THE PATENTS ACT, 1970
(39 of 1970)
COMPLETE SPECIFICATION
[See section 10]


A METHOD OF PREPARATION OF


ANTIVENOM BY PURIFICATION AND

ULTRAFILTRATION;

SERUM INSTITUTE OF INDIA LTD., A COMPANY INCORPORATED UNDER THE COMPANIES ACT, 1956 WHOSE ADDRESS IS 212/2, OFF. SOLI POONAWALLA ROAD, HADAPSAR, PUNE - 411 028, IN THE STATE OF MAHARASHTRA, WITHIN THE UNION OF INDIA;




THE FOLLOWING SPECIFICATION
PARTICULARLY DESCRIBES THE NATURE OF THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFOMED.
1

This invention relates to a method of preparation by fractionation and purification of hyperimmune equine plasma raised against four poisonous snakes for the large-scale production of snake antivenoms.
Though, the demand for antitoxins has been considerably reduced due to production of potent bacterial vaccines and their effective use in immunization programme, the demand and necessity of snake antivenoms will be there forever. Chances of surviving from the poisonous snake bite is directly related to the quality of antivenom being injected both in terms of its neutralising power and purity.
Most protocols for antivenom production are based on ammonium sulphate precipitation with or without pepsin digestion (Latifi, 1978; Russell, 1988; Raw et al, 1991). These procedures have undergone little innovation during the last decade, making the introduction of new methodology that would give products of higher quality a priority.
Due to the presence of high level of euglobulins and also reactogenic proteins the antivenoms hitherto produced are hazy when reconstituted. The protein concentration in these preparations is very high which is again undesirable to load the human body with animal proteins.
Several alternatives have been proposed, such as affinity and ion-exchange
chromatography either alone or in combination with sulfate fractionation and
enzyme digestion (Russell et al, 1985, Dias et al, 1989; Smith et al, 1992). The use
of caprylic acid in the purification of IgG from various sources has also been
described (Steinbush and Andran 1969; Mc Kinney and Parkinson 1987; Perosa et
al, 1990). Although these methods produce high quality product, but are difficult
to adapt to large scale production. Therefore, a need has arisen to develop a
method to remove all unwanted proteins as they are of animal origin while keeping the desired IgG fractions.
2

In order to achieve removal of such proteins the present invention
envisages a method of fractionation and purification of hyperimmune plasma involving ammonium sulphate precipitation and ultra-filtration under controlled physico-chemical conditions. The method will be of immense help for large scale manufacturer of anti-snake venom serum. The pepsin digestion of plasma at pH 2.9 - 4.0 cleaves Fc portion clearly leaving behind Fab2. The Fc portion cleaved in the enzymatic treatment process and other proteins are precipitated with ammonium sulphate at pH 4.0 to 4.8. Toluene precipitates all the lipoproteins. Low molecular weight non-immunoglobulin proteins not precipitated under these conditions are then removed by dia-filtration. The euglobulin responsible for haziness of serum are not precipitated at low pH. For its precipitation, pH is very critical and all the euglobulins settle down when the pH is raised above 4.5 and serum is kept at cold temperature for 24 hours. This is small but critical step responsible for providing a clear colourless serum.
The present invention is a method of removal of euglobulins and other protein components, which contribute to its haziness, allergic reactions and reduced neutralising capacity. By allowing the euglobulins to settle down at a pH of 5.0 - 5.5 at cold temperature for 24 hours, the resultant product is clear and colourless.
Digestion of plasma at a lower pH of 2.9 to 4.0 for 2 hours helps in the
cleaving of Fc regions of Immunoglobulins. pH rises during first hour of and cleaning the digestion /of Fc regions of Immunoglobulins. The rise in pH during first hour of
digestion is indicator to the fact that the cleavage process is over during this time.
To remove lipoproteins, toluene (0.2%) was used to precipitate. Further removal
of lipoproteins anf Fc regions along with high molecular weight impurities are
carried out through a single step precipitation with ammonium sulphate.
Precipitation of serum at this stage enables in the removal of thrombin also which
may be the contributing factor for coagulation. A number of batches of anti-snake
venom serum have been purified employing this method.

Example:
240 litres of horse plasma Lot no. 781 was processed using our innovative method. Horse plasma was diluted to 1.5g% - 3.0g% protein level and digested at 20 -30 Deg.C. using pepsin 0.1g% for 2 hours. After digestion, toluene at 0.2% (V/V) and ammonium sulphate solution was added. The pH was raised to 4.0 - 4.8 and the temperature to 50 -58 Deg.C. and maintained for half an hour. Then, the mixture was cooled rapidly, left overnight and filtered next day. The filtrate was subjected to concentration and ultra filtration. The process is continued till the filtrate is free from ammonium and sulphate ions. The pH was raised to 4.5 - 5.5 and kept overnight at cold temperature. The process was completed within 5 days. No maturation period was required. The resultant anti-venom product had low concentration of protein (4.4g%), with high unitage (15454 mcg/gm protein) and the yield was quite high i.e. 75%. The product was lyophilized thereafter. The reconstituted serum was clear, non-sticky and reconstitution time was 40 seconds.
4

We claim:
1. A method of preparation of antivenom by purifying by single step
ammonium sulphate precipitation and ultra filtration comprising steps of:
(a) subjecting equine plasma containing Immunoglobulins to pepsin treatment, toluene reaction and precipitation with ammonium sulphate.
(b) further concentration and purification of the filtrate using Amicon Hollow Fibre Cartridge to give rise to clear and transparent antivenom and then keeping the at cold temperature for 24 hours for precipitation of euglobulins; and
(c) resultant antivenom is pure (i.e. very low protein concentration of 4.4g%) and highly potent with unitage of 15454 mcg/g protein.

2. A method of preparation of antivenom as claimed in Claim 1, wherein the production yield is as high as 75%.
3. A method of preparation of antivenom as claimed in claim 1, wherein lyophilized product on reconstitution is clear and non-sticky and dissolution time is just 40 seconds.
4. A method as claimed in claim 1, wherein the digestion pH is 2.9-4.0, protein level is 1.5g%-3.0g% at 20-22°.C. with 0.1% W/V pepsin for 2 hours.
5. A method as claimed in claim 1, wherein the toluene concentration is 0.2%
V/V.
6. A method as claimed in claim 1, wherein ammonium sulphate solution was
used to precipitate unwanted proteins and globulins.
5

7. A method as claimed in claim 1, wherein the pH is raised to 4.0-4.8, heated to 50-58° C for 30 mins. and rapidly cooled to 35°C. and the filtrate then subject to ultrafiltration.
8. A method as claimed claims 4 and 5 resulted in completion of production process within 5 days.
9. A product as claimed in claims 3, 4, 5 and 6 is pure (low concentration of 4.4g% protein) and the process yield is quite high (75%) highly potent (unitage of 15454/g. protein).
Dated this 9th day of March,: 2000.