In-process verification of pH probe calibration status

Abstract

The automated system and method for low pH virus inactivation involves adding an elution pool to a first vessel with acid. When the pH probe in the first vessel measures a sufficiently low pH, the pool is transferred to a second vessel, where the pH is checked again, and the pool is held, neutralized, filtered, and transferred to a third vessel for a sufficient time to reduce the virus concentration to a safe level. During this time, the first vessel is filled with a buffer solution of known pH, which is checked against a reading from the pH probe in the first vessel to determine whether recalibration is required. After the pool is transferred to the third vessel, the second vessel is filled with a buffer solution of known pH, which is checked against a reading from the pH probe in the second vessel to determine whether recalibration is required. This process is repeated when the buffer solution of known pH is discarded and a new elution pool is added to the first vessel.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 111,502, entitled "IN - PROCESS VERIFICATION OF CALIBRATION STATUS OF PH PROBES," filed on November 9, 2020, and U.S. Provisional Patent Application No. 63 / 168,608, entitled "IN - PROCESS VERIFICATION OF CALIBRATION STATUS OF PH PROBES," filed on March 31, 2021, the disclosures of each of which are hereby incorporated by reference in their entireties.

[0002] The present disclosure generally relates to virus inactivation, and more specifically to automated virus inactivation techniques that include an automated pH adjustment cycle.

Background Art

[0003] The background description provided herein is for the purpose of generally presenting the context of the present disclosure. Within the scope described in the background art section, the achievements of the inventors listed herein and aspects of the description that would not be prior art as of the filing date without those achievements are not expressly or implicitly admitted as prior art to the present disclosure.

[0004] The manufacture of recombinant biological products for therapeutic use using cell culture processes carries an inherent risk of contamination by viral contaminants. Such contaminants can originate from various sources, including starting materials, the use of animal-derived reagents, and / or contamination of the manufacturing system due to malfunctions in GMP processing. Therefore, regulatory authorities recommend that biomanufacturing processes have dedicated viral inactivation and removal steps, and require manufacturers to verify viral removal and inactivation to ensure the safety of recombinant biological products. The viral inactivation step focuses on enveloped viruses (e.g., retroviruses), while the viral filtration step removes viruses unaffected by the inactivation method (non-enveloped viruses). Several commonly used methods for inactivating enveloped viruses include thermal destruction of the envelope, the use of solvents and / or detergents, and / or low-pH treatment. When viruses are inactivated using inactivators such as detergents, further purification is required to remove the detergents. Advantageously, low-pH viral inactivation does not require further purification to remove the inactivators.

[0005] Viral inactivation can be performed throughout the entire downstream purification process. Inducing factors that help determine the location of the viral inactivation unit operation include the impact of the viral inactivation step on subsequent unit operations, the extent to which inactivators such as detergents or solvents can be removed in subsequent downstream steps, and whether the conditions of a particular unit operation match those of the viral inactivation step. For example, the viral inactivation unit operation is typically performed after the first step of downstream processing, following the collection of cell culture medium from a bioreactor. Typically, this is an affinity chromatography step that removes almost all impurities from the collected fluid. Protein A is commonly used as affinity chromatography for proteins with an Fc region, such as antibodies. Since elution from a protein A chromatography column is typically performed at a lower pH, a low-pH viral inactivation step is a good fit, as the pH of the eluate is already reduced. The acidified eluate is held for a determined time to inactivate the viral concentration by the required number of logs. Following this step, neutralization is typically carried out to pH 5 or higher, because recombinant proteins can be damaged if left at low pH for extended periods, and subsequent purification steps typically require a higher pH.

[0006] The current industry standard for virus inactivation in downstream bioprocessing is manual titration of the eluate pool using a pH probe. With advances in continuous production, the frequency of processing has increased from once per culture to at least once a day throughout the entire production cycle. This requires processing labor and ultimately a significant increase in costs.

[0007] In addition, in typical virus inactivation unit operations performed in a holding container, the pH probes are left dry after the virus inactivation cycle is complete, which can potentially affect their calibration. Therefore, the operating staff must remove the sample and measure the pH using a benchtop probe to verify the calibration status of the pH probes before a new virus inactivation cycle begins. [Overview of the project] [Problems that the invention aims to solve]

[0008] Thus, there is a need for a method to reduce the labor and cost required during virus inactivation, to keep the pH probe moist for virus inactivation unit operations in the manufacturing process, and to automatically verify its calibration status. The present invention, as described herein, satisfies this need through automated in-process verification of virus inactivation and pH probe calibration. [Means for solving the problem]

[0009] In one embodiment, an automated system for low-pH virus inactivation is provided, the system comprising: a first container; a second container; a first pH probe associated with the first container and configured to measure the pH of the contents of the first container; a source of fluid known or suspected to contain at least one enveloped virus, which is transferred to the first container; and an acid pump configured to pump acid into the first container after the fluid has been transferred to the first container, wherein the acid pump is configured to stop pumping acid into the first container in response to the first pH probe measuring a first pH value within an acceptable range of a target pH value for virus inactivation; and in response to the first pH probe measuring a first pH value below a threshold pH value for virus inactivation and the acid pump stopping pumping acid into the first container, The present invention includes a transfer pump configured to pump an acidification pool from one vessel to a second vessel, a first buffer pump configured to pump a first equilibration buffer having a first known pH value into the first vessel in response to the entire acidification pool being pumped from the first vessel, and an alarm generator configured to compare a second pH value measured by a first pH probe with a first known pH value of the first equilibration buffer after the first equilibration buffer has been pumped into the first vessel, determine whether the second pH value measured by the first pH probe differs from the first known pH value of the first equilibration buffer by more than a threshold pH value, and generate a first alarm in response to the second pH value measured by the first pH probe differing from the first known pH of the first equilibration buffer by more than a threshold pH value.

[0010] In some examples, the system includes a supply pump configured to pump fluid from a source to the first container, at least in part, based on a signal indicating that the first container is empty.

[0011] In addition, in some examples, the first buffer pump is configured to pump the first equilibrating buffer into the first container, at least in part, based on a signal indicating that the first container is empty.

[0012] In some examples, an automated system for low-pH virus inactivation includes a second pH probe associated with a second vessel and configured to measure the pH of the contents of the second vessel; a base pump configured to pump bases into the second vessel in response to the elapsed time since the entire acidification pool was pumped into the second vessel exceeding a threshold time for reducing the virus concentration in the acidification pool to a predetermined safe level, and the base pump configured to stop pumping bases into the second vessel in response to the second pH probe measuring a first pH value within a threshold range of neutral pH values; and a discharge configured to pump the neutralized virus inactivation pool from the second vessel to a filter for processing the neutralized virus inactivation pool. The alarm generator may further include a pump and a second buffer pump configured to pump a second equilibration buffer having a second known pH value into the second container in response to the entire pool being pumped from the second container, and the alarm generator may further be configured to compare a second pH value measured by a second pH probe with a second known pH value of the second equilibration buffer after the first equilibration buffer has been pumped into the second container, determine whether the second pH value measured by the second pH probe differs from the second known pH value of the second equilibration buffer by more than a threshold pH value, and generate a second alarm in response to the second pH value measured by the second pH probe differing from the second known pH of the second equilibration buffer by more than a threshold pH value.

[0013] Furthermore, in some examples, the transfer pump is configured to transfer the acidification pool from the first container to the second container, at least in part, based on a signal indicating that the second container is empty.

[0014] In addition, in some examples, the second buffer pump is configured to pump the second equilibrating buffer into the second container, at least in part, based on a signal indicating that the second container is empty.

[0015] Furthermore, in some examples, the automated system for low-pH virus inactivation may further include a third vessel and a recovery pump configured to pump the filtration pool from the filter into the third vessel.

[0016] In some examples, the recovery pump is configured to pump the filtration pool from the second vessel to the third vessel, at least partially based on a signal indicating that the third vessel is empty.

[0017] In addition, in some examples, the automated system for low pH virus inactivation may further include a first pH probe recalibrator configured to automatically recalibrate a first pH probe in response to a first warning. Similarly, in some examples, the automated system for low pH virus inactivation may further include a second pH probe recalibrator configured to automatically recalibrate a second pH probe in response to a second warning.

[0018] Furthermore, in some examples, the automated system for low-pH virus inactivation may further include one or more additional pH probes associated with a first container and configured to measure the pH of the contents of the first container. Similarly, in some examples, the automated system for low-pH virus inactivation may further include one or more additional pH probes associated with a second container and configured to measure the pH of the contents of the second container.

[0019] In addition, in some examples, an automated system for low-pH virus inactivation may further include an operator display configured to show one or more of the first or second warnings to the operator associated with the system.

[0020] Furthermore, in some examples, the acid is selected from formic acid, acidic acid, citric acid, and phosphoric acid at concentrations suitable for ensuring viral inactivation. Additionally, in some examples, the threshold pH for viral inactivation is pH 2–4. Moreover, in some examples, the chromatographic elution pool is exposed to the acid for less than 30 minutes before neutralization. Furthermore, in some examples, the base is Tris base at a concentration of 2 M. Additionally, in some examples, the threshold range for the neutral pH value is pH 4.5–6. Furthermore, in some examples, low-pH viral inactivation is performed at temperatures of 5–25°C.

[0021] Furthermore, in some examples, the neutralized virus-inactivated chromatography eluate pool is transferred from a second container to a retention container. For example, in some examples, the neutralized virus-inactivated chromatography eluate pool is transferred from a second container to a deep filter. In addition, in some examples, after deep filtration, the neutralized virus-inactivated eluate is transferred to a sterile filter. Furthermore, in some examples, the neutralized virus-inactivated chromatography eluate pool is transferred from a second container to a first polish chromatography column.

[0022] In another embodiment, an automated method for low-pH virus inactivation is provided, which includes adding a pool to a first container; adding acid to the first container; measuring a first pH value associated with the first container using a first pH probe associated with the first container; ceasing to add acid to the first container based on the first measured pH value associated with the first container being within an acceptable range for the target pH value of virus inactivation; transferring the pool from the first container to a second container; filling the first container with an equilibration buffer having a known pH value; measuring a second pH value associated with the first container using a first pH probe; comparing the second measured pH value associated with the first container with the known pH value of the equilibration buffer; determining whether the second measured pH value associated with the first container differs from the known pH value of the equilibration buffer by more than a threshold pH value; and generating a first alarm in response to the second measured pH value associated with the first container differing from the known pH value of the equilibration buffer by more than a threshold pH value.

[0023] In some examples, transferring the pool to a first container is at least partially based on receiving a signal indicating that the first container is empty.

[0024] In addition, in some examples, filling the first container with equilibration buffer is at least partially based on receiving a signal indicating that the first container is empty.

[0025] In some examples, an automated method of low pH virus inactivation includes adding a base to a second container after an elapsed time after transfer of the pool to the second container exceeds a threshold time for reducing the virus concentration in the pool to a predetermined safe level, measuring a first pH value associated with the second container using a second pH probe associated with the second container, stopping adding the base to the second container based on the first measured pH value associated with the second container being within a threshold range of neutral pH values, transferring the pool from the second container to a filter to process the neutralized virus inactivation pool, filling the second container with an equilibration buffer having a known pH value, measuring a second pH value associated with the second container using a second pH probe associated with the second container, comparing the second measured pH value associated with the second container to the known pH value of the equilibration buffer, determining whether the second measured pH value associated with the second container differs from the known pH value of the equilibration buffer by more than a threshold pH value, and generating a second alert in response to the second measured pH value associated with the second container differing from the known pH value of the equilibration buffer by more than the threshold pH value.

[0026] For example, in some examples, the transfer of the acidified pool from the first container to the second container is at least partially based on receiving a signal indicating that the second container is empty.

[0027] In addition, in some examples, filling the second container with the equilibration buffer is at least partially based on receiving a signal indicating that the second container is empty.

[0028] Further, in some examples, an automated method of low pH virus inactivation may further include transferring the pool from the filter to a third container.

[0029] For example, in some examples, the transfer of the pool from the filter to the third container is at least partially based on receiving a signal indicating that the third container is empty.

[0030] In addition, in some examples, the automated method for low-pH virus inactivation may further include recalibrating a first pH probe in response to a first alarm. Similarly, in some examples, the automated method for low-pH virus inactivation may further include recalibrating a second pH probe in response to a second alarm.

[0031] In yet another embodiment, a method is provided for inactivating an enveloped virus during the purification of a recombinant protein of interest, the method comprising: obtaining a fluid known or suspected to contain at least one enveloped virus; and adding the fluid to a concentration and time sufficient to cause viral inactivation, the following steps: adding the fluid to a first container; adding acid to the first container; measuring a first pH value associated with the first container using a first pH probe associated with the first container; stopping the addition of acid to the first container based on the first measured pH value associated with the first container being within an acceptable range for the target pH value of viral inactivation; and transferring the fluid from the first container to a second container, and known The procedure includes one or more of the following steps: filling a first container with an equilibration buffer having a pH value; measuring a second pH value associated with the first container using a first pH probe; comparing the second measured pH value associated with the first container with a known pH value of the equilibration buffer; determining whether the second measured pH value associated with the first container differs from the known pH value of the equilibration buffer by more than a threshold pH value; and generating a first alarm in response to the second measured pH value associated with the first container differing from the known pH value of the equilibration buffer by more than a threshold pH value; and subjecting the neutralized virus-inactivating fluid to at least one unit operation including at least a filtration step or a chromatography step.

[0032] In some examples, adding fluid to the first container is partially based on receiving a signal indicating that the first container is empty.

[0033] In addition, in some examples, transferring a fluid from a first container to a second container is partially based on receiving a signal indicating that the second container is empty.

[0034] Furthermore, in some examples, filling the first container with equilibration buffer is partially based on receiving a signal indicating that the first container is empty.

[0035] Furthermore, in some examples, the fluid contains recombinant proteins of interest. Furthermore, in some examples, the fluid is collected host cell culture medium. In addition, in some examples, the fluid is from effluent, eluate, pool, storage, or retainer from a unit operation including a collection step, filtration step, or chromatography step. Furthermore, in some examples, the fluid is eluate collected from deep filtration, microfiltration, affinity chromatography, ion exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography. In addition, in some examples, the fluid is a pool containing collected cell culture medium, eluate from deep filtration, eluate from microfiltration, eluate from affinity chromatography, eluate from ion exchange chromatography, eluate from multimodal chromatography, eluate from hydrophobic interaction chromatography, or eluate from hydroxyapatite chromatography. Furthermore, in some examples, the fluid is a collected host cell culture medium, and the unit operation includes deep filtration. In addition, in some examples, the fluid is a collected host cell culture medium, and the unit operation includes microfiltration. Furthermore, in some examples, the fluid is a collected host cell culture medium, and the unit operation includes protein A affinity chromatography. Furthermore, in some examples, the fluid is a protein A eluent, and the unit operation includes deep filtration.

[0036] Furthermore, in some examples, affinity chromatography is protein A, protein G, protein A / G, or protein L chromatography. Additionally, in some examples, chromatography is selected from affinity chromatography, protein A chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, mixed modal or multimodal chromatography, or hydroxyapatite chromatography.

[0037] In addition, in some examples, the unit operation includes deep filtration. Furthermore, in some examples, the unit operation includes microfiltration.

[0038] In another embodiment, an automated system for low-pH virus inactivation is provided, the system comprising: a first container; a second container; a first pH probe associated with the first container and configured to measure the pH of the contents of the first container; a source of fluid known or suspected to contain at least one enveloped virus, which is transferred to the first container; and an acid pump configured to pump acid into the first container after the fluid has been transferred to the first container, wherein the acid pump is configured to stop pumping acid into the first container in response to the first pH probe measuring a first pH value within an acceptable range of a target pH value for virus inactivation, and the acid pump stops pumping acid into the first container in response to the first pH probe measuring a first pH value below a threshold pH value for virus inactivation. The system includes a transfer pump configured to pump the acidification pool from a first container to a second container in response to stopping; a second pH probe associated with the second container and configured to measure the pH of the contents of the second container; a base pump configured to pump a base to the second container in response to the elapsed time since the entire acidification pool was pumped to the second container exceeding a threshold time for reducing the virus concentration in the acidification pool to a predetermined safe level, and configured to stop pumping the base to the second container in response to the second pH probe measuring a first pH value within a threshold range of neutral pH values; and a discharge pump configured to pump the neutralized virus-inactivated pool from the second container to a filter for processing the neutralized virus-inactivated pool.

[0039] In some examples, the system includes a supply pump configured to pump fluid from a source to the first container, at least in part, based on a signal indicating that the first container is empty.

[0040] Furthermore, in some examples, the transfer pump is configured to transfer the acidification pool from the first container to the second container, at least in part, based on a signal indicating that the second container is empty.

[0041] Furthermore, in some examples, the automated system for low-pH virus inactivation may further include a third vessel and a recovery pump configured to pump the filtration pool from the filter into the third vessel.

[0042] In some examples, the recovery pump is configured to pump the filtration pool from the second vessel to the third vessel, at least partially based on a signal indicating that the third vessel is empty.

[0043] Furthermore, in some examples, the automated system for low-pH virus inactivation may further include one or more additional pH probes associated with a first container and configured to measure the pH of the contents of the first container. Similarly, in some examples, the automated system for low-pH virus inactivation may further include one or more additional pH probes associated with a second container and configured to measure the pH of the contents of the second container.

[0044] Furthermore, in some examples, the acid is selected from formic acid, acidic acids, citric acid, and phosphoric acid at concentrations suitable for ensuring viral inactivation. Additionally, in some examples, the threshold pH for viral inactivation is pH 2–4. Moreover, in some examples, the chromatographic elution pool is exposed to the acid for less than 30 minutes before neutralization. Furthermore, in some examples, the base is Tris base at a concentration of 2 M. Additionally, in some examples, the threshold range for the neutral pH value is pH 4.5–6. Furthermore, in some examples, low-pH viral inactivation is performed at temperatures of 5–25°C.

[0045] Furthermore, in some examples, the neutralized virus-inactivated chromatography eluate pool is transferred from a second container to a retention container. For example, in some examples, the neutralized virus-inactivated chromatography eluate pool is transferred from a second container to a deep filter. In addition, in some examples, after deep filtration, the neutralized virus-inactivated eluate is transferred to a sterile filter. Furthermore, in some examples, the neutralized virus-inactivated chromatography eluate pool is transferred from a second container to a first polish chromatography column.

[0046] In another embodiment, an automated method for low-pH virus inactivation is provided, which includes adding a pool to a first container; adding an acid to the first container; measuring a first pH value associated with the first container using a first pH probe associated with the first container; stopping the addition of the acid to the first container based on the first measured pH value associated with the first container being within an acceptable range of the target pH value for virus inactivation; transferring the pool from the first container to a second container; adding a base to the second container after the time elapsed since the transfer of the pool to the second container has exceeded a threshold time for reducing the virus concentration in the pool to a predetermined safe level; measuring a first pH value associated with the second container using a second pH probe associated with the second container; stopping the addition of the base to the second container based on the first measured pH value associated with the second container being within a threshold range of the neutral pH value; and transferring the pool from the second container to a filter for processing the neutralized virus-inactivated pool.

[0047] In some examples, transferring the pool to a first container is at least partially based on receiving a signal indicating that the first container is empty.

[0048] Furthermore, in some examples, transferring the acidification pool from the first container to the second container is at least partially based on receiving a signal indicating that the second container is empty.

[0049] Furthermore, in some examples, the automated method for low-pH virus inactivation may further include transferring the pool from the filter to a third container.

[0050] For example, in some cases, transferring the pool from the filter to a third container is at least partially based on receiving a signal indicating that the third container is empty.

[0051] In addition, in some examples, the automated method for low-pH virus inactivation may further include recalibrating a first pH probe in response to a first alarm. Similarly, in some examples, the automated method for low-pH virus inactivation may further include recalibrating a second pH probe in response to a second alarm.

[0052] In another embodiment, a method is provided for inactivating an enveloped virus during the purification of a recombinant protein of interest, the method comprising: obtaining a fluid known or suspected to contain at least one enveloped virus; and distributing the fluid at a concentration and time sufficient to cause viral inactivation; the following steps: adding the fluid to a first vessel; adding acid to the first vessel; measuring a first pH value associated with the first vessel using a first pH probe associated with the first vessel; and confirming that the first measured pH value associated with the first vessel is within an acceptable range of the target pH value for viral inactivation. The procedure includes subjecting the addition of acid to the first container to one or more steps based on the above, a step of stopping the addition of acid to the first container, a step of transferring the fluid from the first container to the second container, a step of adding a base to the second container, a step of measuring a second pH value associated with the second container using a second pH probe associated with the second container, and a step of stopping the addition of base to the second container based on the fact that the second measured pH value associated with the second container is within the acceptable range of the target pH value for neutralization, and subjecting the neutralized virus-inactivating fluid to at least one unit operation including at least a filtration step or a chromatography step.

[0053] In some examples, adding fluid to the first container is partially based on receiving a signal indicating that the first container is empty.

[0054] In addition, in some examples, transferring a fluid from a first container to a second container is partially based on receiving a signal indicating that the second container is empty.

[0055] Furthermore, in some examples, the fluid contains recombinant proteins of interest. Furthermore, in some examples, the fluid is collected host cell culture medium. In addition, in some examples, the fluid is from effluent, eluate, pool, storage, or retainer from a unit operation including a collection step, filtration step, or chromatography step. Furthermore, in some examples, the fluid is eluate collected from deep filtration, microfiltration, affinity chromatography, ion exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography. In addition, in some examples, the fluid is a pool containing collected cell culture medium, eluate from deep filtration, eluate from microfiltration, eluate from affinity chromatography, eluate from ion exchange chromatography, eluate from multimodal chromatography, eluate from hydrophobic interaction chromatography, or eluate from hydroxyapatite chromatography. Furthermore, in some examples, the fluid is a collected host cell culture medium, and the unit operation includes deep filtration. In addition, in some examples, the fluid is a collected host cell culture medium, and the unit operation includes microfiltration. Furthermore, in some examples, the fluid is a collected host cell culture medium, and the unit operation includes protein A affinity chromatography. Furthermore, in some examples, the fluid is a protein A eluent, and the unit operation includes deep filtration.

[0056] Furthermore, in some examples, affinity chromatography is protein A, protein G, protein A / G, or protein L chromatography. Additionally, in some examples, chromatography is selected from affinity chromatography, protein A chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, mixed modal or multimodal chromatography, or hydroxyapatite chromatography.

[0057] In addition, in some examples, the unit operation includes deep filtration. Furthermore, in some examples, the unit operation includes microfiltration.

[0058] The drawings described below illustrate various aspects of the systems and methods disclosed herein. Several advantages will become apparent to those skilled in the art from the following description of the multiple embodiments illustrated and described for illustrative purposes. As will be understood below, other different embodiments are possible, and their details are modifiable in various ways. Therefore, the drawings and descriptions should be considered as illustrative and not limiting in nature. Furthermore, wherever possible, the following description will refer to the reference numerals included in the following drawings, and features shown in multiple drawings will be indicated by consistent reference numerals. [Brief explanation of the drawing]

[0059] [Figure 1A] A block diagram of an example of an automated system for low-pH virus inactivation is shown. [Figure 1B] Figure 1A shows an example of an automated system for low-pH virus inactivation, illustrating how a two-container design is used to prevent droplet suspension. [Figure 1C] Figure 1A shows an example of an automated system for low-pH virus inactivation, illustrating how a two-container design is used to prevent droplet suspension. [Figure 2] This shows a piping and instrumentation diagram (P&ID) of an example of an automated system for low-pH virus inactivation. [Figure 3]A flowchart is shown as an example of an automated method for low-pH virus inactivation using a fluid known or suspected to contain at least one enveloped virus. [Figure 4A] A flow chart is shown illustrating an example of an automated method for low-pH virus inactivation using a fluid known or suspected to contain at least one enveloped virus, including an automated cycle of pH probe calibration. [Figure 4B] A flow chart is shown illustrating an example of an automated method for low-pH virus inactivation using a fluid known or suspected to contain at least one enveloped virus, including an automated cycle of pH probe calibration. [Modes for carrying out the invention]

[0060] Inactivation of enveloped viruses known or suspected to be present in fluids can be carried out by many different operations, including heat inactivation / pasteurization, treatment with solvents and / or detergents, ultraviolet and gamma ray irradiation, use of high-intensity broad-field white light, addition of chemical inactivators such as β-propiolactone and / or low-pH virus inactivation.

[0061] This disclosure relates, in general terms, to automated systems and methods for low-pH virus inactivation. These automated systems and methods for low-pH virus inactivation include synchronization with upstream and downstream units through integration with a distributed control system, processing control based on pool pH, and an automated virus inactivation pool filtration system.

[0062] To synchronize upstream and downstream units, communication is required to notify them of the batch status. There are two different types of synchronization methods: synchronous and asynchronous. In the synchronous method, processing is paused until one unit sends a message to a secondary unit and the secondary unit acknowledges receipt of the message. In contrast, the asynchronous method does not require processing to be paused for acknowledgment messages between units, and proceeds to the next step after the first message is sent. The automated systems and methods described herein use a synchronous communication system to prevent the upstream unit from transferring the formulation pool to the downstream unit before the downstream unit is ready. The synchronization method allows the system to vary the number of cycles from upstream chromatography by allowing the system to optionally choose whether to process all eluate pools or to recover multiple pools before processing. The automation is included in a distributed control system and is subject to monitoring and control.

[0063] Generally, a fluid known or suspected to contain at least one enveloped virus is added to a first container, and acid is added to the first container to lower the pH of the elution pool in the first container. When the pH probe in the first container measures a sufficiently low pH, the acidified fluid is transferred to a second container. Using two containers allows the pool to be first lowered to an inactivation pH in the first container and then transferred to the second container where it can be held for an effective inactivation time. This method eliminates the possibility of untreated pool droplets being processed without elution droplets adhering to the upper surface of the container wall and interacting with the acid during the holding time. Using two containers ensures that the entire contents of the pool transferred to the second container are well mixed with the acid. Once the acidified fluid has been held in the second container for an effective inactivation time and the virus has been inactivated to a predetermined safe level, the acidified fluid in the second container is neutralized. Generally, there are two optional choices for the acidification and neutralization methods that can be selected when creating a batch recipe: fixed and variable. Both methods employ incremental dosing, but with fixed elective, the acid / base dosage remains constant, while with variable elective, the next dosage is calculated based on the current pH of the pool and adjusted accordingly.

[0064] In any case, once the acidified fluid in the second container is neutralized, it is filtered through a filtration system combining deep and sterile filtration. Using a discharge pump and a series of valves, the washing solution, preparation buffer, and formulation pool are guided through the filter to the third container. The batch recipe of the distributed control system monitors and proceeds the filtration process without operator intervention unless an alarm requiring attention occurs. In existing systems, it was necessary to manually transfer the inactivated formulation pool to the filtration system. Advantageously, by using the automated systems and methods described herein, a single closed system with connected inactivation and filtration processes can be realized.

[0065] During this time, when the acidified fluid is transferred from the first container to the second container, i.e., when the first container becomes empty, a signal indicating that the first container is empty is sent upstream, and the first container is immediately filled with a known pH equilibration buffer to keep the pH probe wet. The reading of the pH probe in the first container is then compared with the known pH to determine whether any of the pH probes need to be recalibrated. Generally, each container includes at least two probes: a main probe that provides pH readings and a backup probe that can be used as a redundant probe in case the main probe fails. In some cases, if the difference between the reading from the pH probe and the known pH is greater than a threshold, the pH probe is automatically recalibrated; in other cases, an alarm can be issued to the operator prompting them to recalibrate the pH probe.

[0066] When the neutralized virus-inactivating fluid is transferred from the second container to the third container, i.e., when the second container is empty, a signal indicating that the second container is empty is sent upstream, and the second container is immediately filled with equilibration buffer of a known pH, and the pH probe in the second container is compared with the known pH to determine whether recalibration is necessary. The process is then repeated in a new cycle. That is, when the equilibration buffer is removed from the first container, i.e., when the first container is empty again, a signal indicating that the first container is empty is sent upstream, and a new fluid known or suspected to contain at least one enveloped virus is added to the first container. Acid is then added to the first container, and when the equilibration buffer is removed from the second container, i.e., when the second container is empty again, a signal indicating that the second container is empty is sent upstream, and when the pH probe in the first container measures a sufficiently low pH, the acidification pool is added to the second container. In other words, the acidification pool is added from the first container to the second container based on both a signal indicating that the second container is empty and a signal indicating that the pH probe in the first container has measured a pH low enough to inactivate the virus.

[0067] Advantageously, using the automated systems and methods described herein, the pH probes in both containers can be kept immersed and moistened over multiple cycles, and their calibration status can be automatically evaluated and corrected as needed without requiring an operating staff member to always be on standby to manually remove samples and measure pH after each cycle. That is, instead of having a member of the operating staff stand by before and after each cycle to check the calibration status of the pH probes, the operating staff can engage in other tasks as needed and only intervene if an alarm or warning occurs. Beneficially, in some examples, the pH probes in both containers can maintain an accuracy sufficient for use over many consecutive cycles of low-pH virus inactivation without intervention from the operating staff.

[0068] Therefore, the automated system and method can execute cycles independently and iteratively in synchronization with the upstream capture chromatography system, thereby reducing the number of operational staff required. Specifically, the reduction in operational staff can be achieved by enabling the system to automatically initiate cycles by both detecting the amount of formulation recovered from the capture chromatography step and synchronizing communication with the chromatography system.

[0069] Referring here to the drawings, Figure 1A shows a block diagram of an example of an automated system 100 for low-pH virus inactivation. The system 100 includes a first container 102A, a second container 102B, and a third container 102C. The first container 102A and the second container 102B may each be equipped with stirrers 104A and 104B, respectively, configured to mix the substances stored in the first container 102A and the second container 102B, respectively. In addition, the first container 102A and the second container 102B may each be equipped with pH probes 106A and 106B, respectively, configured to measure the pH values ​​associated with the first container 102A and the second container 102B, respectively. Figure 1A shows two pH probes 106A associated with a first container 102A and two pH probes 106B associated with a second container 102B, although in some examples one pH probe 106A or two or more pH probes 106A may be associated with the first container 102A (and in some examples one pH probe 106B or two or more pH probes 106B may be associated with the second container 102B). System 100 further includes a computing device 108 configured to interface with the pH probes 106A and 106B. The computing device 108 may include one or more processors 109 and respective memories 111 (e.g., volatile memory, non-volatile memory) accessible by one or more processors 109 (e.g., via a memory controller), as well as a user interface 113. One or more processors 109 can interact with the memories 111 to execute computer-readable instructions stored in the memories 111. Computer-readable instructions stored in memory 111 can cause one or more processors 109 to execute the pH probe recalibration application 115 and the upstream / downstream notification application 117.

[0070] System 100 further includes a chromatography kit 110, one or more containers 112 or other containers for acids, one or more containers 114 or other containers for bases, one or more filters 116 (e.g., deep filters, sterile-grade filters), and one or more containers 118 or other containers for buffers. In addition, System 100 may include one or more pumps, valves or other means for transferring liquids between these various containers or other containers and through filters. For example, System 100 may include one or more pumps, valves or other means for continuously or intermittently transferring a fluid known or suspected to contain at least one enveloped virus from the chromatography kit 110 to the first container 102A. In some examples, the pumps and / or valves may transfer the fluid from the chromatography kit 110 to the first container 102A only when an upstream signal is received from an upstream / downstream notification application 117 indicating that the first container 102A is currently empty. Furthermore, system 100 may include one or more pumps, valves, or other means for transferring acid from container 112 to the first container 102A. In some examples, the pumps and / or valves may transfer acid from container 112 to the first container 102A only if they receive an upstream signal from the upstream / downstream notification application 117 indicating that the first container 102A currently contains a fluid known or suspected to contain a virus. A stirrer 104A may mix acid with the fluid known or suspected to contain at least one enveloped virus (and / or additional acid may be added to the elution pool) until a pH probe 106A associated with the first container 102A measures a pH value below a predetermined threshold pH value for inactivating enveloped viruses in the fluid (e.g., a pH value of 3.5 to 3.7).

[0071] In addition, the system 100 may include one or more pumps, valves, or other means for transferring the acidifying fluid from the first container 102A to the second container 102B when a pH probe 106A associated with the first container 102A measures a pH value below a predetermined threshold pH value. In some examples, the pump and / or valve may transfer the acidifying fluid from the first container 102A to the second container 102B only if it receives an upstream signal from an upstream / downstream notification application 117 indicating that the second container 102B is currently empty. Once transferred into the second container 102B, the acidifying fluid can remain in the second container 102B for a predetermined time (e.g., 30 minutes or less) sufficient to reduce the virus concentration in the acidified elution pool to below a predetermined safe level (e.g., a level set by regulatory authorities relating to drugs manufactured from fluids known or suspected to contain at least one enveloped virus in addition to a recombinantly manufactured therapeutic protein).

[0072] For example, as shown in Figures 1B and 1C, by transferring the acidified fluid from the first container 102A (shown in Figure 1B) to the second container 102B (shown in Figure 1C) in the manner described above, the pool can be first lowered to an inactivating pH in the first container 102A and then transferred to the second container 102B where it can be held for an effective inactivation time. By holding the pool in the second container 102B rather than in the first container 102A for an effective inactivation time, the system 100 eliminates the possibility of untreated pool droplets being treated without elute droplets adhering to the upper surface of the first container 102A and interacting with the acid during the holding period. In other words, by using two containers 102A and 102B, all contents from the pool being transferred from the first container 102A to the second container 102B are well mixed with the acid.

[0073] Referring again to Figure 1A, one or more pumps or valves in system 100 can transfer the base from container or other container 114 to the second container 102B. In some examples, the pumps and / or valves can transfer the base from container or other container 114 to the second container 102B only if they receive an upstream signal from the upstream / downstream notification application 117 indicating that the second container 102B now contains the acidified (or virus-inactivating) fluid. The agitator 104B can mix the base with the acidified (or virus-inactivating) fluid (and / or add additional acid to the elution pool) until the pH probe 106B associated with the second container 102B measures a neutral pH value (e.g., a pH value of 5.0–6.0). Furthermore, the system 100 may include one or more pumps, valves, or other means for transferring the neutralized virus-inactivating fluid from the second container 102B through one or more filters 116 (such as deep filters and sterile-grade filters) to a third container 102C from which the filtered neutralized virus-inactivating fluid can be recovered for use. In some examples, the pumps and / or valves may transfer the neutralized virus-inactivating fluid from the second container 102B through one or more filters 116 to the third container 102C only if an upstream signal is received from the upstream / downstream notification application 117 indicating that the third container 102C (and / or filters 116) is currently empty.

[0074] During this time, immediately after the acidification fluid is transferred from the first container 102A, the upstream / downstream notification application 117 sends an upstream signal to one or more pumps or valves of the system 100 indicating that the first container 102A is empty, thereby causing equilibration buffer with a known pH to be transferred from container 118 to the first container 102A in order to keep the pH probe 106A wet. At this point, the pH probe 106A measures the pH of the equilibration buffer in the first container 102A and sends a display of the measured pH to the computing device 108, and the pH probe recalibration application 115 can compare the measured pH of the equilibration buffer in the first container 102A with the known pH of the equilibration buffer. If the pH probe recalibration application 115 determines that the difference between the measured pH and the known pH of the equilibration buffer is greater than a threshold pH value (e.g., greater than 0.1 pH units), the pH probe recalibration application 115 may generate an alarm indicating that the pH probe 102A (or a specific one of the pH probes 102A) needs to be recalibrated. The computing device 108 may display the alarm to the operator via the user interface 113 or communicate it in other ways. In addition, in some examples, the pH probe recalibration application 115 may cause the computing device 108 to generate a control signal that causes the pH probe 102A (or a specific one of the pH probes 102A) to automatically recalibrate based on the known pH of the equilibration buffer, for example, causing the pH probe 102A to measure a pH value within ±0.1 pH units of the known pH of the equilibration buffer when measuring the pH of the equilibration buffer.

[0075] Similarly, immediately after the neutralized virus-inactivating fluid has been transferred from the second container 102B, the upstream / downstream notification application 117 sends an upstream signal to one or more pumps or valves of the system 100 indicating that the second container 102B is empty, allowing an equilibration buffer with a known pH to be transferred from one of the containers 118 (which may or may not be the same equilibration buffer used with the first container 102A) to the second container 102B in order to keep the pH probe 106B wet. At this point, the pH probe 106B measures the pH of the equilibration buffer in the second container 102B and sends a display of the measured pH to the computing device 108, so that the pH probe recalibration application 115 can compare the measured pH of the equilibration buffer in the second container 102B with the known pH of the equilibration buffer. If the pH probe recalibration application 115 determines that the difference between the measured pH and the known pH of the equilibration buffer is greater than a threshold pH value (e.g., greater than 0.1 pH units), the pH probe recalibration application 115 may generate an alarm indicating that the pH probe 102B (or a specific one of the pH probes 102B) needs to be recalibrated. The computing device 108 may display the alarm to the operator via the user interface 113 or communicate it in other ways. In addition, in some examples, the pH probe recalibration application 115 may cause the computing device 108 to generate a control signal that causes the pH probe 102B (or a specific one of the pH probes 102B) to automatically recalibrate based on the known pH of the equilibration buffer, for example, causing the pH probe 102B to measure a pH value within ±0.1 pH units of the known pH of the equilibration buffer when measuring the pH of the equilibration buffer.

[0076] Referring here to Figure 2, the Piping and Instrumentation Diagram (P&ID) 200 of an exemplary automated system for low-pH virus inactivation shows the piping and processing equipment of the system together with the instrumentation and control equipment of the system. In Figure 2, fluid-connected elements (i.e., elements through which fluid can flow) are shown with solid lines 246, and interconnected elements are shown with dashed lines. In particular, a short dashed line 242 between two elements indicates that a sensor signal may be transmitted and / or received between the two elements, while a long dashed line 244 between two elements indicates that a control signal may be transmitted and / or received between the two elements.

[0077] As shown in Figure 2, the control system 202 (which in some examples may be or include the computing device 108 shown with reference to Figure 1A, and in some examples may include additional or alternative computing devices) is communicably connected to various elements of the system to receive sensor signals and transmit control signals in order to operate the automated system for low pH virus inactivation in accordance with the information disclosed herein. Specific instructions for the control signals and sensor signals transmitted and received by the control system 202 are shown in Figure 2, however, for the sake of simplicity, Figure 2 does not necessarily show all control signals and sensor signals that the control system 202 may transmit. That is, the control system 202 may transmit and / or receive additional or alternative control signals and / or sensor signals in order to operate the automated system for low pH virus inactivation in accordance with the information provided herein.

[0078] For example, the chromatography kit 204 may be fluid-connected to the first container 206 so that a fluid known or suspected to contain at least one enveloped virus can be transferred from the chromatography kit 204 to the first container 206. A container containing acid or other container 208 may also be fluid-connected to the first container 206. As shown in Figure 2, an acid pump 210 may be fluid-connected to the acid container 208 and the first container 204 to pump acid from the acid container 208 to the first container 204. In some examples, the control system 202 may transmit control signals to the acid pump 210 to control the speed of the acid pump 210 and / or the amount of acid pumped into the first container 204, as described herein, for example. Furthermore, in some examples, a weighing scale 212 may capture the weight indications of the fluids in the first container 206 and the first container 204 and transmit these indications to the control system 202. In some examples, the control system 202 can determine whether the first container 206 is full or empty based on a signal from the weighing scale 212, and based on whether the first container 206 is full or empty, it can control the timing of transferring the enveloped virus from the chromatography kit 204 to the first container 206 (and / or the timing of the acid pump 210 transferring the sugar into the first container 206, the timing of the buffer pump 240 pumping the buffer into the first container 206, etc.). Furthermore, in some examples, the control system 202 can control the speed of the acid pump 210 based on the total weight of the acid in the first container 206 and the fluid known or suspected to contain at least one enveloped virus. In addition, in some examples, the control system 202 may send a control signal to the agitator 214 in the first container 206 so that the agitator 214 mixes the acid in the first container 206 with a fluid known or suspected to contain at least one enveloped virus at the speed and / or position described herein.

[0079] One or more pH probes 216 placed in (or separately associated with) the first container 206 may be configured to measure the pH of the contents of the first container (e.g., an acidified fluid mixed in the first container 206 by a stirrer 214) and transmit sensor signals to the control system 202 indicating one or more pH measurements associated with the first container 206.

[0080] The first container 206 may be fluid-connected to the second container 218 so that the acidified fluid can be transferred from the first container 206 to the second container 218. A transfer pump 220 may be fluid-connected to both the first container 206 and the second container 218 and can pump the acidified fluid from the first container 206 to the second container 218 based on control signals received, for example, from a control system 202. For example, the control system 202 may control the transfer pump 220 to pump the acidified fluid from the first container 206 to the second container 218 based on sensor data received by the control system 202 from other elements (e.g., starting when the pH measured by the pH probe 216 reaches a target pH value that kills the virus, starting when the elapsed time reaches a target total time for acidification, or pumping at a rate or speed based on a target transfer time from the first container 206 to the second container 218).

[0081] The container 222 containing the base or other container 222 may be fluid-connected to the second container 218 so that the base can be transferred from the base container 222 to the second container 218. The base pump 224 may be fluid-connected to the base container 222 and the second container 218 and can pump the base from the first container 206 to the second container 218 based on control signals received, for example, from the control system 202. For example, the control system 202 may transmit control signals that control the speed or rate of the base pump 224 and / or the amount of base pumped into the second container 218, as described herein, when the base pump 224 pumps the base from the base container 222 to the second container 218. Furthermore, in some examples, a weighing scale 226 may capture a reading of the weight of the fluid in the second container 218 and the first container 206 and transmit these readings to the control system 202. In some examples, the control system 202 can determine whether the second container 218 is full or empty based on a signal from the weighing scale 226, and control the timing of the transfer of the acidified fluid from the first container 206 to the second container 218 (and / or the timing of the base pump 224 transferring the base to the second container 218, the timing of the buffer pump 240 pumping the buffer to the second container 218, etc.) based on whether the second container 218 is full or empty. Furthermore, in some examples, the control system 202 can control the speed of the base pump 224 based on the combined weight of the base and the fluid in the second container 218 that is known or suspected to contain at least one enveloped virus. In addition, in some examples, the control system 202 may send a control signal to the agitator 228 in the second vessel 218 to mix the base in the second vessel 218 with a fluid known or suspected to contain at least one enveloped virus at the rate and / or position described herein.

[0082] One or more pH probes 230 placed in (or otherwise associated with) the second container 218 may be configured to measure the pH of the contents of the second container (e.g., a neutralized virus-inactivating fluid mixed in the second container 218 by a stirrer 228) and transmit a sensor signal to the control system 202 indicating the measured pH value or a value associated with the second container 218.

[0083] The second container 218 may be fluidly connected to a series of filters, including a deep filter 232 and a sterilization filter 234. A discharge pump 236 may be fluidly connected to the second container 218 and filters 232, 234, and can pump the neutralized virus-inactivating fluid from the second container 218 through filters 232, 234 to the third container 235 based on a control signal received, for example, from a control system 202. In some examples, the third container 235 may be a collection bag. In addition, in some examples, the third container 235 may include a load cell 237 configured to measure the weight of the load cell and generate an upstream or downstream signal indicating that the third container 235 is full.

[0084] For example, the control system 202 can control the discharge pump 236 to pump the neutralized virus-inactivating fluid from the second container 218 to the filters 232 and 234 based on sensor data received by the control system 202 from other elements (for example, starting when the pH measured by the pH probe 230 reaches the target neutralization pH value, starting when the elapsed time reaches the target total time to neutralize, and / or pumping at a speed or rate based on the target filtration flow rate). In addition, the control system 202 can receive sensor data from sensors associated with the filters 232 and 234 and control the filters 232 and 234 to operate in accordance with the filtration specifications and requirements described herein (i.e., based on the sensor data).

[0085] In addition, the container containing the buffer or other container 238 may be fluidically connected to the first container 206 and / or the second container 218 so that the buffer can be transferred from the buffer container 238 to the first container 206 and / or the second container 218. In some examples, the buffer container 238 may be fluidly connected to the first container 206 and the second container so that the buffer can be transferred from the buffer container to the first container and then to the second container (for example, via a transfer pump 220). The buffer pump 240 may be fluidly connected to the buffer container 238 and the first container 206 and / or the second container 218 and can pump the buffer from the buffer container 238 to the first container 206 and / or the second container 218 based on control signals received from the control system 202. In particular, the control system 202 can control the buffer pump 240 to pump the buffer into the first container 206 and the second container 218 after a fluid known or suspected to contain at least one enveloped virus has been transferred from each of the first container 206 and the second container 218, according to the filtration specifications and requirements. That is, as described above, a buffer that can take on a known pH value can be pumped into the first container 206 after the acidified fluid has been transferred from the first container 206 to the second container 218. Similarly, the buffer can be pumped into the second container 218 after the neutralized virus-inactivating fluid has been pumped from the second container 218 through filters 232 and 234 to the third container 235. pH probes 216 and 230 can measure the pH value of the buffer as it is pumped into the first container 206 and the second container 218, respectively. pH probes 216 and 230 can transmit the measured pH value of the buffer solution to the control system 202, which can compare the measured pH value of the buffer solution with the known pH of the buffer solution to determine whether any recalibration is required for either pH probe 216 or 230. In some cases, the control system 202 can send a control signal to any pH probe that needs recalibration as needed. Furthermore, in some cases, the control system 202 can generate an alarm to the operator if any pH probes need recalibration.

[0086] After any recalibration of probe 216 is complete, the transfer pump 220 can pump buffer from the first container 206 and pump or separately transfer a new fluid known or suspected to contain at least one enveloped virus from the chromatography kit 204 to the first container to initiate a new cycle of automated virus inactivation. Similarly, after any recalibration of probe 230 is complete, the discharge pump 236 can pump buffer from the second container 218, and the transfer pump 220 can pump the newly acidified fluid from the first container 206 to the second container 218. Thus, the system can proceed to a new cycle of automated virus inactivation after recalibrating probes 216 and 230 as needed.

[0087] Figure 3 shows a flow chart associated with an exemplary automated method 300 for low-pH virus inactivation using a fluid known or suspected to contain at least one enveloped virus. Method 300 can be initiated when a chromatographic elution pool is added to a first vessel (block 302). Acid can be added to the first vessel (block 304) and mixed with the fluid known or suspected to contain at least one enveloped virus (e.g., by a stirrer in the first vessel) to acidify the fluid. A first pH probe associated with the first vessel can measure the pH value associated with the first vessel (block 306). The method may include determining whether the measured pH value is below (or within) a threshold pH value associated with virus inactivation (block 308). If the pH value measured by the first pH probe associated with the first container is not below (or outside the range of pH values) the threshold pH value associated with virus inactivation (NO in block 308), additional acid may be added to the first container (block 304), or the acid may be retained in the first container for an additional time before the pH of the first container is measured again (block 306). If the pH value measured by the first pH probe associated with the first container is below (or within the range of pH values) the threshold pH value for virus inactivation (YES in block 308), the addition of acid to the first container may be stopped (block 310), and the acidified fluid may be transferred to the second container (block 312).

[0088] A second pH probe associated with the second container can measure the pH value associated with the second container (block 314). The method may include determining whether the measured pH value is below (or within) a threshold pH value associated with virus inactivation (block 316). If the pH value measured by the second pH probe associated with the second container is not below (or within) the threshold pH value for virus inactivation (NO in block 316), the process may be suspended (block 318), and an alarm may be issued to the operator prompting them to investigate, for example, whether there is any problem with the measured pH. If the pH value measured by the second pH probe associated with the second container is below the threshold pH value (YES in block 316), the method may proceed to block 320 to determine whether the elapsed time after transferring the acidified fluid from the first container to the second container has exceeded the threshold time (e.g., 30 minutes or less) that inactivates the virus concentration in the fluid to a predetermined safe level. If the threshold time is not exceeded (NO in block 320), the determination in block 314 may be repeated after an additional elapsed time. If the threshold time is exceeded (YES in block 320), the method proceeds to block 322, where a base may be added to a second container to neutralize the acidified fluid.

[0089] The second pH probe associated with the second container again measures the pH value associated with the second container (block 324), and a determination may be made as to whether the measured pH value associated with the second container is within the acceptable range of neutral pH (e.g., pH range 5.0 to 6.0). If the measured pH value associated with the second container is not within the acceptable range (NO in block 326), additional base may be added to the container (block 322). If the measured pH value associated with the second container is within the acceptable range (YES in block 326), the addition of base to the second container may be stopped (block 328), and the neutralized virus-inactivating fluid may be transferred to a deep filter (block 330), and then to a sterilization grade filter (block 332).

[0090] Referring here to Figures 4A-4B, a flow chart is shown associated with an example of an automated low-pH virus inactivation method 400, including an automated cycle for pH probe calibration. Method 400 can be initiated when a chromatographic elution pool is added to a first vessel (block 402). Acid can be added to the first vessel (block 404) and mixed with a fluid known or suspected to contain at least one enveloped virus (e.g., by a stirrer in the first vessel) to acidify the fluid. A first pH probe associated with the first vessel can measure the pH value associated with the first vessel (block 406). The method may include determining whether the measured pH value is below (or within) a threshold pH value associated with virus inactivation (block 408). If the pH value measured by the first pH probe associated with the first container is not below (or outside the range of pH values) the threshold pH value associated with virus inactivation (NO in block 408), additional acid may be added to the first container (block 404), or the acid may be retained in the first container for an additional time before the pH of the first container is measured again (block 406). If the pH value measured by the first pH probe associated with the first container is below (or within the range of pH values) the threshold pH value for virus inactivation (YES in block 408), the addition of acid to the first container may be stopped (block 410), and the acidified fluid may be transferred to the second container (block 412). In some examples, method 400 may proceed from block 412 to block 424, as will be described in more detail below with respect to Figure 4B. In any case, method 400 may proceed from block 412 to block 414.

[0091] The first container can be filled with an equilibration buffer having a known pH (block 414), and the first pH probe associated with the first container can measure the pH associated with the first container (block 416). The measured pH value associated with the first container can be compared with the known pH value of the equilibration buffer (block 418) to determine whether the difference between the measured pH value associated with the first container and the known pH value of the equilibration buffer is greater than a threshold pH value (e.g., greater than 0.1 pH units). If the measured pH value associated with the first container is within 0.1 pH units of the known pH value of the equilibration buffer (NO in block 418), method 400 can be terminated, or a new virus inactivation cycle can be started by adding a new fluid known or suspected to contain at least one enveloped virus to the first container (after discarding the equilibration buffer from the first container) and proceeding to block 402.

[0092] If the measured pH value of the pH probe associated with the first container is not within 0.1 pH units of the known pH value of the equilibration buffer (YES in block 418), an alarm may be generated indicating that the pH probe should be recalibrated (block 420). In some examples, method 400 may include displaying or otherwise communicating the alarm to the operator (e.g., via a user interface display) so that the operator can manually recalibrate the pH probe if necessary. Furthermore, in some examples, the method may include automatically recalibrating the pH probe so that the pH probe measures pH within 0.1 pH units of the equilibration buffer (block 422).

[0093] Referring now to Figure 4B, as described above, method 400 may include proceeding from block 412 to block 424.

[0094] A second pH probe associated with the second container can measure the pH value associated with the first container (block 424). The method may include determining whether the measured pH value is below (or within) a threshold pH value associated with virus inactivation (block 426). If the pH value measured by the second pH probe associated with the second container is not below (or within) the threshold pH value for virus inactivation (NO in block 426), the process may be suspended (block 428), and an alarm may be issued to the operator prompting them to investigate, for example, whether there is any problem with the measured pH. If the pH value measured by the second pH probe associated with the second container is below the threshold pH value (YES in block 426), the method may proceed to block 430 to determine whether the elapsed time after transferring the acidified fluid from the first container to the second container has exceeded the threshold time (e.g., 30 minutes or less) that inactivates the virus concentration in the fluid to a predetermined safe level. If the threshold time is not exceeded (NO in block 430), the determination in block 430 may be performed again after an additional elapsed time. If the threshold time is exceeded (YES in block 430), the second pH probe associated with the second container may measure the pH value associated with the first container again (block 432). The method may include determining whether the measured pH value is less than (or within the range of) the threshold pH value associated with virus inactivation (block 434). If the pH value measured by the second pH probe associated with the second container is not less than (or outside the range of) the threshold pH value for virus inactivation (NO in block 434), the process may be suspended (block 436), and an alarm may be issued to the operator prompting them to investigate, for example, whether there is any problem with the measured pH.

[0095] If the pH value measured by the second pH probe associated with the second container is below the threshold pH value (YES in block 434), the method proceeds to block 438, where a base can be added to the second container to neutralize the acidified fluid. The second pH probe associated with the second container can measure the pH value associated with the second container (block 440) and determine whether the measured pH value associated with the second container is within the acceptable range of neutral pH values ​​(e.g., pH range 5.0 to 6.0). If the measured pH value associated with the second container is not within the acceptable range (NO in block 442), additional base can be added to the container (block 438). If the measured pH value associated with the second container is within the acceptable range (YES in block 442), the addition of base to the second container can be stopped (block 444), and the neutralized virus-inactivating fluid can be transferred to a deep filter (block 446) and then to a sterilization grade filter (block 448).

[0096] The second container is filled with an equilibration buffer having a known pH (block 450), and the pH associated with the second container can be measured using a second pH probe associated with the second container (block 452). The measured pH value associated with the second container can be compared with the known pH value of the equilibration buffer (block 454) to determine whether the difference between the measured pH value associated with the second container and the known pH value of the equilibration buffer is greater than a threshold pH value (e.g., greater than 0.1 pH units). If the measured pH value associated with the second container is within 0.1 pH units of the known pH value of the equilibration buffer (NO in block 454), the method 400 can be terminated, or the process can proceed to block 412 (after discarding the equilibration buffer from the second container) and a new acidified fluid can be added to the second container.

[0097] If the measured pH value of the pH probe associated with the second container is not within a 0.1 pH unit range of the known pH value of the equilibration buffer (YES in block 454), an alarm may be generated indicating that the pH probe should be recalibrated (block 456). In some examples, method 400 may include displaying or otherwise communicating the alarm to the operator (e.g., via a user interface display) so that the operator can manually recalibrate the pH probe if necessary. Furthermore, in some examples, the method may include automatically recalibrating the pH probe so that the pH probe measures pH within a 0.1 pH unit range of the equilibration buffer (block 458).

[0098] Fluids known or suspected to contain at least one enveloped virus include effluents, elutes, pools, storage or retained materials from unit operations including a collection step, a filtration step, or a chromatography step. Fluids may be from elutes collected from deep filtration, microfiltration, affinity chromatography, ion exchange chromatography, multimodal chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography. Fluids may be from pools containing collected cell cultures, elutes from deep filtration, elutes from microfiltration, elutes from affinity chromatography, elutes from ion exchange chromatography, elutes from multimodal chromatography, elutes from hydrophobic interaction chromatography, or elutes from hydroxyapatite chromatography. The fluid added to the first tank may be added in its entirety at once, or it may be divided and treated over multiple virus inactivation / neutralization cycles. The fluid can be added in its original form or diluted with a suitable buffer or water to achieve the desired parameters or volume. The fluid in the first tank may be a single pool containing multiple eluent pools.

[0099] The pool added to the first tank can be diluted with a suitable medium such as water. In one embodiment, the pool is diluted to 50-200%. In one embodiment, the pool is diluted to 50-100%. In one embodiment, the pool is diluted to 50-75%. In one embodiment, the pool is diluted to 75-200%. In one embodiment, the pool is diluted to 75-100%. In one embodiment, the pool is diluted to 100-200%.

[0100] The fluid temperature can be in the range of 5 to 25°C. Acidification can be carried out at a temperature of 5 to 25°C. In one embodiment, the temperature is 15 to 25°C. In one embodiment, the temperature is 15 to 20°C, and in one embodiment, the temperature is 20 to 25°C. In one embodiment, the temperature is 20°C.

[0101] In one embodiment, the fluid is added to the first tank at a flow rate of 0.025 to 0.25 kg / min.

[0102] At the minimum operating volume, the pH probe and agitator must be completely immersed in the liquid, and the acid / base inlet must be below the liquid surface. In one embodiment, the operating volume is 1 to 9 liters.

[0103] The acid is added to the fluid and mixed by stirring to acidify the fluid. The fluid may be stirred at 10-30 rpm, or 15-30 rpm in one embodiment. The stirring speed should be appropriate for the fluid level and should not cause splashing or vortex formation.

[0104] Suitable acids for use include formic acid, acidic acid, citric acid, and phosphoric acid at concentrations suitable for ensuring virus inactivation. In one embodiment, the acidic acid is added at a concentration of approximately 70 mL / L.

[0105] The acidified fluid may be retained in the first tank for the time required for the fluid to become sufficiently acidic or for the entire time necessary to achieve the desired degree of virus inactivation, before being transferred to the second tank. The time required for sufficient acidification is 30 minutes or less or more than 30 minutes. The time required for virus inactivation may be 30 minutes to 24 hours or more.

[0106] The pH for virus inactivation is pH 2 to 4. In one embodiment, the pH for virus inactivation is 3 to 4. In one embodiment, the pH for virus inactivation is 3.5 to 4. In one embodiment, the pH is 3.6 to 4. In one embodiment, the pH for virus inactivation is 3.7 to 4. In one embodiment, the pH for virus inactivation is 3.5 to 3.7. In one embodiment, the pH for virus inactivation is 3.5 to 3.7. In one embodiment, the pH for virus inactivation is 3.6.

[0107] The acidified (or virus-inactivated) fluid is then transferred to a second tank. In one embodiment, the fluid is transferred at a rate of 0.025 to 0.25 kg / min.

[0108] The transfer from tank 1 to tank 2 can be completed within 15 minutes.

[0109] At least 1 to 10 liters of acidified (or virus-inactivated) fluid are transferred from tank 1 to tank 2.

[0110] The fluid can be stirred at 10-30 rpm to mix the acid with the fluid, and in one embodiment, stirring is performed at 15-30 rpm. The stirring speed should be appropriate to the fluid level and should not cause splashing or vortex formation. The system should be able to achieve 95% homogeneity within 3 minutes after adding the tracer solution until the water tank is filled (maximum operating volume) within the design stirring range.

[0111] If the acidified fluid is transferred to a second tank before the completion of virus inactivation, the acidified fluid is maintained at a desired pH until the desired degree of inactivation is achieved. It can be determined whether the acidified fluid from the first container has been maintained for a threshold time of virus inactivation, and in one embodiment, the time required for virus inactivation is 30 minutes to 24 hours or more. In one embodiment, the time required for virus inactivation is 60 minutes to 360 minutes. In one embodiment, the time required for virus inactivation may be 60 minutes to 90 minutes. In one embodiment, the time required for virus inactivation is 60 minutes.

[0112] Once virus inactivation is complete, a base is added to the virus inactivation (VI) solution and mixed to neutralize the solution to the desired pH. The base is added in an amount of 1-5% of the working volume of the second tank. Suitable bases for use include Tris base at a concentration of 2 M. In one embodiment, 2 M Tris base is added at a concentration of approximately 55 mL / L. The amount of base added can be verified by mass to ensure an additional precision tolerance of ±2% of the amount added. The time required for neutralization may be 30 minutes or less or more than 30 minutes.

[0113] At least one pH probe associated with the second tank measures the pH value associated with the second tank, and it is possible to determine whether the measured pH value associated with the second tank is within the acceptable range of the neutral pH value. The target pH for neutralization is 4.5 to 6. In one embodiment, the target pH for neutralization is 4.7 to 5.5. In one embodiment, the target pH for neutralization is 4.7 to 5.3. In one embodiment, the target pH for neutralization is 4.7 to 5.1. In one embodiment, the target pH for neutralization is 4.9 to 5.5. In one embodiment, the target pH for neutralization is 4.9 to 5.3. In one embodiment, the target pH for neutralization is 4.9 to 5.1.

[0114] Neutralization can be carried out at temperatures of 5 to 25°C. In one embodiment, neutralization is carried out at 15 to 25°C. In one embodiment, neutralization is carried out at 15 to 20°C. In one embodiment, neutralization is carried out at 20 to 25°C. In one embodiment, neutralization is carried out at 20°C.

[0115] The pH of the solution is monitored during the neutralization process, which is completed within 20 minutes.

[0116] The fluid may be stirred at 10-30 rpm to mix the base and the virus inactivation fluid, and in one embodiment, the stirring is performed at 15-30 rpm. Once neutralization is complete, the neutralized virus inactivation fluid is transferred from the second tank to a holding tank or storage tank or to a filter or chromatography medium.

[0117] The fluid can be transferred at a flow rate of 0.025 to 0.25 kg / min.

[0118] After removing the acidified or virus-inactivated fluid from the first tank (and similarly after removing the neutralized virus-inactivated fluid from the second tank), each tank is filled with an equilibration buffer of a known pH. To keep the pH probes immersed in the liquid and always wet, a suitable buffer is provided, which is acetate with a concentration of 100 mM and a pH of 5.0. To eliminate mixing of the equilibration buffer with the fluid used for virus inactivation or neutralization, the volume of the equilibration buffer must be completely removed from the tank and its associated discharge piping. The pH associated with the equilibration buffer in each tank can be measured by at least one pH probe associated with the tank. By comparing the measured pH value with the known pH value of the equilibration buffer, it can be determined whether the difference between the measured pH value measured by the probe in the tank and the known pH value of the equilibration buffer is greater than a threshold pH value (e.g., greater than ±0.1 pH units).

[0119] If the pH value measured by the pH probe attached to the tank is not within ±0.1 pH units of the known pH value of the equilibration buffer, an alarm can be generated indicating that the pH probe needs to be recalibrated. This may take the form of displaying the alarm to the operator (e.g., via a user interface display) or otherwise communicating it so that the operator can manually recalibrate the pH probe if necessary. In some embodiments, the method may include automatically recalibrating the pH probe so that it measures pH within ±0.1 pH units of the equilibration buffer.

[0120] Viruses are classified into enveloped viruses and non-enveloped viruses. Enveloped viruses have a capsid encased in a lipoprotein membrane, or "envelop." This envelope consists of host cell proteins and phospholipids, as well as viral glycoproteins that coat the virus when it budding from the host cell. This envelope allows the virus to identify, bind to, enter, and infect target host cells. However, due to this membrane, enveloped viruses are vulnerable to inactivation methods, while non-enveloped viruses are difficult to inactivate without risking the proteins produced, although they can be removed by filtration.

[0121] Enveloped viruses include viridae such as herpesviruses, poxviruses, hepadnaviruses, flaviviruses, togaviruses, coronaviruses, orthomyxoviruses, deltaviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, filoviruses, and retroviruses, as well as viruses such as human immunodeficiency virus, Sindbisvirus, herpes simplex virus, pseudorabies virus, Sendai virus, vesicular stomatitis V virus, West Nile virus, bovine viral diarrhea virus, coronaviruses, equine arthritis virus, severe acute respiratory syndrome virus, Moloney's mouse leukemia virus, and vaccinia virus.

[0122] To ensure patient safety, viral inactivation is a necessary component in the purification process when manufacturing protein-based therapeutic drugs. Various methods can be employed for viral inactivation, including heat inactivation / sterilization, ultraviolet and gamma ray irradiation, high-intensity broad-spectrum white light, chemical inactivators, surfactants, solvent / detergent treatment, and low-pH inactivation. Exposure of enveloped viruses to low pH causes viral denaturation.

[0123] Polypeptides and proteins of interest may be of scientific or commercial interest, including therapeutic agents primarily composed of proteins. Proteins of interest include, in particular, secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. Polypeptides and proteins of interest can be produced by recombinant animal cell lines using cell culture methods and may be referred to as “recombinant proteins.” Expressed proteins can be produced or expressed intracellularly and secreted into a culture medium from which the protein can be recovered and / or collected. The term “isolated protein” or “isolated recombinant protein” refers to a polypeptide or protein of interest that has been isolated and purified from a protein or polypeptide that inhibits therapeutic, diagnostic, preventive, research, or other uses, or from other contaminants. Proteins of interest include proteins that exert therapeutic effects by binding to targets, particularly inductive targets, related targets, etc., listed below, and their modifications.

[0124] Proteins of interest include proteins or polypeptides that contain an antigen-binding region or antigen-binding portion that has affinity for another molecule (antigen) to which they bind, i.e., "antigen-binding proteins." Proteins of interest include antibodies, peptide bodies, antibody fragments, antibody derivatives, antibody analogs, fusion proteins, genetically engineered cell surface receptors such as T cell receptors (TCRs) and chimeric antigen receptors (CAR or CAR-T cells, TRUCK (a chimeric antigen receptor that redirects T cells to universal cytokine-mediated killing), and armored CARs (designed to mitigate the immunosuppressive environment)), as well as other proteins containing antigen-binding molecules that interact with target antigens.Multispecific proteins and antibodies comprising proteins recombinantly engineered to simultaneously bind to and neutralize at least two different epitopes of the same antigen, including all forms of bispecific proteins and antibodies, where the bispecific proteins and antibodies include quadroma, nobu-in-hole, closumab, bivariable region IgG (DVD-IgG), IgG-single chain Fv (scFv), scFv-CH3KIH, dual-acting Fab (DAF), half-body exchange, κλ, tandem scFv, scFv-Fc, diabody, single chain diabody, sc-diabody-CH3, triplebody, mini-antibody, mini Body, TriBi minibody, tandem diabody, scdiabody HAS, tandem scFv toxin, biaffinity retargeting molecules (DARTs), nanobody, nanobody HSA, dock and lock (DNL), chain exchange operation region SEED body, Triomab, leucine zipper (LUZ-Y), XmAb (registered trademark), Fab arm exchange, DutaMab, DT-IgG, charge pair, Fcab, orthogonal Fab, IgG(H)-scFv, scFV-(H)IgG, IgG(L)-scFV, IgG(L1H1)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-VV(L)-IgG, KIH This includes, but is not limited to, IgG-scFab, 2scFV-IgG, IgG-2scFv, scFv4-Ig, Zy body, DVI-Ig4 (four-in-one), Fab-scFv, scFv-CH-CL-scFV, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc-diabodyFc, diabodyFc, intrabody, ImmTAC, HSA body, IgG-IgG, Cov-X body, scFv1-PEG-scFv2, single-chain bispecific antibody constructs, single-chain bispecific T cell engagers (BITE®), bispecific T cell engagers, Half-Life extended bispecific T cell engagers (HLEBITE®), hetero-IgBITE®, etc.

[0125] This also includes human and humanized antibodies, as well as human, humanized, and other antigen-binding proteins, that do not cause a significantly potent immune response when administered to humans.

[0126] This also includes modified proteins, such as those chemically modified by non-covalent, covalent, or both covalent and non-covalent bonds. It also includes proteins further comprising one or more post-translational modifications that can be carried out by cellular modification systems, or modifications introduced in vitro by enzymatic and / or chemical methods, or by other means.

[0127] In some embodiments, the protein of interest may include colony-stimulating factors such as granulocyte colony-stimulating factor (G-CSF). Such G-CSF agents include, but are not limited to, Neupogen® (filgrastim) and Neulasta® (pegfilgrastim). Other examples include erythropoiesis-stimulating agents (ESAs) such as Epogen® (epoetin alfa), Aranesp® (darbepoetin alfa), Dynepo® (epoetin delta), Mircera® (methioxypolyethylene glycol-epoetin beta), Hematide®, MRK-2578, INS-22, Retacrit® (epoetin zeta), Neorecormon® (epoetin beta), Silapo® (epoetin zeta), and Binocrit® (epoetin beta). This also includes epoetin alfa, epoetin alfa hexal, Abseamed® (epoetin alfa), Ratioepo® (epoetin theta), Eporatio® (epoetin theta), Biopoin® (epoetin theta), epoetin alfa, epoetin beta, epoetin zeta, epoetin theta, and epoetin delta, epoetin omega, epoetin iota, tissue plasminogen activators, GLP-1 receptor antagonists, and their variants or analogues and any of the biosimilars described above.

[0128] In another embodiment, the protein of interest is absiximab, adalimumab, adekatumumab, aflibercept, alemtuzumab, alirocumab, anakin, atacicept, axictagen siloleucel, basiliximab, belimumab, bevacizumab, biosozumab, blinatumomab, brentuximab vedotin, brodalumab, cantazumab meltansine, canakumab, catumaxomab, cetuximab, certolizumab pegol, konatumab, daclizumab, denozumab, eculizumab, etrecolomab, efalizumab, epratuzumab, ele Numab, erzmaxomab, etanercept, evolocumab, flotezumab (MGD006), galiximab, ganitumab, lutikizumab (ABT981), gemtuzumab, golimumab, ibritumomab, tiyukicetan, infliximab, ipilimumab, lerdolimumab, lumiliximab, lxdkizumab, lymphomun (FBTA05), mapatumumab, motesanib diphosphate, muromonab CD3, natalizumab, nesiritide, nimotuzumab, nivolumab, ocrelizumab, ofatumumab, omalizumab, oprelbequin, ozoraliz Mab (ATN103), palivizumab, panitumumab, pasotuxizumab (AMG112, MT112), pembrolizumab, pertuzumab, pexelizumab, ranivizumab, remtolumab (ABT122), rilotumab, rituximab, romiplostim, romosozumab, sargamostim, sucralostine, solitomab, targomi Rs, tezeperumab, tisagenlecleucel, tricisumab, tositumomab, trastuzumab, ustekinumab, vanucizumab (RG7221), vedolizumab, bicilizumab, boroxiximab, zanolimmab, saltumumab B, AMG211 (MT111, Medi-1565), AMG330, AMG420 (B1836909), AMG-110 (MT110), MDX-447, TF2, rM28, HER2Bi-aATC, GD2Bi-aATC, MGD006, MGD007, MGD009, MGD010, MGD011 (JNJ64052781), IMCgp100, Indium-labeled IMP-205, xm734, LY3164530, OMP-305BB3, REGN1979, COV322, ABT112, ABT165, RG-6013 (ACE910),This includes RG7597 (MEDH7945A), RG7802, RG7813 (RO6895882), RG7386, BITS7201A (RG7990), RG7716, BFKF8488A (RG7992), MCLA-128, MM-111, MM141, MOR209 / ES414, MSB0010841, ALX-0061, ALX0761, ALX0141, BII034020, AFM13, AFM11, SAR156597, FBTA05, PF06671008, GSK2434735, MEDI3902, MEDI0700, MEDI735, and their variants or analogues, as well as any of the biosimilars mentioned above.

[0129] In some embodiments, the proteins of interest may include proteins that bind specifically, alone or in combination, to one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factor, fibroblast growth factor, transforming growth factor (TGF), insulin-like growth factor, bone induction factor, insulin and insulin-related proteins, coagulation and coagulation-related proteins, colony-stimulating factor (CSF), other blood and serum proteins, blood group antigens, receptors, receptor-related proteins, growth hormone, growth hormone receptor, T cell receptor, neurotrophic factors, neurotrophins, relaxin, interferon, interleukin, viral antigens, lipoproteins, integrins, rheumatoid factor, immunotoxins, surface membrane proteins, transport proteins, homing receptors, adresins, regulatory proteins, and immune adhesins.

[0130] In some embodiments, the protein of interest binds alone or in any combination to one or more of the following CD proteins: CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD8, CD8 alpha, CD16, CD19, CD20, CD22, CD25, CD27, CD28, CD28T, CD30, CD33, CD34, CD37, CD38, CD40, CD45, CD49a, CD64, CD70, Igα (CD79a), CD80, CD86, CD123, CD133, CD134, CD137, CD138, CD154, CD171, CD174, and CD247 (B7-H3). For example, HER receptor family proteins including HER2, HER3, HER4 and EGF receptors, EGFRvIII, cell adhesion molecules such as LFA-1, CD1 1a / CD18, Mol, p150, 95, VLA-4, ICAM-1, VCAM and alpha-v / beta-3 integrins, growth factors including but not limited to vascular endothelial growth factor ("VEGF"), VEGFR2, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, Müllerian inhibitor, human macrophage inflammatory protein (MIP-1-alpha), erythropoietin (EPO), nerve growth factors such as NGF-β, platelet-inducible growth factor (PDGF), fibroblast growth factors including aFGF and bFGF, epidermal growth factor (EGF), crypto, transforming growth factor (TGF), etc. This includes TGF-α and TGF-β (including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5), insulin-like growth factor I and II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I) and bone induction factor, insulin, insulin A chain, insulin B chain, proinsulin, insulin-like growth factor binding protein, but is not limited to insulin and insulin-related proteins, particularly coagulation and coagulation-related proteins such as factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1 antitrypsin, urokinase and tissue plasminogen activator ("t-PA"), bombazine, thrombin,Plasminogen activators such as thrombopoietin and thrombopoietin receptors; colony-stimulating factors (CSFs), including particularly M-CSF, GM-CSF, and G-CSF; other blood and serum proteins, including but not limited to albumin, IgE, and blood group antigens; receptor-related proteins, including, for example, flk2 / flt3 receptor, obesity (OB) receptor, growth hormone receptor, and T cell receptor; bone-induced neurotrophic factor (BDNF); and neurotrophin 3, 4, 5, or 6 (NT-3, NT-4, NT-5, or NT-6). Undefined neurotrophic factors, relaxin A chain, relaxin B chain and prorelaxin, interferons including interferon-alpha, -beta and -gamma, interleukins (IL), such as IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, IL-12 / IL-23, IL-2R-alpha, IL-2R-beta, IL-2R-gamma, IL-7R-alpha, IL-1-R1, IL-6 receptor, IL-4 receptor and / or IL-13 receptor, IL-13RA2 or IL-17 receptor, IL-1RAP, AIDS envelope Viral antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor alpha and beta, enkephalinase, BCMA, Ig kappa, ROR-1, ERBB2, mesothelin, RANTES (modulation of action which is normally T cell expression and secretion), mouse gonadotropin-related peptides, DNase, FR alpha, inhibin and activin, but not limited to these, viral antigens, integrins, protein A or D, rheumatoid factor, immunotoxins, bone morphogenetic proteins (BMPs), supero Disidose dismutase, surface membrane protein, disintegration-promoting factor (DAF), AIDS envelope, transport protein, homing receptor, MIC (MIC-a, MIC-B), ULBP1-6, EPCAM, adresin, regulatory protein, immune adhesin, antigen-binding protein, somatotropin, CTGF, CTLA4, eotaxin 1, MUC1, CEA, c-MET, Claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, ganglioside GM2, BAFF, BAFFR,OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, HGF (hepatocyte growth factor), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesothelin dsFv PE38 conjugate, Legionnaires' disease (Ily), IFN gamma, interferon gamma-inducible protein 10 (IP10), IFNAR, TALL-1, TNFα, TNFr, TL1A, thymic stromal lymphapoietin (TSLP), protein converter subtilisin / kexin type 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet-specific platelet glycoprotein Iib / IIIb (PAC-1), tra Forming growth factor beta (TFGβ), STEAP1, zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, platelet-inducible growth factor receptor alpha (PDGFRα), 4-1BB / CD137, ICOS, LIGHT (tumor necrosis factor superfamily member 14, TMFSF14), DAP-10, Fc gamma receptor, MHC class I molecule, signal transduction lymphocyte activating molecule, BTLA, Toll ligand receptor, CDS, GITR, HVEM (LIGHT R), KIRDS, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, ITGA4, VLA1, VLA-6, IA4, CD49D, ITGA6, CD49f, ITGAD, CDl-ld, ITG AE, CD103, ITGAL, CDl-La, LFA-1, ITGAM, CDl-lb, ITGAX, CDl-lc, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TR ANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL 1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT,41-BB, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand, 5T4, AFP, ADAM 17, 17-A, ART-4, Alpha-v-Beta-6 integrin, BAGE, Bcr-abl, BCMA, B7-H3, B7-H6, CAIX, CAMEL, CAP-1, Carbonic anhydrase IX, CASP-8, CDC27m, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70 (CD27L or TNFSF7), CD79a, CD79b, CD123, CD138, CD171, CDK4 / m, Cadherin 19 (CDH19), placental cadherin (CDH3), CEA, CLL-1, CSPG4, CT, Cyp-B, DAM, DDL3, EBV, EGFR, EGFRvIII, EGP2, EGP40, ELF2M, ErbB2 (HER2) , EPCAM, EphA2, EpCAM, ETV6-AML1, FAP, fetal AchR, FLT3, FRα, G250, GAGE, GD2, GD3, 'Glypican-3(GPC3), GNT-V, GP-100, HAGE, HB V, HCV, HER-2 / neu, HLA-A, HPV, HSP70, HST-2, hTERT, iCE, IgE, IL-11Rα, IL-13Rα2, Kappa, KIAA0205, LAGE, Lambda, LDLR / FUT, Lewis-Y, MAGE, MAGE1, MAGEB2, MART-1, / Melan A, MC1R, MCSP, MUM-1, MUM-2, MUM-3, Mesothelin (MSLN), Muc1, Muc16, Myosin / m, NA88-A, NCAM, NKG2D ligand, NY-ESO-1, P15, p190 minor bcr-abl, PML / RARa, PRAME, PSA, PSCA, PSMA, RAGE, ROR1, RU1, RU2, SAGE, SART, SSX-1, SSX-2, SSX-3, Survivin, TAA, TAG72, TEL / AML1, TEMs, TPI, TRP-1, TRP-2, TRP-2 / INT2, VEGFR2, WT1, and any of the above-mentioned bioactive fragments or variants.

[0131] The proteins of interest according to the present invention encompass all of the above, and further include antibodies containing complementarity-determining regions (CDRs) 1, 2, 3, 4, 5, or 6 of any of the antibodies described above. Variants also include those containing regions whose amino acid sequence is identical to that of the reference amino acid sequence of the protein of interest by 70% or more, particularly 80% or more, particularly 90% or more, particularly 95% or more, particularly 97% or more, particularly 98% or more, and particularly 99% or more. This identity can be determined using various publicly known and readily available amino acid sequence analysis software. Preferred software includes those implementing the Smith-Waterman algorithm, which is considered a satisfactory solution to the problem of sequence search and alignment. Other algorithms may also be employed, particularly when speed is a critical requirement. In this regard, commonly used programs for DNA, RNA, and polypeptide alignment and homology matching include FASTA, TFASTA, BLASTN, BLASTP, BLASTX, TBLASTN, PROSRCH, BLAZE, and MPSRCH, the latter being an implementation of the Smith-Waterman algorithm running on a MasPar massively parallel processor.

[0132] "Culturing" means growing and propagating cells outside of a multicellular organism or tissue. Appropriate culture conditions for host cells, such as mammalian cells, are known in the art. Cell media and tissue media are used interchangeably to refer to a medium suitable for host cell growth during in vitro cell culture. Typically, cell media contain buffers, salts, energy sources, amino acids, vitamins, and trace essential elements. Any medium capable of supporting the growth of suitable host cells can be used during culture to maximize cell growth, cell viability, and / or recombinant protein production in specific cultured host cells, and may be further supplemented with other commercially available components. Various types of media can be used over the duration of cell culture. Host cells can be cultured in suspension or in an adherent form attached to a solid substrate. Cell culture can be established with or without microorganisms in fluid-bed bioreactors, hollow fiber bioreactors, roller bottles, shaking flasks, or agitated tank bioreactors.

[0133] Cell cultures can be operated in batch, fed-batch, continuous, semi-continuous, or perfusion modes. Mammalian host cell lines, such as CHO cells, can be cultured in small bioreactors of less than 100 ml to less than 1000 ml. Alternatively, larger bioreactors containing 1000 ml to over 20,000 liters of culture medium can be used. Large-scale cell cultures, such as those used in the clinical and / or commercial biomanufacturing of protein therapeutics, can be maintained for several weeks, or even months, while the cells produce the desired protein.

[0134] Cell culture media containing the expressed recombinant protein can then be collected from the cell culture in a bioreactor. Methods for collecting expressed proteins from suspended cells are known in the art and include, but are not limited to, filtration methods including acid precipitation, accelerated sedimentation such as aggregation, separation by gravity, centrifugation, acoustic wave separation, and membrane filtration using ultrafilters, microfilters, tangential flow filters, deep and alluvial filters. Recombinant proteins expressed by prokaryotes can be recovered from cytoplasmic inclusions by redox folding processes known in the art.

[0135] The recombinant protein of interest in the collected and clarified cell culture medium can then be purified or partially purified using one or more unit operations from residual impurities such as residual cell culture medium, cell extracts, unwanted components, host cell proteins, improperly expressed proteins, contaminants, microorganisms such as bacteria and viruses, and aggregates.

[0136] The term "unit operation" refers to a functional step performed in a process, for example, purifying recombinant proteins from a liquid culture medium. For example, a unit operation may include, but is not limited to, steps such as sampling, capture, purification, polishing, viral inactivation, viral filtration, and / or adjustment of the concentration and formulation of the fluid containing the recombinant protein of interest. A unit operation may also include steps in which the fluid is sampled while in a holding or storage container, chromatography, viral inactivation and neutralization or filtration, followed by the fluid being pooled, held, and / or stored in a capture pool. A single unit operation may be designed to achieve multiple objectives in the same operation, such as sampling and viral inactivation or capture and viral inactivation.

[0137] Capture unit operations include capture chromatography using resins and / or membranes containing agents that bind to and / or interact with recombinant proteins of interest, such as affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography (HIC), and immobilized metal affinity chromatography (IMAC). Such materials are known in the art and are commercially available. Affinity chromatography may include, for example, substrate binding capture mechanisms, antibody or antibody fragment binding capture mechanisms, aptamer binding capture mechanisms, and cofactor binding capture mechanisms. Exemplary affinity chromatography media include protein A, protein G, protein A / G, and protein L. Recombinant proteins of interest can also be purified from IMAC using imidazole after tagging with a polyhistidine tag, or by using specific antibodies targeting epitopes such as FLAG® protein tags after tagging with such epitopes.

[0138] Inactivation of enveloped viruses known or suspected to be present in the fluid can be performed at any point during downstream processing. During the production of a biological product, viral inactivation in a fluid containing recombinant protein of interest can be performed in one or more independent viral inactivation unit operations. In one embodiment, viral inactivation is performed before, as part of, or after a sampling unit operation. In one embodiment, viral inactivation occurs following a sampling unit operation, and in the relevant embodiment, the sampling unit operation includes ultrafiltration and / or microfiltration. In one embodiment, viral inactivation is performed before, as part of, or after a chromatography unit operation. In one embodiment, viral inactivation is performed before, as part of, or after one or more capture chromatography unit operations. In one embodiment, viral inactivation is performed before, as part of, or after one or more affinity chromatography unit operations. In one embodiment, viral inactivation is performed before, as part of, or after one or more protein A chromatography, protein G chromatography, protein A / G chromatography, protein L chromatography, and / or IMAC chromatography. In one embodiment, viral inactivation is performed before, as part of, or after one or more affinity chromatography unit operations. In one embodiment, viral inactivation is performed before, as part of, or after one or more ion exchange chromatography, hydrophobic interaction chromatography, mixed modal or multimodal chromatography, and / or hydroxyapatite chromatography unit operations. In one embodiment, viral inactivation is performed before, as part of, or after one or more ion exchange chromatography. In one embodiment, viral inactivation is performed before, as part of, or after a cation exchange chromatography unit operation. In one embodiment, viral inactivation is performed before, as part of, or after an anion exchange chromatography unit operation. In one embodiment, virus inactivation is performed before, as part of, or after a multimodal or mixed modal chromatography unit operation.In one embodiment, virus inactivation is performed before, as part of, or after a hydrophobic interaction chromatography unit operation. In one embodiment, virus inactivation is performed before, as part of, or after a hydroxyapatite chromatography unit operation. In one embodiment, virus inactivation is performed before, as part of, or after one or more ion exchange chromatography, hydrophobic interaction chromatography, mixed modal or multimodal chromatography, and / or hydroxyapatite chromatography unit operations. In one embodiment, virus inactivation is performed before, as part of, or after a filter unit operation. In one embodiment, virus inactivation is performed before, as part of, or after a virus filtration unit operation. In one embodiment, virus inactivation is performed before, as part of, or after a deep filtration unit operation. In one embodiment, virus inactivation is performed before, as part of, or after a sterile filtration unit operation. In one embodiment, virus inactivation is performed before, as part of, or after one or more deep filtration unit operations and / or sterile filtration unit operations. In one embodiment, virus inactivation is performed before or after one or more ultrafiltration / diafiltration unit operations.

[0139] A filtration and / or chromatography unit operation may be performed following a virus inactivation unit operation. In one embodiment, virus inactivation is performed before, as part of, or after a deep filtration and / or sterile filtration unit operation to remove inactivated viruses, other inactivators such as surfactants and detergents, turbidity and / or precipitates.

[0140] In this specification, the term "polishing" refers to one or more chromatographic steps performed to remove residual contaminants and impurities such as DNA and host cell proteins, formulation-specific impurities, variant formulations and aggregates, and viral adsorbents from a fluid containing recombinant protein that is close to the final desired purity. For example, polishing can be performed in a binding and elution mode by passing the recombinant protein-containing fluid through a chromatographic column or membrane absorber that selectively binds to either the target recombinant protein or any contaminants or impurities present in the recombinant protein-containing fluid. In such an example, the eluate / filtrate from the chromatographic column or membrane absorber contains the recombinant protein.

[0141] Polish chromatography unit operations use chromatographic resins and / or membranes containing agents usable in, for example, flow-through mode, overload or frontal chromatography mode, or binding and elution mode. Suitable chromatographic media for use in such operations include ion exchange chromatography (IEX), such as anion exchange chromatography (AEX) and cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), mixed modal or multimodal chromatography (MM), hydroxyapatite chromatography (HA), reversed-phase chromatography, and gel filtration.

[0142] A method is provided for inactivating enveloped viruses during the purification of recombinant proteins of interest, the method comprising obtaining a fluid known or suspected to contain at least one enveloped virus, and irradiating the fluid with a system or method described herein at a concentration and time sufficient to cause viral inactivation, followed by neutralizing the viral inactivation fluid. The neutralized viral inactivation fluid can be stored for later use. The neutralized viral inactivation fluid can be subjected to at least one unit operation, including at least a filtration step or a chromatography step.

[0143] A method is also provided for inactivating enveloped viruses during the purification of recombinant proteins of interest, the method comprising: obtaining a fluid known or suspected to contain at least one enveloped virus; applying a system or method described herein to the fluid at a concentration and time sufficient to cause viral inactivation; and applying at least one unit operation to the neutralized virus-inactivated fluid, comprising at least a filtration step or a chromatography step. In one embodiment, the filtration step comprises deep filtration. In one embodiment, the filtration step comprises deep filtration and sterile filtration. In one embodiment, the chromatography step comprises affinity chromatography. In one embodiment, affinity chromatography is selected from protein A chromatography, protein G chromatography, protein A / G chromatography, protein L chromatography, or IMAC. In one embodiment, the chromatography step comprises one or more polish chromatography steps. In one embodiment, polish chromatography is selected from ion exchange chromatography, hydrophobic interaction chromatography, multimodal or mixed modal chromatography, or hydroxyapatite chromatography.

[0144] A method is also provided for producing isolated and purified recombinant protein of interest, which includes establishing a cell culture in a bioreactor using host cells expressing the recombinant protein and culturing the cells to express the recombinant protein of interest; collecting a cell culture medium containing the recombinant protein of interest; processing the fluid containing the recombinant protein of interest through at least two unit operations, including a system or method that performs viral inactivation as described herein for a time sufficient to cause enveloped virus inactivation and neutralization; processing the neutralized, virus-inactivated fluid containing the recombinant protein of interest through at least one additional unit operation; and obtaining isolated and purified recombinant protein of interest.

[0145] Isolated and purified recombinant proteins of interest produced using the systems and methods described herein are also provided. Pharmaceutical compositions comprising isolated proteins of interest produced using the systems and methods described herein are also provided.

[0146] While the preceding text provides detailed descriptions of many different embodiments, please understand that the legal scope of the present invention is defined by the language of the claims disclosed at the end of this patent. Since describing every possible embodiment would be impractical, if not impossible, please understand that the detailed descriptions are illustrative and do not describe all possible embodiments. Many alternative embodiments can be realized using either the current technology or technology developed after the filing date of this patent, and these too would be included within the scope of the claims.

[0147] In this patent, unless a term is explicitly defined using the phrase "The term "_" as used herein is defined as meaning..." or similar wording, it is not intended, explicitly or implicitly, to limit the meaning of that term beyond its plain or ordinary meaning, and such term should not be interpreted as having a limited scope based on any description in any chapter of this patent (excluding the wording of the claims). This is done for clarity to avoid confusing the reader, insofar as all terms described in the final claims of this patent are referred to in a manner consistent with a single meaning in this patent, and it is not intended that such terms in the claims be limited to that single meaning, whether implicit or not.

[0148] Throughout this specification, unless otherwise indicated, multiple instances may implement elements, actions, or structures described as a single instance. While individual actions of one or more methods are illustrated and described as separate actions, one or more of these actions may be performed simultaneously, and they do not need to be performed in the illustrated order. Structures and functions shown as separate elements in exemplary configurations may similarly be implemented as combined structures or elements. Similarly, structures and functions shown as single elements may be implemented as separate elements. These and other variations, modifications, additions, and improvements are included within the scope of this specification.

[0149] In addition, certain embodiments described herein include logic, i.e., a plurality of routines, subroutines, applications, or instructions. These include either software (code implemented in a non-temporary tangible machine-readable medium) or hardware. In hardware, routines, etc., are tangible units capable of performing specific operations and may be configured or arranged in a particular manner. In exemplary embodiments, one or more computer systems (e.g., standalone, client, or server computer systems) or one or more hardware modules of a computer system (e.g., a processor or a group of processors) may be configured as hardware modules that operate to perform specific operations as described herein by software (e.g., an application or application portion).

[0150] In various embodiments, hardware modules may be implemented mechanically or electronically. For example, a hardware module may include dedicated circuitry or logic permanently configured to perform a specific operation (e.g., as a dedicated processor such as a field-programmable gate array (FPGA) or application-specific integrated circuit (ASIC)). A hardware module may also include programmable logic or circuitry temporarily configured by software to perform a specific operation (e.g., contained within a general-purpose processor or other programmable processor). It will be understood that the decision to implement a hardware module mechanically, in dedicated and permanently configured circuitry or in temporarily configured circuitry (e.g., configured by software) may be made with cost and time considerations in mind.

[0151] Hardware modules can provide and receive information with other hardware modules. Therefore, the hardware modules described can be considered to be communicatively coupled. When multiple such hardware modules exist simultaneously, communication can be achieved through signal transmission (e.g., via appropriate circuits and buses) connecting the hardware modules. In multiple embodiments where multiple hardware modules are configured or instantiated at different points in time, communication between such hardware modules can be achieved, for example, through the storage and retrieval of information into and from a memory structure accessible to the multiple hardware modules. For example, one hardware module may perform an operation and store the output of that operation in a memory device to which it is communicatively coupled. Further hardware modules can then access the memory device, retrieve the stored output, and process it. Hardware modules can also initiate communication with input or output devices to perform operations on resources (e.g., a set of information).

[0152] Various operations of the exemplary methods described herein may be performed at least in part by one or more processors that are configured (e.g., by software) temporarily or permanently to perform the operations in question. Whether configured temporarily or permanently, such processors may constitute a processor implementation module that operates to perform one or more operations or functions. The modules referred to herein may, in some exemplary embodiments, include modules implemented in a processor.

[0153] Similarly, in some embodiments, the methods or routines described herein may be implemented at least partially by a processor. For example, at least part of the operation of a method may be performed by one or more processors or processor implementation hardware modules. The execution of a particular operation may be distributed across one or more processors and may reside not only within a single machine but also deployed across a number of machines. In some exemplary embodiments, one or more processors or processor implementation modules may be located in a single geographical location (e.g., within a residential environment, an office environment, or a server farm). In other exemplary embodiments, one or more processors or processor implementation modules may be distributed across a number of geographical locations.

[0154] Unless otherwise specifically stated, discussions using words such as “processing,” “computing,” “calculating,” “determining,” “presenting,” and “displaying” in this specification may refer to the operation or processing of a machine (e.g., a computer) that manipulates or transforms data represented as physical (e.g., electronic, magnetic, or optical) quantities in one or more memories (e.g., volatile memory, non-volatile memory, or a combination thereof), registers, or other mechanical elements that receive, store, transmit, or display information.

[0155] Any reference in this specification to “a certain embodiment” or “one embodiment” means that the specific elements, features, structures, or characteristics described in relation to that embodiment are included in at least one embodiment. Any other occurrences of the phrases “in one embodiment” or “in some embodiments” in this specification do not necessarily refer to the same one or more embodiments.

[0156] Some embodiments may be described using the terms “combined,” “connected,” “communicatively connected,” or “communicatively coupled,” along with their derivatives. These terms may refer to direct physical connections or indirect (physical or communication) connections. For example, some implementations may be described using the term “combined” to indicate that two or more elements are in direct physical or electrical contact. However, the term “combined” may also mean that two or more elements do not directly contact each other but cooperate or interact with each other. Unless explicitly stated or required in connection with its use, embodiments are not limited to direct connections.

[0157] The terms “include,” “contain,” “incorporate,” “contain,” “have,” “possess,” or any other variations thereof as used herein are intended to refer to non-exclusive inclusion. For example, a process, method, article, or apparatus that includes a list of elements is not necessarily limited to those elements alone, and may include other elements that are not expressly enumerated or are not specific to such process, method, article, or apparatus. Furthermore, unless expressly stated otherwise, “or” means inclusive “or” and not exclusive “or.” For example, condition A or B is satisfied by any one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0158] In addition, the use of the words “a” or “an” is employed in the description of elements and parts of the embodiments herein. This is done solely for convenience and to give a general meaning to the description. This specification and the subsequent claims should be read as including one or at least one unless otherwise evident from the context, and the singular form also includes the plural form.

[0159] A careful reading of this disclosure will enable those skilled in the art to understand further additional alternative structural and functional designs for automated pH adjustment cycles. Thus, while specific embodiments and applications have been illustrated and described, it will be understood that the embodiments disclosed are not limited to the exact structures and elements disclosed herein. Various modifications, alterations, and variations of the arrangement, operation, and details of the methods and apparatus disclosed herein may be made without departing from the concepts and scope defined in the appended claims, and will be apparent to those skilled in the art.

[0160] Specific features, structures, or properties of any particular embodiment can be combined in any suitable manner, and any suitable combination with one or more other embodiments is also possible, and the use of selected features does not necessarily have to correspond to the use of other features. Many modifications can be made to adapt a particular use, situation, or material to the essential scope and spirit of the invention. It will be understood that other variations and modifications of the embodiments of the invention described and illustrated herein are implementable in light of the teachings herein and are considered to be part of the spirit and scope of the invention.

[0161] Finally, the claims at the end of this patent application are not intended to be construed under Section 112(f) of the U.S. Patent Act unless the claims explicitly contain the conventional means-plus-function language, such as the language of “means for” or “steps for.”

Claims

1. An automated system for low pH virus inactivation, The first container and The second container, A first pH probe associated with the first container and configured to measure the pH of the contents of the first container, A source of fluid known or suspected to contain at least one enveloped virus, which is transferred to the first container, An acid pump configured to pump acid into the first container after the fluid has been transferred to the first container, wherein the acid pump is configured to stop pumping the acid into the first container in response to the first pH probe measuring a first pH value within an acceptable range of the target pH value for virus inactivation, A transfer pump configured to pump an acidified pool from the first container to the second container in response to the first pH probe measuring a first pH value below the threshold pH value for virus inactivation, and in response to the acid pump stopping the pumping of acid to the first container, A first buffer pump is configured to pump a first equilibration buffer having a first known pH value into the first container in response to the entire acidification pool being pumped from the first container, It is an alarm generator, The second pH value measured by the first pH probe after the first equilibration buffer has been pumped into the first container is compared with the first known pH value of the first equilibration buffer, To determine whether the second pH value measured by the first pH probe differs from the first known pH value of the first equilibration buffer by more than a threshold pH value, In response to the second pH value measured by the first pH probe differing from the first known pH of the first equilibration buffer by more than the threshold pH value, a first alarm is generated. An alarm generator configured to perform the following actions An automated system including

2. The automated system for low-pH virus inactivation according to claim 1, further comprising a source pump configured to pump the fluid from the source to the first container based at least in part on a signal indicating that the first container is empty.

3. The automated system for low-pH virus inactivation according to claim 1, wherein the first buffer pump is configured to pump the first equilibrating buffer into the first container based at least in part on a signal indicating that the first container is empty.

4. A second pH probe associated with the second container and configured to measure the pH of the contents of the second container, A base pump configured to pump a base into the second container in response to the elapsed time since the entire acidification pool was pumped into the second container exceeding a threshold time for reducing the virus concentration in the acidification pool to a predetermined safe level, wherein the base pump is configured to stop pumping the base into the second container in response to the second pH probe measuring a first pH value within a threshold range of neutral pH values. To process the neutralized virus-inactivating pool, a discharge pump is configured to pump the neutralized virus-inactivating pool from the second container to a filter. A second buffer pump is configured to pump a second equilibration buffer having a second known pH value into the second container in response to the entire neutralized virus-inactivating pool being pumped from the second container. The alarm generator further includes, The second pH value measured by the second pH probe after the first equilibration buffer has been pumped into the second container is compared with the second known pH value of the second equilibration buffer, To determine whether the second pH value measured by the second pH probe differs from the second known pH value of the second equilibration buffer by more than the threshold pH value, In response to the second pH value measured by the second pH probe differing from the second known pH of the second equilibration buffer by more than the threshold pH value, a second alarm is generated. An automated system for low-pH virus inactivation according to claim 1, further configured to perform the following:

5. The automated system for low-pH virus inactivation according to claim 4, wherein the transfer pump is configured to pump the acidification pool from the first container to the second container at least in part on a signal indicating that the second container is empty.

6. The automated system for low-pH virus inactivation according to claim 4, wherein the second buffer pump is configured to pump the second equilibrating buffer into the second container based at least in part on a signal indicating that the second container is empty.

7. The third container and A recovery pump configured to pump the filtration pool from the filter to the third container, It further includes, The automated system for low-pH virus inactivation according to claim 4, wherein the recovery pump is configured to pump the filtration pool from the second container to the third container at least in part on a signal indicating that the third container is empty.

8. The automated system for low pH virus inactivation according to claim 1, further comprising a first pH probe recalibrator configured to automatically recalibrate the first pH probe in response to the first alarm.

9. The automated system for low pH virus inactivation according to claim 4, further comprising a second pH probe recalibrator configured to automatically recalibrate the second pH probe in response to the second alarm.

10. The automated system for low pH virus inactivation according to claim 4, further comprising an operator display configured to display one or more of the first alarms or the second alarms to an operator associated with the system.

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