text: throughout this disclosure , various publications and patents are referenced . the disclosures of these publications , patents and applications in their entirety are hereby incorporated by reference into this disclosure in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein . the invention provides a series of specific steps , with necessary modifications as may be done by those who are competent with the art , to be used in the upstream and downstream processes to obtain the target protein in a pure , active form . the process starts with culturing the host cells containing the synthetic recombinant gene for expressing the target protein , first in seed inoculum stage and using this seed material to inoculate a bioreactor to produce the cells in high density and then inducing the expression of target protein . this expressed protein aggregates as inclusion bodies inside the host cell and these cells are then broken and the inclusion bodies are separated from the lysate , washed and solubilized using a chaotrophic agent like guanidine hydrochloride at 6 - 8m concentration . the solubilized inclusion bodies are clarified by centrifugation and the supernatant is run through metal affinity chromatography and the positive fractions are pooled and concentrated . the concentrated protein is then refolded in the specific combination of buffer components and conditions used in this process ; the refolded protein is then concentrated and diafiltered to remove the chaotrophic agent and detergents used in the prior steps and run through ion exchange chromatography to purify the monomers from dimers , multimers and other impurities . the pure fractions are then concentrated and sterile filtered before further storage or use . the final yield of the purified protein in this process ranges from 15 % to 25 % at different scales of refolding done and well above the levels achieved for such proteins as described in prior art . this and other objects and advantages of the present invention will be apparent from the following description . it should be understood that the following is merely a description of the preferred embodiments , and is not intended as a description of all possible embodiments . the claims should be looked to determine the full scope of the invention . the preferred embodiments of the invention are described in detail with the aid of following examples and accompanying drawings . various modification ( s ) that may be apparent to one in the art are intended to fall within the scope of this invention . isolation and purification of recombinant pvrii expressed as inclusion body in e . coli the first step in the process sequence is the preparation of inoculum . seed culture from a vial among the stock of vials preserved at or below − 70 deg . c is inoculated into the fully defined fermentation medium to get high cell density . the inoculum preparation can consist of two or three stages of scale - up before the last - stage inoculum is added to the medium sterilized in situ in the fermentor . between each transfer samples are taken and checked for cell density , ph and culture purity , the last of which is done by microscopic examination of wet smear or gram - stained smear of culture , to confirm compliance to specifications ; each stage involves transfer of 5 % to 10 % of inoculum to the succeeding stage , which is grown in a shake - incubator at 150 - 250 rpm for 8 - 12 hours at 33 deg . c - 37 deg . c . the initial conditions of fermentation are set as follows : temperature at 37 ± 2 deg . c , ph at 6 . 9 ± 0 . 1 , stirring speed at 200 - 400 rpm and 100 % availability of the oxygen through compressed air given at 4 to 8 litres . min − 1 . the fermentation is monitored continuously and that parameters are controlled , automatically as per pre - set programmes or manually as required to be within pre - set limits , which are changed as per requirements during fermentation as known to those conversant with the art . once the entire carbon source and other nutrients in the medium are exhausted by growing culture , fed - batch cultivation is started by addition of a fed - batch solution containing modified quantities of the nutrients in the basal medium as known to those conversant with fermentation . during this phase the dissolved oxygen ( do ) level is maintained between 10 % and 50 %, preferably between 10 % and 30 % or 20 % and 40 %. during the entire fermentation periodic samples are taken and in - process parameters as required to monitor growth . this fed batch stage is continued until the desired concentration of cells is reached ; then the culture is induced with 1 - 2 mm of isopropyl β - d thiogalacto pyranoside ( iptg ). during this induction phase the do level is maintained at a higher level than done during the fed - batch stage as required to improve induction of the target protein . this induction phase extends between 3 and 6 hours , preferably not later than that . the final od and pcv after harvest are between 130 - 160 and 18 %- 24 %, respectively . the final wet biomass obtained ranges from 6 kgs . to 8 kgs . for a 50 l batch ( 110 g . to 160 g . litre − 1 ) fermentation . for cell harvesting and washing , a tangential flow filtration ( tff ) cassette module like prostak of millipore corporation , u . s . a , having a porosity of 0 . 45 μm - 0 . 6 μm is connected to the fermentor . the flow - rates of permeate and retentate are controlled to get an optimum cross - flow rate . the cells are concentrated to a level of approximately 20 % to 40 % of their initial volume and then buffer exchanged against washing buffer containing 5 - 10 mm tris buffer , 2 - 5 mm disodium salt of ethylene di - amine tetra - acetic acid ( titriplex ), 50 - 100 mm sodium chloride , ph 7 . 8 ± 0 . 2 to the cells following known procedure as practiced by those experienced . the final volume at this stage is to be maintained between 60 % and 90 % of original harvested cell volumes . the od of the total cell suspension is then checked and adjusted to be between 80 and 120 . during this operation the temperature of the culture is maintained below 20 deg . c , preferably as required to prevent denaturation of proteins by prolonged exposure to higher temperatures . to the cell suspension under stirring are added a detergent like triton - x 100 ( 0 . 5 - 1 . 0 %), protease inhibitor like benzamidine hydrochloride ( 5 - 10 mm ), dithiothreitol ( dtt , 5 - 10 mm ) and lysozyme ( 20 - 30 mg . litre − 1 ). this cell suspension is stirred for 10 to 20 minutes and lysed by passing it , two to four times , through dynomill as known to those experienced in the art . lysis is monitored by checking od and using microscopy of samples after each pass . after required breakage of the cells , of up to 95 % cell lysis , the suspension is centrifuged at 4500 to 5500 × g . for 30 to 60 minutes at 2 to 8 deg . c and the supernatant is collected separately . to one volume of the total pellet about 7 - 10 volumes ( vol . in ml per weight in gms .) of ib wash buffer - 1 containing 5 - 10 mm tris buffer , 2 - 5 mm titriplex , 50 - 100 mm sodium chloride , 2 - 4m urea and 0 . 5 - 1 . 0 % triton x - 100 , ph 7 . 3 ± 0 . 2 is added , stirred for 15 to 30 minutes and centrifuged at 4500 to 5500 × g . for 30 - 60 minutes at 2 - 8 deg . c . the supernatant is collected separately and the pellets are resuspended in the same buffer as before and the process is repeated once more . to the pellet obtained after the second wash , ib wash buffer - 2 containing the constituents as in buffer - 1 above minus urea , edta and detergent but increased concentration of sodium chloride at 0 . 5 - 1m is added at 3 - 5 volumes of the total pellet weight , stirred for 15 - 30 minutes and centrifuged at 4500 - 5500 g . for 30 - 60 minutes at 2 - 8 deg . c . all the washes collected are checked by sds - page analysis for no or not very significant loss of the target protein in the lysate supernatant and ib washes . this novel ‘ two buffer - three step ’ wash resulted in removal of most of the host - cell proteins , which have to be within certain limit in bacterial produced proteins to be used in pre - clinical and clinical evaluations , contaminating the target protein in the inclusion bodies . the final washed ib pellet is then added with 7 - 10 volumes of the buffer ( 5 - 10 mm tris buffer , 50 - 100 mm sodium di - hydrogen phosphate , di - hydrate , 50 - 100 mm sodium chloride , 6 - 8m guanidine - hcl , ph 8 . 0 ) for solubilising them and stirred at room temperature ( 20 - 25 deg . c ) up to overnight . after this the solubilized material is centrifuged at 4500 - 5500 g . for 60 - 90 minutes at 2 - 8 deg . c . the clear supernatant is collected , checked by sds - page for the presence of the target protein and then taken for next step . the next step is the immobilized metal affinity chromatography using the streamline chelating matrix and a streamline column obtained from amarsham pharmacia ( ge healthcare ), u . s . a . the matrix is charged with 50 - 100 mm nickel sulphate , washed with distilled water and equilibrated with 3 - 5 volumes of solubilization buffer . after equilibration , the flow direction is changed to expand the matrix 2 - 3 times of its original packed height with the sample loaded . the sample is passed for totally 3 - 5 times through the column , maintaining the expanded state to a constant level . for further washings and elution , the buffer containing 5 - 10 mm tris , 5 - 100 mm sodium di - hydrogen phosphate di - hydrate , 6 - 8m guanidine - hcl is used with only the change in ph . at the end of the loading , washing of the matrix is done in the expanded state itself using 2 - 3 ( packed bed ) volumes of imac wash buffer - 1 with ph 8 . 0 , followed by washing in the expanded state itself using 5 - 8 ( packed bed ) volumes of imac wash buffer - 2 with ph 6 . 3 . after this washing flow direction is changed and elution is done with 4 - 5 volumes of the elution buffer with ph 4 . 3 . elutes are collected as per requirements to enable fractionation of purified protein using bed - volume as the criteria , in appropriate sterile containers , sampled and analyzed for protein concentration / content and purity by sds - page . protein estimation is done by bradford method and also by checking the sample od at 280 nm and using the molar extinction co - efficient value of 1 . 815 od @ 280 nm = 1 mg / ml of the target protein . imac elutes having more than 60 % target protein are pooled and the protein concentration and content of the pool is checked and calculated . if required , the elutes are concentrated using 1010 tff membrane cassettes to achieve a protein concentration of not less than 3 . 6 mg / ml . the sample is then is added with 2 - 10 mm of dtt , mixed and left up to overnight at 2 - 8 deg . c . the next step of refolding is done using a chilled ( to ˜ 10 deg . c ) buffer having the following components : 50 mm tris buffer , 1 mm titriplex , 1m urea , 0 . 5m arginine - hcl , 2 mm l - cysteine and 0 . 67 mm cystamine dihydrochloride ( the latter two acting as the redox reagents ) and 0 . 57 mm of the detergent triton x - 100 , ph 7 . 2 to 7 . 4 . this refolding system was chosen after many trials with different components and compositions ; the presence of co - solvent like arginine - hcl and redox pair like cysteine - cystamine dihydrochloride were not known to be used along with the detergent - cyclodextrin pair in the total system before . however , when tried in combination only the increases in yields were better . this is probably because of the presence of 12 cysteines and therefore possibly six correct disulphide bonds to be formed in the tertiary structure to confer functionality to the protein . arginine - hcl , the co - solvent , works like an artificial chaperone and this limits the aggregation , the possibility of whose formation is more as the number of cysteines in the molecule increase . the concentration of arginine - hcl tried in the system carried from 0 . 2m to 1 . 0m and there was significant increase in the refolding efficiency up to 0 . 5m concentrations and after that up to 0 . 8m levels there was not much increase and continuing on up to 1m levels resulted in slight drop in the refolding efficiency . the novel redox pair of cysteine - cystamine dihydrochrolide helps in formation and reshuffling of the disulphide bonds . the use of other redox pairs like gssg : gsst ( oxidized and reduced glutathione ), and other detergents tried like ctab ( cetyl - trimethyl ammonium bromide ) in the buffer system did not give similar yields in parallel experiments done to optimize the conditions of refolding ( data not shown ). the ph used was about 1 . 0 to 2 . 0 ph points down from the pi of the target protein ( here the pi of pvrii is ˜ 8 . 9 ). the same refolding buffer - complex was used to check refolding at different temperatures , viz . 4 deg . c , 10 deg . c and 25 deg . c to check for efficiency . the one done at 10 deg . c worked better than the other two done at lower and higher temperatures . the concentration of protein added into the system for refolding was also checked at various levels , from 60 mg . litre − 1 to 240 mg . litre − 1 and level mentioned above was chosen based on the better results obtained in the experiments . two other methods with change of components as follows , but keeping the protein concentration and temperature same as above were tried . the dtt - treated protein at a concentration of at least 3 . 6 mg / ml is added to the buffer under stirring in a vessel , for a final concentration of 120 mg . litre − 1 . after this mixture is stirred for 10 - 20 minutes to ensure complete mixing of the protein in the buffer , 16 mm beta - cyclodextrin solution is added under stirring to it for a final concentration of 4 . 8 mm . the air inside the vessel is flushed - out using nitrogen gas purging for a few minutes to prevent any unwanted effect on the redox potential . this reaction with mild stirring continues for about 20 - 28 hours at ˜ 10 deg . c . after this reaction the refolding mixture is subjected to a concentration and diafiltration step using 10 - kda tff membrane cassettes and to about 15 %- 25 % of the initial volume and then brought into 50 mm phosphate buffer containing 1m urea , ph 7 . 6 to 8 . 0 . the final volume of the concentrated and diafiltered refolded protein would be about 20 %- 30 % of initial volume . this material is filtered through a 20 - 50 μm membrane pre - filter to remove any aggregates , sampled and then taken for the next step . the next step is the anion exchange chromatography , using toyopearl - sp 650c as the matrix and 50 mm phosphate buffer . the refolded , diafiltered and pre - filtered sample is loaded onto the matrix . washing and elution are done using step gradient of nacl concentration , from 50 mm to 500 mm , in phosphate buffer . volumes of all the fractions collected range from 0 . 5 to 2 bed volumes according to the requirement . these fractions including the flow - through and washes are analyzed for protein concentration using od @ 280 nm ( 1 . 834 od @ 280 = mg / ml of native target protein ) and / or bca assay and purity by sds - page . fractions having mostly only the target protein are pooled and then concentrated and diafiltered into 10 mm phosphate buffered saline using 10 - kda tff cassette . the final concentrated protein is checked as above for concentration and purity . the final protein bulk is then sterile - filtered using 0 . 22 μm modified pvdf membranes ( capsule filter ) and stored at 2 - 8 deg . c . in this the protein purified and concentrated after imac run as above in example 1 is taken for refolding . refolding is done using the same conditions as given in example 1 above with the modification of the redox system in refolding buffer constituents as follows : 50 mm tris buffer , 1 mm titriplex , 1m urea , 0 . 5m arginine - hcl , 1 mm cystine , ph 7 . 2 to 7 . 4 . no cyclodextrin is used . the concentrations of the buffer constituents at ± 100 % from above levels were tried and the given level is selected based on the results . in this the protein purified and concentrated after imac run as above in example 1 is taken for refolding . refolding is done using the same conditions as given in example 1 above with the modification of the redox system in refolding buffer constituents as follows : 50 mm tris buffer , 1 mm titriplex , 1m urea , 5 mm lysine , 2 mm proline , ph 7 . 2 to 7 . 4 . no cyclodextrin is used . the concentrations of the buffer constituents at ± 100 % from above levels were tried and the given level is selected based on the results . the results of refolding yields obtained in the three examples given above are given in table - 2 . the final product yield from the levels expressed was about 17 % to 25 %, much higher than the 5 % mentioned for this protein in earlier process shown in the prior art and the final purity of the target protein is more than 98 %. out of the three methods followed for refolding , example 2 gave the highest yield , followed by methods followed in examples 1 and 3 . the final proteins obtained using all the refolding methods above were analyzed for protein content by bca assay and / or od @ 280 nm and / or rp - hplc . the purity was checked by sds - page and rp - hplc assays . each bulk was also checked for extraneous contaminants like host - cell proteins and nucleic acids , endotoxins , residual amount of nickel ( by fluorimetric analysis ) and presence of any free cysteine by ellman test . the most important functional assay done to check the nativity and integrity of the protein was the erythrocyte binding assay . in this assay , about 10 mcg . of the purified protein was bound to washed human erythrocytes for about an hour at room temperature . the erythrocytes were then washed to remove any unbound material and then the bound protein was eluted using 1 . 5m nacl . this eluted fraction was run on sds - page gel , transferred to a nitrocellulose or modified pvdf membrane and then treated with primary and secondary antibodies ( western blotting ); finally after development of the membrane using the appropriate substrate the transferred protein bands could be seen . presence of protein band at the level of the target protein in eluted samples indicated proper functionality . control erythrocytes treated with chymotrypsin to remove the duffy antigen from their surface and so should not bind any pvrii ( duffybinding protein ) present in sample and so should not show any band in western blotting . the purified protein consistently passed all the characterization tests to confirm is purity , strength and activity . three batches of this protein were subjected to stability studies in glass containers at the final concentration ( between 0 . 5 and 1 . 5 mg / ml ) obtained at 2 - 8 deg . c for 24 months and at 25 ± 1 deg . c ( accelerated study ) for 6 months . the parameters analyzed to check stability were physical description , ph , protein content & amp ; purity by reverse phase hplc ( rp - hplc ), protein profile by sds - page , eba , sterility and bacterial endotoxin content by lal assay . in both conditions of storage the protein was found to be stable , maintaining its sterility , purity , integrity and functionality . some profiles of the assay values of the rpvrii bulk antigen are given in fig9 . two formulations of purified protein [ one with alum ( aluminium hydroxide ), the gold - standard adjuvant , and the other a lyophilized one with sugar ] were made . for the lyophilized formulation , the composition and cycle were optimized for the protein . the required concentration of protein was lyophilized with sucrose as the stabilizer . the freezing was done to below − 50 deg . c for up to 6 - 8 hours ; primary drying was done in stages between the temperatures of − 55 deg . c up to + 23 deg . c for a time of 31 - 37 hours ; the secondary drying was done at a temperature between + 23 deg . c and + 25 deg . c for a time of up to 6 - 8 hours . the composition was stable as shown by the study done up to 3 years of storage at 2 - 8 deg . c ( refer fig1 ). this was done to prevent stability issues that have arisen due to pre - mixing of antigens with the new - age oil - based adjuvants mentioned in literature , and to harness the immune - enhancing power of the latter in eliciting memory responses and high antibody titres . both the formulations were checked for storage stability at 2 - 8 deg . c for 36 months and at 25 ± 1 deg . c ( accelerated study ) for 6 months . the parameters analyzed to check stability of alum formulation were physical description , ph , protein content by bca , protein profile by sds - page , western blotting , sterility and pyrogen testing . the parameters analyzed to check stability of lyophilized composition were physical description , ph , protein content & amp ; purity by reverse phase hplc ( rp - hplc ), protein profile by sds - page , eba , moisture content , sterility and bacterial endotoxin content by lal assay . in both conditions of storage , both formulations of the protein were found to be stable , maintaining the sterility , purity , integrity and functionality . some profiles of the assay values of the formulated material are given in fig1 & amp ; 11 . both the above compositions were then tested in various combinations with other adjuvants for evaluating immunogenicity . the alum formulation was kept as the base . a second formulation was alum with the non - cpg molecule ( imt - 504 ). for the other formulations , the lyophilized composition was used with various adjuvants as follows : montanide isa - 720 ( a highly refined emulsifier from the mannide monooleate family in a natural metabolizable oil solution from seppic , france ), mf - 59 ( a type of squalene - oil - water emulsion ), mpl ( mono - phosphoryl lipid a ) and qs - 21 ( a water - soluble triterpene glucoside compound of saponin family extracted from quillaja saponaria ). the alum - based formulations had the protein at the required dosage level in phosphate buffered saline , ph 7 . 2 ± 0 . 2 with alum at a concentration not more than 1 . 25 mg / dose as aloh 3 , all components mixed together by stirring . the alum - imt 504 formulation had the latter added to the alum formulation at 50 to 100 microgram concentration per dose . for other formulations , the lyophilized composition was mixed with the respective oil - based adjuvant so as to form a stable emulsion ; this was obtained using a double - syringe system with inter - connection between the syringes . the protein and the adjuvant were mixed in required ratios and passed under pressure between the syringes to form an emulsion and this formulation was used within a maximum of 12 hours . for these studies , formulations of the bulk / lyophilized rpvrii as appropriate for the adjuvant used were made to give two different final dosage concentrations of 10 μg and 25 μg . inbred balb / c mice were used for the study , chosen based on earlier experimental data . for each formulation one group of 5 mice were used , with one control group which was administered with pbs diluent . two doses of the dosage concentrations in 0 . 5 ml volumes , on days ‘ 0 ’ and ‘ 28 ,’ were give intramuscularly . animals were bled on days − 1 , 21 , 56 and 84 . sera were collected from each sample ; sera from each group and each bleed were separately pooled and stored at − 20 deg . 0 until analysis . elisa method was used to estimate the antibody titres in the serum samples , using plates coated with 2 μg of purified rpvrii antigen . after blocking the coated - plates , several dilutions of the sera starting from ‘ neat sample ( 0 )’ through 1000 , 2000 , 4000 , 8000 and 12000 were added to the assigned wells and incubated for 90 - 120 minutes at 37 deg . c under mild shaking . after washing the wells post - incubation using pbs / tween - 20 , goat anti - mouse hippo - conjugated secondary antibody was added to the wells and again incubated for 60 - 90 minutes at 37 ° c . under mild shaking . after washing the wells post - incubation using pbs / tween - 20 , the substrate solution containing diamino - benzidine ( dab ) was added to each well and incubated under mild shaking at 25 - 30 deg . c for 5 minutes . 2n sulphuric acid was then added to each well to stop colour development and the plates were read at 490 nm using an elisa reader . the end - point titres were then calculated using the readings obtained . the results were then plotted using ms - excel programme to check and compare the immunogenic responses . though most of the oil - based adjuvants , including the montanide isa - 720 mentioned above , have been tried with the many vaccine - candidate molecules , the novelty is in the formulation strategy of using a lyophilized preparation with the adjuvants to enable mixing the adjuvants with the antigen and administering within a short time . this precludes any possible stability issues and also helps in eliciting good immunogenic responses in animal studies as shown by the data given in fig1 . all the formulations were shown to elicit high antibody titres , with the order of response from high to low being as follows : montanide isa720 & gt ; mf59 & gt ; mpl & gt ; qs21 & gt ; alum & gt ; alum + cpg . these responses were consistent with earlier available results of rpvrh produced involving different refolding process . the stability study results of the bulk antigen / formulations and the efficacy results of the formulations reveal that the process developed for the expression and purification of the protein had worked very well and had given a stable and quality protein ; and the formulation strategy used had clearly overcome the stability issues related to the adjuvants used and resulted in very good immune responses to the antigen . isolation and purification of recombinant pff2 expressed as inclusion body in e . coli pff2 is a part ( f2 ) of ‘ region - ii ’ of eba - 175 ( erythrocyte binding antigen - 175 kda ), a p . falciparum surface protein . rii is a 616 amino acid fragment of eba - 175 that consists of two cysteine - rich regions — f1 and f2 . f1 and f2 are homologous to the duffy binding protein of p . vivax and are therefore called duffy binding - like ( dbl ) domains . this pff2 is ˜ 42 kda protein . again a synthetic gene of this rpff2 is cloned similar to the way it was done for rpvrii , expressed as inclusion bodies in e . coli with required changes made as discerned by those experienced in the art . the ib &# 39 ; s are solubilized , and purified through imac . refolding of the purified fraction at 10 deg . c was tried using the three methods mentioned in table - 2 above . the final purification is through ion - exchange step . the final protein is in citrate phosphate buffer ( 50 mm , ph ˜ 5 . 8 ) with maximum of 10 mm nacl . the only difference in the process of purification of pff2 is the buffer used and its ph , during refolding and ion - exchange chromatography steps . due to proteins &# 39 ; s lower pi ( around 6 . 5 ), the buffer used here was citrate - phosphate , ph 5 . 8 instead of the tris buffer used for rpvrii purification . the process resulted in similar yields and purity of the rpff2 protein as obtained for rpvrii . the protein was characterized as done for bulk antigen of rpvrii with similar parameters analyzed , including eba where the chemical used for removing the antigen epitope from rbc &# 39 ; s is neuraminidase instead of chymotrypsin . the lot release results of the three batches proved the consistency of the process . the refolding method given in example 2 above gave the highest refolding yield with rpff2 also , proving that the method works very well for proteins like these having multiple cysteines and so many disulphide bonds . the resulting protein is also checked for its stability since the inherent structure of rpff2 , when compared to rpvrii , is not very stable the storage conditions chosen for the testing were − 70 deg . c for long - term storage ( 24 months ) and 2 - 8 deg . c for accelerated study ( 6 months ). these stability study results of the bulk antigen reveal that the process developed for the expression and purification of the protein has worked very well and has given a stable ( albeit at a lower temperature than rpvrii ) and quality protein .