Title of Invention

"AN IMPROVED PROCESS FOR SEPARATION OF BIOMOLECULES USING DEMIXING TECHNIQUE OF AQUEOUS PHASES GENERATED IN BIOREACTORS"

Abstract The present invention relates to "An improved process for separation of bio molecules using de mixing technique of aqueous phases generated in bioreactors." The process uses "A field assisted process for enhanced separation/ de mixing of aqueous two phase systems." The process relates to a new method to enhance the de mixing of equilibrated dispersions of Aqueous Two Phase Systems (ATPS), generated by adding phase forming macromolecules in combination with polysaccharides and or anionic salts ranging from univalency to tetravalency. The process uses microwave irradiation for de mixing of two aqueous phases, process lies in its efficacy in enhancing the migration velocity of the dispersed phase droplets by decreasing the viscosity of the continuous phase. This uniqueness allows two important improvements: first, a higher rate of phase de mixing; second, elimination of expensive equipment (centrifuge) normally used for phase de mixing.
Full Text The present invention relates to "An improved process for separation of bio molecules using de mixing technique of aqueous phases generated in bioreactors." The present invention particularly relates to "A field assisted process for enhanced separation/ de mixing of aqueous two phase systems." The present invention more particularly relates to a new method to enhance the de mixing of equilibrated dispersions of Aqueous Two Phase Systems (ATPS), generated by adding phase forming macro molecules in combination with polysaccharides and or anionic salts ranging from univalency to tetravalency. More particularly it relates to exposure of the system to microwave irradiation for de mixing of two aqueous phases.
The major application of this invented microwave field assisted de mixing process is in the following aspects of bio processing.
1. In downstream processing of bio molecules (proteins, enzymes etc), especially in the
case of thermostable bio molecules.
2. In the recovery of thermally separated phase forming polymers, where after
extraction, the phase polymer will be separated by increasing the temperature to
certain value and then this separated polymer can be reused
3. A new design of bioreactor for extractive fermentation.
Partitioning in aqueous two-phase systems is an established method for the separation, purification and characterization of biomaterials. Major hindrances for its application in bio industries are; high cost of the phase forming polymers and slow de mixing rates. The former aspect is solved to a great extent by adapting temperature induced phase separation for recovery and recycling of the polymers. While, the latter aspect is not addressed to the same extent. The slow de mixing of the thoroughly mixed

phases is due to small difference in densities between them, high viscosity of the individual phases and low interfac'al tension. Efforts have been directed to speeding up phase separation by various means :.uch as centrifugation, magnetic field assisted demixing and electrokinetic demixing - ;ach having its own drawbacks.
From the economic point of view, centrifugation becomes prohibitively expensively on large scale, and electrokinetic demixing requires fabrication of special equipment and chemical additions, such as salts to the systems and gravity separation alone is a very slow process. The present invention, to perform the same duty, is relatively less expensive when compared to centrifugation and less cumbersome when compared to electrokinetic/magnetic demixing.
Reference may be made to Albertsson, P.A. "Partitioning of cell particles and macromolecules" 3rd Ed., Wiley. New York, 1986. Wherein, a detailed account on fundamental aspects and various applications of ATPS have been dealt. Kinetics of phase demixing under gravity has been highlighted. The drawback of this process is the slow rate of phase demixing. Time range for phase demixing in polymer/polymer systems is 5 min to 12 hours and in polymer/salt systems is 5 to 15 minutes.
Reference may be made to Raghavarao, K.S.M.S., Rastogi, N. K., Karanth, N. G., Gowthaman, M. K. Adv. Appl. Microbiol. 1995, 41, 97-171. Wherein, fundamentals, applications, recent developments and mathematical modeling of ATPS has been explained exhaustively. A brief note on phase demixing aspects in ATPS is included. Slow rate of phase demixing, one of the drawbacks in ATPS, is explained based on the physical properties such density difference between the phases, viscosity of the individual phases and interfacial tension of ATPS. Time required for phase demixing in

polymer/polymer systems ranges from 5 minutes to 12 hours and in polymer/salt systems from 5 to 15 minutes.
Reference may be made to Berggren, K., Voide, A., Nygren, P.-A., Tjemeld, F. Biotechnol. Bioeng. 1999, 62, 135-144. Wherein, one of the drawbacks in ATPS, that is recovery and recycling of phase forming polymers has been addressed by employing temperature sensitive random copolymers of ethylene oxide and propylene oxide. However, demixing kinetics of this temperature induced phase separation has not been addressed in the study.
Reference may be made to Hustedt, A., Kroner, K.H., Papamichaek, N. In: D. Fisher and LA. Sutherland (eds). Separations using aqueous phase systems: Applications in cell biology and biotechnology. Plenum press, New York, 1989. Wherein, application of centrifugation technique has been investigated to enhance the rate of phase demixing in ATPS. The disadvantage of this process is that centrifugation becomes highly expensive at the large scale.
Reference may be made to Vikrostrom P, Flygare S, Grondalen A, Larsson P-0. Anal Biochem. 1987, 167: 331-339. Wherein, researchers have employed magnetic field to enhance the demixing rates in PEG/dextran two phase. The drawbacks of this technique include (1) Addition of micron sized iron particles or ferro-fluids to the system, which may have the chances of getting aggregated in a strong magnetic field, causing the procedure to become inoperative, since the magnetic field would force the particles out of the two-phase system. (2) The technique was not found useful when the PEG phase was dispersed in a PEG/dextran system. (3) Micron-sized particles Ferro-fluids may affect the activity of the enzymes when they are present at low concentrations owing to poorly

coated iron oxide particles, which will segregate into larger entities.
Reference may be made to Raghavarao, K.S.M.S., Stewart, R. M., Todd, P. Sep. Sci. Technol. 1990, 25, 985-991. Wherein, electric field was employed to enhance the phase demixing rates in poly ethyleneglycoi/dexran aqueous two phase systems due to the electrokinetic potential developed across the parse interface owing to unequal distribution of the buffering ions. About 2 to 5 fold increase in demixing rates were observed under the influence of electric field. The major drawback of this process is that it is economically not feasible because the process requires fabrication of special equipment and addition of chemical such as salts to the system. In addition, the process is not applicable when the ATPS is of polymer/salt type.
Reference may be made to Raghavarao, K.S.M.S. Stewart, R. M. Todd, P. Sep. Sci. Technol. 1991, 26, 257-264. Wherein, application of electric field was shown to enhance the phase demixing rates of poly ethyleneglycol/maltodextrin aqueous two-phase system by 2-5 fold. The drawbacks of this study are the same as explained in the above reference.
The main object of the present invention is to provide an improved process for separation of biomolecules using demixing technique of aqueous phases generated in bioreactors which obviates drawbacks as detailed above. Other object of the invention is to achieve seperation using phase forming macromolecules in combination with polysaccharides and/or univalent to tetravalent anionic salts. Still another object is to do demixing. of two aqueous phases by using microwave field irradiation methods to get biomolecules rich phase and recycling the other phase to the system.

Accordingly the present invention provides an improved process for separation of biomolecules using demixing technique of aqueous phases generated in bioreactors, which comprises characterised in that adding phase forming macromolecules of the kind as herein described in the range of 7-35% w/w in combination with polysaccharides in the range of 10-60% w/w and/or univalent to tetravalent anionic salts having valency ranging from 1 to 4 in the range of 10-40% under stirring to a bioreactor till it attains equilibration, subjecting the said mixture to microwave field irradiation for 1-3 minutes at the frequencies of 950 and 2450 MHz, recovering two phases thus formed by known methods to get biomolecules rich phase and recycling the other phase to the system, biomolecules being separated by known methods are -extracellular soluble enzymes/proteins.
In an embodiment of the present invention the phase forming macromolecules used may be polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), polypropylene glycol (PPG) polyvinyl alcohol etc., and polysaccharides used may be maltodextrin (MDX), dextran (DX), hydroxypropyl starch, ficoll, methyl cellulose, hydroxypropyl dextran etc.
In an another embodiment of the present invention the molecular weights of phase forming macromolecules such as PEG, PVP, PPG etc., used may be in the range of 1400 to 20,000 Da and molecular weights of polysaccharides such as MDX, DX, starch etc., used may be in the range of 40,000 to 10,00000 Da.
In an another embodiment of the present invention the phase forming macromolecules used may be polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), polypropylene glycol (PPG) polyvinyl alcohol etc., and anionic salts used may be NaOH, Na2CO3, (NH4)2SO4, FeSO4, K2HPO4, Na3PO4, Na2SiO4, Na4.hydroxyethane diphosphonic acid(HEDPA) etc., having valency ranging from 1 to 4.
In yet another embodiment the biomolecules separated may be such as

extracellular soluble enzymes/proteins, and/or biological products.
The present invention enables to enhance the demixing rate by 1.5-2 fold over the demixing rate in gravity demixing alone. Application of microwave field energy increased the temperature of the system, thereby decreasing the viscosity of the continuous phase. This lower viscosity of the continuous phase offers lesser resistance to migration velocity of the dispersed phase droplets (since it is evident from the Stoke's law that migration velocity of the droplets is inversely proportional to viscosity of the continuous phase), leading to enhanced demixing rates of the phases.
The novelty of the present process lies in its efficacy in enhancing the migration velocity of the dispersed phase droplets by decreasing the viscosity of the continuous phase. This uniqueness allows two important improvements: first, a higher rate of phase demixing; second, elimination of expensive equipment (centrifuge) normally used for phase demixing. The present microwave field assisted demixing process has some similarities to the process of electrokinetic demixing of ATPSs. The electrokinetic process exploits the electrokinetic potential developed across the phase interface due to the unequal distribution of certain buffering ions. However, in contrast to this prior art the process of the present invention is independent of buffer ions and far easier to scale-up and to adopt as continuous process.
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the invention.
EXAMPLE -1
In an experiment, 250g of PEG (MW 6000) / MDX aqueous two-phase system was prepared with a composition of 10 % w/w PEG and 30 % w/w MDX in distilled

water so as to make total weight as 100 % w/w. The system was mixed well with a homogenizer, allowed to separate into two phases in a scparaiing funnel for overnight and the separated phases were collected. The separated and equilibrated phases were again mixed with different volume ratios viz. 70/30, 50/50 and 30/70. To the 100ml of dispersion at each volume ratio 1% w/v of Eschzrichia coli cells (obtained by centrifuging the fermentation broth) was added, mixed for 10 minutes and allowed to demix in the presence of a microwave field (inside a microwave oven) for two minutes and then taken out and allowed to separate under gravity for five minutes. In this way the system was subjected to repeated cycles of exposure to microwave field for two minutes and non-exposure for five minutes until complete demixing was achieved. For all the volume ratios, demixing time under gravity alone was also noted down. It was found that under gravity separation alone, 30/70, 50/50 and 70/30 volume ratios took 165, 50 and 70 minutes, respectively to demix to clarity. When the same volume ratios were again subjected to repeated cycles of exposure to microwave field for 2 minutes and non-exposure for 5 minutes, 30/70 volume ratio took 110 minutes, 50/50 took 36 minutes and 70/30 took 45 nlinutes only to demix to clarity. Thus microwave field assisted demixing process resulted in an about 1.5 fold decrease in the demixing time when compared to gravity demixing alone.
EXAMPLE-2
In another experiment, 250 g of PEG (MW 6000)/potassium phosphate aqueous two-phase system was prepared with a composition of 10 % w/w PEG and 11 % w/w potassium phosphate in distilled water so as to make total weight as 100 % w/w. Top and bottom phases were separately collected. The system was mixed well with a

hoi/.r-getiizcr, allowed to separate into two phases in a separating funnel for overnight and the separated phases were collected. As explained in example 1 demixing experiments were carried out at three volume ratios viz. 70/30. 50/50 and 30/70, in presence of 1% w/v Escherichia coll cells (obtained by ceatrifuging the fermentation broth). The 100ml of the dispersion at each volume ratios were subjected to microwave field for oniy one minute. The dispersions took only about 5.5 minutes to demix to clarity when subjected to microwave field for 1 minute. Where as, the dispersions took around 10 minutes to demix to clarity when subjected to gravity demixing alone. Thus microwave field assisted demixing process resulted in an about 1.8 fold decrease in the demixing time when compared to gravity demixing alone.
Other phase forming macromolecules such as poly vinylpyrrolidone (PVP), poly propylene glycol, polyvinyl alcohol etc., can also be used for top phase formation in the above examples 1 and 2. Similarly, other polysaccharides such as dextran, starch, hydroxypropyl starch and anionic salts such as sodium sulphate, ammonium sulphate, sodium phosphate etc., can be used for bottom phase formation in the above examples 1 and 2.
The main advantages of the present invention are:
The present method provides an enhanced demixing rate over that of gravity method. The present method is simple in comparison with magnetic field and electric field assisted demixing processes and can be carried out without the use of expensive equipment such as centrifuge.
The process is easy to scale up and to adopt for continuous processes.
The process can be adopted for recover}' of thermally separating polymers for recycling
purposes.




We claim:
1. An improved process for separation of bio molecules using de mixing technique of
aqueous phases generated in bioreactors, which comprises characterised in that
adding phase forming macromolecules of the kind as herein described in the range of
7-35% w/w in combination with polysaccharides in the range of 10-60% w/w and/or
univalent to tetravalent anionic salts having valency ranging from 1 to 4 in the range
of 10-40% under stirring to a bioreactor till it attains equilibration, subjecting the said
mixture to microwave field irradiation for 1-3 minutes at the frequencies of 950 and
2450 MHz, recovering two phases thus formed by known methods to get
biomolecules rich phase and recycling the other phase to the system, biomolecules
being separated by known methods are -extracellular soluble enzymes/proteins.
2. An improved process as claimed in claim 1 wherein the phase forming
macromolecules used are polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP),
polypropylene glycol (PPG) polyvinyl alcohol, and polysaccharides used are
maltodextrin (MDX), dextran (DX), hydroxypropyl starch, ficoll, methyl cellulose,
hydroxypropyl dextran.
3. An improved process as claimed in claims 1 and 2 wherein the molecular weights of
phase forming macromolecules such as PEG, PVP, PPG , used are in the range of
1400 to 20,000 Da and molecular weights of polysaccharides such as MDX, DX,
starch, used are in the range of 40,000 to 10,00000 Da.
4. An improved process for the separation of biomolecules using demixing technique of aqueous phases generated in bioreactors substantially as herein described with reference to the examples accompanying this specification.




Documents:

263-del-2000-abstract.pdf

263-del-2000-claims.pdf

263-del-2000-correspondence-others.pdf

263-del-2000-correspondence-po.pdf

263-del-2000-description (complete).pdf

263-del-2000-form-1.pdf

263-del-2000-form-19.pdf

263-del-2000-form-2.pdf


Patent Number 217839
Indian Patent Application Number 263/DEL/2000
PG Journal Number 17/2008
Publication Date 25-Apr-2008
Grant Date 29-Mar-2008
Date of Filing 16-Mar-2000
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110 001, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 KSMS RAGHAVARAO CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE-570 013
2 N.D. SRINIVAS CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE-570 013
PCT International Classification Number B01D 57/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA