Methods for purifying biological products

Abstract

Specifically provided herein are biological manufacturing and downstream purification processes.

Description

[Technical field]

[0001] This application claims the benefit of priority under 35 U.S.C. §119(e) 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 entire contents of each of which are incorporated herein by reference.

[0002] New processes and methods for the production and downstream purification of biological products are provided. Summary of the Invention

[0003] In particular, processes and apparatuses for purifying biological products are provided herein. In an aspect, a process for purifying a biological product is provided herein, the process comprising receiving a heterogeneous mixture comprising a biological product via an input line, and removing impurities from the heterogeneous mixture by filtration with a dynamic filtration module. The biological product is fed to the dynamic filtration module from at least one output head in fluid communication with the input line under negative pressure to remove impurities from the heterogeneous mixture, thereby generating a filtrate containing the biological product.

[0004] The dynamic filtration module includes a dynamic filtration device, a target area configured to receive the heterogeneous mixture from at least one output head, and a membrane support member having a substantially smooth contact surface in communication with a vacuum collection system located between the supply reel and the collection reel. Further, the dynamic filtration device includes a filter membrane extending between the supply reel and the collection reel with at least one support member having a substantially smooth contact surface. Purifying the biological product further includes transmitting the filtrate to a first module capable of separating the solution into two or more fractions, where at least one fraction includes the biological product, and the first module includes 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 the at least one first inlet and the at least one first outlet via a mechanical rotation system. The mechanical rotation system includes a container carousel containing at least one individual container including a suspension of beads. As described herein, the process further includes transmitting the biological product-containing fraction from the at least one outlet of the first module to a second module having at least one inlet for receiving flow from the at least one first outlet of the first module, the second module including at least one free-flow electrophoresis device, the second module including at least one second inlet and at least one second outlet, and configured to allow continuous fluid flow between the second inlet and the second outlet to recover the biological product.

[0005] In an embodiment, the affinity-based purification device further includes a lid system and a collection receptacle system in fluid communication with at least one individual receptacle. For example, the lid system includes at least one lid with a gasket, at least two buffer inlets, a fill inlet, a gas inlet, and an exhaust valve. Additionally, the lid system is movable along a z-axis. The receptacle carousel of the affinity-based purification device is capable of rotational movement in a plane transverse to the z-axis, and the collection receptacle is movable along the z-axis.

[0006] As described herein, the vessel carousel of the affinity-based purification method includes at least one position for binding biological product, at least one position for washing to remove unbound product, at least one position for eluting and collecting the biological product, and at least one regeneration position to allow recycling of the beads.

[0007] For example, the bead surface of the affinity-based purification is linked 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 a separate container at the position of binding to the biological product) is in a concentration range of about 0.01% to about 25% by weight. Alternatively, the initial concentration of the beads is in a range of about 0.01% to about 20% by weight, about 0.01% to about 10% by weight, about 0.01% to about 5% by weight, about 1% to about 20% by weight, or about 5% to about 10% by weight. For example, the diameter of the beads is in a range of about 0.2 μm to about 200 μm. 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.

[0008] In embodiments, the beads (e.g., beads in affinity-based purification) remain mobile during the process to maintain increased surface area available for binding. For example, the beads remain separated (circulating or dispersed) in the solution during the process (e.g., the beads are individual beads). Additionally, mobile beads can also mean that the beads do not clump together, e.g., at least two or more beads do not clump or group together. Additionally, mobile beads can also mean that the beads can form small aggregates that are dispersed and move freely in the solution. Conversely, beads as used herein are not packed but remain mobile and move freely in the solution.

[0009] In an embodiment, the free-flow electrophoresis device comprises electrode channels including an anode electrode channel and a cathode electrode channel in liquid contact with the main separation channel through a wall gap.

[0010] The free-flow electrophoresis device includes at least one electrode channel bubble remover including at least one gas permeable and hydrophobic membrane configured to remove air bubbles via a vacuum system to produce a bubble-free main separation channel, and at least one liquid circuit breaker. In an embodiment, the at least one bubble remover of the free-flow electrophoresis device is configured to remove O 2 generated in the electrode channel under an applied voltage. 2 and H 2 The gas bubble removal system is configured to continuously remove gas bubbles. In some embodiments, removing electrolysis bubbles from the electrode channels is essential to enable continuous operation for substantially long periods of time. For example, the bubble removal system uses a hydrophobic PTFE membrane to create a watertight seal over the electrode channels that allows electrolysis bubbles to be persistently removed at the point of origin by exposure to a vacuum system. For example, the vacuum gauge pressure ranges from about -5 kPa to about -40 kPa (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.

[0011] In an embodiment, a liquid circuit breaker for a free-flow electrophoresis apparatus includes a pressurized vessel configured to maintain a flow rate and generate liquid droplets that break the circuit from a solution coupled to a voltage. In embodiments, the purification process maintains approximately constant flow rates in the dynamic filtration module, the first module, and the second module, for example, in a range of about 0.1 mL / min to about 50 mL / min, or about 5 mL / min to about 10 mL / min.

[0012] In embodiments, the process for purifying a biological product is carried out at a temperature ranging from about 4°C to about 37°C. In further embodiments, the process can include at least two dynamic filtration modules, where each dynamic filtration module has filter membranes containing the same or different pore sizes (e.g., the heterogeneous mixture first contacts a filter membrane with a larger pore size (e.g., 0.45 μm) and then a filter membrane with a smaller pore size (e.g., 0.2 μm)).

[0013] 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 processes described herein further comprise at least two dynamic filtration modules, at least two affinity-based purification modules, or at least two free-flow electrophoresis modules operating in parallel.

[0014] In an embodiment, a dynamic filtration device is provided herein for removing impurities from a biological product in a heterogeneous mixture. The device includes a filter membrane extending between a supply reel and a collection reel, the filter membrane having 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. The membrane support structure of the device has a substantially smooth contact surface for structurally supporting a portion of the filter membrane located between the supply reel and the collection reel to generate the target area. Additionally, the dynamic filtration device has at least one support member having a substantially smooth 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 in communication with the membrane support structure and a vacuum system configured to apply a negative gauge pressure to the dynamic filter membrane, where the negative pressure allows the filtrate containing the biological product to be collected. In another example, the dynamic filtration device includes a wash buffer line.

[0015] In an embodiment, the dynamic filtration device includes a filter membrane that can include polyethersulfone (PES), aqueous polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), aqueous PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), aqueous 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 another example, the pore size is in 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 contain filter membranes of similar or different sizes.

[0016] The dynamic filtration devices described herein include a membrane support structure having a series of parallel slots, for example, from about 1 to about 10 parallel slots. In a specific example, the membrane support structure has 5 parallel slots.

[0017] The dynamic filtration devices described herein include a membrane support structure having a substantially smooth contact surface, where the contact surface is a measure of this static coefficient of friction, such as, for example, from about 0.01 to about 0.1, from about 0.01 to about 0.05, or from about 0.05 to about 0.1. In a specific example, the static coefficient of friction is 0.04.

[0018] In embodiments, the vacuum system of the dynamic filtration module is configured to apply a negative gauge pressure, for example, in the range of about -5 kPa to about -98 kPa (about -0.05 bar to about -0.98 bar).

[0019] In an embodiment, a free-flow electrophoresis device is provided herein for separating a mixture into two or more fractions, at least one of the fractions containing a biological product. The free-flow electrophoresis device includes at least one inlet and at least one outlet configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, at least one fluid channel configured to generate an electric field gradient between two parallel plates and perpendicular to the direction of fluid flow, an electrode channel including an anode electrode channel and a cathode electrode channel, wherein the electrode channel is configured to be connected to the main separation channel by liquid contact through a wall gap located between the electrode channel and the main separation channel, at least one electrode channel bubble remover including at least one gas-permeable and hydrophobic membrane or porous material configured to remove electrolysis bubbles near the point of generation by a vacuum system to generate 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.

[0020] The free-flow electrophoresis devices described herein provide an electrode channel with a bubble remover, where the top of the electrode channel is sealed with at least one gas-permeable and hydrophobic membrane in communication with a vacuum system for removing air bubbles, and the electrode channel is open at the bottom of the channel and configured to allow liquid contact between the main separation channel solution and the electrode solution through a wall gap.

[0021] The free-flow electrophoresis device has at least one electrode channel bubble remover including at least one gas permeable and hydrophobic membrane configured to remove air bubbles via a vacuum system to produce a bubble-free main separation channel, and at least one liquid circuit breaker.

[0022] In an embodiment, the free-flow electrophoresis device comprises a device for detecting O generated in an electrode channel under an applied voltage. 2 and H 2 The method further includes at least one bubble removal system for continuously removing gas bubbles. In some embodiments, removing electrolysis bubbles is essential to enable continuous operation for substantially long periods of time. For example, the bubble removal system uses a hydrophobic PTFE membrane to create a watertight seal over the electrode channel that allows electrolysis bubbles to be persistently removed at the point of generation by exposure to a vacuum system. For example, the vacuum gauge pressure ranges from about -5 kPa to about -40 kPa (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.

[0023] In embodiments, the wall gap (e.g., the space where the electrode channel is open at the bottom of the channel and configured to 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.

[0024] In other embodiments, a liquid circuit breaker in a free-flow electrophoresis apparatus includes a pressurized vessel configured to maintain a flow rate and generate droplets that break the circuit from a solution coupled to a voltage.

[0025] In embodiments, the free-flow electrophoresis device further comprises an in-line sensor, which may include, for example, a flow sensor, a pH sensor, a conductivity sensor, or any combination thereof.

[0026] In embodiments, the free-flow electrophoresis apparatus described herein may comprise at least two free-flow electrophoresis apparatuses connected in series to enable stepwise purification and operated in isoelectric focusing mode, zone electrophoresis mode, isotachophoresis mode, or combinations thereof.

[0027] Further provided herein is the use of the free-flow electrophoresis device for the purification of a biological product from a mixture.The invention further provides the use of the dynamic filtration device for the purification of a biological product from a heterogeneous mixture. In an aspect, provided herein is a process for purifying a biological product. The process comprises receiving a heterogeneous mixture comprising a biological product via an input line. In an embodiment, the process comprises continuously receiving a heterogeneous mixture comprising a biological product via an input line. In an embodiment, the biological product comprises a protein or fragment thereof (polypeptide), an antibody or 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.

[0028] In embodiments, the process includes 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 can be a continuous process for removing large impurities from a heterogeneous mixture. The dynamic filtration process includes at least one dynamic filtration module that continuously feeds a heterogeneous mixture comprising a biological product from at least one output head in fluid communication with an input line under negative pressure to the dynamic filtration module to produce a filtrate comprising the biological product.

[0029] In an embodiment, the process includes conveying the filtrate to a first module capable of separating the solution into two or more fractions, where at least one fraction includes the biological product. In another embodiment, the process includes continuously conveying the filtrate to a first module capable of separating the solution into two or more fractions, where at least one fraction includes the biological product. For example, separating the solution into two or more fractions can include one fraction that includes the biological product and at least one other fraction that includes small impurities (e.g., host cell proteins, undesirable proteins and peptides, undesirable antibodies, undesirable nucleic acids and oligonucleotides, viruses, salts, buffer components, detergents, sugars, metal contaminants, leachables, media components, and / or naturally associated natural organic molecules).

[0030] In an embodiment, the first module comprises an affinity-based magnetic 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 via 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 via a pick-and-place robotic system.

[0031] In an embodiment, the process includes transmitting a fraction comprising the biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving flow from the at least one first outlet of the first module, the second module comprising a charge-based magnetic purification device or an isoelectric focusing-based fluid purification device (also referred to herein as a free-flow electrophoresis device). In another embodiment, the process includes continuously transmitting a fraction comprising the biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving flow from the at least one first outlet of the first module, the second module comprising a charge-based magnetic purification device or an isoelectric focusing-based fluid purification device, also referred to herein as a free-flow electrophoresis device. For example, the second module includes a charge-based magnetic purification device having at least one second inlet and at least one second outlet, and configured to allow continuous fluid flow between the second inlet and the second outlet via a loop conveyor system. In some examples, the second module includes a charge-based magnetic purification device having at least one second inlet and at least one second outlet, and 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 includes a free-flow electrophoresis device having at least one second inlet and at least one second outlet, and configured to allow continuous fluid flow between the second inlet and the second outlet. In embodiments, the processes described herein thereby purify a biological product.

[0032] In embodiments, also provided herein is a process for purifying a biological product, comprising continuously receiving a heterogeneous mixture comprising a biological product via an input line and removing large impurities from the heterogeneous mixture by dynamic filtration. In some embodiments, the dynamic filtration process can be a continuous process for removing large impurities from a heterogeneous mixture. The dynamic filtration process comprises a dynamic filtration module that continuously supplies the biological product from at least one output head in fluid communication with the input line under negative pressure to the dynamic filtration module to produce a filtrate comprising the biological product.

[0033] In an embodiment, the process includes transmitting the filtrate to a first module capable of separating the solution into two or more fractions, where at least one fraction includes the biological product. In another embodiment, the process includes continuously transmitting the filtrate to a first module capable of separating the solution into two or more fractions, where at least one fraction includes the 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 via 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 via a stepped linear system.

[0034] In an embodiment, the process includes transferring a fraction comprising 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 the at least one first outlet of the first module, the second module comprising an isoelectric-based fluid purifier, also referred to herein as a charge-based purification device or a free-flow electrophoresis device. In another embodiment, the process includes continuously transferring a fraction comprising 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 the at least one first outlet of the first module, the second module comprising an isoelectric-based fluid purifier, also referred to herein as a charge-based purification device or a free-flow electrophoresis device. For example, the second module comprises a charge-based purification device having at least one second inlet and at least one second outlet, configured to allow continuous fluid flow between the second inlet and the second outlet via 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 via a stepped 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 processes described herein thereby purify a biological product.

[0035] In embodiments, also provided herein is a process for purifying a biological product, comprising continuously receiving a heterogeneous mixture comprising a biological product via an input line and removing large impurities from the heterogeneous mixture by dynamic filtration. In some embodiments, the dynamic filtration process can be a continuous process for removing large impurities from a heterogeneous mixture. The dynamic filtration process comprises a dynamic filtration module that continuously supplies the biological product from at least one output head in fluid communication with the input line under negative pressure to the dynamic filtration module to produce a filtrate comprising the biological product.

[0036] In an embodiment, the process includes transmitting the filtrate to a first module capable of separating the solution into two or more fractions, where at least one fraction includes the biological product. In another embodiment, the process includes continuously transmitting the filtrate to a first module capable of separating the solution into two or more fractions, where at least one fraction includes the biological product. In an embodiment, the first module includes 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.

[0037] In an embodiment, the process includes transmitting a fraction comprising 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 the at least one first outlet of the first module, the second module comprising an isoelectric-based fluid purifier, also referred to herein as a charge-based fluid purifier or a free-flow electrophoresis device. In an embodiment, the process includes continuously transmitting a fraction comprising 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 the at least one first outlet of the first module, the second module comprising an isoelectric-based fluid purifier, also referred to herein as a charge-based fluid purifier or a free-flow electrophoresis device. For example, the second module comprises a charge-based fluid purifier having at least one second inlet and at least one second outlet, configured to allow continuous fluid flow between the second inlet and the second outlet. In other examples, the second module includes a free-flow electrophoresis device having at least one second inlet and at least one second outlet, configured to allow continuous fluid flow between the second inlet and the second outlet, in embodiments, the processes described herein thereby purify a biological product.

[0038] In an embodiment, the process includes transmitting the filtrate to a first module capable of separating the solution into two or more fractions, where at least one fraction includes the biological product. In another embodiment, the process includes continuously transmitting the filtrate to a first module capable of separating the solution into two or more fractions, where at least one fraction includes the biological product. In an embodiment, the first module includes 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.

[0039] In an embodiment, the process includes transferring a fraction comprising 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, the second module comprising an isoelectric-based fluidic purifier, also referred to herein as a charge-based TFF purification device or a free-flow electrophoresis device. In another embodiment, the process includes continuously transferring a fraction comprising 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, the second module comprising a charge-based TFF purification device or an isoelectric-based fluidic purifier, also referred to herein as a free-flow electrophoresis device. For example, the second module comprises a charge-based TFF purification device having at least one second inlet and at least one second outlet, configured to allow continuous fluid flow between the second inlet and the second outlet. In other examples, the second module includes a free-flow electrophoresis device having at least one second inlet and at least one second outlet, configured to allow continuous fluid flow between the second inlet and the second outlet, in embodiments, the processes described herein thereby purify a biological product.

[0040] As described herein, the process for removing large impurities from a heterogeneous mixture does not include centrifugation, disc stack centrifugation, depth filtration, static filtration, tangential flow filtration, or any combination thereof. Alternatively, the process described herein can receive a heterogeneous mixture comprising a biological product via an input line derived from an input from which any large impurities have been removed by, for example and without intending to be limiting, a centrifuge and depth filtration process.

[0041] As described herein, the process for continuously removing large impurities from a heterogeneous mixture does not include centrifugation, disc stack centrifugation, depth filtration, static filtration, tangential flow filtration, hydrocyclone, or any combination thereof. Alternatively, the process described herein can continuously receive a heterogeneous mixture containing a biological product via an input line derived from any continuously large impurity removed input by, for example, and without intending to be limiting, continuous disc stack centrifuge and depth filtration processes or hydrocyclone processes.

[0042] In embodiments, the processes described herein include purifying a biological product (e.g., a monoclonal antibody) produced in a bioreactor. In some embodiments, the processes described herein include purifying a biological product that is continuously produced in a bioreactor. For example, the bioreactor includes a bioreactor feed line and an output bleed line that allow steady-state cell culture growth conditions, and the output bleed line serves as an input line that allows 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 constantly fed to a downstream purification system. Alternatively, the processes described herein include purifying a biological product (e.g., mRNA) that is not produced in a bioreactor.

[0043] In embodiments, provided herein is a method for purifying a biological product, the method comprising the steps of: receiving a heterogeneous mixture comprising a biological product via an input line; feeding the biological product from at least one output head in fluid communication 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 to generate a filtrate comprising the biological product; and conveying the filtrate to a first module capable of separating a solution into two or more fractions, including at least one fraction comprising the biological product, the first module comprising an affinity-based magnetic purification device, the first module having at least one first inlet and at least one first outlet, the first module being connected to a loop conveyor system or a pipette. and transferring a fraction comprising the biological product from the at least one first outlet of the first module to a second module having at least one inlet for receiving flow from the at least one first outlet of the first module, wherein the second module comprises a charge-based magnetic purification device, the second module having at least one second inlet and at least one second outlet, and configured to allow continuous fluid flow between the second inlet and the second outlet via a loop conveyor system or a pick-and-place robotic system, thereby purifying the biological product.

[0044] In another embodiment, a method of purifying a biological product is provided, the method comprising the steps of receiving a heterogeneous mixture comprising a biological product via an input line; feeding the biological product from at least one output head in fluid communication with the input line under negative pressure to a dynamic filtration module where impurities are removed from the heterogeneous mixture by dynamic filtration in the dynamic filtration module to generate a filtrate comprising the biological product; and conveying the filtrate to a first module capable of separating a solution into two or more fractions, including at least one fraction comprising the biological product, the first module comprising an affinity-based magnetic purification device, the first module having at least one first inlet and at least one first outlet, the filtrate being conveyed by a loop conveyor system or a pick-and-place robot. and transmitting a fraction comprising the biological product from the at least one first outlet of the first module to a second module having at least one inlet for receiving flow from the at least one first outlet of the first module, wherein the second module comprises an isoelectric-based fluid purification device, also referred to herein as a free-flow electrophoresis device, the second module having at least one second inlet and at least one second outlet, and configured to permit continuous fluid flow between the second inlet and the second outlet, thereby purifying the biological product.

[0045] In an embodiment, a method of purifying a biological product is included, the method comprising the steps of receiving a heterogeneous mixture comprising a biological product via an input line; delivering the biological product under negative pressure from at least one output head in fluid communication with the input line to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module to generate a filtrate comprising the biological product; and transmitting the filtrate to a first module capable of separating a solution into two or more fractions, the at least one fraction comprising the biological product, the first module comprising an affinity-based purification device, the first module having at least one first inlet and at least one first filter. and a first inlet and at least one second outlet of the first module configured to allow fluid flow between the first inlet and the first outlet via a mechanical rotation system; and transmitting a fraction comprising the biological product from the at least one first outlet of the first module to a second module having at least one inlet for receiving flow from the at least one first outlet of the first module, wherein the second module comprises a charge-based purification device, the second module having at least one second inlet and at least one second outlet and configured to allow fluid flow between the second inlet and the second outlet via a mechanical rotation system, thereby purifying the biological product.

[0046] In another embodiment, a method for purifying a biological product is provided, the method comprising the steps of receiving a heterogeneous mixture comprising a biological product via an input line; feeding the biological product from at least one output head in fluid communication 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 to generate a filtrate comprising the biological product; and transmitting the filtrate to a first module capable of separating a solution into two or more fractions, the at least one fraction comprising the biological product, the first module comprising an affinity-based purification device, the first module having at least one first inlet and at least one first outlet, the first module being operable to separate the filtrate ... and transmitting a fraction comprising the biological product from the at least one first outlet of the first module to a second module having at least one inlet for receiving flow from the at least one first outlet of the first module, wherein the second module comprises an isoelectric-based fluid purification device, also referred to herein as a free-flow electrophoresis device, the second module having at least one second inlet and at least one second outlet, and configured to permit continuous fluid flow between the second inlet and the second outlet, thereby purifying the biological product.

[0047] In an embodiment, a method of purifying a biological product is included, the method comprising the steps of receiving a heterogeneous mixture comprising a biological product via an input line; delivering the biological product under negative pressure from at least one output head in fluid communication with the input line to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module to generate a filtrate comprising the biological product; and transmitting the filtrate to a first module capable of separating a solution into two or more fractions, the at least one fraction comprising the biological product, the first module comprising an affinity-based purification device, the first module having at least one first inlet and at least one first filter. and configured to allow fluid flow between the first inlet and the first outlet via a stepped linear system; and transmitting a fraction comprising the biological product from the at least one first outlet of the first module to a second module having at least one inlet for receiving flow from the at least one first outlet of the first module, wherein the second module comprises a charge-based purification device, the second module having at least one second inlet and at least one second outlet and configured to allow fluid flow between the second inlet and the second outlet via a stepped linear system, thereby purifying the biological product.

[0048] In another embodiment, a method for purifying a biological product is provided, the method comprising the steps of receiving a heterogeneous mixture comprising a biological product via an input line; feeding the biological product from at least one output head in fluid communication 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 to generate a filtrate comprising the biological product; and transmitting the filtrate to a first module capable of separating a solution into two or more fractions, the at least one fraction comprising the biological product, the first module comprising an affinity-based purification device, the first module having at least one first inlet and at least one first outlet, and transmitting a fraction comprising the biological product from the at least one first outlet of the first module to a second module having at least one inlet for receiving flow from the at least one first outlet of the first module, wherein the second module comprises an isoelectric-based fluid purification device, also referred to herein as a free-flow electrophoresis device, the second module having at least one second inlet and at least one second outlet, and configured to permit continuous fluid flow between the second inlet and the second outlet, thereby purifying the biological product.

[0049] In an embodiment, a method of purifying a biological product is included, the method comprising the steps of receiving a heterogeneous mixture comprising a biological product via an input line; delivering the biological product from at least one output head in fluid communication 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 to generate a filtrate comprising the biological product; and transmitting the filtrate to a first module capable of separating a solution into two or more fractions, the at least one fraction comprising the biological product, the first module comprising an affinity-based fluid purifier, the first module comprising at least one first inlet and a second outlet; and at least one first outlet of the first module, configured to permit fluid flow between the first inlet and the first outlet; and transmitting a fraction comprising the biological product from the at least one first outlet of the first module to a second module having at least one inlet for receiving flow from the at least one first outlet of the first module, wherein the second module comprises a charge-based fluid purification device, the second module having at least one second inlet and at least one second outlet, configured to permit fluid flow between the second inlet and the second outlet, thereby purifying the biological product.

[0050] In another embodiment, a method for purifying a biological product is provided, the method comprising the steps of receiving a heterogeneous mixture comprising a biological product via an input line; delivering the biological product from at least one output head in fluid communication 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 to generate a filtrate comprising the biological product; and transmitting the filtrate to a first module capable of separating a solution into two or more fractions, the at least one fraction comprising the biological product, the first module comprising an affinity-based fluid purification device, the first module having at least one first inlet and at least one first outlet. and transmitting a fraction comprising the biological product from the at least one first outlet of the first module to a second module having at least one inlet for receiving flow from the at least one first outlet of the first module, wherein the second module comprises an isoelectric-based fluid purification device, also referred to herein as a free-flow electrophoresis device, the second module having at least one second inlet and at least one second outlet and configured to permit continuous fluid flow between the second inlet and the second outlet, thereby purifying the biological product.

[0051] In an embodiment, a method of purifying a biological product is included, the method comprising the steps of receiving a heterogeneous mixture comprising a biological product via an input line; delivering the biological product under negative pressure from at least one output head in fluid communication with the input line to a dynamic filtration module to remove impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module to generate a filtrate comprising the biological product; and transmitting the filtrate to a first module capable of separating a solution into two or more fractions, the at least one fraction comprising the biological product, the first module comprising an affinity-based TFF purification device, the first module comprising at least one first inlet and and at least one first outlet of the first module and configured to allow fluid flow between the first inlet and the first outlet; and transmitting a fraction comprising the biological product from the at least one first outlet of the first module to a second module having at least one inlet for receiving flow from the at least one first outlet of the first module, wherein the second module comprises a charge-based TFF purification device, the second module having at least one second inlet and at least one second outlet and configured to allow fluid flow between the second inlet and the second outlet, thereby purifying the biological product.

[0052] In another embodiment, a method for purifying a biological product is provided, the method comprising the steps of receiving a heterogeneous mixture comprising a biological product via an input line; delivering the biological product from at least one output head in fluid communication 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 to generate a filtrate comprising the biological product; and transmitting the filtrate to a first module capable of separating a solution into two or more fractions, the at least one fraction comprising the biological product, the first module comprising an affinity-based TFF purification device, the first module having at least one first inlet and at least one first outlet. and transmitting a fraction comprising the biological product from the at least one first outlet of the first module to a second module having at least one inlet for receiving flow from the at least one first outlet of the first module, wherein the second module comprises an isoelectric-based fluid purification device, also referred to herein as a free-flow electrophoresis device, the second module having at least one second inlet and at least one second outlet and configured to permit continuous fluid flow between the second inlet and the second outlet, thereby purifying the biological product.

[0053] 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, using conventional filtration or tangential flow filtration systems to clarify cells, cell debris, and aggregates from cell culture media typically results in filter membrane fouling or clogging, making these methods unsuitable as a means to continuously remove large impurities from heterogeneous mixtures containing biological products over long periods of time in a continuous process. In contrast, the dynamic filtration devices described herein allow for the continuous removal of large impurities from heterogeneous mixtures containing biological products without membrane fouling because the active target area of ​​the filter membrane is constantly refreshed.

[0054] Additionally, because the entire process of producing and purifying a biological product is continuous and can maintain flow rates ranging from about 0.1 mL / min to about 50 mL / min throughout the entire process, the footprint of the process equipment and the entire process can have a significantly smaller footprint than current standard processes without sacrificing product throughput or yield on a kilogram per year basis. For example, the process of producing and purifying a monoclonal antibody described herein operates in a footprint occupying a maximum of about 30,000 square feet. In comparison, current monoclonal antibody production and downstream processes require a minimum of 200,000 square feet. For example, the flow rate of the process of purifying a biological product ranges from about 1 mL / min to about 10 mL / min. In some examples, the flow rate of the step of continuously removing large impurities from the heterogeneous mixture ranges from about 0.1 mL / min to about 50 mL / min. In other examples, the flow rate of the step of continuously removing large impurities from the heterogeneous mixture is equal to the flow rate from the bioreactor bleed 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 ranges from 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 the biological product-containing fraction from the first outlet to the second module ranges from about 0.1 mL / min to about 50 mL / min.

[0055] Important advantages of the processes and methods using magnetic resin beads (e.g., magnetic agarose) or conventional resin beads (e.g., agarose) described herein include that these systems do not require conventional stationary phase or packed resin columns (e.g., for standard chromatography) for sanitization, recycling, and / or regeneration. For example, these systems provide for recycling and / or regeneration of resin beads (e.g., magnetic or non-magnetic resin beads) to generate infinite surface area of ​​resin beads during operation, resulting in a continuous and cost-effective process.

[0056] In other words, the modules described herein do not have a fixed binding or association capacity. In a specific example, the resin beads used during the purification of biological products described herein are continuously recycled and regenerated, so that they can accept the flow of the previous step of either the dynamic filtration module or the purification module without interrupting the flow from the bioreactor bleed line. In other words, the modules described in the present invention do not need to be left idle for sanitization, regeneration, and / or recycling after a run, because they are continuously undergoing these steps. The method differs from current continuous chromatography methods in that column switching of multiple packed columns is required to accept continuous input flow and allow regeneration and / or recycling of columns that have reached full capacity, because current methods have a column capacity limit limited by resin packing constraints.

[0057] Another advantage of the methods described herein includes that the resin beads are not packed in the stationary phase, but rather the resin beads are mobile. This mobility of the beads increases the surface area available for binding or association, since, for example, more biological products can be bound to the beads, since substantially more of the resin bead surface is exposed and free for binding. Furthermore, resin beads in a typically packed column (e.g., where the beads lack mobility and have reduced surface area) are exposed to high pressure differentials to generate flow through the column. Such high pressure differentials can compromise the integrity of the beads and shorten the life of the column. The mobile resin beads of the invention described herein are exposed to substantially lower pressures and are much softer than the fragile beads, thus extending their life. Furthermore, such mobility increases the likelihood that the beads can be regenerated (e.g., fully regenerated) and returned to their original state. This also increases the cost-effectiveness of the methods described herein, since, for example, the resin is used more efficiently.

[0058] As described herein, the resin beads of the claimed methods and devices are mobile throughout the process. Conventional chromatographic purification methods require, for example, column packing, where the beads are packed together sufficiently to provide a stationary phase and high density. For example, the beads remain separated (circulating or dispersed) in the solution during the process (e.g., the beads are individual beads). Additionally, mobile beads may also mean that the beads do not clump together, e.g., at least two or more beads do not clump or group together. Additionally, mobile beads may also mean that the beads can form small aggregates that are dispersed and move freely in the solution. Conversely, the beads used herein are not packed, but remain mobile and move freely in the solution.

[0059] An important advantage of the processes and methods using free-flow electrophoresis described herein is that the system represents a "no product loss" process, i.e., the product does not need to interact with resins or other purification moieties, since separation occurs in aqueous solution through interaction with an electric field according to the physicochemical properties of the target biological product. Another advantage is observed in the resolution of this approach (e.g., the ability to purify products with a high degree of physicochemical similarity), since higher purity products can be obtained compared to conventional ion exchange chromatography. For example, biological products of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher purity can be obtained using the free-flow electrophoresis modules and methods described herein. Furthermore, the methods and devices described herein can increase the purity (of the biological product) compared to conventional purification and chromatography methods. For example, the term "increased" in relation to levels refers to any % increase over a control level (e.g., the level of purity obtained from purification using conventional methods). In various embodiments, the increased level can be at least or about a 1%, 2%, 3%, 4%, or 5% increase in purity, at least or about a 10% increase, at least or about a 15% increase, at least or about a 20% increase, at least or about a 25% increase, at least or about a 30% increase, at least or about a 35% increase, at least or about a 40% increase, at least or about a 45% increase, at least or about a 50% increase, at least or about a 55% increase, at least or about a 60% increase, at least or about a 65% increase, at least or about a 70% increase, at least or about a 75% increase, at least or about a 80% increase, at least or about a 85% increase, at least or about a 90% increase, or at least or about a 95% increase in purity compared to conventional purification methods.In other examples of the invention, the purity of the biological product obtained from the methods and devices described herein is increased by about 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, or 3.0 fold as compared to the purity of the biological product using standard commercial or chromatographic techniques.

[0060] Furthermore, separation based on intrinsic physicochemical properties of biological products (e.g., isoelectric point, surface charge, net charge, zeta potential, electrophoretic mobility, electrostatic interactions, etc.) extends the utility of such approaches for the purification of a variety of 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 (AAV), or lentiviruses.

[0061] Furthermore, the modular approach provides flexibility in process design to accommodate a range of biological products. In embodiments, during purification by dynamic filtration in the processes described herein, a filtrate comprising the biological product is generated and delivered to a vacuum collection vessel capable of collecting from about 50 mL to about 100 L under negative pressure. For example, the vacuum collection vessel capable of collecting the filtrate is from about 1 L to about 10 L. In other examples, the vacuum collection vessel capable of collecting the filtrate is from about 1 L to about 50 L.

[0062] In embodiments, the dynamic filtration module includes at least one output head for regulating the flow of the heterogeneous mixture and distributing the heterogeneous mixture to an active target area of ​​the filter membrane, for example, the at least one output head is a tube or a slot die.

[0063] In embodiments, the at least one dynamic filtration module may further include at least one additional input line for supplying a wash 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.

[0064] In some embodiments, the dynamic filtration module includes elements known to those skilled in the art, such as, for example and without limitation, active or passive edge guides, tension adjustment devices (e.g., dancers), brakes and tension detectors, or any combination thereof.

[0065] In embodiments, the processes herein include at least one output head (in fluid communication with the input lines to the dynamic filtration module) capable of xy rastering or rθ rastering. For example, at least one output head is capable of xy rastering. In some examples, at least one output head is capable of rθ rastering. In other examples, at least one output head is capable of moving along the z-axis. In yet other examples, at least one output head is capable of xy rastering and is capable of moving along the z-axis.

[0066] In an embodiment, 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.

[0067] In an embodiment, the filter membrane roll comprises a rolled filter membrane, wherein the filter membrane includes, but is not limited to, polyethersulfone (PES), new aqueous polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), new aqueous PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), new aqueous PTFE, or any combination thereof.

[0068] 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 ranges from 0.1 μm to 1 μm. Alternatively, the pore size ranges from about 0.2 μm to about 0.45 μm, or the pore size is less than about 0.45 μm. In other examples, when purifying antibodies, the pore size of the rolled filter membrane ranges from 0.2 μm to about 0.45 μm.

[0069] In an embodiment, the width of the filter membrane roll is from 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 the at least one output head, or the membrane support structure.

[0070] In an embodiment, the filter membrane roll also functions as a supply reel in communication with a collection reel, i.e., the filter membrane starts as a prefabricated roll and is straddled onto an initially empty collection roll, creating a reel-to-reel system.

[0071] In an embodiment, the membrane support structure of the dynamic filtration module includes a mechanically smooth contact surface derived from a material with a low coefficient of static friction (e.g., polytetrafluoroethylene (PTFE)) and an opening in communication with the vacuum line. For example, the coefficient of static friction 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 include a mesh, at least one slot, at least one hole, a frit, a porous material, or any combination thereof.

[0072] In an embodiment, the membrane support structure of the dynamic filtration module includes a temperature control mechanism that maintains a temperature between 4° C. and 37° C. For example, during purification of an antibody, the temperature control mechanism maintains a temperature between 15° C. and 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 proportional integral derivative (PID) controller, a Peltier element, a resistive heating element, and / or a circulating water / propylene glycol jacketed thermal chuck.

[0073] In an embodiment, at least one support rod or roller of the dynamic filtration module has a mechanically smooth contact surface derived from a material with a low coefficient of static friction (e.g., PTFE, perfluoroalkoxyalkane (PFA)). For example, the coefficient of static friction 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 rotating. In some examples, the support rod may further include a bearing, e.g., a sleeve bearing.

[0074] In an embodiment, the vacuum system of the dynamic filtration module maintains a gauge pressure of about -5 kPa to about -98 kPa (about -0.05 bar to about -0.98 bar). In an embodiment, the process of continuously removing large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration includes a multi-stage filtration using at least two separate rolled filter membranes with different pore sizes. For example, these multi-stage dynamic filtration processes include at least one first dynamic filtration device with a large pore size (e.g., 0.45 μm) rolled filter membrane in fluid communication with at least one second dynamic filtration device with a small pore size (e.g., 0.2 μm) rolled filter membrane, thereby producing a filtrate containing a biological product. Alternatively, a similar result can be achieved by a single dynamic filtration device with at least two rolled filter membranes fed by separate feed reels, resulting in a stacked set of filter membranes across a target area (e.g., active target area), where the heterogeneous mixture first contacts a large pore size filter membrane (e.g., 0.45 μm) and then a smaller pore size filter membrane (e.g., 0.2 μm).

[0075] In an embodiment, the process described herein comprises continuously transmitting the filtrate to a first module capable of separating the solution into two or more fractions, including 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 linked to, for example, but not limited to, protein A, protein G, protein L, an antigen protein, a protein, a receptor, an antibody, or an aptamer. For example, the magnetic resin beads can be paramagnetic or superparamagnetic.

[0076] For example, the diameter of the magnetic resin beads in the 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 can vary depending on the biological product being purified and the overall flow rate of the process. For example, purification of monoclonal antibodies can include magnetic resin beads that are about 40 microns to about 90 microns in size. Additionally, the concentration of the magnetic resin beads can range from about 0.01% to about 25% by weight. For example, the concentration of magnetic resin beads can be about 1% by weight. In some examples, purification of monoclonal antibodies can include magnetic resin beads having a concentration of about 1% to about 10% by weight. In other examples, the binding capacity of the magnetic resin beads is a function of bead concentration, surface area to volume ratio, affinity ligand density, or any combination thereof. In still other examples, the magnetic resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

[0077] In an embodiment, the process described herein comprises continuously transmitting a fraction comprising 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 charge and / or negative charge based magnetic purification device), and the charge-based magnetic purification device further comprises magnetic resin beads. For example, the surface of the magnetic resin beads may have cationic functional groups resulting from the attachment of positive charge functional groups to enable purification based on charge or electrostatic interactions. For example, the positive charge functional groups include amines, cationic polymers, net positive charge peptides, net positive charge proteins, or any combination thereof. Alternatively, the surface of the magnetic resin beads may have anionic functional groups resulting from the attachment of negative charge functional groups to enable purification based on charge or electrostatic interactions. For example, the negative charge functional groups include carboxyls, anionic polymers, net negative charge peptides, net negative charge proteins, oligonucleotides, or any combination thereof. For example, the magnetic resin beads may be paramagnetic or superparamagnetic.

[0078] 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 can vary depending on the biological product being purified and the overall flow rate of the process. For example, purification of monoclonal antibodies can include magnetic resin beads having a size of about 40 microns to about 90 microns. Furthermore, the concentration of the magnetic resin beads can range from about 0.01% to about 25% by weight. For example, the concentration of the magnetic resin beads can be about 1% by weight. In some examples, purification of monoclonal antibodies can include magnetic resin beads having a concentration of about 1% to about 10% by weight. In other examples, the charge or electrostatic association capacity of the magnetic resin beads is a function of bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In still other examples, the magnetic resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

[0079] In embodiments, as described herein, one or both of the first (affinity-based magnetic purification) and / or second (charge-based magnetic purification including positive and / or negative charge-based magnetic purification devices) modules may further comprise at least one external magnetic field. For example, the at least one external magnetic field comprises a permanent magnet or an electromagnet. The at least one external magnetic field comprises a magnetic field strength of about 0.01 Tesla to about 1 Tesla (e.g., up to 1 Tesla). Alternatively, the at least one external magnetic field is shielded.

[0080] In an embodiment, 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 subsequently transport the 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 in proximity to an external magnetic field to attract the magnetic resin beads.

[0081] In an embodiment, the pick-and-place robotic system has at least two transport containers filled with magnetic resin beads configured to sequentially receive a mixture containing a biological product and subsequently transport the 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 in proximity to an external magnetic field to attract the magnetic resin beads.

[0082] In embodiments, the first (affinity-based magnetic purification) and / or second (charge-based magnetic purification including positive and / or negative charge-based magnetic purification devices) module further comprises at least one tangential flow filtration system operating in fed-batch or perfusion mode. For example, the tangential flow filtration system may be used to concentrate and buffer exchange fractions containing the biological product.

[0083] In an embodiment, the process described herein comprises continuously transmitting the filtrate to a first module capable of separating the solution into two or more fractions, including at least one fraction containing a biological product, the 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 linked to, for example, but not limited to, protein A, protein G, protein L, an antigen protein, a protein, a receptor, an antibody, or an aptamer.

[0084] 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 can vary depending on the biological product being purified and the overall flow rate of the process. For example, purification of a monoclonal antibody can include resin beads that are about 90 microns in size. Furthermore, the concentration of the resin beads can range from about 0.01% to about 25% by weight. For example, the concentration of the resin beads can be about 1% by weight. In some examples, purification of a monoclonal antibody can include resin beads having a concentration of about 1% to about 10% by weight. In other examples, the binding capacity of the resin beads is a function of bead concentration, surface area to volume ratio, affinity ligand density, or any combination thereof.

[0085] In yet other examples, the resin beads can be solid, porous, nanoporous, microporous, or any combination thereof. In an embodiment, the process described herein comprises continuously transmitting a fraction comprising 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 charge and / or negative charge based purification device), and the charge-based purification device further comprises resin beads. For example, the surface of the resin beads may have cationic functional groups resulting from the attachment of positive charge functional groups to enable purification based on charge or electrostatic interactions. For example, the positive charge functional groups may comprise amines, cationic polymers, net positive peptides, net positive proteins, or any combination thereof. Alternatively, the surface of the resin beads may have anionic functional groups resulting from the attachment of negative charge functional groups to enable purification based on charge or electrostatic interactions. For example, the negative charge functional groups may comprise carboxyls, anionic polymers, net negative peptides, net negative proteins, oligonucleotides, or any combination thereof.

[0086] In embodiments, the diameter of the resin beads in the charge-based purification device is about 0.2 microns to about 200 microns. The diameter of the resin beads can vary depending on the biological product being purified and the overall flow rate of the process. For example, purification of a monoclonal antibody can include resin beads that are about 90 microns in size. Furthermore, the concentration of the resin beads can range from about 0.01% to about 25% by weight. For example, the concentration of the resin beads can be about 1% by weight. In some examples, purification of a monoclonal antibody can include resin beads having a concentration of about 1% to about 10% by weight. In other examples, the charge or electrostatic association capacity of the resin beads is a function of bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In still other examples, the resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

[0087] In an embodiment, a mechanical rotation system (e.g., a system that allows continuous fluid flow between a first and / or second inlet and a first and / or second outlet) has at least two containers containing mobile resin beads (e.g., filled with mobile resin beads) configured to receive (e.g., continuously receive) a mixture containing a biological product and subsequently transport the resulting heterogeneous mixture containing the biological product, resin beads, buffer, or any combination thereof to a designated purification location.

[0088] In other embodiments, the system (e.g., a stepped linear system that allows continuous fluid flow between first and / or second inlets and first and / or second outlets) has at least two vessels that contain (e.g., are filled with) mobile resin beads configured to receive (e.g., sequentially receive) a mixture containing a biological product and subsequently process the resulting mixture containing the biological product, resin beads, buffer, or any combination thereof.

[0089] In embodiments, the first (affinity-based purification) and / or second (charge-based purification including positive and / or negative charge-based purification devices) module further comprises at least one tangential flow filtration system operated in fed-batch or perfusion mode for concentrating and buffer exchanging the fraction containing the biological product.

[0090] In an embodiment, the process described herein includes continuously transmitting the filtrate to a first module capable of separating the solution into two or more fractions, including at least one fraction comprising a biological product, wherein the first module is an affinity-based fluid purification device having at least one hybrid fluidic device or chip. In an embodiment, the at least one hybrid fluidic device or chip includes a cross-flow channel, at least one magnetic field, and at least one mechanical force generator. Further, the at least one mechanical force generator can include an ultrasonic transducer or a piezoelectric component capable of generating a defined unidirectional force. In another example, the at least one external magnetic field includes a permanent magnet, an electromagnet, a patterned magnet, or a combination thereof. For example, the at least one external magnetic field can exhibit a magnetic field strength of about 0.01 Tesla (T) to about 1 Tesla (e.g., up to 1 Tesla). In another example, the magnetic field strength is about 0.01 T, about 0.1 T, or about 1 T. In another embodiment, the at least one hybrid fluidic device or chip includes a cross-flow channel, at least one magnetic field, and at least one dielectrophoretic electrode. The at least one dielectrophoretic electrode can induce a defined unidirectional force. Further, the at least one external magnetic field can include a permanent magnet, an electromagnet, a patterned magnet, or a combination thereof. For example, the at least one external magnetic field can exhibit a magnetic field strength of about 0.01 Tesla to about 1 Tesla (e.g., up to about 1 Tesla).

[0091] In an embodiment, the affinity-based fluid purification device further comprises magnetic resin beads. The surface of the magnetic resin beads is linked to, for example, but not limited to, protein A, protein G, protein L, an antigen protein, a protein, a receptor, an antibody, or an aptamer. For example, the magnetic resin beads can be paramagnetic or superparamagnetic.

[0092] In embodiments, the magnetic resin beads of the affinity-based fluid purification device have a diameter of about 0.2 microns to about 200 microns. For example, purification of monoclonal antibodies may include magnetic resin beads having a size of about 40 microns. Furthermore, the concentration of the magnetic resin beads may range from about 0.01% to about 25% by weight. For example, the initial concentration of the magnetic resin beads may be about 1% by weight. In some examples, purification of monoclonal antibodies may include magnetic resin beads having a concentration of about 1% to about 10% by weight. In other examples, the binding capacity of the magnetic resin beads is a function of bead concentration, surface area to volume ratio, affinity ligand density, or any combination thereof. In still other examples, the magnetic resin beads may be solid, porous, nanoporous, microporous, or any combination thereof.

[0093] In an embodiment, the process described herein includes continuously transmitting a biological product-containing fraction from at least one first outlet of a first module to a second module, wherein the second module includes a charge-based fluid purification device. For example, the charge-based fluid purification device includes at least one hybrid fluidic device or chip. The at least one hybrid fluidic device or chip can include a cross-flow channel, at least one magnetic field, and at least one mechanical force generator. Further, the at least one mechanical force generator includes an ultrasonic transducer or a piezoelectric component capable of generating a defined unidirectional force. In other examples, the at least one external magnetic field includes a permanent magnet, an electromagnet, a patterned magnet, or a combination thereof. For example, the at least one external magnetic field can 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 0.01 T, about 0.1 T, or about 1 T. In another embodiment, the hybrid fluidic device or chip includes a cross-flow channel, at least one magnetic field, and at least one dielectrophoretic electrode, where the at least one dielectrophoretic electrode can induce a force in a defined direction. Further, the at least one external magnetic field includes a permanent magnet or an electromagnet. For example, the at least one external magnetic field includes a magnetic field strength of about 0.01 Tesla to about 1 Tesla (e.g., up to about 1 Tesla).

[0094] In an embodiment, the charge-based fluid purification device (e.g., positive and / or negative charge-based fluid purification device) further comprises a suspension of magnetic resin beads. The surface of the magnetic resin beads has cationic functional groups resulting from the attachment of positively charged functional groups to enable purification based on charge or electrostatic interactions. The positively charged functional groups include amines, cationic polymers, net positive peptides, net positive proteins, or any combination thereof. Alternatively, the magnetic resin bead surface may comprise anionic functional groups resulting from the attachment of negatively charged functional groups to enable purification based on charge or electrostatic interactions. The negatively charged functional groups include carboxyls, anionic polymers, net negative peptides, net negative proteins, oligonucleotides, or any combination thereof. For example, the magnetic resin beads may be paramagnetic or superparamagnetic.

[0095] For example, the diameter of the magnetic resin beads in the charge-based fluid purification device is about 0.2 microns to about 200 microns. The diameter of the magnetic resin beads can vary depending on the biological product being purified and the flow rate of the process. For example, purification of monoclonal antibodies can include magnetic resin beads that are about 40 microns in size. Furthermore, the concentration of the magnetic resin beads can range from about 0.01% to about 25% by weight. For example, the concentration of the magnetic resin beads can be about 1% by weight. In some examples, purification of monoclonal antibodies can include magnetic resin beads having a concentration of about 1% to about 10% by weight. In other examples, the charge or electrostatic association capacity of the magnetic resin beads is a function of bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In still other examples, the magnetic resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

[0096] In an embodiment, the first (affinity-based fluidic purification) module further includes at least one equilibration reservoir that allows binding of the biological product to the magnetic resin bead surface, and at least one low pH equilibration reservoir that allows debinding interactions of the biological product from the magnetic resin bead surface.

[0097] In an embodiment, the second (charge-based fluid purification including positive and / or negative charge-based fluid purification device) module further includes at least one association equilibrium vessel that allows association of the biological product with the magnetic resin bead surface based on charge or electrostatic interaction, and at least one dissociation equilibrium vessel that allows dissociation of the biological product from the magnetic resin bead surface. For example, multiple dissociation equilibrium vessels are used with multiple charge-based fluid purification devices to achieve gradient dissociation, such as, for example, a pH gradient or an ionic strength gradient.

[0098] In embodiments, the magnetic resin beads described herein are recycled and reused. For example, the beads can be reused at least two, three, four or more times to purify biological products. To allow for the recycling and reuse of the magnetic resin beads, at least one regeneration equilibration vessel can be used in conjunction with a tangential flow filtration system to concentrate and buffer exchange the magnetic resin beads and return them to their initial state.

[0099] As described herein, the first (affinity-based fluid purification) and / or second (charge-based fluid purification including positive and / or negative charge based fluid purification devices) modules may comprise hybrid microfluidic, mesofluidic, millifluidic, macrofluidic devices or chips for purifying biological products, or any combination thereof, such as a hybrid microfluidic device including at least one magnetic field and at least one of a piezoelectric component or a dielectrophoretic electrode.

[0100] In an embodiment, the first (affinity-based fluid purification) and / or second (charge-based fluid purification including positive and / or negative charge-based fluid purification devices) module further comprises at least one tangential flow filtration system operating in fed-batch or perfusion mode for concentrating and buffer exchanging the fraction containing the biological product.

[0101] In embodiments, the process described herein comprises continuously transmitting the filtrate to a first module capable of separating the solution into two or more fractions, including at least one fraction comprising a biological product, the first module comprising an affinity-based TFF purification device, for example, the affinity-based TFF purification device has at least three tangential flow filtration systems in fluid communication.

[0102] In embodiments, the affinity-based TFF purification device further comprises a suspension of resin beads, the surface of which is linked to, for example and without limitation, Protein A, Protein G, Protein L, an antigen protein, a protein, a receptor, an antibody, or an aptamer.

[0103] 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 can vary depending on the biological product being purified and the overall flow rate of the process. For example, purification of a monoclonal antibody can include resin beads that are about 90 microns in size. Furthermore, the concentration of the resin beads can range from about 0.01% to about 25% by weight. For example, the concentration of the resin beads can be about 1% to about 20% by weight. In other examples, the binding capacity of the resin beads is a function of bead concentration, surface area to volume ratio, affinity ligand density, or any combination thereof. In yet other examples, the resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

[0104] In embodiments, the processes described herein include continuously transferring a fraction comprising 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 tangential flow filtration systems in fluid communication.

[0105] In an embodiment, the charge-based TFF purification device further comprises a suspension of resin beads. For example, the surface of the resin beads may have cationic functional groups resulting from the attachment of positively charged functional groups to enable purification based on charge or electrostatic interactions. For example, the positively charged functional groups include amines, cationic polymers, net positive peptides, net positive proteins, or any combination thereof. Alternatively, the surface of the resin beads may have anionic functional groups resulting from the attachment of negatively charged functional groups to enable purification based on charge or electrostatic interactions. For example, the negatively charged functional groups include carboxyls, anionic polymers, net negative peptides, net negative proteins, oligonucleotides, or any combination thereof.

[0106] In embodiments, the diameter of the resin beads in the charge-based TFF purification device is about 0.2 microns to about 200 microns. The diameter of the resin beads can vary depending on the biological product being purified and the overall flow rate of the process. For example, purification of a monoclonal antibody can include resin beads that are about 90 microns in size. Furthermore, the concentration of the resin beads can range from about 0.01% to about 25% by weight. For example, the concentration of the resin beads can be about 1% to about 20% by weight. In other examples, the charge or electrostatic association capacity of the resin beads is a function of bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In yet other examples, the resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

[0107] In an embodiment, the first (affinity-based TFF purification) module further comprises at least one equilibration reservoir that allows binding of the biological product to the resin bead surface, and at least one low pH equilibration reservoir that allows debinding interactions of the biological product from the resin bead surface.

[0108] In an embodiment, the second (charge-based TFF purification including positive and / or negative charge-based TFF purification devices) module further includes at least one association equilibrium vessel that allows association of the biological product with the resin bead surface based on charge or electrostatic interaction, and at least one dissociation equilibrium vessel that allows dissociation of the biological product from the resin bead surface. For example, multiple dissociation equilibrium vessels are used with multiple charge-based fluidic purification devices to achieve gradient dissociation, such as, for example, a pH gradient or an ionic strength gradient.

[0109] In embodiments, the resin beads described herein are recycled and reused. For example, the beads can be reused at least two, three, four or more times to purify a biological product. To allow for the recycling and reuse of the resin beads, at least one regeneration equilibration vessel can be used in conjunction with a tangential flow filtration system to concentrate and buffer exchange the resin beads to return them to their initial state.

[0110] In embodiments, the first (affinity-based TFF purification) and / or second (charge-based TFF purification including positive and / or negative charge-based TFF purification devices) module further comprises at least one tangential flow filtration system for concentrating and buffer exchanging fractions containing the biological product.

[0111] In other embodiments, the processes described herein include continuously transmitting a biological product-containing fraction from at least one first outlet of a first module to a second module, wherein said second module includes an isoelectric focusing-based fluid purification device, also referred to herein as a free-flow electrophoresis device. For example, a free-flow electrophoresis device has at least one fluidic element including a fluid channel generated between two parallel plates to operate in an isoelectric focusing mode of operation, an electric field or an electric field gradient perpendicular to the direction of fluid flow, and a pH gradient. In another example, an isoelectric-based fluid purification module includes at least one first fluidic element including a fluidic channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a coarse pH gradient (e.g., pH range of about 2 to about 10) across the main separation channel, and at least one second fluidic element including a fluidic channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a fine pH gradient (e.g., pH range of about 5 to about 8) across the main separation channel. For example, an additional subsequent fluidic element or chip including a fluidic channel generated between two parallel plates and an electric field or electric field gradient orthogonal to the direction of fluid flow can be used to further refine the pH gradient across the main separation channel (e.g., pH range of about 7.1 to about 7.6). Alternatively, the free-flow electrophoresis device has at least one fluidic element that includes a fluid channel generated between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, but without a pH gradient, to operate in a zone electrophoresis or charge separation mode of operation.

[0112] In another example, the isoelectric-based fluid purification module includes at least one first fluidic element including a fluidic channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a constant basic pH (e.g., pH above 7), and at least one second fluidic element including a fluidic channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a constant acidic pH (e.g., pH below 7). Additionally, the free-flow electrophoresis device has at least one fluidic element including a fluidic channel generated between two parallel plates, an electric field or electric field gradient orthogonal 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) to operate in an isotachophoresis mode of operation.

[0113] In some embodiments, an isoelectric-based fluid purification module includes at least one first fluidic element including a fluidic channel generated between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, and at least one second fluidic element including a fluidic channel generated between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, where each element is connected in series and can operate in an independent operating mode to 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 isotachophoresis mode, and can be operated sequentially by series connection to enhance separation resolution.

[0114] In other embodiments, without intending to be limiting, an isoelectric-based fluid purification module includes at least one first fluidic element including a fluidic channel having at least one dielectrophoretic electrode capable of inducing a defined unidirectional force, at least one second fluidic element including a fluidic channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a coarse pH gradient (e.g., pH range of about 2 to about 10) across the main separation channel, and at least one third fluidic element including a fluidic channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a fine pH gradient (e.g., pH range of about 5 to about 8) across the main separation channel. For example, an additional subsequent fluidic element or chip including a fluidic channel generated between two parallel plates and an electric field or electric field gradient orthogonal to the direction of fluid flow can be used to further refine the pH gradient across the main separation channel (e.g., pH range of about 7.1 to about 7.6).

[0115] In yet other embodiments, the isoelectric-based fluid purification apparatus further includes an active cooling system (e.g., Peltier element, circulating water / propylene glycol jacketed thermal chuck) to enable temperature control and Joule heat dissipation. For example, the active cooling system may control cooling and / or Joule heat dissipation to enable operation in the 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.

[0116] In further embodiments, the process of purifying the biological product can also include viral inactivation, viral filtration, tangential flow filtration (TFF), high performance tangential flow filtration (HP-TFF), ultrafiltration / diafiltration (UF / DF), filter sterilization, fill-finish, lyophilization, or any combination thereof, performed semi-continuously and downstream of the second module.

[0117] For example, all processes described herein (processes for purifying biological products) are carried out at temperatures ranging from about 4° C. to about 50° C., preferably from about 4° C. to about 37° C. Furthermore, commercial scale processes for purifying biological products are carried out in systems with footprints that occupy significantly less square feet than current technology, without sacrificing product throughput or yield, on a kilogram per year basis. For example, the processes for producing and purifying monoclonal antibodies described herein are operated in footprints that occupy up to about 30,000 square feet. In comparison, current monoclonal antibody production and downstream processes require a minimum of 200,000 square feet.

[0118] The processes described herein can be used to purify 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, or lentiviruses.

[0119] [Dynamic filtration module] In an embodiment, provided herein is a dynamic filtration module for removing large impurities from a biological product in a heterogeneous mixture, the dynamic filtration module continuously feeding the biological product from at least one output head in fluid communication with an input line under negative pressure to the dynamic filtration module.

[0120] In an embodiment, 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.

[0121] The dynamic filtration module includes a rolled filter membrane extending between a supply reel and a collection reel, the filter membrane having a target area (e.g., an active target area) configured to receive the heterogeneous mixture. For example, the filter membrane of the filter membrane roll is constructed of a suitable material, including, but not limited to, polyethersulfone (PES), aqueous polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), aqueous PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), or aqueous PTFE.

[0122] In an embodiment, 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 an antibody, the pore size of the rolled filter membrane is in the range of 0.2 μm to about 0.45 μm.

[0123] In an embodiment, the width of the filter membrane roll is from about 10 mm to about 600 mm. For example, the width of the filter membrane roll can vary depending on factors such as the size of the dynamic filtration system and the size of the membrane support structure.

[0124] In an embodiment, the filter membrane roll also functions as a supply reel in communication with the 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"), where the filter membrane is continuously moved across the membrane support structure at an appropriate transport speed as a result of the collection reel collecting the used filter membrane portion. For example, the movement of the supply reel is controlled by a servo motor coupled to a gear box that limits the revolutions per minute (RPM) to a ratio of 200:1 to allow for a low membrane transport speed with high torque. The movement of the collection reel is controlled by a servo motor coupled to a gear box that limits the RPM to a ratio of 200:1 to allow for a low membrane transport speed with high torque. Furthermore, the supply reel motor and the collection reel motor are controlled by a closed loop controller that operates a feedback mechanism to ensure a membrane transport speed that is consistent with the diameter of the filter membrane roll, which constantly changes during operation on both the supply reel and the collection reel.

[0125] For example, a thickness monitoring system or rotary encoder can be used to ensure consistent membrane transport speed. For example, the supply reel and collection reel operate at the same speed and in the same direction. In another example, the supply reel and collection reel operate at different speeds and in the same direction. Other methods of transporting the filter membrane from the supply reel to the collection reel can be considered by those skilled in the art of the coating and converting industry. In another example, two dynamic filtration systems are run in parallel. For example, two parallel dynamic filtration systems can allow for sustained flow through the system during replacement of a used filter membrane roll. Additionally, two parallel dynamic filtration systems can allow for equilibration of the entire vacuum collection vessel to atmospheric pressure and allow for fluid flow to the first purification module without interrupting the process of continuously receiving the heterogeneous mixture from the bioreactor bleed line.

[0126] Furthermore, the dynamic filtration module includes a membrane support structure for supporting the target area (e.g., active target area) of the filter membrane when the filter membrane is subjected to negative pressure. The membrane support structure is located between the supply reel and the collection reel, has a mechanically smooth contact surface derived from a material with a low coefficient of static friction (e.g., PTFE), and has an opening in communication with the vacuum line. For example, the opening can include a mesh, at least one slot, at least one hole, a frit, a porous material, or any combination thereof.

[0127] In an embodiment, at least one support rod or roller of the dynamic filtration module has a mechanically smooth 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 with a mechanically smooth contact surface to stabilize the movement of the filter membrane across the membrane support structure.

[0128] In embodiments, the membrane support structure of the dynamic filtration module includes a temperature control mechanism for maintaining a desired temperature in the presence of evaporative cooling. The temperature control mechanism maintains a temperature between about 4° C. and about 37° C. For example, during purification of an antibody, the temperature control mechanism maintains a temperature in the range of about 15° C. to about 37° C.

[0129] In embodiments, the dynamic filtration module includes at least one output head for regulating the flow of the heterogeneous mixture and distributing the heterogeneous mixture to a target area (e.g., an active target area) of the filter membrane, for example, the at least one output head is a tube or a slot die.

[0130] In some embodiments, the dynamic filtration module further includes at least one additional input line for supplying a wash buffer via a coaxial output head, a separate single-axis output head, a separate slot die output head, or a slot die output head having multiple openings.

[0131] In some embodiments, the dynamic filtration module includes elements known in the coating and converting industry, such as, but not limited to, active or passive edge guides, tension adjustment devices (e.g., dancers), brakes and tension detectors, or any combination thereof.

[0132] In embodiments, the dynamic filtration module includes a vacuum system in series with the membrane support structure to apply a negative pressure across a target area (e.g., an active target area) of the filter membrane, where the negative pressure allows the target area (e.g., the active target area) of the filter membrane to cross the membrane support structure and allow collection of filtrate containing the biological product. For example, the vacuum system of the dynamic filtration module maintains a gauge pressure of about -5 kPa to about -98 kPa (about -0.05 bar to about -0.98 bar) for continuous filtration.

[0133] In an embodiment, the dynamic filtration module further includes at least one vacuum collection vessel configured to collect filtrate, and at least one sensor or detector. For example, two parallel dynamic filtration systems are run in staggered time to allow continuous flow through the systems following complete filling and equilibration to atmospheric pressure of a first vacuum collection vessel.

[0134] In an embodiment, the process of continuously removing large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration includes a multi-stage filtration using at least two individual rolled filter membranes with different pore sizes. For example, these multi-stage dynamic filtration processes include at least one first dynamic filtration device with a large pore size (e.g., 0.45 μm) rolled filter membrane in fluid communication with at least one second dynamic filtration device with a small pore size (e.g., 0.2 μm) rolled filter membrane, thereby producing a filtrate containing a biological product. Alternatively, a similar result can be achieved by a single dynamic filtration device with at least two rolled filter membranes fed by separate feed reels, resulting in a stacked set of filter membranes across the active target area, where the heterogeneous mixture first contacts the large pore size filter membrane (e.g., 0.45 μm) and then the smaller pore size filter membrane (e.g., 0.2 μm).

[0135] [Affinity-based magnetic purification module] In an embodiment, provided herein is an affinity-based magnetic purification module for separating a mixture into two or more fractions, where at least one fraction contains a biological product. The affinity-based magnetic purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be consistently constant, for example, during steady-state operation.

[0136] In embodiments, the affinity-based magnetic purification module comprises a suspension of magnetic resin beads, where the surface of the magnetic resin beads is linked to, without intending to be limiting, Protein A, Protein G, Protein L, an antigen protein, a protein, a receptor, an antibody, or an aptamer configured to selectively bind to the biological product. For example, the magnetic resin beads are mobile.

[0137] Further, the affinity-based magnetic purification module includes a loop conveyor system including at least two transport containers filled with magnetic resin beads configured to continuously receive a mixture including a biological product and subsequently transport the resulting heterogeneous mixture including the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0138] Alternatively, the 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 comprising a biological product and subsequently transport the resulting heterogeneous mixture comprising the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0139] In an embodiment, 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 allow washing. Furthermore, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from a heterogeneous mixture to allow elution of the biological product. Alternatively, at least one external magnetic field can be used to allow recycling of the magnetic resin beads. For example, mixing of magnetic resin beads can be achieved by placing at least one transport container between two separate opposing magnetic fields that switch between on and off states.

[0140] In embodiments, the affinity-based magnetic purification module comprises at least one binding / wash buffer system. In embodiments, the affinity-based magnetic purification module comprises at least one elution buffer system.

[0141] In an embodiment, the affinity-based magnetic purification module comprises at least one magnetic resin bead regeneration buffer system. In embodiments, the affinity-based magnetic purification module includes at least one aspirator system for removing waste liquid from at least two transport vessels.

[0142] In an embodiment, the affinity-based magnetic purification module comprises at least one sensor or detector. In embodiments, the affinity-based magnetic purification module includes at least one fluid handling pump.

[0143] [Positive charge-based magnetic purification module] In an embodiment, the present disclosure provides a positive charge-based magnetic purification module for separating a mixture into two or more fractions, where at least one fraction contains a biological product.The positive charge-based magnetic purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be, for example, constant throughout during steady-state operation.

[0144] In embodiments, a positive charge-based magnetic purification module comprises a suspension of magnetic resin beads, where the surface of the magnetic resin beads comprises cationic functional groups configured to selectively associate with the biological product at a particular pH and ionic strength, e.g., the magnetic resin beads are mobile.

[0145] Further, the positive charge-based magnetic purification module includes a loop conveyor system including at least two transport containers filled with magnetic resin beads configured to continuously receive a mixture including a biological product and subsequently transport the resulting heterogeneous mixture including the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0146] Alternatively, the 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 comprising a biological product and subsequently transport the resulting heterogeneous mixture comprising the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0147] In an embodiment, the positive 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 allow washing. Furthermore, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from a heterogeneous mixture to allow dissociation and purification of the biological product. Alternatively, at least one external magnetic field can be used to allow recycling of the magnetic resin beads. For example, mixing of magnetic resin beads can be achieved by placing at least one transport container between two separate opposing magnetic fields that switch between on and off states.

[0148] In an embodiment, the positive charge-based magnetic purification module comprises at least one association / wash buffer system. In embodiments, the positive charge-based magnetic purification module includes at least one dissociation buffer system, for example, multiple dissociation buffers of varying pH, ionic strength, or any combination thereof are utilized in sequence to generate a gradient dissociation effect.

[0149] In an embodiment, the positive charge based magnetic purification module comprises at least one magnetic resin bead regeneration buffer system. In an embodiment, the positive charge-based magnetic purification module includes at least one aspirator system for removing waste liquid from at least two transport vessels.

[0150] In an embodiment, the positive charge-based magnetic purification module comprises at least one sensor or detector. In an embodiment, the positive charge-based magnetic purification module includes at least one fluid handling pump.

[0151] [Negative charge-based magnetic purification module] In an embodiment, the present disclosure provides a negative charge-based magnetic purification module for separating a mixture into two or more fractions, where at least one fraction contains a biological product. The negative charge-based magnetic purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be, for example, constant throughout during steady-state operation.

[0152] In embodiments, a negative charge-based magnetic purification module comprises a suspension of magnetic resin beads, where the surface of the magnetic resin beads comprises anionic functional groups configured to selectively associate with the biological product at a particular pH and ionic strength, e.g., the magnetic resin beads are mobile.

[0153] Further, the negative charge-based magnetic purification module includes a loop conveyor system including at least two transport containers filled with magnetic resin beads configured to continuously receive a mixture including a biological product and subsequently transport the resulting heterogeneous mixture including the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0154] Alternatively, the 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 comprising a biological product and subsequently transport the resulting heterogeneous mixture comprising the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0155] In an embodiment, 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. Furthermore, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from a heterogeneous mixture to enable dissociation and purification of the biological product. Alternatively, at least one external magnetic field can be used to enable recycling of the magnetic resin beads. For example, mixing of magnetic resin beads can be achieved by placing at least one transport container between two separate opposing magnetic fields that switch between on and off states.

[0156] In an embodiment, the negative charge-based magnetic purification module comprises at least one association / wash buffer system. In embodiments, the negative charge-based magnetic purification module includes at least one dissociation buffer system, for example, multiple dissociation buffers of varying pH, ionic strength, or any combination thereof are utilized in sequence to generate a gradient dissociation effect.

[0157] In an embodiment, the negative charge based magnetic purification module comprises at least one magnetic resin bead regeneration buffer system. In an embodiment, the negative charge-based magnetic purification module includes at least one aspirator system for removing waste liquid from at least two transport vessels.

[0158] In an embodiment, the negative charge-based magnetic purification module comprises at least one sensor or detector. In an embodiment, the negative charge-based magnetic purification module includes at least one fluid handling pump.

[0159] [Affinity-based purification module] In an embodiment, provided herein is an affinity-based purification module for separating a mixture into two or more fractions, where at least one fraction includes a biological product. The affinity-based purification module includes at least one inlet and at least one outlet configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be consistently constant, for example, during steady-state operation.

[0160] In embodiments, the affinity-based purification module comprises a suspension of resin beads, where the surface of the resin beads is linked to, without intending to be limiting, Protein A, Protein G, Protein L, an antigen protein, a protein, a receptor, an antibody, or an aptamer configured to selectively bind to the biological product. For example, the resin beads are mobile.

[0161] In embodiments, the affinity-based purification module includes a lid system with at least one gasketed lid, where the at least one gasketed lid has at least one inlet for introducing gas to allow for control of positive head pressure. Additionally, the lid system has at least one vent port to allow for equilibration to atmospheric pressure, at least one inlet for introducing a suspension of resin beads, at least one inlet for receiving filtrate containing a biological product, and / or at least two inlets for introducing a buffer system to disperse resin beads to allow for washing, elution, 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 allow for dispersion of resin beads. For example, the lid system controls movement along the z-axis.

[0162] In embodiments, the affinity-based purification module includes a mechanical rotation system, e.g., a carousel including at least two containers filled with resin beads configured to continuously receive a mixture including a biological product and subsequently transport the resulting heterogeneous mixture including the biological product, resin beads, a buffer, or any combination thereof. For example, the carousel is a rotating structure that holds and transports at least two containers to different process locations. In some examples, the mechanical rotation system is configured to mate with a lid system to allow pressurization and liquid handling. In other examples, the mechanical rotation system controls the movement or rotation in the xy plane.

[0163] In embodiments, at least two vessels of the affinity-based purification module each have a supported base filter or filter membrane. For example, the base filter (or filter membrane) allows retention of the resin beads during binding, debinding, washing, elution, and / or regeneration process steps. For example, the at least two vessels may further include valves for controlling the flow of liquid.

[0164] In other embodiments, the affinity-based purification module includes a stepwise linear system, e.g., at least two containers filled with resin beads configured to sequentially receive a mixture containing a biological product and subsequently process the resulting heterogeneous mixture containing the biological product, resin beads, buffer, or any combination thereof, e.g., the at least two containers are configured to mate with a lid system to allow for pressurization and liquid handling.

[0165] In embodiments, the affinity-based purification module includes a collection system that can interface with at least one of the at least two containers of the mechanical rotation system to collect waste, a fraction containing the biological product, or any combination thereof, e.g., the collection system controls movement along the z-axis.

[0166] In other embodiments, the affinity-based purification module includes a collection system that can interface with at least one of the at least two vessels of the step-wise linear system to collect waste, a fraction containing the biological product, or any combination thereof, e.g., the collection system is coupled to at least one of the at least two vessels.

[0167] In embodiments, the affinity-based purification module includes at least one gas. In some embodiments, without intending to be limiting, the gas includes filtered nitrogen or compressed dry air. For example, the gas generates a pressure head of about 0.689 kPa to about 207 kPa (about 0.1 psi to about 30 psi).

[0168] In embodiments, the affinity-based purification module comprises at least one binding / wash buffer system. In embodiments, the affinity-based purification module includes at least one low pH elution buffer system.

[0169] In embodiments, the affinity-based purification module includes at least one resin bead regeneration buffer system. In embodiments, the affinity-based purification module includes at least one collection vessel.

[0170] In an embodiment, the affinity-based purification module includes at least one sensor or detector. In embodiments, the affinity-based purification module includes at least one fluid handling pump.

[0171] [Positive charge-based purification module] Also provided herein is a positive charge-based purification module for separating a mixture into two or more fractions, where at least one fraction contains a biological product.

[0172] The positive charge-based purification module includes at least one inlet and at least one outlet that are configured to permit continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be consistently constant, for example, during steady-state operation.

[0173] In embodiments, a positive charge-based purification module comprises a suspension of resin beads, where the surface of the resin beads comprises cationic functional groups configured to selectively associate with the biological product at a particular pH and ionic strength, e.g., the resin beads are mobile.

[0174] In embodiments, the positive charge-based purification module includes a lid system having at least one gasketed lid, the at least one gasketed lid including at least one inlet for introducing gas to allow control of positive head pressure, at least one inlet for introducing a suspension of resin beads, at least one vent port to allow equilibration to atmospheric pressure, at least one inlet for receiving filtrate containing a biological product, and at least two inlets for introducing a buffer system to disperse the resin beads to allow washing, dissociation, or regeneration of the resin beads. In some embodiments, the at least one gasketed lid further includes a port for receiving an overhead stirring impeller to allow dispersion of the resin beads. For example, the lid system controls movement along the z-axis.

[0175] In embodiments, the positive charge-based purification module includes a mechanical rotation system, e.g., a carousel including at least two containers filled with resin beads configured to continuously receive a mixture including a biological product and subsequently transport the resulting heterogeneous mixture including the biological product, resin beads, a buffer, or any combination thereof. For example, a carousel is a rotating structure that holds and transports at least two containers to different process locations. In some examples, the mechanical rotation system is configured to mate with a lid system to allow pressurization. In other examples, the mechanical rotation system controls the movement or rotation in the xy plane.

[0176] In an embodiment, at least two containers of the positive charge-based purification module each have a supported base filter or filter membrane. For example, the base filter (or filter membrane) allows retention of the resin beads during association, washing, dissociation, and / or regeneration process steps. For example, the at least two containers may further include valves for controlling the flow of liquid.

[0177] In other embodiments, the positive charge-based purification module includes a stepwise linear system, e.g., at least two containers filled with resin beads configured to sequentially receive a mixture containing a biological product and subsequently process the resulting heterogeneous mixture containing the biological product, resin beads, buffer, or any combination thereof. For example, the at least two containers are configured to mate with a lid system to allow for pressurization and liquid handling.

[0178] In embodiments, the positive charge-based purification module includes a collection system that can interface with at least one of the at least two containers of the mechanical rotation system to collect waste, a fraction containing the biological product, or any combination thereof, for example, the collection system controlling movement along the z-axis.

[0179] In other embodiments, the positive charge-based purification module includes a collection system that can interface with at least one of the at least two vessels of the step-wise linear system to collect waste, a fraction containing a biological product, or any combination thereof, e.g., the collection system is coupled to at least one of the at least two vessels.

[0180] In embodiments, the affinity-based purification module includes at least one gas. In some embodiments, without intending to be limiting, the gas includes filtered nitrogen or compressed dry air. For example, the gas generates a pressure head of about 0.689 kPa to about 207 kPa (about 0.1 psi to about 30 psi).

[0181] In embodiments, the positive charge-based purification module comprises at least one association / wash buffer system. In embodiments, the positive charge-based purification module includes at least one dissociation buffer system, e.g., multiple dissociation buffers of varying pH, ionic strength, or any combination thereof are utilized in series or sequence to generate a gradient dissociation effect.

[0182] In an embodiment, the positive charge-based purification module comprises at least one resin bead regeneration buffer system. In an embodiment, the positive charge-based purification module includes at least one collection vessel.

[0183] In an embodiment, the positive charge-based purification module includes at least one sensor or detector. In embodiments, the positive charge-based purification module includes at least one fluid handling pump.

[0184] [Negative charge-based purification module] In an embodiment, provided herein is a negative charge-based purification module for separating a mixture into two or more fractions, where at least one fraction comprises a biological product. The negative charge-based purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be, for example, constant throughout during steady-state operation.

[0185] In embodiments, the negative charge-based purification module comprises a suspension of resin beads, where the surface of the resin beads comprises cationic functional groups configured to selectively associate with the biological product at a particular pH and ionic strength.

[0186] In embodiments, the negative charge-based purification module includes a lid system having at least one gasketed lid, the at least one gasketed lid including at least one inlet for introducing gas to allow control of positive head pressure, at least one vent port to allow equilibration to atmospheric pressure, at least one inlet for introducing a suspension of resin beads, at least one inlet for receiving filtrate containing a biological product, and at least two inlets for introducing a buffer system to disperse the resin beads to allow washing, dissociation, or regeneration of the resin beads. In some embodiments, the at least one gasketed lid further includes a port for receiving an overhead stirring impeller to allow dispersion of the resin beads. For example, the lid system controls movement along the z-axis.

[0187] In embodiments, the negative charge-based purification module includes a mechanical rotation system, e.g., a carousel including at least two containers filled with resin beads configured to continuously receive a mixture including a biological product and subsequently transport the resulting heterogeneous mixture including the biological product, resin beads, a buffer, or any combination thereof. For example, a carousel is a rotating structure that holds and transports at least two containers to different process locations. In some examples, the mechanical rotation system is configured to mate with a lid system to allow pressurization. In other examples, the mechanical rotation system controls the movement or rotation in the xy plane.

[0188] In an embodiment, at least two containers of the positive charge-based purification module each have a supported base filter or filter membrane. For example, the base filter (or filter membrane) allows retention of the resin beads during association, washing, dissociation, and / or regeneration process steps. For example, the at least two containers may further include valves for controlling the flow of liquid.

[0189] In other embodiments, the positive charge-based purification module includes a stepwise linear system, e.g., at least two containers filled with resin beads configured to sequentially receive a mixture containing a biological product and subsequently process the resulting heterogeneous mixture containing the biological product, resin beads, buffer, or any combination thereof. For example, the at least two containers are configured to mate with a lid system to allow for pressurization and liquid handling.

[0190] In embodiments, the positive charge-based purification module includes a collection system that can interface with at least one of the at least two containers of the mechanical rotation system to collect waste, a fraction containing the biological product, or any combination thereof, for example, the collection system controlling movement along the z-axis.

[0191] In other embodiments, the positive charge-based purification module includes a collection system that can interface with at least one of the at least two vessels of the step-wise linear system to collect waste, a fraction containing a biological product, or any combination thereof, e.g., the collection system is coupled to at least one of the at least two vessels.

[0192] In embodiments, the affinity-based purification module includes at least one gas. In some embodiments, without intending to be limiting, the gas includes filtered nitrogen or compressed dry air. For example, the gas generates a pressure head of about 0.689 kPa to about 207 kPa (about 0.1 psi to about 30 psi).

[0193] In embodiments, the negative charge-based purification module comprises at least one association / wash buffer system. In embodiments, the negative charge-based purification module includes at least one dissociation buffer system, e.g., multiple dissociation buffers of varying pH, ionic strength, or any combination thereof are utilized in series or sequence to generate a gradient dissociation effect.

[0194] In an embodiment, the negative charge-based purification module comprises at least one resin bead regeneration buffer system. In an embodiment, the negative charge-based purification module includes at least one collection vessel.

[0195] In an embodiment, the negative charge-based purification module includes at least one sensor or detector. In embodiments, the negative charge-based purification module includes at least one fluid handling pump.

[0196] [Affinity-based fluid purification module] In an embodiment, provided herein is an affinity-based fluid purification module for separating a mixture into two or more fractions, where at least one fraction includes a biological product. The affinity-based fluid purification module includes at least one inlet and at least one outlet configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be consistently constant, for example, during steady-state operation.

[0197] In embodiments, the affinity-based fluidic purification module comprises a suspension of magnetic resin beads, where the surface of the magnetic resin beads is linked to, without intending to be limiting, Protein A, Protein G, Protein L, an antigen protein, a protein, a receptor, an antibody, or an aptamer configured to selectively bind to the biological product. For example, the magnetic resin beads are mobile.

[0198] In an embodiment, the affinity-based fluid purification module includes at least one equilibration vessel that allows binding of a biological product to the surface of magnetic resin beads, and at least one first hybrid cross-flow fluidic device that includes a cross-flow 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 with the bound biological product from the heterogeneous mixture.

[0199] In an embodiment, the affinity-based fluidic purification module further comprises at least one low pH equilibration vessel to allow for debinding of the biological product from the magnetic resin bead surface, and at least one second hybrid cross-flow fluidic device comprising a cross-flow 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 product and complete their elution.

[0200] In embodiments, the affinity-based fluid purification module further comprises at least one tangential flow filtration system operated in fed-batch or perfusion mode to concentrate and buffer exchange the fraction containing the biological product.

[0201] In an embodiment, the affinity-based fluid purification module includes at least two buffer systems. In an embodiment, the affinity-based fluid purification module includes at least one magnetic resin bead regeneration buffer system.

[0202] In embodiments, the affinity-based fluid purification module includes at least one equilibration vessel configured to allow recycling of said magnetic resin beads. In an embodiment, the affinity-based fluid purification module includes at least one sensor or detector.

[0203] In an embodiment, the affinity-based fluid purification module includes at least one fluid handling pump. [Positive charge-based fluid purification module] In an embodiment, provided herein is a positive charge-based fluid purification module for separating a mixture into two or more fractions, where at least one fraction contains a biological product. The positive charge-based fluid purification module includes at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be consistently constant, for example, during steady-state operation.

[0204] In embodiments, the positive charge-based fluidic purification module comprises a suspension of magnetic resin beads, where the surface of the magnetic resin beads comprises cationic functional groups configured to selectively associate with the biological product at a particular pH and ionic strength, e.g., the magnetic resin beads are mobile.

[0205] In an embodiment, the positive charge based fluid purification module includes at least one association equilibrium vessel that allows association of a biological product with the magnetic resin bead surface, and at least one first hybrid cross-flow fluidic device that includes a cross-flow 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 with which the biological product is associated from the heterogeneous mixture.

[0206] In an embodiment, the positive charge-based fluidic purification module includes at least one dissociation equilibrium vessel that allows dissociation of a biological product from the magnetic resin bead surface, and at least one second hybrid cross-flow fluidic device that includes a cross-flow 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 this purification. For example, multiple dissociation equilibrium vessels containing separate buffers of varying pH, ionic strength, or any combination thereof are utilized in sequence to generate a gradient dissociation effect.

[0207] In embodiments, the positive charge-based fluid purification module further comprises at least one tangential flow filtration system operated in fed-batch or perfusion mode for concentrating and buffer exchanging the fraction containing the biological product.

[0208] In an embodiment, the positive charge-based fluid purification module includes at least two buffer systems. In an embodiment, the positive charge based fluid purification module includes at least one magnetic resin bead regeneration buffer system.

[0209] In an embodiment, the positive charge-based fluid purification module includes at least one equilibration vessel configured to allow recycling of said magnetic resin beads. In an embodiment, the positive charge-based fluid purification module includes at least one sensor or detector.

[0210] In an embodiment, the positive charge-based fluid purification module includes at least one fluid handling pump. [Negative charge-based fluid purification module] In an embodiment, provided herein is a negative charge-based fluid purification module for separating a mixture into two or more fractions, where at least one fraction includes a biological product. The negative charge-based fluid purification module includes at least one inlet and at least one outlet configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be consistently constant, for example, during steady-state operation.

[0211] In embodiments, the negative charge-based fluidic purification module comprises a suspension of magnetic resin beads, where the surface of the magnetic resin beads comprises anionic functional groups configured to selectively associate with the biological product at a particular pH and ionic strength, e.g., the magnetic resin beads are mobile.

[0212] In an embodiment, the negative charge based fluid purification module includes at least one association equilibrium vessel that allows association of a biological product with the magnetic resin bead surface, and at least one first hybrid cross-flow fluidic device that includes a cross-flow 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 with which the biological product is associated from the heterogeneous mixture.

[0213] In an embodiment, the negative charge-based fluidic purification module includes at least one dissociation equilibrium vessel that allows dissociation of a biological product from the magnetic resin bead surface, and at least one second hybrid cross-flow fluidic device that includes a cross-flow 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 this purification. For example, multiple dissociation equilibrium vessels containing separate buffers of varying pH, ionic strength, or any combination thereof are utilized in sequence to generate a gradient dissociation effect.

[0214] In embodiments, the negative charge-based fluid purification module further comprises at least one tangential flow filtration system operated in fed-batch or perfusion mode for concentrating and buffer exchanging the fraction containing the biological product.

[0215] In an embodiment, the negative charge-based fluid purification module includes at least two buffer systems. In an embodiment, the negative charge based fluid purification module includes at least one magnetic resin bead regeneration buffer system.

[0216] In an embodiment, the negative charge-based fluid purification module includes at least one equilibration vessel configured to allow recycling of said magnetic resin beads. In an embodiment, the negative charge-based fluid purification module includes at least one sensor or detector.

[0217] In an embodiment, the negative charge-based fluid purification module includes at least one fluid handling pump. [Affinity-based TFF purification module] In an embodiment, provided herein is an affinity-based TFF purification module for separating a mixture into two or more fractions, where at least one fraction comprises a biological product. The affinity-based TFF purification module comprises at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate is consistently constant during steady-state operation.

[0218] In embodiments, the affinity-based TFF purification module comprises a suspension of resin beads, where the surface of the resin beads is linked to, without intending to be limiting, Protein A, Protein G, Protein L, an antigen protein, a protein, a receptor, an antibody, or an aptamer configured to selectively bind to the biological product. For example, the magnetic resin beads are mobile.

[0219] In embodiments, the affinity-based TFF purification module comprises at least one equilibration vessel that allows binding of a biological product to the surface of resin beads, and at least one first tangential flow filtration system for separating the resin beads bound to the biological product from the heterogeneous mixture.

[0220] In embodiments, the affinity-based TFF purification module further comprises at least one low pH equilibration vessel that allows debinding of the biological product from the resin bead surface, and at least one second tangential flow filtration system to separate the resin beads from the unbound biological product and complete their elution.

[0221] In embodiments, the affinity-based TFF purification module includes at least one regeneration equilibration vessel and at least one third tangential flow filtration system that allows concentration and buffer exchange of the resin beads to return them to their initial state, allowing recycling and reuse of the resin beads.

[0222] In embodiments, the affinity-based TFF purification module includes at least one collection vessel and at least one fourth tangential flow filtration system that allows for concentration and buffer exchange of the biological product to purify the biological product.

[0223] In embodiments, the at least one equilibration vessel, the at least one low pH equilibration vessel, and the at least one regeneration equilibration vessel of an affinity-based TFF purification module may comprise a single vessel that transitions 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 on a parallel flow path.

[0224] In embodiments, regeneration of the resin beads may be achieved by using at least one low pH equilibration vessel and at least one second tangential flow filtration system of an affinity-based TFF purification module configured to contain both a low pH elution buffer and a regeneration buffer to enable purification, concentration and buffer exchange, thereby regenerating the resin beads without the need for a separate regeneration equilibration vessel and corresponding tangential flow filtration system.

[0225] In embodiments, the affinity-based TFF purification module comprises at least two buffer systems. In embodiments, the affinity-based TFF purification module includes at least one resin bead regeneration buffer system.

[0226] In embodiments, the affinity-based TFF purification module comprises at least one hollow fiber membrane filter. In an embodiment, the affinity-based TFF purification module comprises at least one sensor or detector.

[0227] In embodiments, the affinity-based TFF purification module includes at least one fluid handling pump. [Positive charge-based TFF purification module] In an embodiment, the present disclosure provides a positive charge-based TFF purification module for separating a mixture into two or more fractions, wherein at least one fraction comprises a biological product.The positive charge-based TFF purification module comprises at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, and wherein the flow rate is consistently constant during steady-state operation.

[0228] In embodiments, a positive charge-based TFF purification module comprises a suspension of resin beads, where the surface of the resin beads comprises cationic functional groups configured to selectively associate with the biological product at a particular pH and ionic strength, e.g., the magnetic resin beads are mobile.

[0229] In an embodiment, the positive charge-based TFF purification module comprises at least one association equilibration vessel that allows association of a biological product with the resin bead surface, and at least one first tangential flow filtration system for separating the resin beads with associated biological product from the heterogeneous mixture.

[0230] In embodiments, the positive charge-based TFF purification module comprises at least one dissociation equilibrium vessel that allows dissociation of the biological product from the resin bead surface, and at least one second tangential flow filtration system to separate the resin beads from the dissociated biological product and complete their purification. In some aspects, multiple dissociation equilibrium vessels are used with multiple tangential flow filtration systems to achieve gradient dissociation, such as, for example, a pH gradient or an ionic strength gradient.

[0231] In an embodiment, the positive charge-based TFF purification module comprises at least one regeneration equilibration vessel and at least one third tangential flow filtration system that allows concentration and buffer exchange of the resin beads to return them to their initial state, allowing recycling and reuse of the resin beads.

[0232] In an embodiment, the positive charge-based TFF purification module comprises at least one collection vessel and at least one fourth tangential flow filtration system that allows for concentration and buffer exchange of the biological product to purify the biological product.

[0233] In embodiments, at least one association equilibration vessel, at least one dissociation vessel, and at least one regeneration equilibration vessel of a positive charge-based TFF purification module may comprise a single vessel that transitions 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 on a parallel flow path.

[0234] In embodiments, regeneration of the resin beads may be achieved by 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 contain both dissociation and regeneration buffers to enable purification, concentration and buffer exchange, thereby regenerating the resin beads without the need for a separate regeneration equilibration vessel and corresponding tangential flow filtration system.

[0235] In an embodiment, the positive charge-based TFF purification module comprises at least two buffer systems. In an embodiment, the positive charge-based TFF purification module comprises at least one resin bead regeneration buffer system.

[0236] In an embodiment, the positive charge-based TFF purification module comprises at least one hollow fiber membrane filter. In an embodiment, the positive charge-based TFF purification module includes at least one sensor or detector.

[0237] In an embodiment, the positive charge-based TFF purification module includes at least one fluid handling pump. [Negative charge-based TFF purification module] In an embodiment, the present disclosure provides a negative charge-based TFF purification module for separating a mixture into two or more fractions, wherein at least one fraction comprises a biological product.The negative charge-based TFF purification module comprises at least one inlet and at least one outlet, which are configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, and wherein the flow rate is consistently constant during steady-state operation.

[0238] In embodiments, a negative charge-based TFF purification module comprises a suspension of resin beads, where the surface of the resin beads comprises anionic functional groups configured to selectively associate with the biological product at a particular pH and ionic strength.

[0239] In an embodiment, the negative charge-based TFF purification module comprises at least one association equilibration vessel that allows association of a biological product with the resin bead surface, and at least one first tangential flow filtration system for separating the resin beads with associated biological product from the heterogeneous mixture.

[0240] In embodiments, the negative charge-based TFF purification module comprises at least one dissociation equilibrium vessel that allows dissociation of the biological product from the resin bead surface, and at least one second tangential flow filtration system to separate the resin beads from the dissociated biological product to complete their purification. In some aspects, multiple dissociation equilibrium vessels are used with multiple tangential flow filtration systems to achieve gradient dissociation, such as, for example, a pH gradient or an ionic strength gradient.

[0241] In an embodiment, the negative charge-based TFF purification module comprises at least one regeneration equilibration vessel and at least one third tangential flow filtration system that allows concentration and buffer exchange of the resin beads to return them to their initial state, allowing recycling and reuse of the resin beads.

[0242] In an embodiment, the negative charge-based TFF purification module comprises at least one collection vessel and at least one fourth tangential flow filtration system that allows for concentration and buffer exchange of the biological product to purify the biological product.

[0243] In embodiments, at least one association equilibration vessel, at least one dissociation vessel, and at least one regeneration equilibration vessel of a negative charge-based TFF purification module may comprise a single vessel that transitions 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 on a parallel flow path.

[0244] In embodiments, regeneration of resin beads may be achieved by 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 contain both dissociation and regeneration buffers to enable purification, concentration and buffer exchange, to regenerate resin beads without the need for a separate regeneration equilibration vessel and corresponding tangential flow filtration system.

[0245] In an embodiment, the negative charge-based TFF purification module comprises at least two buffer systems. In an embodiment, the negative charge-based TFF purification module comprises at least one resin bead regeneration buffer system.

[0246] In an embodiment, the negative charge-based TFF purification module comprises at least one hollow fiber membrane filter. In an embodiment, the negative charge-based TFF purification module comprises at least one sensor or detector.

[0247] In an embodiment, the negative charge-based TFF purification module includes at least one fluid handling pump. [Isoelectric point-based fluid purification module] In an embodiment, provided herein is an isoelectric-based fluid purification module for separating a mixture into two or more fractions, where at least one fraction includes a biological product. The isoelectric-based fluid purification module includes at least one inlet and at least one outlet configured to allow continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be consistently constant, for example, during steady-state operation.

[0248] In an embodiment, the process described herein comprises continuously transmitting a fraction comprising 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 has at least one fluidic element comprising a fluid channel generated between two parallel plates, an electric field or an 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 ampholytes). For example, the solution contacting surfaces of the two parallel plates comprise glass, ceramic, plastic, or any combination thereof. In some examples, the aqueous ionic solution can generate a pH gradient. In other examples, the aqueous ionic solution can impart a constant pH.

[0249] In an embodiment, the free-flow electrophoresis device has at least one fluidic element including a fluidic channel generated 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-based fluidic purification module includes at least one first fluidic element including a fluidic channel generated 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., the coarse pH gradient can be in the pH range of about 2 to about 10) across the main separation channel, and at least one second fluidic element including a fluidic channel generated between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient (e.g., the fine pH gradient can be in the pH range of about 5 to about 8) across the main separation channel. For example, an additional subsequent fluidic device or chip containing a fluidic channel generated between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow can be used to further regenerate a pH gradient across the main separation channel (e.g., a pH range of about 7.1 to about 7.6).

[0250] In other embodiments, the free-flow electrophoresis device has at least one fluidic element that includes a fluidic channel generated between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, but does not include a pH gradient, for operation in a zone electrophoresis or charge separation mode of operation. For example, an isoelectric-based fluid purification module includes at least one first fluidic element that includes a fluidic channel generated 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., greater than pH 7), and at least one second fluidic element that includes a fluidic channel generated 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., less than pH 7).

[0251] In other embodiments, the free-flow electrophoresis device has at least one fluidic element that includes a fluid channel generated between two parallel plates, 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) to operate in an isotachophoresis operating mode.

[0252] In other embodiments, an isoelectric-based fluid purification module includes at least one first free-flow electrophoresis device including a fluid channel generated 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 electrophoresis device including a fluid channel generated between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, where each element is connected in series and can operate in an independent operating mode to enable purification. For example, the at least one first free-flow electrophoresis device can operate in an isoelectric focusing mode and the at least one second free-flow electrophoresis device can operate in an isotachophoresis mode to enhance separation resolution.

[0253] In other embodiments, the isoelectric-based fluid purification module includes at least one first fluidic device including a fluidic channel having at least one dielectrophoretic electrode capable of inducing a defined unidirectional force, at least one second free-flow electrophoretic device including a fluidic channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a coarse pH gradient (e.g., pH range of about 2 to about 10) across the main separation channel, and at least one third free-flow electrophoretic device including a fluidic channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a fine pH gradient (e.g., pH range of about 5 to about 8) across the main separation channel. For example, the pH gradient across the main separation channel can be further purified using additional subsequent fluidic devices or chips including a fluidic channel generated between two parallel plates and an electric field or electric field gradient orthogonal to the direction of fluid flow (e.g., pH range of about 7.1 to about 7.6).

[0254] In embodiments, the isoelectric-based fluid purification device further comprises at least two electrodes (eg, platinum wire electrodes) to function as an anode or a cathode. In embodiments, the backpressure in an isoelectric-based fluid purifier varies depending on the channel geometry and dimensions, inlet and outlet openings and / or tubing diameter, and input flow rate. For example, the backpressure ranges from about 3.45 kPa to about 68.9 kPa (about 0.5 psi to about 10 psi). In some examples, the backpressure is controlled by, for example, and without intending to be limiting, a needle valve.

[0255] In embodiments, the isoelectric-based fluid purification device detects O generated in the electrode channel under an applied voltage. 2 and H 2 Further comprising at least one bubble removal system for continuously removing gas bubbles. In some embodiments, removing electrolysis bubbles is essential to allow for substantially long-term continuous operation. For example, the bubble removal system utilizes a hydrophobic PTFE membrane to create a watertight seal over the electrode channel that allows electrolysis bubbles to be continuously removed at the point of origin by exposure to a vacuum system. For example, the vacuum gauge pressure is in the range of about -5 kPa to about -40 kPa (about -0.05 bar to about -0.4 bar).

[0256] In embodiments, the isoelectric-based fluid purifier further includes an active cooling system or heat sink to enable temperature control and Joule heat dissipation. For example, an active cooling system includes an aluminum thermal chuck that includes a cooled circulating water / propylene glycol jacket.

[0257] In an embodiment, the isoelectric-based fluid purification module includes at least one buffer or ampholyte system. In embodiments, the isoelectric-based fluid purification module includes at least one electrode solution. In some embodiments, the at least one electrode solution includes an electrolyte solution configured to contact the anode or cathode to enable proper functioning, such as, for example, phosphoric acid and sodium hydroxide, respectively. In other embodiments, the at least one electrode solution includes at least one ampholyte solution configured to contact the anode or cathode to enable proper functioning, such as, for example, Tris-buffered saline, that flows through the main separation channel, the anode channel, and the cathode channel.

[0258] In embodiments, the isoelectric-based fluid purification module includes at least one sensor or detector. For example, the at least one sensor or detector is disposed in-line. In some examples, the 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.

[0259] In an embodiment, the isoelectric-based fluid purification module includes at least one liquid circuit breaker or disconnects the downstream of the device from at least one in-line sensor or detector upstream to enable sensing or detection to be performed in a voltage-free solution.

[0260] In an embodiment, the isoelectric-based fluid purification module includes at least one fluid handling pump. In an embodiment, the isoelectric-based fluid purification module includes at least one collection vessel.

[0261] [method] Provided herein is a method for purifying a biological product from a heterogeneous mixture derived from a bioreactor producing the biological product, comprising utilizing a process described herein. For example, bioreactor types include, but are not limited to, batch bioreactors, fed-batch bioreactors, perfusion bioreactors, chemostat bioreactors, or multi-compartment bioreactors. In some examples, the bioreactor produces the biological product at steady state.

[0262] In embodiments, provided herein is a method for purifying a biological product from a heterogeneous mixture derived from a bioreactor producing the biological product, comprising utilizing 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 fluidic purification module, a positive charge-based fluidic purification module, a negative charge-based fluidic 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 fluidic purification module.

[0263] In some embodiments, provided herein is a method for continuously purifying a biological product from a heterogeneous mixture derived from a bioreactor producing the biological product at steady state, comprising utilizing at least one of the modules described herein, e.g., 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 fluidic purification module, a positive charge-based fluidic purification module, a negative charge-based fluidic 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 fluidic purification module.

[0264] In other embodiments, provided herein is a method of purifying a biological product from a heterogeneous mixture not originating from a bioreactor producing the biological product at steady state, comprising utilizing at least one of the modules described herein, e.g., 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 fluidic purification module, a positive charge-based fluidic purification module, a negative charge-based fluidic 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 fluidic purification module.

[0265] Other aspects of the invention are disclosed below. The patent or application file contains one or more drawings executed in color. Copies of this patent or patent application publication containing color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0266] [Figure 1A] 1A-1D show schematic diagrams of exemplary continuous process flows described herein. Figure 1A shows an exemplary continuous process flow, where step 1 includes a bioreactor producing a biological product at steady state, step 2 includes a continuous dynamic filtration module, step 3 includes an affinity-based magnetic purification module, step 4 includes at least one charge-based magnetic purification module, step 5 includes a standard industrial viral inactivation and filtration process performed, for example, in fed-batch or perfusion mode, and step 6 includes a high performance tangential flow filtration using charged membranes performed, for example, in fed-batch or perfusion mode to prepare for a standard industrial fill-finish process in step 7. [Figure 1B] FIG. 1B shows an exemplary continuous process flow, where step 1 includes a bioreactor producing a biological product at steady state, step 2 includes a continuous dynamic filtration module, step 3 includes an affinity-based magnetic purification module, step 4 includes a positive charge-based magnetic purification module, step 5 includes a negative charge-based magnetic purification module, and step 6 includes high performance tangential flow filtration using charged membranes performed in fed-batch or perfusion mode to prepare for a standard industrial fill-finish process in step 7. [Figure 1C] FIG. 1C shows an exemplary continuous process flow, where step 1 includes a bioreactor producing a biological product at steady state, step 2 includes a continuous dynamic filtration module, step 3 includes an affinity-based magnetic purification module, step 4 includes an isoelectric-based fluidic purification module, step 5 includes a standard industrial viral inactivation and filtration process performed, for example, in fed-batch or perfusion mode, and step 6 includes high performance tangential flow filtration using charged membranes performed in fed-batch or perfusion mode to prepare for a standard industrial fill-finish process in step 7. [Figure 1D]FIG. 1D shows an exemplary continuous process flow, where step 1 includes a bioreactor producing a biological product at steady state, step 2 includes a continuous dynamic filtration module, step 3 includes an affinity-based magnetic purification module, step 4 includes an isoelectric-based fluidic purification module, and step 5 includes high performance tangential flow filtration using charged membranes performed in fed-batch or perfusion mode to prepare for a standard industrial fill-finish process in step 6. [Figure 2A] 2A-2D show schematic diagrams of exemplary continuous process flows described herein. Figure 2A shows an exemplary continuous process flow, where step 1 includes a bioreactor producing a biological product at steady state, step 2 includes a continuous dynamic filtration module, step 3 includes an affinity-based purification module, step 4 includes at least one charge-based purification module, step 5 includes a standard industrial viral inactivation and filtration process performed, for example, in fed-batch or perfusion mode, and step 6 includes a high performance tangential flow filtration using charged membranes performed, for example, in fed-batch or perfusion mode to prepare for a standard industrial fill-finish process in step 7. [Figure 2B] FIG. 2B shows an exemplary continuous process flow, where step 1 includes a bioreactor producing a biological product at steady state, step 2 includes a continuous dynamic filtration module, step 3 includes an affinity-based purification module, step 4 includes a positive charge-based purification module, step 5 includes a negative charge-based purification module, and step 6 includes high performance tangential flow filtration using charged membranes performed in fed-batch or perfusion mode to prepare for a standard industrial fill-finish process in step 7. [Figure 2C]FIG. 2C shows an exemplary continuous process flow, where step 1 includes a bioreactor producing a biological product at steady state, step 2 includes a continuous dynamic filtration module, step 3 includes an affinity-based purification module, step 4 includes an isoelectric-based fluidic purification module, step 5 includes a standard industrial viral inactivation and filtration process performed, for example, in fed-batch or perfusion mode, and step 6 includes high performance tangential flow filtration using charged membranes performed in fed-batch or perfusion mode to prepare for a standard industrial fill-finish process in step 7. [Figure 2D] FIG. 2D shows an exemplary continuous process flow, where step 1 includes a bioreactor producing a biological product at steady state, step 2 includes a continuous dynamic filtration module, step 3 includes an affinity-based purification module, step 4 includes an isoelectric-based fluidic purification module, and step 5 includes high performance tangential flow filtration using charged membranes performed in fed-batch or perfusion mode to prepare for a standard industrial fill-finish process in step 6. [Figure 3A] 3A-3D show schematic diagrams of exemplary continuous process flows described herein. Figure 3A shows an exemplary continuous process flow, where step 1 includes a bioreactor producing a biological product at steady state, step 2 includes a continuous dynamic filtration module, step 3 includes an affinity-based fluid purification module, step 4 includes at least one charge-based fluid purification module, step 5 includes a standard industrial viral inactivation and filtration process performed, for example, in fed-batch or perfusion mode, and step 6 includes a high performance tangential flow filtration using charged membranes performed, for example, in fed-batch or perfusion mode to prepare for a standard industrial fill-finish process in step 7. [Figure 3B]FIG. 3B shows an exemplary continuous process flow, where step 1 includes a bioreactor producing a biological product at steady state, step 2 includes a continuous dynamic filtration module, step 3 includes an affinity-based fluid purification module, step 4 includes a positive charge-based fluid purification module, step 5 includes a negative charge-based fluid purification module, and step 6 includes high performance tangential flow filtration using charged membranes performed in fed-batch or perfusion mode to prepare for a standard industrial fill-finish process in step 7. [Figure 3C] FIG. 3C shows an exemplary continuous process flow, where step 1 includes a bioreactor producing a biological product at steady state, step 2 includes a continuous dynamic filtration module, step 3 includes an affinity-based fluid purification module, step 4 includes an isoelectric point-based fluid purification module, step 5 includes a standard industrial viral inactivation and filtration process performed, for example, in fed-batch or perfusion mode, and step 6 includes high performance tangential flow filtration using charged membranes performed in fed-batch or perfusion mode to prepare for a standard industrial fill-finish process in step 7. [Figure 3D] FIG. 3D shows an exemplary continuous process flow, where step 1 includes a bioreactor producing a biological product at steady state, step 2 includes a continuous dynamic filtration module, step 3 includes an affinity-based fluid purification module, step 4 includes an isoelectric point-based fluid purification module, and step 5 includes high performance tangential flow filtration using charged membranes performed in fed-batch or perfusion mode to prepare for a standard industrial fill-finish process in step 6. [Figure 4A]4A-4D show schematic diagrams of exemplary continuous process flows described herein. Figure 4A shows an exemplary continuous process flow, where step 1 includes a bioreactor producing a biological product at steady state, step 2 includes a continuous dynamic filtration module, step 3 includes an affinity-based TFF purification module, step 4 includes at least one charge-based TFF purification module, step 5 includes a standard industry viral inactivation and filtration process performed, for example, in fed-batch or perfusion mode, and step 6 includes a high performance tangential flow filtration using charged membranes performed, for example, in fed-batch or perfusion mode to prepare for a standard industrial fill-finish process in step 7. [Figure 4B] FIG. 4B shows an exemplary continuous process flow, where step 1 includes a bioreactor producing a biological product at steady state, step 2 includes a continuous dynamic filtration module, step 3 includes an affinity-based TFF purification module, step 4 includes a positive charge-based TFF purification module, step 5 includes a negative charge-based TFF purification module, and step 6 includes high performance tangential flow filtration using charged membranes performed in fed-batch or perfusion mode to prepare for a standard industrial fill-finish process in step 7. [Figure 4C] FIG. 4C shows an exemplary continuous process flow, where step 1 includes a bioreactor producing a biological product at steady state, step 2 includes a continuous dynamic filtration module, step 3 includes an affinity-based TFF purification module, step 4 includes an isoelectric point-based fluidic purification module, step 5 includes a standard industry viral inactivation and filtration process performed, for example, in fed-batch or perfusion mode, and step 6 includes high performance tangential flow filtration using charged membranes performed in fed-batch or perfusion mode to prepare for a standard industrial fill-finish process in step 7. [Figure 4D]FIG. 4D shows an exemplary continuous process flow, where step 1 includes a bioreactor producing a biological product at steady state, step 2 includes a continuous dynamic filtration module, step 3 includes an affinity-based TFF purification module, step 4 includes an isoelectric point-based fluidic purification module, and step 5 includes high performance tangential flow filtration using charged membranes performed in fed-batch or perfusion mode to prepare for a standard industrial fill-finish process in step 6. [Figure 5A] 5A and 5B show an exemplary continuous process flow along with a design schematic for a downstream purification module described herein. [Figure 5B] Same as above. [Figure 6A]6A and 6B show a series of exemplary designs of dynamic filtration apparatus including a single output head for continuously transferring a heterogeneous mixture containing biological product from a steady-state bioreactor bleed output line and a separate output head for supplying a wash buffer. Figure 6A is a schematic diagram of a dynamic filtration apparatus design including a single output head for continuously transferring a heterogeneous mixture containing biological product from a steady-state bioreactor bleed output line, a separate output head for supplying a wash buffer, a rolled filter membrane that functions as a feed reel and a collection reel, two servo motors for controlling the feed reel vs. collection reel system, two support rods with mechanically smooth contact surfaces, a membrane support structure with a mechanically smooth contact surface and an opening that is connected to a vacuum line, a vacuum collection vessel, a diaphragm pump, and a geared pump. FIG. 6B is a schematic diagram of a dynamic filtration device design including a single output head for continuously conveying a heterogeneous mixture containing biological product from the steady-state bioreactor bleed output line, a separate output head for supplying wash buffer, a rolled filter membrane acting as a feed reel and a collection reel, two servo motors for controlling the feed reel vs. collection reel system, two support rods with mechanically smooth contact surfaces, a membrane support structure with a mechanically smooth contact surface and an opening in communication with a vacuum line, a controllable T-valve, two vacuum collection receptacles, a diaphragm pump, and two geared pumps. [Figure 6B] Same as above. [Figure 7A]Figures 7A and 7B show two exemplary designs of a dynamic filtration apparatus including multiple output heads for continuously conveying a heterogeneous mixture containing a biological product from a steady-state bioreactor bleed output line and multiple individual output heads for supplying a wash buffer. Figure 7A shows a schematic diagram of a dynamic filtration apparatus design including multiple output heads for conveying a heterogeneous mixture containing a biological product from a steady-state bioreactor bleed output line, multiple individual output heads for supplying a wash buffer, a rolled filter membrane that functions as a feed reel and a collection reel, two servo motors for controlling the feed reel vs. collection reel system, two support rods with mechanically smooth contact surfaces, a membrane support structure with a mechanically smooth contact surface and an opening that is connected to a vacuum line, a vacuum collection vessel, a diaphragm pump, and a geared pump. FIG. 7B shows a schematic diagram of a dynamic filtration device design including multiple output heads for continuously conveying a heterogeneous mixture containing biological product from a steady-state bioreactor bleed output line, multiple individual output heads for supplying wash buffers, a rolled filter membrane acting as a feed reel and a collection reel, two servo motors for controlling the feed reel vs. collection reel system, two support rods with mechanically smooth contact surfaces, a membrane support structure with a mechanically smooth contact surface and an opening in communication with a vacuum line, a controllable T-valve, two vacuum collection receptacles, a diaphragm pump, and two geared pumps. [Figure 7B] Same as above. [Figure 8] 1 shows an image of an exemplary membrane support structure with five parallel slotted openings. [Figure 9A]9A and 9B are images showing the removal of PolyBeads (0.05% solids; 2 μm (red), 6 μm (red), and 10 μm (blue) diameters) from a heterogeneous mixture in 1×PBS. FIG. 9A is an image showing a visual comparison (from left to right) of an initial heterogeneous mixture of PolyBeads (0.05% solids; 2 μm, 6 μm, and 10 μm diameters) in 1× 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 apparatus (MBM-3 shows a sample dynamically filtered with a 0.45 μm PVDF filter membrane and a flow rate of 0.25 mL / min), and a filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration apparatus (MBM-4 shows a sample dynamically filtered with a 0.45 μm PES filter membrane and a flow rate of 0.25, 0.5, 1.0, 2.0, and 5.0 mL / min). FIG. 9B is a bar graph showing a UV-Vis spectrophotometer comparison of an initial heterogeneous mixture of PolyBeads (0.05% solids; 2 μm, 6 μm, and 10 μm diameters) in 1× 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 having a 0.45 μm PVDF filter membrane and a flow rate of 0.25 mL / min (MBM-3), and a filtrate obtained by purifying the 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 (MBM-4), demonstrating the successful removal of PolyBeads from the heterogeneous mixture using the exemplary dynamic filtration device. [Figure 9B] Same as above. [Figure 10A]10A-10D are a series of data showing the removal of PolyBeads (0.05% solids; 2 μm (red), 6 μm (red), and 10 μm (blue) diameters) from a heterogeneous mixture of PolyBeads suspended in a 0.5 mg / mL solution of BSA-FITC in 1× PBS. FIG. 10A is a graph showing UV-Vis spectrophotometer traces of a serially diluted 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 1× PBS, with the PolyBead signature region indicated, showing the presence of PolyBeads. FIG. 10B is a graph showing UV-Vis spectrophotometer traces of a serially diluted 0.5 mg / mL solution of BSA-FITC in 1× PBS, with the PolyBead signature region indicated, showing the absence of PolyBeads. FIG. 10C shows 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 1×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-3a represents a sample dynamically filtered with a 0.45 μm PVDF filter membrane and a flow rate of 0.25 mL / min), and the filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration device. Shown are images of a visual comparison (from left to right) of filtrates obtained from purifying a heterogeneous mixture with an exemplary dynamic filtration apparatus (MBM-4a represents a sample dynamically filtered with a 0.45 μm PES filter membrane and a flow rate of 0.5 mL / min), filtrates obtained from purifying a heterogeneous mixture with an exemplary dynamic filtration apparatus (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 supernatants collected from purification of a heterogeneous mixture by centrifugation (10,000 x g for 5 min).FIG. 10D is a graph showing a comparison of UV-Vis spectrophotometers of 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 1×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), and the supernatant collected from purification by centrifugation (10,000×g for 5 min), demonstrating the successful purification of BSA-FITC by removing the PolyBeads as indicated by the absence of PolyBeads in the PolyBead signature region using the exemplary dynamic filtration device. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 11A] 11A and 11B show dynamic filtration of a heterogeneous mixture of PolyBeads suspended in a 0.5 mg / mL solution of human polyclonal IgG (hIgG) in 1×PBS at an input flow rate of 10 mL / min. FIG. 11A shows dynamic filtration through a slot die output head. FIG. 11A shows images of an initial heterogeneous mixture of PolyBeads (2 μm, 6 μm, and 10 μm diameters at 1.1×10, 4.2×10, and 1.0×10 particles / mL, respectively) suspended in a 0.5 mg / mL solution of hIgG in 1×PBS, a filtrate obtained by purifying the heterogeneous mixture with an exemplary dynamic filtration device having a 0.45 μm PES filter membrane and a flow rate of 10 mL / min, and a visual comparison of the supernatant collected from purification of the heterogeneous mixture by centrifugation (10,000×g for 5 min). [Figure 11B] FIG. 11B is a graph showing a spectrophotometric comparison of total protein concentration determined by BCA assay of the filtrate obtained by dynamic filtration and the supernatant collected by centrifugation. [Figure 11C]FIG. 11C shows images of a visual comparison (from left to right) of an initial heterogeneous mixture of PolyBeads (0.5 μm, 0.75 μm, 1 μm, 2 μm, 3 μm, and 10 μm diameters at 7.3×10 7 , 1.1×10 8 , 1.1×10 8 , 3.4×10 7 , and 1.0×10 6 particles / mL, respectively) suspended in a 0.5 mg / mL solution of hIgG in 1× PBS, the filtrate obtained by purifying the heterogeneous mixture in an exemplary dynamic filtration device having a 0.45 μm PES filter membrane and a flow rate of 10 mL / min, and the supernatant collected from purification of the heterogeneous mixture by centrifugation (10,000×g for 5 min). [Figure 11D] FIG. 11d is a graph showing a spectrophotometric comparison of total protein concentration as determined by BCA assay of the filtrate obtained by dynamic filtration and the supernatant collected by centrifugation. [Figure 12A] Figures 12A-12D show the protein recovery of dynamic filtration during continuous operation with different proteins, protein concentrations, filter membrane materials and pore sizes, membrane support structures, and different input flow rates. Figure 12A shows the recovery of proteins of different sizes and charges (bovine serum albumin (BSA), lysozyme, and hIgG at 0.5-10 mg / mL, 5 mg / mL, and 0.5 mg / mL, respectively) by BCA assay of filtrate obtained by dynamic filtration through a 0.45 μm PES filter membrane with a transport velocity of 0.5 mm / sec and an input flow rate of 10 mL / min. [Figure 12B] FIG. 12B shows the recovery 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 new aqueous PVDF, 0.22 μm PES) with a transport velocity of 0.5 mm / sec and an input flow rate of 10 mL / min. [Figure 12C]FIG. 12C shows the recovery 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 velocity 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 five parallel slots and a PTFE membrane support structure with a porous aqueous polyethylene (PE) insert). [Figure 12D] FIG. 12D shows the recovery of 0.5 mg / mL lysozyme by BCA assay of filtrates 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 velocity of 0.5 mm / sec. [Figure 13A] Figures 13A-13C show a comparison of cell purification by dynamic filtration and centrifugation of an input heterogeneous mixture containing a suspension cell culture in RPMI medium spiked with hIgG at a final concentration of 1 g / L. Figure 13A shows optical imaging (from left to right) of an initial hIgG-spiked murine myeloma suspension cell culture in RPMI medium (1 g / L hIgG at 2 x 106 cells / mL) and the 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 velocity of 0.5 mm / sec, and the supernatants (C-1, C-2, C-3) collected by centrifugation at 10,000 x g for 5 min. [Figure 13B] FIG. 13B shows SDS-PAGE analysis (from left to right) of initial hIgG-spiked murine myeloma suspension cell cultures in RPMI medium (1 g / L hIgG at 2×106 cells / mL) and the 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 velocity of 0.5 mm / sec, and the supernatants (C-1, C-2, C-3) obtained by centrifugation at 10,000×g for 5 min. [Figure 13C]FIG. 13C shows a comparison of hIgG recovery from a heterogeneous mixture (suspension cell culture in RPMI medium spiked with 1 g / L hIgG) by BCA assay of the filtrate (blue outline bar) obtained by dynamic filtration at an input flow rate of 2 mL / min using a 0.45 μm PES filter membrane with a transport velocity of 0.5 mm / sec, and the supernatant collected after centrifugation at 10,000×g for 5 min. [Figure 14] FIG. 1 shows an exemplary design schematic of an affinity-based magnetic purification device that includes a loop conveyor system and at least one magnetic field that is permanently “on”. [Figure 15] FIG. 1 shows an exemplary design schematic of an affinity-based magnetic purification device that includes a loop conveyor system and at least one magnetic field that can be switched “on / off.” [Figure 16] FIG. 1 shows an exemplary design schematic of a charge-based magnetic purification device that includes a loop conveyor system and at least one magnetic field that is permanently “on”. [Figure 17] FIG. 1 shows an exemplary design schematic of a charge-based magnetic purification device that includes a loop conveyor system and at least one magnetic field that can be switched “on / off.” [Figure 18] FIG. 1 shows an exemplary design schematic of an affinity-based magnetic purification device including a pick-and-place robotic system and at least one magnetic field. [Figure 19] FIG. 1 shows an exemplary design schematic of a charge-based magnetic purification device including a pick-and-place robotic system and at least one magnetic field. [Figure 20A]Figures 20A and 20B show affinity-based magnetic purification performed on an affinity-based magnetic purification device packed with magnetic protein A coated agarose beads for a mixture of hIgG (target, 2 g / L input concentration) and lysozyme (impurity, 1 g / L input concentration). Figure 20A shows total protein analysis by BCA assay on fractions collected from three consecutive cycles of use and recycling of the magnetic affinity beads, demonstrating the ability to reproducibly recycle and reuse the magnetic affinity beads without compromising binding capacity and performance. Figure 20B shows SDS-PAGE analysis on fractions collected from three consecutive cycles of use and recycling of the magnetic affinity beads, demonstrating the ability to reproducibly recycle and reuse the magnetic affinity beads without compromising binding capacity and performance. [Figure 20B] Same as above. [Figure 21A] Figures 21A-21D show exemplary designs of affinity-based purification devices that include a mechanical rotation system: Figure 21A is a schematic diagram of the lid system. [Figure 21B] FIG. 21B is a schematic diagram of a vessel carousel. [Figure 21C] FIG. 21C is a schematic diagram of the collection system. [Figure 21D] FIG. 21D shows how the lid system and collection system interface with the container carousel. [Figure 22A] Figures 22A-22D show an exemplary design of a charge-based purification device that includes a mechanical rotation system: Figure 22A is a schematic of the lid system. [Figure 22B] FIG. 22B is a schematic diagram of a vessel carousel. [Figure 22C] FIG. 22C is a schematic diagram of the collection system. [Figure 22D] FIG. 22D shows how the lid system and collection system interface with the container carousel. [Figure 23A]Figures 23A-23D show 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] FIG. 23B is a schematic diagram of an exemplary gasket lid that includes an air inlet, two buffer inlets configured to generate a circular flow pattern, a vent port, and a fill inlet that are components of the lid system. [Figure 23C] FIG. 23C is a schematic diagram of a vessel including a mesh filter or frit and a valve that are components of a vessel carousel. [Figure 23D] FIG. 23D is a schematic diagram of a collector, a component of the collection system. [Figure 24A] Figures 24A and 24B show an exemplary design of an affinity-based purification device that includes a stepwise linear system: Figure 24A shows the individual system components of an affinity-based purification device. [Figure 24B] FIG. 24B shows the connectivity of an affinity-based purification device that includes a stepwise linear system. [Figure 25A] Figures 25A and 25B show an exemplary design of an affinity-based purification device that includes a stepwise linear system: Figure 25A shows the individual system components of a charge-based purification device. [Figure 25B] FIG. 25B shows the connectivity of a charge-based purification device that includes a stepwise linear system. [Figure 26A]Figures 26A and 26B show affinity-based purification of hIgG (2 g / L input concentration) solution performed on an affinity-based purification device packed with Protein A-coated agarose resin beads. Figure 26A shows total protein analysis by BCA assay on fractions collected from three consecutive cycles of use and recycling of affinity resin beads, demonstrating the ability to reproducibly recycle and reuse affinity resin beads without compromising binding capacity and performance. Figure 26B shows SDS-PAGE analysis on fractions collected from three consecutive cycles of use and recycling of affinity resin beads, demonstrating the ability to reproducibly recycle and reuse affinity resin beads without compromising binding capacity and performance. [Figure 26B] Same as above. [Figure 27A] Figures 27A and 27B show affinity-based purification performed on an affinity-based purification device packed with protein A-coated agarose resin beads for a mixture of hIgG (target, 2 g / L input concentration) and lysozyme (impurity, 1 g / L input concentration). Figure 27A shows total protein analysis by BCA assay on fractions collected from three consecutive cycles of use and recycling of the affinity resin beads, demonstrating the ability to reproducibly recycle and reuse the affinity resin beads without compromising binding capacity and performance. Figure 27B shows SDS-PAGE analysis on fractions collected from three consecutive cycles of use and recycling of the affinity resin beads, demonstrating the ability to reproducibly recycle and reuse the affinity resin beads without compromising binding capacity and performance. [Figure 27B] Same as above. [Figure 28A] Figures 28A-28D are a series of images showing an exemplary design of a hybrid fluidic device: Figure 28A is an image showing a hybrid fluidic device including parallel cross-flow channels, a permanent magnetic field, and two piezoelectric transducers. [Figure 28B] FIG. 28B is an image showing a hybrid fluidic device that includes an angled cross-flow channel, a permanent magnetic field, and two piezoelectric transducers. [Figure 28C]FIG. 28C is an image showing a hybrid fluidic device that includes parallel cross-flow channels, a permanent magnetic field, and two optional dielectrophoretic electrodes. [Figure 28D] FIG. 28D is an image showing a hybrid fluidic device that includes an angled cross-flow channel, a permanent magnetic field, and two selective dielectrophoretic electrodes. [Figure 29] 1 shows an exemplary design schematic of an affinity-based fluid purification device. [Diagram 30] 1 shows an exemplary design schematic of a charge-based fluid purification device. [Diagram 31] FIG. 1 shows an exemplary design schematic of an affinity-based purification device that includes at least one tangential flow filtration system. [Diagram 32] FIG. 1 shows an exemplary design schematic of a charge-based purification device including at least one tangential flow filtration system. [Diagram 33] FIG. 1 shows an exemplary design schematic of an isoelectric-based fluid purification device that includes a fluidic element having a channel generated between two parallel plates, an electric field perpendicular to the direction of fluid flow, and an aqueous ionic solution. [Diagram 34] FIG. 1 shows an exemplary design schematic of a free-flow electrophoresis device comprising a first fluidic element having a channel generated between two parallel plates, an electric field perpendicular to the direction of fluid flow, and a coarse pH gradient connected to a second fluidic element having a channel generated between two parallel plates, an electric field perpendicular to the direction of fluid flow, and a fine pH gradient, the device being capable of operating in isoelectric focusing mode. [Diagram 35] 1 shows an exemplary design schematic of a free-flow electrophoresis device comprising a first fluidic element having channels generated between two parallel plates, an electric field perpendicular to the direction of fluid flow, and a constant basic pH across a main separation channel connected to a second fluidic element having channels generated between two parallel plates, an electric field perpendicular to the direction of fluid flow, and a constant acidic pH across a main separation channel, said device being capable of operating in a zone electrophoresis mode. [Diagram 36]FIG. 1 shows an exemplary design schematic of a free-flow electrophoresis device comprising a first fluidic element having a channel generated between two parallel plates capable of operating in an isoelectric focusing mode and an electric field perpendicular to the direction of fluid flow, coupled to a second fluidic element having a channel generated between two parallel plates capable of operating in an isotachophoresis mode and an electric field perpendicular to the direction of fluid flow. [Figure 37] FIG. 1 shows an exemplary design schematic of a free-flow electrophoresis device comprising a first fluidic element having a channel with selective dielectrophoretic electrodes for pre-sorting mixtures, connected to a second fluidic element having a channel generated between two parallel plates capable of operating in an isoelectric focusing mode and an electric field perpendicular to the direction of fluid flow, connected to a third fluidic element having a channel generated between two parallel plates capable of operating in an isotachophoresis mode and an electric field perpendicular to the direction of fluid flow. [Figure 38] 1 shows an exemplary bubble removal and degassing system design that removes electrolysis bubbles directly from the electrode channels to produce a bubble-free main separation channel. [Figure 39] 1 illustrates an exemplary liquid circuit breaker that creates a break in a solution coupled to an applied voltage flowing from an outlet of a free-flow electrophoresis device to at least one in-line sensor or detector. [Figure 40A] Figures 40A-40E show the isoelectric-based fluidic purification of a mixture of Lodamine 6G (0.25 mg / mL, net charge +1) and Fluorescein (0.25 mg / mL, net charge -1) using an isoelectric-based purification device with an anode channel (H2SO4), a cathode channel (NaOH), and a main separation channel with five inlets and five outlets through which ampholyte solutions flow, where the mixture was introduced into 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] FIG. 40B shows the absorbance spectra of fractions collected from the five outlets at 0 V and 5 mL / min. [Figure 40C]FIG. 40C shows optical images of fractions collected from the five outlets at 1000 V and 10 mL / min in the presence of a pH gradient. [Figure 40D] FIG. 40D shows the absorbance spectra of fractions collected from the five outlets at 1000 V and 10 mL / min in the presence of a pH gradient. [Figure 40E] FIG. 40E shows the purification of the mixture resulting in fractions containing purified Lodamin 6G (outlet 2, towards the cathode) and purified fluorescein (outlet 4, towards the anode). [Figure 41A] Figures 41A-41C show optical imaging of the purification of a mixture of Lodamine 6G (0.25 mg / mL, net charge +1) and Fluorescein (0.25 mg / mL, net charge -1) under different operating conditions using an isoelectric-based purification device with an anode channel (H2SO4), a cathode channel (NaOH), and a main separation channel with five inlets and five outlets through which ampholyte solutions flow, where the mixture was introduced into the center of the inlet (inlet 3) of the device. Figure 41A shows the isoelectric focusing free-flow electrophoresis of a mixture of Lodamine 6G and Fluorescein at 500 V and a flow rate of 3 mL / min. [Figure 41B] FIG. 41B shows free-flow isoelectric focusing of a mixture of Lodamin 6G and fluorescein at 700 V and a flow rate of 5 mL / min. [Figure 41C] FIG. 41C shows free-flow isoelectric focusing of a mixture of Lodamin 6G and fluorescein at 900 V and a flow rate of 10 mL / min. [Figure 42A] Figures 42A and 42B show optical imaging of the purification of a mixture of small molecule dyes using an isoelectric-based purification device, which includes an anode channel (H2SO4), a cathode channel (NaOH), and a main separation channel with five inlets and five outlets through which ampholyte solutions flow, where the mixture was introduced into the center of the inlet (inlet 3) of the device. Figure 42A shows the isoelectric focusing 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] FIG. 42B shows the isoelectric 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. [Figure 43A] Figures 43A-43D show optical imaging of 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-based purification device operated in isoelectric free-flow electrophoresis mode over increasing applied voltages. The mixture was introduced into the central inlet (inlet 3) at a flow rate of 5 mL / min using a device that includes an anode channel, a cathode channel, and a main separation channel with five inlets and ten outlets, where each channel was made to flow the same ampholyte solution at 5 mL / min. When no voltage was applied, the mixture exited the device from the central outlets (outlets 4 and 5) along a laminar flow (Figure 43A). When applying a voltage to the main separation channel with ampholytes at a sample input flow rate of 5 mL / min, a linear pH gradient was established and basic fuchsin and fluorescein migrated to the cathode and anode, respectively, consistent with theoretical electrophoretic mobility predictions (Figures 43B-43D). By increasing the electric field strength by increasing the applied voltage from 600 V (Figure 43B) to 900 V (Figure 43C) to 1100 V (Figure 43D), the separation of the two molecules was observed to increase linearly with the length of the main separation channel. [Figure 43B] Same as above. [Figure 43C] Same as above. [Fig. 43D] Same as above. [Figure 44A]Figures 44A and 44B show optical imaging of the purification of a mixture of small molecule dyes using an isoelectric-based purification device, which includes an anodic channel (H2SO4), a cathodic channel (NaOH), and a main separation channel with five inlets and five outlets through which two ampholyte solutions flow, separated by a spacer solution, where the mixture was introduced into the spacer solution at the center of the inlet (inlet 3) of the device. Figure 44A shows the isotachophoresis 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 concentration of the two dyes into two separate high-resolution lines. [Figure 44B] FIG. 44B shows the UV exposure results of the results shown in FIG. 44A. [Figure 45A] Figures 45A-45E show 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, which includes an anode channel (H2SO4), a cathode channel (NaOH), and a main separation channel with five inlets and five outlets through which ampholyte solutions flow, where the mixture was introduced into the center of the inlet (inlet 3) of the device. Figure 45A shows the spectrophotometer 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] FIG. 45B shows SDS-PAGE analysis of fractions from the five outlets at 0 V and a flow rate of 10 mL / min, showing that the mixture is present in outlet 3. [Figure 45C] FIG. 45C shows spectrophotometric analysis by BCA assay for total protein concentration of fractions from five outlets at 850 V and a flow rate of 10 mL / min, showing that protein is distributed across outlets 2, 3 and 4. [Figure 45D]FIG. 45D shows SDS-PAGE analysis of fractions from the five outlets at 850 V and a flow rate of 10 mL / min, showing that purified lysozyme is present in outlet 2 and purified BSA is present in outlet 4. [Figure 45E] Figure 45E shows the theoretical electrophoretic migration directions of BSA (towards the anode) and lysozyme (towards the cathode). [Figure 46A] Figures 46A-46C show the results of purifying a mixture of hlgG (0.5 mg / mL, pI 6-8) and lysozyme (0.25 mg / mL, pI 11) using an isoelectric point-based purification device, which includes an anode channel (H2SO4), a cathode channel (NaOH), and a main separation channel with five inlets and five outlets through which ampholyte solutions flow, where the mixture was introduced into the center of the inlet (inlet 3) of the device. Figure 46A shows spectrophotometric analysis by BCA assay for total protein concentration of fractions derived from the 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. [Figure 46B] FIG. 46B shows the theoretical electrophoretic migration directions of hIgG (towards the anode, cathode, and center) and lysozyme (towards the cathode). [Figure 46C] FIG. 46C shows SDS-PAGE analysis of fractions 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. [Figure 47A]47A-47D show 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, which includes an anodic channel (H2SO4), a cathodic channel (NaOH), and a main separation channel with five inlets and five outlets through which ampholyte solutions flow, where the mixture was introduced into the center of the inlet (inlet 3) of the device. FIG. 47A shows spectrophotometric analysis by BCA assay of total protein concentration of fractions from the five outlets at a flow rate of 3 mL / min at 0 V and 500 V, showing that the proteins are distributed across outlets 2, 3, and 4 under the applied voltage. FIG. 47B shows spectrophotometric analysis by BCA assay of total protein concentration of fractions from the five outlets at a flow rate of 5 mL / min at 0 V and 700 V, showing that the proteins are distributed across outlets 2, 3, and 4 under the applied voltage. Figure 47C shows spectrophotometer analysis by BCA assay for total protein concentration of fractions from five outlets at 10 mL / min flow rate at 0 V and 850 V, showing that under applied voltage, protein is distributed across outlets 2, 3, and 4. Figure 47D shows SDS-PAGE analysis of fractions from five outlets at (1) 3 mL / min at 0 V or 500 V, (2) 5 mL / min at 0 V or 700 V, or (3) 10 mL / min at 0 V or 850 V. [Figure 47B] Same as above. [Figure 47C] Same as above. [Figure 47D] Same as above. [Figure 48] FIG. 1 shows an exemplary schematic diagram of coupling a charge-based purification module, a magnetic purification module, a charge-based purification module, a charge-based fluidic purification module, a charge-based TFF purification module, or an isoelectric-based purification module to an exemplary semi-continuous process described herein using standard industrial downstream processing equipment operated in fed-batch or perfusion mode to prepare a biological product for fill-finish. [Figure 49]In the absence of a separate viral inactivation and removal process step, an exemplary schematic diagram is shown linking a charge-based purification module, a magnetic purification module, a charge-based purification module, a charge-based fluidic purification module, a charge-based TFF purification module, or an isoelectric-based purification module into an exemplary semi-continuous process described herein using standard industrial downstream processing equipment operated in fed-batch or perfusion mode to prepare a biological product for fill finish. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0267] Provided herein, inter alia, is a continuous process for purifying biological products. The presently claimed process 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, and without intending to be limiting, the process described herein provides a continuous bioprocess for purifying monoclonal antibodies that eliminates the membrane fouling problems inherent in traditional multi-stage filtration processes (e.g., multi-stage tangential flow filtration or depth filtration) by having an initial filtration step that includes at least one dynamic filtration module as described herein to remove large impurities (e.g., cells, cell debris, and aggregates). Furthermore, the continuous process significantly reduces the production facility footprint, the time required for facility construction and validation, the costs associated with facility construction, and capital equipment expenditures when compared to traditional approaches to batch, single-use, or semi-continuous monoclonal antibody manufacturing, while maintaining throughput and yield.

[0268] Continuous bioprocessing as described herein, by its ability to operate continuously, allows for the use of smaller and simplified equipment (e.g., smaller bioreactor volumes and downstream bioprocessing equipment) since the large process equipment required for the centrifugation, depth filtration, and column chromatography steps of traditional downstream bioprocessing (the size of which is dictated by the large bioreactor volumes) is not required. Furthermore, the smaller and simplified equipment that operates continuously allows for the use of very small bioreactors to produce monoclonal antibodies at steady state. Continuous bioprocessing as described herein can also significantly reduce operational costs, overall bioprocess line downtime, and biological product loss when compared to traditional monoclonal antibody manufacturing approaches. Finally, the processes described herein for purifying biological products are performed in systems with footprints that occupy significantly less square feet than current technologies, without sacrificing product throughput or yield on a kilogram per year basis. For example, the processes for producing and purifying monoclonal antibodies described herein are operated in footprints that occupy up to about 30,000 square feet. In comparison, current monoclonal antibody production and downstream processing requires a minimum of 200,000 square feet.

[0269] A continuous process for purifying a biological product using at least one of a dynamic filtration module, an affinity-based magnetic purification module, and a charge-based magnetic purification module or an isoelectric-based fluidic purification module. A continuous process for purifying a biological product is described, the process comprising continuously receiving a heterogeneous mixture comprising a biological product via an input line, the biological product including, but not limited to, a protein or fragment thereof (polypeptide), an antibody or 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 at least 60%, 70%, 80%, 90%, 95%, or 99% by weight of impurities (cells, cell debris, aggregates, host cell proteins, undesirable proteins and peptides, undesirable antibodies, undesirable nucleic acids and oligonucleotides, viruses, salts, buffer components, detergents, sugars, metal contaminants, leachables, media components, and / or naturally associated natural organic molecules) are removed.

[0270] The process includes continuously removing large impurities from a heterogeneous mixture by dynamic filtration. The dynamic filtration process includes at least one dynamic filtration module that continuously supplies a biological product to the dynamic filtration module from at least one output head in fluid communication with an input line under negative pressure to produce a filtrate containing the biological product. The dynamic filtration module may further include at least one additional input line for supplying a wash buffer via a coaxial output head or a separate single-axis output head.

[0271] In embodiments, the processes described herein include purifying a biological product produced continuously in a bioreactor (e.g., fed-batch bioreactors, perfusion bioreactors, and chemostat bioreactors). For example, the bioreactor includes a bioreactor feed line and an output bleed line that allow for steady-state cell culture growth conditions, and the output bleed line acts as an input line that allows for continuous fluid flow from the bioreactor to a dynamic filtration module.

[0272] As described herein, the process of continuously removing large impurities from a heterogeneous mixture (or mixtures) does not include centrifugation, disc stack centrifugation, depth filtration, static filtration, tangential flow filtration, hydrocyclones, or any combination thereof. The term "static filtration" refers to a process in which the heterogeneous mixture being filtered remains static, i.e., for example, a filter membrane (or depth filter) has a limited capacity, and when the membrane reaches its capacity, the filtration rate decreases (e.g., the membrane pores become blocked). In "static" (as opposed to "dynamic") filtration, the filter membrane remains fixed (does not move) and the flow (e.g., the flow of the heterogeneous mixture) passes through the fixed filter membrane. These static filtration methods are conventional, simple, and well known in the art.

[0273] Unlike static filtration methods commonly used in the art, the process herein describes a dynamic filtration module, where the components of the dynamic filtration module move in a coordinated manner (e.g., the membrane moves or advances according to the overall process flow rate) so that filtration can occur continuously in new, unused target areas of the filter membrane, eliminating membrane fouling and clogging and allowing for control of filter cake packing and thickness during operation.

[0274] 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 vessel. In an embodiment, the filter membrane roll comprises a rolled filter membrane, wherein the filter membrane includes, but is not limited to, polyethersulfone (PES), new aqueous polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), new aqueous PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), new aqueous PTFE, or any combination thereof.

[0275] 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 an antibody, the pore size of the rolled filter membrane is in the range of 0.2 μm to about 0.45 μm.

[0276] 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. In an embodiment, the filter membrane roll also functions as a supply reel in communication with the collection reel, meaning that the filter membrane starts from a prefabricated roll and is stretched across an initially empty collection roll, creating a reel-to-reel system. In an aspect, the dynamic filtration module includes a rolled filter membrane extending between the supply reel and the collection reel, said filter membrane having a target area (e.g., an active target area) configured to receive a heterogeneous mixture. For example, the movement of the supply reel is controlled by a servo motor coupled to a gear box that limits the revolutions per minute (RPM) to a ratio of 200:1 to allow for a low membrane transport rate with high torque. The movement of the collection reel is controlled by a servo motor coupled to a gear box that limits the RPM to a ratio of 200:1 to allow for a low membrane transport rate with high torque. Additionally, the supply reel motor and the collection reel motor are controlled by a closed loop controller that operates a feedback mechanism to ensure a consistent speed with the constantly changing diameter of the filter membrane roll on both the supply reel and the collection reel during operation. For example, the supply reel and the collection reel run at the same speed and in the same direction.

[0277] In an embodiment, 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. The membrane support structure of the dynamic filtration module includes a mechanically smooth contact surface derived from a material with a low static friction coefficient (e.g., PTFE) and an opening that is connected to a vacuum line. As used herein, "membrane support structure" refers to a component that is made to provide structural support to the active area of ​​the filter membrane and prevent deformation when passing through a negative pressure area by an opening that is connected to a vacuum line. Furthermore, as used herein, "mechanically smooth contact surface" refers to a surface that has a low static friction coefficient and generates low frictional forces against the transport of the filter membrane, especially when wet. A mechanically smooth contact surface can affect the ease with which the filter membrane moves in a dynamic manner. A mechanically smooth contact surface can also be measured by surface roughness, with the lower the value, the smoother the surface. Furthermore, since a rough surface has a higher frictional force between the surfaces than a smooth surface, a mechanically smooth contact surface as used herein refers to a surface that has low frictional forces (i.e., a low static friction coefficient).

[0278] In an embodiment, 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. Additionally, 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, where each grate is 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 partial or full thickness, and may or may not be in parallel rows. The aperture elements (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, and without intending to be limiting, the aperture may comprise a mesh that is about 0.25 mm to about 5 mm thick.

[0279] The membrane support structure of the dynamic filtration module includes a temperature control mechanism that maintains a temperature from about 4° C. to about 37° C. in the presence of evaporative cooling. For example, during purification of an antibody, the temperature control mechanism maintains a temperature between 15° C. and 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 resistive heating element, and / or a circulating water / propylene glycol jacketed thermal chuck.

[0280] In an embodiment, at least one support rod or roller of the dynamic filtration module has a mechanically smooth contact surface derived from a material with a low coefficient of static friction (e.g., PTFE, PFA). For example, the coefficient of static friction 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 coefficient of static friction is 0.04. For example, the dynamic filtration module includes at least one support rod or roller with a mechanically smooth contact surface to stabilize the movement of the filter membrane across the membrane support structure.

[0281] In embodiments, the dynamic filtration module includes at least one output head for regulating the flow of the 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.

[0282] In some embodiments, the dynamic filtration module further includes at least one additional input line for supplying a wash buffer via a coaxial output head, a separate single-axis output head, a separate slot die output head, or a slot die output head having multiple openings.

[0283] In some embodiments, the dynamic filtration module includes elements known in the coating and converting industry, such as, but not limited to, active or passive edge guides, tension adjustment devices (e.g., dancers), brakes and tension detectors, or any combination thereof.

[0284] In embodiments, the dynamic filtration module includes a vacuum system in communication with the membrane support structure to apply a negative pressure across an active target area of ​​the filter membrane, where the negative pressure allows active transport of the filter membrane across the membrane support structure and allows collection of filtrate containing the biological product. For example, the vacuum system of the dynamic filtration module maintains a gauge pressure of about -5 kPa to about 98 kPa (about -0.05 bar to about 0.98 bar) for continuous filtration.

[0285] In an embodiment, the dynamic filtration module further comprises at least one vacuum collection vessel configured to collect filtrate, and at least one sensor or detector. In an aspect described herein, during purification by dynamic filtration, filtrate containing the biological product is delivered to a vacuum collection vessel capable of collecting from about 50 mL to about 100 L under negative pressure. For example, the vacuum collection vessel capable of collecting filtrate is from about 1 L to about 10 L. In another example, the vacuum collection vessel capable of collecting filtrate is from about 1 L to about 50 L.

[0286] In embodiments, processes for the continuous removal of large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration include multi-stage filtration using at least two individual rolled filter membranes having different pore sizes. For example, these multi-stage dynamic filtration processes include at least one first dynamic filtration device having a large pore size (e.g., 0.45 μm) rolled filter membrane in fluid communication with at least one second dynamic filtration device having a small pore size (e.g., 0.2 μm) rolled filter membrane, thereby producing a filtrate containing a biological product.

[0287] The process described herein includes continuously transmitting the filtrate to a first module capable of separating the solution into two or more fractions, including at least one fraction containing the biological product. For example, separating the solution into two or more fractions can include at least one fraction containing the biological product and at least one other fraction containing small impurities. As described herein, the first module includes an affinity-based magnetic purification device. "Affinity-based magnetic purification device" refers to a purification technology based on structural binding interactions (e.g., ligand-receptor interactions) of molecules in which selective surface-immobilized ligands recognize and bind 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 continuous fluid flow between the first inlet and the first outlet via a loop conveyor system or a pick-and-place robotic system.

[0288] In an embodiment, the affinity-based magnetic purification device further comprises a suspension of magnetic resin beads. The surface of the magnetic resin beads is linked with, for example, but not limited to, Protein A, Protein G, Protein L, antigen protein, protein, receptor, antibody, or aptamer. Continuous purification of biological products (e.g., monoclonal antibodies) using affinity magnetic resin beads can avoid the cumbersome processing steps of traditional affinity column chromatography (e.g., Protein A affinity chromatography).

[0289] In an embodiment, 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 can vary depending on the biological product being purified and the flow rate of the process. Furthermore, the concentration of the magnetic resin beads can range from 0.01% to 25% by weight. For example, the concentration of the magnetic resin beads can be about 1% to about 10% by weight. In another example, the binding capacity of the magnetic resin beads is a function of bead concentration, surface area to volume ratio, affinity ligand density, or any combination thereof. In yet another example, the magnetic resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

[0290] The loop conveyor system may refer to, for example, a continuous or infinite loop. The loop conveyor system is advantageous in that it can move large amounts at high flow rates continuously and efficiently through the process while allowing for a smaller footprint, compared to traditional affinity column chromatography systems (e.g., Protein A affinity chromatography). Since the biological product is directly transported by truck, any object of regular or irregular shape of any size can be configured for transportation. In some embodiments, the object is a transport container with a regular shape (e.g., a cube, a rectangular prism, a cylinder, and a cone).

[0291] In an embodiment, the loop conveyor system has at least two transport containers filled with magnetic resin beads configured to continuously receive filtrate containing a mixture comprising a biological product and subsequently transport the resulting heterogeneous mixture comprising the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0292] A pick-and-place robotic system may refer to, for example, at least one robot or robotic arm. Compared to conventional affinity column chromatography systems (e.g., protein A affinity chromatography), pick-and-place robotic systems are advantageous in that they can move large quantities at high flow rates continuously and efficiently through a process while allowing for a smaller footprint. Biological products contained in transport containers are picked and placed, so that any size of regular shaped object can be configured for transport and stacking, with or without handles. In some embodiments, the object is a transport container with a regular shape (e.g., a cube and a rectangular prism).

[0293] In an embodiment, the pick-and-place robotic system has at least two transport containers filled with magnetic resin beads configured to sequentially receive filtrate containing a mixture comprising a biological product and subsequently transport the resulting heterogeneous mixture comprising the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0294] 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 allow washing in at least one of the at least two transport vessels. Furthermore, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from the heterogeneous mixture to allow elution of the biological product in at least one of the at least two transport vessels. Alternatively, the at least one external magnetic field can be used to allow recycling of the magnetic resin beads in at least one of the at least two transport vessels. For example, mixing of the magnetic resin beads can be achieved by placing at least one transport vessel between two separate opposing magnetic fields that switch between on and off states.

[0295] The process described herein also includes continuously transferring the fraction comprising the biological product from at least one first outlet of the first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module, the second module including a charge-based magnetic purification device. As used herein, "charge-based magnetic purification device" includes, for example, purifying a biological molecule based on its surface charge, ionic properties, electrostatic interaction, or isoelectric point. As described herein, the charge-based magnetic purification device includes 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 continuous fluid flow between the second inlet and the second outlet via a loop conveyor system or a pick-and-place robotic system.

[0296] In embodiments, the charge-based magnetic purification device (e.g., positive 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 comprise cationic or anionic functional groups configured to selectively associate with said biological product at a particular pH and ionic strength to enable positive charge-based magnetic purification or negative charge-based magnetic purification, respectively. Continuous purification of biological products (e.g., monoclonal antibodies) using ionic magnetic resin beads can avoid the cumbersome processing steps of conventional ion exchange column chromatography (e.g., cation exchange or anion exchange chromatography).

[0297] In an embodiment, 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 can vary depending on the biological product being purified and the flow rate of the process. Furthermore, the concentration of the magnetic resin beads can range from 0.01% to 25% by weight. For example, the concentration of the magnetic resin beads can be about 1% to about 10% by weight. In another example, the charge or electrostatic association capacity of the magnetic resin beads is a function of bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In yet another example, the magnetic resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

[0298] The loop conveyor system may refer to, for example, a continuous or infinite loop. The loop conveyor system is advantageous in that it can move large quantities at high flow rates continuously and efficiently through the process while allowing for a smaller footprint, compared to conventional ion exchange column chromatography systems. Since the biological product is directly transported by truck, any object of regular or irregular shape of any size can be configured for transportation. In some embodiments, the object is a transport container having a regular shape (e.g., a cube, a rectangular prism, a cylinder, and a cone).

[0299] In an embodiment, 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 subsequently transport the resulting heterogeneous mixture containing the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0300] A pick-and-place robotic system may refer to, for example, at least one robot or robot arm. Compared to conventional ion exchange chromatography systems, pick-and-place robotic systems are advantageous in that they can move large quantities at high flow rates continuously and efficiently through a process while allowing for a smaller footprint. Biological products contained in transport containers are picked and placed, so that objects of any size and regular shape can be configured for transport and stacking, with or without handles. In some embodiments, the object is a transport container with a regular shape (e.g., a cube and a rectangular prism).

[0301] In an embodiment, the pick-and-place robotic system has at least two transport containers filled with magnetic resin beads configured to sequentially receive filtrate containing a mixture comprising a biological product and subsequently transport the resulting heterogeneous mixture comprising the biological product, magnetic resin beads, a buffer, or any combination thereof.

[0302] 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 in at least one of the at least two transport containers. Furthermore, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from the heterogeneous mixture to enable dissociation and collection of the biological product in at least one of the at least two transport containers. Alternatively, the at least one external magnetic field can be used to enable recycling of the magnetic resin beads in at least one of the 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 on and off states.

[0303] In 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) modules are recycled and reused, e.g., the beads can be reused at least two, three, four or more times to purify a biological product.

[0304] Alternatively, the process described herein includes continuously transmitting a fraction comprising a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving flow from the at least one first outlet of the first module, said second module including a free-flow electrophoresis device. A free-flow electrophoresis device having a fluid channel generated between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an aqueous ionic solution can be used instead of or in addition to a charge-based magnetic purification module for purifying a biological product (e.g., a monoclonal antibody).

[0305] For example, the solution contacting surfaces of the two parallel plates may comprise glass, ceramic, plastic, or any combination thereof. In some examples, the aqueous ionic solution may create a pH gradient across the main separation channel. In other examples, the aqueous ionic solution may impart a constant pH across the main separation channel.

[0306] In an embodiment, the free-flow electrophoresis device has at least one fluidic element including a fluidic channel generated 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-based fluidic purification module includes at least one first fluidic element including a fluidic channel generated 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., the coarse pH gradient can be in the pH range of about 2 to about 10) across the main separation channel, and at least one second fluidic element including a fluidic channel generated between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient (e.g., the fine pH gradient can be in the pH range of about 5 to about 8) across the main separation channel. For example, an additional subsequent fluidic device or chip containing a fluidic channel generated between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow can be used to further regenerate a pH gradient across the main separation channel (e.g., a pH range of about 7.1 to about 7.6).

[0307] In other embodiments, the free-flow electrophoresis device includes a fluid channel generated between two parallel plates and an electric field or electric field gradient orthogonal to the direction of fluid flow for operation in a zone electrophoresis or charge separation mode of operation, and at least one fluidic element without a pH gradient (e.g., constant pH in the main separation channel). For example, an isoelectric-based fluid purification module includes at least one first fluidic element including a fluid channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a constant basic pH (e.g., greater than pH 7), and at least one second fluidic element including a fluid channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a constant acidic pH (e.g., less than pH 7).

[0308] In other embodiments, the free-flow electrophoresis device has at least one fluidic element that includes a fluid channel generated between two parallel plates, 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) to operate in an isotachophoresis operating mode.

[0309] In other embodiments, an isoelectric-based fluid purification module includes at least one first free-flow electrophoresis device comprising a fluid channel generated 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 electrophoresis device comprising a fluid channel generated between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, where each element is connected in series and can operate in independent operating modes to enable purification. For example, the at least one first free-flow electrophoresis device can operate in an isoelectric focusing mode and the at least one second free-flow electrophoresis device can operate in an isotachophoresis mode to enhance separation resolution.

[0310] In other embodiments, the isoelectric-based fluid purification module includes at least one first fluidic device including a fluidic channel having at least one dielectrophoretic electrode capable of inducing a defined unidirectional force, at least one second free-flow electrophoretic device including a fluidic channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a coarse pH gradient (e.g., pH range of about 2 to about 10) across the main separation channel, and at least one third free-flow electrophoretic device including a fluidic channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a fine pH gradient (e.g., pH range of about 5 to about 8) across the main separation channel. For example, the pH gradient across the main separation channel can be further purified using additional subsequent fluidic devices or chips including a fluidic channel generated between two parallel plates and an electric field or electric field gradient orthogonal to the direction of fluid flow (e.g., pH range of about 7.1 to about 7.6).

[0311] In embodiments, the isoelectric-based fluid purification device further comprises at least two electrodes (eg, platinum wire electrodes) to function as an anode or a cathode. In embodiments, the backpressure in an isoelectric-based fluid purifier varies depending on the channel geometry and dimensions, inlet and outlet openings and / or tubing diameter, and input flow rate. For example, the backpressure ranges from about 3.45 kPa to about 68.9 kPa (about 0.5 psi to about 10 psi). In some examples, the backpressure is controlled by, for example, and without intending to be limiting, a needle valve.

[0312] In embodiments, the isoelectric-based fluid purification device detects O generated in the electrode channel under an applied voltage. 2 and H 2Further comprising at least one bubble removal system for continuously removing gas bubbles. In some embodiments, removing electrolysis bubbles is essential to allow for substantially long-term continuous operation. For example, the bubble removal system utilizes a hydrophobic PTFE membrane to create a watertight seal over the electrode channel that allows electrolysis bubbles to be continuously removed at the point of origin by exposure to a vacuum system. For example, the vacuum gauge pressure is in the range of about -5 kPa to about -40 kPa (about -0.05 bar to about -0.4 bar).

[0313] In embodiments, the isoelectric-based fluid purification device further includes an active cooling system or heat sink (e.g., Peltier element, circulating water / propylene glycol jacketed thermal chuck) to enable temperature control and Joule heat dissipation. For example, the active cooling system may 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 separating biological products (e.g., monoclonal antibodies), the temperature is maintained at about 10°C to about 25°C. For example, the active cooling system may include an aluminum thermal chuck with a cooled circulating water / propylene glycol jacket.

[0314] In an embodiment, the isoelectric-based fluid purification module includes at least one buffer or ampholyte system. In embodiments, the isoelectric-based fluid purification module includes at least one electrode solution. In some embodiments, the at least one electrode solution includes an electrolyte solution configured to contact the anode or cathode to enable proper functioning, such as, for example, phosphoric acid and sodium hydroxide, respectively. In other embodiments, the at least one electrode solution includes at least one ampholyte solution configured to contact the anode or cathode to enable proper functioning, such as, for example, Tris-buffered saline, that flows through the main separation channel, the anode channel, and the cathode channel.

[0315] In embodiments, the isoelectric-based fluid purification module includes at least one sensor or detector. For example, the at least one sensor or detector is disposed in-line. In some examples, the 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.

[0316] In an embodiment, the isoelectric-based fluid purification module includes at least one liquid circuit breaker or disconnects the downstream of the device from at least one in-line sensor or detector upstream to enable sensing or detection to be performed in a voltage-free solution.

[0317] The presently claimed process 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, and without intending to be limiting, the process described herein provides a continuous bioprocess for purifying monoclonal antibodies that significantly reduces the production facility footprint, the time required to build and validate the facility, the costs associated with building the facility, and capital equipment expenditures, when compared to traditional approaches to batch, single-use, or semi-continuous monoclonal antibody manufacturing, while maintaining throughput and yield. Continuous bioprocessing as described herein, by its ability to operate continuously, uses smaller and simplified equipment (e.g., smaller bioreactor volumes and downstream bioprocessing equipment) since it does not require the large process equipment (whose size is dictated by the large bioreactor volumes) required for the batch centrifugation, depth filtration, and column chromatography steps of traditional downstream bioprocessing. Additionally, the smaller and simplified equipment operating continuously uses very small bioreactors that produce monoclonal antibodies at steady state. Continuous bioprocessing as described herein can also significantly reduce operational costs, overall bioprocessing line downtime, and biological product loss when compared to traditional monoclonal antibody manufacturing approaches. Finally, the processes described herein for purifying a biological product are performed in systems with footprints that occupy significantly less square feet than current technologies, without sacrificing product throughput or yield on a kilogram / year basis.

[0318] 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 can refer to a process in which heterogeneous mixtures are deposited on the membrane surface or within the membrane pores, causing the membrane to degrade over time, resulting in significant limitations to the usefulness of conventional filtration systems. For example, using conventional filtration or tangential flow filtration systems to clarify cells, cell debris, and aggregates from cell culture media typically results in filter membrane fouling or clogging, making these methods unsuitable as a means to continuously remove large impurities from heterogeneous mixtures containing biological products through long-term continuous processes. In contrast, the dynamic filtration devices described herein allow for the continuous removal of large impurities from heterogeneous mixtures containing biological products without membrane fouling, as the active target area of ​​the filter membrane is continually renewed.

[0319] Furthermore, because the entire process of producing and purifying a biological product can be continuous and can maintain flow rates ranging from about 0.1 mL / min to about 50 mL / min (e.g., from about 5 mL / min to about 10 mL / min) throughout the entire process, the footprint of the process equipment and the entire process can have a significantly smaller footprint than current standard processes without sacrificing product throughput or yield on a kilogram / year basis. For example, the process of producing and purifying a monoclonal antibody described herein operates in a footprint occupying a maximum of about 30,000 square feet. In comparison, current monoclonal antibody production and downstream processes require a minimum of 200,000 square feet. For example, the flow rates of the process of purifying a biological product range from about 1 mL / min to about 10 mL / min. In some examples, the flow rates of the step of continuously removing large impurities from a heterogeneous mixture range 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 the heterogeneous mixture is equal to the flow rate from the bioreactor bleed 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 ranges from 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 the fraction containing the biological product from the first outlet to the second module ranges from about 0.1 mL / min to about 50 mL / min.

[0320] Important advantages of the processes and methods utilizing magnetic resin beads (e.g., magnetic agarose) described herein include that these systems do not require traditional stationary phase or packed resin columns (e.g., for standard chromatography) for sterilization, recycling, and / or regeneration. For example, these systems provide for recycling and / or regeneration of magnetic resin beads to generate infinite surface area of ​​magnetic resin beads during operation, thereby providing a continuous and cost-effective method. In other words, the modules described herein do not have fixed binding or association capabilities. In a specific example, the magnetic resin beads used during purification of biological products as described herein are continuously recycled and regenerated, so that they can receive flow from the previous step of either the dynamic filtration module or the purification module without interrupting the flow from the bioreactor bleed line.

[0321] In other words, the modules described herein undergo these steps continuously and do not need to be left idle after a run to be sanitized, regenerated, and / or recycled. The method differs from current continuous chromatography methods in that current column chromatography methods have a column capacity limit limited by resin packing constraints, and therefore require column switching of multiple packed columns to accept continuous input flow and allow 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 the stationary phase, but rather the magnetic resin beads are mobile. Such bead mobility increases the surface area of ​​the resin beads available for binding or association, since substantially more of the magnetic resin bead surface is exposed and free to bind. Furthermore, the resin beads in the packed column are subjected to high pressure differentials to generate flow through the column, and damage caused by this is one of the reasons for columns having shorter than desired lifetimes. The mobile resin beads in the invention described herein are subjected to substantially lower pressures and are much softer than the fragile beads, thus increasing their lifetime. Furthermore, such mobility increases the likelihood that the magnetic resin beads will be regenerated and return to their initial state. This allows for more efficient use of the magnetic resin, further improving the cost effectiveness of the methods described herein.

[0322] An important advantage of the processes and methods using free-flow electrophoresis described herein is that this system represents a "no product loss" process, i.e., the product does not need to interact with resins or other purification moieties, since separation occurs in aqueous solution and through interaction with an electric field according to the physicochemical properties of the target biological product. Another advantage is observed in the resolution of this approach, since a theoretically higher purity of product can be obtained compared to conventional ion exchange chromatography. Furthermore, separation based on intrinsic physicochemical properties extends the usefulness of such an 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 (AAV), or lentiviruses.

[0323] Furthermore, the modular approach provides flexibility in process design to accommodate a range of biological products. A continuous process for purifying a biological product using at least one of a dynamic filtration module, an affinity-based purification module, and a charge-based or isoelectric-based fluidic purification module. A continuous process for purifying a biological product is described, the process comprising continuously receiving a heterogeneous mixture comprising a biological product via an input line, the biological product including, but not limited to, a protein or fragment thereof (polypeptide), an antibody or 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 at least about 60%, 70%, 80%, 90%, 95%, or 99% by weight of impurities (cells, cell debris, aggregates, host cell proteins, undesirable proteins and peptides, undesirable antibodies, undesirable nucleic acids and oligonucleotides, viruses, salts, buffer components, detergents, sugars, metal contaminants, leachables, media components, and / or naturally associated natural organic molecules) are removed.

[0324] The process includes continuously removing large impurities from a heterogeneous mixture by dynamic filtration. The dynamic filtration process includes at least one dynamic filtration module that continuously supplies a biological product to the dynamic filtration module from at least one output head in fluid communication with an input line under negative pressure to produce a filtrate containing the biological product. The dynamic filtration module may further include at least one additional input line for supplying a wash buffer via a coaxial output head or a separate single-axis output head.

[0325] In embodiments, the processes described herein include purifying a biological product produced continuously in a bioreactor (e.g., fed-batch bioreactors, perfusion bioreactors, and chemostat bioreactors). For example, the bioreactor includes a bioreactor feed line and an output bleed line that allow for steady-state cell culture growth conditions, and the output bleed line acts as an input line that allows for continuous fluid flow from the bioreactor to a dynamic filtration module.

[0326] As described herein, the process of continuously removing large impurities from a heterogeneous mixture does not include centrifugation, disc stack centrifugation, depth filtration, static filtration, tangential flow filtration, hydrocyclones, or any combination thereof. The term "static filtration" refers to a process in which the heterogeneous mixture being filtered remains static, i.e., for example, the filter membrane (or depth filter) has a limited capacity, and when the membrane reaches this capacity, the filtration rate decreases (e.g., the membrane pores become blocked). In "static" (as opposed to "dynamic") filtration, the filter membrane remains fixed (does not move) and the flow (e.g., the flow of the heterogeneous mixture) passes through the fixed filter membrane. These static filtration methods are conventional, simple, and well known in the art.

[0327] Unlike static filtration methods commonly used in the art, the process herein describes a dynamic filtration module, where the components of the dynamic filtration module move in a coordinated manner (e.g., the membrane moves or advances according to the overall process flow rate) so that filtration can occur continuously in new, unused target areas of the filter membrane, eliminating membrane fouling and clogging and allowing for control of filter cake packing and thickness during operation.

[0328] 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 vessel. For example, the filter membrane roll may include a rolled filter membrane, where the filter membrane may be made of, but is not limited to, polyethersulfone (PES), aqueous polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), aqueous PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), aqueous PTFE, or any combination thereof.

[0329] 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 an antibody, the pore size of the rolled filter membrane is in the range of 0.2 μm to about 0.45 μm.

[0330] 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. In an embodiment, the filter membrane roll also functions as a supply reel in communication with the collection reel, meaning that the filter membrane starts from a prefabricated roll and spans an initially empty collection roll, creating a reel-to-reel system. In an aspect, the dynamic filtration module includes a rolled filter membrane extending between the supply reel and the collection reel, the filter membrane having an active target area configured to receive a heterogeneous mixture. For example, the movement of the supply reel is controlled by a servo motor coupled to a gear box that limits the revolutions per minute (RPM) to a ratio of 200:1 to allow for a low membrane transport rate with high torque. The movement of the collection reel is controlled by a servo motor coupled to a gear box that limits the RPM to a ratio of 200:1 to allow for a low membrane transport rate with high torque. Additionally, the supply reel motor and the collection reel motor are controlled by a closed loop controller that operates a feedback mechanism to ensure a consistent speed with the constantly changing diameter of the filter membrane roll on both the supply reel and the collection reel during operation. For example, the supply reel and the collection reel operate at the same speed and in the same direction.

[0331] In an embodiment, 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. The membrane support structure of the dynamic filtration module includes a mechanically smooth contact surface derived from a material with a low static friction coefficient (e.g., PTFE) and an opening that is connected to a vacuum line. As used herein, "membrane support structure" refers to a component that is fabricated to provide structural support to the active area of ​​the filter membrane and prevent deformation when passing through a negative pressure area by an opening that is connected to a vacuum line. Furthermore, as used herein, "mechanically smooth contact surface" refers to a surface that has a low static friction coefficient and generates low frictional forces against the transport of the filter membrane, especially when wet. A mechanically smooth contact surface can affect the ease with which the filter membrane moves in a dynamic manner. A mechanically smooth contact surface can also be measured by surface roughness, with the lower the value, the smoother the surface. Furthermore, since a rough surface has a higher frictional force between the surfaces than a smooth surface, a mechanically smooth contact surface as used herein refers to a surface that has low frictional forces (i.e., a low static friction coefficient).

[0332] In an embodiment, 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. Additionally, 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, where each grate is 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 partial or full thickness, and may or may not be in parallel rows. The aperture elements (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, and without intending to be limiting, the aperture may comprise a mesh having a thickness of about 0.25 mm to about 5 mm.

[0333] The membrane support structure of the dynamic filtration module includes a temperature control mechanism that maintains a temperature from about 4° C. to about 37° C. in the presence of evaporative cooling. For example, during purification of an antibody, 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 circulating water / propylene glycol jacketed thermal chuck.

[0334] In an embodiment, at least one support rod or roller of the dynamic filtration module has a mechanically smooth 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 with a mechanically smooth contact surface to stabilize the movement of the filter membrane across the membrane support structure.

[0335] In embodiments, the dynamic filtration module includes at least one output head for regulating the flow of the 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.

[0336] In some embodiments, the dynamic filtration module further includes at least one additional input line for supplying a wash buffer via a coaxial output head, a separate single-axis output head, a separate slot die output head, or a slot die output head having multiple openings.

[0337] In some embodiments, the dynamic filtration module includes elements known in the coating and converting industry, such as, but not limited to, active or passive edge guides, tension adjustment devices (e.g., dancers), brakes and tension detectors, or any combination thereof.

[0338] In embodiments, the dynamic filtration module includes a vacuum system in communication with the membrane support structure to apply a negative pressure across a target area (e.g., an active target area) of the filter membrane, where the negative pressure allows active transport of the filter membrane across the membrane support structure and allows collection of a filtrate containing the biological product. For example, the vacuum system of the dynamic filtration module maintains a gauge pressure of about -5 kPa to about -98 kPa (about -0.05 bar to about -0.98 bar) for continuous filtration.

[0339] In embodiments, the dynamic filtration module further includes at least one vacuum collection vessel configured to collect filtrate, and at least one sensor or detector. In aspects described herein, during purification by dynamic filtration, filtrate containing the biological product is provided to a vacuum collection vessel capable of collecting from about 50 mL to about 100 L under negative pressure. For example, the vacuum collection vessel capable of collecting filtrate is from about 1 L to about 10 L. In other examples, the vacuum collection vessel capable of collecting filtrate is from about 1 L to about 50 L.

[0340] In embodiments, processes for the continuous removal of large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration include multi-stage filtration using at least two individual rolled filter membranes having different pore sizes. For example, these multi-stage dynamic filtration processes include at least one first dynamic filtration device having a large pore size (e.g., 0.45 μm) rolled filter membrane in fluid communication with at least one second dynamic filtration device having a small pore size (e.g., 0.2 μm) rolled filter membrane, thereby producing a filtrate containing the biological product.

[0341] The process described herein includes continuously transmitting the filtrate to a first module capable of separating the solution into two or more fractions, including at least one fraction containing the biological product. For example, separating the solution into two or more fractions can include at least one fraction containing the biological product and at least one other fraction containing small impurities. As described herein, the first module includes an affinity-based purification device. "Affinity-based purification device" refers to a purification technology based on molecular structural binding interactions (e.g., ligand-receptor interactions) in which selective surface-immobilized ligands recognize and bind 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 continuous fluid flow between the first inlet and the first outlet via a mechanical rotation system including a lid system, a vessel carousel, and a collection system.

[0342] In an embodiment, the affinity-based purification device further comprises a suspension of resin beads, the surface of which is linked with, for example, but not limited to, Protein A, Protein G, Protein L, an antigen protein, a protein, a receptor, an antibody, or an aptamer. Continuous purification of biological products (e.g., monoclonal antibodies) using affinity resin beads can avoid the cumbersome processing steps of traditional affinity column chromatography (e.g., Protein A affinity chromatography).

[0343] In embodiments, the diameter of the resin beads of the affinity-based purification device is from about 0.2 microns to about 200 microns. The diameter of the resin beads can vary depending on the biological product being purified and the flow rate of the process. Furthermore, the concentration of the resin beads can range from 0.01% to 25% by weight. For example, the concentration of the magnetic resin beads can be from about 1% to about 20% by weight. In other examples, the binding capacity of the resin beads is a function of bead concentration, surface area to volume ratio, affinity ligand density, or any combination thereof. In yet other examples, the resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

[0344] In embodiments, the affinity-based purification module includes a lid system having at least one gasketed lid, the at least one gasketed lid including at least one inlet for introducing gas to allow control of positive head pressure, at least one vent port to allow equilibration to atmospheric pressure, at least one inlet for introducing a suspension of resin beads, at least one inlet for receiving filtrate containing a biological product, and at least two inlets for introducing a buffer system to disperse the resin beads to allow washing, elution, or regeneration of the resin beads. In some embodiments, the at least one gasketed lid further includes a port for receiving an overhead stirring impeller to allow dispersion of the resin beads. For example, the lid system controls movement along the z-axis.

[0345] In an embodiment, the affinity-based purification module includes a mechanical rotation system, e.g., a carousel including at least two containers filled with resin beads configured to continuously receive a mixture including a biological product and subsequently transport the resulting heterogeneous mixture including the biological product, resin beads, a buffer, or any combination thereof. For example, the mechanical rotation system is configured to mate with a lid system to allow for pressurization. In another example, the mechanical rotation system controls the movement or rotation in the xy plane.

[0346] In embodiments, at least two vessels of the affinity-based purification module each have a supported base filter or filter membrane that allows retention of the resin beads during the binding, debinding, washing, elution, and regeneration process steps, e.g., at least two vessels further include valves for controlling the flow of liquid.

[0347] In embodiments, the affinity-based purification module includes a collection system that can interface with at least one of the at least two containers of the mechanical rotation system to collect waste, a fraction containing the biological product, or any combination thereof, e.g., the collection system controls movement along the z-axis.

[0348] The process described herein also includes continuously transferring the fraction comprising the biological product from at least one first outlet of the first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module, the second module including a charge-based purification device. As used herein, "charge-based purification device" includes, for example, purifying a biological molecule based on its surface charge, ionic properties, electrostatic interactions, or isoelectric point. As described herein, charge-based purification includes positive charge-based purification devices, negative charge-based purification devices, or combinations thereof. For example, 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 via a mechanical rotation system including a lid system, a vessel carousel, and a collection system.

[0349] 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 comprise cationic or anionic functional groups configured to selectively associate with said biological product at a particular pH and ionic strength to enable positive charge-based purification or negative charge-based purification, respectively. Continuous purification of biological products (e.g., monoclonal antibodies) using ionic resin beads can avoid the cumbersome processing steps of conventional ion exchange column chromatography (e.g., cation exchange or anion exchange chromatography).

[0350] In an embodiment, the diameter of the resin beads of the charge-based purification device is from about 0.2 microns to about 200 microns. The diameter of the resin beads can vary depending on the biological product being purified and the flow rate of the process. Furthermore, the concentration of the resin beads can range from 0.01% to 25% by weight. For example, the concentration of the magnetic resin beads can be from about 1% to about 20% by weight. In other examples, the charge or electrostatic association capacity of the resin beads is a function of bead concentration, surface area to volume ratio, surface charge density, net charge, or any combination thereof. In yet other examples, the resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

[0351] In embodiments, the charge-based purification module includes a lid system having at least one gasketed lid, the at least one gasketed lid including at least one inlet for gas introduction to allow control of positive head pressure, at least one vent port to allow equilibration to atmospheric pressure, at least one inlet for introducing a suspension of resin beads, at least one inlet for receiving filtrate containing a biological product, and at least two inlets for introducing a buffer system to disperse resin beads to allow washing, dissociation, or regeneration of the resin beads. In some embodiments, the at least one gasketed lid further includes a port for receiving an overhead stirring impeller to allow dispersion of resin beads. For example, the lid system controls movement along the z-axis.

[0352] In an embodiment, the charge-based purification module includes a mechanical rotation system, e.g., a carousel including at least two containers filled with resin beads configured to sequentially receive a mixture including a biological product and subsequently transport the resulting heterogeneous mixture including the biological product, resin beads, a buffer, or any combination thereof. For example, the mechanical rotation system is configured to mate with a lid system to allow for pressurization. In another example, the mechanical rotation system controls the movement or rotation in the xy plane.

[0353] In embodiments, at least two vessels of the charge-based purification module each have a supported base filter or filter membrane that allows retention of the resin beads during the association, dissociation, washing, and regeneration process steps, e.g., at least two vessels further include valves for controlling the flow of liquid.

[0354] In embodiments, the charge-based purification module includes a collection system that can interface with at least one of the at least two receptacles of the mechanical rotation system to collect waste, a fraction containing the biological product, or any combination thereof, e.g., the collection system controls movement along the z-axis.

[0355] In embodiments described herein, the resin beads of one or both of the first (affinity-based purification) and / or second (charge-based purification) modules are recycled and reused, e.g., the beads can be reused at least two, three, four, or more times to purify a biological product.

[0356] Alternatively, the process described herein includes continuously transmitting a fraction comprising a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving flow from the at least one first outlet of the first module, the second module including a free-flow electrophoresis device. A free-flow electrophoresis device having a fluid channel generated between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and an aqueous ionic solution can be used instead of or in addition to a charge-based magnetic purification module for purifying a biological product (e.g., a monoclonal antibody).

[0357] For example, the solution contacting surfaces of the two parallel plates may comprise glass, ceramic, plastic, or any combination thereof. In some examples, the aqueous ionic solution may create a pH gradient across the main separation channel. In other examples, the aqueous ionic solution may impart a constant pH across the main separation channel.

[0358] In an embodiment, the free-flow electrophoresis device has at least one fluidic element including a fluidic channel generated 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-based fluidic purification module includes at least one first fluidic element including a fluidic channel generated 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., the coarse pH gradient can be in the pH range of about 2 to about 10) across the main separation channel, and at least one second fluidic element including a fluidic channel generated between two parallel plates, an electric field or electric field gradient perpendicular to the direction of fluid flow, and a fine pH gradient (e.g., the fine pH gradient can be in the pH range of about 5 to about 8) across the main separation channel. For example, an additional subsequent fluidic device or chip containing a fluidic channel generated between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow can be used to further regenerate a pH gradient across the main separation channel (e.g., a pH range of about 7.1 to about 7.6).

[0359] In other embodiments, the free-flow electrophoresis device has at least one fluidic element that includes a fluidic channel generated between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, but does not include a pH gradient, for operation in a zone electrophoresis or charge separation mode of operation. For example, an isoelectric-based fluid purification module includes at least one first fluidic element that includes a fluidic channel generated 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., greater than pH 7), and at least one second fluidic element that includes a fluidic channel generated 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., less than pH 7).

[0360] In other embodiments, the free-flow electrophoresis device has at least one fluidic element that includes a fluid channel generated between two parallel plates, 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) to operate in an isotachophoresis operating mode.

[0361] In other embodiments, an isoelectric-based fluid purification module includes at least one first free-flow electrophoresis device comprising a fluid channel generated 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 electrophoresis device comprising a fluid channel generated between two parallel plates and an electric field or electric field gradient perpendicular to the direction of fluid flow, where each element is connected in series and can operate in an independent operating mode to enable purification. For example, the at least one first free-flow electrophoresis device can operate in an isoelectric focusing mode and the at least one second free-flow electrophoresis device can operate in an isotachophoresis mode to enhance separation resolution.

[0362] In other embodiments, the isoelectric-based fluid purification module includes at least one first fluidic device including a fluidic channel having at least one dielectrophoretic electrode capable of inducing a defined unidirectional force, at least one second free-flow electrophoretic device including a fluidic channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a coarse pH gradient (e.g., pH range of about 2 to about 10) across the main separation channel, and at least one third free-flow electrophoretic device including a fluidic channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a fine pH gradient (e.g., pH range of about 5 to about 8) across the main separation channel. For example, the pH gradient across the main separation channel can be further purified using additional subsequent fluidic devices or chips including a fluidic channel generated between two parallel plates and an electric field or electric field gradient orthogonal to the direction of fluid flow (e.g., pH range of about 7.1 to about 7.6).

[0363] In embodiments, the isoelectric-based fluid purification device further comprises at least two electrodes (eg, platinum wire electrodes) to function as an anode or a cathode. In embodiments, the backpressure in an isoelectric-based fluid purifier varies depending on the channel geometry and dimensions, inlet and outlet openings and / or tubing diameter, and input flow rate. For example, the backpressure ranges from about 3.45 kPa to about 68.9 kPa (about 0.5 psi to about 10 psi). In some examples, the backpressure is controlled by, for example, and without intending to be limiting, a needle valve.

[0364] In embodiments, the isoelectric-based fluid purification device detects O generated in the electrode channel under an applied voltage. 2 and H 2Further comprising at least one bubble removal system for continuously removing gas bubbles. In some embodiments, removing electrolysis bubbles is essential to allow for substantially long-term continuous operation. For example, the bubble removal system utilizes a hydrophobic PTFE membrane to create a watertight seal over the electrode channel that allows electrolysis bubbles to be continuously removed at the point of origin by exposure to a vacuum system. For example, the vacuum gauge pressure is in the range of about -5 kPa to about -40 kPa (about -0.05 bar to about -0.4 bar).

[0365] In embodiments, the isoelectric-based fluid purification device further includes an active cooling system or heat sink (e.g., Peltier element, circulating water / propylene glycol jacketed thermal chuck) to enable temperature control and Joule heat dissipation. For example, the active cooling system may 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 separating biological products (e.g., monoclonal antibodies), the temperature is maintained at about 10°C to about 25°C. For example, the active cooling system may include an aluminum thermal chuck with a cooled circulating water / propylene glycol jacket.

[0366] In an embodiment, the isoelectric-based fluid purification module includes at least one buffer or ampholyte system. In embodiments, the isoelectric-based fluid purification module includes at least one electrode solution. In some embodiments, the at least one electrode solution includes an electrolyte solution configured to contact the anode or cathode to enable proper functioning, such as, for example, phosphoric acid and sodium hydroxide, respectively. In other embodiments, the at least one electrode solution includes at least one ampholyte solution configured to contact the anode or cathode to enable proper functioning, such as, for example, Tris-buffered saline, that flows through the main separation channel, the anode channel, and the cathode channel.

[0367] In embodiments, the isoelectric-based fluid purification module includes at least one sensor or detector. For example, the at least one sensor or detector is disposed in-line. In some examples, the 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.

[0368] In an embodiment, the isoelectric-based fluid purification module includes at least one liquid circuit breaker or disconnects the downstream of the device from at least one in-line sensor or detector upstream to enable sensing or detection to be performed in a voltage-free solution.

[0369] The presently claimed process 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, and without intending to be limiting, the process described herein provides a continuous bioprocess for purifying monoclonal antibodies that significantly reduces the production facility footprint, the time required to build and validate the facility, the costs associated with building the facility, and capital equipment expenditures, when compared to traditional approaches to batch, single-use, or semi-continuous monoclonal antibody manufacturing, while maintaining throughput and yield. The continuous bioprocessing described herein, by its ability to operate continuously, uses smaller and simplified equipment (e.g., smaller bioreactor volumes and downstream bioprocessing equipment) since it does not require the large process equipment required for the centrifugation, depth filtration, and column chromatography steps of traditional downstream bioprocessing (the size of which is dictated by the large bioreactor volumes). Additionally, the smaller and simplified equipment operating continuously uses very small bioreactors that produce monoclonal antibodies at steady state. The continuous bioprocessing described herein can also significantly reduce operational costs, overall bioprocessing line downtime, and biological product loss when compared to traditional monoclonal antibody manufacturing approaches. Finally, the processes described herein for purifying a biological product are performed in systems with footprints that occupy significantly less square feet than current technologies, without sacrificing product throughput or yield on a kilogram / year basis.

[0370] 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 plugging. For example, using conventional filtration or tangential flow filtration systems to clarify cells, cell debris, and aggregates from cell culture media typically results in filter membrane fouling or plugging, making these methods unsuitable as a means to continuously remove large impurities from heterogeneous mixtures containing biological products over long periods of continuous processing. In contrast, the dynamic filtration devices described herein allow for continuous removal of large impurities from heterogeneous mixtures containing biological products without membrane fouling because the active target area of ​​the filter membrane is continually renewed. Furthermore, because the entire process of producing and purifying a biological product is continuous and flow rates ranging from about 0.1 mL / min to about 50 mL / min can be maintained throughout the entire process, the footprint of the process equipment and the entire process can have a significantly smaller footprint than current standard processes without sacrificing product throughput or yield, on a kilogram / year basis. For example, the monoclonal antibody production and purification process described herein operates in a footprint occupying up to about 30,000 square feet. In comparison, current monoclonal antibody production and downstream processes require a minimum of 200,000 square feet. For example, the flow rate of the process for purifying a biological product ranges from about 1 mL / min to about 10 mL / min. In some examples, the flow rate of the step of continuously removing large impurities from the heterogeneous mixture ranges from about 0.1 mL / min to about 50 mL / min. In other examples, the flow rate of the step of continuously removing large impurities from the heterogeneous mixture is equal to the flow rate from the bioreactor bleed line. In other examples, a process is provided in which the flow rate of the step of continuously conveying the filtrate to the first module ranges from 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 transmitting the fraction containing the 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.

[0371] Important advantages of the processes and methods utilizing resin beads (e.g., agarose) described herein include that these systems do not require traditional stationary phase or packed resin columns (e.g., for standard chromatography) for sterilization, recycling, and / or regeneration. For example, these systems provide for recycling and / or regeneration of resin beads to generate infinite surface area of ​​resin beads during operation, resulting in a continuous and cost-effective process. In other words, the modules described herein do not have fixed binding or association capabilities. In a specific example, the resin beads used during the purification of biological products described herein are continuously recycled and regenerated, so that they can accept the flow of the previous step of either the dynamic filtration module or the purification module without interrupting the flow from the bioreactor bleed line. In other words, the modules described in the present invention do not need to be left idle for sterilization, regeneration, and / or recycling after a run, since they are continuously undergoing these steps. The method differs from current continuous chromatography methods in that current column chromatography methods have column capacity limitations limited by column packing constraints, and therefore require multiple packed columns for column switching to accept continuous input flow and allow for 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 the resin beads are mobile. Such bead mobility increases the surface area of ​​the resin beads available for binding or association, since substantially more of the resin bead surface is exposed and free to bind. Furthermore, the resin beads in the packed column are subjected to high pressure differentials to generate flow through the column, and damage caused by this is one of the reasons for columns having shorter than desired lifetimes. The mobile resin beads in the invention described herein are subjected to substantially lower pressures and are much softer than the fragile beads, thus extending their lifetime. Furthermore, such mobility increases the chances that the resin beads will be regenerated and return to their initial state. This also increases the cost-effectiveness of the method described herein, since the resin is used more efficiently.

[0372] An important advantage of the processes and methods using free-flow electrophoresis described herein is that this system represents a "no product loss" process, i.e., the product does not need to interact with resins or other purification moieties, since separation occurs in aqueous solution and through interaction with an electric field according to the physicochemical properties of the target biological product. Another advantage is observed in the resolution of this approach, since a theoretically higher purity of product can be obtained compared to conventional ion exchange chromatography. Furthermore, separation based on intrinsic physicochemical properties extends the usefulness of such an 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 (AAV), or lentiviruses.

[0373] Furthermore, the modular approach provides flexibility in process design to accommodate a range of biological products. A continuous process for purifying a biological product using at least one of a dynamic filtration module, an affinity-based fluidic purification module, and a charge-based or isoelectric-based fluidic purification module. A continuous process for purifying a biological product is described, the process comprising continuously receiving a heterogeneous mixture comprising a biological product via an input line, the biological product including, but not limited to, a protein or fragment thereof (polypeptide), an antibody or 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 at least about 60%, 70%, 80%, 90%, 95%, or 99% by weight of 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, detergents, sugars, metal contaminants, leachables, media components, and / or naturally associated natural organic molecules) are removed.

[0374] The process includes continuously removing large impurities from a heterogeneous mixture by dynamic filtration. The dynamic filtration process includes at least one dynamic filtration module that continuously supplies a biological product to the dynamic filtration module from at least one output head in fluid communication with an input line under negative pressure to produce a filtrate containing the biological product. The dynamic filtration module may further include at least one additional input line for supplying a wash buffer via a coaxial output head or a separate single-axis output head.

[0375] In embodiments, the processes described herein include purifying a biological product produced continuously in a bioreactor (e.g., fed-batch bioreactors, perfusion bioreactors, and chemostat bioreactors). For example, the bioreactor includes a bioreactor feed line and an output bleed line that allow for steady-state cell culture growth conditions, and the output bleed line acts as an input line that allows for continuous fluid flow from the bioreactor to a dynamic filtration module.

[0376] As described herein, the process of continuously removing large impurities from a heterogeneous mixture does not include centrifugation, disc stack centrifugation, depth filtration, static filtration, tangential flow filtration, hydrocyclones, or any combination thereof. The term "static filtration" refers to a process in which the heterogeneous mixture being filtered remains static, i.e., for example, a filter membrane (or depth filter) has a limited capacity, and when the membrane reaches its capacity, the filtration rate decreases (e.g., the membrane pores become blocked). In "static" (as opposed to "dynamic") filtration, the filter membrane remains fixed (does not move) and the flow (e.g., the flow of the heterogeneous mixture) passes through the fixed filter membrane. These stat...

Claims

1. 1. A method for purifying a biological product, the method comprising: receiving a heterogeneous mixture comprising a biological product via an input line; providing the biological product from at least one output head in fluid communication with the input line under negative pressure to a dynamic filtration module to remove impurities from the heterogeneous mixture by filtration in the dynamic filtration module, the dynamic filtration module including a dynamic filtration device having a filter membrane extending between a supply reel and a collection reel with at least one support member having a contact surface with a static friction coefficient of about 0.01 to about 0.1, 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 contact surface with a static friction coefficient of about 0.01 to about 0.1 in communication with a vacuum collection system located between the supply reel and the collection reel, the "about" meaning a variation of ±25% or less from the stated value, to provide the filtrate; transferring the filtrate to a first module capable of separating the solution into two or more fractions, where at least one fraction includes the biological product, the first module including an affinity-based purification device, where the first module has at least one first inlet and at least one first outlet configured to permit fluid flow between the at least one first inlet and the at least one first outlet via a mechanical rotation system including a container carousel containing at least one individual container including a suspension of beads; transferring the biological product-containing fraction from the at least one outlet of the first module to a second module having at least one inlet for receiving flow from the at least one first outlet of the first module, the second module including at least one free-flow electrophoresis device, the second module having at least one second inlet and at least one second outlet, the second module configured to permit continuous fluid flow between the second inlet and the second outlet; and recovering the biological product.

2. The method of claim 1 , wherein the affinity-based purification device further comprises a lid system and a collection container system in fluid communication with at least one individual container.

3. The method of claim 2 , wherein the lid system includes at least one lid having a gasket, at least two buffer inlets, a fill inlet, a gas inlet, and an exhaust valve.

4. The method of claim 2 , wherein the lid system is movable along a z-axis, the container carousel is rotationally movable in a plane transverse to the z-axis, and the collection container is movable along the z-axis.

5. 2. The method of claim 1, wherein the vessel carousel comprises at least one position for binding the biological product, at least one position for washing to remove unbound product, at least one position for eluting and collecting the biological product, and at least one regeneration position to allow recycling of the beads.

6. 2. The method of claim 1, wherein the surface of the bead is linked to protein A, protein G, protein L, an antigen protein, a protein, a receptor, an antibody, or an aptamer configured to selectively bind to the biological product.

7. 2. The method of claim 1, wherein the initial concentration of beads ranges from about 0.01% to about 25% by weight, where "about" means no more than ±25% variation from the stated value.

8. 2. The method of claim 1, wherein the diameter of the beads ranges from about 0.2 μm to about 200 μm, where "about" means no more than ±25% variation from the stated value.

9. The method of claim 1 , wherein the beads remain mobile during the method to maintain an increased surface area available for binding.

10. 2. The method of claim 1, wherein the free-flow electrophoresis device comprises an electrode channel including an anode electrode channel and a cathode electrode channel in liquid contact with a main separation channel through a wall gap, the device further comprising at least one electrode channel bubble remover including at least one gas permeable and hydrophobic membrane configured to remove air bubbles via a vacuum system to produce a bubble-free main separation channel, and at least one liquid circuit breaker.

11. 2. The method of claim 1, wherein the method maintains a substantially constant flow rate in the dynamic filtration module, the first module, and the second module, the flow rate being in the range of about 0.1 mL / min to about 50 mL / min, where "about" means no more than ±25% variation from the stated value.

12. 2. The method of claim 1, wherein the method of purifying a biological product is carried out at a temperature ranging from about 4° C. to about 37° C., where "about" means no more than ±25% variation from the stated value.

13. 10. The method of claim 1, further comprising at least two dynamic filtration modules, wherein each dynamic filtration module has a filter membrane containing the same or different pore sizes.

14. 10. The method of claim 1, further comprising 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.

15. 10. The method of claim 1, further comprising at least two dynamic filtration modules, at least two affinity-based purification modules, or at least two free-flow electrophoresis modules operated in parallel.

16. The method of claim 1, wherein "about" means a variation of less than ±20% from the stated value.

17. The method of claim 1, wherein "about" means a variation of less than ±10% from the stated value.

18. The method described in claim 1, wherein "about" means a variation of less than ±5% from the stated value.

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