Apparatus for using membrane binding and elution chromatography and method of manufacturing the same

Abstract

An integrated chromatography unit has an inlet and an outlet and includes one or more membranes insertable into the internal volume of the unit between the inlet and the outlet. In certain embodiments, each membrane is allocated sufficient space within the unit to expand by placement of one or more spacers. Fluid entering the unit through the fluid inlet passes through the membranes and spacers before exiting the unit through the fluid outlet.

Description

[0001] This application claims priority to U.S. Provisional Application No. 63 / 037,262, filed June 10, 2020, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] In general, the present invention relates to chromatographic units, and more particularly to a single-use or disposable membrane-based chromatographic apparatus, the applications of which include membrane-based binding / elution chromatography. Background Technology

[0003] Good manufacturing practices and government regulations are central to many biopharmaceutical manufacturing processes. These processes typically require mandatory, often lengthy and expensive validation procedures. For example, equipment used to isolate and purify biopharmaceutical products must meet stringent cleanliness requirements for obvious reasons. The associated and repetitive costs of a single cleaning validation for a single device can easily exceed thousands of dollars. To reduce the cost and expense of such cleaning validation and / or minimize the occasions requiring or demanding cleaning, the pharmaceutical and biotechnology industries are increasingly exploring pre-validated, modular, single-use solutions.

[0004] Following these lines of thought, considerable interest has recently arisen in developing disposable solutions for the primary and / or secondary purification of raw pharmaceutical synthetic fluids (e.g., cell culture media) in industrial, laboratory, and clinical volumes. The high-capacity, high-yield requirements of such processes often favor the use of expensive, pre-installed stainless steel equipment, where replaceable cartridges or canisters (e.g., typically comprising numerous lens-like filter elements) are housed within a stainless steel casing or similar container. Significant cost and effort must be invested in cleaning and validating the equipment before reuse, assuming the filtration operation is complete and the used cartridges or canisters are removed.

[0005] Membrane-based devices designed for the biopharmaceutical processing industry are typically constructed entirely of thermoplastic components. This is desirable because the chosen thermoplastics (e.g., polypropylene, polyethylene, polyethersulfone, etc.) are stable in the chemicals and environments they come into contact with. One disadvantage of all thermoplastic devices that utilize secondary molding operations during manufacturing is shrinkage. As the thermoplastic cools, it shrinks, causing the membrane to warp and create undesirable voids within it.

[0006] Scaled-down models can be used to validate filtration procedures, such as virus filtration. For example, a known amount of virus can be added to a sample to simulate contamination, and then removed using a scaled-down device. The performance of the device can be measured to determine its virus removal capability.

[0007] Such devices are typically made of thermoplastics and manufactured using an overmolding process, in which a thermoplastic "window" (usually polypropylene) is injection-molded around a rectangular membrane or medium, and then the sub-assemblies are joined using bonding steps (vibration, hot plates, etc.). In flow-through applications where the separation mechanism is size repulsion or charge-based, the additional voids inside the device due to shrinkage during cooling (and wrinkling of the membrane or medium) do not negatively impact device performance. However, in binding and elution modes used for capture in chromatographic systems, any additional voids created by wrinkled membranes will degrade device performance. This performance degradation can be seen in the sharpness of the breakthrough curve and elution efficiency.

[0008] In addition, even when using wet or semi-wet membranes, it is important to form an overall seal of the membrane within the device.

[0009] Therefore, there is a need for a filtration device, such as a binding and elution chromatography device, which uses one or more membranes coupled to a desired ligand (e.g., protein A ligand), the membranes being kept flat, void-free, and sealed within the device.

[0010] To further understand the nature of the invention and these and other objectives, reference should be made to the following description taken in conjunction with the accompanying drawings. Summary of the Invention

[0011] The embodiments disclosed herein address the problems of the prior art, relating to an integrated chromatographic unit having an inlet and an outlet, and including one or more membranes located in a unit region between the inlet and the outlet. Sufficient space is provided between the membranes to allow membrane expansion. In some embodiments, fluid entering the unit through the inlet passes through one or more membranes before exiting the unit through an outlet spaced apart from the inlet.

[0012] In various embodiments, a chromatographic apparatus is disclosed, comprising a housing having a fluid inlet and a fluid outlet spaced apart from the fluid inlet; an internal volume in a region between the fluid inlet and the fluid outlet; at least a first membrane and a second membrane disposed in the internal volume of the housing; and at least one spacer disposed between the first membrane and the second membrane. The resulting flow path through one or more membranes (and spacers) facilitates the use of the unit in chromatographic applications, including, for example, binding / elution chromatography operations of biopharmaceutical fluids.

[0013] In some embodiments, the at least one spacer is annular; for example, a gasket or annular member. In some embodiments, a filtration zone exists within the internal volume of the housing defined by the active membrane regions of at least the first and second membranes, and the annulus has an opening region disposed within the filtration zone.

[0014] In some embodiments, the first membrane is disposed upstream of the second membrane along the direction of fluid flow during operation of the chromatographic apparatus, and a spacer is disposed between the first and second membranes. In some embodiments, the chromatographic apparatus further includes a porous glass flotation disposed between the fluid inlet and the first membrane. In some embodiments, a porous glass flotation may be disposed between the fluid outlet and the second membrane, the second membrane being disposed downstream of the first membrane along the direction of fluid flow during operation of the chromatographic apparatus. In some embodiments, the glass flotation may be disposed between the fluid inlet and the first membrane, and between the fluid outlet and the second membrane.

[0015] The method for manufacturing this chromatographic unit was also disclosed.

[0016] By appropriately arranging one or more spacers within the internal volume of the housing to provide space for one or more membranes to expand or extend within the housing, the performance loss of the device due to uneven or wrinkled membranes can be minimized or resolved.

[0017] Therefore, a membrane-based binding and elution chromatography unit or device is disclosed, wherein the membrane or multiple membranes are kept flat and uniform in the device to minimize voids, thereby maximizing the active membrane area of ​​the device.

[0018] In some embodiments, a filtration device having an inlet and an outlet is disclosed, the device comprising a polymer frame having a filtration zone and one or more membranes bonded or adhered to the polymer frame in the filtration zone, the filtration zone having one or more spacers separating the membranes in the device.

[0019] In some embodiments, each of the multiple membranes is identical; for example, each membrane has the same chemical and performance characteristics or grades. In other embodiments, each of the multiple membranes has different chemical or performance characteristics to obtain the specific performance characteristics of the resulting filter unit.

[0020] In some embodiments, an integrated unit is disclosed having an inlet and an outlet, and including at least one membrane, wherein fluid entering the disposable integrated unit through the inlet passes through the at least one membrane before leaving the unit through the outlet. One or more spacers are arranged within the integrated unit, preferably in the form of gaskets or rings, to provide extended regions of one or more membranes within the unit. This unit is well-suited for high-yield chromatographic capture of monoclonal antibodies (mAbs) from clarified cell culture media via rapid circulation. It can operate at flow rates much higher than conventional protein A resins. Attached Figure Description

[0021] Figure 1 This is a cross-sectional view of a filter device according to certain embodiments;

[0022] Figure 2 A perspective view of a spacer according to certain implementation schemes;

[0023] Figure 3 A cross-sectional view of the filtration device according to an alternative embodiment;

[0024] Figure 4 Here is an example graph showing the pressure-flow rate relationship of a 1 mL device according to certain implementation schemes;

[0025] Figure 5 This is a schematic diagram of a system setup according to certain implementation schemes, showing the system retention volume between the injection valve and the detector;

[0026] Figure 6 As an exemplary chromatogram according to certain embodiments, a UV280 is shown for measuring a 2% acetone pulse for the system retention volume; and

[0027] Figure 7 The normalized absorbance of a 1 mL device at 280 nm is shown as an exemplary transmission chromatography according to certain embodiments. Detailed Implementation

[0028] The components, methods, and apparatus disclosed herein can be more fully understood by referring to the accompanying drawings. The drawings are merely illustrative representations for convenience and ease of explanation of this disclosure and are therefore not intended to limit or restrict the scope of the exemplary embodiments.

[0029] For clarity, although specific terms are used in the following description, these terms are intended to refer only to the specific structures illustrating the selected embodiments in the drawings and are not intended to define or limit the scope of the invention. It should be understood in the drawings and the following description that the same reference numerals denote parts with the same function.

[0030] The singular forms “a / an” and “the” include plural objects unless the context clearly specifies otherwise.

[0031] As used in this specification, various devices and components may be described as “including” other components. The terms “comprise,” “include,” “having,” “has,” “can,” “contain,” and their variations, as used herein, are open-ended transitional phrases, terms, or words that do not exclude the possibility of additional components.

[0032] Traditionally, scaled-down filtration units are manufactured via a one-step molding operation. One or more membranes are stacked between two plastic components (as well as inlet and outlet), placed in a thermoplastic mold where the components and membranes are mechanically compressed together, and molten thermoplastic is injected around the perimeter to create a seal from the top plastic component to the bottom plastic component. Contact or hot plate welding operations can be used. An integral seal is formed between one or more membranes and the unit, similar to integral welding of the unit housing. A membrane screen arranged beneath each membrane can be included in the unit. Suitable screens include those made of polypropylene, polyethylene, and nylon. A suitable screen is a Naltex extruded mesh from DelStar Technologies, with a thickness of 0.010 inches, a strand count (in) of 13.5 SPI, an out strand count of 13.5 SPI, a 60-degree angle, and a basis weight of 5.50 oz / 10 ft. Separating membranes with a screen reduces elution stress and decreases variability. In some embodiments, one or more spacers 22 may have a membrane screen 25 located within the inner diameter of the spacer 22, for example, by press fitting or adhesive connection. Figure 3 ).

[0033] However, membranes are typically porous hydrogels that require expansion (e.g., typically between 5% and 30%) to achieve optimal function. This expansion results in lower dynamic binding loading and higher pressure drop performance. The embodiments disclosed herein are capable of accommodating expansion and improving device performance.

[0034] Turn now Figure 1 , Figure 1 A filter unit 10 is shown, comprising a housing 12 having a fluid inlet 13 and a fluid outlet 14 spaced apart from the inlet 13. The fluid inlet 13 and fluid outlet 14 may include suitable Luer connectors for easy connection to pipes, etc. The housing is defined by one or more impermeable walls. The inner surface of the housing 12 at the fluid inlet 13 and / or fluid outlet 14 may have a mesh-like surface 8 to enhance fluid distribution within the housing (only when...). Figure 1 (The fluid outlet 13 is shown in the diagram). The mesh-like surface 8 can be integral with the housing, or it can be a separate component attached to the housing.

[0035] In the illustrated embodiment, a plurality of membranes 15a, 15b, 15c, 15d, and 15e are arranged in unit 10. Although five membranes are shown in this embodiment, those skilled in the art will understand that fewer or more membranes may be used. Membrane 15a is arranged as the upstream membrane. Membrane 15b is arranged downstream of membrane 15a along the fluid flow direction from inlet 13 to outlet 14; membrane 15c is arranged downstream of membrane 15b along the fluid flow direction from inlet 13 to outlet 14; membrane 15d is arranged downstream of membrane 15c along the fluid flow direction from inlet 13 to outlet 14; and membrane 15e is arranged downstream of membrane 15d along the fluid flow direction from inlet 13 to outlet 14. Preferably, each of the membranes 15 is sealed to the surface of the impermeable wall of the housing 12, for example, by an overmolding process. The overmolding material may be the same as the material of the housing, such as polyethylene. Therefore, the top and bottom of the housing, along with the membrane, optional screen, and gasket, are positioned in a molding machine, which compresses the stack and, for example, injects molten polypropylene around the periphery to weld the top, bottom, membrane / screen / gasket stack together. Once assembled, each membrane 15 has an active region, the membrane area that can be used for sample flow, and an inactive region, the area sealed to the housing and therefore not usable for sample flow (typically around the membrane periphery). In some embodiments, optionally, a porous glass frit 17 may be located at the fluid inlet 13 and at the most upstream membrane ( Figure 1 Between membranes 15a in the implementation scheme, and / or between the fluid outlet 14 and the downstream membrane ( Figure 1 Between membranes 15e) in the implementation scheme. In some implementations, porous glass frit 17 may be located in the fluid inlet 13 and / or fluid outlet 14 of the unit, such as Figure 3 As shown. That is, the glass frit 17 can be press-fitted into the inner diameter of the fluid inlet 13 and / or the fluid outlet 14, instead of as... Figure 1 As in the implementation scheme, it is overmolded onto the outer shell of the unit. Therefore, as Figure 3 The outer diameter of the glass flotation located at the inlet or outlet is smaller than that shown. Figure 1 The outer diameter of the glass frit is shown. Suitable glass frits include polyolefins, particularly polyethylene. Suitable glass frit thickness can be from about 0.03 inches to about 0.06 inches. Preferably, the glass frit thickness is uniform.

[0036] Suitable membranes include those suitable for binding / elution chromatography and include ligands linked thereto, such as protein A ligands. In some embodiments, membrane 15 may be a non-dryable wet membrane, such as a porous hydrogel. Suitable membranes include those disclosed in U.S. patent applications 7,316,919, 8,206,958, 8,383,782, 8,367,809, 8,206,982, 8,652,849, 8,211,682, 8,192,971, and 8,187,880, the disclosures of which are incorporated herein by reference. Such membranes comprise composite materials comprising a support member and a macroporous crosslinked gel having a plurality of pores extending through the support member, the macroporous crosslinked gel being located in and substantially filling the pores of the support member. In some embodiments, the macroporous gel used is responsive to environmental conditions, thereby providing a responsive composite material. In other embodiments, the microporous gel is used to facilitate chemical synthesis or support the growth of microorganisms or cells.

[0037] In some embodiments, one or more membranes 15 are adhered to and sealed onto a housing 12, which is preferably made of a polymeric material, such as a thermoplastic. Suitable thermoplastics include polyolefins, such as polypropylene and polyethylene, mixtures thereof, and polyethersulfone. The arrangement and sealing of the membranes 15 in the housing 12 are preferably such that all fluid entering the fluid inlet 13 of the device 10 must pass through the active regions of one or more membranes 15 before reaching the fluid outlet 14 of the device 10.

[0038] In various embodiments, spacers or gaskets 20 are arranged between one or more membranes 15. Figure 2 In some embodiments, the spacer or washer 20 is defined by the frame 22 or its periphery, which may be annular and may be continuous or discontinuous. Figure 1 In one embodiment, there are five membranes 15, and four gaskets 20 may be used. Therefore, gasket 20a is arranged between membranes 15a and 15b; gasket 20b is arranged between membranes 15b and 15c; gasket 20c is arranged between membranes 15c and 15d; and gasket 20d is arranged between membranes 15d and 15e. The gaskets 20 have a suitable thickness to provide sufficient space for each membrane 15 to expand or extend within the housing with minimal or no wrinkles or warping. Suitable gasket thicknesses include 0.010 inches, 0.015 inches, 0.020 inches, and 0.030 inches. Other thicknesses of gaskets 20 may be used depending on the required expansion space for one or more membranes 15. In some embodiments, the outer diameter of the gasket 20 is the same as or substantially the same as the outer diameter of the membrane 15. In some embodiments, the outer diameter of the gasket 20 is sufficient to connect and seal it to the housing 12.

[0039] In some embodiments, the device 10 contains a plurality of gaskets, each gasket 20 having the same dimensions. In some embodiments, each gasket 20 has an opening region 21 that, when assembled, is in fluid communication with the active membrane region of the membrane 15. Preferably, the opening region 21 of each gasket is arranged in a filtration zone of the device 10, which is the area within the internal volume of the housing 12 that contains the active membrane region (i.e., the membrane region within the housing 12 that can be used for filtration). Therefore, the gaskets 20 do not impede the flow of fluid within or through the device 10 or the filtration process. In some embodiments, the opening region 21 is aligned with the active region of the membrane 15. The periphery or frame 22 of each gasket 20 may be non-porous and may be used to seal the inner wall of the housing 12. Suitable materials for the gaskets 20 include polyesters, polyolefins such as polypropylene and polyethylene, polystyrene, and polysulfone.

[0040] The device can be implemented at a relatively low cost. Device 10 can be reusable or made as a "single-use" item, meaning that after completing the desired (or predetermined) operation, the device can be disposed of (e.g., after filtering certain environmentally regulated substances, sometimes required by law to be disposed of) or partially or completely reactivated or recycled (e.g., after filtering unregulated substances). The presence of one or more gaskets in the device eliminates variability; that is, data spread is reduced when measuring elution pressure relative to dynamic binding load at 10% penetration and when measuring elution volume relative to elution delay. Example

[0041] Connect to a protein A membrane device containing a 1 mL membrane.

[0042] A chromatographic system with appropriately sized fraction collectors (i.e., ÄKTA) TM Avant 25 or ÄKTA TM Perfuse the device (Pure 25) with equilibration buffer at a flow rate of 10 mL / min until the UV absorbance (UV280), pH, pressure, and conductivity detectors reach constant values. Connect the appropriate tubing to the inlet and outlet of the device, and connect the inlet tubing to the outlet tubing using a zero-volume Luer connector. Flush the tubing with equilibration buffer at a flow rate of 10 mL / min until the UV280, pH, pressure, and conductivity detectors reach constant values. Stop the flow and remove the zero-volume Luer connector. Connect the outlet to the outlet tubing via a Luer connector, while preventing air from entering the device. Reverse flow the equilibration buffer at a flow rate of 1 mL / min (outlet → inlet) to remove any air bubbles, and connect the inlet to the inlet tubing via a Luer connector.

[0043] The device is oriented so that the outlet is at the top, and the outlet cap is removed. The outlet is connected to the outlet tube via a Luer connector, preventing air from entering the device. Equilibrium buffer is backflowed at a flow rate of 1 mL / min (outlet → inlet) to remove any air bubbles; the inlet is connected to the inlet tube via a Luer connector. Equilibrium buffer is then backflowed through the device at a flow rate of 1 mL / min. The flow rate is gradually increased to 10 mL / min, and the pressure drop (δc pressure) is monitored throughout the device. Flow is continued at 10 mL / min until the pressure stabilizes. The pressure drop (δc pressure) should not exceed 100 psi.

[0044] A 50 MV equilibration buffer solution was flowed forward through the device at a flow rate of 10 mL / min. This flow continued until the UV280, pH, pressure, and conductivity detectors reached constant values.

[0045] Pressure flow characterization

[0046] Target pressure reduction

[0047] The flow rate is preferably adjusted to achieve an operating delta column pressure of 2 bar. The observed pressure will depend on the selected buffer solution. To determine the optimal flow rate, blank runs can be performed at flow rates of 7, 8, 9, and 10 MV / min. During blank runs, no mAb solution is loaded onto the device. The remaining steps are the same.

[0048] Blank execution method 1. All buffer solutions are connected via either system pump A or system pump B. Equilibration buffer is flowed through the column bypass at a rate of 10 mL / min until the UV280, pH, pressure, and conductivity detectors reach constant values. The UV280 signal is set to zero. 2. Select the target flow rate (7, 8, 9 or 10 MV / min). 3. Table 1 below also shows the following steps for measuring the operating flow rate: a. Step P1: Perfuse the equilibration buffer with 10 mL of pump wash solution. Equilibrate the device with 10 MV of equilibration buffer at the target flow rate. b. Step P2: Perfuse the device with 10 mL of pump wash solution to obtain high-salt buffer. Rinse the device with 10 MV of high-salt buffer at the target flow rate. c. Step P3: Perfuse the equilibration buffer with 10 mL of pump wash solution. Rinse the device with 10 MV of equilibration buffer at the target flow rate. d. Step P4: Perfuse the elution buffer with 10 mL of wash solution. Flow 15 MV of elution buffer through the device at the target flow rate. e. Step P5: Fill the CIP solution with 10 mL of pump wash fluid. Clean the device with 10 MV of CIP solution at the target flow rate. f. Step P6: Perfuse the equilibration buffer with 10 mL of pump wash solution. Equilibrate the device with 10 MV equilibration buffer at the target flow rate, or until the UV280, pH, pressure, and conductivity detectors reach constant values. 4. If necessary, repeat steps D1 to D6 within the remaining flow range of 7-10 MV / min. 5. If the device is not used in the same phase of a rapid cycle study, remove it from the chromatography system, reinstall the inlet / outlet caps, and store it in a refrigerator. 6. The procedure for determining the appropriate operating flow will be described in the next section. Table 1. Recommended conditions for pressure relative to flow rate when measuring a 1 mL device.

[0049] Flow determination

[0050] To optimize traffic, first determine the maximum operational pressure for each traffic segment. This typically occurs during CIP and will generate similar loads. Figure 4 The pressure-flow curve shown.

[0051] The optimized flow rate can then be determined using linear interpolation, as shown in the following equation: Q op It is a defined operational flow, P op The target operating δ bar pressure drop is 2 bar. P1 and P2 are two observed pressures closest to 2 bar, and Q1 and Q2 are the corresponding flow rates.

[0052] It is recommended to conduct further experiments at the stated flow rate.

[0053] System retention volume

[0054] Description of system retention volume

[0055] The system retention volume is the volume between the injection valve and the detector. Figure 5 This can be determined by equilibrating the device with a equilibration buffer and then injecting a pulse of tracer solution (2% acetone, high-salt solution). The retention volume of the observed peak is the system retrieval volume.

[0056] Measurement of system retention volume 1. Equilibrate the buffer solution through the device at the target flow rate and monitor the UV280 or conductivity detector to establish a baseline signal. 2. Load the tracer solution into a 100 μL sample loop. 3. Inject the tracer solution while continuing to flow the equilibration buffer at the target flow rate. During this injection, the time / volume ratio will be set to zero. 4. If the peaks are split or severely distorted, the apparatus may be incomplete and the evaluation should be stopped. 5. The observed maximum peak volume is the system retention volume. Figure 6 An example of a chromatogram produced during the measurement of the system retention volume using a 1 mL device is shown. 6. The system retention volume is typically 2-6 mL. The device and the chromatographic system will each contribute 1-3 mL. If the measured system retention volume is significantly larger, the system retention volume can be measured separately by replacing the device with the zero-volume connector.

[0057] Dynamic combined load capacity

[0058] Dynamic combined loading feed preparation

[0059] Preferably, the dynamic binding loading is determined using a pre-purified mAb solution. mAb penetration can then be observed by monitoring the UV280 signal. The purified mAb solution should have a similar concentration, pH, and conductivity to the mAb feed used for rapid cycling studies. The device should be loaded to approximately 50 g / L to adequately observe penetration behavior.

[0060] If purified mAb solution is unavailable, clarified mAb feed can also be used to determine dynamic binding load. When using clarified cell culture medium, a longer wavelength, such as 300 nm, should be used in this case because the UV detector is saturated at 280 nm. Alternatively, higher accuracy can be obtained using the method described by Swinnen et al., in which the volume fraction of mAb breakthrough is collected and the corresponding mAb concentration is measured offline by analyzing protein A chromatography (J. Chromatograph. B, 848 (2007) 97–107).

[0061] Dynamic combined load method 1. Prepare buffer solution and mAb feed for dynamic binding load measurement. Table 1 shows the approximate amount of buffer solution required for a single dynamic binding load measurement using the device: Table 1. Buffer volumes required for dynamic binding capacity and rapid cycling experiments using a single 1 mL device. 2. mAb feed is connected via the sample pump, and all buffer solutions are connected via system pump A or system pump b. 3. Flow the equilibration buffer through the column bypass at a rate of 10 mL / min until the UV280 / UV300, pH, and conductivity detectors reach constant values. Set the UV280 / UV300 signal to zero. 4. The mAb feed was flowed through the column bypass at a rate of 1 mL / min until the UV280 / UV300 signal reached a stable value. This value was recorded as the 100% breakthrough value and will be used to calculate DBC in the next section. 10 . 5. Flow the equilibration buffer through the column bypass at a rate of 10 mL / min until the UV280 / UV300, pH, and conductivity detectors reach constant values. The UV280 / 300 signal should return to zero. 6. The following steps for measuring dynamic combined load are also shown in Table 3. a. Step D1: Perfuse the equilibration buffer with 10 mL of pump wash solution. Equilibrate the device with 10 MV of equilibration buffer at the target flow rate. b. Step D2: Load 50 mg mAb into a 1 mL device at the target flow rate. c. Step D3: Clean the device with 10 MV of equilibration buffer at the target flow rate. d. Step D4: Perfuse the device with high-salt buffer using 10 mL of pump wash solution. Rinse the device with 10 MV of high-salt buffer at the target flow rate. e. Step D5: Perfuse the equilibration buffer with 10 mL of pump wash solution. Clean the device with 10 mV of equilibration buffer at the target flow rate. f. Step D6: Fill the eluent with 10 mL of wash buffer using the pump. Elute the mAb from the device at the target flow rate using 15 MV of elution buffer. g. Step D7: Fill the CIP solution with 10 mL of pump cleaning fluid. Clean the device with 10 MV of CIP solution at the target flow rate. h. Step D8: Perfuse the equilibration buffer with 10 mL of pump wash solution. Equilibrate the device with 10 MV equilibration buffer at the target flow rate, or until the UV280, pH, pressure, and conductivity detectors reach constant values. 7. If the device is not used in the same phase of a rapid cycle study, remove it from the chromatography system, install the inlet / outlet caps, and store it in a refrigerator. 8. DBC 10 The value will be calculated by analyzing the chromatogram of the UV280 signal, as described in the next section. Table 3. Dynamic binding loading method for 1 mL devices.

[0062] Calculate DBC10 1. The UV signal is normalized by dividing it by the 100% transmittance value measured in step 4 of the "Dynamic Combined Loading Method" section. Figure 7 ). 2. Locate the load volume when the UV signal on the penetration curve reaches 10%. The dynamic combined load at 3.10% penetration is calculated using the following formula: DBC 10 The dynamic binding load at 10% penetration, V 10%BT V is the buffer volume when the UV signal reaches 10%. HU For the system's residual volume, C 进料 V represents the concentration of the mAb feed. 膜 Let be the volume of the membrane in the device. 4. For example, if 10% breakthrough is 12.5 mL, system retention volume is 2.47 mL, mAb feed titer is 3 g / L, and membrane volume is 1 mL, then DBC 10 It is 30.1 g / L.

[0063] Rapid Cyclic Research

[0064] Length of study

[0065] Preferably, the apparatus is evaluated with 100 cycles. One binding / elution cycle typically takes 8-15 minutes, depending on the load density, feed concentration, operating flow rate, and the time required for LC unit pump washing. Therefore, completing 100 cycles takes 13-25 hours.

[0066] Calculate the required mAb feed volume

[0067] The feed volume required for rapid cycle studies is calculated using the following formula: Where V 进料 V is the volume of feed required. 膜 Let LD be the volume of the membrane in the device, C be the loading density, and LD be the density of the membrane. 进料 The concentration of mAb feed, N 循环 This represents the number of iterations.

[0068] Note that the load density is calculated using the following formula: For example, if the device's DBC is detected 10If the feed concentration is 30.1 g / L, the unit load will be 30.1 g / L × 80%, or 24.08 g / L. When the feed concentration is 1 g / L, each cycle will require a load of 24.08 mL, and 100 cycles will require 2,408 mL. Note that an additional amount of feed should be prepared to fill the system, and the feed container should be avoided from being completely emptied.

[0069] Fast Loop Method 1. Prepare the mAb feed and buffer for rapid cycling studies. Table 1 above shows the amount of buffer to prepare for 100 cycles using a 1 mL apparatus. 2. Preferably, the mAb feed is kept below 10°C throughout the cycle study. 3. mAb feed is connected via the sample pump, and all buffer solutions are connected via system pump A or system pump b. 4. Describe a single binding / elution cycle in the following steps and show them in Table 4. The cycle process should be performed automatically on the chromatography system software. For example, on the AKTA system, this is done by constructing the method using the "Search" function in the "Method Editor" or by using the "Method Queue" when setting up the experiment. a. Step R1: Perfuse the equilibration buffer with 10 mL of pump wash solution. Equilibrate the equipment at the target flow rate using 20 MV of equilibration buffer. b. Step R2: Load the device with mAb-clarified cell culture medium to the calculated loading density (80% × DBC). 10 Before starting the first cycle, the feed valve should be filled. Subsequent cycles do not require filling. c. Step R3: Clean the device with 10 MV of equilibration buffer at the target flow rate. d. Step R4: Perfuse the device with 10 mL of pump wash solution to high-salt buffer. Rinse the device with 10 MV of high-salt buffer at the target flow rate. e. Step R5: Perfuse the equilibration buffer with 10 mL of pump wash solution. Rinse the device with 10 MV of equilibration buffer at the target flow rate. f. Step R6: Perfuse the elution buffer with 10 mL of wash solution using the pump. Elute the mAb from the device at the target flow rate using 15 MV of elution buffer. Preferably, when UV... 280 When the elution peak is above 100 mAU, collect the eluent in a 15 mL tube every 10 cycles. The 100 mAU value may need to be adjusted for specific feeds. g. Step R7: Fill the CIP solution with 10 mL of pump wash fluid. Clean the device with 10 MV of CIP solution at the target flow rate. 5. After completing 100 cycles, equilibrate the device at the target flow rate with 35 MV equilibration buffer, or until the UV280, pH, pressure, and conductivity detectors reach constant values. 6. If all 100 cycles cannot be completed in one operation, flush the device with equilibration buffer until the UV280, pH, pressure, and conductivity detectors reach constant values. Remove the device from the chromatography system, reinstall the inlet / outlet caps, and store it in a refrigerator.

[0070] Table 4. Rapid circulation method for 1 mL apparatus.

Claims

1. A chromatographic apparatus, comprising, The housing has a fluid inlet and a fluid outlet spaced apart from the fluid inlet; The internal volume is located within the outer shell; At least a first membrane and a second membrane are disposed in the internal volume of the housing in the region between the fluid inlet and the fluid outlet, the first membrane and the second membrane each having an active membrane region through which fluid introduced into the fluid inlet can flow. as well as At least one spacer is disposed between the first membrane and the second membrane, the at least one spacer having a periphery defining a continuous opening in fluid communication with the active membrane region, the at least one spacer not impeding the flow of fluid within or through the device; Between the first membrane and the second membrane, fluid flows only from the first membrane to the second membrane, wherein a filtration zone exists within the internal volume defined by the active membrane regions of the at least first membrane and the second membrane, wherein the opening of the at least one spacer is arranged within the filtration zone, and wherein the at least one spacer is annular. The at least one spacer includes a gasket disposed between the first membrane and the second membrane, the gasket having an opening region aligned with the active membrane regions of the first membrane and the second membrane and disposed within the filtration zone, and the gasket having a thickness that provides sufficient space for the first membrane and the second membrane to allow the first membrane and the second membrane to expand or extend within the housing with minimal or no wrinkles or warping; and The first and second membranes are porous hydrogel-bonded and eluting chromatographic membranes, the first and second membranes expand by 5% to 30%, and the thickness of the gasket is selected from 0.010 inches, 0.015 inches, 0.020 inches, and 0.030 inches to provide expansion space so that the expansion is minimal or without wrinkles or warping.

2. The chromatographic apparatus of claim 1, wherein the first membrane is disposed upstream of the second membrane along the direction of fluid flow during operation of the chromatographic apparatus, and the chromatographic apparatus further comprises a porous glass frit disposed between the fluid inlet and the first membrane.

3. The chromatographic apparatus according to claim 1 further includes a porous glass frit disposed between the fluid outlet and the second membrane.

4. The chromatographic apparatus of claim 1, wherein the at least first membrane and the second membrane and the at least one spacer are arranged in the housing such that, during operation of the apparatus, fluid enters the fluid inlet and passes continuously through the first membrane, the at least one spacer and the second membrane before exiting the housing through the fluid outlet.

Images