| Title of Invention | PROCESS FOR PRODUCING RECOMBINANT HUMAN SERUM ALBUMIN IN PICHIA PASTORIS BY A NOVEL GENE AND ITS PHARMACEUTICAL USES |
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| Abstract | A process for preparation and purification of a recombinant human serum albumin and a novel clone thereof, which comprises of: Culturing of recombinant Pichia pastoris in complex / defined medium to build up high cell mass. Culturing of recombinant Pichia pastoris in complex / defined medium to produce human serum albumin protein. Culturing of recombinant Pichia pastoris in charcoal containing complex media to produce human serum albumin protein of therapeutically acceptable quality and color. Culturing of recombinant Pichia pastoris in a two step fermentation process. Purification of recombinant human serum albumin (r-HSA) from fermentation broth of above mentioned recombinant Pichia pastoris. Formulation of purified r-HSA with various pharmaceutically accepted ingredients. Use of the r-HSA for various medical purposes. |
| Full Text | FORM 2 The PATENT ACT, 1970 (39 of 1970) Complete Specification PROCESS FOR PRODUCING RECOMBINANT HUMAN SERUM ALBUMIN IN Pichia pastoris BY A NOVEL GENE AND ITS PHARMACEUTICAL USES. CADILA HEALTH CARE LTD, Zydus Research Center 'Zydus Tower', Satellite Crossroads, Sarkhej-Gandhinagar Highway, Ahmedabad-380015, Gujarat. (An Indian company registered under the Company Act 1950) The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed. FIELD OF THE INVENTION: The present invention relates to human serum albumin, a major constituent of human plasma protein useful in the treatment of numerous diseases such as hyperbilirubinaemia, hypoproteinaemia, burns, blood volume loss, priming of heart lung machine during cardiopulmonary bypass surgery and related diseases. The invention further relates to the cloning of a novel gene encoding Human Serum Albumin (HSA) using novel oligonucleotide as primer. Insertion of the same in an Expression vector producing a recombinant Pichia pastoris culture, capable of expression of the heterologous polypeptide similar to HSA and its secretion. The disclosure also relates to the process of high cell density fermentation, a suitable process for its purification and therapeutic application. Particularly the invention relates to the preparation of recombinant human serum albumin (hereinafter referred to as r-HSA) using a novel clone of Pichia pastoris containing a novel gene encoding the same, by culturing the said recombinant strain of Pichia pastoris with inducible promoter having high transcriptional activity. The general principles embedded in this invention can easily be applied to prepare various other heterologous proteins in a similar way. BACKGROUND OF THE INVENTION: Human serum albumin is the most abundant human plasma protein of adults. The concentration of albumin is 40 mg / ml or 160 g circulating throughout the human body for an adult male of 70 kg body weight. This protein maintains osmotic pressure and functions in the binding and transport of copper, nickel, calcium (weakly, at 2-3 binding sites), bilirubin, protoporphyrin, long chain fatty acids, prostaglandins, steroid hormones (weak binding with these hormones promotes their transport across the membranes), thyroxine, triiodothyronine, cystine and glutathione. According to Peters and Reed {Peters and Reed, Albumin:Structure,Biosynthesis and Function. Pp.11-20,1977), over 10,000 kilograms of purified albumin are required to be administered annually in the United States alone to patients. Currently the only commercial source of HSA is from fractionated blood. Considering the possible dangers, of blood borne contaminants and pathogens, it would be a considerable advantage to use HSA prepared as recombinant protein. With the advent of recombinant DNA technology, it is now possible to produce recombinant HSA and use this as an alternate source. There are considerable ongoing efforts to increase heterologous expression of genes and hence, the yields of desired proteins in expression systems, such as a methylotrophic yeast, like Pichia pastoris as a host. HSA has also been expressed in Saccharomyces cerevisiae as disclosed by Etcheverry et al (Etchevery et al; In Biotechnology. August 1976: 726). Etchevery disclosed HSA expression intracellularly in a concentration of approximately 6 mg / L and the secretion of HSA which remained cell associated. Arjun Singh also disclosed the expression of HSA in Saccharomyces cerevisiae in combination with the alpha factor promoter and signal sequence/ Singh Arjun; EP-A 123544) Singh appears to have achieved, an intracellular production level of 25 mg / L and a secreted production level of 3 mg / L. Pichia pastoris has also been used to express HSA as is disclosed in EPA 344, 459 wherein the concentration of HSA produced in Pichia pastoris is about 89 ng HSA / mg of protein. Although the process for producing HSA in recombinant expression system has been established by these experiments it would be desirable to optimize these processes to achieve the maximum possible HSA production. Hence, it would be of a significant interest to provide a process for good yield of HSA in microorganisms. In recent years, numerous methods for producing a heterologous protein using regulatory region in AOX gene has been reported ( Yeast; 1989; 5 :167-177). But a promoter having a stronger transcription activity for an increased expression has still been desired. In US 5707828 the recombinant Pichia pastoris has altered mode of methanol consumption. The preferred clone mentioned in the above patent utilized methanol poorly, as a carbon source, during fermentation. The genetically engineered Pichia pastoris has been used in European patent application no. 344459, where r-HSA structural gene has been isolated, manipulated and inserted in vector. In another method addition of linkers or blunt ending the fragment, yields one strain having 8.9 % yield of r-HSA. US patent 5610036 involves a mutation at AOX2 promoter in Pichia pastoris, wherein yields are about 120 mg / ml. US patent 5643792 discloses a mutant strain, which has been characterized and the strain of this invention grows on methanol as well as glucose. In US patent no. 5707827 mutation occurring at a different AOX2 promoter site resulting in mutant strain that grows on methanol preferably has been described. The mutant Pichia pastoris described in EP 0504823 grow on methanol albeit poorly and have been subsequently adopted to grow on methanol. US patent 5330901 employ use of AOX promoter region in conjugation with the expression of r-HSA in which Pichia pastoris grows very slowly at pH of 5.7 to 6.4. Despite all above efforts there is no commercially viable process to produce r-HSA. The main factors in realization of this goal are cost of single dose and its purity. Further the r-HSA must be equivalent to the plasma derived HAS in all respects. As far as activity is concerned there are stringent quality requirements to be fulfilled. Thus to commercially produce r-HSA, the question of yield, cost and the quantity of r-HSA produced is to be considered. OBJECTS OF THE INVENTION: The present invention aims to provide a novel clone of Pichia pastoris and a method for producing the same having a novel DNA sequence, which encodes human serum albumin. A process for the fermentation of recombinant clone having this novel DNA yielding high cell density and expression of HSA, a process for its purification and formulation to yield a pharmaceutically acceptable composition for various therapeutic applications as mentioned herein. SUMMARY OF THE INVENTION: The present invention describes a novel clone of Pichia pastoris having a unique DNA sequence, encoding for human serum albumin. Novel oligonucleotide primers have been used to isolate the said gene. This gene on isolation has been inserted into a plasmid, propagated and isolated. Appropriate changes have been incorporated in this fragment, which is then cloned into Pichia pastoris shuttle vector. Later this novel segment of DNA has been incorporated into methylotrophic yeast Pichia pastoris host cells. The gene of choice, which encodes for HSA is driven by an AOX promoter. A high cell density fermentation protocol has been devised by regulating appropriate fermentation parameters. Later the recombinant Pichia pastoris cells are induced to produce and secrete the said protein in high yield. The fermentation process and expression is designed so as to yield r-HSA of acceptable color. The r-HSA is then purified by a novel process to yield highly pure, pharmaceutically acceptable form of r-HSA protein. DETAILED DESCRIPTION OF THE INVENTION: A. Overview: The present invention will now be described in greater detail with referance to the accompanying drawings wherein: Figure 1 shows the HSA nucleotide sequence (Sequence ID 1) of the present invention,where at 1668lh nucleotide there is 'T' instead of'C . Figure 2 shows the HSA nucleotide sequence (Sequence ID 2) obtained from NCBI Genebank database, where at 16681'1 nucleotide there is 'C. Figure 3 shows the Amino acid sequence of HSA protein (r-HSA of this invention) which is similar to those reported from NCBI data bank. Figure 4 shows the SDS-PAGE stained with Coomassie blue showing production of two proteins, one having ~ 67 kDa (similar to wild type HSA) and another ~ 43 kDa in fermentation samples at 48, 72 and 96 hours incubation time post induction in defined salt medium. Lane 1: Plasma derived Human Serum Albumin (Commercial HSA, Centeon) Lane 2: Fermentation broth sample at 48 hrs post induction. Lane 3: Fermentation broth sample at 72 hrs post induction. Lane 4: Fermentation broth sample at 96 hrs post induction. Figure 5 shows the SDS-PAGE of purified r-Human Serum Albumin stained with Coomassie blue in which from the left Lane 1: Low Molecular Weight Marker (Amersham Pharmacia) Lane 2: r-HSA of present invention. Lane 3: r-HSA of present invention in formulated sample. Lane 4: Plasma derived Human Serum Albumin (Commercial. HSA, Centeon) Figure 6 shows the SDS-PAGE of purified r-Human Serum Albumin stained with silver nitrate, in which from the left: Lane 1: r-HSA of present invention. Lane 2: r-HSA of present invention in formulated sample. Lane 3: Plasma derived Human Serum Albumin (Commercial HSA, Centeon) Lane 4: Low Molecular Weight Markers (Amersham Pharmacia) Figure 7 shows the Western Blotting of purified r-Human Serum Albumin developed with anti-HSA antibodies (Sigma) in which from the left: Lane 1: Plasma derived Human Serum Albumin (Commercial HSA, Centeon) Lane 2: r-HSA of present invention. Lane 3: r-HSA of present invention in formulated sample. Figure 8 shows thelsoelectric focussing of recombinant human serum albumin showing pi of4.8. Lane 1: pi marker ( Pharmacia) Lane 2: Plasma derived Human Serum Albumin (Commercial HSA, Centeon) Lane 3: r-HSA of present invention. Lane 4: r-HSA of present invention in formulated sample. Figure 9 shows the purity of recombinant HSA of this invention as detected by Size Exclusion Chromatography-HPLC. Figure 10 shows the Molecular Weight determination of recombinant HSA of this invention, by MALDI-TOF. The present invention provides a novel clone of Pichia pastoris, process to obtain a novel structural gene encoding human serum albumin, comprising of the base sequence as set forth in sequence I.D no. 1, Fig. 1, and the amino acid sequence as described in sequence I.D no. 2, Fig. 3. As per the gene sequence homology search the novel DNA of the present invention was found to have 99 % homology with the native HSA gene sequence deposited at NCBI Genbank database, except at 1668th position wherein instead of 'C in the present invention there is 'T'.However there was no difference in the amino acid sequence. The present invention discloses the design of an expression / secretion system to optimize the production of HSA. According to the present invention, the mutant mature human serum albumin gene is immediately down stream to the AOX promoter. The HSA producing host to be used in the present invention which is prepared by genetic engineering is subject to no particular limitation as long as it is prepared by genetic engineering and those disclosed in known literature or those to be developed in the future. Specific examples can include cells imparted with HSA producing ability by genetic engineering such as E.coli, B.subtilis, Yeast etc. or animal cells such as CHO etc. Particularly in the present invention yeast and more specifically a methylotrophic yeast such as Pichia pastoris is used as a host of choice. Thus the r-HSA may either be produced as intra cellular or extra cellular expression protein. Another aspect of the invention discloses the step of removing coloring contaminants from human serum albumin. The term "coloring contaminants" as used herein includes, within the meaning thereof, not only culture medium derived coloring contaminants, but also any substance capable imparting color to HSA. Preparation of host cells capable of producing the r-HSA, production of r-HSA by culturing the host cells, the isolation and recovery of r-HSA from the resulting culture broth may be effected using known techniques or modified procedures thereof. For example, preparation of a HSA producing host (or strain) may be effected using a process in which a wild type HSA gene has been used, a process in which a modified HSA gene is used, a.process in which a synthetic signal sequence is used, a process in which a serum albumin signal sequence is used, a process in which a recombinant plasmid is introduced into chromosome, a process in which hosts are fused, a process in which mutation is generated in methanol utilization, a process in which a mutant AOX2 promoter is used, a process in which HSA is expressed in B.subtilis, a process in which HSA is expressed in yeast, and a process in which HSA is expressed in Pichia ( US Patent No. 5,691,451). The method for producing a novel sequence of DNA expressing HSA, a method for producing Pichia pastoris expressing the same, the method of its large-scale cultivation and a process for its purification has been disclosed herein. B. Cloning process: The HSA producing yeast strain, Pichia pastorisGBSl was obtained by transformation of Pichia pastoris GS115 with plasmid carrying a novel HSA gene, which is integrated into its genome. The plasmid carrying novel HSA gene can be prepared by appropriate techniques known in the prior art, specifically the plasmid contains a novel HSA gene, a promoter, a signal sequence, a terminator and so forth. 1. The HSA structural genes have been sequenced by Lawn et al (Lawn et al (1981) Nucleic Acid research 9: 6105.) and Dugaiczyk et al (Dugaiczyk et al. PNAS, 1982; 79;71) The gene may be obtained by isolation as per the technique of Lawn et al and Dugaiczyk et al. Alternate procedures such as in vitro synthesis by custom gene manufacturer may be carried out. Another possible method of obtaining HSA gene would be to screen a liver c-DNA library or genomic library with oligonucleotide probe or can be prepared as cDNA in a conventional manner using m-RNA. In the present invention the HSA gene has been obtained by conventional manner by preparing cDNA from m-RNA. Total RNA has been isolated from a liver biopsy sample. The m-RNA from this RNA was used to synthesis the first strand c-DNA using reverse transcriptase. Gene specific second strand synthesis was carried out using the following primers: Information for Sequence ID No.4: (i) Sequence Characteristics: (a) Length: 21 bases (b) Type: nucleic acid (c) Strandedness: single (d) Topology: linear (ii) Molecular type: other nucleic acid (a) Description: forward primer (iii) Sequence description: Sequence ID No.4 5'-ATG AAG TGG GTA ACC TTT ATT-3' Information for Sequence ID No.5: (i) Sequence Characteristics: (a) Length: 21 bases (b) Type: nucleic acid (c) Strandedness: single (d) Topology: linear (ii) Molecular type: other nucleic acid (a) Description: reverse primer (iii) Sequence description: Sequence ID No.5 5'-TTA TAA GCC TAA GGC AGC TTG-3' This PCR product was end filled by Klenow reaction and cloned into M13 mp!8 vector which was dephosphorylated after linearizing with HlndW restriction enzyme digestion. This ligated M13 mpl8 construct having HSA gene called M13GHSA was transformed into E. coll JM109. The transformants were selected by blue-white screening and called E coll GHSA, 1-26. The HSA gene so isolated was sequenced by Sanger's method. It was established that it was a novel sequence (SEQ ID No. 1; Fig. 1) having 99% homology with the wild type gene of NCBI GenBank database (SEQ ID No.2; Fig.2) when nucleotide sequence of present HSA gene was compared. The novelty was in the nucleotide sequence ,wherein in the present invention there is 'T' instead of 'C.However the primary amino acid sequence remained the same (SEQ ID No.3; Fig 3). However we have incorporated nucleotide sequence needed to code for preprotein sequence of HSA. It is needless to mention that the r-HSA protein produced does not carry preprotein sequence and is released as mature r-HSA in this heterologous expression system. The DNA of this novel HSA gene was reamplified using primers SEQ ID No. 6 & 7 and template DNA obtained from E.coli GHSA.2. Information for Sequence ID No.6: (i) Sequence Characteristics: (a) Length: 36 bases (b) Type: nucleic acid (c) Strandedness: single (d) Topology: linear (ii) Molecular type: other nucleic acid (a) Description: forward primer (iii) Sequence description: Sequence ID No.6 5' CCC TCA CAC GCC TTT GAA TTC ATG AAG TGG GTA ACC 3' Information for Sequence ID No.7: (i) Sequence Characteristics: (a) Length: 39 bases (b) Type: nucleic acid (c) Strandedness: single (d) Topology: linear (ii) Molecular type: other nucleic acid (a) Description: reverse primer (iii) Sequence description: Sequence ID No.7 5' GAC TCT ACG AAA ATT TGA GAA TTC TTA TAA CGG TAA GGC 3' This novel gene may be now cloned into an expression vector such as pBR322, pBR325, pUC12. PUC13 of E.coli, pUBHO, pTP5, pC194 etc. of B.subtilis, pSH19, pSH15, pPICZ alphaA, pPIC9 etc. of yeast, pATll, pXTl, pRc/CMV, pRc/RSV etc. of mammalian cells such as CHO. The preferred expression vector of the present invention is pPIC9 and the host cell chosen is Pichia pas tor is GS115. The promoter may be derived from the genomic DNA of yeast as is known to one skilled in the art. Suitable promoter sequences include those that regulate the TRP 1 gene, ADH 1 gene, acid phosphatase (PH03 or PH05) gene, GAL 1, GAL 10 or GAL 7 that is galactose matabolizing gene, the invertase gene (SUC 2), the AOX 1, AOX 2 gene (alcohol oxidase). A mutant AOX promoter has been used herein whereby the host can not utilize methanol as a carbon source for its growth. In a further preferred mode of gene manipulation, a signal sequence is incorporated into the plasmid construct. Signal sequence used could be derived from yeast sequences such as those associated with the invertase and alpha factor gene, or of native HSA. Alternatively synthetic signal sequence may also be designed. In the present invention the vector pPIC 9 of Invitrogen Inc. has been used wherein the vector has a Multiple Cloning Site (MCS) flanked by an AOX promoter and yeast mating type signal sequence. The said HSA gene obtained as mentioned above and detailed in example 1 & 2 of this document has been inserted into the expression cassette. The pPIC9 vector was amplified in E.coll Topi OF' host. This DNA was purified and used for constructing the novel vector of this invention. The DNA of novel HSA gene of this invention obtained from E.coll GHSA2 and the vector DNA of pPIC9 were both digested with EcoRl restriction enzyme. The pPIC9 vector DNA digested with EcoBJ was dephosphorylated as per standard protocol. The EcoRl digested pPIC9 vector DNA and DNA of novel HSA gene from GHAS.2 were ligated and this novel construct now called pPICGHSA2 was transformed into E.coll Top 10 F'. The resulting transformants were screened with P32 labelled HSA specific probes. The recombinant clones were hereafter called E.coll PICGHSA.l to 12. The DNA isolated from E.coll PICGHSA.1-4 were characterized by restriction mapping and gene specific PCR amplification. The desired gene may be introduced into the host cell such as yeast Saccharomyces cerevisiae, Klyuveroinyces lactis, or Pichia pastoris and preferred host herein being Pichia pastoris. The gene may be introduced directly as a plasmid as multicopy or may be integrated in the genome of the host chromosome. The transformation of Pichia pastoris to introduce plasmid directly may be carried out by any one of the known methods such as calcium phosphate, microprecipitation, polyethylene glycol treatment of protoplasts or by electroporation. In the present invention as detailed in example 3 below, it was preferred to transform Pichia pastoris GS115 by electroporation and allowing the integration of the linearized DNA stabley into the host genome. An HSA producing Pichia pastoris strain with the plasmid integrated in the yeast genome is used preferably in practice of this invention. The plasmid contains a DNA sequence of part of a gene occurring naturally in the host yeast genome for example Leu 2, TRP 1, HIS 4, URA 3 etc. In the present invention the HIS 4 region has been selected. The homologous sequence enhances the likelihood of the whole plasmid or a linear fragment thereof integrated suitably into the host genome. The modification enables the culture of descendant cells stably retaining the introduced genetic material during proliferation in the absence of a selective pressure. Thus, a plasmid containing a sequence of naturally occurring yeast chromosomal gene together with the HSA gene can be integrated into the locus of said chromosomal gene and retained in a stable manner. More specifically, it is desirable that the plasmid be cleaved at a site in the sequence that is homologous to the host yeast cell genome by restriction enzyme digestion and the linearized plasmid be introduced into the host. The linearized plasmid is integrated into the region on the host cell chromosome that is homologous to the region that is inserted into the plasmid. The linearized plasmid is integrated into the host chromosome with increased frequency as compared to a circular plasmid. Homologous sequence to yeast chromosome are as mentioned above that of Leu, His, Ura etc. and preferably the 'His' sequence has been used, wherein the host used is also an auxotroph for histidine. The DNA from the clone pPlCGHSA.l was linearized with Sad restriction enzyme and transformed into Pichia pastoris GS115 his- mut+ and plated onto minimal dextrose medium plates without histidine. The histidine prototrophy is the indication of Pichia pastoris GS115 transformant. These transformants were subjected to a secondary selection by plating on minimal dextrose plates with and without methanol. The colonies growing on minimal dextrose plate and not on minimal dextrose +methanol were selected. These recombinants were now called Pichia pastoris GBS 1-20. Flask level experiments were carried out to evaluate the clones for the secretion of HSA. One such novel clone is Pichia pastoris GBS 1 which secretes the putative HSA and this clone was used for all subsequent work. C. Fermentation and expression of heterologous protein: The unique fermentation process of this invention comprises of producing a high cell density culture of novel Pichia pastoris GBS 1, and expression of heterologous protein under suitable conditions. Different types of fermentation techniques like batch, fed batch and continuous fermentation processes are well known to those of skill in the art. The equipment, materials and necessary parameters to be controlled to carry out fermentation by any conventional fermenter are well known. Also, standard instrumentation is used to monitor various parameters such as temperature, pH, dissolve oxygen concentration, amount of nutrients such as carbon and nitrogen source. All the equipment and additives are sterilized as per suitable methods known in prior art. The novel fermentation process of the present invention provides conditions to build up high density cell mass. The protocol is a typical fed batch process suitably modified. The rate of addition of nutrient is related with the growth rate of cells, rate at which carbon and nitrogen are assimilated and also with C / H / N content of the cells. Transformed Pichia pastoris cells can be.selected, by using appropriate techniques of selection such as, culturing previously auxotrophic cells in the absence of the nutritional requirement, or in the presence of an antibiotic wherein the resistance gene is present in the transformants. The mutant strain of this invention does not utilize methanol as a carbon source, therefore can be selected on suitable growth media with and without methanol. The genetically engineered Pichiapastorls GBS1 developed in the present invention can utilize glucose, sucrose and glycerol but more specifically has been grown on glycerol as a sole carbon source. The present invention also discloses a process for producing r-HSA by a two-stage fermentation (vide infra) of Pichia pastoris GBS1 containing a novel human albumin gene driven by an AOX promoter. The strain used in the present invention is an inducible, which means that the expression and production are subject to be controlled by catabolites, metabolites and inducers. The factors affecting growth of the above recombinant and expression of said protein include type of medium, oxygen saturation in growth medium and presence of inducer. Further in the process of this invention oxygen supply is proportional to the growth of the cells. The fermentor design also affects oxygen transfer rate and its utilization (Rosen et al ; Process Biochemistry ; 1977; 12(3) : 10-12). Hence fermentor design parameters such as optimal Length to Diameter (L/D) ratio, sparger design and agitator diameter in relation to vessel diameter of the reactor plays crucial role in the fermentation during growth phase and production of protein during production phase. Although the growth requirements of yeast are not complex and they can be readily grown in defined media, the mode of yeast growth is extremely sensitive to change in the growth conditions. Generally Pichia pastoris is optimally grown at about pH 4.8 to 5.2. At this pH range, Pichia pastoris provided with a suitable nutrient medium exhibits robust growth. This pH range appears to result in high level of expressions of several foreign proteins such as hepatitis B surface antigen. The temperature of fermentation should generally range from 20° C to 35° C and preferably it should be around 30° C. The dissolved oxygen concentration in the batch-fed fermentation process may range from 20 % to 80 % saturation, preferably from 30 % to 60 %. The cultivation of transformed Pichia pastoris can be conducted in an aqueous continuous or batch-fed manner, utilizing a variety of carbon-energy sources and/or nutrient sources. A suitable nutrient source or media for growing Pichia pastoris would include the following: one nitrogen source, phosphate source source of minerals such as iron, copper, zinc, magnesium, manganese, calcium and other trace elements, and vitamins such as biotin, pantothenic acid and thiamine. In the present invention, fed batch process of fermentation is preferred. Suitable carbon-energy sources can be methanol, glycerol, sorbitol, glucose, fructose and combination of any two or more carbon sources thereof. The suitable source for nitrogen may be peptone, yeast extracts or corn steep liquor or may be well-defined media consisting of specified amino acids. Trace elements utilized in the present invention are those which include but are not limited to cobalt, molybdenum, iron, copper, zinc, and manganese. However, the clone Pichia pastoris GBS1 constructed and used in the present invention behaves differently form the one disclosed in the prior art in that it does not grow on methanol and requires a critical pH of 6.5 ± 0.3 during expression phase. In order to maintain stringent pH required as mentioned above, we have prepared the medium in Phosphate buffer enabling minimum pH shift and any shift is regulated by addition of ammonia by automated pH control system. The use of phosphate buffer as the medium base enables: a. Reduction of cell mass build-up time. b. It maintains pH in a stringent window/range During fermentation numerous steps have been taken to ensure the increase in cell mass, as listed below: 1. The genetically engineered Pichia pastoris GBS1 developed in the present invention does not grow on methanol. It can be grown on glucose, sucrose and glycerol but more specifically has been grown on glycerol as carbon source. 2. The concentrations of the nutrients in medium are of vital significance. Therefore, there is continual increment of carbon substrate input corresponding to the biomass built up and its utilization along with suitable nitrogen feed at regular intervals. Such mode of addition of carbon source at an incremental rate and nitrogen source in a fractionated dose, results into high cell mass. 3. The high dissolve oxygen concentration required during high cell density cultivation of recombinant Pichia pastoris GBS1 was provided by controlling agitation, aeration and further mixing of pure oxygen with air as required 4. The cell mass has been built up to 200 to 250 g/L on wet wt. basis. The fractionated addition of nitrogen supplement is made with 10 X medium, so that a minimum volume would provide adequate nitrogen and avoiding the dilution effect. 5. The fermentation is continued for 66-72 hours till the maximum growth is achieved. At the end of growth stage the cells are aseptically separated to remove traces of unutilized glycerol, and any metabolic products secreted during growth stage and again suspended in production medium aseptically. The production medium contains charcoal so as to adsorb coloring contaminants produced during the fermentation process. Cells are induced with methanol to synthesize and secrete r-HSA. Methanol is added at a controlled rate as 50% aqueous solution to increase the efficiency of membrane transport in aqueous solutions. Further at critical methanol concentration a rate limiting concentration of glycerol is added to keep cells metabolically active,. 6. During this stage pH is maintained above 6.00, more specifically between 6.20 to 6.80 by addition of ammonia. This increases the yield of recombinant HSA. 7. The expression of r-HSA is monitored by densitometric analysis of commassie blue stained SDS-PAGE gels using Zero-D scan software of Eagle Eye gel documentation system. (Strategene) 8. At the end of production phase the culture broth is harvested and cell separation carried out using suitable technique such as centrifugation or cross flow Alteration to obtain clear filtrate. 9. Further the present invention describes a purification process comprising of 5 to 6 steps to obtain a pure r-HSA which is free from any contaminating protein, polysaccharide and nucleic acids. The purified r-HSA was further characterized and comparatively evaluated with native HSA by various analytical techniques confirming its molecular purity, similarity to native protein in terms of its physical, chemical, molecular and immunological characteristics. Thus the present invention describes the production of r-HSA, in genetically engineered Pichia pastoris GBS, by a two-stage fermentation process followed by a novel purification process to yield pure r-HSA of pharmaceutically acceptable color and quality. TABLE 1: Different media used to culture recombinant Pichia pastoris are as follows: No Composition of the medium ■ 1A BGY (complex medium) Peptone 20 g, Yeast Extract 10 g, and phosphate buffer 0.5 M, pH 6.0 (100 ml) was added to each liter of medium prepared in demineralized water. BGY used for seed preparation contained 10 g / L glycerol. IB BMY (complex medium) Peptone 20 g, Yeast Extract 10 g, Propylene Glycol 40 ml, and phosphate buffer 1 M, pH 6.0 (100 ml) was added to each liter of medium prepared in demineralized water.. Methanol added separtely as described vide infra. 1C BGY (10X) (complex medium) Peptone 200 g, Yeast Extract 100 g, antifoam (10 % dilution) 20 ml and phosphate buffer 1.0 M, pH = 6.0 (500 ml), was added to each liter of medium prepared in demineralized water. ID IE Defined salt Medium: To each liter of medium prepared phosphoric Acid 2.7 ml, Calcium sulfate 0.09 g, Potassium sulfate 1.8 g, Potassium hydroxide 2.065 g, Magnesium sulfate.7H2O 1.5 g, trace metal solution A and B, 4.4 ml each was added and pH was adjusted to 5.5 with 25 % aqueous ammonia solution prior to sterilization. Composition of trace metal solution A (PTM A) was Copper sulfate.5H20 3.0 g, Maganese sulfate. 2H20 3.0 g, Ammonium Molybdate 0.2 g, Boric acid 0.02 g, Cobalt chloride 0.5 g, Zinc chloride 2.0 g and Sulfuric acid 5.0 ml dissolved in D.M water to final volume 1.0 L. Composition of trace metal solution B (PTM B) was Ferrous sulfate 6.5 g, and 2-3 drops of Sulfuric acid dissolved in D.M water to final volume 1.0 L. YNB medium Peptone 20 g, Yeast Extract 10 g, Yeast Nitrogen Base without amino acid 6.7 g, Glycerol 20 ml and phosphate buffer 0.5 M, pH 6.0 (100 ml) was added to each liter of medium prepared in demineralized water. IF YPD agar Peptone 20 g, Yeast Extract 10 g, Dextrose 20 g, and agar agar 20 g (pH = 4.5) was added to each liter of medium prepared in demineralized water.. 1G Luria Broth Casein Hydrolysate 10 g, Yeast extract 5 g, sodium chloride 5 g, ( pH = 7.0 ± 0.2) was added to each liter of medium prepared in demineralized water.. Stock solutions of certain compounds are prepared in appropriate strengths as given below,: Biotin (0.2 g / L), Ammonia (50 % v/v aqueous), Antifoam (10 % v/v aqueous), Methanol (50 % v/v aqueous solution), Glycerol (65 % v/v aqueous solution), Ampicillin stock solution (50 mg / ml) and Tetracycline stock solution (5 mg / ml in ethanol). ' Methanol and Glycerol solution used with defined media contained 12.0 ml each of PTM A & B per liter and 8.8 ml each of PTM A & B per liter respectively. Each medium is sterilized by autoclaving, while the biotin, ampicillin, tetracycline, ammonia and methanol stock solutions are sterilized by filtration. Biotin stock solution is added at 2 ml / L concentration after sterilization to each of above medium 1A, IB, 1C, and ID. D. Purification of Heterologous Protein obtained from Transformed Pichia Number of methods to isolate the recombinant proteins obtained from transformed yeast cells are described in prior art [(Romanos M., et al. DNA Cloning 2: Expression Systems, IRL Press, 2nd ed. Pp.123-167. 799J);(Trotta P.P. et al. Developments in Industrial Microbiology, Elsevier, Amsterdam. Pp. 53-64,1987); and (Nagabhushan T.L. and Trotta P.P. Ullman's Encyclpedia of Industrial Chemistry,Al4, VCH,Weinheim,Federal Republic of Germany. Pp.372-374, 1989)) Standard techniques, such as affinity chromatography, size exclusion chromatography, ion exchange chromatography, HPLC and the like can be used to purify protein of interest. Typically, secreted proteins can have purity anywhere in between 30 - 80 %. The expressed protein can be further purified to at least about 90 % purity; or also to about 95 % purity; or even greater than 95 % purity with respect to contaminating macromolecules, particularly other proteins, nucleic acids, and freedom from infectious and pyrogenic agents. Proteins expressed by methylptrophic yeast may also be purified to a pharmaceutically pure state, which is greater than 99.9 % pure. In cases where proteins are secreted into culture media, the advantage is of relatively lower contaminating substances, and the supernatant can be collected by known methods to isolate proteins [(Berg K., Acta Path.Microbiol.Immunol.Scand, Section C 1982, suppl.279:1-136) and ( Pestka S and Rubinstein M.;US Patent No.4,289,690)], the relevant techniques are incorporated herein by reference. Culture supernatant containing expressed protein can be purified by any one or more than one of the methods or a combination of methods disclosed below. Different types of protein purification methods are based on differences in the physicochemical characteristics of the proteins, such as : . i) solubility difference: examples of which include methods such as salting out, sedimentation with solvents ii) Differences in molecular size or weight: examples include dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis iii) Difference in the electric charge: examples include ion-exchange chromatography, iv) Methods utilizing specific affinity: examples include affinity chromatography, v) Methods utilizing a difference in the hydrophobic property: examples include reversed-phase high-performance liquid chromatography, and vi) Methods utilizing a difference in the isoelectric point: examples include isoelectric electrophoresis, etc. In some process immunoaffinity chromatography is used initially, but the cost of the reagents (especially matrix) is prohibitively high and hence such a method is unsuitable for large scale protein purification. The prior art also describes various methods using combination of classical chromatography, monoclonal antibody, to achieve different levels of purity, particularly when it is desired to isolate single subspecies of particular protein. These techniques were found to be unsuitable for large-scale purification and lacked consistency in purity of product obtained. In order to obtain the native protein in its correctly folded state, it is preferable to use processes which avoid denaturation and precipitation steps. Accordingly, the present invention relates to a purification process of r-HSA consisting of 5 to 6 steps to obtain a pure form of r-HSA which is free from protein and polysaccharide contaminants, using chromatographic techniques particularly ion exchange and concentrating the purified protein by ultrafiltration and finally to achieve homogeneity by gel filtration chromatography. Thus purified r-HSA was further characterized and comparatively evaluated with native HSA by various analytical techniques confirming its molecular purity, similarity to native protein in terms of its physical, chemical, molecular and Immunological characteristics. The objective of present invention was to provide a large-scale protein purification process to achieve high degree of purity in an economical way. Suitable chromatographic media used for protein purification include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like, including PEI, DEAE, QAE, and Q derivatives. Examples of chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like, or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica- based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross- linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. Optionally, one can modify these supports with reactive groups such that proteins can link with amino, carboxyl, sulfhydryl, hydroxyl and/or carbohydrate moieties from the protein. Also, it is also possible to engineer a tag onto the amino- or carboxyl-terminus of the recombinant protein to allow purification by affinity chromatography [(Morgantl L, et al.Biotechno.Appl.Biochem. 1996,23.67) and (Luo W. et al. Arch.Biochem.Biophys. 1996,329:215)]. In cases where the protein thus obtained is in a free form, the free protein can be converted into a salt thereof by known methods or method analogous thereto. In case, where the protein thus obtained is in a salt form the protein salt can be converted into a free form or into any other salt thereof by known methods or method analogous thereto. The suitable buffer solution may contain a protein-denaturing agent such as urea or guanidine hydrochloride or a surfactant such as Triton X-100 TM. The process of this invention preferably elutes the r-HSA from ion exchange columns by increasing or decreasing the pH. Such pH changes can be obtained by applying a buffer solution to the column. Such process may involve applying a solution of the said crude r-HSA onto an column, such as eluting the adsorbed r-HSA from said column using a buffer solution, wherein suitable known techniques of the chromatography may be used, which may include for example salt gradient or pH gradient; concentrating the elute obtained from previous step in a suitable way; further subjecting it to anion exchange chromatography; gel filtration chromatography and recovering the r-HSA obtained. The objects of the purification process will be clear from the following description of the process, which comprises of the following steps: a) reconditioning of the heat treated supernatant containing protease inhibitors by dilution, pH adjustment, and filtration; b) applying a solution of said crude r-HSA onto cation exchange column, and eluting the adsorbed r-HSA from said column using aqueous buffer solution; c) applying the eluate resulting from step b) onto an anion exchange column, and eluting the adsorbed human serum albumin from said column using a different aqueous buffer solution; d) concentrating the eluate resulting from step c) by ultrafiltration. e) passing the concentrated protein through gel filtration column, wherein the said column is equilibrated with phosphate buffered saline. f) concentrating the elute resulting from step e) by ultrafiltration. The first filtrate is heat-treated at 40 to 75° C in the presence of protease inhibitors such as PMSF, EDTA, EGTA etc. for 10 to 300 minutes. This specified treatment is sufficient for inactivation of proteases. Reconditioning of heated supernatant by diluting it with suitable buffer to yield resulting sample (second filtrate) of desired pH and conductivity. The second filtrate is allowed to contact with a cationic exchanger at a pH of 4.0 to 4.7 and a conductivity of 5 to 15 ms/cm, and then exposing the cationic exchanger to a pH of 5.0 to 6.0 and a salt concentration of 0.1 to 0.25 M to yield a first elute. It is further reconditioned by suitable buffer to desired pH and conductivity. The reconditioned first eluate is allowed to contact with an anionic exchanger at a pH of 5.0 to 6.0 and a conductivity of 2 to 6 ms/cm and then eluting absorbed fraction of albumin with sodium acetate buffer of pH 4.5 to 4.9 (second eluate). Allowing second eluate to concentrate with an ultrafiltration membrane having a molecular weight exclusive limit of from 10,000 to 50,000 to yield concentrated r-HSA sample, having protein contents of about 50 - 100 mg/mL. Resulting concentrated sample from above step is allowed to pass through gel filtration chromatography to obtain highly purified monomeric form of recombinant r-HSA. The process of purification to obtain pure recombinant human serum albumin is depicted in Example 13 and its method of evaluation is described in test example. The purified r-HSA is found to be identical to native protein in terms of its physical, chemical, molecular and immunological characteristics. , The total protein content was determined according to the Bradford's method (Current Protocols in Protein Science, Vol.1). Determinatipn of specific protein, r-HSA carried out by gel densitometry using Strategene Eagle eye Video documentation system. % Purity of r-HSA protein carried out by SEC-HPLC using TSK Gel G3000 SWXL column (Toshohaas) (Dodsworth Met al. Biotechnol. Appl. Biochem.1996; 24:171-176). The purified protein of the present invention has been characterized for its physicochemical, immunological and biological characteristics as follows: Characterzation of r-HSA protein: Molecular weight and purity determination by SDS-PAGE: The said protein has been resolved on SDS-PAGE and stained with coomassie blue method as described by Oakley et al (Oakley BR et al. Anal. Biochemistry.1980; 105;361). The results are summarized in FIG. 5, and 6 demonstrating its purity and molecular weight to be ~ 67.0 kDa. Western Blotting lor Immunological identity: Various methods described in literature and standard instruction manuals were used [(Ames CFL.J.Biol.Chem.l974;249;634) and (Towbin H.et al.Proc.Matl.Acad.Sci.l979;76;4350)]. The results are depicted in FIG. 7. The anti HSA antibody detects / reacts with the resolved human serum albumin produced as per present invention, at similar intensity to that of plasma derived human serum albumin (Centeon). Thus the immunological activity and epitope presentation during final folding of the said protein is not altered. This establishes the similarity of r-HSA produced as per present invention with that of plasma derived HSA protein (Centeon). Isoelcctro focussing of human serum albumin protein: The pi of r-HSA protein of the present invention was determined by IEF (Westermeier R., Electrophoresis in Practice,2nd.,VCH,fVinheim,Germany, J997) and the results as depicted in FIG.8 was found to be ~ 4.8 which is the expected pi range of HSA protein (Dodsworth N.et al Biotechnol. Appl. Biochem. 1996;24:171-176). LCMS of r-HSA protein: To determine the Mol. Wt. of the r-HSA protein of the present invention, LCMS analysis was carried out & the Mol. Wt. as depicted in FIG. 10 was found to be 66.2 to 66.8 KDa, which is as per expected molecular weight of HSA. Amino acid Sequence analysis: Amino acid sequence analysis of the human serum albumin produced as per present invention was based on the Edman degradation method (Edman P.Begg G.Eur.J.Blochem.,1976;l;80-91). The first 15 amino terminal amino acid residues match with that of known plasma derived HSA. The remaining amino acids have been deduced from nucleotide sequence which shows 100 % match with that of known plasma derived HSA. The results are as summarized in FIG. 3. E. Utility of the protein The therapeutic applications of r-HSA are for serious bums, to prevent haemoconcentration and to combat fluid and sodium losses. It is used as blood substituent, plasma volume expander in a variety of emergency conditions or in a case when suitable donor is unavailable. In the emergency treatment of shock and in conditions where restoration of blood volume is urgent. It is also indicated for clinical situations associated with low plasma protein (hypoproteinaemia) with or without oedema. It is also used as an adjunct to exchange transfusion for the treatment of hyperbilirubinaemia in hemolytic disease of newborn. Another application of HSA is in priming heart lung machine for cardiopulmonary bypass. In addition HSA is usually used as a stabilizer in various biologicals such as proteins, vaccines, etc. F. Pharmaceutical Compositions The r-human serum albumin of the present invention can be formulated by addition of capryillic acid, etc. In general, the compositions of the subject invention will facilitate the administration of the HSA. The compositions used in these therapies may also be in a variety of forms but is preferable as infusible solutions and the like. The compositions also preferably include conventional pharmaceutical^ acceptable carriers and adjuvants that are known to those of skill in the art. Preferably, the compositions of the invention include any suitable dosage forms known in the prior art. The present invention, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention. Example 1: Isolation of human serum albumin gene: Total KNA was isolated from 100 mg of human liver biopsy sample using the Trizol method. The first strand DNA synthesis was carried out using 1.0 micro gm of total RNA isolated from the said tissue in reaction mixture that contained 50 units RNAse inhibitor, 20 units AMV reverse transcriptase, dNTP mix and oligo dT 18 in a 20 u.1 reaction volume and was incubated at 42 °C for 60 min. In next step double stranded DNA was prepared using novel primers with SEQ. ID No. 4 and 5. The reaction mixture consists of cDNA synthesized above, along with 150 nM of each primer (SEQ ID 4 and 5), 150 uM of dNTP mixture and 1.5 mM MgCl2 in PCR reaction buffer and PCR amplification was carried out as per standard protocol. The PCR product obtained was resolved on l % agarose gel containing ethedium bromide (0.5 ug/ml) in 1 x TAE buffer at 50 V for 2 hrs. The PCR product was observed at the expected molecular weight range of- 1830 bp in comparison to standard molecular weight marker. The 1.0 ug human serum albumin gene PCR product of example 1 was treated with 1.0 - 2.0 units klenow enzyme to end fill the overhang created during PCR. Example 2: Cloning of human serum albumin gene in M13mpl8 vector: The M13mpl8 vector of E. coli was isolated from 10.0 ml of overnight cultures grown at 37 °C using known method. The vector DNA was resolved on 1.5 % agarose gel and quantified. The size of the vector was determined by digestion with restriction endonucleases Hind II and resolved on agarose gel. The digested vector was dephosphorylated using 1.0 unit of Shrimp alkaline phosphatase enzyme in 20 ml reaction volume. The vector DNA was treated with phenol: chloroform and precipitated with ethanol. The dephosphorylated linearized M13 mpl8 plasmid was blunt end ligated with human serum albumin gene obtained in example 1, in 1:3 molar ratio and 3 units of T4 DNA ligase, 1 x ligation buffer and 1 mM riboATP in 20 ul of reaction mixture. This ligated construct was transformed in E.coli JM 109 competent cells by CaCl2 method (Current Protocols in Protein Science.Vol.J). The transformants were grown on Luria agar containing ampicillin, X-gal and IPTG, from which 26 white recombinant colonies were isolated and named as E. coli GHSA1 through GHSA26. The RF DNA was isolated from of recombinant E.coli GHSA1 and 2 to 5 (.tg of RF DNA was subjected to double restriction digestion. The reaction mixture had 2 units each of £coRl and Hind III restriction enzymes, 1 x universal buffer in 50 ul solution and was incubated at 37 °C for 4 to 5 hrs. The product was resolved on 1 % agarose gel having 0.5 ug/ml of ethidium bromide. The size of the released fragment was confirmed as -1845 bp when compared with standard molecular weight markers. The HSA gene was sequenced using Sanger's dideoxy chain termination method with the fluorescent dye chemistry. DNA sequence is as shown in SEQ ID. No. 1; Fig. 1. Example 3: Cloning of human serum albumin gene in pPIC9 vector: The expression vector pPIC9, was obtained from Invitrogen and propagated in E. coli TOP 10F'. The plasmid DNA was isolated by alkali lysis method (Current Protocols in Protein Science, Vol.1). The human serum albumin gene was amplified using primers, having SEQ. ID No. 6 & 7 as forward & reverse primers respectively from pM13GHSAl DNA. The reaction mixture contained 100 ng of template pMBGHSAl DNA, 1 x PCR buffer, 1.5 mM MgCl2, 150 (.iM dNTP mixture, 150 nM of each primer and 2 units of Taq polymerase (MBI Fermentas) according to previously described procedure. An aliquot of amplified DNA was resolved on 1.5 % w/v agarose gel containing,0.5 u.g / ml of ethedium bromide along with the standard 1 kb ladder marker (MBI Fermentas). Two to five ug of PCR product obtained above was restriction digested in a reaction containing 2-5 units each of £coRl enzyme, in 1 x universal buffer in a 50 u.1 solution. The sample was resolved on agarose gel having 0.5 (.ig / ml of ethidium bromide. The size of the released fragment was confirmed as -1875 bp when compared to standard molecular weight markers in the adjacent lane. Similarly, the pPIC9 vector was restriction digested using EcoRl restriction endonuclease, 1 x universal buffer in 50 ul solution and dephosphorylated using 1.0U of shrimp alkaline phosphatase enzyme. The treated vector DNA was extracted with phenol and ethanol precipitated. The purified digested serum albumin gene was ligated to pPIC9 vector DNA in 1: 3 molar ratio. This ligated DNA was then used to transform E. coli"TOP 10F' by electroporation method. The competent E coli cells were mixed with ~ 100 ng of purified ligated DNA in chilled electroporation cuvette and cells were transformed using Electroporetor 1000 (Strategene) (Current Protocols in Protein Science, Vol.1).The transformed cells were plated on a Luria agar containing 100 (.ig/ml of ampicillin and 25 ug/ml tetracycline, plates were incubated at 37 °C overnight. The transformants, (hereinafter called E coli PICGHSA1-12) were verified for the presence of the human serum albumin gene as described by PCR method utilizing the gene specific primers and combination of vector specific and gene specific primers. The positive clones or the recombinant clones having the right orientation of serum albumin gene were called E coli PICGHSA 1,2,3,4. and E coli PICGHSA 1 clone was sequenced using the dideoxy termination method. The sequence data confirmed that the gene is novel human serum albumin gene as described in SEQ ID No. 1; Fig .1. The homology search was carried out with sequences at NCBI GenBank database using "BALST-N" and it was concluded that the gene of present invention had a nearest match (>99%) with published human serum albumin gene sequence. It had mutation at 1668th position where the novel HSA gene obtained in the present invention has "T" nucleotide and other nearly matched sequence (GenBank No. 76863) had "C" nucleotide, coding the same amino acid. EXAMPLE 4: Transformation of yeast Pichia pastoris with expression construct: Transformation of yeast Pichia pastoris GS115 was carried out according to method described in literature using LiCl (Higgins D.R. and Cregg J.M.feds.) Pichia Protocols, ethods in Molecular Biology Humana Press,(Totowa,NJ),103,pp.249-261,1998). A single isolated colony of Pichia pastoris GS1T5 was grown in 10 ml of YPD media. The cells were harvested, washed twice with distilled water and in 10 ml of 100 mM LiCl. Competent cells were recovered by brief centrifugation. Yeast pellet was mixed with 240 p.1 of 50 % PEG, 36 p.1 of 1 M LiCl, 25 p.1 of 2 mg / ml SS DNA and 50 p.1 of the Sacl linearized construct (1-10 pg) and construct was introduced into yeast cell by heat shock. The transformed cells were plated on minimal dextorose plate and incubated for 2 to 4 days at 30°C for His+ transformants. Ten His+ transformants / clones were selected and patched on the minimal methanol plate for the Muts phenotype. Out often, six are Mut and these were screened for the production of the r-human serum albumin protien of-68 kDa at flask level.(Fig. 5) These novel clones were named as Pichia pastoris GBS.1-6. EXAMPLE 5: Development of inoculum using BGY & YNB medium for fermentation process: 50 ml of BGY medium (composition Table 1) was taken in 250 ml Erlenmeyer flask and was inoculated with 1 ml inoculum of Pichia pastoris GBS1 (as described in example 4) from thawed glycerol stock (stored at - 70° C). The flask was incubated at 30 ± 0.5° C, on rotary shaker at about 240 rpm, with 1" displacement for 24-48 hours. 2 % v/v of the above inoculum was further diluted in 200 ml YNB medium (composition Table 1) into a IL flask, which was incubated for about 24-48 hrs on rotary shaker under the similar conditions. The inoculum prepared was used for fermentation and production of desired protein. ; EXAMPLE 6: Development of inoculum using BGY medium for fermentation process. In an another method, the inoculum was developed using BGY medium (Table: 1). To the medium biotin stock solution was added (0.4 mg / L of biotin was present in the medium). The seed stage 1 was initiated by inoculating 50 ml BGY medium with 1 ml of thawed glycerol stock of Pichia pastoris GBS1 stored at - 70° C, under aseptic condition. The flask was incubated at 30 ± 0.5 °C for about 48 hours on rotary shaker at about 240 rpm, with 1" displacement. After 24-48 hrs of fermentation, the purity of seed was confirmed by microscopy. Later for seed stage II, 200 ml BGY contained in a 1 L Erlenmeyer flask, was inoculated with 2 % v/v concentration of cells from seed stage 1. This seeded flask was incubated on rotary shaker and cultivated under similar conditions. The inoculum prepared was used for fermentation to produce recombinant human serum albumin protein. EXAMPLE 7: Flask experiment-1 with complex medium: Effect of glycerol concentration on growth: The inoculum for this flask experiment was developed as per above example: 6. Flasks containing BGY medium with varying concentration of glycerol were inoculated with inoculum seed II and incubated at 30 ± 0.5 °C for 72 hours on rotary shaker at about 240 rpm, with 1" displacement. Samples were collected aseptically at different time intervals and analyzed for pH and growth by measuring optical density at 600 nm. The results are as tabulated below in table:2. TABLE - 2 Effect of glycerol concentration on biomass Glycerol added Hrs. of growth at time t0 24 48 72 Concentration (% v/v) pH /OD units pH /OD units pH/OD units 1.0 4.95/20 5.12/24 5.25/26 1.5 4.92/19 4.53/30 4.69/34 2.0 4.93/20 4.40/35 4.00/45 2.5 4.92/20 4.37/37 3.98/44 3.0 4.96/20 4:40/35 4.02/46 It is evident from the above results that increased concentration of glycerol with progression of time results in the drop of pH from 5.0 at 24 hours to about 4.0 at 72 hours incubation. Proportionate increase in growth has not resulted with increase of glycerol concentration. The inventors then hypothesized that the drop of pH is unfavorable to obtain concurrent increase in biomass and utilization of glycerol present. EXAMPLE 8: Flask experiment 2 with complex medium: Effect of pH control and glycerol concentration on growth In yet another experiment carried out as per previous experiment described in example: 7, the drop in pH was compensated by addition of ammonia. It was observed that the pH control has resulted in increase of biomass. Thus the inventors conclude that for high cell density fermentation, regulated glycerol feed and maintenance of pH is required. The results are as depicted in the table 3 and 4. TABLE -3 Effect of pH control and glycerol concentration on growth Glycerol added Hrs. of growth Series at time t0 24 48 72 96 120 Conc(%) pH/ODunits pH/ODunits pH/ODunits pH/ODunits pH/ODunits Series I = No alkali added, Series II = Alkali added Table: 4 Quantity of 12.5% v/v ammonia added in series II flask of Table 3 I 1 5.20/28 5.68/28 5.88/30 5.80/27 6.00/28 2 5.13/32 4.50/44 4.64/31 4.60/38 4.78/40 ' 3 5.18/31 4.46/45 4.08/50 3.94/44 4.12/51 4 5.17/31 4.46/53 4.00/53 3.64/60 3.61/62 ■ II 1 5.20/28 5.98/30 6.15/28 6.07/30 6.25/29 ' 2 5.13/32 4.76/45 5.90/41 5.93/45 6.02/44 3 5.18/31 4.77/46 4.61/54 5.57/55 5.77/56 4 5.17/31 4.70/45 4.58/60 4.30/69 4.54/67 Series Glycerol added 12.5% v/v ammonia added at time t0 Concn (%) 24hrs 48hrs 72hrs 96hrs II 1 0.2 ml 0.4 ml Nil Nil 2 0.2 ml 0.4 ml Nil Nil 3 0.2 ml 0.4 ml 0.4 ml Nil 4 0.2 ml 0.4 ml 0.4 ml 0.6 ml The series II flasks where pH drop was compensated by addition of 12.5% v/v ammonia resulted in higher biomass production as compare to series I flasks. EXAMPLE 9: Fed-batch Fermentation with complex medium: The fermentation process was carried out under submerged aerobic conditions. The fermentor was equipped with automatic pH, temperature and dissolved oxygen controls. In 20 L fermenter, 8 to 10 L of complex medium was prepared, sterilized and inoculated with the inoculum prepared as described in example 6. The agitation rate varied between 200 to 550 rpm. The aeration rates varied from about 0.4 to 1.0 volume (at about atmospheric pressure and about 25° C) per volume of ferment per minute of air supplied. The air supplied was mixed with sufficient oxygen whenever required, in order to maintain dissolved oxygen at about 20 to 60 % saturation. The complex media (BGY) used in the present fermentation process was prepared as described in Table 1, after sterilization the media was further supplemented with 2 ml/L of biotin stock solution. The stock biotin solution was prepared (Table 1), filter-sterilized and stored at +4 °C. The fermentation was carried out at 30 °C at about atmospheric pressure in fed batch protocol, wherein glycerol was added in rate limiting concentration and was later slowly increased. At the end of about 60 to 65 hours of fermentation process, when growth had plateaued, broth was harvested from fermenter aseptically. The cells were separated by centrifugation at 6000-8000 g, resuspended in 10 litre of BMY medium (described in Table 1) and were transferred back to the fermenter under aseptic conditions. The cells were induced with sterile methanol (50 % aqueous solution), wherein the concentration of methanol was maintained between 0.5 to 3.0 % v/v. Fermentation was monitored by withdrawing samples aseptically at regular time intervals. The methanol concentration in the ferment was monitored by gas chromatography. Multiple inductions were carried out. The concentration of expressed r-human serum albumin protein obtained was ~ 1.2 to 1.5 g/L as monitored by densitometric analysis of coommassie blue stained SDS-PAGE gel. EXAMPLE 10: Modified Fed-batch Fermentation Process 1: In the present example it is demonstrated that to obtain maximum cell mass, the addition of carbon at a controlled rate and nitrogen as 10 X BGY (Table: 1) in a fractionated dose at specific time point is required for continued increase in cell mass. The present example is different from the Example 9 in that carbon and nitrogen are added in a regulated manner to obtain high cell density. Seed I and II are developed as described in Example 6. In a 20 L fermenter containing 10 L of batch culture medium (Medium composition as per Table 1) 2 % v/v seed II is transferred. Glycerol was fed as 65% v/v aqueous solution at controlled rate. The agitation and aeration was increased in proportion to increase in biomass. In case of control batch no nitrogen supplementation was given, while in the case of experimental batch nitrogen supplementation was provided in a fractionated manner so as to get maximum biomass during growth phase. Results obtained are as depicted in Table: 5. TABLE -5 Effect of Nitrogen supplementation on Biomass Hrs. of Control batch Experimental batch > fermentation Biomass(OD units) Biomass(OD units) 0 4.1 0~9 12 10.2 7.7 24 63.2 66.0 36 71.0 48 93.5 60 117.0 72 135.0 107.0 153.0 202.0 226.0 EXAMPLE 11: Modified Fed-batch Fermentation Process 2: In the present example inventors have demonstrated a process wherein activated charcoal is used, it is possible to reduce the color index of the finished product which comes from coloring components present in the raw material as well as some impurities produced and secreted during production of r-HSA by using activated charcoal. It is possible that r-HSA is adsorbed by activated charcoal and that the addition of charcoal during fermentation may result in loss of total r-HSA produced. Thus it is decided to remove color of medium prior to the synthesis of r-HSA and to evaluate the relative impact on color removal and total yield. Thus two methods of charcoal treatment has been designed. The present example demonstrates comparison of reduction in degree of coloration of medium and purified product, after treating expression medium with activated charcoal by two different methods. Seed I and seed II is developed as described in Example 6. 2% v/v seed II is transferred into a 20 L fermenter containing 10 L of batch culture medium (Medium composition as per Table 1). Batch cultivation is carried out as described previously in Example-10. At the end of growth phase cells are starved for about 2 hours. Cell separation is carried out under aseptic condition by using any of the known method. Three different expression media herein called as Control, Experimental medium A and Experimental medium B (Composition as per Table 6) were prepared. Extent of coloration in the medium was measured as absorbance at the wavelength of 350nm and 405nm and results are as depicted in the Table 7. The harvested broth is processed by purification protocol described to obtain a pure form of r-HSA. Color index of purified r-HSA is given in the Table 8. Thus in the present instant it is demonstrated that by following two-step charcoal treatment protocol the color of the final product is reduced and the color of the purified product is within the acceptable range. TABLE-6 Composition of Expression media Components Concentration (g/L) Control Medium A Medium B Yeast extract 15 15 15 Peptone 30 30 30 Biotin 0.0004 0.0004 0.0004 0.1 M K-P04 buffer, pH 6.0 100 ml 100 ml 100 ml Activated Charcoal Nil 10* 15** Dist. water To make 1 L To make 1 L To make 1 L * To expression medium 10 g/L activated charcoal was added prior to sterilization ** To expression medium 5 g/L activated charcoal was added and medium was heated up to 65°C with constant stirring for 30 min. After cooling medium was filtered and 10 g/L activated charcoal was added prior to sterilization. TABLE -7 Comparison of reduction in degree of coloration in medium Medium Absorbance 350 nm 405 nm Control 0.328 0.156 Exp. medium A 0.198 0.075 Exp. medium B 0.110 0.043 TABLE - 8 Comparison of reduction in degree of coloration in purified product Medium Ratio Ratio 350 nm/280nm 405 nm/280nm Control 0.091 0.069 Exp. medium A 0.051 0.024 Exp. medium B 0.017 0.009 EXAMPLE 12: Modified Fed-batch Fermentation Process 3: In yet another fermentation process defined salt medium was used in place of complex medium. Inoculum was developed as described in above example 5. Defined salt medium was prepared and sterilized in fermenter and inoculated vising seed stage II at 4.0 % v/v concentration. Fed batch fermentation was carried out by providing glycerol at controlled incremental rate. pH was monitored and regulated at 5.0 to 5.5 w'm automatic addition of sterile ammonia solution through pH controller during growth phase. 0>ther parameters like temperature and dissolve oxygen were same as per above example 11. The process differs from the above process described for complex media (Examples 9,10 & 11) in that sterile cell separation was not carried out at the end of growth stage and the cells were induced with Sterile methanol (50 % aqueous solution). The concentration of methanol was maintained between 0.5 to 3.0 % v/v. The methanol concentration was monitored by measuring the methanol content in the ferment by gas chromatography. Multiple inductions were carried out. The concentration of expressed r-human serum albumin protein obtained was ~ 2.0 to 3.0 g/L as monitored by densitometries analysis of coommassie blue stained SDS-PAGE gel. It was further observed that apart from HSA a protein having 43 kDa molecular weight was cosynthesized (Fig. 4). This protein was also copurified along with HSA indicating its physico-chemical similarity to HSA. It was also found to be cross reacting with anti-HSA antibodies suggesting its immunological similarity with HSA- This protein however was not desired protein. Various purification protocols tried were unable to remove this 43 kDa protein. Also purified r-HSA was of green color i.e. pharmaceutically unacceptable. Example 13: Purification of r-HSA The first filtrate was obtained by remova' of biomass and activated charcoal from fermentation broth by any of the known method described in prior art (i.e. centrifugation, cross flow microfiltration with ceramic cartridge etc.). About 10 L of the first filtrate was he^ed to 65 degree C in the presence of protease inhibitor for 90 minutes. Next, the heated solution was quickly cooled to about 8 degree C and diluted and reconditioned to get pH & conductivity of filtrate similar to that of start buffer (second filtrate). The second filtrate was allowed to contact with a cationic exchanger (CM Sepharose FF, Amersham Pharmacia, Sweden) at a pH of 4.0 - ^.6 and a conductivity of about 4 -7 ms/cm at 25°C, and then exposing the cationic exchanger to a pH of 5.0 to 5.5 and a salt concentration of 0.01 to 0.25 M to yield a first elute. It was further reconditioned by suitable buffer to desired pH and conductivity. The reconditioned first elute was allowed to contact with an anionic exchanger (DEAE Sepharose FF, Amersham Pharmacia, Sweden) at a pH of 5.0 to 6.0 and a conductivity of 2 to 5 ms/cm at 25°C and then eluting absorbed fraction of albumin with sodium acetate buffer of pH 4.5 to 4.9 .(second elute). Second elute was further concentrated using Amicon stirred cell provided with an ultrafiltration membrane having a molecular weight exclusive limit of 10,000 to 30,000 Dalton to yield concentrated r-HSA sample, having protein contents of about 50 - 100 mg/ml. Resulting concentrated sample from above step was allowed to pass through gel filtration chromatography (Sephacryl HR-200) to obtain highly purified monomeric form of recombinant r-HSA (Third elute). Pure recombinant human serum albumin was again concentrated by using Amicon stirred cell provided with an ultrafiltration membrane having molecular weight cutoff of 10,000 to 30,000 Dalton and subjected to buffer replacement (Purified bulk). Sample removed from the concentrated purified bulk containing homogeneous r-HSA taken for analysis by SDS-PAGE, SEC-HPLC. Necessary stabilizers such as sodium caprylate and acetyltryptophan are added and then sterilized by 0.22u filterartion. The resulting r-HSA solution can be used for injection. TABLE: 9 Step recovery of r-HSA following various purification stages Step % Step recovery % Total recovery First filtrate 100.0 Second filtrate 66.89 66.89 First elute 88.15 58.97 Second elute 85.13 50.20 Concentration 97.75 49.07 Third elute 96.98 47.59 Purified bulk 98.41 46.83 Final purified bulk r-HSA protein by SDS-PAGE = 99.5 % pure. Final purified bulk r-HSA protein by SEC-HPLC = 99.15 % pure. We claim: 1. A process for preparation and purification of a recombinant human serum albumin and a novel clone thereof, which comprises of: Culturing of recombinant Pichia pastoris in complex / defined medium to build up high cell mass. Culturing of recombinant Pichia pastoris in complex / defined medium to produce human serum albumin protein. Culturing of recombinant Pichia pastoris in charcoal containing complex media to produce human serum albumin protein of therapeutically acceptable quality and color. Culturing of recombinant Pichia pastoris in a two step fermentation process. Purification of recombinant human serum albumin (r-HSA) from fermentation broth of above mentioned recombinant Pichia pastoris. Formulation of purified r-HSA with various pharmaceutically accepted ingredients. Use of the r-HSA for various medical purposes. 2. A process as claimed in claim 1 wherein culture medium for culturing recombinant Pichia pastoris could .hp/selected from complex media like BGY, BY, BMY, YPD, preferably BGY. 3. A process as claimed in claim 2 wherein culture medium for culturing recombinant Pichia pastoris could also be defined salt media. 4. A process as claimed in claim 1 to 3 wherein culture medium contains phosphate buffer to regulate said pH range of 6.2 to 6.8. 5. A process as claimed in claim 1 to 3 wherein culture medium contains at least one carbon source, at least one nitrogen source, minerals and vitamins. 6. A process as claimed in claim 1 to 5 wherein said carbon source comprises one or more of the group selected from methanol, glycerol, sorbitol, glucose and fructose, preferably glycerol and methanol. 7. A process as claimed in claim 1 to 6 wherein said nitrogen source is selected from the group consisting of ammonium salts, nitrates, casein, meat extracts, protein hydrolysates, urea, ammonium hydroxide, one or more peptone, yeast extract, corn steep liquor and amino acids preferably yeast extract and peptone. 8. A process as claimed in claims 1 to 7 wherein said minerals are selected from iron, copper, zinc, magnesium, manganese, calcium and trace elements. 9. A process as claimed in claims 1 to 8 wherein said vitamins are selected from one or more thiamine, biotin and pantothenic acid. 10. A process as claimed in claims 1 to 9 wherein said trace elements are selected from cobalt, molybdanum, iron, copper, magnesium and zinc. 11. A process as claimed in claims 1 to 10 wherein said carbon source is added in an incremental rate and said nitrogen source is added in a fractionated rate to result in said high biomass, where the carbon source can be methanol, glycerol,, sorbitol, glucose and fructose, preferably methanol and glycerol and the nitrogen source may be any one of ammonium salts, nitrates, casein, meat extracts, protein hydrolysates, urea, ammonium hydroxide, one or more peptone, yeast extract, corn steep liquor and amino acids, preferably yeast extract and peptone. 12. A process as claimed in any preceding claim wherein said fermentation is carried out on for (a) Time: 66 to 72 hours for complex media and 90-110 hours for defined salt medium. (b) pH : 4.5 to 6.5 for complex media and 3.5 to 4.5 for defined salt medium 13. A process as claimed in any preceding claim wherein said purification comprises: (a) Separation of cells from the fermented broth to obtain the supernatant (b) heating the supernatant from said fermentation broth,diluting it and subjecting to filteration to get crude HSA. (c) The crude HSA is subjected to a cation exchange column and eluting the absorbed HSA from said column. (d) applying the eluate from step (c) on to an anion exchange column and eluting the absorbed HSA. (e) Concentrating the eluate from step (d) by ultrafiltration. (f) Passing the ultrafiltered product of step (e) through gel filteration. (g) The purification of crude HSA can also be achieved by any combination of processes mentioned in steps (c) to (f). (h) Concentrating the eluate resulting from step (f) to produce pure HSA . 14. A process as claimed in any preceding claim wherein the source of said gene is human liver cells. 15. A process as claimed in any preceding claims wherein said human serum albumin gene comprises of the following sequence: Nucleotide sequence of r-HSA gene: gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60 gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120 aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtcagctgaa 180 aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240 cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300 tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360 gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat . 420 gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480 tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540 aagctcgatg aacttcggga tgaagggaag gcttcgtctg ccaaacagag actcaagtgt 600 gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660 cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720 gtccacacgg aatgctgcca tggagatctg cttgaatgtg ctgatgacag ggcggacctt 780 gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840 aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct . 900 gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960 gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020 tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080 tgtgccgctg cagatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140 gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200 tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260 ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gcagcaaatg ttgtaaacat 1320 cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380 tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440 ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg atgaaacata cgttcccaaa 1500 gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560 agacaaatca agaaacaaac tgcacttgtt gagcttgtga aacacaagcc caaggcaaca 1620 aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680 gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740 gctgccttag gcttataa 1758 16. A process as claimed in any preceding claim wherein said gene is isolated by first isolating total RNA from a human liver biopsy samples, synthesizing first strand c-DNA from m-RNA of the total RNA by reverce transription, synthesizing gene specific second strand of DNA by polymerase chain reaction in the presence of primers having Seq ID # 3 and Seq.ID # 4,1 cloning the PCR product so obtained in to a vector and isolating said gene. 17. A process as claimed in claim 16 wherein said isolated gene is reamplified in the presence of primers having Seq ID # 5 and Seq.ID # 6. 18. A process as claimed in claim 16 or 17 wherein said PCR product is cloned in to a vector downstream to AOX promoter and alpha mat signal sequence. 19. A pharmaceutical composition comprising of Human serum albumin of the present invention and a pharmaceutically acceptable carrier, diluent or excipient. 20. A human albumin gene comprising of the following sequence: Nucleotide sequence of r-HSA gene: gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60 gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120 aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtcagctgaa 180 aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240 cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300 tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360 gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420 gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480 tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540 aagctcgatg aacttcggga tgaagggaag gcttcgtctg ccaaacagag actcaagtgt 600 gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660 cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720 gtccacacgg aatgctgcca tggagatctg cttgaatgtg ctgatgacag ggcggacctt 780 gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840 aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900 gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960 gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020 tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080 tgtgccgctg cagatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140 gtggaagagc ctcagaattt aatcaa'acaa aattgtgagc tttttgagca gcttggagag 1200 tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260 ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gcagcaaatg ttgtaaacat 1320 cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380 tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440 ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg atgaaacata cgttcccaaa 1500 gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560 agacaaatca agaaacaaac tgcacttgtt gagcttgtga aacacaagcc caaggcaaca 1620 aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680 gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa i 1740 gctgccttag gcttataa 1758 Dated this the 14th day of November 2002 (H.SURAMANIAM) OF SUBRAMXNIAM, NATARAJ & ASSOCIATES ATTORNEY FOR THE APPLICANTS |
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993-mum-2002-cancelled pages(10-12-2007).pdf
993-mum-2002-claims(15-11-2002).doc
993-mum-2002-claims(15-11-2002).pdf
993-mum-2002-correspondence(12-12-2007).pdf
993-mum-2002-correspondence(ipo)-(30-05-2007).pdf
993-mum-2002-drawing(15-11-2002).pdf
993-mum-2002-form 1(15-11-2002).pdf
993-mum-2002-form 18(15-11-2002).pdf
993-mum-2002-form 2(granted)-(15-11-2002).doc
993-mum-2002-form 2(granted)-(15-11-2002).pdf
993-mum-2002-form 3(15-11-2002).pdf
993-mum-2002-power of authority(01-10-2002).pdf
| Patent Number | 213698 | |||||||||||||||||||||
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| Indian Patent Application Number | 993/MUM/2002 | |||||||||||||||||||||
| PG Journal Number | 12/2008 | |||||||||||||||||||||
| Publication Date | 21-Mar-2008 | |||||||||||||||||||||
| Grant Date | 10-Jan-2008 | |||||||||||||||||||||
| Date of Filing | 15-Nov-2002 | |||||||||||||||||||||
| Name of Patentee | CADILA HEALTHCARE LIMITED | |||||||||||||||||||||
| Applicant Address | ZYDUS TOWER, SATELLITE CROSS ROADS, AHMEDABAD 380 015, GUJARAT, INDIA. | |||||||||||||||||||||
Inventors:
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| PCT International Classification Number | A61K 38/00 | |||||||||||||||||||||
| PCT International Application Number | N/A | |||||||||||||||||||||
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