Abstract:
Sample preparation device and method particularly useful for large volume capture and small volume elution. The device comprising a manifold, a sample holder or reservoir, and a filter unit containing chromatography media such that a filtration path is established between the sample holder, the filter unit and the manifold. Upon subjecting the sample in the sample holder to a driving force such as vacuum, the sample flows through the chromatography media in the filter unit. Molecules of interest bind to the media and any unwanted molecules can be washed under vacuum mode. Elution can then be carried out by removing the filter unit and subjecting the media to a driving force such as vacuum or centrifugation. In another embodiment, molecules of interest can be eluted into a low volume centrifugal spinner directly in the manifold.

Description:
[0001]     This application is a divisional of U.S. Ser. No. 11/051,047 filed Feb. 4, 2005, which claims priority of U.S. Provisional Appln. Ser. No. 60/545,671 filed Feb. 18, 2004, the disclosures of which are incorporated herein by reference. 
     
    
     BACKGROUND OF THE INVENTION  
       [0002]     Numerous laboratory devices have been developed to carry out filtration, chromatography and centrifugation in order to concentrate, separate and/or purify laboratory samples.  
         [0003]     Researchers routinely need to concentrate their sample prior to other investigative research. There are two primary approaches to sample concentration: specific capture, (chromatography resin and affinity chemistries) or size exclusion filters.  
         [0004]     For researchers using size exclusion filters, there are limitations they must accommodate in their work. The format of these devices can be either small sample volume centrifugal filters or preparative scale tangential flow separation systems. The preparation systems typically include pumps and gauges and require the user to be skilled and to monitor the process during the separation. A further limitation for the preparative systems is that the final concentrate volume can be larger (50 or more mls). Considering that the researcher&#39;s starting sample may be 250 to 1000 mls, a 50 mls concentrate is a low concentration factor. The centrifugal devices are small in scale and the sample they can conveniently process is small (less then 100 ml) because of the size limitations of the centrifuge rotor. The centrifuge devices but the small starting volume would require the research to monitor the separation and repeatedly refill the filter unit to concentrate the entire starting sample.  
         [0005]     In some cases, the centrifuge device is prepared with a packed column of separation media, chromatography or affinity. These devices similarly suffer by starting volume limitations and require repeated fillings to concentrate samples greater than 100 mls.  
         [0006]     The same limitation for process UF applies to the preparative scale chromatography media systems, in which high cost for pumps and gauges and skilled operators are required and final concentrate volumes are larger than desired.  
         [0007]     Another low cost approach to specific capture systems is to use gravity columns. When using these systems, the researcher has to set up the system so as to achieve a head pressure sufficient to process the sample through the column. This is achieved by placing the sample to be processed in a tank and positioning it above the separation column. Tubes and connectors are used to assemble the typical gravity set up. These set ups may be inexpensive to run, but a major limitation is the processing time which can be long enough so as to require that the process be performed in a cold room to protect the sample from thermal degradation.  
         [0008]     U.S. Pat. No. 4,755,301 discloses a centrifugal method and apparatus for concentrating macromolecules without filtering to dryness. A semi-permeable ultrafiltration membrane separates a sample reservoir from a filtrate cup, and filtrate ducts below the membrane are offset sufficiently inward from the edge of the membrane so that when the apparatus is used in a fixed angle centrifuge rotor, filtration stops once the retentate meniscus reaches the centrifugal radial level of the outermost edge of the outermost filtrate duct.  
         [0009]     Conventional sample preparation devices are limited to relatively small sample volumes, generally about 0.5-80 milliliters.  
         [0010]     These devices have had chromatography media added to them, typically by removing the membrane and replacing it with a more openly porous filter or frit and adding the chromatography media upstream of that filter. In some instances, a top layer of frit may be used to hold the media in the tube during storage and handling. A small sample containing a mixture of components including the desired component (generally a peptide or protein) is added to the sample reservoir and the device is then centrifuged. The desired component typically binds (is captured) to the selected media and all other material and fluid passes through the device to the filtrate reservoir. The device is removed, the filtrate is either dumped or used in further testing and a wash solution is added to the device that removes any unbound material that may have been trapped between the media or left on the surface as the fluid level dropped. This device is removed, the filtrate is dumped (generally a new filtrate cup is attached) and an elution fluid (typically a buffer at different pH or ionic strength that causes the bound desired component to release from the media) is added and spun through the device in the centrifuge. The eluent in the filtrate cup is collected as it contains the released desired component.  
         [0011]     A problem with this type of device is that the volume that can be filtered is limited by the size of the device that can fit within the centrifuge. To filter/capture a large sample such as a liter of serum or tissue culture, one needs to carry out the above process (at least the binding step and often the washing step) multiple times.  
         [0012]     An alternative is to use a chromatography column such as a bench scale or preparative column to process the higher volume. Such devices are expensive, and require columns, holders, pumps and sample and filtrate, wash and eluent tanks. Additionally, many of these columns are prepacked with selected media, limiting one&#39;s choices of the media available. Further, the capacity of the media used in such columns is typically much greater than what is needed for the amount of material to be captured. This means that one often wastes the extra capacity inherent in the column that is an expensive exercise. Alternatively, one can clean and store the column and use it a second time. This involves a large amount of effort to ensure that the media is fully cleaned so that no residual material (desired or contaminant) from the last run is left behind that might adversely affect the results of the next use of the column. Also most media needs to be kept refrigerated and must be kept from being contaminated during storage.  
         [0013]     It would be desirable to provide a device and method for rapid high-quality separations or purifications of samples in a convenient and reliable manner, which can handle sample volumes considerably higher than that handled by conventional centrifugation devices and which effectively uses the correct amount of media and eliminates the need for the washing and storage of columns between runs.  
         [0014]     It is therefore an object of the present invention to provide a sample preparation device that can conveniently and rapidly process relatively large volumes of sample, particularly in a single pass with excellent capture.  
         [0015]     It is another object of the present invention to provide a sample preparation device that can conveniently and rapidly process relatively large volumes of sample and elute the component of interest at a high concentration factor, greater then 50×.  
         [0016]     It is still another object of the present invention to provide a sample preparation device that can conveniently and rapidly process relatively large volumes of sample, without expensive equipment and to provide a system that can reliably process the sample unattended.  
         [0017]     It is a still further object of the present invention to provide a sample preparation device that can process relatively large volumes fast enough so as to not require refrigeration.  
       SUMMARY OF THE INVENTION  
       [0018]     The problems of the prior art have been overcome by the present invention, which provides a sample preparation device and method particularly useful for large volume capture and small volume elution. The device of the present invention combines the favorable aspects of both vacuum filtration and centrifugation to provide a low volume (e.g., less than or equal to about 10 mls) of highly purified sample from a large volume (e.g., about 100 mls to 1 liter or more) of sample, such as tissue culture supernatant typically from a hybridoma monoclonal antibody producing cell line.  
         [0019]     In one embodiment, the present invention provides a sample preparation device comprising a manifold, a sample holder or reservoir, and a filter unit containing chromatography media such that a filtration path is established between the sample holder, the filter unit and the manifold. Upon subjecting the sample in the sample holder to a driving force such as vacuum, the sample flows from the large reservoir through the chromatography media in the filter unit. Molecules of interest bind to the media and can be washed under vacuum mode. Elution can then be carried out by removing the filter unit and subjecting the media to a driving force such as vacuum or centrifugation and collecting the eluted sample. In another embodiment, molecules of interest can be eluted into a low volume centrifugal spinner directly within the manifold and then further concentrated by centrifugation. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0020]      FIG. 1  is a perspective view of the sample preparation assembly in accordance with the present invention;  
         [0021]      FIG. 2  is an exploded view of the assembly of  FIG. 1 , having an ultrafiltration device positioned downstream of the separation device;  
         [0022]      FIG. 3  is a partial exploded view of the assembly of  FIG. 1  ;  
         [0023]      FIG. 4  is a cross-sectional view of a portion of the sample preparation assembly in accordance with an embodiment of the present invention;  
         [0024]      FIG. 5  is a cross-sectional view of a portion of the sample preparation assembly in accordance with another embodiment of the present invention;  
         [0025]      FIG. 6  is a perspective exploded view of the underside of the sample holder in accordance with the present invention;  
         [0026]      FIG. 7  is a perspective view of the assembled underside of the sample holder in accordance with the present invention;  
         [0027]      FIG. 8  is an exploded view of a sample preparation assembly in accordance with an alternative embodiment of the present invention having an ultrafiltration concentrator positioned downstream of the filtration device;  
         [0028]      FIG. 9  is a cross-sectional view of the assembly of FIG.  8 , shown in an assembled condition;  
         [0029]      FIG. 10  is a cross-sectional view of the assembly of  FIG. 8  with the ultrafiltration concentrator removed;  
         [0030]      FIG. 11  is an exploded view of a sample preparation assembly in accordance with an alternative embodiment of the present invention having a collection tube positioned downstream of the filtration device;  
         [0031]      FIG. 12  is a perspective view of the assembly of  FIG. 11  in an assembled condition; and  
         [0032]      FIG. 13  is a cross-sectional view of the assembly of  FIG. 11 . 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0033]     Turning first to  FIG. 1 , in accordance with one embodiment of the present invention there is shown a vacuum manifold  12  having a standard connector  13  for connection to a source of vacuum via suitable hosing or the like. The manifold  12  also can include a vent  14 . The vent  14  can be used by the researcher to throttle back the flow rate through the filtration device  30  to optimize the sample capture. As can be best seen in  FIGS. 4 and 5 , the manifold  12  of this embodiment is generally a solid body having an internal bore  15  that is open at the top  15 A. The bore  15  communicates through passageway  16  with a driving force such as a vacuum source. The bore  15  also communicates through passageway  17  with vent  14 . Preferably the bore  15  is stepped at shoulders  15 B and  15 C as shown, in order to support a filtration device  30  (as discussed in greater detail below). The manifold  12  is preferably made of a material that is sufficiently rigid and strong to withstand the vacuum applied to the device. Further the manifold material should be compatible with the materials being processed. Suitable materials include metal, ceramics and plastics. Preferably the manifold is made from plastics and more preferably the manifold is made from polyolefins such as polypropylene or polyethylene.  
         [0034]     Turning back to  FIGS. 1-3 , the sample holder  20  is a housing having an open top end  21  as shown. In the embodiment shown, the sample holder  20  is a generally cylindrical one-piece housing that can hold relatively large volumes of sample, preferably at least about 50 milliliters, more preferably at least about 100 milliliters, most preferably at least about 500 milliliters. Preferably the sample holder  20  is made of a plastic such as a polyolefin, particularly polypropylene, but is commonly made from polystyrene. The top end  21  preferably has a wide diameter opening in order to facilitate sample transfer into the holder  20 . Preferably the sample holder  20  may include a filter  31  such as a membrane and/or glass fibers for pre-filtering the sample in order to minimize or prevent fouling of the chromatography media in the filtration device downstream of the holder  20 . This is particularly advantageous where it is desired to re-use the filtration device  30 .  
         [0035]     The bottom of the sample holder  20  mates with a collar  25 . Typically, the collar  25  and holder  20  are bonded together as an integral unit. Bonding can be made by a heat bond, sonic weld, adhesive and the like. The collar  25  is preferably cylindrical, and is configured to mate with the vacuum manifold  12  on annular shoulder  12 A ( FIG. 4 ). Collar  25  includes an inner annular ring  26 , preferably centrally located in the collar  25 , which attaches to the protruding member  27  of the manifold  12 . Any suitable means of attachment can be used, such as threads. Preferably the attachment is not permanent, so that the manifold  12  can be re-used with other sample holders and collars. The collar also includes an adaptor  33 , best seen in  FIGS. 2 and 3 . The adaptor  33  is preferably cylindrical and centrally located in the collar  25 , preferably circumscribed by inner annular ring  26 . It includes a sealing element  34  such as an O-ring, for sealing about filtration device  30 . Alternatively, a gasket  36  ( FIG. 6 ) can be used for sealing, which provides a face seal with the open end of the filter unit. The sample holder  20  and collar  25  combination is commercially available from Millipore Corporation and is sold under the Stericup™ or Steritop™ name.  
         [0036]     The filtration device  30  is preferably a one-piece housing made from a plastic material such as a polyolefin, particularly polypropylene. It is generally cylindrical, with an upper portion  38  defining a sample chamber, larger enough to contain the required elution volume, that converges to a smaller, generally cylindrical lower portion that contains the chromatography media. The lower portion  39  terminates in a spout  40  defining a fluid outlet for the device.  
         [0037]     The device  30  preferably includes chromatography media  45  ( FIGS. 4 , and  5 ), the formats of which are not particularly limited. Basically, any media used in peptide and/or protein recovery can be used in this device.  
         [0038]     For example, suitable media include functional composite structures comprising resin particles derivatized with functional groups including styrenedivinyl-benzene-based media (unmodified or derivatized with e.g., sulphonic acids, quaternary amines, etc.); silica-based media (unmodified or derivatized with C 2 , C 4 , C 6 , C 8 , or C 18  or ion exchange functionalities), to accommodate a variety of applications for peptides, proteins, nucleic acids, and other organic compounds.  
         [0039]     Additionally, media formed of polysaccharides such as agarose, crosslinked agarose or dextran or of trisacyrl polymers can be used alone or can be used with various capture chemistries attached to them such as ligands including but limited to Protein A, Protein G, Protein G and the like. Further examples include paramagnetic particles that contain a capture chemistry. Likewise one can utilize controlled pore glass alone or with a ligand such as Protein A (including ProSep®A controlled pore glass media available from Millipore Corporation of Billerica, Mass.).  
         [0040]     Those skilled in the art will recognize that a wide variety of matrices with alternative selectivities (e.g., hydrophobic interaction media, ion exchange media, reverse phase media, affinity media (e.g., Protein A, Protein G, Protein L, boronate affinity resins), etc.) also can be used, especially for classes of molecules other than peptides.  
         [0041]     Stacked membranes are also suitable as chromatography media. Suitable devices may incorporate a plurality of composite porous structures having materials with different functional groups to fractionate analytes that vary by charge, size, affinity and/or hydrophobicity, and include stacked filters such as glass fiber disc, surface charged membranes or membrane with affinity molecules coupled to the membrane surface.  
         [0042]     The term “particles” as used herein is intended to encompass particles having regular (e.g., spherical) or irregular shapes, as well as shards, fibers and powders and optionally including capture chemistries as mentioned above. These particles may be contained between glass, metal or plastic frit or glass mats or plastic non-wovens as is well known in chromatography packing. Alternatively, they may be packed into a chromatography packet that can then be inserted into the device. The filtration device  30  is configured to be slidably received in the bore  15  of the manifold  12 . The top rim  37  of the filtration device may include an annular flange that sits on the top shoulder of the manifold  12 , as seen in  FIGS. 4 and 5 . The base of the upper portion  38  of the device  30  seats on a sealing means  46  positioned on shoulder  15 B of the manifold  12 . Suitable sealing means include an o-ring, flat elastomeric gaskets, or the like, to couple the filtration device  30  and the manifold  12  to create a flow path for the sample. When the device  30  is so positioned in the bore  15  of the manifold, there is sufficient space below the outlet of the device to allow fluid to flow to waste (as in the embodiment of  FIGS. 3 and 4 ), or to allow for the positioning of a second device such as a centrifugal filter unit (as in the embodiment of  FIGS. 2 and 5 ) to further concentrate and desalt the eluted sample, for example. In this latter embodiment, a suitable centrifugal filter unit  50 , such as an Amicon® Ultra unit containing an Ultracel® membrane, commercially available from Millipore Corporation, can be used. The centrifuge device is placed below the filter unit after the binding and the washing steps are complete. It is positioned to collection the eluant that may be further concentrated prior to analysis or use.  
         [0043]     The flow rate of sample through the device can be controlled in a number of ways. For example, an air leak can be introduced, such as via vent  14  to reduce the vacuum pressure applied to the filter device  30  thereby slowing the flow rate through the device. Alternatively, the packing of the chromatographic media in the filtration device  30 , and/or the filter in the sample holder  20 , can be modified to control flow.  
         [0044]     In operation, the device is assembled with a filtration device  30  positioned in the manifold and the sample holder  20  and collar  25  sealingly positioned over the manifold. Sample is added to the sample holder, and vacuum is applied to the device. The sample flows into the filtration device  30  (preferably after passing through the pre-filter in the sample holder  20 ), and molecules of interest bind to the media in the filtration device. Molecules that are not of interest pass through the device and are directed to waste or a collection vessel. The bound molecules optionally can then be washed by introducing a suitable wash solution into the sample holder  20 , again with the application of vacuum. The filtration device can be removed from the manifold and subjected to further processing, such as centrifugation to elute the molecules of interest.  
         [0045]     In another embodiment, a centrifugal device  60  ( FIG. 5 ), such as an ultrafiltration centrifugual device, is positioned in the manifold downstream of the filtration device such as after the bind and wash steps and prior to the elution step. Fluid flowing out of the filtration device is received by the centrifugal device for further processing and analysis.  
         [0046]      FIGS. 8-13  illustrate another embodiment wherein the manifold  12 ′ is a bottle or similar housing adapted to be used in communication with a source of vacuum. The sample reservoir  20 , (which includes a fitting  44  connectable to a vacuum source), and the filtration device  30 , as well as the optional centrifugal device  60 , are as described above and are shown in the assembled condition in  FIG. 12 . The manifold  12 ′ includes a volume sufficient to contain the filtration device  30  and optional centrifugal device  60 , as best seen in  FIG. 9 . In the embodiment shown, the manifold  12 ′ includes an upper lip  62  having external threads  63  for sealingly engaging corresponding inner threads in the inner annular ring  26  of the collar  25 . Those skilled in the art will appreciate that other means of sealing attaching the manifold  12 ′ to the holder  20  are within the scope of the present invention. The upper lip  62  has an outer diameter smaller that the outer diameter of the annular flange  31  of the filtration device  30 , so that the annular flange  31  can seat on the top surface of the upper lip  62  as shown in  FIG. 9 . An adapter  33 ′ seals the filtration device  30  to the sample holder  20  as shown.  
         [0047]     In the particular embodiment of  FIG. 10 , the centrifugal device  60  is not used, and filtrate from the filtration device  30  simply collects in the manifold  12 ′. Depending on the application, this filtrate may be used or discarded.  
         [0048]     Suitable materials for the manifold  12 ′ include stainless steel, glass, plastics preferably polyolefins such as polyethylene and polypropylene, but most typically polystyrene.  
         [0049]      FIGS. 11 and 13  illustrate an embodiment wherein the eluant from the filtration device  30  is collected in a collection tube  70 . The eluted sample so collected can be stored or further processed.