadotropin is classified as a drug and available by prescription only.
Crude hCG is prepared from the urine of first trimester pregnant women. Urine being the source, extract contains the high load of endotoxins approx. in the range of 15-20 EU/IU.

Endotoxins, also referred as lipopolysaccharides (LPS), are major contaminants found in commercially available proteins or biologically active substances, which often complicate biological effects of the main ingredient. Due to such complications, it is essential to remove endotoxins from drugs, injectables, and other biological and pharmaceutical products.
The pharmaceutically acceptable limit as prescribed by the Indian Pharmacopeia the load of endotoxin in final preparation of hCG should be not more than 0.03EU/IU. In order to meet the required standards endotoxin load needs to be reduced to 0.03EU/IU. The currently known techniques for reducing of endotoxin levels in protein products are not satisfactory, either they are not effective and do not reduce the endotoxins to the desired level or may result in substantial loss of end product. Besides methods are not commonly applicable, what is applicable for one protein is not applicable for another protein. Also, such processes are both expensive and time consuming, thereby resulting in an increased cost of the end-product. Reducing endotoxin levels in hCG, especially when it is derived from the biological source like urine, which inherently has very high load of endotoxin of 15 -20 EU/IU thus remains critical and challenging.
Summary of the Invention
The present invention is directed to a process for reducing the endotoxin levels to pharmacopeially acceptable levels in hCG for human consumption.

In one aspect, the invention provides a process for reducing the endotoxin levels in hCG, comprising steps of:
a) subjecting hCG containing solution to atleast one or more precipitation steps including addition of organic or inorganic salt(s) and separating precipitate(s) by suitable means to obtain hCG solution with reduced endotoxin levels,
b) subjecting the hCG solution with reduced endotoxin level to atleast one filtration step, preferably ultrafiltration step,
c) subjecting ultrafiltered hCG Solution to purification process for further reducing endotoxin levels and
d) recovering hCG with reduced endotoxin levels of less than 0.03 EU/IU.
In another aspect, the present invention further provides a purification process for further reducing endotoxin level of the ultrafiltered hCG solution.
In exemplary embodiments, the purification process includes chromatography by hydrophobic-interaction chromatography. Additional steps such as diafiltration and lyophilization are also contemplated tc, be part of purification process.
Description of the Invention
The present invention provides a process for reducing the endotoxin levels to pharmacopeially acceptable levels in hSG for human consumption, which is effective, possible to carry out in a simple setup, cost effective and easily adaptable

from Lab scale to commercial production.
In an embodiment, the present invention provides a process for reducing the endotoxin levels in hCG, comprising steps of:
a) subjecting hCG containing solution to atleast one or more precipitation steps including addition of organic or inorganic salt(s) and separating precipitate(s) by suitable means to obtain hCG solution with reduced endotoxin levels,
b) subjecting the hCG solution with reduced endotoxin level to atleast one filtration step, preferably ultrafiltration step,
c) subjecting ultrafiltered hCG solution to purification process for further reducing endotoxin levels and
d) recovering hCG with reduced endotoxin levels of less than 0.03 EU/IU.
The hCG solution to be used in the process of the present invention may be obtained by dissolving the crude hCG with a suitable solvent and / or water. Preferably alcohol is used to dissolve the crude hCG in water. It is known that HCG contained in urine from pregnant women is extracted by a purification procedure using an adsorbent to thereby obtain several hundreds international units (IU)/mg (protein) of crude HCG. Alternately, filtered urine of pregnant women may be used as a source to prepare hCG solution.
As per the present invention, the precipitation step involves addition of organic or

inorganic salts to the crude hCG solution, the salts may be selected from the group consisting of but not limited to ammonium acetate, calcium acetate, tri-sodium phosphate, or the like. The precipitate is removed by a suitable method, for example by centrifugation so as to get the hCG solution with reduced endotoxin levels of about less than 5EU/IU. The supernatant is subjected to further precipitation step, by addition of same or different salt(s) and separating the precipitate for example by centrifugation to further reduce the endotoxin load to less than 1EU/IU.
The hCG solution with such reduced endotoxin level is subjected to one or more cycles of ultrafiltration with suitable membranes to remove soluble micelles and vesicles of endotoxin to further reduce the load of endotoxin to less than 0.05 EU/IU.
Further reduction in endotoxin levels to less than 0.03 EU/IU is achieved by subjecting the ultrafiltered hCG solution to purification process.
In an embodiment, the purification process includes chromatography for example by hydrophobic-interaction chromatography. Further, additional steps such as diafiltration step before chromatography and lyophilization step after chromatography are also contemplated to be part of purification process.
Final ultrafiltered solution is diafiltered with EOTA and sodium citrate buffer of neutral pH(7.0) before subjecting to chromatography.
Hydrophobic-interaction chromatography efficiently removes endotoxin and other

impurities using either bind and elute methods or flow-through methods.
hCG with the required pharmacopeial limit of endotoxin is obtained by recovering the hCG from the chromatography eluent by subjecting it to series of steps. The chromatography eluent is subjected to precipitation, followed by vacuum drying and lyophilization steps to yield purified hCG with endotoxin level of less than 0.02EU/IU.
In one embodiment the present invention provides a process for reducing the endotoxin levels in hCG, comprising steps of:
a) subjecting hCG containing solution to first precipitation step including addition of organic or inorganic salt(s) and separating precipitate by suitable means, preferably centrifugation under suitable condition and appropriate speed for pre-determined period to obtain hCG solution with reduced endotoxin levels of about less than 1EU/IU,
b) subjecting the hCG solution with initially reduced endotoxin level to second precipitation step including addition of organic or Inorganic salt(s) and separating precipitate by suitable means, preferably centrifugation to obtain hCG solution with reduced endotoxin levels of about less than 0.1EU/IU,
c) subjecting the hCG solution with reduced endotoxin level to first ultrafiltration cycle for removing soluble micelles and vesicles of endotoxin by retaining them on 500kDa - 300kDa ultrafiltration membrane, subjecting permeate to another cycle of ultrafiltration on 30Kda - 10Kda membrane to

yield the hCG with endotoxin load less than 0.05 EU/IU,
d) subjecting the ultrafiltered hCG solution to purification process for further reducing endotoxin levels and recovering hCG with reduced endotoxin levels of less than 0.02 EU/IU,
e) recovering hCG by precipitation with addition of ethanol in specific ratio followed by centrifugation under suitable condition and appropriate speed for pre-determined period, subjecting precipitate to primary drying at 20-30 C under vacuum, holding the semi-dried precipitate at -10 C to -30C for 10 hrs to 30 hrs, subjecting the precipitate to secondary drying at 20C - 30C for 30-50 hrs to obtain lyophilized HCG with reduced endotoxin level of less than 0.01EU/IU.
Any suitable technique may be employed for the separation of precipitates, such as centrifugation under suitable condition and appropriate speed for pre-determined period. The centrifugation may be carried out for example at 5,000rpm- 20,000rpm and at temperature 20 - 30 C for 10 min to 45 min.
Final ultrafiltered solution from the crude urine extract of pregnant women urine is diafiltered with EDTA and sodium citrate buffer of neutral pH(7.0) before subjecting to chromatography.
The ultrafiltered solution of hCG is subjected to a purification process comprising chromatography for example by hydrophobic-interaction chromatography.

Hydrophobic-interaction chromatography adsorbents with ligands such as methyl, ethyl, propyl, butyl, phenyl or any other hydrophobic ligand may be used effectively. Selected adsorbent/resin is first sanitized with alkali, for example 0.5M NaOH, before washing it with ultrapure(endotoxin free) water. Column is then equilibrated with a buffer containing ammonium sulphate and sodium citrate. Sample containing hCGis then loaded onto the equilibrated column. After loading column is again washed with equilibration buffer to remove impurities/unwanted protein except the target protein hCG, which is eluted with ultrapure water with recovery of more than 90% and potency of more than 7500IU/mg.
Elution sample is concentrated and diafiltered with Sodium phosphate buffer before subjecting it to Lyophilization technique known to a person skilled in the art.
As per the pharmacopeially prescribed limit for endotoxin in final preparation of hCG should be not more than 0.03EU/IU. With the process of the present invention it is possible to reduce endotoxin levels in hCG to less than 0.01 EU/IU, which is even below the pharmacopeially acceptable limit. Thus, the process of the present invention provides hCG with much better purity making it suitable for various pharmaceutical applications, including those for parenteral administration.
It is contemplated that as per the process of the present invention specific combinations or sequences of steps are particularly advantageous. Such process provides a product of very high activity, whose degree of purity is very high with

endotoxin levels less than 0.01 EU/IU. The yield in relation to the starting material is more than 90%.
Additional aspects and details of the invention will be apparent from the following examples, which are intended to be illustrative rather than limiting.
EXAMPLES
Example 1
Endotoxin removal from hCG:
25 liters of pregnant women urine was collected and filtered through muslin cloth to remove any particle matters or fiber if present. It was then stirred on magnetic stirrer and equal amount of ethanol was added while stirring followed by addition of 4gm of ammonium acetate. After stirring for 30 min, 5ml of 0.3M calcium acetate was added followed by drop wise addition of 5ml of 0.2M tri-sodium orthophosphate after lhr, the solution was stirred again for lhr and 1ml of 5M NaOH added under continued stirring for one more hr. The solution was centrifuged at 12000g for 30min at RT. To separate supernatant and precipitate, to the supernatant 5ml of 0.3M calcium acetate was added and stirred for lhr and 5ml of 0.2M tri-sodium orthophosphate was added and solution was again stirred for lhr and centrifuged for 30min at 12000g at RT. The supernatant was filtered through 300KDa polyether sulphone diafiltration membrane and the permeate was collected. Collected permeate is then ultrafiltered through lOkDa filtration membrane before concentrating the sample to 5 folds of volume.

Example 2:
Endotoxin removal from hCG:
100gm of crude urine extract was dissolved in 2500ml of distilled water stirred on magnetic stirrer and add equal amount of ethanol while stirring followed by addition of 400gm of ammonium acetate. After stirring for 30min 500ml of 0.3M calcium acetate was added followed by drop wise addition of 500ml of 0.2M tri-sodium orthophosphate after lhr, solution was again stirred for lhr and 100ml of 5M NaOH was added and stirring was continued for one more hr. the solution was centrifuged at 12000g for 30min at RT. Supernatant was separated, to the supernatant 500ml of 0.3M calcium acetate was added and stirred for lhr and 500ml of 0.2M tri-sodium orthophosphate added and again stirred for lhr and centifuged for 30min at 12000g at RT. The supernatant collected through 300KDa diafiltration membrane was filtered and the permeate was collected. Collected permeate was then ultrafiltered through lOkDa filtration membrane before concentrating the sample to 5 folds of volume.
Example 3:
Purification of hCG;
Final ultrafiltered sample from the crude urine extract/pregnant women urine was diafiltered with 1M EDTA and 20mMsodium citrate buffer of neutral pH(7.0).

Approx 1500ml of p-HIC adsorbents was packed in a glass column of diameter 10cm, which was first sanitized with 5CV of 0.5M NaOH, before washing it with ultrapure(endotoxin free) water. Column was then equilibrated with 10CV of buffer containing 1M ammonium sulphate and 20mM sodium citrate at a flowrate of 40ml/min. Diafiltered sample containing hCG was loaded onto the equilibrated column. After loading column was again washed with equilibration buffer to remove impurities/unwanted protein except the target protein hCG, which was eluted with ultrapure water. Elution sample was concentrated and diafiltered with 10mM Sodium phosphate buffer, pH 7 before subjecting it to lyophilization. Powder sample was analyzed with 2-point and 3-point biological assay prescribed by IP and USP respectively for chorionic gonadotropins and by HPLC method. Biological activity of the final sample was 6480.23IU/mg and endotoxin limit as determined by LAL method was less than 0.01EU/IU. Final recovery of process was about 90.2%.
Numerous modifications and variations in the invention as set forth in the above illustrative examples are expected to occur to those skilled in the art. Such embodiments are construed to be within the scope of the present invention. Consequently only such limitations as appear in the appended claims should be placed on the invention

WE CLAIM
1. A process for reducing the endotoxin levels in hCG, comprising steps of:
a) subjecting the hCG containing solution to a first precipitation step including addition of organic or inorganic salt(s) and separating precipitate by suitable means to obtain hCG solution with reduced endotoxin levels of about less than 1EU/IU,
b) subjecting the hCG solution with initially reduced endotoxin level to second precipitation step including addition of organic or inorganic salt(s) and separating precipitate by suitable means, to obtain hCG solution with reduced endotoxin levels of about less than 0.1EU/IU,
c) subjecting the hCG solution with reduced endotoxin level to first ultrafiltration cycle for removing soluble micelles and vesicles of endotoxin by retaining them on 500kDa - 300kDa ultrafiltration membrane, subjecting permeate to another cycle of ultrafiltration on 30Kda - lOKda membrane to yield the hCG with endotoxin load less than 0.05 EU/IU,
d) subjecting the ultrafiltered hCG solution to purification process for further reducing endotoxin levels and recovering hCG with reduced endotoxin levels of less than 0.02 EU/IU,
e) recovering hCG by precipitation with addition of ethanol in specific ratio followed by centrifugation subjecting precipitate to primary drying at 20-30 C

under vacuum, holding the semi-dried precipitate at -10 C to -30C for 10 hrs to 30 hrs, subjecting the precipitate to secondary drying at 20C - 30C for 30-50 hrs to obtain lyophilized HCG with reduced endotoxin level of less than 0.01EU/IU.
2. The process as claimed in claim 1, wherein said hCG solution is obtained by dissolving a crude hCG in alcohol and / or water,
3. The process, as claimed in claim 2, wherein said crude hCG is obtained from urine of pregnant women.
4. The process as claimed in claim 1, wherein said means for separating precipitate is centrifugation at 5,000rpm- 20,000rpm speed and temperature of 20 - 30 C for 10 mins to 45 mins.
5. The process as claimed in claim 1, wherein said salt is selected from the group consisting of but not limited to ammonium acetate, calcium acetate, tri-sodium and phosphate.
6. The process as claimed in claim 1, wherein said purification process comprises of hydrophobic-tnteractlon chromatography, diafiltration step before chromatography and lyophilization step after chromatography step.

7. The process as claimed in claim l, wherein said diafiltration is carried out with EDTA and sodium citrate buffer of neutral pH of 7.0.