Antibody / resin binding apparatus and method

Abstract

To provide antibody-resin coupling apparatus and methods.SOLUTION: The present disclosure relates to devices and methods for activating resins and coupling antibodies to the resin. An antibody-resin coupling apparatus quickly and efficiently activates resin beads and couples them to antibodies, while preventing breakdown and crosslinking of the beads, thereby improving downstream column purification processes, extending the usable life of the resin beads, and increasing molecule capture efficiency of the resultant resin-antibody complexes, to allow improved isolation and purification of factor VIII molecules or other drug compounds.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of and priority to U.S. Application No. 62 / 541,601, filed August 4, 2017, the contents of which are incorporated by reference.

[0002] The present disclosure relates to devices and methods for activating resins and binding antibodies to resins. [Background technology]

[0003] Hemophilia is a genetic bleeding disorder that prevents blood from clotting normally and is characterized by bleeding that can occur spontaneously or after minor trauma. It is often associated with a deficiency of an essential blood clotting protein, factor VIII, also known as antihemophilic factor (AHF). In humans, factor VIII is encoded by the F8 gene. Defects in this gene result in hemophilia A ("classical" hemophilia), a recessive X-linked coagulation disorder that results in prolonged plasma clotting times. Another type of hemophilia is acquired hemophilia A (AHA), which occurs in patients who have a normal F8 gene but who develop autoantibodies that inhibit factor VIII, resulting in a functional defect that prevents clotting.

[0004] Providing intravenous factor VIII to patients with hemophilia can temporarily improve clotting. Many treatments for hemophilia involve recombinant factor VIII protein that is synthesized and purified in-house, such as those sold under the trade names RECOMBINATE, ADVATE, ADYNOVATE, HEMOFIL, and OBIZUR and available from Shire Plc (Lexington, MA).

[0005] However, a major limiting factor in the production of such therapeutics is the ability to isolate and purify the factor VIII molecule. One method is to use an antibody to factor VIII to capture the molecule in a column. The antibody is bound to a resin and loaded into a column, and the medium containing the expressed factor VIII molecule is passed through it.

[0006] This purification process requires high precision to properly manipulate the resin. This can be extremely expensive to implement because the resin is expensive and can easily be damaged or discarded if not handled accurately. If the resin is not sufficiently bound to the antibody, cross-linking can occur between the resin beads. Also, the beads can break if the resin is not handled carefully. Broken or cross-linked resin beads can clog the column, creating excessive backpressure and causing the extraction process to fail. Thus, the difficulties in resin handling procedures and the limitations of resin / antibody binding technology make the manufacturing process for hemophilia drugs expensive and complex. Summary of the Invention [Means for solving the problem]

[0007] The device described herein efficiently activates resin beads and binds them to antibodies while preventing bead breakage and cross-linking, thereby improving the downstream column purification process, extending the useful life of the resin beads, and increasing the molecular capture efficiency of the resulting resin / antibody complex, allowing for improved isolation and purification of a wide variety of biological compounds, including factor VIII molecules.

[0008] While the device is generally described herein in connection with its use in purifying FVIII, it should be understood that the device and associated methods are useful for binding any type of antibody to a porous resin. The disclosed invention can be applied to other affinity resins, particularly for other enzyme replacement therapy products or other drugs or biomolecules of interest. It is applicable to cross-linking chemistries between proteins or amine-containing ligands and the resin support. The resin may be agarose, glass, or other known porous resins of various densities. The antibodies used may be either polyclonal or monoclonal. While the device is described herein primarily as being useful for binding antibodies to a resin, the invention is compatible with any chemistry in which a hydroxyl group is linked to an amino group. It should be understood that the invention is useful with any known antibody that can be used for affinity chromatography purification of any biological compound, including peptides, nucleic acids, carbohydrates, and any ligand with an amino group.

[0009] The apparatus is generally a mixing device that includes a container with an inlet for introducing resin beads and various fluids, a dispersion tube for precise application of the activation solution, a mesh screen for supporting the resin beads and allowing fluid to flow through, an agitator for mixing the beads as the fluid flows through, and an outlet directly below the screen for draining the fluid. The components of the apparatus can be made of stainless steel or other elastic materials. In operation, resin beads in a buffer solution are poured into the container before being activated by dispersion of CNBr and acetonitrile through the dispersion tube. The beads are agitated using the agitator for a short period (approximately 3 minutes or less) before the fluid is drained through the outlet. The beads may be washed with water or other buffer before adding the binding solution and monoclonal antibody. The beads and antibody are incubated with agitation for a set period while the device maintains a low temperature and monitors the pH of the solution. The antibody-bound resin is captured on the screen and removed from the device for use. The device can be used for a variety of resin and antibody types. The system allows large amounts of resin to be activated quickly and uniformly without damaging the resin beads and avoiding cross-linking between beads, providing more effective resin / antibody binding while controlling chemistry and extending the useful life of the resin beads.

[0010] A key aspect of this device is the dispersion tube, which is optimized for distribution of the CNBr activation solution to the resin. This allows for uniform dispersion of CNBr over a controlled time period and helps achieve rapid addition of the activation solution, preferably in less than about 3 minutes. These factors are important for homogeneous conjugation of antibody and resin because they reduce variability in the addition of solutions to the resin / buffer mixture, which may transiently exceed the buffer capacity of the buffer. Such variability would result in undesirable isourea crosslinks that hydrolyze and increase antibody leaching, or potentially interact with other nucleophiles, contaminating the resin or causing increased leaching. Controlling the timing of the activation solution aids in downstream process control for antibody conjugation, as it allows for optimal resin activation followed by rapid addition of antibody prior to the loss of a significant number of activated groups on the resin.

[0011] In one aspect, the present disclosure relates to a device for binding an antibody to a resin. The device includes a mixing vessel divided into an upper section and a lower section by a mesh screen stretched across the vessel, the upper section having at least one inlet and the lower section having at least one outlet, the mesh screen having a pore size of 5 to 80 μm. The device further includes an agitator disposed within the upper section and a dispersion device above the agitator. The dispersion device includes an elongated tubular structure forming a lumen. The tubular structure has a proximal section extending vertically outside the mixing vessel with an upwardly opening inlet, a closed end, a distal section with a plurality of downwardly facing holes positioned horizontally within the upper section, and a bend connecting the distal and proximal sections.

[0012] The agitator may include a rotor and a rotating impeller, which may include a hub and at least two blades extending perpendicularly in opposite directions from the hub. Each blade has an axis generally transverse to the axis of the rotor on which the hub is configured to rotate. The agitator is configured to rotate and impart a force on the fluid in the vessel, providing lift and keeping the beads moving across the screen. The screen may also have support beams configured to support the screen and prevent it from sagging. The screen may be configured to be removable from the vessel.

[0013] The agitator rotates at a speed sufficient to rapidly mix the resin beads without damaging them. The agitator rotation speed may be about 10-50 RPM, and in certain embodiments, the speed is 20 or 35 RPM. In embodiments, the blades have a fixed pitch and rounded edges.

[0014] In a related aspect, the present disclosure relates to a dispersion tube device including an elongated tubular structure forming a lumen with a circular cross-section. The tubular structure includes a proximal portion with an upwardly facing inlet, a closed end, and a distal portion with 4 to 100, preferably about 8 to 30, downwardly facing holes arranged in two or more rows parallel to the axis of the distal portion. The rows are positioned about 15 to about 60 degrees apart from each other on the distal portion. The tubular structure also includes a bend between the distal and proximal portions, comprising a bend in the tubular structure for orienting the inlet to open in a direction generally perpendicular to the axis of the distal portion.

[0015] In some embodiments, the distal portion of the dispersion device has three parallel rows of downwardly facing holes. In some embodiments, there are 12 to 25 downwardly facing holes, and in preferred embodiments, there are 21 holes (rows of 10, 8, and 3 holes). In some embodiments, the rows have different numbers of holes, while in others, they have the same number of holes. The rows of holes may be positioned approximately 15 to 105 degrees apart. The bend within the bend orients the distal and proximal portions relative to each other at 60 to 120 degrees, in some embodiments, approximately 80 to 100 degrees, and in preferred embodiments, approximately 90 degrees. The tubular structure can be made of stainless steel. The dispersion device may also include a valve for opening and closing the inlet and a funnel connectable to the inlet for holding the dispersion fluid prior to use.

[0016] In a related aspect, the present disclosure relates to a method for activating resin. The method involves inserting resin beads suspended in water into a mixing vessel containing a dispersing device, a mesh screen with holes smaller than the resin beads, and an agitator positioned above the mesh screen. The resin beads may comprise agarose, such as CL-4B or CL-2B beads, or agarose beads sold under the trade name CAPTO by GE Healthcare Life Sciences (Marlborough, MA). The method further includes dispersing an activation solution containing CNBr and acetonitrile onto the resin beads via the dispersing device and agitating with the agitator for less than 5 minutes. The method then involves draining the activation solution through the mesh screen, thereby leaving the activated resin beads supported on the screen.

[0017] In some embodiments, the method also involves washing the activated resin beads with a fluid. Washing may include filling a mixing vessel with the fluid, incubating with an agitator, and draining the fluid from the mixing vessel. The fluid may be a buffer, water, or a solution containing sodium bicarbonate and sodium chloride. The stirring may be at 10 to 40 RPM, preferably about 20 or 35 RPM. The stirring and incubation may last for less than 4 minutes, preferably less than 3 minutes. The present invention provides, for example, the following. (Item 1) A dispersion device comprising: 1. An elongated tubular structure forming a lumen having a circular cross section, the tubular structure comprising: (a) a proximal portion with an upwardly facing inlet; (b) a distal portion with a closed end, the distal portion comprising a plurality of downwardly facing holes arranged in three rows parallel to an axis of the distal portion, the three rows being positioned on the distal portion at about 15 to about 60 degrees from each other; and (c) a bend between the distal portion and the proximal portion, the bend comprising a bend in the tubular structure that orients the inlet so that it opens in a direction that is generally perpendicular to the axis of the distal portion. A dispersion device comprising: (Item 2) Item 1. The dispersion device of item 1, wherein the distal portion comprises 8 to 30 downwardly facing holes. (Item 3) Item 3. The dispersion device of item 2, wherein the distal portion comprises exactly 21 downwardly facing holes. (Item 4) Item 4. The dispersion device of item 3, wherein a first of the three rows has 10 holes, a second of the three rows has 8 holes, and a third of the three rows has 3 holes. (Item 5) Item 1, wherein the three rows have different numbers of holes. (Item 6) Item 1, wherein the three rows have the same number of holes. (Item 7) Item 2. The dispersion device according to item 1, wherein the three rows are positioned 30 to 60 degrees apart from each other. (Item 8) 8. The dispersion device according to item 7, wherein the three rows are positioned 45 degrees apart. (Item 9) Item 1. The dispersion device of item 1, wherein the bend in the bend orients the distal portion and the proximal portion at 60 to 120 degrees relative to each other. (Item 10) Item 10. The dispersion device of item 9, wherein the bend in the bend orients the distal portion and the proximal portion at 80-120 degrees relative to each other. (Item 11) Item 11. The dispersion device of item 10, wherein the bend in the bend orients the distal and proximal portions at exactly 90 degrees relative to each other. (Item 12) Item 10. The dispersion device of item 1, wherein the tubular structure comprises stainless steel. (Item 13) Item 10. The dispersion device of item 1, further comprising a valve for opening and closing the inlet. (Item 14) Item 10. The dispersion device of item 1, further comprising a funnel connectable to the inlet. (Item 15) A resin washing device, a vessel comprising a cylinder divided into an upper section and a lower section by a screen stretched across the cylinder, the upper section comprising an inlet and the lower section comprising an outlet; an agitator disposed within the upper portion, the agitator comprising a rotor and a rotating impeller, the rotating impeller comprising a hub, a first blade, and a second blade, the first and second blades extending perpendicularly in opposite directions from the hub, each blade having an axis transverse to an axis of the rotor on which the hub is configured to rotate; Equipped with the screen comprises a mesh material with a pore size of about 5 μm to about 80 μm; the agitator is configured to impart a force to fluid in the vessel, the force being directed away from the screen. Resin cleaning equipment. (Item 16) Item 16. The resin washing apparatus according to item 15, wherein each blade has a fixed pitch. (Item 17) Item 16. The resin washing device according to item 15, wherein the blade has rounded edges. (Item 18) Item 16. The resin washing apparatus of item 15, wherein the agitator rotates at a speed sufficient to rapidly mix the resin beads without damaging them. (Item 19) Item 16. The resin washing device according to item 15, wherein the agitator rotates at 10 to 50 RPM. (Item 20) 20. The resin washing apparatus of claim 19, wherein the agitator rotates at 20 RPM. (Item 21) 20. The resin washing apparatus of claim 19, wherein the agitator rotates at 35 RPM. (Item 22) Item 20. The resin washing apparatus according to item 19, wherein the mesh pore size is 30 μm. (Item 23) Item 16. The resin washing apparatus according to item 15, further comprising a support beam, the support beam being configured to support the screen and prevent the screen from bending. (Item 24) Item 16. The resin washing apparatus according to item 15, wherein the screen is removable from the vessel. (Item 25) 1. A device for binding an antibody to a resin, said device comprising: a mixing vessel divided into an upper section and a lower section by a mesh screen extending across the vessel, the upper section having an inlet and the lower section having an outlet, the mesh screen having a pore size of about 5 μm to about 80 μm; an agitator disposed within the upper portion; a dispersion device comprising an elongated tubular structure forming a lumen, the tubular structure comprising: a proximal portion extending vertically outside the mixing vessel with an upwardly opening inlet; a distal portion positioned horizontally within the upper portion above the agitator, the distal portion comprising a closed end and a plurality of downwardly facing holes; and a bend connecting the distal portion and the proximal portion; A device comprising: (Item 26) Item 26. The device of item 25, wherein the agitator comprises a rotor and a rotating impeller. (Item 27) Item 27. The device of item 26, wherein the impeller comprises a hub, a first blade, and a second blade, the first and second blades extending perpendicularly in opposite directions from the hub, each blade having an axis transverse to the axis about which the hub is configured to rotate. (Item 28) Item 28. The device of item 27, wherein each blade has a fixed pitch. (Item 29) Item 28. The device of item 27, wherein the blade has rounded edges. (Item 30) Item 26. The device of item 25, wherein the agitator is configured to impart a force to fluid in the vessel, the force being directed away from the screen. (Item 31) Item 26. The device of item 25, wherein the tubular structure has a circular cross section. (Item 32) 26. The device of claim 25, wherein the distal portion has 8 to 30 holes. (Item 33) 26. The device of claim 25, wherein the distal portion comprises exactly 21 downwardly facing holes. (Item 34) Item 26. The device of item 25, wherein the holes are arranged in three rows parallel to the axis of the distal portion. (Item 35) Item 35. The device of item 34, wherein the rows each have the same number of holes. (Item 36) Item 35. The device of item 34, wherein the rows each have a different number of holes. (Item 37) Item 37. The device of item 36, wherein a first of the three rows has 10 holes, a second of the three rows has 8 holes, and a third of the three rows has 3 holes. (Item 38) Item 35. The device of item 34, wherein the three rows are positioned 30 to 60 degrees apart. (Item 39) Item 35. The device of item 34, wherein the three rows are positioned 45 degrees apart. (Item 40) 26. The device of item 25, further comprising a valve for opening and closing the inlet. (Item 41) 26. The device of claim 25, further comprising a funnel connectable to the inlet. (Item 42) Item 26. The device of item 25, further comprising a support beam, the support beam configured to support the mesh screen and prevent the mesh screen from sagging. (Item 43) 26. The device of claim 25, wherein the screen is removable from the mixing vessel. (Item 44) Item 26. The device of item 25, wherein the bend comprises a bend in the tubular structure, the bend orienting the inlet so that the inlet opens in a direction that is generally perpendicular to the axis of the distal portion. (Item 45) Item 26. The device according to item 25, wherein the mesh pore size is 20 to 50 μm. (Item 46) 26. The device of claim 25, wherein the mesh pore size is 30 μm. (Item 47) 1. A method for activating a resin, the method comprising: Inserting resin beads suspended in water into a mixing vessel, the mixing vessel comprising a dispersing device, a mesh screen with holes smaller than the resin beads, and an agitator disposed above the mesh screen; dispersing an activation solution comprising CNBr and acetonitrile onto the resin beads via the dispersing device; allowing the agitator to agitate for less than 5 minutes; draining the activation solution through the mesh screen, thereby leaving activated resin beads supported on the screen; A method comprising: (Item 48) 48. The method of claim 47, further comprising washing the activated resin beads with a fluid. (Item 49) Item 49. The method of item 48, wherein washing comprises filling the mixing vessel with the fluid, stirring and incubating, and draining the fluid from the mixing vessel. (Item 50) Item 48. The method according to item 47, wherein the fluid is water or a solution containing sodium bicarbonate and sodium chloride. (Item 51) Item 48. The method according to item 47, wherein the stirring is carried out at 10 RPM to 40 RPM. (Item 52) 52. The method of claim 51, wherein the stirring is performed at 20 RPM. (Item 53) 52. The method of claim 51, wherein the stirring is performed at 35 RPM. (Item 54) Item 48. The method of item 47, wherein the agitating of the agitator is for less than 4 minutes. (Item 55) Item 55. The method of item 54, wherein the agitating of the agitator is for less than 3 minutes. (Item 56) 48. The method of claim 47, wherein the resin beads comprise agarose. [Brief explanation of the drawings]

[0018] [Figure 1A] Figures 1A-C show views of the mixing vessel: Figure 1A shows a side cross-sectional view, Figure 1B shows a top cross-sectional view, and Figure 1C shows a front cross-sectional view. [Figure 1B] Figures 1A-C show views of the mixing vessel: Figure 1A shows a side cross-sectional view, Figure 1B shows a top cross-sectional view, and Figure 1C shows a front cross-sectional view. [Figure 1C] Figures 1A-C show views of the mixing vessel: Figure 1A shows a side cross-sectional view, Figure 1B shows a top cross-sectional view, and Figure 1C shows a front cross-sectional view.

[0019] [Figure 2A] Figures 2A-G show the sparging tube and a preferred arrangement of holes for the sparging tube: Figure 2A shows a side view of the sparging tube. [Figure 2B] Figures 2A-G show the sparging tube and a preferred arrangement of holes for the sparging tube. Figure 2B shows a side view of the sparging tube with a particular configuration of holes. [Figure 2C] Figures 2A-G show the sparging pipe and the preferred arrangement of holes relative to the sparging pipe, and Figure 2C shows a radial cross section of the sparging pipe showing the angle at which the holes are drilled. [Figure 2D] Figures 2A-G show the sparging tube and the preferred arrangement of holes on the sparging tube. Figure 2D shows a side view of the sparging tube with the location of the row of eight holes. [Figure 2E] Figures 2A-G show the sparging tube and a preferred arrangement of holes for the sparging tube. Figure 2E shows a radial cross section of the sparging tube showing the angle at which the holes of Figure 2D are drilled. [Figure 2F]Figures 2A-G show the sparging tube and the preferred arrangement of holes on the sparging tube. Figure 2F shows a side view of the sparging tube with the location of the row of 10 holes. [Figure 2G] Figures 2A-G show the sparging pipe and a preferred arrangement of holes for the sparging pipe. Figure 2G shows a radial cross section of the sparging pipe showing the angle at which the holes of Figure 2F are drilled.

[0020] [Figure 3] FIG. 3 shows a removable holding vessel or funnel for the activation solution and a valve connecting the funnel to the dispersion tube.

[0021] [Figure 4A] Figures 4A-C show views of the agitator: Figure 4A is a side cross-sectional view of the agitator. [Figure 4B] Figures 4A-C show views of the agitator, and Figure 4B is an upside down perspective view of the agitator and blades. [Figure 4C] Figures 4A-C show views of the agitator, and Figure 4C is an enlarged view of the hub and blades of Figure 4B. DETAILED DESCRIPTION OF THE INVENTION

[0022] Devices for isolating and purifying molecules from culture media are essential for drug manufacturing. Many drugs involve recombinant proteins grown in culture and then must be purified and extracted from the media. Purification of these compounds requires precise control of reagents and materials, as well as specialized equipment. Due to the complexity of preparing and using these materials, devices are needed to generate purification matrices for drug extraction in a more efficient, accurate, and cost-effective manner. Such devices would improve the availability of recombinant proteins needed to treat various diseases.

[0023] Many drugs involve recombinant proteins that, when introduced into a patient, deliver the desired biochemical response. For example, hemophilia drugs such as RECOMBINATE® and ADVATE® are intravenously injectable factor VIII molecules that improve blood clotting and control and prevent bleeding episodes in patients with hemophilia. Treatment with these drugs increases plasma levels of factor VIII, temporarily correcting the coagulation disorder in these patients and normalizing clotting times over an effective dosing cycle. Factor VIII replacement drugs can be used for routine prevention and reduction of bleeding, or they can be administered before, during, and after surgery to manage blood clotting. The goal of such treatment is to maintain plasma factor VIII activity levels at or above the desired level. For example, for minor bleeding episodes, such as early joint bleeding, minor muscle bleeding, or minor oral bleeding episodes, it may be desirable to administer a dose to achieve approximately 20–40% of normal factor VIII levels. For moderate bleeding, such as muscle bleeding, bleeding into the mouth, obvious joint bleeding, and known trauma, 30-60% of normal factor VIII activity may be required. To treat major bleeding, such as serious gastrointestinal bleeding, intracranial, intraperitoneal, or intrathoracic bleeding, central nervous system bleeding, bleeding in the retropharyngeal or retroperitoneal space or iliopsoas sheath, fractures, or head trauma, factor VIII levels of 60-100% may be required.

[0024] RECOMBINATE®, ADVATE®, ADYNOVATE®, HEMOFIL®, and OBIZUR® are examples of recombinant factor VIII molecules that can be used to temporarily replace deficient factor VIII in patients and achieve hemostasis. For example, ADVATE® is a purified glycoprotein consisting of 2,332 amino acids synthesized by a genetically engineered Chinese hamster ovary cell line. ADYNOVATE® is a purified ADVATE® molecule covalently conjugated with one or more molecules of polyethylene glycol, which reduces binding to the physiological factor VIII clearance receptor (LRP1) and exhibits a prolonged terminal half-life. OBIZUR® is a recombinant analog of porcine factor VIII. The B domain present in naturally occurring porcine factor VIII is replaced with a 24-amino acid linker. Once activated, the resulting drug has activity comparable to endogenous human factor VIII.

[0025] To manufacture these and other drugs involving recombinant proteins, the molecule must be purified from the culture medium. Typically, a recombinant cell line expresses and secretes the Factor VIII protein into the cell culture medium. The molecule is then purified from the culture medium. The purification process involves introducing the medium into one or more immunoaffinity chromatography columns, in which a purification matrix prepared by immobilizing a monoclonal antibody to a resin selectively isolates Factor VIII. The method may also involve one or more filtration steps. For example, HEMOFIL® is a Factor VIII protein isolated from pooled human plasma by immunoaffinity chromatography using a mouse monoclonal antibody to Factor VIII, followed by an ion-exchange chromatographic step for further purification.

[0026] Monoclonal antibodies are particularly useful for capturing factor VIII and other target molecules. Purification matrices that selectively purify target molecules are created by conjugating antibodies to resins. Preparing resins and conjugating antibodies is challenging because even slight changes in chemistry affect the conjugation process and the resulting purification matrix. The device disclosed herein provides improved conjugation of antibodies to resin beads. The chemistry of the resin conjugation process is controlled to produce antibody / resin conjugates with greater efficacy and longer lifespans. With proper manufacturing and handling, resins can last for years and be reused hundreds of times before needing to be replaced. Because resin beads can cost tens of thousands of dollars per liter, it is important for manufacturers to have a reliable process for producing long-lasting antibody / resin conjugates without wasting large amounts of resin.

[0027] As explained above, slight anomalies in the chemistry can reduce the functionality of the resin product. Resins must be prepared quickly and precisely to achieve large quantities of homogeneous product. The disclosed device enables rapid, homogeneous reactions that result in reproducible, standardized products. Without such a device, the resulting resin product would have microheterogeneities within the resin, creating leaching problems. If the resin is not homogeneously activated, the beads can cross-link with each other, creating clumps within the resin. Aggregation and heterogeneity within the resin can lead to plugging problems in downstream purification protocols. For example, resins that aggregate together are not permeable to fluid passage and can cause pressure buildup within the column. Heterogeneous resins can result in inefficient capture of target molecules, or they may simply be unusable and need to be discarded.

[0028] The disclosed device also prevents excessive bead breakage during processing steps. If more than about 2 percent of the beads are broken, the resulting resin will be too dense and over-compressed when loaded into a column. Thus, the broken beads cause backpressure within the column. If too much resin is broken during production, some or all of the resin will need to be discarded, resulting in excessive waste and expense. The device disclosed herein avoids that problem and others. The device optimizes resin preparation and ensures proper filling of the resin in downstream columns.

[0029] In addition to improved column fluid dynamics, there are additional advantages to binding resins using the disclosed apparatus. The present apparatus allows for greater reproducibility of resins than prior art devices. The resulting resins are more uniform in terms of ligand density, bead cross-linking, and bead integrity. This provides greater stability, reduced variability in the manufacturing process, and higher yields. The result is a product with a longer useful life that can be used over a greater number of production cycles without antibody / ligand leaching and loss of binding capacity. This reduces the cost of producing enzymes or other biochemical products.

[0030] This device can be used for many types of conjugation chemistries. For antibodies, the conjugation agent is often amine-based. Antibodies may have 20-30 amino groups and another 20-30 carboxyl groups. The conjugation techniques disclosed herein can be used for conjugating any antibody or homogenous smaller molecule. In some embodiments, monoclonal antibodies are conjugated, and in other embodiments, polyclonal antibodies are conjugated to the resin. This technique may also be used to conjugate peptides. In either case, the goal of this technique is to achieve homogenous conjugation of molecules (antibodies, peptides, or other) to the resin beads. Homogeneous linking of molecules to the resin provides the resulting product with predictable leaching behavior and longer shelf life.

[0031] The binding device shown in the accompanying figures rapidly and effectively activates resin and binds antibody, reduces waste, and improves binding efficiency and drug recovery. As explained above, the device generally includes a mixing vessel, a dispersion tube, an agitator, and a screen.

[0032] 1A-C show a binding apparatus 100. FIG. 1A shows a side cross-sectional view, FIG. 1B shows a top cross-sectional view, and FIG. 1C shows a front cross-sectional view. Apparatus 100 includes a mixing vessel 110, which houses other elements described below and serves as a mixing vessel for the various mixing steps. Vessel 110 has multiple inlets 120 and an outlet 130. A hinged hatch 150 is present at the top of vessel 110 through which resin beads can be inserted. Resin beads are available from GE Healthcare Life Sciences (Marlborough, MA) and are typically agarose beads such as CAPTO®, CL-4B, or CL-2B beads, which are inserted into vessel 110 in an aqueous suspension. Up to 50 liters of resin beads, typically about 22-39 liters, can be activated in vessel 110 at a time. Once inside the vessel 110, the beads are supported by a screen 310 made of a mesh material with holes of about 10-80 μm, small enough to prevent the resin beads from entering the lower portion of the vessel 110. In a preferred embodiment, the holes are about 30 μm. During the activation and binding process, the beads are mixed by an agitator 410 (shown in FIGS. 4A-C), which prevents the beads from settling on the screen and helps maintain homogeneity.

[0033] The vessel contains a dispersion tube 210, illustrated in more detail in Figures 2A-G, through which an activation solution of CNBr and acetonitrile is added to the beads. The dispersion tube 210 is designed to evenly distribute the activation solution over the beads, while an agitator 410 keeps them moving and prevents the resin from clumping. As explained below, the agitator 410 provides lift to the beads and rotates at a speed sufficient to keep them moving, but slight enough to prevent bead breakage. After activation, the activation solution can be drained through a waste outlet 130, leaving the activated beads on the screen 310.

[0034] Various fluids and buffers can be inserted into the vessel 110 through the sparger tube 210, the inlet 120, or the hatch 150, as desired. Different solutions are required for the activation, washing, and binding processes. An exemplary resin binding process begins with the resin being poured into the vessel 110 through the hatch 150. The resin contains resin beads in a water suspension. An activation solution of CNBr and acetonitrile is dispersed onto the resin through the sparger tube 210, while the agitator 410 mixes the resin and keeps the beads moving above the screen 310. A small amount of acetonitrile may be used to chase the activation solution in the sparger tube. The beads may be washed with the activation solution for up to five minutes to activate the resin. Preferably, activation occurs in less than three minutes. Various buffers and binding solutions may be added, in addition to the antibody suspended in the binding solution, to bind them to the beads. As during activation, during all washing and binding steps, an agitator rotates to keep the beads in constant motion, preventing them from sticking to the screen, and uniformly dispersing the various fluids and buffers to ensure that all surfaces of the beads are contacted. Washing and other mixing steps may be continuous, with fluids flowing continuously in and out of the inlets, or the vessel may be filled, and mixing can occur over a set time period, ranging from a few seconds to minutes or even hours. The vessel 110 also contains a temperature control so that the contents can be incubated at a desired temperature during mixing. The device 100 may also include one or more probes 190, such as a temperature gauge and / or pH probe, for monitoring conditions inside the vessel during operation.

[0035] The screen 310 is designed to be removable and is held in place by a series of clamps 330. The vessel 110 also includes a trunnion 175 so that the device can be pivotally mounted. This allows the device to be tilted so that the user can fill the vessel or pour liquid out of it as needed. The tilting feature allows the processed resin to be poured out the hatch rather than manually scooped, which prevents additional stress on the beads and screen. The trunnion 175 can be locked in place during use so that it remains steady while mixing. It is important that the screen 310 and agitator 410 remain approximately horizontal during mixing to achieve proper movement of the beads within the vessel and ensure that the beads are evenly exposed to the various fluid mixtures.

[0036] 2A-G show the sparging tube 210. As shown in FIG. 2A, the sparging tube 210 is a generally hollow pipe with a long horizontal section 230, a vertical section 220, and a bend 240. The horizontal section 230 is generally a length of metal tubing perforated at regular or semi-regular intervals. The length of the horizontal section 230 extends across the vessel. In the embodiment shown, the sparging tube 210 has a plurality of holes 231 whose positions are marked (in FIGS. 2B, 2D, and 2F) along their distance along the length of the tube 210. The holes 231 allow the CNBr activation mixture to flow through. Together, they provide a reproducible and uniform distribution of the activation mixture onto the resin below. The dispersion tube 210, especially in combination with the agitator 410, prevents inconsistent distribution of CNBr and avoids local pH spikes that can lead to incorrect binding chemistry and increased ligand leaching.

[0037] The vertical portion 220 of the dispersion tube 210 is configured to extend out of the vessel 110, while the horizontal portion 230 is positioned within the vessel 110 above the agitator 410 and screen 310. The vertical portion 220 includes an inlet 225 through which CNBr can be flowed and introduced into the mixing vessel 110. The horizontal portion 230 has a closed end 235 and a plurality of holes 231. One possible arrangement of the holes is shown in Figures 2B-G, although other similar arrangements of holes are also contemplated. The holes should be distributed along the horizontal portion of the dispersion tube to achieve sufficient coverage of the resin being mixed below.

[0038] Figures 2B-C show the locations of three holes drilled along the bottom edge of the horizontal section 230. Figure 2B shows a radial cross section of the dispersion pipe. As depicted, the holes are located at 11.75 inches, 15.75 inches, and 26.50 inches, measured from the axis 229 of the vertical section 220. Figure 2C shows a cross section of the horizontal section. As shown in Figure 2C, holes are drilled through the bottom within the horizontal section.

[0039] Figures 2D-E show the locations of eight holes drilled at a 45 degree angle from the bottom edge of the horizontal section 230. Figure 2D shows a radial cross section of the dispersion pipe showing the measurements of the holes from the axis 229 of the vertical section 220. As depicted, the holes are located at 3.00 inches, 4.00 inches, 6.00 inches, 8.00 inches, 19.50 inches, 21.50 inches, 23.50 inches, and 25.00 inches. Figure 2E shows a cross section of the horizontal section showing the angle at which the set of holes is drilled.

[0040] Figures 2F-G show the locations of another 10 holes drilled at 45 degree angles on the other side of the sparging pipe 210 from the set of 8 holes (shown in Figure 2D). Figure 2F shows a radial cross section of the sparging pipe showing measurements of the holes from the axis 229 of the vertical portion 220. As depicted, the holes are located at 2.50 inches, 3.50 inches, 4.50 inches, 6.50 inches, 9.50 inches, 11.50 inches, 21.00 inches, 23.00 inches, 24.00 inches, and 26.00 inches. Figure 2G shows a cross section of the horizontal portion showing the angle at which this set of holes is drilled.

[0041] As depicted in Figures 2B-G, the set of holes is arranged in three parallel rows drilled into the lower half of the horizontal section 230. Each of the three rows has a different number of holes 231, and each hole is drilled at a different distance along the horizontal section 230 from all the other holes. The location of the holes is an important consideration to ensure uniform distribution of the activation solution on the resin. This arrangement of holes was found to be optimal based on salt modeling experiments. However, other arrangements of holes could also be used. The exact number of holes, their relationship to each other, the number of rows, the angle at which the holes are drilled, the size of the holes, and other factors can be adjusted as needed.

[0042] Adding the activation mixture using a sparger tube is superior to manual addition because it achieves greater uniformity in the resulting resin product. As explained above, resins produced by the disclosed binding apparatus perform differently in a column compared to resins produced using other methods. Using a sparger tube can add 100 or more cycles to the useful life of the resin beads.

[0043] Another important advantage of the sparger tube is that it allows for homogeneous distribution of the activation mixture in a very short time period. Activation of the resin with CNBr needs to occur quickly to reduce the occurrence of cross-linking. In most cases, it should be completed in less than 5 minutes, and ideally, it should be completed in less than about 3 minutes. If the activation process takes significantly longer than 3 minutes, binding efficiency is reduced, resulting in excessive cross-linking. The sparger tube, especially in conjunction with the agitator system described below, allows the activation process to occur quickly to produce high-quality resin. CNBr can be added to up to about 40 liters of resin, mixed, and removed in less than 3 minutes, and in some cases, less than 2 minutes.

[0044] FIG. 3 shows a removable holding vessel or funnel 290 for the CNBr / acetonitrile solution that can be included in the apparatus. The outlet 291 of the funnel 290 is in fluid communication with the inlet 225 on the vertical portion 220 of the sparging tube. A valve 280 controls the flow of fluid from the funnel 290 into the sparging tube 210. In use, the removable holding vessel 290 is filled with the CNBr solution and attached to the sparging tube. The removable funnel 290 allows the CNBr to be handled more safely and avoids spillage. The CNBr / acetonitrile mixture is corrosive and hazardous, so the removable funnel allows the mixture to be mixed under a hood before being connected to the coupling device.

[0045] Once the resin is prepared for activation, valve 280 is opened, allowing the solution to proceed onto the resin through dispersion tube 210. Dispersion typically takes 2-4 minutes. The solution can be chased with a small amount of acetonitrile solution to rinse any remaining CNBr out of funnel 290 and tube 210.

[0046] Inside the vessel 110, the resin beads are agitated by an agitator 410, which keeps them moving across the screen 310. A side cross-sectional view of the agitator is shown in FIG. 4A. The agitator includes a rotor 420, which is a vertical rod attached to an agitation motor (not shown). The agitator also includes a blade 440 attached to the rod at a hub 445. In FIG. 4A, the blade 440 is shown in cross-section. An upside-down perspective view of the agitator is shown in FIG. 4B, providing a clearer view of the configuration of the blade 440. The blade 440 rotates and lifts the resin beads away from the screen 310. The agitator also includes a circular frame 450 that circumscribes the outermost point of the blade 440, which further includes spokes 460 for additional support. Referring again to FIG. 4A, the bottom edge of the frame 450 is configured to be parallel to a screen (not shown) that is positioned directly below the agitator 410 .

[0047] The blade 440 shown in FIG. 4 has a width of 3.0 inches, a thickness of 0.25 inches, and a length of 28.0 inches. However, the dimensions of the blade 440 may vary for different uses. In some embodiments, the agitator 410 may include more than two blades. As shown, each blade 440 has a pitch of 40 degrees relative to the horizontal. In different embodiments, the blades may have different pitch angles, such as 10 degrees, 20 degrees, 30 degrees, 50 degrees, 60 degrees, 70 degrees, 80 degrees, and the like. The blade 440 may have rounded edges 449, as shown in FIG. 4C, which help prevent breakage of the beads. The agitator may be made of stainless steel or another resilient material. In the embodiment shown in FIGS. 4A-C, the agitator is configured to rotate clockwise so that the blades create an upward lift force in the fluid in the vessel (rather than pushing the fluid downward into the screen).

[0048] The agitator's rotation speed can be adjusted based on the type of resin beads being used and the amount of mixing required. The agitator must provide enough lift to keep the suspended beads moving across the screen and rapidly mix them while avoiding breaking or crushing the beads. Because different beads settle at different rates, the agitator speed should be adjustable to account for different types of beads. For example, CAPTO® beads settle more rapidly than CL-4B or CL-2B beads. In various embodiments, the agitator may rotate at up to 60 RPM. A preferred agitation speed is 35 RPM. Another preferred agitation speed is 20 RPM. The speed may be determined based on the type of bead, the type of liquid, the temperature of the liquid, and the length of incubation time. Based on these factors, the agitator is designed to thoroughly mix the resin during the resin activation process and the antibody conjugation process. This allows for proper mixing for chemical dispersion and avoids crushing / breaking the resin beads during the conjugation process. The agitator also increases resin recovery as it prevents the resin from sticking to the screen.

[0049] In some embodiments, the two blades 440 of the agitator are angled in opposite directions to provide dynamic resin agitation and suspension in the solution inside the vessel. In other words, one blade is angled downward to smack the resin downward onto the screen, while the other is angled upward to create a lift force. In other embodiments, both agitator blades are angled in the same direction. The agitator is configured to create a flow pattern as the agitator is rotated that causes thorough mixing and suspension of the resin slurry.

[0050] During the resin activation process, the dispersion tube 210 described above can be used to distribute the activation mixture over the beads while they are moving. According to various methods, buffers and other fluids can be pumped through the vessel during the activation, washing, and binding processes. The fluids pass through the screen and drain out through an outlet at the bottom of the vessel, while the agitator 410 keeps the beads moving above the screen.

[0051] The screen shown in Figures 1A-C has a 30-inch diameter with a 30 μm mesh pore size to retain resin and allow different solutions and buffers to filter through the mesh during the binding process. The mesh may have a pore size as small as about 5 μm and up to about 80 μm. In either case, the pore size should be small enough that beads cannot pass through the screen. The screen may be a single mesh screen, or it may be two, three, or more sintered layers. Topmesh TM3-BM30 is a mesh compatible with the present invention supplied by G. BOPP USA, INC. (Wappingers Falls, NY).

[0052] In one embodiment, the top layer is a 30 μm filtration mesh sintered with a 0.850 mm square mesh and a 2.0 mm square mesh for support. The screen is welded to a stainless steel 30-inch frame. The screen may be held in place within the container by a gasket, which may be removable from the screen.

[0053] As discussed above, the agitator is designed to thoroughly mix the resin while preventing it from breaking down, which would clog the screen and prevent efficient resin recovery. However, the screen is removable so that it can be cleaned or replaced after normal wear and tear. The bond activation process requires rapid buffer exchange, on the order of minutes, and screen blockage can lead to a slower buffer exchange rate and therefore cause process incompatibility and potential rejection of the lot.

[0054] The screen is resilient and resistant to tearing or breakage, preventing resin from escaping through the screen. Additionally, the screen structure is strong enough not to sag or bend under the weight of the resin. Sagging can lead to non-uniform mixing or insufficient buffer flow through the device, causing the mesh to block. Sagging can also block the release valve 130 for the container 110 located a short distance below the level of the screen. In some embodiments, perforated support plates may be positioned between layers for added rigidity and durability. Additionally, or alternatively, the screen may include a stainless steel support beam positioned directly below the screen to support the center. The inclusion of a support structure helps prevent non-uniformity in the distribution of beads across the screen. Non-uniform distribution would lead to resin clumping and changes in the chemistry and leaching properties of the resin. (Cited by reference)

[0055] Any and all references and citations made throughout this disclosure to patents, patent applications, patent publications, journals, books, articles, and other documents, such as web content, are incorporated herein by reference in their entirety for all purposes. (Equivalent)

[0056] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics, and the foregoing embodiments are therefore to be considered in all respects illustrative and not restrictive of the invention described herein.

Claims

1. 1. A method for activating resin beads, the method comprising: Inserting resin beads suspended in water into a mixing vessel comprising a dispersing device, a mesh screen having holes smaller than the resin beads, and an agitator disposed above the mesh screen, the mixing vessel being divided into upper and lower sections by the mesh screen extending across the mixing vessel, the agitator being disposed in the upper section, and the dispersing device being disposed above the agitator; uniformly dispersing and supplying an activation solution comprising CNBr and acetonitrile onto the resin beads through the dispersing device, the dispersing device including an elongated tubular structure forming a lumen, the elongated tubular structure including a plurality of downwardly facing holes; agitating the agitator for less than 5 minutes, the agitator being configured to rotate to impart a force on fluid in the mixing vessel, thereby providing lift and keeping the beads moving across the mesh screen; draining the activation solution through the mesh screen, thereby leaving activated resin beads supported on the mesh screen; A method comprising:

2. The method of claim 1 , wherein the method further comprises washing the activated resin beads with a fluid.

3. 3. The method of claim 2, wherein washing comprises filling the mixing vessel with the fluid, stirring and incubating, and draining the fluid from the mixing vessel.

4. 3. The method of claim 2, wherein the fluid is water or a solution containing sodium bicarbonate and sodium chloride.

5. 10. The method of claim 1, wherein the stirring is performed at between 10 and 40 RPM.

6. 6. The method of claim 5, wherein the stirring is performed at 20 RPM.

7. 6. The method of claim 5, wherein the stirring is performed at 35 RPM.

8. 10. The method of claim 1, wherein the agitator agitates for less than four minutes.

9. 9. The method of claim 8, wherein the agitation is for less than 3 minutes.

10. The method of claim 1 , wherein the resin beads comprise agarose.

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