DESC:FORM 2

THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003

COMPLETE

Specification
(See section 10 and rule 13)

Title: Purification of Polyclonal Anti Snake Venom using High Affinity Chromatography

The following specification particularly describes the nature of this invention and the manner in which it is to be performed.

Purification of Polyclonal Anti Snake Venom using High Affinity Chromatography
Field of Invention
The present invention relates to a technique for the preparation of protein G and/or fragments thereof with the capability of binding immunoglobulin G (IgG). Moreover, this invention generally relates to processes for purifying antibodies. More specifically it relates to antibody purification by affinity chromatography processes using the IgG binding property of Protein G in a high affinity chromatography for producing Anti Snake Venom.
Background of the Invention
Up to now the method used for the preparation of protein G is based on liberating the protein from the surface of streptococcal bacteria with the help of proteolytic enzymes, where after the protein is isolated. This method has serious disadvantages. For example, only a part of the protein G molecule is liberated from the bacterial surface, and even if this part is clearly capable of binding IgG, it would still be advantageous to isolate the whole molecule. Moreover, this method is restricted to the use of streptococci as a starting material, and bearing in mind that these are pathogens and difficult to cultivate on a large scale, it would be desirable to find a method which can apply to other starting materials. Thanks to the advent of modern genetic engineering, and specifically hybrid DNA technology, improved techniques have become available for the preparation of proteins with particularly attractive properties. The technique begins by locating the genetic information which codes for the desired protein. This genetic information is transferred using vectors from one cell to another. As a result of this transformation, the transformed cell, hitherto unable to synthesize the desired protein, may become capable of producing the desired protein. Such a method has been used for the synthesis of protein A (a cell wall protein of S. aureus) and is described in two Swedish patent applications, Nos. 8204810-9 and 8204811-7, and two European patent applications, Nos. 0 107 509 and 0 124 374.
Compared with protein A, protein G has substantial advantages, especially as a therapeutic agent for removal of antigen-antibody complexes from the blood during extracorporeal blood treatment in connection with certain autoimmune sicknesses. For example, protein G binds to all IgG subclasses, whereas protein A lacks the ability to bind human IgG 3. Furthermore, protein G is a more selective Fc receptor than protein A, since it does not bind immunoglobulin A and immunoglobulin M.
It is therefore an object of the present invention to provide, using hybrid DNA technology, a method for the preparation of protein G and/or fragments of protein G with substantially the same properties as protein G insofar as the capability of binding IgG is concerned. Moreover, this invention generally relates to processes for purifying antibodies. More specifically it relates to antibody purification by affinity chromatography processes using the IgG binding property of Protein G in a high affinity chromatography for producing Anti Snake Venom.
Summary of the Invention
In accordance with the present invention, these and other objects have now been attained by the presence of a hybrid DNA molecule which is capable of cellular expression comprising the DNA sequence for protein G. said sequence codes for a protein with substantially the same IgG binding properties as naturally occurring protein G. In a preferred embodiment, the molecule may be comprised of DNA sequences coding for fragments of protein G. Moreover, the DNA sequence may code for both fragments of protein G and protein G. The DNA sequence can be isolated from streptococcal DNA.
In accordance with another embodiment of the present invention, a cell transformed by a hybrid DNA molecule encoding the protein G binding site for IgG is provided. The cell may also encode fragments of protein G as well as protein G itself. The type of cell used may vary but can include Gram negative bacteria, Gram positive bacteria, yeast cells and plant cells. Specifically, bacteria of the species E. coli can be used. The encoding DNA molecule may be derived from streptococcal DNA.
In accordance with the method of the present invention, the IgG binding site of protein G is prepared. The method comprises extracting DNA fragments coding for the IgG binding site of protein G by disrupting bacterial cell walls. The extracted DNA is cleaved using restriction enzymes, into fragments of a size enabling introduction of the DNA into designated vector. A cell, not normally capable of producing the IgG binding site of protein G, is transformed with the use of this vector. The cell is cultivated and, subsequently, the IgG binding site of the protein G synthesized by the cell is isolated. As a result, the cell is transformed into a cell which produces the IgG binding site for protein G wherein the binding site has substantially the same binding properties as naturally occurring protein G IgG binding sites. The DNA fragments extracted for use in this transformation may be derived from streptococcal DNA. A variety of cells may be used in the transformation including Gram negative bacteria, Gram positive bacteria, yeast cells and plant cells. Specifically, bacteria of the species E. coli can be used. The DNA fragments may encode the entire protein G or fragments thereof. The IgG binding sites of protein G may be enzymatically or radioactively marked and said binding sites are capable of binding peroxidase marked IgG.
Another embodiment of present invention discloses processes for purifying antibodies. More specifically it relates to antibody purification by affinity chromatography processes using the IgG binding property of Protein G in a high affinity chromatography for producing Anti Snake Venom.
Detailed Description
A hybrid DNA molecule is produced comprising a vector into which has been introduced a DNA sequence coding for a protein capable of being expressed in a cell. The hybrid DNA molecule is characterized in that the DNA sequence codes for protein G and/or fragments of protein G with substantially the same properties as naturally occurring protein G insofar as the ability to bind IgG is concerned.
A cell is also produced which has been transformed by a vector comprising a hybrid DNA molecule into which has been introduced a DNA sequence encoding a polypeptide capable of expression in the cell. Specifically, the polypeptide encoded by the DNA sequence is protein G and/or fragments of protein G which comprise the binding site for IgG. The properties of the IgG binding site are substantially the same, insofar as the capacity of IgG binding is concerned, as is found in naturally occurring protein G.
Moreover, a method is provided for the preparation of protein G and/or fragments of protein G with substantially the same properties as protein G, again insofar as the capability of binding IgG is concerned. The method involves the incorporation into a host cell, in the currently preferred embodiment of a microbial cell, of a hybrid DNA molecule using a vector into which has been introduced a DNA sequence encoding a protein capable of being expressed in the cell, and which is characterized in that the DNA sequence codes for protein G and/or fragments of protein G with substantially the same properties as protein G, insofar as the capability of binding IgG is concerned.
In accordance with the invention, a DNA fragment is isolated from streptococcal bacteria. By way of example, DNA from group A, C or G streptococci may be used, but preferably group C and/or group G streptococcal DNA is used. Conventional techniques are used including application of restriction enzymes which cleave the isolated DNA into suitably sized DNA fragments. The size of the DNA fragments may vary and depend, among other things, on the type of vector selected to accomplish the transformation.
Examples of vectors which can be used for the cloning in this invention include bacterial plasmids (e.g. plasmids from E. coli), phage DNA (e.g. phage lambda or derivatives of lambda, such as EMBL 3 and gt 11), vectors obtained from combinations of plasmids and phage DNA, yeast plasmids, and the like. The selection of vectors is made in consideration of the host cell to be used for expression. Such selection can readily be made by those versed in the art. The method of introducing the proper DNA fragment into the vector is the conventional method and is accomplished using ligating enzymes.
Suitable host cells which can be transformed by the hybrid DNA molecule of this invention, and which consequently become capable of producing the said protein G and/or fragments thereof, comprise Gram negative bacteria, specifically E. coli in the currently prefered embodiment, Gram positive bacteria, yeast cells, plant cells, and the like.
The IgG binding property of Protein G is being used in High affinity chromatography to purify the polyclonal anti snake venom antibodies obtained from horse serum. Horse serum comprising of IgG is passed through a Sapharose 4 fast flow column for purification that ensured by employing Protein G which selectively binds the required IgG from the horse serum. Protein G achieved from the present technique comprises of a large number of active sites thus a large number of IgG molecules attached to it.
To purify the high titer Anti snake venom in a single step the Protein-G required for affinity chromatography is expressed and purified from E.coli and the purified protein-G was conjugated to sepharose-4B and used as an affinity resin for the purification of ASV.
The invention having been disclosed in connection with the foregoing embodiments, additional variations will now be apparent to persons skilled in the art. Various modifications and variations to the above described development of anti-snake venom of high potent can be made without departing from the scope of the invention.
Experiment 1: 20 animals for immunization were used with the nano particle based montanide adjuvant incombination with reduced doses of venom. Animals were tested, evaluated, and assessed their health condition prior to immunization. After the immunization, blood samples from the immunized animals were collected at different time points for checking the titer and potency of the ASV in mice models. Additional boosters were given once in 15 days till the titer value reached a plateau. At this point, blood was collected for the plasma isolation. Blood collection was done once a month after establishing the titers. Plasma was separated and pooled to obtain pooled plasma. Each time pooling was done, samples were tested for titer value to ensure the high titer value of the pooled lot.
The serum collected from 20 animals was subjected to purification through the following steps (Refer Figure 2): It was first centrifuged and was loaded on to single step affinity chromatography column that contains the in-house developed resin. For this step, 30 L batch fermentation scale were produced to yield 8 -10 gm of purified protein G, which was sufficient for the activation and preparation of 10 liter of resin using the same procedure mentioned under section development of a single step affinity chromatography. The developed resin was sufficient to purify 35-45 liters of plasma protein in a single run. The resulted affinity purified plasma protein was subjected to pepsin treatment followed by caprylic acid treatment for precipitation of the protein molecule. After precipitation the plasma protein was subjected to 55 degree Celsius for one hour and was centrifuged. The resultant supernatant was concentrated using tangential flow filtration and dia-filtered to obtain the formulated drug substance. In the final step, DS was formulated and bulk was vial filled and subjected to lyo cycles to obtain the lyophilized powder.
Preclinical Protocol: Abnormal Toxicity Test
The main objective of the abnormal toxicity test is to reveal a product’s toxicity. The test is carried out on two animal models: on 5 healthy white mice with a body weight of 18 to 20 g and two guinea-pigs with a weight of 250 to 300 g. The animals weight is determined on the first day of the experiment. The test should be conducted on healthy animals previously unused in experiments. The housing and feeding conditions should ensure normal vital functions of experimental animals.
Test on Swiss Albino Mice
The test product is administered 1.0ml to each of 5 experimental mice of Swiss albino strain intraperitoneally. The observation period should last 7 days. The product passes the test if the following conditions are fulfilled throughout the observation period:
 No experimental animal dies
 None of the animals present with manifestations of intoxication
 No weight reduction occurs in experimental animals compared with baseline.
If more than one animal dies, the product fails this test. If one animal dies, manifestations of intoxication, or a reduction in body weight is observed, the test should be repeated on twice as many animals. The product passes the test if no animal dies in the second group, no manifestations of intoxication develop, and no reduction in body weight is observed throughout the observation period.
Test on guinea-pigs
The test product shall be administered to two Dunken Hartley guinea pigs animals intraperitoneally, at a dose 5.0ml respectively. The observation period shall last 7 days. The product passes the test if the following conditions are fulfilled throughout the observation period:
 No experimental animal dies and none of the animals develops visible signs of disease
 No weight reduction occurs in any of the experimental animals on the observation final day, as compared with baseline
 None of the animals administered the test product develops a necrosis or abscess at the administration.
The medicinal product passes the test if no animal develops manifestations of intoxication and no body weight reduction is observed.
Plasma from these 10 animals is separated through apheresis and subjected to purification steps using in house developed protein-G based affinity chromatography.
Development of affinity resin
Recombinant protein-G was cloned and expressed in E.coli and purified recombinant protein-G was coupled to CL-sepharose resin.
Cloning and expression of protein-G is depicted in Figure 1.
Purification of ASV by using protein-G affinity resin is depicted in Figure 2.
Potency test
We have observed the increased potencywith protein-G affinity purified ASV
Table No: 1 Potency evaluation in between conventional ASV purified bulk and High titer ASV.
Potency assay with purified concentrated bulk of conventional ASV
S.No Concentrated bulk
Volume
(1:2 diluted) 0.1% venom solution (ml) Saline (ml) Total volume Concentration
(mcg/ml) No.of mice injected Observation
after 48h
Survival
1 2 3 4
1 1.0 0.100 0.900 2.0 100 4 - - - - 4/4
2 1.0 0.150 0.850 2.0 150 4 - - - - 4/4
3 1.0 0.200 0.800 2.0 200 4 - - - - 4/4
4 1.0 0.250 0.750 2.0 250 4 - - - - 4/4
5 1.0 0.300 0.700 2.0 300 4 - - - - 4/4
6 1.0 0.350 0.650 2.0 350 4 - - - - 4/4
7 1.0 0.400 0.600 2.0 400 4 - - - - 4/4
8 1.0 0.450 0.550 2.0 450 4 D D D D 0/4
9 1.0 0.500 0.500 2.0 500 4 D D D D 0/4
10 1.0 0.550 0.450 2.0 550 4 D D D D 0/4
11 1.0 0.600 0.400 2.0 600 4 D D D D 0/4
12 1.0 0.650 0.350 2.0 650 4 D D D D 0/4
13 1.0 0.700 0.300 2.0 700 4 D D D D 0/4
14 1.0 0.750 0.250 2.0 750 4 D D D D 0/4
15 1.0 0.800 0.200 2.0 800 4 D D D D 0/4
16 1.0 0.850 0.150 2.0 850 4 D D D D 0/4
17 1.0 0.900 0.100 2.0 900 4 D D D D 0/4
18 1.0 0.950 0.50 2.0 950 4 D D D D 0/4
19 1.0 100 0.00 2.0 1000 4 D D D D 0/4

Potency assay with purified concentrated bulk of high titer ASV
S.No Concentrated bulk
Volume
(1:2 diluted) 0.1% venom solution (ml) Saline (ml) Total volume Concentration
(mcg/ml) No.of mice injected Observation
after 48h
Survival
1 2 3 4
1 1.0 0.100 0.900 2.0 100 4 - - - - 4/4
2 1.0 0.150 0.850 2.0 150 4 - - - - 4/4
3 1.0 0.200 0.800 2.0 200 4 - - - - 4/4
4 1.0 0.250 0.750 2.0 250 4 - - - - 4/4
5 1.0 0.300 0.700 2.0 300 4 - - - - 4/4
6 1.0 0.350 0.650 2.0 350 4 - - - - 4/4
7 1.0 0.400 0.600 2.0 400 4 - - - - 4/4
8 1.0 0.450 0.550 2.0 450 4 - - - - 4/4
9 1.0 0.500 0.500 2.0 500 4 - - - - 4/4
10 1.0 0.550 0.450 2.0 550 4 - - - - 4/4
11 1.0 0.600 0.400 2.0 600 4 - - - - 4/4
12 1.0 0.650 0.350 2.0 650 4 - - - - 4/4
13 1.0 0.700 0.300 2.0 700 4 - - - - 4/4
14 1.0 0.750 0.250 2.0 750 4 - - - - 4/4
15 1.0 0.800 0.200 2.0 800 4 D D D D 0/4
16 1.0 0.850 0.150 2.0 850 4 D D D D 0/4
17 1.0 0.900 0.100 2.0 900 4 D D D D 0/4
18 1.0 0.950 0.50 2.0 950 4 D D D D 0/4
19 1.0 100 0.00 2.0 1000 4 D D D D 0/4

From the foregoing it will be understood that the embodiments of the present invention described above are well suited to provide the advantages set forth, and since many possible embodiments may be made of the various features of this invention and as the process herein described may be varied in various parts, all without departing from the scope of the invention, it is to be understood that all matter hereinbefore set forth or shown in the accompanying process is to be interpreted as illustrative and that in certain instances some of the features may be used without a corresponding use of other features, all without departing from the scope of the invention.
The following specific and non-limiting steps for functioning need to be construed as merely illustrative, and do not limit the present disclosure in any way whatsoever.
,CLAIMS:We Claim,
1. A high potent Anti Snake venom, wherein the Anti snake venom is purified using high affinity chromatography wherein the presence of a hybrid DNA molecule.
2. A high potent Anti Snake venom according to claim 1, wherein the process of purification comprises of a cell transformed by a hybrid DNA molecule encoding the protein G binding site for IgG and fragments of protein G as well as protein G itself.
3. A high potent Anti Snake venom according to claim 1 and 2, wherein the type of cell used may include, but not limited to, Gram negative bacteria, Gram positive bacteria, yeast cells and plant cells and combinations thereof.
4. High potent Anti Snake venom according to claim 3, wherein the cell can be of species E.coli.
5. The process of purification of ASV using High affinity chromatography comprises of a single step wherein, the Protein G required for affinity chromatography is expressed and purified from E-coli and the purified Protein G was conjugated to Sepharose-4B and used as an affinity resin for the purification of ASV.