Patent Application: US-39050709-A

Abstract:
the invention is a large - scale production and purification of beta - poly , biodegradable natural polyester of l - malic acid of molecular weight 30 , 000 to 300 , 000 from plasmodia of the physarum family , especially physarum polycephalum . this will replace previous laborious and uncontrolled production by bioreactor methods of high productivity and quality , especially to obtain pure polymer of high molecular weight . the described 20 - liter bioreactor method can be scaled up for industrial purpose at optimal production and minimum growth time to prevent degradation the invention includes the method of isolation of polymalic acid of 100 , 000 mn from the culture broth optimized towards high yields of extremely pure polymalic acid . the non - hygroscopic , water and organic solvent - soluble polymer is endotoxin and agglutinin free and ready to use in chemical syntheses . because of its high molecular weight and purity it offers a wide spectrum of applications in pharmacy and medicine .

Description:
the following description is provided to enable any person skilled in the art to make and use the invention and sets forth the best modes contemplated by the inventor of carrying out his invention . various modifications , however , will remain readily apparent to those skilled in the art , since the general principles of the present invention have been defined herein specifically to provide an improved method to produce and isolate polymalic acid . the following example of pmla production is given for a 20 - liter biostat ® c bioreactor from braun biotech international . the solution without d - glucose and hemin is brought to ph 4 . 5 with 5 m naoh and sterilized at 120 ° c . the remaining ingredients at a 10 fold higher concentration are sterilized separately at 120 ° c . and combined with the first solution . six 1 liter aliquots of culture medium ( each contained in a 5 liter erlenmeyer flask ) are each inoculated with 40 ml of settled bed plasmodia of any yellow strain physarum polycephalum ( e . g . strain m3cvii ( atcc204388 ) and then grown for 40 hours at 25 ° c . in the dark . the cells are allowed to settle . the supernatant of settled cells must be clear . otherwise , the flask contents are discarded . the settled cells are washed 3 × with sterile salt solution containing 0 . 2 % ( w / v ) kh 2 po 4 , 0 . 35 % ( w / v ) citric acid , 0 . 1 % ( w / v ) cacl 2 , 0 . 085 g / liter mncl 2 - tetrahydrate , 0 . 085 g / liter feso 4 - hepthydrate , and 0 . 035 g / liter znso 4 - heptahydrate . the solution was adjusted to ph 4 . 5 with 5 m naoh , and 18 liters of the solution were sterilized in the bioreactor at 121 ° c . for 30 min at 1 bar . a sterile suspension of 45 % ( w / v ) caco 3 and 10 - fold concentration of a sterile solution of hemin were pumped into the bioreactor to give final concentrations of 3 . 5 % ( w / v ) caco 3 and 0 . 0025 g / liter hemin . because of the caco 3 , the ph increased to ph 5 . 5 . pumping was continued , and 750 ml settled bed microplasmodia of the inoculation culture were added to the biorector under sterile conditions with the help of a type mcp ismatec ® ism 404 peristaltic pump with tubing of 6 . 4 mm diameter set at 69 - 80 rpm that corresponds to a flow rate of 300 ml / min . the culturing of plasmodia was carried out for 75 h at 25 ° c . with a 10 liter / min air flow and 150 rpm constant stirring by a 3 - leaf segment stirrer c20 - 2 ( 3 etages ) biostat ® by braun biotech international . a minimum amount of sigma antifoam a ( a - 5758 ) at a dilution of 1 : 30 ( distilled water ) was added as needed . the fermentation was terminated when the ph showed a plateau at ph 4 . 78 - 4 . 80 signifying the end of pmla - production . pmla content was then determined by the hydroxamate / fe ( iii )- color assay which indicated an a 540 reading of 1 . 0 for a 160 μl sample . at the end of the fermentation , the culture broth in the bioreactor was immediately cooled to 10 ° c . and adjusted to ph 7 . 5 by the addition of 2 m naoh . the invention combines bioreactor technology with microbilogical techniques with the aim of polymalic acid production from physarum polycephalum strain m3cvii ( atcc 204388 ); an increase in capacity and ready to scale up ; sustainable instrumentation ; readily scalable methodology for carrying out pmla production in stainless steel bioreactors ; optimization of production reproducibility and polymalate quality by control of fermentation conditions ; increased productivity for polymalic acid by supplying simultaneously d - glucose and caco 3 ; protection of high molecular weight polymalic acid by minimizing spontaneous hydrolytic chain scission through shortening the time of fermentation , lowering the ph of the culture broth to ph 5 . 5 , and by rapid adjustment of the broth to ph 7 . 5 with 2 m naoh with cooling to 10 ° c . during fermentation termination ; low enzymatic cleavage activity of polymalatase in the culture broth due to ph increased to ph 5 . 5 ( away from enzyme optimum ) and reduced polymalate production time from 96 hours to 75hrs ; high yield ( 20 g polymalate / 18 liter bioreactor ); fixed working scheme and reproducible time schedule ; less laborious than previous methods and completion in less time ; controlled culturing in a closed reactor conditions reducing cell death under unfavorable culture conditions and contamination like in open culture vessels culture broth was adjusted to ph 7 . 5 with 2 m naoh while cooled to 10 ° c ., and plasmodia were allowed to settle by gravity . the broth was removed by passing over a cheese cloth or a similar filter device or by low speed centrifugation ( 500 × g ). the clear broth was then diluted 1 : 1 by volume with 0 . 05 m tris - hcl ph 7 . 5 and pumped at a speed of 10 liter per hour in the reverse flow mode ( i . e ., from the bottom up ) through streamline - deae - cellulose ( 1 . 5 liter bed volume per 10 liter of culture broth in a column of a 12 cm diameter ). the adsorbed pmla was washed with 5 column volumes of 20 mm tris - hcl buffer containing 0 . 2 m nacl entering from the bottom of the column until all yellow pigment , sugars , proteins and nucleic acids ( all by standard text book assays ) had disappeared . pmla was then eluted from the top of the column with 2 - 3 column volumes of 0 . 7 m nacl in the same 20 mm tris - hcl buffer ph 7 . 5 . a 5 . 0 m solution of cacl 2 was stirred into the eluted fractions containing pmla to give a final concentration of 0 . 1 m cacl 2 . addition was followed by slow addition of non - expensive , ice - cold technical grade ethanol ( 96 %) to give a final ethanol concentration of 70 - 80 % ( v / v ). precipitation was completed overnight at − 20 ° c . the precipitated pmla , ca - salt , was harvested by centrifugation or filtration and washed with 2 - 3 volumes of 80 % ( v / v ) ice - cold aqueous ethanol . at this stage , the material could be stored for several months at − 20 ° c . without measurable loss of quality . the precipitate was dissolved in a minimum of distilled water and processed further in either of the two following ways : ( a ) without further purification , the material was converted to pmla - acid by passage over amberlite 120 h + . the resulting pmla - acid was instantaneously freeze - dried and dissolved into water - free 1 : 1 ( v / v ) mixture of acetone and isobutylmethylketone . insolubles were removed , and pmla - acid was precipitated with diethylether . any up to then resisting pigment remained in the mother liquor . the pure pmla - acid is inhomogenous over a wide range of molecular weights . ( b ) crude fractionation according to molecular weight and removal of traces of pigment as well of residual inorganic salt was achieved by gel permeation chromatography on sephadex - g25 fine grade ( 5 × 100 cm ). fractionation resulted in three equally sized pools of molecular mass 60 - 300 kda , 30 - 50 kda , and 10 - 30 kda . the fractions of each range were combined yielding preparations of polymalic acid : mn 100000 ( major fraction , typically 7 - 10 g ), mn 50000 ( typically 5 - 8 g ), mn 15000 . ( typically 3 - 5 g ). mn denotes number - averaged molecular weight . polydispersity factors were in the range 1 . 2 - 1 . 4 . an example of there preparations is shown in fig3 . if endotoxin removal is desired , fractions were then diluted to 1 mg / ml pmla in phosphate buffer ( 150 ml portions in 150 mm buffer , ph 6 . 8 ) and made 1 % ( v / v ) in triton x - 114 before 5 min vigorous mixing . mixtures were allowed to clear on ice and then incubated for 10 min under shaking at 37 ° c . after 5 min centrifugation at 5000 × g , the upper layer was removed and concentrated by membrane filtration . residual triton was removed by gel filtration over pd - 10 column ( ge healthcare ) prewashed and eluted with lal reagent water ( lonza ). all fractions were then passed over amberlite 120 h + ( 3 × 20 cm ), immediately collected on ice , and polymalic acid instantaneously freeze - dried . method ( b ) is the preferred one , because it achieves substantial fractionation by size size being fast , reproducible , and convenient . the purities of polymalic acid obtained by either methods are similar . in either case , the prepared pmla - acid was stored at − 20 ° c . the overall labor time for the fermentative production and isolation of highly purified pmla - acid from 20 liter culture broth was eight days . the yield of highly purified polymalic acid , mn 100000 , was 30 - 40 % of polymalic acid in the culture broth . the kind of polymalic acid lost during purification was mainly of low molecular weight (& lt ; 20 , 000 ). the preparations were of extreme purity as indicated by optical rotation ( 7 , 10 ), absorbance —( 7 ), infra red —( 7 ), c13 / h1 — nmr - spectra ( 7 , 10 , 11 ), elementary analysis ( 7 ), melting analysis ( 11 ), and the ability of polymalic acid to crystallize [ ref . ( 1 ), and unpublished data ]. according to the results of conventional tests , the preparations of polymalic acid with mn 100 , 000 and with mn 50 , 000 after endotoxin removal by the triton method were devoid of endotoxins (& lt ; 0 . 1 eu / mg by the lal turbidity kinetic method ) and of agglutinins , which do not copurify under the conditions of the purification methods . preparations of polymalic acid and polymalate are white , non - hygroscopic powders after lyophilisation . the acid tends to become crystalline when concentrated from a solution of anhydrous acetone . polymalic acid and polymalate solubilize extremely well in water , the acid strongly acidifying the solution , the dissolved salt remaining neutral . polymalic acid dissolved in water is spontaneously cleaved under acidic and basic conditions . the hydrolysis at neutral ph is almost completed after 24 hrs at room temperature , ultimately forming l - malic acid , while the salt is stable for several days at ph 7 . 0 . solutions should be kept at low temperature & lt ; 0 . 4 ° c . or better frozen at & lt ;(−) 20 ° c . in order to slow down cleavage . the lyophilized polymer salt and acid is substantially more stable and can be stored for any period at − 20 to − 80 ° c . the lyophilized salt ( usually ca or na salt ) of polymalic acid withstands transportation for several days to probably weeks at temperatures below 40 ° c . the pendant carboxyl groups are chemically substituted after activation using carbodiimide derivatives ( 12 ). in these reactions , the main chain ester bonds remain intact if synthesis is carried out in anhydrous organic solvents . the carboxyl substitution may stabilize the main chain ester bond . the invention is to combine several biotechniques to increase the capacity to purify polymatae from the culto broth ready to be scaled up ; to employ sustainable instrumentation ( regenerative and reusable chromatographic materials and columns , reusable setups of chromatographic columns , pumping devices , lyophilizer ); to achieve reproducible quality by following the purification via polymalic acid , not via the salt . various salt forms of polymalate can then be obtained by neutralization of polymalic acid with appropriate metal hydroxide or carbonate ; by choosing ethanol precipitation of the polymer in the form of calcium - polymalate as an improved method ; precipitation being a robust method where technical ethanol is sufficient ; efficient and fast size fractionation using a large sephadex g25 column ; high yields of polymalic acid mn 100 , 000 in the order of 30 - 40 %. achievement of high grade pure polymalic acid mn 100 , 000 . complete removal of contaminants . on demand , removal of endotoxin by the triton method . after isolation from the culture broth , polymalic acid and various salts are obtained with purity and stability superior to chemically synthesized polymalic acid . because of the relative high costs of fermentation / purification , polymalaic acid has not been commercially available . a scale - up of the invented bioreactor production and invented purification protocol should allow industrial production and commercial availablility of polymalic acid and its salts . a variety of applications are possible . pmla is non - toxic , non - immunogenic , and biodegradable . it has a high value for syntheses in chemistry , especially for pharmaceutical chemistry and nanochemistry ( 12 , 13 ). due to its biocompatibility and the high number of reactive carboxylic groups and their straight - forward chemistry , it offers an effective platform for covalent conjugation of a wide range of pharmaceutically active compounds , unmatched by any other polymers known to date having the high level of biocompatibility . polymalic acid is an optimal material to be used in many kinds of applications , due to its solubility in aqueous and organic solvents and its chemical ease for conjugation with interesting compounds especially by condensation reactions involving carbodiimide reagents . in addition , pmla can be used as starting material for the design of micelles , microspheres , biocapsules , biogels , liquid crystals , and coat materials . on a larger scale , a field of application could be the use of ( biodegradable ) adhesives , plastics , dispersion media , packaging for controlled release , or for hydrophilic coating . the following claims are thus to be understood to include what is specifically illustrated and described above , what is conceptually equivalent , what can be obviously substituted and also what essentially incorporates the essential idea of the invention . those skilled in the art will appreciate that various adaptations and modifications of the just - described preferred embodiment can be configured without departing from the scope of the invention . the illustrated embodiment has been set forth only for the purposes of example and that should not be taken as limiting the invention . therefore , it is to be understood that , within the scope of the appended claims , the invention may be practiced other than as specifically described herein . 1 . b .- s . lee , m . vert , and e . holler ( 2002 ) in biopolymers ( doi , y . and steinbüchel , a ., eds .) pp 75 - 103 , new york , weinheim ( bergstrasse ). 2 . e . holler and b .- s . lee ( 2002 ) recent res . devel . anal . chem . 2 , 177 - 192 . 3 . a . schmidt , c . windisch , and e . holler . nuclear accumulation and homeostasis of the unusual polymer b - poly ( l - malate ) in plasmodia of physarum polycephalum . eur . j . biochem . 70 , 373 - 380 . 1996 . 4 . k . rathberger , h . reisner , b . h . willibald , h .- p . molitoris , and e . holler . comparative synthesis and hydrolytic degradation of poly ( l - 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