AUTOMATED DEVICE AND METHOD FOR PURIFYING BIOMATERIALS FROM A MIXTURE BY USING MAGNETIC PARTICLES AND DISPOSABLE PRODUCT CONTACT MATERIALS - Patent application

Abstract

The present invention relates to a purification device and method for using the device that separates a substance of interest from contaminants using non-porous magnetic particles that contact the substance of interest and single-use or disposable materials. The process includes multiple cycles of a single batch to reduce the cost of the magnetic particles. The method can be run in a fully automated manner with a controller managing the various inputs and outputs of the system hardware.

Description

[Technical field]

[0001] [Priority Declaration] This application claims priority to U.S. patent application Ser. No. 17 / 163,472, filed Jan. 31, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to methods and systems used to separate macromolecules, including cells, cellular components, proteins, lipids, carbohydrates, and biological materials such as tissue, from mixtures. [Background technology]

[0003] Biopharmaceuticals are revolutionizing healthcare worldwide. Biopharmaceuticals are generally complex molecules or particles compared to traditional chemical-based simple molecular drugs. Many of the biopharmaceuticals are recombinant proteins such as antibodies because they can exhibit very high affinity for therapeutic targets, fewer side effects, lower immunogenic rejection, and long life spans. Some other biopharmaceuticals include viruses, bacteria, whole cells or parts of cells (e.g., exosomes), carbohydrates, lipids, DNA, or RNA.

[0004] Biopharmaceuticals are typically more expensive to manufacture than chemical-based drugs, with much of the manufacturing cost coming from complex downstream purification processes (MAbs. 2009 Sep-Oct;1(5):443-452).

[0005] Recombinant proteins can be produced using a variety of host expression systems, including bacteria, yeast, and mammalian cells. Recombinant antibodies are generally produced in mammalian cells, since some other host systems lack the sophisticated post-translational machinery required for the production of functional antibodies.

[0006] Proteins are complex and therefore recombinantly produced in a host organism, either intracellularly (e.g., bacterial host expression systems) or by extracellular secretion (e.g., yeast and mammalian expression systems). When mammalian expression systems are used to produce proteins, the cell culture harvest is clarified by separating the cells from the supernatant using centrifugation and / or filtration techniques. Harvest clarification requires expensive equipment and / or single-use filter modules, large manufacturing floor space, large amounts of buffers, and expensive utility costs. Harvest clarification also requires at least an extra day of processing time. Increasing cell density in culture can improve productivity and reduce the overall cost of therapeutics. At the same time, increasing cell density poses several challenges for traditional harvest clarification techniques. Disc stack centrifuges are commonly used in harvest clarification to clarify cell cultures and discharge unwanted cells in the form of a slurry. Although some of the product is lost with the discharged slurry during this process, the amount of discharge cycles is limited, so it does not significantly affect the overall product yield when cell densities are low. Increasing cell concentration also increases product loss due to the increased number of discharge cycles with increasing cell density. In addition, disc stack centrifuges exert high shear forces on the cells, causing the release of host cell proteins and proteolytic enzymes in the clarified harvest (Subramanian, G. (2014). Continuous Processing in Pharmaceutical Manufacturing.: Wiley‐VCH Verlag GmbH&Co. KGaA). This not only poses challenges for downstream purification but also negatively impacts product quality. Also, filtration-based techniques are impractical for high cell density cultures as they require very large systems and surface areas, as well as cause significant product loss and high levels of host cell protein contamination (Subramanian, G. (2014). Continuous Processing in Pharmaceutical Manufacturing.: Wiley‐VCH Verlag GmbH&Co. KGaA).

[0007] High cell density poses challenges not only for upstream processes but also for downstream processes, as purification requires very large chromatography columns, making removal of host cell proteins difficult. Current chromatography techniques rely on porous beads that create a large chromatographic surface area in a relatively smaller volume. Because the pore size of the beads is very small, the penetration of the molecules to be purified requires significant diffusion time, which increases the processing time. In chromatography columns packed with such beads, the small pore channels of the beads increase the flow resistance, which also requires high pressure delivery of liquid. In addition, because of the smaller pore size, some of the non-specific material is retained after recovery of the product of interest, thus necessitating purification and regeneration steps in each cycle. This not only increases the processing time, but also requires large amounts of additional buffer for washing and regeneration of the chromatography resin. If the affinity ligand coupled to the resin is sensitive to the purification agent, the chromatography material can only be used for one cycle. Also, therapeutic or vaccine macromolecules (e.g., viruses, bacteria, whole cells, or exosomes) that have a size larger than the pores of the chromatography resin cannot be purified using current chromatography processes.

[0008] Regarding the materials used in chromatography systems, most systems use stainless steel equipment that requires inter-batch cleaning to reduce cross contamination. Cleaning validation of the system is not only expensive but also causes significant delays between batches. In addition, the cleaning process requires a large infrastructure and the equipment is hard-piped and cannot be moved once installed. Single-use systems have gained attention in recent years due to their low capital requirements, flexibility, reduced turnaround time, no risk of cross contamination between batches, and environmental friendliness. Although most single-use chromatography systems use disposable product contact surfaces, columns are still reused for multiple batches due to their high cost. Simulated moving bed technology attempts to make columns disposable by increasing the number of cycles at the expense of additional processing time. However, all such systems use porous chromatography media / resins, and the challenge of porous chromatography resins remains.

[0009] Purified and unpurified biomaterials derived from body fluids are also valuable and are used for many different purposes. For example, immunoglobulins purified from donated human plasma (intravenous immunoglobulin or IVIG) have been used to treat several diseases, including life-threatening sepsis. Antihemophilic factor VIII purified from donated human plasma has been used to treat hemophilia in factor VIII-deficient patients. Immunoglobulins purified from animals immunized with venom have been used to treat poisoning from venomous snakebites. During pathogen epidemics, in the absence of vaccines or therapeutics, convalescent serum from recovered healthy donors can be used as a therapeutic agent to treat critically ill patients or as a preventative treatment for at-risk populations (Casadevall A, Scharff MD. Return to the past: the case for antibody-based therapies in infectious diseases. Clin Infect Dis. 1995; 21(1): 150-161; Nat Rev Microbiol. 2004; 2(9): 695-703).

[0010] The use of convalescent serum has several drawbacks. Generally, recently recovered donors are unable to donate large amounts of serum and may be prone to hypovolemic shock. In addition, donated unpurified serum contains many biological materials (including infectious agents) other than therapeutic immunoglobulins, which may be potentially harmful to the critically ill patient who receives it (Gajic O, et al. Transfusion-related acute lung injury in the critically ill: prospective nested case-control study. Am J Respir Crit Care Med. 2007; 176(9): 886-891). For example, viruses may be unintentionally transmitted from the donor's serum to the recipient. Serum-induced immune reactions, such as serum sickness, are also common (medlineplus.gov / ency / article / 000820.htm).

[0011] In serum donations, each donor cannot donate more than a certain amount (unless sacrificed), so the amount of the biomolecule of interest collected from each donor is limited. In many applications of plasma (including factor VIII, IVIG, and antitoxins), plasma from several donors is shipped to a processing facility, pooled, and the biomaterial of interest is isolated. This process typically takes months to complete, during which time the pathogen may mutate and render the purified therapeutic ineffective if the goal is to combat a novel pathogen during an epidemic or pandemic. Summary of the Invention

[0012] The device, product, and method described herein solves many problems associated with biopharmaceutical purification processes. The method significantly reduces total processing time and simplifies downstream processes by eliminating or reducing the need for pre-chromatographic clarification. All product contact surfaces are suitable for biopharmaceutical manufacturing (e.g., meet USP Class VI requirements) and are replaced after each batch, but can support multiple cycles of processing within the same batch. The methods described herein use particles that are attracted to magnets (ferromagnetic, paramagnetic, or superparamagnetic), have a small size and therefore a large surface area to volume ratio (per unit volume), are preferably non-porous, and have specific binding properties. Such particles may be various forms of iron or cobalt oxides (e.g., Fe3O4) or mixtures thereof. The surface to area ratio per unit volume is greater than 2, e.g., greater than 3, greater than 4, greater than 4.8, or greater than 5, 10, 15, 20, or more. The magnetic core of the particle is rigid and non-porous, but the tethered ligands or linkers can create a porous structure around the particle. The volume of this porous structure is smaller than the volume of the rigid core of the particle. In some embodiments, the volume of the porous structure is less than 90 percent of the volume of the rigid core of the particle. In some embodiments, the volume of the porous structure is less than 80, 70, 60, or 50 percent of the volume of the rigid core of the particle. Particles with a porous structure volume that is less than 90 percent of the volume of the rigid core of the particle are considered to be "nearly non-porous." Such particles exhibit very short binding residence times because most of the binding sites are on the surface. The diameter or side of such particles is 10 to 10,000 nm, which is up to 20,000 times smaller than standard porous chromatography resin beads. In some embodiments, the size is less than 1000 nm, for example, less than 900, 800, 700, 600, 500, or 400 nm. In some embodiments, the size is in the range of 10 to 5,000 nm, for example, in the range of 50 to 1,000, 50 to 800, 100 to 500, or 200 to 400 nm.In some embodiments, the particles are non-spherical. In some embodiments, the particles are cubic or tetrahedral in shape. The surface of the particles is modified to have properties such as affinity for a particular type of molecule (e.g., modification with protein A ligand in the case of antibody purification, or modification with an antibody that specifically binds to the biomaterial), positive or negative charge, hydrophobic interaction, or a combination of two or more of these properties. Such particles may be referred to as magnetic particles or beads in the present invention. The biomolecule or biomaterial to be purified from the crude mixture may be an antibody, a protein, a nucleic acid (e.g., DNA and RNA), a cellular organelle, a virus, a viral vector (e.g., adeno-associated virus, lentivirus, poxvirus, etc.), a cell, an exosome, a bacterium, a yeast, a pathogen, or another organic entity.

[0013] The particles of the invention are mixed with the starting material, which may be a cell culture, partially clarified broth, blood or other body fluid, or other biomolecule-containing mixture. An external magnetic field is applied to recover such particles from the mixture. If the magnetic particles have low magnetic susceptibility due to their small size, a high gradient magnetic field is used to isolate the particles mixed with the mixture. A high gradient magnetic field is generated by applying a magnetic field to an array of magnetically soft metal (e.g., steel wool). This results in a local high magnetic field around the soft metal, allowing the separation of particles that would otherwise be weakly attracted to the magnet. Under challenging test conditions of a mixture of Fe3O4 particles (300 nm, cubic shape) and a high concentration of Saccharomyces cerevisiae in a medium, >99% of the particles were captured after application of a high gradient magnetic field generated by a magnetic field ranging from 0.1 to 1.7 Tesla. Small-scale batch chromatography methods for purifying proteins (microgram or nanogram scale) using magnetic beads are currently available, but most of these methods use porous magnetic beads that are not reused and are manually manipulated. Commercially available magnetic particles are spherical with diameters equal to or greater than 1 micron and are only used in a single cycle at a very small scale (e.g., Sera-Mag from GE Healthcare Life Sciences and Dynabeads from ThermoFisher Scientific). These particles are not well suited for the present invention due to their porosity, large size, and reusability.

[0014] The magnetic particles of the present invention have a high volume to surface area ratio. For example, the volume to surface area ratio of tetrahedral and cubic particles is at least 25% greater than that of spherical particles. The attachment of the ligand to the surface of the magnetic particle can be achieved directly with the ligand or by a linker that can bind to the inorganic surface of the ligand and the magnetic particle. In one embodiment, a linker with a silane group is used to chemically bind to the magnetic particle. The other end of the linker is selected so that it can be easily bound to the ligand used for purification. For example, the epoxide group at the other end of the linker can react with primary amine groups, sulfhydryl groups, or carboxyl groups that are commonly found in various amino acids of proteins. 3-glycidoxypropyltrimethoxysilane or 3-glycidoxypropyltriethoxysilane are two examples of such linkers. In our studies, 0.5-3% wt / wt treatment with 3-glycidoxypropyltrimethoxysilane resulted in excellent conjugation to Fe3O4 cubic particles with an average size of 300 nm.

[0015] The ligand may be a protein (e.g., antibody, single chain antibody, affinity protein, polylysine, polyarginine), carbohydrate (e.g., heparin), matrix (e.g., natural secreted polymer), polynucleotide (e.g., oligo dT), nucleic acid (e.g., cDNA, primer), or a molecule with strong affinity for a particular macromolecule or particle (e.g., primary, secondary, and tertiary amine groups, sulfonic acid groups).

[0016] In affinity chromatography, macromolecules of interest are bound to magnetic particles that have surface affinity for such macromolecules. The collected magnetic particles are washed multiple times to remove impurities, and then elution is performed by introducing an elution buffer that dissociates the binding of the macromolecules of interest from the magnetic particles. In one embodiment, a low pH buffer is used to dissociate the antibodies captured by the Protein A-coupled magnetic particles. Similarly, imidazole is used to elute histidine-tagged proteins bound to Ni-NTA particles, and a high salt buffer is used to remove charged proteins bound to ion-exchange particles. The eluted macromolecules pass through, while the magnetic particles are retained by an external magnetic field. If the affinity performance of the particles decreases after a certain number of cycles due to non-specific blockage of specific binding sites by impurities, the particles can be regenerated using a regeneration solution (e.g., NaOH, or chaotropic agents such as urea or guanidine).

[0017] In flow-through chromatography, impurities are reduced by binding the impurities to magnetic particles and allowing the macromolecules of interest to pass through. The surface of the magnetic particles is modified to have affinity for the impurities but not for the macromolecules of interest. Such impurities can be DNA, host cell proteins, or other unwanted macromolecules. In one embodiment, positively charged quaternary ammonium magnetic particles can be used to remove contaminating negatively charged DNA from the proteins of interest. The contaminating impurities bind to the magnetic particles, while the macromolecules of interest pass through and are collected. After each cycle, the magnetic particles are regenerated to remove bound contaminants so that they can be reused in subsequent cycles.

[0018] These and other aspects of the invention are set forth in greater detail in the description of the invention below. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows an example of an affinity purification method. [Diagram 2]FIG. 1 shows an overview of the process for purifying immunoglobulins from human blood. [Diagram 3] FIG. 1 shows an example of purifying biological material from a body fluid. [Figure 4A] FIG. 1 shows the dynamic binding capacity of protein A coupled magnetic particles and purified polyclonal IgG. [Figure 4B] FIG. 1 shows the dynamic binding capacity of protein A-coupled magnetic particles and polyclonal IgG derived from whole blood. [Diagram 5] FIG. 1 illustrates an example of a chamber having rigid and flexible parts. [Figure 6A] FIG. 1 shows the binding kinetics of Protein A coupled magnetic particles. [Figure 6B] FIG. 1 shows the reusability of Protein A coupled magnetic particles. [Figure 7] FIG. 1 illustrates an example of a chamber having multiple magnet cavities. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Binding of macromolecules or impurities (in flow-through mode) is performed in a mixing vessel where the magnetic particles are mixed with a starting solution (e.g., cell culture or partially clarified cell culture) containing a mixed population of macromolecules and impurities. Mixing is performed to improve the binding kinetics between the target molecules and the magnetic particles. Mixing can be performed by using standard mixing techniques or by recirculation of the fluid via a single pump or multiple pumps (e.g., P1 in Figure 1). When a pump is used for mixing, the material moves through the separation chamber (Figure 1). The mixing caused by the pump can be enhanced by intermittently reversing the flow, by using very high flow rates, or by starting the mixing before removing the magnetic field. After binding of the macromolecules to the magnetic particles is completed, a magnetic field is introduced by switching on an electromagnet or by physically bringing a permanent magnet into the separation chamber. To capture small particles with low magnetic susceptibility, a high gradient magnetic field can be generated by using ferromagnetic stainless steel wool or other ferromagnetic support material in the separation chamber. Multiple permanent magnets can also be used to improve the retention efficiency of the magnetic particles. In one embodiment, a series of magnets are placed (or a magnetic field is applied) near the inlet to capture the incoming magnetic particles, and another series of magnets are placed (or a magnetic field is applied) near the outlet to capture any remaining particles not captured by the magnets placed near the inlet. The magnetic particles with bound target molecules are retained, while the remaining material is pumped out of the system to waste. The retention efficiency of the magnetic particles can be increased again by using standard mixing techniques (such as a rotating impeller) or by simply recirculating the fluid through a single pump or multiple pumps. Mixing also not only improves the retention efficiency, but also provides homogeneous capture of the magnetic particles on the ferromagnetic support material. Without mixing, the magnetic particles may be prone to clogging the ferromagnetic support material and may cause overpressure. Since increasing mixing would prevent complete capture of the particles, the mixing can be gradually reduced to completely capture the magnetic particles.If an overpressure is detected by the pressure sensor, a fluid path is created (e.g., V3 is opened and the remaining valves are closed) and the liquid in the separation chamber is recirculated by a pump (e.g., P1) without applying a magnetic field. A gradually increasing or constant magnetic field can then be applied so that the particles are homogeneously trapped in the separation chamber.

[0021] Magnetic particles may require resuspension during initial binding and washing. Particle resuspension is achieved by turning off the magnetic field and recirculating the liquid at high flow rates with a pump (between the inlet and outlet of the separation chamber) or by mixing in the separation chamber using standard rotating, reciprocating, or oscillating impellers. The shape and material of the chamber affect particle resuspension.

[0022] In affinity purification, the bound particles are washed either with or without magnetic force to remove any non-specifically bound impurities. The bound macromolecules are eluted from the magnetic particles by introducing an elution buffer into the separation chamber with or without an external magnetic force. If the binding performance of the particles decreases after multiple cycles, the particles may be purified using a purification buffer (e.g., sodium hydroxide, urea, or guanidine hydrochloride). The entire cycle can be repeated multiple times until the batch is fully purified.

[0023] In flow-through purification, the macromolecules of interest (e.g., recombinant proteins) do not bind to the magnetic particles and pass through, while impurities (e.g., DNA) are captured by the magnetic particles, which can be regenerated using purification buffer for additional cycles.

[0024] One advantage of using non-porous magnetic beads is that, unlike porous chromatography resins, impurities do not clog the pores and significantly less purification is required. Porous chromatography media requires purification with high concentrations of sodium hydroxide (e.g., 0.5 M) after every cycle. 50 purification cycles were performed to isolate immunoglobulins from human whole blood using Protein A-coupled magnetic particles. Purification with a low sodium hydroxide concentration of 0.1 N was performed after every 5 cycles. The results (Figure 6B) show that the binding capacity of the magnetic particles did not change significantly over the 50 cycles. In each cycle, a specific volume of starting material (from the batch) is pumped into the system. This is called the cycle volume and is calculated based on the amount of magnetic particles loaded into the system, the estimated amount of material to be purified (product or impurity), the desired number of cycles or total processing time, and the reusability of the magnetic particles. For example, if a 2000 L batch with an antibody concentration of 5 g / L is affinity purified using Protein A magnetic particles with a capacity of 50 g / L (grams of antibody captured per liter of magnetic particles), a single cycle would require 200 L of magnetic particles. On the other hand, if the magnetic particles are reused for 50 cycles, only 4 L of magnetic particles would be needed. Although the time required to run 50 cycles is roughly 50 times longer than running a single cycle, the savings realized by using less magnetic particles and the smaller size of the equipment far outweigh the longer processing time. If a single cycle takes 10 minutes, then 50 cycles will take a little over 8 hours. This is a reasonable processing time for a 2000 L batch, as traditional methods require 2-4 days of processing.

[0025] Depending on the binding affinity of the ligand and the concentration of the molecule to be purified, the chromatography media can show a maximum binding capacity when the concentration of the molecule to be purified is much higher than the binding capacity of the time media. In this case, the dynamic binding capacity (when most of the molecules are bound and some are not) will be lower than the maximum binding capacity. The inventors' results with purified polyclonal human IgG show that there was no difference between the maximum binding capacity and the dynamic binding capacity of magnetic particles coupled with protein A (Figure 4A). However, the results with male human blood show that the maximum binding capacity was above 50 g / L (grams of purified antibody per liter of magnetic particles), while the dynamic binding capacity was about 40 g / L (Figure 4B).

[0026] With reference to FIG. 5, for the separation chamber 10, the shape has an inlet 12 and an outlet 14, which are designed to be separate or spaced apart from each other (e.g., positioned on opposite sides of the chamber). The spaced inlet and outlet allow for maximizing the residence time of the material entering the chamber within the chamber. The inner diameter of the tube is large enough (e.g., greater than 0.125 inches in diameter) to prevent the magnetic particles from clogging before being fully suspended. In one embodiment, a cylindrical separation chamber with a conical bottom (apex at the bottom) is used to isolate the magnetic particles in different parts of the process. In another embodiment, the top of the separation chamber is conical (apex at the top). A fully cylindrical chamber can also be used. Another alternative is a rectangular shaped separation chamber, which can improve mixing and result in better resuspension of the particles. The separation chamber is made of a material that is less attracted to magnetic forces (e.g., LDPE, high durometer silicone, polypropylene, polystyrene, EVA, PVC, etc.). The chamber may be made of rigid or flexible materials or a combination of both (Figure 5). If the entire separation chamber is constructed of rigid materials and the magnetic field is removed from the separation chamber, the magnetic particles tend to clump together and stick to the walls even after strong mixing. This is problematic because it leads to incomplete cleaning, resulting in an increase in impurities in the eluate, and reduced media capacity. If the outer shell or body 16 of the separation chamber 10 is rigid (e.g., polycarbonate or polypropylene) and the shell (magnet cavity) 18 that holds the magnet is flexible (e.g., silicone), experimental results show that the concentrated magnetic particles mix more quickly after removal of the magnetic field. In addition, results show that the separation of the magnetic particles from the fluid is much faster. The increase in separation kinetics is due to the flexible material being able to protrude into the chamber, resulting in an increase in the exposed magnetic surface area to which the particles are attracted, and the flexible material between the particles and the magnet follows the shape of the magnet to result in a higher magnetic field. The magnet cavity is shaped as a sleeve or cover for a magnet (temporary or permanent) (see, for example, M in Figures 1 and 3).The chamber may include steel wool or wire mesh 20 to enhance the magnetic field by high gradient field generation to capture small and weakly magnetic particles. In one embodiment, the material of the steel wool or wire mesh (1 micron to 1000 microns) is ferromagnetic stainless steel (e.g., 430 or 410 stainless steel) to provide corrosion resistance. The ferromagnetic steel wool can also be coated with metal (e.g., nickel or chromium) or plastic to significantly reduce or eliminate corrosion. The steel wool or wire mesh can be placed anywhere in the chamber, preferably around one or more magnet cavities. When scaling up the process, the chamber can be appropriately scaled up, or multiple chambers can be used in parallel, or multiple magnet cavities can be added to an appropriately scaled up chamber (Figure 7). For example, if 50 mL of magnetic chromatography media is used in one chamber with a single magnet cavity for purification from 10 L of starting material, a scale-up process for purifying 40 L of material may include a chamber four times larger in volume with one large magnet cavity, or four chambers connected in parallel, or a chamber four times larger in volume with four magnet cavities (FIG. 7). The separation chambers of FIG. 5 or FIG. 7 can be used in the systems of FIG. 1-FIG. 3 described below.

[0027] An external magnetic field, typically in the range of 0.1 to 1.7 Tesla, is applied to the separation chamber either by moving a permanent magnet nearby, or by turning on an electromagnet surrounding the chamber, or by other means. Depending on the needs of the purification process, a controller controls the magnetic force applied to the separation chamber by turning it on or off, or by reducing or increasing its strength.

[0028] In one embodiment, the separation efficiency and throughput of the process is increased by arranging multiple separation chambers in parallel and / or in series. With multiple separation chambers, the processing flow rate increases significantly due to parallel processing. Arranging separation chambers in series significantly improves particle retention at higher flow rates. Arranging separation chambers in series has another potential advantage that less amount of chromatographic magnetic particles can be used for separation if the particles have a maximum binding capacity much higher than the dynamic binding capacity. An excess amount of starting material is loaded into the first chamber with an amount of chromatographic medium calculated based on its maximum binding capacity. This allows some macromolecules to remain unbound, and these macromolecules will move to the second chamber in series. The second chamber contains an amount of chromatographic medium calculated based on the dynamic binding capacity, so that almost all of the remaining unbound macromolecules will be bound. This will result in an increase in the amount of purified material per unit of chromatographic medium without any significant addition to purification time.

[0029] The complete disposable manifold, including tubing, separation chambers (with or without steel wool), mixing bag, pH / conductivity flow cell, optical density and absorbance measurement flow cell, and filter, is pre-assembled and generally sterilized before use and is capable of withstanding multiple cycles of batches. Tubing or pipes with single-use product contact surfaces are used to circulate the various fluids through the system. For example, flexible silicone, C-flex, or PVC tubing can be used to connect the various parts of the system to conduct the fluids. The disposable manifold is connected by connectors or welding of thermoplastic tubing to bags or containers containing the magnetic particle slurry, various buffers, starting materials (e.g., bioreactor), and final purified materials. The connectors can be sterile or non-sterile.

[0030] Product contact surfaces that come into contact with the substance of interest include tubing, connectors, all flow cells (pH / conductivity, OD, etc.), pressure sensors, and the inside surfaces of the separation chamber. Product contact surfaces also include the outer surfaces of particles.

[0031] A controller with an HMI (human machine interface) is used by a user to automatically or manually control and receive information from multiple parts of the system, including pumps (speed, direction, start / stop, feedback, etc.), valves (open, close, feedback), occlusion / pressure sensors, optical density and absorbance detectors, air bubble sensors, in-line pH / conductivity meters, temperature sensors, and magnetic field control systems. This information is provided as analog or digital signals to and from the controller. Commercial automation systems from various vendors, such as Siemens, Backhoff, or Allen-Bradley, are available as complete solutions including controllers, input / output digital or analog cards, and HMI. Examples include the Siemens SIMATIC S7-1200 kit, the Beckhoff TwinCAT3 automation platform, and Delta V.

[0032] The user inputs various process parameters into a process screen that is part of the HMI. The process parameters include cycle volumes, incubation times, mixing times, washing times, and recirculation times, pH, conductivity, and optical density and absorbance values ​​to start or stop a step. When the user initiates a process by providing input to the HMI, the controller can follow the process screen and automatically execute the process using the input process parameters. A number of valves (FIG. 1, V1-V10) are controlled to form various fluid paths at various aspects of the purification process. In one embodiment, pinch valves are used to open and close fluid paths in flexible tubing.

[0033] The present invention will now be described in more detail with reference to the accompanying drawings, in which some embodiments of the invention are shown. However, the present invention may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0034] Like numbers refer to like components throughout. In the drawings, certain line thicknesses, layers, components, elements or features may be exaggerated for clarity.

[0035] The terms used herein are merely for the purpose of describing particular embodiments and are not intended to limit the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. It will be further understood that the terms "includes", "include", "including", "comprises" and / or "comprising" as used herein specify the presence of stated features, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0036] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the present specification and related art, and should not be interpreted in an idealized or overly formal sense unless expressly defined as such in the present specification. Well-known functions or structures may not be described in detail for brevity and / or clarity.

[0037] When an element is referred to as "on," "attached," "connected," "coupled," "in contact," etc., with another element, it will be understood that it may be directly on, attached, connected, coupled, or in contact with the other element, or that intervening elements may be present. In contrast, when an element is referred to, for example, as "directly on," "directly attached," "directly connected," "directly coupled," or "in direct contact with" another element, there are no intervening elements present. Additionally, one of ordinary skill in the art will understand that reference to a structure or feature that is located "adjacent" to another feature may have overlapping or underlying portions with the adjacent feature.

[0038] The fluid may be a liquid or air, although the embodiments using a liquid are also applicable to air.

[0039] Having described the invention, the invention will be described in further detail in the following examples, which are included herein for illustrative purposes only and are not intended to limit the invention.

[0040] The scheme shown in Figure 1 is an example for introducing various fluids at various process steps during the purification process. Two examples of automated methods for purifying biopharmaceuticals and an example of an automated method for purifying from body fluids are described below. In affinity purification methods, the substance of interest binds to particles, whereas in flow-through purification methods, the substance of interest passes through and the substance bound to the particles is generally discarded. EXAMPLES

[0041] [Example 1] Affinity purification Affinity purification methods can be used to purify various macromolecules or particles from a mixture. Magnetic particles can successfully separate viruses, viral vectors (e.g., lentivirus, AAV, poxvirus). In this process, magnetic particles coupled to negatively charged matrices or molecules (e.g., quaternary or tertiary ammonium, primary amines, polyamines, polylysine, polyarginine) or specific affinity ligands (e.g., proteins, antibodies, antibody fragments, single-chain antibodies) are used to isolate viruses or viral vectors from cell cultures or other starting materials. This process does not require the removal of cells from the culture prior to purification. Protein A-coupled magnetic particles were used to purify monoclonal antibodies (mAbs) from CHO cell cultures. Results show that the mAbs are 100% recovered and have a similar SDS-PAGE profile to commercially available purified human antibodies.

[0042] Affinity purification methods are also used to purify nucleic acids. In one embodiment, magnetic particles coupled to oligo-dT are used to purify mRNA with poly-A tail from reaction mixtures or crude extracts. Specific mRNA can be purified by magnetic particles coupled with either the complementary sequence of the mRNA or one or more complementary nucleotides together with oligo-dT. This method can be used to purify mRNA for vaccines in one step.

[0043] In FIG. 1, V1-V10 are valves, P1 indicates a bidirectional pump, F / R indicates the forward / reverse direction of the pump, OD / Abs indicates optical density / absorbance measurements or sensors, pH / cond indicates pH and conductivity sensors, BS1-4 indicate air bubble sensors, N or S indicates the polarity of the magnet M, and B1-B2 indicate bags or containers.

[0044] The various steps of the affinity purification process are detailed below. At the end of each step, the pumps are turned off and all valves are closed. All flexible bags or containers containing the various liquids are connected via tubing or other pathways as shown in Figure 1. The bag or container holding the magnetic particles is attached to a tube connected to valve V10. At this stage, no starting material is connected to the system.

[0045] Step 1. Loading of magnetic particles After loading the disposables into the system, valves V1 and V10 are opened, while the remaining valves remain closed. Pump P1 is turned on to move the magnetic particles in liquid suspension from the container or flexible bag B1 to the separation chamber. When air bubble sensor BS2 detects air (because container B1 is empty), pump P1 is stopped after the residual material in the tubing between container B1 and pump P1 enters the separation chamber. Valve V3 is then opened, valves V1 and V10 are closed, and pump P1 is turned on to recirculate the material in the separation chamber. At the same time, the magnetic force is turned on to capture the magnetic particles while recirculating the loop. The capture of the magnetic particles by the separation chamber can be monitored by measuring the particle-specific optical density and absorbance with an OD sensor (e.g., at 370 nm). The flow rate of pump P1 can be reduced to allow better capture of the magnetic particles. Once the magnetic particles have been captured, pump P1 is reversed, valve V3 is closed, and valves V1 and V10 are opened to fill container B1 with the liquid in which the magnetic particles were suspended. Bag B1 can be shaken manually or mechanically to suspend any magnetic particles that may remain in the bag. Once air bubble sensor BS2 detects an air bubble, pump P1 switches direction to forward, moving the liquid back to the separation chamber. Once air bubble sensor BS2 detects an air bubble, valve V10 is closed and valve V3 is opened to recirculate again in the loop until the optical density or absorbance is significantly reduced. The flow rate of pump P1 can be reduced to allow better capture of the magnetic particles. At this stage, the direction of pump P1 is reversed, valve V9 is opened, and the supernatant is discarded by closing valve V3. Pump P1 is stopped after air bubble sensor BS3 detects air.

[0046] Step 2. Washing magnetic particles using a magnetic field After closing valve V9, open valves V5 and V1 and introduce the equilibration buffer by running pump P1 in the forward direction until air bubble sensor BS1 detects liquid. The equilibration buffer is used to wash the particles and reduce non-specific binding to the particles. Examples of equilibration buffers are phosphate buffered saline (PBS) and Tris-based buffers. At this stage, open valve V3 and close valve V5. Washing progress can be assessed by monitoring data from the in-line pH / conductivity sensor pH / cond. Typically, within one minute of washing the magnetic particles in this way, reverse the direction of pump P1, open valve V9, and close valve V3. Once air bubble sensor BS3 detects air, briefly open valve V3 to allow residual liquid in the loop to empty. This washing process can be repeated one to two times.

[0047] Cleaning / Regeneration Step Whenever regeneration, purification or robust purification of the magnetic particles is required, a purification or purification buffer connected to valve V8 can be used. The magnetic field is turned off, valves V8 and V1 are opened, and the equilibration buffer is introduced by running pump P1 in the forward direction until the air bubble sensor BS1 detects liquid. At this stage, valve V3 is opened and valve V8 is closed. After recirculating the magnetic particles to create a mix, the magnetic field is turned on to collect the magnetic particles in the separation chamber. A reduction in the flow rate of the pump can allow for better capture of the magnetic particles. Once the optical density has been significantly reduced, the direction of pump P1 is reversed, valve V9 is opened, and valve V3 is closed. Once the air bubble sensor BS3 detects air, valve V3 is temporarily opened to allow any residual liquid in the loop to empty. Finally, pump P1 is stopped and all valves are closed. In general, the regeneration step is followed by step 5 or step 2 to remove any traces of regeneration buffer from the system.

[0048] Step 3. Binding of magnetic particles to macromolecules Bag B1 is removed and a starting material source is attached to the tubing connected to valve V10. The starting material source may be a bioreactor or bag or container containing starting material. Starting material is introduced into the system by opening valves V1 and V10, turning off the magnetic field, and running pump P1 in the forward direction until air bubble sensor BS1 detects liquid. At this stage, valve V1 is closed, valve V2 is opened, and pump P1 continues to run in the forward direction for a set time until the total volume of starting material loaded into the system is equal to the specified cycle volume. If mixing tank B2 is a rigid container, a vent with a 0.2 micron filter is attached to the top of the container to displace air with the incoming liquid. Once the set amount of cycle volume has been pumped, valve V4 is opened and valves V10 and V1 are closed to mix the magnetic beads with the starting material. The flow direction can be switched back and forth to improve mixing. Once the incubation time required for the macromolecules to bind to the beads is complete, the magnetic field is turned on and the magnetic beads are captured in the separation chamber. The flow rate of pump P1 can be reduced to allow better capture of the magnetic particles. Most of the beads can be captured by this recirculation process and the capture efficiency can be measured by the optical density sensor OD. The pump direction is then reversed to capture the remaining magnetic particles, valve V4 is closed and valve V9 is opened to discard the macromolecule-depleted starting material to waste. When air bubble sensor BS3 detects air, valve V2 is closed and valves V3 and V4 are opened for a few seconds to allow the residual liquid in the loop to empty.

[0049] Step 4. Washing magnetic particles using a magnetic field Follow step 2 above.

[0050] Step 5. Washing magnetic particles without a magnetic field Close valve V9, turn off the magnetic field, open valves V5 and V1, and introduce the equilibration buffer by running pump P1 in the forward direction until the air bubble sensor BS1 detects liquid. At this stage, open valve V3 and close valve V5. After recirculating the magnetic particles to create a mix, turn on the magnetic field to collect the magnetic particles in the separation chamber. The flow rate of pump P1 can be reduced to allow better capture of the magnetic particles. Once the OD370 or the optical density that specifically detects the particles is significantly reduced, reverse the direction of pump P1, open valve V9, and close valve V3. Once the air bubble sensor BS3 detects air, briefly open valve V3 to allow any residual liquid in the loop to empty. Repeat this washing process 1-2 times.

[0051] Step 6. Elution: Step 6.1: Close valve V9, open valves V6 and V1, and run pump P1 in the forward direction briefly to fill the tubing up to pump P1. Then close valve V6, open valve V9, and reverse pump P1. This step clears the tubing with elution buffer.

[0052] Step 6.2: To fill the separation chamber with elution buffer, turn off the magnetic field, open valve V6, and introduce elution buffer by operating pump P1 in the forward direction until the air bubble sensor BS1 detects liquid. At this stage, open valve V3 and close valve V6. After recirculating the magnetic particles to create a mix, turn on the magnetic field to collect the magnetic particles in the separation chamber. Reducing the flow rate of pump P1 can allow for better capture of the magnetic particles. Once the OD for detecting the magnetic particles is significantly reduced and the OD for detecting the eluted material (OD at 280 nm for proteins) is maximized, open valve V7, close valve V3, reverse the direction of the pump, and operate until the air bubble sensor BS4 detects air. To empty the residual liquid in the loop, briefly open valve V3 while keeping pump P1 operating, then close valves V3 and V7. This elution process from step 6.2 is repeated 1-2 times to collect the eluted molecules. If this process is repeated, the step of purging the tubing with elution buffer (step 6.1) is not necessary.

[0053] After step 6, repeat steps 2-6 until the end of the bioreactor is detected by air at the BS2 sensor. At this point, the final cycle has been completed by performing steps 2-6 and the disposable assembly can be discarded.

[0054] [Example 2] Flow-through purification method In FIG. 1, V1-V10 are valves, P1 indicates a bidirectional pump, F / R indicates the forward / reverse direction of the pump, OD / Abs indicates optical density / absorbance measurements or sensors, pH / cond indicates pH and conductivity sensors, BS1-4 indicate air bubble sensors, N or S indicates the polarity of the magnet M, and B1-B2 indicate bags or containers.

[0055] The various steps of the flow-through purification process are detailed below. At the end of each step, the pump and all valves are closed. All the flexible bags or containers containing the various liquids are connected via tubing or other pathways as shown in Figure 1. The bag or container holding the magnetic particles is attached to a tube connected to valve V10. At this stage, no starting material is connected to the system.

[0056] Step 1. Loading of magnetic particles After loading the disposables into the system, valves V1 and V10 are opened, while the remaining valves remain closed. Pump P1 is turned on to move the magnetic particles in liquid suspension from the container or flexible bag B1 to the separation chamber. When air bubble sensor BS2 detects air (because container B1 is empty), pump P1 is stopped after the residual material in the tubing between container B1 and pump P1 enters the separation chamber. Valve V3 is then opened, valves V1 and V10 are closed, and pump P1 is started to recirculate the material in the separation chamber. At the same time, the magnetic force is turned on to capture the magnetic particles while recirculating the loop. The capture of the magnetic particles by the separation chamber can be monitored by measuring the particle-specific optical density and absorbance with an OD sensor (e.g., at 370 nm). Once the magnetic particles are captured, pump P1 is reversed, valve V3 is closed, and valves V1 and V10 are opened to fill container B1 with the liquid in which the magnetic particles were suspended. Bag B1 can be manually shaken to suspend any magnetic particles that may remain in bag B1. If air bubble sensor BS2 detects an air bubble, switch the direction of pump P1 to forward and move the liquid back into the separation chamber. If air bubble sensor BS2 detects an air bubble, close valve V10, open valve V3 and recirculate again in the loop until the optical density or absorbance drops significantly. At this stage, discard the supernatant by reversing the pump direction, opening valve V9 and closing valve V3. If air bubble sensor BS3 detects air, stop pump P1.

[0057] Step 2. Washing magnetic particles using a magnetic field After closing valve V9, valves V5 and V1 are opened and the equilibration buffer is introduced by running pump P1 in the forward direction until air bubble sensor BS1 detects liquid. At this stage, valve V3 is opened and valve V5 is closed. Washing progress can be assessed by monitoring the data from the in-line pH / conductivity sensor pH / cond. Within one minute of washing the magnetic particles in this way, the pump direction is reversed, valve V9 is opened and valve V3 is closed. Once air bubble sensor BS3 detects air, valve V3 is briefly opened to allow any residual liquid in the loop to empty. This washing process can be repeated 1-2 times.

[0058] Cleaning / Regeneration Step Whenever regeneration, purification or robust purification of the magnetic particles is required, a purification or purification buffer connected to valve V8 can be used. The magnetic field is turned off, valves V8 and V1 are opened, and the equilibration buffer is introduced by running pump P1 in the forward direction until the air bubble sensor BS1 detects liquid. At this stage, valve V3 is opened and valve V8 is closed. After recirculating the magnetic particles to create a mix, the magnetic field is turned on to collect the magnetic particles in the separation chamber. The flow rate of pump P1 can be reduced to allow better capture of the magnetic particles. Once the optical density specific to the particles is significantly reduced, the pump direction is reversed, valve V9 is opened, and valve V3 is closed. Once the air bubble sensor BS3 detects air, valve V3 is temporarily opened to allow any residual liquid in the loop to empty. Finally, pump P1 is stopped and all valves are closed. In general, the regeneration step is followed by step 6 or step 2 to remove any traces of regeneration buffer from the system.

[0059] Step 3. Binding of magnetic particles with impurities Valve V10 is opened, the magnetic field is turned off, and the starting material is introduced into the system by running pump P1 in the forward direction for a set time while valve V2 is open. If the mixing tank B2 is a rigid vessel, a vent with a 0.2 micron filter is attached to the top of the vessel to displace air with the incoming liquid. Once a set amount of cycle volume equal to the cycle volume has been pumped, valve V4 is opened and valve V10 is closed to mix the magnetic beads with the starting material. Once the incubation time required for impurities to bind to the beads is complete, the magnetic field is turned on to capture the magnetic beads in the separation chamber. The flow rate of pump P1 is reduced to allow better capture of the magnetic particles. The majority of the beads can be captured by this recirculation process. The pump direction is then reversed, valve V4 is closed, and valve V7 is opened to capture the remaining magnetic particles and collect the purified material. Once air is detected by the air bubble sensor BS4, valve V2 is closed and V3 is opened temporarily to allow the remaining liquid in the loop to empty.

[0060] Step 4. Washing magnetic particles using a magnetic field A wash with equilibration buffer can be used to improve yield by recovering remaining starting material from the fluid path. This step may be omitted to reduce total processing time. Equilibration buffer is introduced by opening valves V5 and V1 and running pump P1 in the forward direction until air bubble sensor BS1 detects liquid. At this stage, valve V3 is opened and valve V5 is closed. Less than a minute after washing the magnetic particles in this way, the pump direction is reversed, valve V7 is opened and valve V3 is closed. When air bubble sensor BS4 detects air, valve V3 is briefly opened to allow residual liquid in the loop to empty.

[0061] Step 5. Cleansing / Regeneration Regenerate the magnetic particles by removing bound impurities by washing with a regeneration, purification, or cleansing buffer connected to valve V8. Turn off the magnetic field, open valves V8 and V1, and introduce the regeneration buffer by running pump P1 in the forward direction until air bubble sensor BS1 detects liquid. At this stage, open valve V3 and close valve V8. After recirculating the magnetic particles to create a mix, turn on the magnetic field to collect the magnetic particles in the separation chamber. Reducing the flow rate of pump P1 can allow better capture of the magnetic particles. Once the optical density (e.g., OD370) specific to the particles is significantly reduced, reverse the pump direction, open valve V9, and close valve V3. Once air bubble sensor BS3 detects air, open valve V3 momentarily to allow the residual liquid in the loop to empty.

[0062] Step 6. Washing magnetic particles without a magnetic field Turn off the magnetic field, open valves V5 and V1, and introduce the equilibration buffer by running pump P1 in the forward direction until air bubble sensor BS1 detects liquid. At this stage, open valve V3 and close valve V5. After recirculating the magnetic particles to create a mix, turn on the magnetic field to collect the magnetic particles in the separation chamber. The flow rate of pump P1 can be reduced to allow better capture of the magnetic particles. Once the particle-specific optical density (e.g., OD370) has been significantly reduced, reverse the pump direction, open valve V9, and close valve V3. Once air bubble sensor BS3 detects air, briefly open and close valve V3 to allow residual liquid in the loop to empty. Finally, stop pump P1 and close all valves. The washing progress can be assessed by monitoring the data from the in-line pH / conductivity sensor pH / cond. This washing process is repeated 1-2 times.

[0063] After step 6, repeat steps 3-6 until the end of the bioreactor is detected by air at the BS2 sensor. At that point, the final cycle has been completed by performing steps 3-6 and the disposable assembly can be discarded.

[0064] [Example 3] Method for purifying biological materials from body fluids - Patents.com The device and method for using the device described in the present invention are not only useful for biomanufacturing, but also allow purification of biomaterials from crude animal or human body fluids. The device and method described herein allow the purification of the desired biomaterial at the blood donation facility, and after capturing the desired biomaterial, the remaining material can be returned to the donor. By returning the biomaterial-depleted body fluid and repeating the process multiple times, much larger amounts of the desired biomaterial can be purified from a single blood donation, while eliminating the risk of hypovolemic shock in the donor. This also significantly reduces the processing, shipping, storage, and logistics costs compared to existing methods. Another advantage of this method is that blood and other body fluids can be used directly without the need for pretreatment (e.g., removal of cells, etc.).

[0065] Multiple cycles of blood collection, which can treat multiple recipients without inducing hypovolemic shock in the donor, allow for the purification of significantly larger quantities of immunoglobulin. Another advantage of this new purification method is that the recipient of the purified material has a significantly lower risk of serum disease compared to convalescent serum, and blood type matching is no longer required. This device and process has the potential to save millions of lives during epidemics or pandemics. Because the antibodies purified from blood donations will be against the most recent circulating virus strains, this process can efficiently manage current and emerging strains of SARS-CoV-2. These purified antibodies can then be injected into recipients exposed to the same virus strains in a short period of time. Isolated immunoglobulins derived from convalescent serum can be screened against pathogens to identify neutralizing antibodies, and this information can be used for the development of recombinant antibodies and vaccines.

[0066] In some diseases, harmful or toxic biomaterials accumulate in the patient's blood. Using this method, harmful or toxic biomaterials can be targeted and their circulating concentrations reduced. For example, tumor necrosis factor (TNF) concentrations are elevated in rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, plaque psoriasis, Crohn's disease, and ulcerative colitis. Several antibody-drug treatments that target TNF are available, but with repeated administration, most patients develop neutralizing antibodies against the drugs, necessitating switching to a different drug. Using this method, it is also possible to reduce circulating TNF by specifically capturing and removing it from the blood, improving the condition. Because no external drug is infused into the patient, the patient does not suffer from drug side effects, and the treatment method can be used effectively for the patient's lifetime. This process is similar to a flow-through purification process.

[0067] Similar to the effects of repeated administration of anti-TNF antibody therapeutics, repeated administration of other protein therapeutics also leads to the development of neutralizing antibodies against the administered protein therapeutics, thereby reducing their effectiveness over time. In contrast, this method of treatment can be used effectively for the patient's lifetime.

[0068] In another embodiment, this method is used to remove excess drugs from the blood to reduce side effects. For example, high doses of drugs are required to treat cancer or other indications to achieve maximum effectiveness. After a drug specifically binds to a target, some free drug may remain circulating in the blood and cause side effects. Using this technology, unbound or free drug remaining in the blood can be specifically removed to reduce side effects.

[0069] The device uses single-use or disposable kits and particles with specific properties. All product-contact surfaces of the single-use kits are suitable for medical devices (e.g., meet USP Class VI requirements) and can be replaced after each blood donation, but can support multiple cycles of the process within the same blood donation. In the case of collection from animals, the disposable kits can potentially be reused for more than one collection, after a washing cycle with purification and equilibration buffers to purify and clean the single-use surfaces that come into contact with the bodily fluid. The method described herein uses particles that are attracted to magnets (ferromagnetic, paramagnetic, or superparamagnetic), have a large surface area due to their small size, are preferably nonporous, and have specific binding properties. Such particles exhibit very short binding residence times because most of the binding sites are on the surface. The size of such particles is between 10 and 10,000 nm, up to 20,000 times smaller than standard porous chromatography resin beads. The surface of such particles is modified with ligands with specific binding properties (e.g., surface attachment of Protein A in the case of antibody purification, or attachment of antibodies with affinity for specific biomaterials, or attachment of positively or negatively charged ligands, or attachment of hydrophobic ligands, or attachment of ligands with mixed properties).

[0070] The body fluid containing the target biomaterial is taken into and mixed with the bag containing the particles having affinity for the target biomaterial, either directly or indirectly. Other buffers or reagents may be added to the collection bag for conditioning. For example, citrate or heparin may be added to the collection bag to manage blood clotting. An external magnetic field is applied to recover these particles from the body fluid. If the magnetic particles have low magnetic susceptibility due to their small size, a high gradient magnetic field is used to isolate the particles mixed with the body fluid. Small-scale batch chromatography methods for purifying proteins (microgram scale) using magnetic beads are currently available, but most of these methods use porous magnetic beads that are not reused and are manually operated. Commercially available magnetic particles are spherical with diameters equal to or greater than 1 micron and are only used in a single cycle at a very small scale (e.g., Sera-Mag from GE Healthcare Life Sciences and Dynabeads from ThermoFisher Scientific). These particles are not well suited for the present invention due to their porosity, large size, and reusability.

[0071] Porous affinity chromatography media have slow binding kinetics and result in long residence times due to narrow pore channels leading to poor mass transfer of macromolecules. Our results show that nonporous magnetic particles exhibit very fast binding kinetics even in highly viscous human whole blood, with most of the immunoglobulins bound in 30 seconds (Figure 6A).

[0072] The magnetic particles described in the present invention have a high volume to surface area ratio. For example, the volume to surface area ratio of tetrahedral and cubic particles is at least 25% greater than that of spherical particles. Attachment of the ligand to the surface of the magnetic particle is achieved by a linker that can bind to the ligand and to the inorganic surface of the magnetic particle. In one embodiment, a linker with a silane group is used to chemically bind to the magnetic particle. The other end of the linker is selected so that it can be directly or indirectly bound to the ligand used for purification. For example, the epoxide group at the other end of the linker can react with primary amine groups, sulfhydryl groups, or carboxyl groups commonly found in various amino acids of the ligand protein. 3-glycidoxypropyltrimethoxysilane or 3-glycidoxypropyltriethoxysilane are two examples of such linkers. In a study by the inventors, 0.5-3% w / w treatment with 3-glycidoxypropyltrimethoxysilane resulted in excellent conjugation to Fe3O4 cubic particles with an average size of 300 nm.

[0073] When the donor's body fluid is mixed with magnetic particles that have a surface affinity for the biomaterials of interest, the biomaterials bind to the magnetic particles. The magnetic particles are captured in a separation chamber by applying an external magnetic force. The flow-through material can be collected and infused back to the donor. The captured magnetic particles are then washed multiple times to remove impurities, and then elution is performed by introducing an elution buffer that dissociates the binding of the macromolecules of interest from the magnetic particles. In one embodiment, a low pH buffer (typically below pH 4.0) is used to dissociate the antibodies or immunoglobulins captured by the Protein A-coupled magnetic particles. In another embodiment, a low or high pH buffer (below 6.5 or above 8.4) is used to dissociate the macromolecules bound to the antibody-coupled magnetic particles. Similarly, changes in pH and / or salt concentration can be used to elute Factor VIII bound to quaternary ammonium particles with anion exchange properties. If specific cells are the target material, the elution buffer may contain peptides or molecules that compete with the interaction (between the affinity magnetic particles and receptors or molecules on the cell surface) or enzymes that destabilize the interaction. The eluted biomaterial passes through and is collected or discarded (depending on the application) while the magnetic particles are retained by an external magnetic field.

[0074] During affinity purification, the bound particles are washed with or without magnetic force to remove any non-specifically bound impurities. The bound macromolecules are eluted from the magnetic particles by introducing an elution buffer into the separation chamber with or without an external magnetic force. The entire cycle can be repeated multiple times until the desired amount of target biomaterial is purified or removed.

[0075] If the affinity performance of the particles decreases after a certain number of cycles due to non-specific blocking of the specific binding sites by impurities, the particles can be regenerated using a regeneration solution (e.g., NaOH, or a solution having a chaotropic agent such as urea or guanidine).

[0076] In each cycle, a specific volume of bodily fluid is collected in a bag attached to the system. This volume is based on guidelines regarding the amount of bodily fluid that can be taken from a subject at one time. This is called the cycle volume. For example, a typical blood donation range for humans is 250-500 mL. If multiple cycles are performed, 250 mL (the cycle volume) of blood can be taken per cycle, and another 250 mL blood take-up is started as soon as the bodily fluid donation bag is emptied. Figure 2 shows an overview of the process of purifying immunoglobulins from human blood.

[0077] The magnetic beads with affinity for the target molecule and a conditioning buffer or reagent (e.g., anticoagulant) can either be preloaded into the bag, or added later from a separate bag that can be attached aseptically, or injected aseptically from a syringe. The mixture in the bag is mixed by recirculation, impeller, or other mechanical means. The magnetic beads are reused and generally do not require replenishment after the first cycle. However, it may be necessary to inject the anticoagulant or another suitable buffer before each cycle of fluid collection.

[0078] In various parts of the process, a cylindrical separation chamber with a conical bottom (apex at the bottom) is used to isolate the magnetic particles. In another embodiment, the top of the separation chamber is conical (apex at the top). The separation chamber is made of a material that is not highly attracted to magnetic forces (e.g., LDPE, high durometer silicone, polypropylene, polystyrene, EVA, PVC, etc.). The chamber may contain steel wool or wire mesh to enhance the magnetic field by generating a high gradient field and capture small, weakly magnetic particles. In one embodiment, the material of the steel wool or wire mesh (1 micron to 1000 microns) is ferromagnetic stainless steel (e.g., 430 or 410 stainless steel) to provide corrosion resistance. The ferromagnetic steel wool can also be coated with a metal (e.g., nickel or chromium) or plastic to significantly reduce or eliminate corrosion.

[0079] An external magnetic field, typically in the range of 0.1 to 1.7 Tesla, is applied to the separation chamber either by moving a permanent magnet nearby, by turning on an electromagnet surrounding the chamber, or by other means. Depending on the needs of the purification process, a controller controls the magnetic force applied to the separation chamber by turning it on or off, or by reducing or increasing its strength.

[0080] The complete disposable manifold, including tubing, connectors, separation chambers (with or without steel wool), mixing bag, pH / conductivity flow cell, optical density measurement flow cell, pressure sensor, absorbance measurement flow cell, and filter, is preassembled and sterilized before use. The complete assembly is designed to withstand multiple cycles of a batch. Tubing or pipes with single-use product contact surfaces are used to circulate the various fluids through the system. For example, flexible silicone, C-flex, or PVC tubing can be used to connect the various parts of the system to conduct the fluids. The disposable manifold is connected to bags or containers containing magnetic particles, various buffers, donor body fluids, and final purified substances by means of sterile connectors or welding of thermoplastic tubing.

[0081] A controller with an HMI (human machine interface) is used by the user to automatically or manually control and receive information from multiple parts of the system, including pumps (speed, direction, start / stop, feedback, etc.), valves (open, close, feedback), occlusion / pressure sensors, optical density and absorbance detectors, air bubble sensors, in-line pH / conductivity meters, temperature sensors, weight load cells, and magnetic field control systems (Figure 2). This information is provided as analog or digital signals to and from the controller. Commercial automation systems from various vendors, such as Siemens, Backhoff, or Allen-Bradley, are available as complete solutions including controllers, input / output digital or analog cards, and HMI. Examples include the Siemens SIMATIC S7-1200 kit, Beckhoff TwinCAT3, and Delta V automation platforms. During the process, such platforms, which meet the requirements of Title 11 of the Code of Federal Regulations, allow the process data to be electronically stored or exported (to an external data management system).

[0082] The user inputs various process parameters into a process screen that is part of the HMI. This can be converted into a recipe that is executed each time the process is performed. The process parameters include cycle volumes, incubation times, mixing times, washing times, and recirculation times, pH, conductivity, and optical density and absorbance values ​​to start or stop a step. Once the process is started by the user providing input to the HMI, the controller can automatically execute the process according to the process screen and using the input process parameters. A number of valves (FIG. 1, V1-V13) are controlled to form different fluid paths at different aspects of the purification process. In one embodiment, pinch valves are used to open and close fluid paths in flexible tubing. The scheme shown in FIG. 2 is an example of introducing different fluids at different process steps during the purification process. An automated method for processing body fluids is described below.

[0083] Process Steps In Figure 3, V1-V13 are valves, P1 indicates a bidirectional pump, F / R indicates the forward / reverse direction of the pump, OD / Abs indicates optical density / absorbance measurement, pH / cond indicates pH and conductivity sensors, BS1-2 indicate air bubble sensors, M indicates a magnet, N or S indicates the polarity of the magnet, and B1-B2 indicate bags or containers.

[0084] At the end of each step, the pump is turned off and all valves are closed. All flexible bags or containers containing various liquids are connected as in Figure 3. A set amount of fluid (cycle volume) is collected in the fluid donor bag (e.g., 250 mL of blood). Sterile magnetic particles and appropriate conditioning agents (e.g., citrate or heparin for blood) are either injected directly into the fluid donor bag or transferred from the bag containing the magnetic particles. The donor and return bags are placed or hung on load cells so that accurate weight can be measured. Real-time weight information is continuously transmitted to the controller. With the supply connected and valve V12 open, fluid begins to flow into the fluid donor bag. For example, insert the needle of the IV tubing into one arm of the donor for blood collection and hang the blood donor bag at a height lower than the body. Once the cycle volume (corresponding to the weight detected by the load cell) has been collected, close valve V12 and start step 1.

[0085] Step 1. Binding of magnetic particles to target macromolecules Fluid is introduced into the rest of the system by opening valves V1 and V10, turning off the magnetic field, and running pump P1 forward until air bubble sensor BS1 detects liquid. At this stage, valve V1 is closed, valve V2 is opened, and pump P1 continues to run forward until the fluid donor bag is empty and air bubble sensor BS2 detects air. If mixing bag B2 is a rigid container (rather than a flexible bag), a vent with a 0.2 micron filter is attached to the top of the container to displace air with the incoming liquid. Once air bubble sensor BS2 detects air, valve V4 is opened and valve V10 is closed to mix the magnetic beads with the starting material.

[0086] If another cycle of fluid donation is required, a set amount of anticoagulant or other required buffer is injected into the donation bag at this point, and then valve V12 is opened to allow another collection of fluid for the second cycle. Once the cycle volume (corresponding to the weight detected by the load cell) has been collected, valve V12 is closed.

[0087] The flow direction of pump P1 is changed multiple times to improve mixing. Once the incubation time required for the macromolecules to bind to the beads is complete (typically 0.5-10 min), the magnetic field is turned on to capture the magnetic beads in the separation chamber. The flow rate of pump P1 can be reduced to allow better capture of the magnetic particles. The majority of the beads can be captured by this recirculation process, and the capture efficiency can be monitored by an optical density / absorbance sensor (e.g., OD370 for Fe3O4 particles). The pump direction is then reversed, valve V4 is closed, and valve V11 is opened to capture the remaining magnetic particles and collect the body fluid (without the macromolecules of interest) in the return bag. When air bubble sensor BS2 detects air, valves V3 and V4 are opened and closed once to remove the residual liquid in the loop. Valves V2 and V11 are then closed and pump P1 is stopped. After this step, valve V13 is opened to return the body fluid (without the target macromolecules) to the donor. To ensure that no air can get into the donor return tubing, valve V13 is closed as soon as a set weight of fluid has been infused back to the patient (typically below 10% of the cycle volume). To avoid hypovolemic shock, the controller is programmed to infuse at least 50% of the collected volume back to the donor before collecting fluid for the second cycle.

[0088] Step 2. Washing magnetic particles using a magnetic field In this step, the magnetic field is left on and the magnetic particles are washed with equilibration buffer to remove impurities. The equilibration buffer is chosen so as not to interfere with the binding between the target macromolecules and the magnetic particles (e.g., PBS or PlasmaLyte solution). After closing valve V11, valves V5 and V1 are opened and the equilibration buffer is introduced into the system by running pump P1 in the forward direction until air bubble sensor BS1 detects liquid. At this stage, valve V3 is opened and valve V5 is closed. The washing progress can be assessed by monitoring the data from the in-line pH / conductivity sensor pH / cond. Within one minute of washing the magnetic particles in this way, the pump direction is reversed, valve V9 is opened and valve V3 is closed. Once air bubble sensor BS2 detects air, valve V3 is briefly opened and then closed to allow residual liquid in the loop to empty. This washing process can be repeated 1-2 times.

[0089] Step 3. Washing magnetic particles without a magnetic field After closing valve V9, turn off the magnetic field, open valves V5 and V1, and introduce the equilibration buffer by operating pump P1 in the forward direction until the air bubble sensor BS1 detects liquid. At this stage, open valve V3 and close valve V5 to generate mixing by recirculation for 0.5-2 min. Once recirculation is complete, turn on the magnetic field to collect the magnetic particles in the separation chamber. Reducing the flow rate of pump P1 can allow better capture of the magnetic particles. Once the optical density specific to the particles (e.g., OD370) has been significantly reduced, reverse the direction of the pump, open valve V9, and close valve V3. Once the air bubble sensor BS2 detects air, open and close valve V3 to allow the residual liquid in the loop to empty. Repeat this washing process 1-2 times.

[0090] Step 4. Elution: Use an elution buffer to dissociate the target macromolecules from the magnetic particles. For example, a low pH buffer (100 mM sodium citrate buffer with pH<4) can be used to dissociate both the antibody-antigen interaction and the Protein A-IgG interaction. Step 4.1: After closing valve V9, open valves V6 and V1 and run pump P1 in the forward direction for a short time to fill the tubing up to pump P1. Then close valve V6, open valve V9 and reverse the pump direction. This step cleanses the tubing with elution buffer.

[0091] Step 4.2: To fill the separation chamber with elution buffer, turn off the magnetic field, open valve V6, and introduce the elution buffer by operating pump P1 in the forward direction until the air bubble sensor BS1 detects liquid. At this stage, open valve V3 and close valve V6. After recirculating the magnetic particles to create a mix, turn on the magnetic field to collect the magnetic particles in the separation chamber. Reducing the flow rate of pump P1 can allow for better capture of the magnetic particles. Once the OD for detecting the particles (e.g., OD370) is significantly reduced and the OD for detecting the eluted molecules (e.g., OD at 280 nm for protein-based macromolecules) is maximized, open valve V7, close valve V3, and operate the pump in reverse direction until the air bubble sensor BS2 detects air. To empty the residual liquid in the loop, open and close valve V3 while pump P1 is operating, and then close valves V3 and V7. This elution process from step 4.2 is repeated 1-2 times to collect the eluted macromolecules or biomaterials. When repeating this process, the step of rinsing the tube with elution buffer (4.1) is not necessary.

[0092] Step 5. Washing magnetic particles without a magnetic field First, turn off the magnetic field, open valves V5 and V1, and introduce the equilibration buffer by running pump P1 in the forward direction until air bubble sensor BS1 detects liquid. At this stage, open valve V3 and close valve V5. After recirculating the magnetic particles to create a mix, turn on the magnetic field to collect the magnetic particles in the separation chamber. The flow rate of pump P1 can be reduced to allow better capture of the magnetic particles. Once the particle-specific optical density (e.g., OD370) has been significantly reduced, reverse the pump direction, open valve V9, and close valve V3. Once air bubble sensor BS3 detects air, briefly open and close valve V3 to allow residual liquid in the loop to empty. Finally, stop pump P1 and close all valves. The washing progress can be assessed by monitoring the data from the in-line pH / conductivity sensor pH / cond. This washing process is repeated 1-2 times.

[0093] After step 5, repeat steps 1-4 until sufficient target macromolecule has been purified. At that point, the final cycle is complete and the disposable assembly can be discarded.

[0094] The purified material is discarded if it is not usable (e.g. to remove autoantibodies or overproduced cytokines). Otherwise, the purified material is stored. If immunoglobulin is the product, the purified immunoglobulin is maintained at a low pH for viral inactivation, then a set amount of neutralizing buffer is added (from a syringe or a sterile connected external bag) to raise the pH to the physiological range (approximately pH 6-8) and make the final solution isotonic. The purified macromolecule can be exposed to UV or nanofiltered to remove or reduce contaminating viral particles. Depending on the amount of macromolecule collected, the final product can be aliquoted into several bags or vials and stored at a lower temperature. The appropriate dose can be administered to the patient in need of it. The purified macromolecule can also be lyophilized to extend the storage period and reduce transportation and logistics costs.

[0095] In addition to applications in the medical and biomanufacturing fields, there are numerous other industrial applications for this platform. For example, in the food and beverage industry, this platform can be used to remove unwanted impurities to reduce toxicity or enhance flavor. The device and method can be used to extract specific components that may be of value. This method can also isolate components that are at very low concentrations in a mixture.

[0096] The foregoing is illustrative of the present invention and is not to be construed as limiting the present invention, which is defined by the following claims, with equivalents of the claims to be included therein.

Claims

1. A method for separating a substance of interest from a starting material, comprising the steps of: (a) mixing the starting material with a plurality of non-porous magnetic particles to form a mixture, each of the non-porous magnetic particles having an outer surface with an affinity for the substance of interest, binding the substance of interest to the non-porous magnetic particles; (b) exposing the mixture to a magnetic field to capture the non-porous magnetic particles with the bound substance of interest; (c) washing the mixture with an equilibration buffer in the presence of the magnetic field to remove starting material not bound to the non-porous magnetic particles; (d) mixing the non-porous magnetic particles with an elution buffer in the absence of a magnetic field to elute the substance of interest from the non-porous magnetic particles; The method includes:

2. The method of claim 1, further comprising the step of washing the non-porous magnetic particles by recirculating the equilibration buffer in the absence of a magnetic field and then circulating the equilibration buffer in the presence of the magnetic field.

3. The method of claim 1, comprising repeating steps (c) and (d), thereby improving separation of the desired material from the starting material.

4. The method of claim 1, wherein the plurality of non-porous magnetic particles have a cubic or tetrahedral shape.

5. The method of claim 1, wherein the plurality of non-porous magnetic particles have an average size in the range of about 10 nanometers to about 5,000 nanometers.

6. The method of claim 1, wherein the magnetic field is a high gradient magnetic field generated by a magnetizable material or a ferromagnetic material.

7. The method of claim 1, wherein the starting material is an unpurified or partially purified cell culture or microbial culture.

8. The method of claim 1, wherein the starting material is an unpurified or partially purified body fluid derived from an animal or human.

9. The method of claim 1, wherein the plurality of non-porous magnetic particles are regenerated by exposing the plurality of non-porous magnetic particles to a regeneration buffer in the absence of a magnetic field, followed by exposing the plurality of non-porous magnetic particles to a regeneration buffer in the presence of the magnetic field to remove non-specific impurities bound to the non-porous magnetic particles.

10. The method of claim 1, wherein the target substance is an organic substance, an inorganic substance, a virus, a bacterium, a cell, an organelle, a protein, a carbohydrate, a lipid, DNA, or RNA.

11. The method of claim 1, wherein the target substance is one or more impurities in the starting material.

12. The method of claim 1, wherein the volume of the starting material is greater than 50 milliliters.

13. The method of claim 1, wherein the volume of the starting material is greater than 1 liter.

14. A method for separating a substance of interest from a starting material, comprising the steps of: (a) mixing the starting material with a plurality of non-porous magnetic particles to form a mixture, each of the non-porous magnetic particles having an outer surface with an affinity for the substance of interest, binding the substance of interest to the non-porous magnetic particles; (b) exposing the mixture to a magnetic field to capture the non-porous magnetic particles with the bound substance of interest; (c) washing the captured non-porous magnetic particles with an equilibration buffer in the presence of the magnetic field to remove any remaining starting material not bound to the non-porous magnetic particles; (d) mixing the non-porous magnetic particles with an elution buffer in the absence of a magnetic field to elute the substance of interest from the non-porous magnetic particles; (e) recirculating the equilibration buffer in the absence of a magnetic field and then further washing the non-porous magnetic particles in the presence of the magnetic field. (f) repeating steps (c) and (d) to improve separation of the target material from the starting material. The method includes:

15. The method of claim 14, wherein the plurality of non-porous magnetic particles have a cubic or tetrahedral shape.

16. The method of claim 14, wherein the magnetic field is a high gradient magnetic field generated by a magnetizable material or a ferromagnetic material.

17. The method of claim 14, wherein the equilibration buffer is phosphate buffered saline (PBS) or a Tris-based buffer.

18. A method for separating a substance of interest from a starting material, comprising the steps of: (a) providing a plurality of non-porous magnetic particles into a separation chamber, each of the non-porous magnetic particles having a cubic or tetrahedral shape and an outer surface having an affinity for the substance of interest; (b) washing the plurality of non-porous magnetic particles with an equilibration buffer in the presence of a magnetic field; (c) mixing the starting material with the plurality of non-porous magnetic particles in the separation chamber to form a mixture, wherein the target material is bound to the non-porous magnetic particles in the separation chamber; and (d) exposing the mixture to a magnetic field to capture the non-porous magnetic particles with the bound substance of interest; (e) removing unwanted impurities bound to the non-porous magnetic particles by introducing a regeneration buffer into the mixture in the absence of a magnetic field, followed by exposure to the regeneration buffer in the presence of the magnetic field; (f) washing the plurality of non-porous magnetic particles with an equilibration buffer in the presence of the magnetic field; (g) repeating step (e) and step (f) one or more times, thereby improving separation of the desired material from the starting material. The method includes:

19. The method of claim 18, further comprising the step of mixing the non-porous magnetic particles with an elution buffer in the absence of a magnetic field to elute the target substance from the non-porous magnetic particles.

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