Continuous multistage purification with vessel pressure control

Abstract

Methods and compositions for multistage purifications that are capable of being run continuously are provided. Flow rates for individual steps can be controlled, at least in part, by measuring changes of volume of material in airtight intermediate vessels and maintaining flow to be within a desired range.

Description

CONTINUOUS MULTISTAGE PURIFICATION WITH VESSEL PRESSURE CONTROLCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 683,757, filed August 16, 2024, which application is incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Award No. R43TR003975 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Biopharmaceuticals, such as monoclonal antibodies, interferons and others, are used to treat a myriad of diseases including viral infections, cancer, multiple sclerosis and more. Biopharmaceutical processes are operated over a range of scales from process development volumes of around 1 L of cell culture to commercial scales of over 20,000 L. The biopharmaceutical process has historically been time intensive and costly. Production of these therapeutics requires cell culture in a bioreactor, harvest of the cell culture material to separate the protein of interest from the cells and cellular debris, and purification of the harvested material to isolate the target protein from DNA, host cell proteins, and product related impurities. The protein therapeutic is then transferred to a solution suitable for injection into patients. Primary cost drivers include cell culture media and chromatography resins, in particular protein A resins.

[0004] Recent efforts have been made to reduce both the time and cost of the manufacture by introducing continuous processes. Continuous processing in the biopharmaceutical industry is often compared to the assembly line for the manufacture of cars. In comparison to a traditional process where each step is performed one-at-a-time, a continuous process operates all steps simultaneously with fluid connection between the steps at all times. Continuous biopharmaceutical manufacturing provides over four-fold productivity increase over traditional manufacturing methods. By performing multiple purification steps simultaneously, the process can be performed in a reduced amount of time (e.g. four times faster if four steps are performed at the same time) or result in significantly more production over the same amount of time (e.g. four times more output if four steps are performed simultaneously).-1- S&W Docket No. PAK-003.PCT

[0005] For smaller-scale purifications, current methods Biopharmaceuticals, such as monoclonal antibodies, interferons and others, are used to treat a myriad of diseases including viral infections, cancer, multiple sclerosis and more. Biopharmaceutical processes are operated over a range of scales from process development volumes of around 1 L of cell culture to commercial scales of over 20,000 L. The biopharmaceutical process has historically been time intensive and costly. Production of these therapeutics requires cell culture in a bioreactor, harvest of the cell culture material to separate the protein of interest from the cells and cellular debris, and purification of the harvested material to isolate the target protein from DNA, host cell proteins, and product related impurities. The protein therapeutic is then transferred to a solution suitable for injection into patients. Primary cost drivers include cell culture media and chromatography resins, in particular protein A resins.

[0006] Recent efforts have been made to reduce both the time and cost of the manufacture by introducing continuous processes. Continuous processing in the biopharmaceutical industry is often compared to the assembly line for the manufacture of cars. In comparison to a traditional process where each step is performed one-at-a-time, a continuous process operates all steps simultaneously with fluid connection between the steps at all times. Continuous biopharmaceutical manufacturing provides over four-fold productivity increase over traditional manufacturing methods. By performing multiple purification steps simultaneously, the process can be performed in a reduced amount of time (e.g. four times faster if four steps are performed at the same time) or result in significantly more production over the same amount of time (e.g. four times more output if four steps are performed simultaneously).

[0007] For smaller-scale purifications, currently known systems do not comprise measurement devices with acceptable noise levels to monitor the level of vessels between each step of the process and control flow rates to prevent overflow or drainage due to inconsistencies. Thus, there is a need for such systems.INCORPORATION BY REFERENCE

[0008] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.-2- S&W Docket No. PAK-003.PCTBRIEF DESCRIPTION OF THE DRAWINGS

[0009] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0010] Figure 1 shows components and steps of an exemplary continuous biopharmaceutical purification process that utilizes pressure to determine flow.

[0011] Figure 2A shows a first side of an exemplary flow kit.

[0012] Figure 2B shows a second side of an exemplary flow kit.

[0013] Figure 3 shows a schematic of the testing system for pressure level control. The system comprises a product pump (PP), a first pressure sensor (PT), a product valve (VP), a vessel (FK), a second pressure sensor (PT), and a second pressure sensor for each stage. Each stage is fluidly connected to a upstream feed solution or stage output and to a downstream stage or collection vessel.

[0014] Figure 4 shows an exemplary vessel configured with a pressure sensor and sealed with PTFE tape.

[0015] Figure 5 shows a chart showing vessel pressure over time during system startup, illustrating stabilization at a 5 psi set point within approximately 45 minutes and subsequent fluctuation between 4.7 and 5.2 psi.

[0016] Figure 6 shows a chart showing vessel pressure over time across five days of continuous operation, illustrating stable maintenance of the 5 psi set point with fluctuations between 4.7 and 5.2 psiDETAILED DESCRIPTION

[0017] Provided herein are methods and compositions to ensure consistent flow rates across a multi- step continuous biopharmaceutical purification system using intermediate vessel pressure as an indicator of flow rates. A continuous purification system, for example, a continuous biopharmaceutical purification system, is capable of performing two or more purification steps, also referred to herein as stages, in series simultaneously. Any suitable purification component / method may be used in a stage of the process. Although the compositions and methods provided herein may be used in any suitable continuous multistage purification process, for convenience they will be described for a continuous multistage biopharmaceutical purification process. In such a process, purification steps may include-3- S&W Docket No. PAK-003.PCTchromatography, dead-end filtration, tangential flow filtration, and virus inactivation, among others, as described more fully herein. Fluid flows from an upstream step into an intermediate break vessel, also referred to herein simply as an intermediate vessel, to a downstream step. The same flow rate is maintained through all process steps, either continuously or with periodic adjustments. Consistent flow rates ensure that intermediate break vessels do not overflow or drain. A control scheme can ensure that all stages run at the same rate as a rate specified for a stage in the series, such as the last stage, thus allowing a continuous flow multistage production without user intervention for days or weeks on end.

[0018] One strategy to accomplish consistent flow rates is to monitor the liquid level or weight in the intermediate vessel and adjust the flow rate so that the level or weight of the vessel remains constant. In this scenario, the intermediate vessel is typically vented. Provided herein are methods and compositions that adjust flow rate based on the intermediate vessel head pressure, which indirectly indicates the vessel level. The vessel is not vented, i.e., is airtight, so the pressure increases if liquid level in the vessel increases and decreases if liquid level in the vessel decreases. Control may be achieved with any suitable control mechanism, such as a proportional-integral-derivative (PID) control loop. This strategy can be used where measurement of vessel level or weight is impractical, such as for vessel volumes less than 100 ml, and where noise from vibration or physical impact can disrupt the process control. Specifically, this mechanism has been proven to control a four-step continuous process with a vessel volume between 5 and 50 ml over 5 days. Although particularly useful for apparatus and methods where intermediate vessels contain relatively low volumes, the methods and compositions provided herein may be used with larger volumes and flow rates.

[0019] Methods and compositions disclosed herein utilize intermediate vessels between purification stages, where the intermediate vessel is airtight and comprises a pressure sensor to measure pressure in the air space above a liquid in the vessel. An “air space,” as that term is used herein, includes a space occupied by any suitable gas or mixture of gases; typically this will be air, but it need not be. In general, a multistage process with n steps will have n-1 intermediate vessels between stages.

[0020] Liquid material to be purified is flowed through a first purification component to produce a first liquid purification product which enters a first airtight intermediate vessel that is partially filled with the first liquid purification product to leave an air space above the first liquid purification product; a pressure sensor in contact with the air in the air space measures information regarding pressure in the air space and communicates the information to a first-4- S&W Docket No. PAK-003.PCTcontroller configured to adjust one or more flow rates to maintain air pressure in the first intermediate vessel that prevents the vessel from overflowing or draining. The first liquid purification product then flows to a second purification component to produce a second liquid purification product. This cycle can be utilized for any suitable number of purification stages, for example, two stages, three stages, four stages, etc. In certain embodiments, two or more purification systems are used in series, where at least the first system is a multistage purification system, with product from the first system used as starting material for the second system. Thus, a first, multistage, purification system may have 2, 3, or 4 stages, for example, 4 stages, and a second purification system which receives product from the first may have 1, 2, 3, or 4 stages, with a final purified product from the final stage of the second system. The purification product of the final stage may be directed to a collection vessel or other suitable arrangement to finalize purification. It will be appreciated that a liquid purification product from one component may be modified, e.g., by addition of one or more other liquids, e g., buffers and the like, before or after it is flowed to another component. Thus, a “liquid purification product,” as that term is used herein, includes a liquid comprising a product produced by a purification component and does not necessarily have to comprise only that product.

[0021] The intermediate vessel may have any suitable volume, shape and material. In certain embodiments, the intermediate vessel may have a total volume of at least 1, 2, 5, 12, 15, 20, 25, 30, 35, 40, 45, 50, 70, 100, 150, 200, 300, 400, 500, 700, or 1000 ml and / or not more than 2, 5, 12, 15, 20, 25, 30, 35, 40, 45, 50, 70, 100, 150, 200, 300, 400, 500, 700, 1000 or 5000 ml, such as 1-5000 ml, 2-1000 ml, 2-500 ml, 10-200 ml, 10-100 ml, 20-200 ml, or 20- 100 ml. The size of an intermediate vessel and the amount of liquid it can contain can depend size of the purification operation, the ability to control flow rates, and the like. For example, in a system for purification IL to 10L of cell culture per day, intermediate vessel volume may be, e.g., 50 ml. This is merely exemplary, and any suitable intermediate vessel volume for the specific purification system, capacity, and flow rates may be used. It will be appreciated that the liquid capacity of an intermediate vessel for use in methods disclosed herein will be greater than the actual liquid volume in the vessel during operation to allow for an air space in which pressure can be measured. Tubing and other connections can be selected to allow a flow rate into an intermediate vessel suitable for the purification process and for adjustments as necessary to maintain air pressure above liquid in the vessel in a predetermined range, for example, tubing and connections allowing flow rates of of up to-5- S&W Docket No. PAK-003.PCT0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 50, 70, or 100 ml / min, such as up to 1 ml / min, or up to 2 ml / min, or up to 5 ml per min, or up to 10 ml / min, or up to 50 ml / min. In a process utilizing a plurality of intermediate vessels, the vessels may have the same volume or different volumes.

[0022] The pressure in the air space may be any suitable pressure and maintained by adjusting flow rates to keep the pressure within an absolute or percentage range, which keeps flow rate to within a range suitable for the process being used. In certain embodiments, pressure in one or more intermediate vessels is maintained within 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.5, 0.7, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 7.0, or 10 psi of a set point pressure, preferably within 1.0 psi, more preferably within 0.7 psi, yet more preferably within 0.5 psi, still more preferably within 0.2 psi of a set point pressure. In certain embodiments, pressure in one or more intermediate vessels is maintained within 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or 0.1% of a set point pressure, preferably within 10%, more preferably within 7%, even more preferably within 6%, still more preferably within 4%. In certain embodiments the set point pressure is a specific value from a range of at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, or 30 psi and / or not more than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, or 50 psi, preferably a specific value in the range of 1-20, more preferably 1-10 psi, such as a value of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 psi, for example, 5 psi. Advantageously, intermediate vessels are not necessarily fixed in position and can be moved as desired or needed.

[0023] Figure 1 shows an exemplary flow chart of components and steps in a process provided herein. A product feed (101) delivers product to a product pump and valve (102), which directs flow to a first purification component, e.g., a first filter (103). The output of the first purification component, e.g., first filter (103) proceeds into a first intermediate vessel (104). A first pressure sensor (105) is arranged in communication with the first vessel (104). A dashed line connecting the first pressure sensor (105) and the product pump and valve (102) represents a feedback control system configured to adjust pump flow rate based on pressure data from the vessel (104). The product stream continues along a product line (106) into a second purification stage comprising a second product pump and valve (107), a second purification component, e.g., second filter (108), and a second intermediate vessel (109). A second pressure sensor (110) is arranged in communication with the second vessel (109), with a dashed line between the second pressure sensor (110) and the second pump and valve (107) representing a feedback control system for regulating flow based on vessel pressure.-6- S&W Docket No. PAK-003.PCTFrom the second intermediate vessel (109), the treated product proceeds to downstream operations (111). The system can be extended to accommodate two or more purification steps, each comprising a pump and valve, a purification component (e.g., filter in this example), an intermediate vessel, and a pressure sensor arranged to provide feedback control of flow rates to maintain consistent operation across all steps.

[0024] Purification stages may utilize any suitable purification component or components. In biopharmaceutical purification processes, stages can include virus inactivation, dead-end filtration, tangential filtration for ultrafiltration and diafiltration (UFDF), chromatography, and any other suitable process. These steps may be selected in any suitable order. A common biopharmaceutical purification process will operate a bind and elute capture chromatography step, a virus inactivation step, followed by an anion exchange flow through chromatography step and then a bind and elute cation exchange chromatography step. Lastly, a UFDF step with excipient addition will be performed before the drug substance is suitable for patients. However, the advanced automation is not limited to a certain sequence of steps. Further details regarding purification components and stages, hardware configurations, and other aspects of continuous multistage pharmaceutical purifications systems may be found in US Patent Publication No. 20220168668.

[0025] Virus inactivation A virus inactivation step involves the addition of a solution that destroys viral particles, commonly with a low pH or detergent, followed by a hold of the of the product with the inactivating solution for a specified amount of time. In continuous manufacturing, the hold is achieved with continuous flow through a residence chamber. Several residence chamber designs are used in the industry, including a chromatography column filled with size exclusion (SEC) or other inert resin, a coiled tube or “jib”, and other tortuous paths. Continuous flow through a residence chamber such as a chromatography column, jib or other residence chamber device provides the specified hold time to achieve the required virus log reduction values (LRV). In certain embodiments a virus inactivation stage performs an inline pH adjustment and monitors time in the residence chamber. If a batch of material is held within the residence chamber beyond a preset time, the system will dump all such material to waste until the residence time of exiting material is within specification. The flow is continuously moving, with the automated valves switching to direct the flow to the waste line or collection vessel as programmed. The system also allows for inline pH, conductivity, or volumetric ratio buffer addition at the outlet of the virus inactivation step to meet target ranges for the subsequent stage. Upon exiting the residence-7- S&W Docket No. PAK-003.PCTchamber, the product stream may be pH adjusted, commonly to a neutral pH to improve stability of the product. Other modes of continuous virus inactivation involve multiple hold tanks that alternate between fill and hold and drain modes at staggered times.

[0026] Dead-end filtration. Dead end filtration steps may include sterile (e.g. 0.2 pm filter), virus, depth (e.g. diatomaceous earth) pre-filters, or others. Filters are often sized to ensure that pressure limits are not exceeded at maximum throughput (L / m2) within a desired lifetime (e.g. hours). In a traditional process a single filter may be used for the entire batch. In comparison, for the continuous process a filter is typically sized for changeout over a specific duration such as every 24 or 48 hours. This ensures minimal hold-up volume in the system, which reduces the total processing time for a discrete amount of product and can improve product quality for an unstable molecule. In traditional processes, the filtration step may be controlled based on a flow rate set point or a pressure set point. For pressure control, the flow rate is changed to reach the desired pressure and process the material as quickly as possible. In a continuous process, flow control is typically used (although in some instances pressure control may be used). The flow rate is based on the mass flow control of the process as a whole, which contains multiple unit operations. The pressure is expected to slowly increase over time as the filter capacity is reached but not exceeding the limit.

[0027] Diafiltration. A diafiltration step exchanges the buffer salts in a solution and is commonly used to change the solution properties prior to a chromatography step or to place the product into a preferred solution prior to injection into patients. The product containing solution is run over a membrane such that the direction of flow is tangential to the membrane surface. A pressure differential forces the buffer solution through the membrane while the protein product is retained due to the pore size. In a traditional ultrafiltration / diafiltration (UFDF) process, material is re-circulated between the membrane and a retentate tank and flow continues in this loop until a desired amount of buffer is removed and the product is concentrated to a desired set point. In a continuous process the concentration is achieved in a single step with a series of tangential flow filtration (TFF) membranes or a membrane length sufficient that the concentration can be achieved in a single pass (single pass TFF). The membranes are commonly four times as long as the traditional TFF membranes to achieve this goal. Diafiltration buffer is added to the product, typically at the same volume that the buffer solution is removed through the membrane. A series of concentration and buffer addition steps can achieve the desired buffer exchange. For example, a lOx concentration and lOx dilution achieves 90% buffer exchange. Repeating this dilution and-8- S&W Docket No. PAK-003.PCTconcentration step twice results in 99% buffer exchange and repeating a third time results in 99.9% buffer exchange, which is within typical ranges for a final UFDF step of a biopharmaceutical process.

[0028] Chromatography , Chromatography steps use chromatography resin or membranes to bind either the solubilized product or impurities, while allowing the non-bound components to be directed to waste. Suitable resins for use in the biopharmaceutical industry include protein A resins, blue Sepharose, anion exchange, cation exchange, hydrophobic interaction and others. Chromatography steps that bind the impurities but not the product are referred to as “flow through chromatography”. Chromatography steps that bind the product are typically referred to as “bind and elute”. Buffer solutions with a selective pH and / or conductivity range are run through the column to selectively remove certain impurities and separate them from the product. A typical bind and elute chromatography step will include an equilibration buffer passed over the column before and after the product stream is loaded onto the column. Then, one or more wash solutions are used before an eluate solution that removes the product of interest. Strip steps are used to remove any remaining proteinaceous material from the resin before a sanitization step. One full sequence of the buffer steps is considered a cycle and these cycles may be repeated over one hundred times before the resin has degraded to a condition where binding of the product is significantly reduced due to sanitization buffers and other reasons. In a traditional mode of processing, three to five cycles are performed with a relatively large column for a given batch. In continuous processing, fifty or even one hundred cycles may be performed with a small column in a single batch. The number of cycles is maximized in order to use the minimal size column and save on expensive chromatography resins. Continuous manufacturing removes the need to run the chromatography step quickly to enable the product to be forward processed by the subsequent step, as these subsequent steps can be performed simultaneously while maintaining or reducing the total run time.

[0029] One or more dual chromatography steps may be utilized. _A dual column chromatography step enables bind and elutes chromatography methods to be performed in a semi-continuous manner. Two stages of a system can be dedicated to a dual column chromatography step. The first product pump of the two stages continuously feeds product intermediate from the upstream step onto one of the two columns where it binds to the resin in the packed column. A sequence of buffers is flushed through the other column to remove impurities and / or elute product intermediately. After the buffer sequence and product load step are completed, the-9- S&W Docket No. PAK-003.PCTfirst and second valves of each chromatography stage switch positions so that the column that was previously in the product load state is now flushed with a sequence of buffers and the column that was previously flushed with buffers now receives product. The columns continue to switch roles in this manner for the duration of the process. The sequence of the added buffers can be adjusted depending on the material being processed, and are taken from the group comprising equilibration, wash, elution, strip, and sanitization buffers. Each such buffer will flow through the column and then to waste, except elution buffer, which removes the product. This elution buffer, and the product intermediate eluted off the column, can be collected in an auxiliary vessel associated with the system since the volume to be collected is larger than the intermediate vessel volume. The product in the auxiliary vessel is then fed either to the subsequent stage or directly to the final stage in the process. The auxiliary vessel holds approximately five elution volumes and is mixed to ensure there is no spike in pH, conductivity, or concentration of the product fed to the subsequent stage.

[0030] A flow kit designed for multi- or single-use can be installed to manage fluid flow through the stages of the system. An airtight intermediate vessel comprising a pressure sensor, such as disclosed herein, can be used as part of a flow kit, either integral to the flow kit, or attached thereto. One flow kit is installed for each stage that will be operated. In certain embodiments, a flow kit comprises connectors, tubing, one or more static mixers, one or more pH probes, one or more conductivity probes, one or more sample ports, and an airtight intermediate vessel comprising a pressure sensor. The intermediate vessel can be any suitable size, depending on the process, such as a 50 ml conical tube with an inlet, outlet, optional vent and pressure sensor. Tubing line sizes for the flow kit can be chosen based on the volume of the vessel and expected flow rates, e.g., a 50 ml vessel tubing can be 1 / 16” and 1 / 32” to manage flow rates up to 10 mL / min. Feed material is supplied to the flow kit from the feed pump. The feed material may mix with buffer through a tee on the flow kit. Material then travels through a first static mixer to a pH probe, a conductivity sensor and a pressure sensor. The product then leaves the flow kit and proceeds to the first product valve where it is directed either to a purification component, e.g., filter or column, or to waste. From the outlet of the purification component, e.g., filter or column, the product proceeds back to the flow kit where it may mix with additional buffer through a tee, is directed through a second static mixer, a second pH probe, a second conductivity sensor and a second pressure sensor. The fluid stream then leaves the flow kit and enters the second product valve which directs the flow to waste or to an intermediate vessel located on the flow kit or a-10- S&W Docket No. PAK-003.PCTseparate collection vessel (e.g. for chromatography eluate or final stage in the process). This flow path is repeated for each of four stages.

[0031] Figure 2A shows a first side of an exemplary flow kit and directions of fluid flow. Feed material (206) is supplied to a first side (200) of the flow kit from a feed pump (not shown). The feed material may be combined with buffer material (207) supplied from a buffer pump (not shown) through a tee (208) positioned on the flow kit. The resulting mixture is directed through a first static mixer (209), followed by a first pH probe (210), a first conductivity sensor (211), and a first pressure sensor (212). The processed fluid then exits the flow kit (213) and is directed to a first product valve, which selectively routes the stream to a first purification stage, e.g., filter or column, or to waste.

[0032] Figure 2B shows the product stream returning (215) from the outlet of the purification stage, e.g., filter or column, to a second side (214) of the flow kit. At this stage, the product may be combined with additional buffer (216) supplied through a second tee (217). The stream then passes through a second static mixer (218), a second pH probe (219), a second conductivity sensor (220), a UV sensor (221), and a second pressure sensor (222). The fluid stream exits the flow kit (223) and proceeds to a second product valve, which directs the stream either to waste or to an intermediate vessel (201) located on, or integrated into, the flow kit or to a separate collection vessel (e.g., for chromatography eluate or a final-stage product). This flow path is repeated for each of up to four stages. The intermediate vessel receives the fluid stream through an inlet (202) which then flows out through an outlet (203) to a second purification stage. Pressure in the intermediate vessel is detected by a pressure sensor (205). The intermediate vessel may include an optional sample port (204). All flow kit components can mounted onto a scaffold (224), which can be modular in design to allow user customization.

[0033] Control strategies to operate multiple complex steps simultaneously can be deployed on systems of all scales. The control strategies operate each step at the same rate to ensure that a constant feed is supplied to the downstream step and intermediate vessels do not drain or overflow. These control strategies control not only the rate through a given step but the rate of the process as a whole and enable an entire process to increase or decrease in rate with a single set point update by the user. While maintaining these process rates, the control strategies ensure that all process parameters are met for any given step. These process parameters can include pH set points for inline titration, conductivity ranges after a buffer adjustment, linear velocities through a chromatography column, pressures on a filter, and the-11- S&W Docket No. PAK-003.PCTlike. The high degree of automation employed enables production without user intervention for days or weeks on end.

[0034] The control scheme used to achieve a process where all stages of the process run at the same rate, sets the following:1. The feed pump for the last stage in series is controlled to a flow rate set point2. The feed pumps for the upstream stages are controlled by a proportional-integral- derivative (PID) control loop set to maintain the downstream vessel level.

[0035] The overall flow rate may be any suitable flow rate. In certain embodiments, the overall flow rate is at least 0.1, 0.2, 0.5, 0.7, 1, 1.2, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 70, 100, 150, or 200 mL / min and / or not more than 0.2, 0.5, 0.7, 1, 1.2, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 70, 100, 150, 200 or 500 mL / min, such as 0.1-500 mL / min, or such as 0.2-200 mL / min, or such as 0.2-100 mL / min, or such as 0.1-50 mL / min, or such as 0.1-20 mL / min, or such as 0.5-20 mL / min, or such as 0.5-15 mL per minute, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15 mL per minute. In certain embodiments the physical response of each individual intermediate vessel level changes causes that vessel level control to “respond” and results in overarching control without direct communication, e.g., digital communication, between various controllers.

[0036] This control scheme ensures that all stages run at the same rate as the rate specified for the last stage in series. If a stage runs slower than the overall process, the downstream intermediate vessel drains, and the PID control loop increases the speed of the feed pump to bring the intermediate vessel level back to the target volume. If a stage runs faster, the downstream intermediate vessel fills and the PID control loop decreases the speed of the feed pump to compensate. In addition to maintaining a consistent rate across steps, this control strategy allows for the rate of the entire process to be increased or decreased based on a single set point change, the flow rate of the last feed pump in series. The flow rate change cascades upstream to the remaining steps as a result of the vessel level control.

[0037] Another level of the control strategy allows any stage, and not solely the last stage, to define the rate of the entire process. This is done to ensure that the critical parameters are met for all process steps and ensure that the step that is the most sensitive to the process rate sets the target for that rate. The sensitivity to the process rate is defined by the step type and the criticality is typically ranked in the order of virus inactivation (most critical), chromatography, and lastly filtration. The throughput of each step is monitored and the automation controls the flow rate set point of the last step in the process to meet the target-12- S&W Docket No. PAK-003.PCTthroughput of the most critical step. The automation will also ensure the throughput through all other steps is within their allowable ranges and not exceeded.

[0038] The system can thus run continuously, without the need for user intervention until, e.g., purification is complete, for example, for hours or days of continuous operation without user intervention.EXAMPLE

[0039] Continuous protein purification using pressure-based control.

[0040] The purpose of this study was to evaluate a continuous purification system configured with up to four flow-through purification stages and vessels. The experiment sought to demonstrate that pressure-based vessel level monitoring and feedback control could maintain continuous operation.

[0041] Figure 3 illustrates the purification system. The system comprises a product pump (PP), a first pressure sensor (PT), a product valve (VP), a vessel (FK), a second pressure sensor (PT), and a second pressure sensor for each stage. Each stage is fluidly connected to a upstream feed solution or stage output and to a downstream stage or collection vessel.

[0042] For this test water was used as an aqueous fluid to simulate buffered and product solutions. The purification process flow includes directed a feed stream through a series of product pumps and valves, each pump delivering material into a respective vessel. Pressure sensors were arranged in communication with the vessels and integrated into feedback control loops for regulating flow rates based on vessel level pressure.

[0043] Process conditions were as follows: max feed pressure - 20 PSI; max delta pressure - 20 PSI; swap time - 96 hours; chase volume 0.25 L; 5 PSI setpoint volume; target flow rates - 1 mL / min; target vessel pressure level - 5 PSI.

[0044] Reservoir sealing integrity was ensured by positioning a gasket within the reservoir cap groove and applying wo layers of PTFE thread seal tape to the connectors. The pressure controller was mounted above the reservoirs to minimize risk of backflow. Hydrostatic pressure contributions (~1 mbar per cm of liquid column) were taken into account. Prior to opening any pressurized reservoir, pressure was released to atmospheric conditions to prevent spills or equipment damage. An exemplary reservoir with fluid connections is shown in Figure 4.

[0045] Results Continuous purification was successfully maintained for 96 hours. Pressure sensors in each vessel provided reproducible correlations between headspace pressure and liquid level, allowing for accurate vessel level control at the 5 psi setpoint. No leaks were observed-13- S&W Docket No. PAK-003.PCTduring operation, and the PTFE sealing strategy proved effective. The system operated within the defined feed pressure and differential pressure limits. Figure 5 is a chart showing vessel pressure (Y-axis) as a function of time (X-axis) during continuous operation of the purification system. At initiation of system operation, pressure increases toward the vessel level control set point. Within approximately 45 minutes, the measured pressure stabilizes at the 5 psi set point. Following stabilization, vessel pressure remains within a narrow control range, fluctuating between about 4.7 psi and 5.2 psi over the duration of the run. This result demonstrates the effectiveness of pressure-based vessel level control in maintaining stable operation of the continuous purification process. Figure 6 is a chart showing vessel pressure (Y-axis) as a function of time (X-axis) over five days of continuous operation of the purification system. Throughout the entirety of the five-day run, the measured pressure remained within a narrow operating range, fluctuating between about 4.7 psi and 5.2 psi. The pressure values for each of the 3 second pressure sensors are shown to be maintained within these values. These results confirm that the pressure-based vessel level control strategy maintained stable operation of the purification process over extended continuous operation.

[0046] Table 1 illustrates vessel level measurements obtained from the three intermediate vessels (FK1-FK3) during continuous operation of the purification system. At the initiation of operation, vessel volumes were between 13.5 and 15 mL. Over the course of operation, vessel levels increased steadily in response to controlled flow through the purification stages. By 3.25 hours, FK1, FK2, and FK3 levels increased to 18.5 mL, 17.0 mL, and 16.5 mL, respectively. At 25.6 hours, vessel levels reached 22.5 mL (FK1), 19.5 mL (FK2), and 18.0 mL (FK3). Levels continued to rise over subsequent days, with FK1 achieving 32.0 mL, FK2 reaching 26.5 mL, and FK3 reaching 22.5 mL by 88.2 hours.

[0047] These results demonstrate that vessel volumes can be reproducibly controlled and tracked by pressure monitoring across multiple vessels. The progressive and consistent increase in vessel levels over nearly six days of continuous operation validates the robustness of the pressure-based vessel level control strategy.-14- S&W Docket No. PAK-003.PCTTable 1. Vessel levels (mL) measured over time for three intermediate vessels (FK1-FK3) during continuous purification.

[0048] This example demonstrate that a continuous biopharmaceutical purification system can be operated using pressure-based vessel level control without requiring buffer additions or inline analytical sensors. The use of modular flow kits enables flexibility in configuration, while maintaining long-duration, leak-free operation. These results validate the system’s suitability for continuous protein purification in a simplified format.EMBODIMENTS

[0049] In embodiment 1 provided is a purification system comprising at least two purification stages, wherein the system comprises (i) a first purification component; (ii) a first intermediate vessel fluidly connected to the first purification component; and (iii) a second purification component fluidly connected to the intermediate vessel; wherein the first intermediate vessel is airtight and is configured to receive material produced by the first purification component and to release material to the second purification component, and wherein the first intermediate vessel comprises a pressure sensor configured to communicate a first intermediate vessel pressure to a first controller. In embodiment 2 provided is the system of embodiment 1 wherein the first controller is configured to adjust one or more flow rates to maintain the first intermediate vessel pressure in a range that prevents the first intermediate vessel from overflowing or draining. In embodiment 3 provided is the system of embodiment 1 or 2 further comprising (iv) a second intermediate vessel fluidly connected to the second purification component wherein the second intermediate vessel is airtight and is configured to receive material produced by the second purification component and to release material to a third purification component, and wherein the second intermediate vessel comprises a pressure sensor configured to communicate a second intermediate vessel pressure to a second controller. In embodiment 4 provided is the system of embodiment 3 wherein the second controller is configured to adjust one or more flow rates to maintain the second intermediate vessel pressure in a range that prevents the second intermediate vessel-15- S&W Docket No. PAK-003.PCTfrom overflowing or draining. In embodiment 5 provided is the system of embodiment 3 or 4 further comprising (v) a third intermediate vessel fluidly connected to the third purification component wherein the third intermediate vessel is airtight and is configured to receive material produced by the third purification component and to release material to a fourth purification component, wherein the third intermediate vessel comprises a pressure sensor configured to communicate a third intermediate vessel pressure to a third controller. In embodiment 6 provided is the system of embodiment 5 wherein the third controller is configured to adjust one or more flow rates to maintain the third intermediate vessel pressure in a range that prevents the third intermediate vessel from overflowing or draining. In embodiment 7 provided is the system of any previous embodiment further comprising a collection vessel for collecting material from the final purification stage. In embodiment 8 provided is the system of any previous wherein the first, second, and / or third controllers operate in tandem with a fourth controller that sets an overall flow rate for the entire system. In embodiment 9 provided is the system of embodiment 8 wherein the fourth controller is not in digital communication with the first, second, and / or third controllers. In embodiment 10 provided is the system of any previous embodiment wherein one or more of the controllers comprise a proportional-integral-differential (PID) control loop. In embodiment 11 provided is the system of any previous embodiment wherein the purification components comprise one or more components to perform virus inactivation, dead-end filtration, tangential filtration, or chromatography. In embodiment 12 provided is the system of any previous embodiment wherein one or more intermediate vessel volumes are in the range of 10-200 mb. In embodiment 13 provided is the system of embodiment 12 wherein one or more intermediate vessel volumes are in the range of 20-100 m . In embodiment 14 provided is a composition comprising (i) an intermediate vessel for use in a multistage biopharmaceutical purification system, wherein the intermediate vessel is airtight and comprises (a) an inlet line to allow liquid from a first component of the purification process to enter the intermediate vessel and flow into a pool of liquid that partially fills the intermediate vessel;(b) an outlet line to allow liquid from the pool of liquid to flow from the intermediate vessel to a second stage of the purification process; and(c) a pressure sensor in contact with air above the pool of liquid to measure information regarding air pressure and transmit the information to a controller. In embodiment 15 provided is the composition of embodiment 14 further comprising the controller. In embodiment 16 provided is the composition of embodiment 15 wherein the controller comprises a PID control loop. In embodiment 17 provided is the-16- S&W Docket No. PAK-003.PCTcomposition of any one of embodiment 14 through 16 wherein the intermediate vessel has a volume of 10-200 mL. In embodiment 18 provided is the composition of embodiment 17 wherein the intermediate vessel has a volume of 20-100 mL. In embodiment 19 provided is the composition of any one of embodiments 14 through 18 further comprising a static mixer fluidly connected to the intermediate vessel. In embodiment 20 provided is the composition of any one of embodiments 14 through 19 further comprising a pH probe. In embodiment 21 provided is the composition of any one of embodiments 14 through 20 further comprising a UV sensor. In embodiment 22 provided is the composition of any one of embodiments 14 through 21 further comprising a conductivity sensor. In embodiment 23 provided is a flow kit for use in a multistage purification process comprising(i) a first side comprising (a) a first conduit for feed material and a second conduit, different from the first, for buffer; (b) a tee connector fluidly connected to the first and second conduits and to a static mixer to mix buffer and feed to produce mixed buffer and feed; and(c) an outlet to direct the mixed buffer and feed to a purification stage that produces a purification product; and(ii) a second side comprising (a) a third conduit to flow the purification product and a fourth conduit, different from the third, to flow buffer;(b) a tee connector fluidly connected to the first and second conduits and to a static mixer to mix buffer and the purification product, to produce mixed buffer and product, and(c) a valve fluidly connected to the static mixer to send the mixed buffer and product to either waste or to(iii) an airtight intermediate vessel comprising a pressure sensor operably connected to a control system for controlling one or more flows into or out of the intermediate vessel. In embodiment 24 provided is a multistage purification method comprising(i) flowing liquid material to be purified through a first purification component to produce a first liquid purification product;(ii) flowing the first liquid purification product into a first intermediate vessel, wherein the first intermediate vessel is airtight and wherein the first liquid purification product partially fills the first intermediate vessel to leave an air space above the first liquid purification product, and wherein the first intermediate vessel comprises a first pressure sensor in contact with the air in the air space that measures information regarding pressure in the air space; and(iii) flowing the first liquid purification product from the first intermediate vessel to a second purification component to produce a second liquid purification product, wherein the first pressure sensor communicates the information regarding pressure in the air space to a first controller configured to adjust one or more flow rates to maintain air pressure in the first intermediate vessel in a range that prevents the vessel from overflowing or draining. In-17- S&W Docket No. PAK-003.PCTembodiment 25 provided is the method of embodiment 24 comprising(iv) flowing the second liquid purification product, optionally combined with further material, into a second intermediate vessel, wherein the second intermediate vessel is airtight and wherein the second liquid purification product partially fills the second intermediate vessel to leave an air space above the second liquid purification product, and wherein the second intermediate vessel comprises a second pressure sensor in contact with the air in the air space that measures information regarding pressure in the air space; and (v) flowing the second liquid purification product from the second intermediate vessel to a third purification component to produce a third liquid purification product wherein the second pressure sensor communicates the information regarding pressure in the air space to a second controller configured to adjust one or more flow rates to maintain air pressure in the second intermediate vessel in a range that prevents the vessel from overflowing or draining. In embodiment 26 provided is the method of embodiment 25 comprising(vi) flowing the third liquid purification product, optionally combined with further material, into a third intermediate vessel, wherein the third intermediate vessel is airtight and wherein the third liquid purification product partially fills the third intermediate vessel to leave an air space above the third liquid purification product, and wherein the third intermediate vessel comprises a third pressure sensor in contact with the air in the air space that measures information regarding pressure in the air space; and(vii) flowing the third liquid purification product from the third intermediate vessel to a fourth purification component wherein the third pressure sensor communicates the information regarding pressure in the air space to a third controller configured to adjust one or more flow rates to maintain air pressure in the second intermediate vessel in a range that prevents the vessel from overflowing or draining. In embodiment 27 provided is the method of any one of 24 through 26 wherein air pressure in the intermediate container or containers is maintained within 5 psi of a set point pressure. In embodiment 28 provided is the method of embodiment 27 wherein air pressure in the intermediate container or containers is maintained within 1 psi of a set point pressure. In embodiment 29 provided is the method of embodiment 27 wherein air pressure in the intermediate container or containers is maintained within 0.5 psi of a set point pressure. In embodiment 30 provided is the method of any one of embodiments 24 through 29 wherein the final purification component is a collection vessel. In embodiment 31 provided is the method of any one of embodiments 24 through 30 wherein the first, second, and / or third controllers operate in tandem with a fourth controller that sets an overall flow rate for the entire system. In embodiment 32 provided is the method-18- S&W Docket No. PAK-003.PCTof embodiment 31 wherein the fourth controller is not in digital communication with the first, second, and / or third controllers. In embodiment 33 provided is the method of embodiment 31 or 32 wherein the overall flow rate is 0.2-200 mL / min. In embodiment 34 provided is the method of embodiment 33 wherein the overall flow rate is 0.5-100 mL / min. In embodiment 35 provided is the method of embodiment 33 wherein the overall flow rate is 0.5-15 mL / min. In embodiment 36 provided is the method of any one of embodiments 24 through 35 wherein one or more of the controllers comprise a proportional-integral- differential (PID) control loop. In embodiment 37 provided is the method of any one of embodiments 24 through 36 wherein the purification components comprise one or more components to perform virus inactivation, dead-end filtration, tangential filtration, or chromatography.

[0050] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.-19- S&W Docket No. PAK-003.PCT

Claims

What is claimed is:1 . A purification system comprising at least two purification stages, wherein the system comprises(i) a first purification component;(ii) a first intermediate vessel fluidly connected to the first purification component; and(iii) a second purification component fluidly connected to the intermediate vessel; wherein the first intermediate vessel is airtight and is configured to receive material produced by the first purification component and to release material to the second purification component, and wherein the first intermediate vessel comprises a pressure sensor configured to communicate a first intermediate vessel pressure to a first controller.

2. The system of claim 1 wherein the first controller is configured to adjust one or more flow rates to maintain the first intermediate vessel pressure in a range that prevents the first intermediate vessel from overflowing or draining.

3. The system of claim 1 or 2 further comprising(iv) a second intermediate vessel fluidly connected to the second purification component wherein the second intermediate vessel is airtight and is configured to receive material produced by the second purification component and to release material to a third purification component, and wherein the second intermediate vessel comprises a pressure sensor configured to communicate a second intermediate vessel pressure to a second controller.

4. The system of claim 3 wherein the second controller is configured to adjust one or more flow rates to maintain the second intermediate vessel pressure in a range that prevents the second intermediate vessel from overflowing or draining.

5. The system of claim 3 or 4 further comprising(i) a third intermediate vessel fluidly connected to the third purification component-20- S&W Docket No. PAK-003.PCTwherein the third intermediate vessel is airtight and is configured to receive material produced by the third purification component and to release material to a fourth purification component, wherein the third intermediate vessel comprises a pressure sensor configured to communicate a third intermediate vessel pressure to a third controller.

6. The system of claim 5 wherein the third controller is configured to adjust one or more flow rates to maintain the third intermediate vessel pressure in a range that prevents the third intermediate vessel from overflowing or draining.

7. The system of any previous claim further comprising a collection vessel for collecting material from the final purification stage.

8. The system of any previous wherein the first, second, and / or third controllers operate in tandem with a fourth controller that sets an overall flow rate for the entire system.

9. The system of claim 8 wherein the fourth controller is not in digital communication with the first, second, and / or third controllers.

10. The system of any previous claim wherein one or more of the controllers comprise a proportional-integral-differential (PID) control loop.

11. The system of any previous claim wherein the purification components comprise one or more components to perform virus inactivation, dead-end filtration, tangential filtration, or chromatography.

12. The system of any previous claim wherein one or more intermediate vessel volumes are in the range of 10-200 mL.

13. The system of claim 12 wherein one or more intermediate vessel volumes are in the range of 20-100 mL.-21- S&W Docket No. PAK-003.PCT14. A composition comprising(i) an intermediate vessel for use in a multistage biopharmaceutical purification system, wherein the intermediate vessel is airtight and comprises(a) an inlet line to allow liquid from a first component of the purification process to enter the intermediate vessel and flow into a pool of liquid that partially fills the intermediate vessel;(b) an outlet line to allow liquid from the pool of liquid to flow from the intermediate vessel to a second stage of the purification process; and(c) a pressure sensor in contact with air above the pool of liquid to measure information regarding air pressure and transmit the information to a controller.

15. The composition of claim 14 further comprising the controller.

16. The composition of claim 15 wherein the controller comprises a PID control loop.

17. The composition of any one of claim 14 through 16 wherein the intermediate vessel has a volume of 10-200 mb.

18. The composition of claim 17 wherein the intermediate vessel has a volume of 20-100 mb.

19. The composition of any one of claims 14 through 18 further comprising a static mixer fluidly connected to the intermediate vessel.

20. The composition of any one of claims 14 through 19 further comprising a pH probe.

21. The composition of any one of claims 14 through 20 further comprising a UV sensor.

22. The composition of any one of claims 14 through 21 further comprising a conductivity sensor.-22- S&W Docket No. PAK-003.PCT23. A flow kit for use in a multistage purification process comprising(i) a first side comprising(a) a first conduit for feed material and a second conduit, different from the first, for buffer;(b) a tee connector fluidly connected to the first and second conduits and to a static mixer to mix buffer and feed to produce mixed buffer and feed; and(c) an outlet to direct the mixed buffer and feed to a purification stage that produces a purification product; and(ii) a second side comprising(a) a third conduit to flow the purification product and a fourth conduit, different from the third, to flow buffer;(b) a tee connector fluidly connected to the first and second conduits and to a static mixer to mix buffer and the purification product, to produce mixed buffer and product, and(c) a valve fluidly connected to the static mixer to send the mixed buffer and product to either waste or to(iii) an airtight intermediate vessel comprising a pressure sensor operably connected to a control system for controlling one or more flows into or out of the intermediate vessel.

24. A multistage purification method comprising(i) flowing liquid material to be purified through a first purification component to produce a first liquid purification product;(ii) flowing the first liquid purification product into a first intermediate vessel, wherein the first intermediate vessel is airtight and wherein the first liquid purification product partially fills the first intermediate vessel to leave an air space above the first liquid purification product, and wherein the first intermediate vessel comprises a first pressure sensor in contact with the air in the air space that measures information regarding pressure in the air space; and(iii) flowing the first liquid purification product from the first intermediate vessel to a second purification component to produce a second liquid purification product, wherein the first pressure sensor communicates the information regarding pressure in the air space to a first controller configured to adjust one or more flow rates to maintain air pressure-23- S&W Docket No. PAK-003.PCTin the first intermediate vessel in a range that prevents the vessel from overflowing or draining.

25. The method of claim 24 comprising(i) flowing the second liquid purification product, optionally combined with further material, into a second intermediate vessel, wherein the second intermediate vessel is airtight and wherein the second liquid purification product partially fills the second intermediate vessel to leave an air space above the second liquid purification product, and wherein the second intermediate vessel comprises a second pressure sensor in contact with the air in the air space that measures information regarding pressure in the air space; and(ii) flowing the second liquid purification product from the second intermediate vessel to a third purification component to produce a third liquid purification product wherein the second pressure sensor communicates the information regarding pressure in the air space to a second controller configured to adjust one or more flow rates to maintain air pressure in the second intermediate vessel in a range that prevents the vessel from overflowing or draining.

26. The method of claim 25 comprising(i) flowing the third liquid purification product, optionally combined with further material, into a third intermediate vessel, wherein the third intermediate vessel is airtight and wherein the third liquid purification product partially fills the third intermediate vessel to leave an air space above the third liquid purification product, and wherein the third intermediate vessel comprises a third pressure sensor in contact with the air in the air space that measures information regarding pressure in the air space; and(ii) flowing the third liquid purification product from the third intermediate vessel to a fourth purification component wherein the third pressure sensor communicates the information regarding pressure in the air space to a third controller configured to adjust one or more flow rates to maintain air pressure in the second intermediate vessel in a range that prevents the vessel from overflowing or draining.-24- S&W Docket No. PAK-003.PCT27. The method of any one of 24 through 26 wherein air pressure in the intermediate container or containers is maintained within 5 psi of a set point pressure.

28. The method of claim 27 wherein air pressure in the intermediate container or containers is maintained within 1 psi of a set point pressure.

29. The method of claim 27 wherein air pressure in the intermediate container or containers is maintained within 0.5 psi of a set point pressure.

30. The method of any one of claims 24 through 29 wherein the final purification component is a collection vessel.

31. The method of any one of claims 24 through 30 wherein the first, second, and / or third controllers operate in tandem with a fourth controller that sets an overall flow rate for the entire system.

32. The method of claim 31 wherein the fourth controller is not in digital communication with the first, second, and / or third controllers.

33. The method of claim 31 or 32 wherein the overall flow rate is 0.2-200 mL / min.

34. The method of claim 33 wherein the overall flow rate is 0.5-100 mL / min.

35. The method of claim 33 wherein the overall flow rate is 0.5-15 mL / min.

36. The method of any one of claims 24 through 35 wherein one or more of the controllers comprise a proportional-integral-differential (PID) control loop.

37. The method of any one of claims 24 through 36 wherein the purification components comprise one or more components to perform virus inactivation, dead-end filtration, tangential filtration, or chromatography.-25- S&W Docket No. PAK-003.PCT

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