Patent Description:
This invention relates to chromatography columns and methods of manufacture and use.

Column chromatography is a separation and/or purification technique in which a stationary "bed" of a packing medium is contained within a rigid tube. The packing medium can be in the form of particles of a solid ("stationary phase") or a solid support material coated with a liquid stationary phase. Either way, the packing medium typically fills the inside volume of the column tube.

In separation chromatography, as a liquid sample ("mobile phase") passes through the column, different compounds in the sample can associate differentially with the stationary phase such that they are slowed relative to the mobile phase and move through the column at different speeds. Thus, those compounds that associate more with the stationary phase move more slowly through the column than those that associate less, and this speed differential results in the compounds being separated from one another as they pass through and exit the column. Features of the stationary phase that promote differential association can be ionic charge (ion exchange chromatography), hydrophobicity (hydrophobic interaction chromatography), and porosity (size exclusion chromatography).

In yet another type of column chromatography, affinity chromatography, the packing medium includes binding agents, such as antigens, antibodies, or ligands, that specifically bind to one or more desired compounds or molecules in the liquid sample. Thus, as the liquid sample flows through the packing medium only the desired compounds or molecules remain in the column. A subsequent flow through the packing medium of an eluting liquid separates the desired compounds or molecules from the binding agents attached to the packing medium, or separates the binding agents from the packing medium. Either way, the desired compounds or molecules are rinsed out of the column and collected in the eluting fluid. Affinity chromatography can be used in a number of applications, including nucleic acid purification, protein purification from cell free extracts, and purification from blood.

The main components of a chromatography column are the tube, which is often made of a metal, glass, or highly rigid plastic material, and a pair of flow distributors, which are typically inserted into the two ends of the tube to form a space or chamber in the tube between the flow distributors into which the packing medium is loaded. In such columns a small space or void can form between the outer edge of the flow distributors and the inner wall of the column tube up to the point at which the O-ring is creating a seal between the flow distributors and the inner wall of the column tube. This void creates a so-called "dead zone" or "dead space" into which fluids and contaminants can enter and become entrapped and stagnant, rather than flowing through the medium within the column tube. In addition, such dead zones can become contaminated and are difficult to clean when the columns are to be reused.

The current invention comprises a method according to claim <NUM>.

The invention is based, at least in part, on the discovery that if one manufactures chromatography column tubes from elastic plastic/thermoplastic and/or composite materials (such as polypropylene (PP), polyethylene (PE), polyamides, acetals, or glass-filled or carbon-filled plastics, e.g., glass-fiber and carbon-fiber plastics) and secures at least one of the two flow distributors within the column tube with a tight interference fit or press fit, the induced hoop tension opposing the interference fit between the flow distributor and the tube wall provides a sufficiently tight seal to prevent leakage and the resulting chromatography columns can be manufactured with significantly reduced dead zones around the press fit flow distributors. A second useful feature of the invention is that columns made in accordance with the description herein have an infinitely adjustable packing medium volume, also known as the medium "bed height.

In one aspect, the disclosure features methods of making and packing chromatography columns. These methods include: <NUM>) selecting a column tube, e.g., of plastic or another appropriately elastic material, that has an appropriate inner diameter and length to accommodate a desired volume of packing medium; <NUM>) selecting appropriately sized first and second flow distributors, wherein at least the second flow distributor (or both the first and the second flow distributors) has a diameter that is larger than the inner diameter of the tube, e.g., about <NUM> to <NUM>% larger than the inner diameter of the tube; <NUM>) permanently securing the first flow distributor to a first end of the tube; <NUM>) adding a packing medium into the column tube; <NUM>) inserting the second flow distributor into a second end of the tube by applying an axial force to drive the second flow distributor into the column tube to establish an interference fit, e.g., to thereby induce a hoop tension, that is sufficiently effective to from a sealed, e.g., a hydrostatically sealed, chamber within the tube between the first and second flow distributors; <NUM>) adjusting the longitudinal position of the second flow distributor within the tube by (i) applying an additional axial force to the second flow distributor until it reaches a desired location within the column tube, or (ii) forcing liquid into the chamber to apply a hydraulic force to move the second flow distributor back towards the second end of the tube, or any combination of (i) and (ii); and <NUM>) when the second flow distributor is properly positioned, permanently securing the second flow distributor within the tube.

The new methods can include securing both the first and second flow distributors into the tube by applying an axial force to drive the flow distributors into the tube to establish an induced hoop tension that is sufficient to produce a hydrostatic seal. This induced hoop tension, created by the interference fit between the flow distributor and the tube wall reduces or avoids the formation of any gaps between an outer circumferential surface of the first and/or second flow distributors and an inner surface of the tube. In some embodiments, the first and/or second flow distributors can be permanently secured within the tube, for example by welding or other means.

In some embodiments, the axial force to drive the second flow distributor into the tube to establish the interference fit within the tube is about <NUM> kN (<NUM> lbf) to about <NUM> kN (<NUM>,<NUM> lbf).

In some embodiments an inner surface of the tube includes a chamfer formed around at least one end of the tube to aid in inserting and centering the flow distributor into the column tube. In some of the new methods, the first flow distributor can be formed as an integral component of the tube.

In certain embodiments, wherein the packing medium can include a slurry that comprises about <NUM>% to about <NUM>% solids.

In another aspect, the disclosure features chromatography columns that include a plastic tube having a first end and a second end and an inner diameter DTi, wherein the inner diameter DTi is gradually increased at the second end of the tube to an end diameter DTe to form a chamfer; a first flow distributor secured to a first end of the plastic tube; and a second flow distributor having an external diameter Dfd that is greater than DTi (e.g., at least <NUM>%, e.g., about <NUM> to about <NUM>, <NUM>. <NUM>, <NUM>. <NUM>, <NUM> or <NUM>% greater); wherein the second flow distributor is secured within the second end of the tube with an interference fit directly resulting in sufficient induced hoop tension to form a hydrostatically sealed chamber within the tube between the first and second flow distributors.

In some embodiments, the plastic tube further has an increased end diameter DTe to form a chamfer at the first end, wherein the first flow distributor has an external diameter Dfd that is greater than DTi, and wherein the first flow distributor is secured within the first end of the tube with an interference fit directly resulting in sufficient induced hoop tension. In certain embodiments, the first flow distributor is permanently bonded to the tube or both the first and second flow distributors can be secured to the inner wall of the tube with a permanent bond such as a welded joint.

In certain embodiments, the new chromatography columns can include a packing medium within the chamber. In some embodiments, the chamber is hydrostatically sealed. In certain embodiments, the chamber is constructed to withstand an internal pressure that is at least <NUM> kPa (<NUM> pounds per square inch).

In some embodiments, all three of these features are present.

In some embodiments the plastic tube and the second flow distributor are made of the same type of plastic and the first flow distributor is an integral feature of the tube.

As used herein, the term "bed height" refers to the linear height of the bed of packed chromatography media particles contained within a completed chromatography column.

As used herein, a "packed bed" refers to the final state of chromatography media particles within a chromatography column. This final state is achieved in a variety of ways. For example, one method is to combine fluid flow followed by axial compression of the bed by one or both of the flow distributors. Other methods known in the art include gravity settling of particles, vibration settling, and/or mechanical axial compression alone.

As used herein, a "flow distributor" is a component, e.g., a cylindrical component, which is secured at or near each end of a chromatography column. The flow distributors can be multi-part assemblies that serve multiple purposes. One function is to convey liquid into/out of the column by means of a port that can mate with different pipes/tubing that feed liquids into or out of the column. Another function is to direct inflow of liquid from one or multiple smaller channels to spread the liquid as evenly as possible over the entire cross-sectional area of the packed bed. Conversely the flow distributor on the outlet side of the column must efficiently gather liquid spread across the entire cross-sectional area and convey it out of the column through one or multiple smaller channels (e.g., a <NUM> column can have inlet/outlet ports of <NUM> diameter).

As used herein, a "bed support" is a net, screen, mesh, or frit that allows the passage of various liquids yet retains the small particles of packing medium that comprises the packed bed. These bed supports can be directly connected to the flow distributors.

As used herein, the terms "permanent bond" and "permanently bonded" are used to indicate that such a bond between two components cannot be separated other than by breaking the bond or one or both of the bonded components (e.g., a tube and a flow distributor).

As used herein, the term "induced hoop tension" refers to the circumferential stress generated in the wall of the tube by the insertion of a flow distributor with an outer diameter that is larger than the inner diameter of the tube. The diametrical difference between these values is referred herein as the interference fit. The induced hoop tension is triggered by internal stresses due to the interference fit as the flow distributor is forced to compress and deflect inward and the tube wall is stretched outward.

Due to the tight fit between the flow distributor and the tube wall resulting from the induced hoop tension, the new chromatography columns avoid the formation of dead zones in the vicinity of the flow distributors, which provides significant advantages in terms of flow efficiency and the ability to adequately clean the columns for reuse.

Another unique advantage of the new methods of manufacture is the ability to construct pre-packed, disposable columns with fully customizable and variable bed heights and diameters. The resolution of the specific column bed heights is limited only by the available press and linear actuator technologies used to press-fit the flow distributors through the length of column tubes. Current technologies are capable of resolving the exact location of the flow distributors within a tube to a few thousandths of an inch or better. Once a flow distributor is moved into a desired position, the induced hoop tension allows the column to withstand significant operational pressures and maintain a hydraulic seal without being permanently fixed in position. This initial seal provides the opportunity to test the performance of the column prior to permanently securing the second flow distributor. If testing reveals that column performance can be improved by an axial adjustment of the flow distributor position, such adjustment can be made and the column retested. Once the desired position has been established, the flow distributor can be permanently secured in place. To permanently secure the flow distributors in place, in some embodiments, the flow distributors can be robustly welded to the tube wall. Other methods of permanently fixing the flow distributors can be used.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features, objects, and advantages will be apparent from the description and drawings.

The new chromatography columns described herein can be made of relatively inexpensive plastic materials, and can thus be considered disposable, yet are specifically designed to be sufficiently robust to permit repeated cleaning and reuse. The new methods of manufacture described herein reduce and/or avoid the formation of dead zones around the press fit flow distributors, thus making the new chromatography columns far more effective, useful, and easier to clean than presently available chromatography columns.

The aim of the invention is a pre-packed chromatography column for use in biopharmaceutical applications made entirely from widely available plastic/thermoplastics and/or composites (such as polypropylene (PP), polyethylene (PE), polyamides (such as various nylons), acetals, or glass-filled or carbon-filled plastics, e.g., glass-fiber and carbon-fiber plastics) or elastomeric components. The column's design is such that it can be packed with various types of chromatography packing media, or resins, to a "bed height" with infinite variability between <NUM> and <NUM> and longer within a given internal diameter that can be, for example, but not limited to, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> or larger, up to about <NUM>, <NUM>, or <NUM>, or larger.

The chromatography columns described herein consist primarily of a column tube and a pair of flow distributors (or one flow distributor and one end cap). The flow distributors include a cylindrical disc and one or more inlet/outlet pipes that enable liquids to flow into and through the disc. In addition, the flow distributors can include a bed support, screen, and/or filter that are attached to the packing medium side of the flow distributor disc. The column also may or may not incorporate O-rings between the flow distributors and column tube, but the present invention can generally be used to avoid the need for O-rings entirely.

The flow path of the flow distributors can be designed according to standard practices and known designs, and the flow distributors themselves can be made, for example, of the same or a similar plastic material as the tubes, but can also be made of metal, ceramics, and other materials that are inert to the liquids and reagents that are to be flowed through the columns.

The tubes are hollow, cylindrical members, which are typically round cylinders that permit a fluid (e.g., a liquid) to flow from a first end (e.g., an upper end) to a second end (e.g., a lower end). The inner diameter of the tubes are sized and configured to receive the flow distributors for delivering fluid to and removing fluid from the tube. Based on various chromatography column performance specifications, the tubes can be made in a variety of different sizes and configurations. In some embodiments, the tubes are sized and configured to maintain structural integrity under the induced internal operating pressures of the system while being able to withstand internal pressures up to as much as about <NUM> psi (e.g., about <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> psi; <NUM> psi = <NUM> kPa). In some embodiments, the tubes are typically cylindrical members having an inner diameter that is about <NUM> to about <NUM> and a length that is about <NUM> to about <NUM>. The tubes are initially selected to be about twice as long as the desired final bed height, and are cut shorter once both flow distributors are secured in place within the column tube.

In general, the overall induced hoop tension of the tube, based on a variety of factors, can vary based on an end user's specification, such as expected internal pressure to which the chromatography column will be subjected. For example, the tube must have sufficiently thick or otherwise robust walls to avoid yielding of the tube during the insertion of the flow distributors. For example, the wall thickness of the tube can be large enough such that it can withstand adequate factors of safety above the maximum operating pressure via deriving desired induced hoop tension. For example, depending on the nature of the material, e.g., for polypropylene, a <NUM> column has a tube that has a nominal inner diameter of <NUM> and a nominal wall thickness of <NUM>. A <NUM> polypropylene column has a tube that has a nominal inner diameter of <NUM> and a nominal wall thickness of <NUM>. In some examples, depending on the nature of the material, a tube that has an inner diameter of <NUM> should have a wall thickness of from about <NUM> to <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. A tube having a diameter of <NUM> should have a wall thickness of about <NUM> to <NUM>, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. The wall thickness of the tube can be specified so that the tube has suitable strength to withstand internal pressure during use (e.g., about <NUM> psi to about <NUM> psi, e.g., <NUM>, <NUM>, <NUM>, or <NUM> psi; <NUM> psi = <NUM> kPa).

Furthermore, adequate wall thickness helps to maintain the column geometry (e.g., volume) throughout the intended range of operating pressure, thereby limiting the amount of deflection of the column walls, which will help to ensure proper function of the columns. Walls may be thinner in tubes made from thermoplastics that are reinforced with additional structural materials such as glass or carbon fibers or particles.

In some examples, a tube should have an induced hoop tension of <NUM> PSI to <NUM> PSI, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> PSI (<NUM> PSI = <NUM> kPa). The induced hoop tension of the tube can be specified so that the tube has suitable material properties to withstand internal pressure during use (e.g., about <NUM> psi to about <NUM> psi, e.g., <NUM>, <NUM>, <NUM>, or <NUM> psi; <NUM> psi = <NUM> kPa). Furthermore, adequate induced hoop tension helps to maintain the column geometry (e.g., volume) throughout the intended range of operating pressure, thereby limiting the amount of deflection of the column walls, which will help to ensure proper function of the claims. Adequate induced hoop tension also allows the column to withstand significant operational pressures and maintain a hydraulic seal without being permanently fixed in position.

In addition, the inner wall of the tube may be thinned or reduced in thickness at the ends, or at least at one end, to form a ramp or chamfer of from about <NUM> to about <NUM> degrees, e.g., about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> degrees, which can facilitate the insertion of the flow distributors. The chamfer should run from the end of the tube inwards from about <NUM> to about <NUM>. As discussed in detail below, the flow distributor has an outer diameter that is greater than the inner diameter of the tube, and the chamfers help align the flow distributor into the tube during manufacturing.

As shown in <FIG>, in some embodiments, a tube <NUM> is a cylinder having a chamfer <NUM> formed along the inner surface at each end of the tube <NUM>. In this example, the tube <NUM> has a length that is about <NUM> long, an inner diameter that is about <NUM>, and a wall thickness that is about <NUM>. The chamfer <NUM>, in this example, is about <NUM> degrees and runs from the end of the tube inwards about <NUM>.

Flow distributors that are sized and configured to be received in the tube <NUM> have an inlet hole that is hydraulically connected to an outlet hole and a network of fluid distribution conduits, such as grooves that extend from the inlet hole to the packing medium side of the flow distributor. Thus, the flow distributors are configured to receive fluid at one or more inlet locations from a first side of the flow distributor and distribute the fluid outward radially along a second side of the flow distributor that faces the packing medium when inserted into the tube. Additionally, typically by reversing the flow direction, the flow distributors can receive fluid along their entire second side and direct the fluid inward towards the one or more outlet locations on the first side.

Typically, the flow distributors are round, disc-like members that have an outer diameter that is slightly larger than the inner diameter of the tube into which they are to be inserted, such that the insertion thereof will produce an interference fit sufficient to induce a hoop tension effective to prevent leaking up to desired internal pressures. Because the flow distributor is relatively incompressible and the tube wall is relatively compliant, the interference fit causes the tube to distend leading to the formation of a liquid-tight seal. For example, for a polypropylene tube having an inner diameter of <NUM>, the polypropylene flow distributor can have an outer diameter of between <NUM> and <NUM> (e.g., about <NUM>). For an inner diameter of <NUM>, the outer diameter of the flow distributor can be about <NUM> to <NUM>. Both the tube <NUM> and the flow distributors <NUM> are designed such that the induced hoop tension during assembly is less than the yield strength of the materials. Thus, the tube walls, and in many embodiments to a lesser extent the flow distributors, experience plastic deformation and maintain their hoop tension during the life of the column. It is this hoop tension value that assures a leak-proof seal at the tube <NUM> and flow distributor <NUM> interface and limits the maximum operating pressure of the column.

The value of the hoop tension is directly related to the magnitude of the press-fit interference, the thickness of the tube wall, and the specific Young's Modulus and Poisson's Ratio of the tube <NUM> and flow distributor <NUM> materials as shown in Equation (<NUM>). <MAT> where σhoop tension is the induced hoop tension, δint is the interference fit (which is the difference between the outer diameter of the flow distributor and the inner diameter of the tube), Dfd is the outer diameter of the flow distributor, Dtube,o is the outer diameter of the tube, εtube is the Young's Modulus of the tube material, εfd is the Young's Modulus of the flow distributor material, νtube is the Poisson's Ratio for the tube material, and νfd is the Poisson's Ratio for the flow distributor material. Considering continuum mechanics, the induced hoop tension is simply a stress that is created in the body of the tube wall and/or flow distributor, which are only produced during the application of an external force and the subsequent deformation of the tube wall and flow distributor.

For example, in one particular implementation using polypropylene, it was found that to provide an adequate hoop tension to ensure upwards of <NUM> kPa (<NUM> PSI) operating conditions without a leak considering the available part tolerances due to various fabrication methods, a <NUM> column would need a <NUM> nominal inner diameter tube <NUM> with a <NUM> nominal tube wall thickness. The nominal diameter of the flow distributors <NUM> would need to be <NUM>. This would ensure at worst case interference conditions an <NUM> kPa (80PSI) induced hoop tension at each flow distributor <NUM>. At the other end of the spectrum considering maximum interference conditions with the allowable tolerances for the tube <NUM> inner diameter tolerances and flow distributor <NUM> outer diameters, this would create up to <NUM> MPa (<NUM> PSI) induced hoop stress at each flow distributor <NUM>.

<FIG> illustrate that in some implementations a flow distributor <NUM> is a disc-like member having a fitting hole <NUM> formed at a central region along a first side <NUM> and a system of multiple grooves and channels <NUM> formed along a second side <NUM>. The fitting hole <NUM> is a blind hole that is sized and configured to receive a fitting. The fitting hole <NUM> includes one or more features to receive the fitting. In one specific implementations, the fitting hole <NUM> is threaded to receive a threaded fitting (e.g., an M30x3. <NUM> threaded fitting). In some embodiments, the fitting is connected to the flow distributor <NUM> in various other ways, such as adhesives, welding, bayonet or luer connections, or other sufficient connection techniques. In some embodiments, the fitting is manufactured as an integral component of the flow distributor <NUM>. The flow distributor <NUM> also includes a fluid passage <NUM> to hydraulically connect the fitting hole <NUM> to the second side <NUM> of the flow distributor <NUM> so fluid can pass between the second side <NUM> of the flow distributor <NUM> and a fitting inserted into the fitting hole <NUM>.

As shown, the multiple grooves and channels <NUM> extend substantially radially from the fluid passage <NUM> to direct fluid flow inward and outward radially, depending on the location of the flow distributor <NUM>. The height or depth of the grooves is tapered from a center/feed port (e.g., the fluid passage <NUM>) to a lower height at an outer periphery region of the flow distributor <NUM>. An aspect ratio of this taper is the subject of various publications which provide general design guidelines (see e.g., <NPL>). This tapered profile can help to minimize pressure gradients radially and axially, which can negatively impact column performance (e.g., efficiency) by dispersion of target molecules travelling through the packed bed.

In some embodiments, the flow distributor <NUM> defines a recess <NUM> along its outer diameter. The recess <NUM> can be sized and configured to receive a sealing member (e.g., an O-ring).

The flow distributor <NUM> can be formed by any various manufacturing techniques, such as molding, casting, machining, or other methods, and can be obtained commercially. In some embodiments, a general shape of the flow distributor <NUM> is cast or molded and the grooves and channels <NUM> are machined from the general shape. To closely mate with the inner diameter of the tube, in some embodiments, an outer diameter of the flow distributor is formed using a lathe to ensure that the outer edge is round and to tolerance.

The fittings are mechanical attachments that can be fastened or secured to the flow distributor to deliver fluid to or remove fluid from a flow distributor and the tube in which the flow distributor is arranged. To deliver fluid, the fittings have a fluid delivery hole formed through the fitting along its central axis. The fittings also include one or more features to be received in the fitting hole of the flow distributor to retain the fitting. As shown in <FIG>, <FIG>, in this example, fittings <NUM> have a threaded end <NUM> (e.g., an M30x3. <NUM> threaded end) to engage the fitting hole <NUM>. The fittings <NUM> also have a nut portion <NUM> that can be gripped by a tool (e.g., a torque wrench) for turning and securing the fitting <NUM> within the fitting hole <NUM>. In some embodiments, the fitting <NUM> includes other types of connection mechanisms, such as adhesives, welding, bayonet or luer connections, or other sufficient connection techniques.

Fittings <NUM> can have different additional features based on their installed location. For example, an inlet fitting 38a that is installed on a top flow distributor 24a can have a connection feature at an end of the fitting opposite the threaded end. The connection feature, such as a hose connection, permits hose or tubing to be connected to the fitting in an easy manner. In this example, the inlet fitting 24a defines a recess <NUM> that is sized and configured to be received in a hose fitting, such as a sanitary fitting (e.g., a tri-clamp connection or a cam lock) style hose fitting.

Alternatively, an outlet fitting 38b that is connected to the bottom flow distributor 24b can have a different style connection than the inlet fitting. In this example the outlet fitting 38b is secured to a hose <NUM> to hydraulically connect the outlet fitting 38b to a remote quick disconnect outlet fitting <NUM>. The remote quick disconnect outlet fitting <NUM> can be mounted or arranged in a region that can be more conveniently accessed by a user than the outlet fitting 38b.

The chromatography components described (e.g., the tube <NUM>, the flow distributors 24a, 24b, the fittings 38a, 38b, and other components) can be made from any of various structurally and chemically suitable materials. For example, the components can be made from various plastics, such as thermoplastics (e.g., acrylonitrile butadiene styrene (ABS), acrylic (PMMA), polypropylene (PP), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), other thermoplastics, or composites) and thermosetting plastics (e.g., epoxy resins, and fiber reinforced plastics. Material selection considerations can include the specific mechanical properties of the materials and if the materials will withstand the induced internal operating pressures of the system.

In certain specific embodiments, the tubes can be made of sufficiently elastic metals that provide an effective induced hoop tension, such as certain steels, beryllium copper alloys, titanium alloys, nickel alloys, cobalt chrome, other types of metals, or alloys of these or other metals. While metals or other materials can be used, forming the tube from plastic materials can result in producing a lower cost, and in some cases, a disposable chromatography column.

In some examples, most of the components (e.g., the tube, the flow distributors, and the fittings) are made from a thermoplastic and/or polyolefin material (e.g., such as polypropylene (PP), polyethylene (PE), polyamides, acetals, or glass-filled or carbon-filled plastics, e.g., glass-fiber and carbon-fiber plastics). Some of the components, such as the tube and flow distributors can be made from the same type of thermoplastic and can thus be welded to one another. For example, USP Class VI certified polypropylene (e.g., Product No. P9G1Z-<NUM> from Flint Hills) or an equivalent can be used. The chromatography column components can be manufactured by any of a number of manufacturing processes known in the art, such as molding, casting, machining, composite tape laying, or other methods.

A chromatography column <NUM> can further include a base, e.g., a bottom end cap <NUM> that is sized and configured to suitably support and arrange the tube <NUM> and the other components in a generally vertical orientation. The base <NUM> includes features (e.g., holes or recesses) to receive and secure a portion (e.g., the lower portion) of the tube <NUM>.

Foot-like protrusions extending from a lower surface of the base <NUM> can be included to provide a substantially level supporting surface for the chromatography column <NUM>. The bottom end cap or base <NUM> can also include casters in the case of larger column diameters that cannot be easily lifted and/or carried. The base <NUM> is made from any various structurally suitable materials, such as metals, plastics, or composite materials. In this example, the base is made from ABS, PE, PP, or glass-filled or carbon-filled plastics, e.g., glass-fiber and carbon-fiber plastics, composite PP. In some cases, the base includes non-skid materials or features (e.g., soft rubber foot-like protrusions) to increase stability.

The chromatography column <NUM> can also further include a top end cap <NUM> that encloses the tube <NUM> and upper flow distributor 24a. The top cap <NUM> includes features (e.g., holes, recesses, or gripping elements) that receive and secure a portion (e.g., the upper portion) of the tube <NUM>. The top cap <NUM> includes an inlet fitting hole <NUM> and an outlet fitting hole <NUM> that are sized and configured to receive the inlet fitting 38a and remote quick disconnect outlet fitting <NUM>, respectively. The top cap <NUM> can also include one or more handles <NUM> that can be used to pick up and carry the chromatography column <NUM> or used to steer/direct larger columns that have integral casters or once placed on rolling carts/dollies. The top cap <NUM> is made from any various structurally suitable materials, such as metals, plastics, or composite materials that can support the weight of the chromatography column when it is lifted by the handle. In this example, the top cap is made from ABS, PE, PP, or glass-filled, e.g., glass-fiber, plastic.

A shroud or side-guard piece <NUM> can also be further included. The shroud piece <NUM> can be sized and configured to extend from the base <NUM> to the top cap <NUM> and cover some of the inner components of the chromatography column <NUM> (e.g., the hose <NUM> connecting the outlet fitting 38b to the remote outlet fitting <NUM>). The shroud <NUM> can be formed of any various suitable materials such as metals, plastics, or composite materials.

Top and bottom flow distributors 24a, 24b are installed (e.g., press-fit) into the top and bottom of the tube <NUM> during the manufacturing and packing of the column. In some embodiments, the tube <NUM> and one or both of the flow distributors 24a, 24b are permanently bonded prior to insertion of the top flow distributor 24a and packing of the tube <NUM> with medium material. Following satisfactory testing of the column, the second, e.g., top, flow distributor 24a is permanently bonded in place.

Such permanent bonds cannot be readily separated other than by breaking the bond or the bonded items (e.g., the tube <NUM> and flow distributor 24a, 24b). At an upper end, an additional cap (e.g., the top cap) <NUM> can optionally be seated on and secured to the tube <NUM> and aligned so that the inlet fitting 38a installed on the flow distributor 24a at the top of the column passes through the inlet fitting hole <NUM> of the additional top end cap <NUM>. Such optional top cap <NUM>, which is primarily an aesthetic feature, can be secured to the tube <NUM> using various securement mechanisms, such as fasteners, adhesives, friction between the tube and the top cap, or other mechanisms.

At a lower end, the tube <NUM> can optionally be seated on and secured to the bottom cap (e.g., base) <NUM>. The base <NUM> can be secured to the tube <NUM> using various securement mechanisms, such as fasteners, adhesives, friction between the tube and the bottom cap, or other mechanisms. When an optional base <NUM> is used, the outlet fitting 38b installed on the flow distributor 24b at the bottom of the tube <NUM> can extend into a cavity in the optional base <NUM> and the hose <NUM> connected to the outlet fitting 38b from the bottom flow distributor 24b is directed outward toward a region outside the periphery of the tube <NUM>. As shown, the hose <NUM> can be routed out of the optional base <NUM> and upward along the side of the tube <NUM> to connect to the remote quick disconnect outlet fitting <NUM> that is fixed at or near the top of the column <NUM>. By using the hose <NUM> and arranging the remote outlet fitting <NUM> near the top of the column <NUM>, a user need not have access to the underside of the tube <NUM>, which results in an easier to use chromatography column <NUM>.

The tubes of the chromatography columns described herein can be packed with any solid phase medium material that is used in column chromatography as specified by the end-user. This diversity of potential packing medium materials extends to both the composition of base particles as well as their functional chemistries (e.g., affinity, ion exchange, and hydrophobic interaction). Packing medium materials can include a slurry of stationary phase particles added to an eluent solvent. Stationary phase particles can include silica gel (SiO<NUM>), alumina (Al<NUM>O<NUM>), cellulose, and other suitable materials in various mesh sizes. Eluents can include one or more of various solvents, such as deionized water, ethanol or acetone.

Examples of packing media include, but are not limited to, agarose (e.g., Sepharose® Fast Flow and Capto™ from GE Health Care) controlled pore glass (ProSep® from Millipore), ceramic hydroxyapatite, polymethacrylate (e.g., ToyoPearl® media from Tosoh Bioscience), and other synthetic polymeric resins (e.g., Life Technologies' Poros™ media and Fractogel™ media from EMD).

One known characteristic of certain plastics/thermoplastics is their inherent compliance or ability to deform without fracturing with the application of force. The new chromatography columns are made using an assembly process that takes advantage of the "flow-ability," e.g., elasticity, of the plastics as defined by the induced hoop tension, used to make the column tube <NUM>. The column tube <NUM> are made from extruded, cast, molded (injection, roto, or other), or machined plastic/thermoplastic or tape laid composite materials of specified internal and external dimensions. The designs and methods described herein for the flow distributors <NUM> include an outside diameter that is larger than the nominal internal diameter of the column tubes <NUM>, described henceforth as the interference fit.

When used with cylindrical column tube <NUM>, the flow distributors <NUM> must also be round, with as few (e.g., no) non-uniformities as possible on the outer surface, to ensure a uniform induced hoop tension and a sufficiently liquid-tight mating and sealing of the flow distributor <NUM> against the surface of the inner wall of the tube <NUM> when press fit into the tube <NUM>. A sufficient degree of uniform roundness or circularity can readily be achieved by turning the flow distributor <NUM> on a lathe, but other methods of achieving this degree of uniform roundness are known to those skilled in the art.

The degree of acceptable interference-fit is determined by the mechanical properties, i.e., the elasticity or flow-ability, of the particular plastic/thermoplastic or composite components encompassing the tube <NUM> and flow distributor <NUM>, and therefore, in the case of polypropylene, the thickness, of the tube <NUM> wall, but in all cases, the outer diameter of the flow distributor <NUM> exceeds the nominal inner diameter of the tube <NUM> to produce the required interference fit to assure satisfactory induced hoop tension when the flow distributor <NUM> is driven into the tube <NUM>.

This assembly process provides unique advantages to the new chromatography columns. Traditional columns constructed of more dimensionally stable materials (steel, glass, etc.) are designed such that the flow distributor <NUM> is slightly smaller than the column tube, which is necessary to allow this component to be easily inserted and moved to the desired position within the column tube during assembly. An O-ring or similar sealing mechanism is employed around the flow distributor <NUM> to achieve a liquid-tight seal between the flow distributor <NUM> and the tube <NUM> wall. In these traditional designs, the combination of a flow distributor with smaller outer diameter than the tube inner diameter and the necessity to include an O-ring necessarily results in an area that is referred to as a "dead space" between the flow distributor <NUM> and the tube <NUM> wall up to the point at which the O-ring is seated. These "dead spaces" are difficult to expose to column flow and therefore pose a risk to column cleanability and resulting cleanliness. The interference fit design eliminates or greatly reduces the "dead space" of traditional columns thereby minimizing risk of carry-over contamination between column uses. The interference fit can, in some embodiments, also allow the elimination of O-rings altogether, thereby minimizing column complexity, cost, and risk to integrity due to seal failure. Another advantage of this feature is to reduce the exposure of a valuable product being purified by column chromatography to contaminants that may be released from such O-rings (typically elastomerics) that require costly and time consuming risk assessments in the form of studies of the extractables and leachables.

As shown in <FIG>, the methods of making the new chromatography columns <NUM> include several steps.

First, specify a plastic column tube <NUM> that has the appropriate diameter and length to accommodate the volume of medium material that is desired for the final column (<NUM>), as well an appropriate elasticity, as described elsewhere herein. The length of the tube should be about twice the length or "bed height" of the medium material in the final column. The final length of the tube <NUM> can be about the same as the inner diameter, e.g., <NUM> and/or <NUM> inner diameter tube <NUM> might have a final length of about <NUM> to <NUM>, e.g., about <NUM>. The chamfer formed along the inner surface of each end of the tube is also selected. This chamfer is required to align and assist in inserting the flow distributors <NUM> to be driven into the interior of the column tube <NUM>.

Second, an appropriately sized flow distributor <NUM> should be specified to have an outer diameter that is slightly larger, e.g., about. <NUM>%, <NUM> to about <NUM>, <NUM>. <NUM>, <NUM>. <NUM>, <NUM> or <NUM>% larger than the inner diameter ("ID") of the tube (<NUM>). For example, for a polypropylene tube having an inner diameter of and/or <NUM>, the flow distributor <NUM> should have an outer diameter ("OD") greater than <NUM>, e.g., of between <NUM> and <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>). The flow distributor <NUM> is designed to a specific nominal OD such that it will induce sufficient hoop tension in the tube <NUM> wall. When selecting the appropriate nominal OD account factors to consider include the physical properties of the materials of construction (e.g., coefficient of friction, Young's modulus, modulus of elasticity, and elongation at yield) in combination with the geometries including tolerances of both the column tube's ID and its wall thickness and the tolerance of the flow distributor <NUM> OD. The forces required to press-fit the assembly together can be theoretically determined (e.g., via advanced analytical tools, such as Finite Element Analysis) and, as an alternative, this assessment may be carried out by empirical studies with specific materials of construction.

In some embodiments, the flow distributors can be made of the same material as the tube, to ensure compatibility in use and to simplify the securing of the flow distributor to the interior wall of the tube, e.g., during welding.

Third, as shown in <FIG>, a first, e.g., bottom, flow distributor 24b is secured to a first end, e.g., the bottom end, of the tube <NUM> (<NUM>). This can be done by any known means, or the interference fit methods described herein can be used to help avoid or reduce any dead space associated with the first flow distributor. For example, the first flow distributor 24b can be secured using metal clamps, threading cut into the tube <NUM> (either on the inner wall or on the outer wall) and flow distributor peripheral wall, adhesives, and various types of welding. The main point is that this first flow distributor 24b need not be moved once it is secured to the first end of the tube <NUM>. In some embodiments, the first flow distributor 24b is formed as an integral part of the tube <NUM>. For example, the first flow distributor can be molded as a feature of the tube <NUM> using known techniques.

If the interference fit method is used for the first, e.g., bottom, flow distributor, it can be initially held in place at the desired location by an induced hoop tension to provide an effective hydraulic seal at the required pressures, and then permanently secured at that location using any known means, including welding, screws, or adhesive. In particular, to establish an appropriate interference fit, the flow distributor <NUM> is aligned with the chamfered bottom end of the tube and then an axial force of about <NUM> lbf to <NUM>,<NUM> lbf (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>,<NUM> lbf; <NUM> lbf = <NUM> N) is applied on the flow distributor <NUM> to drive it into the column tube <NUM>, thereby expanding the inner diameter of the tube. For example, while the flow distributor <NUM> is inserted into the tube <NUM>, both the tube <NUM> and the flow distributor <NUM> are plastically deforming to fit together, the magnitude of the tube <NUM> deformation is larger than the magnitude of the flow distributor <NUM> deformation.

The force required to drive the flow distributor into the tube depends on, amongst other factors, the angle of the chamfer formed into the tube, and other physical characteristics specific to the materials of construction (mentioned above) in combination with their geometric dimensions. For example, the axial force to drive the second flow distributor into the tube to establish the interference fit within the tube is a function of the interference fit, tube wall thickness, and specific mechanical properties of the tube and flow distributor materials. The force required to drive the flow distributor into either end of the tube can be measured by a load cell, or similar tensile testing instrument, and should be inspected during each assembly to assure adequate interference fit between the flow distributor and the tube wall. The axial force required to drive the flow distributor into the tube must be greater than and opposite to opposing forces resulting from adhesion and deformation friction forces between the tube wall and the flow distributor outer circumferential edges.

Equation <NUM> below describes the insertion force further. <MAT> where Fapplied is the axial force necessary to overcome the friction forces opposing the insertion of the flow distributor into the tube, Ffriction,insertion is the friction force due to adhesion between the flow distributor and tube wall materials, Ffriction,deformation is the friction force due to deformation of the flow distributor and/or tube wall, and Ffriction,net is the net frictional force. If necessary, one can differentiate the two opposing friction forces by applying a lubricant to remove the adhesion friction forces and subtracting the resulting axial force required to insert a flow distributor from the total axial force required to insert a flow distributor without the lubricant.

Alternatively, one can determine a minimum axial force to drive the flow distributor into the tube to produce a sufficient resulting induced hoop tension. This induced hoop tension acts as a radial force that holds the flow distributor at a specified location inside the tube. Considering well-known interference fit equations, an expression was derived to represent the induced hoop tension for all tube and flow distributor sizes, represented by Equation <NUM> above.

The induced hoop tension can be related to a total radial force exerted by the tube wall on the walls of the flow distributor by multiplying it by the circumferential area of the flow distributor in contact with the tube wall. Equation <NUM> below explains this further. <MAT> where Fradial is the radial force equally distributed around the tube walls acting radially inward to the flow distributor walls and Acontact,fd is the area of the flow distributor in contact with the tube wall. It can further be scene that this radial force is directly related to the perpendicular friction force, Ffriction,net, between the flow distributor and the inner wall of the tube. Thus, one can relate the force required to overcome the friction force, Fapplied, to drive the flow distributor into the tube to an induced hoop tension, σhoop tension, that will hold the flow distributor at a desired location inside the tube. Equations <NUM>, <NUM>, and <NUM> below describe this relationship further. <MAT> <MAT> and <MAT> where µfriction is the friction coefficient between the flow distributor material and the tube wall material.

As a result of this correlation, as long as empirical testing can assure that a given induced hoop tension will provide a leak proof seal up to adequate factors of safety above the recommended maximum operating pressure, e.g., 2x, 3x, or 4x, one can assure, and check during assembly with a load cell or similar instrument, the adequate operating pressure of the column. It is important to note that dust, humidity, oxide films, surface finish, velocity of sliding, temperature, vibration, and extent of contamination to the column and flow distributor walls can contribute to variation in the value for the coefficient of friction, µfriction, thus affecting the recorded insertion force. In an attempt to reduce this error, it is recommended that all initial testing to determine the accurate coefficient of friction (µfriction) and subsequent applied load (Fapplied) to achieve the required induced hoop tension be performed in a stable, repeatable manufacturing/ laboratory environment, i.e., clean room. Ultimately, it is preferred that the facility has very little dust, low humidity, minimal UV light (that could affect the mechanical properties of the materials), minimal vibrations, constant temperatures (close to room temperature conditions), low extent of contamination, and a constant insertion velocity.

In addition, the following equation was used to determine the magnitude of the surface finish on the resulting interference fit and it was shown that the surface finish (for the materials in our case) are negligible on the overall interference fit. <MAT> where δeff is the effective interference and Δδ is the Correction to the Measured Interference considering the surface finish of the inner tube wall and the circumferential surface of the flow distributor. <MAT> where Rz,tube is the surface finish of the inner wall of the tube and Rz,fd is the surface finish of the outer wall of the flow distributor.

To guarantee sufficient induced hoop stress to contain this pressure, experiments can first be carried out to develop a relationship between the amount of interference between the flow distributor and the tube wall in order to prevent leaks up to a certain pressure. Equation (<NUM>) shows that the induced hoop tension is directly responsible for creating a leak-proof seal between the flow distributor and the tube wall. Three major variables, considering constant tube and flow distributor materials, will contribute to the magnitude of the induced hoop tension: the interference fit δinit, outer diameter of the tube Dtube,o, and the outer diameter of the flow distributor Dfd. Once two of these values are chosen, varying the third variable will allow one to test several cases of applied force to insert the flow distributor Fapplied versus the internal pressure to leaking. Once an adequate internal pressure is attained without any leaks past the flow distributors, the value of applied force can be used to back calculate the induced hoop tension necessary to contain the desired pressures. Once the necessary induced hoop tension is found for a certain chromatography column size (tube internal diameter), the three major variables that contribute to the induced hoop tension can once again be modified to optimize the design as long as they ultimately attain the same final induced hoop tension value.

<FIG> show schematic free body diagrams of the forces generated while a flow distributor <NUM> is initially driven into the tube <NUM> before it reaches a chamfer <NUM>. As the flow distributor <NUM> first enters the tube <NUM>, the tube <NUM> has not yet expanded. The interference between the flow distributor <NUM> and the tube <NUM> wall will force the tube <NUM> to enlarge and the flow distributor <NUM> to compress. Since the wall thickness of the tube <NUM> is smaller than the diameter and thickness of the flow distributor <NUM>, the overall net force will result in expansion of the tube wall (note that the flow distributor <NUM> may correspondingly undergo a small amount of compression). For this to occur, the force in the axial direction must be large enough to overcome the force created due to the induced hoop tension. The axial force is from the linear actuator and the horizontal or radial force is from the induced hoop stress. The axial force is simply overcoming the frictional force. The frictional force is directly related to the value of the force from the induced hoop.

<FIG> show schematic, free body diagrams of the forces generated while the flow distributor <NUM> is driven along the axial length of the tube <NUM> after it passes the chamfer <NUM>. Although some component of the axial force is contributing to expanding the tube <NUM>, the stress is distributed <NUM>-<NUM> characteristic dimensions away from the initial contact point between the flow distributor <NUM> and the tube <NUM> and the tube <NUM> is already expanding in front of the flow distributor <NUM>. Thus, as the flow distributor <NUM> is inserted axially further along the length of the tube <NUM>, the axial force to push the flow distributor <NUM> is larger to overcome the higher induced hoop tension occurring not only at the point of contact with the flow distributor <NUM>, but also <NUM>-<NUM> characteristic dimensions in front of the flow distributor <NUM>. In some embodiments, the chamfer begins at the very end of the tube wall and e.g., can extend along the entire length of the tube.

<FIG> shows a chart illustrating the axial force required to press the flow distributor <NUM> into the tube <NUM> as the flow distributor <NUM> travels into the tube <NUM> in one embodiment. As shown, the force initially increases to a peak while a first portion of the flow distributor <NUM> enters and passes the beginning of the tube chamfer <NUM>. Initially, the flow distributor <NUM> and tube wall are experiencing static friction and the force to overcome the static friction is greatest. Once the deformation of the flow distributor <NUM> and tube <NUM> wall give way to sliding of the flow distributor <NUM> into the tube <NUM>, the force required to continue pressing the flow distributor <NUM> into the tube drops since it is experiencing dynamic friction. Dynamic friction is significantly less than static friction to overcome. Two additional peaks are also present in this graph. The first peak at about <NUM> corresponds to when a bottom of the chamfer <NUM> is in an O-ring groove <NUM> of the flow distributor <NUM> (shown in <FIG>). The second peak corresponds to the point at which the entire flow distributor <NUM> is engaged in the region of the tube <NUM> beyond the chamfer. As shown, in this example, the maximum axial force is about <NUM> - <NUM> kN (<NUM> - <NUM> lbf).

For certain embodiments, the seal can be improved by the use of an O-ring arranged within an O-ring groove <NUM> in the outer wall of the flow distributor <NUM>. In certain embodiments, the press-fit or interference fit is sufficient to hold the flow distributor in place, but in other embodiments, a more permanent bond is desired.

Once the flow distributor <NUM> has been driven about <NUM> to <NUM>, e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>, into the first, e.g., bottom, end of the tube, the flow distributor <NUM> can be permanently secured in place, for example by welding, e.g., if the flow distributor <NUM> and tube are made of the same or sufficiently similar materials. Various welding techniques can be employed to form the weld between flow distributor and column tube including, but not limited to, hot tool welding, hot gas welding (e.g., at <NUM>), ultrasonic, extrusion, laser, conductive, high frequency, etc. If the two pieces are made of different materials, they can be connected using mechanical clamps, such as metal hose clamps, applied externally to compress the tube and apply a force that will anchor the flow distributor within the tube at that location, or by adhesives or by mechanical fasteners that pass through the tube wall and into the flow distributor.

Fourth, the inlet and outlet fittings 38a, 38b are attached to the first (e.g., bottom) and second (e.g., top) flow distributors 24a, 24b (<NUM>). The inlet and outlet fittings 38a, 38b have threaded regions <NUM> that are screwed into threaded fitting holes <NUM> in top and bottom flow distributors 24a, 24b. A recess (e.g., an O-ring gland) can be formed either at a bottom end of the each fitting (i.e., an end that mates with a flow distributor) or in a terminal end of the threaded fitting hole <NUM> of the flow distributor. In this example, an O-ring is arranged between the fittings <NUM> and the flow distributors <NUM> to form a seal (e.g., a liquid-tight seal) between the fittings <NUM> and the flow distributors when they are threaded together. A torque wrench can be used to ensure adequate compression of the O-ring to create sufficient seal at this interface.

Fifth, the packing medium in the form of a liquid slurry is loaded into the column tube <NUM> in the space (chamber) above the bottom flow distributor 24b (<NUM>).

Sixth, as shown in <FIG> and <FIG>, once the second, e.g., top, flow distributor 24a is plumbed with tubing (and optionally already connected to a liquid source) it is inserted into the tube <NUM> in much the same way as the first flow distributor 24b is inserted when using the interference fit method (<NUM>). It is important that the interference fit method is used for the second flow distributor, because the initial location to which this second (e.g., top) flow distributor 24a is driven into the tube <NUM> should not be immediately fixed, because it may be desirable to readjust the initial position of the second flow distributor following testing. Thus, the interference fit method is used, so that the second, e.g., top, flow distributor 24a can be moved internally within the tube <NUM> to make final adjustments. It is also important that the interference fit be designed and implemented such that it ensures a liquid-tight seal at the pressures used during testing of the column.

At this point, the packing medium can be actively settled into a packed bed using a method suitable for the particular medium, for example, flow with an appropriately formulated solution ("mobile phase" or "packing buffer") or suction applied from the column outlet fitting 38b, or any other suitable known techniques or methods. The second, e.g., top, flow distributor can be driven further into the tube by applying an additional axial force to the flow distributor until it contacts the packing medium and may compress the packing medium to reach a desired position. Such compression can range from none at all to <NUM>% or more of the packed bed height depending on the nature of the packing medium. The performance of the column as measured by HETP (Height Equivalent to a Theoretical Plate) testing and asymmetry analysis will be a function, in part, of the compression of the bed. If appropriate, it is also possible to move the inserted flow distributor 24a out towards the end of the tube to reduce bed compression. This is done using hydrostatic pressure by applying a force to the liquid inside the chamber created between the first and second flow distributors. Since the first flow distributor 24B is permanently secured, the second flow distributor 24A, which is secured using a press fit, will move once a force sufficient to overcome the press fit is exerted against it by the liquid within the column tube.

Seventh, suitability of the column packing medium can be tested by a pulse injection of an unretained and readily detectible test article (e.g., acetone via UV monitoring or sodium chloride via conductivity monitoring) (<NUM>). Based on the outcome of the packing test, the top flow distributor 24a can travel down (e.g., can be driven) further into the packed bed and the packing test can be repeated. If the top flow distributor is moved too far into the tube, which can result in over compressing the packed bed, liquid can be forced into the chamber through the inlet fitting with the outlet fitting sealed shut thereby using hydraulic force to move the top flow distributor 24a back towards the top end of the tube and reducing compression of the packed bed. Once suitability of column packing is determined, the column can then be sanitized and/or flushed with a bacteriostatic storage solution per end-user specifications.

Eighth, when the second, e.g., top, flow distributor 24a is properly positioned, it can be permanently secured, such as by welding or other means as noted above for securing the first flow distributor (<NUM>). In some embodiments, the interference fit may suffice to secure the top (or second) flow distributor 24a to the inner wall of the tube <NUM>.

In some embodiments, the loaded final chromatography column can then be fitted with a top cap, a base, and/or a side guard. The chromatography column can then undergo final sterilization and be used or packaged for shipping.

Evaluation of packed columns can include an HETP (Height Equivalent to a Theoretical Plate) test and asymmetry analysis. The HETP/asymmetry tests measure the quality of the packed bed using injection of a small volume of a readily detectable chemical test article (e.g., acetone, NaCl) that does not interact with the column resin. In a well-packed bed, the test article will move through the column uniformly and will elute as a narrow symmetrical peak. The results are expressed as plates per meter (N/m).

The number of plates (N) in a column is given by: <MAT> where Wh is the peak width at half height of a retention volume peak response curve and Ve is the retention volume.

Plates per meter (N/m) is calculated as: <MAT> where L(m) is the packed bed height expressed in meters. where L is the packed bed height and N is the number of theoretical plates as calculated above.

Asymmetry (As) is defined as b/a, where "a" is a horizontal distance from a point at <NUM>% of the leading edge of a retention volume peak response curve to a vertical center line at the peak, and "b" is a horizontal distance from the vertical center line to a point at <NUM>% of the trailing edge of the retention volume peak response curve.

For additional, general details regarding chromatography, please refer to <NPL>.

The systems and methods described herein provide end-users with disposable, pre-packed, and pre-qualified chromatography columns that are comparable in performance to other chromatography columns that typically exist in a durable hardware installation requiring significant capital expenditure. The column tube's construction of polymeric materials enables it to be manufactured quickly, easily, and less expensively while maintaining robust form and function and simple operation for up to <NUM>-<NUM> or more usage cycles. The new columns are used in the same manner as other known chromatography columns, but given the disposability, the new columns are especially useful for separating and purifying reagents that are toxic or otherwise hazardous, e.g., viruses, pathogens, and explosives.

However, the new chromatography columns are surprisingly robust and can be used repeatedly. In addition, the design of the new chromatography columns provides easy cleaning for such reuse, and the new chromatography columns will provide at least <NUM> to <NUM> cycles of use.

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

The purpose of this example is to pressure test press-fit assemblies of extruded polypropylene (PP) column tubes and machined PP end pieces (e.g., to simulate flow distributors) without O-rings. After testing the sealing ability of the press-fit, the columns were welded on both ends and re-pressurized to challenge the strength of the hot-gas welding attachment method.

All machined column tubes were measured at <NUM> points to obtain a minimum, maximum, and average inner diameter.

The first ("lower") end pieces were press-fit into column tubes using an Exlar linear actuator (Exlar, Minnesota).

For all column assemblies the lower press fit pieces were hot gas welded in place, filled with water, and second ("upper") end piece was press-fit into column tubes again using the Exlar linear actuator.

The column tube upper end was restrained with the hydraulic press and hydrostatic pressure was incrementally increased until water was visibly observed to bypass the press-fit seal.

Following breach of press-fit seal, the assemblies were de-pressurized and "upper" end piece was hot gas welded in place.

Welded pressure assemblies were again subjected to hydrostatic pressure testing to assess effectiveness of weld to perform hydraulic sealing as well as mechanical strength.

Table <NUM> summarizes the data obtained from this series of experiments (<NUM> psi = <NUM> kPa).

During pressurization, the internal pressure was measured while the tube to end piece joint area was visibly observed for leaks.

Hydraulic sealing of the press-fit increased linearly between <NUM> and <NUM> of diametric interference, with each additional millimeter imparting approximately <NUM> psi (~<NUM> Bar, <NUM> kPa) improvement in sealing ability. Increasing interference from <NUM> to <NUM> increased sealing ability by approximately <NUM> psi (~<NUM> Bar, <NUM> kPa). <FIG> is a plot of leak pressure based on the amount of diametric interference and graphically represents the observed sealing trend.

Table <NUM> also summarizes the data obtained when fully welded (i.e., top and bottom end pieces welded in place) press-fit assemblies were re-pressurized to challenge weld strength. A portion of weld in the column tube with the <NUM> end pieces did yield at <NUM> psi (<NUM> Bar, <NUM> MPa).

The welds of the other assemblies could not be tested to failure. Excessive leaking past weld gaps and/or threaded inputs/outputs did not allow pressurization beyond the points reported in table <NUM>. Furthermore, in all columns but the test subject with an end cap diameter of <NUM>, the welds did not prevent weld seams from leaking at/around the leak pressures observed prior to welding. <FIG> graphically represents the observed weld strengths of the four assemblies.

<FIG> is plot of observed weld strength based on various degrees of interference. The test subject with an end cap diameter of <NUM> was the only assembly yielded to pressure. Excessive leaking past weld gaps and/or threaded inputs/outputs in other assemblies did not allow further pressurization.

Based upon the data collected in this study, press-fitting alone can achieve a leak pressure (hydrostatic sealing pressure) of <NUM> psi (<NUM> Bar, <NUM> kPa) at <NUM> of diametric interference distance and even <NUM> psi (<NUM> kPa) at <NUM> of diametric interference distance, which are both more than sufficient to provide an initial attachment of a flow distributor that is effective for column testing under normal operating conditions. Weld strength tests (welded pressure hold) show that seals over <NUM> psi can be achieved and support this approach as a viable method to permanently secure the flow distributors to the inner wall surface of the column tubes to provide a significant safety factor well above normal operating pressures that might arise during use of these columns.

The purpose of this example is to assemble pucks (solid cylindrical discs with outer dimensions similar to flow distributors), with a range of outer diameters, with tubes. The pucks were machined from blocks of polypropylene (PP) and fitted with ports to permit the introduction of liquid. All of the tubes were PP manufactured by extrusion to a nominal inner diameter and wall thickness. These tests were conducted with pucks that did not contain O-rings or O-ring grooves in an effort to attain an accurate induced hoop tension value for each interference fit. Pucks were axially forced into tubes (two pucks per tube, one at each end) and each column was pressurized with water and observed for leaks. After testing the sealing ability of the interference fit, the pucks were welded to the tubes on both ends and re-pressurized to challenge the strength of the hot-gas welding attachment method.

All PP tubes were measured at <NUM> points to obtain a minimum, maximum, and average inner diameter along the axial length of the tube.

One puck was axially forced into one end of a tube using the Exlar linear actuator. This first puck was hot gas welded in place for all assemblies. The columns were filled with water, and then a second end piece (with identical nominal outer diameter) was forced into the opposite end of the tube using the Exlar linear actuator. The hydraulic press was then lined up with the second puck, which had not been welded in place to assure that the puck did not experience any axial movement while the column was pressurized. The hydraulic press helped to minimize sources of leaking and assure that leaking was a direct result of overcoming the induced hoop tension between the puck and the tube wall.

After leaking was observed, the columns were depressurized and the second puck was welded in place. Finally the column was pressurized a second time to check the new pressure at which leaking was observed. During all pressure testing, the internal pressure was ramped up from ambient pressures by <NUM> PSI (<NUM> kPa) increments and allowed to stabilize at each new pressure for <NUM> seconds before checking for leaks and increasing the internal pressure again if no leaks were detected.

Table <NUM> along with <FIG>, <FIG> summarizes the data obtained from this series of experiments (<NUM> psi = <NUM> kPa).

Hydraulic sealing of the press-fit increased linearly between <NUM> and <NUM> of diametric interference, with each additional millimeter imparting approximately <NUM> psi (~<NUM> Bar) improvement in sealing ability. Increasing interference from <NUM> to <NUM> increased sealing ability by approximately <NUM> psi (~<NUM> Bar, <NUM> kPa). <FIG> is a plot of leak pressure based on the amount of diametric interference and graphically represents the observed sealing trend while <FIG> describes the induced hoop tension (PSI) versus the observed leak pressure and required applied axial load to insert the puck. This aspect of the experiment is important in determining operating conditions. For example, this work illustrated that to attain an operating pressure of up to <NUM> kPa (<NUM> PSI), one would need to design the <NUM> column to contain an induced hoop tension greater or equal to <NUM> MPa (<NUM> PSI).

To assure that this value of induced hoop tension has been attained, <FIG> shows that during assembly, one would want to see an axial load greater or equal to <NUM> N to insert the puck into the tube. It is important to note from Equation <NUM> that the radial force exerted as a result of the induced hoop tension is linearly related to the applied force that is required to force the puck into the tube by the coefficient of friction. For the two materials used in this work, an initial experiment was carried out to determine an accurate coefficient of friction between these materials as <NUM>. Exploring this further, a range of columns can be built with varying tube wall thickness, tube inner diameter, and puck (flow distributor) outer diameters while the considering material properties as long as the induced hoop tension and, as a direct result, the axial force to insert the puck (flow distributor) is greater than a known value.

Table <NUM> also summarizes the data obtained when fully welded (i.e., top and bottom pucks both welded to the tube wall) press-fit assemblies were re-pressurized to challenge weld strength. A portion of weld in the column tube with the <NUM> end pieces did yield at <NUM> psi (<NUM> Bar, <NUM> MPa).

<FIG> is a plot of observed weld strength based on various magnitudes of interference. The test subject with an end cap diameter of <NUM> was the only assembly that yielded to pressure. Excessive leaking past weld gaps and/or threaded inputs/outputs in other assemblies did not allow further pressurization.

Based upon the data collected in this study, the induced hoop tension created by an interference fit alone can achieve a leak pressure (hydrostatic sealing pressure) of <NUM> PSI (<NUM> Bar, <NUM> kPa) at <NUM> of diametric interference and upwards of <NUM> PSI (<NUM> kPa) at <NUM> of diametric interference, which are both more than sufficient to provide an initial attachment of a flow distributor that is effective for column testing under normal operating conditions. Weld strength tests (welded pressure hold) show that seals over <NUM> PSI (<NUM> MPa) can be achieved and support this approach as a viable method to permanently secure the flow distributors to the inner wall surface of the column tubes to provide a significant safety factor well above normal operating pressures that might arise during use of these columns.

A column tube was packed using a commercially available packing medium material based on synthetic polymer (e.g., polymethacrylate) particles functionalized to have hydrophobic interaction (HIC) properties. The axial forces required to achieve a suitable degree of induced hoop tension as a result of the magnitude of the interference fit between the column tube and flow distributors were recorded and plotted. Column packing evaluation tests are in-line with functional requirements of this particular media type.

The column was packed according to sequence discussed herein with reference to <FIG>. The continuous axial forces required to force the second flow distributor into the tube after the packing medium was packed into the tube were measured and recorded. Also, a column packing efficiency evaluation was conducted after fixing a location of the second flow distributor by detecting pulse injections of NaCl as they were flushed through the length of the packed column bed.

<FIG> is a plot of observed axial forces needed to press the flow distributor into the packed tube. <FIG> zooms in on the displacement from <NUM>-<NUM> inside the tube from <FIG>. Two important conclusions can be drawn from <FIG>:.

<FIG> is a chromatogram plot of a <NUM>/hr packing evaluation performed on the packed tube.

Table <NUM> summarizes the data obtained from the column packing study.

The data collected reinforces that the induced hoop tension as a result of the interference fit connection methods described herein provide suitable performance characteristics for packing high performing chromatography columns. The observed axial forces required for packing do not exceed forces that can be generated using conventional methods and coincide with the fuctional requirements as set forth by the media manufacturer.

Cleanability and sanitization of a <NUM> internal diameter (ID) polypropylene column was assessed for small molecules, endotoxins, and bacteria.

Inorganic phosphate was used as a small molecule tracer. A <NUM> x <NUM> polypropylene column packed with Sepharose® 6FF was loaded with <NUM> column volume of <NUM> sodium phosphate at a flow rate of <NUM>/h. The phosphate was re-circulated for a total of <NUM> column volumes to ensure saturation. The column was then washed with deionized water for <NUM> column volumes to remove any traces of phosphate. Samples were collected during load, recirculation, and wash, and then assayed for phosphate. A sensitive colorimetric method was performed capable of detecting phosphate to µM levels. This method is known in the art and for additional details, please refer to <NPL>.

A quantitative cleaning investigation was performed to demonstrate the effectiveness of sanitization using sodium hydroxide as a cleaning agent. Sanitization procedure:.

Samples of pre and post inoculation and sanitization were collected and assayed for microbial colony forming units (CFU) and endotoxin.

Microbial testing was performed by filtering <NUM> of the sample through a <NUM> filter unit, washing the filter with <NUM> of <NUM>% peptone water, removing the filter from the unit, and placing it on a Tryptic Soy Agar (TSA) plate. The flow-through after the overnight incubation was diluted <NUM>:<NUM><NUM> prior to filtration, while the post-sanitization water rinse was filtered without dilution. The TSA plate was placed in an incubator at <NUM> for <NUM> days, and colonies are counted at day <NUM> and day <NUM>.

Endotoxin testing was performed using gel-clotting limulus amebocyte lysate (LAL) test with a sensitivity of <NUM> EU/mL.

<FIG> is a graph that demonstrates that a small molecule can easily be removed from a polypropylene column as a result of the well-engineered column design and packing procedures. A reduction of <NUM> logs is achieved in less than <NUM> column volumes of wash, and undetectable levels of phosphate are achieved in less than <NUM> column volumes.

Results for bioburden and endotoxin levels from the microbial challenge are outlined in Table <NUM>, which shows the sanitization procedure completely removed bioburden from millions of CFU to zero CFU in the post-sanitization water rinse. In addition, endotoxin levels were brought below the limit of detection (<NUM> EU/mL) for the assay.

Through the phosphate removal experiments, the innovative design of the columns described herein has been qualified for cleaning applications required in downstream processing. The results of the cleaning experiments demonstrate the absence of significant dead-spaces in the column design and the ease of cleaning a pre-packed polypropylene column. Such columns are therefore suitable for use in standard downstream processing applications and can withstand the cleaning protocols required in today's downstream processing applications.

To test effectiveness of sanitization on a polypropylene column made in accordance with the present description, a worst case scenario was devised where the column was loaded with an excess of E. coli culture (a gram-negative, endotoxin producing bacteria). The results of the sanitization protocol demonstrate the effective removal of bioburden and endotoxin contamination.

Claim 1:
A method of making and loading a chromatography column, the method comprising:
selecting a column tube which has an elasticity, and an inner surface having an inner diameter and a length to accommodate a volume of packing medium,
selecting first and second flow distributors each in the form of a disc having an outer diameter, which outer diameter is larger than the inner diameter of the inner surface of the column tube,
permanently securing the first flow distributor to a first end of the column tube;
loading a packing medium into the column tube;
inserting the second flow distributor into a second end of the column tube by applying an axial force to drive the second flow distributor into the column tube to establish an interference fit between the second flow distributor and the inner surface of the column tube to form a sealed chamber within the column tube between the first and second flow distributors,
wherein the interference fit induces a hoop tension sufficient to provide a liquid-tight seal allowing the column to withstand operational pressures up to <NUM>.5kPa (<NUM> psi) and maintain a hydrostatic seal without the second flow distributor being permanently fixed in position, and
adjusting a longitudinal position of the second flow distributor within the column tube by
(i) applying an additional axial force to move the second flow distributor further into the column tube, or
(ii) forcing liquid into the sealed chamber to apply a hydraulic force to move the second flow distributor back towards the second end of the column tube, or
any combination of (i) and (ii); and
when the second flow distributor has reached a final longitudinal position,
permanently securing the second flow distributor within the column tube at the final longitudinal position.