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

Field of invention:
The present invention relates to a therapeutic composition comprising antibodies against tetanus virus. The invention also relates to a process of preparing the composition comprising antibodies obtained from egg yolk of birds immunized with antigens of tetanus virus.
Background of invention:
Tetanus is a disease cause by neurotoxin of Clostridium tetani and ranks among major lethal infection. It is estimated that 1 million deaths occur annually in the world, mainly from neonatal tetanus. The disease is characterized by stiffness of jaw, followed by stiffness in neck and back. Once the toxins are released in circulation, antitoxin is the only therapy of choice to avoid fatality. Antitoxin is also used to protect patient for a limited period of time.
Artificial passive immunization is used when it is considered necessary to protect a patient at short notice like in life threatening envenomations or toxemia due to systemic circulation of bacterial toxins in infection. Thus, readymade antibodies, which may be antivenin, antitoxin or antiviral, 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.
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 type of adverse reaction like serum sickness and occasionally anaphylactic shock or delayed type of adverse reactions. The housing and care of mammals, especially large animals like equines for antibody
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production is expensive and labor intensive and hence the antisera / antitoxins may become unaffordable to people in country like India..
Apart from above, the repeated blood harvesting at period intervals is an invasive technique and hence appear inhuman, which is being widely criticized in the present socio political climate by animal activists.
Hence, one of the alternatives is the use of antibodies generated in poultry birds (IgY). As this method does not involve frequent blood harvesting, it is a noninvasive method and thus it is acceptable to animal welfare activists. However, the currently employed methods do not result in high yield of antibodies. Further, none of these processes have been sealed up and since the experimental products suffer from one deficiency or the other, none have reached the market. The present invention addresses these needs and provides a therapeutical composition useful against 4 deadly poisonous snakes which can be marketed on a large scale.
Objects of the present invention:
The main object of the present invention is to generate a composition comprising antibodies against tetanus toxins, the antibodies being obtained from egg yolk.
Accordingly the invention provides a composition comprising antibodies against tetanus toxins, the antibodies being obtained from egg yolk. The invention also provides a process for preparing the said therapeutic composition such that the composition conforms to Indian Pharmacopoeia requirements.
The process comprises of following steps:
1. Immunization of specific pathogen free (SPF) hens maintained under conditions where they are exposed to antigens against which antibody production is desired, by injecting a predetermined antigen of specific concentration in combination with an adjuvant.
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The amount of antigen injected in the bird may vary from few micrograms to few milligrams.
The immunization of birds is started at pre laying period, say at 10-12 weeks of age. The doses are given in gradually increasing doses 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 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 adjutants, in an equal amount of antigen.
2. Repeating the process of immunization by giving booster doses of different antigens, of specific concentrations in combination with an adjuvant.
3. The invention also provides for isolating the egg yolk antibodies from a pool of egg yolks which comprise of the following steps:

a) Separating egg yolks from eggs and diluting it with phosphate buffer saline.
b) Precipitation of phospholipids, lipids and proteins other than egg yolk antibodies using a precipitant such as polyethylene glycol( PEG) in a concentration of about 2 to 8 % or Caprylic Acid in a concentration of 8-10%.
c) Subsequent purification of the precipitated egg yolk antibodies using salt such as ammonium sulphate or Sodium sulphate and finally concentrating the precipitated antibodies by dialysis or ultra filtration.
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.
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Example 1:
Immunization of birds
SPF Hens are immunized with Tetanus toxoid for Tetanus Antitoxin. The present invention contemplates all types of venom modifications like heat inactivation, chemical inactivation and Gamma ray irradiation.
The present invention is not limited to any particular mode of immunization and commonly uses intramuscularly and subcutaneous routes.
The invention also contemplates immunization using adjutants and not limited to using any particular type.
The present invention contemplates various different immunization schedules concerning dose of antigen, frequency of injections, ratio of modified antigens and type of adjutants used.
Example 2:
Purification
PEG purification:
Egg yolk from hyper-immunized eggs is mixed with 3-4 times phosphate buffered saline (PBS). Then polyethylene glycol (PEG 8000) is added to a concentration of about 5 % slowly while continuously stirring the mixture. The mixture is then centrifuged at 10000 rpm for 10-12 min, and supernatant is filtered. To the filtrate obtained is added 10-15 % of polyethylene glycol 8000 is slowly added. The mixture is then centrifuged at 10000 rpm for 60 min. The supernatant is discarded and precipitate is dissolved in PBS. The mixture is stirred on a magnetic stirrer and 70% ammonium sulphate/ sodium sulphate is added. It is then centrifuged at 10000 rpm for 30 min. and the precipitate is dissolved in PBS. It is then dialyzed/ ultra filtered to separate the salts.
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(B) Caprylic Acid purification:
Yolk of hyper immune hens is diluted about 7-8 times with PBS. Then about 8-10% Caprylic Acid is added and pH is brought down to between 4-6. The mixture is centrifuged at 15000 rpm for about 45 minutes. The precipitate is discarded and supernatant is subjected to 25% Ammonium sulphate at pH 6-7. The precipitate is dialyzed / ultra filtered to obtain IgY.
Dated this 18th Day of November, 2006
(RAJESHWARI H.)
Of K and S PARTNERS AGENT FOR THE APPLICANTS
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