CN116568697A

CN116568697A - In-process validation of pH probe calibration status

Info

Publication number: CN116568697A
Application number: CN202180074864.6A
Authority: CN
Inventors: V·纳塔拉詹; M·德利索; J·A·贝扎尔; A·卡维利; J·S·康纳; J·亨特; S·佩尔森; S·惠斯通
Original assignee: Amgen Inc
Current assignee: Amgen Inc
Priority date: 2020-11-09
Filing date: 2021-11-09
Publication date: 2023-08-08

Abstract

An automated low pH virus inactivation system and method includes adding an elution pool with an acid to a first container. Once the first vessel pH probe measures a sufficiently low pH, the pool is transferred to the second vessel, at which point the pH is checked again and the pool is maintained for a period of time sufficient to reduce the virus concentration to a safe level, and neutralized, filtered and transferred to the third vessel. At the same time, the first container is filled with a buffer of known pH, which is checked against the reading from the first container pH probe to determine if recalibration is required. After transferring the pool to the third container, the second container is filled with a buffer of known pH, which is checked against the reading from the second container pH probe to determine if recalibration is required. The process is repeated as buffer of known pH is poured out and a new elution pool is added to the first container.

Description

In-process validation of pH probe calibration status

Cross Reference to Related Applications

The present application claims provisional application No. 63/111,502 entitled "pH Probe calibration State IN-process validation (IN-PROCESS VERIFICATION OF CALIBRATION STATUS OF PH PROBES)", filed 11/9/2020; and provisional application number 63/168,608 entitled "pH Probe calibration State IN-process validation (IN-PROCESS VERIFICATION OF CALIBRATION STATUS OF PH PROBES)" filed on 3/31/2021, the respective disclosures of which are incorporated herein by reference IN their entirety.

Technical Field

The present disclosure relates generally to viral inactivation, and more particularly to techniques for automated viral inactivation (including automated cycling of pH adjustment).

Background

The background description provided herein is for the purpose of generally presenting the context of the disclosure. Work of the presently named inventors, to the extent it is described in this background section, as well as aspects of the description that may not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art against the present disclosure.

The use of cell culture methods to manufacture therapeutic recombinant biological products carries an inherent risk of transmission of viral contaminants. Such contaminants may come from a variety of sources, including starting materials, use of reagents of animal origin, and/or contamination of manufacturing systems due to malfunctions in the GMP process. Thus, regulatory authorities recommend that the biological manufacturing process have specialized viral inactivation and viral removal steps and require manufacturers to verify the removal and inactivation of viruses to ensure the safety of the recombinant biological product. The virus inactivation step is focused on enveloped viruses (e.g., retroviruses) and the virus filtration step removes those viruses that are not affected by the inactivation method (non-enveloped viruses). Some commonly used methods of inactivating an enveloped virus include disrupting the envelope by heating, using solvents and/or detergents, and/or low pH treatments. When inactivating viruses using an inactivating agent (e.g., a detergent), further purification is required to remove the detergent. Advantageously, low pH viral inactivation does not require further purification to remove the inactivating agent.

Viral inactivation may be performed throughout the downstream purification process. Guidance factors that aid in determining the location of a viral inactivation unit operation include the effect of the viral inactivation step on subsequent unit operations, and if an inactivating agent (e.g., a detergent or solvent) is used, how effective the agent is in subsequent downstream steps, and whether the conditions of the particular unit operation are compatible with the viral inactivation step. For example, the virus inactivation unit operation is typically performed after the first step of the downstream process after harvesting the cell culture broth from the bioreactor. Typically, this is an affinity chromatography step that removes almost all impurities from the harvested fluid. Protein a is a common method of affinity chromatography, directed against proteins (e.g., antibodies) having an Fc region. Since elution from the protein a column is typically performed at a lower pH, the low pH virus inactivation step works well because the eluate is already at a reduced pH. The acidified eluate is maintained for an amount of time to determine the inactivated virus concentration based on the number of recordings required. This step is after neutralization (typically to a pH of 5 or higher), because if left at a reduced pH for too long, the recombinantly expressed protein may be damaged, and the subsequent purification step typically requires a higher pH.

The current industry standard for viral inactivation in downstream biological processes is manual titration of eluate collections using pH probes. As continuous manufacturing progresses, the frequency of running the process has increased from once per culture run to at least once per day throughout the production period. This requires a significant increase in labor and ultimately process costs.

In addition, in a typical virus inactivation unit operation performed in a holding vessel, the pH probe is kept dry after the virus inactivation cycle is completed, which may affect its calibration state. Thus, before a new virus inactivation cycle can begin, the operator must draw a sample and measure the pH using a bench probe to verify the calibration status of the pH probe.

Thus, there is a need for a method for reducing the labor and costs required during viral inactivation as well as keeping pH probes wet and automatically verifying their calibration status for viral inactivation unit operation during manufacturing. The invention described herein meets this need by verifying in the course of automated virus inactivation and pH probe calibration.

Disclosure of Invention

In one aspect, an automated low pH viral inactivation system is provided, the system comprising: a first container; a second container; a first pH probe associated with the first container and configured to measure a pH of a content of the first container; a fluid source to be transferred to the first container, the fluid source known or suspected to contain at least one enveloped virus; an acid pump configured to pump acid into the first container after transferring the fluid into the first container, and configured to stop pumping acid into the first container in response to the first pH probe measuring a first pH value within a target pH value tolerance range (tolerance band) 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 below a threshold pH for viral inactivation and in response to the acid pump stopping pumping acid into the first container; a first buffer pump configured to pump a first equilibration buffer having a first known pH into the first container in response to the entire acidified pool being pumped out of the first container; and an alert generator configured to: after the first equilibration buffer is pumped into the first vessel, comparing a second pH measured by the first pH probe to the first known pH of the first equilibration buffer; determining whether the difference between the second pH measured by the first pH probe and the first known pH of the first equilibration buffer is greater than a threshold pH; and generating a first alert in response to a second pH measured by the first pH probe that differs from the first known pH of the first equilibration buffer by more than the threshold pH.

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

Additionally, in some examples, the first buffer pump is configured to pump the first equilibration buffer into the first container based at least in part on a signal indicating that the first container is empty.

In some examples, the automated low pH viral inactivation system may further comprise: a second pH probe associated with the second container and configured to measure a pH of a content of the second container; an alkaline pump configured to pump alkaline into the second container in response to an elapsed time from the entire acidified pool being pumped into the second container exceeding a threshold amount of time to reduce the concentration of virus in the acidified pool to a predetermined safe level, and configured to stop pumping alkaline into the second container in response to the second pH probe measuring a first pH value within a threshold range of neutral pH values; a drain pump configured to pump the neutralized virally inactivated collection from the second container to a filter to process the neutralized virally inactivated collection; a second buffer pump configured to pump a second balanced buffer having a second known pH into the second container in response to the entire pool being pumped out of the second container; and the alert generator may be further configured to: after the first equilibration buffer is pumped into the second vessel, comparing a second pH measured by the second pH probe to the second known pH of the second equilibration buffer; determining whether a difference between the second pH measured by the second pH probe and the second known pH of the second equilibration buffer is greater than the threshold pH; and generating a second alert in response to a second pH measured by the second pH probe that differs from the second known pH of the second equilibration buffer by more than the threshold pH.

Further, in some examples, the transfer pump is configured to pump the acidified pool from the first container to the second container based at least in part on a signal indicating that the second container is empty.

Additionally, in some examples, the second buffer pump is configured to pump the second balancing buffer into the second container based at least in part on a signal indicating that the second container is empty.

Moreover, in some examples, the automated low pH viral inactivation system may further comprise a third container; and a collection pump configured to pump the filtered collection from the filter to the third container.

In some examples, the collection pump is configured to pump the filtered pool from the second container to the third container based at least in part on a signal indicating that the third container is empty.

Additionally, in some examples, the automated low pH virus inactivation system may further comprise a first pH probe recalibrator configured to automatically recalibrate the first pH probe in response to the first alarm. Similarly, in some examples, the automated low pH virus inactivation system may further comprise a second pH probe recalibrator configured to automatically recalibrate the second pH probe in response to the second alarm.

Further, in some examples, the automated low pH viral inactivation system may further comprise one or more additional pH probes associated with the first container and configured to measure the pH of the contents of the first container. Similarly, in some examples, the automated low pH viral inactivation system may further comprise one or more additional pH probes associated with the second container and configured to measure the pH of the contents of the second container.

Additionally, in some examples, the automated low pH virus inactivation system may further comprise an operator display configured to display one or more of the first alarm or the second alarm to an operator associated with the system.

Moreover, in some examples, the acid is selected from formic acid, acidic acid, citric acid, and phosphoric acid at concentrations suitable to ensure viral inactivation. Furthermore, in some examples, the threshold pH for viral inactivation is pH 2 to 4. Additionally, in some examples, the chromatographic elution pool is exposed to acid for less than 30 minutes prior to neutralization. Moreover, in some examples, the base is Tris base at a concentration of 2M. Further, in some examples, the threshold value for neutral pH ranges from pH4.5 to 6. In addition, in some examples, low pH virus inactivation is performed at a temperature of 5 ℃ to 25 ℃.

Furthermore, in some examples, the neutralized virus-inactivated chromatography elution pool is transferred from the second vessel to a holding vessel. For example, in some examples, the neutralized virus-inactivated chromatographic eluate collection is transferred from the second vessel to a depth filter. Additionally, in some examples, the neutralized virus-inactivated eluate is transferred to a sterile filter after depth filtration. Also, in some examples, the neutralized virus-inactivated chromatography elution pool is transferred from the second vessel to a first refining chromatography column.

In another aspect, an automated low pH viral inactivation method is provided, the method comprising: adding the pool to a first container; adding an acid to the first container; measuring a first pH associated with the first container by a first pH probe associated with the first container; stopping the addition of acid to the first container based on a first measured pH associated with the first container that is within an allowable range of a target pH for viral inactivation; transferring the pool from the first container to a second container; filling the first container with an equilibration buffer having a known pH; measuring a second pH associated with the first container by the first pH probe; comparing a second measured pH associated with the first container to the known pH of the equilibration buffer; determining whether a difference between the second measured pH associated with the first container and the known pH of the equilibration buffer is greater than a threshold pH; and generating a first alert in response to a second measured pH associated with the first container that differs from the known pH of the equilibration buffer by more than the threshold pH.

In some examples, transmitting the collection to the first container is based at least in part on receiving a signal indicating that the first container is empty.

Additionally, in some examples, filling the first container with the equilibration buffer is based at least in part on receiving a signal indicating that the first container is empty.

In some examples, the automated low pH virus inactivation method may further comprise adding a base to the second container after an elapsed time after transferring the collection to the second container exceeds a threshold for an amount of time to reduce the concentration of virus in the collection to a predetermined safe level; measuring a first pH associated with the second container by a second pH probe associated with the second container; stopping the addition of base to the second vessel based on a first measured pH associated with the second vessel that is within a threshold range of neutral pH values; transferring the pool from the second vessel to a filter to treat the neutralized virally inactivated pool; filling the second container with an equilibration buffer having the known pH; measuring a second pH associated with the second container by a second pH probe associated with the second container; comparing a second measured pH associated with the second container to the known pH of the equilibration buffer; determining whether a difference between the second measured pH associated with the second container and the known pH of the equilibration buffer is greater than a threshold pH; and generating a second alert in response to a second measured pH associated with the second container that differs from the known pH of the equilibration buffer by more than the threshold pH.

For example, in some examples, the acidified aggregate is transferred from the first container to the second container based at least in part on receiving a signal indicating that the second container is empty.

Additionally, in some examples, filling the second container with the equilibration buffer is based at least in part on receiving a signal indicating that the second container is empty.

Moreover, in some examples, the automated low pH viral inactivation method may further comprise transferring the collection from the filter to the third container.

For example, in some examples, transmitting the collection from the filter to the third container is based at least in part on receiving a signal indicating that the third container is empty.

Additionally, in some examples, the automated low pH virus inactivation method may further comprise recalibrating the first pH probe in response to the first alarm. Similarly, in some examples, the automated low pH virus inactivation method may further comprise recalibrating the second pH probe in response to the second alarm.

In still another aspect, during purification of the recombinant protein of interest, a method for inactivating an enveloped virus is provided, the method comprising: obtaining a fluid known or suspected to contain at least one enveloped virus; subjecting the fluid to one or more of the following steps at a concentration and for a time sufficient to cause viral inactivation: adding the fluid to a first container; adding an acid to the first container; measuring a first pH associated with the first container by a first pH probe associated with the first container; stopping the addition of acid to the first container based on a first measured pH associated with the first container that is within an allowable range of a target pH for viral inactivation; transferring the fluid from the first container to a second container; filling the first container with an equilibration buffer having a known pH; measuring a second pH associated with the first container by the first pH probe; comparing a second measured pH associated with the first container to the known pH of the equilibration buffer; determining whether a difference between the second measured pH associated with the first container and the known pH of the equilibration buffer is greater than a threshold pH; and generating a first alert in response to a second measured pH associated with the first container that differs from the known pH of the equilibration buffer by more than the threshold pH; and subjecting the neutralized virally inactivated fluid to at least one unit operation comprising at least a filtration step or a chromatography step.

In some examples, adding the fluid to the first container is based in part on receiving a signal indicating that the first container is empty.

Additionally, in some examples, transferring the fluid from the first container to the second container is based in part on receiving a signal indicating that the second container is empty.

Also, in some examples, filling the first container with the equilibration buffer is based in part on receiving a signal indicating that the first container is empty.

Further, in some examples, the fluid comprises a recombinant protein of interest. Moreover, in some examples, the fluid is a harvested host cell culture fluid. Additionally, in some examples, the fluid is from an effluent stream, eluate, pool, storage or holding vessel from a unit operation comprising a harvesting step, a filtration step, or a chromatography step. Furthermore, in some examples, the fluid is an eluate collected from depth filtration, microfiltration, affinity chromatography, ion exchange chromatography, multi-mode chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography. Additionally, in some examples, the fluid is a collection containing harvested cell culture fluid, an eluate from depth filtration, an eluate from microfiltration, an eluate from affinity chromatography, an eluate from ion exchange chromatography, an eluate from multi-mode chromatography, an eluate from hydrophobic interaction chromatography, or an eluate from hydroxyapatite chromatography. Furthermore, in some examples, the fluid is a harvested host cell culture fluid and the unit operation includes depth filtration. Additionally, in some examples, the fluid is a harvested host cell culture fluid and the unit operation includes microfiltration. Moreover, in some examples, the fluid is a harvested host cell culture fluid and the unit operation comprises protein a affinity chromatography. Further, in some examples, the fluid is a protein a eluate, and the unit operation includes depth filtration.

Also, in some examples, the affinity chromatography is protein a, protein G, protein a/G, or protein L chromatography. Additionally, in some examples, the chromatography is selected from affinity chromatography, protein a chromatography, ion exchange chromatography, anion exchange 20 chromatography, cation exchange chromatography; hydrophobic interaction chromatography; mixed mode or multimode chromatography or hydroxyapatite chromatography.

Additionally, in some examples, the unit operation includes depth filtration. Further, in some examples, the unit operation includes microfiltration.

In another aspect, an automated low pH viral inactivation system is provided, the automated system comprising: a first container; a second container; a first pH probe associated with the first container and configured to measure a pH of a content of the first container; a fluid source to be transferred to the first container, the fluid source known or suspected to contain at least one enveloped virus; an acid pump configured to pump acid into the first container after transferring the fluid into the first container, and configured to stop pumping acid into the first container in response to the first pH probe measuring a first pH value within an allowable range of a 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 below a threshold pH for viral inactivation and in response to the acid pump stopping pumping acid into the first container; a second pH probe associated with the second container and configured to measure a pH of a content of the second container; an alkaline pump configured to pump alkaline into the second container in response to an elapsed time from the entire acidified pool being pumped into the second container exceeding a threshold amount of time to reduce the concentration of virus in the acidified pool to a predetermined safe level, and configured to stop pumping alkaline into 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 drain pump configured to pump the neutralized virally inactivated collection from the second container to a filter to process the neutralized virally inactivated collection.

In yet another aspect, an automated low pH viral inactivation method is provided, the method comprising: adding the pool to a first container; adding an acid to the first container; measuring a first pH associated with the first container by a first pH probe associated with the first container; stopping the addition of acid to the first container based on a first measured pH associated with the first container that is within an allowable range of a target pH for viral inactivation; transferring the pool from the first container to a second container; adding base to the second container after the elapsed time after transferring the collection to the second container exceeds a threshold value of an amount of time that reduces the concentration of virus in the collection to a predetermined safe level; measuring a first pH associated with the second container by a second pH probe associated with the second container; stopping the addition of base to the second vessel based on a first measured pH associated with the second vessel that is within a threshold range of neutral pH values; and transferring the pool from the second vessel to a filter to treat the neutralized virally inactivated pool.

Further, in some examples, the acidified aggregate is transferred from the first container to the second container based at least in part on receiving a signal indicating that the second container is empty.

In another aspect, during purification of the recombinant protein of interest, a method for inactivating an enveloped virus is provided, the method comprising: obtaining a fluid known or suspected to contain at least one enveloped virus; subjecting the fluid to one or more of the following steps at a concentration and for a time sufficient to cause viral inactivation: adding the fluid to a first container; adding an acid to the first container; measuring a first pH associated with the first container by a first pH probe associated with the first container; stopping the addition of acid to the first container based on a first measured pH associated with the first container that is within an allowable range of a target pH for viral inactivation; transferring the fluid from the first container to a second container; adding a base to the second vessel; measuring a second pH associated with the second container by a second pH probe associated with the first container; stopping adding base to the second vessel based on a second measured pH associated with the second vessel that is within an allowable range of a neutral target pH; and subjecting the neutralized virally inactivated fluid to at least one unit operation comprising at least a filtration step or a chromatography step.

Drawings

The figures described below depict aspects of the systems and methods disclosed therein. The advantages will become more readily apparent to those of ordinary skill in the art from the following description of the preferred embodiments, which have been shown and described by way of illustration. As will be realized, the embodiments of the invention are capable of other and different embodiments and its details are capable of modification in various respects. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive. Further, the following description refers to the reference numerals included in the following drawings, where possible, wherein features depicted in the various drawings are designated by consistent reference numerals.

FIG. 1A illustrates a block diagram of an example automated system for low pH viral inactivation.

FIGS. 1B and 1C illustrate examples of how a two-vessel design may be used to prevent hanging drops in an exemplary automated system for low pH viral inactivation of FIG. 1A.

FIG. 2 illustrates tubing and instrumentation diagrams (P & ID) of an example automated system for low pH viral inactivation.

FIG. 3 illustrates a flow chart associated with an exemplary automated method for low pH viral inactivation using a fluid known or suspected of containing at least one enveloped virus.

Figures 4A-4B illustrate a flow chart associated with an exemplary automated method (including an automated cycle of pH probe calibration) for low pH virus inactivation using a fluid known or suspected to contain at least one enveloped virus.

Detailed Description

Inactivation of enveloped viruses known or suspected to be contained in a fluid may be performed by a number of different procedures including heat inactivation/pasteurization, treatment with solvents and/or detergents, UV and gamma irradiation, use of broad spectrum white light of high intensity, addition of chemical inactivating agents (such as B-propiolactone) and/or low pH virus inactivation.

The present disclosure relates generally to automated low pH viral inactivation systems and methods. Automated low pH viral inactivation systems and methods include synchronization with upstream and downstream units through their integration with a distributed control system, process control based on pool pH, and an automated viral inactivation pool filtration system.

For synchronization between upstream and downstream units, communication is required to signal the status of the batch. There are two different types of synchronization strategies; synchronous and asynchronous. The synchronization policy involves a unit sending a message to the secondary unit and suspending the process until the secondary unit acknowledges the message and sends an acknowledgement back. In contrast, an asynchronous policy does not require that the process be stopped by acknowledgement messages between units and will continue to perform its next step after the initial message is sent. In the automated system and method described herein, a synchronous communication system is used to prevent an upstream unit from transmitting a product collection to a downstream unit until it is ready. The synchronization strategy also enables the system to allow for a variable number of cycles from upstream chromatography by providing the option of processing each eluate pool or collecting multiple pools prior to processing. Automation is contained in a distributed control system and allows supervisory control.

In general, a fluid known or suspected of containing at least one enveloped virus is added to a first vessel and an acid is added to the first vessel to reduce the pH of an elution pool in the first vessel. Once the pH probe in the first container measures a sufficiently low pH, the acidified fluid is transferred to a second container. The use of two containers allows the pool to first drop to the inactivation pH in the first container and then transfer to the second container to maintain a validated inactivation time. This method eliminates the option for droplets of eluate to stick to the upper side of the container wall during holding and miss interactions with the acid, which would allow for the transfer of untreated pool droplets throughout the process. Using two containers, the contents of all the pools from the transfer to the second container were thoroughly mixed with the acid. Once the acidified fluid is maintained in the second container for a validated inactivation time, the virus is inactivated to a predetermined safe level and the acidified fluid in the second container is neutralized. In general, there are two options for the acidification and neutralization strategies, which can be selected when creating a batch recipe: fixed and variable. Incremental dosing was used in both strategies, but when a fixed option was used, the acid/base dose was constant, and when a variable option was used, the next dose was calculated based on the current pH of the pool and adjusted based on the results.

In any event, once the acidified fluid in the second vessel is neutralized, it is filtered by a combination of depth filtration and a sterile filtration system. A drain pump and a series of valves are used to direct the cleaning solution, preparation buffer and product collection through the filter to a third container. Batch recipe monitoring and advancing filtration processes on a distributed control system do not require operator involvement unless there are alarms that require confirmation. In existing systems, the inactivated product collection will have to be manually transferred to the filtration system. Advantageously, the use of the automated systems and methods described herein allows for a single closed system with connected inactivation and filtration processes.

At the same time, once the acidified fluid is transferred from the first container to the second container, i.e. once the first container is empty, a signal is sent upstream indicating that the first container is empty, causing the first container to be immediately filled with an equilibration buffer of known pH, keeping the pH probes wet, and the reading of the pH probes in the first container is checked against the known pH to determine if any one of the pH probes needs to be recalibrated. Generally, each container contains at least two probes: a primary probe and a backup probe providing pH readings, the backup probe being used as a backup probe in the event of a failure of the primary probe. In some cases, if the pH probe's reading differs from the known pH by more than a threshold amount, the pH probe may be automatically recalibrated, while in other cases an alert may be generated to an operator that will recalibrate the pH probe.

Once the neutralized virally inactivated fluid is transferred from the second container to the third container, i.e., once the second container is empty, a signal is sent upstream indicating that the second container is empty, causing the second container to be immediately filled with an equilibration buffer of known pH, and the pH probe of the second container is checked against the known pH to determine if recalibration is required. The process is then repeated in a new cycle. That is, once the equilibration buffer is removed from the first container, i.e., once the first container is again empty, a signal is sent upstream indicating that the first container is empty, resulting in a new fluid known or suspected to contain at least one enveloped virus being added to the first container. An acid is then added to the first container and once the equilibration buffer is removed from the second container, i.e., once the second container is again empty, a signal is sent upstream indicating that the second container is empty, resulting in the acidified pool being added to the second container once the pH probe in the first container measures a sufficiently low pH. In other words, based on two signals, an acidified pool from the first container is added to the second container: a signal indicating that the second container is empty, and a signal indicating that the pH probe in the first container measured a sufficiently low virus inactivation pH.

Advantageously, using the automated system and method described herein, the pH probes of both containers can remain submerged and wet for multiple cycles and their calibration status can be automatically assessed and corrected as needed without the need for an operator to manually withdraw samples at hand and measure the pH after each cycle. In other words, rather than having a member of the operator ready and waiting to check the calibration status of the pH probe before or after each cycle, the operator may engage in other activities as needed and may only need to intervene in generating one or more alarms. Advantageously, in some examples, the pH probes of the two containers can remain accurate for use in many consecutive cycles of low pH virus inactivation without intervention from an operator.

Thus, use of the automated system and method may facilitate a reduction in operator demand because it can be synchronized with the upstream capture chromatography system to cycle independently and repeatedly. In other words, the operator can be reduced by allowing the system to automatically initiate a cycle, by detecting the amount of product collected from the capture chromatography step, and by communicating synchronously with the chromatography system.

Referring now to the drawings, FIG. 1A illustrates a block diagram of an example automated system 100 for low pH viral 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 a respective stirrer 104A and 104B configured to mix the substances stored in the first container 102A and the second container 102B, respectively. In addition, the first and second containers 102A and 102B may each be equipped with a respective pH probe 106A and 106B configured to measure pH values associated with the first and second containers 102A and 102B, respectively. Although fig. 1A illustrates two pH probes 106A associated with the first container 102A and two pH probes 106B associated with the second container 102B, in some examples, there may be one pH probe 106A or more pH probes 106A associated with the first container 102A (and in some examples, there may be one pH probe 106B or more pH probes 106B associated with the second container 102B). The 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 memory 111 (e.g., volatile memory, non-volatile memory) accessed by the one or more processors 109 (e.g., via a memory controller), and a user interface 113. The one or more processors 109 may interact with the memory 111 to execute computer readable instructions stored in the memory 111. Computer readable instructions stored in the memory 111 may cause the one or more processors 110 to execute a pH probe recalibration application 115 and an upstream/downstream signaling application 117.

The system 100 further includes a chromatographic skid 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., depth filters, sterile grade filters, etc.), one or more containers 118, or other containers for buffers. In addition, the system 100 may include one or more pumps, valves, or other devices for transferring liquid between these various containers or other containers and through a filter. For example, the system 100 may include one or more pumps, valves, or other devices for continuously or intermittently transferring fluid known or suspected of containing at least one enveloped virus from the chromatographic skid 110 to the first container 102A. In some examples, the pump and/or valve may transfer fluid from the chromatographic slide 110 to the first container 102A only upon receiving an upstream signal from an upstream/downstream signaling application 117 indicating that the first container 102A is currently empty. In addition, the system 100 may include one or more pumps, valves, or other devices for transferring acid from the container 112 to the first container 102A. In some examples, the pump and/or valve may only transmit acid from the container 112 to the first container 102A upon receiving an upstream signal from an upstream/downstream signaling application 117 indicating that the first container 102A currently contains a fluid known or suspected to contain a virus. The agitator 104A may mix the acid with a fluid known or suspected to contain at least one enveloped virus (and/or additional acid may be added to the elution pool) until one or more pH probes 106A associated with the first vessel 102A measure a pH below a predetermined threshold pH (e.g., pH 3.5-3.7) for enveloped viruses in the inactivated fluid.

Additionally, the system 100 may include one or more pumps, valves, or other devices for transferring the acidified fluid from the first vessel 102A to the second vessel 102B once one or more pH probes 106A associated with the first vessel 102A measure a pH below a predetermined threshold pH. In some examples, the pump and/or valve may transfer acidified fluid from the first container 102A to the second container 102B only upon receiving an upstream signal from an upstream/downstream signaling application 117, the signal indicating that the second container 102B is currently empty. Once transferred into the second container 102B, the acidified fluid may remain in the second container 102B for a predetermined period of time (e.g., a period of time of 30 minutes) sufficient to reduce the concentration of virus in the acidified elution pool below a predetermined safe level (e.g., a level set by regulatory authorities associated with a drug made from a fluid known or suspected to contain at least one enveloped virus in addition to recombinantly produced therapeutic protein).

For example, as shown in fig. 1B and 1C, transferring acidified fluid from the first container 102A (as shown in fig. 1B) to the second container 102B (as shown in fig. 1C) in this manner allows the pool to first drop to an inactivation pH in the first container 102A and then to the second container 102B to maintain a validated inactivation time. By maintaining the pool in the second container 102B for a verified inactivation time, rather than maintaining the pool in the first container 102A for a verified inactivation time, the system 100 eliminates the option of droplets of eluate adhering to the upper side of the first container 102A walls and missing interactions with acid during this hold, which would allow untreated pool droplets to be transported through the process. In other words, by using two containers 102A and 102B, all of the contents from the collection transferred from the first container 102A to the second container 102B are thoroughly mixed with the acid.

Referring back to fig. 1A, one or more pumps or valves of the system 100 may transfer the base from the container or other vessel 114 to the second container 102B. In some examples, the pump and/or valve may only transmit base from the container or other vessel 114 to the second container 102B upon receiving an upstream signal from an upstream/downstream signaling application 117 indicating that the second container 102B currently contains an acidified (or virally inactivated) fluid. The agitator 104B may mix the base with the acidified (or virus inactivated) fluid (and/or additional acid may be added to the elution pool) until one or more pH probes 106B associated with the second vessel 102B measure a neutral pH (e.g., a pH of 5.0-6.0). In addition, the system 100 may include one or more pumps, valves, or other devices for transferring the neutralized virally inactivated fluid from the second vessel 102B through one or more filters 116 (e.g., a depth filter and a sterile grade filter) and transferring the filtered neutralized virally inactivated fluid to the third vessel 102C where it may be collected for use. In some examples, the pump and/or valve may transmit the neutralized virally inactivated fluid from the second container 102B to the third container 102C through one or more filters 116 only upon receiving an upstream signal from the upstream/downstream signaling application 117, the signal indicating that the third container 102C (and/or the filters 116) is currently empty.

Meanwhile, after the acidified fluid has been immediately transferred out of the first vessel 102A, the upstream/downstream signaling application 117 may send an upstream signal to one or more pumps or valves of the system 100 indicating that the first vessel 102A has been empty, resulting in an equilibration buffer having a known pH being transferred from the vessel 118 into the first vessel 102A such that the pH probe 106A remains wet. At this point, the pH probe 106A may measure the pH of the equilibration buffer in the first vessel 102A and send an indication of the measured pH to the computing device 108, wherein the pH probe recalibration application 115 may compare the measured pH of the equilibration buffer in the first vessel 102A to a known pH of the equilibration buffer. If the pH probe recalibration application 115 determines that the measured pH differs from the known pH of the equilibration buffer by more than a threshold pH value (e.g., more than 0.1 pH units), the pH probe recalibration application 115 may generate an alert indicating that the pH probe 102A (or a particular one of the pH probes 102A) needs to be recalibrated. The computing device 108 may display or otherwise communicate an alert to an operator via the user interface 113. Additionally, 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 particular one of the pH probes 102A) to recalibrate automatically based on a known pH of the equilibration buffer, e.g., cause an adjustment such that the pH probe 102A measures a pH value within +/-0.1 pH units of the known pH of the equilibration buffer when measuring the pH of the equilibration buffer.

Similarly, immediately after the neutralized virally inactivated fluid has been transferred out of the second container 102B, the upstream/downstream signaling application 117 may send an upstream signal to one or more pumps or valves of the system 100 indicating that the second container 102B has been empty, resulting in the transfer of an equilibration buffer having a known pH from one of the containers 118 (which may or may not be the same equilibration buffer as used by the first container 102A) into the second container 102B such that the pH probe 106B remains wet. At this point, the pH probe 106B may measure the pH of the equilibration buffer in the second vessel 102B and send an indication of the measured pH to the computing device 108, wherein the pH probe recalibration application 115 may compare the measured pH of the equilibration buffer in the second vessel 102B to a known pH of the equilibration buffer. If the pH probe recalibration application 115 determines that the measured pH differs from the known pH of the equilibration buffer by more than a threshold pH value (e.g., more than 0.1 pH units), the pH probe recalibration application 115 may generate an alert indicating that the pH probe 102B (or a particular one of the pH probes 102B) needs to be recalibrated. The computing device 108 may display or otherwise communicate an alert to an operator via the user interface 113. Additionally, 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 particular one of the pH probes 102B) to recalibrate automatically based on the known pH of the equilibration buffer, e.g., cause an adjustment such that the pH probe 102B measures a pH value within +/-0.1 pH units of the known pH of the equilibration buffer when measuring the pH of the equilibration buffer.

Referring now to fig. 2, a piping and instrumentation diagram (P & ID) 200 of an exemplary automated system for low pH virus inactivation illustrates piping and process equipment of the system and instrumentation and control devices of the system. Fig. 2 shows the components of the fluid connection (i.e., the components between which fluid may flow) in solid lines 246 and the components of the communication connection in dashed lines. In particular, a short dashed line 242 between two components indicates that a sensor signal may be sent and/or received between the two components, while a long dashed line 244 between the two components indicates that a control signal may be sent and/or received between the two components.

As shown in fig. 2, a control system 202 (which may be or may include the computing device 108 described with respect to fig. 1A in some examples, and may include additional or alternative computing devices in some examples) is communicatively connected to the different components of the system to receive sensor signals and transmit control signals to facilitate operating an automated low pH viral inactivation system according to the information disclosed herein. While some indications of control signals and sensor signals transmitted and received by the control system 202 are shown in fig. 2, fig. 2 may not necessarily show every control signal and sensor signal that may be transmitted by the control system 202 for simplicity of the drawing. In other words, the control system 202 may send and/or receive additional or alternative control signals and/or sensor signals to facilitate operating an automated low pH viral inactivation system according to the information provided herein.

For example, the chromatographic slide 204 can be fluidly connected to the first container 206 such that a fluid known or suspected of containing at least one enveloped virus can be transferred from the chromatographic slide 204 to the first container 206. An acid-containing container or other vessel 208 may also be fluidly connected to the first container 206. As shown in fig. 2, an acid pump 210 may be fluidly connected to the acid container 208 and the first container 204, and may pump acid from the acid container 208 to the first container 204. In some examples, the control system 202 may send control signals to the acid pump 210, for example, in order to control the speed of the acid pump 210 and/or the amount of acid pumped into the first container 204 as described herein. Further, in some examples, the weight scale 212 may capture an indication of the weight of the first container 206 and the fluid within the first container 204 and may send such indication to the control system 202. In some examples, the control system 202 may determine whether a first container 206 is full or empty based on a signal from the weighing scale 212, and may control when enveloped viruses are transferred from the chromatographic slide 204 to the first container 206 (and/or when the acid pump 210 transfers acid into the first container 206, when the buffer pump 240 pumps buffer into the first container 206, etc.) based on whether the first container 206 is full or empty. Further, in some examples, the control system 202 may control the speed of the acid pump 210 based on the combined weight of acid and fluid known or suspected to contain at least one enveloped virus within the first container 206. . Additionally, in some examples, the control system 202 can send a control signal to the agitator 214 within the first container 206 such that the agitator 214 mixes the acid with a fluid known or suspected to contain at least one enveloped virus at a rate and/or location as described herein in the first container 206.

One or more pH probes 216 positioned within (or otherwise associated with) the first container 206 may be configured to measure the pH of the first container's contents (e.g., by the agitator 214, mixing an acidified fluid in the first container 206) and send a sensor signal to the control system 202 that indicates the measured pH value or a value associated with the first container 206.

The first container 206 may be fluidly connected to the second container 218 such that acidified fluid may be transferred from the first container 206 to the second container 218. A transfer pump 220 may be fluidly connected to the first container 206 and the second container 218, and may pump acidified fluid from the first container 206 to the second container 218, e.g., based on control signals received from the control system 202. For example, the control system 202 may control the transfer pump 220 to pump acidified fluid from the first container 206 to the second container 218 based on sensor data received by the control system 202 from other components (e.g., starting at a time based on the pH measured by the pH probe 216 reaching a target pH value for killing viruses, starting at a time based on an elapsed time reaching a target total time of acidification, and/or pumping at a rate or speed based on a target transfer time from the first container 206 to the second container 218).

A container or other vessel 222 containing a base may be fluidly connected to the second container 218 such that the base may be transferred from the base container 222 to the second container 218. A base pump 224 may be fluidly connected to the base reservoir 222 and the second reservoir 218 and may pump base from the first reservoir 206 to the second reservoir 218, for example, based on control signals received from the control system 202. For example, the control system 202 may send control signals to control the base pump 224 because the base pump pumps base from the base container 222 to the second container 218, e.g., to 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. Further, in some examples, the weight scale 226 may capture an indication of the weight of the second container 218 and the fluid within the first container 218 and may send such indications to the control system 202. In some examples, the control system 202 may determine whether the second container 218 is full or empty based on the signal from the weighing scale 226, and may control when acidified fluid from the first container 206 is transferred into the second container 218 (and/or when the alkaline pump 224 transfers alkaline into the second container 218, when the buffer pump 240 pumps buffer into the second container 218, etc.) based on whether the second container 218 is full or empty. Further, in some examples, the control system 202 may control the speed of the alkaline pump 224 based on the combined weight of alkaline and fluid known or suspected to contain at least one enveloped virus within the second vessel 218. Additionally, in some examples, the control system 202 can send a control signal to the agitator 228 within the second container 218 such that the agitator 228 mixes the base with a fluid known or suspected to contain at least one enveloped virus in the second container 218 at a speed and/or location as described herein.

One or more pH probes 230 positioned within (or otherwise associated with) the second container 218 may be configured to measure the pH of the contents of the second container (e.g., mix the neutralized virally inactivated fluid in the second container 218 by the agitator 228) and send a sensor signal to the control system 202 that is indicative of the measured pH value or a value associated with the second container 218.

The second vessel 218 may be fluidly connected to a series of filters, including a depth filter 232 and a sterile filter 234. A drain pump 236 may be fluidly connected to the second vessel 218 and the filters 232, 234, and may pump the neutralized virus-inactivated fluid from the second vessel 218 through the filters 232, 234 into a third vessel 235, e.g., based on control signals received from the control system 202. In some examples, the third container 235 may be a collection bag. Additionally, in some examples, the third container 235 may include a load cell 237 configured to measure a weight of the load cell and generate an upstream or downstream signal indicating that the third container 235 is full.

For example, the control system 202 may control the evacuation pump 236 to pump the neutralized virally inactivated fluid from the second vessel 218 to the filters 232, 234 based on sensor data received by the control system 202 from other components (e.g., starting at a time based on the pH measured by the pH probe 230 reaching a target neutralization pH value, starting at a time based on an elapsed time reaching a target total time of neutralization, and/or pumping at a rate or speed based on a target filtration flow rate). Additionally, the control system 202 can receive sensor data from sensors associated with the filters 232, 234 and can control the filters 232, 234 (i.e., based on the sensor data) to operate according to the filter specifications and requirements described herein.

Additionally, a container or other vessel 238 containing a buffer may be fluidly connected to the first container 206 and/or the second container 218 such that buffer may 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 such that buffer may be transferred from the buffer container to the first container and then subsequently transferred to the second container (e.g., via the transfer pump 220). A buffer pump 240 may be fluidly connected to the buffer reservoir 238 and the first reservoir 206 and/or the second reservoir 218 and pump buffer from the buffer reservoir 238 to the first reservoir 206 and/or the second reservoir 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 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 out of each of the first container 206 and the second container 218, respectively, according to filtration specifications and requirements. In other words, as described above, a buffer having a known pH may be provided and may be pumped into the first container 206 after the acidified fluid is pumped from the first container 206 to the second container 218. Similarly, after passing the neutralized virally inactivated fluid from the second container 218 through the filters 232 and 234 and into the third container 235, a buffer may be pumped into the second container 218. The pH probes 216 and 230 may each measure the pH of the buffer as the buffer is pumped into the respective first container 206 and second container 218. The pH probes 216 and 230 may send an indication of their respective measured buffer pH values to the control system 202, which may compare the measured pH values of the buffer to the known pH of the buffer to determine if any recalibration is required by either of the pH probes 216 or 230. In some cases, the control system 202 may send control signals to any pH probes that need to be recalibrated as needed to facilitate recalibration of the probes. Also, in some cases, the control system 202 may generate an alarm for the operator indicating which pH probes (if any) need to be recalibrated.

After any recalibration of the probe 216 is complete, the transfer pump 220 may pump buffer out of the first container 206 and may pump or otherwise transfer a new fluid known or suspected to contain at least one enveloped virus from the chromatographic slide 204 into the first container in order to begin a new cycle of automated virus inactivation. Similarly, after any recalibration of the probe 230 is complete, the drain pump 236 may pump buffer out of the second container 218, and the transfer pump 220 may pump freshly acidified fluid from the first container 206 into the second container 218. Thus, the system can be performed by automating a new cycle of virus inactivation after recalibrating the probes 216 and 230 as needed.

FIG. 3 illustrates a flow chart associated with an exemplary automated method 300 of low pH viral inactivation using a fluid known or suspected of containing at least one enveloped virus. The method 300 may begin when a chromatography elution pool is added (block 302) to a first container. An acid may be added (block 304) to the first container and mixed with a fluid known or suspected to contain at least one enveloped virus (e.g., by a stirrer of the first container) to acidify the fluid. A first pH probe associated with the first container may measure (block 306) a pH associated with the first container. The method may include determining (block 308) whether the measured pH is below a threshold pH (or within a range of pH) associated with viral inactivation. If the pH measured by the first pH probe associated with the first container is not below the threshold pH for virus inactivation (block 308, no) (or is not within the range of pH values), additional acid may be added to the first container (block 304), or acid may be held in the first container for an additional period of time before the pH of the first container is again measured (block 306). If the pH measured by the first pH probe associated with the first container is below (or within a range of) the threshold pH value for virus inactivation (block 308, yes), the addition of acid to the first container may be stopped (block 310) and the acidified fluid may be transferred (block 312) to the second container.

A second pH probe associated with the second container may measure (block 314) a pH associated with the first container. The method may include determining (block 316) whether the measured pH is below a threshold pH (or within a range of pH) associated with viral inactivation. If the measured pH of the second pH probe associated with the second container is not below (or is not within) the threshold pH for virus inactivation (block 316, no), the process may remain (block 318) and an alert may be generated to the operator, e.g., to motivate the operator to investigate any problems related to the measured pH. If the pH measured by the second pH probe associated with the second container is below the threshold pH (block 316, yes), the method may proceed to block 320 where a determination may be made as to whether the elapsed time after transferring the acidified fluid from the first container to the second container has exceeded a threshold (e.g., +.30 minutes) for the amount of time that the concentration of virus in the fluid has been inactivated to a predetermined safe level. If not (no at block 320), the determination at block 314 may be made again after additional elapsed time. If so (block 320, yes), the method may proceed to block 322, where a base may be added to the second vessel to neutralize the acidified fluid.

A second pH probe associated with the second container may again measure (block 324) the pH associated with the second container, and a determination may be made as to whether the measured pH associated with the second container is within an acceptable range of neutral pH values (e.g., a pH range of 5.0-6.0). If the measured pH associated with the second vessel is not within an acceptable range (block 326, NO), additional base may be added (block 322) to the vessel. If the measured pH associated with the second vessel is within an acceptable range (block 326, yes), the addition of base to the second vessel may be stopped (block 328), and the neutralized virally inactivated fluid may be transferred (block 330) to a depth filter and then to a disinfection grade filter.

Referring now to fig. 4A-4B, a flowchart associated with an example automated method 400 of low pH virus inactivation is illustrated that includes an automated cycle of pH probe calibration. The method 400 may begin when a chromatography elution pool is added (block 402) to a first container. An acid may be added (block 404) to the first container and mixed with a fluid known or suspected to contain at least one enveloped virus (e.g., by a stirrer of the first container) to acidify the fluid. A first pH probe associated with the first container may measure (block 406) a pH associated with the first container. The method may include determining (block 408) whether the measured pH is below a threshold pH (or within a range of pH) associated with viral inactivation. If the pH measured by the first pH probe associated with the first container is not below the threshold pH for virus inactivation (block 408, no) (or is not within the range of pH values), additional acid may be added to the first container (block 404), or acid may be held in the first container for an additional period of time before the pH of the first container is again measured (block 406). If the pH measured by the first pH probe associated with the first container is below (or within a range of) the threshold pH value for virus inactivation (block 408, yes), the addition of acid to the first container may be stopped (block 410) and the acidified fluid may be transferred (block 412) to the second container. In some examples, the method 400 may proceed from block 412 to block 424, as discussed in more detail below with respect to fig. 4B. In any case, the method 400 may proceed from block 412 to block 414.

The first container may be filled with an equilibration buffer having a known pH (block 414), and the pH associated with the first container may be measured (block 416) by a first pH probe associated with the first container. This measured pH associated with the first container may be compared to a known pH of the equilibration buffer (block 418) to determine if the measured pH associated with the first container differs from the known pH of the equilibration buffer by more than a threshold pH (e.g., more than 0.1 pH units). If the measured pH associated with the first container is within 0.1 pH units of the known pH of the equilibration buffer (block 418, NO), the method 400 may end or may proceed to block 402 to begin a new viral inactivation cycle by adding a new fluid known or suspected of containing at least one enveloped virus to the first container (after the equilibration buffer has been poured from the first container).

If the measured pH of the pH probe associated with the first container is not within 0.1 pH units of the known pH of the equilibration buffer (block 418, yes), an alarm may be generated indicating that the pH probe is to be recalibrated (block 420). In some examples, the method 400 may include displaying or otherwise communicating an alert to an operator (e.g., via a user interface display) so that the operator may manually recalibrate the pH probe as desired. Also, in some examples, the method may include automatically recalibrating (block 422) the pH probe such that the pH measured by the pH probe is within 0.1 pH units of the equilibration buffer.

Referring now to fig. 4B, as discussed above, the method 400 may include proceeding from block 412 to block 424.

A second pH probe associated with the second container may measure (block 424) a pH associated with the first container. The method may include determining (block 426) whether the measured pH is below a threshold pH (or within a range of pH) associated with viral inactivation. If the measured pH of the second pH probe associated with the second container is not below (or is not within) the threshold pH for virus inactivation (block 426, no), the process may remain (block 428) and an alert may be generated to the operator, e.g., to motivate the operator to investigate any problems related to the measured pH. If the pH measured by the second pH probe associated with the second container is below the threshold pH (block 426, yes), the method may proceed to block 430, where a determination may be made as to whether the elapsed time after transferring the acidified fluid from the first container to the second container has exceeded a threshold (e.g., 30 minutes) for the amount of time that the concentration of virus in the fluid has been inactivated to a predetermined safe level. If not (block 430, no), the determination at block 430 may be made again after additional elapsed time. If so (block 430, yes), the second pH probe associated with the second container may again measure (block 432) the pH associated with the first container. The method may include determining (block 434) whether the measured pH is below a threshold pH (or within a range of pH values) associated with virus inactivation. If the measured pH of the second pH probe associated with the second container is not below (or is not within the range of) the threshold pH value for virus inactivation (block 434, no), the process may remain (block 436) and an alert may be generated to the operator, e.g., to motivate the operator to investigate any problems related to the measured pH.

If the pH measured by the second pH probe associated with the second vessel is below the threshold pH (block 434, yes), the method may proceed to block 438 where a base may be added to the second vessel to neutralize the acidified fluid. The second pH probe associated with the second container may measure (block 440) a pH associated with the second container and a determination may be made as to whether the measured pH associated with the second container is within an acceptable range of neutral pH values (e.g., a pH range of 5.0-6.0). If the measured pH associated with the second vessel is not within an acceptable range (block 442, no), additional base may be added (block 438) to the vessel. If the measured pH associated with the second vessel is within an acceptable range (block 442, yes), the addition of base to the second vessel may be stopped (block 444), and the neutralized virally inactivated fluid may be transferred (block 446) to a depth filter and then to a disinfection grade filter.

The second container may be filled with an equilibration buffer having a known pH (block 450), and the pH associated with the second container may be measured (block 452) by the second pH probe associated with the second container. This measured pH associated with the second container may be compared to the known pH of the equilibration buffer (block 454) to determine if the measured pH associated with the second container differs from the known pH of the equilibration buffer by greater than a threshold pH (e.g., greater than 0.1 pH units). If the measured pH associated with the second container is within 0.1 pH units of the known pH of the equilibration buffer (block 454, no), the method 400 may end or may proceed to block 412 where a new acidified fluid is added to the second container (after the equilibration buffer is poured from the second container).

If the measured pH of the pH probe associated with the second container is not within 0.1 pH units of the known pH of the equilibration buffer (block 454, yes), an alarm may be generated indicating that the pH probe is to be recalibrated (block 456). In some examples, the method 400 may include displaying or otherwise communicating an alert to an operator (e.g., via a user interface display) so that the operator may manually recalibrate the pH probe as desired. Also, in some examples, the method may include automatically recalibrating (block 458) the pH probe such that the pH measured by the pH probe is within 0.1 pH units of the equilibration buffer.

Fluids known or suspected to contain at least one enveloped virus include harvested host cell culture fluid, fluid from effluent streams, eluate, collections, storage or holding containers from unit operations comprising a harvesting step, a filtration step or a chromatography step. The fluid may be derived from an eluate collected from depth filtration, microfiltration, affinity chromatography, ion exchange chromatography, multi-mode chromatography, hydrophobic interaction chromatography or hydroxyapatite chromatography. The fluid may be derived from a collection containing harvested cell culture fluid, an eluate from depth filtration, an eluate from microfiltration, an eluate from affinity chromatography, an eluate from ion exchange chromatography, an eluate from multimode chromatography, an eluate from hydrophobic interaction chromatography or an eluate from hydroxyapatite chromatography. The fluid added to the first tank may be added as a single volume or may be added in portions and treated in multiple virus inactivation/neutralization cycles. The fluid may be added neat or diluted with an appropriate buffer or water to achieve the desired parameters or volumes. The fluid in the first tank may be a pool comprising a plurality of eluate pools.

The collection added to the first tank may be diluted in a suitable medium, such as water. In one embodiment, the pool is diluted 50% to 200%. In one embodiment, the pool is diluted 50% to 100%. In one embodiment, the pool is diluted 50% to 75%. In one embodiment, the pool is diluted 75% to 200%. In one embodiment, the pool is diluted 75% to 100%. In one embodiment, the pool is diluted 100% to 200%.

The temperature of the fluid may range from 5 ℃ to 25 ℃. Acidification may be carried out at a temperature of 5-25 ℃. In one embodiment, the temperature is 15 ℃ to 25 ℃. In one embodiment, the temperature is from 15 ℃ to 20 ℃, and in one embodiment, the temperature is from 20 ℃ to 25 ℃. In one embodiment, the temperature is 20 ℃.

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

At minimum working volume, the pH probe and agitator must be completely immersed in the fluid and the acid/base inlet must be below the liquid level. In one embodiment, the working volume is 1 to 9 liters.

Acid is added to the fluid and mixed by stirring to acidify the fluid. The fluid may be agitated at 10-30rpm, in one embodiment 15-30 rpm. The stirring rate should be adapted to the liquid level and not lead to splashing or vortex formation.

Suitable acids for use include formic acid, acid acids, citric acid and phosphoric acid in concentrations suitable to ensure viral inactivation. In one embodiment, the acid is added at a concentration of about 70 mL/L.

The acidified fluid may remain in the first tank for a period of time until the fluid is sufficiently acidified, or until the desired degree of viral inactivation is reached, all of the time, before being transferred to the second container. The time for sufficient acidification is 30 minutes or less, or longer. The time for virus inactivation may be 30 minutes to 24 hours or more.

The pH of virus inactivation is pH 2 to 4. In one embodiment, the virus inactivation pH is 3 to 4. In one embodiment, the virus inactivation pH is from 3.5 to 4. In one embodiment, the pH is 3.6 to 4. In one embodiment, the virus inactivation pH is 3.7 to 4. In one embodiment, the virus inactivation pH is from 3.5 to 3.7. In one embodiment, the virus inactivation pH is from 3.5 to 3.7. In one embodiment, the virus inactivation pH is 3.6.

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

The transfer from tank 1 to tank 2 may be completed in 15 minutes or less.

At least 1 to 10 litres of acidified (or virally inactivated) fluid is transferred from tank 1 to tank 2.

The fluid may be agitated at 10-30rpm to mix the acid with the fluid, and in one embodiment, at 15-30 rpm. The stirring rate should be adapted to the liquid level and not lead to splashing or vortex formation. Within the designed stirring range, the system should be able to reach a homogeneity of 95% within 3 minutes after adding the tracer solution to the full (maximum working volume) water tank.

If an acidified fluid is transferred to the second tank before viral inactivation is complete, the acidified fluid is maintained at the desired pH until the desired degree of inactivation has been achieved. A determination may be made as to whether the acidified fluid from the first container has been maintained at a threshold amount of time for viral inactivation, in one embodiment, from 30 minutes to 24 hours or more. In one embodiment, the time for viral inactivation is 60 to 360 minutes. In one embodiment, the time for virus inactivation may be 60 to 90 minutes. In one embodiment, the time for viral inactivation is 60 minutes.

Once virus inactivation is complete, a base is added to the Virus Inactivation (VI) fluid and mixed to neutralize the fluid to the desired pH. Alkali is added at 1% -5% of the working volume of the second tank. Suitable bases for use include Tris base at a concentration of 2M. In one embodiment, 2M Tris base is added at a concentration of about 55 mL/L. The amount of base added can be verified by mass to ensure additional tolerance of accuracy for the added volume of + -2%. The neutralization time may be 30 minutes or longer.

At least one pH probe associated with the second tank measures a pH associated with the second tank, and a determination may be made as to whether the measured pH associated with the second tank is within an acceptable range of neutral pH values. The target pH for neutralization is 4.5-6. 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.

The neutralization may be carried out at a temperature of 5 ℃ to 25 ℃. In one embodiment, the neutralization is performed at 15 ℃ to 25 ℃. In one embodiment, the neutralization is performed at 15 ℃ to 20 ℃. In one embodiment, the neutralization is performed at 20 ℃ to 25 ℃. In one embodiment, the neutralization is performed at 20 ℃.

The pH of the fluid is monitored during the neutralization process, which may take 20 minutes or less.

The fluid may be agitated at 10-30rpm to mix the base and virus-inactivated fluid, and in one embodiment, at 15-30 rpm. Once neutralization is complete, the neutralized virally inactivated fluid is transferred out of the second tank and into a holding tank or storage tank, or onto a filter or chromatographic medium.

The fluid may be delivered at a flow rate of 0.025 to 0.25 kg/min.

After the acidified or virally inactivated fluid is removed from the first tank (and similarly after the neutralized virally inactivated fluid is removed from the second tank), each tank is filled with an equilibration buffer at a known pH. Suitable buffers include acetate at a concentration of 100mM at a pH of 5.0 to keep the pH probe immersed in the liquid and wet at all times. The volume of equilibration buffer must be completely purged from the tank and associated outlet tubing to eliminate mixing between the equilibration buffer and the fluid used for the viral inactivation or neutralization process. The pH associated with the equilibration buffer in each tank may be measured by at least one pH probe associated with the tank. This measured pH value may be compared to a known pH value of the equilibration buffer to determine if the measured pH value measured by a probe in the tank differs from the known pH value of the equilibration buffer by more than a threshold pH value (e.g., more than ± 0.1 pH units).

If the measured pH of the pH probe associated with the tank is not within + -0.1 pH units of the known pH of the equilibration buffer, an alarm may be generated indicating that the pH probe is to be recalibrated. This may take the form of displaying or otherwise communicating an alert to an operator (e.g., via a user interface display) so that the operator may manually recalibrate the pH probe as desired. In some embodiments, the method may include automatically recalibrating the pH probe such that the pH measured by the pH probe is within ±0.1 pH units of the equilibration buffer.

Viruses are classified into enveloped viruses and non-enveloped viruses. Enveloped viruses have a capsid that is enclosed by a lipoprotein membrane or "envelope". The envelope is composed of host cell proteins and phospholipids, and viral glycoproteins that coat the viral surface as the virus buds from the host cell. Such an envelope allows the virus to recognize, bind, enter and infect target host cells. However, due to this membrane, enveloped viruses are sensitive to inactivation methods, whereas non-enveloped viruses are more difficult to inactivate without risk to the manufactured proteins but they can be removed by filtration methods.

Enveloped viruses include the family of viruses such as the herpesviridae (herpesviridae virus), poxviridae (poxviridae), hepadnaviridae (hepadnaviridae virus), flaviviridae (flaviviridae virus), togaviridae (togaviridae virus), coronaviridae (coronaviridae virus), orthomyxoviridae (orthomyxoviridae virus), hepatitis delta viridae (deltavirus), paramyxoviridae (paramyxoviridae virus), rhabdoviridae (rhabdoviridae virus), bunyaviridae (bunyaviridae virus), filoviridae (filoviridae virus), retrovirus (retroviridae virus); and viruses such as human immunodeficiency virus (human immunodeficiency virus), sindbis virus, herpes simplex virus (herpes simplex virus), pseudorabies virus (pseudorabies virus), sendai virus, vesicular stomatitis 5virus (vesicular stomatitis virus), west Nile virus (West Nile virus), bovine viral diarrhea virus (bovine viral diarrhea virus), coronavirus, equine arthritis virus (equine arthritis virus), severe acute respiratory syndrome virus (severe acute respiratory syndrome virus), moloney murine leukemia virus (Moloney murine leukemia virus), and vaccinia virus (vaccinia virus).

In order to ensure patient safety, viral inactivation is an essential component of the purification process when manufacturing protein therapeutics. Various methods can be used to inactivate viruses and include heat inactivation/pasteurization, pH inactivation, UV and gamma irradiation, use of high intensity broad spectrum white light, addition of chemical inactivating agents, surfactants, and solvent/detergent treatments. Exposure of enveloped viruses to low pH conditions can lead to denaturation of the virus.

The polypeptides and proteins of interest may be of scientific or commercial interest, including protein-based therapeutics. The proteins of interest include, inter alia, secreted proteins, non-secreted proteins, intracellular proteins or membrane-bound proteins. The polypeptides and proteins of interest may be produced by recombinant animal cell lines using cell culture methods and may be referred to as "recombinant proteins". The expressed protein or proteins may be produced intracellularly or secreted into the medium from which the protein may be recovered and/or collected. The term "isolated protein" or "isolated recombinant protein" refers to a polypeptide or protein of interest that is purified from a protein or polypeptide or other contaminant that would interfere with its therapeutic, diagnostic, prophylactic, research or other uses. Proteins of interest include proteins that exert a therapeutic effect by binding to a target, particularly those listed below, including targets derived therefrom, targets associated therewith, and modifications thereof.

The protein of interest includes a protein or polypeptide comprising an antigen binding region or antigen binding portion having affinity for another molecule (antigen) to which it bindsForce, is "antigen binding protein". Proteins of interest include antibodies; a peptide body; an antibody fragment; an antibody derivative; an antibody analog; a fusion protein; genetically engineered cell surface receptors such as T Cell Receptors (TCRs) and chimeric antigen receptors (CARs or CAR-T cells, TRUCK (chimeric antigen receptor that redirects T cells for universal cytokine-mediated killing), and armored CARs (designed to regulate the immunosuppressive environment)); and other proteins comprising antigen binding molecules that interact with the antigen being targeted. Also included are multispecific proteins and antibodies, including bispecific proteins and antibodies, including proteins recombinantly engineered to bind and neutralize at least two different antigens or at least two different epitopes on the same antigen simultaneously, including all forms of bispecific proteins and antibodies, including but not limited to four-source hybridomas (quadromas), knob-in-holes, cross monoclonal antibodies (cross-Mabs), double variable domain IgG (DVD-IgG), igG-single chain Fv (scFv), scFv-CH3 KIH, dual-action Fab (DAF), semi-molecular exchange, kappa lambda-bodies, tandem scFv, scFv-Fc, diabodies, single chain diabodies (sc diabodies), sc diabody-CH 3, triabodies, minibodies, tri-minibodies, tandem diabodies, sc diabodies-toxin, tandem scFv-toxin, dual and heavy targeting molecules (dial-affinity retargeting molecules, DART), nanohasbs, nanobodies, locked-and locked-chain (dded), SEZ-engineered diabodies (SEZ), and triple-chain (SEZ-engineered diabodies) Fab-arm exchange, dutaMab, DT-IgG, charge pair (charged pair), fcab, orthogonal Fab, igG (H) -scFv, scFV- (H) IgG, igG (L) -scFv, igG (L1H 1) -Fv, igG (H) -V, V (H) -IgG, igG (L) -V V (L) -IgG, KIH IgG-scFab, 2scFv-IgG, igG-2scFv, scFv4-Ig, zy body, DVI-Ig4 (quadby body), fab-scFv, scFv-CH-CL-scFv, F (ab') 2-scFv2, scFv-KIH, fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, intracellular antibody, immTAC, HSA body (body), igG-IgG, cov-X body, scFv1-PEG-scFv2, single chain diabodySpecific antibody construct, single-stranded bispecific T-cell adapter->Bispecific T-cell adaptors, half-life extended bispecific T-cell adaptors (HLE +.>) And HeteroIg->

Also included are human, humanized and other antigen binding proteins, such as human antibodies and humanized antibodies, that do not produce a significantly detrimental immune response when administered to a human.

Also included are modified proteins, such as proteins chemically modified via non-covalent bonds, or both covalent and non-covalent bonds. Also included are proteins further comprising one or more post-translational modifications that may be made by cellular modification systems or modifications introduced ex vivo or otherwise by enzymatic and/or chemical methods.

In some embodiments, the protein of interest may include a colony stimulating factor, such as granulocyte colony stimulating factor (G-CSF). Such G-CSF agents include, but are not limited to(febuxostat) and->(pefegelsemine). Also included are Erythropoiesis Stimulating Agents (ESA), such as +.>(ebastine alpha), ->(dapoxetine alpha),(ebastin delta), ->(methoxy polyethylene glycol-ebastine beta), ->MRK-2578、INS-22、/>(ebastine ζ)>(ebastine beta),>(ebastine ζ),(ebastine alpha), epoetin alfa Hexal, < >>(ebastine alpha), ->(ebastine θ), ->(ebastine θ), ->(ebitemtθ), ebitemtα, ebitemtβ, ebitemtζ, ebitemtθ and ebitemtδ, ebitemtω, ebitemtiota; tissue plasminogen activator; GLP-1 receptor agonists; and variants or analogs thereof, and the biomimetic pharmaceutical of any one of the preceding claims.

In a further embodiment of the present invention, the target protein comprises Acximab, adalimumab, aldrib (adecatumumab), abelmoschus, albizumab, abamectin, albizepine, albizipran, albiziram (axicabtagene ciloleucel), baricximab, bellevimumab, bevacizumab, bei Suozhu monoclonal antibody (biosozumab), bonauzumab, rituximab (brentuximab vedotin) brucellab (brodalumab), mecamylab (cantuzumab mertansine), kanakiab (canakiumab), katuzumab (cataxomab), cetuximab (certolizumab pegol), colamumab (concatumumab), dalizumab, denomumab, eculizumab, exenatide, ibritumomab, efalizumab, apamulab, rapamycin, certolizumab, ceruzumab, other combinations Ai Nushan antibody (erenmab), ai Tuma antibody (ertumaxomab), etanercept, exemestane, volcanic antibody (flotuzmab) (MGD 006), gliximab (galiximab), gurney antibody (ganitumab), lu Tizhu antibody (lutikizumab) (ABT 981), gemtuzumab (gemtuzumab), golimumab, timomumab (ibritumomab tiuxetan), infliximab, eplimumab, le Demu antibody (lerdiltiazem), lu Xishan antibody (lumiriximab), lu Kezhu antibody (lxdkizumab), lizumab (lymphoman) (FBTA), ma Pamu antibody (mapapumab), diproteizumab Sha Ni, molluscab-CD 3, natalizumab, ceririn, nitenpyram, wutuzumab, oxuzumab (oxymevaluzumab), rezucclizumab, ofatumumab, omalizumab, opregumumab, ozoliumumab (ATN 103), panizumab, panitumumab (pam 112, MT 112), panitumumab (pembrolizumab), pertuzumab, petuzumab, pekelizumab, ranibizumab, lanitumumab (remtuumab) (ABT 122), rilotumumab (rilotumumab), rituximab, romideputyzib, luo Mozhu monoclonal antibody (romazumab), sagamostim, sclerostin (sclerostin), sosmallumab, targomumab, tezesamumab, tisamel, tolucel, tolukumab, tolizumab, toluximab, tabuzumab, temab, temozokermanab, temozokerumab; vanulizumab (RG 7221), wei Dezhu mab (vedolizumab), wei Saizhu mab (visilizumab), wo Luo mab (volociximab), zanolizumab (zanoliumab), zanolimumab (zanolimumab), zanolimumab (zalutumumab), AMG211 (MT 111, medi-1565), AMG330, AMG420 (B1836909), AMG-110 (MT 110), MDX-447, TF2, rM28, HER2 Bi-aaCT, GD2 Bi-aaCT, MGD006, MGD007, MGD009, MGD010, MGD011 (MGJ 64052781), IMCgp100, indium labeled IMP-205, xm734, LY3164530, OMP-305BB3, REGN1979, COV322, ABT112, ABT165, RG-6013 (ACE), 7597 (MEDH 7945A), RG 3, RG 86, RG 3, RG 7788, RG 7790, RG 3, RG 7780, RG 7790, RG 111-7790, MGD007, MGD009, MGD011, OMP-305, and OMP-305 MM141, MOR209/ES414, MSB0010841, ALX-0061, ALX0761, ALX0141; BII034020, AFM13, AFM11, SAR156597, FBTA05, PF06671008, GSK2434735, MEDI3902, MEDI0700, MEDI735, and variants or analogues thereof, as well as biomimetic pharmaceuticals of any of the foregoing.

In some embodiments, the protein of interest may include a protein that specifically binds, alone or in combination, to one or more of the following: CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming Growth Factors (TGFs), insulin-like growth factors, osteoinductive factors, insulin and insulin-related proteins, blood clotting and blood clotting-related proteins, colony Stimulating Factors (CSF), other blood and serum protein blood group antigens; receptors, receptor-related proteins, growth hormone receptors, and T cell receptors; neurotrophic factors, neurotrophins, relaxins (relaxins), interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressees, regulatory proteins and immunoadhesins.

In some embodiments, the protein of interest binds, alone or in any combination, one or more of the following: CD proteins, including but not limited to CD2, CD3 (α, β, δ, ε, γ, ζ), CD4, CD5, CD7, CD8 α, CD16, CD19, CD20, CD22, CD25, CD27, CD28T, CD, CD33, CD34, CD37, CD38, CD40, CD45, CD49a, CD64, CD70, igα (CD 79 a), CD80, CD86, CD123, CD133, CD134, CD137, CD138, CD154, CD171, CD174, CD247 (B7-H3). HER receptor family proteins including, for example, HER2, HER3, HER4, and EGF receptor, egfrvlll, cell adhesion molecules, for example L FA-1, CD1 1a/CD18, mol, p150,95, VLA-4, ICAM-1, VCAM and αv/β3 integrins, growth factors including, but not limited to, for example, vascular endothelial growth factor ("VEGF"); VEGFR2, growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, miller's tube inhibiting substance (mullerian-inhibiting substance), human macrophage inflammatory protein (MIP-1-. Beta.1), erythropoietin (EPO), nerve growth factors (such as NGF-. Beta.0), platelet-derived growth factor (PDGF), fibroblast growth factors (including, for example, aFGF and bFGF), epidermal Growth Factor (EGF), cripto, transforming Growth Factor (TGF) (including, inter alia, TGF-. Beta.2 and TGF-. Beta.3 (including TGF-. Beta.41, TGF-. Beta.2, TGF-. Beta.3, TGF-. Beta.4, or TGF-. Beta.5)), insulin-like growth factor-I and insulin-like growth factor-II (IGF-I and IGF-II), des (1-3) -IGF-I and bone inducing factors, insulin and insulin-related proteins (including, but not limited to insulin, insulin A chain, insulin B chain, proinsulin and insulin-like growth factor binding protein; (coagulation proteins and coagulation-related proteins such as, inter alia, factor VIII, tissue factor, van wilford (von Willebrand) factor, protein C, beta 5-1-antitrypsin, plasminogen activator (such as urokinase and tissue plasminogen activator ("T-PA")), bangbacin (combazine), thrombin, thrombopoietin and thrombopoietin receptor, colony Stimulating Factor (CSF) (including, inter alia, M-CSF, GM-CSF and G-CSF), other blood and serum proteins (including, but not limited to, albumin, igE and blood group antigens), receptor and receptor-related proteins (including, for example, flk2/flt3 receptor, obesity (OB) receptor, growth hormone receptor and T cell receptor); neurotrophins, including but not limited to bone-derived neurotrophins (BDNF) and neurotrophin-3, neurotrophin-4, neurotrophin-5 or neurotrophin-6 (NT-3, NT-4, NT-5 or NT-6), relaxin A-chain, relaxin B-chain and relaxin pro-1, interferons, including, for example, interferon-alpha, interferon-beta and interferon-gamma, interleukins (IL), such as IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, IL-12/IL-23, IL-2Ra, IL-2 Rbeta, IL-2 Rgamma, IL-7 Ralpha, IL1-R1, IL-6 receptor, IL-4 receptor and/or IL-13 receptor, IL-13RA2 or IL-17 receptor Body, IL-1RAP; viral antigens, including but not limited to AIDS envelope viral antigen; lipoproteins, calcitonin, glucagon, atrial natriuretic factors, pulmonary surfactants, tumor necrosis factor-alpha and tumor necrosis factor-beta, enkephalinase, BCMA, igκ, ROR-1, ERBB2, mesothelin, RANTES (modulation of activation of normal T cell expression and secretion), mouse gonadotropin-associated peptides, dnase, FR- β0, inhibins and activins, integrins, protein a or D, rheumatoid factors, immunotoxins, bone Morphogenic Proteins (BMP), superoxide dismutase, surface membrane proteins, decay Accelerating Factors (DAF), AIDS envelopes, transport proteins, homing receptors, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, addressee, regulatory protein, immunoadhesin, antigen binding protein, growth hormone, CTGF, CTLA4, eosinophil chemokine-1, MUC1, CEA, c-MET, sealing protein-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, hepatocyte Growth Factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligand, PD1 and 1, mannose receptor/hCG beta, hepatitis c virus, mesothelin fv [ PE38 conjugate, pneumophila (ilv), interferon gamma, induction protein gamma (ifl 10), nalyte 10, nald 1, TNFa, TNFr, TL1A, thymic Stromal Lymphopoietin (TSLP), proprotein convertase subtilisin/Kexin type 9 (PCSK 9)), stem cell factor, flt-3, calcitonin Gene-related peptide (CGRP), OX40L, α4β7, platelet specificity (platelet glycoprotein Iib/IIIb (PAC-1), transforming growth factor β (TFG β), STEAP1, zona pellucida sperm binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor α (PDGFRα), 4-1BB/CD137, ICOS, LIGHT (tumor necrosis factor superfamily member 14; TMFSF 14), DAP-10, fc gamma receptor, MHC class I molecule, signaling lymphocyte activating molecule, BTLA, toll ligand receptor, CDS, GITR, HVEM (LIGHT R), KIRDS, SLAMF7, NKp80 (KLRF 1), NKp44, NKp30, NKp46, ITGA4, VLA1, VLA-6, IA4, CD49D, ITGA6, CD49f, ITGAD, CDl-ld, ITGAE, CD, ITGAL, CDl-la, LFA-1, ITGAM, C Dl-lb, ITGAX, CDl-lc, ITGBl, CD, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD 226), SLAMF4 (CD 244, 2B 4), CD84, CD96 (Tactive), CEACAM1, CRT AM, ly9 (CD 229), CD160 (BY 55), PSGL1, CD100 (SEMA 4D), CD69, SLAMF6 (NTB-A, lyl 08), SLAM (SLAMF 1, CD150, IPO-3), BLAMME (SLAMF 8), SELPLG (CD 162), LTBR, LAT, 41-BB, GADS, SLP-76, PAG/Cbp, CD19a, CD83 ligand, 5T4, AFP, ADAM 17, 17-A, ART-4, alpha _v β ₆ Integrins, BAGE.Bcr-abl, BCMA, B-H3, B7-H6, CAIX, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8, CDC27m, CD19, CD20, CD22, CD30, CD33, CD44v6, CD44v7/8, CD70 (CD 27L or TNFSF 7), CD79a, CD79B, CD123, CD138, CD171, CDK4/m, cadherin 19 (CDH 19), placenta-cadherin (CDH 3), CEA, CLL-1, CSPG4, CT, cyp-B, DAM, DDL, CD123 EBV, EGFR, EGFRvIII, EGP, EGP40, ELF2M, erbB2 (HER 2), EPCAM, ephA2, epCAM, ETV6-AML1, FAP, fetal AchR, FLT3, FR alpha, G250, GAGE, GD2, GD3,' -phosphatidylinositol glycan-3 (GPC 3), GNT-V, GP-100, HAGE, HBV, HCV, HER-2/neu, HLa-6272, HST-2, hTERT, iCE, igE, IL-11R alpha, IL-13R alpha 2, 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 small bcr-abl, PML/RARa, PRAME, PSA, PSCA, PSMA, RAGE, ROR1, RU2, SAGE, SART, SSX-1, SSX-2, SSX-3, survivin, TAA, TAG72, TEL/AML1, TEMs, TPI, TRP-1, TRP-2/INT2, VEGFR2, WT1, biologically active fragments or variants of any of the foregoing.

The protein of interest according to the invention encompasses all of the foregoing and further includes antibodies comprising 1, 2, 3, 4, 5 or 6 Complementarity Determining Regions (CDRs) of any of the antibodies described above. Also included are variants that include regions of the amino acid sequence that have 70% or more, particularly 80% or more, more particularly 90% or more, still more particularly 95% or more, particularly 97% or more, more particularly 98% or more, still more particularly 99% or more identity to a reference amino acid sequence of a protein of interest. Identity in this regard can be determined using a variety of well known and readily available amino acid sequence analysis software. Preferred software includes those that implement the Smith-Waterman algorithm, which is considered a satisfactory solution to the search and alignment sequence problem. Other algorithms may also be employed, particularly where speed is an important consideration. Common procedures for alignment and homology matching of DNA, RNA and polypeptides that may be used in this regard include FASTA, TFASTA, BLASTN, BLASTP, BLASTX, TBLASTN, PROSRCH, BLAZE and MPSRCH, the latter being an embodiment of the smith-whatman algorithm for execution on a massively parallel processor manufactured by MasPar.

"culture" or "culturing" refers to the growth and propagation of cells outside a multicellular organism or tissue. Suitable culture conditions for host cells (e.g., mammalian cells) are known in the art. Cell culture medium and tissue culture medium are used interchangeably to refer to a medium suitable for growth of host cells during in vitro cell culture. Typically, the cell culture medium contains buffers, salts, energy sources, amino acids, vitamins and trace essential elements. Any medium capable of supporting the growth of a suitable host cell in culture may be used and may be further supplemented with other components that maximize cell growth, cell viability, and/or recombinant protein production in the particular cultured host cell, which are commercially available. Different media formulations may be used during the life cycle of the cell culture. The host cells may be cultured in suspension or in an adherent form, attached to a solid substrate. Cell culture can be established in a fluidized bed bioreactor, hollow fiber bioreactor, roller bottle, shake flask or stirred bioreactor with or without microcarriers

Cell culture can be performed in batch, fed-batch, continuous, semi-continuous, or perfusion mode. Mammalian host cell lines (e.g., CHO cells) can be cultured in bioreactors on smaller scales of from less than 100ml to less than 1000 ml. Alternatively, larger scale bioreactors containing 1000ml to 20,000 liters or more of medium may be used. Large scale cell cultures, such as those used for clinical and/or commercial scale biological manufacture of protein therapeutics, can be maintained for weeks or even months during which time the cells produce the desired protein or proteins.

The cell culture broth containing the expressed recombinant protein can then be harvested from the cell culture in the bioreactor. Methods for harvesting expressed proteins from suspended cells are known in the art and include, but are not limited to, acid precipitation, accelerated sedimentation (e.g., flocculation), use of gravity separation, centrifugation, sonic separation, filtration (including membrane filtration using ultra-filters, micro-filters, tangential flow filters, depth filters, and alluvial filters). Recombinant proteins expressed by prokaryotes can be recovered from inclusion bodies in the cytoplasm by methods known in the art for redox folding processes.

The recombinant protein of interest in the clarified harvested cell culture fluid may then be purified or partially purified from any impurities, such as residual cell culture medium, cell extracts, unwanted components, host cell proteins, incorrectly expressed proteins, contaminants, microorganisms (such as bacteria and viruses), aggregates, and the like, using one or more unit operations.

The term "unit operation" refers to a functional step performed during purification of a recombinant protein, such as from a liquid medium. For example, unit operations may include steps such as, but not limited to, harvesting, capturing, purifying, refining, virus inactivation, virus filtration, and/or adjusting the concentration and formulation of the fluid containing the recombinant protein of interest. The unit operations may also include the step of pooling, holding and/or storing the fluid (e.g., capturing the pooling after harvesting, chromatography, virus inactivation, and neutralization or filtration), wherein the fluid is placed in a holding or storage vessel. A single unit operation may be designed to accomplish multiple objectives in the same operation, such as harvesting and virus inactivation or capture and virus inactivation.

The capture unit operations include capture chromatography using resins and/or membranes containing reagents that bind and/or interact with recombinant proteins of interest, e.g., affinity chromatography, size exclusion chromatographySpectrum, ion exchange chromatography, hydrophobic Interaction Chromatography (HIC), solid phase metal affinity chromatography (IMAC), and the like. 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 be tagged with a polyhistidine tag and subsequently purified by IMAC using imidazole, or using an epitope (such asProtein tag) followed by purification using specific antibodies against the epitope.

The enveloped viruses known or suspected to be contained in the fluid may be inactivated at any time in a downstream process. During manufacture of biopharmaceutical substances, viral inactivation in the fluid containing the recombinant protein of interest may occur in one or more separate viral inactivation unit operations. In one embodiment, viral inactivation occurs before, as part of, or after the harvesting unit operation. In one embodiment, viral inactivation occurs after a harvesting unit operation, which in related embodiments includes ultrafiltration and/or microfiltration. In one embodiment, viral inactivation occurs before, as part of, or after the chromatographic unit operation. In one embodiment, viral inactivation occurs prior to, as part of, or after operation of one or more capture chromatography units. In one embodiment, viral inactivation occurs before, as part of, or after operation of one or more affinity chromatography units. In one embodiment, viral inactivation occurs before, as part of, or after one or more of protein a chromatography, protein G chromatography, protein a/G chromatography, protein L chromatography, and/or IMAC chromatography. In one embodiment, viral inactivation occurs prior to, as part of, or after operation of one or more of the refining chromatographic units. In one embodiment, virus inactivation is by one or more ion exchange chromatography, hydrophobic interaction chromatography; mixed mode or multimode chromatography, and/or hydroxyapatite chromatography unit operation, as part of, or after. In one embodiment, viral inactivation occurs prior to, as part of, or after one or more ion exchange chromatography. In one embodiment, viral inactivation occurs before, as part of, or after operation of the cation exchange chromatography unit. In one embodiment, viral inactivation occurs before, as part of, or after operation of the anion exchange chromatography unit. In one embodiment, viral inactivation occurs before, as part of, or after operation of the multi-mode or mixed-mode chromatography unit. In one embodiment, viral inactivation occurs before, as part of, or after operation of the hydrophobic interaction chromatography unit. In one embodiment, viral inactivation occurs before, as part of, or after the hydroxyapatite chromatography unit operation. In one embodiment, virus inactivation is by one or more ion exchange chromatography, hydrophobic interaction chromatography; mixed mode or multimode chromatography, and/or hydroxyapatite chromatography unit operation, as part of, or after. In one embodiment, viral inactivation occurs before, as part of, or after operation of the filter unit. In one embodiment, viral inactivation occurs before, as part of, or after operation of the viral filtration unit. In one embodiment, viral inactivation occurs before, as part of, or after the depth filtration unit operation. In one embodiment, viral inactivation occurs before, as part of, or after operation of the sterile filtration unit. In one embodiment, viral inactivation occurs before, as part of, or after one or more depth filtration unit operations and/or sterile filtration unit operations. In one embodiment, viral inactivation occurs and/or occurs before or after operation of one or more ultrafiltration/diafiltration units.

The virus inactivation unit operation may be performed after the filtration and/or chromatography unit operation. In one embodiment, virus inactivation occurs before, as part of, or after depth filtration and/or sterile filtration unit operation to remove inactivated virus, other inactivating agents (e.g., surfactants and detergents), turbidity, and/or precipitation.

The term "refining" is used herein to refer to one or more chromatographic steps performed to remove residual contaminants and impurities (e.g., DNA, host cell proteins; product specific impurities, variant products and aggregates, and virus adsorption from fluids (including recombinant proteins of near final desired purity)). For example, the purification can be performed in a binding and elution mode by passing the fluid comprising the recombinant protein through one or more chromatographic columns or one or more membrane absorbents, which selectively bind the recombinant protein of interest or contaminants or impurities present in the fluid comprising the recombinant protein. In this example, the eluate/filtrate of the one or more chromatographic columns or membrane absorbers comprises recombinant proteins.

For example, the polishing chromatographic unit operation uses chromatographic resins and/or membranes containing reagents that can be used in flow-through mode, overload or front-end chromatography mode, or binding and elution mode. Chromatographic media suitable 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 mode or multimode chromatography (MM), hydroxyapatite chromatography (HA); reversed phase chromatography and gel filtration.

Methods for inactivating an enveloped virus during purification of a recombinant protein of interest are provided, the methods comprising obtaining a fluid known or suspected to contain at least one enveloped virus; the fluid is subjected to the systems or methods described herein at a concentration and for a time sufficient to cause viral inactivation, followed by neutralization of the virally inactivated fluid. The neutralized virus-inactivated fluid may be stored for later use. The neutralized virally inactivated fluid may be subjected to at least one unit operation comprising at least a filtration step or a chromatography step.

Also provided are methods for inactivating an enveloped virus during purification of a recombinant protein of interest, the method comprising obtaining a fluid known or suspected to contain at least one enveloped virus; subjecting the fluid to a system or method described herein at a concentration and for a time sufficient to cause viral inactivation; and subjecting the neutralized virally inactivated fluid to at least one unit operation comprising at least a filtration step or a chromatography step. In one embodiment, the filtering step comprises depth filtration. In one embodiment, the filtering step includes depth filtration and aseptic filtration. In one embodiment, the chromatography step comprises affinity chromatography. In one embodiment, the 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 refining chromatography steps. In one embodiment, the purification chromatography is selected from ion exchange chromatography, hydrophobic interaction chromatography, multi-mode or mixed-mode chromatography, or hydroxyapatite chromatography.

Also provided are methods for producing an isolated, purified recombinant protein of interest, the methods comprising establishing a cell culture in a bioreactor with a host cell expressing the recombinant protein and culturing a cell expressing the recombinant protein of interest; harvesting a cell culture broth containing the recombinant protein of interest; treating a fluid comprising a recombinant protein of interest by at least two unit operations, wherein at least one unit operation comprises a viral inactivation system or method described herein for a time sufficient to cause inactivation and neutralization of the enveloped virus; treating the neutralized virus-inactivated fluid containing the recombinant protein of interest by at least one additional unit operation; and obtaining the isolated, purified recombinant protein of interest.

Also provided are isolated, purified, recombinant proteins of interest prepared using the systems and methods described herein. Also provided are pharmaceutical compositions comprising the isolated proteins of interest prepared using the systems and methods described herein.

While the disclosure herein sets forth a detailed description of a number of different embodiments, it should be understood that the legal scope of the invention is defined by the words of the claims set forth at the end of this patent and their equivalents. This detailed description is to be construed as exemplary only and does not describe every possible embodiment since describing every possible embodiment would be impractical, if not impossible. Numerous alternative embodiments could be implemented, using either current technology or technology developed after the filing date of this patent, which would still fall within the scope of the claims.

It will be further understood that the term '_______' is defined herein to mean … … "or a similar sentence to explicitly define the term, unless the sentence is used in this patent, that is not intended to clearly or implicitly limit the meaning of the term (beyond its plain or ordinary meaning), and that such term should not be interpreted as limiting in scope based on any statement made in any part of this patent (except in the language of the claims). Any terms set forth in the claims at the end of this patent are referred to in this patent in a manner consistent with a single meaning, and are only done for sake of clarity so as to not confuse the reader, and it is not intended that such claim term by limited, by implication or otherwise, to that single meaning.

Throughout this specification, multiple instances may implement a component, operation, or structure described as a single instance. Although individual operations of one or more methods are illustrated and described as separate operations, one or more of the individual operations may be performed concurrently and nothing requires that the operations be performed in the order illustrated. Structures and functions presented as separate components in the example configuration may be implemented as a combined structure or component. Similarly, structures and functions presented as a single component may be implemented as separate components. These and other variations, modifications, additions, and improvements may fall within the scope of the subject matter herein.

In addition, certain embodiments are described herein as comprising logic or many routines, subroutines, applications, or instructions. These may constitute software (code presented on a non-transitory, tangible machine-readable medium) or hardware. In hardware, routines and the like are tangible units capable of performing certain operations and may be configured or arranged in some manner. In example embodiments, one or more computer systems (e.g., stand-alone client or server computer systems) or one or more hardware modules of a computer system (e.g., a processor or a set of processors) may be configured by software (e.g., an application or application part) as hardware modules that operate to perform certain operations as described herein.

In various embodiments, the hardware modules may be implemented mechanically or electronically. For example, a hardware module may include specialized circuitry or logic that is permanently configured (e.g., as a special purpose processor such as a Field Programmable Gate Array (FPGA), or as an application-specific integrated circuit (ASIC)) to perform certain operations. A hardware module may also include programmable logic or circuitry (e.g., as contained within a general-purpose processor or other programmable processor) that is temporarily configured by software to perform particular operations. It will be appreciated that the decision to mechanically implement a hardware module in dedicated and permanently configured circuitry or in temporarily configured circuitry (e.g., configured by software) may be driven by cost and time considerations.

A hardware module may provide information to and receive information from other hardware modules. Thus, the described hardware modules may be considered to be communicatively coupled. When a plurality of such hardware modules are present at the same time, communication may be achieved by signal transmission (e.g., through appropriate circuitry and buses) connecting the hardware modules. In embodiments in which multiple hardware modules are configured or instantiated at different times, communication between such hardware modules may be implemented, for example, by storing and retrieving information in memory structures accessible to the multiple hardware modules. For example, a hardware module may perform an operation and store an output of the operation in a storage device to which the hardware module is communicatively coupled. Another hardware module may then access the storage device at a later time to retrieve and process the stored output. The hardware module may also initiate communication with an input or output device and may operate on a resource (e.g., a collection of information).

Various operations of the example methods described herein may be performed, at least in part, by one or more processors that are temporarily configured (e.g., via software) or permanently configured to perform the relevant operations. Such a processor, whether temporarily configured or permanently configured, may constitute a processor-implemented module that operates to perform one or more operations or functions. In some example embodiments, the modules referred to herein may comprise processor-implemented modules.

Similarly, in some embodiments, the methods or routines described herein may be implemented at least in part by a processor. For example, at least some operations of the method may be performed by one or more processors or hardware modules implemented by a processor. The execution of certain of the operations may be distributed among one or more processors, residing not only within a single machine, but also across multiple machines. In some example embodiments, one or more processors or processor-implemented modules may be located in a single geographic location (e.g., within a home environment, an office environment, or a server farm). In other embodiments, one or more processors or processor-implemented modules may be distributed across multiple geographic locations.

Unless specifically stated otherwise, discussions utilizing terms such as "processing," "computing," "calculating," "determining," "presenting," "displaying," or the like, may refer to the action or processes of a machine (e.g., a computer) that manipulates and transforms data represented as physical (e.g., electronic, magnetic, or optical) quantities within the machine's storage (e.g., volatile memories, non-volatile memories, or combinations thereof) or registers or other machine components.

As used herein, any reference to "one embodiment" or "an embodiment" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearances of the phrase "in one embodiment" or "in some embodiments" in various places in the specification are not necessarily all referring to the same embodiment or embodiments.

The terms "coupled," "connected," "communicatively connected," or "communicatively coupled," along with their derivatives, may be used to describe some embodiments. These terms may refer to either a direct physical connection or an indirect (physical or communication) connection. For example, some embodiments may be described using the term "coupled" to indicate that two or more elements are in direct physical or electrical contact. The term "coupled," however, may also mean that two or more elements are not in direct contact with each other, but yet still co-operate or interact with each other. Embodiments are not limited to direct connections unless their use context clearly indicates or requires.

As used herein, the terms "comprises," "comprising," "includes," "including," "having" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Furthermore, unless explicitly stated to the contrary, "or" refers to an inclusive or rather than an exclusive or. For example, condition a or B satisfies any one of the following: a is true (or present) and B is false (or absent), a is false (or absent) and B is true (or present), and both a and B are true (or present).

Furthermore, the word "a" or "an" is used to describe elements and components of embodiments herein. This is done merely for convenience and to give a general sense of description. The specification and claims which follow should be construed to include one or at least one of the following unless the context clearly dictates otherwise, and the singular also includes the plural.

Those skilled in the art will appreciate alternative structural and functional designs for additional automated cycles of pH adjustment after reading this disclosure. Thus, while specific embodiments and applications have been illustrated and described, it is to be understood that the disclosed embodiments are not limited to the precise construction and components disclosed herein. Various modifications, changes, and variations apparent to those skilled in the art may be made in the arrangement, operation, and details of the methods and apparatus disclosed herein without departing from the spirit and scope defined in the appended claims.

The particular features, structures, or characteristics of any specific embodiment may be combined in any suitable manner and in any suitable combination with one or more other embodiments, including the use of selected features without the corresponding use of other features. In addition, many modifications may be made to adapt a particular application, situation or material to the essential scope and spirit of the present invention. It is to be understood that other variations and modifications of the embodiments of the invention described and illustrated herein are possible in light of the teachings herein and are to be considered as part of the spirit and scope of the invention.

Patent claims at the end of this patent application are not intended to be construed in accordance with 35u.s.c. ≡112 (f), unless traditional means plus functional language is explicitly recited, such as the language of "means for … …" or "steps for … …" explicitly recited in the claim(s).

Claims

1. An automated low pH viral inactivation system, the automated system comprising:

a first container;

a second container;

a first pH probe associated with the first container and configured to measure a pH of a content of the first container;

a fluid source to be transferred to the first container, the fluid source known or suspected to contain at least one enveloped virus;

an acid pump configured to pump acid into the first container after transferring the fluid into the first container, and configured to stop pumping acid into the first container in response to the first pH probe measuring a first pH value within an allowable range of a 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 below a threshold pH for viral inactivation and in response to the acid pump stopping pumping acid into the first container;

A first buffer pump configured to pump a first equilibration buffer having a first known pH into the first container in response to the entire acidified pool being pumped out of the first container; and

an alert generator configured to:

after the first equilibration buffer is pumped into the first vessel, comparing a second pH measured by the first pH probe to the first known pH of the first equilibration buffer;

determining whether the difference between the second pH measured by the first pH probe and the first known pH of the first equilibration buffer is greater than a threshold pH; and

a first alert is generated in response to a second pH measured by the first pH probe that differs from the first known pH of the first equilibration buffer by more than the threshold pH.

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

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

4. The automated low pH viral inactivation system of claim 1, further comprising:

a second pH probe associated with the second container and configured to measure a pH of a content of the second container;

an alkaline pump configured to pump alkaline into the second container in response to an elapsed time from the entire acidified pool being pumped into the second container exceeding a threshold amount of time to reduce the concentration of virus in the acidified pool to a predetermined safe level, and configured to stop pumping alkaline into the second container in response to the second pH probe measuring a first pH value within a threshold range of neutral pH values;

a drain pump configured to pump the neutralized virally inactivated collection from the second container to a filter to process the neutralized virally inactivated collection;

A second buffer pump configured to pump a second balanced buffer having a second known pH into the second container in response to the entire pool being pumped out of the second container; and is also provided with

Wherein the alert generator is further configured to:

after the first equilibration buffer is pumped into the second vessel, comparing a second pH measured by the second pH probe to the second known pH of the second equilibration buffer;

determining whether a difference between the second pH measured by the second pH probe and the second known pH of the second equilibration buffer is greater than the threshold pH; and

a second alert is generated in response to a second pH measured by the second pH probe that differs from the second known pH of the second equilibration buffer by more than the threshold pH.

5. The automated low pH viral inactivation system of claim 4, wherein the first and second equilibration buffers are the same equilibration buffer.

6. The automated low pH viral inactivation system of claim 4, wherein the first and second equilibration buffers are different equilibration buffers.

7. The automated low pH viral inactivation system of claim 4, wherein the transfer pump is configured to pump the acidified pool from the first container to the second container based at least in part on a signal indicating that the second container is empty.

8. The automated low pH viral inactivation system of claim 4, wherein the second buffer pump is configured to pump the second equilibration buffer into the second container based at least in part on a signal indicating that the second container is empty.

9. The automated low pH viral inactivation system of claim 4, further comprising:

a third container; and

a collection pump configured to pump filtered collection from the filter to the third container.

10. The automated low pH viral inactivation system of claim 9, wherein the collection pump is configured to pump the filtered pool from the second container to the third container based at least in part on a signal indicating that the third container is empty.

11. The automated low pH viral inactivation system of claim 1, further comprising:

A first pH probe recalibrator configured to recalibrate the first pH probe automatically in response to the first alarm.

12. The automated low pH virus inactivation system of claim 1, further comprising one or more additional pH probes associated with the first container and configured to measure the pH of the contents of the first container.

13. The automated low pH viral inactivation system of claim 4, further comprising one or more additional pH probes associated with the second container and configured to measure the pH of the contents of the second container.

14. The automated low pH viral inactivation system of claim 4, further comprising:

a second pH probe recalibrator configured to automatically recalibrate the second pH probe in response to the second alarm.

15. The automated low pH viral inactivation system of claim 4, further comprising:

An operator display configured to display one or more of the first alert or the second alert to an operator associated with the system.

16. The automated low pH viral inactivation system according to claim 1, wherein the acid is selected from the group consisting of formic acid, acidic acid, citric acid, and phosphoric acid at a concentration suitable to ensure viral inactivation.

17. The automated low pH viral inactivation system of claim 1, wherein the threshold pH for viral inactivation is pH 2 to 4.

18. The automated low pH viral inactivation system according to claim 1, wherein the chromatographic elution pool is exposed to acid for less than 30 minutes prior to neutralization.

19. The automated low pH viral inactivation system of claim 1, wherein the base is Tris base at a 2M concentration.

20. The automated low pH viral inactivation system according to claim 1, wherein the threshold range of neutral pH values is pH 4.5 to 6.

21. The automated low pH viral inactivation system of claim 1, wherein the low pH viral inactivation is performed at a temperature of 5 ℃ to 25 ℃.

22. The automated low pH viral inactivation system of claim 1, wherein a neutralized virally inactivated chromatographic elution pool is transferred from the second container to a holding container.

23. The automated low pH viral inactivation system according to claim 1, wherein the neutralized virally inactivated chromatographic elution pool from the second container is transferred to a depth filter.

24. The automated low pH viral inactivation system of claim 23, wherein the neutralized virally inactivated eluate is transferred to a sterile filter after depth filtration.

25. The automated low pH viral inactivation system according to claim 1, wherein the neutralized virally inactivated chromatographic elution pool from the second container is transferred to a first refining chromatographic column.

26. An automated low pH viral inactivation method, the method comprising:

adding the pool to a first container;

adding an acid to the first container;

measuring a first pH associated with the first container by a first pH probe associated with the first container;

stopping the addition of acid to the first container based on a first measured pH associated with the first container that is within an allowable range of a target pH for viral inactivation;

transferring the pool from the first container to a second container;

Filling the first container with an equilibration buffer having a known pH;

measuring a second pH associated with the first container by the first pH probe;

comparing a second measured pH associated with the first container to the known pH of the equilibration buffer;

determining whether a difference between the second measured pH associated with the first container and the known pH of the equilibration buffer is greater than a threshold pH; and

a first alert is generated in response to a second measured pH associated with the first container that differs from the known pH of the equilibration buffer by more than the threshold pH.

27. The automated low pH viral inactivation method of claim 26, wherein transmitting the pool to the first container is based at least in part on receiving a signal indicating that the first container is empty.

28. The automated low pH viral inactivation system of claim 26, wherein filling the first container with the equilibration buffer is based at least in part on receiving a signal indicating that the first container is empty.

29. The automated low pH virus inactivation method of claim 26, further comprising:

Adding base to the second container after the elapsed time after transferring the collection to the second container exceeds a threshold value of an amount of time that reduces the concentration of virus in the collection to a predetermined safe level;

measuring a first pH associated with the second container by a second pH probe associated with the second container;

stopping the addition of base to the second vessel based on a first measured pH associated with the second vessel that is within a threshold range of neutral pH values;

transferring the pool from the second vessel to a filter to treat the neutralized virally inactivated pool;

filling the second container with an equilibration buffer having the known pH;

measuring a second pH associated with the second container by a second pH probe associated with the second container;

comparing a second measured pH associated with the second container to the known pH of the equilibration buffer;

determining whether a difference between the second measured pH associated with the second container and the known pH of the equilibration buffer is greater than a threshold pH; and

A second alert is generated in response to a second measured pH associated with the second container that differs from the known pH of the equilibration buffer by more than the threshold pH.

30. The automated low pH viral inactivation method of claim 29, wherein the acidified pool is transferred from the first container to the second container based at least in part on receiving a signal indicating that the second container is empty.

31. The automated low pH viral inactivation method of claim 29, wherein filling the second container with the equilibration buffer is based at least in part on receiving a signal indicating that the second container is empty.

32. The automated low pH virus inactivation method of claim 29, further comprising:

transferring the collection from the filter to the third container.

33. The automated low pH viral inactivation method of claim 32, wherein transferring the collection from the filter to the third container is based at least in part on receiving a signal indicating that the third container is empty.

34. The automated low pH virus inactivation method of claim 26, further comprising:

Recalibrating the first pH probe in response to the first alarm.

35. The automated low pH viral inactivation method of claim 34, wherein the recalibration is an automatic recalibration.

36. The automated low pH viral inactivation method of claim 34, wherein the recalibration is manual recalibration.

37. The automated low pH virus inactivation method of claim 29, further comprising:

recalibrating the second pH probe in response to the second alarm.

38. A method for inactivating an enveloped virus during purification of a recombinant protein of interest, the method comprising:

obtaining a fluid known or suspected to contain at least one enveloped virus;

subjecting the fluid to one or more of the following steps at a concentration and for a time sufficient to cause viral inactivation:

adding the fluid to a first container;

adding an acid to the first container;

Transferring the fluid from the first container to a second container;

filling the first container with an equilibration buffer having a known pH;

generating a first alert in response to a second measured pH associated with the first container that differs from the known pH of the equilibration buffer by more than the threshold pH; and

subjecting the neutralized virally inactivated fluid to at least one unit operation comprising at least a filtration step or a chromatography step.

39. The method of claim 38, wherein adding the fluid to the first container is based in part on receiving a signal indicating that the first container is empty.

40. The method of claim 38, wherein transferring the fluid from the first container to the second container is based in part on receiving a signal indicating that the second container is empty.

41. The method of claim 38, wherein filling the first container with the equilibration buffer is based in part on receiving a signal indicating that the first container is empty.

42. The method of claim 38, wherein the fluid comprises a recombinant protein of interest.

43. The method of claim 38, wherein the fluid is a harvested host cell culture fluid.

44. The method of claim 38, wherein the fluid is from an effluent stream, eluate, pool, storage or holding vessel from a unit operation comprising a harvesting step, a filtration step, or a chromatography step.

45. A method according to claim 44, wherein the fluid is an eluate collected from depth filtration, microfiltration, affinity chromatography, ion exchange chromatography, multi-mode chromatography, hydrophobic interaction chromatography or hydroxyapatite chromatography.

46. A method according to claim 44, wherein the fluid is a collection comprising harvested cell culture fluid, eluate from depth filtration, eluate from microfiltration, eluate from affinity chromatography, eluate from ion exchange chromatography, eluate from multimode chromatography, eluate from hydrophobic interaction chromatography or eluate from hydroxyapatite chromatography.

47. The method of claim 46, wherein the affinity chromatography is protein A, protein G, protein A/G or protein L chromatography.

48. The method of claim 38, wherein the chromatography is selected from affinity chromatography, protein a chromatography, ion exchange chromatography, anion exchange 20 chromatography, cation exchange chromatography; hydrophobic interaction chromatography; mixed mode or multimode chromatography or hydroxyapatite chromatography.

49. The method of claim 38, wherein the fluid is a harvested host cell culture fluid and the unit operation comprises depth filtration.

50. The method of claim 38, wherein the fluid is a harvested host cell culture fluid and the unit operation comprises microfiltration.

51. The method of claim 38, wherein the fluid is a harvested host cell culture fluid and the unit operation comprises protein a affinity chromatography.

52. The method of claim 38, wherein the fluid is a protein a eluate and the unit operation comprises depth filtration.

53. The method of claim 38, wherein the unit operation comprises depth filtration.

54. The method of claim 38, wherein the unit operation comprises microfiltration.

55. An automated low pH viral inactivation system, the automated system comprising:

a first container;

a second container;

An alkaline pump configured to pump alkaline into the second container in response to an elapsed time from the entire acidified pool being pumped into the second container exceeding a threshold amount of time to reduce the concentration of virus in the acidified pool to a predetermined safe level, and configured to stop pumping alkaline into 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 drain pump configured to pump the neutralized virally inactivated collection from the second container to a filter to process the neutralized virally inactivated collection.

56. The automated low pH viral inactivation system of claim 55, further comprising a source pump configured to pump the fluid from the source into the first container based at least in part on a signal indicating that the first container is empty.

57. The automated low pH viral inactivation system of claim 55, wherein the transfer pump is configured to pump the acidified pool from the first container to the second container based at least in part on a signal indicating that the second container is empty.

58. The automated low pH viral inactivation system of claim 55, further comprising:

a third container; and

59. The automated low pH viral inactivation system of claim 58, wherein the collection pump is configured to pump the filtered pool from the second container to the third container based at least in part on a signal indicating that the third container is empty.

60. The automated low pH viral inactivation system of claim 55, further comprising one or more additional pH probes associated with the first container and configured to measure the pH of the contents of the first container.

61. The automated low pH viral inactivation system of claim 55, further comprising one or more additional pH probes associated with the second container and configured to measure the pH of the contents of the second container.

62. The automated low pH viral inactivation system according to claim 55, wherein the acid is selected from the group consisting of formic acid, acidic acid, citric acid, and phosphoric acid at a concentration suitable to ensure viral inactivation.

63. The automated low pH viral inactivation system of claim 55, wherein the threshold pH for viral inactivation is pH 2 to 4.

64. The automated low pH viral inactivation system according to claim 55, wherein the chromatographic elution assembly is exposed to acid for less than 30 minutes prior to neutralization.

65. The automated low pH viral inactivation system of claim 55, wherein the base is Tris base at a 2M concentration.

66. The automated low pH viral inactivation system according to claim 55, wherein the threshold range of neutral pH is pH 4.5 to 6.

67. The automated low pH viral inactivation system of claim 55, wherein the low pH viral inactivation is performed at a temperature of 5 ℃ to 25 ℃.

68. The automated low pH viral inactivation system of claim 55, wherein the neutralized virally inactivated chromatographic elution pool is transferred from the second container to a holding container.

69. The automated low pH viral inactivation system according to claim 55, wherein the neutralized virally inactivated chromatographic elution pool from the second container is transferred to a depth filter.

70. An automated low pH viral inactivation system according to claim 58, wherein the neutralized virally inactivated eluate is transferred to a sterile filter after depth filtration.

71. The automated low pH viral inactivation system according to claim 55, wherein the neutralized virally inactivated chromatographic elution pool from the second container is transferred to a first refining chromatographic column.

72. An automated low pH viral inactivation method, the method comprising:

adding the pool to a first container;

adding an acid to the first container;

transferring the pool from the first container to a second container;

stopping the addition of base to the second vessel based on a first measured pH associated with the second vessel that is within a threshold range of neutral pH values; and

transferring the pool from the second vessel to a filter to treat the neutralized virally inactivated pool.

73. The automated low pH viral inactivation method of claim 72, wherein transmitting the collection to the first container is based at least in part on receiving a signal indicating that the first container is empty.

74. The automated low pH viral inactivation method of claim 72, wherein filling the second container with the equilibration buffer is based at least in part on receiving a signal indicating that the second container is empty.

75. The automated low pH viral inactivation method of claim 72, further comprising:

transferring the collection from the filter to the third container.

76. The automated low pH viral inactivation method of claim 75, wherein transferring the collection from the filter to the third container is based at least in part on receiving a signal indicating that the third container is empty.

77. A method for inactivating an enveloped virus during purification of a recombinant protein of interest, the method comprising:

obtaining a fluid known or suspected to contain at least one enveloped virus;

adding the fluid to a first container;

adding an acid to the first container;

transferring the fluid from the first container to a second container;

adding a base to the second vessel;

measuring a second pH associated with the second container by a second pH probe associated with the first container;

stopping adding base to the second vessel based on a second measured pH associated with the second vessel that is within an allowable range of a neutral target pH; and

78. The method of claim 77, wherein adding the fluid to the first container is based in part on receiving a signal indicating that the first container is empty.

79. The method of claim 77, wherein transferring the fluid from the first container to the second container is based in part on receiving a signal indicating that the second container is empty.

80. The method of claim 77, wherein filling the first container with the equilibration buffer is based in part on receiving a signal indicating that the first container is empty.

81. The method of claim 77, wherein said fluid comprises a recombinant protein of interest.

82. The method of claim 77, wherein said fluid is a harvested host cell culture fluid.

83. The method of claim 77, wherein said fluid is from an effluent stream, eluate, pool, storage or holding vessel from a unit operation comprising a harvesting step, a filtration step, or a chromatography step.

84. The method of claim 83, wherein the fluid is an eluate collected from depth filtration, microfiltration, affinity chromatography, ion exchange chromatography, multi-mode chromatography, hydrophobic interaction chromatography, or hydroxyapatite chromatography.

85. The method of claim 83, wherein the fluid is a collection comprising harvested cell culture fluid, an eluate from depth filtration, an eluate from microfiltration, an eluate from affinity chromatography, an eluate from ion exchange chromatography, an eluate from multi-mode chromatography, an eluate from hydrophobic interaction chromatography, or an eluate from hydroxyapatite chromatography.

86. The method of claim 77, wherein the affinity chromatography is protein a, protein G, protein a/G, or protein L chromatography.

87. The method of claim 77, wherein said chromatography is selected from the group consisting of affinity chromatography, protein a chromatography, ion exchange chromatography, anion exchange 20 chromatography, cation exchange chromatography; hydrophobic interaction chromatography; mixed mode or multimode chromatography or hydroxyapatite chromatography.

88. The method of claim 77, wherein said fluid is harvested host cell culture fluid and said unit operation comprises depth filtration.

89. The method of claim 77, wherein said fluid is a harvested host cell culture fluid and said unit operation comprises microfiltration.

90. The method of claim 77, wherein said fluid is a harvested host cell culture fluid and said unit operation comprises protein a affinity chromatography.

91. The method of claim 77, wherein said fluid is a protein a eluate and said unit operation comprises depth filtration.

92. The method of claim 77, wherein said unit operations comprise depth filtration.

93. The method of claim 77, wherein said unit operation comprises microfiltration.