FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION (See section 10, rule 13)
"POLYVALENT SNAKE ANTIVENOM (CHICKEN EGG YOLK
ORIGIN)"
VENKYS (INDIA) LIMITED, SPF Egg Division, Venkateshwara House, S.No.l 14/A/2, Pune - 411 030 and HAFFKINE BIO PHARMACEUTICALS CORPORATION LIMITED, Antitoxin and Sera Dept. Pimpri, Pune - 411 018
The following specification particularly describes the invention and the manner in which it isto be performed.
1909/MUM/2008
29JAN 2009

Field of invention:
The present invention relates to a composition useful as polyvalent snake antivenin. The invention also provides a process for preparing the said composition. The said composition comprises polyvalent antibodies obtained from chicken egg yolk (IgY).
Background of invention:
Amongst all the venomous animals, snake bites represent the major cause of envenoming in the world. Estimates of global mortality from snake bites have been reported to range from 50,000 to 1,00,000 per year.
It is estimated that, there are 2,50,000 cases of snake bites every year in India, however, these largely hospital based figures are likely to be underestimates as many snake bite victims seek traditional treatment or may die at home unreported. Of these over 30,000 people die and a substantial number suffer from post bite complications, possibly due to inadequate availability of equine origin snake antivenin, the presently known specific therapy.
Artificial passive immunization is used when it is considered necessary to protect a patient at short notice like in life threatening envenomations. Thus, readymade antibodies, in the form of antivenin, are raised either in humans or animals for use to give temporary protection. Human preparations (homologous) are less likely to give adverse reactions, but its production on commercial scale has ethical issues apart from possibility of viral transmissions, even though they confer longer protection. Animal preparations (heterologous), although convenient for commercial production, may trigger occasional adverse reactions.
The present regimen of treatment of snakebites is administration of equine origin polyvalent snake antivenin because once the venom enters in the circulation, only antivenin can neutralize it. In India, all commercial antisera available are prepared invariably in equines. Unfortunately mammalian antibodies have several inherent
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disadvantages. When used for treatment purpose, they can cause compliment-mediated side effects; the serum proteins can cause immediate or delayed type of adverse reactions.
The housing and care of mammals, especially large animals like equines for antibody production is expensive and labor intensive and hence the cost of antisera/ antitoxins may become unaffordable to people in developing countries like India.
Apart from above, the repeated blood harvesting of donor animals at periodic intervals is an invasive technique and hence appears cruel, which is widely criticized in the present socio political situation by animal activists.
A simple and convenient alternate antibody source is found in the avian eggs. Hens immunized with antigens secrete the antibodies in the egg yolk (Yolk Immunoglobulin- IgY) in comparatively higher levels than present in the blood. The yield of antibodies from egg yolk is higher in comparison to the plasma from immunized mammals. Around 100 -200 mg of antibody can be obtained from every egg (i.e. around 15 ml of yolk) in comparison to 5 mg of antibodies per ml of horse blood.
Since poultry farming is comparatively easy and highly organized, it is economical to keep hens in large numbers and collect eggs for harvesting of antibodies. This method does not involve frequent blood harvesting, thus it is a non-invasive method and hence it could be acceptable to animal welfare activists. However, current methods do not result in high yield of specific antibodies. Further, none of the processes have been scaled up and since these products suffer from one deficiency or the other, and hence none have reached the market. The present invention addresses these needs and provides a therapeutic composition useful against snake envenomations.
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Objects of the present invention:
The main object of the present invention is to generate a composition useful as polyvalent snake antivenin comprising antibodies against snake venoms, the antibodies being obtained from chicken egg yolk( IgY).
Detailed description:
Accordingly, the invention provides a composition useful as polyvalent snake antivenin said composition comprising anti-snake venom antibodies together with acceptable preservatives and stabilizers, wherein the antibodies are obtained from chicken egg yolk.
Further, the invention provides a process for preparing the aforesaid composition, comprising the steps of:
a) Immunizing specific pathogen free (SPF) hens by injecting natural snake
venom antigen at a starting dose 200 µg for Cobra & Russell's viper venom and
40 u,g for Krait & Saw scaled viper venom in combination with an adjuvant;
b) Repeating the step of immunization with gradually increasing higher doses
of natural and/or modified antigen for a period of 40-50 weeks;
c) Collecting the eggs laid by the hens and isolating the egg yolk and antigen specific polyvalent antibodies therefrom.
The venoms employed for immunization may comprise natural/ modified venoms obtained from 4 commonly found major Indian venomous snakes, namely Indian Cobra ( Naja naja) and Indian Common Krait (Bungarus caeruleus) both from Family Elapidae and Russell's Viper ( Vipera russellii) and Saw Scaled Viper ( Echis carinatus) both from Family Viperidae, either singly or as a cocktail. The amount of antigen injected in the bird may vary from few micrograms to few milligrams.
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The immunization of birds is started in growing period, say at 10-12 weeks of age. The doses are given in gradually increasing concentrations at periodic intervals say every 2-4 weeks so as to make them hyper immune by the time their laying starts, so that a higher concentration of antigen specific IgY antibodies are available in eggs for about a year.
The adjuvant employed for immunization may be such as Freund's adjuvant, or Montannide group of adjuvants, in an equal amount of antigen.
In another embodiment, the invention provides for purification of egg yolk antibodies from a pool of egg yolks, which comprise of following steps:
a) Separating egg yolk from eggs and diluting it with phosphate buffer saline;
b) Separating phospholipids, lipids and proteins other than egg yolk antibodies using different methods;
c) Concentrating egg yolk antibodies by dialysis or ultra filtration and finally purifying the egg yolk. antibodies by immunoaffinity chromatography to obtain antigen specific antibodies.
The invention is now illustrated by the following examples, which are provided only for illustration and are not be read as limitations on the scope of the inventive concept.
Example 1: Immunization of birds
1. SPF Hens were immunized with snake venoms either singly or in
combination.
The 1% solution of lyophilised venoms of four different venomous species of snakes commonly found in India, viz. Indian Cobra, Indian common Krait, Russell's viper and Saw scaled viper was prepared in sterile Normal Saline and filtered through 0.22 u membrane filter. The sterile solution of venoms was stored at 2°C to 8°C till further use.
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2. The present invention contemplates all types of venom modifications like heat inactivation, chemical inactivation and Gamma ray irradiation and their use in combination with natural venom for immunization purpose.
Venom modification methods:
a) Heat inactivation: This was done by heating the venoms at 50° to 90°C
for 30 minutes b) Chemical inactivation: This was done by adding 1 % Glutaraldehyde to
1% solution of natural venoms and incubating the mixture at 37°C for
14 days. Free Glutaraldehyde was removed by dialysis and was stored
at 2 - 8°C till further use. c) Irradiation: The venoms at 0.2% concentration were exposed to
ionizing radiations from CO60 source either at 0.5, 1.0 or 2.0 KGy and
were stored at 2 - 8°C till further use.
3. The present invention is not limited to any particular mode of immunization
and commonly uses intramuscular and subcutaneous routes.
4. The invention also contemplates immunization using adjuvants and not
limited to using any particular type.
5. The present invention contemplates various different immunization schedules
concerning dose of antigen, monovalent or polyvalent, frequency of injections,
ratio of modified antigens and type of adjuvants used.
Example 2:
Purification and concentration of monovalent and / or polyvalent snake antivenin antibodies (IgY)
Eggs Of immunized hens were collected on a daily basis and stored at 2 - 8°C after three inoculations. A pooled sample of 30 eggs was drawn from the eggs collected every week. These eggs were then further processed. The eggs were cracked and the egg yolk was separated from egg white. The yolk was washed
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with chilled buffer to remove traces of albumin. The yolk was then rolled on filter paper / tissue paper to dry it completely and the yolk sac ruptured with a needle. The yolk was then collected and further processed as follows:
(A) PEG Method:
One part of egg yolk from hyper-immunized eggs was mixed with 3-4 parts of phosphate buffered saline (PBS). Then polyethylene glycol (PEG) 6000 was added to a concentration of about 5 %. The mixture was then centrifuged at 10000 rpm for 10-12 min, and supernatant was filtered. To the filtrate obtained was added 10-15 % of PEG slowly. The mixture was then centrifuged at 10000 rpm for 60 min. The supernatant was discarded and precipitate was dissolved in PBS. The mixture was stirred and 70% ammonium sulphate or 14% sodium sulphate was added. It was then centrifuged at 10000 rpm for 30 min. and the precipitate was dissolved in PBS. It was then dialyzed/ ultra filtered to separate the salts.
(B) Chloroform Method:
200 ml of egg yolk was mixed with 200 ml of PBS. Chloroform was added to the mixture and it was kept at 2-8°C for I hour. It was then centrifuged at 3000 rpm for 30 minutes. The top aqueous layer was separated and stored at -20°C till further use.
(C) Caprylic Acid purification:
Egg yolk of hyper immune hens was diluted about 7-8 times with PBS. Then about 8-10% Caprylic Acid was added and pH was adjusted to 4-6. The mixture was centrifuged at 15000 rpm for about 45 minutes. The precipitate was discarded and supernatant was subjected to 25% Ammonium sulphate at pH 6-7. The precipitate was dialyzed / ultra filtered to obtain IgY.
(D) pH change Method
200 ml of yolk was mixed with 800 ml of PBS at pH 7.4 and the mixture was kept at 2-8°C for 3 hours. The pH was then lowered to 5.0 by adding 1 N HCL. This
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was kept at 4°C for 6 hrs. It was then centrifuged at 10,000 rpm for 10 min. The pH of supernatant was adjusted to 7 by 1 M NaOH and the mixture was heated at 53°C for 30 min followed by cooling. The filtrate obtained was treated with Ammonium Sulphate and precipitate obtained was dialyzed/ ultra filtered to concentrate the IgY.
(E) Ammonium Sulphate Method;
200 ml of yolk was mixed with 1400 ml of distilled water. 0.1% pepsin was added to the mixture and it was held at 37°C for 2 hrs. The mixture was heated to 55°C for 30 min. The supernatant was centrifuged at 10,000 rpm for 30 min. 20 % ammonium sulphate was added to the supernatant and precipitate was dialyzed/ ultra filtered to obtain IgY
Immunoaffinity Chromatography
Affinity Chromatography was undertaken only on PEG purified IgY samples so as to remove the non-antigen specific IgY from the pool of antibodies. Affinity purification helped in producing antigen specific IgY, thus reducing total protein burden in the product. The specific antigen (i.e. single or cocktail of snake venoms) was bound to the activated matrix. IgY sample to be purified was applied in a solution to the antigen matrix. The matrix was then washed with wash buffer to remove the unbound protein. Thereafter the bound antigen specific IgY was dissociated and eluted with a suitable elution buffer. The biological activity of the affinity purified antibody was high.
The purified and concentrated IgY samples were stored at either 2-8°C in liquid form or at ambient temperature in lyophilized form till further use.
SDS-PAGE Analysis Results:
SDS-PAGE analysis of purified samples was done at 10% gel concentration. Standard marker proteins 14 Kda to 200 Kda (Banglore Genei) were run with the
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samples. 4x Lameilli running buffer was used. 10 (ill of each sample was loaded in the wells. A current of 50 mA was applied for about 2 hours.
The SDS-PAGE analysis of purified samples showed that IgY purified by PEG-Affinity method was the most pure without any traces of contaminant proteins.
Example 3: In vivo tests Neutralization Test;
To determine the Median LD50 of each venom, graded dilutions of each venom solution were prepared, such that middle diiution (in 0.25ml) contained the expected value Total volume was made up to 4.0 ml by normal saline, and incubated at 37°C for 30 minutes. 0.5ml of each dilution was injected in six mice (Swiss Albino mice weighing 18-20 gms.) intravenously. Death and survival was recorded after 48 hours and the LD50 values were estimated statistically using Reed and Muench (1938) method. The median lethal dose of Cobra venom (CV) was 7.3 ± 2.0 p,g, and 8.0± 2.0 µ.g for Russell's viper venom (RV), 2.3 ± 1.0 µ.g for Krait venom (KV), and 15.0 ± 3.0 jug for Saw Scaled Viper venom (EV) respectively.
The neutralizing antibody level of each sample was analyzed as per B. P. (1998) method as standardized by Central Drug Laboratory, India for equine origin Snake Antivenin. The mouse neutralization assay was performed by taking constant amount of venom (3LD50 challenge for each venom) prepared in sterile normal saline, mixed with varying dilutions of IgY sample (dilution factorl.5) and incubated at 37°C for 30 min. The reaction mixture was prepared in such manner that, 0.5ml of the reaction mixture contained amount of venom corresponding to 3LD50 and injected intravenously into groups of six mice (Swiss albino, 18-20 gm). Deaths were recorded over a period of 48 hrs and 50% end point (Effective dose 50, ED50) was estimated statistically by using Reed and Muench method, (1938). Neutralization was expressed as number LD50's of venom neutralized by 1.0 ml of IgY sample.
Example 4: In vitro tests
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Assessment of Antivenin specificity by Immunodiffusion
3.5 ml of 1 % noble agar was poured on a microscope slide and allowed tc solidify. Wells were punched on the slide with a standard punch with one central well and six peripheral wells. The test was run for each of the purified IgY sample against each venom separately and observations were made after 24 hours and 48 hours. Each of the purified IgY showed precipitation lines against the respective venoms indicating presence of specific antibodies.
Example 5:
Monovalent Indian Cobrs antivenin
This example describes method of production of cobra antivenin.
The immunization of hens and purification of antivenin was carried out as described in Example 1 and 2 respectively.
Table: 1
Yield of IgY obtained by various methods:
Sr. No Method of purification Yield of IgY mg / ml of yolk
1 PEG- Affinity 7.9
2 Chloroform 4.8
3 Caprylic acid 6.2 4 pH change 4.5
5 Ammonium sulphate 3.2
Since samples purified by PEG-affinity method showed a greater yield, neutralization test was carried out using those samples only.
In vivo neutralization capacity of monovalent Indian cobra antivenin derived from egg yolk of SPF hens hyper immunised with Indian cobra venom (CV) was demonstrated as described in Example 3 and the results of In vivo neutralization are as follows:
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Table: 2
Monovalent Indian Cobra Antivenin
Neutralization of Venom In vivo
Antivenin Type Challenge Dose No. of No. of
mice dead mice alive
IgY 3LD50 3 7
Std. Monovalent IgG2 3LD50 0 10
Control CV 3LD50 10 0

1 -- Neutralization against Cobra venom
2 -- Standard Monovalent Equine Sera against Cobra venom
Example 6:
Monovalent Russell's viper antivenin
This example describes method of production of Russell's viper antivenin.
The immunization of hens and purification of antivenin was carried out as described in Example 1 and 2 respectively.
Table: 3
Yield of IgY mg / ml of yolk
8.1 5.2
6.4
4.8
3.5
Yield of IgY obtained by various methods: Sr. No Method of purification
1. PEG-Affinity
2. Chloroform
3. Caprylicacid

4. pH change
5. Ammonium sulphate
Since samples purified by PEG-affinity method showed a greater yield, neutralization test was carried out using those samples only.
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In vivo neutralization capacity of monovalent Russell's viper antivenin derived
from egg yolk of SPF hens hyper immunised with Russell's viper venom (RV)
• >
was demonstrated as described in Example 3 and the results of In vivo
neutralization are as follows:
Table: 4
Monovalent Russell's viper Antivenin
Neutralization of Venom In vivo
Antivenin Type Challenge Dose No. of No. of
micedead micealive
IgY1 3LD50 4 6
Std. Monovalent IgG2 3LD50 0 10
Control RV 3LD50 10 0

1 — Neutralization against Russell's viper venom
2 - Standard Monovalent Equine Sera against Russell's viper venom
Example 7:
Monovalent Indian Common Krait antivenin:
This example describes method of production of Krait antivenin.
The immunization of hens and purification of antivenin was carried out as described in Example 1 and 2 respectively.
Table: 5
of IgY mg / ml of yolk
7.5 4.5 6.0 4.3 3.0
Yield of IgY obtained by various methods: Sr. No Method of purification Yield
1. PEG-Affinity
2. Chloroform
3. Caprylicacid
4. pH change
5. Ammonium sulphate
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Since samples purified by PEG-affmity method showed a greater yield, neutralization test was carried out using those samples only.
In vivo neutralization capacity of monovalent Indian common Krait antivenin derived from egg yolk of SPF hens hyper immunised with Indian Common Krait venom (KV) was demonstrated as described in Example 3 and the results of In vivo neutralization are as follows:
Table: 6
Monovalent Indian Common Krait Antivenin
Neutralization of Venom In vivo
Antivenin Type Challenge Dose No. of No, of
mice dead micealive
IgY1 3LD50 3 7
Std. Monovalent IgG2 3LD50 0 10
Control KV 3LD50 10 0
1 ~ Neutralization against Krait venom
2 - Standard Monovalent Equine Sera against Krait venom
Example 8:
Monovalent Saw Scaled Viper antivenin
This example describes method of production of Saw Scaled Viper antivenin.
The immunization of hens and purification of antivenin was carried out as described in Example 1 and 2 respectively.
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Table: 7
Yield of IgY obtained by-various methods:
1. PEG- Affinity
2. Chloroform
3. Caprylic acid
4. pH change
5. Ammonium sulphate
Sr. No Method of purification Yield of IgY mg / ml of yolk
8.4
5.5
6.7
5.0
3.5
Since samples purified by PEG-affmity method showed a greater yield, neutralization test was carried out using those samples only.
In vivo neutralization capacity of monovalent Saw Scaled Viper antivenin derived from egg yolk of SPF hens hyper immunised with Saw Scaled Viper venom (EV) was demonstrated as described in Example 3 and the results of In vivo neutralization are as follows:
Table: 8
Monovalent Saw Scaled Viper Antivenin

Neutralization of Venom In vivo
Antivenin Type Challenge Dose No. of mice dead No. of mice alive
IgY1 3LD50 4 6
Std. Monovalent IgG2 3LD50 0 10
Control EV 3LD50 10 0
1 — Neutralization against Saw Scaled Viper venom
2 ---Standard Monovalent Equine Sera against Saw Scaled Viper venom
14

Example 9:
Bivalent Viper antivenin
This example describes method of production of bivalent Viper antivenin.
The immunization of hens and purification of antivenin was carried out as described in Example 1 and 2 respectively.
Table: 9
Yield of lgY obtained by various methods:
Sr. No Method of purification Yield of IgY mg / ml of yolk
1. PEG-Affinity 9.5
2. Chloroform 5.8
3. Caprylic acid 7.2
4. pH change 5.6
5. Ammonium sulphate 3.8
Since samples purified by PEG-affmity method showed a greater yield, neutralization test was carried out using those samples only.
In vivo neutralization capacity of bivalent Viper antivenin derived from egg yolk of SPF hens hyper immunised with Russell's Viper (RV) and Saw Scaled Viper venom(EV) was demonstrated as described in Example 3 and the results of In vivo neutralization are as follows:
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Table: 10
Bivalent Viper Antivenin

Neutralization of Venom In vivo
Antivenin Type Challenge Dose No. of No. of
mice dead mice alive
IgY1 3LD50 1 9
IgY2 3LD50 0 10
Std. Monovalent IgG3 3LD50 0 10
Std. Monovalent IgG4 3LD50 0 10
Control RV 3LD50 10 0
Control EV 3LD5o 10 0
1 — Neutralization against Russell's viper venom
2 ~ Neutralization against Saw Scaled Viper venom
3 - Standard Monovalent Equine Sera against Russell's viper venom
4 - Standard Monovalent Equine Sera against Saw Scaled Viper venom
Example : 10
Bivalent Elapid antivenin
This example describes method of production of bivalent Elapid antivenin.
The immunization of hens and purification of antivenin was carried out as
described in Example 1 and 2 respectively.

Table: 11
Yield of IgY obtained by various methods:
10.2
6.0
7.5
5.7
4.4
Sr. No Method of purification Yield of IgY mg / ml of yolk
1. PEG-Affinity
2. Chloroform
3. Caprylic acid
4. pH change
5. Ammonium sulphate

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Since samples purified by PEG-affmity method showed a greater yield, neutralization test was carried out using those samples only.
In vivo neutralization capacity of bivalent elapid antivenin derived from egg yolk of SPF hens hyper immunised with Indian cobra venom(CV) and Indian Common krait venom (KV) was demonstrated as described in Example 3 and the results of In vivo neutralization are as follows:
Table: 12
Bivalent Elapid Antivenin
Neutralization of Venom In vivo
Antivenin Type Challenge Dose No. of No. of
mice dead mice alive
IgY1 3LD50 2 8
IgY2 3LD50 0 10
Std. Monovalent IgG3 3LD50 0 10
Std. Monovalent IgG4 3LD50 0 10
Control CV 3LD50 10 0
Control KV 3LD50 10 0
1--- Neutralization against Cobra venom 2 — Neutralization against Krait venom
3 ----Standard Monovalent Equine Sera against Cobra venom
4 ----Standard Monovalent Equine Sera against Krait venom
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Example 11:
Polyvalent antivenin
This example describes method of production of polyvalent antivenin.
The immunization of hens and purification of antivenin was carried out as described in Example 1 and 2 respectively.
Table: 13
Yield of IgY obtained by various methods:
Sr. No Method of purification Yield of IgY mg / ml of yolk
1. PEG-Affinity 12.5
2. Chloroform 6.8
3. Caprylic acid 8.7
4. pH change 6.3
5. Ammonium sulphate 4.8
Since samples purified by PEG-affinity method showed a greater yield, neutralization test was carried out using those samples only.
In vivo neutralization capacity of polyvalent antivenin derived from egg yolk of SPF hens hyper immunised simultaneously with Indian Cobra(CV), Russell's viper(RV), Indian Common Krait(KV) and Saw Scaled Viper(EV) venom was demonstrated as described in Example 3 and the results of In vivo neutralization are as follows:
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Table: 14
Polyvalent Antivenin
Neutralization of Venom In vivo
Antivenin Type Challenge Dose No. of No. of
mice dead mice alive

IgY1 3LD50 1 9
IgY2 3LD50 0 10
IgY3 3LD50 0 10
IgY4 3LD50 0 10
Std. Monovalent IgG5 3LD50 0 10
Std. Monovalent IgG6 3LD50 0 10
Std. Monovalent IgG7 3LD50 0 10
Std. Monovalent IgG8 3LD50 0 10
Control CV 3LD50 10 0
Control RV 3LD50 10 0
Control KV 3LD50 10 0
Control EV 3LD50 10 0
1 — Neutralization against Cobra venom
2 - Neutralization against Russell's viper venom
3 -- Neutralization against Krait venom
4 - Neutralization against Saw Scaled Viper venom
5 - Standard Monovalent Equine Sera against Cobra venom
6 - Standard Monovalent Equine Sera against Russell's viper venom
7 - Standard Monovalent Equine Sera against Krait venom
8 - Standard Monovalent Equine Sera against Saw Scaled Viper venom
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The above results clearly indicated that Chicken egg yolk origin Polyvalent Snake Antivenin has given higher yield as well as In vivo neutralization capacity than any of monovalent or bivalent antivenin.
Example 12: Freeze drying (lvonhilization)
The antibodies to be freeze dried were prepared and handled as an aqueous solution or suspension. The aqueous antibody preparation as obtained in Example 2 was frozen rapidly and cooled to an experimentally determined temperature below its eutectic point in a lyophilizer at temperature of-40° or lower.
When the product was properly cooled and completely frozen the chamber was sealed and evacuated. The ice in the frozen antibody product gradually sublimed from the frozen surface and it was collected in lyophilizer condenser chamber. As the vapors leave the antibody product, the drying residues maintain essentially its original volume and become porous.
The product was processed until there was less then 1 % moisture in the dried antibody product. After completion of drying cycle, re-absorption of moisture was prevented by immediate removal of product from the chamber and sealing as rapidly as possible under controlled low humidity conditions.
The freeze dried product essentially maintains its biological activity and also extends its shelf life apart from the advantage of storage of product at ambient temperature.
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WE CLAIM:
1. A composition useful as polyvalent snake antivenin, comprising anti snake
venom antibodies together with preservatives and stabilizers wherein the antibodies are obtained from chicken egg yolk ( IgY).
2. A composition as claimed in claim 1, wherein the antibodies present in the
composition are able to protect mice at a challenge dose of 3 LD50 of each venom.
3 . A composition as claimed in claim 1, wherein the composition is in the form of an injection or oral dosage form.
4 . A process for preparing egg yolk antibodies comprising the steps of,
a) immunizing SPF hen by injecting a pre-determined
Snake venom antigen either singly or in combination together with appropriate adjuvant,
b) repeating immunizations over a period of 40-50 weeks,
c) collecting eggs of the immunized hens and purifying antigen specific antibodies from egg yolk.

5. A process as claimed in claim 4, wherein the antigen employed for immunization comprises of natural and/or modified snake venom antigen.
6. A process as claimed in claim 4, step a) wherein the amount of natural antigen is injected at a starting dose of 150-250 µg for Cobra & Russell's viper venom and 30-50 µg for Krait & Saw scaled viper venom and repeating the step of immunization with gradually increasing higher doses of natural and/ or modified antigen for a period of 40-50 weeks.
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1. A process as claimed in claim 4, wherein the adjuvant employed for immunizatiort is selected from Freund's adjuvant or Montnnide group of adjuvants.
8. A process as claimed in claim 4, wherein the antibodies are purified from
egg yolk by:
a) Separating egg yolk from egg, diluting the yolk with phosphate buffer and processing using different methods;
b) Concentrating the antibodies with an appropriate buffer by using dialysis or ultra filtration followed by purification of antigen specific antibodies using immunoaffinity chromatography.
9 . A composition and a process of preparing the same substantially as herein described with reference to the foregoing examples.

(RAJESHWARI H.)
Of K & S PARTNERS
ATTORNEY FOR THE APPLICANTS
Dated this 20th day of November, 2006
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