Abstract:
An apparatus and method for dehydrating biological materials, such as vaccines and microorganism cultures, in which the materials are dehydrated in an evacuated container which is in a microwave waveguide that is open to the atmosphere. The apparatus comprises means for freezing the container of biological material, a microwave generator, a waveguide, means for introducing the container into the waveguide, means for applying a vacuum to the container and means for removing the dehydrated material from the waveguide. In the method of the invention, the container of biological material is put in a microwave waveguide open to the atmosphere, a vacuum is applied to the container, the material is frozen and is radiated to dehydrate it. The dehydrated material is then removed from the waveguide.

Description:
FIELD OF THE INVENTION 
       [0001]    The invention pertains to apparatuses and methods for microwave vacuum-drying of biological materials, such as vaccines, antibiotics, antibodies enzymes, proteins and microorganism cultures. 
       BACKGROUND OF THE INVENTION 
       [0002]    Many biologically-active materials, such as vaccines, microbial cultures, etc., are dehydrated for purposes of storage. Methods used in the prior art include freeze-drying and air-drying methods such as spray-drying. Dehydration generally lowers the viability of the materials. Freeze-drying allows higher viability levels than air-drying but it requires long processing times and is expensive. It also causes some level of loss of viability in the dried materials. 
         [0003]    It is also known in the art to dehydrate biological and other materials using a resonance chamber type of microwave vacuum dehydrator. This directs microwave energy into a vacuum chamber that serves as a resonance cavity for microwaves. However, particularly where the quantity of material being dried is relatively small, which is commonly the case with biomaterials, controlling the temperature of the material can be difficult. When microwaves are reflected within a resonance chamber, as the material dries the microwave energy output of the apparatus must be absorbed by less and less water and material in the sample. The mass of the material to be processed also has to be matched with the microwave power of the apparatus; quantities of material that are small relative to the microwave power of the apparatus may reach high temperatures when drying because of the abundance of microwave energy absorbed by the material. 
       SUMMARY OF THE INVENTION 
       [0004]    The invention provides an apparatus and method for dehydrating biological materials, in which the materials are dehydrated in an evacuated container which is in a microwave waveguide that is open to the atmosphere. Being open, the waveguide can be air-cooled to avoid overheating of the material. Since the dehydration is done under vacuum, i.e. at a pressure that is less than atmospheric pressure, the boiling point of water is reduced so the evaporation occurs at lower temperatures, minimizing damage to the biological activity of the material being dried. More control of the temperature of the material can be achieved using the invention than using a resonance chamber type of microwave vacuum dehydrator. Very small quantities of material can be processed without overheating. 
         [0005]    According to one embodiment of the invention, the apparatus comprises means for freezing a container of biological material, a microwave generator, a waveguide that is open to the atmosphere, means for introducing the container of biological material into the waveguide, means for applying a vacuum to the container, and means for removing the dehydrated material from the waveguide. 
         [0006]    The apparatus may optionally include means for effecting relative movement between the sample in the waveguide and the microwave field. This may comprise means for moving the container through the waveguide, or means for moving the generator, or means for moving the biological material within the container. The apparatus may optionally include means for removing a cap from the container, and means for sealing the container. 
         [0007]    According to another embodiment of the invention, the apparatus has a waveguide with an input end for the introduction of a microwave-transparent container of a biological material and a discharge end for removal of the container. The apparatus includes means for introducing the container into the input end, means for removing a cap from the container and means for applying a high vacuum (sufficient to cause and/or maintain freezing of the material) to the container. It includes means for moving the evacuated container through the microwave guide from the input end to the discharge end, means for replacing the cap onto the container and means for removing the container from the microwave guide at the discharge end. The apparatus may include a microwave absorbing sink at the end of the waveguide opposite to the generator. 
         [0008]    According to another embodiment of the invention, there is provided a method for dehydrating biological materials. A container is provided holding the biological material to be dehydrated, the container being transparent to microwave radiation. The container is put in a microwave waveguide that is open to the atmosphere. A vacuum is applied to the container. The material is frozen, either by the application of the vacuum or before being put into the waveguide. Microwave radiation is applied to dehydrate the biological material. The dehydrated material is removed from the waveguide. Optionally, the container of dehydrated material is sealed before removal from the waveguide or from the vacuum. 
         [0009]    Where the container of material is capped before it is put into the microwave guide, the method includes removing the cap before applying microwave radiation. The method may optionally include the step of effecting relative movement between the sample in the waveguide and the microwave field. This may be either the step of moving the evacuated container through the microwave waveguide while applying the microwave radiation, or the step of moving the generator. 
         [0010]    The invention accordingly produces containers of dehydrated biological material, having a moisture content as low as, for example, three to four percent or lower. It is particularly suitable for the dehydration of proteins, for example monoclonal antibodies, enzymes and polypeptides. 
         [0011]    These and other features of the invention will be apparent from the following description and drawings of the preferred embodiments. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0012]      FIG. 1  is a side elevation view, partly in section, of an apparatus according to one embodiment of the invention. 
           [0013]      FIG. 2  is a top plan view thereof. 
           [0014]      FIG. 3  is a cross-sectional view of part of the apparatus at the input end, prior to removal of the cap from the vial. 
           [0015]      FIG. 4  is a cross-sectional view of part of the apparatus at the discharge end, prior to replacement of the cap on the vial. 
           [0016]      FIGS. 5 and 6  are flow diagrams of methods of dehydration according to the invention. 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The Dehydrating Apparatus 
       [0017]    The dehydrating apparatus  10  has a support platform  12  with a microwave generator  14 , a circulator  73  and a water sink  16  positioned below the platform  12 . A microwave waveguide  18  above the platform extends between the circulator  73 , and the water sink  16 , passing through spaced-apart bores  20 ,  22  in the platform  12 . The waveguide  18  is supported on the platform  12  by a frame  25 . The waveguide  18  includes a longitudinally-extending section, referred to herein as the treatment section  24 , through which the material to be dehydrated is moved, as described below. 
         [0018]    The treatment section  24  has a bottom wall  40 , side walls  42 ,  44  and an upper wall  46 . A longitudinal slot  49  extends through the upper wall  46 . The interior of the waveguide  18  is accordingly open to the atmosphere. The opening of the slot  49  is surrounded by a microwave choke  51 , for reducing the escape of microwave radiation through the slot. There is a moveable cover (not shown) above the slot and choke to reduce the escape of radiation. The treatment section  24  has a product input end  26 , into which the container of material to be dehydrated is introduced, and a product discharge end  28 , from which the container of dehydrated material is removed. For purposes of the present description of the preferred embodiment, the container is a microwave-transparent vial  38  for containing, for example, a protein. 
         [0019]    A vial-lifting mechanism  30  is affixed to the support platform  12  under the input end  26  of the treatment section  24  of the waveguide. The mechanism comprises an air cylinder  32  with a vial-lifting piston  34 , mounted on the underside of the platform  12 , with the piston  34  extending through a bore in the platform  12 , and a vial-holding platform  36  on the upper end of the piston  34  for holding the vial  38  of material. The treatment section  24  of the waveguide  18  has a port  48  in its bottom wall  40  above the vial-holding platform  34 , for entry of the vial  38  and the vial-lifting platform  36  into the treatment section  24 . 
         [0020]    A vial-lowering mechanism  50  is affixed to the support platform  12  under the product discharge end  28  of the treatment section  24 . This mechanism is structurally the same as the vial-lifting mechanism  30 , and comprises an air cylinder  52  with a vial-lowering piston  54 , extending through a bore in the support platform  12 , and a vial-holding platform  56  on the upper end of the piston  54 . The treatment section  24  of the waveguide  18  has a port  55  in its bottom wall  40  above the vial-holding platform  56 , for removal of the vial from the treatment section  24  after dehydration of the material. A tube  57  extends downwardly around each of the ports  48 ,  55  to reduce leakage of radiation from the waveguide. 
         [0021]    A vial pickup head  58  provides for the transport of the vial  38  through the treatment section  24 . The pickup head  58  has a body  60  affixed to a movable support platform  62 . The platform  62  is arranged for movement along the treatment section  24  of the waveguide by a pickup head moving mechanism  64 . This mechanism comprises a belt drive  66  supported on the frame  25 , parallel to the treatment section  24 , and driven by a motor  68 . The moveable support platform  62  is affixed to the belt drive  66  for movement thereon, such that actuation of the belt drive  66  moves the pickup head  58  along the length of the treatment section  24 . The cover for the waveguide slot can be affixed to, or be an extension of, the support platform  62 . 
         [0022]    The structure of the vial pickup head  58 , best seen in  FIG. 1 , has a body  60  with an upper part  61  and a base part  63 . The upper part  61  has ports which lead respectively to a condenser  65 , a temperature sensor  67  and a vacuum sensor  69  (omitted from  FIGS. 2 to 4  for clarity). The condenser  65  contributes to the condensation of moisture given off from the material during dehydration. The temperature sensor  67  and vacuum sensor  69  respectively measure the temperature and pressure within the vial. The upper part  61  is rotatable on the base part  63  of the pickup head body  60  about a vertical axis, in order to permit the vertical alignment of the respective sensors with the vial, when a measurement is desired. 
         [0023]    The body  60  of the pickup head has a vacuum cavity  70  therein in the form of a cylindrical bore. A vacuum source, condenser and vacuum line (not shown) are connected to a vacuum port  71  in the base part  63  of body  60  of the vial pickup head to provide for the evacuation of the vacuum cavity  70  and removal and condensation of moisture from the material. A vial pickup sleeve  72  is mounted in the vacuum cavity  70  with its upper portion in the vacuum cavity  70  and its lower portion extending through a bore in the pickup head support platform  62  and through the longitudinal slot  49  in the upper wall  46 . The sleeve  72  thus extends into the treatment section  24  of the waveguide  18 . A sealing surface  76  is provided at the bottom edge of the sleeve  72  for airtight sealing engagement with the vial  38 . 
         [0024]    An air cylinder  78  is affixed to the upper part  61  of the pickup head body  60 . It has a piston  80  which extends through a bore  82  in the upper end of the body  60  and into the pickup sleeve  72 . A cap holder  84  at the bottom end of the piston  80  has a circumferential flange  86  shaped and adapted to engage and hold a cap  88  of the vial  38 . 
         [0025]    In order to provide for air-cooling of the vial during the dehydration process, a compressed air line (not shown) may be attached to the pickup head support platform  62 , directing compressed air at the vial  38  through the slot  49  in the upper wall  46  of the treatment section. Alternatively, air vanes may be provided on the lower part of the pickup sleeve  72  to blow air in the waveguide against the vial as it is being spun. 
         [0026]    For freezing of the biological material prior to microwaving, the vacuum system that is provided is one capable of evacuating the container to a pressure less than about 4 mm of mercury, more accurately 4.58 mm of mercury, the triple point pressure of water. Typically, pressures of about 2.5 mm of mercury or less are required, because solutions of biological materials have a lower freezing point than pure water. Alternatively, a freezer such as a liquid nitrogen bath or low temperature freezer (not shown in the drawings) is provided. 
         [0027]    It will be understood that the apparatus  10  also includes appropriate air lines and controls to actuate the air cylinders, a vacuum line and controls to evacuate the vacuum chamber  70 , and controls to operate the drive motor. 
         [0028]    In an alternative embodiment of the apparatus (not shown in the drawings) the microwave generator is mounted on a moveable stand so it can be moved, relative to the sample, during microwaving. In this case, the sample of material is stationary within the waveguide and relative movement between the sample and the microwave field is achieved by moving the generator rather than the sample. Such relative movement evens out the energy field experienced by the sample. 
         [0029]    In another alternative embodiment of the apparatus (not shown in the drawings) the container remains within the waveguide and the biological material is moved through the container. The container is stationary and the material is moved by means such as vibration or gravity. 
       The Methods of Dehydrating 
       [0030]    At the start of a cycle of operation of the dehydrating apparatus  10 , the vial-lifting piston  34  and the vial-lowering piston  54  are both in their retracted positions, such that the vial-holding platforms  36 ,  56  are on the support platform  12 . The pickup head piston  80  is also in its retracted position, such that the cap holder  84  is in its raised position within the body  60  of the pickup head  58 . The pickup head support platform  62  is at the inlet end  26  of the treatment section  24  of the waveguide  18 , with the pickup head  58  vertically aligned with the vial entry port  48 . The vial  38  with material to be dehydrated, e.g. a protein, covered by a cap  88  and at atmospheric pressure, is placed on the vial-holding platform  36 . 
         [0031]    The vial-lifting cylinder  32  is actuated to raise the piston  34  and the vial-holding platform  36 , lifting the vial  38  through the vial entry port  48  into the treatment section  24  of the waveguide, until the shoulder of the vial abuts the sealing surface  76  at the lower end of the vial pickup sleeve  72 . The pickup head air cylinder  78  is then actuated, to lower the pickup head piston  80  and cap holder  84  to engage the cap  88  of the vial. This position of the apparatus is shown in  FIG. 8 . A high vacuum is then applied to the vacuum chamber  70  by means of the vacuum source and line, reducing the absolute pressure in the vacuum chamber to less than about 2.5 mm of mercury, alternatively less than about 0.2 mm of mercury. 
         [0032]    The pickup head air cylinder  78  is then actuated, lifting the cap holder  84  and removing the cap  88  from the vial  38 . This removal is facilitated by the pressure differential between the inside of the vial, which is at atmospheric pressure, and the partial vacuum of the vacuum chamber  70  and pickup sleeve  72 . The cap removal causes a vacuum to be applied to the vial  38 . The vacuum applied through the pickup sleeve  72  causes a seal between the vial and the pickup sleeve  72  at the sealing surface  76 , permitting the vial to be held securely by the pickup sleeve  72 . The vial-lifting cylinder  32  is then actuated to lower the vial-lifting piston  34 , withdrawing the vial-holding platform  36  from the waveguide  18 . 
         [0033]    The application of high vacuum to the container cools the sample below its freezing point. 
         [0034]    The microwave generator  14  is then actuated, causing microwave energy to travel through the waveguide  18  to the water sink  16 . The circulator  73  prevents microwave energy from re-entering the generator. The belt drive motor  68  is actuated, to move the belt drive  66  and accordingly the pickup head support platform  62 . The direction of movement of the support platform  62  is towards the discharge end  28  of the treatment section  24 . The vial  38  remains evacuated. The heating of the biological material by the microwave energy causes dehydration of the material. If desired, the pressure and temperature in the vial can be measured during the dehydration process by means of the sensors  69 ,  67 . The dehydration of the sample is by sublimation, as the ice turns directly to gas. 
         [0035]    At the discharge end  28 , the vial  38  is brought into alignment with the vial removal port  55  in the bottom wall  40  of the treatment section  24  and the belt drive motor  68  is stopped. The microwave generator  14  is deactivated. The air cylinder  52  is actuated to raise the vial-lowering piston  54 , extending the vial-holding platform  56  through the port  55  into the treatment section  24  of the microwave guide so it engages the bottom of the vial  38 . This position is shown in  FIG. 4 . The pickup head air cylinder  78  is actuated to lower the pickup head piston  80 , pushing the cap  88  back onto the vial  38 . The vacuum in the vacuum chamber  70  is then released. This breaks the seal between the pickup sleeve  72  and the vial  38  at the sealing surface  76 , releasing the vial from the grip of the sleeve. The release of vacuum also results in a pressure differential between the inside of the vial, which is at reduced pressure, and the vacuum chamber  70  and pickup sleeve  72 , which are now at atmospheric pressure. The pickup head air cylinder  78  is then actuated, to lift the piston  80  and the cap holder  84 . Due to the pressure differential, the reduced pressure in the vial holds the cap  88  in place on the vial  38  as the cap holder  84  is retracted. The air cylinder  52  is then actuated to lower the vial-holding platform  56 , and with it the vial  38 , withdrawing the vial from the waveguide  18 . The vial can then be manually removed from the apparatus  10 . It is a vacuum sealed, capped vial containing dehydrated material. 
         [0036]    To return the apparatus to the starting condition for processing of a further vial of material, the drive motor  68  is actuated to return the pickup head  58  to the input end  26  of the treatment section  24 . 
         [0037]    The foregoing method can be understood in general terms as comprising the following steps, as illustrated in the flow diagram of  FIG. 5 . In step  100 , the capped container of biological material is loaded into the waveguide. In step  102 , the cap is removed and a high vacuum is applied to the container, causing freezing of the material in step  104 . In step  106 , microwave energy is directed through the waveguide. In step  108 , the container is moved through the waveguide to the outlet end. In step  110 , the container is capped. In step  112  the evacuated container of dehydrated material is removed from the waveguide. 
         [0038]    Instead of capping the container of dehydrated material in the waveguide, the container may alternatively be removed uncapped. Capping would then be done subsequently, after removal of the container from the apparatus. 
         [0039]    Alternatively, the container of material is frozen before processing, for example by placing it in a bath of liquid nitrogen or low temperature freezer. The frozen material is then processed in the dehydrating apparatus  10 . The step of freezing in this method is thus a preliminary step before dehydrating the biological material in the apparatus. This method is illustrated in the flow diagram of  FIG. 6 . In step  99 , the container of material is frozen in liquid nitrogen. The frozen material is then loaded into the waveguide in step  101 . In step  103 , the cap is removed and a vacuum is applied, typically less than 2.5 mm of mercury. This low pressure keeps the material frozen during microwaving. The material is then processed with steps  106 ,  108 ,  110  and  112 . 
         [0040]    Alternatively, the vial may be kept stationary while the microwave field is moved about it, for example by moving the microwave generator relative to the sample. 
         [0041]    Dehydration of biological materials can also be achieved without the step of moving the container through the waveguide, or moving the generator. Movement equalizes the field to which the material is exposed. Without such movement, it is necessary that the intensity of microwave energy at the fixed position of the container in the waveguide be appropriate for the sample. The steps of this method can comprise the steps illustrated in the flow charts of  FIG. 5  or  6 , omitting step  108  of moving the container. 
       Example 1 
       [0042]    An apparatus according to the invention has a microwave generator having a power output of 900 watts, a water sink and a microwave guide extending between them. The guide has a treatment section approximately 33 cm long, with a channel that is rectangular in cross-section approximately 5.25 cm high and 10.9 cm wide. The slot in the upper wall of the treatment section is approximately 2.8 cm wide and is surrounded by a microwave choke. 
       Example 2 
       [0043]      Lactobacillus salivarius  stationary phase cells were mixed with 10% skim milk powder and divided into aliquots of 0.5 ml and were frozen at −80° C. freezer for one day and then dried in accordance with the invention (100-700 W, 19-21 minutes, vacuum of 2 mm mercury). The final viable cells were counted by plating dilutions series on petrifilm after 48 hours anaerobic incubation at 37° C. The percent of colony-forming units that survived dehydration were 52.2±9.67%. The moisture content of the dehydrated material was 3.48±1.23%. 
       Example 3 
       [0044]    A 10% lysozyme solution was prepared using powder enzyme and sterile distilled water. An aliquot of 0.5 ml of 10% enzyme was poured into a container and was frozen at −80° C. for two hours. Frozen samples were dried in accordance with the invention (800 W, vacuum 2 mm mercury, 27 minutes dehydration time). The activity of enzyme before and after drying was measured using Shugar method. 
         [0045]    Activity and moisture of 10% lysozyme before and after dehydration 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 Enzyme activity Shugar unit/mg 
                   
               
             
          
           
               
                 Before 
                   
                 Final Moisture 
               
               
                 Treatment 
                 After Treatment 
                 Content 
               
               
                   
               
               
                 13232 ± 876 
                 14133 ± 2584 
                 2-5% 
               
               
                   
               
             
          
         
       
     
         [0046]    Although the invention has been described in terms of various embodiments, it is not intended that the invention be limited to these embodiments. Various modifications within the scope of the invention will be apparent to those skilled in the art. 
       LIST OF COMPONENTS IN THE DRAWINGS 
       [0000]    
       
           10  dehydrating apparatus 
           12  support platform 
           14  microwave generator 
           16  water sink 
           18  microwave waveguide 
           20 ,  22  bores in platform  12  for the waveguide 
           24  treatment section of the waveguide 
           25  frame 
           26  input end of treatment section 
           28  discharge end of treatment section 
           30  vial-lifting mechanism 
           32  vial-lifting air cylinder 
           34  vial-lifting piston 
           36  vial-holding platform 
           38  vial 
           40  bottom wall of treatment section 
           42 ,  44  side walls of treatment section 
           46  upper wall of treatment section 
           48  vial entry port 
           49  longitudinal slot in upper wall of treatment section 
           50  vial-lowering mechanism 
           51  microwave choke 
           52  vial-lowering air cylinder 
           54  vial-lowering piston 
           55  vial-removal port 
           56  vial-holding platform 
           57  tubes below vial ports 
           58  vial-pickup head 
           60  body of vial-pickup head 
           61  swivelling part of  60   
           62  pickup head support platform 
           63  base part of  60   
           64  pickup head moving mechanism 
           65  condenser 
           66  belt drive 
           67  temperature sensor 
           68  belt drive motor 
           69  vacuum sensor 
           70  vacuum cavity in vial-pickup head 
           71  vacuum port in body  60   
           72  vial-pickup sleeve 
           73  circulator 
           76  sealing surface of pickup sleeve 
           78  air cylinder on pickup head 
           80  piston for air cylinder on pickup head 
           82  bore in top of body  60   
           84  cap holder 
           86  flange on cap holder 
           88  cap of vial