|Title of Invention||
A NOVEL PROCESS FOR THE PREPARATION AND PURIFICATION OF RECOMBINANT PROTEINS
|Abstract||A novel process for the purification of recombinant protein expressed as protein or particle is herewith described. In this purification process, the protein is purified by hydrophobic interaction. The interaction of this protein step resulted in an increase in recovery and purity from 15%-18%. The protein further purified has its application in vaccines and pharmaceuticals.|
A Novel Process for the Preparation and Purification of Recombinant Proteins.
The present invention further relates to a novel process for the preparation and purification of viral antigenic proteins and other recombinant therapeutic proteins produced in either prokaryotic or eukaryotic cell systems.
BACKGROUND OF INVENTION
Use of prokaryotic and eukaryotic cell systems for the production of various therapeutic protein molecules is a common method in present day Biotechnology. In this process, the protein of interest is expressed in the said cell system by suitably engineering the molecular genetics of the expression system to incorporate a plasmid to promote the production of the desired proteins when suitably induced during the growth of the cells.
Similarly, the use of various cell substrates for the multiplication of viruses for the production of viral antigens is also a common practice. In this process, the cells are multiplied to large volumes and then they are "infected" with the required virus to facilitate the growth of the viruses. Alternately, transfected cells can also be grown. The viral harvests are obtained from the culture supemates or by cell lysis.
In both the cases as above, the proteins of interest is then concentrated , purified and further treated suitably( inactivated or cleaved) to prepare a therapeutic preparation or vaccine as the case may be.
The major challenges in any of the above processes are the following.
a) Recovery of the protein or antigen of interest in a most economic way.
b) Purification of the protein of interest to eliminate the contaminating substances like the host cell proteins, media components and any other materials used in the process.
c) Concentration of the purified protein to enable further processing.
d) Maintenance of the fimctional structure and activity of the protein during various stages of purification and the efficiency of recovery.
e) Preparation of a product of therapeutic value at the end of the process which shows equal or better performance as that of the reference product.
In order to achieve the above objectives, various processes are adapted. Recombinant molecules can be expressed as heterologous proteins in yeasts such as Sacharomyces cerevisiae, Pichia pastoris or E.coli and other organisms. Many biopharmaceuticals and other polypeptides such as Hepatitis B, Insulin, Streptokinase, Erythropoeitin, Human Growth hormone have been produced by recombinant DNA technology. The expressed proteins are purified from the culture of expression host to obtain the product. Similarly several viral vaccines are also produced by culture in different types of primary or continuous cell lines. The virus grown thus is then suitably purified, concentrated and inactivated/ or used as such for the preparation of vaccines.
Several steps of purification are generally adapted like clarification, centrifugation, filtration, and ultra-filtration, ammonium sulphate precipitation, use of silica beads.
continuous centrifugation , rate zonal gradient centrifligation, various methods of chromatography like gel permeation, size exclusion, affinity and Ion-exchange etc.
The purification processes named above have several draw-backs such as multiple steps, product loss, costly equipments and consumables and some times use of harmful chemicals like Cesium chloride etc and some of the processes make the product nonviable due to high cost of the "down stream process".
BRIEF DESCRIPTION OF THE INVENTION
According to the present invention as herein described, the recombinant proteins are made to be expressed in the vectors like E.coli, Yeast, Eukaryotic cell etc., extracted And purified by using HIMAX technology. It is to be understood that the word "HIMAX" is coined by us and refers to only the technology developed for this invention as explained here under.
OBJECTS OF THE INVENTION
1) The first object of the invention is to provide a method for the preparation and purification of recombinant proteins from the vectors by using HIMAX technology.
2) The second object of the invention is to prepare recombinant proteins which are highly purified without loss of biological activity.
3) The third object of the invention during the preparation of recombinant proteins is to achieve negligible interference of the nucleic acid or other contaminants if any.
4) The fourth object of the invention is to provide a process for simultaneous concentration and purification of various recombinant proteins, viral antigens, and biotherapeutic molecules.
5) The fifth object of the invention is to provide a process of protein purification which is less time consuming and cost effective.
6) Another embodiment of the invention is to provide a process of purification of live and inactivated viral antigens from cell lysate and fluid.
7) The seventh object of the invention is to purify the recombinant proteins by using divalent cations like Zn , Ca, Mg etc., in combination with anions like Acetate, Phosphate and chlorides.
DETAILED DESCRIPTION OF THE INVENTION
Now the details of the present invention:
a) The desired protein obtained through recombinant expression method or by culture in suitable tissue culture is obtained in a clarified harvest after various steps like cell lysis, cell debris removal and clarification etc.
b) A primary capture of the protein or antigen is carried out using the HIMAX method. Briefly the method involves using the addition of a divalent ionic salt
ranging from 0.2% to 10%, with counter ions of either phosphate, chlorides or acetate solution to form an insoluble matrix. The insoluble matrix thus obtained is then gently centrifliged to separate the bound antigen mass .The pellet thus obtained is then desorbed repeatedly with either Tris buffer of pH 8.0 to 8.5 or Tris buffer with EDTA at pH 7.0 to 8.0.
c) The desorbate containing the desired antigen is then further processed . In case of viral antigens , the process involved could be an inactivation followed by chromatography ( ion exchange). In case of other antigens the desorbate is directly taken on to chromatography purification to obtain highly pure protein.
d) The final bulk product is obtained afler pooling of the chromatographically purified fractions containing the desired proteins followed by diafiltration and or sterile filtration steps.
The above steps of invention are more clearly depicted in the following examples for some recombinant and cell culture proteins.
The examples provided herein are only for the explanation of the invention in detail and
is not to be construed that the provided examples limits the scope of the alleged
Varying options which are within the scope of the invention but are not covered in the
description that are available to the persons skilled in the art are to be taken as included in
the present invention.
Hepatitis B antigen production from a recombinant pathway
The cell lysate after fermentation is subjected to centrifugation and the insoluble fraction is treated with detergent. The supematent after centrifugation was either subjected to Aerosil adsorption and desorption (traditional technology) (table 1) or to primary capturing of HBsAg by a batch procedure in which salts of divalent cations such as Calcium Magnesium and Zinc are added at 0.2% to 10% (w/v) in the presence of phosphates, Chlorides or Acetates to form white insoluble matrix. The insitu formation of the matrix further interact with the antigen and this process of protein capturing is referred as HIMAX technology, (tasble 2) This matrix was separated by centrifugation between 7000g to 10,000g and the bound antigen was desorbed repeatedly with this buffer of pH 8.5.
The desorbate was further purified using an anion exchange matrix namely the DEAE.
The HbsAg activity in all the intermediate steps are given in table I and table 11)
In another strategy the cell lysate is directly subkjected to primary capturing of the
antigen by cations at 0.2% to 10% in the presence of phosphates, chlorides and acetates .
All subsequent steps were similar to earlier procedure.
The HBsAg activity in all the intermediate steps is given in table in
The major difference between table 2 and table 3 is the usage of detergent,
In the table 2, the insoluble fraction is treated with detergent, and further processing
Is carried with Adsorption and desorption technology.
While in the experiments represented in table 3, the cell lysate is directly
subjected to adsorption and desorption by HIMAX technology.
Rabies antigen production from a cell culture pathway (FIG 2)
The large scale virus culture facilitates obtaining Rabies virus in the culture supemates. Traditionally the harvests of virus thus obtained are concentrated by ultrafilteration and then purified using the gradient ultracentrifiigation on sucrose in a continuous or batch mode zonal centrifuge. In the present invention the culture supematants are initially purified by the use of HIMAX for primary capturing of rabies antigen by a batch procedure in which salts of divalent cations such as Calcium Magnisium and Zinc are added to yield a final concentration of 8 to 10 fold (WA^) resulting in the formation of white insoluble matrix fiirther interacts. The insitu formation of the matrix fiirther interact with the antigen and this process of protein capuring is referred as HIMAX technology. This matrix was separated by centrifiigation between 7000g to 10,000g and the bound antigen was desorbed repeatedly with tris EDTA buffer of pH 7.2. The concentrated antigen so obtained is then inactivated by usual methods and fiirther purified using an anion exchange matrix to obtain purified rabies antigen. The antigen is then diafiltered and blended as vaccine
The HIMAX purification yeilds with rabies antigen in all the intermediate steps are given in table IV.
Flow chart for HIMAX in Rabies Vaccine production
Hepatitis A antigen production from a cell culture pathway
The large scale virus culture facilitates obtaining Hepatitis A virus in the culture as cell bound virus. Traditionally the harvests of virus are obtained as cell lysates which are clarified , inactivated and then purified using the gradient ultracentrifugation on sucrose in a continuous or batch mode zonal centrifuge. In the present invention the culture lysates are initially purified by the use of HIMAX for primary capturing of Hepatitis A antigen by a batch procedure in which salts of divalent cations such as Calcium Magnisium and Zinc are added to yield a final concentration of 8 to 10 fold (WA^) resulting in the formation of white insoluble matrix fiirther interacts. The insitu formation of the matrix fiirther interact with the antigen and this process of protein capuring is referred as HIMAX technology. This matrix was separated by centrifugation between 7000g to 10,000g and the bound antigen was desorbed repeatedly with tris EDTA buffer of pH 7.2.
The concentrated antigen so obtained is then inactivated by usual methods and further purified using an anion exchange matrix to obtain purified Hepatitis A antigen. The antigen is then diafiltered and blended as vaccine
The HIMAX purification yeilds with Hepatitis A antigen in all the intermediate steps are given in table V.
1. A novel process for the purification of recombinant protein, expressed as either
protein, or as particle or viral particle such as herein described, expressed in
genetically engineering yeast, wherem the said yeast;
(a) Cells are subjected to lysis wherein the process of lysis is carried out in the
absence of any detergent to obtain cell lysate:
(b) solution of step (a) is subjected to centrifugation at ranging fi-om 1000 to 10,000g
(c) obtaining the solid of step (b) by decantation, wherein the solid contain the
recombinant protein expressed as protein, particles or viral particles
(d) suspending the said solid in buffer ranging from pH 6 to 7.5 and optimally
treating this, with a detergent such as herein described to solubulize the minute
(e) captxuing the recombinant protein, or particle of step (d) with divalent ions like
Zn, Ca, Mg in concentration ranging from 0.2% to 3%.
(f) recovering the said recombinant protein or particle with Tris buffer ranging from
0.1 to 1.5M, pH ranging from 8 to 8.5
(g) recovering the said protein through Ultrafiltration, Chromatography on colloidal
silica and or ion exchange, hydrophobic and or affinity chromatography.
2. The process as claimed in claim 1, of step (d), wherein the detergent is non ionic
3. The process wherein the steps of Ultrafiltration is carried out using membrane
filters of 100-300K molecular weight cut off
4. The process as claimed in claim 1, the solid obtained in step (c) is treated with
divalent ions without treating with detergent
5. The process wherein the ion exchange matrices are selected from anionic
exchange resins such as sulphated cellulose/DEAE matrics.
|Indian Patent Application Number||1061/CHE/2003|
|PG Journal Number||05/2007|
|Date of Filing||30-Dec-2003|
|Name of Patentee||M/S. BHARAT BIOTECH INTERNATIONAL LIMITED|
|Applicant Address||GENOME VALLEY, TURKAPALLI, SHAMEERPET MANDAL RANGAREDDY DISTRICT, HYDERABAD 500 078|
|PCT International Classification Number||C07K 001/18|
|PCT International Application Number||N/A|
|PCT International Filing date|