Patent Abstract:
The invention provides an automated crossflow filtration method and system for separating a component of interest from one or more other components in a solution. The invention is of particular use in the field of protein separations and concentration, where specific proteins must be separated and purified from cell lysates and cultures. The system may be under the control of a computer software programme.

Full Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a filing under 35 U.S.C. § 371 and claims priority to international patent application number PCT/EP2007/002588 filed Mar. 23, 2007, published on Oct. 4, 2007, as WO 2007/110203, which claims priority to patent application number 0606144.4 filed in Great Britain on Mar. 28, 2006. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to an automated crossflow filtration method and system for separating a component of interest from one or more other components in a solution. The invention is of use in the field of protein separations, where specific proteins must be separated and purified from cell lysates and cultures. The invention finds particular utility in concentrating proteins which are present at low concentrations in a solution containing one or more components. 
       BACKGROUND OF THE INVENTION 
       [0003]    Separation of target molecules is of great commercial interest in the chemical and biotechnological fields, such as the production of novel biological drugs and diagnostic reagents. Furthermore, the isolation and purification of proteins is of great significance due to advances in the field of proteomics, wherein the function of proteins expressed by the human genome is studied. Proteins of interest are often present at very low concentrations within a biological sample and so it is very important to develop isolation and separation techniques which can handle low volumes of such samples with minimal wastage. This is particularly true in research laboratories which are concerned with the early stage purification and characterisation of proteins which are present in low concentrations of source material. 
         [0004]    In general, proteins are produced in cell culture, where they are either located intracellularly or secreted into the surrounding culture media. Since the cell lines used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, growth factors, etc. Separation and purification of a desired protein from the complex mixture of nutrients and cellular by-products, to a level sufficient for characterisation, poses a formidable challenge. 
         [0005]    Semi-permeable membrane filtration is often used in the purification of proteins, microfiltration and ultrafiltration being the most commonly practised techniques. Microfiltration membranes exhibit permselective pores ranging in diameter from between 0.01 and 10 μm. Micro-filtration is defined as a low pressure membrane filtration process which removes suspended solids and colloids generally larger than 0.1 μm in diameter. Such processes can be used to separate particles or microbes that can be seen with the aid of a microscope such as cells, macrophage, large virus particles and cellular debris. 
         [0006]    Ultra-filtration membranes are characterized by pore sizes which enable them to retain macromolecules having a molecular weight ranging between 500 and 1,000,000 daltons, and thus are often used for concentrating proteins. Ultra-filtration is a low-pressure membrane filtration process which separates solutes up to 0.1 μm in size. Thus, for example, a solute of molecular size significantly greater than that of the solvent molecule can be removed from the solvent by the application of a hydraulic pressure, which forces only the solvent to flow through a suitable membrane (usually one having a pore size in the range of 0.001 to 0.1 μm). Ultra-filtration is capable of removing bacteria and viruses from a solution. 
         [0007]    Many automated systems exist for the separation of proteins using such ultra- and microfiltration membranes (e.g. GE Healthcare Life Sciences, Uppsala, Sweden). 
         [0008]    Crossflow filtration (sometimes referred to a ‘tangential flow filtration’) systems are widely used in industry; typical examples include manufacturing process separations, waste treatment plants and water purification systems where they extend the lifetime of filtration membranes by removing and preventing the build up of contaminants (e.g. WO 2005/081627) and promote consistency of the filtration process with time. 
         [0009]    The most commonly used crossflow membrane processes are microfiltration and ultrafiltration. These processes are pressure driven and depend upon the ‘membrane flux’, defined as the flow volume over time per unit area of membrane, across the microfiltration or ultrafiltration membrane. At low pressures, the transmembrane flux is proportional to pressure. Thus by varying the transmembrane pressure difference driving force and average pore diameter, the membrane may serve as a selective barrier by permitting certain components of a mixture to pass through while retaining others. This results in two phases, the permeate and retentate phases, each of which is enriched in one or more of the components of the mixture. 
         [0010]    Crossflow filtration systems are commercially available from a number of manufacturers for a range of applications, including the separation of biological materials (e.g. GE Infrastructure, Water and Process Technologies, Fairfield, Conn., USA; Millipore, Billerica, M, USA; SciLog, Wis., USA; GEA filtration, MG Technologies, Frankfurt, Germany). 
         [0011]    However, one major disadvantage of existing systems which are used to purify biological materials is that they require relatively large volumes of sample (typically &gt;25 mls), due to the internal configuration of the pumps, and have significant ‘dead volumes’. This can be extremely wasteful of material which, in the case of proteins which are often only present in relatively low concentrations in biological samples, can be very expensive and resource consuming. 
         [0012]    Another disadvantage associated with conventional crossflow systems is that of foaming, caused by air within the system, which also leads to losses of material. 
         [0013]    U.S. Pat. No. 5,935,437 describes a single-use, manually operated crossflow filtration system for preparing plasma samples from patients&#39; blood during surgery. The system disclosed is capable of handling a small volume (e.g. less than 10 ml of blood) under aseptic conditions. While this system is clearly suitable for use in an operating theatre, it is not suitable for use in a research or industrial laboratory where users require automated systems which are robust, reliable, environmentally regulated and precise. 
         [0014]    Spectrum Labs (Spectrum Laboratories Inc., USA) provide the components for making a simple cross flow separation system for use in processing small volumes of samples containing biological materials. The disposable MICROKROS® modules comprise hollow fibre membranes in a polysulfone housing. These modules can be operated manually using conventional syringes to handle volumes as low as 2 ml of sample. Alternatively, the modules can be used with a peristaltic pump, such as the Spectrum MICROKROS® System, to process sample volumes ranging from 10 to 200 ml. Although this system can accommodate small volumes of solution (i.e. from 10 to 200 ml), the precision of separation can be variable as the system is controlled by a peristaltic pump. 
         [0015]    There is therefore a need within the research communities of the chemical and biotechnological industries for an automated crossflow filtration system which can handle small volumes of solution, under carefully regulated conditions, with a high level of precision and minimal wastage of sample. Further cost savings could be achieved if it were possible to wash and reuse the membranes employed in such a system. 
         [0016]    The present invention addresses these problems and provides a method and system for separating a first component of interest from one or more components in a solution. To improve consistency and efficiency, the system of the invention may be under the control of a computer software programme. 
       SUMMARY OF THE INVENTION 
       [0017]    In a first aspect of the invention, there is provided an automated crossflow filtration method for separating a component of interest from one or more other components in 50 ml or less of a solution comprising the steps of
       i) transferring said solution from a sample container into a receiving chamber of a first pump, said chamber being in fluid communication via one or more flow-directing valves with a receiving chamber of a second pump, wherein both said chambers have a moveable wall for altering the volume of the chamber;   ii) passing the solution through a filter unit, said filter unit comprising
           i. a first inlet and a second inlet in fluid communication with each other   ii. an outlet   iii. a filtration membrane separating the inlets from the outlet, by simultaneously driving the solution from the chamber of the first pump through the filtration membrane and aspirating the first retentate produced into the chamber of said second pump;   
           iii) collecting the first permeate produced which has passed through the filtration membrane;   iv) reversing the direction of flow across the filtration membrane by simultaneously driving the first retentate from the chamber of the second pump back through the filter unit and the filtration membrane and aspirating the second retentate produced into the chamber of the first pump;   v) collecting the second permeate produced and/or the second retentate;
 
wherein a predetermined membrane flux or pressure is maintained across the filtration membrane by controlling the differential rate of movement of the wall in the first and second receiving chamber of the first and second pump.
       
 
         [0026]    A component of interest may be chemical compound, or a biological entity or a biological molecule. Examples of chemical compounds include naturally occurring and synthetic compounds such as drugs and therapeutic agents. Biological entities include, for instance, cells (e.g. blood cells and animal cells), microbes (e.g. bacteria and fungi), and sub-cellular particles (e.g. mitochondria, viruses etc). Biological molecules may include proteins, peptides, polynucleotides, and polysaccharides. The method is of particular utility in separating proteins and in concentrating proteins which are present at low concentrations in a solution containing one or more components. 
         [0027]    Membranes may include ultrafiltration membranes, affinity membranes (i.e. membranes which are derivitized to bind to ligands in a specific or non-specific manner), microfiltration membranes, ion exchange resins and reverse phase membranes. Such membranes are well known in the art and are available from a range of suppliers (e.g. GE Healthcare Life Sciences, Sweden; Sartorius AG; Germany; Meissner Inc., USA). The membranes may be of flat or hollow configuration. 
         [0028]    A second aspect of the invention relates an automated crossflow filtration system for separating a component of interest from one or more other components in 50 ml or less of a solution comprising
       i) a first pump having a receiving chamber and a moveable wall for altering the volume of said chamber, said moveable wall being operable by a first drive motor, the chamber being in fluid communication via a first flow-directing valve with a sample container and a first inlet of a filter unit;   ii) said filter unit comprising
           a. a first inlet and a second inlet in fluid communication with each other   b. an outlet   c. a filtration membrane separating the inlets from the outlet,   
           iii) the second inlet of the filter unit being in fluid communication via a second flow-directing valve with a receiving chamber of a second pump;   iv) said second pump comprising said receiving chamber and a moveable wall for altering the volume of the chamber, said moveable wall being operable by a second drive motor;   v) the first flow-directing valve comprising one or more ports enabling fluid communication of the chamber of the first pump with one or more containers for aspiration of solution therefrom and/or the collection of retentate therein; optionally, enabling the aspiration of buffer therefrom;   vi) the second flow-directing valve comprising one or more ports enabling fluid communication of the chamber of the second pump with a plurality of containers for aspiration of washing fluid therefrom and/or collection of retentate or waste therein;
 
characterised in that a predetermined membrane flux or pressure is maintained across the filtration membrane by controlling the differential rate of movement of the wall in the first and second receiving chamber of the first and second pump.
       
 
         [0038]    A third aspect of the invention relates to a computer programme arranged to perform the method of the invention. 
         [0039]    A fourth aspect of the invention relates to a data carrier in which the computer programme is stored. 
         [0040]    Further features and advantages of the invention will become apparent from the following description of preferred embodiments of the invention, given by way of example only, which is made with reference to the accompanying drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0041]    The transverse section in  FIG. 1  shows one embodiment of the invention in which the crossflow filtration system has a series of filter units which each comprise a microfiltration membrane. 
           [0042]      FIG. 2  depicts a transverse section of one embodiment of the invention in which the crossflow filtration system has a series of filter units which each comprise an ultrafiltration membrane. 
           [0043]      FIG. 3  illustrates, in transverse section, an embodiment of the invention in which the crossflow filtration system has a filter unit containing a microfiltration membrane, a filter unit comprising an ultrafiltration membrane, and an affinity membrane. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0044]    One embodiment of an automated crossflow system  1  according to the invention, utilising a microfiltration membrane is shown in transverse section in  FIG. 1 . The system can be used to separate components present in a solution, such as are commonly found in biological samples. For example, depending upon the pore size of the membrane used, cells (such as blood cells) can be washed with buffers prior to lysis to remove contaminants, cellular debris can be separated from soluble materials, and/or proteins can be purified for characterisation. 
         [0045]    The system  1  comprises a first pump  10  and second pump  20  which are in fluid connection with one another through one or more filter units  30 ,  40 ,  50 ,  60  connected through a first flow-directing valve  70  and a second flow directing valve  80 . Each pump comprises a receiving chamber  12 ,  22  and a moveable wall  14 ,  24  connected through a drive shaft  16 ,  26  to independent drives  18 ,  28 . A solution can be drawn into or expulsed from the receiving chamber  12 ,  22  by the axial movement of the wall  14 ,  24  relative to the body of the pump  10 ,  20  (e.g. in the direction of the arrow shown in  FIG. 1 ) when the drive  18 ,  28  is activated. The walls of the receiving chamber  12 ,  22  are made of an inert material, such as glass, ceramics, stainless steel or an appropriate plastic polymer which can withstand high operational pressures and not react with any components within the solution. 
         [0046]    In use, solutions  91 ,  92 ,  93 ,  94  which each comprise a component of interest and one or more other components, are sequentially aspirated from their respective sample containers into the receiving chamber  12  of the first pump  10  by movement of the wall  14  in the opposite direction to the arrow shown in the figure. The use of the system  1  will be described in relation to separating components of interest from a single solution  91  but it will be understood that the system can be used to sequentially separate components from other components within a plurality of solutions (e.g. from solutions  92 ,  93 ,  94 ). 
         [0047]    The solution  91  is drawn from its sample container into the receiving chamber  12  of the first pump  10  via the flow directing valve  70  by means of tubing  71 . The tubing  71  and valve  70  are made of conventional materials, such as metals or plastics, which do not react with any components in the solution. The valve  70 , comprises one or more ports (not shown) which can be used to allow the valve  70  to act as a filter unit  30  selecting valve and/or an inlet/outlet valve. 
         [0048]    In the first half of the cycle, solution  91  is driven from the receiving chamber  12  of the first pump  10 , by movement of the wall  14  in the direction of the arrow shown in  FIG. 1 , through the valve  70  and into the filter unit  30  by means of tubing  76 . The first pump  10  thus controls or regulates the flowrate of ‘feed’ solution  91  (i.e. the solution prior to filtration) moving into and through the filter unit  30 . 
         [0049]    The filter unit  30  comprises a first inlet  32  in fluid communication with a second inlet  34 , the inlets being connected to the first and second flow-directing valves  70 ,  80 , respectively, by inert tubing  76 ,  86 . The inlets  32 ,  34  are separated from an outlet  36  by a membrane  38  within the filter unit  30  which is selectively permeable to the component of interest. The membrane  38  in  FIG. 1  is a microfiltration membrane but it will be understood that, depending upon the nature of the separation to be effected, an ultrafiltration membrane could be used. A microfiltration membrane will be chosen which has pore sizes such that the component of interest within the solution will pass through the membrane whereas larger components will be retained by it. The solution passing through the membrane is known as the permeate, while the material retained by the membrane is called the retentate. 
         [0050]    As described above and shown in  FIG. 1 , the second pump  20  is in fluid communication with the first pump  10  by means of the first and second flow-directing valves  70 ,  80 . The pumps  10 ,  20  are independently driven such that the receiving chamber  12  of the first pump  10  empties at a faster rate than the receiving chamber  22  of the second pump  20  fills. The higher speed of the wall  14  in emptying the first chamber  12  compared to the speed of the wall  24  in filling the second chamber  22  creates a permeate flux across the membrane  38 . Thus the permeate flux, which determines the rate of separation of components across the membrane, is controlled by the differential speed of the walls  14 ,  24  of the first  12  and second  22  receiving chambers. This permeate flux may be monitored by pressure sensors  101 ,  103 . Other sensors ( 102 ,  104 ,  105 ) may be employed to monitor other physical parameters (e.g. temperature, conductivity, pH, oxygen concentration, ultraviolet light absorption) within the system. 
         [0051]    In the embodiment shown in  FIG. 1 , the filter unit  30  contains a microfiltration membrane  38  and permeate passing through the membrane  38  is collected from the outlet  36  as product  111 . The retentate is collected in the receiving chamber  22  of the second pump  20 . 
         [0052]    When the wall  14  reaches the end position of the stroke in emptying the solution  91  from the chamber  12 , the first half of the cycle is complete and the movement of both drives  18 ,  28  is reversed. In this half of the cycle, the ‘feed control’ pump (initially the first pump  10  in the first half of the cycle) becomes the retentate control pump and the retentate control pump (the second pump  20  in the first half of the cycle) becomes the feed control pump. The direction of flow is thus reversed such that retentate is driven from the second receiving chamber  22  back into the filter unit  30  and across the membrane  38  to further remove components of interest from the retentate. Once again, the slower speed of filling the retentate control pump (first pump  10  in this phase of the cycle) relative to the speed of emptying the feed control pump (i.e. second pump  20 ) creates a permeate flux across the membrane  38 . The permeate passing through the membrane  38  is collected as further product  111  and the resulting retentate aspirated into the first receiving chamber  12 . In this way, components of interest are sequentially removed from the solution  91 . The cycle can be repeated, either using the same retentate or by aspirating fresh solution  91  into the first chamber  12  (or second chamber  22 ) to maintain the volume of solution within the system by means of the flow-directing valve  70 ,  80  at the start of each new stroke. By replenishing the system with fresh solution  91 ,  121  in this way, the system is not limited to simply processing volumes equivalent to the volume of the receiving chamber  12 ,  22 . At the end of a complete cycle, waste materials can be removed from the system via the second flow-directing valve  80  as waste  124 . 
         [0053]    By means of the flow-directing valves (e.g.  80 ) equipped with inlet/outlet ports, the membrane  38  can be cleaned with washing fluid/buffers  122 ,  123  at the end of a complete cycle to remove any contaminants (such as solids, particles, etc) which adsorb to the membrane surface and block the pores. In this way, the operational lifetime of the membrane can be increased and its efficiency maintained. 
         [0054]    It will be understood by the person skilled in the art that other samples  92 ,  93 ,  94  can be sequentially filtered in a similar manner either through the same filter unit  30  or different filter units  40 ,  50 ,  60  which either contain the same or different membranes (e.g. one having a different pore size). Following filtration in the filter units  40 ,  50 ,  60 , permeate can be collected from outlets (see shorter arrows) as product  112 ,  113  and  114 . It will also be understood that the system can be used in combination with ultrafiltration membranes, as described below. 
         [0055]    All materials used in the construction of the system which come into contact with the solution, retentate and/or permeate are selected to avoid any chemical interaction and to minimise physical adsorption with the components within the solution. Typically, the walls of the receiving chamber and the valves are made of glass, ceramics or stainless steel and the tubing of an inert plastic polymer. 
         [0056]      FIG. 2  is a transverse section showing a second embodiment of an automated crossflow system  2  according to the invention. This embodiment can be used to ultrafiltrate samples, for example, the system can be used to concentrate particular components present in a sample, such as proteins, for further characterisation. 
         [0057]    The system  2  has a similar configuration to that described in  FIG. 1  above. Thus a first pump  110  and second pump  120  are in fluid connection with one another through one or more filter units  130 ,  140 ,  150 ,  160  connected through a first and second flow-directing valve  170 ,  180 . Each pump comprises a receiving chamber  112 ,  122  and a moveable wall  114 ,  124  connected through a drive shaft  116 ,  126  to independent drives  118 ,  128 . A solution  191  can be drawn into or expulsed from the receiving chamber  112 ,  122  by the axial movement of the wall  114 ,  124  relative to the body of the pump  110 ,  120  (e.g. in the direction of the arrow shown in  FIG. 2 ) when the drive  118 ,  128  is activated. The walls of the receiving chamber  12 ,  22  are made of an inert material, such as glass, ceramics, stainless steel or an appropriate plastic polymer which can withstand high operational pressures and not react with any components within the solution. 
         [0058]    In use, solutions  191 ,  192 ,  193 ,  194  (which each comprise a component of interest in mixture with other components) are sequentially aspirated from their respective sample containers into the receiving chamber  112  of the first pump  110  by movement of the wall  114  in the opposite direction to the arrow shown in the figure. The use of the system  2  will be described in relation to separating components of interest from a single solution  191  but it will be understood that the system can be used sequentially to separate components from other components within a plurality of solutions (e.g. from solutions  192 ,  193 ,  194 ). In the present example, the solution  191  contains a protein of interest which is to be separated from other components present in the solution and concentrated by ultrafiltration. 
         [0059]    As described in  FIG. 1  above, the first step in the process is for the solution  191  to be drawn from its sample container into the receiving chamber  112  of the first pump  110  via the flow directing valve  170  by means of tubing  171 . The tubing  171  and valve  170  are made of conventional materials, such as metals or plastics, which do not react with any components in the solution. The valve  170 , comprises one or more ports (not shown) which can be used to allow the valve  170  to act as a filter unit  130  selecting valve and/or an inlet/outlet valve. 
         [0060]    In the first half of the cycle, solution  191  is driven from the receiving chamber  112  of the first pump  110 , by movement of the wall  114  in the direction of the arrow shown in  FIG. 2 , through the valve  170  and into the filter unit  130  via tubing  176 . The first pump  110  thus controls or regulates the flowrate of ‘feed’ solution  191  (i.e. the solution prior to filtration) moving into and through the filter unit  130 . 
         [0061]    The filter unit  130  comprises a first inlet  132  in fluid communication with a second inlet  134 , the inlets being connected to the first and second flow-directing valves  170 ,  180 , respectively, by inert tubing  176 ,  186 . The inlets  132 ,  134  are separated from an outlet  231  by a membrane  138  which is selectively impermeable to the component of interest. An ultrafiltration membrane will be chosen which has pore sizes such that the component of interest within the solution (in this case a protein) will be retained by the membrane (i.e. the retentate) whereas smaller components will pass through it (i.e. the permeate). The membrane may be hollow or flat in configuration; in the example shown a hollow membrane is used such that permeate passing through the membrane may then be expulsed from the system through outlet  136  as waste. 
         [0062]    As shown in  FIG. 2 , the second pump  120  is in fluid communication with the first pump  110  by means of the first and second flow-directing valves  170 ,  180 . The pumps  110 ,  120  are independently driven such that the receiving chamber  112  of the first pump  110  empties at a faster rate than the receiving chamber  122  of the second pump  120  fills. The higher speed of the wall  114  in emptying the first chamber  112  compared to the speed of the wall  124  in filling the second chamber  122  creates a pressure difference across the membrane  138 . This pressure difference determines the rate of separation of components across the membrane and is controlled by the differential speed of the walls  114 ,  124  of the first  112  and second  122  receiving chambers. This pressure difference is monitored by pressure sensors  201 ,  203 . Other sensors ( 202 ,  204 ,  205 ) may be employed to monitor other physical parameters (e.g. temperature, conductivity, pH, oxygen concentration, ultraviolet light absorption) within the system. 
         [0063]    In the embodiment shown in  FIG. 2 , the retentate following filtration is collected in the receiving chamber  122  of the second pump  120  and the permeate passing through the membrane  138  is discarded from the outlet  136  as waste. 
         [0064]    When the wall  114  reaches the end position of the stroke in emptying the solution  191  from the chamber  112 , the first half of the cycle is complete and the movement of both drives  118 ,  128  is reversed. In this half of the cycle, the ‘feed control’ pump (initially the first pump  10  in the first half of the cycle) becomes the retentate control pump and the retentate control pump (the second pump  120  in the first half of the cycle) becomes the feed control pump. The direction of flow is thus reversed such that retentate is driven from the second receiving chamber  122  back into the filter unit  130  and across the membrane  138  to further remove contaminating components from the retentate. Once again, the slower speed of filling the retentate control pump (first pump  110  in this phase of the cycle) relative to the speed of emptying the feed control pump (i.e. second pump  120 ) creates a pressure differential across the membrane  138 . The resulting retentate is aspirated into the first receiving chamber  112 . Permeate containing low molecular weight components passing through the membrane  138  is discarded as waste from outlet  136 . 
         [0065]    In this way, contaminating components are sequentially removed from the solution  191  and the component of interest (e.g. a protein) is concentrated in the retentate. The retentate can be collected as product  211  at the end of the cycle. 
         [0066]    The cycle can be repeated, either using the same retentate, or by aspirating fresh solution  191  into the first chamber  112  (or second chamber  122 ) to maintain the volume of solution within the system by means of the flow-directing valve  170 ,  180  at the start of each new stroke. By replenishing the system with fresh solution  191  in this way, the system is not limited to simply processing volumes equivalent to the volume of the receiving chamber  112 ,  122 . At the end of a complete cycle, the retentate is collected as product  211  and low molecular weight contaminating components are effluxed from the system via outlet  136 . 
         [0067]    It will be understood that if diafiltration is desired, the retentate can be diluted with dialysis buffer at the end of either or both halves of the cycle by the addition of the appropriate buffer solution  230  into either or both receiving chambers  112 ,  122  to maintain a constant sample volume. The retentate can thus be washed with buffer  230  at a suitable pH and/or having an appropriate ionic strength, either once or repeatedly, to ensure removal of low molecular weight contaminants. The resulting retentate can be collected as product  211  and can be further diluted, if required, in the dialysis buffer ready for characterisation. 
         [0068]    Following the final collection of retentate as product  211 , the membrane  138  can be cleaned with washing fluid/buffers  221 ,  223  at the end of a complete cycle to remove any contaminants (such as solids, particles, etc) which adsorb to the membrane surface and block the pores. In this way, the operational lifetime of the membrane can be increased and its efficiency maintained. 
         [0069]    All materials used in the construction of the system which come into contact with the solution, retentate and/or permeate are selected to avoid any chemical interaction and to minimise physical adsorption with the components within the solution. Typically, the walls of the receiving chamber and valves are made of glass, ceramics or stainless steel and the tubing of an inert plastic polymer. 
         [0070]    It will be understood by the person skilled in the art that other samples  192 ,  193 ,  194  can be sequentially filtered in a similar manner either through the same filter unit  130  or different filter units  140 ,  150 ,  160  which either contain the same or different membranes (e.g. microfiltration membranes having different pore sizes). Following filtration in the filter units  140 ,  150 ,  160 , retentate can be collected from the outlets (see shorter arrows) as product  212 ,  213  and  214 . 
         [0071]    The skilled person will also understand that other forms of separation membranes can be used in the system and method of the invention, either alone or in combination. Thus, for example, the system can be used to separate components on interest on the basis of size, charge, chirality by selection of the appropriate membrane. A combination of different types of membranes (e.g. ultrafiltration, microfiltration, affinity membranes, reverse phase membranes, ion exchange membranes, hydrophobic membranes) can be employed in the system, as illustrated in the embodiment depicted in  FIG. 3 . The transverse section in  FIG. 3  shows a system according to the invention utilising three different forms of separation—i.e. affinity chromatography, ultrafiltration and microfiltration. Such a system is particularly suitable for the separation of proteins from biological samples. 
         [0072]    The system  3  has a similar configuration to that described in  FIGS. 1 and 2  above and operates in a similar manner. A first pump  310  and second pump  320  are in fluid connection with one another through one or more filter units  330 ,  340 ,  350  connected through a first and second flow-directing valve  370 ,  380 . Filter unit  330  contains an affinity membrane (not shown), unit  340  a microfiltration membrane  348  and unit  350  an ultrafiltration membrane  358 . 
         [0073]    Each pump comprises a receiving chamber  312 ,  322  and a moveable wall  314 ,  324  connected through a drive shaft  316 ,  326  to independent drives  318 ,  328 . A solution  391  can be drawn into or expulsed from the receiving chamber  312 ,  322  by the axial movement of the wall  314 ,  324  relative to the body of the pump  310 ,  320  when the drive  318 ,  328  is activated. The walls of the receiving chamber  312 ,  322  are made of an inert material, such as glass, ceramics, stainless steel or an appropriate plastic polymer which can withstand high operational pressures and not react with any components within the solution. 
         [0074]    In the example shown, the solution  391  contains a protein of interest which is to be separated from other components present in the solution by affinity chromatography and microfiltration, followed by washing and diafiltration. 
         [0075]    Solution  391 , which comprises a protein of interest in mixture with other components, is aspirated from its container into the receiving chamber  312  of the first pump  310  via tubing  371  and valve  370  by the upward movement of the wall  314  (i.e. in the opposite direction to the arrow shown in  FIG. 3 ). The tubing  371  and valve  370  are made of conventional materials, such as metals or plastics, which do not react with any components in the solution. The valve  370 , comprises one or more ports (not shown) which can be used to allow the valve  370  to act as a filter unit  330  selecting valve and/or an inlet/outlet valve. 
         [0076]    In the first half of the affinity separation cycle, solution  391  is driven from the receiving chamber  312  of the first pump  310 , by movement of the wall  314  in the direction of the arrow shown in  FIG. 3 , through the valve  370  and into the filter unit  330  (via tubing  376 ). As described in  FIGS. 1 and 2  above, the first pump  310  controls or regulates the flowrate of ‘feed’ solution  391  (i.e. the solution prior to filtration) moving into and through the filter unit  330 . 
         [0077]    The filter unit  330  comprises a first inlet  332  in fluid communication with an outlet  334 , the inlet and outlet being connected to the first and second flow-directing valves  370 ,  380 , respectively, by inert tubing  376 ,  386 . The inlet  332  is separated from the outlet  334  by an affinity membrane (not shown) to which the protein of interest in the solution selectively binds. Affinity membranes are well known in the art (see for example ‘Affinity Membranes: Their Chemistry and Performance in Adsorptive Separation Processes’, E Klein, 1991) and are commercially available from a number of suppliers (e.g. GE Healthcare Life Sciences). An affinity membrane will be chosen or prepared such that the protein of interest is bound to the membrane while other components in the sample pass through the membrane and are collected in the receiving chamber  322 . The contents of the receiving chamber  322  are then discarded as waste  336  in the second half of the cycle following reversal of the flow (as described in  FIGS. 1 and 2  above). 
         [0078]    Bound protein is released from the affinity membrane in the second cycle by washing with an appropriate affinity buffer  431  and collecting the protein-enriched fraction in the receiving chamber  322  (the process may be repeated using an additional affinity buffer  432  as required to ensure complete removal of the protein from the affinity membrane). This fraction may be purified by passage across microfiltration membrane  348  in the second half of the cycle, to remove any high molecular weight contaminants, the resulting permeate  345  being collected. 
         [0079]    The permeate  345  can then be concentrated further or subjected to diafiltration by passage across ultrafiltration membrane  358  in a third cycle. If diafiltration is desired, the permeate  345  is diluted with a dialysis buffer  430  and the retentate obtained by passage across the membrane in the first half of the cycle is collected as product  411 , either directly or following further dilution with dialysis buffer  430 , the permeate being discarded as waste  355 . Alternatively, the retentate may be purified still further by reversing the direction of flow across the ultrafiltration membrane  358  (as described in  FIGS. 1 and 2  above) to remove any remaining low molecular weight components and collecting the retentate in the first receiving chamber  312  (the permeate from the ultrafiltration being discarded as waste  355 ). The retenate can then be collected directly as product  411  by expulsion from chamber  312  (via valves  370 / 380 ) or diluted further with diafiltration buffer  430  prior to collection as product  411  (via valves  370 / 380 ). 
         [0080]    If the user simply wishes to concentrate the protein, then the permeate  345  is subjected to the ultrafiltration steps described above without the addition of the diafiltration buffer  430 . The retentate produced is then collected as product  411 . 
         [0081]    Washing fluids  421 ,  422 ,  423  can be used to clean the membranes and filter units  330 ,  340 ,  350  at the end of a complete cycle. 
         [0082]    It will be understood that the skilled person may wish to carry out variations in the separation process described in  FIG. 3  above. Thus, for example, it is possible to carry out the same process but in a different sequence (e.g. microfiltration first, followed by ultrafiltration/diafiltration and then affinity separation). Such variations are clearly possible, the order in which each of the separation steps are conducted depending upon the objective of the skilled person. 
         [0083]    The above embodiments are to be understood as illustrative examples of the invention. Further embodiments of the invention are envisaged. It is to be understood that any feature described in relation to any one embodiment may be used alone, or in combination with other features described, and may also be used in combination with one or more features of any other of the embodiments, or any combination of any other of the embodiments. Furthermore, equivalents and modifications not described above may also be employed without departing from the scope of the invention, which is defined in the accompanying claims.

Technology Classification (CPC): 1