Patent Application: US-56934205-A

Abstract:
significant research is being done to develop and improve delivery mechanisms for biopharmaceuticals and vaccines , including pulmonary , nasal , transdermal , and oral alternatives . market projections indicate that the delivery of proteins and vaccines by inhalation and oral formulation has become and will continue to be increasingly important . these delivery mechanisms , to be effective , will require better stabilization of the biologicals so that they can maintain potency and effectiveness at ambient temperatures for extended periods of time . the novel preservation by vaporization technology described herein provides cost - effective and efficient industrial scale stabilization of proteins , viruses , bacteria , and other sensitive biologicals , thereby allowing a production of products that are not possible to be produced by existing methods . the suggested new pbv process comprises primary drying under vacuum from a partially frozen state at near subzero temperatures followed by stability drying at elevated temperatures . the new suggested method can be performed aseptically in unit doze format and / or in bulk format . the drying can be performed as a continuous load process in a manifold vacuum dryer comprising a plurality of vacuum chambers attached to a condenser during the drying .

Description:
the novel methods and equipment of the present invention allow for extended storage and transportation of bioactive materials at ambient temperatures . it has been commonly known to avoid or minimize freezing of biologicals because freezing is considered by many to be a damaging process . however , freezing at near − 0 ° c . temperatures may be not damaging ( or at least is less damaging compared to freezing at or to − 20 ° c . or below ) because the ph change associated with crystallization hydrolysis is proportional to the surface of ice crystals divided by the volume of liquid phase remaining between the ice crystals . this ratio will be small during freezing near 0 ° c . at the same time , vaporization of water from a partially frozen material at temperatures close to ice melting point ( for example at − 5 ° c . or above ) can be very efficient if performed under vacuum , e . g ., below 3 torr , which is the equilibrium pressure of water vapor above ice at − 5 ° c . at such temperatures , which are considerably higher than t g ′, the subject material will be a “ slush ,” a two phase system of ice crystals and a concentrated solution that remains between ice crystals . because the chemical potential of water in the slush is equal to the chemical potential of ice , the equilibrium pressure of water vapor above the liquid portion in the slush is equal to that of ice . if the vacuum pressure is below that of the equilibrium pressure , the liquid in the slush overheats and boils . therefore , subjecting a slush to a vacuum will result in quick vaporization of water from the slush by sublimation from ice crystals , by boiling of the unfrozen solution between ice crystals , and by evaporation from the slush surface simultaneously . preservation by vaporization ( pbv ) is a preservation process that comprises primary drying and stability drying . primary drying is performed by intensive vaporization ( sublimation , boiling and evaporation ) of water at temperatures significantly ( approximately 10 ° c . or more ) higher than t g ′ from a partially frozen and at the same time overheated ( vacuum pressure is below the equilibrium pressure of water vapor ) material . during pbv , the boiling in the course of the primary drying does not produce a lot of splattering because the equilibrium pressure at subzero temperatures above the slush is low and ice crystals on the surface of the slush prevent or inhibit the splattering . typically , a material ( e . g ., frozen solutions or suspensions ), which has been subjected to pbv drying , looks like a foam partly covered with a skim of a thin freeze - dried cake . preventing eruptions ( splattering ) during the boiling step is important for more effective bulk drying . it is particularly important when lyoguard tray or other bags covered by water - permeable membranes are used . if splattering takes place , it negatively effects vapor flow through the membrane because the membrane is covered with drops of the material splattered onto its surface . splattering also negatively affects the appearance of the material after drying in vials . elimination of splattering also obviates the need for a complex and unreliable “ two dimensional ” drying protocol discussed above and simplifies the execution of the drying step . in addition , unlike preservation by foam formation ( pff ), preservation by vaporization ( pbv ) can be very effective for preserving biologicals contained or incorporated within an alginate gel formulation and other gel formulations . a pbv process can be performed by drying frozen gel particles under a vacuum at small negative ( on the celsius scale ) temperatures . for such hydrogel systems , vaporization comprises simultaneous sublimation of ice crystals , boiling of water inside unfrozen microinclusions , and evaporation from the gel surface . pbv is different from freeze - drying because freeze - drying suggests the product processing temperature to be at or below t g ′ ( which , typically , is below − 25 ° c .) during primary drying and because freeze - drying suggests avoiding the “ collapse ” phenomenon during both primary and secondary drying . pbv comprises drying at temperatures substantially higher than t g ′, i . e ., higher than − 15 ° c ., better higher than − 10 ° c ., and yet better higher than − 5 ° c . the pff methods disclosed in u . s . pat . no . 5 , 766 , 520 ( bronshtein ) or in wo 96 / 040077 ( roser and gibbon )) suggest that freezing of the material to be dried should be avoided during the primary drying . stability drying differs from secondary drying , which is part of a freeze - drying process . without secondary drying , freeze - dried material will collapse . on the other hand , at the end of primary pbv drying step the material is mechanically stable ( i . e ., it does not collapse ) at room temperature under vacuum . the stability drying is performed ( 1 ) to further increase the glass transition temperature of the dry material , ( 2 ) to make it mechanically stable at ambient temperatures without vacuum , and ( 3 ) to preserve the potentcy ( and , therefore , efficiency ) of the biological during a long - term storage at ambient temperatures . to increase t g of the material to for example 37 ° c . and to thereby ensure stabilization at this temperature , the stability drying step should be performed at temperatures significantly higher than 37 ° c . over many hours to remove water from inside of already dried material . the process of dehydration of biological specimens at elevated temperatures may be very damaging to the subject biologicals if the temperature used for drying is higher than the applicable protein denaturation temperature . to protect the sample from the damage that can be caused by elevated temperatures , the stability dehydration process ( i . e ., stabililty drying ) may need to be performed in steps . the first step ( either in air or vacuum ) should be performed at a starting temperature to ensure dehydraion without a siginicant loss of a biological &# 39 ; s viability and potency . after such first drying step , the process of dehydration may be continued in subsequent steps by drying at a gradually higher temperature during each subsequent step . each step will allow simultaneous increases in the extent of the achievable dehydration and the temperature used for drying during the following step . for example , in the case of enzyme preservation , it was shown that after drying at a room temperature the drying temperature may be increased to at least 50 degrees celsius without a loss of enzymatic activity . the extent of dehydration obtained after drying at 50 degrees celsius will allow a further increase in the drying temperature without a loss of activity , any given specimen to be preserved is characterized by a maximum temperature it can withstand during the preservation process . however , various protectants and protective co - solutes may provide additional protection to materials during the drying process . pbv process is scalable because evaporative area of the material increases many hundreds of times during formation of a dry mechanically stable specimen . this evaporative area is created because of sublimation of ice crystals and formation of vapor bubbles inside the material . this is true for both drying a hydrogel and for drying a biological solution or suspension . drying of a solution or suspension by a pbv process can be performed effectively in 3 to 5 ml vials ( 0 . 5 ml fill ), 200 ml ( 10 - 30 ml fill ) vials , small cylindrical lyoguard rtm cup containers , and in lyoguard rtm trays ( 250 - 300 ml fill ). at the end of the primary drying by vaporization from the slush state , the material looks like dry foam partially covered with a skim of a freeze - dried cake . at the end of primary drying , the material becomes mechanically stable if stored under a vacuum . high evaporative area of this material allows then to effectively perform a stability drying step under a vacuum by evaporation at elevated temperatures . drying of hydrogels is more effective when the hydrogel particles are small ( about 1 mm , or below ). the reason for that may be that the growth of vapor bubbles nucleated inside gel particles is limited by high viscosity inside the gel . an efficient pbv drying step can be performed when particle size is about or below 0 . 2 mm . thus , it has been shown that primary pbv drying of 1 . 5 kg of an industrial enzyme encapsulated inside alginate gel spherical particles with diameter below 0 . 2 mm can be done within about six ( 6 ) hours . to accomplish that , the gel particles are placed and dried on an open steel tray used in conventional freeze - drying . the pbv process is beneficial as compared to freeze - drying not only because it is faster , but also because it can be efficiently performed at higher vacuum pressures . for example at − 5 ° c . or above the pbv primary drying can be effectively pertormed at several ( 1 to 3 ) torrs in the chamber . vacuum pressure during freeze - drying should be significantly below 0 . 476 torrs , which is the equilibrium pressure above ice at temperatures below − 25 ° celsius . the process is even more efficient if the pressure is below 0 . 1 torr . because of this , the bags used for balk freeze - drying processing should be carachterized by a very high coefficient of permeation for water vapor . an example of such gas - permeable bag is a product called lyoguard . rtm , which has been developed by w . l . gore for bulk lyophilization . . . . the lyoguard rtm lyophilization bag is a heat sealable flexible bag . one of its sides is made of plastic that is not permeable to water vapor . its other side is made of a gore - tex rtm membrane . this membrane is expanded polytetrafluoroethylene ( ptfe ), nominally containing 0 . 2 micron pore size , hydrophobic and not permeable to liquid water , but permeable to water vapor . because the lyoguard bag can pass water vapor while still preventing a liquid product from penetrating the membrane and leaking out , it provides a way to process products that require some sterility . a tray could also be applied to animal health products , probiotics , food , etc . any product for which enclosed container processing may present an advantage can potentially benefit from the use of lyoguard bags in the preservation by vaporization process . such advantages may be derived where sterility , ease of handling , isolation of pathogens ( e . g ., bacteria ) from the manufacturing personnel , or enhanced contamination control are desirable . lyoguard trays may be used in an industrial - scale pbv processing equipment . however , lyoguard rtm trays are characterized by several shortcomings that need to be addressed : ( 1 ) the lyoguard rtm trays are expensive , and ( 2 ) the 0 . 2 micron pores in expanded polytetrafluoroethylene membranes do not ensure an adequate barrier for viruses , toxins and other dangerous chemicals . because pbv ( and pff process ) process can be performed at pressures that are considerably higher than those required for freeze - drying , expensive expanded polytetrafluorethylene membranes are not necessary and can be replaced by membranes made of less expensive materials . at the same time , such less expensive membranes can provide for better barriers to prevent viruses , toxins and other dangerous chemicals from leaving the containers used for drying . for example , bags covered with relatively inexpensive sartorius membranes used for ultrafiltration can be effectively used in industrial - scale pbv drying pursuant to the methods disclosed herein . other medical grade membranes that can be used to replace expanded polytetrafluoroethylene membranes are 10 to 50 micron polypropylene or polyurethane breathable membranes such as the medfilm x medical films manufactured by mylan technologies inc . and various inspire wound dressing films ( e . g ., inspire 1101 ( 10 μm ), 2202 ( 20 μm ), etc .) made by intelicoat technologies . it should be understood that many other membranes can be used to implement the disclosed herein methods and apparatus designs . using the membranes as described above makes the proposed drying method a barrier aseptic process . a similar approach for using breathable membranes has been disclosed for freeze - drying ( see e . g ., u . s . pat . no . 5 , 309 , 649 ). however , that approach has not been used in the industry because permeation of not porous breathable membranes is too slow for the execution of effective freeze - drying . therefore performance of pbv or pff drying in a container ( e . g ., a bag ) covered with a breathable ( e . g ., polypropylene or polyurethane ) membrane without pores presents a novel opportunity for aseptic industrial scale preservation by drying . because such membranes ( which are characterized by a thickness in the range of 20 - 50 micron ) have limited mechanical strength , the design can be reinforced by using a “ sandwich ” that contains a breathable membrane between two low - cost porous membranes ( e . g ., sartorius membranes ) that are characterized by a permeability to water vapor and by a higher mechanical strength . containers ( e . g ., bags ) for drying pursuant to the inventive methods disclosed herein require suitable connectors that allow to : ( 1 ) aseptically introduce a fluid , such as biological solution or / and viral or cellular suspension , into a container without collapsing the container , ( 2 ) aseptically dry the fluid , ( 3 ) aseptically store the dry specimen in the container , and ( 4 ) transfer the finished dry product from the container to other devices for downstream processing ( e . g ., milling , mixing , packaging , transportation , etc .). preferably , the containers used in the disclosed novel methods should be less expensive than the costly lyoguard trays covered with expensive membranes . now , an embodiment of equipment for industrial - scale implementation of the novel methods for preservation of biologicals will be described . it should be understood that the described embodiments are presented herein only for illustration purposes . a person skilled in the art of preservation of biologicals will be able to easily use the same ideas to design other equipment and processes which should be considered within the spirit and scope of the invention disclosed herein . there are several barriers that water vapor has to go through on the way from the specimen to be dried to the condenser . first , the vapor passes the membrane that covers the specimen container ( e . g ., a bag or tray used for bulk drying ) or leaves the vials through holes under the stoppers during the unit doze drying . second , the vapor must travel from the drying chamber to condenser . because freeze - drying is performed at very low pressures , the vapor flow between the drying chambers and the condenser “ chokes ,” thereby limiting the speed of the drying process . for this reason , a typical industrial freeze - dryer that can condense many liters of water from the subject material in a given time is currently built as a one - chamber apparatus in which the diameter of the connector between the chamber and the condenser must be relatively large ( typically , about 1 meter or more ). this is the reason why industrial scale vacuum freeze - dryers cannot be built utilizing a manifold design , i . e . an apparatus with a number of large chambers . unlike the equipment that uses a freeze - drying process , equipment that uses a pbv process makes it possible to build manifold - based pbv or manifold - based pff equipment because the primary drying for those processes can be performed at considerably higher vacuum pressures ( for example 1 to 3 torrs ), thereby allowing for considerably higher flows between the chambers and the condenser . for example it has been established in practice that primary pbv drying of 4 . 5 kg of an industrial enzyme incorporated inside alginate beads inside a vacuum chamber of modified genesis freeze - dryer ( from virtis co ) can be accomplished within about six hours . the apparatus used in that process has a connector between the drying chamber and the condenser with a diameter of only about 0 . 1 meter . likewise , it has been reported ( u . s . pat . nos . 6 , 306 , 345 and 6 , 692 , 695 ) that an apparatus that utilizes a pff process is capable of removing a similar amount of water from a chamber to a condenser through a connector with a similar diameter within several hours . therefore , equipment that utilizes either a pbv or pff process can be built using a manifold drying apparatus design , thereby providing for a continues load ( and , thus , faster and more efficient ) production . a manifold dryer can be designed and built as a large condenser , which communicates through a plurality of connectors with a plurality of drying chambers . the connectors may optionally be can equipped with vacuum valves ( or other suitable devices ) to control or close the flow of air or water vapors from the chamber into the condenser . the material to be dried is placed in the chambers . a single or a plurality of heat sources are provided for conveying heat to the chambers in order to compensate for a loss of energy due to evaporation during the drying process . additionally , a cooling device is provided to allow cooling of the material before the drying step to about − 10 ° c . the cooling device may utilize any known conventional design for refrigeration equipment , i . e . it may comprise a compressor and a heat exchanger . the chambers can , for example , be cylindrical as disclosed in u . s . pat . no . 6 , 692 , 695 . the chambers also may be flat to accommodate a tray filled with vials , or bags like lyoguard bag . the heat can be delivered by conduction , infrared radiation , using low frequency 50 hz - 500 hz ), radio frequency ( 5 mhz - 60 mhz ) electromagnetic heating of the material in the slush state during primary drying , or by any other known source or method of heat generation and transfer . the apparatus may also be equipped with an optional control system that will control the various processes . for example , the control system can provide automatic or other control of the heating , gas flows , cooling , and other functions of the apparatus . additionally , the control system could be programmable . it also may be programmed to advantageously schedule the progress of the drying processes in various chambers of the apparatus . this feature will allow to connect a new chamber to the manifold and disconnect a chamber where the drying process is finished from the dryer within predetermined periods of time , e . g ., every hour , or 30 minutes . thus , if the chamber capacity is 5 kg and a new chamber is attached every 30 minutes , the production throughput of one chamber will equate to about 240 kg per day , i . e ., 10 kg per hour . it will be appreciated by those skilled in the art that such throughputs are considerably higher than those achievable by conventional freeze - drying equipment . the manifold - type equipment design described above can provide a production rate that is limited only by the capacity of the condenser . if instead of using a mechanical refrigeration device , liquid nitrogen is used to cool the condenser , considerably higher production rates can be achieved . even more importantly from the industrial production standpoint , manifold drying equipment that utilizes either a pff or a pbv process allows to perform continuous load high - speed manufacturing that , as discussed above , is impossible using a freeze - drying process . it shall be understood that there is a large plurality of ways to design and build the novel equipment disclosed herein that will be consistent with novel principles of the invention hereof . for example , the mechanical design of the drying chambers , the design and configuration of the connectors , the geometry of the manifold , etc . can take many different forms . all such various design and engineering solutions shall be understood to be within the spirit and scope of the invention disclosed herein . a number of feasibility experiments have been conducted which demonstrate the methods disclosed herein are functional and effective . for example , a live viral enveloped vaccine has been stabilized with no loss of activity after drying pursuant to a pbv method and subsequent storage at 40 ° c . for one month . in addition , enzymes and ice nucleating proteins have been preserved using the pbv method with no loss of activity . after a preservation by vaporization , the material can be milled or otherwise processed to make it suitable for specific modes of delivery . the following examples are offered only to illustrate , but not to limit the claimed invention . to obtain ice nucleating bacteria ( inb ), a preservation mixture of 180 g of concentrated suspension of ice nucleating bacteria pseudomonas syringae atcc 53543 were mixed with 108 g of sucrose and 12 g of maltrin . the mixture was then mixed until sugars were completely dissolved . the resulting mixture was placed in 100 ml serum vials . thus , 16 . 66 g of the mixture was placed inside each vial . the mixture in the vials then was dried inside an “ ultra ” freeze - drying machine made by virtis corporation and modified for better vacuum pressure control in the drying chamber . the vials were placed on the surface of a stainless steel shelf inside the drying chamber . the shelf temperature was maintained by circulating ethylene glycol / water antifreeze at a controlled temperature inside the shelf . specimens were preserved using a preservation by foam formation process described in u . s . pat . no . 5 , 766 , 520 . no freezing was observed in the vials during the preservation . after stable dry foams formed inside the vials , the foams were dried at 50 ° c . for 24 hours under high vacuum . after that , the vials were closed with rubber stoppers under vacuum and sealed with aluminum seals . a lot of splattering was observed on the walls of the vials . the intensive splattering took place during the boiling of the material inside the vials . all glass walls of the vials were covered with small ( 1 mm or less in diameter ) drops of the mixture . the dry material in vials was irradiated to sterilize the material . sterility was tested by plating of reconstituted bacteria on different agar media . no bacterial growth was observed . ice nucleating activity of preserved inb was measured after the sample reconstitution with 100 ml of 0 . 01m phosphate buffer . ice nucleating activity was measured as a concentration of ice nucleating centers that can nucleate an ice crystal in a 10 μl buffer drop during 5 minutes at − 5 degrees celsius . the results of the assay show that more than 50 % of ice nucleating activity remained in the preserved samples even after the radiation treatment . no decrease in the activity of the inb was observed during the subsequent storage at room temperature and 37 ° c . five vials with preserved inb bacteria from example 1 above were reconstituted with 9 g of water . the vials were placed in the “ ultra ” freeze - drying machine ( made by virtis corporation ). before application of vacuum , the shelf was precooled to − 10 ° c . and the material inside the vials froze . than vacuum was applied simultaneously with increasing the shelf temperature to 35 ° c . the vacuum pressure was controlled so that the temperature inside the vials was maintained between − 5 ° c . and − 10 ° c . after about 3 hours , the primary drying was complete with no visible splattering of the material on the walls of the vials . the dry material looked like foam in partly covered with a skim of thin freeze - dried cake . after a stable dry material formed inside the vials , the material was dried at 50 ° c for 24 hours under a high vacuum . after that , the vials were closed with rubber stoppers and sealed with aluminum seals . the results of the assay performed after reconstituting the vials with 100 ml of 0 . 01m phosphate buffer show no statistically significant decrease of activity after the pbv drying . an aqueous 50 % glycerol isocitrate dehydrogenase solution from sigma chemical co . was dialyzed for 5 hours in 0 . 1 m tris hcl buffer ( ph 7 . 4 ). the activity of the isocitrate dehydrogenase ( idh ) in the 0 . 1 m tris hcl solution after dialysis was 23 ± 1 units per ml . the dialyzed idh was mixed 1 : 1 with preservation solution containing 30 % sucrose and 15 % raffinose , filled in 5 ml vials ( 0 . 5 ml per vial ) and placed on the shelf of “ genesis ” ( made by virtis corporation ) freeze - dryer modified to provide better vacuum pressure control in the drying chamber . the material was first frozen by cooling the shelf to − 15 ° c . then a vacuum ( 1 torr ) was applied simultaneously with raising the shelf temperature to 45 ° c . after about 1 hour the vacuum was increased ( the pressure was decreased below 0 . 2 torrs ). then the material was dried overnight and sealed with rubber stoppers ; vacuum was released and the vials were places at 37 ° c . after drying , the material looked like a foam partly covered with a skim of freeze - dried cake . no splattering was observed on the walls of the vials . after one month of storage at 37 ° c ., the specimens were reconstituted with 0 . 375 ml of water and activity of idh was measured . the reconstituted samples were tested for activity by assaying ability to reduce nadp , measured spectrophotometrically at 340 nm . the reaction mix included : 2 ml 0 . 1 m tris hcl buffer , ph 7 . 4 ; 10 μl of 0 . 5 % by weight nadp +; 10 μl of 10 mm mnso 4 ; 10 μl of 50 mm 1 - isocitrate ; and 10 μl of reconstituted idh solution . the activity was 11 ± 0 . 5 units / ml , which means there was no significant loss of activity during drying and during the month of subsequent storage at 37 ° c . lactobacillus acidophilus atcc 4356 is the type strain of this commercially significant species . l . acidophilus ( probiotics ) grows by fermentation of lactose , glucose and a range of carbohydrates . the end product of this fermentation is almost 1 . difco mrs broth containing 0 . 05 % cysteine and 0 . 1 % of ca c12 ; 2 . mrs agar ; 3 . 10 % cysteine , dubecco &# 39 ; s phosphate buffered saline ( dpbs ); 4 . preservation solution - 1 ( ps - 1 ): 20 % sucrose , 10 % msg ; 0 . 1 % of reconstituted inb from experiment 1 . 5 . preservation solution - 2 ( ps - 2 ): 20 % sucrose , 10 % msg , 1 % alginate ; 0 . 1 % of reconstituted inb from experiment 1 . 6 . edta - 1 solution for reconstitution of dry gel powder comprising 45 g of 0 . 2m kh2po4 + 30 g of 0 . 3m k2hpo4 + 75 g of water + 15 g of standard edta solution from j . t . baker . 1 . l . acidophilus , strain 4356 , obtained from atcc was used to inoculate 150 ml of mrs broth + 0 . 05 % cysteine . 2 . the preculture were grown in belco spin flask at 37 ° c . incubator with gently stirring overnight . at the end the optical density a ( 600 ) in preculture was 3 . 027 and ph was 4 . 03 . fermentation was performed using a new brunswick scientific company bioflo 2000 fermentor with a 2 l working capacity . 1 . 2 l of mrs broth was prepared from commercial difco powder and autoclaved inside bioflo 2000 fermentor for 30 min . ( liq . cycle ) 2 . 10 ml of 10 % cysteine were added into fermentor to obtain final concentration of cysteine = 0 . 05 % 3 . 20 ml of preculture were inoculated into the fermentor . 4 . fermentor was operated at 37 c , 50 rpm agitation , with no ph regulation . 5 . optical density a ( 600 ) and ph after 9 hrs of fermentation remained stable . a ( 600 )= 2 . 9 − 3 . 0 ; ph = 4 . 08 − 4 . 00 . 6 . after 10 . 5 hrs of fermentation the fermentor was harvested and culture distributed into 8 × 250 ml centrifuge tubes , 250 g of culture into each tube . 7 . the tubes were centrifuged at 4 c , 300 rpm , for 15 min . 8 . supernatant was decanted off . 1 . to prepare preservation mixture - 1 ( pm - 1 ) the pellets in 7 tubes were reconstituted with ps - 1 ( 50 g of ps - 1 into each tube ). the mixtures were thoroughly vortexes and merged together . 2 . to prepare the preservation mixture - 2 ( pm - 2 ) the pellet in remaining 8 th tube was reconstituted with 50 g of ps - 2 . the mixture was thoroughly vortexes 3 . pm - 1 was distributed into 50 × 5 ml serum vials , 0 . 5 g per vial ( formulation 1 ). 4 . pm - 1 was filled into each of 5 small cylindrical lyoguard containers ( caps ), 10 g per a cap ( formulation 2 ). 5 . 250 g of pm - 1 was placed in a lyoguard tray ( formulation 3 ). 6 . 10 g of pm - 2 released through the 20g needle in a bath containing 2 % cacl2 dissolved in 90 % ps - 1 to form gel particles looking like spaghetti . the gel particles were collected using 90 micron sieve . the liquid outside gel particles was sacked out using laboratory vacuum pump . the particles were placed in a small cylindrical 200 ml lyoguard containers ( caps ). 5 caps were filled ( formulation 4 ). 7 . activity of bacteria in pm - 1 and pm - 2 before drying was determined by plating of 0 . 1 ml of a pm diluted millions times on mrs agar + 0 . 05 % cysteine . 8 . all plates were stored under anaerobic condition at 37 ° c . incubator for 48 hrs . 1 . initial shelf temperature was set to 0 ° c ; 2 . specimens froze after the temperature inside the specimens decreased to approximately − 4 ° c . as a result evaporation after vacuum application . the freezing began at the surface of the specimens . after that the shelf temperature was increased to 35 ° c . 3 . the primary drying was performed by keeping the temperature inside the specimens between − 5 ° c . and − 10 ° c . to obtain mechanically stable dehydrated state . after drying the material in the vials and in lyoguard tray looked foamy with a skim of freeze - dried cake above the foam . the alginate particles looked like dry spaghetti . 4 . the stability drying was performed under complete vacuum first at 25 ° c . overnight . then shelf temperature was raised to 50 ° c . for additional 48 hrs . 5 . after drying in glass vials the mass of dry material in a vial was approximately 0 . 15 g 6 . after drying , materials from caps and tray were milled in dry room ( at 15 % relative humidity ) using virtis laboratory homogenizer . the milled powders were filled in 5 ml glass vials ( 0 . 15 g per vial ). the vials were sealed with robber stoppers covered with aluminum seals . 7 . to measure the activity of 0 . 15 g of dry material in each vial was reconstituted with 4 . 85 ml of dpbs and than diluted an additional 100 , 000 times before 0 . 1 ml was plated on mrs agar + 0 . 05 % cysteine . 8 . to measure the activity of 0 . 15 g of dry material containing alginate was reconstituted with 4 . 85 ml of edta - 1 solution and than diluted an additional 100 , 000 times before 0 . 1 ml was plated on mrs agar + 0 . 05 % cysteine . it shall be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof that may be suggested to persons skilled in the art are within the spirit and are to be included within the purview of this disclosure and scope of the claims that follow . while the foregoing invention has been described in some detail for purposes of clarity and understanding , it will be clear to one skilled in the art from a reading of this disclosure that various changes in the form and detail can be made without departing from the true scope of this invention . for example , the formulations , techniques , apparatus and specific process parameters described herein can be used in various combinations and / or adjusted to suit a specific biological without departing from the scope of the disclosed and claimed inventions . all publications , patents , patent applications , and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication , patent , patent application and / or other document were individually indicated to be incorporated by reference for all purposes . 1 . annear , “ preservation of leptospir . ae butted . by drying ” j . path . bact . vol ., 72 , pp . 322 - 323 , 1956 : 2 . annear , “ observations of the preservation by drying of leotospirae and some other bacteria ”, austral j . exp . biol . vol ., 36 , pp . 1 - 4 :, 1958 . 3 . annear , “ observation on drying bacteria from the frozen and from the liquid state ” austral . j . exp biol . vol . 36 , pp . 211 - 221 , 1958 . 4 . annear , “ recovery of strigomonas oncopeltii after drying from the liquid state ” aust . j . exp . biol ., vol . 39 , pp . 295 - 303 , 1961 . 5 . annear , “ preservation of the reiter treponeme by drying from the liquid state ”, j . bact ., vol . 83 , pp . 932 - 933 , 1962 . 6 . annear , “ the preservation of leptospires by drying from the liquid state ”, j . gen . microbiol ., vol . 27 , pp . 341 - 343 , 1962 . 7 . annear , “ recoveries of bacteria after drying on cellulose fibres a method for the routine preservation of bacteria ”, austral . j . exp . biol ., vol . 40 , pp . 1 - 8 , 1962 . 8 . annear , “ preservation of yeast by drying ”, austral . j . exp . biol ., vol . 41 , pp . 575 - 580 ., 1963 . 9 . annear , “ recoveries of bacteria after drying in glutamate and other substances ”, aust . j . exp . biol . med . sci ., vol . 42 , pp . 717 - 722 , 1964 . 10 . annear , recoveries of bacteria after drying in vacuo at a bath temperature of 100 . degree . c ., nature , no . 5050 , p . 761 , aug . 13 , 1966 . 11 . annear , survival of bacteria in desiccates at 100 . degree . c . in dry atmospheres , nature vol . 206 no . 4991 , pp . 1373 - 1374 , jun . 26 , 1965 . 12 . bronshtein , v . 1998 . preservation by foam formation . u . s . pat . no . 5 , 766 , 520 . 13 . burke , m . j . 1986 . the glassy state and survival of anhydrous biological systems . in “ membranes , metabolism , and dry organisms ”, p . 358 - 363 , ed . c . leopold , cornell university press , ithaca , n . y . 14 . annear , d . i . 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