# US 12,760,262 B2 · In-process verification of calibration status of pH probes > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,760,262 B2 - Document ID: US12760262B2 - Status: Granted - Kind: B2 - Application number: 18/035,316 - Filed: 2021-11-09 - Published: 2023-12-28 - Granted: 2026-10-13 - Earliest claimed: 2020-11-09 - Assignees: Amgen Inc. - Applicants: Amgen Inc. - Inventors: Venkatesh Natarajan; Marco DeLiso; Jeremy Aaron Bezaire; Andrew Cavigli; Jeremy S. Conner; Jon Hunter; Sidney Pehrson; Sarah Whetstone - Web page: https://patentgrep.com/patent/US12760262B2 ## CPC - C12M 37/00: {Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination (C12M23/38 takes precedence; filtration in general and filters per se B01D24/00-B01D41/00; autoclaves B01J3/04; treatment of microorganisms with electrical or wave energy C12N13/00)} - C12M 37/06: {Means for testing the completeness of the sterilization (testing for sterility conditions C12Q1/22)} - C12M 41/26: {of pH} - C12M 47/10: {Separation or concentration of fermentation products (bioreactors combined with means for distillation or extraction of liquid fuel C12M43/02)} - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Automated systems and methods for low-pH viral inactivation include adding an elution pool to a first vessel with an acid. Once first vessel pH probes measure sufficiently low pH, the pool is transferred to a second vessel, where the pH is checked again, and the pool is held for a time sufficient to reduce virus concentration to a safe level, and neutralized, filtered, and transferred to a third vessel. Meanwhile, the first vessel is filled with a known-pH buffer, which is checked against readings from first vessel pH probes to determine whether recalibration is needed. After the pool is transferred to the third vessel, the second vessel is filled with a known-pH buffer, which is checked against readings from second vessel pH probes to determine whether recalibration is needed. The process repeats when the known-pH buffer is dumped and a new elution pool is added to the first vessel. ## Claims ### Claim 1 An automated system for low pH viral inactivation, wherein the pH is from 2 to 4, comprising: a first vessel; a second vessel; a first pH probe associated with the first vessel and configured to measure the pH of contents of the first vessel; a source of a fluid known or suspected to contain at least one enveloped virus to be transferred to the first vessel; an acid pump configured to pump acid into the first vessel after the fluid is transferred into the first vessel to form an acidified pool and configured to cease pumping acid into the first vessel responsive to the first pH probe measuring a first pH value that is within a tolerance band of a target pH value for viral inactivation; a transfer pump configured to pump the acidified pool from the first vessel to the second vessel responsive to the first pH probe measuring the first pH value that is below the target pH value for viral inactivation, and responsive to the acid pump ceasing to pump acid into the first vessel; a first buffer pump configured to pump a first equilibration buffer, having a first known pH value, into the first vessel responsive to the entire acidified pool being pumped out of the first vessel; and an alert generator configured to: compare a second pH value, measured by the first pH probe after the first equilibration buffer is pumped into the first vessel, to the first known pH value of the first equilibration buffer; determine whether the second pH value measured by the first pH probe is different from the first known pH value of the first equilibration buffer by greater than a threshold pH value; and generate a first alert responsive to the second pH value measured by the first pH probe being different from the first known pH of the first equilibration buffer by greater than the threshold pH value. ### Claim 2 (depends on 1) The automated system for low pH viral inactivation of claim 1, further comprising a source pump configured to pump the fluid into the first vessel from the source based at least in part on a signal indicating that the first vessel is empty. ### Claim 3 (depends on 1) The automated system for low pH viral inactivation of claim 1, wherein the first buffer pump is configured to pump the first equilibration buffer into the first vessel based at least in part on a signal indicating that the first vessel is empty. ### Claim 4 (depends on 1) The automated system for low pH viral inactivation of claim 1, further comprising: a second pH probe associated with the second vessel and configured to measure the pH of contents of the second vessel; a base pump configured to pump base into the second vessel responsive to an elapsed time, from the entire acidified pool being pumped into the second vessel, exceeding a threshold amount of time for reducing a concentration of virus in the acidified pool to a predetermined safe level, and configured to cease pumping base into the second vessel responsive to the second pH probe measuring a first pH value that is within a threshold range of neutral pH values, wherein the base neutralizes the acidified pool to form a neutralized viral inactivated pool; a discharge pump configured to pump the neutralized viral inactivated pool from the second vessel into a filter for treatment of the neutralized viral inactivated pool; a second buffer pump configured to pump a second equilibration buffer, having a second known pH value, into the second vessel responsive to the entire neutralized viral inactivated pool being pumped out of the second vessel; and wherein the alert generator is further configured to: compare a second pH value, measured by the second pH probe after the second equilibration buffer is pumped into the second vessel, to the second known pH value of the second equilibration buffer; determine whether the second pH value measured by the second pH probe is different from the second known pH value of the second equilibration buffer by greater than a threshold pH value; and generate a second alert responsive to the second pH value measured by the second pH probe being different from the second known pH of the second equilibration buffer by greater than the threshold pH value. ### Claim 5 (depends on 4) The automated system for low pH viral inactivation of claim 4, wherein the first equilibration buffer and the second equilibration buffer are the same equilibration buffer. ### Claim 6 (depends on 4) The automated system for low pH viral inactivation of claim 4, wherein the first equilibration buffer and the second equilibration buffer are distinct equilibration buffers. ### Claim 7 (depends on 4) The automated system for low pH viral inactivation of claim 4, wherein the transfer pump is configured to pump the acidified pool from the first vessel to the second vessel based at least in part on a signal indicating that the second vessel is empty. ### Claim 8 (depends on 4) The automated system for low pH viral inactivation of claim 4, wherein the second buffer pump is configured to pump the second equilibration buffer into the second vessel based at least in part on a signal indicating that the second vessel is empty. ### Claim 9 (depends on 4) The automated system for low pH viral inactivation of claim 4, further comprising: a third vessel; and a collection pump configured to pump the filtered pool from the filter to the third vessel. ### Claim 10 (depends on 9) The automated system for low pH viral inactivation of claim 9, wherein the collection pump is configured to pump the filtered pool from the second vessel to the third vessel based at least in part on a signal indicating that the third vessel is empty. ### Claim 11 (depends on 4) The automated system for low pH viral inactivation of claim 4, further comprising one or more additional pH probes associated with the second vessel and configured to measure the pH of contents of the second vessel. ### Claim 12 (depends on 4) The automated system for low pH viral inactivation of claim 4, further comprising: a second pH probe recalibrator configured to automatically recalibrate the second pH probe responsive to the second alert. ### Claim 13 (depends on 4) The automated system for low pH viral inactivation 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. ### Claim 14 (depends on 4) The automated system of low pH viral inactivation of claim 4, further comprising a holding vessel, wherein the neutralized viral inactivated pool from the second vessel is transferred to the holding vessel. ### Claim 15 (depends on 4) The automated system of low pH viral inactivation of claim 4, further comprising a depth filter, wherein the neutralized viral inactivated pool from the second vessel is transferred to the depth filter configured to perform depth filtration to form a neutralized viral inactivated eluate. ### Claim 16 (depends on 15) The automated system of low pH viral inactivation of claim 15, further comprising a sterile filter, wherein following depth filtration, the neutralized viral inactivated eluate is transferred to the sterile filter. ### Claim 17 (depends on 4) The automated system of low pH viral inactivation of claim 4, further comprising a first polish chromatography column, wherein the neutralized viral inactivated pool from the second vessel is transferred to the first polish chromatography column. ### Claim 18 (depends on 1) The automated system for low pH viral inactivation of claim 1, further comprising: a first pH probe recalibrator configured to automatically recalibrate the first pH probe responsive to the first alert. ### Claim 19 (depends on 1) The automated system for low pH viral inactivation of claim 1, further comprising one or more additional pH probes associated with the first vessel and configured to measure the pH of contents of the first vessel. ### Claim 20 An automated method of low pH viral inactivation, wherein the pH is from 2 to 4, the method comprising: adding a pool comprising fluid known or suspected to contain at least one enveloped virus to a first vessel; adding acid to the first vessel; measuring, by a first pH probe associated with the first vessel, a first pH value associated with the first vessel; ceasing, based on the first measured pH value associated with the first vessel being within a tolerance band of a target pH value for viral inactivation, the addition of acid to the first vessel; transferring the pool from the first vessel to a second vessel; filling the first vessel with an equilibration buffer having a known pH value; measuring, by the first pH probe, a second pH value associated with the first vessel; comparing the second measured pH value associated with the first vessel to the known pH value of the equilibration buffer; determining whether the second measured pH value associated with the first vessel is different from the known pH value of the equilibration buffer by greater than a threshold pH value; and generating a first alert responsive to the second measured pH value associated with the first vessel being different from the known pH value of the equilibration buffer by greater than the threshold pH value.