A process for the preparation and purification of recombinant proteins

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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%-80%. The protein further purified has its application in vaccines and pharmaceuticals.

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.

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 the1 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 supernates 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 functional 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, Streptokihase, Erytnropoeitin, 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 centrifugation, 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 non-viable due to high cost of the 'down stream process'. BRIEF DESCRIPTION OF TH EINVENTION According to the present invention as herein described, the recombinant proteins are made to be expressed in the vectors like E.coli, yeast, Eukaiyotic cell, etc., extracted and purified by using HIMAX technology. It is understood that the word Η AX' is coined by the inventors and refers to only the technology developed for this invention as explained hereunder, 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 HEVIAX 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 is to achieve negligible interference of the nucleic acid or other contaminants if any during the preparation of recombinant proteins. 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. Accordingly the present invention relates to a process for the preparation and purification of protein(s) such as viral antigenic proteins, other recombinant therapeutic proteins characterized in that the purification is carried out by a novel technique termed as HEVIAX technology which is as herein described and recovering the said protein(s).