FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
(See section 10, rule 13)


"DIPHTHERIA ANTITOXIN (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 is to be performed.

1908/mum/2006
20/10/2006

Field of invention:
Background of invention:

Diphtheria is caused by Corynebacterium diphtheriae and it is an anfection of
the local tissue of upper respiratory tract with producUon of a tox, that causes acute, toxin mediated disease. The toxin is responsible for major complications like myocarditis and neuritis and can also cause low platelet counts and proteinuria. Diphtheria has virtually disappeared from the developed countries by mass vaccination, but it still remains endemic in India. Diphtheria Anti toxin is the specific therapy in clinical cases because antibiotics have no cffccl on preformed toxins, which rapidly diffuse and can only be neutralized by antiserum before it becomes irreversibly bound to tissues.
Artificial passive immunization is used when it is considered necessary to protect a patient at a short notice like in life threatening toxemia due to systemic circulation of bacterial toxins in infection. Thus, ready made antibodies in the form of antitoxin are raised either in human or animal 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.
In clinical cases of diphtheria, equine origin antitoxin/ antiserum is administered systemically. In India, all commercial antisera available are prepared invariably in equines. Unfortunately mammalian antibodies have

several inherent 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 antisera / antitoxins may become unaffordable to people in developing countries like India.
Apart from above, the repeated blood harvesting at periodic intervals is an invasive technique and hence appears inhuman, which is being widely criticized in the present socio political climate by animal activists.
A simple and convenient alternate antibody source is found in the avian egg. Hens immunized with antigens secrete the antibodies in the egg yolk (Yolk Immunoglobulin- IgY) in comparatively higher levels than present in the blood. Since poultry farming is comparatively easy and highly organized, it is economical to keep hens in large numbers and collect eggs. As this method does not involve frequent blood harvesting, it is a non-invasive method and thus it could be acceptable to animal welfare activists. However, current methods do not result in high yield of 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 diphtheria.
Objects of the present invention:
The main object of the present invention is to generate a composition comprising antibodies against diphtheria, the antibodies being obtained from chicken egg yolk.

Detailed description:
Accordingly, the invention provides a composition useful against diphtheria, said composition comprising anti-diphtheria antibodies together with acceptable preservatives and stabilizers, wherein the antibodies are obtained from 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 either
natural and/or modified diphtheria antigen starting at 100 Lf in combination
with an adjuvant;
b) Repeating the step of immunization with higher doses of antigen for a
period of 40-50 weeks;
c) Collecting the egg laid by the hens; and separation of egg yolk.
d) Purification of diphtheria antibodies there from.
The immunization of birds is started during the 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 buffered 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 are immunized with diphtheria antigen either in natural or in modified form.
2. The present invention contemplates antigen modification like chemical inactivation and use of the modified toxins (toxoid) in combination with natural toxin for immunization purpose.
Toxin Modification / Toxoid Preparation:
Formaldehyde at a concentration of 0.6% was added to diphtheria toxin and incubated for 45 days. Free formaldehyde was removed by dialysis and the toxoid was 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, frequency of injections, ratio of
modified antigens and type of adjuvants used.
Example 2:
Purification and concentration of Diphtheria Antitoxin
After three inoculations, eggs of immunized hens were collected on a daily basis and stored at 2 - 8°C. 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 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 on a magnetic stirrer 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 in equal volume to the nv.xture and it was kept at 2-8°C for 1 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 centrifiiged 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 was kept at 4°C for 6 firs. 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.
Immunoaffinitv 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. diphtheria toxin / toxoid) 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 seen to be higher. 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.
The yield of IgY obtained by various methods is shown in table Table No. 1
Table: 1
Yield of Diphtheria antitoxin IgY obtained by various methods:
Sr. No Method of purification Yield of IgY mg / ml of yolk
1 PEG-Affinity 10.7
2 Chloroform 6.1
3 Caprylicacid 9.1
4 pH change 5.7
5 Ammonium sulphate 5.3
Since samples purified by PEG-affinity method showed a greater yield, subsequent tests were carried out with those samples only.
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 samples. 4x Lameilli running buffer was used. 10 ul 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 diphtheria antitoxin samples showed that IgY purified by PEG-Affinity method was the most pure without any traces of contaminant proteins. Example 3: In vivo test Neutralization Test:
First the test dose of toxin was determined by Lr/100 dose method which was the amount of the toxin present in 0.2 ml of the mixture which caused at the site of injection a small characteristic reaction in the skin of the Guinea pig or Rabbit. The solution of National Standard was prepared in normal saline such that 1 ml contained 0.1 units. Serial dilutions of 2.0 ml were prepared in such a manner that each contained 1.0 ml of National Standard Antitoxin contained 0.1 Unit and graded volumes of test toxin, the volume made to 2.0 ml by adding normal saline. This mixture after incubation for 60 Min at room temp in dark conditions, 0.2 ml of each was injected intracutaneously at suitably spaced sites into the shaven or depilated flanks of two animals and observed for 48 hours.
The Lr/100 of the test toxin was 0.00166 IU.
To determine of potency of purified IgY samples, a solution of 0.5 units per ml of National Standard Antitoxin was prepared. A solution of test toxin was prepared such that 1.0 ml contained ten times the Lr/100 dose. Dilution mixtures were prepared such that each tube contained 1.0 ml of test toxin and graded volumes of antitoxin containing IgY to be tested, the final volume of which was made up to 2.0 ml Similar dilution mixtures were prepared containing 1.0 ml of test toxin and graded volumes of National Standard Antisera containing 0.1 Unit, the final volume being made upto 2.0 ml. This mixture after incubation for 60 Min. at room temperature in dark, 0.2 ml of each was injected intracutaneously at suitably spaced sites into the shaven or

depilated flanks of two animals and observed for 48 hours. The potency of IgY samples tested by different methods is as given in Table 2.
Example 4: In vitro test
Assessment of Antitoxin specificity by Immunodiffusion
3.5 m\ of 1 % noble agar was poured on a microscope slide and allowed to solidify. Wells were punched on the slide with a standard punch with one central well and six peripheral wells. The test was performed with purified IgY sample in central well and placing diphtheria antigen and tetanus antigen in 3 alternate peripheral wells. Observations were made after 24 hours and 48 hours. The purified IgY showed precipitation lines against only diphtheria antigen indicating high specificity of purified IgY.
Example 5 : In vitro Test Indirect ELISA
The toxoid (stock 1000 Lf / ml ) was diluted 1:9000 in Phosphate Buffer Saline (PBS), Nunc Immuno Plates ( Maxisorp ) were coated overnight at 4°C in a humidified chamber with 200 μl / well of the antigen.
Next day the plates were washed with washing buffer (0.1% Tween 80, in 0.8% Nacl in Distilled water ) four times and blocked with 1% Bovine Serum Albumin, 200 μl / well for one hour at room temperature. To perform the ELISA, sera samples collected from birds ( pre and post immunization ) at every 7 days interval were diluted in PBST ( 0.1% Tween 80 in PBS). 100 μl / well of each sample were added along with positive and negative control and plates were incubated for 30 minutes, after which they were washed with washing buffer. HRPO conjugated to rabbit anti chicken IgG ( diluted 1:10,000 in PBST ) was added to the plates @ 100 μl / well. The plates were incubated for 30 minutes at room temperature after which they were washed and 100 μl /

well of TMB substrate ( 0.01 % ) was added to each well. The plates were incubated for 15 minutes and then read at 450 nm.
The ELISA results showed a clear differentiation between pre and post immunization sera. There was a gradual e.evation in the titres with peak tttres seen after 12 weeks of immunisation.
Example 6: In vitro Test
Limes flocculationis lest (Lf Test)
The Limes flocculationis of diphtheria antitoxin was determined by incubating together the IgY sample and the standard diphtheria test toxin in appropriate concentrations. When the concentration of the IgY preparation is kept constant and the concentration of the test toxin varied in mixtures of constant volume, the mixture flocculating first is that which contains the most nearly equivalent quantities of IgY and the test toxin.
Preliminary tests were carried out to determine the range of concentrations to be used. To each of a series of small tubes containing equal volumes of graded concentration of the Standard Test Toxin, a constant volume of IgY sample was added. In successive tubes there was an increase in Standard Test Toxin concentration by amounts not greater than one-tenth of the concentration in the middle of the range of concentrations. The range was chosen in such a manner that the optimum flocculating mixture occurred in the middle of the range. The tubes were heated in a water-bath at 45°C to 50°C with half the fluid column immersed to obtain convection currents. Heating was continued and the tubes were observed until the most rapidly flocculating mixture was determined. The number of Lf in the quantity of the IgY sample was numerically equal to the number of Lf equivalents of standard test toxin in the mixture.

Table 2: Potency of Diphtheria Antitoxin IgY by different tests
SrNo* Tests Activity
_ IgY IgG1
1 Neutralization (IU / ml) 1000 1200
2 Limes flocculationis (Lf / mi) 900 1100
1 -Commercially available Diphtheria Antitoxin of Equine Origin.
Advantages of the present invention:
a) Minimizes the risk of adverse reactions occasionally associated with equine origin antibodies.
b) A non-invasive technique for the production of therapeutic antibodies.
c) The egg yolk antibodies of the invention are of sufficiently higher titer and specificity comparable to currently available mammalian antibodies in general and equine origin in particular.
Example 7: Freeze drvinff (lynphilization)
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 was collected in lyophmzer condense chamber. As the vapors leave the antibody product, the drying residues maintain essentially its original volume and became 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.

WE CLAIM:
1. A composition useful against diphtheria toxin, comprising anti-diphtheria antibodies together with acceptable preservatives and stabilizers wherein the antibodies are obtained from chicken egg yolk( IgY).
2. A composition as claimed in claim 1, wherein the amount of antibodies present in the composition is in the range of 900 to 1000 IU/ml.
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 Diphtheria antigen 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 diphtheria toxin.
6. A process as claimed in claim 4, step a) wherein the amount of antigen injected is in the range of 100 to 450 Lf.
7. A process as claimed in claim 4, wherein the adjuvant employed for immunization is selected from Freund's adjuvant or Montannide 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 antigen specific antibodies with an appropriate buffer by using dialysis or ultra filtration followed by purification using immunoaffinity chromatography.
9. A composition and a process of preparing the same substantially as
herein described with reference to the foregoing examples.