|Title of Invention||
METHOD FOR THE PRODUCTION AND PURIFICATION OF POLYSIALIC ACID
|Abstract||The present invention provides a method for the production and purification of PSA with high molecular weight from 70 KD to 100 KD comprising (1) fermenting an Escherichia coli K1 LP 1674 strain capable of produsing Polysialic acid using an appropriate carbon source, in order to maintains microorganism growth effective to support Polysialic acid production; and (2) purifying the Polysialic acid from the culture by single chromatographic step of anion exchange (DEAE) coupled with diafilteration and concentration removing portion, lipid and DNA impurities to give highly purified Polysialic acid.|
|Full Text||FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
THE PATENTS RULES, 2003
[See section 10, Rule 13]
METHOD FOR THE PRODUCTION AND PURIFICATION OF POLYSIALIC ACID;
SERUM INSTITUTE OF INDIA LIMITED, A COMPANY INCORPORATED UNDER THE COMPANIES ACT, 1956, WHOSE ADDRESS IS "SAROSH BHAVAN", 16-B/1, DR. AMBEDKAR ROAD, PUNE - 411 001, MAHARASHTRA, INDIA
THE FOLLOWING SPECIFICATION
DESCRIBES THE NATURE OF THIS INVENTION.
FIELD OF THE INVENTION
The present invention relates to a production and purification of the Polysialic acid. More particularly the present invention relates to a production of polysialic acid by a microbial fermentation followed by the purification step to yield high concentration of purified Polysialic acid.
BACKGROUND OF THE INVENTION
Polysialic acid (hereinafter referred as PSA in forthcoming specification). Polysialic acid is also called as colominic acid. Barry et al. discovered a colominic acid in 1957, which made the configuration unit the sialic acid (N-acetyl neuraminic acid) macromolecule homopolymer. Since this colominic acid has many physiological functions, it is important as a raw material for drugs and cosmetics. Sialic acid exists in many animal and plant bodies as well as in several microbial cell surfaces. Sialic acid and its derivatives have been shown to be involved in the regulation of many physiological processes and play important biological roles like inter cellular recognition, immunological response and metastasis of cancer cells.
Polysialic acid, a polymer of sialic acid, is a natural constituent of the human body and also of certain bacteria from which it can be easily produced in large quantities and with pre-determined physical characteristics. Being chemically identical to polysialic acid in the human body, bacterial polysialic acid, by virtue of this structural mimicry, is completely non-immunogenic, even when coupled to proteins. Unlike, other polymers (eg. PEG), polysialic acid is biodegradable. This
is particularly important where a polymer is used for therapeutics given chronically or in large dosages.
Conventionally the production of the Polysialic acid carried out by microorganism by fermentation method. JP, 1-144989A discloses the production of colominic acid by E.coli and the downstream processing involves the ion exchange resin wherein the proper elution of the colominic acid require repeated process of elution with formic acid - soda which takes maximum time to elute the desire end product. The major drawback of this procedure is low yield of colominic acid i.e 40%-50%.
Further, the JP patent JP 06245786A2 discloses the use of liquid raw material wherein the E.coli was used to ferment said raw material in order to yield the colominic acid and further purified by affinity column chromatography by using lectin as a stationary phase. The colominic acid was recoverable, and yield was 83% and showed the result of having excelled to the yield by the conventional method being 40 - 50%. Moreover, the purity of the obtained colominic acid was 95%. However, the drawback of the above method was the presence of traced impurities in the final end product and the molecular weight of the colominic acid was also 36 Kd.
Generally, the conventional culture conditions for the production of colominic acid as disclosed in the Patents JP.47-26319B, JP1-144989A by using E.coli are the culture temperature of 37 degrees C, and pH 7-8, and the following media constituents are used as a culture medium.
NaHP04 0.6 % KH2P04 0.3 % NaCI 0.5 % NH4CI 0.1 % MgS04 1mM vitamin B1 Source of 4mgC In a sorbitol, a glucose, etc. However the lacuna behind using the above-mentioned media for the fed batch production of colominic acid was the low yield of the end product, which is about 40 mg/L and the purity of the final product was also less.
The JP patent JP-05084091 discloses the use of L-malic acid as a source of C and the ammonium sulfate as a source of N, in order to increase the production of the colominic acid. However the said patent does not disclosed about the purification of the fermentation product.
The JP patent JP-08070882 discloses the method of production of highly purified sialic acid. However the method yield only 98% of the purity and it has not further proceeded for the polysialic acid or colominic acid production or purification on large scale. Since sialic acid is a raw material which is used for several other application.
In order to meet the increasing demand for the commercial sialic acid, economical large - scale production is necessary. For the industrial scale preparation of sialic acid sources such as swallows nests, whey from cheese production, chalaze, egg membranes and the products of microbial fermentation processes have been explored. E.coli is the major and cost-effective and eco-friendly source for the large-scale production of polysialic acid on an industrial scale.
The present invention mainly overcomes the drawbacks as mentioned in the above prior arts in terms of the procedure followed for the production and the
purification of the final product i.e colominic acid / polysialic acid. However the present invention specifically uses two-anion exchange method specifically to generate colominic acid, which is a mono dispersed polymer, and not a monomer i.e sialic acid as disclosed in JP-08070882.
DESCRIPTION OF THE INVENTION
The object of the present invention is to provide an eco-friendly and cost effective method for the higher production of the purified PSA / colominic acid.
Another object of the present invention is fed batch fermentation for the production of Polysialic acid by using E.coli LP 1674.
Another object of the present invention is to devise a downstream processing in order to achieve the highly purified polydispersed polysialic acid with high molecular size.
Yet another object of the present invention is to achieve the purified polysialic acid with high molecular size ranging from 70 KD to 100 KD MW.
Another object of the present invention is to achieve the purity of the Polysialic acid, to comply with the predetermined specifications, and quality attributes like test for purity, identity, protein content, lipid content, DNA content, as the PSA produced can be used directly in human beings especially in conjugation with several therapeutic drugs.
Strain of Escherichia coli K1 LP1674 producing polysialic acid was isolated from a female patient with urinary tract infection in the early 1970s. LP1674 has shown no evidence of O-acetylation of polysialic acid hence LP 1674 was preferred over
the other strains for Polysialic Acid manufacturing. One of the reasons for selection was also that they grow well in standard nutrient media and there is no need for any further special requirements. The low protein impurity content during the downstream processing in purification step is the distinct feature and characteristic of production method of PSA with E. coli strain LP1674 coupled with fermentation strategy of DO based substrate feeding in sorbitol-casamino acid media.
A fed batch fermentation method is preferred for the production of the Polysialic acid from E.coli LP -1674. The optimization of the media component was carried out with various shake flask and fermentor level to observe the effect on the biomass and productivity to finalize media components with carbon source for higher PSA productivity in terms of polysialic acid yield and biomass at fermentation stage.
Biomass was analyzed by spectroscopic method by measuring absorbance at 600 nm. After fermentation, cells were separated by centrifugation approximately at 4000 RPM for 10 min and supernatant obtained. Diafiltration using 10 KD centrifugal filters was done in order to remove media ingredients that might interfere in sialic acid estimation. Resorcinol method was used for estimation of sialic acid content.
For the standardization of the carbon source different Glucose, glycerol, sorbitol and succinic acid were studied in synthetic salt media as well as terrific broth complex media at shake flask level. Various concentration of the media ingredient was tried for the maximum production of the PSA.
After the standardization results on shake flask level it has been seen that the terrific broth basal media is better than synthetic salt media in terms of biomass accumulation. Among the carbon sources, sorbitol gives better PSA yield irrespective of media. Sorbitol with casamino acid and yeast extract media gives highest PSA yield in terms of quantity (3.1 g/L) as well as productivity per biomass in shake flask experiments. It is better than sorbitol in terrific broth media with respect to yield and productivity.
Similarly the standardization protocol for the Fermentor scale devised in order to verify the shake flask results for Sorbitol and succinic acid in synthetic salt media, glucose in terrific broth media and sorbitol in casmino acid/yeast extract media were tested at fermentor scale. Fed batch mode fermentation was followed, as it gives better results in terms of biomass formation.
The fermentation studies revealed that the sorbitol with casamino acid/yeast extract supplementation is better than all other media in terms of PSA total yield and productivity per biomass. This is similar to shake flask experiment results, conforming the method of fermentation. For glucose in terrific broth media, although the biomass is higher, productivity per biomass is relatively lower. It has been seen that the sorbitol with casamino acid and yeast extract supplementation in DO based substrate fed batch mode is found to be optimal for the PSA manufacturing process with yield of 3.1 g/L.
Further, the harvesting procedure is employed and further purification of the said
biomass in Order to remove the small molecular weight impurities, media
. ingredients and interfering substances from PSA sample, which further undergo
purification procedure with anion exchange chromatography. This partial purification step also reduces the volume of PSA sample to be processed by cross flow ultrafiltration achieving its concentration.
As the main object of this invention to fractionate DNA, protein and polysialic acid in order to obtain the purified high molecular weight PSA; an anion exchange using DEAE of ultrafiltration retentate is done in order to remove DNA and any protein/lipid contamination. This strategy is based on the exploitation of difference in pi values and charge intensity differences between DNA/protein and PSA molecules at pH 7.5.
Therefore the downstream processing involves single chromatographic step of anion exchange (DEAE) coupled with diafiltration and concentration steps. It is able to remove protein, DNA and lipid impurities to give high quality polysialic acid in the high molecular size range of 70-100 KD.
Dated this 13th day of July, 2006.
|Indian Patent Application Number||1110/MUM/2006|
|PG Journal Number||14/2013|
|Date of Filing||13-Jul-2006|
|Name of Patentee||SERUM INSTITUTE OF INDIA LIMITED|
|Applicant Address||"SAROSH BHAVAN",16-B/1,DR.AMBEDKAR ROAD,PUNE-411 001,|
|PCT International Classification Number||C07K9/00|
|PCT International Application Number||N/A|
|PCT International Filing date|