Abstract:
An apparatus to lyophilize, store, transport, rehydrate, and process aqueous biological materials in a container which maintains sterility of its contents, allows container shrinkage after lyophilization, and optimally permits filtration or dialysis of the contents in situ, without the need for a second or series of additional containers. These benefits are met by a microporous container constructed of a membrane that is water vapor permeable, yet water impermeable.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    1. Field of the Invention  
           [0002]    The invention relates to the use of an integrated container for lyophilization, storage, distribution, and processing of fluids, cells or tissues.  
           [0003]    2. Description of Related Art  
           [0004]    Distribution of materials requires storage under conditions suitable for optimum product stabilization, minimum storage cost, and simple operation at site of use. This is of particular importance in the field of biopharmaceuticals because of the propensity of such products to lose their biological activity in the liquid, aqueous state. Cooling below −20 degrees Celsius (° C.) is a popular approach, but costs of maintaining the materials at reduced temperatures (−20°, −80° or −196° (C.)) for extended periods and during transport are high. Additionally, many important biological products, such as blood and plasma, have high mass (weight), which in turn creates the shipping challenges of logistics and expense.  
           [0005]    A typical example of a biological material susceptible to the above challenges is blood plasma. With whole blood having a limited storage life, blood plasma and its ability to keep for two years (either frozen or lyophilized) has long been an important medical product, particularly in hospitals and military operations. Transporting blood plasma per se is problematic due to the need for temperature controls and due to the high mass of its primary constituent, water. Even when blood plasma is lyophilized to remove the water and its attendant disadvantages, storage, transport and processing for use become no easier because the container is fixed prior to lyophilization and because of the documentation and rehydration requirements of such products.  
           [0006]    Lyophilization is a useful mode of storing many biological products, and involves the processes of freezing, removal of water as vapor (under a vacuum), storage, and rehydration prior to use. Existing methods for lyophilizing aqueous biological materials depend upon the use of a rigid container, which can withstand the vacuum imposed within the container to sublimate the water for removal as water vapor. The water vapor is removed via a connection to the neck of the container, with storage and shipment resulting in large amounts of wasted space, namely, the space formerly occupied by the water. If further processing is required after hydration with pyrogen-free water, such as removal of certain constituents by filtration or dialysis, the product must be transferred to a new container. Aseptic conditions are essential, but many manipulative steps can compromise sterility.  
           [0007]    Some examples of materials that undergo such processing include: vaccines; extracts from animal, vegetable, bacterial, yeast sources; proteins and carbohydrates sensitive to heat; oligonucleotides; organometallics; liposomes; antibiotics; and blood products. In such applications, during the manufacturing process, the products are lyophilized for later rehydration as needed. Additional applications in genetic engineering, biochemistry, biotechnology, cell biology, and medicine include storage of bacterial, mammalian, yeast, and plant cells. In such situations, a “cryostabilizing” agent, such as mannitol or trehalose, is added to the cell suspension before freezing. After storage and rehydration, these agents should be removed before the cells can be used for direct therapeutic application.  
           [0008]    Cost-effective lyophilization requires a confined container that does not hinder the processing of the biological material. At a minimum, such processing requires: a simple means of applying a vacuum to the frozen solution; the use of a container with mechanically strong walls to withstand the pressures created during the vacuum; provision of a maximum surface to volume ratio for the frozen materials in order to facilitate egress of water vapor from the frozen matrix; and simple removal of the product when needed. Both rigid bottles and pliable bags for storage of fluids and cells are widely used, often featuring compartments separated by a common wall. Common wall materials available for such units range in their water vapor permeability from zero to high permeability.  
           [0009]    One common lyophilization approach involves “shell freezing” materials within wide mouth glass flasks that are attached to a vacuum system. The water vapor exits from the mouth by sublimation and when completed, the vacuum is released and the flask is sealed and removed for storage. The disadvantages of this approach include the large size of the container to be stored, the fragility of the glass container, and the difficulty of maintaining aseptic conditions during the process.  
           [0010]    W.L. Gore &amp; Associates recently introduced a system that addresses many of these disadvantages (Genetic Engineering News 22: pp. 22 and 26, Jan. 01, 2002). Their approach involves the use of a disposable lightweight tray composed of a filling port on one of five rigid walls with a permeable Gore-Tex® expanded polyytetrafluoroethylene (ePTEE) laminate developed for this process. The laminate material has a microporous “body” to which a large mesh cover is attached for structural stability. The material was designed to provide a high vapor transfer rate and integrity to prevent passage of microorganisms, such as  Bacillus subtillis  and  Bacillus licheniformis.  The process involves filling of the container with the material to be lyophilized by freezing, placing the tray into a vacuum chamber, transferring of the water through the ePTEE membrane, returning the container to atmospheric conditions, and removing of the tray from the vacuum chamber for storage.  
           [0011]    Despite the advances accorded by this approach, several critical features either have not been addressed or have been specifically excluded. First, containers are not provided in a sterile condition but contain specific instructions that any such sterilization is the sole responsibility of the user. Steam sterilization can be used, if necessary, but other useful procedures are either not recommended (e.g., radiation techniques) or are not addressed (e.g., ethylene oxide, gas plasma, formaldehyde gas, hydrogen peroxide vapor). Second, after removal of the water, the container is returned to the atmosphere, and the product cannot be stored under a vacuum due to the “open” nature of the ePTEE materials. This allows interaction of the lyophilized materials with oxygen and atmospheric water vapor during storage. Third, the ePTEE surface must be sealed with a foil barrier pouch or other vapor barrier enclosure to prevent product rehydration. This requires additional post-processing steps. Fourth, the rigid nature of the tray system precludes the integration and use of such a system into processes that involve centrifugation and decantation of fluids and cell suspensions (e.g., blood cell fractionation). Finally, the approach does not allow for facile exchange of the solution after lyophilization (e.g., removal of mannitol). This precludes the use of a single package for the storage and delivery of cells by infusion.  
           [0012]    Accordingly, a need remains for a lyophilization method and apparatus that overcomes the prior art problems of wasted storage space, potential compromise of sterility, and multiple method steps in more than one container.  
         SUMMARY OF THE INVENTION  
         [0013]    The present invention is an apparatus to lyophilize, store, transport, rehydrate, and process aqueous biological materials in a container which maintains sterility of its contents, allows container shrinkage after lyophilization, and optimally permits filtration or dialysis of the contents in situ, without the need for a second or series of additional containers. These benefits are met by a microporous container constructed of a membrane that is water vapor permeable, yet water impermeable. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0014]    [0014]FIG. 1 is a top perspective view, partially in section, of an integrated container for lyophilization and rehydration in accordance with the present invention;  
         [0015]    [0015]FIG. 2 is a top perspective view, partially in section, of a collapsible container with a lyophilization compartment sub-component made in accordance with the present invention;  
         [0016]    [0016]FIG. 3 is a top perspective view, partially in section, of the embodiment shown in FIG. 2 in a collapsed state;  
         [0017]    [0017]FIG. 4 is a top perspective view, partially in section, of another embodiment of the present invention;  
         [0018]    [0018]FIG. 5 is a top perspective view, partially in section, of another embodiment of the present invention;  
         [0019]    [0019]FIG. 6 is a top perspective view, partially in section, of another embodiment of the present invention;  
         [0020]    [0020]FIG. 7 is a top perspective view, partially in section, of another embodiment of the present invention;  
         [0021]    [0021]FIG. 8 is a top perspective view, partially in section, of another embodiment of the present;  
         [0022]    [0022]FIG. 9 is a top perspective view, partially in section, of another embodiment of the present invention;  
         [0023]    [0023]FIG. 10 is a top perspective view, partially in section, of another embodiment of the present invention;  
         [0024]    [0024]FIG. 11 is a top perspective view, partially in section, of another embodiment of the present invention;  
         [0025]    [0025]FIG. 12 is a top perspective view, partially in section, of another embodiment of the present invention; and  
         [0026]    [0026]FIG. 13 is a top perspective view, partially in section, of a further embodiment of the present invention. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0027]    The present invention is a container having a lyophilization compartment and one or more vacuum-processing compartments. The common wall between the lyophilization compartment and one adjacent vacuum-processing compartment is fabricated with a flexible controlled pore membrane with hydrophobic surfaces that is water vapor permeable, yet water impermeable, with the remaining walls of the container fabricated with a pliable material capable of compression upon vacuum pressure and of maintaining a barrier between an internal vacuum and the external atmosphere. The lyophilization compartment contains an access port to allow entry of biological materials or water. Each vacuum-processing compartment contains an exit port that connects to a vacuum and condenser system (not shown). All ports contain a barrier therein to maintain sterility of the material being processed. Mechanical strength sufficient to retain the compartments from collapse under vacuum pressure can be achieved either by external or internal mechanical restraints. External mechanical restraints are located on the external surfaces of the container and are attached to tabs located on the external surfaces of the container. Internal mechanical restraints are located between the lyophilization compartment and the vacuum-processing compartment and can be made of either a thin honeycomb-like, open cell plastic structure that lies adjacent to the pore membrane, or a pattern of crisscrossing raised plastic “bumps” that are placed on top of the pore membrane.  
         [0028]    One way to address some of the disadvantages of the prior art is to use membranes of any material that meet the requirements for sterility and selective permeate flow (gas permeable, yet water impermeable). An example of such a material is RoTrac® Capillary Pore Membranes provided by Oxyphen AG. These membranes are made of a polyester film that is exposed to a controlled beam of heavy ions, such as krypton. When the accelerated ions pass through the polymer film, they break the polymer chains and the tracks are accessible for chemical etching. The cylindrical pores that are formed can have a diameter between 0.03 μm and 10 μm, with the number of pores per unit area adapted to the requirements of the particular system. The separation membrane can then be laminated to various non-woven materials that differ according to their water-attractant characteristics (e.g., polypropylene that is hydrophobic; polyester-terephtalate that is hydrophilic), in order to achieve mechanical stability. The pores of the support material are much larger than the membrane pores in order to allow unhindered permeate flow. The resultant laminate membrane can be sterilized several times, without shrinking, using a variety of sterilization methods, including autoclave, steam-sterilization, formaldehyde, hydrogen peroxide, percarbonic acid, ethylene oxide, or gamma radiation. Biological materials differ widely in their capacity to tolerate “residual” materials incident to sterilization, thus emphasizing the need for use of such membranes that are capable of sterilization by a variety of methods.  
         [0029]    RoTrac® Capillary Pore Membranes differ from other microfiltration membranes in several critical ways. Most membranes have a layer with an irregular spongeous structure that does not define an exact pore diameter. However, RoTrac® Capillary Pore Membranes have a well-defined geometry, i.e., a known pore size (diameter) with a diameter tolerance of a maximum 10%, and a defined number of pores per unit area. They have a high porosity, homogeneous area density and defined pore diameter, and are highly gas permeable. The unique properties of these membranes allow simple incorporation into customized membrane products by direct injection molding, ultrasonic welding, heat, or use of adhesives, thus making them suitable for biomedical applications, although they have had, to date, limited biomedical use.  
         [0030]    Referring now to FIG. 1, the container  10  is a semi-collapsible closed structure having a cavity therein. The cavity is divided equally into two compartments: a lyophilization compartment  12  and vacuum-processing compartment  14 . Each compartment  12 ,  14  is bounded by six walls comprised of an upper face  16 , a lower face  18 , and four lateral faces  20 . Although the four lateral faces  20  may be pleated, they could also be flat, yet flexible enough to collapse. Also it is possible that the two walls are straight lateral faces  20 , while opposite lateral faces  20  are pleated. The upper face  16  of the lyophilization compartment  12  is fabricated entirely or in part with a flexible controlled pore membrane with hydrophobic surfaces, and the four lateral faces  20  are fabricated with a flexible material that is capable of compression under vacuum pressure and of maintaining a barrier between an internal vacuum and the external atmosphere. Examples of materials that provide both flexibility and resistance to vacuum pressure are plastics, such as polyethylene, polyurethane and polyester/polyether block copolymers. An access port  22  is located on a lateral face  20  of the lyophilization compartment  12 . The access port  22  allows for the entrance of biological materials or water therein. The access port  22  is sealed with a microporous hydrophobic membrane barrier  24  to maintain sterility. The upper face  16  of the lyophilization compartment  12  and the lower face  18  of the vacuum-processing compartment  14  serve as a common wall between the lyophilization compartment  12  and the vacuum-processing compartment  14 . The upper face  16  and lateral faces  20  of the vacuum-processing compartment  14  and the lower face  18  of the lyophilization compartment  12  are fabricated with a rigid material, such as acrylic, polycarbonate, polypropylene or ABS. The vacuum-processing compartment  14  contains an exit port  26  therein that is sealed with a barrier  28  to maintain sterility. The exit port  26  is connected to a vacuum and condenser system (not shown), commonly known and used by those skilled in the art, for removal of water vapor from the lyophilization compartment  12 . During compression of the lyophilization compartment  12 , the vacuum can be replaced by inert gases according to the needs of the user. An additional feature of the container  10  is the versatility of the lyophilization compartment  12 , allowing it to serve as a subcomponent of a more complex system, such as for blood fractionation. Furthermore, the entire container  10  can be sterilized before use.  
         [0031]    [0031]FIG. 2 is an embodiment of a collapsible container  100  with a lyophilization compartment subcomponent that can be used in a pre-existing vacuum-processing system. The container  100  is composed of a lyophilization compartment  112  bounded by six walls comprised of an upper face  116 , a lower face  118 , and four lateral faces  120 . The upper face  116  of the lyophilization compartment  112  is fabricated entirely or in part with a flexible controlled pore membrane with hydrophobic surfaces, and the four lateral faces  120  are fabricated with a flexible material that is capable of compression under vacuum pressure and of maintaining a barrier between an internal vacuum and the external atmosphere. An access port  122  is located on a lateral face  120  of the lyophilization compartment  112 . The access port  122  is sealed with a microporous hydrophobic membrane barrier  124  to maintain sterility.  
         [0032]    [0032]FIG. 3 illustrates the collapsible container  100  with a lyophilization compartment subcomponent in its collapsed state after completion of vacuum processing.  
         [0033]    [0033]FIG. 4 is an embodiment of a fully collapsible container  200 , in which the cavity therein is divided into two compartments: a lyophilization compartment  212  and a vacuum-processing compartment  214 . Each compartment  212 ,  214  is bounded by six walls comprised of an upper face  216 , a lower face  218 , and four lateral faces  220 . The upper face  216  of the lyophilization compartment  212  is fabricated entirely or in part with a flexible controlled pore membrane with hydrophobic surfaces, and the four lateral faces  220  are fabricated with a flexible material that is capable of compression under vacuum pressure and of maintaining a barrier between an internal vacuum and the external atmosphere. An access port  222  is located on a lateral face  220  of the lyophilization compartment  212 . The access port  222  is sealed with a microporous hydrophobic membrane barrier  224  to maintain sterility. The upper face  216  of the lyophilization compartment  212  and the lower face  218  of the vacuum-processing compartment  214  serve as a common wall between the lyophilization compartment  212  and the vacuum-processing compartment  214 . The upper face  216  of the vacuum-processing compartment  214  and the lower face  218  of the lyophilization compartment  212  are fabricated with a rigid material as described above. The lateral faces  220  of the vacuum-processing compartment  214  are fabricated with a flexible material as described above. The vacuum-processing compartment  214  contains an exit port  226  therein and is sealed with a barrier  228  to maintain sterility. In order to provide mechanical strength sufficient to retain the vacuum-processing compartment  214  from collapse under the vacuum pressure, the outside of each lateral face  220  of the vacuum-processing compartment  214  has a tabular structure  230  affixed thereto capable of attaching reversibly to an external mechanical restraint  232 . After lyophilization, the external mechanical restraints  232  can be released, allowing the container  200  to compress to a minimal volume for storage or transport.  
         [0034]    Two alternatives to external restraints  232 , described above, are to use internal restraints that are capable of providing mechanical strength sufficient to prevent the lower face  218  of the vacuum-processing compartment  214  from coming in contact with the flexible controlled pore membrane of the lyophilization compartment  216 .  
         [0035]    [0035]FIG. 5 is an embodiment of a fully collapsible closed container  300  that uses an internal honeycomb-like open cell plastic structure to prevent contact between the upper face  316  and the lower face  318  of the vacuum-processing compartment  314 . The mesh shape need not be honeycomb-like but can have any mesh configuration. The collapsible closed container  300  contains a cavity therein divided into two compartments: a lyophilization compartment  312  and a vacuum-processing compartment  314 . Each compartment  312 ,  314  is bounded by six walls comprised of an upper face  316 , a lower face  318 , and four lateral faces  320 . The upper face  316  of the lyophilization compartment  312  is fabricated entirely or in part with a flexible controlled pore membrane with hydrophobic surfaces, and the four lateral faces  320  are fabricated with a flexible material that is capable of compression under vacuum pressure and of maintaining a barrier between an internal vacuum and the external atmosphere. An access port  322  is located on a lateral face  320  of the lyophilization compartment  312  and is sealed with a microporous hydrophobic membrane barrier  324  to maintain sterility. The upper face  316  of the lyophilization compartment  312  and the lower face  318  of the vacuum-processing compartment  314  serve as a common wall between the lyophilization compartment  312  and the vacuum-processing compartment  314 . The upper face  316  of the vacuum-processing compartment  314  and the lower face  318  of the lyophilization compartment  312  are fabricated with a rigid material as described above. The lateral faces  320  of the vacuum-processing compartment  314  are fabricated with a flexible material as described above. The vacuum-processing compartment  314  contains an exit port  326  therein and is sealed with a barrier  328  to maintain sterility. An internal restraint  330 , fabricated from a honeycomb-like open cell plastic structure, is attached to the lower face  318  of the first distal vacuum-processing compartment  314 .  
         [0036]    [0036]FIG. 6 is an embodiment of a fully collapsible closed container  400  that uses raised crisscrossing plastic bumps to prevent contact between the upper and lower faces  416 ,  418  of the vacuum-processing compartment  414 . The collapsible closed container  400  contains a cavity therein divided into two compartments: a lyophilization compartment  412  and a vacuum-processing compartment  414 . Each compartment  412 ,  414  is bounded by six walls comprised of an upper face  416 , a lower face  418 , and four lateral faces  420 . The upper face  416  of the lyophilization compartment  412  is fabricated entirely or in part with a flexible controlled pore membrane with hydrophobic surfaces, and the four lateral faces  420  are fabricated with a flexible material that is capable of compression under vacuum pressure and of maintaining a barrier between an internal vacuum and the external atmosphere. An access port  422  is located on a lateral face  420  of the lyophilization compartment  412  and is sealed with a microporous hydrophobic membrane barrier  424  to maintain sterility. The upper face  416  of the lyophilization compartment  412  and the lower face  418  of the vacuum-processing compartment  414  serve as a common wall between the lyophilization compartment  412  and the vacuum-processing compartment  414 . The upper face  416  of the vacuum-processing compartment  414  and the lower face  418  of the lyophilization compartment  412  are fabricated with a rigid material as described above. The lateral faces  420  of the vacuum-processing compartment  414  are fabricated with a flexible material as described above. The vacuum-processing compartment  414  contains an exit port  426  therein and is sealed with a barrier  428  to maintain sterility. A raised internal restraint  430 , composed of a pattern of crisscrossing plastic “bumps,” is attached to the lower face  418  of the distal vacuum-processing compartment  414 .  
         [0037]    [0037]FIG. 7 is an embodiment of a semi-collapsible closed container  500 , in which the cavity therein is divided into three compartments: a central, lyophilization compartment  512  and two distal vacuum-processing compartments  514 . Each compartment  512 ,  514  is bounded by six walls comprised of an upper face  516 , a lower face  518 , and four lateral faces  520 . The upper face  516  and lower face  518  of the central lyophilization compartment  512  are fabricated entirely or in part with a flexible controlled pore membrane with hydrophobic surfaces and the four lateral faces  520  are fabricated with a flexible material. An access port  522  is located on a lateral face  520  of the central lyophilization compartment  512  and is sealed with a microporous hydrophobic membrane barrier  524  to maintain sterility. The upper face  516  and lower face  518  of the central lyophilization compartment  512 , the lower face  518  of the first distal vacuum-processing compartment  514 , and the upper face  516  of the second distal vacuum-processing compartment  514  serve as a common wall between the first and second distal vacuum-processing compartments  514  and the central lyophilization compartment  512 . The lateral faces  520  of the distal vacuum-processing compartments  514 , the upper face  516  of the first distal vacuum-processing compartment  514 , and the lower face  518  of the second distal vacuum-processing compartment  514  are fabricated with a rigid material as described above. Each distal vacuum-processing compartment  512 ,  514  contains an exit port  526  therein and is sealed with a barrier  528  to maintain sterility. Each exit port  526  is connected to a vacuum and condenser system (not shown).  
         [0038]    [0038]FIG. 8 is an embodiment of a fully collapsible integrated three-compartment container  600  having two external restraints  632 . The container  600  has a cavity therein comprised of a central lyophilization compartment  612  and two distal vacuum-processing compartments  614 . Each compartment  612 ,  614  is bounded by six walls comprised of an upper face  616 , a lower face  618 , and four lateral faces  620 . The upper face  616  and lower face  618  of the central lyophilization compartment  612  are fabricated entirely or in part with a flexible controlled pore membrane with hydrophobic surfaces and the four lateral faces  620  are fabricated with a flexible material. An access port  622  is located on a lateral face  620  of the central lyophilization compartment  612  and is sealed with a microporous hydrophobic membrane barrier  624  to maintain sterility. The upper face  616  and lower face  618  of the central lyophilization compartment  612 , the lower face  618  of the first distal vacuum-processing compartment  614 , and the upper face  616  of the second distal vacuum-processing compartment  614  serve as a common wall between the first and second distal vacuum-processing compartments  614  and the central lyophilization compartment  612 . The lateral faces  620  of the distal vacuum-processing compartments  614 , the upper face  616  of the first distal vacuum-processing compartment  614 , and the lower face  618  of the second distal vacuum-processing compartment  614  are fabricated with a rigid material as described above. Each distal vacuum-processing compartment  614  contains an exit port  626  therein and is sealed with a barrier  628  to maintain sterility. Each exit port  626  is connected to a vacuum and condenser system (not shown). The outside of each lateral face  620  of the distal vacuum-processing compartments  614  has a tabular structure  630  affixed thereto capable of attaching reversibly to a mechanical external restraint  632  that provides mechanical strength sufficient to retain the distal vacuum-processing compartments  614  from collapse under the vacuum pressure. After lyophilization, the mechanical restraints  632  can be released allowing the container  600  to compress to a minimal volume for storage or transport.  
         [0039]    [0039]FIG. 9 is an embodiment of a fully collapsible integrated three-compartment container  700  having four external restraints  732 . The container  700  has a cavity therein comprised of a lyophilization compartment  712  and two distal vacuum-processing compartments  714 . Each compartment  712 ,  714  is bounded by six walls comprised of an upper face  716 , a lower face  718 , and four lateral faces  720 . The upper face  716  and the lower face  718  of the central lyophilization compartment  712  are fabricated entirely or in part with a flexible controlled pore membrane with hydrophobic surfaces and the four lateral faces  720  are fabricated with a flexible material. An access port  722  is located on a lateral face  720  of the central lyophilization compartment  712  and is sealed with a microporous hydrophobic membrane barrier  724  to maintain sterility. The upper face  716  and the lower face  718  of the central lyophilization compartment  712 , the lower face  718  of the first distal vacuum-processing compartment  714 , and the upper face  716  of the second distal vacuum-processing compartment  714  serve as a common wall between the first and second distal vacuum-processing compartments  714  and the central lyophilization compartment  712 . The lateral faces  720  of the distal vacuum-processing compartments  714 , the upper face  716  of the first distal vacuum-processing compartment  714 , and the lower face  718  of the second distal vacuum-processing compartment  714  are fabricated with a rigid material as described above. Each distal vacuum-processing compartment  714  contains an exit port  726  therein and is sealed with a barrier  728  to maintain sterility. Each exit port  726  is connected to a vacuum and condenser system (not shown). The outside of each lateral face  720  of each distal vacuum-processing compartment  714  has affixed thereto two tabular structures  730 . Each pair of tabular structures  730  is capable of attaching reversibly at its end to a mechanical restraint  732  that provides mechanical strength sufficient to retain the distal vacuum-processing compartments  714  from collapse under the vacuum pressure.  
         [0040]    [0040]FIG. 10 is an embodiment of a fully collapsible integrated three-compartment container  800  having an internal restraint  830  that can be fabricated of a honeycomb-like open cell plastic structure. The container  800  has a cavity therein comprised of a lyophilization compartment  812  and two distal vacuum-processing compartments  814 . Each compartment  812 ,  814  is bounded by six walls comprised of an upper face  816 , a lower face  818 , and four lateral faces  820 . The upper face  816  and the lower face  818  of the central lyophilization compartment  812  are fabricated entirely or in part with a flexible controlled pore membrane with hydrophobic surfaces, and the four lateral faces  820  are fabricated with a flexible material. An access port  822  is located on a lateral face  820  of the central, lyophilization compartment  812  and is sealed with a microporous hydrophobic membrane barrier  824  to maintain sterility. The upper face  816  and the lower face  818  of the central lyophilization compartment  812 , the lower face  818  of the first distal vacuum-processing compartment  814 , and the upper face  816  of the second distal vacuum-processing compartment  814  serve as a common wall between the first and second distal vacuum-processing compartments  814  and the central lyophilization compartment  812 . The lateral faces  820  of the distal vacuum-processing compartments  814 , the upper face  816  of the first distal vacuum-processing compartment  814 , and the lower face  818  of the second distal vacuum-processing compartment  814  are fabricated with a rigid material as described above. Each distal vacuum-processing compartment  814  contains an exit port  826  therein and is scaled with a barrier  828  to maintain sterility. Each exit port  826  is connected to a vacuum and condenser system (not shown). One internal restraint  830  is attached to the lower face  818  of the first distal vacuum-processing compartment  814 , and another internal restraint  830  is attached to the upper face  816  of the second distal vacuum-processing compartment  814 .  
         [0041]    [0041]FIG. 11 is an embodiment of a fully collapsible integrated three-compartment container  900  having an internal restraint  930  made of raised crisscrossing plastic “bumps.”The container  900  has a cavity therein comprised of a lyophilization compartment  912  and two distal vacuum-processing compartments  914 . Each compartment  912 ,  914  is bounded by six walls comprised of an upper face  916 , a lower face  918 , and four lateral faces  920 . The upper face  916  and the lower face  918  of the central lyophilization compartment  912  are fabricated entirely or in part with a flexible controlled pore membrane with hydrophobic surfaces and the four lateral faces  920  are fabricated with a flexible material. An access port  922  is located on a lateral face  920  of the central lyophilization compartment  912  and is sealed with a microporous hydrophobic membrane barrier  924  to maintain sterility. The upper face  916  and the lower face  918  of the central lyophilization compartment  912 , the lower face  918  of the first distal vacuum-processing compartment  914 , and the upper face  916  of the second distal vacuum-processing compartment  914  serve as a common wall between the first and second distal vacuum-processing compartments  914  and the central lyophilization compartment  912 . The lateral faces  920  of the distal vacuum-processing compartments  914 , the upper face  916  of the first distal vacuum-processing compartment  914 , and the lower face  918  of the second distal vacuum-processing compartment  914  are fabricated with a rigid material as described above. Each distal vacuum-processing compartment  914  contains an exit port  926  therein and is sealed with a barrier  928  to maintain sterility. Each exit port  926  is connected to a vacuum and condenser system (not shown). One raised internal restraint  930  lies on top of the lower face  918  of the distal vacuum-processing compartment  914  and can be composed of a pattern of crisscrossing plastic “bumps.” 
         [0042]    [0042]FIG. 12 is an embodiment of a non-collapsible closed container  1000  in which the cavity therein is divided into three compartments: a central lyophilization compartment  1012  and two distal vacuum-processing compartments  1014 . Each compartment  1012 ,  1014  is bounded by six walls comprised of an upper face  1016 , a lower face  1018 , and four lateral faces  1020 . The upper face  1016  and the lower face  1018  of the central lyophilization compartment  1012  are fabricated entirely or in part with a flexible controlled pore membrane with hydrophobic surfaces. An access port  1022  is located on a lateral face  1020  of the central lyophilization compartment  1012  and is sealed with a microporous hydrophobic membrane barrier  1024  to maintain sterility. The upper face  1016  and the lower face  1018  of the central lyophilization compartment  1012 , the lower face  1018  of the first distal vacuum-processing compartment  1014 , and the upper face  1016  of the second distal vacuum-processing compartment  1014  serve as a common wall between the first and second distal vacuum-processing compartments  1014  and the central lyophilization compartment  1012 . The lateral faces  1020  of the three compartments  1012  and  1014 , the upper face  1016  of the first distal vacuum-processing compartment  1014 , and the lower face  1018  of the second distal vacuum-processing compartment  1014  are fabricated with a rigid material as described above. Each distal vacuum-processing compartment  1014  contains an exit port  1026  therein and is sealed with a barrier  1028  to maintain sterility. Each exit port  1026  is connected to a vacuum and condenser system (not shown).  
         [0043]    Depending on the needs of the user, additional sample processing steps might be needed before sample use. For example, successful lyophilization of cells often requires high concentrations of materials, such as mannitol or trehalose, for their cryosurvival. It often would be useful to remove those low molecular weight materials from the desired cells before use.  
         [0044]    To accomplish this, a further embodiment of the present invention is illustrated in FIG. 13 in which a semi-collapsible integrated closed container  1100  is comprised of five compartments: a central lyophilization compartment  1112 , two distal vacuum-processing compartments  1114 , and two external compartments  1134 . Each compartment  1112 ,  1114 ,  1134  is bounded by six walls comprised of an upper face  1116 , a lower face  1118 , and four lateral faces  1120 . The upper face  1116  and the lower face  1118  of the central lyophilization compartment  1012  are fabricated entirely or in part with a flexible controlled pore membrane with hydrophobic surfaces and the four lateral faces  1120  are fabricated with a flexible material. The upper face  1116  and the lower face  1118  of the central lyophilization compartment  1112 , the lower face  1118  of the first distal vacuum-processing compartment  1114 , and the upper face  1116  of the second distal vacuum-processing compartment  1114  serve as a common wall between the first and second distal vacuum-processing compartments  1114  and the central lyophilization compartment  1112 . The lateral faces  1120  of the distal vacuum-processing compartments  1114 , the upper face  1116  of the first distal vacuum-processing compartment  1114  and the external compartments  1134 , and the lower face  1118  of the second distal vacuum-processing compartment  1114  and the external compartments  1134  are fabricated with a rigid material as described above. Two lateral faces  1120  of the central lyophilization compartment  1112  are fabricated with a flexible material. An access port  1122  is located on the outer lateral faces  1120  of the external compartments  1134  and is sealed with a microporous hydrophobic membrane barrier  1124  to maintain sterility. The inner lateral faces  1120  of the external compartments  1134  serve as common walls between the external compartments  1134  and the two lateral faces  1120  of the central lyophilization compartment  1112  and are fabricated with a porous surface containing pores which are initially filled with an erodible or otherwise removable pore plugging substance as further described in U.S. Pat. Nos. 5,026,342 and 5,261,870, incorporated herein by reference.  
         [0045]    Many simple variations of this invention will be apparent to those skilled in the art. For example, increasing or decreasing the number of vacuum-processing compartments and alteration of the hydrophobic membrane materials will affect the rate of water removal and change the surface area through which water vapor will pass. A wide variety of materials can be used for construction of the container, allowing fabrication of unique containers with exceptional pliability, low weight, chemical reactivity, enhanced compatibility with biological materials, optical properties, and other physical properties. Depending upon the needs of the user, for instance, if storage and transport of the biological material is not required, the outer walls of the container can be fabricated with a rigid material instead of more pliable materials, thus eliminating the use of mechanical restraints during lyophilization.  
       EXAMPLE 1  
     Lyophilization and Storage of Blood Plasma under Vacuum or Inert Gas Conditions  
       [0046]    The lower compartment is filled via its access port with 15 ml of bovine blood plasma recovered from the blood fractionation process, after which the access port is sealed. External mechanical restraints are attached to tabs located on the exterior surfaces of the compartments in order to provide mechanical strength sufficient to retain the compartments from collapse under the vacuum pressure. The filled lower compartment is cooled to −20° C. or colder to freeze the water within. The exit port of the upper compartment, protected by barriers to assure maintenance of sterility, is connected to a vacuum and condenser system (not shown), and the water is removed as vapor without thawing the blood plasma in the lower compartment. After the water is removed, the exit port from the upper compartment to the vacuum is sealed. Alternatively, the lower compartment can be filled with inert gases according to the needs of the user while under vacuum. The external restraints are then released, thus allowing the compartments to collapse to a minimal volume. The sample is stored under conditions suitable for blood plasma. When appropriate, the lyophilized blood plasma in the lower compartment is rehydrated by the addition of sterile water via the access port, in which the vacuum therein allows “self filling” to the maximum volume of the compartment.  
       EXAMPLE 2  
     Container for Lyophilization, Storage, and Processing of Cell Suspensions after Rehydration  
       [0047]    The lower compartment is filled via its access port with a cell suspension containing cryoprotectant materials that are essential for cellular survival during lyophilization, such as mannitol or trehalose, after which the access port is sealed. External mechanical restraints are attached to tabs located on the exterior surfaces of the compartments in order to provide mechanical strength sufficient to retain the compartments from collapse under the vacuum pressure. The filled lower compartment is cooled to −20° C. or colder to freeze the water within. The exit port of the upper compartment, protected by barriers to assure maintenance of sterility, is connected to a vacuum and condenser system (not shown), and the water is removed as vapor without thawing the blood plasma in the lower compartment. After the water is removed, the exit port from the upper compartment to the vacuum is sealed. The external restraints are then released, thus allowing the compartments to collapse to a minimal volume. The sample is stored under conditions suitable for the specific cell suspension. When appropriate, the lyophilized cell suspension in the lower compartment is rehydrated by the addition of sterile water or buffer medium via the access port in which the vacuum therein allows “self filling” to the maximum volume of the compartment. In order to remove the cryoprotectant materials after storage and before use, the external compartments are activated and used as further described in U.S. Pat. No. 6,065,294, incorporated herein by reference.  
         [0048]    Although the invention has been described with particularity above, the invention is only to be considered limited insofar as is set forth in the accompanying claims.