Abstract:
An aseptic sampling system includes substantially sealed sampling lines connecting one or more sample container to a vessel containing the fluid to be sampled. A flow control system includes valves and/or clamps placed strategically along the sampling lines and filtered vent lines are provided at strategic locations relative to a reversible fluid pump in communication with the sampling lines. The clamps are selectively actuated, the vent lines are selectively opened or closed, and the pump is operated in a forward or reverse direction to permit a volume of sample fluid to be drawn from the vessel and into a sample container and to permit fluid to be purged from the sampling lines either into the sample container or back into the vessel. Thus, sampling and purging can be accomplished without opening the system to potentially contaminating agents and without wasting or having to properly dispose the fluid.

Description:
PRIORITY CLAIM 
       [0001]    This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application No. 61/161,831, filed Mar. 20, 2009, the disclosure of which is hereby incorporated by reference in its entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The invention relates to mammalian cell culture or microbial fermentation and, more particularly, to systems and methods for aseptically withdrawing discrete samples of culture material from a vessel containing such material. 
       BACKGROUND 
       [0003]    Cell culture (mammalian cell culture) procedures using a bioreactor or fermentation (microbial cell culture) procedures using a fermentor or fermentation vessel embody techniques for growing and proliferating unit cells separate from an organism and is widely used in biology, medical science, pharmacy, and agriculture. Additionally, the use of biological cultivation procedures has expanded into other disciplines, such as the treatment of waste water or oil. 
         [0004]    Apparatuses designed for cultivation of microbial organisms or eukaryotic cells, known as bioreactors or fermentors, have been used for production of various biological or chemical products in the pharmaceutical, biotechnological and beverage industry. A typical bioreactor includes a vessel for containing culture medium in a sterile environment that provides the various nutrients required to support growth of the homogeneous biological agents of interest. 
         [0005]    Effective cell culture process requires appropriate supplies of nutrient substances, such as glutamine, glucose, and other medium components, and gas, such as oxygen and carbon dioxide, for the growing cells in a bioreactor. In addition, timely control of physiological conditions, such as appropriate pH, temperature, and osmolarity is required for mass cell culture production. In order to provide optimal culture conditions in a bioreactor, rapid and effective mixing in the culture medium is prerequisite, and cells should be uniformly dispersed throughout the culture medium without aggregation in any portion of the cultivation vessel. 
         [0006]    During a cell culture process, aseptic withdrawal of a culture broth sample that is representative of the overall cell culture condition is critical for monitoring the performance of the cell culture or fermentation process and for troubleshooting any process problems. The aseptic sampling step is also applicable in medium batching and holding vessels, for which maintaining the desired dissolved carbon dioxide level can be critical to ensuring the proper pH of the cell culture medium. Conventional sample withdrawal from a bioreactor, fermentor, or medium holding vessel, however, is typically performed by a series of manual operations, including purging the sampling line, connecting a sample device aseptically to the line, removing the sample from the bioreactor, and closing the line. The purge step is usually required at the beginning of each sampling step to flush the residual sample in the sampling line from the previous sampling into a waste reservoir. The conventional sample withdrawal procedure results in waste of sample held up in the main sampling line and requires an additional step to switch the sampling line between the waste reservoir and the actual sample container. The conventional sampling procedure also creates the additional step of properly disposing the flushed material. 
       SUMMARY 
       [0007]    Aspects of the invention are embodied in a system for withdrawing discrete fluid samples from a vessel. The system includes a main sampling line in fluid communication with the vessel, a pump in fluid communication with the main sampling line and adapted to selectively pump fluid from in the main sampling line in a first direction away from the vessel or a second direction toward the vessel, a first vent port in fluid communication with the main sampling line and disposed on a first side of the pump, a second vent port in fluid communication with the main sampling line and disposed on a second side of the pump, one or more sample containers in fluid communication with a portion of the main sampling line on the second side of the pump, and a flow control system adapted to be selectively configured to open or close each of the first and second vent ports, open or close one or more portions of the main sampling line, and open or close each sample container. When the flow control system is in a first configuration, the first and second vent ports are closed, the main sampling line is open on the first and second sides of the pump, and at least one sample container is open, so that the pump can be operated in a first direction to move an amount of fluid from the vessel, through a portion of the main sampling line, and into the open sample container. When the flow control system is in a second configuration, the first vent port is closed, the second vent port is open, each of the one or more sample containers is closed, and a portion of the main sampling line on the first side of the pump is open so that the pump can be operated in a second direction to move fluid disposed in the main sampling line into the vessel without withdrawing fluid from the at least one sample container. When the flow control system is in a third configuration, the first vent port is open, the second vent port is closed, the main sampling line is closed on the first side of the pump and opened on the second side of the pump, and the at least one sample container is open so that the pump can be operated in the first direction to move fluid disposed in the main sampling line and into the open sample container without withdrawing additional fluid from the main sample container. 
         [0008]    Other aspects of the invention are embodied in a method for aseptically removing a sample portion of a fluid from a vessel containing the fluid. A fluid flow connection is provided between the vessel and a sample container, and fluid is pumped in a first direction from the vessel to the sample container through the fluid flow connection. The vessel is then disconnected from the fluid flow connection, a vent is opened upstream from the pump, and fluid is pumped in the first direction through the fluid flow connection into the sample container without pumping any additional fluid from the vessel. The upstream vent is then closed, the vessel is reconnected to the fluid flow connection, the sample container is disconnected from the fluid flow connection, a vent is opened downstream from the pump, and fluid is pumped in a second direction opposite the first direction through the fluid flow connection and into the vessel without pumping any fluid from the sample container. 
         [0009]    These and other features, aspects, and advantages of the present invention will become apparent to those skilled in the art after considering the following detailed description, appended claims and accompanying drawings. 
     
    
     
       DESCRIPTION OF THE DRAWING 
         [0010]      FIG. 1  is a schematic drawing of an aseptic sample withdrawal system embodying aspects of the invention. 
           [0011]      FIG. 2  is a schematic drawing of an alternative embodiment of an aseptic sample withdrawal system embodying aspects of the invention. 
           [0012]      FIG. 3  is a schematic drawing of another alternative embodiment of an aseptic sample withdrawal system embodying aspects of the invention. 
           [0013]      FIG. 4  is a flow chart showing steps performed in an aseptic sampling procedure embodying aspects of the invention. 
           [0014]      FIG. 5  is a schematic diagram of a control system for automation of the sample withdrawal system and sampling procedure. 
           [0015]      FIG. 6  is a schematic view of a sample manifold for selectively connecting individual sample containers to a main sampling line in accordance with an embodiment of the invention. 
       
    
    
     DETAILED DESCRIPTION 
       [0016]    As used herein, unless noted otherwise, the words “a” and “an” mean “one or more.” Furthermore, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein. 
         [0017]    An aseptic withdrawal system embodying aspects of the invention is shown in  FIG. 1 . The system includes a vessel  20 , a dip tube  22  extending into the vessel  20 , a sampling line  24 , a bi-directional pump  30  placed in line with the sampling line  24 , vent lines  32 ,  34  branching from the sampling line  24 , filters  26 ,  28  disposed on the vent lines  32 ,  34 , respectively, a flow control system which, in the illustrated embodiment, comprises a plurality of valves  1 - 14 , and sample containers B- 1 -B- 5  each connected to a respective secondary line branching from the main sampling line  24 . Vessel  20  may be the fluid holding container of a bioreactor for mammalian cell culture, a fermentor or fermentation vessel for microbial cell culture, or fermentation, or a medium batching/holding vessel. A bioreactor with which the aseptic withdrawal system may be incorporated is described by Lee, “Bioreactor Apparatus,” U.S. Patent Publication No. 2009-0269849, the disclosure of which is hereby incorporated by reference. A “medium batching/holding vessel” refers to a tank designated for either batching or holding cell culture medium at a set temperature, pressure, and agitation prior to transfer into a bioreactor or a fermentor. Such batching vessels are used in many biotech facilities to shorten the manufacturing run time, by allowing medium to be batched and held for a few days, up to a week, while a previous batch is still running in the bioreactor or fermentor. Medium batching is typically a non-sterile process, and batched medium is transferred through a sterilizing-grade filter into a medium holding tank, where it is then held under sterile conditions. Samples are typically taken during medium batching to ensure the medium has the desired pH and dissolved CO 2  level and may be taken after medium hold to ensure it was held as expected. 
         [0018]    Vessel  20  may comprise a bag formed from a suitable plastic film operatively supported by a rigid frame or housing. Valves  1 - 14  may comprise control pinch valves, but any suitable valve can be used. For example, since valves  1 - 14  are simply used for automating the opening and closing of tubing, they may be replaced with any number and/or combination of clamps, hemostats, or stopcocks for manually pinching off tubing. 
         [0019]    Pinch valves, if used, by be electronically actuated or pneumatically actuated. Suitable pinch valves are available from BioChem Fluidics Part No. 100P-2-NC-24-05 S Q. A suitable pump is the Watson Marlow  114  pump No. 010.5E20.00A. Exemplary volumes for sample containers B- 1 -B- 5  are up to 50 mL for R&amp;D applications and up to 1.0 L for cGMP (current good manufacturing practice) applications. 
         [0020]    The sampling system includes the main sampling line  24  with two vent lines  32 ,  34  that split off from the main sampling line  24  on opposite sides of the pump  30 . Each vent line  32 ,  34  has a vent filter  26 ,  28 , respectively (preferably a sterilizing grade gas filter with, e.g., a 0.2 μm membrane) on its distal end. Filter  26  will be referred to as the “vessel side filter,” and filter  28  will be referred to as the “sample side filter.” Similarly, vent line  32  may be referred to as the “vessel side vent line,” and vent line  34  will be referred to as the “sample side vent line.” With the exception of the vent lines  32 ,  34 , which are open to ambient conditions through filters  26  and  28 , respectively, vessel  20 , the main sampling line  24 , the secondary sampling lines, and the sample containers B- 1  through B- 5  are preferably closed to ambient conditions (although a filtered gas exhaust may be provided in the vessel), thereby maintaining an aseptic fluid transfer path between the vessel  20  and the sample containers. 
         [0021]    One end of the sampling line  24  is connected to the dip tube  22  that extends into the vessel  20 , and the other end of the sampling line  24  is connected to a series of sample containers (e.g., bags) B- 1 -B- 5  attached to the sampling line  24  via secondary sampling lines branching from the main sampling line  24 . If the vessel  20  comprises a plastic bag, the dip tube  22  may be inserted into the vessel  20  through a port disc (not shown) heat-sealed to the bag film. If vessel  20  is made out of rigid plastic material, the dip tube  22  may also be inserted into the vessel  20  through plastic port that is molded as part of the top plate of the vessel. In an alternative embodiment, shown in  FIG. 2 , the end  22 ′ of the sampling line  24  may be connected to the bottom of the vessel  20 , for example, through a port disc  36  heat-sealed to the bag film. If vessel  20  is made out of rigid plastic material, the end  22 ′ of the sampling line  24  may also be introduced to the bottom of the vessel  20  as a molded plastic channel that runs vertically on the inside of the vessel  20  from the top plate of the vessel. The number of sample containers is customizable and can be specified ahead of time, but more vessels can be added aseptically by the user through sterile tube welding. To ensure that the sampling system is sterile, i.e., free of live bacteria, other microorganisms, or bioactive DNA, the entire sampling system can be either pre-assembled and pre-sterilized (e.g., by gamma irradiation) with the vessel  20  or sterilized separately and attached to the existing sampling line through sterile tube welding or through aseptic connection devices. Sterilization may also be performed by autoclaving if the sampling system consists of material that can withstand a typical autoclave cycle. 
         [0022]    The portion of the sampling line  24  between the two filtered lines  32 ,  34  is fitted to a bi-directional pump  30  (e.g., a peristaltic pump) to help drive the fluid either into one of the sample containers B- 1 -B- 5  or back into the vessel  20 , depending on the open/closed position of the valves  1 - 14  and the rotational direction of the pump head  30 . 
         [0023]    Other, alternative arrangements may be used. For example,  FIG. 3  shows an alternative to the arrangement shown in  FIG. 1  in which the sample containers B- 1 -B- 5  are connected to a common node with a rotary valve  36  disposed at the node for selectively opening one of the sample containers connected to the node while the other sample containers remain closed. 
         [0024]    The aseptic sample withdrawal system described herein includes the dual-filter design that allows the system to be selectively opened to atmosphere without jeopardizing the sterility of the closed system, thereby allowing the culture fluid trapped in the sampling line  24  to be pumped back into the vessel  20  or into one of the sample containers B- 1 -B- 5  by alternating the flow direction within the sampling line  24  after the desired amount has been captured in the selected sample container. This eliminates the wasteful purge step, which can affect the final harvest volume, especially for smaller working volumes, as well as the unsanitary waste reservoir, where cells can die and lyse. The added advantage is that any bi-directional (e.g., peristaltic) pump can be used with this system, since there is no purge step and therefore no limitation in tubing size. The flexibility in tubing size used in the system also enables the user to choose from a wide variety of tube welding or aseptic connection options. 
         [0025]    A sampling procedure for the system as shown in  FIGS. 1-3 , which can be automated by a computer controller or manipulated manually, is shown by flow chart  50  in  FIG. 4  and described below. The procedure is described with respect to the sampling system shown in  FIG. 1 . 
         [0026]    In step  52 , the sampling line  24  is purged to ensure there is no residual fluid in the sampling line  24  by pumping residual fluid from the sampling line  24  back into the vessel  20 . In the system of  FIG. 1 , step  52  is performed by: (a) closing sampling line  24  on the sampling side of the pump by closing valve  4 , (b) venting the sample side of the sampling line  24  by opening valve  3  of the sample side vent line  34 , (c) opening the sampling line  24  on the vessel side of the pump  30  by opening valve  1 , (d) closing the sample side venting line  32  by closing valve  2 , and then (e) operating pump  30  in direction B to pump any residual fluid contained in the sampling line  24  into the vessel  20 . Steps (a) through (d) do not necessarily need to be performed in the order listed, but step (e), operating the pump, should not be performed until all the valves are opened or closed, as required. Although valve  4  closes off the sampling line to all of the sampling containers B- 1  through B- 5 , valves  5 - 14 , which are downstream of valve  4 , may also be closed to redundantly close off the sample containers 
         [0027]    In step  54 , a prescribed volume of sample material is withdrawn from the vessel  20  and is deposited in one of the sample containers B- 1  through B- 5 . In the system of  FIG. 1 , step  54  is performed by: (a) opening the vessel side of the sampling line  24  by opening valve  1 , (b) opening the sample side of the sampling line  24  by opening valve  4 , (c) closing both vessel side vent line  32  and sample side vent line  34  by closing valves  2  and  3 , respectively, (d) opening the first sample container B- 1  by opening valve  5 , (e) closing the remaining sample containers B- 2  through B- 5  by closing valve  6 , and then (f) operating the pump  30  in the direction A for a prescribed period of time (or a prescribed number of revolutions) to transfer a prescribed volume of sample material from the vessel  20  into the container B- 1 . Steps (a) through (e) do not necessarily need to be performed in the order listed, but step (f), operating the pump, should not be performed until all the valves are opened or closed, as required. 
         [0028]    In step  56 , any residual sample fluid in the sampling line  24  is pumped back into the vessel  20 . In the system of  FIG. 1 , step  56  is performed by: (a) closing off the sample side of the sampling line  24  by closing valve  4  and/or valve  5 , (b) venting the sample side of the sampling line  24  by opening valve  3  of sample side vent line  34 , (c) closing valve  2  of the vessel side vent line  32 , (d) opening the vessel side of the sampling line  24  by opening valve  1 , and (e) operating pump  30  in direction B to move fluid from the sampling line  24  back into the vessel  20  without removing any fluid from container B- 1 . Again, the order in which the steps are performed, other than operation of the pump, is not necessarily critical. 
         [0029]    In step  58 , any remaining residual fluid downstream of valve  4  and/or valve  5  is purged from the sampling line  24 . In the system of  FIG. 1 , step  58  is performed by: (a) closing off the vessel side of the sampling line  24  by closing valve  1 , (b) venting the vessel side of the sampling line  24  by opening valve  2  of vessel side vent line  32 , (c) closing sample side vent  34  by closing valve  3 , (d) opening the container B- 1  by opening valves  4  and  5 , (e) closing the remaining sample containers B- 2  through B- 5  by closing valve  6 , and then (f) operating pump  30  in direction A to remove any remaining fluid from the sampling line  24  into the sample container B- 1  without withdrawing any additional fluid from the vessel  20 . Again, the order in which the steps are performed, other than operation of the pump, is not necessarily critical. 
         [0030]    In step  60 , the just-filled sample container B- 1  is removed from the system. Step  60  is performed by closing the valve  5  and cutting or otherwise removing sample container B- 1  from the system. 
         [0031]    Steps  52  to  62  are repeated for each of the other sample containers to be filled. Implementation of the steps differs somewhat in that different valves must be operated to fill different sample containers. For example, to fill sample container B- 2 , valves  6  and  7  are opened while valves  5  and  8  remain closed. To fill sample vessel B- 3 , valves  6 ,  8 , and  9  are opened while valves  5 ,  7 , and  10  are closed. To fill sample container B- 4 , valves  6 ,  8 ,  10 , and  11  are opened while valves  5 ,  7 ,  9 , and  12  are closed. And to fill sample container B- 5 , valves  6 ,  8 ,  10 ,  12 , and  13  are opened while valves  5 ,  7 ,  9 ,  11 , and  14  are closed. 
         [0032]    In an alternative procedure, the order of steps  56  and  58  can be reversed. That is, after performing step  54 , step  58  can be performed with the system of  FIG. 1  by: (a) closing off the vessel side of the sampling line  24  by closing valve  1  and (b) venting the vessel side of the sampling line  24  by opening valve  2  of vessel side vent line  32  while (d) continuing to operate pump  30  in direction A to pump remaining fluid from the sampling line  24  into the sample container B- 1  without withdrawing any additional fluid from the vessel  20 . After step  58 , step  56  can be performed with the system of  FIG. 1  by: (a) closing off the sample side of the sampling line  24  by closing valve  4  and/or valve  5 , (b) venting the sample side of the sampling line  24  by opening valve  3  of sample side vent line  34 , (c) closing valve  2  of the vessel side vent line  32 , (d) opening the vessel side of the sampling line  24  by opening valve  1 , and (e) operating pump  30  in direction B to move fluid from the sampling line  24  back into the vessel  20  without removing any fluid from container B- 1 . 
         [0033]      FIG. 5  schematically shows the sampling system incorporated with an automated control system  70  configured to automatically control operation of the valves of the flow control system and the pump  30 . The automated control system  70  includes a programmable automation controller (“PAC”)  72 . A suitable PAC is available from National Instruments Model No. sbRIO 9641. The PAC  72  includes a 24 volt digital output  74 , which is connected to each of the valves  1 - 14  (the connection of digital output  74  to valves  7 - 14  is not shown in  FIG. 5  so as to avoid obscuring the drawing). The pump  30  is connected to the digital output via a DPDT (double pole double throw) relay  78  and a DPST (double pole single throw) relay  76 . In the illustrated embodiment, the PAC  72  and the relays  76 ,  78  are connected to a 24 volt power source. 
         [0034]    The PAC  72  is programmed with a sequencing algorithm, such as an algorithm that will implement the process shown in  FIG. 4 . Initiation of the sampling sequence may be programmed into the PAC  72  so as to be automatic, or the sampling sequence may be initiated by a user via a user interface. In one embodiment, each of the valves  1 - 14  is an electronic pinch valve that is normally in a closed state and may be opened by a signal generated by the PAC  72  and output by the digital output  74 . As shown in the illustrated embodiment, the PAC  72  and digital output  74  generate a dedicated signal V 1 , V 2 , V 3 , V 4 , V 5 , V 6 , etc., for each of the valves  1 - 14 , respectively. The P 2  output, along with the controls of the DPST relay  76 , govern whether power gets to the DC motor of the pump  30 . The P 1  output, along with the controls of the DPDT relay  78 , changes the polarity of the power that gets to the DC motor of the pump  30  thereby controlling the direction of the pump. In an embodiment of the invention, the PAC effects a timing-based control of the valves and the pump to transfer a desired volume fluid, in one direction or the other, based on the volume of the sampling lines and the flow rate of the pump. 
         [0035]    In an alternate embodiment, valves  5 - 14  of the flow control system are not connected to the PAC  72  and are not automatically controlled. In such an embodiment, the appropriate valves are open and closed manually. In still other embodiments, valves  5 - 14  are omitted from the flow control system altogether, and the appropriate flow control is achieved using clamps or hemostats at pinch points in the main and secondary sample tubing corresponding to the locations of valves  5 - 14  shown in  FIGS. 1 and 2 . In a still further embodiment, shown in  FIG. 6 , the flow control system includes a manifold  80  connected to the main sampling line  24 , and each of the sample containers B- 1 , B- 2 , B- 3 , B- 4 , and B- 5  is coupled to the sample manifold  80  via an associated three-way stopcock  82 ,  84 ,  86 ,  88 , and  90 . Reference characters A, C, E, and G indicate pinch points in the secondary sampling lines were clamps are placed when the associated sample containers are removed. 
         [0036]    An exemplary sampling sequence using the automated system  70  is described below. 
         [0037]    Before the sampling sequence commences, each of the automatically-controlled valves  1 - 4  is closed and the pump  30  is not operating. Digital output  74  outputs an “off” or null signal for outputs V 1 , V 2 , V 3 , V 4 , P 1 , and P 2 . Valves  5 - 6  are closed, either manually or via an “off” or null signal from the digital output  74  of the PAC, or main and secondary sampling lines are clamped at pinch points corresponding to the locations indicated by valves  5 - 14 . The sampling sequence is initiated by a user at a user interface, or a prescheduled sampling sequence may be programmed into the PAC  72  for automatic initiation by the PAC  72 . To perform the sampling sequence, generally corresponding to step  54  in  FIG. 4 , the PAC  72  opens valve  1  by changing signal V 1  to “on” and opens valve  4  by changing signal V 4  to “on” to open the sampling line  24 . Valve  5  is opened, either automatically or manually, or the clamp is removed from the pinch point  5 . Thus, the sampling lines are open from the vessel  20  to the first sample container B- 1 . 
         [0038]    Signal P 1  is changed to “on” and signal P 2  is changed to “on” to operate the pump  30  in a forward direction to pump fluid from the vessel  20 , through the main and secondary sampling lines, and into the sample container B- 1 . At a first prescribe time lapse (ΔTI) following the initiation of the sample pumping sequence, the sampling sequence is terminated, and a forward flush (corresponding to step  58  in  FIG. 4 ) is performed. PAC  72  changes signal V 1  to “off” to close valve  1 , thereby terminating sample flow from the vessel  20 , and changes signal V 2  to “on” to open the valve  2  and vessel side vent line  32  to vent the vessel side of sampling line  24 . Signals P 1  and P 2  remain “on” to continue forward flow of the pump  30 , V 3  remains “off” and V 4  remains “on” to continue flow into the sample container B- 1  without drawing additional fluid from the vessel  20  thereby clearing fluid from the sampling line  24 . The length of the time lapse ΔT 1  for beginning the flush sequence is calculated from the tubing volume in the system, the desired sample volume (which may be input by the user), and the pump flow rate. 
         [0039]    At a second prescribed time lapse (ΔT 2 ) following initiation of the sampling sequence a reverse flush (corresponding to step  56  in  FIG. 4 ) is performed. PAC  72  changes signal V 1  to “on” to open valve  1 , thereby connecting vessel  20  to the sampling line  24 , changes signal V 2  to “off” to close valve  2 , thereby closing sample side vent line  32 , changes signal V 3  to “on” to open valve  3  to open the valve  3  and sample side vent line  34  to vent the sample side of sampling line  24 , changes signal V 4  to “off” to close valve  4 , thereby closing off the sample containers, and changes signal P 1  to “off” while keeping signal P 2  at “on” to cause a reverse flow of the pump  30  to pump any fluid remaining in sampling line  24  and dip tube  22  into the vessel  20  without pumping any fluid from the sample container B- 1 . 
         [0040]    The sample sequence is terminated at a third prescribed time lapse (ΔT 3 ) from initiation of the sampling sequence by turning all signals to “off”, thereby closing valves  1 - 4  and stopping pump  30 . The second and third time lapses, ΔT 2  and ΔT 3 , are calculated from the tubing volume and the pump flow rate. 
         [0041]    Sample container B- 1  is then removed by closing valve  5 , either manually or automatically, or by clamping the secondary sampling line at the pinch point corresponding to the location of valve  5 , and then cutting the secondary sampling line below the valve or clamp. To take the next sample, valves  6  and  7  are opened, or the user opens the clamps at pinch points corresponding to the locations of valves  6  and  7 , and valve  8  is closed, or the user clamps the tubing at a pinch point corresponding to the location of valve  8 . The sampling sequence is repeated as described above, and, after sample container B- 2  is filled and the sampling lines are flushed out, valve  7  is closed, or the secondary sample tubing is clamped at the pinch point corresponding to valve  7 , and the secondary sampling line connecting container B- 2  is cut below the valve or clamp. 
         [0042]    A similar process is performed for each of the remaining sample containers B- 3 , B- 4 , and B- 5 . 
         [0043]    Thus, exemplary embodiments have been fully described above with reference to the drawing figures. Although the invention has been described based upon these exemplary embodiments, it would be apparent to those of skill in the art that certain modifications, variations, and alternative constructions could be made to the described embodiments within the spirit and scope of the invention.