Patent Publication Number: US-6904701-B2

Title: Flask and method for drying biological materials

Description:
STATEMENT REGARDING FEDERAL SPONSORED RESEARCH AND DEVELOPMENT 
   Embodiments of this invention were made with Government support under Grant No. 6600100C8048, awarded by the Department of Defense Advanced Research Projects Agency (DARPA). The Government has certain rights to embodiments of this invention. 

   BACKGROUND OF THE INVENTION 
   1. Field of the Invention 
   The present invention is related to a flask or container which is preferably used for drying biological materials. More specifically, embodiments of the present invention provide a flask for receiving biological materials, and method for treating or processing the biological materials while contained in the flask. 
   2. Description of the Prior Art 
   Dried biological materials are becoming increasingly useful in agriculture, biotechnology and medicine. For instance, freeze-dried human blood products, vaccines and the like are already in use, or are proposed to be in use, in clinical settings for both animal and human applications. In the field of biotechnology, biosensors have wide spread applications. In all such cases, long term storage under sterile conditions, and often under unfavorable environmental circumstances, is a requirement. 
   Conventional devices in the market do not optimally provide drying of biological materials under sterile conditions, while allowing for certain desired contamination-free processing of the biological materials under defined conditions, such as during drying, storage and re-hydration. More particularly, because biological materials show much improved survival if they are exposed to water vapor prior to immersion in liquid water, it is desired to pre-hydrate biological samples with water vapor without contaminating the biological samples. 
   A patentability investigation was conducted to determine the state of the art with respect to solving problems of contamination while processing biological materials during drying, storage and re-hydration, and the following U.S. Patents were discovered: U.S. Pat. No. 4,232,453 to Edelmann; U.S. Pat. No. 4,275,511 to Parkinsen, et al.; U.S. Pat. No. 4,966,469 to Fraser, et al.; U.S. Pat. No. 5,154,007 to Piunno, et al.; U.S. Pat. No. 5,689,595 to Sutherland, et al.; and U.S. Pat. No. 6,122,836 to Tenedini, et al. 
   U.S. Pat. No. 4,232,453 to Edelmann discloses a tray for holding a biological specimen and a submersible container for freeze-drying the specimen. A heating element is taught for melting a synthetic resin for embedding the specimen therein. 
   U.S. Pat. No. 4,275,511 to Parkinsen, et al. discloses an evaporator/sublimator flask having a straight sided cylinder, preferably made of borosilicate glass tubing of sufficient wall thickness to prevent implosion when subjected to a high vacuum. The straight sided cylinder is open at one end and sealed at the other. An elastomer cap is disposed over the open end of the cylinder. 
   U.S. Pat. No. 4,966,469 to Fraser, et al. discloses a flask for freeze-drying. A positioning device engages the top of the flask and comprises a generally circular stopper having an opening. An annular tube extends through the stopper and into the flask. A thermocouple is coiled around the lower part of the annular tube. 
   U.S. Pat. No. 5,154,007 to Piunno, et al. discloses an apparatus and describes a method for distillation drying of one or more biological samples. The apparatus includes a retaining assembly, a vacuum assembly, a cooling assembly, a monitoring assembly and a control assembly for actively regulating the temperature and pressure conditions of biological tissue so that biological samples may be dried without damage. 
   U.S. Pat. No. 5,689,895 to Sutherland, et al. discloses a device for positioning a probe (e.g., a temperature sensor) in a flask for freeze-drying. The device includes a stopper secured to an open end of the flask. The stopper has a center opening and at least one radial opening spaced from the center opening. The radial opening allows for fluid communication between inside and outside of the flask when the stopper is secured to the open end of the flask. The center opening receives a guide tube which extends into the flask and receives the probe. 
   U.S. Pat. No. 6,122,836 to Tenedini, et al. discloses a freeze-drying apparatus and associated lyophilization procedures employing vapor flow detection and/or vacuum control for monitoring and control of a lyophilization process. The vapor flow detector (e.g., a windmill sensor) is disposed to monitor vapor flow from product undergoing lyophilization. 
   None of the foregoing patents teach a flask, device or container which permits drying (freeze-drying, air-drying, foam drying) of biological materials under sterile conditions and which allows for processing under defined conditions during drying, storage and re-hydration. Therefore, what is needed and what has been invented is a flask and method which overcomes the contamination deficiencies of the prior art. What is more specifically needed and what has been invented is a flask for drying (e.g., freeze-drying) substances under sterile conditions, and method for processing a substance under sterile conditions, including drying, storage, and rehydration. In the method for processing, the flask is placed on a shelf of a freeze-drying and re-hydration apparatus where substances contained in the flask are freeze-dried and re-hydrated without contamination. 
   SUMMARY OF THE INVENTION 
   Embodiments of the present invention broadly provide a device for holding substances during drying. The device includes a flask having a structure defining an opening, a first filter member disposed in the opening, and a second filter member disposed in the opening juxtaposedly to the first filter member. The first filter member comprises at least one aperture sized to preclude the passing of bacteria there through. Preferably, first filter member comprises a plurality of apertures having an average opening with an average maximum dimension (e.g. diameter, maximum diagonal distance across opening, etc.) ranging from about 0.10 um to about 0.22 um. In one embodiment of the invention the first filter member has a higher flexibility than the second filter member. In another embodiment of the invention, the difference in average permeability or average maximum dimension of openings (i.e., the openings that permit gases or liquids to pass through) between the first and second filter members ranges from about 0.00 um to about 0.90 um. Thus, if one filter member has an average aperture opening of about 0.22 um, the other filter member may have an average aperture opening ranging from about 0.60 um to about 0.90 um. A retainer ring is engaged to the flask for retaining the first and second filter members in the opening. The structure of the flask additionally comprises a second opening wherein a third filter member maybe disposed. A temperature-conductive member passes through a side of the flask. The device or flask may be disposed in a freeze-drying apparatus where substances contained in the device are processed by freeze-drying and prehydration. 
   Embodiments of the present invention also provide a method for processing a substance under sterile conditions comprising disposing a substance in a flask, positioning the flask in a drying apparatus, and passing a drying medium through a first filter member and through a second filter member juxtaposed to the first filter member for drying the substance. The method may additionally comprise contacting the substance with a temperature-conductive member for monitoring the temperature of the substance. The temperature-conductive member typically passes through a side of the flask and has a thermocouple coupled thereto. The flask may be exposed to water vapor as desired for prehydration purposes. 
   These provisions together with the various ancillary provisions and features which will become apparent to those skilled in the art as the following description proceeds, are attained by the methods and flask(s) of the present invention, preferred embodiments thereof being shown with reference to the accompanying drawings, by way of example only, wherein: 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
       FIG. 1  is a top plan view of the device or flask for holding biological materials; 
       FIG. 2A  is a side elevational view of the flask of  FIG. 1  illustrating the filter cover and filter cap removed therefrom; 
       FIG. 2B  is a front elevational view of the neck taken in direction of the arrows and along the plane of line  2 B— 2 B in  FIG. 2A ; 
       FIG. 3  is an enlarged side elevational partial sectional view of the flask illustrating the top aperture with the filters and covering removed therefrom; 
       FIG. 4  is a top plan view of the flask illustrating the thermocouple probes which are to pass through a side of the flask and contact any substance therein; 
       FIG. 5  is a front elevational of the removable filter cap having a partially exposed filter; 
       FIG. 6  is an enlarged partial vertical sectional view of the two superimposed filters disposed over the flask opening with the cover or retainer ring coupled to the top of the flask such as to keep the two filters sandwiched over the flask opening; 
       FIG. 7  is the view of  FIG. 6  with the filter cover in place and represented by dashed lines; 
       FIG. 8  is a vertical sectional view of the filter cover; 
       FIG. 9  is a partial perspective view of a freeze-drying apparatus containing the flask for treating or processing any biological materials contained in the flask; 
       FIG. 10  is a graph of freeze-drying sample temperatures during three simultaneous freeze-drying runs; and 
       FIG. 11  is an image of dried cells for the Example taken on film using an inverted microscope, showing the dried cells encased within strands of the drying matrix. 
   

   DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION 
   Referring in detail now to the drawings for various embodiments of the invention, there is seen a flask, generally illustrated as  10 , having a bottom  12 , a rear wall  14  and side walls  16  and  18  bound to the bottom  12 , and a front wall assembly  20  bound to the bottom  12  and the side walls  16  and  18 . A top  22  is secured to the rear wall  14 , to the side walls  16  and  18 , and to the front wall assembly  20 . The flask  10  may be manufactured from any suitable material, preferably a transparent plastic (polyethylene, polypropylene, polystyrene, etc.). 
   The top  22  of the flask  10  includes an opening  30  with a perimeter  32 . Filters  34  and  36  are superimposedly disposed over the opening  30  such that the respective perimeters  34   a  and  36   a  associated with filters  34  and  36  extend beyond the perimeter  32  of opening  30  for structural support purposes, as best shown in  FIGS. 6 and 7 . A cover or retainer ring  40  is conveniently coupled to the top  22  for holding the filters  34  and  36  in place superimposedly. The retainer ring  40  has in inwardly protruding lip  42  for defining an internal opening  44  to expose filters  34  and  36  and for protruding or lapping over the perimetrical fringes of filters  34  and  36  for posturing same in their filtering position, as best shown in  FIGS. 6 and 7 . A removable filter cap  50  may be conveniently disposed within the ring  40  to cover internal opening  44  and superimposed filters  34  and  36  (see FIG.  7 ). As best shown in  FIG. 8 , filter cap  50  includes a continuous bottom  54  for immediately covering opening  44  and the filters  34  and  36 . Bottom  54  terminates in shoulder  58  and an upstanding wall  60  which forms an opening  62  to expose the bottom  54 . A depending ridge  64  is integrally secured to shoulder  58  for resting on, and being supported by, protruding lip  42  when the bottom  54  spacedly covers the superimposed filters  34  and  36 . The ridge  64  allows the bottom  54  to be spaced or separated from filter  36 . 
   The front wall assembly  20  is formed with a protruding hollow neck  70  to provide an opening  74  (see  FIG. 2B ) for placing and removing material  80  inside the flask. Material  80  may be any desired substance to be processed, such as biological materials. Removably secured to neck  70  is a cap  86 , preferably a cap  86  including an exposable filter  88  (see hydrophobic filter in  FIG. 5 ) upon suitable rotation or manipulation. The cap  86  may be any suitable cap which is capable of being removably disposed around the neck  70 . In one preferred embodiment of the invention, the cap  86  is that manufactured under the trade name “Nunc EasYFlask closure” by Nalge Nunc International Corp. of Naperville, Ill. This particular cap  86  is available as a filter cap (with hydrophobic filter  88 ) or as a vent/close cap. The Nunc EasYFlask closure system of Nalge Nunc International Corp. allows one to open or close the flask  10  by merely a ⅓ rd turn of the cap  86 . Once one removes the cap  86 , the opening  74  of the angled neck  70  allows easy access to the entire growth surface of the flask  10  with both pipettes and cell scrapers. The slope and the design of the neck  70  allow complete drainage, and yet minimal risk of any medium within the flask  10  splashing into the opening  74  of the neck  70  when disposing the flask  10  horizontally. 
   In a preferred embodiment of the invention, one or more temperature probes  90  maybe disposed such as to be in contact with substance or material  80  (see FIG.  1 ). Preferably, the probes  90  pass through side of side wall  16  and couple to thermocouple conductors  94 — 94  which extend to an indicator (not shown) for displaying temperature indicia of the substance or material  80 . The temperature of the substance or material  80  is preferably monitored during processing, especially the drying process. The progress of the temperature of the material  80  during freeze-drying, air drying, or foam drying provides valuable information about the progress of treatment or processing, especially drying. For this purpose, the temperature probes  90  are placed in contact with the sample or material  80 . The thermocouple conductor  94  is coupled to the probe  90  to monitor sample temperature. 
   Filter  34  is preferably a bacteria-filtering filter which precludes the entry of bacteria into the flask  10 . Filter  36  is a prefilter, preferably for filtering large foreign particles, such as dust. Filter  36  is preferably a support membrane type filter which increases the structural rigidity of the combination of the superimposed filters  34  and  36 . In operation, filter  34  would typically move or flex toward and/or against filter  36 . Preferably, filter  34  has a higher flexibility than filter  36 . Preferably further, filter  34  comprises a plurality of apertures having an average opening with an average maximum dimension (e.g. diameter, maximum diagonal distance across the opening, etc.) ranging from about 0.10 um to about 0.65 um, more preferably from about 0.10 um to about 0.45 um, and most preferably from about 0.10 um to about 0.22 um. Filter  36  comprises a plurality of apertures having an average opening with an average maximum dimension (e.g. diameter, maximum diagonal distance across the opening, etc.) ranging from about 0.60 um to about 1.0 um, more preferably from about 0.65 um to about 0.95 um, and most preferably from about 0.70 um to about 0.90 um. Stated alternatively, and in another embodiment of the invention, the difference in average size of filter openings between the filters  34  and  36  ranges from about 0.00 um to about 0.90 um, more preferably from about 0.45 um to about 0.75 um, and most preferably from about 0.50 um to about 0.70 um. Thus, if the average size openings in filter  34  is about 0.22 microns, the average size openings in filter  36  may range anywhere from about 0.60 um to about 0.90 um. Filters  34  and  36  may be manufactured from any suitable material, such as polyvinylidene fluoride, cellulose, fiberglass, etc. Preferably filter  34  is manufactured of polyvinylidene fluoride, while filter  36  is manufactured from fiberglass. 
   In a method for processing a substance or the material  80  under sterile conditions, the material  80  is disposed in the flask  10  via the opening  74  in the neck  70 . The flask  10  containing the material  80  is then placed in a suitable apparatus for processing the material  80  with the flask  10 . The apparatus for processing, generally illustrated as  100  in  FIG. 9 , may be any suitable apparatus for drying and/or rehydrating the material  80 . A suitable apparatus  100  is that which is manufactured by Kinetics Group, Inc., and sold under the trade name FTS Systems Lyostar. 
   After the flask  10  containing the material  80  has been suitably disposed in apparatus  100 , the material  80  is frozen within the flask  10  at a rate that is considered optimal for the sample and as can be controlled by an apparatus, such as apparatus  100 . Once frozen to low temperature, such as −60° C. for example, the sample can be exposed to a strong vacuum as produced by the drying apparatus  100 . The applied vacuum draws water out of the samples by sublimation of the frozen water, making the ice change directly into liquid vapor. The water vapor leaves the material  80  and passes serially through the filters  34  and  36  and is collected within the apparatus  100  by a condenser. This drying continues as the material  80  is slowly heated back to ambient room temperature under vacuum. 
   After freeze-drying, the flask  10  containing the freeze-dried sample may be removed from the apparatus  100  and for storage purposes. Storing in a sterile manner may be at room temperature in a suitable dry location while the freeze-dried material  80  remains in the flask. During storage, the filter cap  50  may be placed within the confines of the ring  40  to cover internal opening  44  of the retainer ring  40  and the superimposed filters  34  and  36 . When it is desired to use material  80 , the flask  10  including the material  80  is removed from storage and is subsequently disposed within a suitable humid chamber for rehydration of the freeze-dried material  80 . During rehydration, minute particulates of water vapor pass through filters  36  and  34  while contamination is prevented from entering the inside of flask  10  by the filters  34  and  36 . The water vapor is typically at a temperature ranging from about 20° C. to about 37° C. The remaining rehydration is then achieved by the addition of water, and/or a preferred resuspension solution, such as a cell growth medium, via the opening  74  in neck  70  of the flask  10 . 
   Embodiments of the present invention will be illustrated by the following set forth example which is being given to set forth the presently known best mode and by way of illustration only and not by way of any limitation. All parameters such as concentrations, mixing proportions, temperatures, rates, compounds, etc., submitted in these examples are not to be construed to unduly limit the scope of the invention. 
   EXAMPLE 
   The flask  10  may be loaded with a sterile sample, in a sterile hood, sealed, and then transferred to the drying apparatus (a freeze-dryer)  100 , such as that manufactured by Kinetics Group, Inc. The sterile sample may be 293H human embryonic kidney cell line in 2.5 ml buffer solution. When freeze-drying is complete, the flask  10  containing the freeze-dried sample may be stored without precautions against contamination since the flask  10  is a closed system. Vapor phase re-hydration may be accomplished simply by exposing the flask  10  to water vapor within a humidified incubator. Since the water vapor contacts the sample by passage through the bacterial filter  34 , there is no risk of contamination. It is preferable to monitor sample temperature during the drying process. The progress of sample temperature during freeze drying, air drying, or foam drying provides valuable information about the progress of drying. For this purpose, the end of the temperature probe  90  may be placed in contact with the sample or material  80 . The thermocouple wire  94  is coupled to temperature probes  90 . 
   Material  80  used in the example consisted of a 2.5 ml sample buffer solution containing 293H cells placed within flasks  10  via the opening  74  in neck  70 . The flasks  10  were placed within the apparatus  100  for freeze-drying. The flasks  10  and samples  80  were frozen at 1° C. per minute to −60° C. and held at that temperature for 1 hour. The vacuum was initiated during this time interval while the samples were held at −60° C. As the vacuum was applied by apparatus  100 , the change in sample temperature was observed via port  90  due to the heat released by the material  80  during loss of water, as indicated by FIG.  10 . Next, under vacuum, the samples were held at −25° C. for an additional 6 hours. Following this time interval, the samples were subsequently slowly heated back to room temperature of +22° C. over an 8 hour period while still under vacuum.  FIG. 10  depicts the process in its entirety and displays flask temperatures as monitored through temperature port  90  of each flask and shelf temperatures, both as monitored by apparatus  100 .  FIG. 10  more specifically shows sample temperatures during three simultaneous freeze-drying runs, illustrating repeatable processing. All came to minimal water contents simultaneously. Residual water content was≦3% by weight. Maintenance of sterility was assessed during processing. Thus, starting with sterile sample materials  80 , the flasks  10  were subjected to freeze-drying, followed by re-hydration in culture media, in a sterile hood. Over the following nine days sterility was assessed by microscopy and pH change. Based on these criteria, no contamination was evident.  FIG. 11  is an image of the dried cells for this Example. The  FIG. 11  image was taken on film using an inverted microscope and shows the dried cells encased within strands of the drying matrix. 
   Therefore, due to the inherent design, having a set of filters  34  and  36  covering a smaller area than the top surface  22  of the flask and having a flask  10  that is of rigid, transparent plastic, one can view a sample  80  disposed within the flask  10  (prior to or after drying) directly using a  1   b  microscope (preferably an inverted type or by inversion of the flask  10  on a standard scope) without any risk of contamination to a sterile sample  80 . In other words, by having the filers  34  and  36  only covering a portion of the upper surface  22 , there is a free visual path through the plane of the bottom surface  12  (via top surface  22 ) of the flask  10  for allowing microscopic (visible, fluorescence, etc.) viewing. With a microscopy, valuable photographs (micrographs, digital images, video, etc.) can be taken of the samples  80  during any stage of the process. These images can help one ascertain the quality of drying, the integrity of a sample, sample structure, and allow viability assays, while the samples  80  are still contained within the flask  10 . 
   While the present invention has been described herein with reference to particular embodiments thereof, a latitude of modification, various changes and substitutions are intended in the foregoing disclosure, and it will be appreciated that in some instances some features of the invention will be employed without a corresponding use of other features without departing from the scope and spirit of the invention as set forth. Therefore, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope and spirit of the present invention. It is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments and equivalents falling within the scope of the appended claims.