Process for purification of biological product

Abstract

To provide novel processes for biological product manufacturing and downstream purification.SOLUTION: The present invention provides a process that includes a step of receiving a heterogeneous mixture containing a biological product via an input line, and a step of removing impurities from the heterogeneous mixture by filtration in a dynamic filtration module. Impurities are removed from the heterogeneous mixture by supplying the biological product to the dynamic filtration module from at least one output head in fluid communication with the input line under negative pressure, thereby generating a filtrate containing the biological product.SELECTED DRAWING: Figure 5A

Description

Technical Field

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 077,766, filed on September 14, 2020, U.S. Provisional Application No. 63 / 154,108, filed on February 26, 2021, and U.S. Provisional Application No. 63 / 154,109, filed on February 26, 2021, under 35 U.S.C. § 119(e), 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, via an input line, a heterogeneous mixture comprising the biological product, and removing impurities from the heterogeneous mixture by filtration in a dynamic filtration module. The impurities are removed from the heterogeneous mixture by supplying the biological product to the dynamic filtration module from at least one output head in fluid communication with the input line under negative pressure, thereby producing a filtrate containing the biological product.

[0004] The dynamic filtration module includes a dynamic filtration device, a target area configured to receive a non-uniform mixture from at least one output head, and a membrane support member having a substantially smooth contact surface that communicates with a vacuum collection system located between a supply reel and a collection reel. Further, the dynamic filtration device includes a filter membrane extending between a supply reel and a collection reel together 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, wherein at least one fraction contains 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 at least one first inlet and at least one first outlet via a mechanical rotation system. The mechanical rotation system includes a container carousel containing at least one individual container containing a suspension of beads. As described herein, the process further includes transmitting a fraction containing the biological product from at least one outlet of the first module to a second module having at least one inlet for receiving the flow from at least one first outlet of the first module. The second module includes at least one free-flow electrophoresis device, and the second module is provided with 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 so that the biological product can be recovered.

[0005] In an embodiment, the affinity-based purification apparatus further includes a lid system and a collection vessel system that is in fluid communication with at least one individual vessel. For example, the lid system includes at least one lid with a gasket, at least two buffer inlets, a filling inlet, a gas inlet, and an exhaust valve. Further, the lid system is movable along the z-axis. The container carousel of the affinity-based purification apparatus is rotatable within a plane transverse to the z-axis, and the collection vessel is movable along the z-axis.

[0006] As described herein, the container carousel of the affinity-based purification method includes at least one position for binding a 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 that enables recycling of the beads.

[0007] For example, the bead surface of the affinity-based purification 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. The initial concentration of the beads (e.g., within an individual vessel at the position where it binds to the biological product) is in the concentration range of about 0.01 wt% to about 25 wt%. Alternatively, the initial concentration of the beads is in the range of about 0.01 wt% to about 20 wt%, about 0.01 wt% to about 10 wt%, about 0.01 wt% to about 5 wt%, about 1 wt% to about 20 wt%, or about 5 wt% to about 10 wt%. For example, the diameter of the beads is in the range of about 0.2 μm to about 200 μm. In 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 an embodiment, the beads (e.g., beads for affinity-based purification) maintain mobility during the process to maintain an increased surface area available for binding. For example, the beads are maintained in a separated (circulated or dispersed) state in solution during the process (e.g., the beads are individual beads). Further, mobile beads may mean that the beads do not aggregate together, e.g., at least two or more beads do not aggregate or group together. Further, mobile beads may mean that the beads can form small aggregates that are dispersed and freely move within the solution. Conversely, the beads used herein are not packed, but maintain mobility and freely move within the solution.

[0009] In an embodiment, the free-flow electrophoresis device includes an electrode channel that includes 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 that includes at least one gas-permeable and hydrophobic membrane configured to remove bubbles via a vacuum system to produce a bubble-free main separation channel, and at least one liquid circuit breaker. In an embodiment, at least one bubble remover of the free-flow electrophoresis device is configured to continuously remove O2 and H2 gas bubbles generated within the electrode channel under an applied voltage. In some embodiments, removing electrolysis bubbles from the electrode channel is essential to enable substantially long-term continuous operation. For example, the bubble removal system uses a hydrophobic PTFE membrane to create a waterproof seal over the electrode channel that can continuously remove electrolysis bubbles at the point of generation by exposure to the 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 processes described herein remove gas bubbles before they enter the main separation channel.

[0011] In an embodiment, the liquid circuit breaker of the free-flow electrophoresis apparatus includes a pressurized container configured to maintain a flow rate and generate droplets that isolate the circuit from a solution connected to a voltage. In an embodiment, the purification process maintains a substantially constant flow rate in the dynamic filtration module, the first module, and the second module. For example, the flow rate ranges from about 0.1 mL / min to about 50 mL / min, or from about 5 mL / min to about 10 mL / min.

[0012] In an embodiment, the process of purifying a biological product is performed at a temperature in the range of about 4°C to about 37°C. In a further embodiment, the process can include at least two dynamic filtration modules, where each dynamic filtration module has a filter membrane with the same or different pore sizes (e.g., a heterogeneous mixture first contacts a filter membrane with a large pore size (e.g., 0.45 μm) and then contacts a filter membrane with a smaller pore size (e.g., 0.2 μm)).

[0013] In an embodiment, the process includes at least two free-flow electrophoresis modules configured to operate in an isoelectric focusing electrophoresis mode, a zone electrophoresis mode, an isotachophoresis mode, or a combination thereof. The processes described herein further include at least two dynamic filtration modules operating in parallel, at least two affinity-based purification modules, or at least two free-flow electrophoresis modules.

[0014] In one aspect, provided herein is a dynamic filtration device for removing impurities from biological products in a heterogeneous mixture. The device includes a filter membrane extending between a supply reel and a collection reel, and the filter membrane has a target region configured to receive the heterogeneous mixture from at least one output head configured to distribute the heterogeneous mixture to the target region. 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 region. Further, the dynamic filtration device has at least one support member having a substantially smooth contact surface to stabilize the transport of the filter membrane across the membrane support structure. The dynamic filtration device has a system configured to control the transport speed of the filter membrane. The dynamic filtration device has at least one vacuum line in communication with the membrane support structure and has a vacuum system configured to apply a negative gauge pressure to the dynamic filter membrane, where the negative pressure enables the collection of the filtrate containing the biological product. In other examples, the dynamic filtration device includes a wash buffer line.

[0015] In embodiments, the dynamic filtration device has a filter membrane that can include polyethersulfone (PES), novel hydrophilic polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), novel hydrophilic PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), novel hydrophilic PTFE, or any combination thereof. The pore size of the filter membrane ranges from about 0.1 μm to about 1 μm. In other examples, the pore size ranges from 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 can include filter membranes of similar or different sizes.

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

[0017] The dynamic filtration device described herein includes a membrane support structure having a substantially smooth contact surface, where the contact surface has a coefficient of static friction, e.g., 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.

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

[0019] In an aspect, provided herein is a free-flow electrophoresis device for separating a mixture into two or more fractions, where at least one of the fractions contains a biological product. The free-flow electrophoresis device includes at least one inlet and at least one outlet configured to permit a continuous fluid flow between the at least one inlet and the at least one outlet, at least one fluid channel formed between two parallel plates and configured to generate an electric field gradient orthogonal to the direction of fluid flow, an electrode channel including an anode electrode channel and a cathode electrode channel (where 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 generation point by a vacuum system to generate a bubble-free main separation channel, at least one liquid circuit breaker configured to isolate the solution connected to the voltage before interacting with at least one sensor or detector, an active cooling system, and at least one collection container.

[0020] The free-flow electrophoresis device described in this specification provides an electrode channel having a bubble remover, wherein the upper part of the electrode channel is sealed with at least one gas-permeable and hydrophobic membrane communicating with a vacuum system for removing bubbles, the electrode channel is open at the bottom of the channel, and is configured to enable 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 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 further includes at least one bubble removal system for continuously removing O2 and H2 gas bubbles generated in the electrode channel under an applied voltage. In some embodiments, removing electrolysis bubbles is essential to enable substantially long-term continuous operation. For example, the bubble removal system uses a hydrophobic PTFE membrane to create a waterproof seal over the electrode channel capable of continuously removing electrolysis bubbles at the point of generation by exposure to the 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). Different from current methods, the process described in this specification removes gas bubbles before they enter the main separation channel.

[0023] In an embodiment, the wall gap (e.g., the space where the electrode channel is open at the bottom of the channel and is configured to enable liquid contact between the main separation channel solution and the electrode solution) is from about 0.01 mm to about 0.25 mm. For example, the wall gap is from about 0.01 mm to about 0.2 mm, from about 0.01 mm to about 0.015 mm, or from about 0.01 mm to about 0.01 mm.

[0024] In other embodiments, the liquid circuit breaker of the free-flow electrophoresis apparatus includes a pressurized container configured to maintain a flow rate and generate droplets that isolate the circuit from a solution connected to a voltage.

[0025] In embodiments, the free-flow electrophoresis apparatus further includes an in-line sensor. For example, the in-line sensor may include a flow sensor, a pH sensor, a conductivity sensor, or any combination thereof.

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

[0027] Furthermore, provided herein is the use of a free-flow electrophoresis apparatus for purifying a biological product from a mixture. The present invention further provides the use of a dynamic filtration apparatus for purifying a biological product from a heterogeneous mixture. In an aspect, provided herein is a process for purifying a biological product. The process includes receiving a heterogeneous mixture containing a biological product via an input line. In embodiments, the process includes continuously receiving a heterogeneous mixture containing a biological product via an input line. In embodiments, the biological product includes a protein or a fragment thereof (polypeptide), an antibody or a fragment thereof, a cytokine, a chemokine, a growth factor, an enzyme, an oligonucleotide, a virus, an adenovirus, an adeno-associated virus (AAV), or a lentivirus.

[0028] In an embodiment, 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 supplies a heterogeneous mixture containing 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 generate a filtrate containing the biological product.

[0029] In an embodiment, the process includes transmitting the filtrate to a first module that can separate the solution into two or more fractions, where at least one fraction contains a biological product. In other embodiments, the process includes continuously transmitting the filtrate to a first module that can separate the solution into two or more fractions, where at least one fraction contains a biological product. For example, separating the solution into two or more fractions can include one fraction containing a biological product and at least one other fraction containing small impurities (e.g., host cell proteins, unwanted proteins and peptides, unwanted antibodies, unwanted nucleic acids and oligonucleotides, viruses, salts, buffer components, surfactants, sugars, metal contaminants, leachates, medium components, and / or naturally occurring organic molecules bound naturally).

[0030] In an embodiment, the first module includes 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 a continuous fluid flow between the first inlet and the first outlet via a loop conveyor system. In other examples, the first module has at least one first inlet and at least one first outlet and is configured to allow a continuous fluid flow between the first inlet and the first outlet via a pick-and-place robotic system.

[0031] In an embodiment, the process includes conveying a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module, wherein the second module includes a charge-based magnetic purification device or an isoelectric point-based fluid purification device (also referred to herein as a free-flow electrophoresis device). In other embodiments, the process includes continuously conveying a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module, wherein the second module includes a charge-based magnetic purification device or an isoelectric point-based fluid purification device, also referred to herein as a free-flow electrophoresis device. For example, the second module includes a charge-based magnetic purification device having at least one second inlet and at least one second outlet and is configured to permit a 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 is configured to permit a continuous fluid flow between the second inlet and the second outlet via a pick-and-place robot 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 permit a continuous fluid flow between the second inlet and the second outlet. In an embodiment, the process described herein thereby purifies the biological product.

[0032] In an embodiment, the present specification also provides a process for purifying a biological product that includes continuously receiving a heterogeneous mixture containing the 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 the heterogeneous mixture. The dynamic filtration process includes a dynamic filtration module that generates a filtrate containing the biological product by continuously supplying the biological product to the dynamic filtration module from at least one output head in fluid communication with the input line under negative pressure.

[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 contains the biological product. In other embodiments, 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 contains 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 a continuous fluid flow between the first inlet and the first outlet via a mechanical rotation system. In other examples, the first module has at least one first inlet and at least one first outlet and is configured to allow a continuous fluid flow between the first inlet and the first outlet via a step-linear system.

[0034] In an embodiment, the process includes conveying a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module, wherein the second module includes a charge-based purification device or an isoelectric point-based fluid purification device, also referred to herein as a free-flow electrophoresis device. In other embodiments, the process includes continuously conveying a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module, wherein the second module includes a charge-based purification device or an isoelectric point-based fluid purification device, also referred to herein as a free-flow electrophoresis device. For example, the second module includes a charge-based purification device having at least one second inlet and at least one second outlet and is configured to allow a continuous fluid flow between the second inlet and the second outlet 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 a continuous fluid flow between the second inlet and the second outlet via a step-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 a continuous fluid flow between the second inlet and the second outlet. In an embodiment, the process described herein thereby purifies the biological product.

[0035] In an embodiment, the present specification also provides a process for purifying a biological product, which includes continuously receiving a heterogeneous mixture containing the 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 the heterogeneous mixture. The dynamic filtration process includes a dynamic filtration module that generates a filtrate containing the biological product by continuously supplying the biological product to the dynamic filtration module from at least one output head in fluid communication with the input line under negative pressure.

[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 contains the biological product. In other embodiments, 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 contains 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 a continuous fluid flow between the first inlet and the first outlet.

[0037] In an embodiment, the process includes transmitting a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module, wherein the second module includes a charge-based fluid purification device or an isoelectric point-based fluid purification device, also referred to herein as a free-flow electrophoresis device. In an embodiment, the process includes continuously transmitting a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module, wherein the second module includes a charge-based fluid purification device or an isoelectric point-based fluid purification device, also referred to herein as a free-flow electrophoresis device. For example, the second module includes a charge-based fluid purification device having at least one second inlet and at least one second outlet and is configured to permit a continuous fluid flow between the second inlet and the second outlet. In another example, 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 permit a continuous fluid flow between the second inlet and the second outlet. In an embodiment, the process described herein thereby purifies a biological product.

[0038] In an embodiment, the process includes transmitting a filtrate to a first module capable of separating a solution into two or more fractions, wherein at least one fraction contains a biological product. In other embodiments, the process includes continuously transmitting a filtrate to a first module capable of separating a solution into two or more fractions, wherein at least one fraction contains a 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 permit a continuous fluid flow between the first inlet and the first outlet.

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

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

[0041] As described herein, the process of continuously removing large impurities from a heterogeneous mixture does not include centrifugation, disk stack centrifugation, depth filtration, static filtration, tangential flow filtration, hydrocyclones, or any combination thereof. Alternatively, the processes described herein can continuously receive a heterogeneous mixture containing a biological product via an input line obtained, for example, without intending to limit, from an input in which large impurities have been continuously removed by a continuous disk stack centrifuge and a depth filtration process or a hydrocyclone process.

[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 continuously produced in a bioreactor. For example, the bioreactor includes a bioreactor feed line and an output bleed line that enable steady-state cell culture growth conditions, and the output bleed line functions as an input line that permits a continuous fluid flow from the bioreactor to a dynamic filtration module. For example, the type of bioreactor includes, but is not limited to, a fed-batch bioreactor, a perfusion bioreactor, a chemostat bioreactor, or a multi-compartment bioreactor. For example, the flow from the bioreactor bleed line is always supplied to a downstream purification system. Alternatively, the processes described herein include purifying a biological product (e.g., mRNA) not produced in a bioreactor.

[0043] In an embodiment, the present specification provides a method for purifying a biological product, the method comprising receiving, via an input line, a heterogeneous mixture containing the biological product; supplying 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 produce a filtrate containing the biological product; 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, wherein the first module includes an affinity-based magnetic purification device, the first module has at least one first inlet and at least one first outlet, and is configured to allow a fluid flow between the first inlet and the first outlet via a loop conveyor system or a pick-and-place robot system; transmitting a fraction containing 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 at least one first outlet of the first module, wherein the second module includes a charge-based magnetic purification device, the second module has at least one second inlet and at least one second outlet, and is configured to allow a continuous fluid flow between the second inlet and the second outlet via a loop conveyor system or a pick-and-place robot system; and thereby purifying the biological product.

[0044] In other embodiments, a method for purifying a biological product is provided. The method includes receiving, via an input line, a heterogeneous mixture comprising the biological product; supplying the biological product from the input line to a dynamic filtration module from at least one output head in fluid communication with the input line under negative pressure, such that a filtrate comprising the biological product is produced by removing impurities from the heterogeneous mixture by dynamic filtration in the dynamic filtration module; transmitting the filtrate to a first module capable of separating the solution into two or more fractions including at least one fraction comprising the biological product, wherein the first module includes an affinity-based magnetic purification device, the first module has at least one first inlet and at least one first outlet, and is configured to allow a flow of fluid between the first inlet and the first outlet via a loop conveyor system or a pick-and-place robotic system; transmitting a 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 a flow from at least one first outlet of the first module, wherein the second module includes an isoelectric point-based fluid purification device, also referred to herein as a free-flow electrophoresis device, the second module has at least one second inlet and at least one second outlet, and is configured to allow a continuous flow of fluid between the second inlet and the second outlet; and thereby purifying the biological product.

[0045] In an embodiment, a method for purifying a biological product is included, the method comprising receiving, via an input line, a heterogeneous mixture containing the biological product; supplying 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 containing the biological product; 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, wherein the first module includes an affinity-based purification device, the first module has at least one first inlet and at least one first outlet, and is configured to allow a fluid flow between the first inlet and the first outlet via a mechanical rotation system; transmitting a fraction containing 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 at least one first outlet of the first module, wherein the second module includes a charge-based purification device, the second module has at least one second inlet and at least one second outlet, and is configured to allow a fluid flow between the second inlet and the second outlet via a mechanical rotation system; and thereby purifying the biological product.

[0046] In other embodiments, a method for purifying a biological product is provided, the method comprising: receiving, via an input line, a heterogeneous mixture comprising the biological product; supplying 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 produce a filtrate comprising the biological product; transmitting the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising the biological product, wherein the first module comprises an affinity-based purification device, the first module having at least one first inlet and at least one first outlet and being configured to permit a flow of fluid between the first inlet and the first outlet via a mechanical rotation system; transmitting a 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 at least one first outlet of the first module, wherein the second module comprises an isoelectric point-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 being configured to permit a continuous flow of fluid between the second inlet and the second outlet; and thereby purifying the biological product.

[0047] In an embodiment, a method for purifying a biological product is included, the method comprising receiving, via an input line, a heterogeneous mixture containing a biological product; supplying 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 containing the biological product; 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, wherein the first module includes an affinity-based purification device, the first module has at least one first inlet and at least one first outlet, and is configured to allow fluid flow between the first inlet and the first outlet via a stepwise linear system; transmitting a fraction containing 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 at least one first outlet of the first module, wherein the second module includes a charge-based purification device, the second module has at least one second inlet and at least one second outlet, and is configured to allow fluid flow between the second inlet and the second outlet via a stepwise linear system; and thereby purifying the biological product.

[0048] In other embodiments, a method for purifying a biological product is provided, the method comprising receiving, via an input line, a heterogeneous mixture comprising the biological product; supplying 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 produce a filtrate comprising the biological product; transmitting the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising the biological product (wherein the first module comprises an affinity-based purification device, the first module having at least one first inlet and at least one first outlet and being configured to permit a flow of fluid between the first inlet and the first outlet via a stepwise linear system); transmitting a 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 at least one first outlet of the first module (wherein the second module comprises an isoelectric point-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 being configured to permit a continuous flow of fluid between the second inlet and the second outlet); and thereby purifying the biological product.

[0049] The embodiment includes a method for purifying a biological product, the method comprising: receiving, via an input line, a heterogeneous mixture containing the biological product; supplying 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 containing the biological product; 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, wherein the first module includes an affinity-based fluid purification device, the first module has at least one first inlet and at least one first outlet, and is configured to allow fluid flow between the first inlet and the first outlet; transmitting a fraction containing 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 at least one first outlet of the first module, wherein the second module includes a charge-based fluid purification device, the second module has at least one second inlet and at least one second outlet, and is configured to allow fluid flow between the second inlet and the second outlet; and thereby purifying the biological product.

[0050] In other embodiments, a method for purifying a biological product is provided, the method comprising receiving, via an input line, a heterogeneous mixture comprising the biological product; supplying 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 produce a filtrate comprising the biological product; transmitting the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising the biological product, wherein the first module comprises an affinity-based fluid purification device, the first module having at least one first inlet and at least one first outlet and being configured to permit a flow of fluid between the first inlet and the first outlet; transmitting a 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 at least one first outlet of the first module, wherein the second module comprises an isoelectric point-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 being configured to permit a continuous flow of fluid between the second inlet and the second outlet; and thereby purifying the biological product.

[0051] The embodiment includes a method for purifying a biological product, the method comprising receiving, via an input line, a heterogeneous mixture containing the biological product; supplying 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 produce a filtrate containing the biological product; 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, wherein the first module includes an affinity-based TFF purification device, the first module has at least one first inlet and at least one first outlet, and is configured to allow a fluid flow between the first inlet and the first outlet; transmitting a fraction containing 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 at least one first outlet of the first module, wherein the second module includes a charge-based TFF purification device, the second module has at least one second inlet and at least one second outlet, and is configured to allow a fluid flow between the second inlet and the second outlet; and thereby purifying the biological product.

[0052] In other embodiments, a method for purifying a biological product is provided, the method comprising receiving, via an input line, a heterogeneous mixture comprising the biological product; supplying 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 such that a filtrate comprising the biological product is produced; transmitting the filtrate to a first module capable of separating the solution into two or more fractions comprising at least one fraction comprising the biological product, wherein the first module comprises an affinity-based TFF purification device, the first module having at least one first inlet and at least one first outlet and being configured to permit a flow of fluid between the first inlet and the first outlet; transmitting a 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 at least one first outlet of the first module, wherein the second module comprises an isoelectric point-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 being configured to permit a continuous flow of fluid between the second inlet and the second outlet; and 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 fouling or clogging of the membrane. For example, purifying cells, cell debris, and aggregates from cell culture media using conventional filtration or tangential flow filtration systems typically results in fouling or clogging of the filter membrane, and these methods are not suitable as a means of continuously removing large impurities from heterogeneous mixtures containing biological products by long-term continuous processes. In contrast, the dynamic filtration device described herein enables continuous removal of large impurities from heterogeneous mixtures containing biological products without fouling the membrane because the active target region of the filter membrane is constantly refreshed.

[0054] Furthermore, since the entire process of producing and purifying biological products is continuous and a flow rate in the range of about 0.1 mL / min to about 50 mL / min can be maintained throughout the process, the footprint of the process equipment and the entire process can have a significantly smaller footprint than current standard processes without sacrificing the product throughput or yield on a kilogram / year basis. For example, the process of producing and purifying monoclonal antibodies described herein operates with a footprint that occupies up to about 30,000 square feet. In contrast, current monoclonal antibody production and downstream processes require at least 200,000 square feet. For example, the flow rate of the process of purifying biological products is in the range of 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 is in the range of 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 transferring the filtrate to the first module is in the range of about 0.1 mL / min to about 50 mL / min. In yet other examples, 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 is in the range of about 0.1 mL / min to about 50 mL / min.

[0055] An important advantage of the processes and methods using the magnetic resin beads (e.g., magnetic agarose) or conventional resin beads (e.g., agarose) described herein is that these systems do not require conventional stationary phases or packed resin columns (e.g., for standard chromatography) for sterilization, recycling, and / or regeneration. For example, these systems provide recycling and / or regeneration of resin beads (e.g., magnetic or non-magnetic resin beads) to generate an infinite surface area of the resin beads during operation, resulting in a continuous and cost-effective method.

[0056] In other words, the modules described herein do not have a fixed binding or association ability. In a specific example, the resin beads used during the purification of the biological products described herein are continuously recycled and regenerated, so that they can receive the flow of any step before 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 are continuously receiving these steps, so there is no need to leave them idle for sterilization, regeneration, and / or recycling after execution. The method is different from the current continuous chromatography method in that the current method has a limited column capacity limit due to the constraints of resin packing, and column switching of multiple packed columns is required to receive a continuous input flow and enable regeneration and / or recycling of the column that has reached its full capacity.

[0057] Another advantage of the methods described herein involves the resin beads not being packed into a stationary phase, but rather the resin beads being mobile. This mobility of the beads exposes substantially more resin bead surface area and allows for free binding, such that, for example, more biological products can bind to the beads, increasing the surface area available for binding or association. Further, resin beads within a typically packed column (e.g., where bead mobility is lacking and surface area is reduced) are exposed to high pressure differentials to create flow through the column. Such high pressure differentials compromise bead integrity and shorten column life. The mobile resin beads of the invention described herein are exposed to substantially lower pressures and are much gentler on fragile beads, extending their life. Further, such mobility increases the likelihood that the beads can be regenerated (e.g., fully regenerated) and returned to their initial state. This also increases the cost - effectiveness of the methods described herein, for example, as the resin is used more efficiently.

[0058] As described herein, the resin beads of the claimed methods and apparatus are mobile throughout the process. Conventional chromatography purification methods require, for example, column packing where beads are well - packed together to achieve a high density with the stationary phase. For example, the beads are maintained in a separated (circulated or dispersed) state in solution during the process (e.g., the beads are individual beads). Further, mobile beads may mean that the beads do not aggregate together, for example, at least two or more beads do not aggregate or group together. Further, mobile beads may mean that the beads can form small aggregates that disperse and move freely within the solution. Conversely, the beads used herein are not packed, yet maintain mobility and move freely within the solution.

[0059] An important advantage of the processes and methods using free-flow electrophoresis described herein is that this system represents a "product-loss-free" process, i.e., since separation occurs in an aqueous solution through interaction with an electric field according to the physicochemical properties of the target biological product, the product does not need to interact with a resin or other purification moiety. Another advantage is observed in the resolution of this approach (e.g., the ability to purify products with a high degree of physicochemical similarity) because a higher purity product can be obtained compared to conventional ion-exchange chromatography. For example, biological products with a purity of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher can be obtained using the free-flow electrophoresis modules and methods described herein. Further, the methods and apparatuses described herein can increase the purity (of biological products) compared to conventional purification and chromatography methods. For example, the term "increased" in relation to a level refers to any % increase rate that exceeds a control level (e.g., the level of purity obtained from purification using a conventional method). In various embodiments, the increased level can be an increase in purity of at least or about 1%, 2%, 3%, 4%, or 5%, at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95% compared to a conventional purification method.In other examples of the present invention, the purity of the biological product obtained from the methods and apparatuses described herein is about 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, or 3.0 times higher than the purity of the biological product using standard commercial or chromatographic techniques.

[0060] Furthermore, separating based on the essential physicochemical properties of the biological product (e.g., isoelectric point, surface charge, net charge, zeta potential, electrophoretic mobility, electrostatic interactions, etc.) 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, growth factors, enzymes, oligonucleotides, viruses, adenoviruses, adeno-associated viruses (AAV), or lentiviruses.

[0061] Furthermore, the modular approach provides flexibility in process design for accommodating a wide range of biological products. In embodiments, during purification by dynamic filtration in the processes described herein, a filtrate containing the biological product is generated and supplied 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 the active target region of the filter membrane. For example, the at least one output head is a tube or a slot die.

[0063] In an embodiment, at least one dynamic filtration module may further include at least one additional input line for supplying a wash buffer by a coaxial output head, a separate single-axis output head, a separate slot die output head, a slot die output head having a plurality of 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, without intending to be limiting, active or passive edge guides, tension adjustment devices (such as dancer), brakes and tension detectors, or any combination thereof.

[0065] In an embodiment, the process herein includes that at least one output head (in fluid communication with the input line to the dynamic filtration module) is 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 movable along the z-axis. In still other examples, at least one output head is capable of xy rastering and is movable 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 container.

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

[0068] In an embodiment, the pore size of the rolled filter membrane varies depending on the biological product to be 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 an antibody, 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 can vary depending on the size of the dynamic filtration system, the size of 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 from a prehub roll and initially spans an 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 continuous with a vacuum line. For example, the coefficient of static friction ranges 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. 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. The temperature control mechanism maintains a temperature of 4°C to 37°C. For example, during antibody purification, the temperature control mechanism maintains a temperature of 15°C to 37°C. Exemplary temperature control mechanisms include, but are not limited to, a single-loop controller, a multi-loop controller, a closed-loop controller, a proportional-integral-derivative (PID) controller, a Peltier element, a resistive heating element, and / or a thermal chuck with a circulating water / propylene glycol jacket.

[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, perfluoroalkoxy alkane (PFA)). For example, the coefficient of static friction ranges 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. For example, the support rod or roller may be fixed or may rotate. In some examples, the support rod may further include a bearing, such as 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, a process for continuously removing large impurities (e.g., cells, cell debris, and aggregates) from a heterogeneous mixture by dynamic filtration involves multi-stage filtration using at least two separate 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, whereby a filtrate containing a biological product is produced. Alternatively, a similar result can be achieved by a single dynamic filtration device having at least two rolled filter membranes supplied by separate supply reels, such that a stacked set of filter membranes is produced across a target region (e.g., an active target region), where the heterogeneous mixture first contacts a filter membrane with a large pore size (e.g., 0.45 μm) and then contacts a filter membrane with a smaller pore size (e.g., 0.2 μm).

[0075] In an embodiment, the process described herein includes continuously transferring 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 including an affinity-based magnetic purification device. For example, the affinity-based magnetic purification device further includes a suspension of magnetic resin beads. The surface of the magnetic resin beads is linked to, for example, without intending to be limiting, 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 of an affinity-based magnetic purification apparatus is from about 0.2 microns to about 200 microns. In other examples, the diameter of the beads is from about 0.2 μm to about 100 μM, from about 1 μm to about 200 μm, from about 10 μm to about 200 μm, from about 20 μm to about 200 μm, from about 30 μm to about 200 μm, from about 50 μm to about 200 μm, or from about 150 μm to about 200 μm. Alternatively, the diameter of the beads is from about 1 μm to about 100 μm, or from 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, the purification of monoclonal antibodies can involve magnetic resin beads having a size from about 40 microns to about 90 microns. Further, the concentration of the magnetic resin beads can range from about 0.01 wt% to about 25 wt%. For example, the concentration of the magnetic resin beads can be about 1 wt%. In some examples, the purification of monoclonal antibodies can involve magnetic resin beads having a concentration from about 1 wt% to about 10 wt%. 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 includes continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module, where the second module includes 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 includes magnetic resin beads. For example, the surface of the magnetic resin beads may have cationic functional groups derived from the attachment of positive charge functional groups to enable purification based on charge or electrostatic interaction. 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 derived from the attachment of negative charge functional groups to enable purification based on charge or electrostatic interaction. For example, the negative charge functional groups include carboxyl, anionic polymers, net negative charge peptides, net negative charge proteins, oligonucleotides, or any combination thereof. For example, the magnetic resin beads can be paramagnetic or superparamagnetic.

[0078] In an embodiment, 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 to be purified and the overall flow rate of the process. For example, the purification of monoclonal antibodies can include magnetic resin beads having a size of about 40 microns to about 90 microns. Further, the concentration of the magnetic resin beads can be in the range of about 0.01 wt% to about 25 wt%. For example, the concentration of the magnetic resin beads can be about 1 wt%. In some examples, the purification of monoclonal antibodies can include magnetic resin beads having a concentration of about 1 wt% to about 10 wt%. In other examples, the charge or electrostatic association capacity of the magnetic resin beads is a function of 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 an embodiment, as described herein, one or both of the first (affinity-based magnetic purification) and / or the second (charge-based magnetic purification including a magnetic purification device based on positive and / or negative charges) modules may further include at least one external magnetic field. For example, the at least one external magnetic field includes a permanent magnet or an electromagnet. 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 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, buffer, or any combination thereof. For example, at least one of the at least two transport containers is located within or proximate to an external magnetic field to attract the magnetic resin beads.

[0081] In an embodiment, the pick and place robot 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, buffer, or any combination thereof. For example, at least one of the at least two transport containers is located within or proximate to an external magnetic field to attract the magnetic resin beads.

[0082] In an embodiment, the first (affinity-based magnetic purification) and / or the second (charge-based magnetic purification including a magnetic purification device based on positive and / or negative charges) module further includes at least one tangential flow filtration system operating in a fed-batch or perfusion mode. For example, the tangential flow filtration system can be used to concentrate and buffer exchange a fraction containing a biological product.

[0083] In an embodiment, the process described herein includes continuously transmitting a filtrate to a first module that can separate a solution into two or more fractions including at least one fraction containing a biological product, and the first module includes an affinity-based purification device. For example, the affinity-based purification device further includes a suspension of resin beads. The surface of the resin beads is linked to, for example, Protein A, Protein G, Protein L, an antigen protein, a protein, a receptor, an antibody, or an aptamer without intending to limit.

[0084] For example, 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 to be purified and the overall flow rate of the process. For example, the purification of a monoclonal antibody can include resin beads having a size of about 90 microns. Further, the concentration of the resin beads can be in the range of about 0.01 wt% to about 25 wt%. For example, the concentration of the resin beads can be about 1 wt%. In some examples, the purification of a monoclonal antibody can include resin beads having a concentration of about 1 wt% to about 10 wt%. 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 includes continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module, where the second module includes a charge-based purification device (e.g., a positive charge and / or negative charge-based purification device), and the charge-based purification device further includes resin beads. For example, the surface of the resin beads may have cationic functional groups derived from the attachment of positive charge functional groups to enable purification based on charge or electrostatic interaction. 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 resin beads may have anionic functional groups derived from the attachment of negative charge functional groups to enable purification based on charge or electrostatic interaction. For example, the negative charge functional groups include carboxyl, anionic polymers, net negative charge peptides, net negative charge proteins, oligonucleotides, or any combination thereof.

[0086] 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 to be purified and the overall flow rate of the process. For example, the purification of monoclonal antibodies may include resin beads having a size of about 90 microns. Further, the concentration of the resin beads can be in the range of about 0.01 wt% to about 25 wt%. For example, the concentration of the resin beads can be about 1 wt%. In some examples, the purification of monoclonal antibodies may include resin beads having a concentration of about 1 wt% to about 10 wt%. 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 a continuous fluid flow between a first and / or second inlet and a first and / or second outlet) includes at least two containers filled with (e.g., filled with) mobile resin beads configured to receive (e.g., continuously receive) a mixture containing a biological product and subsequently transport a 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 stepwise linear system that allows a continuous fluid flow between a first and / or second inlet and a first and / or second outlet) includes at least two containers filled with (e.g., filled with) mobile resin beads configured to receive (e.g., continuously receive) a mixture containing a biological product and subsequently process a resulting mixture containing the biological product, resin beads, buffer, or any combination thereof.

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

[0090] In an embodiment, the process described herein includes continuously transmitting a filtrate to a first module that can separate a solution into two or more fractions, including at least one fraction containing a biological product, where the first module is an affinity-based fluid purification device having at least one hybrid fluid element or chip. In an embodiment, the at least one hybrid fluid element or chip has 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 that can generate 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 from about 0.01 tesla (T) to about 1 tesla (e.g., up to 1 tesla). In other examples, the magnetic field strength is about 0.01 T, about 0.1 T, or about 1 T. In other embodiments, the at least one hybrid fluid element or chip has 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 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 from 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 includes magnetic resin beads. The surface of the magnetic resin beads is linked to, for example, without intending to limit, 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 an embodiment, the diameter of the magnetic resin beads of the affinity-based fluid purification apparatus is from about 0.2 microns to about 200 microns. For example, the purification of monoclonal antibodies can involve magnetic resin beads having a size of about 40 microns. Further, the concentration of the magnetic resin beads can range from about 0.01 wt% to about 25 wt%. For example, the initial concentration of the magnetic resin beads can be about 1 wt%. In some examples, the purification of monoclonal antibodies can involve magnetic resin beads having a concentration of from about 1 wt% to about 10 wt%. 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.

[0093] In an embodiment, the process described herein includes continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module, where the second module includes a charge-based fluid purification device. For example, the charge-based fluid purification device includes at least one hybrid fluid element or chip. The at least one hybrid fluid element 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 that can generate 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 other embodiments, the hybrid fluid element 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 defined unidirectional force. 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., a positive and / or negative charge-based fluid purification device) further includes a suspension of magnetic resin beads. The surface of the magnetic resin beads has cationic functional groups derived from the attachment of positive charge functional groups to enable purification based on charge or electrostatic interaction. The positive charge functional groups include amines, cationic polymers, net positive charge peptides, net positive charge proteins, or any combination thereof. Alternatively, the magnetic resin bead surface may include anionic functional groups derived from the attachment of negative charge functional groups to enable purification based on charge or electrostatic interaction. 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 can be paramagnetic or superparamagnetic.

[0095] For example, the diameter of the magnetic resin beads of the charge-based fluid purification device is from about 0.2 microns to about 200 microns. The diameter of the magnetic resin beads can vary depending on the biological product to be purified and the flow rate of the process. For example, the purification of monoclonal antibodies can involve magnetic resin beads having a size of about 40 microns. Further, the concentration of the magnetic resin beads can be in the range of about 0.01 wt% to about 25 wt%. For example, the concentration of the magnetic resin beads can be about 1 wt%. In some examples, the purification of monoclonal antibodies can involve magnetic resin beads having a concentration in the range of about 1 wt% to about 10 wt%. In other examples, the charge or electrostatic association capacity of the magnetic resin beads is a function of 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 fluid purification) module further includes at least one equilibration vessel that enables the binding of the biological product to the surface of the magnetic resin beads, and at least one low pH equilibration vessel that enables the dissociation interaction of the biological product from the surface of the magnetic resin beads.

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

[0098] In an embodiment, the magnetic resin beads described herein are recycled and reused. For example, the beads can be reused at least 2, 3, 4, or more times for purifying a biological product. To enable the recycling and reuse of the magnetic resin beads, at least one regeneration equilibrium vessel can be used together with a tangential flow filtration system for concentrating and buffer-exchanging the magnetic resin beads to return the magnetic resin beads to their initial state.

[0099] As described herein, the first (affinity-based fluid purification) and / or the second (charge-based fluid purification including a fluid purification device based on positive and / or negative charges) module includes a hybrid microfluidic, mesofluidic, millifluidic, macrofluidic element or chip for purifying a biological product, or any combination thereof, for example, a hybrid microfluidic element including at least one of at least one magnetic field and a piezoelectric component or a dielectrophoretic electrode.

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

[0101] In an embodiment, the process described herein includes continuously transmitting a filtrate to a first module that can separate a solution into two or more fractions including at least one fraction containing a biological product, and the first module includes an affinity-based TFF purification device. For example, the affinity-based TFF purification device includes at least three tangential flow filtration systems in fluid communication.

[0102] In an embodiment, the affinity-based TFF purification device further includes a suspension of resin beads. The surface of the resin beads is linked to, for example, Protein A, Protein G, Protein L, an antigen protein, a protein, a receptor, an antibody, or an aptamer, without intending to be limiting.

[0103] In an embodiment, the diameter of the resin beads of the affinity-based TFF purification device is from about 10 microns to about 200 microns. The diameter of the resin beads can vary depending on the biological product to be purified and the overall flow rate of the process. For example, the purification of a monoclonal antibody can include resin beads having a size of about 90 microns. Further, the concentration of the resin beads can be in the range of about 0.01 wt% to about 25 wt%. For example, the concentration of the resin beads can be from about 1 wt% to about 20 wt%. 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 still other examples, the resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

[0104] In an embodiment, the process described herein includes continuously transmitting a fraction containing a biological product from at least one first outlet of the first module to a second module, where the second module includes 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 includes at least three tangential flow filtration systems in fluid communication.

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

[0106] In an embodiment, the diameter of the resin beads of the charge-based TFF purification apparatus is from about 0.2 microns to about 200 microns. The diameter of the resin beads may vary depending on the biological product to be purified and the overall flow rate of the process. For example, the purification of monoclonal antibodies may include resin beads having a size of about 90 microns. Further, the concentration of the resin beads may be in the range of about 0.01 wt% to about 25 wt%. For example, the concentration of the resin beads may be from about 1 wt% to about 20 wt%. 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 may be solid, porous, nanoporous, microporous, or any combination thereof.

[0107] In an embodiment, the first (affinity-based TFF purification) module further includes at least one equilibration vessel that enables binding of the biological product to the resin bead surface and at least one low pH equilibration vessel that enables the dissociation interaction of the biological product from the resin bead surface.

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

[0109] In an embodiment, the resin beads described herein are recycled and reused. For example, the beads can be reused at least 2 times, 3 times, 4 times, or more for purifying biological products. To enable the recycling and reuse of the resin beads, at least one regeneration equilibrium vessel can be used together with a tangential flow filtration system for concentrating and buffer-exchanging the resin beads to return the resin beads to their initial state.

[0110] In an embodiment, the first (affinity-based TFF purification) and / or the second (charge-based TFF purification including a positive charge and / or negative charge-based TFF purification device) module further includes at least one tangential flow filtration system for concentrating and buffer-exchanging the fraction containing the biological product.

[0111] In other embodiments, the processes described herein include continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module, where the second module includes an isoelectric point-based fluid purification device, also referred to herein as a free-flow electrophoresis device. For example, a free-flow electrophoresis device includes at least one fluid element that includes a fluid channel created between two parallel plates for operating in an isoelectric focusing mode of operation, an electric field or electric field gradient orthogonal to the direction of fluid flow, and a pH gradient. In another example, the isoelectric point-based fluid purification module includes at least one first fluid element that includes a fluid channel created 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., a pH range of about 2 to about 10) across the main separation channel, and at least one second fluid element that includes a fluid channel created 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., a pH range of about 5 to about 8) across the main separation channel. For example, an additional subsequent fluid element or chip that includes a fluid channel created 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., a pH range of about 7.1 to about 7.6). Alternatively, a free-flow electrophoresis device includes at least one fluid element that includes a fluid channel created between two parallel plates and an electric field or electric field gradient orthogonal to the direction of fluid flow for operating in a zone electrophoresis or charge separation mode of operation and that has no pH gradient.

[0112] In other examples, an isoelectric point-based fluid purification module includes a fluid channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and at least one first fluid element including a constant basic pH (e.g., greater than pH 7), and a fluid channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and at least one second fluid element including a constant acidic pH (e.g., less than pH 7). Further, a free-flow electrophoresis device includes at least one fluid element including both an acidic pH gradient and a basic pH gradient separated by a spacer solution (e.g., an NaCl solution) for operating in an isotachophoresis operation mode, a fluid channel generated between two parallel plates, and an electric field or electric field gradient orthogonal to the direction of fluid flow.

[0113] In some embodiments, an isoelectric point-based fluid purification module includes at least one first fluid element including a fluid channel generated between two parallel plates and an electric field or electric field gradient orthogonal to the direction of fluid flow, and at least one second fluid element including a fluid channel generated between two parallel plates and an electric field or electric field gradient orthogonal to the direction of fluid flow, where each element is connected in series and can operate in an independent operation mode enabling purification. For example, at least one first free-flow electrophoresis device can operate in an isoelectric focusing mode, at least one second free-flow electrophoresis device can operate in an isotachophoresis mode, and can operate sequentially by series connection to enhance separation resolution.

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

[0115] In still other embodiments, the isoelectric point-based fluid purification apparatus further includes an active cooling system (e.g., a Peltier element, a thermal chuck with a circulating water / propylene glycol jacket) to enable temperature control and Joule heat dissipation. For example, the active cooling system can control cooling and / or Joule heat dissipation to enable operation in 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 a biological product can also include virus inactivation, virus filtration, tangential flow filtration (TFF), high-performance tangential flow filtration (HP-TFF), ultrafiltration / diafiltration (UF / DF), filter sterilization, filling-finishing, lyophilization, or any combination thereof, which are 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 a temperature in the range of about 4°C to about 50°C, preferably about 4°C to about 37°C. Further, a commercial-scale process for purifying biological products is carried out in a system that occupies significantly fewer square feet than current technology, on a kilogram / year basis, without sacrificing product throughput or yield. For example, the process for producing and purifying monoclonal antibodies described herein operates with a footprint that occupies up to about 30,000 square feet. In contrast, current monoclonal antibody production and downstream processes require at least 200,000 square feet.

[0118] The processes described herein are used to purify biological products, which include, but are 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 aspect, a dynamic filtration module is provided herein for removing large impurities from biological products in a heterogeneous mixture. The dynamic filtration module 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.

[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 container.

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

[0122] In an embodiment, the pore size of the rolled filter membrane varies depending on the biological product to be 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 an antibody, the pore size of the rolled filter membrane ranges from 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 that communicates with a collection reel to create a reel-to-reel system. During operation, the heterogeneous mixture is applied to a new, unused target region of the filter membrane, also referred to herein as the "target region" (or "active target region"), where the filter membrane continuously moves 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 gearbox that limits the revolutions per minute (RPM) to a ratio of 200:1 in order to allow for high torque and a low membrane transport speed. The movement of the collection reel is controlled by a servo motor coupled to a gearbox that limits the RPM to a ratio of 200:1 in order to allow for high torque and a low membrane transport speed. Further, 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 membrane transport speed for the filter membrane roll that is constantly changing in diameter at both the supply reel and the collection reel during operation.

[0125] For example, a thickness monitoring system or a rotary encoder can be used to ensure a consistent membrane transport speed. For example, the supply reel and the collection reel operate at the same speed and in the same direction. In other examples, the supply reel and the 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 contemplated by those skilled in the art of the coating and converting industries. In other examples, two dynamic filtration systems are run in parallel. For example, two parallel dynamic filtration systems can allow for a continuous flow through the system while the used filter membrane roll is being replaced. Further, two parallel dynamic filtration systems can allow for the equilibration of the entire vacuum collection vessel to atmospheric pressure and allow for the flow of fluid 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 a target region (e.g., an active target region) of the filter membrane when the filter membrane is under negative pressure. The membrane support structure is located between a supply reel and a 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 continuous with a vacuum line. For example, the opening may include a mesh, at least one slot, at least one hole, a frit, a porous material, or any combination thereof.

[0127] In embodiments, 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 having 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 when there is evaporative cooling. The temperature control mechanism maintains a temperature of about 4°C to about 37°C. For example, during antibody purification, the temperature control mechanism maintains a temperature 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 a heterogeneous mixture and distributing the heterogeneous mixture to a target region (e.g., an active target region) 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 uniaxial output head, a separate slot die output head, or a slot die output head having a plurality of openings.

[0131] In some embodiments, the dynamic filtration module includes elements known in the coating and converting industries, such as, without intending to limit, active or passive edge guides, tension adjustment devices (e.g., dancer), brakes and tension detectors, or any combination thereof.

[0132] In an embodiment, the dynamic filtration module includes a vacuum system that is continuous with the membrane support structure to apply a negative pressure across a target region (e.g., an active target region) of the filter membrane, where the negative pressure permits a target region (e.g., an active target region) of the filter membrane across the membrane support structure and enables collection of a filtrate containing biological products. 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 the filtrate and at least one sensor or detector. For example, two parallel dynamic filtration systems are run with a time shift to allow for a continuous flow through the system 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 involves 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 rolled filter membrane with a large pore size (e.g., 0.45 μm) in fluid communication with at least one second dynamic filtration device having a rolled filter membrane with a small pore size (e.g., 0.2 μm), whereby a filtrate containing a biological product is produced. Alternatively, a similar result can be achieved by a single dynamic filtration device having at least two rolled filter membranes supplied by separate supply reels, resulting in a stacked set of filter membranes across the active target area, where the heterogeneous mixture first contacts a filter membrane with a large pore size (e.g., 0.45 μm) and then contacts a filter membrane with a smaller pore size (e.g., 0.2 μm).

[0135] [Affinity-based magnetic purification module] In an aspect, 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 permit a continuous flow of fluid between the at least one inlet and the at least one outlet, where the flow rate can be, for example, consistent and constant during steady operation.

[0136] In an embodiment, the affinity-based magnetic purification module includes a suspension of magnetic resin beads, where the surface of the magnetic resin beads 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, without intending to be limiting. For example, the magnetic resin beads are mobile.

[0137] Furthermore, 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 containing a biological product and subsequently transport a resulting heterogeneous mixture containing the biological product, magnetic resin beads, buffer, or any combination thereof.

[0138] Alternatively, the affinity-based magnetic purification module includes a pick-and-place robot system including at least two transport containers filled with magnetic resin beads configured to continuously receive a mixture containing a biological product and subsequently transport a resulting heterogeneous mixture containing the biological product, magnetic resin beads, buffer, or any combination thereof.

[0139] In embodiments, the affinity-based magnetic purification module includes at least one external magnetic field that can be used to attract and separate the magnetic resin beads from the heterogeneous mixture to enable washing. Further, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from the heterogeneous mixture to enable elution 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 the magnetic resin beads can be achieved by placing at least one transport container between two separate opposing magnetic fields that switch between an on and an off state.

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

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

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

[0143] [Positive charge-based magnetic purification module] In an aspect, provided herein is 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 a continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be, for example, consistent and constant during steady operation.

[0144] In an embodiment, the positive charge-based magnetic purification module includes a suspension of magnetic resin beads, where the surface of the magnetic resin beads includes cationic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength. For example, the magnetic resin beads are mobile.

[0145] Furthermore, 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 containing a biological product and subsequently transport a resulting heterogeneous mixture containing the biological product, magnetic resin beads, buffer, or any combination thereof.

[0146] Alternatively, a positive-charge-based magnetic purification module includes a pick-and-place robot system that continuously receives a mixture containing a biological product and subsequently transports a resulting heterogeneous mixture containing the biological product, magnetic resin beads, buffer, or any combination thereof, filled with magnetic resin beads configured to transport the mixture.

[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 the heterogeneous mixture to enable washing. Further, 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 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 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.

[0148] In an embodiment, the positive-charge-based magnetic purification module includes at least one association / washing buffer system. In an embodiment, the positive-charge-based magnetic purification module includes at least one dissociation buffer system. For example, multiple dissociation buffers that vary pH, ionic strength, or any combination thereof are sequentially utilized to generate a gradient dissociation effect.

[0149] In an embodiment, the positive-charge-based magnetic purification module includes 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 containers.

[0150] In an embodiment, the positive-charge-based magnetic purification module includes 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 aspect, provided herein is 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 a continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be, for example, consistent and constant during steady operation.

[0152] In an embodiment, the negative charge-based magnetic purification module includes a suspension of magnetic resin beads, where the surface of the magnetic resin beads includes anionic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength. For example, the magnetic resin beads are mobile.

[0153] Furthermore, 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 containing a biological product and subsequently transport a resulting heterogeneous mixture containing the biological product, magnetic resin beads, buffer, or any combination thereof.

[0154] Alternatively, the negative charge-based magnetic purification module includes a pick-and-place robot system including at least two transport containers filled with magnetic resin beads configured to continuously receive a mixture containing a biological product and subsequently transport a resulting heterogeneous mixture containing the biological product, magnetic resin beads, buffer, or any combination thereof.

[0155] In an embodiment, the negatively charged magnetic purification module includes 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. Further, 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 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 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.

[0156] In an embodiment, the negatively charged magnetic purification module includes at least one association / washing buffer system. In an embodiment, the negatively charged magnetic purification module includes at least one dissociation buffer system. For example, multiple dissociation buffers that vary pH, ionic strength, or any combination thereof are sequentially utilized to generate a gradient dissociation effect.

[0157] In an embodiment, the negatively charged magnetic purification module includes at least one magnetic resin bead regeneration buffer system. In an embodiment, the negatively charged magnetic purification module includes at least one aspirator system for removing waste liquid from at least two transport containers.

[0158] In an embodiment, the negatively charged magnetic purification module includes at least one sensor or detector. In an embodiment, the negatively charged magnetic purification module includes at least one fluid handling pump.

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

[0160] In an embodiment, the affinity-based purification module contains a suspension of resin beads, where the surface of the resin beads 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 without intending to limit. For example, the resin beads are mobile.

[0161] In an embodiment, the affinity-based purification module includes a lid system with at least one gasket lid, where the at least one gasket lid has at least one inlet for introducing gas so as to be able to control a positive head pressure. Further, the lid system has at least one vent port to enable equilibration to atmospheric pressure, at least one inlet for introducing a suspension of resin beads, at least one inlet for receiving a filtrate containing a biological product, and / or at least two inlets for introducing a buffer system for dispersing the resin beads to enable washing, elution, or regeneration of the resin beads. In some embodiments, the at least one gasket lid also includes a port for receiving an overhead stirring impeller to enable dispersion of the resin beads. For example, the lid system controls movement along the z-axis.

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

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

[0164] In other embodiments, the affinity-based purification module includes a step linear system, such as at least two containers filled with resin beads configured to continuously receive a mixture containing a biological product and subsequently process a 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 enable pressurization and liquid handling.

[0165] In an embodiment, the affinity-based purification module includes a collection system that interfaces with at least one of the at least two containers of the mechanical rotation system to collect waste, fractions containing biological products, or any combination thereof. For example, the collection system controls movement along the z-axis.

[0166] In other embodiments, the affinity-based purification module includes a collection system that interfaces with at least one of at least two vessels of a stepwise linear system to collect waste, fractions containing biological products, or any combination thereof. For example, the collection system is connected 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 from about 0.689 kPa to about 207 kPa (from about 0.1 psi to about 30 psi).

[0168] In embodiments, the affinity-based purification module includes at least one binding / washing 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 embodiments, 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, wherein at least one fraction contains a biological product.

[0172] The positively charged purification module includes at least one inlet and at least one outlet, which are configured to allow a continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be consistent and constant, for example, during steady operation.

[0173] In an embodiment, the positively charged purification module includes a suspension of resin beads, where the surface of the resin beads contains cationic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength. For example, the resin beads are mobile.

[0174] In an embodiment, the positively charged purification module includes a lid system having at least one gasketed lid, where the at least one gasketed lid includes at least one inlet for introducing gas to control 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 a filtrate containing the biological product, and at least two inlets for introducing a buffer system to disperse the resin beads to enable 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 agitation impeller to enable dispersion of the resin beads. For example, the lid system controls movement along the z-axis.

[0175] In an embodiment, the positively charged purification module includes a mechanical rotation system, for example, a carousel including at least two containers filled with resin beads configured to continuously receive a mixture containing a biological product and subsequently transport a resulting heterogeneous mixture containing the biological product, resin beads, buffer, or any combination thereof. For example, the carousel is a rotating structure that holds at least two containers and transports them to different process positions. In some examples, the mechanical rotation system is configured to mate with a lid system to enable pressurization. In other examples, the mechanical rotation system controls movement or rotation in the xy plane.

[0176] In an embodiment, at least two containers of the positively charged purification module each have a supported base filter or filter membrane. For example, the base filter (or filter membrane) enables retention of the resin beads during the 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 positively charged purification module includes a step linear system, for example, a carousel including at least two containers filled with resin beads configured to continuously receive a mixture containing a biological product and subsequently process a 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 enable pressurization and liquid handling.

[0178] In an embodiment, the positively charged 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, fractions containing biological products, or any combination thereof. For example, the collection system controls movement along the z-axis.

[0179] In other embodiments, the positive charge-based purification module includes a collection system that interfaces with at least one of at least two containers of a stepwise linear system to collect waste, fractions containing biological products, or any combination thereof. For example, the collection system is connected to at least one of the at least two containers.

[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 from about 0.689 kPa to about 207 kPa (from about 0.1 psi to about 30 psi).

[0181] In embodiments, the positive charge-based purification module includes at least one association / washing buffer system. In embodiments, the positive charge-based purification module includes at least one dissociation buffer system. For example, multiple dissociation buffers that vary pH, ionic strength, or any combination thereof are utilized continuously or sequentially to generate a gradient dissociation effect.

[0182] In embodiments, the positive charge-based purification module includes at least one resin bead regeneration buffer system. In embodiments, the positive charge-based purification module includes at least one collection container.

[0183] In embodiments, 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 aspect, the present specification provides a negative charge-based purification module for separating a mixture into two or more fractions, where at least one fraction contains a biological product. The negative charge-based purification module includes at least one inlet and at least one outlet, which are configured to allow a continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be consistent, for example, during steady operation.

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

[0186] In an embodiment, the negative charge-based purification module includes a lid system having at least one gasketed lid, where the at least one gasketed lid includes at least one inlet for introducing gas to control 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 a filtrate containing the biological product, and at least two inlets for introducing a buffer system to disperse the resin beads to enable 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 enable dispersion of the resin beads. For example, the lid system controls movement along the z-axis.

[0187] In an embodiment, the negatively charged purification module includes a mechanical rotation system, for example, a carousel including at least two containers filled with resin beads configured to continuously receive a mixture containing a biological product and subsequently transport a resulting heterogeneous mixture containing the biological product, resin beads, buffer, or any combination thereof. For example, the carousel is a rotating structure that holds at least two containers and transports them to different process positions. In some examples, the mechanical rotation system is configured to mate with a lid system to enable pressurization. In other examples, the mechanical rotation system controls movement or rotation in the xy plane.

[0188] In an embodiment, at least two containers of the positively charged purification module each have a supported base filter or filter membrane. For example, the base filter (or filter membrane) enables retention of the resin beads during the 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 positively charged purification module includes a step linear system, for example, a carousel including at least two containers filled with resin beads configured to continuously receive a mixture containing a biological product and subsequently process a 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 enable pressurization and liquid handling.

[0190] In an embodiment, the positively charged purification module includes a collection system that interfaces with at least one of the at least two containers of the mechanical rotation system to collect waste, fractions containing biological products, or any combination thereof. For example, the collection system controls movement along the z-axis.

[0191] In other embodiments, the positive charge-based purification module includes a collection system that interfaces with at least one of at least two containers of a stepwise linear system to collect waste, fractions containing biological products, or any combination thereof. For example, the collection system is connected to at least one of the at least two containers.

[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 from about 0.689 kPa to about 207 kPa (from about 0.1 psi to about 30 psi).

[0193] In embodiments, the negative charge-based purification module includes at least one association / washing buffer system. In embodiments, the negative charge-based purification module includes at least one dissociation buffer system. For example, a plurality of dissociation buffers that vary the pH, ionic strength, or any combination thereof are used continuously or sequentially to generate a gradient dissociation effect.

[0194] In embodiments, the negative charge-based purification module includes at least one resin bead regeneration buffer system. In embodiments, the negative charge-based purification module includes at least one collection container.

[0195] In embodiments, 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 aspect, the present specification provides an affinity-based fluid purification module for separating a mixture into two or more fractions, where at least one fraction contains a biological product. The affinity-based fluid purification module includes at least one inlet and at least one outlet, which are configured to allow a continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be consistent and constant, for example, during steady operation.

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

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

[0199] In an embodiment, the affinity-based fluid purification module further includes at least one low-pH equilibrium vessel that allows the dissociation of the biological product from the surface of the magnetic resin beads, and a cross-flow channel, at least one magnetic field, and at least one second hybrid cross-flow fluid element including 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 an embodiment, the affinity-based fluid purification module further includes at least one tangential flow filtration system operating in a fed-batch or perfusion mode to concentrate and buffer-exchange a fraction containing a 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 an embodiment, the affinity-based fluid purification module includes at least one equilibration vessel configured to enable recycling of the 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 aspect, provided herein is a positive charge-based fluid purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The positive charge-based fluid purification module includes at least one inlet and at least one outlet, which are configured to permit a continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be consistent and constant, for example, during steady operation.

[0204] In an embodiment, the positive charge-based fluid purification module includes a suspension of magnetic resin beads, wherein the surface of the magnetic resin beads includes cationic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength. For example, the magnetic resin beads are mobile.

[0205] In an embodiment, the positive charge-based fluid purification module includes at least one association equilibrium vessel that enables the association between the surface of magnetic resin beads and a biological product, and a cross-flow channel, at least one magnetic field, and at least one first hybrid cross-flow fluid element including at least one of a piezoelectric component or a dielectrophoresis electrode configured to generate or induce a unidirectional force to separate the magnetic resin beads associated with the biological product from the heterogeneous mixture.

[0206] In an embodiment, the positive charge-based fluid purification module includes at least one dissociation equilibrium vessel that allows the dissociation of a biological product from the surface of magnetic resin beads, and a cross-flow channel, at least one magnetic field, and at least one second hybrid cross-flow fluid element including at least one of a piezoelectric component or a dielectrophoresis electrode configured to separate the magnetic resin beads from the dissociated biological product and generate or induce a unidirectional force to complete this purification. For example, a plurality of dissociation equilibrium vessels including a separate buffer for changing the pH, ionic strength, or any combination thereof to generate a gradient dissociation effect are sequentially utilized.

[0207] In an embodiment, the positive charge-based fluid purification module further includes at least one tangential flow filtration system operating in a fed-batch or perfusion mode to concentrate and buffer-exchange a fraction containing a 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 equilibrium vessel configured to enable the recycling of the 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 aspect, provided herein is a negative charge-based fluid purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The negative charge-based fluid purification module includes at least one inlet and at least one outlet, which are configured to permit a continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be consistent and constant, for example, during steady operation.

[0211] In an embodiment, the negative charge-based fluid purification module includes a suspension of magnetic resin beads, wherein the surface of the magnetic resin beads includes anionic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength. For example, the magnetic resin beads are mobile.

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

[0213] In an embodiment, the negative charge-based fluid purification module includes at least one dissociation equilibrium vessel that allows dissociation of biological products from the surface of magnetic resin beads, and a cross-flow channel, at least one magnetic field, and at least one second hybrid cross-flow fluid element including at least one of a piezoelectric component or a dielectrophoretic electrode configured to separate the magnetic resin beads from the dissociated biological products and generate or induce a unidirectional force to complete this purification. For example, multiple dissociation equilibrium vessels including separate buffers that vary pH, ionic strength, or any combination thereof are sequentially utilized to generate a gradient dissociation effect.

[0214] In an embodiment, the negative charge-based fluid purification module further includes at least one tangential flow filtration system operating in a fed-batch or perfusion mode to concentrate and buffer-exchange a fraction containing biological products.

[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 equilibrium vessel configured to enable recycling of the 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 aspect, provided herein is an affinity-based TFF purification module for separating a mixture into two or more fractions, wherein at least one fraction contains a biological product. The affinity-based TFF purification module includes at least one inlet and at least one outlet, which are configured to permit a continuous fluid flow between the at least one inlet and the at least one outlet, wherein the flow rate is consistent and constant during steady operation.

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

[0219] In an embodiment, the affinity-based TFF purification module includes at least one equilibration vessel that permits binding of the biological product to the resin bead surface, 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 an embodiment, the affinity-based TFF purification module further includes at least one low pH equilibration vessel that enables dissociation of the biological product from the resin bead surface, and at least one second tangential flow filtration system for separating the resin beads from the unbound biological product and completing their elution.

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

[0222] In an embodiment, an affinity-based TFF purification module includes at least one collection container and at least one fourth tangential flow filtration system that enables concentration and buffer exchange of a biological product to purify the biological product.

[0223] In an embodiment, at least one equilibration container, at least one low pH equilibration container, and at least one regeneration equilibration container of the affinity-based TFF purification module may include a single container that transitions between corresponding tangential flow filtration systems to enable purification and regeneration of resin beads using appropriate buffers while maintaining continuous flow of filtrate through at least one additional container in a parallel flow path.

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

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

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

[0227] In an embodiment, an affinity-based TFF purification module includes at least one fluid handling pump. [Positive charge-based TFF purification module] In an aspect, provided herein is a positive charge-based TFF purification module for separating a mixture into two or more fractions, where at least one fraction contains a biological product. The positive charge-based TFF purification module includes at least one inlet and at least one outlet, which are configured to allow a continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate is consistent and constant during steady operation.

[0228] In an embodiment, the positive charge-based TFF purification module contains a suspension of resin beads, where the surface of the resin beads contains cationic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength. For example, the magnetic resin beads are mobile.

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

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

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

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

[0233] In an embodiment, at least one association equilibrium container, at least one dissociation container, and at least one regeneration equilibrium container of the positive charge-based TFF purification module may include a single container that transitions between corresponding tangential flow filtration systems to enable purification and regeneration of resin beads using an appropriate buffer while maintaining a continuous flow of filtrate through at least one additional container in a parallel flow path.

[0234] In an embodiment, regeneration of the resin beads is achieved by regenerating the resin beads without the need for a separate regeneration equilibrium container and corresponding tangential flow filtration system using at least one second tangential flow filtration system of the positive charge-based TFF purification module configured to include both a dissociation buffer and a regeneration buffer to enable dissociation and purification, concentration, and buffer exchange.

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

[0236] In an embodiment, the positive charge-based TFF purification module includes 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 aspect, provided herein is a negative charge-based TFF purification module for separating a mixture into two or more fractions, where at least one fraction contains a biological product. The negative charge-based TFF purification module includes at least one inlet and at least one outlet, which are configured to allow a continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate is consistent and constant during steady operation.

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

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

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

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

[0242] In an embodiment, the negatively charged-based TFF purification module includes at least one collection container and at least one fourth tangential flow filtration system that enables concentration and buffer exchange of a biological product to purify the biological product.

[0243] In an embodiment, at least one association equilibrium container, at least one dissociation container, and at least one regeneration equilibrium container of the negatively charged-based TFF purification module may include a single container 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 container in a parallel flow path.

[0244] In an embodiment, regeneration of the resin beads can be achieved by regenerating the resin beads without the need for a separate regeneration equilibrium container and corresponding tangential flow filtration system using at least one dissociation container and at least one second tangential flow filtration system of the negatively charged-based TFF purification module configured to include both a dissociation buffer and a regeneration buffer to enable purification, concentration, and buffer exchange.

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

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

[0247] In an embodiment, the negatively charged-based TFF purification module includes at least one fluid handling pump. [Isoelectric point-based fluid purification module] In an aspect, the present specification provides an isoelectric point-based fluid purification module for separating a mixture into two or more fractions, where at least one fraction contains a biological product. The isoelectric point-based fluid purification module includes at least one inlet and at least one outlet, which are configured to permit a continuous fluid flow between the at least one inlet and the at least one outlet, where the flow rate can be, for example, consistent and constant during steady operation.

[0248] In embodiments, the process described herein includes continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module, where the second module includes a free-flow electrophoresis device. For example, a free-flow electrophoresis device includes a fluid channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and at least one fluid element including an aqueous solution (e.g., an ionic solution, or a solution providing a buffer or ampholyte). For example, the solution contact surfaces of the two parallel plates include glass, ceramic, plastic, or any combination thereof. In some examples, the aqueous ionic solution can create a pH gradient. In other examples, the aqueous ionic solution can impart a constant pH.

[0249] In an embodiment, a free-flow electrophoresis device includes at least one fluid 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 pH gradient. For example, an isoelectric point-based fluid purification module includes at least one first fluid 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 coarse pH gradient across the main separation channel (for example, the coarse pH gradient can be in a pH range of about 2 to about 10), and at least one second fluid 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 fine pH gradient across the main separation channel (for example, the fine pH gradient can be in a pH range of about 5 to about 8). For example, an additional subsequent fluid element or chip including a fluid 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 (for example, in a pH range of about 7.1 to about 7.6).

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

[0251] In other embodiments, a free-flow electrophoresis apparatus has, for operating in an isotachophoresis mode of operation, at least one fluid element that includes a fluid 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., an NaCl solution).

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

[0253] In other embodiments, an isoelectric point-based fluid purification module includes at least one first fluid element that includes a fluid channel having at least one dielectrophoretic electrode that can induce a defined unidirectional force, at least one second free-flow electrophoresis apparatus that includes a fluid 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., a pH range of about 2 to about 10) across the main separation channel, and at least one third free-flow electrophoresis apparatus that includes a fluid 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., a pH range of about 5 to about 8) across the main separation channel. For example, an additional subsequent fluid element or chip that includes a fluid 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., a pH range of about 7.1 to about 7.6).

[0254] In an embodiment, the isoelectric point-based fluid purification device further includes at least two electrodes (e.g., platinum wire electrodes) to function as an anode or a cathode. In an embodiment, the back pressure in the isoelectric point-based fluid purification device varies depending on the channel shape and dimensions, the openings of the inlet and outlet, and / or the tubing diameter, and the input flow rate. For example, the back pressure ranges from about 3.45 kPa to about 68.9 kPa (about 0.5 psi to about 10 psi). In some examples, the back pressure is controlled, for example, without intending to limit, by a needle valve.

[0255] In an embodiment, the isoelectric point-based fluid purification device further includes at least one bubble removal system for continuously removing O2 and H2 gas bubbles generated in the electrode channel under an applied voltage. In some embodiments, removing electrolysis bubbles is essential to enable substantially long-term continuous operation. For example, the bubble removal system utilizes a hydrophobic PTFE membrane to create a waterproof seal on the electrode channel that can continuously remove electrolysis bubbles at the generation point 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).

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

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

[0258] In an embodiment, an isoelectric point-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 backpressure sensor.

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

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

[0261] [Method] Provided herein is a method for purifying a biological product from a heterogeneous mixture derived from a bioreactor that produces the biological product, including using the processes described herein. For example, the type of bioreactor includes, but is not limited to, a batch bioreactor, a fed-batch bioreactor, a perfusion bioreactor, a chemostat bioreactor, or a multi-compartment bioreactor. In some examples, the bioreactor produces the biological product in a steady state.

[0262] In embodiments, provided herein is a method for purifying a biological product from a heterogeneous mixture derived from a bioreactor that produces the biological product, including using at least one of the modules described herein, such as, for example, a dynamic filtration module, an affinity-based magnetic purification module, a positive charge-based magnetic purification module, a negative charge-based magnetic purification module, an affinity-based purification module, a positive charge-based purification module, a negative charge-based purification module, an affinity-based fluid purification module, a positive charge-based fluid purification module, a negative charge-based fluid purification module, an affinity-based TFF purification module, a positive charge-based TFF purification module, a negative charge-based TFF purification module, and / or an isoelectric point-based fluid purification module.

[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 in a steady state, including using at least one of the modules described herein, such as, for example, a dynamic filtration module, an affinity-based magnetic purification module, a positive charge-based magnetic purification module, a negative charge-based magnetic purification module, an affinity-based purification module, a positive charge-based purification module, a negative charge-based purification module, an affinity-based fluid purification module, a positive charge-based fluid purification module, a negative charge-based fluid purification module, an affinity-based TFF purification module, a positive charge-based TFF purification module, a negative charge-based TFF purification module, and / or an isoelectric point-based fluid purification module.

[0264] In other embodiments, provided herein is a method for purifying a biological product from a heterogeneous mixture not derived from a bioreactor producing the biological product in a steady state, including using at least one of the modules described herein, such as, for example, a dynamic filtration module, an affinity-based magnetic purification module, a positive charge-based magnetic purification module, a negative charge-based magnetic purification module, an affinity-based purification module, a positive charge-based purification module, a negative charge-based purification module, an affinity-based fluid purification module, a positive charge-based fluid purification module, a negative charge-based fluid purification module, an affinity-based TFF purification module, a positive charge-based TFF purification module, a negative charge-based TFF purification module, and / or an isoelectric point-based fluid purification module.

[0265] Other aspects of the invention are disclosed below. This patent or application file contains one or more drawings created in color. Copies of this patent or patent application publication with color drawings will be provided by the Patent Office upon request and payment of the necessary fees.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0267] Disclosed herein is, in particular, a continuous process for purifying a biological product. The presently claimed process offers numerous advantages over current downstream methods and processes for purifying biological products such as, for example, proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, growth factors, oligonucleotides, viruses, adenoviruses, adeno-associated viruses, or lentiviruses. For example, without intending to be limiting, the process described herein has 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), thereby eliminating the membrane fouling problems inherent in conventional multi-stage filtration processes (e.g., multi-stage tangential flow filtration or depth filtration) for purifying monoclonal antibodies. Further, the continuous process significantly reduces the footprint of the production facility, the time required for facility construction and validation, the cost associated with facility construction, and the capital equipment expenditure compared to conventional approaches for manufacturing batch, single-use, or semi-continuous monoclonal antibodies while maintaining throughput and yield.

[0268] Continuous bioprocessing as described herein enables the use of smaller and simplified equipment (e.g., smaller bioreactor volumes and downstream bioprocessing equipment) because the ability to operate continuously obviates the need for large process equipment (the size of which is dictated by the volume of large bioreactors) required for conventional downstream bioprocessing centrifugation, depth filtration, and column chromatography steps. Further, smaller and simplified equipment that operates continuously enables the use of very small bioreactors that produce monoclonal antibodies in steady state. The continuous bioprocess as described herein can also significantly reduce operating costs, overall bioprocess line downtime, and loss of biological product when compared to conventional monoclonal antibody manufacturing approaches. Finally, the processes described herein for purifying biological products are performed in systems with footprints that occupy significantly fewer square feet than current technology without sacrificing product throughput or yield on a kilogram / year basis. For example, the process for producing and purifying monoclonal antibodies described herein operates with a footprint that occupies up to approximately 30,000 square feet. In contrast, current monoclonal antibody production and downstream processes require at least 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 point-based fluid purification module] A continuous process for purifying a biological product is described, the process including continuously receiving a heterogeneous mixture containing the biological product via an input line, where the biological product includes, but is not limited to, a protein or fragment thereof (polypeptide), an antibody or fragment thereof, a cytokine, a chemokine, an enzyme, or a growth factor. In purification, a biological product (e.g., a monoclonal antibody) is substantially pure when at least 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, or 99 wt% of impurities (cells, cell debris, aggregates, host cell proteins, unwanted proteins and peptides, unwanted antibodies, unwanted nucleic acids and oligonucleotides, viruses, salts, buffer components, surfactants, sugars, metal contaminants, leachates, media components, and / or naturally bound native organic molecules) are removed.

[0270] The process includes continuously removing large impurities from the heterogeneous mixture by dynamic filtration. The dynamic filtration process includes at least one dynamic filtration module that generates a filtrate containing the biological product by continuously supplying the biological product to the dynamic filtration module from at least one output head in fluid communication with the input line under negative pressure. 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 process described herein includes purifying a biological product continuously produced in a bioreactor (e.g., a fed-batch bioreactor, a perfusion bioreactor, and a chemostat bioreactor). For example, the bioreactor includes a bioreactor supply line and an output bleed line that enable steady-state cell culture growth conditions, and the output bleed line functions as an input line that permits a continuous fluid flow from the bioreactor to the dynamic filtration module.

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

[0273] Unlike the 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 manner adjusted so that filtration can occur continuously in a new target area of the filter membrane that has not been used (e.g., the membrane moves or advances according to the flow rate of the entire process). This eliminates membrane fouling and blockage and allows control of the packing and thickness of the operating filter cake.

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

[0275] The pore size of the rolled filter membrane varies depending on the biological product to be 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 an antibody, the pore size of the rolled filter membrane ranges from 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 can 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 that communicates with a collection reel, which means that the filter membrane starts from a prehub roll and initially spans an empty collection roll, creating a reel-to-reel system. In an aspect, the dynamic filtration module includes a rolled filter membrane that extends between the supply reel and the collection reel, and the filter membrane has a target region (e.g., an active target region) configured to receive a heterogeneous mixture. For example, the movement of the supply reel is controlled by a servo motor connected to a gearbox that limits the revolutions per minute (RPM) to a ratio of 200:1 to enable high torque and a low membrane transport speed. The movement of the collection reel is controlled by a servo motor connected to a gearbox that limits the RPM to a ratio of 200:1 to enable high torque and a low membrane transport speed. Further, 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 continuously changing diameter of the filter membrane roll at 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.

[0277] In an embodiment, the transport speed of the filter membrane ranges from about 0.1 mm / second to about 100 mm / second, preferably from about 0.1 mm / second to about 10 mm / second. 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., PTFE), and an opening that is continuous with the vacuum line. As used herein, "membrane support structure" refers to a component fabricated to provide structural support to the active area of the filter membrane and prevent deformation when passing through the negative pressure area by an opening that is continuous with the vacuum line. Further, as used herein, "mechanically smooth contact surface" refers to a surface with a low coefficient of static friction and, in particular, a low frictional force generated to oppose the transport of the filter membrane when wet. The mechanically smooth contact surface can affect the ease with which the filter membrane moves in a dynamic manner. The mechanically smooth contact surface can also be measured by surface roughness, and the lower the value, the smoother the surface. Further, since a rough surface has a greater frictional force between surfaces than a smooth surface, the mechanically smooth contact surface used herein refers to a surface with a low frictional force (i.e., a low coefficient of static friction).

[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). Further, the opening may include regularly spaced elements, for example, the opening may include a series of parallel slots with the same spacing. Further, the opening may include one grate (e.g., a series of regularly or irregularly spaced elements as described above). In other examples, the opening may include one or more grates, where each grate is perpendicular. 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 thickness or full thickness and may or may not be in parallel rows. The elements of the opening (e.g., a mesh, at least one slot, at least one hole, a frit, a porous material, or any combination thereof) may be of any desired thickness. For example, without intending to be limiting, the opening may include a mesh having a thickness of about 0.25 mm to about 5 mm.

[0279] The membrane support structure of the dynamic filtration module includes a temperature control mechanism. The temperature control mechanism maintains a temperature from about 4 °C to about 37 °C in the presence of evaporative cooling. For example, during the purification of an antibody, the temperature control mechanism maintains a temperature of 15 °C to 37 °C. Exemplary temperature control mechanisms include, but are not limited to, a single loop controller, a multi-loop controller, a closed loop controller, a PID controller, a Peltier element, a resistive heating element, and / or a thermal chuck with a circulating water / propylene glycol jacket.

[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 ranges 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 coefficient of static friction is 0.04. For example, the dynamic filtration module includes at least one support rod or roller having a mechanically smooth contact surface to stabilize the movement of the filter membrane across the membrane support structure.

[0281] In an embodiment, 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 region (e.g., an active target region) of the 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 cleaning buffer via a coaxial output head, a separate uniaxial output head, a separate slot die output head, or a slot die output head having a plurality of openings.

[0283] In some embodiments, the dynamic filtration module includes elements known in the coating and converting industries, e.g., without intending to be limiting, active or passive edge guides, tension adjustment devices (e.g., dancers), brakes and tension detectors, or any combination thereof.

[0284] In an embodiment, the dynamic filtration module includes a vacuum system that is continuous with the membrane support structure to apply a negative pressure across the active target region of the filter membrane, where the negative pressure allows for active transport of the filter membrane across the membrane support structure and enables collection of the 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 includes at least one vacuum collection container configured to collect filtrate, and at least one sensor or detector. In the aspects described herein, during purification by dynamic filtration, the filtrate containing the biological product is supplied to a vacuum collection container capable of collecting from about 50 mL to about 100 L under negative pressure. For example, the vacuum collection container capable of collecting filtrate is from about 1 L to about 10 L. In other examples, the vacuum collection container capable of collecting filtrate is from about 1 L to about 50 L.

[0286] 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 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, whereby a filtrate containing the biological product is produced.

[0287] The process described herein includes continuously transferring the filtrate to a first module capable of separating a 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 technique based on the structural binding interaction of molecules (e.g., ligand-receptor interaction) that recognize and bind to the biological product purified by a selective surface-fixed ligand. 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 robot system.

[0288] In an embodiment, the affinity-based magnetic purification apparatus further includes a suspension of magnetic resin beads. The surface of the magnetic resin beads is linked to, for example, Protein A, Protein G, Protein L, antigen protein, protein, receptor, antibody, or aptamer without intending to limit. By continuous purification of biological products (e.g., monoclonal antibodies) using affinity magnetic resin beads, cumbersome processing steps of conventional affinity column chromatography (e.g., Protein A affinity chromatography) can be avoided.

[0289] In an embodiment, the diameter of the magnetic resin beads of the affinity-based magnetic purification apparatus is from about 0.2 microns to about 200 microns. The diameter of the magnetic resin beads can vary depending on the biological product to be purified and the flow rate of the process. Further, the concentration of the magnetic resin beads can be in the range of 0.01 wt% to 25 wt%. For example, the concentration of the magnetic resin beads can be from about 1 wt% to about 10 wt%. 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.

[0290] The loop conveyor system can refer to, for example, a continuous or infinite loop. The loop conveyor system is advantageous in that it allows a smaller footprint while enabling a large amount to move continuously and efficiently at a high flow rate throughout the process compared to a conventional affinity column chromatography system (e.g., Protein A affinity chromatography). Since biological products are transported directly on the track, objects of any size and regular or irregular shape can be configured for transportation. In some aspects, the object is a transport container having a regular shape (e.g., cube, rectangular prism, cylinder, and cone).

[0291] In an embodiment, the loop conveyor system continuously receives a filtrate containing a mixture including a biological product, and subsequently transports a generated heterogeneous mixture including the biological product, magnetic resin beads, a buffer, or any combination thereof, and there are at least two transport containers filled with magnetic resin beads configured to do so.

[0292] The pick-and-place robot system can refer to, for example, at least one robot or robot arm. The pick-and-place robot system is advantageous in that it allows a smaller footprint while enabling a large amount to move continuously and efficiently at a high flow rate throughout the process compared to a conventional affinity column chromatography system (e.g., protein A affinity chromatography). Since the biological product contained in the transport container is picked and placed, objects of any size and regular shape can be configured for transport and stacking, with or without a handle. In some embodiments, the object is a transport container having a regular shape (e.g., a cube and a rectangular prism).

[0293] In an embodiment, the pick-and-place robot system continuously receives a filtrate containing a mixture including a biological product, and subsequently transports a generated heterogeneous mixture including the biological product, magnetic resin beads, a buffer, or any combination thereof, and there are at least two transport containers filled with magnetic resin beads configured to do so.

[0294] The affinity-based magnetic purification module further includes at least one external magnetic field that can be used to attract and separate the magnetic resin beads from the heterogeneous mixture in order to enable washing within at least one of the at least two transport vessels. Further, the at least one external magnetic field can be used to attract and separate the magnetic resin beads from the heterogeneous mixture in order to enable elution of the biological product within at least one of the at least two transport vessels. Alternatively, the at least one external magnetic field can be used to enable recycling of the magnetic resin beads within 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 a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module, the second module including a charge-based magnetic purification device. As used herein, a "charge-based magnetic purification device" includes, for example, purifying biological molecules based on their surface charge, ionic properties, electrostatic interactions, or isoelectric point. As described herein, a charge-based magnetic purification device 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 a 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 an embodiment, a charge-based magnetic purification apparatus (e.g., positive charge and / or negative charge-based magnetic purification) further includes a suspension of magnetic resin beads. For example, the surface of the magnetic resin beads may each include a cationic or anionic functional group configured to selectively associate with the biological product at a specific pH and ionic strength to enable positive charge-based magnetic purification or negative charge-based magnetic purification. By continuous purification of biological products (e.g., monoclonal antibodies) using ionic magnetic resin beads, cumbersome processing steps of conventional ion exchange column chromatography (e.g., cation exchange or anion exchange chromatography) can be avoided.

[0297] In an embodiment, the diameter of the magnetic resin beads of the charge-based magnetic purification apparatus is from about 0.2 microns to about 200 microns. The diameter of the magnetic resin beads can vary depending on the biological product to be purified and the flow rate of the process. Further, the concentration of the magnetic resin beads can be in the range of 0.01 wt% to 25 wt%. For example, the concentration of the magnetic resin beads can be from about 1 wt% to about 10 wt%. In other examples, the charge or electrostatic association capacity of the magnetic resin beads is a function of 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.

[0298] The loop conveyor system can refer to, for example, a continuous or infinite loop. The loop conveyor system is advantageous in that it allows a smaller footprint while enabling continuous and efficient movement of large quantities at high flow rates throughout the process compared to conventional ion exchange column chromatography systems. Since biological products are transported directly on the track, objects of any size and regular or irregular shape can be configured for transportation. In some aspects, the object is a transport container having a regular shape (e.g., cube, rectangular prism, cylinder, and cone).

[0299] In an embodiment, the loop conveyor system includes at least two transport containers filled with magnetic resin beads configured to continuously receive a mixture containing a biological product and subsequently transport a resulting heterogeneous mixture containing the biological product, magnetic resin beads, buffer, or any combination thereof.

[0300] The pick-and-place robot system can refer to, for example, at least one robot or robotic arm. The pick-and-place robot system is advantageous in that it allows a smaller footprint while enabling a large amount to move continuously and efficiently at a high flow rate throughout the process compared to conventional ion exchange chromatography systems. Since the biological product contained in the transport container is picked and placed, objects of any size and regular shape can be configured for transport and stacking, with or without a handle. In some embodiments, the object is a transport container having a regular shape (e.g., a cube and a rectangular prism).

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

[0302] The charge-based magnetic purification module further includes at least one external magnetic field that can be used to attract and separate the magnetic resin beads from the heterogeneous mixture to enable washing within at least one of the at least two transport containers. Further, 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 within 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 within 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 the embodiments described herein, the magnetic resin beads of one or both of the first (affinity-based magnetic purification) and / or second (charge-based magnetic purification) modules are recycled and reused. For example, the beads can be reused at least 2, 3, 4, or more times to purify biological products.

[0304] Alternatively, the process described herein includes continuously transferring a fraction containing a 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 at least one first outlet of the first module, wherein the second module includes 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 orthogonal to the direction of fluid flow, and an aqueous ionic solution can be used instead of or in addition to the charge-based magnetic purification module for purifying biological products (e.g., monoclonal antibodies).

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

[0306] In an embodiment, a free-flow electrophoresis device has at least one fluid 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 pH gradient. For example, an isoelectric point-based fluid purification module includes at least one first fluid 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 coarse pH gradient across the main separation channel (for example, the coarse pH gradient can be in a pH range of about 2 to about 10), and at least one second fluid 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 fine pH gradient across the main separation channel (for example, the fine pH gradient can be in a pH range of about 5 to about 8). For example, an additional subsequent fluid element or chip including a fluid 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 (for example, in 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 operating in zone electrophoresis or charge separation operation mode, and there is at least one fluid element without a pH gradient (e.g., constant pH in the main separation channel). For example, an isoelectric point-based fluid purification module includes a fluid channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and at least one first fluid element including a constant basic pH (e.g., greater than pH 7), and a fluid channel generated between two parallel plates, an electric field or electric field gradient orthogonal to the direction of fluid flow, and at least one second fluid element including a constant acidic pH (e.g., less than pH 7).

[0308] In other embodiments, the free-flow electrophoresis device includes at least one fluid element including an acidic pH gradient and a basic pH gradient separated by a spacer solution (e.g., NaCl solution) for operating in isotachophoresis operation mode, a fluid channel generated between two parallel plates, and an electric field or electric field gradient orthogonal to the direction of fluid flow.

[0309] In other embodiments, an isoelectric point-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 orthogonal 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 orthogonal to the direction of fluid flow, where each element is connected in series and can operate in an independent operation mode enabling purification. For example, at least one first free-flow electrophoresis device can operate in isoelectric focusing mode, and at least one second free-flow electrophoresis device can operate in isotachophoresis mode to enhance separation resolution.

[0310] In other embodiments, the isoelectric point-based fluid purification module includes at least one first fluid element including a fluid channel having at least one dielectrophoretic electrode capable of inducing a defined unidirectional force, at least one second free-flow electrophoresis device 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 coarse pH gradient (e.g., a pH range of about 2 to about 10) across the main separation channel, and at least one third free-flow electrophoresis device 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 fine pH gradient (e.g., a pH range of about 5 to about 8) across the main separation channel. For example, an additional subsequent fluid element or chip including a fluid 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., a pH range of about 7.1 to about 7.6).

[0311] In embodiments, the isoelectric point-based fluid purification device further includes at least two electrodes (e.g., platinum wire electrodes) to function as an anode or a cathode. In embodiments, the back pressure within the isoelectric point-based fluid purification device varies depending on the channel shape and dimensions, the inlet and outlet openings and / or tubing diameter, and the input flow rate. For example, the back pressure ranges from about 3.45 kPa to about 68.9 kPa (about 0.5 psi to about 10 psi). In some examples, the back pressure is controlled, for example, without intending to limit, by a needle valve.

[0312] In an embodiment, the isoelectric point-based fluid purification apparatus further includes at least one bubble removal system for continuously removing O2 and H2 gas bubbles generated in the electrode channels under an applied voltage. In some embodiments, removing electrolysis bubbles is essential to enable substantially long-term continuous operation. For example, the bubble removal system utilizes a hydrophobic PTFE membrane to create a waterproof seal over the electrode channels that can continuously remove electrolysis bubbles 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).

[0313] In an embodiment, the isoelectric point-based fluid purification apparatus further includes an active cooling system or a heat sink (e.g., a Peltier element, a thermal chuck with a circulating water / propylene glycol jacket) to enable temperature control and Joule heat dissipation. For example, the active cooling system can control cooling and / or heat dissipation in the range of about 4 °C to about 50 °C, preferably about 4 °C to about 37 °C. Ideally, when 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 includes an aluminum thermal chuck with a cooled circulating water / propylene glycol jacket.

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

[0315] In an embodiment, the isoelectric point-based fluid purification module includes at least one sensor or detector. For example, the at least one sensor or detector is disposed inline. 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 point-based fluid purification module includes at least one liquid circuit breaker or isolates the downstream of the device and the upstream of at least one inline sensor or detector so that sensing or detection can be performed with a non-voltage solution.

[0317] The presently claimed process offers numerous advantages compared to current downstream methods and processes for purifying biological products such as, for example, proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, or growth factors. For example, without intending to be limiting, the process described herein provides a continuous bioprocess for purifying monoclonal antibodies that, while maintaining throughput and yield, significantly reduces the footprint of the production facility, the time required for facility construction and validation, the cost associated with facility construction, and capital equipment expenditures compared to conventional approaches for manufacturing batch, single-use, or semi-continuous monoclonal antibodies. Continuous bioprocessing as described herein, by virtue of its ability to operate continuously, does not require large process equipment (the size of which is dictated by the volume of large bioreactors) required for batch centrifugation, depth filtration, and column chromatography steps of conventional downstream bioprocessing, and thus uses smaller and simplified equipment (e.g., smaller bioreactor volumes and downstream bioprocessing equipment). Further, the smaller and simplified equipment that operates continuously uses very small bioreactors that produce monoclonal antibodies in steady state. Continuous bioprocessing as described herein can also significantly reduce operating costs, overall bioprocess line downtime, and loss of biological product compared to conventional monoclonal antibody manufacturing approaches. Finally, the process described herein for purifying biological products is carried out in a system that has a footprint that occupies significantly fewer square feet than current technology, on a kilogram / year basis, without sacrificing product throughput or yield.

[0318] The advantages of the processes and methods described herein include the ability to remove large impurities (e.g., cells, cell debris, and aggregates) without fouling or plugging of the membrane. Membrane fouling can refer to a process in which a heterogeneous mixture accumulates on the membrane surface or within the pores of the membrane, leading to a decline in membrane performance over time and imposing significant limitations on the usefulness of conventional filtration systems. For example, purifying cells, cell debris, and aggregates from a cell culture medium using a conventional filtration or tangential flow filtration system typically results in fouling or plugging of the filter membrane, and these methods are inappropriate as a means of continuously removing large impurities from a heterogeneous mixture containing biological products by a long-term continuous process. In contrast, the dynamic filtration device described herein enables the continuous removal of large impurities from a heterogeneous mixture containing biological products without fouling the membrane because the active target region of the filter membrane is continuously renewed.

[0319] Furthermore, the entire process of producing and purifying the biological product can be continuous, and a flow rate in the range of about 0.1 mL / min to about 50 mL / min (e.g., about 5 mL / min to about 10 mL / min) can be maintained throughout the entire process. Therefore, the process equipment and the footprint of the entire process can have a footprint that is significantly smaller than the current standard process on a kilogram / year basis without sacrificing product throughput or yield. For example, the process of producing and purifying the monoclonal antibodies described herein operates with a footprint that occupies up to about 30,000 square feet. In contrast, the 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 the biological product is in the range of 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 is in the range of 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 where the flow rate of the step of continuously transferring the filtrate to the first module is in the range of about 0.1 mL / min to about 50 mL / min. In yet other examples, a process is provided where the flow rate of the step of continuously transferring 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.

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

[0321] In other words, since the module according to the present invention continuously receives these steps, there is no need to leave it in an idle state for sterilization, regeneration, and / or recycling after execution. Since the current column chromatography method has limitations in column capacity due to resin packing constraints, the method receives a continuous input flow and requires column switching of multiple packed columns to enable regeneration and / or recycling of columns that have reached full capacity, which is different from the current continuous chromatography method. Another advantage of the method described herein includes the fact that the magnetic resin beads are not packed in the stationary phase, but rather the magnetic resin beads move. Such mobility of the beads increases the surface area of the resin beads that can be used for binding or association, since substantially more surface of the magnetic resin beads is exposed and can bind freely. Furthermore, the resin beads in a packed column are subjected to a high pressure difference to generate a flow through the column, and the resulting damage is one of the reasons for shortening the column life compared to the desired life. The mobile resin beads in the invention described herein are subjected to substantially lower pressures and are much softer with respect to the fragile beads, thus extending the life. Furthermore, such mobility increases the likelihood that the magnetic resin beads will complete regeneration and return to their initial state. This further improves the cost efficiency of the method described herein, since the magnetic resin is used more efficiently.

[0322] An important advantage of the processes and methods using free-flow electrophoresis described herein is that this system represents a "product-lossless" process, i.e., since separation occurs through interaction with an electric field in an aqueous solution according to the physicochemical properties of the target biological product, the product does not need to interact with a resin or other purification moiety. Compared with conventional ion-exchange chromatography, another advantage in the resolution of this approach is that a product of theoretically higher purity can be obtained. Furthermore, separation based on essential physicochemical properties extends the usefulness of such an approach for the purification of various biological products including, but not limited to, proteins or their fragments (polypeptides), antibodies or their fragments, 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 wide 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 purification module or an isoelectric point-based fluid purification module] A continuous process for purifying a biological product is described, the process including continuously receiving a heterogeneous mixture containing the biological product via an input line, where the biological product includes, but is not limited to, a protein or its fragment (polypeptide), an antibody or its fragment, a cytokine, a chemokine, an enzyme, or a growth factor. When purified, the biological product (e.g., a monoclonal antibody) is substantially pure when at least about 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, or 99 wt% of the impurities (cells, cell fragments, aggregates, host cell proteins, unwanted proteins and peptides, unwanted antibodies, unwanted nucleic acids and oligonucleotides, viruses, salts, buffer components, surfactants, sugars, metal contaminants, leachates, media components, and / or naturally bound 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 generates a filtrate containing a biological product by continuously supplying the biological product to the dynamic filtration module from at least one output head in fluid communication with an input line under negative pressure. 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 process described herein includes purifying a biological product continuously produced in a bioreactor (e.g., a fed-batch bioreactor, a perfusion bioreactor, and a chemostat bioreactor). For example, the bioreactor includes a bioreactor supply line and an output bleed line that enable steady-state cell culture growth conditions, and the output bleed line functions as an input line that permits a continuous flow of fluid from the bioreactor to the dynamic filtration module.

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

[0327] Unlike the static filtration methods commonly used in the art, the processes herein describe dynamic filtration modules, where the components of the dynamic filtration module move in a manner adjusted so that filtration can occur continuously in a new target area of the filter membrane (e.g., the membrane moves or advances according to the flow rate of the entire process). This eliminates membrane fouling and clogging and allows control of the packing and thickness of the filter cake 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 container. For example, the filter membrane roll includes a rolled filter membrane, where the filter membrane includes, but is not limited to, polyethersulfone (PES), novel hydrophilic polysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF), novel hydrophilic PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), novel hydrophilic PTFE, or any combination thereof.

[0329] The pore size of the rolled filter membrane varies depending on the biological product to be 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 an antibody, the pore size of the rolled filter membrane ranges from 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 can 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 that communicates with a collection reel, which means that the filter membrane starts from a prehub roll and initially spans an empty collection roll to create a reel-to-reel system. In an aspect, the dynamic filtration module includes a rolled filter membrane that extends between the supply reel and the collection reel, and the filter membrane has an active target region configured to receive a heterogeneous mixture. For example, the movement of the supply reel is controlled by a servo motor coupled to a gearbox that limits the revolutions per minute (RPM) to a ratio of 200:1 to enable a high torque and a low membrane transport speed. The movement of the collection reel is controlled by a servo motor coupled to a gearbox that limits the RPM to a ratio of 200:1 to enable a high torque and a low membrane transport speed. Further, 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 changing diameter of the filter membrane roll constantly at 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 ranges from about 0.1 mm / second to about 100 mm / second, preferably from about 0.1 mm / second to about 10 mm / second. 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., PTFE) and an opening that is continuous with the vacuum line. As used herein, a "membrane support structure" refers to a component fabricated to provide structural support to the active region of the filter membrane and prevent deformation when passing through the negative pressure region by an opening that is continuous with the vacuum line. Further, as used herein, a "mechanically smooth contact surface" refers to a surface with a low coefficient of static friction and, in particular, generates a low frictional force that opposes the transport of the filter membrane when wet. The mechanically smooth contact surface can affect the ease with which the filter membrane moves in a dynamic manner. The mechanically smooth contact surface can also be measured by surface roughness, and the lower the value, the smoother the surface. Further, since a rough surface has a greater frictional force between surfaces than a smooth surface, the mechanically smooth contact surface used herein refers to a surface with a low frictional force (i.e., a low coefficient of static friction).

[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). Further, the opening can include regularly spaced elements. For example, the opening may include a series of parallel slots with the same spacing. Further, the opening may include one grate (e.g., a series of regularly or irregularly spaced elements as described above). In other examples, the opening can include one or more grates, where each grate is perpendicular. The opening can be a collection of irregular or regular elements (e.g., a series of parallel slots). The opening may also include a mesh, which can be partial thickness or full thickness and may or may not be in parallel rows. The elements of the opening (e.g., a mesh, at least one slot, at least one hole, a frit, a porous material, or any combination thereof) can be of any desired thickness. For example, without intending to be limiting, the opening may include a mesh having a thickness of from about 0.25 mm to about 5 mm.

[0333] The membrane support structure of the dynamic filtration module includes a temperature control mechanism. The temperature control mechanism maintains a temperature from about 4 °C to about 37 °C in the presence of evaporative cooling. For example, during antibody purification, the temperature control mechanism maintains a temperature of from about 15 °C to about 37 °C. Exemplary temperature control mechanisms include, but are not limited to, a single loop controller, a multi-loop controller, a closed loop controller, a PID controller, a Peltier element, and / or a thermal chuck with a circulating water / propylene glycol jacket.

[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 having a mechanically smooth contact surface to stabilize the movement of the filter membrane across the membrane support structure.

[0335] In an embodiment, the dynamic filtration module includes at least one output head for regulating the flow of the heterogeneous mixture and distributing the heterogeneous mixture to the active target region of the 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 cleaning buffer via a coaxial output head, a separate uniaxial output head, a separate slot die output head, or a slot die output head having a plurality of openings.

[0337] In some embodiments, the dynamic filtration module includes elements known in the coating and converting industries, such as, without intending to limit, active or passive edge guides, tension adjustment devices (e.g., dancer), brakes and tension detectors, or any combination thereof.

[0338] In an embodiment, the dynamic filtration module includes a vacuum system that is continuous with the membrane support structure to apply a negative pressure across the target region (e.g., active target region) of the filter membrane, where the negative pressure permits active transport of the filter membrane across the membrane support structure and enables collection of the filtrate containing biological products. 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 an embodiment, the dynamic filtration module further includes at least one vacuum collection container configured to collect filtrate, and at least one sensor or detector. In the aspects described herein, during purification by dynamic filtration, the filtrate containing the biological product is supplied to a vacuum collection container capable of collecting from about 50 mL to about 100 L under negative pressure. For example, the vacuum collection container capable of collecting filtrate is from about 1 L to about 10 L. In other examples, the vacuum collection container capable of collecting filtrate is from about 1 L to about 50 L.

[0340] 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 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, whereby a filtrate containing the biological product is produced.

[0341] The process described herein includes continuously transferring the filtrate to a first module capable of separating a 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 may 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 technique based on the structural binding interaction of molecules (e.g., ligand-receptor interaction) that recognize and bind to the biological product purified by a selective surface-fixed ligand. For example, the first module has at least one first inlet and at least one first outlet, and is configured to allow a continuous fluid flow between the first inlet and the first outlet through a mechanical rotation system including a lid system, a container carousel, and a collection system.

[0342] In an embodiment, the affinity-based purification apparatus further includes a suspension of resin beads. The surface of the resin beads is linked to, for example, Protein A, Protein G, Protein L, antigen protein, protein, receptor, antibody, or aptamer without intending to limit. By continuous purification of biological products (e.g., monoclonal antibodies) using affinity resin beads, the cumbersome processing steps of conventional affinity column chromatography (e.g., Protein A affinity chromatography) can be avoided.

[0343] In an embodiment, the diameter of the resin beads of the affinity-based purification apparatus is from about 0.2 microns to about 200 microns. The diameter of the resin beads can vary depending on the biological product to be purified and the flow rate of the process. Further, the concentration of the resin beads can be in the range of 0.01 wt% to 25 wt%. For example, the concentration of the magnetic resin beads can be from about 1 wt% to about 20 wt%. 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 still other examples, the resin beads can be solid, porous, nanoporous, microporous, or any combination thereof.

[0344] In an embodiment, the affinity-based purification module includes a lid system having at least one gasket lid, and the at least one gasket lid includes at least one inlet for introducing gas to control 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 a filtrate containing a biological product, and at least two inlets for introducing a buffer system for dispersing the resin beads to enable washing, elution, or regeneration of the resin beads. In some embodiments, the at least one gasket lid further includes a port for receiving an overhead stirring impeller to enable dispersion of the resin beads. For example, the lid system controls movement along the z-axis.

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

[0346] In an embodiment, at least two containers of the affinity-based purification module each have a supported base filter or filter membrane that enables retention of the resin beads during binding, unbinding, washing, elution, and regeneration process steps. For example, the at least two containers further include valves for controlling the flow of liquid.

[0347] In an embodiment, the affinity-based purification module includes a collection system that interfaces with at least one of the at least two containers of the mechanical rotation system to collect waste, fractions containing biological products, or any combination thereof. For example, the collection system controls movement along the z-axis.

[0348] The processes described herein also include continuously transferring a fraction containing a biological product from at least one first outlet of a first module to a second module having at least one inlet for receiving the flow from the at least one first outlet of the first module, wherein the second module includes a charge-based purification device. As used herein, a "charge-based purification device" includes, for example, purifying biological molecules based on their surface charge, ionic properties, electrostatic interactions, or isoelectric point. As described herein, charge-based purification includes a positive charge-based purification device, a negative charge-based purification device, or a combination thereof. For example, the second module has at least one second inlet and at least one second outlet and is configured to allow a continuous fluid flow between the second inlet and the second outlet via a 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 includes a suspension of resin beads. For example, the surface of the resin beads can include cationic or anionic functional groups configured to selectively associate with the biological product at a specific pH and ionic strength to enable positive charge-based purification or negative charge-based purification, 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 to be purified and the flow rate of the process. Further, the concentration of the resin beads can be in the range of 0.01 wt% to 25 wt%. For example, the concentration of the magnetic resin beads can be from about 1 wt% to about 20 wt%. 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.

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

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

[0353] In an embodiment, at least two containers of the charge-based purification module each have a supported base filter or filter membrane that enables retention of resin beads during the association, dissociation, washing, and regeneration process steps. For example, the at least two containers further include valves for controlling the flow of liquid.

[0354] In an embodiment, the charge-based purification module includes a collection system that interfaces with at least one of the at least two containers of the mechanical rotation system and can collect waste, fractions containing biological products, or any combination thereof. For example, the collection system controls movement along the z-axis.

[0355] In the 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. For example, the beads can be reused at least 2, 3, 4, or more times for purifying biological products.

[0356] Alternatively, the process described herein includes continuously transferring a fraction containing a 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 at least one first outlet of the first module, wherein the second module includes 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 orthogonal 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 biological products (e.g., monoclonal antibodies).

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

[0358] In an embodiment, the free-flow electrophoresis device has at least one fluid 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 pH gradient. For example, an isoelectric point-based fluid purification module includes at least one first fluid 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 coarse pH gradient across the main separation channel (e.g., the coarse pH gradient can be in a pH range of about 2 to about 10), and at least one second fluid 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 fine pH gradient across the main separation channel (e.g., the fine pH gradient can be in a pH range of about 5 to about 8). For example, an additional subsequent fluid element or chip including a fluid 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., in a pH range of about 7.1 to about 7.6).

[0359] In other embodiments, the free-flow electrophoresis device includes at least one fluid element that 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 operating in zone electrophoresis or charge separation operation mode, and does not include a pH gradient. For example, an isoelectric point-based fluid purification module includes at least one first fluid element that includes 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 fluid element that includes 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).

[0360] In other embodiments, the free-flow electrophoresis device includes at least one fluid element that includes a fluid 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) for operating in isotachophoresis operation mode.

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

[0362] In other embodiments, the isoelectric point-based fluid purification module includes at least one first fluid element including a fluid channel having at least one dielectrophoretic electrode capable of inducing a defined unidirectional force, at least one second free-flow electrophoresis device 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 coarse pH gradient (e.g., a pH range of about 2 to about 10) across the main separation channel, and at least one third free-flow electrophoresis device 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 fine pH gradient (e.g., a pH range of about 5 to about 8) across the main separation channel. For example, an additional subsequent fluid element or chip including a fluid 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., a pH range of about 7.1 to about 7.6).

[0363] In embodiments, the isoelectric point-based fluid purification device further includes at least two electrodes (e.g., platinum wire electrodes) to function as an anode or a cathode. In embodiments, the backpressure within the isoelectric point-based fluid purification device varies depending on the channel shape and dimensions, the inlet and outlet openings and / or tubing diameter, and the 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 a needle valve, for example, without intending to be limiting.

[0364] In an embodiment, the isoelectric point-based fluid purification apparatus further includes at least one bubble removal system for continuously removing O2 and H2 gas bubbles generated in the electrode channels under an applied voltage. In some embodiments, removing electrolysis bubbles is essential to enable substantially long-term continuous operation. For example, the bubble removal system utilizes a hydrophobic PTFE membrane to create a waterproof seal over the electrode channels that can continuously remove electrolysis bubbles at the generation points 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).

[0365] In an embodiment, the isoelectric point-based fluid purification apparatus further includes an active cooling system or a heat sink (e.g., a Peltier element, a thermal chuck with a circulating water / propylene glycol jacket) to enable temperature control and Joule heat dissipation. For example, the active cooling system can control cooling and / or heat dissipation in the range of about 4°C to about 50°C, preferably about 4°C to about 37°C. Ideally, when 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 includes an aluminum thermal chuck with a cooled circulating water / propylene glycol jacket.

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

[0367] In an embodiment, the isoelectric point-based fluid purification module includes at least one sensor or detector. For example, at least one sensor or detector is disposed inline. 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 point-based fluid purification module includes at least one liquid circuit breaker or isolates the downstream of the device from the upstream of at least one inline sensor or detector so that sensing or detection can be performed with a non-voltage solution.

[0369] The presently claimed process offers numerous advantages compared to current downstream methods and processes for purifying biological products such as, for example, proteins or fragments thereof (polypeptides), antibodies or fragments thereof, cytokines, chemokines, enzymes, or growth factors. For example, without intending to be limiting, the process described herein provides a continuous bioprocess for purifying monoclonal antibodies that, while maintaining throughput and yield, significantly reduces the footprint of the production facility, the time required for facility construction and validation, the cost associated with facility construction, and capital equipment expenditures compared to conventional approaches for manufacturing batch, single-use, or semi-continuous monoclonal antibodies. The continuous bioprocessing described herein uses smaller and simplified equipment (e.g., smaller bioreactor volumes and downstream bioprocessing equipment) because the ability to operate continuously obviates the need for large process equipment (the size of which is dictated by the volume of large bioreactors) required for centrifugation, depth filtration, and column chromatography steps of conventional downstream bioprocessing. Further, the smaller and simplified equipment that operates continuously uses very small bioreactors that produce monoclonal antibodies in steady state. The continuous bioprocess described herein can also significantly reduce operating costs, overall bioprocess line downtime, and loss of biological product compared to conventional monoclonal antibody manufacturing approaches. Finally, the process described herein for purifying biological products is carried out in a system that, on a kilogram / year basis, has a footprint that occupies significantly less square feet than current technology without sacrificing product throughput or yield.

[0370] The advantages of the processes and methods described herein include the ability to remove large impurities (e.g., cells, cell debris, and aggregates) without fouling or clogging of the membrane. For example, purifying cells, cell debris, and aggregates from a cell culture medium using conventional filtration or tangential flow filtration systems typically results in fouling or clogging of the filter membrane, and these methods are not suitable as a means for continuously removing large impurities from a heterogeneous mixture containing biological products by a long-term continuous process. In contrast, the dynamic filtration device described herein enables continuous removal of large impurities from a heterogeneous mixture containing biological products without fouling the membrane because the active target region of the filter membrane is continuously renewed. Further, since the entire process of producing and purifying biological products is continuous and a flow rate in the range of about 0.1 mL / min to about 50 mL / min can be maintained throughout the entire process, the footprint of the process device and the entire process can have a significantly smaller footprint than current standard processes on a kilogram / year basis without sacrificing product throughput or yield. For example, the process for producing and purifying monoclonal antibodies described herein operates with a footprint that occupies up to about 30,000 square feet. In contrast, current monoclonal antibody production and downstream processes require at least 200,000 square feet. For example, the flow rate of the process for purifying biological products is in the range of 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 is in the range of 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 transferring the filtrate to the first module is in the range of about 0.1 mL / min to about 50 mL / min. In still other examples, 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 is in the range of about 0.1 mL / min to about 50 mL / min.

[0371] An important advantage of the processes and methods utilizing the resin beads (e.g., agarose) described herein is that these systems do not require conventional stationary phases or packed resin columns (e.g., for standard chromatography) for sterilization, recycling, and / or regeneration. For example, these systems provide for the recycling and / or regeneration of the resin beads to generate an infinite surface area of the resin beads during operation, resulting in a continuous and cost-effective method. 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 the biological products described herein are continuously recycled and regenerated, so that they can receive the flow from 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 continuously receive these steps and thus do not need to be left idle for sterilization, regeneration, and / or recycling after execution. The method is different from the current continuous chromatography method in that the current column chromatography method has a limited column capacity limited by column packing constraints, so column switching of multiple packed columns is required to receive a continuous input flow and enable the regeneration and / or recycling of the column that has reached its full capacity. Another advantage of the method described herein is that the resin beads are not packed in a stationary phase, rather the resin beads move. This mobility of such beads increases the surface area of the resin beads available for binding or association as substantially more of the resin bead surface is exposed and free to bind. Further, the resin beads in a packed column are subjected to a high pressure differential to generate a flow through the column, and the resulting damage is one reason for shortening the column's desired lifespan. The mobile resin beads in the invention described herein are subjected to a substantially lower pressure and are much softer on the fragile beads, thus extending their lifespan. Further, such mobility increases the likelihood that the resin beads will complete regeneration and return to their initial state. This also increases the cost efficiency of the method described herein as 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 "product-loss-free" process, i.e., since separation occurs in an aqueous solution through interaction with an electric field according to the physicochemical properties of the target biological product, the product does not need to interact with a resin or other purification moiety. Compared with conventional ion-exchange chromatography, another advantage is also observed in the resolution of this approach because a product of theoretically higher purity can be obtained. 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 their fragments (polypeptides), antibodies or their fragments, 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 wide range of biological products. [A continuous process for purifying a biological product using at least one of a dynamic filtration module, an affinity-based fluid purification module, and a charge-based fluid purification module or an isoelectric point...

Claims

Claim 1 A method for purifying a biological product, the method comprising: receiving a heterogeneous mixture containing a biological product via an input line; generating a first aqueous solution containing the biological product by removing impurities from the heterogeneous mixture by a cell purification process by at least one of a dynamic filtration module, a centrifugation module, a depth filtration module, and a hydrocyclone module by supplying the biological product from at least one output in fluid communication with the input line; generating a residual liquid containing the biological product by transferring the first aqueous solution to a second module capable of exchanging the first aqueous solution with a second aqueous solution, the second module including at least one of a tangential flow filtration device, a cross-flow filtration device, and a diafiltration device, wherein the second module has at least one second inlet and at least one second outlet configured to allow a fluid flow between the at least one second inlet and the at least one second outlet, the step of generating the residual liquid; transferring the residual liquid containing the biological product from at least one outlet of the second module to a third module having at least one inlet for receiving the flow from at least one second outlet of the second module, the third module including at least one free-flow electrophoresis device, wherein the third module has at least one third inlet and at least one third outlet and is configured to allow a continuous fluid flow between the third inlet and the third outlet, the free-flow electrophoresis device including 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 free-flow electrophoresis device further including at least one gas-permeable and hydrophobic membrane configured to generate a bubble-free main separation channel by removing bubbles via a vacuum system, and at least one liquid circuit breaker, the step of transferring to the third module; recovering the biological product. A method comprising. **Claim 2**: The method according to claim 1, wherein the tangential flow filtration device further comprises a hollow fiber filter, a flat plate, or a combination thereof. **Claim 3**: The method according to claim 2, wherein the hollow fiber filter or the flat plate comprises a membrane having a pore size in the range of about 10 kDa to about 1 μm. **Claim 4**: The method according to claim 2, wherein the hollow fiber filter comprises an inner diameter in the range of about 0.5 mm to about 5 mm. **Claim 5**: The method according to claim 2, wherein the hollow fiber filter or the flat plate comprises a membrane selected from polyethersulfone (PES), modified polyethersulfone (mPES), polysulfone, mixed cellulose ester, hydrophilic PVDF, or a combination thereof. **Claim 6**: The method according to claim 1, wherein the method maintains a substantially constant flow rate in the dynamic filtration module, the second module, and the third module, and the flow rate is in the range of about 0.1 mL / min to about 50 mL / min. **Claim 7**: The method according to claim 1, wherein the method for purifying the biological product is carried out at a temperature in the range of about 4°C to about 37°C. **Claim 8**: The method according to claim 1, further comprising at least two dynamic filtration modules, each dynamic filtration module having a filter membrane with the same or different pore sizes. **Claim 9**: The method according to claim 1, further comprising at least two free-flow electrophoresis modules configured to operate in isoelectric focusing mode, zone electrophoresis mode, isotachophoresis mode, or a combination thereof. **Claim 10**: The method according to claim 1, further comprising at least two dynamic filtration modules, at least two tangential flow filtration modules, or at least two free-flow electrophoresis modules operating in parallel. **Claim 11**: A method for purifying a biological product, the method comprising: receiving a heterogeneous mixture containing a biological product via an input line; Generating a first aqueous solution comprising the biological product by removing impurities from the heterogeneous mixture by a cell purification process using a dynamic filtration module by supplying the biological product from at least one output in fluid communication with the input line, wherein the dynamic filtration module comprises a filter membrane extending between a supply reel and a collection reel together with at least one support member having a substantially smooth contact surface, a target region of the filter membrane configured to receive the heterogeneous mixture from at least one output head, and a membrane support member having a substantially smooth contact surface communicating with a vacuum collection system located between the supply reel and the collection reel, the step of generating the first aqueous solution; Transferring the first aqueous solution to a second module capable of exchanging the first aqueous solution with a second aqueous solution to generate a residual liquid comprising the biological product, wherein the second module comprises at least one of a tangential flow filtration device, a cross-flow filtration device, and a diafiltration device, and wherein the second module has at least one second inlet and at least one second outlet configured to allow a flow of fluid between the at least one second inlet and the at least one second outlet, the step of generating the residual liquid; Transferring the residual liquid comprising the biological product from at least one outlet of the second module to a third module having at least one inlet for receiving the flow from at least one second outlet of the second module, wherein the third module comprises at least one free-flow electrophoresis device, and wherein the third module has at least one third inlet and at least one third outlet and is configured to allow a continuous flow of fluid between the third inlet and the third outlet, the step of transferring to the third module; Recovering the biological product, a method comprising. **Claim 12** The method according to claim 11, wherein the tangential flow filtration device further comprises a hollow fiber filter, a flat plate, or a combination thereof. **Claim 13** The method according to claim 12, wherein the hollow fiber filter or the flat plate comprises a membrane having a pore size in the range of about 10 kDa to about 1 μm. **Claim 14**: The method according to claim 12, wherein the hollow fiber filter has an internal diameter in the range of about 0.5 mm to about 5 mm. **Claim 15**: The method according to claim 12, wherein the hollow fiber filter or flat sheet comprises a membrane selected from polyethersulfone (PES), modified polyethersulfone (mPES), polysulfone, mixed cellulose ester, hydrophilic PVDF, or combinations thereof. **Claim 16**: The free-flow electrophoresis apparatus according to claim 11, further comprising at least one electrode channel bubble remover including an electrode channel in liquid contact with the main separation channel through a wall gap, the free-flow electrophoresis apparatus including at least one gas-permeable and hydrophobic membrane configured to generate a bubble-free main separation channel by removing bubbles through a vacuum system, and at least one liquid circuit breaker. **Claim 17**: The method according to claim 11, wherein the method maintains a substantially constant flow rate in the dynamic filtration module, the second module, and the third module, and the flow rate is in the range of about 0.1 mL / min to about 50 mL / min. **Claim 18**: The method according to claim 11, wherein the method of purifying the biological product is performed at a temperature in the range of about 4°C to about 37°C. **Claim 19**: The method according to claim 11, further comprising at least two dynamic filtration modules, each dynamic filtration module having a filter membrane with the same or different pore sizes. **Claim 20**: The method according to claim 11, further comprising at least two free-flow electrophoresis modules configured to operate in isoelectric focusing mode, zone electrophoresis mode, isotachophoresis mode, or combinations thereof. **Claim 21**: The method according to claim 11, further comprising at least two dynamic filtration modules, at least two tangential flow filtration modules, or at least two free-flow electrophoresis modules operating in parallel.

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