Improved column for expanded adsorption fluidized bed and method of use thereof

Abstract

The present disclosure provides chromatography columns and methods of chromatographic separation using the same. In particular, the disclosure provides an improved column in which the cross-sectional area of ​​the column flares in regions near the ends of the column. The improved column can be used in an Expanded Bed Absorption (EBA) system.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This disclosure claims the benefit of U.S. Provisional Application No. 62 / 944,579, filed December 6, 2019, the contents of which are incorporated by reference in their entirety herein.

[0002] Field This disclosure relates generally to columns for expanded adsorption fluidized beds and methods of chromatographic separation using same. In particular, this disclosure relates to an improved column in which the cross-sectional area of ​​the column is flared and increases in a region near the end of the column. [Background technology]

[0003] Expanded bed adsorption (EBA) is a chromatographic process in which desired proteins are purified from a feed material. A column containing adsorbent particles is expanded by applying an upward liquid flow to the column, so that a stable fluidized bed is formed with the adsorbent particles suspended in equilibrium between the particle settling velocity and the upward liquid flow rate. The feed material is applied to the expanded bed and the target protein binds to the adsorbent while cellular debris, cells and contaminants pass through.

[0004] Simulated moving bed (SMB) chromatography is a chromatographic technique based on a liquid flow (mobile phase) moving counter to a constant flow of solids (stationary phase). Countercurrent flow increases the separation possibilities, which enhances the efficiency of the process compared to traditional batch chromatography. It also becomes possible to separate a continuous flow of feed material. As a result, large quantities of highly purified materials can be produced at significantly reduced costs.

[0005] Traditional EBA columns require manual manipulation, including raising and lowering the outlet collector as the fluidized bed height rises and falls to avoid particles exiting the column, which can make them difficult to use in a continuous SMB process. For example, an improved version of the EBA column that can be used in a continuous SMB process is desirable. Summary of the Invention

[0006] Improved chromatography columns with variable cross-sectional area have been developed. The columns can be used in expanded adsorption fluidized bed (EBA) and / or simulated moving bed (SMB) chromatography processes. In particular, the improved columns have a uniform cross-sectional area for a portion of the column, and then the area near the top of the column "flares" and increases, so that the end of the column has a large cross-sectional area. This design can reduce the linear flow rate of the chromatographic media, and particles can be minimized from exiting the column.

[0007] Accordingly, the present disclosure provides a chromatography column comprising: a tubular housing for containing a chromatographic medium, the tubular housing including a vertical major axis, a lower end and an upper outwardly flared region, the lower end and the upper outwardly flared region being separated by the major axis; an inlet for providing fluid to the lower end; and An outlet for collecting eluted material.

[0008] In one embodiment, the outlet extends from the upper outwardly flared region.

[0009] In another embodiment, the upper outwardly flared region has a base end and a top end, the top end having a larger diameter than the base end. Optionally, the diameter of the base end is the same as the diameter of the shaft, and the diameter of the top end is at least 1.1, 1.2, 1.3, 1.4, or 1.5 times the diameter of the shaft.

[0010] In one embodiment, the chromatography column further comprises a narrowing flared region extending from an upper end of the outward flared region.

[0011] In one embodiment, the narrowed flared region extends at an angle of 5 to 20 degrees from the horizontal, optionally 8 to 12 degrees from the horizontal.

[0012] In another embodiment, the outlet extends from a narrowed flared region.

[0013] In another embodiment, the upper outwardly flared region extends at an angle of 1-50° from the major axis, optionally 2-12°, 3-10°, 15-50°, or 20-45° from the major axis.

[0014] In another embodiment, the inlet is operably connected to a fluid distributor. Optionally, the fluid distributor is a static fluid distributor.

[0015] In another embodiment, the chromatographic medium comprises a fluidized, or expanded, particle bed.

[0016] In another embodiment, the outlet is operably connected to a collector for collecting eluted material, the collector being positioned at a fixed location within the column.

[0017] The present disclosure also provides an expanded adsorption fluidized bed (EBA) system comprising the chromatography columns described herein.

[0018] The present disclosure also provides a simulated moving bed system (SMB) comprising the chromatography columns described herein.

[0019] The present disclosure also provides a method for purifying a target molecule from a mixture containing the target molecule and undesired components, comprising: a) providing a fluid containing the target molecule and undesired components to a chromatography column described herein; b) contacting the fluid with a chromatographic medium; and c) Eluting the target molecule from the chromatographic medium.

[0020] In one embodiment, the chromatographic medium comprises particles and, prior to providing the fluid, the particles are fluidized to expand the medium from a settled bed to an expanded bed.

[0021] In another embodiment, the particles are fluidized by providing a fluidization buffer to the column using a static distributor.

[0022] In another embodiment, the expansion ratio of the height of the expanded bed to the height of the settled bed is maintained throughout steps a) to c) at 1.8 to 2.5, optionally 2 to 2.3.

[0023] In another embodiment, the fluid is provided to the column at a variable rate.

[0024] In another embodiment, elution comprises collecting an eluate containing the target molecule from a fixed location within the column.

[0025] The present disclosure also provides the use of a chromatography column as described herein for purifying a target molecule from a mixture containing the target molecule and undesired components.

[0026] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples indicating embodiments of the present disclosure are given by way of example only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0027] [Figure 1] The original commercial scale EBA column design is shown. [Diagram 2] 1 shows a modified commercial scale EBA column with a flared top. [Diagram 3] 1 shows the order of buffers used in Example 1. [Figure 4] The steady state expansion ratios are shown for a standard EBA column, a modified EBA column with a 3° flare, and a modified EBA column with a 10° flare. [Diagram 5] 1 shows the expansion ratio of the resin bed and the fine particle cloud caused during liquid switching for a modified EBA column with a 3° flare angle. [Figure 6]1 shows the expansion ratio of the resin bed and the fine particle cloud caused during liquid switching for a modified EBA column with a 10° flare angle. [Figure 7] 4 shows the expansion ratio of the resin bed and the particulate cloud caused during liquid switching for a standard EBA column. [Figure 8] Conductivity curves are shown for a modified EBA column with a 3° flare angle (top), a modified EBA column with a 10° flare angle (middle), and a standard EBA column (bottom). The estimated AUC represents the time or volume of buffer required for the high conductivity buffer to be exchanged with WFI. [Figure 9] The integrated AUC for the 3° and 10° flare modified columns and the standard column are shown. [Figure 10] Flared Top - Shows a modified EBA column with a 3° flare angle. Volume of the upper cone section = 77.29 mL. [Figure 11] (AL) Area Under the Curve (AUC) calculations for conductivity during liquid switch. FIG. 11A shows the AUC for 1M NaOH in a 3° flare modified column. FIG. 11B shows the AUC for 1M NaCl in a 3° flare modified column. FIG. 11C shows the AUC for 2M NaCl in a 3° flare modified column. FIG. 11D shows the AUC for 3M NaCl in a 3° flare modified column. FIG. 11E shows the AUC for 1M NaCl in a 10° flare modified column. FIG. 11F shows the AUC for 2M NaCl in a 10° flare modified column. FIG. 11G shows the AUC for 1M NaOH in a 10° flare modified column. FIG. 11H shows the AUC for 3M NaCl in a 10° flare modified column. FIG. 11I shows the AUC for 1M NaOH in a standard column. Figure 11J shows the AUC for 1M NaCl in the standard column, Figure 11K shows the AUC for 2M NaCl in the standard column, and Figure 11L shows the AUC for 3M NaCl in the standard column. [Figure 12]The flow-expansion relationship is shown for solutions common to columns A, PreD, and D. (Regeneration=1M NaCl, CIP=1M NaOH, Storage=0.1M NaOH). [Figure 13] Flared Top - Shows modified EBA column with 10° flare angle. Volume of upper cone region = 35.15 mL. [Figure 14] 1 shows a modified EBA column with a second narrowed flared region. [Figure 15] A typical process on a standard EBA column is shown. [Figure 16] Two static fluid distributors are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] I. Definition Unless otherwise stated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the disclosure described herein that are suitable, as would be understood by one of ordinary skill in the art.

[0029] In understanding the scope of the present disclosure, the term "comprise" and its derivatives are intended herein to be open-ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers, and / or steps. The same also applies to words with similar meanings, such as the terms "contain" and "have" and their derivatives. The term "consisting of" and its derivatives are intended herein to be closed terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers, and / or steps. The term "essentially consisting of" is intended herein to specify the presence of stated features, elements, components, groups, integers, and / or steps, as well as those that do not substantially affect the basic and novel property(s) of the features, elements, components, groups, integers, and / or steps.

[0030] Terms of degree, such as "substantially," "about," and "approximately," are used herein to mean a reasonable amount of deviation from the modified term that does not materially change the end result. These terms of degree should be construed to include deviations of at least ±5% of the modified term, unless such deviations would negate the meaning of the word they modify.

[0031] In this application, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise.

[0032] II. Column In the field of expanded adsorbent bed (EBA) chromatography, the chromatographic media can be sensitive to linear flow rate. In particular, as the chromatographic media expands (increases in volume), the fluid level in the column can rise. A "flared" end of the column can reduce the change in fluid level height when expansion occurs. Such a design can minimize the amount of operator interference required during chromatography. It can also reduce the likelihood of resin and / or fines (low density and / or high mobility small or fragmented resin) exiting the column.

[0033] Thus, the present disclosure provides improved columns for performing chromatography (also referred to herein as "chromatography columns"). In particular, the improved columns have a uniform cross-sectional area for a portion of the column, and then the cross-sectional area "flares" toward the end of the column, so that the end of the column has a larger cross-sectional area than the remainder of the column. The portion of the column having the increased cross-sectional area may have a reduced internal linear flow velocity.

[0034] Figure 2 shows an embodiment of a column of the present disclosure. Thus, in one embodiment, a chromatography column 1 includes a tubular housing 2 that defines a housing interior 3 for containing a chromatographic medium.

[0035] Tubular housing 2 includes a vertical main axis 4, a lower end 5, and an upper outwardly flared region 6 (also referred to herein as the "outwardly flared region"), which are separated by main axis 4. Main axis 4 has a consistent diameter, while the diameter of upper outwardly flared region 6 increases as the outwardly flared region extends from the main axis, resulting in a "flared" or "conical" shape.

[0036] The outwardly flared region 6 extends from the vertical major axis 4 at a flare angle A. The larger the flare angle, the more rapidly the diameter of the upper outwardly flared region increases as it extends from the major axis. In one embodiment, the flare angle A ranges from 1 to 50°, optionally 2 to 12°, 3 to 10°, 15 to 50°, or 20 to 45°. In other embodiments, the flare angle A is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50°.

[0037] In another embodiment, the diameter D3 of the outwardly flared region 6 at its maximum point (i.e., the upper end of the outwardly flared region) is 1.1 to 5 times the diameter D1 of the major shaft, optionally 1.2 to 2 times, 1.1 to 4 times, or 1.1 to 3 times the diameter of the major shaft.

[0038] In one particular embodiment, the diameter D3 of the outwardly flared region at its maximum point is 1.4 times the diameter of the major axis 4 and extends at an angle of 30° from the major axis.

[0039] Various heights of the main shaft 4 are contemplated herein. In one embodiment, the height H1 of the main shaft 4 is between 10 and 100 centimeters, optionally between 15 and 45 centimeters or between 20 and 30 centimeters.

[0040] Various diameters of the main shaft 4 are contemplated herein. In one embodiment, the diameter D1 of the main shaft 4 is between 2 and 50 centimeters, optionally between 2 and 8 centimeters or between 15 and 35 centimeters.

[0041] Various heights of the outwardly flared region 6 are also contemplated herein. In one embodiment, the height H2 of the outwardly flared region 6 is between 2.5 and 20 centimeters, optionally between 5 and 15 centimeters.

[0042] The diameter of the outwardly flared region increases from the bottom end of the outwardly flared region (where it joins the major shaft) to the top end of the outwardly flared region. The diameter of the bottom end of the outwardly flared region is the same as or similar to the diameter of the major shaft 4. In one embodiment, the diameter D2 of the bottom end of the outwardly flared region 6 is between 3 and 50 centimeters, optionally between 15 and 35 centimeters.

[0043] The diameter D3 of the outwardly flared region is greater than the diameter of the main axis 4. In one embodiment, the diameter D3 of the outwardly flared region 6 is between 8 and 70 centimeters, optionally between 25 and 45 centimeters.

[0044] The column is designed to hold a chromatographic medium, usually in the form of a resin. The resin can include particles (or beads) of various materials and sizes. Suitable resins are known in the art. Examples of resins include, but are not limited to, MABDirect resin (tungsten carbide-agarose beads; median particle size distribution range: 90-110 μm; density: 2.8-3.2 g / mL; ligand: p-aminobenzoic acid) and FastLine DEAE resin (tungsten carbide-agarose beads; median particle size distribution range: 110-140 μm; density: 2.8-3.2 g / mL; ligand: DEAE).

[0045] The resin can exist within the column in one of two forms: a packed, or settled, form (also referred to herein as a "settled bed") and an expanded, or fluidized, form. In the fluidized form, the resin particles are suspended in a fluid, such as a buffer. When fluidized, the particles form an "expanded bed" (also referred to herein as a "fluidized bed") within the column.

[0046] In one embodiment, the height H3 of the settled bed is 30-60% of the height H1 of the main axis, optionally 35%-55% or 40-50% of the height H1 of the main axis.

[0047] In another embodiment, the height of the expanded bed is 75-100% of the column height H5, optionally 85-99% or 85-95% of the column height H5.

[0048] As used herein, the term "expansion ratio" refers to expanded bed height / settled bed height. In one embodiment, the chromatographic medium has an expansion ratio of 1.8 to 2.5, optionally 2 to 2.3.

[0049] The settled bed 20 is shown in Figure 2 and has a height H3. The area above that bed is called the headspace 21. The headspace height H4 is optionally 1-10 centimeters above the extended bed and 10-40 centimeters above the settled bed.

[0050] In one embodiment, the headspace height H4 does not change beyond the outwardly flared region height H2 during operation of the column.

[0051] Examples of buffers that can be used to fluidize the particles include, but are not limited to, 1M NaOH, WFI, 1M NaCl, 2M NaCl, and 3M NaCl.

[0052] 14, a narrowing flare region 30 extends from the top of the outward flare region 6. The narrowing flare region can redirect the flow to the upper port 8.

[0053] Various heights H6 of the narrowed flared region 30 are contemplated herein.

[0054] The diameter of the narrowed flare region decreases from the bottom end of the narrowed flare region (where it joins the top end of the outward flare region) to the top end of the narrowed flare region, and the diameter of the bottom end of the narrowed flare region is the same as or similar to the diameter of the top end of the outward flare region.

[0055] Narrowed flare-like region 30 extends at a flare angle D from the horizontal. In one embodiment, flare angle D ranges from 5 to 20 degrees from the horizontal, optionally 8 to 12 degrees from the horizontal. In other embodiments, flare angle D is 8, 9, 10, 11 or 12 degrees from the horizontal.

[0056] The column in FIG. 2 also includes a lower port 7 (also called inlet 7) and an upper port 8 (also called outlet 8).

[0057] The lower port 7 can be used, for example, to provide fluids to the column. Examples of fluids that can be provided through the lower port include buffers, such as buffers for expanding the chromatographic medium and elution buffers, wash fluids and raw materials to be processed.

[0058] The top port 8 is used, for example, to collect eluted material.

[0059] The bottom port 7 is optionally operably connected to a fluid distributor 10, which distributes fluid (e.g., mobilization buffer, elution buffer, and / or feedstock) to the columns. In one embodiment, the fluid distributor 10 is a static distributor. In another embodiment, the fluid distributor 10 is a non-static distributor. Static (non-moving) distributors are distinguished from non-static distributors, such as motorized rotary distributors. In one embodiment, the fluid distributor is adapted to deliver a uniform flow of fluid to the column. The fluid split optionally includes multiple arms projecting from a central distributor line, each adapted to provide fluid to a column. The arms are optionally straight (e.g., as shown in FIG. 16, top) or curved (as shown in FIG. 16, bottom).

[0060] The top port 8 can extend from an upper end of the outwardly flared region 6. If the column includes a narrowing flared region 30, the top port 8 can extend from an upper end of the narrowing flared region 30. The top port 8 can be operably connected to a collector 11.

[0061] Collector 11 is optionally positioned at a fixed height within the column, in one embodiment, collector 11 is positioned at a fixed height of 1 to 6 centimeters, optionally 2 to 5 centimeters, above the expanding bed.

[0062] The columns of the present disclosure are optionally adapted for use in an Expanded Bed Absorption (EBA) system. Accordingly, the present disclosure also provides an EBA system that includes the columns described herein.

[0063] The columns of the present disclosure are optionally adapted for use in simulated moving bed (SMB) systems. Thus, the present disclosure also provides an SMB system comprising the columns described herein.

[0064] III. Methods and Uses The present disclosure also includes a method for purifying a target molecule from a mixture containing the target molecule and undesired components, comprising: a) providing a fluid containing the target molecule and undesired components to a chromatography column of the present disclosure; b) contacting the fluid with a chromatographic medium; c) Eluting the target molecule from the chromatographic medium.

[0065] In one embodiment, the target molecule is a protein.

[0066] In another embodiment, the unwanted components include cells, cell debris, containment and / or unwanted proteins.

[0067] In one embodiment, the fluid is plasma, optionally human plasma, and the target molecule is a plasma protein, optionally IVIG, albumin or alpha-1 antitrypsin (AAT).

[0068] In another embodiment, the target molecule is IgA, IgM, IgD, IgE, alpha-1-proteinase inhibitor, blood coagulation promoting proteins, blood anticoagulant proteins, thrombolytic agents, antiangiogenic proteins, alpha-2-antiplasmin factor, C-1 esterase inhibitor, apolipoproteins, HDL, LDL, fibronectin, beta-2-glycoprotein I, fibrinogen, plasminogen, plasmin, plasminogen activators, plasminogen inhibitors, plasma protease inhibitors, thrombin, antithrombin III, streptoproteins, alpha-2-antiplasmin factor, C-1 esterase inhibitor ... kinase, inter-alpha-trypsin inhibitor, alpha-2-macroglobulin, amyloid protein, ferritin, prealbumin, GC-globulin, hemopexin, C3-complement, transferrin, urokinase, alpha-1-acid-glycoprotein, and a coagulation or anticoagulation factor selected from the group consisting of factor II, factor V, factor VII, factor VIII, von Willebrand factor, factor VIII-von Willebrand factor complex, factor IX, factor X, factor XI, C1 inhibitor, protein C, and protein S.

[0069] In one embodiment, the chromatographic medium comprises a resin. Optionally, the method further comprises fluidizing the resin and providing an expanded bed (e.g., expanding a settled bed to an expanded bed) prior to providing the fluid. The resin is optionally fluidized by providing a fluidization buffer to the column. A variety of mobilization buffers are known in the art, including: 1M NaOH; WFI; 1M NaCl; 2M NaCl; 3M NaCl; 0.1M NaOH; 0.5M NaCl; 20mM NaCl; 20mM NaCl, 0.6M NaCl; 0.1M KH2PO4; 5mM KH2PO4; 10mM NCP, 10mM EACA; 0.2M KH2PO4, 0.5M NaCl, 10mM EACA (pH 7.80); 0.2M KH2PO4, 0.5M NaCl, 10mM EACA (pH 4.50); ​​265mM glycine (pH 4.15); 5mM NaOAc (pH 5.75); 5mM NaOAc, 1M NaCl; 0.5M NaOAc (pH 5.25); 0.15M NaOAc (pH 5.25); 10mM These include, but are not limited to, NaOAc, 300 mM NaCl; 25 mM NaOAc (pH 5.20); 5 mM NaAce (pH 4.50); ​​10 mM KH2PO4, 80 mM NaCl; 0.1 M NaOH, 1 M NaCl; 50 mM NaCl and 60 mM NaHCO3.

[0070] In one embodiment, the method further comprises rinsing the chromatography column to remove at least some of the undesired material before eluting the target molecule. This step can be performed after contacting the fluid with the chromatographic medium and before eluting the target molecule from the chromatographic medium. Examples of rinsing fluids that can be used to rinse the column include, but are not limited to, 20 mM NaCl; 5 mM KH2PO4; 10 mM NCP, 10 mM EACA; 0.15 M NaOAc (pH 5.25) and 25 mM NaOAc (pH 5.20).

[0071] In one embodiment, "eluting the target molecule" includes providing a suitable elution buffer to the column and then collecting an eluate containing the target molecule from the column. The eluate can be collected from an outlet collector, such as outlet collector 8 of FIG. 2. In one embodiment, the eluate is collected from a fixed position within the column. In other words, a collector with an adjustable height is not required.

[0072] In this method, liquids including but not limited to mobilization buffer, rinsing fluid, elution buffer and fluids containing target molecules and undesired components are provided to the column using a distributor, optionally a static distributor.

[0073] The columns of the present disclosure can allow for operation of the column with a constant, or relatively constant, expansion ratio (expanded bed height / settled bed height). In one embodiment, the expansion ratio of the chromatographic medium is maintained at 1.8-2.5, optionally 2-2.3 during operation.

[0074] Maintaining a constant, or relatively constant, height of the expanded bed means that the height of the collector can be maintained at a fixed height throughout the process. Thus, in another embodiment, the collector 8 is maintained at a fixed height of 1 to 6 centimeters, optionally 2 to 5 centimeters, above the expanded bed.

[0075] The flow rate of the liquid provided to the column can be varied so that the expansion ratio remains constant, or relatively constant, throughout the separation process.

[0076] In another embodiment, a buffer is provided to the column to fluidize the chromatographic medium prior to providing the fluid.

[0077] In one embodiment, the method is performed continuously. For example, the method can be performed without manual adjustment of the height of the collector. For example, the method can optionally be performed as part of a simulated moving bed (SMB) system.

[0078] As is known in the art, SMB systems combine a series of columns in close proximity flow paths, whereby feedstocks are added to the system and target molecules are collected in a continuous run, which can be achieved by timed injection and flow path splitting of products and buffers in a sequential manner through the series of columns.

[0079] The following non-limiting examples illustrate the present disclosure.

[0080] Working Example Flared top expanded adsorbent fluidized bed (EBA) chromatography columns have been found to reduce the probability of chromatographic media (resin) and fines entering the exhaust line during operation and to reduce the risk of overexpansion when there is an increase in flow rate or an increase in the viscosity of the fluid flowing through the column. The effect of different headspace volumes for buffer exchange was also investigated.

[0081] I. Introduction Figure 1 is an example of a large scale EBA column design used for clinical manufacturing of PlasmaCap IG. The outlet collector is submerged in the liquid above the expanded bed. Throughout the process, liquids with different physical and chemical properties are passed through the column. The expanded bed height is allowed to increase and decrease as the liquid properties change.

[0082] The drain is manually raised and lowered to maintain approximately 3-5 cm of liquid above the varying bed height. If the outlet is not raised with the expanded bed, the chromatographic media will exit through the column via the outlet and be included in the product pool. If the outlet is not lowered with the expanded bed, a higher volume will be required to collect the product, resulting in excessive dilution of the product. Additionally, chromatographic media fines of the lowest size and density may also exit the column if there is a disturbance in the bed. This is of particular concern during liquid switchover when the buffers used have different viscosities. A cloud of fines may persist above the main bed height for a longer period of time and they may enter the drain line.

[0083] An improved version of the original EBA column is desirable for use in a continuous simulated moving bed (SMB) system. In order to use an EBA column in an SMB system, two main issues need to be addressed. First, the manual nature of the EBA column operation needs to be changed to an automatic nature. Second, the probability of resin potentially entering the discharge line needs to be reduced.

[0084] In Figure 2, a modified commercial scale EBA column with a flared top design can be seen. The top of the column is a closed system, where the top is physically closed off from the outside environment and directed into a closed tubing flow path without exposure to air. This eliminates the need for a second pump on the discharge line.

[0085] Operating the column with a constant expansion and variable flow, rather than a variable expansion, also eliminates the need for a collector with an adjustable height. It is easier to operate the column without having to manually increase or decrease the height of the exhaust line during operation, minimizing the risk of human error in adjustments. This design is more suitable for use on a continuous SMB system, since there are no parts that need to be manually adjusted.

[0086] The upper flared region of the column (also referred to herein as the "upper outward flared region") is shaped like a truncated cone with a larger diameter at the top than at the bottom.

[0087] As described below, the increase in diameter caused a decrease in linear flow from the axial region to the top of the column where collection occurs. It was also shown that with the decrease in linear flow velocity, the possibility of resin fines rising to the aperture region was reduced. In addition, the introduction of horizontal flow vectors reduced the risk of overexpansion due to changes in flow or fluid properties. It was also shown that a smaller head space leads to better conversion efficiency.

[0088] procedure Standard and flared column designs were compared using available lab-scale columns and EBA DEAE resin. The standard EBA column was a 2 cm diameter column and the improved column was a 2.2 cm diameter column with a 3.3 cm diameter flared head area and flare angles of 3° and 10° (specifications are shown in Figures 10 and 13).

[0089] procedure: 1. The expansion ratios of DEAE EBA resin on a standard EBA column, a modified EBA column with a 3° flare angle, and a modified EBA column with a 10° flare angle are evaluated using a series of liquids with various physical properties.

[0090] 2. For all column designs, monitor the height and duration of the overexpanded fine resin cloud caused by liquid switching.

[0091] 3. Compare various columns to evaluate the effect of different headspace volumes on exchange efficiency.

[0092] Materials and equipment Tables 1-3 detail the materials and equipment used in the study. [Table 1] [Table 2] [Table 3]

[0093] method 1. Follow SOP-E-015 Column Packing and Unpacking, but pack columns to a settled bed volume of 20±0.5 cm using 1 M NaOH as the packing buffer. The modified 3° and 10° flare angle columns were packed to 20 cm, and the standard columns were packed to 19.5 cm.

[0094] 2. An in-line conductivity meter was installed in the outlet line of the column.

[0095] 3. The flow rate was set to the desired flow rate and the resin was allowed to expand 2x (with 1M NaCl): 30 mL / min for the modified column and 27 mL / min for the standard column.

[0096] 4. The buffer was run through the column at the flow rate described above.

[0097] 5. The resin bed height was recorded at regular intervals throughout the expansion to determine the expansion ratio. Conductivity data was recorded.

[0098] 6. Sufficient time was allowed for expansion to reach its new steady state.

[0099] 7. If a fine resin cloud was seen during the change, its height was recorded along with the time it took for the cloud to reduce to the main resin bed height. Photographs were taken of any remaining fines that were present in the exit beaker to qualitatively measure how much resin was lost.

[0100] 8. Steps 4-7 were repeated until all of the specified buffer had been run through the column.

[0101] Buffers were used in the order outlined in Figure 3. We started with 1M NaOH since this was the one the resin was loaded with first. WFI followed NaOH and NaCl so that the less concentrated buffers could follow the more concentrated buffers. Significant lifting of the resin was expected to occur and so this was noted to characterize the differences in the fine resin cloud between the three column designs. If resin entered the drain line it was noted and the resin was pumped back into the column at the end of the sequence.

[0102] The modified columns had a fixed headspace of 3 cm for the 10° flare column and 10 cm for the 3° flare column. A fixed headspace of 10 cm was used for the standard EBA column (2× above the resin expansion line).

[0103] Results and Discussion The following sections examine the expansion ratios of DEAE EBA resin on a standard EBA column, a modified EBA column with a 3° flare angle, and a modified EBA column with a 10° flare angle, using a series of liquids with various physical properties. They also examine the height and duration of the overexpanded fine resin cloud caused by liquid switching. The effect of different headspace volumes on the exchange efficiency of the various columns is also evaluated.

[0104] Resin bed expansion ratio In Figure 4, the steady state expansion ratios for the standard EBA column and the modified EBA columns with 3° and 10° flares can be seen. The standard EBA column had the highest expansion ratio level, as indicated by the circular markers. The modified EBA column with a 3° flare angle, indicated by the square markers, had the second highest expansion ratio. The modified EBA column with a 10° flare angle, indicated by the triangular markers, had the lowest expansion ratio. The higher the flare angle, the faster the column diameter increases and the smaller the resin bed expansion ratio. Some characteristics of the succession of buffers used can be seen in Table 4 below. The most reliable factor for determining how much the resin expands was the conductivity. As the conductivity increased, the expansion ratio steadily increased. Density and viscosity also had a clear linear increasing trend with bed height when 1M NaCl to 3M NaCl were analyzed, but with 1M NaOH the trend was less reliable. [Table 4]

[0105] Resin fine powder cloud expansion ratio In Figure 5, we can see data for the resin bed and dust cloud expansion ratios induced during liquid switchover for a modified EBA column with a 3° flare angle. Looking at the black and grey data sets, which represent the resin bed height and conductivity, respectively, a trend can be seen. The trend is that as the conductivity increases, the bed height also increases. The higher the conductivity, the higher the bed height. If the conductivity is low, the bed height is also low. However, when a high conductivity buffer is immediately followed by a low conductivity buffer, an interesting phenomenon occurs at the resin bed / liquid interface in the column. Small dust particles of resin are swept up in the upward flow and circulate around the top of the column. The dust cloud (visualized by the yellow dots) persists for 7-8 minutes before gradually thinning and dissipating.

[0106] In Figure 6, data can be seen for the expansion ratio of the resin bed and the fines cloud caused during liquid switch for the modified EBA column with a 10° flare angle. Similar to Figure 5, during liquid switch from high conductivity liquid to WFI, the fines cloud rose to the top of the column for 7-8 minutes. The additional flare angle did not appear to have a significant difference in terms of the amount of resin in the cloud.

[0107] In Figure 7 you can see the data for the expansion ratio of the resin bed and the fines cloud caused during liquid switchover for the standard EBA column. The conductivity data set is slightly corrupted at some points due to bubbles entering the system. However, the trend is the same as with the previous column design. The fines cloud rose to the top of the column during liquid switchover.

[0108] The amount of fines generated on the way from the column to the waste beaker was also observed. There was minimal difference between the 3° flare and 10° flare columns as similar amounts of resin were generated on the way out of the drain line for the improved column. For the standard column, slightly more resin was found in the drain vessel. Without being bound by theory, it is possible that the flare design reduced the amount of resin and fines that could make it into the drain line.

[0109] Headspace volume and buffer equilibration The 3° flare angle column had a headspace of 10 cm and 77.29 mL, the 10° flare angle had a headspace of 3 cm and 35.15 mL, and the standard column had a maintained headspace of 10 cm and 31.42 mL.

[0110] The method used for analysis was to compare the estimated area under the curve (AUC) for conductivity during liquid switching. The AUC represents the time or amount of buffer required for equilibration, as shown in Figure 9. An integration of the AUC was performed and then plotted in Figure 10. The calculation can be seen in Figure 11.

[0111] The AUC for any given liquid was shown to be lower using the 10° flare compared to the 3° flared and standard columns. The difference in headspace of 3 cm versus 10 cm had the greatest effect on equilibration time. Overall, the 10° flare column had a total AUC that was 34% less than the 3° flare column and 36% less than the standard column. In practice, it should be noted that when the standard column is used, only 3-5 cm of headspace is manually maintained, so the exchange efficiency will be similar to that of the 3° flare column.

[0112] overview The flare design was shown to reduce the risk of overexpansion when there is an increase in flow rate or an increase in fluid viscosity flowing through the column (Figure 4). The larger the flare angle, the better the column reduced overexpansion. The 3° flare design was better than the standard design, and the 10° flare design was better than the 3° flare design. The flare design was shown to be better than the standard EBA design in reducing the probability of fines entering the exhaust line during operation. The effect of different headspace volumes on buffer exchange was also investigated, showing that the smaller the headspace, the more efficient the buffer exchange. In a more typical use of a standard EBA column, the headspace is maintained at 3-5 cm, which increases the buffer exchange efficiency, however, it will also increase the amount of resin loss.

[0113] Overall, the Flare Design column is an improvement over the original EBA column and can be used in a continuous simulated moving bed (SMB) system.

Claims

1. a tubular housing for containing a chromatographic medium, said tubular housing including a vertical major axis, a lower end and an upper outwardly flared region, said lower end and said upper outwardly flared region being positionally separated by said major axis; an inlet for providing a fluid to said lower end; and An outlet for collecting eluted material Including, A chromatography column, wherein said main vertical axis has a height of 10 to 100 centimeters and a diameter of 2 to 50 centimeters, and said upper outwardly flared region extends from said main vertical axis at an angle of 20 to 45 degrees from said main vertical axis.

2. 2. The chromatography column of claim 1, wherein the outlet extends from the upper outwardly flared region.

3. 3. The chromatography column of claim 1 or 2, wherein the upper outwardly flared region has a bottom end and a top end, the top end having a larger diameter than the bottom end.

4. 4. The chromatography column of claim 3, wherein the diameter of the bottom end is the same as the diameter of the shaft and the diameter of the top end is at least 1.1, 1.2, 1.3, 1.4, or 1.5 times the diameter of the shaft.

5. 5. The chromatography column of claim 3 or 4, further comprising a narrowing flared region extending from said upper end of said upper outward flared region.

6. 6. The chromatography column of claim 5, wherein the narrowed flared region extends at an angle of 5 to 20 degrees from the horizontal.

7. The chromatography column of claim 5, wherein the narrowed flared region extends at an angle of 8 to 12 degrees from the horizontal.

8. 7. A chromatography column according to claim 5 or 6, wherein the outlet extends from the narrowed flared region.

9. The chromatography column of any one of claims 1-8, wherein the inlet is operably connected to a fluid distributor.

10. 10. The chromatography column of claim 9, wherein the fluid distributor is a static fluid distributor.

11. The chromatography column of any one of claims 1-10, wherein the chromatographic medium comprises an expanded bed.

12. A chromatography column according to any one of claims 1 to 11, wherein the outlet is operatively connected to a collector for collecting the eluted material, the collector being disposed at a fixed position within the column.

13. An expanded bed adsorption (EBA) system comprising a chromatography column according to any one of claims 1 to 12.

14. A simulated moving bed system (SMB) comprising a chromatography column according to any one of claims 1 to 12.

15. 1. A method for purifying a target molecule from a mixture containing said target molecule and undesired components, comprising the steps of: a) providing a fluid comprising said target molecule and said undesired components to a chromatography column according to any one of claims 1 to 12, b) contacting said fluid with said chromatographic medium; and c) eluting the target molecule from the chromatographic medium. A method comprising:

16. 16. The method of claim 15, wherein the chromatographic medium comprises particles, and prior to providing the fluid, the particles are fluidized to expand the medium from a settled bed to an expanded bed.

17. 17. The method of claim 16, wherein the particles are fluidized by providing a fluidization buffer to the column using a distributor.

18. 18. The method of claim 16 or 17, wherein the expansion ratio of the height of the expanded bed to the height of the settled bed is maintained throughout steps a) to c) at 1.8 to 2.5, or 2 to 2.

3.

19. The method of any one of claims 15-18, wherein the fluid is provided to the column at a variable rate.

20. The method of any one of claims 15-19, wherein elution comprises collecting an eluate containing the target molecule from a fixed position within the column.

21. Use of a chromatography column according to any one of claims 1 to 12 for purifying a target molecule from a mixture comprising said target molecule and undesired components.

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