Process technology for biological product manufacturing and downstream purification

Abstract

In particular, biological manufacturing and downstream purification processes are provided in this specification.

Description

Technology Field

[0001] Related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 077,766, filed September 14, 2020, U.S. Provisional Application No. 63 / 154,108, filed February 26, 2021, and U.S. Provisional Application No. 63 / 154,109, filed February 26, 2021, the full contents of each of which are incorporated herein by reference.

[0003] Field of the present invention

[0004] A new process and method for the preparation and downstream purification of a biological product are provided.

[0005] In particular, a process and apparatus for purifying a biological product are provided herein. In aspects, a process for purifying a biological product is provided herein, the process comprising the steps of receiving a heterogeneous mixture containing a biological product through an input line, and removing impurities from the heterogeneous mixture by filtration in a dynamic filtration module. Impurities are removed from the heterogeneous mixture by supplying the biological product to the dynamic filtration module from at least one output head fluidly communicating with the input line under negative pressure, thereby producing a filtrate containing the biological product.

[0006] The dynamic filtration module comprises a dynamic filtration device, a target area configured to receive a heterogeneous mixture from at least one output head, and a membrane support member having a substantially flat contact surface communicating with a vacuum collection system located between a feed reel and a collection reel. Additionally, the dynamic filtration device comprises a filter membrane extending between the feed reel and the collection reel together with at least one support member having a substantially flat contact surface. Purifying the biological product further comprises transferring the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains the biological product; the first module comprises an affinity-based purification device. The affinity-based purification device has at least one first inlet and at least one first outlet configured to allow fluid flow between at least one first inlet and at least one first outlet through a mechanical rotation system. The mechanical rotation system comprises a container carousel containing at least one individual container containing a suspension of beads. As described herein, the process further comprises transferring a fraction containing a biological product from at least one outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module. The second module includes at least one free-flow electrophoresis device, and the second module has at least one second inlet and at least one second outlet, and is configured to allow a continuous fluid flow between the second inlet and the second outlet to recover the biological product.

[0007] In embodiments, the affinity-based purification device further comprises a lid system and a collection container system fluidly communicating with at least one individual container. For example, the lid system includes at least one lid with a gasket, at least two buffer inlets, a filling inlet, a gas inlet, and a venting valve. Additionally, the lid system is movable along the z-axis. The container carousel of the affinity-based purification device is rotatable in a plane across the z-axis, and the collection container is movable along the z-axis.

[0008] As described herein, the container carousel of the affinity-based purification method comprises at least one location for binding with a biological product, at least one location for washing to remove unbound products, at least one location for eluting and collecting the biological product, and at least one regeneration location to enable recycling of the beads.

[0009] For example, the surface of the beads of the affinity-based purification is connected to Protein A, Protein G, Protein L, antigen protein, protein, receptor, antibody, or aptamer configured to selectively bind to the biological product. The initial concentration of the beads (e.g., in individual containers at the binding site to the biological product) is within a concentration range of about 0.01 wt% to about 25 wt%. Alternatively, the initial concentration of the beads is within a range of about 0.01 wt% to about 20 wt%, about 0.01 wt% to about 10 wt%, about 0.01 wt% to about 5 wt%, about 1 wt% to about 20 wt%, or about 5 wt% to about 10 wt%. For example, the diameter of the beads is in the range of about 0.2 μm to about 200 μm. In another example, the diameter of the bead is about 0.2 μm to about 100 μm, about 1 μm to about 200 μm, about 10 μm to about 200 μm, about 20 μm to about 200 μm, about 30 μm to about 200 μm, about 50 μm to about 200 μm, or about 150 μm to about 200 μm. Alternatively, the diameter of the bead is about 1 μm to about 100 μm, or about 50 μm to about 100 μm.

[0010] In embodiments, beads (e.g., beads of affinity-based tablets) maintain mobility during the process to maintain an increased surface area available for binding. For example, the beads are kept separated (circulated or dispersed) from the solution during the process (e.g., the beads are individual beads). Additionally, mobility may mean that the beads do not aggregate together, for example, that at least two beads do not aggregate or group together. Furthermore, mobility may mean that the beads can form small aggregates that are dispersed and move freely within the solution. Conversely, the beads used herein are not packed but maintain mobility and move freely within the solution.

[0011] In embodiments, the free-flow electrophoresis device includes electrode channels comprising an anode electrode channel and a cathode electrode channel that are in liquid contact with the main separation channel through a wall gap.

[0012] The free-flow electrophoresis apparatus comprises at least one electrode channel de-bubbler comprising at least one gas-permeable and hydrophobic membrane configured to remove bubbles by a vacuum system to create a bubble-free main separation channel, and at least one liquid circuit breaker. In embodiments, at least one de-bubbler of the free-flow electrophoresis apparatus is configured to continuously remove O2 and H2 gas bubbles generated in the electrode channel under an applied voltage. In some embodiments, removing electrolytic bubbles from the electrode channel is essential to enable continuous operation for substantially long periods. For example, the de-bubbler system uses a hydrophobic PTFE membrane to create a water-tight seal over the electrode channel that can continuously remove electrolytic bubbles at the point of generation by exposure to the vacuum system. For example, the vacuum gauge pressure is in the range of about -0.05 bar to about -0.4 bar. Unlike current methods, the process described herein removes gas bubbles before they enter the main separation channel.

[0013] In embodiments, the liquid circuit breaker of the free-flow electrophoresis device includes a pressurized vessel configured to maintain the flow rate and generate a droplet that disconnects the circuit from the solution connected to the voltage.

[0014] In embodiments, the purification process maintains a roughly constant flow rate in the dynamic filtration module, the first module, and the second module. For example, the flow rate is in the range of about 0.1 mL / min to about 50 mL / min, or about 5 mL / min to about 10 mL / min.

[0015] In embodiments, the process of purifying the biological product is carried out at a temperature in the range of about 4°C to about 37°C.

[0016] In a further embodiment, the process may include at least two dynamic filtration modules, each of which has a filter membrane having the same or different pore sizes (e.g., a heterogeneous mixture first contacts a filter membrane with a larger pore size (e.g., 0.45 μm) and then contacts a filter membrane with a smaller pore size (e.g., 0.2 μm)).

[0017] In embodiments, the process comprises at least two free-flow electrophoresis modules configured to operate in an isoelectric focusing mode, a zone electrophoresis mode, an isotachophoresis mode, or a combination thereof. The process described herein further comprises at least two dynamic filtration modules, at least two affinity-based purification modules, or at least two free-flow electrophoresis modules operating in parallel.

[0018] In aspects, the present specification provides a dynamic filtration device for removing impurities from biological products within a heterogeneous mixture. The device comprises a filter membrane extending between a feed reel and a collect reel, wherein the filter membrane has a target area configured to receive the heterogeneous mixture from at least one output head configured to distribute the heterogeneous mixture to the target area. A membrane support structure of the device has a substantially flat contact surface for structurally supporting a portion of the filter membrane located between the feed reel and the collect reel to create the target area. Additionally, the dynamic filtration device has at least one support member having a substantially flat contact surface for stabilizing the transport of the filter membrane across the membrane support structure. The dynamic filtration device has a system configured to control the transport rate of the filter membrane. The dynamic filtration device has at least one vacuum line communicating with the membrane support structure and a vacuum system configured to apply a negative pressure gauge pressure to the dynamic filter membrane, wherein the negative pressure enables the collection of the filtrate containing the biological product. In another example, the dynamic filtration device includes a wash buffer line.

[0019] In embodiments, the dynamic filtration device has a filter membrane that may comprise polyethersulfone (PES), hydrophilic polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), hydrophilic PTFE, or any combination thereof. The pore size of the filter membrane is in the range of about 0.1 μm to about 1 μm. In other examples, the pore size is within the range of about 0.1 μm to about 0.9 μm, about 0.1 μm to about 0.8 μm, about 0.1 μm to about 0.7 μm, about 0.1 μm to about 0.6 μm, about 0.1 μm to about 0.5 μm, about 0.1 μm to about 0.4 μm, about 0.1 μm to about 0.3 μm, or about 0.1 μm to about 0.2 μm. As described herein, when two or more dynamic filtration devices are used, they may include filter membranes of similar or different sizes.

[0020] The dynamic filtration device described herein includes a membrane support structure having a series of parallel slots, for example, about 1 to about 10 parallel slots. In a specific example, the membrane support structure has 5 parallel slots.

[0021] The dynamic filtration device described herein comprises a membrane support structure having a substantially flat contact surface, wherein the contact surface is a measure of its static coefficient of friction, for example, about 0.01 to about 0.1, about 0.01 to about 0.05, or about 0.05 to about 0.1. In a specific example, the static coefficient of friction is 0.04.

[0022] In embodiments, the vacuum system of the dynamic filtration module is configured to apply a negative pressure gauge pressure in the range of, for example, about -0.05 bar to about -0.98 bar.

[0023] In aspects, the present specification provides a free-flow electrophoresis apparatus for separating a mixture into two or more fractions, wherein at least one of the fractions contains a biological product. The free-flow electrophoresis apparatus comprises: at least one inlet and at least one outlet configured to allow continuous fluid flow between at least one inlet and at least one outlet; at least one fluid channel configured to create an electric field gradient between two parallel plates and perpendicular to the direction of fluid flow; electrode channels including a positive electrode channel and a negative electrode channel—wherein the electrode channels are configured to be connected to the main separation channel by liquid contact through a wall gap located between the electrode channels and the main separation channel—; at least one electrode channel bubble remover comprising at least one gas-permeable and hydrophobic membrane or porous material configured to remove electrolytic bubbles near the point of creation by a vacuum system to create a bubble-free main separation channel; at least one liquid circuit breaker configured to disconnect the solution connected to the voltage before interacting with at least one sensor or detector; an active cooling system; and at least one collection vessel.

[0024] The free-flow electrophoresis apparatus described herein provides electrode channels having a bubble remover, wherein the upper portion of the electrode channels is sealed with at least one gas-permeable and hydrophobic membrane communicating with a vacuum system for removing bubbles, and the electrode channels are open at the bottom of the channels and configured to enable liquid contact between the main separation channel solution and the electrode solution through a wall gap.

[0025] The free-flow electrophoresis device includes at least one electrode channel bubble remover comprising at least one gas-permeable and hydrophobic membrane configured to remove bubbles by a vacuum system to create a bubble-free main separation channel, and at least one liquid circuit breaker.

[0026] In embodiments, the free-flow electrophoresis apparatus further comprises at least one bubble removal system for continuously removing O2 and H2 gas bubbles generated in the electrode channel under an applied voltage. In some embodiments, removing electrolytic bubbles is essential to enable continuous operation for substantially long periods. For example, the bubble removal system uses a hydrophobic PTFE membrane to create a waterproof seal over the electrode channel that can continuously remove electrolytic bubbles at the point of generation by exposure to a vacuum system. For example, the vacuum gauge pressure is in the range of about -0.05 bar to about -0.4 bar. Unlike the current method, the process described herein removes gas bubbles before they enter the main separation channel.

[0027] In embodiments, the wall gap (e.g., a space configured such that electrode channels are open at the bottom of the channels and allow liquid contact between the main separation channel solution and the electrode solution) is about 0.01 mm to about 0.25 mm. For example, the wall gap is about 0.01 mm to about 0.2 mm, about 0.01 mm to about 0.015 mm, or about 0.01 mm to about 0.01 mm.

[0028] In another embodiment, the liquid circuit breaker of the free-flow electrophoresis device includes a pressurized vessel configured to maintain the flow rate and generate a droplet that disconnects the circuit from the solution connected to the voltage.

[0029] In embodiments, the free-flow electrophoresis device further includes an inline sensor. For example, the inline sensor may include a flow sensor, a pH sensor, a conductivity sensor, or any combination thereof.

[0030] In embodiments, the free-flow electrophoresis apparatus described herein may include at least two free-flow electrophoresis apparatuses connected in series to enable stepwise purification and operated in an isoelectric focusing mode, a band electrophoresis mode, an isokinetic electrophoresis mode, or a combination thereof.

[0031] Additionally, the present specification provides for the use of a free-flow electrophoresis device for purifying a biological product from a mixture. The present invention further provides for the use of a dynamic filtration device for purifying a biological product from a heterogeneous mixture.

[0032] In aspects, the present specification provides a process for purifying a biological product. The process comprises receiving a heterogeneous mixture containing the biological product through an input line. In embodiments, the process comprises continuously receiving a heterogeneous mixture containing the biological product through an input line. In embodiments, the biological product comprises a protein or a fragment thereof (polypeptide), an antibody or a fragment thereof, a cytokine, a chemokine, a growth factor, an enzyme, an oligonucleotide, a virus, an adenovirus, an adeno-associated virus (AAV), or a lentivirus.

[0033] In embodiments, the process comprises removing impurities (e.g., large impurities such as cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration. In some embodiments, the dynamic filtration process may be a continuous process for removing large impurities from a heterogeneous mixture. The dynamic filtration process comprises at least one dynamic filtration module that continuously feeds a heterogeneous mixture containing a biological product from at least one output head fluidly communicating with an input line under negative pressure to a dynamic filtration module to produce a filtrate containing the biological product.

[0034] In embodiments, the process comprises transferring the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains a biological product. In another embodiment, the process comprises continuously transferring the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains a biological product. For example, separating the solution into two or more fractions may include one fraction containing a biological product and at least one other fraction containing small impurities (e.g., host cell proteins, undesirable proteins and peptides, undesirable antibodies, undesirable nucleic acids and oligonucleotides, viruses, salts, buffer components, surfactants, sugars, metal contaminants, leachables, medium components, and / or naturally occurring organic molecules).

[0035] In embodiments, the first module comprises an affinity-based magnetic purification apparatus. For example, the first module has at least one first inlet and at least one first outlet and is configured to allow continuous fluid flow between the first inlet and the first outlet through a loop conveyor system. In another example, the first module has at least one first inlet and at least one first outlet and is configured to allow continuous fluid flow between the first inlet and the first outlet through a pick-and-place robotics system.

[0036] In embodiments, the process comprises transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, said second module comprising a charge-based magnetic purification device or an isoelectric point-based fluid purification device (also referred to herein as a free-flow electrophoresis device). In another embodiment, the process comprises continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, said second module comprising a charge-based magnetic purification device or an isoelectric point-based fluid purification device, also referred to herein as a free-flow electrophoresis device. For example, said second module comprises a charge-based magnetic purification device having at least one second inlet and at least one second outlet and is configured to allow a continuous fluid flow between the second inlet and the second outlet through a loop conveyor system. In some examples, the second module comprises a charge-based magnetic purification device having at least one second inlet and at least one second outlet, and is configured to allow continuous fluid flow between the second inlet and the second outlet via a pick-and-place robotic system. In other examples, the second module comprises a free-flow electrophoresis device having at least one second inlet and at least one second outlet, and is configured to allow continuous fluid flow between the second inlet and the second outlet. In embodiments, the process described herein thereby purifies a biological product.

[0037] In embodiments, the present specification also provides a process for purifying a biological product, comprising continuously receiving a heterogeneous mixture containing a biological product through an input line and removing large impurities from the heterogeneous mixture by dynamic filtration. In some embodiments, the dynamic filtration process may be a continuous process for removing large impurities from a heterogeneous mixture. The dynamic filtration process includes a dynamic filtration module that produces a filtrate containing a biological product by continuously feeding the biological product to a dynamic filtration module from at least one output head that fluidly communicates with the input line under negative pressure.

[0038] In embodiments, the process comprises delivering the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains a biological product. In another embodiment, the process comprises continuously delivering the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains a biological product. For example, the first module includes an affinity-based purification device. For example, the first module has at least one first inlet and at least one first outlet and is configured to allow continuous fluid flow between the first inlet and the first outlet through a mechanical rotation system. In another example, the first module has at least one first inlet and at least one first outlet and is configured to allow continuous fluid flow between the first inlet and the first outlet through a staged linear system.

[0039] In embodiments, the process comprises transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, said second module includes an isoelectric point-based fluid purification device, also referred to herein as a charge-based purification device or a free-flow electrophoresis device. In another embodiment, the process comprises continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, said second module includes an isoelectric point-based fluid purification device, also referred to herein as a charge-based purification device or a free-flow electrophoresis device. For example, said second module includes a charge-based purification device having at least one second inlet and at least one second outlet and is configured to allow a continuous fluid flow between the second inlet and the second outlet through a mechanical rotation system. In some examples, the second module has at least one second inlet and at least one second outlet and is configured to allow continuous fluid flow between the second inlet and the second outlet through a stepwise linear system. In other examples, the second module includes a free-flow electrophoresis device having at least one second inlet and at least one second outlet and is configured to allow continuous fluid flow between the second inlet and the second outlet. In embodiments, the process described herein thereby purifies a biological product.

[0040] In embodiments, the present specification also provides a process for purifying a biological product, comprising continuously receiving a heterogeneous mixture containing a biological product through an input line and removing large impurities from the heterogeneous mixture by dynamic filtration. In some embodiments, the dynamic filtration process may be a continuous process for removing large impurities from a heterogeneous mixture. The dynamic filtration process includes a dynamic filtration module that produces a filtrate containing a biological product by continuously feeding the biological product to a dynamic filtration module from at least one output head that fluidly communicates with the input line under negative pressure.

[0041] In embodiments, the process comprises delivering the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains a biological product. In another embodiment, the process comprises continuously delivering the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains a biological product. In embodiments, the first module comprises an affinity-based fluid purification device. For example, the first module has at least one first inlet and at least one first outlet and is configured to allow continuous fluid flow between the first inlet and the first outlet.

[0042] In embodiments, the process comprises transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of a first module, said second module includes an isoelectric point-based fluid purification device, also referred to herein as a charge-based fluid purification device or a free-flow electrophoresis device. In embodiments, the process comprises continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of a first module, said second module includes an isoelectric point-based fluid purification device, also referred to herein as a charge-based fluid purification device or a free-flow electrophoresis device. For example, said second module includes a charge-based fluid purification device having at least one second inlet and at least one second outlet and is configured to allow a continuous fluid flow between the second inlet and the second outlet. In another example, the second module comprises a free-flow electrophoretic device having at least one second inlet and at least one second outlet, and is configured to allow continuous fluid flow between the second inlet and the second outlet. In embodiments, the process described herein thereby purifies a biological product.

[0043] In embodiments, the process comprises delivering the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains a biological product. In another embodiment, the process comprises continuously delivering the filtrate to a first module capable of separating the solution into two or more fractions, wherein at least one fraction contains a biological product. In embodiments, the first module comprises an affinity-based tangential flow filtration (TFF) purification device. For example, the first module has at least one first inlet and at least one first outlet and is configured to allow continuous fluid flow between the first inlet and the first outlet.

[0044] In embodiments, the process comprises transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, said second module includes an isoelectric point-based fluid purification device, which is also referred to herein as a charge-based TFF purification device or a free-flow electrophoresis device. In another embodiment, the process comprises continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, said second module includes an isoelectric point-based fluid purification device, which is also referred to herein as a charge-based TFF purification device or a free-flow electrophoresis device. For example, said second module includes a charge-based TFF purification device having at least one second inlet and at least one second outlet and is configured to allow a continuous fluid flow between the second inlet and the second outlet. In another example, the second module comprises a free-flow electrophoretic device having at least one second inlet and at least one second outlet, and is configured to allow continuous fluid flow between the second inlet and the second outlet. In embodiments, the process described herein thereby purifies a biological product.

[0045] As described herein, the process for removing large impurities from a heterogeneous mixture does not include centrifugation, disk-stack centrifugation, deep filtration, static filtration, tangential flow filtration, or any combination thereof. Alternatively, the process described herein may receive a heterogeneous mixture containing a biological product through an input line derived from an input from which any large impurities have been removed by centrifugation and deep filtration processes, for example, without limitation.

[0046] As described herein, the process for continuously removing large impurities from a heterogeneous mixture does not include centrifugation, disk-stack centrifugation, deep filtration, static filtration, tangential flow filtration, a hydrocyclone, or any combination thereof. Alternatively, the process described herein may continuously receive a heterogeneous mixture containing a biological product through an input line derived from an input from which any continuous large impurities have been removed by a continuous disk-stack centrifugation and deep filtration process or a hydrocyclone process, for example, without limitation.

[0047] In embodiments, the process described herein comprises purifying a biological product (e.g., monoclonal antibody) produced in a bioreactor. In some embodiments, the process described herein comprises purifying a biological product continuously produced in a bioreactor. For example, the bioreactor includes a bioreactor feed line and an output bleed line that enable steady-state cell culture growth conditions, and said output bleed line functions as an input line allowing continuous fluid flow from the bioreactor to a dynamic filtration module. For example, bioreactor types include, but are not limited to, fed-batch bioreactors, perfusion bioreactors, chemostat bioreactors, or multi-compartment bioreactors. For example, the flow from the bioreactor bleed line is always supplied to a downstream purification system. Alternatively, the process described herein comprises purifying a biological product (e.g., mRNA) not produced in the bioreactor.

[0048] In embodiments, the present specification provides a method for purifying a biological product, the method comprising: receiving a heterogeneous mixture containing the biological product through an input line; supplying the biological product from at least one output head fluidly communicating with the input line under negative pressure to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module, thereby producing a filtrate containing the biological product; and transferring the filtrate to a first module capable of separating the solution into two or more fractions, each fraction containing the biological product, wherein the first module comprises an affinity-based magnetic purification device, the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet via a loop conveyor system or a pick-and-place robot system. The method comprises the step of transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, wherein the second module comprises a charge-based magnetic purification device, and the second module has at least one second inlet and at least one second outlet, and is configured to allow a continuous fluid flow between the second inlet and the second outlet via a loop conveyor system or a pick-and-place robot system; and thereby purifying the biological product.

[0049] In another embodiment, a method for purifying a biological product is provided. The method comprises the steps of: receiving a heterogeneous mixture containing a biological product through an input line; supplying the biological product from at least one output head fluidly communicating with the input line under negative pressure to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module, thereby producing a filtrate containing the biological product; and transferring the filtrate to a first module capable of separating the solution into two or more fractions, each fraction containing at least one fraction containing the biological product— wherein the first module comprises an affinity-based magnetic purification device, and the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet via a loop conveyor system or a pick-and-place robot system. The method comprises the step of transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, wherein the second module comprises an isoelectric point-based fluid purification device, which is also referred to herein as a free-flow electrophoresis device, and the second module has at least one second inlet and at least one second outlet and is configured to allow a continuous fluid flow between the second inlet and the second outlet; and thereby purifying the biological product.

[0050] In embodiments, a method for purifying a biological product comprises the steps of: receiving a heterogeneous mixture containing the biological product through an input line; supplying the biological product from at least one output head fluidly communicating with the input line under negative pressure to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module, thereby producing a filtrate containing the biological product; and transferring the filtrate to a first module capable of separating the solution into two or more fractions, each fraction containing the biological product, wherein the first module comprises an affinity-based purification device, and the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet through a mechanical rotation system. The method comprises the step of transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, wherein the second module includes a charge-based purification device, and the second module has at least one second inlet and at least one second outlet and is configured to allow fluid flow between the second inlet and the second outlet through a mechanical rotation system; and thereby purifying the biological product.

[0051] In another embodiment, a method for purifying a biological product is provided, the method comprising: receiving a heterogeneous mixture containing the biological product through an input line; supplying the biological product from at least one output head fluidly communicating with the input line under negative pressure to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module, thereby producing a filtrate containing the biological product; and transferring the filtrate to a first module capable of separating the solution into two or more fractions, each fraction containing the biological product, wherein the first module comprises an affinity-based purification device, and the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet through a mechanical rotation system. The method comprises the step of transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, wherein the second module comprises an isoelectric point-based fluid purification device, which is also referred to herein as a free-flow electrophoresis device, and the second module has at least one second inlet and at least one second outlet and is configured to allow a continuous fluid flow between the second inlet and the second outlet; and thereby purifying the biological product.

[0052] In embodiments, a method for purifying a biological product comprises: receiving a heterogeneous mixture containing the biological product through an input line; supplying the biological product from at least one output head fluidly communicating with the input line under negative pressure to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module, thereby producing a filtrate containing the biological product; and transferring the filtrate to a first module capable of separating the solution into two or more fractions, each fraction containing the biological product, wherein the first module comprises an affinity-based purification device, and the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet through a stepwise linear system. The method comprises the step of transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, wherein the second module includes a charge-based purification device, and the second module has at least one second inlet and at least one second outlet and is configured to allow fluid flow between the second inlet and the second outlet through a stepwise linear system; and thereby purifying the biological product.

[0053] In another embodiment, a method for purifying a biological product is provided, the method comprising: receiving a heterogeneous mixture containing the biological product through an input line; supplying the biological product from at least one output head fluidly communicating with the input line under negative pressure to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module, thereby producing a filtrate containing the biological product; and transferring the filtrate to a first module capable of separating the solution into two or more fractions, each fraction containing the biological product, wherein the first module comprises an affinity-based purification device, and the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet through a stepwise linear system. The method comprises the step of transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, wherein the second module comprises an isoelectric point-based fluid purification device, which is also referred to herein as a free-flow electrophoresis device, and the second module has at least one second inlet and at least one second outlet and is configured to allow a continuous fluid flow between the second inlet and the second outlet; and thereby purifying the biological product.

[0054] In embodiments, a method for purifying a biological product is included, the method comprising: receiving a heterogeneous mixture containing the biological product through an input line; supplying the biological product from at least one output head fluidly communicating with the input line under negative pressure to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module, thereby producing a filtrate containing the biological product; and transferring the filtrate to a first module capable of separating the solution into two or more fractions, each fraction containing the biological product, wherein the first module comprises an affinity-based fluid purification device, and the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet. The method comprises the step of transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, wherein the second module includes a charge-based fluid purification device, and the second module has at least one second inlet and at least one second outlet and is configured to allow fluid flow between the second inlet and the second outlet; and thereby purifying the biological product.

[0055] In another embodiment, a method for purifying a biological product is provided, the method comprising: receiving a heterogeneous mixture containing the biological product through an input line; supplying the biological product from at least one output head fluidly communicating with the input line under negative pressure to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module, thereby producing a filtrate containing the biological product; and transferring the filtrate to a first module capable of separating the solution into two or more fractions, each fraction containing the biological product, wherein the first module comprises an affinity-based fluid purification device, and the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet. The method comprises the step of transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, wherein the second module comprises an isoelectric point-based fluid purification device, which is also referred to herein as a free-flow electrophoresis device, and the second module has at least one second inlet and at least one second outlet and is configured to allow a continuous fluid flow between the second inlet and the second outlet; and thereby purifying the biological product.

[0056] In embodiments, a method for purifying a biological product is included, the method comprising: receiving a heterogeneous mixture containing the biological product through an input line; supplying the biological product from at least one output head fluidly communicating with the input line under negative pressure to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module, thereby producing a filtrate containing the biological product; and transferring the filtrate to a first module capable of separating the solution into two or more fractions, each fraction containing the biological product, wherein the first module comprises an affinity-based TFF purification device, and the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet. The method comprises the step of transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, wherein the second module includes a charge-based TFF purification device, and the second module has at least one second inlet and at least one second outlet and is configured to allow fluid flow between the second inlet and the second outlet; and thereby purifying the biological product.

[0057] In another embodiment, a method for purifying a biological product is provided, the method comprising: receiving a heterogeneous mixture containing the biological product through an input line; supplying the biological product from at least one output head fluidly communicating with the input line under negative pressure to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module, thereby producing a filtrate containing the biological product; and transferring the filtrate to a first module capable of separating the solution into two or more fractions, each fraction containing the biological product, wherein the first module comprises an affinity-based TFF purification device, and the first module has at least one first inlet and at least one first outlet and is configured to allow fluid flow between the first inlet and the first outlet. The method comprises the step of transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, wherein the second module comprises an isoelectric point-based fluid purification device, which is also referred to herein as a free-flow electrophoresis device, and the second module has at least one second inlet and at least one second outlet and is configured to allow a continuous fluid flow between the second inlet and the second outlet; and thereby purifying the biological product.

[0058] Advantages of the processes and methods described herein include the ability to remove large impurities (e.g., cells, cell debris, and aggregates) without membrane fouling or clogging. For example, clarifying cells, cell debris, and aggregates from cell culture media using conventional filtration or tangential flow filtration systems typically results in fouling or clogging of the filter membrane, making these methods unsuitable as means for continuously removing large impurities from heterogeneous mixtures containing biological products over long continuous processes. In contrast, the dynamic filtration device described herein enables the continuous removal of large impurities from heterogeneous mixtures containing biological products without contaminating the membrane, because the active target area of ​​the filter membrane is continuously refreshed.

[0059] Additionally, because the entire process of producing and purifying biological products can be continuous and a flow rate ranging from about 0.1 mL / min to about 50 mL / min can be maintained throughout the entire process, the process equipment and the overall process footprint can occupy a much smaller footprint than current standard processes on a kilogram-year basis without sacrificing product throughput or yield. For example, the process for producing and purifying monoclonal antibodies described herein operates on a footprint of up to about 30,000 square feet. In contrast, current monoclonal antibody production and downstream processes require at least 200,000 square feet. For example, the flow rate of the process for purifying biological products ranges from about 1 mL / min to about 10 mL / min. In some examples, the flow rate of the step for continuously removing large impurities from heterogeneous mixtures ranges from about 0.1 mL / min to about 50 mL / min. In another example, the flow rate of the step of continuously removing large impurities from a heterogeneous mixture is the same as the flow rate from the bioreactor discharge line. In another example, a process is provided in which the flow rate of the step of continuously transferring the filtrate to the first module is in the range of about 0.1 mL / min to about 50 mL / min. In yet another example, a process is provided in which the flow rate of the step of continuously transferring a fraction containing a biological product from the first outlet to the second module is in the range of about 0.1 mL / min to about 50 mL / min.

[0060] A significant advantage of the process and method using the magnetic resin beads (e.g., magnetic agarose) or conventional resin beads (e.g., agarose) described herein is that such a system does not require a conventional stationary phase or packed resin column (e.g., for standard chromatography) for sanitization, recycling, and / or regeneration. For example, such a system provides recycling and / or regeneration of resin beads (e.g., magnetic or non-magnetic resin beads) to create an infinite surface area of ​​the resin beads during operation, and consequently provides a continuous and cost-effective method.

[0061] In other words, the module described herein does not possess a fixed coupling or association capability. As a specific example, since the resin beads used during the purification of biological products as described herein are continuously recycled and regenerated, the flow from the previous stage can be accommodated in either the dynamic filtration module or the purification module without interrupting the flow from the bioreactor discharge line. In other words, since the module described in the present invention undergoes these steps sequentially, there is no need to leave it idle for sterilization, regeneration, and / or recycling after execution. The method differs from current continuous chromatography methods in that, because the current method has a limited column capacity due to resin packing constraints, column switching of multiple packed columns is required to accommodate a continuous input flow and enable the regeneration and / or recycling of a column that has reached full capacity.

[0062] Another advantage of the method described herein includes that the resin beads are not packed into the stationary bed, but rather move. This mobility of the beads increases the surface area available for binding or association, as substantially more resin bead surface is exposed and binding becomes free, allowing, for example, more biological products to bind to the beads. Additionally, resin beads in a conventionally packed column (e.g., where bead mobility is lacking and surface area is reduced) are exposed to high pressure differences to generate flow through the column. Such high pressure differences compromise the integrity of the beads and shorten the column life. The mobile resin beads of the present invention are subjected to substantially lower pressures and are much softer than brittle beads, thereby extending their life. Furthermore, this mobility increases the likelihood that the beads will regenerate (e.g., fully regenerate) and return to their initial state. This also increases the cost-effectiveness of the method described herein, for example, because the resin is utilized more efficiently.

[0063] As described herein, the resin beads of the claimed method and apparatus are mobile throughout the process. Conventional chromatographic purification methods require, for example, column packing to sufficiently pack the beads together to create a high density with the stationary phase. For example, the beads are kept separated (circulated or dispersed) from the solution during the process (e.g., the beads are individual beads). Additionally, mobile beads may mean that the beads do not aggregate together, for example, that at least two beads do not aggregate or group together. Furthermore, mobile beads may mean that the beads can form small aggregates that are dispersed within the solution and move freely. Conversely, the beads used herein are not packed but maintain mobility and move freely within the solution.

[0064] A significant advantage of the process and method using free-flow electrophoresis described herein is that this system exhibits a "no product loss" process, meaning that since separation occurs through interaction with an electric field based on the physicochemical properties of the target biological product within an aqueous solution, the product does not need to interact with a resin or other purification moiety. Another advantage is observed in the resolution of this approach (e.g., the ability to purify products with a high degree of physicochemical similarity) because higher purity products can be obtained compared to conventional ion exchange chromatography. For example, using the free-flow electrophoresis module and method described herein, biological products of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher purity can be obtained. Furthermore, the method and apparatus described herein can increase the purity (of the biological product) compared to conventional purification and chromatography methods. For example, in relation to levels, the term "increased" refers to any percentage increase that exceeds the control level (e.g., the purity level obtained after purification using conventional methods).In various embodiments, the increased level may be an increase in purity of at least or about 1%, 2%, 3%, 4%, or 5% compared to a conventional purification method, at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, or at least or about 95%. In another example of the present invention, the purity of the biological product resulting from the method and apparatus described herein is increased by about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, or 3.0 times compared to the purity of the biological product using standard commercial or chromatographic techniques.

[0065] Additionally, separation based on the intrinsic physicochemical properties of the biological products (e.g., isoelectric point, surface charge, net charge, zeta potential, electrophoretic mobility, electrostatic interactions, etc.) extends the utility of this approach for the purification of various biological products, including but not limited to proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, growth factors, enzymes, oligonucleotides, viruses, adenoviruses, adeno-associated viruses (AAVs), or lentiviruses.

[0066] In addition, the modular approach provides flexibility in process design to accommodate a diverse range of biological products.

[0067] In embodiments, in the process described herein, during purification by dynamic filtration, a filtrate containing a biological product is produced and supplied to a vacuum collection vessel capable of collecting about 50 mL to about 100 L under negative pressure. For example, the vacuum collection vessel capable of collecting the filtrate is about 1 L to about 10 L. In another example, the vacuum collection vessel capable of collecting the filtrate is about 1 L to about 50 L.

[0068] In embodiments, the dynamic filtration module includes at least one output head for controlling the flow of a heterogeneous mixture and distributing the heterogeneous mixture to an active target area of ​​a filter membrane. For example, the at least one output head is a tube or a slot die.

[0069] In embodiments, at least one dynamic filtration module may additionally include at least one additional input line for supplying a washing buffer through a coaxial output head, a separate single-axis output head, a separate slot die output head, a slot die output head with multiple openings, or any combination thereof.

[0070] In some embodiments, the dynamic filter module includes elements known to those skilled in the art, such as, for example, without limitation, an active or passive edge guide, a tension regulator (e.g., a dancer), a brake and a tension detector, or any combination thereof.

[0071] In embodiments, the process of this specification comprises at least one output head (fluidly communicating with an input line for a dynamic filtration module) capable of xy rasterizing or rθ rasterizing. For example, at least one output head is capable of xy rasterizing. In some examples, at least one output head is capable of rθ rasterizing. In other examples, at least one output head is movable along the z-axis. In yet another example, at least one output head is capable of xy rasterizing and is movable along the z-axis.

[0072] In embodiments, the dynamic filtration module includes a filter membrane roll, a membrane support structure, at least one support rod or roller, at least one vacuum line, a vacuum system, and at least one vacuum collection vessel.

[0073] In embodiments, the filter membrane roll comprises a rolled filter membrane, wherein the filter membrane comprises, but is not limited to, polyethersulfone (PES), hydrophilic polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), hydrophilic PTFE, or any combination thereof.

[0074] In embodiments, the pore size of the rolled filter membrane varies depending on the biological product being purified. For example, the pore size of the rolled filter membrane is in the range of 0.1 μm to 1 μm. Alternatively, the pore size is in the range of about 0.2 μm to about 0.45 μm, or the pore size is less than about 0.45 μm. In another example, when purifying antibodies, the pore size of the rolled filter membrane is in the range of 0.2 μm to about 0.45 μm.

[0075] In embodiments, the width of the filter membrane roll is about 10 mm to about 600 mm. For example, the width of the filter membrane roll may vary depending on the size of the dynamic filtration system, the size of at least one output head, or the membrane support structure.

[0076] In embodiments, the filter membrane roll also functions as a supply roll communicating with a collection reel, that is, the filter membrane starts from a prefabricated roll and extends to an initially empty collection roll to create a reel-to-reel system.

[0077] In embodiments, the membrane support structure of the dynamic filtration module comprises a mechanically flat contact surface derived from a material with a low static friction coefficient (e.g., polytetrafluoroethylene (PTFE)) and an opening continuous with a vacuum line. For example, the static friction coefficient is in the range of about 0.01 to about 0.1, about 0.01 to about 0.05, or about 0.05 to about 0.1. For example, the membrane support structure of the dynamic filtration module includes an opening. For example, the opening may comprise a mesh, at least one slot, at least one hole, a frit, a porous material, or any combination thereof.

[0078] In embodiments, the membrane support structure of the dynamic filtration module includes a temperature control mechanism. The temperature control mechanism maintains a temperature of 4°C to 37°C. For example, during antibody purification, the temperature control mechanism maintains a temperature of 15°C to 37°C. Exemplary temperature control mechanisms include, but are not limited to, a single loop controller, a multiloop controller, a closed loop controller, a proportional-integral-derivative (PID) controller, a Peltier element, a resistive heating element, and / or a thermal chuck with a circulating water / propylene glycol jacket.

[0079] In embodiments, at least one support rod or roller of the dynamic filtration module has a mechanically flat contact surface derived from a material with a low static friction coefficient (e.g., PTFE, perfluoroalkoxyalkanes (PFA)). For example, the static friction coefficient is in the range of about 0.01 to about 0.1, about 0.01 to about 0.05, or about 0.05 to about 0.1. For example, the support rod or roller may be stationary or rotatable. In some examples, the support rod may further include a bearing, for example, a sleeve bearing.

[0080] In the embodiments, the vacuum system of the dynamic filtration module maintains a gauge pressure of about -0.05 bar to about -0.98 bar.

[0081] In embodiments, a process for continuously removing large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration comprises multistage filtration using at least two individual rolled filter membranes with different pore sizes. For example, this multistage dynamic filtration process comprises at least one first dynamic filtration device having a rolled filter membrane with a large pore size (e.g., 0.45 μm) in fluid communication with at least one second dynamic filtration device having a rolled filter membrane with a small pore size (e.g., 0.2 μm), thereby producing a filtrate containing biological products. Alternatively, a similar result can be achieved by a single dynamic filtration device having at least two rolled filter membranes supplied by separate supply reels, resulting in a stacked set of filter membranes across a target area (e.g., active target area), wherein the heterogeneous mixture first comes into contact with a filter membrane with a larger pore size (e.g., 0.45 μm) and then comes into contact with a filter membrane with a smaller pore size (e.g., 0.2 μm).

[0082] In embodiments, the process described herein comprises continuously transferring a solution to a first module capable of separating the solution into two or more fractions, each fraction comprising at least one fraction containing a biological product, said first module comprising an affinity-based magnetic purification device. For example, the affinity-based magnetic purification device further comprises a suspension of magnetic resin beads. The surface of the magnetic resin beads is connected to, for example, without limitation, protein A, protein G, protein L, antigen protein, protein, receptor, antibody, or aptamer. For example, the magnetic resin beads may be paramagnetic or superparamagnetic.

[0083] For example, the diameter of the magnetic resin beads of an affinity-based magnetic purification device is about 0.2 microns to about 200 microns. In other examples, the diameter of the beads is about 0.2 μm to about 100 μm, about 1 μm to about 200 μm, about 10 μm to about 200 μm, about 20 μm to about 200 μm, about 30 μm to about 200 μm, about 50 μm to about 200 μm, or about 150 μm to about 200 μm. Alternatively, the diameter of the beads is about 1 μm to about 100 μm, or about 50 μm to about 100 μm. The diameter of the magnetic resin beads may vary depending on the biological product being purified and the overall flow rate of the process. For example, the purification of the monoclonal antibody may include magnetic resin beads having a size of about 40 microns to about 90 microns. Additionally, the concentration of the magnetic resin beads may be in the range of about 0.01 wt% to about 25 wt%. For example, the concentration of the magnetic resin beads may be about 1 wt%. In some examples, the purification of the monoclonal antibody may include magnetic resin beads having a concentration of about 1 wt% to about 10 wt%. In other examples, the binding ability of the magnetic resin beads is a function of the bead concentration, the surface area to volume ratio, the affinity ligand density, or any combination thereof. In yet another example, the magnetic resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

[0084] In embodiments, the process described herein comprises continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module, wherein the second module comprises a charge-based magnetic purification device (e.g., a positive and / or negative charge-based magnetic purification device), and said charge-based magnetic purification device further comprises magnetic resin beads. For example, the surface of the magnetic resin beads may have cationic functional groups derived from the binding of positively charged functional groups to enable purification based on charge or electrostatic interactions. For example, said positively charged functional groups include amines, cationic polymers, purely positively charged peptides, purely positively charged proteins, or any combination thereof. Alternatively, the surface of the magnetic resin beads may have anionic functional groups derived from the binding of negatively charged functional groups to enable purification based on charge or electrostatic interactions. For example, the negatively charged functional group comprises a carboxyl, anionic polymer, a purely negatively charged peptide, a purely negatively charged protein, an oligonucleotide, or any combination thereof. For example, the magnetic resin beads may be paramagnetic or superparamagnetic.

[0085] In embodiments, the magnetic resin beads of the charge-based magnetic purification device have a diameter of about 0.2 microns to about 200 microns. The diameter of the magnetic resin beads may vary depending on the biological product being purified and the overall flow rate of the process. For example, the purification of a monoclonal antibody may include magnetic resin beads having a size of about 40 microns to about 90 microns. Additionally, the concentration of the magnetic resin beads may be in the range of about 0.01 wt% to about 25 wt%. For example, the concentration of the magnetic resin beads may be about 1 wt%. In some examples, the purification of the monoclonal antibody may include magnetic resin beads having a concentration of about 1 wt% to about 10 wt%. In other examples, the charge or electrostatic association capacity of the magnetic resin beads is a function of the bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In another example, the magnetic resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

[0086] In embodiments, as described herein, one or both of the first (affinity-based magnetic purification) and / or second (charge-based magnetic purification including a positive and / or negative charge-based magnetic purification device) module(s) may also include at least one external magnetic field. For example, at least one external magnetic field includes a permanent magnet or an electromagnet. At least one external magnetic field includes a magnetic field strength of about 0.01 Tesla to about 1 Tesla (e.g., up to 1 Tesla). Alternatively, at least one external magnetic field is shielded.

[0087] In embodiments, the loop conveyor system has at least two transport containers filled with magnetic resin beads configured to continuously receive a mixture containing a biological product and then transport a resulting heterogeneous mixture containing the biological product, magnetic resin beads, a buffer, or any combination thereof. For example, at least one of the at least two transport containers is located within or near an external magnetic field to attract the magnetic resin beads.

[0088] In embodiments, the pick-and-place robotic system has at least two transport containers filled with magnetic resin beads configured to continuously receive a mixture containing a biological product and then transport a resulting heterogeneous mixture containing the biological product, magnetic resin beads, a buffer, or any combination thereof. For example, at least one of the at least two transport containers is placed in or near an external magnetic field to attract the magnetic resin beads.

[0089] In embodiments, the first (affinity-based magnetic purification) and / or second (charge-based magnetic purification including a positive and / or negative charge-based magnetic purification device) module further comprises at least one tangential flow filtration system operating in a fed-batch or perfusion mode. For example, the tangential flow filtration system may be used to concentrate a fraction containing a biological product and to buffer exchange.

[0090] In embodiments, the process described herein comprises continuously transferring a solution to a first module capable of separating the solution into two or more fractions, each fraction comprising at least one fraction containing a biological product, said first module comprising an affinity-based purification device. For example, the affinity-based purification device further comprises a suspension of resin beads. The surface of the resin beads is connected to, for example, without limitation, protein A, protein G, protein L, antigen protein, protein, receptor, antibody, or aptamer.

[0091] For example, the diameter of the resin beads in an affinity-based purification device is about 0.2 microns to about 200 microns. The diameter of the resin beads may vary depending on the biological product being purified and the overall flow rate of the process. For example, the purification of a monoclonal antibody may include resin beads with a size of about 90 microns. Additionally, the concentration of the resin beads may be in the range of about 0.01 wt% to about 25 wt%. For example, the concentration of the resin beads may be about 1 wt%. In some examples, the purification of a monoclonal antibody may include resin beads having a concentration of about 1 wt% to about 10 wt%. In other examples, the binding ability of the resin beads is a function of the bead concentration, the surface area to volume ratio, the affinity ligand density, or any combination thereof.

[0092] In another example, the resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

[0093] In embodiments, the process described herein comprises continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module, wherein the second module comprises a charge-based purification device (e.g., a positive and / or negative charge-based purification device), and said charge-based purification device further comprises resin beads. For example, the surface of the resin beads may have a cationic functional group derived from the binding of a positively charged functional group to enable purification based on charge or electrostatic interaction. For example, said positively charged functional group comprises an amine, a cationic polymer, a purely positively charged peptide, a purely positively charged protein, or any combination thereof. Alternatively, the surface of the resin beads may have an anionic functional group derived from the binding of a negatively charged functional group to enable purification based on charge or electrostatic interaction. For example, said negatively charged functional group comprises a carboxyl, anionic polymer, a purely negatively charged peptide, a purely negatively charged protein, an oligonucleotide, or any combination thereof.

[0094] In embodiments, the diameter of the resin beads of the charge-based purification device is about 0.2 microns to about 200 microns. The diameter of the resin beads may vary depending on the biological product being purified and the overall flow rate of the process. For example, the purification of a monoclonal antibody may include resin beads with a size of about 90 microns. Additionally, the concentration of the resin beads may be in the range of about 0.01 wt% to about 25 wt%. For example, the concentration of the resin beads may be about 1 wt%. In some examples, the purification of a monoclonal antibody may include resin beads having a concentration of about 1 wt% to about 10 wt%. In other examples, the charge or electrostatic associative capacitance of the resin beads is a function of the bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In another example, the resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

[0095] In embodiments, a mechanical rotating system (e.g., a system allowing continuous fluid flow between a first and / or second inlet and a first and / or second outlet) has at least two containers comprising (e.g., filled with mobile resin beads) configured to receive (e.g., continuously receive) a mixture containing a biological product, and then transport a resulting heterogeneous mixture containing the biological product, resin beads, a buffer, or any combination thereof to a designated purification location.

[0096] In another embodiment, the system (e.g., a stepwise linear system allowing continuous fluid flow between a first and / or second inlet and a first and / or second outlet) has at least two containers comprising (e.g., filled with mobile resin beads) configured to receive (e.g., continuously receive) a mixture containing a biological product, and then process a resulting mixture containing a biological product, resin beads, a buffer, or any combination thereof.

[0097] In embodiments, the first (affinity-based purification) and / or second (charge-based purification including a positive and / or negative charge-based purification device) module further comprises at least one tangential flow filtration system operating in fed-batch or perfusion mode to concentrate a fraction containing a biological product and buffer exchange.

[0098] In embodiments, the process described herein comprises continuously transferring a solution to a first module capable of separating it into two or more fractions, each fraction comprising at least one fraction containing a biological product, wherein the first module is an affinity-based fluid purification device having at least one hybrid fluidic device or chip. In embodiments, the at least one hybrid fluidic device or chip has an orthogonal current channel, at least one magnetic field, and at least one mechanical force generator. Additionally, the at least one mechanical force generator may include an ultrasonic transducer or a piezoelectric component capable of generating a defined unidirectional force. In other examples, at least one external magnetic field comprises a permanent magnet, an electromagnet, a patterned magnet, or a combination thereof. For example, at least one external magnetic field may exhibit a magnetic field strength of about 0.01 Tesla (T) to about 1 Tesla (e.g., up to 1 Tesla). In other examples, the magnetic field strength is about 0.01 T, about 0.1 T, or about 1 T. In other embodiments, at least one hybrid fluid element or chip has an orthogonal current channel, at least one magnetic field, and at least one dielectrophoretic electrode. At least one dielectrophoretic electrode can induce a defined unidirectional force. Additionally, at least one external magnetic field comprises a permanent magnet, an electromagnet, a patterned magnet, or a combination thereof. For example, at least one external magnetic field may exhibit a magnetic field strength of about 0.01 Tesla to about 1 Tesla (e.g., up to about 1 Tesla).

[0099] In embodiments, the affinity-based fluid purification device further comprises magnetic resin beads. The surface of the magnetic resin beads is connected to, for example, protein A, protein G, protein L, antigen protein, protein, receptor, antibody, or aptamer, without being limited. For example, the magnetic resin beads may be paramagnetic or superparamagnetic.

[0100] In embodiments, the diameter of the magnetic resin beads of the affinity-based fluid purification device is about 0.2 microns to about 200 microns. For example, the purification of a monoclonal antibody may include magnetic resin beads with a size of about 40 microns. Additionally, the concentration of the magnetic resin beads may be in the range of about 0.01 wt% to about 25 wt%. For example, the initial concentration of the magnetic resin beads may be about 1 wt%. In some examples, the purification of the monoclonal antibody may include magnetic resin beads having a concentration of about 1 wt% to about 10 wt%. In other examples, the binding ability of the magnetic resin beads is a function of the bead concentration, the surface area to volume ratio, the affinity ligand density, or any combination thereof. In yet another example, the magnetic resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

[0101] In embodiments, the process described herein comprises continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module, wherein the second module comprises a charge-based fluid purification device. For example, the charge-based fluid purification device comprises at least one hybrid fluid element or chip. The at least one hybrid fluid element or chip may have an orthogonal current channel, at least one magnetic field, and at least one mechanical force generator. Additionally, the at least one mechanical force generator comprises an ultrasonic transducer or a piezoelectric component capable of generating a defined unidirectional force. In other examples, at least one external magnetic field comprises a permanent magnet, an electromagnet, a patterned magnet, or a combination thereof. For example, at least one external magnetic field may exhibit a magnetic field strength of about 0.01 Tesla (T) to about 1 Tesla (e.g., up to about 1 Tesla). In other examples, the magnetic field strength is about 0.01T, about 0.1T, or about 1T. In another embodiment, the hybrid fluid device or chip may have an orthogonal current channel, at least one magnetic field, and at least one dielectrophoretic electrode, wherein the at least one dielectrophoretic electrode may induce a defined unidirectional force. Additionally, at least one external magnetic field comprises a permanent magnet or an electromagnet. For example, at least one external magnetic field comprises a magnetic field strength of about 0.01 Tesla to about 1 Tesla (e.g., up to about 1 Tesla).

[0102] In embodiments, a charge-based fluid purification device (e.g., a positive charge and / or negative charge-based fluid purification device) further comprises a suspension of magnetic resin beads. The surface of the magnetic resin beads has a cationic functional group derived from the binding of a positively charged functional group to enable purification based on charge or electrostatic interactions. The positively charged functional group includes an amine, a cationic polymer, a purely positively charged peptide, a purely positively charged protein, or any combination thereof. Alternatively, the surface of the magnetic resin beads may include an anionic functional group derived from the binding of a negatively charged functional group to enable purification based on charge or electrostatic interactions. The negatively charged functional group includes a carboxyl, anionic polymer, a purely negatively charged peptide, a purely negatively charged protein, an oligonucleotide, or any combination thereof. For example, the magnetic resin beads may be paramagnetic or superparamagnetic.

[0103] For example, the diameter of the magnetic resin beads in a charge-based fluid purification device is about 0.2 microns to about 200 microns. The diameter of the magnetic resin beads may vary depending on the biological product being purified and the flow rate of the process. For example, the purification of a monoclonal antibody may include magnetic resin beads with a size of about 40 microns. Additionally, the concentration of the magnetic resin beads may be in the range of about 0.01 wt% to about 25 wt%. For example, the concentration of the magnetic resin beads may be about 1 wt%. In some examples, the purification of the monoclonal antibody may include magnetic resin beads having a concentration of about 1 wt% to about 10 wt%. In other examples, the charge or electrostatic associative capacitance of the magnetic resin beads is a function of the bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In another example, the magnetic resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

[0104] In embodiments, the first (affinity-based fluid purification) module further comprises at least one equilibration vessel enabling binding of a biological product to a magnetic resin bead surface, and at least one low pH equilibration vessel enabling de-binding interaction of the biological product from the magnetic resin bead surface.

[0105] In embodiments, the second module (charge-based fluid purification including a positive and / or negative charge-based fluid purification device) further comprises at least one association equilibrium vessel enabling association based on charge or electrostatic interaction between the magnetic resin bead surface and the biological product, and at least one dissociation equilibrium vessel enabling dissociation of the biological product from the magnetic resin bead surface. For example, a plurality of dissociation equilibrium vessels are used in conjunction with a plurality of charge-based fluid purification devices to achieve gradient dissociation, such as, for example, a pH gradient or an ionic strength gradient.

[0106] In embodiments, the magnetic resin beads described herein are recycled and reused. For example, the beads may be reused at least two, three, four, or more times to purify a biological product. To enable the recycling and reuse of the magnetic resin beads, at least one regeneration equilibrium vessel may be used in conjunction with a tangential flow filtration system to concentrate the magnetic resin beads and buffer exchange them to return the magnetic resin beads to their initial state.

[0107] As described herein, the first (affinity-based fluid purification) and / or second (charge-based fluid purification including a positive and / or negative charge-based fluid purification device) module for purifying biological products comprises a hybrid microfluidic, mesofluidic, millifluidic, or macrofluidic device or chip, or any combination thereof, for example, a hybrid microfluidic device comprising at least one magnetic field and at least one of a piezoelectric component or a dielectrophoretic electrode.

[0108] In embodiments, the first (affinity-based fluid purification) and / or second (charge-based fluid purification including a positive and / or negative charge-based fluid purification device) module further comprises at least one tangential flow filtration system operating in a fed-batch or perfusion mode to concentrate a fraction containing a biological product and buffer exchange.

[0109] In embodiments, the process described herein comprises continuously transferring a filtrate to a first module capable of separating a solution into two or more fractions, each fraction comprising at least one fraction containing a biological product, said first module comprising an affinity-based TFF purification device. For example, the affinity-based TFF purification device comprises at least three fluidly communicating tangential flow filtration systems.

[0110] In embodiments, the affinity-based TFF purification device further comprises a suspension of resin beads. The surface of the resin beads is connected to, for example, protein A, protein G, protein L, antigen protein, protein, receptor, antibody, or aptamer, without being limited.

[0111] In embodiments, the diameter of the resin beads of the affinity-based TFF purification device is about 10 microns to about 200 microns. The diameter of the resin beads may vary depending on the biological product being purified and the overall flow rate of the process. For example, the purification of a monoclonal antibody may involve resin beads with a size of about 90 microns. Additionally, the concentration of the resin beads may be in the range of about 0.01 wt% to about 25 wt%. For example, the concentration of the resin beads may be about 1 wt% to about 20 wt%. In another example, the binding ability of the resin beads is a function of the bead concentration, the surface area to volume ratio, the affinity ligand density, or any combination thereof. In yet another example, the resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

[0112] In embodiments, the process described herein comprises continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module, wherein the second module comprises a charge-based TFF purification device (e.g., a positive and / or negative charge-based TFF purification device). For example, the charge-based TFF purification device has at least three fluidly communicating tangential flow filtration systems.

[0113] In embodiments, the charge-based TFF purification device further comprises a suspension of resin beads. For example, the surface of the resin beads may have a cationic functional group derived from the binding of a positively charged functional group to enable purification based on charge or electrostatic interactions. For example, the positively charged functional group includes an amine, a cationic polymer, a purely positively charged peptide, a purely positively charged protein, or any combination thereof. Alternatively, the surface of the resin beads may have an anionic functional group derived from the binding of a negatively charged functional group to enable purification based on charge or electrostatic interactions. For example, the negatively charged functional group includes a carboxyl, anionic polymer, a purely negatively charged peptide, a purely negatively charged protein, an oligonucleotide, or any combination thereof.

[0114] In embodiments, the diameter of the resin beads of the charge-based TFF purification device is about 0.2 microns to about 200 microns. The diameter of the resin beads may vary depending on the biological product being purified and the overall flow rate of the process. For example, the purification of a monoclonal antibody may involve resin beads with a size of about 90 microns. Additionally, the concentration of the resin beads may be in the range of about 0.01 wt% to about 25 wt%. For example, the concentration of the resin beads may be about 1 wt% to about 20 wt%. In another example, the charge or electrostatic associative capacitance of the resin beads is a function of the bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In yet another example, the resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

[0115] In embodiments, the first (affinity-based TFF purification) module further comprises at least one equilibrium vessel enabling binding of the biological product to the surface of a resin bead, and at least one low pH equilibrium vessel enabling debinding interaction of the biological product from the surface of the resin bead.

[0116] In embodiments, the second module (charge-based TFF purification including a positive and / or negative charge-based TFF purification device) further comprises at least one association equilibrium vessel enabling association based on charge or electrostatic interaction between the resin bead surface and the biological product, and at least one dissociation equilibrium vessel enabling dissociation of the biological product from the resin bead surface. For example, a plurality of dissociation equilibrium vessels are used in conjunction with a plurality of charge-based fluid purification devices to achieve gradient dissociation, such as, for example, a pH gradient or an ionic strength gradient.

[0117] In embodiments, the resin beads described herein are recycled and reused. For example, the beads may be reused at least two, three, four, or more times to purify a biological product. To enable the recycling and reuse of the resin beads, at least one regeneration equilibrium vessel may be used with a tangential flow filtration system to concentrate the resin beads and buffer exchange them to return the resin beads to their initial state.

[0118] In embodiments, the first (affinity-based TFF purification) and / or second (charge-based TFF purification including a positive and / or negative charge-based TFF purification device) module further comprises at least one tangential flow filtration system for concentrating and buffering a fraction containing a biological product.

[0119] In another embodiment, the process described herein comprises continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module, wherein the second module comprises an isoelectric point-based fluid purification device, also referred to herein as a free-flow electrophoresis device. For example, the free-flow electrophoresis device comprises at least one fluid element comprising a fluid channel created between two parallel plates to operate in an isoelectric focusing operation mode, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a pH gradient. In another example, the isoelectric point-based fluid purification module comprises at least one first fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a coarse pH gradient (e.g., a pH range of about 2 to about 10) across a main separation channel; and at least one second fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient across the main separation channel (e.g., a pH range of about 5 to about 8). For example, the pH gradient across the main separation channel can be further refined using additional subsequent fluid elements or chips comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow (e.g., a pH range of about 7.1 to about 7.6). Alternatively, the free-flow electrophoresis device has at least one fluid element that has no pH gradient, comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, to operate in band electrophoresis or charge separation operating mode.

[0120] In another example, an isoelectric point-based fluid purification module comprises at least one first fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant basic pH (e.g., pH greater than 7); and at least one second fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant acidic pH (e.g., pH less than 7). Additionally, a free-flow electrophoresis device has at least one fluid element comprising a fluid channel created between two parallel plates to operate in an isokinetic electrophoresis operating mode, an electric field or electric field gradient perpendicular to the direction of fluid flow, and both an acidic pH gradient and a basic pH gradient separated by a spacer solution (e.g., NaCl solution).

[0121] In some embodiments, the isoelectric point-based fluid purification module comprises at least one first fluid element comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, and at least one second fluid element comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, wherein each element is connected in series and can operate in independent operating modes that enable purification. For example, at least one first free-flow electrophoresis device can operate in an isoelectric focusing mode and at least one second free-flow electrophoresis device can operate in an isokinetic electrophoresis mode and can be operated sequentially through a series connection to increase separation resolution.

[0122] In another embodiment, without limitation, the isoelectric point-based fluid purification module comprises: at least one first fluid element comprising a fluid channel having at least one dielectrophoretic electrode capable of inducing a defined unidirectional force; at least one second fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across a main separation channel (e.g., a pH range of about 2 to about 10); and at least one third fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient across a main separation channel (e.g., a pH range of about 5 to about 8). For example, the pH gradient across the main separation channel can be further refined using additional subsequent fluid elements or chips comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow (e.g., a pH range of about 7.1 to about 7.6).

[0123] In another embodiment, the isoelectric point-based fluid purification device further includes an active cooling system (e.g., a Peltier element, a thermal chuck with a circulating water / propylene glycol jacket) to enable temperature control and Joule heat dissipation. For example, the active cooling system can control cooling and / or Joule heat dissipation to enable operation in a range of about 4°C to about 50°C, preferably about 4°C to about 37°C. For example, when isolating a biological product (e.g., a monoclonal antibody), the temperature is maintained at about 4°C to about 37°C.

[0124] In an additional embodiment, the process for purifying the biological product may also include virus inactivation, virus filtration, tangential flow filtration (TFF), high-performance tangential flow filtration (HP-TFF), ultrafiltration / decongestion filtration (UF / DF), filter sterilization, fill-finish, freeze-drying, or any combination thereof, which are performed semi-continuously and downstream of the second module.

[0125] For example, the entire process described herein (the process for purifying biological products) is carried out at a temperature within the range of about 4°C to about 50°C, preferably about 4°C to about 37°C. Additionally, the commercial production-scale process for purifying biological products is carried out in a system having a footprint occupying much less square feet than current technology, without sacrificing product throughput or yield on a kilogram / year basis. For example, the process for producing and purifying monoclonal antibodies described herein operates in a footprint occupying up to about 30,000 square feet. In contrast, current monoclonal antibody production and downstream processes require at least 200,000 square feet.

[0126] The process described herein is used to purify a biological product, the biological product comprising, but not limited to, a protein or fragment thereof (polypeptide), an antibody or fragment thereof, a cytokine, a chemokine, an enzyme, a growth factor, an oligonucleotide, a virus, an adenovirus, an adeno-associated virus, or a lentivirus.

[0127] Dynamic filtration module

[0128] In some aspects, the present specification provides a dynamic filtration module for removing large impurities from a biological product in a heterogeneous mixture. The dynamic filtration module continuously feeds the biological product to the dynamic filtration module from at least one output head that fluidly communicates with an input line under negative pressure.

[0129] In embodiments, the dynamic filtration module includes a filter membrane roll, a membrane support structure, at least one support rod or roller, at least one vacuum line, a vacuum system, and at least one vacuum collection vessel.

[0130] The dynamic filtration module comprises a rolled filter membrane extending between a feed reel and a collect reel, said filter membrane having a target region (e.g., an active target region) configured to accommodate a heterogeneous mixture. For example, the filter membrane of the filter membrane roll is composed of a suitable material comprising, but not limited to, polyethersulfone (PES), hydrophilic polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), or hydrophilic PTFE.

[0131] In embodiments, the pore size of the rolled filter membrane varies depending on the biological product being purified. For example, the pore size of the rolled filter membrane is in the range of 0.1 μm to 1 μm. Alternatively, the pore size is in the range of about 0.2 μm to about 0.45 μm, or the pore size is less than about 0.45 μm. In another example, when purifying antibodies, the pore size of the rolled filter membrane is in the range of 0.2 μm to about 0.45 μm.

[0132] In embodiments, the width of the filter membrane roll is about 10 mm to about 600 mm. For example, the width of the filter membrane roll may vary depending on factors such as the size of the dynamic filtration system and the size of the membrane support structure.

[0133] In embodiments, the filter membrane roll also functions as a supply reel communicating with a collection reel to create a reel-to-reel system. During operation, the heterogeneous mixture is applied to a new, unused target area of ​​the filter membrane, also referred to herein as a "target area" (or "active target area"), whereby the filter membrane continues to move across the membrane support structure at an appropriate transport speed as a result of the collection reel collecting the used portion of the filter membrane. For example, the supply reel movement is controlled by a servo motor connected to a gearbox that limits the rotational speed per minute (RPM) to a ratio of 200:1 to enable low membrane transport speeds with high torque. The collection reel movement is controlled by a servo motor connected to a gearbox that limits the RPM to a ratio of 200:1 to enable low membrane transport speeds with high torque. In addition, the supply reel motor and the collection reel motor are controlled by a closed-loop controller that operates a feedback mechanism to ensure a constant membrane transport speed and the diameter of the filter membrane rolls that constantly change in both the supply reel and the collection reel during operation.

[0134] For example, a consistent membrane transport rate can be ensured using a thickness monitoring system or a rotary encoder. For instance, the feed reel and the collection reel operate in the same direction at the same speed. In another example, the feed reel and the collection reel operate in the same direction at different speeds. Other methods of transporting filter membranes from the feed reel to the collection reel may be considered by experts in the coating and converting industry. In another example, two dynamic filtration systems are operated in parallel. For instance, two parallel dynamic filtration systems can allow for a continuous flow through the system while replacing used filter membrane rolls. Additionally, two parallel dynamic filtration systems can bring the entire vacuum collection vessel into equilibrium with atmospheric pressure, allowing fluid to flow to the first purification module without interrupting the process of continuously receiving heterogeneous mixtures from the bioreactor discharge line.

[0135] Additionally, the dynamic filtration module includes a membrane support structure for supporting a target area (e.g., an active target area) of the filter membrane when the filter membrane experiences negative pressure. The membrane support structure is located between the supply reel and the collection reel, has a mechanically flat contact surface derived from a material with a low coefficient of static friction (e.g., PTFE), and has an opening continuous with the vacuum line. For example, the opening may include a mesh, at least one slot, at least one hole, a frit, a porous material, or any combination thereof.

[0136] In embodiments, at least one support rod or roller of the dynamic filtration module has a mechanically flat contact surface derived from a material with a low coefficient of static friction (e.g., PTFE, PFA). For example, the dynamic filtration module includes at least one support rod or roller having a mechanically flat contact surface to stabilize the movement of a filter membrane across a membrane support structure.

[0137] In embodiments, the membrane support structure of the dynamic filtration module includes a temperature control mechanism for maintaining a desired temperature when evaporative cooling occurs. The temperature control mechanism maintains a temperature of about 4°C to about 37°C. For example, during antibody purification, the temperature control mechanism maintains a temperature within the range of about 15°C to about 37°C.

[0138] In embodiments, the dynamic filtration module includes at least one output head for controlling the flow of a heterogeneous mixture and distributing the heterogeneous mixture to a target area (e.g., an active target area) of a filter membrane. For example, the at least one output head is a tube or a slot die.

[0139] In some embodiments, the dynamic filtration module further includes at least one additional input line for supplying a cleaning buffer through a coaxial output head, a separate single-axis output head, a separate slot die output head, or a slot die output head with multiple openings.

[0140] In some embodiments, the dynamic filtration module includes elements known in the coating and converting industry, for example, without limitation, active or passive edge guides, tension regulators (e.g., dancers), brakes and tension detectors, or any combination thereof.

[0141] In embodiments, the dynamic filtration module comprises a vacuum system continuous with a membrane support structure to apply a negative pressure across a target area (e.g., an active target area) of the filter membrane, wherein the negative pressure allows the target area (e.g., an active target area) of the filter membrane across the membrane support structure to enable the collection of filtrate containing biological products. For example, the vacuum system of the dynamic filtration module maintains a gauge pressure of about -0.05 bar to about -0.98 bar for continuous filtration.

[0142] In embodiments, the dynamic filtration module further comprises at least one vacuum collection vessel configured to collect the filtrate, and at least one sensor or detector. For example, two parallel dynamic filtration systems are operated with a time delay to allow the first vacuum collection vessel to be fully filled and equilibrated with atmospheric pressure, after which it can flow continuously through the system.

[0143] In embodiments, a process for continuously removing large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration comprises multistage filtration using at least two individual rolled filter membranes with different pore sizes. For example, this multistage dynamic filtration process comprises at least one first dynamic filtration device having a rolled filter membrane with a large pore size (e.g., 0.45 μm) in fluid communication with at least one second dynamic filtration device having a rolled filter membrane with a small pore size (e.g., 0.2 μm), thereby producing a filtrate containing biological products. Alternatively, a similar result can be achieved by a single dynamic filtration device having at least two rolled filter membranes supplied by separate supply reels, resulting in a stacked set of filter membranes across an active target area, wherein the heterogeneous mixture first comes into contact with a filter membrane of a larger pore size (e.g., 0.45 μm) and then comes into contact with a filter membrane of a smaller pore size (e.g., 0.2 μm).

[0144] Affinity-based magnetic purification module

[0145] In aspects, the present specification provides an affinity-based magnetic purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The affinity-based magnetic purification module comprises at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between at least one inlet and at least one outlet, wherein the flow rate may be consistent and constant, for example, during steady-state operation.

[0146] In embodiments, the affinity-based magnetic purification module comprises a suspension of magnetic resin beads, wherein the surface of the magnetic resin beads is connected to a protein A, protein G, protein L, antigen protein, protein, receptor, antibody, or aptamer configured to selectively bind to the biological product without limitation. For example, the magnetic resin beads are mobile.

[0147] Additionally, the affinity-based magnetic purification module includes a loop conveyor system comprising at least two transport containers filled with magnetic resin beads configured to continuously receive a mixture containing a biological product and then transport a generated heterogeneous mixture containing the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0148] Alternatively, an affinity-based magnetic purification module comprises a pick-and-place robotic system comprising at least two transport containers filled with magnetic resin beads configured to continuously receive a mixture containing a biological product and then transport a generated heterogeneous mixture containing the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0149] In embodiments, the affinity-based magnetic purification module includes at least one external magnetic field that can be used to attract and separate the magnetic resin beads from a heterogeneous mixture to enable washing. Additionally, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from the heterogeneous mixture to enable the elution of the biological product. Alternatively, at least one external magnetic field may be used to enable the recycling of the magnetic resin beads. For example, mixing of the magnetic resin beads can be achieved by placing at least one transport container between two separate opposing magnetic fields that switch between an on state and an off state.

[0150] In embodiments, the affinity-based magnetic purification module includes at least one coupling / washing buffer system.

[0151] In embodiments, the affinity-based magnetic purification module includes at least one elution buffer system.

[0152] In embodiments, the affinity-based magnetic purification module includes at least one magnetic resin bead regeneration buffer system.

[0153] In embodiments, the affinity-based magnetic purification module includes at least one aspirator system for removing waste solution from at least two transport containers.

[0154] In embodiments, the affinity-based magnetic purification module includes at least one sensor or detector.

[0155] In embodiments, the affinity-based magnetic purification module includes at least one fluid handling pump.

[0156] Positive charge-based magnetic purification module

[0157] In aspects, the present specification provides a positive charge-based magnetic purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The positive charge-based magnetic purification module comprises at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between at least one inlet and at least one outlet, wherein the flow rate may be consistent and constant, for example, during steady-state operation.

[0158] In embodiments, a positive charge-based magnetic purification module comprises a suspension of magnetic resin beads, wherein the surface of the magnetic resin beads comprises cationic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength. For example, the magnetic resin beads are mobile.

[0159] Additionally, the positive charge-based magnetic purification module includes a loop conveyor system comprising at least two transport containers filled with magnetic resin beads configured to continuously receive a mixture containing a biological product and then transport a generated heterogeneous mixture containing the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0160] Alternatively, a positive charge-based magnetic purification module comprises a pick-and-place robotic system comprising at least two transport containers filled with magnetic resin beads configured to continuously receive a mixture containing a biological product and then transport a generated heterogeneous mixture containing the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0161] In embodiments, a positive charge-based magnetic purification module comprises at least one external magnetic field that can be used to attract and separate the magnetic resin beads from a heterogeneous mixture to enable washing. Additionally, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from the heterogeneous mixture to enable the dissociation and purification of the biological product. Alternatively, at least one external magnetic field may be used to enable the recycling of the magnetic resin beads. For example, mixing of the magnetic resin beads can be achieved by placing at least one transport container between two separate opposing magnetic fields that switch between an on state and an off state.

[0162] In embodiments, the positive charge-based magnetic purification module includes at least one assembly / washing buffer system.

[0163] In embodiments, the positive charge-based magnetic purification module comprises at least one dissociation buffer system. For example, several dissociation buffers with different pH, ionic strength, or any combination thereof are used sequentially to produce a gradient dissociation effect.

[0164] In embodiments, the positive charge-based magnetic purification module includes at least one magnetic resin bead regeneration buffer system.

[0165] In embodiments, the positive charge-based magnetic purification module includes at least one suction system for removing waste liquid from at least two transport containers.

[0166] In embodiments, the positive charge-based magnetic purification module includes at least one sensor or detector.

[0167] In embodiments, the positive charge-based magnetic purification module includes at least one fluid handling pump.

[0168] Negative charge-based magnetic purification module

[0169] In aspects, the present specification provides a negative charge-based magnetic purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The negative charge-based magnetic purification module comprises at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between at least one inlet and at least one outlet, wherein the flow rate may be consistent and constant, for example, during steady-state operation.

[0170] In embodiments, a negative charge-based magnetic purification module comprises a suspension of magnetic resin beads, wherein the surface of the magnetic resin beads comprises an anionic functional group configured to selectively associate with the biological product at a specific pH and ionic strength. For example, the magnetic resin beads are mobile.

[0171] Additionally, the negative charge-based magnetic purification module includes a loop conveyor system comprising at least two transport containers filled with magnetic resin beads configured to continuously receive a mixture containing a biological product and then transport a generated heterogeneous mixture containing the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0172] Alternatively, a negative charge-based magnetic purification module comprises a pick-and-place robotic system comprising at least two transport containers filled with magnetic resin beads configured to continuously receive a mixture containing a biological product and then transport a generated heterogeneous mixture containing the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0173] In embodiments, the negative charge-based magnetic purification module includes at least one external magnetic field that can be used to attract and separate the magnetic resin beads from a heterogeneous mixture to enable washing. Additionally, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from the heterogeneous mixture to enable the dissociation and purification of the biological product. Alternatively, at least one external magnetic field may be used to enable the recycling of the magnetic resin beads. For example, mixing of the magnetic resin beads can be achieved by placing at least one transport container between two separate opposing magnetic fields that switch between an on state and an off state.

[0174] In embodiments, the negative charge-based magnetic purification module includes at least one assembly / washing buffer system.

[0175] In embodiments, the negative charge-based magnetic purification module comprises at least one dissociation buffer system. For example, several dissociation buffers with different pH, ionic strength, or any combination thereof are used sequentially to produce a gradient dissociation effect.

[0176] In embodiments, the negative charge-based magnetic purification module includes at least one magnetic resin bead regeneration buffer system.

[0177] In embodiments, the negative charge-based magnetic purification module includes at least one suction system for removing waste liquid from at least two transport containers.

[0178] In embodiments, the negative charge-based magnetic purification module includes at least one sensor or detector.

[0179] In embodiments, the negative charge-based magnetic purification module includes at least one fluid handling pump.

[0180] Affinity-based purification module

[0181] In aspects, the present specification provides an affinity-based purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The affinity-based purification module comprises at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between at least one inlet and at least one outlet, wherein the flow rate may be consistent and constant, for example, during steady-state operation.

[0182] In embodiments, the affinity-based purification module comprises a suspension of resin beads, wherein the surface of the resin beads is connected to a protein A, protein G, protein L, antigen protein, protein, receptor, antibody, or aptamer configured to selectively bind to the biological product without limitation. For example, the resin beads are mobile.

[0183] In embodiments, the affinity-based purification module comprises a lid system having at least one gasketed lid, wherein the at least one gasketed lid has at least one inlet for introducing gas to control positive head pressure. Additionally, the lid system has at least one vent port to enable equilibrium with atmospheric pressure, at least one inlet for introducing a suspension of resin beads, at least one inlet for receiving a filtrate containing a biological product, and / or at least two inlets for introducing a buffer system for dispersing the resin beads to enable washing, elution from, or regeneration of the resin beads. In some embodiments, the at least one gasketed lid also includes a port for receiving an overhead stirring impeller to enable dispersion of the resin beads. For example, the lid system controls movement along the z-axis.

[0184] In embodiments, the affinity-based purification module comprises a mechanical rotation system, for example, a carousel comprising at least two containers filled with resin beads configured to continuously receive a mixture containing a biological product and then transport a resulting heterogeneous mixture containing the biological product, resin beads, a buffer, or any combination thereof. For example, the carousel is a rotating structure that holds at least two containers and transports them to different process locations. In some examples, the mechanical rotation system is configured to mate with a lead system to enable pressurization and liquid handling. In other examples, the mechanical rotation system controls movement or rotation in the xy plane.

[0185] In embodiments, each of the at least two vessels of the affinity-based purification module has a supported basement filter or filter membrane. For example, the basement filter (or filter membrane) enables the retention of resin beads during bonding, debonding, washing, elution, and / or regeneration process steps. For example, the at least two vessels may further include a valve for controlling liquid flow.

[0186] In another embodiment, the affinity-based purification module comprises at least two containers filled with resin beads configured to sequentially receive a mixture containing a biological product, for example, a stepwise linear system, and then process a resulting heterogeneous mixture containing the biological product, resin beads, a buffer, or any combination thereof. For example, the at least two containers are configured to be matched with a lead system to enable pressurization and liquid handling.

[0187] In embodiments, the affinity-based purification module includes a collection system capable of collecting waste, fractions containing biological products, or any combination thereof by interfacing with at least one of at least two containers of a mechanical rotating system. For example, the collection system controls movement along the z-axis.

[0188] In another embodiment, the affinity-based purification module includes a collection system capable of collecting waste, fractions containing biological products, or any combination thereof by interfacing with at least one of at least two vessels of a stepwise linear system. For example, the collection system is connected to at least one of the at least two vessels.

[0189] In embodiments, the affinity-based purification module comprises at least one gas. In some embodiments, without limitation, the gas comprises filtered nitrogen or compressed dry air. For example, the gas generates a pressure head of about 0.1 psi to about 30 psi.

[0190] In the embodiments, the affinity-based purification module includes at least one binding / washing buffer system.

[0191] In embodiments, the affinity-based purification module includes at least one low pH elution buffer system.

[0192] In embodiments, the affinity-based purification module includes at least one resin bead regeneration buffer system.

[0193] In the embodiments, the affinity-based purification module includes at least one collection container.

[0194] In embodiments, the affinity-based purification module includes at least one sensor or detector.

[0195] In embodiments, the affinity-based purification module includes at least one fluid handling pump.

[0196] Positive charge-based purification module

[0197] Additionally, the present specification provides a positive charge-based purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product.

[0198] The positive charge-based purification module includes at least one inlet and at least one outlet, configured to allow continuous fluid flow between at least one inlet and at least one outlet, wherein the flow rate can be consistent and constant, for example, during steady-state operation.

[0199] In embodiments, the positive charge-based purification module comprises a suspension of resin beads, wherein the surface of the resin beads comprises cationic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength. For example, the resin beads are mobile.

[0200] In embodiments, a positive charge-based purification module comprises a lead system having at least one gasketed lead, wherein the at least one gasketed lead comprises: at least one inlet for introducing gas to control a positive pressure head pressure; at least one inlet for introducing a suspension of resin beads; at least one vent port to equilibrate with atmospheric pressure; at least one inlet for receiving a filtrate containing a biological product; and at least two inlets for introducing a buffer system for dispersing the resin beads to enable washing, dissociation from, or regeneration of the resin beads. In some embodiments, the at least one gasketed lead further comprises a port for receiving an overhead stirring impeller to enable dispersion of the resin beads. For example, the lead system controls movement along the z-axis.

[0201] In embodiments, the positive charge-based purification module comprises a mechanical rotation system, for example, a carousel comprising at least two containers filled with resin beads configured to continuously receive a mixture containing a biological product and then transport a resulting heterogeneous mixture containing the biological product, resin beads, a buffer, or any combination thereof. For example, the carousel is a rotating structure that holds at least two containers and transports them to different process locations. In some examples, the mechanical rotation system is configured to be coupled with a lead system to enable pressurization. In other examples, the mechanical rotation system controls movement or rotation in the xy plane.

[0202] In embodiments, each of at least two vessels of the positive charge-based purification module has a supported base filter or filter membrane. For example, the base filter (or filter membrane) enables the retention of resin beads during assembly, washing, dissociation, and / or regeneration process steps. For example, at least two vessels may further include a valve for controlling liquid flow.

[0203] In another embodiment, the positive charge-based purification module comprises at least two containers filled with resin beads configured to sequentially receive a mixture containing a biological product, for example, a stepwise linear system, and then process a resulting heterogeneous mixture containing the biological product, resin beads, a buffer, or any combination thereof. For example, the at least two containers are configured to be matched with a lead system to enable pressurization and liquid handling.

[0204] In embodiments, the positive charge-based purification module includes a collection system capable of collecting waste, fractions containing biological products, or any combination thereof by interfacing with at least one of at least two vessels of a mechanical rotation system. For example, the collection system controls movement along the z-axis.

[0205] In another embodiment, the positive charge-based purification module includes a collection system capable of collecting waste, fractions containing biological products, or any combination thereof by interfacing with at least one of at least two vessels of a stepwise linear system. For example, the collection system is connected to at least one of the at least two vessels.

[0206] In embodiments, the affinity-based purification module comprises at least one gas. In some embodiments, without limitation, the gas comprises filtered nitrogen or compressed dry air. For example, the gas generates a pressure head of about 0.1 psi to about 30 psi.

[0207] In embodiments, the positive charge-based purification module includes at least one assembly / washing buffer system.

[0208] In embodiments, the positive charge-based purification module includes at least one dissociation buffer system. For example, to produce a gradient dissociation effect, several dissociation buffers with different pH, ionic strength, or any combination thereof are used sequentially or in series.

[0209] In embodiments, the positive charge-based purification module includes at least one resin bead regeneration buffer system.

[0210] In embodiments, the positive charge-based purification module includes at least one collection container.

[0211] In embodiments, the positive charge-based purification module includes at least one sensor or detector.

[0212] In embodiments, the positive charge-based purification module includes at least one fluid handling pump.

[0213] Negative charge-based purification module

[0214] In aspects, the present specification provides a negative charge-based purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The negative charge-based purification module comprises at least one inlet and at least one outlet, wherein the inlet and outlet are configured to allow continuous fluid flow between the at least one inlet and at least one outlet, wherein the flow rate may be consistent and constant, for example, during steady-state operation.

[0215] In embodiments, the negative charge-based purification module comprises a suspension of resin beads, wherein the surface of the resin beads comprises a cationic functional group configured to selectively associate with the biological product at a specific pH and ionic strength.

[0216] In embodiments, the negative charge-based purification module comprises a lead system having at least one gasketed lead, said at least one gasketed lead comprises: at least one inlet for introducing gas to control a static pressure head pressure; at least one vent port to equilibrate with atmospheric pressure; at least one inlet for introducing a suspension of resin beads; at least one inlet for receiving a filtrate containing a biological product; and at least two inlets for introducing a buffer system for dispersing the resin beads to enable washing, dissociation from, or regeneration of the resin beads. In some embodiments, the at least one gasketed lead further comprises a port for receiving an overhead stirring impeller to enable dispersion of the resin beads. For example, said lead system controls movement along the z-axis.

[0217] In embodiments, the negative charge-based purification module comprises a carousel comprising at least two containers filled with resin beads configured to continuously receive a mixture containing, for example, a biological product, and then transport a resulting heterogeneous mixture containing the biological product, resin beads, a buffer, or any combination thereof. For example, the carousel is a rotating structure that holds at least two containers and transports them to different process locations. In some examples, the mechanical rotating system is configured to be coupled with a lead system to enable pressurization. In other examples, the mechanical rotating system controls movement or rotation in the xy plane.

[0218] In embodiments, each of at least two vessels of the positive charge-based purification module has a supported base filter or filter membrane. For example, the base filter (or filter membrane) enables the retention of resin beads during assembly, washing, dissociation, and / or regeneration process steps. For example, at least two vessels may further include a valve for controlling liquid flow.

[0219] In another embodiment, the positive charge-based purification module comprises at least two containers filled with resin beads configured to sequentially receive a mixture containing a biological product, for example, a stepwise linear system, and then process a resulting heterogeneous mixture containing the biological product, resin beads, a buffer, or any combination thereof. For example, the at least two containers are configured to be matched with a lead system to enable pressurization and liquid handling.

[0220] In embodiments, the positive charge-based purification module includes a collection system capable of collecting waste, fractions containing biological products, or any combination thereof by interfacing with at least one of at least two vessels of a mechanical rotation system. For example, the collection system controls movement along the z-axis.

[0221] In another embodiment, the positive charge-based purification module includes a collection system capable of collecting waste, fractions containing biological products, or any combination thereof by interfacing with at least one of at least two vessels of a stepwise linear system. For example, the collection system is connected to at least one of the at least two vessels.

[0222] In embodiments, the affinity-based purification module comprises at least one gas. In some embodiments, without limitation, the gas comprises filtered nitrogen or compressed dry air. For example, the gas generates a pressure head of about 0.1 psi to about 30 psi.

[0223] In embodiments, the negative charge-based purification module includes at least one assembly / wash buffer system.

[0224] In embodiments, the negative charge-based purification module includes at least one dissociation buffer system. For example, several dissociation buffers with different pH, ionic strength, or any combination thereof are used sequentially or in succession to produce a gradient dissociation effect.

[0225] In embodiments, the negative charge-based purification module includes at least one resin bead regeneration buffer system.

[0226] In embodiments, the negative charge-based purification module includes at least one collection container.

[0227] In embodiments, the negative charge-based purification module includes at least one sensor or detector.

[0228] In embodiments, the negative charge-based purification module includes at least one fluid handling pump.

[0229] Affinity-based fluid purification module

[0230] In aspects, the present specification provides an affinity-based fluid purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The affinity-based fluid purification module comprises at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between at least one inlet and at least one outlet, wherein the flow rate may be consistent and constant, for example, during steady-state operation.

[0231] In embodiments, the affinity-based fluid purification module comprises a suspension of magnetic resin beads, wherein the surface of the magnetic resin beads is connected to a protein A, protein G, protein L, antigen protein, protein, receptor, antibody, or aptamer configured to selectively bind to the biological product without limitation. For example, the magnetic resin beads are mobile.

[0232] In embodiments, the affinity-based fluid purification module comprises at least one equilibrium vessel that enables the binding of a biological product to the surface of a magnetic resin bead; and at least one first hybrid orthogonal fluid element comprising an orthogonal fluid channel, at least one magnetic field, and at least one of a piezoelectric component or a dielectrophoretic electrode configured to generate or induce a unidirectional force to separate the magnetic resin beads to which the biological product is bound from the heterogeneous mixture.

[0233] In embodiments, the affinity-based fluid purification module further comprises at least one low pH equilibrium vessel that allows debinding of biological products from the surface of magnetic resin beads; and at least one second hybrid orthogonal fluid element comprising an orthogonal current channel, at least one magnetic field, and at least one of a piezoelectric component or a dielectrophoretic electrode configured to generate or induce a unidirectional force to separate the magnetic resin beads from the unbound biological products and complete their elution.

[0234] In embodiments, the affinity-based fluid purification module further includes at least one tangential flow filtration system operating in fed-batch or perfusion mode to concentrate a fraction containing a biological product and buffer exchange.

[0235] In embodiments, the affinity-based fluid purification module includes at least two buffer systems.

[0236] In embodiments, the affinity-based fluid purification module includes at least one magnetic resin bead regeneration buffer system.

[0237] In embodiments, the affinity-based fluid purification module includes at least one equilibrium vessel configured to enable the recycling of the magnetic resin beads.

[0238] In embodiments, the affinity-based fluid purification module includes at least one sensor or detector.

[0239] In embodiments, the affinity-based fluid purification module includes at least one fluid handling pump.

[0240] Positive charge-based fluid purification module

[0241] In aspects, the present specification provides a positive charge-based fluid purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The positive charge-based fluid purification module comprises at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between at least one inlet and at least one outlet, wherein the flow rate may be consistent and constant, for example, during steady-state operation.

[0242] In embodiments, a positive charge-based fluid purification module comprises a suspension of magnetic resin beads, wherein the surface of the magnetic resin beads comprises cationic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength. For example, the magnetic resin beads are mobile.

[0243] In embodiments, the positive charge-based fluid purification module comprises at least one association equilibrium vessel that enables association between a magnetic resin bead surface and a biological product; and at least one first hybrid orthogonal fluid element comprising an orthogonal current channel, at least one magnetic field, and at least one of a piezoelectric component or a dielectrophoretic electrode configured to generate or induce a unidirectional force to separate the magnetic resin beads to which the biological product is associated from the heterogeneous mixture.

[0244] In embodiments, a positive charge-based fluid purification module comprises at least one dissociation equilibrium vessel allowing the dissociation of a biological product from the surface of magnetic resin beads; and at least one second hybrid orthogonal fluid element comprising an orthogonal current channel, at least one magnetic field, and at least one of a piezoelectric component or a dielectrophoretic electrode configured to generate or induce a unidirectional force to separate the magnetic resin beads from the dissociated biological product and complete its purification. For example, several dissociation equilibrium vessels comprising distinct buffers with different pH, ionic strength, or any combination thereof are used sequentially to create a gradient dissociation effect.

[0245] In embodiments, the positive charge-based fluid purification module further comprises at least one tangential flow filtration system operating in fed-batch or perfusion mode to concentrate a fraction containing a biological product and buffer exchange.

[0246] In embodiments, the positive charge-based fluid purification module includes at least two buffer systems.

[0247] In embodiments, the positive charge-based fluid purification module includes at least one magnetic resin bead regeneration buffer system.

[0248] In embodiments, the positive charge-based fluid purification module includes at least one equilibrium vessel configured to enable the recycling of the magnetic resin beads.

[0249] In embodiments, the positive charge-based fluid purification module includes at least one sensor or detector.

[0250] In embodiments, the positive charge-based fluid purification module includes at least one fluid handling pump.

[0251] Negative charge-based fluid purification module

[0252] In aspects, the present specification provides a negative charge-based fluid purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The negative charge-based fluid purification module comprises at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between at least one inlet and at least one outlet, wherein the flow rate may be consistent and constant, for example, during steady-state operation.

[0253] In embodiments, a negative charge-based fluid purification module comprises a suspension of magnetic resin beads, wherein the surface of the magnetic resin beads comprises anionic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength. For example, the magnetic resin beads are mobile.

[0254] In embodiments, the negative charge-based fluid purification module comprises at least one association equilibrium vessel that enables association between a magnetic resin bead surface and a biological product; and at least one first hybrid orthogonal fluid element comprising an orthogonal current channel, at least one magnetic field, and at least one of a piezoelectric component or a dielectrophoretic electrode configured to generate or induce a unidirectional force to separate the magnetic resin beads to which the biological product is associated from the heterogeneous mixture.

[0255] In embodiments, the negative charge-based fluid purification module comprises at least one dissociation equilibrium vessel allowing the dissociation of a biological product from the surface of magnetic resin beads; and at least one second hybrid orthogonal fluid element comprising an orthogonal current channel, at least one magnetic field, and at least one of a piezoelectric component or a dielectrophoretic electrode configured to generate or induce a unidirectional force to separate the magnetic resin beads from the dissociated biological product and complete its purification. For example, several dissociation equilibrium vessels comprising distinct buffers having different pH, ionic strength, or any combination thereof are used sequentially to create a gradient dissociation effect.

[0256] In embodiments, the negative charge-based fluid purification module further comprises at least one tangential flow filtration system operating in fed-batch or perfusion mode to concentrate a fraction containing a biological product and buffer exchange.

[0257] In embodiments, the negative charge-based fluid purification module includes at least two buffer systems.

[0258] In embodiments, the negative charge-based fluid purification module includes at least one magnetic resin bead regeneration buffer system.

[0259] In embodiments, the negative charge-based fluid purification module includes at least one equilibrium vessel configured to enable the recycling of the magnetic resin beads.

[0260] In embodiments, the negative charge-based fluid purification module includes at least one sensor or detector.

[0261] In embodiments, the negative charge-based fluid purification module includes at least one fluid handling pump.

[0262] Affinity-based TFF purification module

[0263] In aspects, the present specification provides an affinity-based TFF purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The affinity-based TFF purification module comprises at least one inlet and at least one outlet, wherein the inlet and outlet are configured to allow continuous fluid flow between the at least one inlet and at least one outlet, wherein the flow rate is consistent and constant during steady-state operation.

[0264] In embodiments, the affinity-based TFF purification module comprises a suspension of resin beads, wherein the surface of the resin beads is connected to a protein A, protein G, protein L, antigen protein, protein, receptor, antibody, or aptamer configured to selectively bind to the biological product without limitation. For example, the magnetic resin beads are mobile.

[0265] In embodiments, the affinity-based TFF purification module comprises at least one equilibrium vessel allowing the binding of a biological product to a resin bead surface; and at least one first tangential flow filtration system for separating the biological product-bound resin beads from the heterogeneous mixture.

[0266] In embodiments, the affinity-based TFF purification module further comprises at least one low pH equilibrium vessel that enables the debinding of biological products from the surface of resin beads; and at least one second tangential flow filtration system for separating the resin beads from the unbound biological products and completing the elution thereof.

[0267] In embodiments, the affinity-based TFF purification module includes at least one regenerative equilibrium vessel; and at least one third tangential flow filtration system that allows for the concentration and buffer exchange of resin beads to return the resin beads to their initial state, thereby enabling the recycling and reuse of the resin beads.

[0268] In embodiments, the affinity-based TFF purification module comprises at least one collection vessel; and at least one fourth tangential flow filtration system that purifies the biological product by enabling concentration and buffer exchange of the biological product.

[0269] In embodiments, at least one equilibrium vessel, at least one low pH equilibrium vessel, and at least one regeneration equilibrium vessel of the affinity-based TFF purification module may comprise a single vessel that switches between corresponding tangential flow filtration systems to enable purification and regeneration of resin beads using appropriate buffers while maintaining a continuous flow of filtrate through at least one additional vessel in a parallel flow path.

[0270] In embodiments, regeneration of resin beads can be achieved by regenerating the resin beads without requiring a separate regeneration equilibrium vessel and a corresponding tangential flow filtration system by using at least one low pH equilibrium vessel and at least one second tangential flow filtration system of an affinity-based TFF purification module configured to include both a low pH elution buffer and a regeneration buffer to enable purification, concentration and buffer exchange.

[0271] In the embodiments, the affinity-based TFF purification module includes at least two buffer systems.

[0272] In embodiments, the affinity-based TFF purification module includes at least one resin bead regeneration buffer system.

[0273] In embodiments, the affinity-based TFF purification module includes at least one hollow fiber membrane filter.

[0274] In embodiments, the affinity-based TFF purification module includes at least one sensor or detector.

[0275] In embodiments, the affinity-based TFF purification module includes at least one fluid handling pump.

[0276] Positive charge-based TFF purification module

[0277] In some aspects, the present specification provides a positive charge-based TFF purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The positive charge-based TFF purification module comprises at least one inlet and at least one outlet, wherein the inlet and outlet are configured to allow continuous fluid flow between the inlet and outlet, wherein the flow rate is consistent and constant during steady-state operation.

[0278] In embodiments, the positive charge-based TFF purification module comprises a suspension of resin beads, wherein the surface of the resin beads comprises cationic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength. For example, the magnetic resin beads are mobile.

[0279] In embodiments, the positive charge-based TFF purification module comprises at least one association equilibrium vessel that enables association between the resin bead surface and the biological product; and at least one first tangential flow filtration system for separating the biological product-associated resin beads from the heterogeneous mixture.

[0280] In embodiments, the positive charge-based TFF purification module comprises at least one dissociation equilibrium vessel that enables the dissociation of a biological product from the surface of a resin bead; and at least one second tangential flow filtration system for separating the resin beads from the dissociated biological product and completing the purification thereof. In some aspects, for example, a plurality of dissociation equilibrium vessels are used together with a plurality of tangential flow filtration systems to achieve gradient dissociation, such as a pH gradient or an ionic strength gradient.

[0281] In embodiments, the positive charge-based TFF purification module comprises at least one regenerative equilibrium vessel; and at least one third tangential flow filtration system that allows for the concentration and buffer exchange of resin beads to return the resin beads to their initial state, thereby enabling the recycling and reuse of the resin beads.

[0282] In embodiments, the positive charge-based TFF purification module comprises at least one collection vessel; and at least one fourth tangential flow filtration system that purifies the biological product by enabling concentration of the biological product and buffer exchange.

[0283] In embodiments, at least one assembling equilibrium vessel, at least one dissociation vessel, and at least one regeneration equilibrium vessel of a positive charge-based TFF purification module may comprise a single vessel that switches between corresponding tangential flow filtration systems to enable purification and regeneration of resin beads using appropriate buffers while maintaining a continuous flow of filtrate through at least one additional vessel in a parallel flow path.

[0284] In embodiments, regeneration of resin beads can be achieved by regenerating the resin beads without requiring a separate regeneration equilibrium vessel and a corresponding tangential flow filtration system using at least one dissociation vessel and at least one second tangential flow filtration system of a positive charge-based TFF purification module configured to include both a dissociation buffer and a regeneration buffer to enable purification, concentration and buffer exchange.

[0285] In embodiments, the positive charge-based TFF purification module includes at least two buffer systems.

[0286] In embodiments, the positive charge-based TFF purification module includes at least one resin bead regeneration buffer system.

[0287] In embodiments, the positive charge-based TFF purification module includes at least one hollow fiber membrane filter.

[0288] In embodiments, the positive charge-based TFF purification module includes at least one sensor or detector.

[0289] In embodiments, the positive charge-based TFF purification module includes at least one fluid handling pump.

[0290] Negative charge-based TFF purification module

[0291] In aspects, the present specification provides a negative charge-based TFF purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The negative charge-based TFF purification module comprises at least one inlet and at least one outlet, wherein the inlet and outlet are configured to allow continuous fluid flow between the inlet and outlet, wherein the flow rate is consistent and constant during steady-state operation.

[0292] In embodiments, the negative charge-based TFF purification module comprises a suspension of resin beads, wherein the surface of the resin beads comprises an anionic functional group configured to selectively associate with the biological product at a specific pH and ionic strength.

[0293] In embodiments, the negative charge-based TFF purification module comprises at least one association equilibrium vessel that enables association between a resin bead surface and a biological product; and at least one first tangential flow filtration system for separating the biological product-associated resin beads from the heterogeneous mixture.

[0294] In embodiments, the negative charge-based TFF purification module comprises at least one dissociation equilibrium vessel that enables the dissociation of a biological product from the surface of a resin bead; and at least one second tangential flow filtration system for separating the resin beads from the dissociated biological product and completing the purification thereof. In some aspects, for example, a plurality of dissociation equilibrium vessels are used together with a plurality of tangential flow filtration systems to achieve gradient dissociation, such as a pH gradient or an ionic strength gradient.

[0295] In embodiments, the negative charge-based TFF purification module comprises at least one regenerative equilibrium vessel; and at least one third tangential flow filtration system that allows for the concentration and buffer exchange of resin beads to return the resin beads to their initial state, thereby enabling the recycling and reuse of the resin beads.

[0296] In embodiments, the negative charge-based TFF purification module comprises at least one collection vessel; and at least one fourth tangential flow filtration system that purifies the biological product by enabling concentration and buffer exchange of the biological product.

[0297] In embodiments, at least one assembling equilibrium vessel, at least one dissociation vessel, and at least one regeneration equilibrium vessel of a negative charge-based TFF purification module may comprise a single vessel that switches between corresponding tangential flow filtration systems to enable purification and regeneration of resin beads using appropriate buffers while maintaining a continuous flow of filtrate through at least one additional vessel in a parallel flow path.

[0298] In embodiments, regeneration of resin beads can be achieved by regenerating the resin beads without requiring a separate regeneration equilibrium vessel and a corresponding tangential flow filtration system using at least one dissociation vessel and at least one second tangential flow filtration system of a negative charge-based TFF purification module configured to include both a dissociation buffer and a regeneration buffer to enable purification, concentration and buffer exchange.

[0299] In embodiments, the negative charge-based TFF purification module includes at least two buffer systems.

[0300] In embodiments, the negative charge-based TFF purification module includes at least one resin bead regeneration buffer system.

[0301] In embodiments, the negative charge-based TFF purification module includes at least one hollow fiber membrane filter.

[0302] In embodiments, the negative charge-based TFF purification module includes at least one sensor or detector.

[0303] In embodiments, the negative charge-based TFF purification module includes at least one fluid handling pump.

[0304] Isoelectric point-based fluid purification module

[0305] In aspects, the present specification provides an isoelectric point-based fluid purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The isoelectric point-based fluid purification module comprises at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between at least one inlet and at least one outlet, wherein the flow rate may be consistent and constant, for example, during steady-state operation.

[0306] In embodiments, the process described herein comprises continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module, wherein the second module comprises a free-flow electrophoresis device. For example, the free-flow electrophoresis device comprises at least one fluid element comprising a fluid channel formed between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an aqueous solution (e.g., an ionic solution, or a solution providing a buffer or ampholyte). For example, the solution contact surfaces of the two parallel plates comprise glass, ceramic, plastic, or any combination thereof. In some examples, the aqueous ionic solution may create a pH gradient. In other examples, the aqueous ionic solution may impart a constant pH.

[0307] In embodiments, the free-flow electrophoresis device has at least one fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a pH gradient. For example, an isoelectric point-based fluid purification module comprises at least one first fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across a main separation channel (e.g., the approximate pH gradient may be in the pH range of about 2 to about 10); and at least one second fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient across a main separation channel (e.g., the fine pH gradient may be in the pH range of about 5 to about 8). For example, the pH gradient across the main separation channel can be further refined using additional subsequent fluid elements or chips comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow (e.g., a pH range of about 7.1 to about 7.6).

[0308] In another embodiment, the free-flow electrophoresis device has at least one fluid element that has no pH gradient, comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow to operate in band electrophoresis or charge separation operating mode. For example, an isoelectric point-based fluid purification module comprises at least one first fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant basic pH (e.g., pH greater than 7); and at least one second fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant acidic pH (e.g., pH less than 7).

[0309] In another embodiment, the free-flow electrophoresis device has at least one fluid element comprising a fluid channel created between two parallel plates to operate in an isometric electrophoresis operating mode, an electric field or electric field gradient perpendicular to the direction of fluid flow, and both an acidic pH gradient and a basic pH gradient separated by a spacer solution (e.g., NaCl solution).

[0310] In another embodiment, the isoelectric point-based fluid purification module comprises at least one first free-flow electrophoretic device comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, and at least one second free-flow electrophoretic device comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, wherein each element is connected in series and can operate in independent operating modes that enable purification. For example, at least one first free-flow electrophoretic device can operate in an isoelectric focusing mode, and at least one second free-flow electrophoretic device can operate in an isokinetic electrophoretic mode to increase separation resolution.

[0311] In another embodiment, the isoelectric point-based fluid purification module comprises at least one first fluid element comprising a fluid channel having at least one dielectrophoretic electrode capable of inducing a defined unidirectional force; at least one second free-flow electrophoretic device comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across a main separation channel (e.g., a pH range of about 2 to about 10); and at least one third free-flow electrophoretic device comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient across a main separation channel (e.g., a pH range of about 5 to about 8). For example, the pH gradient across the main separation channel can be further refined using additional subsequent fluid elements or chips comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow (e.g., a pH range of about 7.1 to about 7.6).

[0312] In embodiments, the isoelectric point-based fluid purification device further includes at least two electrodes (e.g., platinum wire electrodes) to function as an anode or a cathode.

[0313] In embodiments, the backpressure within the isoelectric-based fluid purification device depends on the channel shape and dimensions, the inlet and outlet openings and / or tubing diameters, and the input flow rate. For example, the backpressure is in the range of about 0.5 psi to about 10 psi. In some examples, the backpressure is controlled by a needle valve, for example, without any intention of limiting it.

[0314] In embodiments, the isoelectric point-based fluid purification device further comprises at least one bubble removal system for continuously removing O2 and H2 gas bubbles generated in the electrode channel under an applied voltage. In some embodiments, removing electrolytic bubbles is essential to enable continuous operation for substantially long periods. For example, the bubble removal system uses a hydrophobic PTFE membrane to create a waterproof seal over the electrode channel that can continuously remove electrolytic bubbles at the point of generation by exposure to a vacuum system. For example, the vacuum gauge pressure is in the range of about -0.05 bar to about -0.4 bar.

[0315] In embodiments, the isoelectric point-based fluid purification device further includes an active cooling system or a heat sink to enable temperature control and Joule heat dissipation. For example, the active cooling system includes an aluminum thermal chuck comprising a cooled circulating water / propylene glycol jacket.

[0316] In embodiments, the isoelectric point-based fluid purification module includes at least one buffer or amphoteric electrolyte system.

[0317] In embodiments, the isoelectric point-based fluid purification module comprises at least one electrode solution. In some embodiments, the at least one electrode solution comprises an electrolyte solution configured to enable proper function in contact with an anode or a cathode, for example, phosphoric acid and sodium hydroxide, respectively. In other embodiments, the at least one electrode solution comprises at least one amphoteric electrolyte solution configured to enable proper function in contact with an anode or a cathode, for example, Tris buffered saline, flowing through a main separation channel, an anode channel, and a cathode channel.

[0318] In embodiments, the isoelectric point-based fluid purification module includes at least one sensor or detector. For example, at least one sensor or detector is placed inline. In some examples, at least one sensor or detector includes, but is not limited to, a flow sensor, a temperature sensor, a conductivity sensor, a pH sensor, a refractive index detector, a UV detector, or a back pressure sensor.

[0319] In embodiments, the isoelectric point-based fluid purification module includes at least one liquid circuit breaker, or disconnects the downstream of the device from the upstream of at least one inline sensor or detector to enable detection or sensing in a voltage-free solution.

[0320] In embodiments, the isoelectric point-based fluid purification module includes at least one fluid handling pump.

[0321] In embodiments, the isoelectric point-based fluid purification module includes at least one collection container.

[0322] method

[0323] A method for purifying a biological product from a heterogeneous mixture derived from a bioreactor producing the biological product is provided herein, comprising using the processes described herein. For example, the bioreactor type includes, but is not limited to, a batch bioreactor, a fed-batch bioreactor, a perfusion bioreactor, a chemostat bioreactor, or a multi-compartment bioreactor. In some examples, the bioreactor produces the biological product under steady conditions.

[0324] In embodiments, the present specification provides a method for purifying a biological product from a heterogeneous mixture derived from a bioreactor producing a biological product, comprising using at least one of the modules described herein, such as, for example, a dynamic filtration module, an affinity-based magnetic purification module, a positive charge-based magnetic purification module, a negative charge-based magnetic purification module, an affinity-based purification module, a positive charge-based purification module, a negative charge-based purification module, an affinity-based fluid purification module, a positive charge-based fluid purification module, a negative charge-based fluid purification module, an affinity-based TFF purification module, a positive charge-based TFF purification module, a negative charge-based TFF purification module, and / or an isoelectric point-based fluid purification module.

[0325] In some embodiments, the present specification provides a method for continuously purifying a biological product from a heterogeneous mixture derived from a bioreactor that produces the biological product in a steady state, comprising using at least one of the modules described herein, such as, for example, a dynamic filtration module, an affinity-based magnetic purification module, a positive charge-based magnetic purification module, a negative charge-based magnetic purification module, an affinity-based purification module, a positive charge-based purification module, a negative charge-based purification module, an affinity-based fluid purification module, a positive charge-based fluid purification module, a negative charge-based fluid purification module, an affinity-based TFF purification module, a positive charge-based TFF purification module, a negative charge-based TFF purification module, and / or an isoelectric point-based fluid purification module.

[0326] In another embodiment, the present specification provides a method for purifying a biological product from a heterogeneous mixture not derived from a bioreactor producing a biological product in a steady state, comprising using at least one of the modules described herein, such as, for example, a dynamic filtration module, an affinity-based magnetic purification module, a positive charge-based magnetic purification module, a negative charge-based magnetic purification module, an affinity-based purification module, a positive charge-based purification module, a negative charge-based purification module, an affinity-based fluid purification module, a positive charge-based fluid purification module, a negative charge-based fluid purification module, an affinity-based TFF purification module, a positive charge-based TFF purification module, a negative charge-based TFF purification module, and / or an isoelectric point-based fluid purification module.

[0327] Other aspects of the present invention are disclosed below. Brief explanation of the drawing

[0328] This patent or application file contains one or more drawings produced in color. A copy of this patent or patent application publication containing the color drawing(s) will be provided by the Office upon request and payment of the necessary fees. Figs. 1a-1dFIG. 1a illustrates an exemplary continuous process flow described herein. FIG. 1a shows an exemplary continuous process flow, wherein step 1 comprises a bioreactor producing a biological product in a steady state, step 2 comprises a continuous dynamic filtration module, step 3 comprises an affinity-based magnetic purification module, step 4 comprises at least one charge-based magnetic purification module, step 5 comprises a standard industrial virus inactivation and filtration process performed, for example, in a fed-batch or perfusion mode, and step 6 comprises high-performance tangential flow filtration using a charged membrane performed, for example, in a fed-batch or perfusion mode to prepare for a standard industrial charge-finishing process of step 7. FIG. 1b illustrates an exemplary continuous process flow, wherein step 1 comprises a bioreactor producing a biological product in a steady state, step 2 comprises a continuous dynamic filtration module, step 3 comprises an affinity-based magnetic purification module, step 4 comprises a positive charge-based magnetic purification module, step 5 comprises a negative charge-based magnetic purification module, and step 6 comprises high-performance tangential flow filtration using a charged membrane performed in a fed-batch or perfusion mode to prepare, for example, a standard industrial fill-and-finish process of step 7. FIG. 1c illustrates an exemplary continuous process flow, wherein step 1 comprises a bioreactor producing a biological product in a steady state, step 2 comprises a continuous dynamic filtration module, step 3 comprises an affinity-based magnetic purification module, step 4 comprises an isoelectric point-based fluid purification module, step 5 comprises a standard industrial virus inactivation and filtration process performed, for example, in a fed-batch or perfusion mode, and step 6 comprises high-performance tangential flow filtration using a charged membrane performed in a fed-batch or perfusion mode to prepare for the standard industrial fill-and-finish process of step 7.FIG. 1d illustrates an exemplary continuous process flow, wherein step 1 comprises a bioreactor producing a biological product in a steady state, step 2 comprises a continuous dynamic filtration module, step 3 comprises an affinity-based magnetic purification module, step 4 comprises an isoelectric point-based fluid purification module, and step 5 comprises high-performance tangential flow filtration using a charged membrane performed in a fed-and-dive or perfusion mode to prepare for a standard industrial fill-and-finish process of step 6. FIGS. 2a to 2dFIG. 2a illustrates a schematic diagram of an exemplary continuous process flow described herein. FIG. 2a illustrates an exemplary continuous process flow, wherein step 1 comprises a bioreactor producing a biological product in a steady state, step 2 comprises a continuous dynamic filtration module, step 3 comprises an affinity-based purification module, step 4 comprises at least one charge-based purification module, step 5 comprises a standard industrial virus inactivation and filtration process performed, for example, in a fed-batch or perfusion mode, and step 6 comprises high-performance tangential flow filtration using a charged membrane performed, for example, in a fed-batch or perfusion mode to prepare for a standard industrial charge-finishing process of step 7. FIG. 2b illustrates an exemplary continuous process flow, wherein step 1 comprises a bioreactor producing a biological product in a steady state, step 2 comprises a continuous dynamic filtration module, step 3 comprises an affinity-based purification module, step 4 comprises a positive charge-based purification module, step 5 comprises a negative charge-based purification module, and step 6 comprises high-performance tangential flow filtration using a charged membrane performed in a fed-batch or perfusion mode to prepare, for example, a standard industrial charge-finish process of step 7. FIG. 2c illustrates an exemplary continuous process flow, wherein step 1 comprises a bioreactor producing a biological product in a steady state, step 2 comprises a continuous dynamic filtration module, step 3 comprises an affinity-based purification module, step 4 comprises an isoelectric point-based fluid purification module, step 5 comprises a standard industrial virus inactivation and filtration process performed, for example, in a fed-batch or perfusion mode, and step 6 comprises high-performance tangential flow filtration using a charged membrane performed in a fed-batch or perfusion mode to prepare for the standard industrial fill-and-finish process of step 7.FIG. 2d illustrates an exemplary continuous process flow, wherein step 1 comprises a bioreactor producing a biological product in a steady state, step 2 comprises a continuous dynamic filtration module, step 3 comprises an affinity-based purification module, step 4 comprises an isoelectric point-based fluid purification module, and step 5 comprises high-performance tangential flow filtration using a charged membrane performed in a fed-batch or perfusion mode to prepare for the standard industrial fill-and-finish process of step 6. FIGS. 3a to 3dFIG. 3a illustrates an exemplary continuous process flow described herein. FIG. 3a illustrates an exemplary continuous process flow, wherein step 1 comprises a bioreactor producing a biological product in a steady state, step 2 comprises a continuous dynamic filtration module, step 3 comprises an affinity-based fluid purification module, step 4 comprises at least one charge-based fluid purification module, step 5 comprises a standard industrial virus inactivation and filtration process performed, for example, in a fed-batch or perfusion mode, and step 6 comprises high-performance tangential flow filtration using a charged membrane performed, for example, in a fed-batch or perfusion mode to prepare for a standard industrial charge-finish process of step 7. FIG. 3b illustrates an exemplary continuous process flow, wherein step 1 comprises a bioreactor producing a biological product in a steady state, step 2 comprises a continuous dynamic filtration module, step 3 comprises an affinity-based fluid purification module, step 4 comprises a positive charge-based fluid purification module, step 5 comprises a negative charge-based fluid purification module, and step 6 comprises high-performance tangential flow filtration using a charged membrane performed in a fed-batch or perfusion mode to prepare, for example, a standard industrial fill-and-finish process of step 7. FIG. 3c illustrates an exemplary continuous process flow, wherein step 1 comprises a bioreactor producing a biological product in a steady state, step 2 comprises a continuous dynamic filtration module, step 3 comprises an affinity-based fluid purification module, step 4 comprises an isoelectric point-based fluid purification module, step 5 comprises a standard industrial virus inactivation and filtration process performed, for example, in a fed-batch or perfusion mode, and step 6 comprises high-performance tangential flow filtration using a charged membrane performed in a fed-batch or perfusion mode to prepare for the standard industrial fill-and-finish process of step 7.FIG. 3d illustrates an exemplary continuous process flow, wherein step 1 comprises a bioreactor producing a biological product in a steady state, step 2 comprises a continuous dynamic filtration module, step 3 comprises an affinity-based fluid purification module, step 4 comprises an isoelectric point-based fluid purification module, and step 5 comprises high-performance tangential flow filtration using a charged membrane performed in a fed-batch or perfusion mode to prepare for the standard industrial fill-and-finish process of step 6. FIGS. 4a to 4dFIG. 4a illustrates an exemplary continuous process flow described herein. FIG. 4a illustrates an exemplary continuous process flow, wherein step 1 comprises a bioreactor producing a biological product in a steady state, step 2 comprises a continuous dynamic filtration module, step 3 comprises an affinity-based TFF purification module, step 4 comprises at least one charge-based TFF purification module, step 5 comprises a standard industrial virus inactivation and filtration process performed, for example, in a fed-batch or perfusion mode, and step 6 comprises high-performance tangential flow filtration using a charged membrane performed, for example, in a fed-batch or perfusion mode to prepare for a standard industrial charge-finish process of step 7. FIG. 4b illustrates an exemplary continuous process flow, wherein step 1 comprises a bioreactor producing a biological product in a steady state, step 2 comprises a continuous dynamic filtration module, step 3 comprises an affinity-based TFF purification module, step 4 comprises a positive charge-based TFF purification module, step 5 comprises a negative charge-based TFF purification module, and step 6 comprises high-performance tangential flow filtration using a charged membrane performed in a fed-batch or perfusion mode to prepare, for example, a standard industrial charge-finish process of step 7. FIG. 4c illustrates an exemplary continuous process flow, wherein step 1 comprises a bioreactor producing a biological product in a steady state, step 2 comprises a continuous dynamic filtration module, step 3 comprises an affinity-based TFF purification module, step 4 comprises an isoelectric point-based fluid purification module, step 5 comprises a standard industrial virus inactivation and filtration process performed, for example, in a fed-batch or perfusion mode, and step 6 comprises high-performance tangential flow filtration using a charged membrane performed in a fed-batch or perfusion mode to prepare for the standard industrial fill-and-finish process of step 7.FIG. 4d illustrates an exemplary continuous process flow, wherein step 1 comprises a bioreactor producing a biological product in a steady state, step 2 comprises a continuous dynamic filtration module, step 3 comprises an affinity-based TFF purification module, step 4 comprises an isoelectric point-based fluid purification module, and step 5 comprises high-performance tangential flow filtration using a charged membrane performed in a fed-batch or perfusion mode to prepare for the standard industrial fill-and-finish process of step 6. Figures 5a and 5b It shows an exemplary continuous process flow along with design schematics for the downstream purification module described herein. Figures 6a and 6bFigure 6 illustrates a series of exemplary designs for a dynamic filtration device comprising a single output head for continuously delivering a heterogeneous mixture containing biological products from a steady-state bioreactor discharge output line, and a separate output head for supplying a wash buffer. Figure 6a is a schematic diagram of a dynamic filtration device design comprising: a single output head for continuously delivering a heterogeneous mixture containing biological products from a steady-state bioreactor discharge output line; a separate output head for supplying a wash buffer; a rolled filter membrane functioning as a feed reel and a collection reel; two servo motors for controlling a feed reel-to-collection reel system; two support rods having mechanically flat contact surfaces; a membrane support structure having a mechanically flat contact surface and an opening continuous with a vacuum line; a vacuum collection vessel; a diaphragm pump; and a peristaltic pump. Figure 6b is a single output head for continuously delivering a heterogeneous mixture containing biological products from a steady-state bioreactor discharge output line; This is a schematic diagram of a dynamic filtration device design comprising: a separate output head for supplying a washing buffer; a rolled filter membrane functioning as a supply reel and a collection reel; two servo motors for controlling a supply reel-to-collection reel system; two support rods having mechanically flat contact surfaces; a membrane support structure having a mechanically flat contact surface and an opening continuous with a vacuum line; a controllable T-valve; two vacuum collection vessels; a diaphragm pump; and two peristaltic pumps. Figures 7a and 7bFigure 7 illustrates two exemplary designs of a dynamic filtration device comprising a plurality of output heads for continuously delivering a heterogeneous mixture containing biological products from a steady-state bioreactor discharge output line, and a plurality of individual output heads for supplying a wash buffer. Figure 7a shows a schematic diagram of a dynamic filtration device design comprising: a plurality of output heads for delivering a heterogeneous mixture containing biological products from a steady-state bioreactor discharge output line; a plurality of individual output heads for supplying a wash buffer; a rolled filter membrane functioning as a feed reel and a collection reel; two servo motors for controlling a feed reel-to-collection reel system; two support rods having mechanically flat contact surfaces; a membrane support structure having a mechanically flat contact surface and an opening continuous with a vacuum line; a vacuum collection vessel; a diaphragm pump; and a peristaltic pump. Figure 7b shows a plurality of output heads for continuously delivering a heterogeneous mixture containing biological products from a steady-state bioreactor discharge output line; a plurality of individual output heads for supplying a wash buffer; A schematic diagram of a dynamic filtration device design is shown, comprising: a rolled filter membrane functioning as a feed reel and a collection reel; two servo motors for controlling a feed reel-to-collection reel system; two support rods having mechanically flat contact surfaces; a membrane support structure having a mechanically flat contact surface and an opening continuous with a vacuum line; a controllable T-valve; two vacuum collection vessels; a diaphragm pump; and two peristaltic pumps. Fig. 8 It shows an image of an exemplary membrane support structure having an opening with five parallel slots. Figures 9a and 9bFigure 9a is an image showing the removal of PolyBeads (0.05% solids; 2 μm (red), 6 μm (red), and 10 μm (blue) diameters) from a heterogeneous mixture in 1X PBS. Figure 9a shows the initial heterogeneous mixture of PolyBeads (0.05% solids; 2 μm, 6 μm, and 10 μm diameters) in 1X PBS; the filtrate obtained by purifying the heterogeneous mixture with a 0.2 μm PTFE syringe filter; and the filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration device (MBM-3 represents a sample dynamically filtered with a 0.45 μm PVDF filter membrane at a flow rate of 0.25 mL / min). This is an image showing a visual comparison (from left to right) of the filtrate obtained by purifying a heterogeneous mixture with an exemplary dynamic filtration device (MBM-4 represents samples dynamically filtered with a 0.45 μm PES filter membrane and flow rates of 0.25, 0.5, 1.0, 2.0, and 5.0 mL / min). Fig. 9b is an initial heterogeneous mixture of PolyBeads (0.05% solids; 2 μm, 6 μm, and 10 μm diameters) in 1X PBS; the filtrate obtained by purifying the heterogeneous mixture with a 0.2 μm PTFE syringe filter; and the filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration device having a 0.45 μm PVDF filter membrane and a flow rate of 0.25 mL / min (MBM-3). A bar graph showing UV-Vis spectrophotometric comparison of the filtrate (MBM-4) obtained by purifying a heterogeneous mixture with an exemplary dynamic filtration device having a 0.45 μm PES filter membrane and flow rates of 0.25, 0.5, 1.0, 2.0, and 5.0 mL / min, which demonstrates that PolyBeads are successfully removed from the heterogeneous mixture using the exemplary dynamic filtration device. FIGS. 10a to 10dFigure 10a is a series of data showing the removal of PolyBeads (0.05% solids; 2μm (red), 6μm (red), and 10μm (blue) diameters) from a heterogeneous mixture of PolyBeads suspended in a 0.5mg / mL solution of BSA-FITC in 1X PBS. Figure 10a is a graph showing the UV-Vis spectrophotometric trace of a serially diluted heterogeneous mixture of PolyBeads (0.05% solids; 2μm, 6μm, and 10μm diameters) suspended in a 0.5mg / mL solution of BSA-FITC in 1X PBS, with PolyBead signature regions indicated, showing the presence of PolyBeads. Fig. 10b is a graph showing UV-Vis spectrophotometric traces of serially diluted 0.5 mg / mL solutions of BSA-FITC in 1X PBS, where a PolyBead signature region is indicated, showing the absence of PolyBeads. Fig. 10c is an initial heterogeneous mixture of PolyBeads (0.05% solids; 2 μm, 6 μm, and 10 μm diameters) suspended in a 0.5 mg / mL solution of BSA-FITC in 1X PBS; a filtrate obtained by purifying the heterogeneous mixture with a 0.2 μm PTFE syringe filter; and a filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration device (MBM-3a represents a sample dynamically filtered through a 0.45 μm PVDF filter membrane at a flow rate of 0.25 mL / min). Images showing a visual comparison (from left to right) of the filtrate obtained by purifying a heterogeneous mixture with an exemplary dynamic filtration device (MBM-4a represents a sample dynamically filtered with a 0.45 μm PES filter membrane and a flow rate of 0.5 mL / min); the filtrate obtained by purifying a heterogeneous mixture with an exemplary dynamic filtration device (MBM-5a represents a sample dynamically filtered with a 0.45 μm PES filter membrane and a flow rate of 2.0 mL / min); and the supernatant collected from the purification of the heterogeneous mixture by centrifugation (5 min at 10,000 x g).FIG. 10d is an initial heterogeneous mixture of PolyBeads (0.05% solids; 2 μm, 6 μm, and 10 μm diameters) suspended in a 0.5 mg / mL solution of BSA-FITC in 1X PBS; a filtrate obtained by purifying the heterogeneous mixture with a 0.2 μm PTFE syringe filter; a filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration device (MBM-3a); a filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration device (MBM-4a); a filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration device (MBM-5a); A graph showing a UV-Vis spectrophotometric comparison of the supernatant collected from purification by centrifugation (5 min at 10,000xg) and demonstrates that BSA-FITC is successfully purified by removing PolyBeads using an exemplary dynamic filtration device, as indicated by the absence of PolyBeads in the PolyBead signature region. Figures 11a and 11b Figure 11 shows the dynamic filtration of a heterogeneous mixture of PolyBeads suspended in a 0.5 mg / mL solution of human polyclonal IgG (hIgG) in 1X PBS at an input flow rate of 10 mL / min. Figure 11a shows dynamic filtration through a slot die output head. Figure 11a shows PolyBeads (1.1 x 10⁻¹⁴, each) suspended in a 0.5 mg / mL solution of hIgG in 1X PBS. 8 , 4.2x10 6 , and 1.0x10 6Images showing a visual comparison (from left to right) of an initial heterogeneous mixture of particles (2 μm, 6 μm, and 10 μm diameters) at 1 / mL; the filtrate obtained by purifying the heterogeneous mixture using an exemplary dynamic filtration device with a 0.45 μm PES filter membrane and a flow rate of 10 mL / min; and the supernatant collected from the purification of the heterogeneous mixture by centrifugation (5 min at 10,000 x g). Fig. 11b is a graph showing a spectrophotometric comparison of the total protein concentration determined by the BCA assay of the filtrate obtained by dynamic filtration and the supernatant collected by centrifugation. Fig. 11c shows PolyBeads (7.3 x 10⁶, each) suspended in a 0.5 mg / mL solution of hIgG in 1X PBS. 7 , 1.1x10 8 , 1.1x10 8 , 1.1x10 8 , 3.4x10 7 , and 1.0x10 6 Figure 11d shows visual comparison images (from left to right) of an initial heterogeneous mixture of particles (0.5 μm, 0.75 μm, 1 μm, 2 μm, 3 μm, and 10 μm diameters) at 0.5 μm, 0.75 μm, 1 μm, 2 μm, 3 μm, and 10 μm diameters) at 0.45 μm PES filter membrane and an exemplary dynamic filtration device with a flow rate of 10 mL / min, and a supernatant collected from the purification of the heterogeneous mixture by centrifugation (5 min at 10,000 x g). Figure 11d is a graph showing a spectrophotometric comparison of the total protein concentration determined by BCA assay of the filtrate obtained by dynamic filtration and the supernatant collected by centrifugation. FIGS. 12a to 12dFigure 12a shows the protein recovery of dynamic filtration during continuous operation at different input flow rates for different proteins, protein concentrations, filter membrane materials and pore sizes, and membrane support structures. Figure 12a shows the recovery rates of proteins of different sizes and charges (bovine serum albumin (BSA), lysozyme, and hIgG, 0.5-10 mg / mL, 5 mg / mL, and 0.5 mg / mL, respectively) by BCA assay of the filtrate obtained by dynamic filtration with a 0.45 μm PES filter membrane with a transport rate of 0.5 mm / sec and an input flow rate of 10 mL / min. Figure 12b shows the recovery rate of 0.5 mg / mL hIgG by BCA assay of the filtrate obtained by dynamic filtration using filter membranes of different materials and pore sizes (0.45 μm PES, 0.45 μm hydrophilic PVDF, 0.22 μm PES) with a transport rate of 0.5 mm / sec and an input flow rate of 10 mL / min. Figure 12c shows the recovery rate of 0.5 mg / mL hIgG by BCA assay of the filtrate obtained by dynamic filtration using a 0.45 μm PES filter membrane with a transport rate of 0.5 mm / sec and an input flow rate of 10 mL / min, and different membrane support structures (a PTFE membrane support structure with 5 parallel slots and a PTFE membrane support structure with a porous hydrophilic polyethylene (PE) insert). Figure 12d shows the recovery rate of 0.5 mg / mL of lysozyme by BCA assay of the filtrate obtained by dynamic filtration at different flow rates (5 and 10 mL / min) during long-term continuous operation using a 0.45 μm PES filter membrane with a transport rate of 0.5 mm / sec. Figs. 13a-13c Figure 13a compares the cell purification by dynamic filtration and centrifugation of an input heterogeneous mixture containing a suspended cell culture in RPMI medium spiked with hIgG at a final concentration of 1 g / L. Figure 13a shows an initial hIgG-spikeged murine myeloma suspended cell culture (2 x 10⁻¹⁰) in RPMI medium.6 1 g / L hIgG at 1 g cells / mL; filtrate (DF-1, DF-2, DF-3) obtained by dynamic filtration at an input flow rate of 2 mL / min using a 0.45 μm PES filter membrane with a transport rate of 0.5 mm / s; and supernatant (C-1, C-2, C-3) collected by centrifugation at 10,000 x g for 5 minutes; are shown (from left to right). Fig. 13b shows the initial hIgG spiked murine myeloma suspension cell culture (2 x 10⁶) in RPMI medium. 6 Shows SDS-PAGE analysis (from left to right) of 1 g / L hIgG at canine cells / mL); filtrates (DF-1, DF-2, DF-3) obtained by dynamic filtration at an input flow rate of 2 mL / min using a 0.45 μm PES filter membrane with a transport rate of 0.5 mm / s; and supernatants (C-1, C-2, C-3) obtained by centrifugation at 10,000 xg for 5 minutes. Fig. 13c shows the filtrate obtained by dynamic filtration at an input flow rate of 2 mL / min using a 0.45 μm PES filter membrane with a transport rate of 0.5 mm / s (blue outline bar); This shows a comparison of hIgG recovery rates from a heterogeneous mixture (cell cultures suspended in RPMI medium spiked with 1 g / L hIgG) by BCA assay of the supernatant collected by centrifugation at 10,000 xg for 5 minutes. Fig. 14 Figure 1 shows an exemplary design schematic of an affinity-based magnetic purification device comprising a loop conveyor system and at least one magnetic field that is permanently "on". Fig. 15 Figure 1 shows an exemplary design schematic of an affinity-based magnetic purification device comprising a loop conveyor system and at least one magnetic field capable of "on / off" toggling. Fig. 16 This shows an exemplary design schematic of a charge-based magnetic purification device comprising a loop conveyor system and at least one magnetic field that is permanently "on." Fig. 17Figure [Illegible] shows an exemplary design schematic of a charge-based magnetic purification device comprising a loop conveyor system and at least one magnetic field capable of "on / off" switching. Fig. 18 Figure [Illegible] shows an exemplary design schematic of an affinity-based magnetic purification device comprising a pick-and-place robot system and at least one magnetic field. Fig. 19 Figure 1 shows an exemplary design schematic of a charge-based magnetic purification device comprising a pick-and-place robot system and at least one magnetic field. Figs. 20a and 20b Figure 20 shows affinity-based magnetic purification performed on a mixture of hIgG (target, 2 g / L input concentration) and lysozyme (impurity, 1 g / L input concentration) using an affinity-based magnetic purification device filled with magnetic protein A-coated agarose beads. Figure 20a shows total protein analysis via BCA assay on fractions collected from three consecutive cycles of magnetic affinity bead use and recycling, demonstrating the ability of magnetic affinity beads to be reproducibly recycled and reused without impairing binding ability and performance. Figure 20b shows SDS-PAGE analysis on fractions collected from three consecutive cycles of magnetic affinity bead use and recycling, demonstrating the ability of magnetic affinity beads to be reproducibly recycled and reused without impairing binding ability and performance. FIGS. 21a to 21d Figure 21 shows an exemplary design of an affinity-based tablet device including a mechanical rotation system. Figure 21a is a schematic diagram of a lead system. Figure 21b is a schematic diagram of a container carousel. Figure 21c is a schematic diagram of a collection system. Figure 21d shows how the lead system and the collection system interface with the container carousel. FIGS. 22a to 22dFigure 22 shows an exemplary design of a charge-based purification device including a mechanical rotation system. Figure 22a is a schematic diagram of a lead system. Figure 22b is a schematic diagram of a container carousel. Figure 22c is a schematic diagram of a collection system. Figure 22d shows how the lead system and the collection system interface with the container carousel. FIGS. 23a to 23d Figure 23 shows individual system components of an affinity-based purification or charge-based purification device. Figure 23a is a schematic diagram of an exemplary gasketed lid, vessel, and collector assembly. Figure 23b is a schematic diagram of an exemplary gasketed lid comprising an air inlet, two buffer inlets configured to generate a circular flow pattern, a vent port, and a filling inlet, which are components of the lid system. Figure 23c is a schematic diagram of a vessel comprising a mesh filter or frit and a valve, which are components of the vessel carousel. Figure 23d is a schematic diagram of a collector, which is a component of the collection system. Figs. 24a and 24b Figure 24 illustrates an exemplary design of an affinity-based purification device including a stepwise linear system. Figure 24a shows the individual system components of the affinity-based purification device. Figure 24b shows the connectivity of the affinity-based purification device including a stepwise linear system. FIGS. 25a and 25b Figure 25 illustrates an exemplary design of an affinity-based purification device including a stepwise linear system. Figure 25a shows the individual system components of a charge-based purification device. Figure 25b shows the connectivity of a charge-based purification device including a stepwise linear system. Figures 26a and 26bFigure 26 shows the affinity-based purification of an hIgG (2 g / L input concentration) solution performed with an affinity-based purification device filled with protein A-coated agarose resin beads. Figure 26a shows the total protein analysis via BCA assay on fractions collected from three consecutive cycles of affinity resin bead use and recycling, demonstrating the ability of affinity resin beads to be reproducibly recycled and reused without impairing binding ability and performance. Figure 26b shows the SDS-PAGE analysis on fractions collected from three consecutive cycles of affinity resin bead use and recycling, demonstrating the ability of affinity resin beads to be reproducibly recycled and reused without impairing binding ability and performance. FIGS. 27a and 27b Figure 27 shows affinity-based purification performed on a mixture of hIgG (target, 2 g / L input concentration) and lysozyme (impurity, 1 g / L input concentration) using an affinity-based purification device filled with protein A-coated agarose resin beads. Figure 27a shows total protein analysis via BCA assay on fractions collected from three consecutive cycles of affinity resin bead use and recycling, demonstrating the ability of affinity resin beads to be reproducibly recycled and reused without impairing binding ability and performance. Figure 27b shows SDS-PAGE analysis on fractions collected from three consecutive cycles of affinity resin bead use and recycling, demonstrating the ability of affinity resin beads to be reproducibly recycled and reused without impairing binding ability and performance. FIGS. 28a to 28dFigure 28a is an image showing exemplary designs of hybrid fluidic devices. Figure 28b is an image showing a hybrid fluidic device including a parallel orthogonal current channel, a permanent magnetic field, and two piezoelectric transducers. Figure 28c is an image showing a hybrid fluidic device including a parallel orthogonal current channel, a permanent magnetic field, and two piezoelectric transducers. Figure 28d is an image showing a hybrid fluidic device including a parallel orthogonal current channel, a permanent magnetic field, and two optional dielectrophoretic electrodes. Fig. 29 Figure 1 shows an exemplary design schematic of an affinity-based fluid purification device. Fig. 30 This shows an exemplary design schematic of a charge-based fluid purification device. Fig. 31 Figure [] shows an exemplary design schematic of an affinity-based purification device including at least one tangential flow filtration system. Fig. 32 This shows an exemplary design schematic of a charge-based purification device including at least one tangential flow filtration system. Fig. 33 Figure 1 shows an exemplary design schematic of an isoelectric point-based fluid purification device comprising a channel created between two parallel plates, an electric field perpendicular to the direction of fluid flow, and a fluid element having an aqueous ionic solution. Fig. 34 This shows an exemplary design schematic of a free-flow electrophoretic device comprising a first fluid element having a channel formed between two parallel plates, an electric field perpendicular to the direction of fluid flow, and an approximate pH gradient, which is connected to a second fluid element having a channel formed between two parallel plates, an electric field perpendicular to the direction of fluid flow, and a fine pH gradient, wherein the device can operate in an isoelectric focusing mode. Fig. 35This shows an exemplary design schematic of a free-flow electrophoresis device comprising a channel formed between two parallel plates, an electric field perpendicular to the direction of fluid flow, and a first fluid element having a constant basic pH across the main separation channel, which is connected to a second fluid element having a channel formed between two parallel plates, an electric field perpendicular to the direction of fluid flow, and a constant acidic pH across the main separation channel, wherein the device can operate in band electrophoresis mode. Fig. 36 Figure 1 shows an exemplary design schematic of a free-flow electrophoretic device comprising a first fluid element having a channel created between two parallel plates and an electric field perpendicular to the direction of fluid flow, which is connected to a second fluid element having an electric field perpendicular to the direction of fluid flow and a channel created between two parallel plates that can operate in an isoelectric focusing mode. Fig. 37 It shows an exemplary design schematic of a free-flow electrophoretic device comprising a first fluid element having a channel with a selective dielectrophoretic electrode for pre-sorting a mixture, which is connected to a third fluid element having a channel created between two parallel plates capable of operating in an isoelectric focusing mode and an electric field perpendicular to the direction of fluid flow, and which is connected to a second fluid element having a channel created between two parallel plates capable of operating in an isoelectric electrophoretic mode and an electric field perpendicular to the direction of fluid flow. Fig. 38 This shows the design of an exemplary bubble removal and degassing system that removes electrolytic bubbles directly from electrode channels to create a bubble-free main separation channel. Fig. 39 It represents an exemplary liquid circuit breaker that creates a disconnection in a solution connected to an applied voltage flowing from the outlet of a free-flow electrophoresis device to at least one inline sensor or detector. FIGS. 40a to 40eFigure 40 illustrates the isoelectric-point-based fluid purification of a mixture of Rhodamine 6G (0.25 mg / mL, net charge +1) and Fluorescein (0.25 mg / mL, net charge -1) using an isoelectric-point-based purification device having a positive channel (H2SO4), a negative channel (NaOH), and a main separation channel having five inlets and five outlets through which an amphoteric electrolyte solution flows, wherein the mixture was introduced from the center of the inlet (inlet 3) of the device. Figure 40a shows optical images of fractions collected from the five outlets at 0 V and 5 mL / min. Figure 40b shows the absorbance spectra of fractions collected from the five outlets at 0 V and 5 mL / min. Fig. 40c shows optical images of fractions collected from five outlets at 1000 V and 10 mL / min in the presence of a pH gradient. Fig. 40d shows absorbance spectra of fractions collected from five outlets at 1000 V and 10 mL / min in the presence of a pH gradient. Fig. 40e shows the purification of a mixture in which fractions including purified Rhodamine 6G (outlet 2, facing the cathode) and purified Fluorescein (outlet 4, facing the anode) are produced. FIGS. 41a to 41cFigure 41 shows optical imaging regarding the purification of a mixture of Rhodamine 6G (0.25 mg / mL, net charge +1) and Fluorescein (0.25 mg / mL, net charge -1) under different operating conditions using an isoelectric point-based purification device having an anode channel (H2SO4), a cathode channel (NaOH), and a main separation channel having five inlets and five outlets through which an amphoteric electrolyte solution flows, wherein the mixture was introduced from the center of the inlet (Inlet 3) of the device. Figure 41a shows the isoelectric focused free-flow electrophoresis of the mixture of Rhodamine 6G and Fluorescein at 500 V and a flow rate of 3 mL / min. Figure 41b shows the isoelectric focused free-flow electrophoresis of the mixture of Rhodamine 6G and Fluorescein at 700 V and a flow rate of 5 mL / min. Figure 41c shows the isoelectric focused free-flow electrophoresis of a mixture of Rhodamine 6G and fluorescein at 900 V and a flow rate of 10 mL / min. Figs. 42a and 42b Figure 42a shows optical imaging regarding the purification of a mixture of small molecule dyes using an isoelectric point-based purification device, wherein the device comprises an anode channel (H2SO4), a cathode channel (NaOH), and a main separation channel having five inlets and five outlets through which an amphoteric electrolyte solution flows, wherein the mixture was introduced at the center of the inlet (inlet 3) of the device. Figure 42a shows the isoelectric focused free-flow electrophoresis of a mixture of basic fuchsin (0.05 mg / mL, net charge +3) and fluorescein (0.25 mg / mL, net charge -1) at 500 V and a flow rate of 5 mL / min. Figure 42b shows the isoelectric focused free-flow electrophoresis of a mixture of crystal violet (0.05 mg / mL, net charge +3) and fluorescein (0.25 mg / mL, net charge -1) at 500 V and a flow rate of 5 mL / min. FIGS. 43a to 43dFigure 43a illustrates optical imaging regarding the purification of a mixture of basic fuchsin (0.005 mg / mL, net charge +3) and fluorescein (0.25 mg / mL, net charge -1) using an isoelectric point-based purification device operating in isoelectric focused free-flow electrophoresis mode over increasing applied voltage. The mixture was introduced into a central inlet (inlet 3) at a flow rate of 5 mL / min using a device comprising an anode channel, a cathode channel, and a main separation channel having 5 inlets and 10 outlets, with each channel flowing the same amphoteric electrolyte solution at 5 mL / min. When no voltage is applied, the mixture exits the device through central outlets (outlets 4 and 5) along a laminar flow. When voltage is applied to the main separation channel having an amphoteric electrolyte with a sample input flow rate of 5 mL / min, a linear pH gradient is established, and basic fuchsine and fluorescein migrate to the cathode and anode, respectively, which is consistent with theoretical electrophoretic mobility predictions (Figs. 43b to 43d). As the electric field strength is increased by raising the applied voltage from 600 V (Fig. 43b) to 900 V (Fig. 43c) and to 1100 V (Fig. 43D), it was observed that the separation of the two molecules increases in proportion to the length of the main separation channel. Figs. 44a and 44bFigure 44 shows optical imaging regarding the purification of a mixture of small molecule dyes using an isoelectric point-based purification device, wherein the device comprises a main separation channel having five inlets and five outlets through which two amphoteric electrolyte solutions are separated by an anode channel (H2SO4), a cathode channel (NaOH), and a spacer solution, and the mixture is introduced into the spacer solution at the center of the device's inlet (Inlet 3). Figure 44a shows the isokinetic electrophoresis of a mixture of Rhodamine 6G (0.25 mg / mL, net charge +1) and Fluorescein (0.25 mg / mL, net charge -1) at 250 V and a flow rate of 5 mL / min, resulting in the two dyes being concentrated into two separate high-resolution lines. Figure 44b shows the UV irradiation results of the results shown in Figure 44a. FIGS. 45a to 45eFigure 45 shows the results of purifying a mixture of BSA (0.5 mg / mL, pI 4-5) and lysozyme (0.25 mg / mL, pI 11) using an isoelectric point-based purification device, wherein the device comprises an anode channel (H2SO4), a cathode channel (NaOH), and a main separation channel having five inlets and five outlets through which an amphoteric electrolyte solution flows, and the mixture was introduced from the center of the inlet (inlet 3) of the device. Figure 45a shows spectrophotometric analysis by BCA assay for the total protein concentration of fractions derived from the five outlets at 0 V and a flow rate of 10 mL / min. Figure 45b shows SDS-PAGE analysis of fractions derived from the five outlets at 0 V and a flow rate of 10 mL / min, showing that the mixture is present at outlet 3. Figure 45c shows the spectrophotometric analysis by BCA assay for the total protein concentration of fractions derived from five outlets at 850 V and a flow rate of 10 mL / min, showing that the protein is distributed across outlets 2, 3, and 4. Figure 45d shows the SDS-PAGE analysis of fractions derived from five outlets at 850 V and a flow rate of 10 mL / min, showing that purified lysozyme is present at outlet 2 and purified BSA is present at outlet 4. Figure 45e shows the theoretical electrophoretic migration directions of BSA (facing the anode) and lysozyme (facing the cathode). FIGS. 46a to 46cFigure 46a shows the results of purifying a mixture of hlgG (0.5 mg / mL, pl 6-8) and lysozyme (0.25 mg / mL, pl 11) using an isoelectric point-based purification device, wherein the device comprises an anode channel (H2SO4), a cathode channel (NaOH), and a main separation channel having five inlets and five outlets through which an amphoteric electrolyte solution flows, wherein the mixture was introduced into the center of the inlet (inlet 3) of the device. Figure 46a shows spectrophotometric analysis by BCA assay for the total protein concentration of fractions derived from the five outlets at (1) 0V, 5mL / min, (2) 1000V, 5mL / min, (3) 1500V, 5mL / min, (4) 0V, 10mL / min, or (5) 1000V, 10mL / min. Figure 46b shows the theoretical electrophoretic migration directions of hIgG (facing the anode, cathode, and center) and lysozyme (facing the cathode). Figure 46c shows the SDS-PAGE analysis of fractions originating from five outlets at (1) 0 V, 5 mL / min, (2) 1000 V, 5 mL / min, (3) 1500 V, 5 mL / min, (4) 0 V, 10 mL / min, or (5) 1000 V, 10 mL / min. FIGS. 47a to 47dFigure 47a shows the results of purifying a mixture of BSA (0.5 mg / mL, pI 4-5) and lysozyme (0.25 mg / mL, pI 11) using an isoelectric point-based purification device, wherein the device comprises an anode channel (H2SO4), a cathode channel (NaOH), and a main separation channel having five inlets and five outlets through which an amphoteric electrolyte solution flows, wherein the mixture was introduced from the center of the inlet (inlet 3) of the device. Figure 47a shows spectrophotometric analysis by BCA assay for the total protein concentration of fractions derived from the five outlets at a flow rate of 3 mL / min at 0 V and 500 V, showing that the protein is distributed across outlets 2, 3, and 4 under applied voltage. Figure 47b shows a spectrophotometric analysis by BCA assay of the total protein concentration of fractions derived from five outlets at a flow rate of 5 mL / min at 0 V and 700 V, showing that the protein is distributed across outlets 2, 3, and 4 under the applied voltage. Figure 47c shows a spectrophotometric analysis by BCA assay of the total protein concentration of fractions derived from five outlets at a flow rate of 10 mL / min at 0 V and 850 V, showing that the protein is distributed across outlets 2, 3, and 4 under the applied voltage. Figure 47d shows an SDS-PAGE analysis of fractions derived from five outlets at (1) 0 V or 500 V at 3 mL / min, (2) 0 V or 700 V at 5 mL / min, or (3) 0 V or 850 V at 10 mL / min. Fig. 48 The figure shows an exemplary schematic diagram of connecting a charge-based purification module, a magnetic purification module, a charge-based purification module, a charge-based fluid purification module, a charge-based TFF purification module, or an isoelectric-point-based purification module to an exemplary semi-continuous process described herein using standard industrial downstream processing equipment running in fed-batch or perfusion mode to prepare a biological product for filling finish. Fig. 49It shows an exemplary schematic diagram of connecting a charge-based purification module, a magnetic purification module, a charge-based purification module, a charge-based fluid purification module, a charge-based TFF purification module, or an isoelectric-point-based purification module to the exemplary semi-continuous process described herein using standard industrial downstream processing equipment running in fed-batch or perfusion mode to prepare a biological product for filling finish in the absence of an independent virus inactivation and removal process step. Specific details for implementing the invention

[0329] In this specification, a continuous process for purifying biological products is provided, in particular. The process currently claimed offers many advantages over current downstream methods and processes for purifying biological products, such as, for example, proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, growth factors, oligonucleotides, viruses, adenoviruses, adeno-associated viruses, or lentiviruses. For example, without intent to be limited, the process described herein provides a continuous bioprocess for purifying monoclonal antibodies that eliminates the membrane contamination problems inherent in conventional multi-stage filtration processes (e.g., multi-stage tangential flow filtration or deep filtration) by having an initial filtration step comprising at least one dynamic filtration module as described herein to remove large impurities (e.g., cells, cell debris, and aggregates). In addition, the above continuous process significantly reduces production facility footprint, time required for facility setup and validation, facility setup costs, and capital equipment expenditures compared to existing approaches for manufacturing batch, single-use, or semi-continuous monoclonal antibodies while maintaining throughput and yield.

[0330] Continuous bioprocessing as described herein allows for the use of smaller and more streamlined equipment (e.g., smaller bioreactor volumes and downstream bioprocess equipment) because, due to its ability to operate continuously, it eliminates the need for the large process equipment required for the centrifugation, deep filtration, and column chromatography steps of conventional downstream bioprocessing—the size of which depends on the large bioreactor volume. Furthermore, smaller and more streamlined equipment operating continuously allows for the use of much smaller bioreactor(s) for producing monoclonal antibodies under steady conditions. Continuous bioprocessing as described herein can also significantly reduce operating costs, overall bioprocess line downtime, and biological product loss compared to conventional monoclonal antibody manufacturing approaches. Finally, the process described herein for purifying the biological product is performed in a system having a footprint of much less than current technology, without sacrificing product throughput or yield on a kilogram-per-year basis. For example, the process for producing and purifying monoclonal antibodies described herein operates on a footprint of up to approximately 30,000 square feet. In contrast, current monoclonal antibody production and downstream processes require at least 200,000 square feet.

[0331] A continuous process for purifying a biological product using at least one of a dynamic filtration module, an affinity-based magnetic purification module, a charge-based magnetic purification module, or an isoelectric point-based fluid purification module.

[0332] A continuous process for purifying a biological product is described, said process comprising continuously receiving a heterogeneous mixture containing a biological product through an input line, said biological product comprising, but not limited to, a protein or a fragment thereof (polypeptide), an antibody or a fragment thereof, a cytokine, a chemokine, an enzyme, or a growth factor. Upon purification, the biological product (e.g., a monoclonal antibody) is substantially pure when impurities (cells, cell debris, aggregates, host cell proteins, undesirable proteins and peptides, undesirable antibodies, undesirable nucleic acids and oligonucleotides, viruses, salts, buffer components, surfactants, sugars, metal contaminants, leachates, medium components, and / or naturally occurring organic molecules) are removed at least 60% by weight, 70% by weight, 80% by weight, 90% by weight, 95% by weight, or even 99% by weight.

[0333] The above process includes continuously removing large impurities from a heterogeneous mixture through dynamic filtration. The dynamic filtration process includes at least one dynamic filtration module that produces a filtrate containing biological products by continuously feeding biological products from at least one output head that fluidly communicates with an input line under negative pressure to the dynamic filtration module. The dynamic filtration module may additionally include at least one additional input line for supplying a wash buffer through a coaxial output head or a separate single-axis output head.

[0334] In embodiments, the process described herein comprises purifying a biological product continuously produced in a bioreactor (e.g., fed-batch bioreactor, perfusion bioreactor, and chemostat bioreactor). For example, the bioreactor includes a bioreactor feed line and an output bleed line that enable steady-state cell culture growth conditions, and the output bleed line functions as an input line that allows continuous fluid flow from the bioreactor to a dynamic filtration module.

[0335] As described herein, a process for continuously removing large impurities from a heterogeneous mixture (or mixture) does not include centrifugation, disk-stack centrifugation, deep filtration, static filtration, tangential flow filtration, a hydrocyclone, or any combination thereof. The term “static filtration” refers to a process in which the heterogeneous mixture being filtered remains in a static state, that is, for example, a filter membrane (or deep filter) has a limited capacity, and when the membrane reaches its capacity, the filtration rate decreases (for example, the membrane pores become clogged). In “static” (as opposed to “dynamic”) filtration, the filter membrane remains in a stationary state (does not move), and the flow (for example, the flow of the heterogeneous mixture) passes through the stationary filter membrane. Such static filtration methods are common, simple, and well known in the art.

[0336] Unlike static filtration methods commonly used in the industry, the process described herein describes a dynamic filtration module, wherein the components of the dynamic filtration module move in a coordinated manner so that filtration can occur continuously in new, unused target areas of the filter membrane (e.g., the membrane moves or advances according to the flow rate of the entire process). This prevents membrane fouling or clogging and allows for control of filter cake packing and thickness during operation.

[0337] The dynamic filtration module includes a filter membrane roll, a membrane support structure, at least one support rod or roller, a vacuum line, a vacuum system, and at least one vacuum collection container.

[0338] In embodiments, the filter membrane roll comprises a rolled filter membrane, wherein the filter membrane comprises, but is not limited to, polyethersulfone (PES), hydrophilic polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), hydrophilic PTFE, or any combination thereof.

[0339] The pore size of the rolled filter membrane varies depending on the biological product being purified. For example, the pore size of the rolled filter membrane is in the range of 0.1 μm to 1 μm. Alternatively, the pore size is in the range of about 0.2 μm to about 0.45 μm, or the pore size is less than about 0.45 μm. In another example, when purifying antibodies, the pore size of the rolled filter membrane is in the range of 0.2 μm to about 0.45 μm.

[0340] The filter membrane roll has a width of about 10 mm to about 600 mm. For example, the width of the filter membrane roll may vary depending on the size of the dynamic filtration system or the membrane support structure.

[0341] In embodiments, the filter membrane roll also functions as a feed reel communicating with the collection reel, meaning that the filter membrane starts from the assembly roll and extends to the collection reel, which is initially empty, to create a reel-to-reel system. In aspects, the dynamic filtration module includes a rolled filter membrane extending between the feed reel and the collection reel, said filter membrane having a target area (e.g., an active target area) configured to accommodate a heterogeneous mixture. For example, feed reel movement is controlled by a servo motor connected to a gearbox that limits the rotational speed per minute (RPM) to a ratio of 200:1 to enable low membrane transport speeds with high torque. Collection reel movement is controlled by a servo motor connected to a gearbox that limits the RPM to a ratio of 200:1 to enable low membrane transport speeds with high torque. In addition, the feed reel motor and the collect reel motor are controlled by a closed-loop controller that operates a feedback mechanism to ensure a constant speed and the diameter of the filter membrane rolls that change constantly on both the feed reel and the collect reel during operation. For example, the feed reel and the collect reel operate in the same direction at the same speed.

[0342] In embodiments, the transport speed of the filter membrane is in the range of about 0.1 mm / sec to about 100 mm / sec, preferably about 0.1 mm / sec to about 10 mm / sec.

[0343] The membrane support structure of the dynamic filtration module comprises a mechanically flat contact surface derived from a material with a low coefficient of static friction (e.g., PTFE) and an opening continuous with the vacuum line. As used herein, "membrane support structure" refers to a component manufactured to provide structural support to the active area of ​​the filter membrane to prevent deformation when passing through a negative pressure region due to the opening continuous with the vacuum line. Additionally, as used herein, "mechanically flat contact surface" refers to a surface with a low coefficient of static friction, which generates low frictional forces opposing the transport of the filter membrane, particularly when wet. The mechanically flat contact surface can affect the ease with which the filter membrane moves in a dynamic manner. The mechanically flat contact surface can also be measured by surface roughness, where a lower value indicates a flatter surface. In addition, since rough surfaces have greater frictional forces between surfaces than flat surfaces, the mechanically flat contact surface used herein refers to a surface with low friction (i.e., a low coefficient of static friction).

[0344] In embodiments, the membrane support structure of the dynamic filtration module includes an opening. The opening may include, for example, a mesh, at least one slot, at least one hole, a frit, a porous material, or any combination thereof. For example, the opening may include a series of regularly or irregularly spaced elements (e.g., a mesh, at least one slot, at least one hole, or any combination thereof). Additionally, the opening may include regularly spaced elements, for example, the opening may include a series of equally spaced parallel slots. Furthermore, the opening may include a grate (e.g., a series of regularly or irregularly spaced elements as described above). In another example, the opening may include one or more grates, each grate being vertical. The opening may be a collection of irregular or regular elements (e.g., a series of parallel slots). The opening may also include a mesh, which may be split-thickness or full thickness, and may or may not be in parallel rows. The elements of the opening (e.g., mesh, at least one slot, at least one hole, frit, porous material, or any combination thereof) may be of any desired thickness. For example, without being intended to be limiting, the opening may include a mesh with a thickness of about 0.25 mm to about 5 mm.

[0345] The membrane support structure of the dynamic filtration module includes a temperature control mechanism. The temperature control mechanism maintains a temperature of about 4°C to about 37°C when evaporative cooling is present. For example, during antibody purification, the temperature control mechanism maintains a temperature of 15°C to 37°C. Exemplary temperature control mechanisms include, but are not limited to, a single-loop controller, a multi-loop controller, a closed-loop controller, a PID controller, a Peltier element, a resistance heating element, and / or a thermal chuck with a circulating water / propylene glycol jacket.

[0346] In embodiments, at least one support rod or roller of the dynamic filtration module has a mechanically flat contact surface derived from a material with a low static friction coefficient (e.g., PTFE, PFA). For example, the static friction coefficient is in the range of about 0.01 to about 0.1, about 0.01 to about 0.05, or about 0.05 to about 0.1. In a specific example, the static friction coefficient is 0.04. For example, the dynamic filtration module includes at least one support rod or roller having a mechanically flat contact surface to stabilize the movement of a filter membrane across a membrane support structure.

[0347] In embodiments, the dynamic filtration module includes at least one output head for controlling the flow of a heterogeneous mixture and distributing the heterogeneous mixture to a target area (e.g., an active target area) of a filter membrane. For example, the at least one output head is a tube or a slot die.

[0348] In some embodiments, the dynamic filtration module further includes at least one additional input line for supplying a cleaning buffer through a coaxial output head, a separate single-axis output head, a separate slot die output head, or a slot die output head with multiple openings.

[0349] In some embodiments, the dynamic filtration module includes elements known in the coating and converting industry, for example, without limitation, active or passive edge guides, tension regulators (e.g., dancers), brakes and tension detectors, or any combination thereof.

[0350] In embodiments, the dynamic filtration module includes a vacuum system continuous with a membrane support structure to apply a negative pressure across an active target area of ​​the filter membrane, wherein the negative pressure allows for active transport of the filter membrane across the membrane support structure and enables the collection of filtrate containing biological products. For example, the vacuum system of the dynamic filtration module maintains a gauge pressure of about -0.05 bar to about 0.98 bar for continuous filtration.

[0351] In embodiments, the dynamic filtration module further comprises at least one vacuum collection vessel configured to collect the filtrate, and at least one sensor or detector. In the aspect described herein, during purification by dynamic filtration, the filtrate containing the biological product is fed under negative pressure to a vacuum collection vessel capable of collecting about 50 mL to about 100 L. For example, the vacuum collection vessel capable of collecting the filtrate is about 1 L to about 10 L. In another example, the vacuum collection vessel capable of collecting the filtrate is about 1 L to about 50 L.

[0352] In embodiments, a process for continuously removing large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration comprises multistage filtration using at least two individual rolled filter membranes with different pore sizes. For example, this multistage dynamic filtration process comprises at least one first dynamic filtration device having a rolled filter membrane with a large pore size (e.g., 0.45 μm) in fluid communication with at least one second dynamic filtration device having a rolled filter membrane with a small pore size (e.g., 0.2 μm), thereby producing a filtrate containing biological products.

[0353] The process described herein comprises continuously transferring a solution to a first module capable of separating it into two or more fractions, each containing at least one fraction containing a biological product. For example, separating the solution into two or more fractions may include at least one fraction containing a biological product and at least one other fraction containing small impurities. As described herein, the first module comprises an affinity-based magnetic purification device. An "affinity-based magnetic purification device" refers to a purification technique based on molecular structural binding interactions (e.g., ligand-receptor interactions) in which a selective surface-immobilized ligand recognizes and binds to the biological product to be purified. For example, the first module has at least one first inlet and at least one first outlet and is configured to allow a continuous fluid flow between the first inlet and the first outlet via a loop conveyor system or a pick-and-place robotic system.

[0354] In embodiments, the affinity-based magnetic purification apparatus further comprises a suspension of magnetic resin beads. The surfaces of the magnetic resin beads are connected to, for example, protein A, protein G, protein L, antigen protein, protein, receptor, antibody, or aptamer, without any intention of limitation. Continuous purification of a biological product (e.g., monoclonal antibody) with affinity magnetic resin beads can avoid the cumbersome processing steps of conventional affinity column chromatography (e.g., protein A affinity chromatography).

[0355] In embodiments, the diameter of the magnetic resin beads of the affinity-based magnetic purification device is about 0.2 microns to about 200 microns. The diameter of the magnetic resin beads may vary depending on the biological product being purified and the flow rate of the process. Additionally, the concentration of the magnetic resin beads may be in the range of 0.01 wt% to 25 wt%. For example, the concentration of the magnetic resin beads may be about 1 wt% to about 10 wt%. In another example, the binding ability of the magnetic resin beads is a function of the bead concentration, the surface area to volume ratio, the affinity ligand density, or any combination thereof. In yet another example, the magnetic resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

[0356] A loop conveyor system can refer, for example, to a continuous or infinite loop. A loop conveyor system is advantageous in that it allows for the continuous and efficient movement of large volumes at high flow rates through the process while allowing for a smaller footprint compared to conventional affinity column chromatography systems (e.g., protein A affinity chromatography). Since biological products are transported directly on the track, objects of all sizes and shapes, whether regular or irregular, can be configured for transport. In some respects, the object is a transport container having a regular shape (e.g., a cube, a rectangular prism, a cylinder, and a cone).

[0357] In embodiments, the loop conveyor system has at least two transport containers filled with magnetic resin beads configured to continuously receive a filtrate containing a mixture containing a biological product, and then transport a generated heterogeneous mixture containing a biological product, magnetic resin beads, a buffer, or any combination thereof.

[0358] A pick-and-place robotic system may refer, for example, to at least one robot or robotic arm. A pick-and-place robotic system is advantageous in that it allows for the continuous and efficient transfer of large volumes at high flow rates through the process while allowing for a smaller footprint compared to conventional affinity column chromatography systems (e.g., protein A affinity chromatography). Since biological products contained in transport containers are picked up and placed, regular-shaped objects of all sizes, with or without handles, can be configured for transport and loading. In some aspects, the object is a transport container having a regular shape (e.g., a cube, and a rectangular prism).

[0359] In embodiments, the pick-and-place robot system has at least two transport containers filled with magnetic resin beads configured to continuously receive a filtrate containing a mixture containing a biological product, and then transport a generated heterogeneous mixture containing the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0360] The affinity-based magnetic purification module further comprises at least one external magnetic field that can be used to attract and separate the magnetic resin beads from the heterogeneous mixture to enable washing within at least one of at least two transport containers. Additionally, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from the heterogeneous mixture to enable the elution of the biological product within at least one of at least two transport containers. Alternatively, the at least one external magnetic field can be used to enable the recycling of the magnetic resin beads within at least one of at least two transport containers. For example, mixing of the magnetic resin beads can be achieved by placing at least one transport container between two separate opposing magnetic fields that switch between an on state and an off state.

[0361] The process described herein also comprises continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving the flow from at least one first outlet of the first module, said second module comprising a charge-based magnetic purification device. A "charge-based magnetic purification device" as used herein comprises, for example, purifying biological molecules based on their surface charge, ionic properties, electrostatic interactions, or isoelectric point. As described herein, a charge-based magnetic purification device comprises a positive charge-based magnetic purification device, a negative charge-based magnetic purification device, or a combination thereof. For example, the second module has at least one second inlet and at least one second outlet and is configured to allow a continuous fluid flow between the second inlet and the second outlet via a loop conveyor system or a pick-and-place robot system.

[0362] In embodiments, a charge-based magnetic purification device (e.g., positive charge and / or negative charge-based magnetic purification) further comprises a suspension of magnetic resin beads. For example, the surface of the magnetic resin beads may each comprise a cationic or anionic functional group configured to selectively associate with the biological product at a specific pH and ionic strength to enable positive charge-based magnetic purification or negative charge-based magnetic purification. Continuous purification of a biological product (e.g., a monoclonal antibody) with ionic magnetic resin beads can avoid the cumbersome processing steps of conventional ion exchange column chromatography (e.g., cation exchange or anion exchange chromatography).

[0363] In embodiments, the diameter of the magnetic resin beads of the charge-based magnetic purification device is about 0.2 microns to about 200 microns. The diameter of the magnetic resin beads may vary depending on the biological product being purified and the flow rate of the process. Additionally, the concentration of the magnetic resin beads may be in the range of 0.01 wt% to 25 wt%. For example, the concentration of the magnetic resin beads may be about 1 wt% to about 10 wt%. In another example, the charge or electrostatic associative capacitance of the magnetic resin beads is a function of the bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In yet another example, the magnetic resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

[0364] A loop conveyor system can be referred to, for example, as a continuous or infinite loop. A loop conveyor system is advantageous in that it allows for the continuous and efficient movement of large volumes at high flow rates through the process while allowing for a smaller footprint compared to conventional ion exchange column chromatography systems. Since biological products are transported directly on the track, objects of all sizes and shapes, whether regular or irregular, can be configured for transport. In some respects, the object is a transport container having a regular shape (e.g., a cube, a rectangular prism, a cylinder, and a cone).

[0365] In embodiments, the loop conveyor system has at least two transport containers filled with magnetic resin beads configured to continuously receive a mixture containing a biological product and then transport a resulting heterogeneous mixture containing the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0366] A pick-and-place robotic system may refer, for example, to at least one robot or robotic arm. A pick-and-place robotic system is advantageous in that it allows for the continuous and efficient movement of large volumes at high flow rates through the process while allowing for a smaller footprint compared to conventional ion exchange chromatography systems. Since biological products contained in transport vessels are picked up and placed, regular-shaped objects of all sizes, with or without handles, can be configured for transport and loading. In some aspects, the object is a transport vessel having a regular shape (e.g., a cube, and a rectangular prism).

[0367] In embodiments, the pick-and-place robot system has at least two transport containers filled with magnetic resin beads configured to continuously receive a filtrate containing a mixture containing a biological product, and then transport a generated heterogeneous mixture containing the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0368] The charge-based magnetic purification module further comprises at least one external magnetic field that can be used to attract and separate the magnetic resin beads from the heterogeneous mixture to enable washing within at least one of at least two transport containers. Additionally, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from the heterogeneous mixture to enable the dissociation and collection of the biological product within at least one of at least two transport containers. Alternatively, the at least one external magnetic field can be used to enable the recycling of the magnetic resin beads within at least one of at least two transport containers. For example, mixing of the magnetic resin beads can be achieved by placing at least one transport container between two separate opposing magnetic fields that switch between an on state and an off state.

[0369] In the embodiments described herein, the magnetic resin beads of one or both of the first (affinity-based magnetic purification) and / or second (charge-based magnetic purification) module(s) are recycled and reused. For example, the beads may be reused at least two, three, four, or more times to purify biological products.

[0370] Alternatively, the process described herein comprises continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, said second module comprising a free-flow electrophoresis device. A free-flow electrophoresis device having a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an aqueous ionic solution may be used instead of or in addition to charge-based magnetic purification module(s) for purifying a biological product (e.g., a monoclonal antibody).

[0371] For example, the solution contact surfaces of two parallel plates include glass, ceramic, plastic, or any combination thereof. In some examples, the aqueous ionic solution can create a pH gradient across the main separation channel. In other examples, the aqueous ionic solution can impart a constant pH across the main separation channel.

[0372] In embodiments, the free-flow electrophoresis device has at least one fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a pH gradient. For example, an isoelectric point-based fluid purification module comprises at least one first fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across a main separation channel (e.g., the approximate pH gradient may be in the pH range of about 2 to about 10); and at least one second fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient across a main separation channel (e.g., the fine pH gradient may be in the pH range of about 5 to about 8). For example, the pH gradient across the main separation channel can be further refined using additional subsequent fluid elements or chips comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow (e.g., a pH range of about 7.1 to about 7.6).

[0373] In another embodiment, the free-flow electrophoresis device comprises a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow to operate in band electrophoresis or charge separation operating mode, and at least one fluid element without a pH gradient (e.g., constant pH in the main separation channel). For example, an isoelectric point-based fluid purification module comprises at least one first fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant basic pH (e.g., pH greater than 7); and at least one second fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant acidic pH (e.g., pH less than 7).

[0374] In another embodiment, the free-flow electrophoresis device has at least one fluid element comprising a fluid channel created between two parallel plates to operate in an isometric electrophoresis operating mode, an electric field or electric field gradient perpendicular to the direction of fluid flow, and both an acidic pH gradient and a basic pH gradient separated by a spacer solution (e.g., NaCl solution).

[0375] In another embodiment, the isoelectric point-based fluid purification module comprises at least one first free-flow electrophoretic device comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, and at least one second free-flow electrophoretic device comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, wherein each element is connected in series and can operate in independent operating modes that enable purification. For example, at least one first free-flow electrophoretic device can operate in an isoelectric focusing mode, and at least one second free-flow electrophoretic device can operate in an isokinetic electrophoretic mode to increase separation resolution.

[0376] In another embodiment, the isoelectric point-based fluid purification module comprises at least one first fluid element comprising a fluid channel having at least one dielectrophoretic electrode capable of inducing a defined unidirectional force; at least one second free-flow electrophoretic device comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across a main separation channel (e.g., a pH range of about 2 to about 10); and at least one third free-flow electrophoretic device comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient across a main separation channel (e.g., a pH range of about 5 to about 8). For example, the pH gradient across the main separation channel can be further refined using additional subsequent fluid elements or chips comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow (e.g., a pH range of about 7.1 to about 7.6).

[0377] In embodiments, the isoelectric point-based fluid purification device further includes at least two electrodes (e.g., platinum wire electrodes) to function as an anode or a cathode.

[0378] In embodiments, the backpressure within the isoelectric-based fluid purification device depends on the channel shape and dimensions, the inlet and outlet openings and / or tubing diameter, and the input flow rate. For example, the backpressure is in the range of about 0.5 psi to about 10 psi. In some examples, the backpressure is controlled by a needle valve, for example, without the intention of limiting it.

[0379] In embodiments, the isoelectric point-based fluid purification device further comprises at least one bubble removal system for continuously removing O2 and H2 gas bubbles generated in the electrode channel under an applied voltage. In some embodiments, removing electrolytic bubbles is essential to enable continuous operation for substantially long periods. For example, the bubble removal system uses a hydrophobic PTFE membrane to create a waterproof seal over the electrode channel that can continuously remove electrolytic bubbles at the point of generation by exposure to a vacuum system. For example, the vacuum gauge pressure is in the range of about -0.05 bar to about -0.4 bar.

[0380] In embodiments, the isoelectric point-based fluid purification device further comprises an active cooling system or heat sink (e.g., a Peltier element, a thermal chuck with a circulating water / propylene glycol jacket) to enable temperature control and Joule heat dissipation. For example, the active cooling system can control cooling and / or heat dissipation in the range of about 4°C to about 50°C, preferably about 4°C to about 37°C. Ideally, when isolating biological products (e.g., monoclonal antibodies), the temperature is maintained at about 10°C to about 25°C. For example, the active cooling system comprises an aluminum thermal chuck containing a cooled circulating water / propylene glycol jacket.

[0381] In embodiments, the isoelectric point-based fluid purification module includes at least one buffer or amphoteric electrolyte system.

[0382] In embodiments, the isoelectric point-based fluid purification module comprises at least one electrode solution. In some embodiments, the at least one electrode solution comprises an electrolyte solution configured to enable proper function in contact with an anode or a cathode, for example, phosphoric acid and sodium hydroxide, respectively. In other embodiments, the at least one electrode solution comprises at least one amphoteric electrolyte solution configured to enable proper function in contact with an anode or a cathode, for example, Tris buffered saline, flowing through a main separation channel, an anode channel, and a cathode channel.

[0383] In embodiments, the isoelectric point-based fluid purification module includes at least one sensor or detector. For example, at least one sensor or detector is placed inline. In some examples, at least one sensor or detector includes, but is not limited to, a flow sensor, a temperature sensor, a conductivity sensor, a pH sensor, a refractive index detector, a UV detector, or a back pressure sensor.

[0384] In embodiments, the isoelectric point-based fluid purification module includes at least one liquid circuit breaker, or disconnects the downstream of the device from the upstream of at least one inline sensor or detector to enable detection or sensing in a voltage-free solution.

[0385] The process currently claimed offers many advantages over current downstream methods and processes for purifying biological products, such as proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, or growth factors. For example, without limitation, the process described herein provides a continuous bioprocess for purifying monoclonal antibodies that significantly reduces production facility footprint, time required for facility setup and validation, facility setup costs, and capital expenditures compared to existing approaches for manufacturing batch, single-use, or semi-continuous monoclonal antibodies while maintaining throughput and yield. Continuous bioprocessing as described herein enables the use of smaller and more streamlined equipment (e.g., smaller bioreactor volumes and downstream bioprocess equipment) because, due to its ability to operate continuously, it does not require the large process equipment—whose size depends on the large bioreactor volume—that is required for the batch centrifugation, deep filtration, and column chromatography steps of conventional downstream bioprocessing. Furthermore, smaller and more streamlined equipment operating continuously enables the use of much smaller bioreactor(s) for producing monoclonal antibodies under steady conditions. Continuous bioprocesses as described herein can also significantly reduce operating costs, total bioprocess line downtime, and biological product loss compared to conventional monoclonal antibody manufacturing approaches. Finally, the process described herein for purifying the biological product is performed in a system having a footprint of much less than current technology, without sacrificing product throughput or yield on a kilogram-per-year basis.

[0386] Advantages of the processes and methods described herein include the ability to remove large impurities (e.g., cells, cell debris, and aggregates) without membrane fouling or clogging. Membrane fouling may refer to a process in which heterogeneous mixtures are deposited on the membrane surface or within the membrane pores, causing a decrease in membrane performance over time and placing significant limitations on the use of conventional filtration systems. For example, purifying cells, cell debris, and aggregates from cell culture media using conventional filtration or tangential flow filtration systems typically results in fouling or clogging of the filter membrane, making these methods unsuitable as a means of continuously removing large impurities from heterogeneous mixtures containing biological products over long-term continuous processes. In contrast, the dynamic filtration device described herein enables the continuous removal of large impurities from heterogeneous mixtures containing biological products without contaminating the membrane, because the active target area of ​​the filter membrane is continuously refreshed.

[0387] Additionally, because the entire process for producing and purifying biological products can be continuous and maintain a flow rate in the range of about 0.1 mL / min to about 50 mL / min (e.g., about 5 mL / min to about 10 mL / min) throughout the entire process, the process equipment and the overall process footprint can occupy a much smaller footprint than current standard processes on a kilogram / year basis without sacrificing product throughput or yield. For example, the process for producing and purifying monoclonal antibodies described herein operates on a footprint of up to about 30,000 square feet. In contrast, current monoclonal antibody production and downstream processes require at least 200,000 square feet. For example, the flow rate of the process for purifying biological products ranges from about 1 mL / min to about 10 mL / min. In some examples, the flow rate of the step for continuously removing large impurities from heterogeneous mixtures ranges from about 0.1 mL / min to about 50 mL / min. In another example, the flow rate of the step of continuously removing large impurities from a heterogeneous mixture is the same as the flow rate from the bioreactor discharge line. In another example, a process is provided in which the flow rate of the step of continuously transferring the filtrate to the first module is in the range of about 0.1 mL / min to about 50 mL / min. In yet another example, a process is provided in which the flow rate of the step of continuously transferring a fraction containing a biological product from the first outlet to the second module is in the range of about 0.1 mL / min to about 50 mL / min.

[0388] A significant advantage of the process and method using the magnetic resin beads (e.g., magnetic agarose) described herein is that such a system does not require a conventional stationary bed or packed resin column (e.g., for standard chromatography) for sterilization, recycling, and / or regeneration. For example, such a system provides the recycling and / or regeneration of magnetic resin beads to create an infinite surface area of ​​the magnetic resin beads during operation, and consequently provides a continuous and cost-effective method. In other words, the module described herein does not have a fixed coupling or associative capability. As a specific example, since the magnetic resin beads used during the purification of biological products as described herein are continuously recycled and regenerated, the flow from the previous stage can be accommodated in either the dynamic filtration module or the purification module without interrupting the flow from the bioreactor discharge line.

[0389] In other words, since the module described in the present invention undergoes these steps sequentially, it does not need to be left idle for sterilization, regeneration, and / or recycling after execution. The method differs from current continuous chromatography methods in that, because current column chromatography methods have limited column capacity due to resin packing constraints, column switching of multiple packed columns is required to accommodate a continuous input flow and enable the regeneration and / or recycling of columns that have reached full capacity. Another advantage of the method described herein includes that the magnetic resin beads are not packed into a stationary phase, but rather move. This mobility of the beads increases the surface area of ​​the resin beads available for bonding or aggregation, as substantially more of the magnetic resin bead surface is exposed to allow for free bonding. Additionally, resin beads within a packed column are exposed to high pressure differences to generate a flow through the column, and damage resulting from this is one of the reasons the column's life is shortened beyond the desired life. The movable resin beads of the present invention are subjected to substantially lower pressure, making them much softer for brittle beads and thus extending their lifespan. Additionally, this mobility increases the likelihood that the magnetic resin beads will be fully regenerated and return to their initial state. This further enhances the cost-effectiveness of the method described herein because the magnetic resin is utilized more efficiently.

[0390] A significant advantage of the process and method utilizing free-flow electrophoresis described herein is that this system represents a "product loss-free" process, meaning that the product does not need to interact with a resin or other purification moiety because separation occurs through interaction with an electric field based on the physicochemical properties of the target biological product in an aqueous solution. Another advantage in the resolution of this approach is observed because theoretically higher purity products can be obtained compared to conventional ion exchange chromatography. Additionally, separation based on intrinsic physicochemical properties extends the utility of this approach for the purification of various biological products, including but not limited to proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, growth factors, oligonucleotides, viruses, adenoviruses, adeno-associated viruses (AAVs), or lentiviruses.

[0391] In addition, the modular approach provides process design flexibility to accommodate a diverse range of biological products.

[0392] A continuous process for purifying a biological product using at least one of a dynamic filtration module, an affinity-based purification module, a charge-based purification module, or an isoelectric point-based fluid purification module.

[0393] A continuous process for purifying a biological product is described, said process comprising continuously receiving a heterogeneous mixture containing a biological product through an input line, said biological product comprising, but not limited to, a protein or a fragment thereof (polypeptide), an antibody or a fragment thereof, a cytokine, a chemokine, an enzyme, or a growth factor. Upon purification, the biological product (e.g., a monoclonal antibody) is substantially pure when impurities (cells, cell debris, aggregates, host cell proteins, undesirable proteins and peptides, undesirable antibodies, undesirable nucleic acids and oligonucleotides, viruses, salts, buffer components, surfactants, sugars, metal contaminants, leachates, medium components, and / or naturally occurring organic molecules) are removed at least about 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, or even 99 wt%.

[0394] The above process includes continuously removing large impurities from a heterogeneous mixture through dynamic filtration. The dynamic filtration process includes at least one dynamic filtration module that produces a filtrate containing biological products by continuously feeding biological products from at least one output head that fluidly communicates with an input line under negative pressure to the dynamic filtration module. The dynamic filtration module may additionally include at least one additional input line for supplying a wash buffer through a coaxial output head or a separate single-axis output head.

[0395] In embodiments, the process described herein comprises purifying a biological product continuously produced in a bioreactor (e.g., fed-batch bioreactor, perfusion bioreactor, and chemostat bioreactor). For example, the bioreactor includes a bioreactor feed line and an output bleed line that enable steady-state cell culture growth conditions, and the output bleed line functions as an input line that allows continuous fluid flow from the bioreactor to a dynamic filtration module.

[0396] As described herein, the process for continuously removing large impurities from a heterogeneous mixture does not include centrifugation, disk-stack centrifugation, deep filtration, static filtration, tangential flow filtration, hydrocyclone, or any combination thereof. The term “static filtration” refers to a process in which the heterogeneous mixture being filtered remains in a static state, that is, for example, a filter membrane (or deep filter) has a limited capacity, and when the membrane reaches its capacity, the filtration rate decreases (for example, the membrane pores become clogged). In “static” (as opposed to “dynamic”) filtration, the filter membrane remains in a stationary state (does not move), and the flow (for example, the flow of the heterogeneous mixture) passes through the stationary filter membrane. Such static filtration methods are common, simple, and well known in the art.

[0397] Unlike static filtration methods commonly used in the industry, the process described herein describes a dynamic filtration module, wherein the components of the dynamic filtration module move in a coordinated manner so that filtration can occur continuously in new, unused target areas of the filter membrane (e.g., the membrane moves or advances according to the flow rate of the entire process). This prevents membrane fouling or clogging and allows for control of filter cake packing and thickness during operation.

[0398] The dynamic filtration module includes a filter membrane roll, a membrane support structure, at least one support rod or roller, a vacuum line, a vacuum system, and at least one vacuum collection container.

[0399] For example, a filter membrane roll comprises a rolled filter membrane, wherein the filter membrane comprises, but is not limited to, polyethersulfone (PES), hydrophilic polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), hydrophilic PTFE, or any combination thereof.

[0400] The pore size of the rolled filter membrane varies depending on the biological product being purified. For example, the pore size of the rolled filter membrane is in the range of 0.1 μm to 1 μm. Alternatively, the pore size is in the range of about 0.2 μm to about 0.45 μm, or the pore size is less than about 0.45 μm. In another example, when purifying antibodies, the pore size of the rolled filter membrane is in the range of 0.2 μm to about 0.45 μm.

[0401] The filter membrane roll has a width of about 10 mm to about 600 mm. For example, the width of the filter membrane roll may vary depending on the size of the dynamic filtration system or the membrane support structure.

[0402] In embodiments, the filter membrane roll also functions as a feed reel communicating with the collection reel, meaning that the filter membrane starts from the assembly roll and extends to an initially empty collection reel to create a reel-to-reel system. In aspects, the dynamic filtration module includes a rolled filter membrane extending between the feed reel and the collection reel, and the filter membrane has an active target area configured to accommodate a heterogeneous mixture. For example, feed reel movement is controlled by a servo motor connected to a gearbox that limits the rotational speed per minute (RPM) to a ratio of 200:1 to enable low membrane transport speeds with high torque. Collection reel movement is controlled by a servo motor connected to a gearbox that limits the RPM to a ratio of 200:1 to enable low membrane transport speeds with high torque. In addition, the feed reel motor and the collect reel motor are controlled by a closed-loop controller that operates a feedback mechanism to ensure a constant speed and the diameter of the filter membrane rolls that change constantly on both the feed reel and the collect reel during operation. For example, the feed reel and the collect reel operate in the same direction at the same speed.

[0403] In embodiments, the transport speed of the filter membrane is in the range of about 0.1 mm / sec to about 100 mm / sec, preferably about 0.1 mm / sec to about 10 mm / sec.

[0404] The membrane support structure of the dynamic filtration module comprises a mechanically flat contact surface derived from a material with a low static friction coefficient (e.g., PTFE) and an opening continuous with the vacuum line. As used herein, "membrane support structure" refers to a component manufactured to provide structural support to the active area of ​​the filter membrane to prevent deformation when passing through a negative pressure region due to the opening continuous with the vacuum line. Additionally, as used herein, "mechanically flat contact surface" refers to a surface with a low static friction coefficient, which generates low frictional forces opposing the transport of the filter membrane, particularly when wet. A mechanically flat contact surface can affect the ease with which the filter membrane moves in a dynamic manner. A mechanically flat contact surface can also be measured by surface roughness, where a lower value indicates a flatter surface. Furthermore, because rough surfaces generate greater frictional forces between surfaces than flat surfaces, as used herein, a mechanically flat contact surface refers to a surface with low frictional forces (i.e., a low static friction coefficient).

[0405] In embodiments, the membrane support structure of the dynamic filtration module includes an opening. The opening may include, for example, a mesh, at least one slot, at least one hole, a frit, a porous material, or any combination thereof. For example, the opening may include a series of regularly or irregularly spaced elements (e.g., a mesh, at least one slot, at least one hole, or any combination thereof). Additionally, the opening may include regularly spaced elements, and for example, the opening may include a series of equally spaced parallel slots. Furthermore, the opening may include a grate (e.g., a series of regularly or irregularly spaced elements as described above). In another example, the opening may include one or more grates, each grate being vertical. The opening may be a collection of irregular or regular elements (e.g., a series of parallel slots). The opening may also include a mesh, which may be split-thickness or full thickness, and may or may not be in parallel rows. The elements of the opening (e.g., mesh, at least one slot, at least one hole, frit, porous material, or any combination thereof) may be of any desired thickness. For example, without being intended to be limiting, the opening may include a mesh with a thickness of about 0.25 mm to about 5 mm.

[0406] The membrane support structure of the dynamic filtration module includes a temperature control mechanism. The temperature control mechanism maintains a temperature of about 4°C to about 37°C when evaporative cooling is present. For example, during antibody purification, the temperature control mechanism maintains a temperature of about 15°C to about 37°C. Exemplary temperature control mechanisms include, but are not limited to, a single-loop controller, a multi-loop controller, a closed-loop controller, a PID controller, a Peltier element, and / or a thermal chuck with a circulating water / propylene glycol jacket.

[0407] In embodiments, at least one support rod or roller of the dynamic filtration module has a mechanically flat contact surface derived from a material with a low coefficient of static friction (e.g., PTFE, PFA). For example, the dynamic filtration module includes at least one support rod or roller having a mechanically flat contact surface to stabilize the movement of a filter membrane across a membrane support structure.

[0408] In embodiments, the dynamic filtration module includes at least one output head for controlling the flow of a heterogeneous mixture and distributing the heterogeneous mixture to an active target area of ​​a filter membrane. For example, the at least one output head is a tube or a slot die.

[0409] In some embodiments, the dynamic filtration module further includes at least one additional input line for supplying a cleaning buffer through a coaxial output head, a separate single-axis output head, a separate slot die output head, or a slot die output head with multiple openings.

[0410] In some embodiments, the dynamic filtration module includes elements known in the coating and converting industry, for example, without limitation, active or passive edge guides, tension regulators (e.g., dancers), brakes and tension detectors, or any combination thereof.

[0411] In embodiments, the dynamic filtration module comprises a vacuum system continuous with a membrane support structure to apply a negative pressure across a target area (e.g., an active target area) of the filter membrane, wherein the negative pressure allows for active transport of the filter membrane across the membrane support structure and enables the collection of filtrate containing biological products. For example, the vacuum system of the dynamic filtration module maintains a gauge pressure of about -0.05 bar to about -0.98 bar for continuous filtration.

[0412] In embodiments, the dynamic filtration module further comprises at least one vacuum collection vessel configured to collect the filtrate, and at least one sensor or detector. In the aspect described herein, during purification by dynamic filtration, the filtrate containing the biological product is fed under negative pressure to a vacuum collection vessel capable of collecting about 50 mL to about 100 L. For example, the vacuum collection vessel capable of collecting the filtrate is about 1 L to about 10 L. In another example, the vacuum collection vessel capable of collecting the filtrate is about 1 L to about 50 L.

[0413] In embodiments, a process for continuously removing large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration comprises multistage filtration using at least two individual rolled filter membranes with different pore sizes. For example, this multistage dynamic filtration process comprises at least one first dynamic filtration device having a rolled filter membrane with a large pore size (e.g., 0.45 μm) in fluid communication with at least one second dynamic filtration device having a rolled filter membrane with a small pore size (e.g., 0.2 μm), thereby producing a filtrate containing biological products.

[0414] The process described herein comprises continuously transferring a solution to a first module capable of separating it into two or more fractions, each containing at least one fraction containing a biological product. For example, separating the solution into two or more fractions may include at least one fraction containing a biological product and at least one other fraction containing small impurities. As described herein, the first module comprises an affinity-based purification device. An "affinity-based purification device" refers to a purification technique based on molecular structural binding interactions (e.g., ligand-receptor interactions) in which a selective surface-immobilized ligand recognizes and binds to the biological product to be purified. For example, the first module has at least one first inlet and at least one first outlet and is configured to allow a continuous fluid flow between the first inlet and the first outlet through a mechanical rotation system comprising a lead system, a vessel carousel, and a collection system.

[0415] In embodiments, the affinity-based purification apparatus further comprises a suspension of resin beads. The surfaces of the resin beads are connected to, for example, protein A, protein G, protein L, antigen protein, protein, receptor, antibody, or aptamer, without any intention of limitation. Continuous purification of a biological product (e.g., monoclonal antibody) with affinity resin beads can avoid the cumbersome processing steps of conventional affinity column chromatography (e.g., protein A affinity chromatography).

[0416] In embodiments, the diameter of the resin beads of the affinity-based purification device is about 0.2 microns to about 200 microns. The diameter of the resin beads may vary depending on the biological product being purified and the flow rate of the process. Additionally, the concentration of the resin beads may be in the range of 0.01 wt% to 25 wt%. For example, the concentration of the magnetic resin beads may be about 1 wt% to about 20 wt%. In another example, the binding ability of the resin beads is a function of the bead concentration, the surface area to volume ratio, the affinity ligand density, or any combination thereof. In yet another example, the resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

[0417] In embodiments, the affinity-based purification module comprises a lead system having at least one gasketed lead, wherein the at least one gasketed lead comprises: at least one inlet for introducing gas to control a static pressure head pressure; at least one vent port to equilibrate with atmospheric pressure; at least one inlet for introducing a suspension of resin beads; at least one inlet for receiving a filtrate containing a biological product; and at least two inlets for introducing a buffer system for dispersing the resin beads to enable washing, elution from, or regeneration of the resin beads. In some embodiments, the at least one gasketed lead further comprises a port for receiving an overhead stirring impeller to enable dispersion of the resin beads. For example, the lead system controls movement along the z-axis.

[0418] In embodiments, the affinity-based purification module comprises a carousel comprising at least two containers filled with resin beads configured to continuously receive a mixture containing, for example, a biological product, and then transport a resulting heterogeneous mixture containing the biological product, resin beads, a buffer, or any combination thereof. For example, the mechanical rotation system is configured to be coupled with a lead system to enable pressurization. In another example, the mechanical rotation system controls movement or rotation in the xy plane.

[0419] In embodiments, each of at least two vessels of the affinity-based purification module has a supported base filter or filter membrane that enables retention of resin beads during bonding, debonding, washing, elution, and regeneration process steps. For example, at least two vessels further include a valve for controlling liquid flow.

[0420] In embodiments, the affinity-based purification module includes a collection system capable of collecting waste, fractions containing biological products, or any combination thereof by interfacing with at least one of at least two containers of a mechanical rotating system. For example, the collection system controls movement along the z-axis.

[0421] The process described herein also comprises continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving the flow from at least one first outlet of the first module, said second module comprising a charge-based purification device. A “charge-based purification device” as used herein comprises, for example, purifying biological molecules based on their surface charge, ionic properties, electrostatic interactions, or isoelectric point. As described herein, charge-based purification comprises a positive charge-based purification device, a negative charge-based purification device, or a combination thereof. For example, said second module has at least one second inlet and at least one second outlet and is configured to allow a continuous fluid flow between the second inlet and the second outlet through a mechanical rotational system comprising a lead system, a container carousel, and a collection system.

[0422] In embodiments, the charge-based purification device (e.g., positive and / or negative charge-based purification) further comprises a suspension of resin beads. For example, the surface of the resin beads may each include a cationic or anionic functional group configured to selectively associate with the biological product at a specific pH and ionic strength to enable positive charge-based purification or negative charge-based purification. Continuous purification of the biological product (e.g., monoclonal antibody) with ionic resin beads can avoid the cumbersome processing steps of conventional ion exchange column chromatography (e.g., cation exchange or anion exchange chromatography).

[0423] In embodiments, the diameter of the resin beads of the charge-based purification device is about 0.2 microns to about 200 microns. The diameter of the resin beads may vary depending on the biological product being purified and the flow rate of the process. Additionally, the concentration of the resin beads may be in the range of 0.01 wt% to 25 wt%. For example, the concentration of the magnetic resin beads may be about 1 wt% to about 20 wt%. In another example, the charge or electrostatic associative capacitance of the resin beads is a function of the bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In yet another example, the resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

[0424] In embodiments, the charge-based purification module comprises a lead system having at least one gasketed lead, said at least one gasketed lead comprises: at least one inlet for gas introduction to enable control of a static pressure head pressure; at least one vent port to enable equilibrium with atmospheric pressure; at least one inlet for introducing a suspension of resin beads; at least one inlet for receiving a filtrate containing a biological product; and at least two inlets for introducing a buffer system for dispersing the resin beads to enable washing, dissociation from, or regeneration of the resin beads. In some embodiments, the at least one gasketed lead further comprises a port for receiving an overhead stirring impeller to enable dispersion of the resin beads. For example, said lead system controls movement along the z-axis.

[0425] In embodiments, the charge-based purification module comprises a carousel comprising at least two containers filled with resin beads configured to continuously receive a mixture containing, for example, a biological product, and then transport a generated heterogeneous mixture containing the biological product, resin beads, a buffer, or any combination thereof. For example, the mechanical rotation system is configured to be coupled with a lead system to enable pressurization. In another example, the mechanical rotation system controls movement or rotation in the xy plane.

[0426] In embodiments, each of the at least two vessels of the charge-based purification module has a supported base filter or filter membrane that enables retention of resin beads during assembly, dissociation, washing, and regeneration process steps. For example, the at least two vessels further include a valve for controlling liquid flow.

[0427] In embodiments, the charge-based purification module includes a collection system capable of collecting waste, fractions including biological products, or any combination thereof by interfacing with at least one of at least two vessels of a mechanical rotation system. For example, the collection system controls movement along the z-axis.

[0428] In the embodiments described herein, resin beads of one or both of the first (affinity-based purification) and / or second (charge-based purification) module(s) are recycled and reused. For example, the beads may be reused at least two, three, four, or more times to purify a biological product.

[0429] Alternatively, the process described herein comprises continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving a flow from at least one first outlet of the first module, said second module comprising a free-flow electrophoresis device. A free-flow electrophoresis device having a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an aqueous ionic solution may be used instead of or in addition to charge-based magnetic purification module(s) for purifying a biological product (e.g., a monoclonal antibody).

[0430] For example, the solution contact surfaces of two parallel plates include glass, ceramic, plastic, or any combination thereof. In some examples, the aqueous ionic solution can create a pH gradient across the main separation channel. In other examples, the aqueous ionic solution can impart a constant pH across the main separation channel.

[0431] In embodiments, the free-flow electrophoresis device has at least one fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a pH gradient. For example, an isoelectric point-based fluid purification module comprises at least one first fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across a main separation channel (e.g., the approximate pH gradient may be in the pH range of about 2 to about 10); and at least one second fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient across a main separation channel (e.g., the fine pH gradient may be in the pH range of about 5 to about 8). For example, the pH gradient across the main separation channel can be further refined using additional subsequent fluid elements or chips comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow (e.g., a pH range of about 7.1 to about 7.6).

[0432] In another embodiment, the free-flow electrophoresis device has at least one fluid element that has no pH gradient, comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow to operate in band electrophoresis or charge separation operating mode. For example, an isoelectric point-based fluid purification module comprises at least one first fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant basic pH (e.g., pH greater than 7); and at least one second fluid element comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a constant acidic pH (e.g., pH less than 7).

[0433] In another embodiment, the free-flow electrophoresis device has at least one fluid element comprising a fluid channel created between two parallel plates to operate in an isometric electrophoresis operating mode, an electric field or electric field gradient perpendicular to the direction of fluid flow, and both an acidic pH gradient and a basic pH gradient separated by a spacer solution (e.g., NaCl solution).

[0434] In another embodiment, the isoelectric point-based fluid purification module comprises at least one first free-flow electrophoretic device comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, and at least one second free-flow electrophoretic device comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, wherein each element is connected in series and can operate in independent operating modes that enable purification. For example, at least one first free-flow electrophoretic device can operate in an isoelectric focusing mode, and at least one second free-flow electrophoretic device can operate in an isokinetic electrophoretic mode to increase separation resolution.

[0435] In another embodiment, the isoelectric point-based fluid purification module comprises at least one first fluid element comprising a fluid channel having at least one dielectrophoretic electrode capable of inducing a defined unidirectional force; at least one second free-flow electrophoretic device comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an approximate pH gradient across a main separation channel (e.g., a pH range of about 2 to about 10); and at least one third free-flow electrophoretic device comprising a fluid channel created between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient across a main separation channel (e.g., a pH range of about 5 to about 8). For example, the pH gradient across the main separation channel can be further refined using additional subsequent fluid elements or chips comprising a fluid channel created between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow (e.g., a pH range of about 7.1 to about 7.6).

[0436] In embodiments, the isoelectric point-based fluid purification device further includes at least two electrodes (e.g., platinum wire electrodes) to function as an anode or a cathode.

[0437] In embodiments, the backpressure within the isoelectric-based fluid purification device depends on the channel shape and dimensions, the inlet and outlet openings and / or tubing diameter, and the input flow rate. For example, the backpressure is in the range of about 0.5 psi to about 10 psi. In some examples, the backpressure is controlled by a needle valve, for example, without the intention of limiting it.

[0438] In embodiments, the isoelectric point-based fluid purification device further comprises at least one bubble removal system for continuously removing O2 and H2 gas bubbles generated in the electrode channel under an applied voltage. In some embodiments, removing electrolytic bubbles is essential to enable continuous operation for substantially long periods. For example, the bubble removal system uses a hydrophobic PTFE membrane to create a waterproof seal over the electrode channel that can continuously remove electrolytic bubbles at the point of generation by exposure to a vacuum system. For example, the vacuum gauge pressure is in the range of about -0.05 bar to about -0.4 bar.

[0439] In embodiments, the isoelectric point-based fluid purification device further comprises an active cooling system or heat sink (e.g., a Peltier element, a thermal chuck with a circulating water / propylene glycol jacket) to enable temperature control and Joule heat dissipation. For example, the active cooling system can control cooling and / or heat dissipation in the range of about 4°C to about 50°C, preferably about 4°C to about 37°C. Ideally, when isolating biological products (e.g., monoclonal antibodies), the temperature is maintained at about 10°C to about 25°C. For example, the active cooling system comprises an aluminum thermal chuck containing a cooled circulating water / propylene glycol jacket.

[0440] In embodiments, the isoelectric point-based fluid purification module includes at least one buffer or amphoteric electrolyte system.

[0441] In embodiments, the isoelectric point-based fluid purification module comprises at least one electrode solution. In some embodiments, the at least one electrode solution comprises an electrolyte solution configured to enable proper function in contact with an anode or a cathode, for example, phosphoric acid and sodium hydroxide, respectively. In other embodiments, the at least one electrode solution comprises at least one amphoteric electrolyte solution configured to enable proper function in contact with an anode or a cathode, for example, Tris buffered saline, flowing through a main separation channel, an anode channel, and a cathode channel.

[0442] In embodiments, the isoelectric point-based fluid purification module includes at least one sensor or detector. For example, at least one sensor or detector is placed inline. In some examples, at least one sensor or detector includes, but is not limited to, a flow sensor, a temperature sensor, a conductivity sensor, a pH sensor, a refractive index detector, a UV detector, or a back pressure sensor.

[0443] In embodiments, the isoelectric point-based fluid purification module includes at least one liquid circuit breaker, or disconnects the downstream of the device from the upstream of at least one inline sensor or detector to enable detection or sensing in a voltage-free solution.

[0444] The process currently claimed offers many advantages over current downstream methods and processes for purifying biological products, such as, for example, proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, or growth factors. For example, without limitation, the process described herein provides a continuous bioprocess for purifying monoclonal antibodies that significantly reduces production facility footprint, time required for facility setup and validation, facility setup costs, and capital expenditures compared to existing approaches for manufacturing batch, single-use, or semi-continuous monoclonal antibodies while maintaining throughput and yield. Continuous bioprocessing as described herein enables the use of smaller and more streamlined equipment (e.g., smaller bioreactor volumes and downstream bioprocess equipment) because, due to its ability to operate continuously, it does not require the large process equipment—whose size depends on the large bioreactor volume—that is required for the centrifugation, deep filtration, and column chromatography steps of conventional downstream bioprocessing. Furthermore, smaller and more streamlined equipment operating continuously enables the use of much smaller bioreactor(s) for producing monoclonal antibodies under steady conditions. Continuous bioprocesses as described herein can also significantly reduce operating costs, total bioprocess line downtime, and biological product loss compared to conventional monoclonal antibody manufacturing approaches. Finally, the process described herein for purifying the biological product is performed in a system having a footprint of much less than current technology, without sacrificing product throughput or yield on a kilogram-per-year basis.

[0445] Advantages of the processes and methods described herein include the ability to remove large impurities (e.g., cells, cell debris, and aggregates) without membrane fouling or clogging. For example, purifying cells, cell debris, and aggregates from cell culture media using conventional filtration or tangential flow filtration systems typically results in fouling or clogging of the filter membrane, making these methods unsuitable as means for continuously removing large impurities from heterogeneous mixtures containing biological products over long continuous processes. In contrast, the dynamic filtration device described herein enables the continuous removal of large impurities from heterogeneous mixtures containing biological products without contaminating the membrane, because the active target area of ​​the filter membrane is continuously refreshed. Additionally, because the entire process of producing and purifying biological products can be continuous and a flow rate ranging from about 0.1 mL / min to about 50 mL / min can be maintained throughout the entire process, the process equipment and the overall process footprint can occupy a much smaller footprint than current standard processes on a kilogram / year basis without sacrificing product throughput or yield. For example, the process for producing and purifying monoclonal antibodies described herein operates in a footprint of up to about 30,000 square feet. In contrast, current monoclonal antibody production and downstream processes require at least 200,000 square feet. For example, the flow rate of the process for purifying biological products ranges from about 1 mL / min to about 10 mL / min. In some examples, the flow rate of the step for continuously removing large impurities from heterogeneous mixtures ranges from about 0.1 mL / min to about 50 mL / min. In another example, the flow rate of the step for continuously removing large impurities from a heterogeneous mixture is the same as the flow rate from the bioreactor discharge line. In another example, a process is provided in which the flow rate of the step for continuously transferring the filtrate to the first module is in the range of about 0.1 mL / min to about 50 mL / min.In another example, a process is provided in which the flow rate of the step of continuously transferring a fraction containing a biological product from a first outlet to a second module is in the range of about 0.1 mL / min to about 50 mL / min.

[0446] A significant advantage of the process and method using the resin beads (e.g., agarose) described herein is that such a system does not require a conventional stationary bed or packed resin column (e.g., for standard chromatography) for sterilization, recycling, and / or regeneration. For example, such a system provides the recycling and / or regeneration of resin beads to create an infinite surface area of ​​the resin beads during operation, and consequently provides a continuous and cost-effective method. In other words, the module described herein does not have a fixed coupling or associative capability. As a specific example, since the resin beads used during the purification of biological products as described herein are continuously recycled and regenerated, the flow from the previous stage can be accommodated in either the dynamic filtration module or the purification module without interrupting the flow from the bioreactor discharge line. In other words, because the module described in the present invention undergoes these steps continuously, it does not need to be left idle for sterilization, regeneration, and / or recycling after operation. The above method differs from current continuous chromatography methods in that, since current column chromatography methods have limited column capacity due to column packing constraints, column switching between multiple packed columns is required to accommodate a continuous input flow and enable the regeneration and / or recycling of columns that have reached full capacity. Another advantage of the method described herein includes that the resin beads are not packed into the stationary phase but rather move. This mobility of the beads increases the surface area of ​​the resin beads available for bonding or aggregation, as substantially more resin bead surfaces are exposed to allow for free bonding. Additionally, resin beads within a packed column are exposed to high pressure differences to generate a flow through the column, and damage resulting from this is one of the reasons why the column's life is shortened beyond the desired life.The movable resin beads of the present invention are subjected to substantially lower pressure, making them much softer for brittle beads and thus extending their lifespan. Additionally, this mobility increases the likelihood that the resin beads will be fully regenerated and return to their initial state. This also enhances the cost-effectiveness of the method described herein because the resin is utilized more efficiently.

[0447] A significant advantage of the process and method utilizing free-flow electrophoresis described herein is that this system represents a "product loss-free" process, meaning that the product does not need to interact with a resin or other purification moiety because separation occurs through interaction with an electric field based on the physicochemical properties of the target biological product in an aqueous solution. Another advantage in the resolution of this approach is observed because theoretically higher purity products can be obtained compared to conventional ion exchange chromatography. Additionally, separation based on intrinsic physicochemical properties extends the utility of this approach for the purification of various biological products, including but not limited to proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, growth factors, oligonucleotides, viruses, adenoviruses, adeno-associated viruses (AAVs), or lentiviruses.

[0448] In addition, the modular approach provides process design flexibility to accommodate a diverse range of biological products.

[0449] A continuous process for purifying biological products using at least one of a dynamic filtration module, an affinity-based fluid purification module, and a charge-based fluid purification module or an isoelectric point-based fluid purification module.

[0450] A continuous process for purifying a biological product is described, said process comprising continuously receiving a heterogeneous mixture containing a biological product through an input line, said biological product comprising, but not limited to, a protein or a fragment thereof (polypeptide), an antibody or a fragment thereof, a cytokine, a chemokine, an enzyme, or a growth factor. Upon purification, the biological product (e.g., a monoclonal antibody) is substantially pure when impurities (e.g., cells, cell debris, aggregates, host cell proteins, undesirable proteins and peptides, undesirable antibodies, undesirable nucleic acids and oligonucleotides, viruses, salts, buffer components, surfactants, sugars, metal contaminants, leachates, medium components, and / or naturally occurring organic molecules) are removed at least about 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, or even 99 wt%.

[0451] The above process includes continuously removing large impurities from a heterogeneous mixture through dynamic filtration. The dynamic filtration process includes at least one dynamic filtration module that produces a filtrate containing biological products by continuously feeding biological products from at least one output head that fluidly communicates with an input line under negative pressure to the dynamic filtration module. The dynamic filtration module may additionally include at least one additional input line for supplying a wash buffer through a coaxial output head or a separate single-axis output head.

[0452] In embodiments, the process described herein comprises purifying a biological product continuously produced in a bioreactor (e.g., fed-batch bioreactor, perfusion bioreactor, and chemostat bioreactor). For example, the bioreactor includes a bioreactor feed line and an output bleed line that enable steady-state cell culture growth conditions, and the output bleed line functions as an input line that allows continuous fluid flow from the bioreactor to a dynamic filtration module.

[0453] As described herein, the process for continuously removing large impurities from a heterogeneous mixture does not include centrifugation, disk-stack centrifugation, deep filtration, static filtration, tangential flow filtration, hydrocyclone, or any combination thereof. The term “static filtration” refers to a process in which the heterogeneous mixture being filtered remains in a static state, that is, for exampl...

Claims

Claim 1 A method for purifying a biological product, the method comprising: receiving a heterogeneous mixture containing the biological product through an input line; supplying the biological product from at least one output fluidly communicating with the input line to remove impurities from the heterogeneous mixture by a clarification method using at least one of a dynamic filtration module, a centrifugation module, a deep filtration module, and a hydrocyclone module, thereby generating a first aqueous solution containing the biological product; and transferring the first aqueous solution to a second module capable of exchanging the first aqueous solution with a second aqueous solution to generate a retentate containing the biological product, wherein the second module comprises at least one of a tangential flow filtration device, a cross-flow filtration device, and a diafiltration device, wherein the second module comprises at least one second inlet configured to allow fluid flow between at least one second inlet and at least one second outlet, and There is at least one second outlet -; a step of transferring a residual liquid containing the biological product from at least one outlet of the second module to a third module having at least one inlet for receiving a flow from at least one second outlet of the second module - the third module includes at least one free-flow electrophoresis device, wherein the third module has at least one second inlet and at least one third outlet, and the third module is configured to allow a continuous fluid flow between the third inlet and the third outlet;The above free-flow electrophoresis device comprises electrode channels including an anode electrode channel and a cathode electrode channel that are in liquid contact with a main separation channel through a wall gap, and the device further comprises at least one electrode channel de-bubbler comprising at least one gas-permeable and hydrophobic membrane configured to remove bubbles by a vacuum system to create a bubble-free main separation channel, and at least one liquid circuit breaker; and a step of recovering the biological product; a method comprising; Claim 2 A method according to claim 1, wherein the tangential flow filtration device further comprises a hollow fiber filter, a flat plate, or a combination thereof. Claim 3 The method according to paragraph 2, wherein the hollow fiber filter or plate comprises a membrane having a pore size in the range of about 1 kDa to about 1 μm. Claim 4 The method according to paragraph 2, wherein the hollow fiber filter comprises an inner diameter in the range of about 0.5 mm to about 5 mm. Claim 5 A method according to paragraph 2, wherein the hollow fiber filter or plate comprises a membrane selected from polyethersulfone (PES), modified polyethersulfone (mPES), polysulfone, mixed cellulose ester, hydrophilic PVDF, or a combination thereof. Claim 6 The method of claim 1, wherein the method maintains a constant flow rate in the dynamic filtration module and the second module, and the flow rate is in the range of about 0.1 mL / min to about 50 mL / min. Claim 7 The method of claim 1, wherein the method of purifying the biological product is performed at a temperature in the range of about 4°C to about 37°C. Claim 8 The method of claim 1, wherein the method further comprises at least two dynamic filtration modules, each dynamic filtration module having a filter membrane having the same or different pore sizes. Claim 9 The method of claim 1, wherein the method further comprises at least two free-flow electrophoresis modules configured to operate in an isoelectric focusing mode, a zone electrophoresis mode, an isotachophoresis mode, or a combination thereof. Claim 10 The method of claim 1, wherein the method further comprises at least two dynamic filtration modules, at least two tangential flow filtration modules, or at least two free-flow electrophoresis modules operating in parallel. Claim 11 A method for purifying a biological product, the method comprising: receiving a heterogeneous mixture containing the biological product through an input line; supplying the biological product at at least one output fluidly communicating with the input line to remove impurities from the heterogeneous mixture by a cellular clarification method by a dynamic filtration module to produce a first aqueous solution containing the biological product, wherein the dynamic filtration device comprises a filter membrane extending between a feed reel and a collection reel with at least one support member having a substantially flat contact surface, a target area of ​​the filter membrane configured to receive the heterogeneous mixture from at least one output head, and a membrane support member having a substantially flat contact surface communicating with a vacuum collection system located between the feed reel and the collection reel; and transferring the first aqueous solution to a second module capable of exchanging the first aqueous solution for a second aqueous solution, thereby producing a solution containing the biological product A step in which a retentate is generated - wherein the second module comprises at least one of a tangential flow filtration device, a cross-flow filtration device, and a diafiltration device, and wherein the second module has at least one second inlet and at least one second outlet configured to allow fluid flow between at least one second inlet and at least one second outlet -;A method comprising the step of transferring a residual liquid containing the biological product from at least one outlet of the second module to a third module having at least one inlet for receiving a flow from at least one second outlet of the second module, wherein the third module comprises at least one free-flow electrophoresis device, wherein the third module has at least one second inlet and at least one third outlet, and the third module is configured to allow a continuous fluid flow between the third inlet and the third outlet; and the step of recovering the biological product. Claim 12 A method according to claim 11, wherein the tangential flow filtration device further comprises a hollow fiber filter, a flat plate, or a combination thereof. Claim 13 A method according to claim 12, wherein the hollow fiber filter or plate comprises a membrane having a pore size in the range of about 1 kDa to about 1 μm. Claim 14 In claim 12, the method wherein the hollow fiber filter comprises an inner diameter in the range of about 0.5 mm to about 5 mm. Claim 15 A method according to claim 12, wherein the hollow fiber filter or plate comprises a membrane selected from polyethersulfone (PES), modified polyethersulfone (mPES), polysulfone, mixed cellulose ester, hydrophilic PVDF, or a combination thereof. Claim 16 The method of claim 11, wherein the free-flow electrophoresis device comprises electrode channels including an anode electrode channel and a cathode electrode channel that are in liquid contact with a main separation channel through a wall gap, and the device further comprises at least one electrode channel de-bubbler comprising at least one gas-permeable and hydrophobic membrane configured to remove bubbles by a vacuum system to create a bubble-free main separation channel, and at least one liquid circuit breaker. Claim 17 In claim 11, the method maintains a constant flow rate in the dynamic filtration module and the second module, wherein the flow rate is in the range of about 0.1 mL / min to about 50 mL / min. Claim 18 In claim 11, the method of purifying the biological product is performed at a temperature in the range of about 4°C to about 37°C. Claim 19 The method of claim 11, wherein the method further comprises at least two dynamic filtration modules, each dynamic filtration module having a filter membrane having the same or different pore sizes. Claim 20 The method of claim 11 further comprises at least two free-flow electrophoresis modules configured to operate in an isoelectric focusing mode, a zone electrophoresis mode, an isotachophoresis mode, or a combination thereof. Claim 21 The method of claim 11, wherein the method further comprises at least two dynamic filtration modules, at least two tangential flow filtration modules, or at least two free-flow electrophoresis modules operating in parallel. Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete

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