Patent Publication Number: US-2021170303-A1

Title: Packing of Chromatography Columns

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     The present application is a continuation of U.S. application Ser. No. 14/201,175, filed Mar. 7, 2014, which is a continuation-in-part of U.S. application Ser. No. 13/518,029, now U.S. Pat. No. 9,958,421, issued May 1, 2018, which is a filing under 35 U.S.C. § 371 and claims priority to international application number PCT/SE2010/051424 filed Dec. 20, 2010, which claims priority to application No. 0951003-3 filed in Sweden on Dec. 22, 2009, the entire contents of which are incorporated herein by reference in their entirety. 
    
    
     TECHNICAL FIELD OF THE INVENTION 
     The present invention relates to packed bed columns useful for liquid chromatography separation of biomolecules, to a method of packing such columns and to methods of separating biomolecules with the columns. 
     BACKGROUND OF THE INVENTION 
     Columns used in liquid chromatography typically comprise a tubular body enclosing a packed bed of porous chromatography medium through which a carrier liquid flows, with separation taking place by partitioning between the carrier liquid and solid phase of the porous medium. 
     Prior to any separation process, the bed has to be prepared by starting from the particulate medium that is to be introduced into the column. The process of bed formation is called ‘the packing procedure’ and a correctly packed bed is a critical factor influencing the performance of a column containing a packed bed. Typically, the packed bed is prepared by slurry packing, i.e. consolidating a suspension of discrete particles in liquid, known as slurry that is pumped, poured, or sucked into the column. Once the predetermined volume of slurry has been delivered into the column it needs to be further consolidated and compressed by moving a movable adapter down the longitudinal axis of the column towards the bottom of the column, normally at a constant speed. The excess liquid during this procedure is expelled at the column outlet, while the media particles are retained by means of a filter material, a so-called ‘bed support’, with pores too small to allow the media particles to pass though. The packing process is complete once the packed bed has been compressed by the optimum degree of compression. Another approach for column slurry packing is the flow packing method, where compression of the porous structure is primarily achieved by applying a high flow rate over the column, hereby forming a porous structure starting at the outlet bed support. The resulting drag force on the particles in the porous structure causes eventually a pressure drop and a compression of the bed. The compressed bed is finally confined by bringing the adapter into position. 
     The efficiency of subsequent chromatographic separation relies strongly on 1) the liquid distribution and collection system at the fluid inlet and outlet of the packed bed, 2) the special orientation (also know as the packing geometry) of the media particles in the packed bed, and 3) the compression of the packed bed. If the compression of the packed bed is too low then chromatographic separations performed on that bed suffer from “tailing” and, generally, such insufficiently compressed beds are unstable. If the compression of the packed bed is too high then chromatographic separations performed by the bed suffer from “leading” and such over-compressed beds can affect throughput and binding capacity, and, in general, give much higher operating pressures. If the compression is optimum, then the separation peaks formed during use exhibit much less leading or tailing and are substantially symmetrical. The optimum degree of compression is also crucial for achieving good long-term stability of the porous structure, hereby securing optimal performance throughout a number of process cycles. The optimum degree of compression required for a column is determined experimentally for each column size (width or diameter), bed height, and media type. 
     An alternative packing method is called “dry packing”, where the column is filled with dry particles of the porous medium and liquid is introduced in the column afterwards. This has advantages in prepacked columns that can be delivered dry to the customer without having to add any preservatives to the packing liquid and minimizing weight during transport. Dry packing is typically used for silica media aimed at separation of small molecules, as described e.g. by G Guiochon J Chromatogr A 704 (1995) 247-268, although fairly poor column efficiencies are obtained. For swellable chromatography media, such as dextran or agarose-based media commonly used in separation of biomolecules, dry packing has however been avoided due to a perception that the swelling of the particles will cause poor performance of the packed bed. U.S. Pat. No. 4,353,801 mentions dry packing of solvent-swellable styrene-divinylbenzene particles as being inferior to wet packing. 
     SUMMARY OF THE INVENTION 
     One aspect of the present invention is to provide a packing method for a high efficiency chromatography column starting from dry swellable particles. This is achieved with a method comprising the steps of a) transferring to a column chamber  2  in a chromatography column  1  an amount of said dry swellable particles  5  sufficient to give a swollen volume Vs in a liquid of about 105-120% of the column chamber volume, b) closing the column and c) providing said liquid to the column. In other words, the method comprises metering out (e.g. by weighing) an aliquot of dry swellable particles  5 , providing said aliquot to a column chamber  2  in a chromatography column  1 , closing the column chamber and supplying a liquid to the column chamber, wherein the swollen volume Vs of said aliquot is about 105-120% of the column chamber volume. 
     A specific aspect of the invention is a storage stable prepacked column that is easily shipped to the user and equilibrated by the user. This is achieved by a column comprising dry swellable particles, wherein the amount of particles is sufficient to give a swollen volume Vs in an aqueous liquid of about 105-120% of the column chamber volume. 
     Another aspect is to provide a method for preparation of a plurality of columns connected in parallel from dry particles of a chromatography medium. This is achieved by a method as defined in the claims, which allows the preparation of columns with remarkably low differences in permeability. This is important in order to avoid overall band broadening due to flow rate differences between the columns. 
     A further aspect is to provide a method for manufacturing a chromatography subsystem comprising a plurality of chromatography columns connected in parallel, using dry particles of a chromatography medium to pack the columns. This is achieved by a method as defined in the claims and enables a chromatography system which is scalable by the addition of further chromatography columns connected in parallel. 
     Other aspects are a plurality of columns connected in parallel as described above and their use for separation of biomolecules, where their unique scalability is advantageous. 
     One or more of the aspects above may be achieved by the present invention as defined by the appended claims. Additional aspects, details and advantages of the invention will appear from the detailed description and claims that follow. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  i) shows a schematic drawing of a column  1  with fluid distributors  3  and a column chamber  2  delimitated by porous screens  4 . 
         FIG. 1  ii) shows the column with dry swellable particles  5  in the column chamber. 
         FIG. 1  iii) shows the column after application of liquid with the now swollen particles  6  filling the column chamber. 
         FIG. 2  i) shows a schematic drawing of a flexible container  7  with dry swellable particles  5 . 
         FIG. 2  ii) shows the flexible container  7  placed in the column chamber  2  with porous top and bottom plates  13 . 
         FIG. 2  iii) shows the column after application of liquid with the now swollen particles  6  filling and expanding the flexible container in the column chamber. 
         FIG. 3  shows a schematic drawing of a column  14  with dry swellable particles  5  in a tubular column chamber  8  with a rigid tube wall  9 , flexible porous endpieces  10  and fitted in a rigid housing  11  with fluid distributors  3  and porous top and bottom plates  13  in contact with the porous endpieces  10 . 
         FIG. 4  is a flow diagram of an embodiment where step a) comprises transferring dry particles to the column, step b) comprises closing the column and step c) comprises supplying liquid to the column. 
         FIG. 5  is a flow diagram of an embodiment where step a) is preceded by a step a′) comprising determining the liquid uptake of the dry particles and from the liquid uptake deciding the amount of particles to transfer to the column. 
         FIG. 6  is a flow diagram of an embodiment where step c) is preceded by a step c′) comprising vibrating the column. 
         FIG. 7  illustrates the calculation of reduced plate height h and asymmetry factor As for a column. 
         FIG. 8  shows an arrangement with two columns coupled in parallel. 
     
    
    
     DEFINITIONS 
     The term “swellable particles” means herein particles that increase in size upon immersion in a liquid. This can be observed as a sediment volume in the liquid which is significantly larger than the bulk volume of the same amount of dry particles. 
     The term “swollen volume” (Vs) means herein the sediment volume of an aliquot of particles suspended and equilibrated in a liquid. The sediment volume can be measured by suspending the aliquot of particles, equilibrating for up to 24 h, resuspending the particles if needed, letting the particles sediment and measuring the sediment volume in a graded vessel (e.g. a measuring cylinder). 
     The term “liquid uptake” (Vs/md) means herein the ratio between the swollen volume Vs of an aliquot of particles as defined above and the dry weight and of the particle aliquot before immersion in the liquid. 
     DETAILED DESCRIPTION OF EMBODIMENTS 
     In one embodiment of the invention, illustrated in  FIGS. 1 and 4 , an amount of dry swellable particles  5  is transferred to a column chamber  2  in a chromatography column  1 , wherein the amount of particles is sufficient to give a swollen volume Vs in a liquid of about 105-120% of the column chamber volume, the column is closed and the liquid is supplied to the column chamber. Since the swollen volume is higher than the column chamber volume, the particle bed will be compressed to a corresponding degree (the compression factor CF for a packed column is usually defined as Vs/Vcol, where Vcol is the column chamber volume), which provides for a high column efficiency and low peak asymmetry. The column efficiency can be expressed as the reduced plate height h, which is the plate height divided by the average diameter of the particles. The plate height is measured by methods well known in the art. The peak asymmetry can be expressed as the asymmetry factor As, which is the ratio between the distance from the back slope of a peak to the center of the peak and the distance from the front slope to the peak center (all measured at 10% of the peak height). Both the plate height and the asymmetry factor are suitably measured for small molecule non-retained species. The column may be designed for axial or radial flow and it may comprise fluid distributors  3 . The inlet and outlet of the column chamber may be delimitated by porous screens  4  to retain the particles and let liquids pass. The porous screens may be prepared from woven meshes, sintered frits or any other type of porous material. 
     In a further embodiment, depicted in  FIG. 5 , an initial step in the method is to determine the liquid uptake Vs/md of the dry swellable particles and to decide from said liquid uptake the amount of dry swellable particles to be transferred to the column chamber. The liquid uptake can be determined from a separate sample aliquot of the particles, using established methods for taking representative samples. The sample is weighed dry (i.e. in equilibrium with the ambient atmosphere) and is then suspended in a test liquid. The test liquid can be the same liquid to be used in the packing and operation of the column or a liquid of similar composition; typically an aqueous buffer or salt solution of similar ionic strength/conductivity and pH. The particles in the test liquid are left to equilibrate, typically for about one hour and in any case not more than 24 hours. The particles are resuspended and left to sediment. The sediment volume is measured e.g. in a measuring cylinder. The liquid uptake is then calculated as the ratio Vs/md between the sediment volume Vs and the weight and of the dry particle sample. An advantage of initially measuring the liquid uptake is that the swollen volume of the particles in the column can be predicted, resulting in better control of the compression and column efficiency. In a specific embodiment the liquid uptake is determined with less than 5% coefficient of variation or even less than 2% coefficient of variation to allow for high precision in the control of the column performance. 
     In an alternative embodiment, the liquid uptake is determined on basis of dry particle volume instead of using the weight of the dry particles as reference. Equally, preparation of aliquots to be filled into at least one column of a given specific column may be conducted on basis of dry particle volume instead of dry particle mass. 
     In certain embodiments the dry swellable particles in the column chamber can be manipulated to allow for a more even spatial distribution before the providing of liquid to the column. In one embodiment, illustrated in  FIG. 6 , the column is subjected to vibration before the providing of liquid and in another embodiment gas (e.g. air) is injected from the bottom end of the column before the providing of liquid. Both of these actions can cause at least partial fluidisation of the dry particles, leading to a more even distribution of the particles over the horizontal bottom surface of the column chamber. An advantage of this is that a more even spatial distribution of the swollen particles after application of the liquid may be achieved. The vibration or gas injection may be performed at any stage after the transfer of the dry swellable particles to the column chamber or a flexible container. In one embodiment an inert gas may be injected into the column to provide protection against oxidative degradation reactions during storage. 
     In one embodiment the liquid is provided to the column in the upflow direction. The liquid will then rise evenly in a vertical direction from the bottom inlet through the dry particle bed, resulting in a more even particle distribution than if the liquid is pouring down from the top of the column. Once the particles have been swollen by the liquid, the column may then be operated in any direction (upflow, downflow, tilted etc.) In a specific embodiment the rate of liquid addition to the dry swellable particles does not exceed the rate of capillary suction by the particles. The rate of capillary suction can be determined e.g. by placing a column with dry swellable particles in a trough containing liquid up to the lower end of the dry bed and measuring the rate of liquid rising through the bed by optical, gravimetric or other methods. Keeping the rate of liquid addition lower or equal to the rate of capillary suction has the advantage of giving a more homogeneous particle distribution after swelling and hence better column performance. The rate of liquid addition can be expressed as the liquid velocity in the column chamber (the liquid flow rate divided by the internal cross section area of the column chamber). In a specific embodiment the liquid velocity in the column chamber is less than 100 cm/h, such as between 5 and 70 cm/h, during the addition of liquid to the dry particles. In one embodiment the liquid is aqueous and it can then comprise a wetting agent (an additive that decreases the surface tension), such as a lower alcohol, e.g. ethanol, or a surfactant. An advantage of this is that a more uniform distribution of the particles after swelling can be obtained. 
     In one embodiment, depicted in  FIG. 2 , the step of transferring the swellable dry particles to a column chamber in a chromatography column comprises first transferring the dry swellable particles  5  to a flexible container  7  and then placing the flexible container in the column chamber  2 . This has the advantage that the particles can be supplied to the user packaged in an easily transported low-cost flexible container and the user can place the container in the column and apply liquid. The walls of the flexible container can have porous portions to be fitted against the porous top and bottom plates of the column chamber. The flexible container can be e.g. a plastic bag construction or be assembled from molded and/or extruded parts. In one embodiment the flexible container is expanded by the swollen particles to fit the column chamber. 
     In one embodiment the method comprises, after the transfer of dry swellable particles to the column chamber  8  and before the closure of the column, a step of transferring the column chamber  8  to a rigid housing  11  providing dimensional stability for the column chamber during swelling of the gel and/or operation of the column. In this context, the free-standing column chamber  8  may deflect somewhat during swelling of the gel or due to pressurization during operation of the column. A rigid housing  11  will prevent any such deflection and any associated deterioration of the column performance. 
     In one embodiment the method comprises before the addition of liquid to the column a step of radiation sterilising the column or the flexible container with the dry swellable particles. This step may be carried out at any stage of the method. The radiation sterilisation can be carried out through gamma or electron beam irradiation. 
     One embodiment of the invention is a column comprising dry swellable particles, wherein the amount of particles is sufficient to give a swollen volume Vs in an aqueous liquid of about 105-120% of the column chamber volume. In one embodiment this liquid is a 0.1 mol/l solution of sodium chloride (NaCl) in water at 20° C. Such a solution has a conductivity of approx. 10 mS/cm which is in the range of many buffers used in biomolecule/biopharmaceutical separations. 
     In one embodiment the liquid uptake Vs/md of the dry swellable particles (in 0.1 M aqueous NaCl at 20 C) is between 5 and 25 ml/g, such as between 5 and 15 ml/g. An advantage of this embodiment is a good control of the compression factor and minimised variability in swelling (and column performance) with composition of the liquids applied during operation of the column. In one embodiment the liquid uptake of the dry swellable particles in distilled water is less than 1.5 times the liquid uptake in 0.1M aqueous NaCl. An advantage of this is that the variability in swelling with liquid composition is kept low. 
     In one embodiment the column volume is fixed. A fixed volume column can be constructed without any moving parts (adaptors etc), which simplifies construction and is advantageous from a cost point of view. In one embodiment the column comprises molded or extruded parts. Molding and extrusion methods such as injection molding, blow molding, rotation molding, compression molding etc are convenient and cost-efficient methods to manufacture column parts from e.g. thermoplastic materials. 
     In one embodiment the column chamber volume is at least one litre. Columns with more than one litre bed volume are frequently used in industrial biopharmaceutical purification. They are often complicated and expensive steel constructions with moving parts, so solutions allowing use of simple plastic constructions are desirable. 
     In one embodiment illustrated by  FIG. 2 , the column  1 , comprises a flexible container  7  comprising the dry swellable particles  5 . This has the advantage that the particles can be supplied to the user packaged in an easily transported low-cost flexible container and the user can place the container in the column and apply liquid. The flexible container can comprise at least one flexible wall, i.e. a wall that can deflect enough to cause at least 1% volume change of the container when an overpressure of 1 bar is applied to the unsupported container. The walls of the flexible container can have porous portions fitted against the porous top and bottom plates  13  of a column chamber  2 . The flexible container can be e.g. a plastic bag construction or be assembled from molded and/or extruded parts. In one embodiment the flexible container is expandable to fit the column chamber upon swelling of the particles. In another embodiment, porous top and bottom plates segments that retain the particles and provide liquid distribution may be incorporated into the flexible container. In this case, the flexible container is a closed system and not in fluid contact with the housing or other parts supporting the flexible container mechanically from the outside. 
     In one embodiment illustrated by  FIG. 3 , the column comprises at least one tubular column chamber  8 , with e.g. circular, elliptical or rectangular cross section, with at least one rigid tube wall  9  and at least one flexible endpiece  10 . The flexible endpiece can be porous and placed in immediate contact with a porous top or bottom plate  13  in a rigid housing  11 . The top and bottom ends of the rigid housing can be held together by a generic clamping structure well known in the art (not shown in  FIG. 3 ). 
     In one embodiment the dry swellable particles comprise a polysaccharide, such as crosslinked agarose. Crosslinked agarose particles are highly useful for biomolecule and biopharmaceutical purification due to their high throughput and low non-specific adsorption. They are normally supplied in aqueous ethanol solution and an advantage of using dry agarose particles is that no flammable ethanol has to be handled. Drying of agarose particles can be done using e.g. freeze drying, spray drying or vacuum drying. 
     In embodiment the dry swellable particles comprise charged ligands. Such ligands are predominantly used in ion exchange and multimodal separations, both of which are highly useful in biomolecule and biopharmaceutical separations. Both cation exchangers, anion exchangers and multimodal ion exchangers are contemplated. 
     In one embodiment the dry swellable particles comprise ligands comprising hydrophobic functionality. Such ligands are used in hydrophobic interaction chromatography and many multimodal separations, both of which are highly useful in biomolecule and biopharmaceutical separations. Examples of ligands and ligand parts with hydrophobic functionality are alkyl and aryl groups. 
     In one embodiment the dry swellable particles comprise affinity ligands. Such ligands are highly useful for high selectivity separation of biomolecules and biopharmaceuticals. Examples of affinity ligands are protein A, protein G, protein L, lectins, enzyme substrates, lectins, biotin, avidin, antibodies, antibody fragments, antigens etc. 
     In one embodiment the liquid provided to the column with the dry swellable particles comprises at least one biomolecule, such as a biopharmaceutical. Examples of such liquids include protein solutions and virus suspensions, e.g. feeds to be purified in bioprocessing operations. An advantage of swelling the dry particles directly with a feed is that a separate swelling step can be eliminated and that the swelling in itself can provide a separation and concentration effect for species not penetrating into the swollen structure. 
     One embodiment of the invention is a column comprising swollen particles, packed according to the previously described embodiments. In a specific embodiment the column has a reduced plate height h less than 15, such as less than 10 for a test species. The test species can be a non-retained small molecule, such as an inorganic salt or acetone. In a further embodiment the asymmetry factor As of the column is less than 3, such as less than 2.5 for a test species selected as above. 
     One embodiment is at least two columns which are prepared from the same batch of dry swellable particles. In a specific embodiment the columns are connected in parallel. Connection in parallel of columns with the same bed volume, particle type and bed height is a convenient way of increasing the total cross-sectional area and throughput. When columns connected in parallel are used, it is however of great importance that the fluid velocities are identical in the columns to ensure that the same separation is achieved in each column. This puts high demands on the reproducibility of the columns with respect to hydraulic resistance and pressure-flow behavior. With dry swellable particles from the same batch in the columns, it is possible to control the hydraulic resistance and pressure-flow behavior well enough to allow parallel use. 
     In one embodiment the column comprising swollen particles is used for separation of at least one biomolecule such as a biopharmaceutical. Suitable biomolecules can be proteins, peptides, nucleic acids, carbohydrates, virus particles etc. Suitable biopharmaceuticals can be immunoglobulins (e.g. monoclonal antibodies), immunoglobulin fragments and other constructs, insulin and other therapeutic peptides, erythropoietin, plasma proteins, oligonucleotides, plasmids, vaccines etc. In a specific embodiment the biomolecule or biopharmaceutical is a protein. 
     In one embodiment the biomolecule or biopharmaceutical binds to the particles and at least one impurity is removed by washing with a washing liquid. The biomolecule or biopharmaceutical may then be eluted from the particles with an elution liquid. This mode is often called bind-elute separation and is particularly useful when the amount of impurities is significant and/or when a very high separation selectivity is required. 
     In another embodiment at least one impurity binds to the particles and the biomolecule or biopharmaceutical is recovered in the flow-through of the column. This mode is often called flow-through separation and provides a very high throughput, particularly when the amount of impurities is relatively low. 
     In a further aspect the invention discloses a method for preparing a connected plurality of packed chromatography columns, comprising the steps of:
         a) providing dry chromatography medium particles and preparing a plurality of aliquots of said particles, which aliquots differ from each other by less than 5.0 percent by weight;   b) providing a plurality of substantially identical chromatography columns and packing one of said aliquots into each chromatography column to obtain a plurality of packed chromatography columns;   c) fluidically connecting said plurality of packed chromatography columns in parallel.       

     The chromatography medium particles can e.g. be the dry swellable particles discussed above, but they can also be other types of chromatography media. The aliquots can suitably be prepared by weighing, which easily allows the provision of aliquots which differ from each other by less than 2.0 percent by weight, such as by less than 1.0 percent or even less than 0.5 percent by weight. In contrast, the preparation of aliquots from chromatography media suspended in liquids is much less accurate due to sedimentation and segregation effects. The weighing and any determination of the weight differences can suitably be done when the particles are in equilibrium with the surrounding atmosphere, to avoid any effects of differences in moisture uptake after drying processes. The aliquots can e.g. be prepared from a single homogeneous batch of dry chromatography medium particles. This batch can either be a single manufacturing batch or a homogeneous blend of several manufacturing batches. The homogeneity can for example be ensured by tumbling or other type of mixing. If any segregation effects should be observed, for example in cases of very wide particle size distributions or density distributions, the homogeneity of the aliquots might be improved by using for example a riffler or rotary sample divider to divide the particles into rough aliquots, weighing the aliquots and making fine adjustments of their weights. 
     Alternatively, the aliquots can be prepared from different batches. In this case, the batches have suitably been tested and found to be sufficiently similar to allow for use in parallel columns. Suitable testing parameters include volume average particle size (diameter), width of the particle size distribution and liquid uptake as discussed above. The width of the particle size distribution can for example be measured as a standard deviation or as a ratio between the volume and number average sizes (diameters). The acceptance values for the testing parameters can be selected such that the permeability or flow velocity differences between columns packed from the different batches are less than 20, 10, 5, 2 or 1 percent. 
     In one embodiment the method further comprises, after step b), c) or d), a step b′) of supplying a reswelling liquid to said plurality of packed chromatography columns. This step allows the swelling of the particles and can provide compression of the column as described in the embodiments above. 
     In one embodiment the method comprises, after step a) and before step b), a step a′) of separately suspending the aliquots in a packing liquid. This embodiment allows a wet packing of the columns from resuspended particles. As the aliquots are prepared with a low weight difference from dry particles, this embodiment still allows the preparation of columns with low differences in permeability and other properties. 
     In one embodiment step c) of the method further comprises fluidically connecting the plurality of chromatography columns to a single sample inlet. The connection may be as disclosed in  FIG. 8 , where the columns are connected via a manifold to a valve and further to a sample inlet. This allows the injection of one single sample to all columns in the plurality at the same time and suitably, all the flow paths between the sample inlet and the columns have the same lengths and volumes to avoid any system contributions to bandspreading between the columns. A pump system can suitably be fluidically connected to the manifold and arranged to convey liquids through the plurality of columns via the manifold. The pump system may comprise a single pump or, e.g. if gradients of liquids are to be provided, a plurality of pumps. The columns can further be coupled to a single sample outlet, e.g. via a second manifold and a valve as shown in  FIG. 8 . The connection to a single sample inlet allows scaling of a process chromatography step through addition of further chromatography columns coupled in parallel. 
     In a further aspect the invention discloses a method for manufacturing a chromatography subsystem comprising a plurality of chromatography columns connected in parallel, said method comprising the steps of:
         a) providing dry chromatography medium particles and preparing a plurality of aliquots of said particles, which aliquots differ from each other by less than 5.0 percent by weight;   b) providing a plurality of substantially identical chromatography columns and packing one of said aliquots into each chromatography column to obtain a plurality of packed chromatography columns;   c) providing a subsystem support structure and mounting said plurality of packed chromatography columns in said support structure;   d) fluidically connecting said plurality of packed chromatography columns in parallel.       

     The embodiments discussed above apply equally to this method. The subsystem support structure may e.g. be a rack or skid arranged to receive a plurality of chromatography columns. The chromatography columns can be individual self-supporting columns as shown e.g. in  FIG. 1 , but they can also comprise flexible containers or column chambers with support structures as depicted in  FIG. 2 or 3 . The support structure for such containers/chambers can e.g. be provided by the subsystem support structure. Examples of columns/containers/chambers and subsystem support structures are shown in WO2011152788, US20120118807 and US20120097591, which are hereby incorporated in their entireties. If sterile columns/containers/chambers are desired, they can be sterilized by radiation or high temperatures according to known methods or they can alternatively be packed under aseptic conditions. 
     In one embodiment all the columns are connected in parallel. In other embodiments, the subsystem comprises a plurality of serial column trains connected in parallel. 
     In another aspect the invention discloses a chromatography subsystem comprising a plurality of chromatography columns connected in parallel, manufactured by the above method. This subsystem can be used for separation of at least one biomolecule, such as a biopharmaceutical. 
     In one embodiment the biomolecule or biopharmaceutical binds to the particles, at least one impurity is removed by washing with a washing liquid and the biomolecule or biopharmaceutical is recovered from the column by elution with an elution liquid (bind-elute mode), while in another embodiment at least one impurity binds to the particles and the biomolecule or biopharmaceutical is recovered in the flow-through of the column or in a wash fraction (flowthrough mode). In both these embodiments it is advantageous with no or minimal flow velocity difference between the columns. In bind-elute mode it is advantageous to avoid premature breakthrough of the biomolecule/biopharmaceutical, which can occur if the flow velocity is higher in one particular column. It is also advantageous to have the same flow velocity in all columns during elution, to get a narrow elution peak and a correspondingly high concentration of the biomolecule/biopharmaceutical in the elution pool. In flowthrough mode it is advantageous to avoid premature breakthrough of the impurity. 
     In some embodiments the (relative) flow velocity difference between any two of said plurality of packed columns is less than 20 or less than 10 percent, such as less than 5, less than 2 or less than 1 percent. The flow velocity difference may be measured directly by flow meters in line with the different columns, but it can also be assessed indirectly e.g. by measuring the difference in retention time of a substance, such as a substance which does not bind to the chromatography medium. The flow velocity difference can be a direct function of the difference in permeability between the different columns and hence, the (relative) permeability difference between any two of said plurality of packed columns can be less than 20 or less than 10 percent, such as less than 5, less than 2 or less than 1 percent. The permeability may be calculated as the flow velocity through a column multiplied by the viscosity of the liquid and divided by the back pressure over the column. 
     Other features and advantages of the invention will be apparent from the following examples and from the claims. 
     EXAMPLES 
     Column Efficiency Test 
     A tracer (acetone 2% v/v in DI water) is applied as tracer with a volume of 1-1.5% of the column volume to analyze the residence time distribution at the column outlet (UV signal @ 280 nm). The residence time distribution should be represented as a single peak, compare  FIG. 7 . The evaluation is described below. 
     Column efficiency is typically defined in terms of two parameters:
         Peak broadening over the column is described by an equivalent number of theoretical plates (equilibrium stages)   Peak symmetry is described by a peak asymmetry factor A s          

     The relative peak width is defined as number of (theoretical) plates N, height equivalent of a theoretical plate HETP or preferably as reduced plate height h: 
     
       
         
           
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     The asymmetry factor A s  describes the deviation from an ideal Gaussian peak shape and calculates from the peak width at 10% of peak height: 
     
       
      
       A 
       S 
       =b/a  
      
     
     Nomenclature 
     Plate number N 
     Mean residence time 1  μ 1    
     Variance 1  σ 2    
     Retention time 2  t R    
     Retention volume 2  V R    
     Peak width at half peak height w h    
     Bed height L 
     Particle diameter d p    
     Height equivalent of a theoretical plate HETP 
     Reduced plate height h=HETP/d p    
     Asymmetry factor A s    1  first and second moments in residence time distribution (RTD) curve derived from integration of tracer signal 2  retention time (retention volume) corresponding to time (or eluted volume) at maximum peak height 
     Example 1 
     The dry swellable particles used were Q SEPHAROSE HP (GE Healthcare, Sweden), an anion exchanger with tetramethylammonium group ligands coupled to crosslinked agarose beads of 34 micron average diameter when swollen in water. The Q SEPHAROSE HP particles had been transferred to acetone and then vacuum-dried at room temperature. 
     129 mg dry particles per ml column chamber volume were transferred to two vertically standing columns of different types: an XK26 column (GE Healthcare, Sweden) having a column chamber of diameter 26 mm, height 30 mm and volume 15.93 ml and an XK50 column (GE Healthcare, Sweden) having a column chamber of diameter 50 mm, height 31 mm and volume 60.87 ml. 
     A water solution of 20% ethanol was pumped into the bottom inlet of each column with a fluid velocity of 60 cm/h. The pumping was continued until three column chamber volumes had passed the column and three column volumes of distilled water were then pumped through at a fluid velocity of 200 cm/h. The columns were conditioned by 4×3 column chamber volumes of distilled water at 300 cm/h, pumped in alternating upflow and downflow mode. 
     The performance of the columns was evaluated by the column efficiency test described above. The performance results are given in Table 1. 
     
       
         
           
               
               
               
               
               
             
               
                   
                   
               
               
                   
                 Run 
                 N/m 
                 h (—) 
                 As (—) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 XK26down002 
                 15746 
                 1.87 
                 2.03 
               
               
                   
                 XK26down003 
                 15739 
                 1.87 
                 2.05 
               
               
                   
                 XK26up002 
                 15969 
                 1.84 
                 1.96 
               
               
                   
                 XK26up003 
                 15882 
                 1.85 
                 2.01 
               
               
                   
                 XK50down003 
                 10672 
                 2.76 
                 1.27 
               
               
                   
                 XK50down004 
                 10818 
                 2.72 
                 1.31 
               
               
                   
                 XK50up003 
                 11020 
                 2.67 
                 1.25 
               
               
                   
                 XK50up004 
                 10645 
                 2.76 
                 1.28 
               
               
                   
                   
               
            
           
         
       
     
     Example 2 
     The dry swellable particles used were CAPTO S (GE Healthcare, Sweden), a cation exchanger with sulfonate group ligands coupled to dextran-extended crosslinked agarose beads of 90 micron average diameter when swollen in water. The CAPTO S particles had been transferred to acetone and then vacuum-dried at room temperature. 
     3.34 g dry particles were transferred to two a vertically standing XK26 column (GE Healthcare, Sweden) having a column chamber of diameter 26 mm, height 30 mm and volume 15.93 ml 
     A water solution of 20% ethanol was pumped into the bottom inlet of the column with a fluid velocity of 30 cm/h. The pumping was continued until three column chamber volumes had passed the column and 1.5 column volumes of distilled water were then pumped through at a fluid velocity of 100 cm/h. 
     The performance of the columns was evaluated by the column efficiency test described above. A stability test was also run with 340 cm/h distilled water in upflow mode for 20 h. The performance and stability results are given in Table 2. 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
               
                   
                 N/m 
                 h (—) 
                 As (—) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Run 
                   
                   
                   
               
               
                   
                 Down001 
                 3602 
                 3.27 
                 1.57 
               
               
                   
                 Up005 
                 4587 
                 2.56 
                 1.38 
               
               
                   
                 Down002 
                 4855 
                 2.42 
                 1.49 
               
               
                   
                 Up006 
                 5161 
                 2.28 
                 1.48 
               
               
                   
                 Down003 
                 4966 
                 2.37 
                 1.56 
               
               
                   
                 Up007 
                 5229 
                 2.25 
                 1.51 
               
               
                   
                 Down004 
                 5035 
                 2.34 
                 1.53 
               
               
                   
                 Up008 
                 5269 
                 2.23 
                 1.54 
               
               
                   
                 Stability test 
               
               
                   
                 Down001 
                 5066 
                 2.32 
                 1.57 
               
               
                   
                 Up001 
                 5251 
                 2.24 
                 1.58 
               
               
                   
                 Down002 
                 5010 
                 2.35 
                 1.58 
               
               
                   
                   
               
            
           
         
       
     
     Example 3 
     As dry swellable particles, a single batch of the chromatography medium CAPTO Q (GE Healthcare Bio-Sciences AB, Sweden) was used. This is an anion exchanger with tetramethylammonium group ligands coupled to crosslinked agarose beads of 90 micron average diameter when swollen in water. The medium was obtained as a slurry in 20% aqueous ethanol solution and was dried by sequential washing on a glass filter with distilled water, 99.5% ethanol and acetone, followed by vacuum drying at 20° C. for 3 days. 
     Two aliquots of 12.70 g (calculated to give a free swollen volume of 66 ml) each were weighed out from the air-dry particles. Each of these was poured into an empty HISCALE 50 column (GE Healthcare Bio-Sciences AB, Sweden), empty column with high pressure stability vertically arranged with the adaptor positioned in the middle of the column. The adaptor in each column was then moved downwards to a position corresponding to a bed height of 30 mm. The columns were wetted with an upwards flow of 30 cm/h distilled water. After 6 column volumes had passed each column, the flow was raised to 300 cm/h for an additional 20 column volumes. A downward flow of 1000 cm/h distilled water was then applied for 60 column volumes. 
     The two columns were arranged in parallel, as shown in  FIG. 8 , in an AKTA Pilot (GE Healthcare Bio-Sciences AB, Sweden) protein purification chromatography system with a separate UV detector for each column. Efficiency tests as described above were performed on the assembly at different flow rates and the efficiency (N/m), asymmetry (As) and retention time (min) for the acetone peak were determined separately for the two columns denoted C1 and C2. The results are given in Table 3. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                   
               
               
                 Flow rate 
                   
                   
                   
                   
                   
                   
               
               
                 (cm/h) 
                 N/m C1 
                 N/m C2 
                 As C1 
                 As C2 
                 min C1 
                 min C2 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 30 
                 5070 
                 4613 
                 1.30 
                 1.36 
                 5.95 
                 5.82 
               
               
                 100 
                 3360 
                 2268 
                 1.29 
                 1.82 
                 1.82 
                 1.78 
               
               
                 500 
                 1712 
                 1544 
                 1.39 
                 1.50 
                 0.42 
                 0.40 
               
               
                   
               
            
           
         
       
     
     The relative difference in retention time of the acetone peak is a measure of the relative flow rate difference between the two columns and is within the 2.2-2.3% range for all three global flow rates. 
     The columns were then conditioned with three column volumes of 50 mM Tris buffer, pH 8.00 and the feed was switched to 5 mg/ml bovine serum albumin (BSA) dissolved in 50 mM Tris buffer, pH 8.00. About 80 column volumes of BSA solution was pumped in, before the BSA was eluted with 10 column volumes of 50 mM Tris+1 M NaCl, pH 8.00. This test was repeated at three different flow rates and the dynamic binding capacity at 10% breakthrough (Qb10) was calculated individually for each column according to standard methods. The results are presented in Table 4. 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Flow rate 
                 Qb 10 
                 Qb 10 
               
               
                 (cm/h) 
                 (mg BSA/ml gel) C1 
                 (mg BSA/ml gel) C2 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 180 
                 154 
                 148 
               
               
                 500 
                 130 
                 124 
               
               
                 1000 
                 106 
                 101 
               
               
                   
               
            
           
         
       
     
     The relative capacity differences between the two columns are within the 4-6% interval. 
     Example 4 
     The packing and efficiency tests of Example 3 were repeated with a single batch of CAPTO ImpRes Q (GE Healthcare Bio-Sciences AB, Sweden) as the chromatography medium, dried under the same conditions as in Example 3. Aliquots of 11.60 g (calculated to give a free swollen volume of 66 ml) air-dry particles were weighed out and otherwise the conditions were the same. The efficiency results are presented in Table 5. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                   
               
               
                 Flow rate 
                   
                   
                   
                   
                   
                   
               
               
                 (cm/h) 
                 N/m C1 
                 N/m C2 
                 As C1 
                 As C2 
                 min C1 
                 min C2 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 30 
                 8573 
                 10170 
                 1.55 
                 1.15 
                 6.03 
                 5.99 
               
               
                 100 
                 7262 
                 7915 
                 1.82 
                 1.15 
                 1.84 
                 1.84 
               
               
                   
               
            
           
         
       
     
     In this case the relative retention time difference between the two columns is less than 0.7%. 
     This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims. 
     All patents, patent publications, and other published references mentioned herein are hereby incorporated by reference in their entireties as if each had been individually and specifically incorporated by reference herein. While preferred illustrative embodiments of the present invention are described, one skilled in the art will appreciate that the present invention can be practiced by other than the described embodiments, which are presented for purposes of illustration only and not by way of limitation. The present invention is limited only by the claims that follow.