Title of Invention

"A PROCESS OF SYNTHESIZING HIGH QUANTITIES OF HUMAN INTERFERON ALPHA 2a PROTEIN"

Abstract

This invention provides a process for the production of recombinant human interferon alpha 2a (rhIFN-a 2a) protein from recombinant E.coli comprising the steps of: isolating Interferon alpha 2a gene from human Genomic DNA using PGR .technique,- ligating said Interferon alpha 2a gene downstream of T7 promoter in an expression plasmid pRSETA to form a construct called pRSETA-IFNα, transforming E.coli BL21 (DE3) codonplus (RIL) cells with the said construct, growing the transformed cells to high cell density in a fermentor at 37°C with optimized media and an exponential feed of concentrated media for 3-6 hours to achieve a high concentration of cells, inducing said cells with IPTG to start synthesis of rhIFN-a 2a protein, continuing the feed post induction for 6-12 hours to get high level build up of rhIFN-a 2a protein inside the cells in the form of inclusion bodies, harvesting said cells by centrifugation to separate said cells from the culture media, breaking the cells by standard physio-chemical methods to get cell lysate, centrifuge said cell lysate to get the inclusion bodies containing rhlFN- α 2 a in the pellet, washing said pellet with a washing buffer, solubilizing washed pellet in denaturation buffer to get pure rhIFN-a 2a protein.

Full Text

This invention relates to a process of synthesizing high quantities of human Interferon-. alpha 2a (hlFN-α 2a) protein. BACKGROUND The Interferons are a heterogeneous group of proteins with similar biological activities. The three main human Interferons are known as Interferon-alpha (DFN-α), Interferon-beta (IFN-ß), and Interferon-gamma (IFN-), although there are others. The broad classes of action currently recognized for Interferons are characterized as 1) antiviral, 2) antiproliferative, 3) regulator of differentiation, 4) modulator of lipid metabolism, 5) inhibitor of angiogenesis, 6) antitumoral, and 7) immunoregulator etc. Alpha Interferons are used to treat several diseases, including some types of cancer, such as hairy cell leukemia, melanoma, renal cell carcinoma, follicular non-Hodgkin's lymphoma, chronic myelogenous leukemia, and AIDS-related Kaposi's sarcoma. They are also used to treat genital warts and some kinds of hepatitis. Used in combination with retinoids, IFN-a 2 has induced regression in advanced squamous carcinomas of the skin and cervix, suggesting that the cytokine may influence cell differentiation. It also inhibits vascular and endothelial cell proliferation and thus has a place in treatment of melanomas, hypemephromas, and hemangiomas. Because it can increase the intensity of antigen expression on certain tumors (ovarian and colorectal carcinomas), IFN-α 2 has potential for diagnostics (imaging) and therapeutics (monoclonal antibodies). IFN-α genes have a length of 1-2 kb and are clustered on human chromosome 9p22. There are several different molecular species of Interferon alpha (IFN-alpha-1, IFN-alpha-2, IFN-alpha-4 etc), and several recombinant products are available commercially. These proteins have molecular masses between 19-26 kDa and consist of proteins with lengths of 156-166 and 172 amino acids. IFN-a 2 was the first pure human protein found to be effective in the treatment of cancer. The two principal subtypes of IFN-a 2 are designated alpha-2a and alpha-2b. IFN-a 2a contains 165 amino acids, and it has an approximate molecular weight of 19,000 daltons. Using human cells in culture, recombinant IFN-a 2a, has been shown to have antiproliferative and immunomodulatory activities. Large-scale production and recovery of human leukocyte Interferon from peripheral blood 1 eukocytes h as been reported by Horowitz et al (1986). However, the process is laborious, tedious and time consuming. The yields are very low. The specific activity of the purified Interferon is only 2xl06 lU/mg; much below the WHO recommended specific activity of 2x108 lU/mg. Lee Jae Ho et al, (1989) have reported production of human leukocyte Interferon in E.coli by control of growth rate in fed-batch fermentation. The yield however, is low (1 x 109 IU/L). Production of recombinant human IFN-α by E.coli using a computer control cultivation process has been reported by Xiao-Ming Yang et al, (1992). The expression levels are onlyl.25x109IU/L. Secretion of a fusion protein of human IFN-a and bacterial endoglucanase from Asperigillus nidulans has been reported by Gwynne et al., (1987). The incorrect cleavage of the IFN-a from the fusion protein is a drawback of this process and the yields are very low (Img/L). Regulated high efficiency expression of human IFN-a has been reported in Saccharomyces cerevisiae by Tiute et al., (1982). The process involves the expression and secretion of human IFN-a as a fusion protein. The problems of cleaving the fusion protein and purification are not addressed in this process. The expression is only about 15 mg/L. In Indian patent no. 153751 the process comprises of producing recombinant DNA molecules coding for Interferon sequence and cloning the said sequence to produce alpha type Interferon. In Indian patent no. 162268 the process comprises of submerged cultivation of Pseudomonas species VG84 carrying a plasmid PVG3 with an inserted gene of human leukocyte Interferon a-2, in a nutrient medium under aeration in the presence of streptomycin followed by isolation and purification of IFN-α 2a. In US patent No 5,710,027 the process comprises cloning of the IFN-a gene downstream of a signal sequence (the heat stable enterotoxin signal sequence- ST II) and the phoA promoter. This permits export to the periplasm and hence production of correctly folded IFN-a The drawback is that the specific yields are low. The drawbacks of producing a human IFN-a from natural sources are as follows: • The contamination of the blood by an unidentified infectious agent cannot be eliminated. • The process uses a costly medium and is tedious. • Gives low yields and low specific activities. The major problems in producing human IFN-a by recombinant DNA based process that have been reported so far are as follows: • Low specific yield of the protein- the specific yield is less than 1 -2 % of total cellular protein, which causes problems in purification. • The volumetric yields in gm/lit of culture have been low The object of the present invention is to overcome the above drawbacks and produce the recombinant human Interferon a-2a (rhIFN-α 2a) by using a genetically engineered E. coli strain. Further object of this invention is to produce high volumetric yields of rhIFN-a 2a simultaneous with high specific yields which not only serves to provide high quantities of the product but also a cost effective recovery by simplifying downstream processing (purification). To achieve the said objective this invention provides a process of synthesizing high quantities of rhIFN-α 2a protein comprising the steps of: isolating Interferon alpha 2a gene from human Genomic DNA using PCR technique, ligating said Interferon alpha 2a gene downstream of T7 promoter in an expression plasmid pRSETA to form a construct called pRSETA-IFNa, transforming E.coli BL21 (DE3) codonplus (RIL) cells with the said construct, growing the transformed cells to high cell density in a fermentor at 37°C with optimized media and an exponential feed of concentrated media for 3-6 hours to achieve a high concentration of cells, inducing said cells with IPTG to start synthesis of rhIFN-a 2a protein, continuing the feed post induction for 6-12 hours to get high level build up of rhIFN-a 2a protein inside the cells in the form of inclusion bodies, harvesting said cells by centrifugation to separate said cells from the culture media, breaking the cells by standard physio-chemical methods to get cell lysate, centrifuge said cell lysate to get the inclusion bodies containing rhlFN- a 2a in the pellet, washing said pellet with a washing buffer, solubilizing washed pellet in denaturation buffer to get pure rhIFN-a 2a protein. The said washing buffer contains 10 mM Ethylene Diamine Tetra Acetate, 2% Triton X100 and 2% Deoxycholate. The said denaturation buffer contains either 8M urea or 6M guanidine hydrochloride. The optimized media composition is as follows: TABLE 1(Table Removed)The said exponential feed of concentration media is as given below: TABLE 2A(Table Removed) TABLE 2B ** Following are the composition of trace elements (1000X) per liter(Table Removed) The exponential feed pattern is 10-15 ml/h per liter of culture medium at the start of said feeding and is changed every hour at values equivalent to OD600 till the OD600 reaches 40. The feed media is started when the cell concentration reached an OD600 of 1 0-15 and when OD600 reaches a value between 20-30; the cells are induced with 1 mM IPTG for protein synthesis. Brief Description of the Drawings: The invention will now be described with reference to the accompanying drawings and foregoing examples: Fig. 1 shows the construction of the recombinant E.coli cells for producing rhIFN-a 2a according to this invention. Fig. 2 shows plot of fermentor parameters against time for the production of rhIFN-a 2a in E.coli. Fig. 3 shows the plot of specific growth rate (µ) of the E.coli cells vs. time for the production of IFN-α 2a in the fermentor. It also shows the volumetric activity of IFN-a 2a 6h and 12h after induction Fig. 4 shows the standard curve for calculating rhIFN-a 2a concentration by ELISA. Fig. 5 shows the SDS-PAGE showing induced bands of rhIFN-a 2a. Lane no. M: protein molecular wt markers; lane no.l: uninduced sample, lane no.2 to 1 4: induced samples (0.5 to 12 hrs post induction) Fig. 6 shows the SDS-PAGE showing comparison between known amount of BSA with different amount of rhIFN-a 2a. Lane 1 to 7: 1 to 7 µl.of OD600 =5 (12 hr post induction samples). Lane 8 to 14: BSA, 200ng to 800ng. Lanes 15 to 21: 1 to 7 yl of OD600 =5 (6 hr post induction samples). Fig. 7 shows the Western Blot showing induced bands of rhIFN-a 2a. Lane no. M: protein molecular wt markers; lane no.l: uninduced sample, lane no.2 to 13: induced samples (0.5 to 12 hrs post induction). Detailed Description: 1. The cloning of the gene of human Interferon-α 2a as shown in Fig. 1. Isolating genomic DNA from peripheral blood mononuclear cells (1). Amplification of hlFN-a 2a gene from human cells (PBMCs) using genomic PCR (2). Digestion o f the hlFN-a 2a gene and the plasmid vector p RSETA with restriction enzymes Ndel and BamHI (3). Ligating the digested hlFN-a 2a gene downstream of a strong "T7" promoter present in the plasmid vector pRSETA to produce the plasmid pRSETA-IFNa (4). Sequencing of the gene to confirm its nucleotide sequence (5). Transformation of E. coli BL21 (DE3) CodonPlus (RIL) cells by pRSETA-IFNa (6). 2. High level expression of rhIFN-a 2a during scale up in a fermentor. Growing the above cells in optimized media (TABLE 1) to get maximum specific activity of rhIFN-α. 2a. Using an exponential feeding strategy (TABLE 2A and 2B) to achieve high cell density. Induction o f p rotein s ynthesis i n t he c ells, w ith I sopropylthiogalactoside (IPTG), once high cell density has been achieved (Figs 2 &3). Harvesting the cells after 6-12 hours post induction in order to allow significant build up of the rhIFN-a 2a in the form of inclusion bodies (Fig.5). Partial p urification b y t he i solation o f i nclusion b odies. T his is done by lysing the cell pellet (by standard physico-chemical techniques) and pelleting the inclusion bodies by centrifugation. This is followed by washing the said pellet by buffer to get rid of contaminating biomolecules on the surface of the inclusion bodies. Dissolving the washed inclusion body pellet in denaturing buffer. The production of rhIFN-a 2a is enhanced using a combination of genetic and bioprocess strategies. Genetic Strategy: The IFN-ot 2a gene is cloned in a pRSETA plasmid, which has the advantage of driving heterologous gene-expression from a strong IPTG-inducible T7 promoter. The hlFN-a 2a gene is cloned in the restriction site Ndel/BamHl (fig 1). However, the hlFN-a 2a gene has some codons, which are rarely used in E. coli, which lowers the rate of synthesis of the protein. This plasmid is therefore used to transform E. coli BL21 (DE3) CodonPlus (RIL) cells which carry genome-integrated T7 RNA polymerase gene (used for high level gene expression from T7 promoters) along with plasmid borne extra copies of the argU, IleY and leuW tKNA genes that help in the high level expression of genes which use the rare codons AGA, AGG, AUA and CUA. Bioprocess Strategies: The bioprocess parameters like pH, D.O., temperature and the like are optimized, the feed composition of the medium and the initial batch medium are also optimized and feeding strategy fine-tuned leading to significant improvements in the volumetric and specific yields of rhIFN-a 2a (Fig.2). The initial batch media composition is as follows. TABLE 1(Table Removed)The composition for feed media (500ml, 15X) is as given below. TABLE 2A(Table Removed)Yeast Extract and Peptone are autoclaved in 478 ml of water, Glycerol is autoclaved separately, and 1M stocks of K2HPO4 and MgSO4 are prepared separately. TABLE 2B ** Following are the composition of trace elements (1000X) per liter(Table Removed)A l00µg/ml ampicillin and 50µg/ml chloramphenicol concentration is also used in the beginning (batch). The temperature, pH and DO are set at 37°C, 7.0 and 40% respectively. The initial flow rate of the feed is calculated from the following equation, Where F = flow rate of feed (1/h), µ = specific growth rate of the cells (h-1), S'0 = initial substrate concentration in batch culture (g/1), V = volume of the reactor (1), S0 = substrate concentration in feed (g/1). The feed is incrementally increased according to the following equation. F = F0eµt Harvesting of the cells is done after 6-12 hours post induction. RhIFN-a 2a is purified in the form of inclusion bodies from the harvested cells. First the harvested cells are lysed by standard physicochemical methods. The lysate is centrifuged to pellet cell debris along with inclusion bodies. Said pellet is washed with a buffer having 10 mM Ethylene Diamine Terra Acetate, 2% Triton XI00 and 2% Deoxycholate to remove cell debris and other contaminating molecules from the inclusion bodies. The washed pellet comprising of inclusion bodies are finally solubilized in denaturation buffer containing either 8M Urea or 6M Guanidine Hydrochloride. EXAMPLE A single colony (E.coli: BL21 (DE3) CodonPlus (RIL) cells containing the plasmid (pRSETA-IFα) is inoculated from Luria Broth (LB) plate in 10 ml Terrific Broth (TB)+ glycerol containing proper antibiotics (l00µg/ml ampicillin and 50(µg/ml chloremphenicol) (primary seed). It is grown overnight (16 hrs approximately) in rotary shaker at about 200 rpm at 37°C. Then 1ml of the culture is used to inoculate 100 ml of TB+ glycerol containing proper antibiotics (secondary seed) so that effective inoculum is 2% approximately. This secondary seed is allowed to grow on a rotary shaker at 250 rpm at 37°C for approximately 6 hours till the cell density reaches mid-log phase (i.e. about 5.0 g/1 of wet weight of cells or OD600- 2.0-2.2). These cells are used for inoculation of fermentor. The feed is started when the cells reach an ODeoo of 15 in the fermentor at the rate of 15 ml/h (The value of F is calculated from the equation F= (µS'0V/S0). When the OD600 reaches a value of 20 the culture is induced with 1 mM IPTG. After this the feed is changed every hour at values equivalent to the ODeoo (For example when OD600 reaches 26, the feed is fixed at 26 ml/h). This is continued till the culture reaches an OD600 = 40. From this point the feed is kept constant at 40 ml/h till it is completely used up. The specific growth rate is monitored and is found to continuously increase till it reaches a value of 0.767 h"1 when the culture is induced (Fig.3). Immediately following the induction we see a drastic fall in the value of µ. This fall continues till about 4 hours after induction (Fig.3.) and becomes as low as 0.0858 h-1. The agitation rate is simultaneously affected significantly after induction and a sharp fall in the RPM value can be seen from the graph (Fig.2). This is concomitant with a steep rise in DO levels, which is expected during metabolic shocks. After 4 hours, the cells recover from the induction shock, as can be seen from the rise in the value of µ and RPM and a high demand for oxygen (Figs.2.). After 6 hours, the culture is induced again with 1 mM IPTG. At this point the OD600 is about 87.75 and the value of µ, is 0.253 h"1. Once again, the same pattern is noticeable. We see a sharp fall in the values of µ. and RPM. The fall in the value of RPM and the simultaneously high DO value after 10 hours (Fig.2.) is definitely due to the fact the feed is over at that time point. This demonstrates that the residual concentration of nutrients is quite low in the culture medium implying efficient uptake of the nutrients by the cells. ESTIMATION OF rhIFN-a 2a CONCENTRATION OF THE FERMENTOR SAMPLES POST INDUCTION: a) ByELISA: The concentrations of rhIFN-a 2a for the 2 representative time points 6hr and 12hr post induction of the fermentor study are estimated by comparing the 00450 value obtained by ELISA with the corresponding value of the standard curve within the linear range (Fig.4). A 5 x 104 dilution of the cell lysate (12th hr sample where 1ml of 1 OD equivalent cells are taken) gives an ELISA reading of 0.89 (OD at 450nm). Thus the effective rhIFN-a 2a amount is calculated from the standard curve given in figure 4. An OD450 of 0.89 corresponds to 600pg/ml of WFN-alpha Thus the effective volumetric activity is = (600 x 5xl04x 129) pg/ml = 387xl07 pg/ml = (387xl07) /106 mg/ml = 3.87 g/1 of rhIFN-a 2a (Fig. 3.). b) By comparative visual estimation with known amounts of standard protein Bovine Serum Albumin (BSA). Figure 6 shows an SDS-PAGE where different amount of whole cell lysate are loaded and compared with the known amount of BSA. The amount of BSA loaded is 200 to 800 ng in 7 different wells. Fermentor samples (whole cell lysate) of 6hr and 12 hr post induction are loaded in 7 wells each. OD600 for 6 hr and 12 hr post induction sample is 87.75 and 129 respectively. Thus the cell lysates are diluted 87.75/5 and 129/5 times respectively and 1 to 7 µ1 of this diluted lysate is loaded in 7 wells as shown. It is important to note that the rhIFN-a 2a band constitutes the major band in the total lysate implying a very high specific activity. The intensity of the rhlFN-α 2a band is compared with the intensity of the BSA bands of known concentration to provide further confirmation of the attained yields. Thus a simple calculation shows: 4 µl of diluted cell lysate has a rhIFN-a 2a band corresponding to 600 ng of BSA. Thus rhlFN-α 2a yield = (600/4) x (129/5) ng/ul = 3.8 g/1. SDS-PAGE and Western blot analysis: The whole cell lysate is analysed on Sodium Dodecyl Sulfate- Poly Acrylamide Gel Electrophoresis (SDS-PAGE) (Fig.5.) and Western Blot (Fig.7.). The Western Blot is used to confirm that the induced band is indeed that of rhlFN-a 2a. From the SDS-PAGE it is observed that there is negligible expression pre-induction, however the expression levels rise quickly to reach their maximum values within 4 hrs of induction. After that it remains constant for 2-3 hrs. After 7 hrs there is a slow decline in specific activities, which however is compensated by the increase in biomass. Thus volumetric activities increase steadily to reach the maximum value 12 hrs after induction. We claim: 1. A process for the production of recombinant human interferon alpha 2a (rhlFN- α 2a) protein from recombinant E.coli comprising the steps of: isolating Interferon alpha 2a gene from human Genomic DNA ligating said Interferon alpha 2a gene downstream of T7 promoter in an expression plasmid pRSETA to form a construct called pRSETA-IFNα, transforming E.coli BL21 (DE3) codonplus (RIL) cells with the said construct, growing the transformed cells to high cell density in a fermentor at 37°C with optimized media and an exponential feed of concentrated media for 3-6 hours to achieve a high concentration of cells, inducing said cells with IPTG to start synthesis of rhIFN-a 2a protein, continuing the feed post induction for 6-12 hours to get high level build up of rhIFN-a 2a protein inside the cells in the form of inclusion bodies, harvesting said cells by centrifugation to separate said cells from the culture media, breaking the cells by standard physio-chemical methods to get cell lysate, centrifuge said cell lysate to get the inclusion bodies containing rhlFN- a 2a in the pellet, washing said pellet with a washing buffer, solubilizing washed pellet in denaturation buffer to get pure rhIFN-a 2a protein. 2. A process as claimed in claim 1 wherein said washing buffer contains 10 mM Ethylene Diamine Tetra Acetate, 2% Triton XI00 and 2% Deoxycholate. 3. A process as claimed in claim 1 wherein said denaturation buffer contains either 8M urea or 6M guanidine hydrochloride. 4. A process as claimed in claim 1 wherein the optimized media composition contains the following ingredients: Ingredients Amount i) Yeast extract 24g ii) Tryptone 12g iii) Glycerol 4ml Said ingredients are autoclaved in 900ml of H2O in reactor i) K2HPO4 12.54g ii) KH2PO4 2.3 1g Said ingredients are autoclaved in 100ml of H2O in flask. The above autoclaved ingredients are mixed before fermentation. 5. A process as claimed in claim 1 wherein the said exponential feed contains the following ingredients: Ingredients Amount i) Yeast extract 45g ii) Peptone 45g iii) Glycerol 62.5 ml iv) K2HPO4(1M) 5ml v) MgSO4(lM) 10ml vii) Trace Elements (1000X) 500 µl as herein described vii) Ampicillin (l00mg/ml) 5ml viii) Chloramphenicol 1.2ml 6. A process as claimed in claim 5 wherein said trace elements contains the following composition (1000X) per liter: Cupric sulphate-5H2O 2mg Aluminum sulfate l0mg Manganese Chloride-4 H2O 20mg Sodium molybdate-2H20 50mg Boric Acid 1mg Cobalt Chloride 2.50mg Zinc Sulphate 5mg Ferrous Sulfate 50mg Nickel Chloride-6H2O 1mg 7. A process as claimed in claim 1 wherein feed media is started when the cell concentration reaches an OD eoo of 10-15 and when OD 600 reaches a value between 20-30, the cells are induced with 1 mM IPTG for recombinant protein synthesis. 8. A process for the production of recombinant human interferon alpha 2a (rhlFN- 2a) protein from recombinant E.coli substantially as herein described with reference to the accompanying drawings and foregoing examples.

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1276-del-2002-abstract.pdf

1276-del-2002-claims.pdf

1276-del-2002-correspondence-others.pdf

1276-del-2002-correspondence-po.pdf

1276-del-2002-description (complete).pdf

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1276-del-2002-form-1.pdf

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1276-del-2002-gpa.pdf

1276-del-2002-petition-124.pdf