Method for ph meter calibration and correction

Abstract

This disclosure relates to a system and method for detecting and correcting errors in pH meter calibration or function when the pH of a sample is changed. The system includes measuring an initial pH, adding two or more amounts of titrant, measuring the corresponding pH value, and applying a model to determine whether the measured pH value corresponds to a pH value predicted by the model. These systems and methods can be used to control pH during virus inactivation of protein samples.

Description

Technical Field

[0001] [Cross-reference to related applications]

[0002] This application claims priority and interest in U.S. Provisional Application No. 63 / 253,336, filed on October 7, 2021, the contents of which are incorporated herein by reference in their entirety. Prior Technology

[0003] Conventional pH measurement methods may be problematic. The method of sterilizing the pH probe and inserting it into the sample often leads to pH meter calibration errors. Therefore, there is a need in this art for a system and method for determining pH meter calibration errors. Summary of the Invention

[0004] This disclosure provides a method for determining and correcting errors in pH meter calibration during the process of changing the pH of a sample.

[0005] This disclosure provides a method comprising: (a) measuring the initial pH of a sample (pH initial); (b) adding a first amount of titrant (titrant n) to the sample and measuring a first additional pH value (pH n), wherein titrant n is the amount of titrant added to the sample to achieve pH n, and wherein pH n is different from pH initial; (c) applying a model to determine a standardized initial amount of titrant (titrant initial) and standardized titrant n, wherein the model correlates the standardized titrant added to the sample with the pH of the sample; (d) adding a second amount of titrant to the sample to achieve a second pH value (pH n+1), wherein the second amount of titrant is determined by pH initial, pH n, standardized initial titrant, and standardized titrant n; (e) measuring the pH of the sample after adding the second amount of titrant to determine the measured pH n+1; and (f) when the measured pH n+1 is compared with the pH from the model... The difference between n+1 indicates the calibration error in the pH meter used to measure the pH of the sample, and the pH meter should be calibrated.

[0006] In some embodiments of the method disclosed herein, the difference between the measured pH n+1 and the pH n+1 from the model is > 0.01 pH units, > 0.02 pH units, > 0.03 pH units, > 0.04 pH units, > 0.05 pH units, > 0.06 pH units, > 0.07 pH units, > 0.08 pH units, > 0.09 pH units, or > 0.10 pH units. In some embodiments, the difference between the measured pH n+1 and the pH n+1 from the model is > 0.05 pH units. In some embodiments, the method includes adding a third amount of titrant (titer n+2) to the sample and measuring a third additional pH (pH n+2), and repeating steps (c) through (e), wherein the difference between the measured pH n+2 and the pH n+2 from the model indicates a calibration error in the pH meter. In some embodiments, the method includes repeating steps (d) through (f) until a final pH is reached. In some embodiments, the method includes repeating steps (d) through (f) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, a difference of >0.01 pH units, >0.02 pH units, >0.03 pH units, >0.04 pH units, >0.05 pH units, >0.06 pH units, >0.07 pH units, >0.08 pH units, >0.09 pH units, or >0.10 pH units between the pH measured at any repetition and the pH from the model indicates a calibration error in the pH meter.

[0007] In some embodiments, calibrating a pH meter includes sending a signal to the operator indicating that the pH probe is faulty. In some embodiments, calibrating a pH meter includes recalibrating or replacing the pH probe.

[0008] In some embodiments of the methods disclosed herein, the methods include generating models. In some embodiments, the methods include: (i) generating at least one reference titration curve from at least one reference sample, relating the amount of titrant added to the reference sample to the pH of the reference sample; (ii) normalizing the at least one reference titration curve; and (iii) generating a model to fit the at least one reference titration curve. In some embodiments, the model includes a polynomial relating the normalized titrant to pH.

[0009] This disclosure provides an apparatus configured for use in the method of the present invention.

[0010] This disclosure provides an apparatus for controlling pH in a process of altering the pH of a sample. In some embodiments, the sample comprises a protein. In some embodiments, the apparatus may include a reactor and a pH flow channel including a pH probe disposed therein, the pH flow channel being fluidly coupled to the reactor. The pH flow channel may receive a slip stream for sampling from the reactor and contains the pH probe disposed therein, the pH probe measuring the pH of the slip stream. The apparatus includes an acid titrant supply fluidly coupled to the reactor. The acid titrant supply provides acid titrant to the reactor to lower the pH in the reactor. The apparatus further includes an alkaline titrant supply fluidly coupled to the reactor. The alkaline titrant supply provides alkaline titrant to the reactor to increase the pH in the reactor. In some embodiments, the apparatus may further include a sampling rod that delivers the slip stream from the reactor to the pH flow channel. In some embodiments, the apparatus may include a waste receiver for receiving effluent from the pH flow channel. Simple Explanation of the Diagram

[0011] Figure 1 shows 11 titration curves generated using five different proteins. The pH was lowered by adding acid solution to achieve a target pH of 3.6. The pH is displayed on the Y-axis, while the X-axis indicates the amount of acid titrant added (rot / kg) in pump rotations per kilogram of dissolving agent. A time shift was applied to the pump speed data to account for the delay between acid addition and pH reaction.

[0012] Figure 2 shows the titration curves in Figure 1 after linear transformation of the X-axis and Y-axis using Equation 1 (Y-axis) and Equation 2 (X-axis).

[0013] Figure 3 is a diagram showing the application of pH modeling in one embodiment of this disclosure.

[0014] Figure 4 shows 12 titration curves generated using 7 different proteins. pH was increased by adding alkaline solution to achieve a target pH between 7.5 and 8.0, depending on the protein. pH is displayed on the Y-axis, while the X-axis indicates the amount of acid titrant added (rot / kg) in pump rotations per kilogram of dissolving agent. A time shift was applied to the pump speed data to account for the delay between acid addition and pH reaction.

[0015] Figure 5 shows the titration curves in Figure 4 after linear transformation of the X and Y axes using Equations 5 (Y-axis) and 6 (X-axis).

[0016] Figure 6 shows the titration curves in Figure 4 after linear transformation of the X and Y axes using Equations 7 (Y-axis) and 8 (X-axis).

[0017] Figure 7 shows a graph of the standardized titration curves from Figure 6, color-coded using the difference between the initial online and offline measurements of the pH probe used to collect data for each titration curve. The curve with the largest deviation from the model (black line) also has the largest difference between the initial online and offline measurements.

[0018] Figure 8 shows the standardized titration curve of Figure 6 after correcting the pH line using Equation 9.

[0019] Figure 9 shows the titration curves of Figure 4 after forced convergence at two fixed pH values ​​(pH 3.70 and pH 7.60) and a linear transformation of the X-axis using Equation 10. The pH values ​​on the line (Y-axis) are corrected using Equation 9.

[0020] Figure 10 shows the titration curves of Figure 4 after forced convergence at two fixed pH values ​​(pH 3.70 and pH 7.60) and a linear transformation of the X-axis using Equation 10. The pH values ​​on the line (Y-axis) are corrected using Equation 9. The sixth-order polynomial of the data is shown as a solid line.

[0021] Figure 11 is a list of components of an exemplary system for controlling pH while changing the pH of a sample.

[0022] Figure 12 is a flowchart illustrating an exemplary control strategy for reducing the pH of a protein sample used to inactivate the virus. VI: Virus inactivation.

[0023] Figure 13 is a flowchart illustrating an exemplary control strategy for increasing the pH of protein samples after virus inactivation. VIP: Virus inactivation pool (sample after alkali addition).

[0024] Figure 14 shows an exemplary calibration curve of a pH meter used in an exemplary method of this disclosure.

[0025] Figure 15 is a table showing the results of five test operations for lowering and raising pH using the apparatus and method of one embodiment of this disclosure.

[0026] Figure 16 is a pair of graphs showing the difference in pH between the slipstream and offline (ΔpH) (top graph) and the dosing error (bottom graph). The formulas used to calculate ΔpH and dosing error % are shown in Figure 15.

[0027] Figure 17 is a diagram comparing pH probe conditions in a virus inactivation test operation using the apparatus and method of one embodiment of this disclosure.

[0028] Figure 18 shows the real-time process monitoring of the virus inactivation process in the sample.

[0029] Figure 19 is a block diagram of a device for pH control according to one embodiment.

[0030] Figure 20 is a schematic diagram of a device for pH control according to an embodiment.

[0031] Figure 21 shows the difference between the actual pH (measured by an offline reference probe) and the target pH for 18 batches of protein after pH adjustment using 3 (circles) or 4 (crosses) additions of acid or base. The left x-axis shows the difference between the pH measured after lowering the pH to between 3.50 and 3.60 (depending on the protein) to inactivate the virus and the target pH. The right x-axis shows the difference between the actual pH and the target pH after raising the pH to between 5.50 and 8.00 (depending on the protein). The dashed line indicates the target final pH after addition, which is within 0.10 pH units of the target pH.

[0032] Figure 22 shows the difference between the pH measured by the online control probe and the target pH for 18 batches of protein after pH adjustment using 3 (circles) or 4 (crosses) additions of acid or alkali. The left x-axis shows the difference between the pH measured after lowering the pH to 3.6 to inactivate the virus and the target pH. The right x-axis shows the difference between the actual pH and the target pH after raising the pH to 7.7 to 8.0 (depending on the protein). The dashed line indicates the target final pH after addition, which is within 0.05 pH units of the target pH.

[0033] Figure 23 shows the difference (ΔpH) between the pH measured by an offline reference probe and the pH measured by an online control probe installed in the flow-through tank at each addition step for 18 batches of protein.

[0034] Figure 24 shows the percentage of additional volume error (sometimes called feed error) for each addition step for 18 batches of protein (added a total of 133 times). The formula for the percentage of additional volume error is shown in Figure 15. Implementation

[0035] This disclosure relates to a method for determining the presence of errors in pH meter calibration. Many manufacturing processes involve altering the pH of a sample, and errors in pH meter calibration, reflected by the sample's pH, can lead to sample damage. One example of such a process is the large-scale manufacture of biologics such as antibodies or other therapeutic proteins. The manufacture of many therapeutic proteins involves culturing cells that express the therapeutic protein, followed by purification of the protein from the cultured cells and / or cell culture medium. Controlling the pH of the cell culture medium during cell culture and controlling the pH of the sample during protein purification are both important for the production of therapeutic proteins. pH meter calibration errors and the resulting sample pH errors can lead to protein denaturation, requiring sample discard if certain minimum or maximum pH values ​​are exceeded. Even if pH errors do not cause protein degradation, samples exceeding the acceptable pH range may still require costly and time-consuming quality control measures to ensure the protein remains unaffected.

[0036] pH control is crucial during multiple processes in the production of therapeutic proteins. Most mammalian cells have a specific pH range that supports optimal cell growth, metabolism, and protein production. If pH meter calibration errors result in cell culture conditions outside this optimal range, it can negatively impact cell growth and protein production. Furthermore, cells used to manufacture therapeutic proteins may carry viruses, which could be harmful if they contaminate the active pharmaceutical ingredient or drug substance. One method for inactivating potentially harmful viruses involves briefly lowering the pH during the purification of the therapeutic protein. Many viruses undergo irreversible denaturation and are effectively destroyed at pH levels of approximately 5.0 to 5.5. Some enveloped viruses are effectively inactivated at pH levels of approximately 3.5 to 4.0. However, excessively lowering or raising the pH of protein samples carries the risk of denaturing the therapeutic protein, potentially leading to the destruction of a batch and increased manufacturing costs. Therefore, there is a need to measure and control pH during the manufacture of therapeutic proteins, both during cell culture and protein purification.

[0037] Conventional methods for measuring pH during protein purification are unreliable and lead to waste of protein products. One approach involves measuring pH during protein purification by directly inserting a sterile pH probe into the reaction vessel containing the protein solution. However, maintaining sterility and probe accuracy can be challenging with this method. pH probes are typically calibrated, sealed in a bag with a bellows connector for insertion into the reaction vessel, and sterilized by autoclave or gamma irradiation. However, this results in a time gap between calibration and use when the pH probe is dry, which can affect probe accuracy. Furthermore, pH probes are made of glass and may break during insertion into the vessel. Maintaining sterility while inserting the probe can be difficult. Another method for indirectly measuring the pH of the protein solution involves removing a "slip" from the main protein solution and measuring the pH of the slip using a pH probe. However, without directly measuring the main protein solution pool, direct feedback control to adjust the pH during titration is impossible. Additionally, any protein sampled from the main pool for pH measurement is not returned to the main pool and is ultimately wasted. While statistical titration models can be used to predict the amount of acid or base added when adjusting the pH of protein solutions during manufacturing, these models require manual input of protein concentrations by the user, and each titration type (acid or base) requires a large historical dataset to generate the model. Furthermore, these models are not universally accurate for all protein processes and types. Finally, without independent measurements, such as from a second pH probe, these methods may fail to detect pH meter calibration errors early in the manufacturing process.

[0038] Therefore, there is a need for a method to detect errors in pH meter calibration that does not require direct insertion of the pH probe into the protein solution pool or continuous aspiration of material from the protein pool. This disclosure provides a method and system for detecting errors in pH meter calibration during processes involving changes in the pH of a sample (e.g., a protein sample). The method of this disclosure is accurate across a wide range of proteins, requires no operator input or offline concentration measurements, and does not require extensive historical data. The method of this disclosure can also be used to infer the amount of acid or base titrant needed to adjust the pH during the manufacturing process. Furthermore, the method and system disclosed herein can reproducibly and accurately achieve pH values ​​within 0.05 to 0.10 pH units of the desired target pH during processes involving changes in sample pH. Only 3 to 4 additions of titrant are needed to accurately and reliably achieve the final target pH, such as the target acidic pH for virus inactivation of protein samples, or the target alkaline pH after inactivation. The methods and systems disclosed herein can accurately determine and add the amount of acid or base titrant to a sample, adding the required volume of titrant with an accuracy of 10% or less volumetric error per titrant addition. Because the methods and systems disclosed herein can reproducibly and accurately achieve the target pH value, they also provide superior effectiveness in detecting and improving errors in pH meter calibration. For example, if the pH value measured during a process that changes the sample pH does not conform to the expected (or target) pH value of the model described herein, a person skilled in this technique can stop the process and recalibrate the pH meter before the sample is damaged due to pH exceeding an acceptable range.

[0039] This disclosure provides a method comprising measuring the initial pH of a sample, adding a first conservative amount of titrant, such as an acidic or alkaline solution, measuring a first intermediate pH, adding a second amount of titrant and measuring a second intermediate pH, applying a model to standardize the first and second amounts of titrant added to the sample, and calibrating a pH meter when the second measured pH differs from the value predicted by the model for the standardized second amount of titrant added to the sample. The invention further provides an apparatus for performing the method of this disclosure. Therefore, this disclosure provides a method comprising: (a) measuring the initial pH of a sample (pH initial); (b) adding a first amount of titrant (titrant n) to the sample and measuring a first additional pH value (pH n), wherein titrant n is the amount of titrant added to the sample to achieve pH n, and wherein pH n is different from pH initial; (c) applying a model to determine a standardized titrant n, wherein the model correlates the standardized titrant added to the sample with the pH of the sample; (d) adding a second amount of titrant (titrant n+1) to the sample and measuring a second additional pH value (pH n+1) and repeating step (c); and (e) calibrating the pH meter when the difference between pH n+1 and the model indicates a calibration error in the pH meter used to measure the pH of the sample. definition

[0040] As used herein, the term "initial pH" refers to the pH of the sample before the addition of a titrant to change the pH, i.e., before a solution with an initial pH of acidity or alkalinity is used.

[0041] As used herein, "final pH" refers to the desired pH of the sample. For example, a sample may have a pH of 3.6, but for a specific purpose, a pH of 7.5 is required, and the method used herein is to detect errors in pH meter calibration while controlling the addition of an alkaline titrant to change the pH from 3.6 to 7.5. In this case, 3.6 is the initial pH, and 7.5 is the final or target pH. Those skilled in this technique will understand that the initial and final pH values ​​of any given sample can differ depending on the sample, sample conditions, and application. Those generally skilled in this technique will understand that when performing a method to change pH, the method can encompass multiple steps, each with a relevant target pH before reaching the sample's final pH (or final target pH).

[0042] As used herein, “total titrant” (total titrant) refers to the amount of titrant added to the sample to change the pH from the initial pH to the final pH.

[0043] As used herein, "pH n" refers to the pH of the sample after adding a certain amount of titrant (requiring a change in pH from a previous pH (pH n-1) to pH n). Therefore, the amount of titrant required to change the pH from, for example, the initial pH to pH n is referred to herein as titrant n. Those skilled in this art will understand that the measured pH value and the corresponding amount of titrant added to the sample to change the sample pH to that measured pH value can be iterative. That is, another amount of titrant can be added to a sample at pH n to change the sample pH to pH n+1, and the amount of titrant added to the sample to change the pH from the initial pH to pH n+1 is referred to as titrant n+1. Similarly, a certain amount of titrant is added to a sample at pH n+1 to change the sample pH to pH n+2, and so on, until the target pH is reached.

[0044] The term "sample" refers to a sample whose pH has been altered by the methods described herein. In some cases, the sample contains proteins, such as purified or partially purified proteins in a liquid solution. However, other types of samples are considered within the scope of this disclosure and include DNA, RNA, and pharmaceuticals. Those skilled in the art will understand that, as used herein, a sample refers to a liquid solution, such as a liquid solution containing a plurality of biomolecules (DNA, RNA, or protein) or an analyte (compound, pharmaceutical, or analogue). Samples may be at any suitable concentration or initial pH and may include any suitable buffers or carriers.

[0045] The term "reference sample" refers to a reference sample that has similar or identical properties to the sample, undergoes pH changes similar to those of the sample, and from which data on pH and titrant addition, as well as the relationship between the two, has been collected. The reference sample can be identical to the sample; for example, it can be a reference sample obtained from a larger sample (i.e., a subsample used as a reference sample). However, if its behavior is similar to that of the sample upon titrant addition, the reference sample does not need to be identical to the sample. For example, the sample and reference sample can be different batches of the same protein produced and purified using the same or similar processes. As another example, the sample and reference sample can be similar but not identical proteins, such as two antibodies, or two proteins with similar glycosylation patterns, that behave similarly when subjected to similar titration processes.

[0046] As used herein, the term "titer curve" refers to a graph (or series of measurements) relating the volume of titrant added to the sample as the independent variable to the pH of the solution as the dependent variable. Titer curves can be generated by continuous measurements, for example, by directly inserting a pH probe into the sample and performing continuous measurements. Alternatively, titter curves can be generated from discontinuous measurements, and then an appropriate curve can be fitted to the measured data points.

[0047] As used in this article, "standardization" refers to adjusting values ​​measured at different scales to a common scale.

[0048] As used herein, "titrant" refers to a solution having a known pH and preferably a known concentration, which is added (titrated) to another solution to change the pH of that solution.

[0049] "Acid titrant" refers to a titrant with a pH that is more acidic than the sample. Generally, the pH of an acid titrant will be less than 7.0. Commonly used acid titrants include phosphoric acid (H₃PO₄), glycine salt (C₂H₆ClNO₂), acetic acid (CH₃COOH), hydrochloric acid (HCl), perchloric acid (HClO₄), and sulfuric acid (H₂SO₄). Acid titrant solutions can be prepared by diluting commercially available concentrated stock solutions and their concentrations are determined by standardization against a standard weak base. Exemplary acid titrants include phosphoric acid with concentrations between 0.20 M and 2.0 M, 0.25 M and 1.5 M, or 0.5 M and 1.0 M. For example, phosphoric acid at concentrations of 0.10 M, 0.20 M, 0.25 M, 0.30 M, 0.35 M, 0.40 M, 0.45 M, 0.50 M, 0.60 M, 0.70 M, 0.80 M, 0.90 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, or 2.0 M can be used as an acid titrant. Other exemplary acid titrants include glycine HCl at concentrations between 0.1 M and 1.0 M, between 0.2 M and 0.75 M, between 0.25 M and 0.75 M, or between 0.25 M and 0.5 M. For example, glycine HCl at concentrations of 0.10 M, 0.20 M, 0.25 M, 0.30 M, 0.35 M, 0.40 M, 0.45 M, 0.50 M, 0.60 M, 0.70 M, 0.80 M, 0.90 M, or 1.0 M is used as an acid titrant. Other exemplary acid titrants include acetic acid at concentrations between 0.5 M and 3.0 M, 1.0 M and 2.5 M, 1.0 M and 2.0 M, or 1.5 M and 2.0 M. For example, acetic acid at concentrations of 0.50 M, 0.60 M, 0.70 M, 0.80 M, 0.90 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, 2.1 M, 2.2 M, 2.3 M, 2.4 M, 2.5 M, 2.6 M, 2.7 M, 2.8 M, 2.9 M, or 3.0 M is used as an acid titrant.

[0050] "Alkaline titrant" or "basic titrant" refers to a titrant with a pH that is more alkaline than the sample. Commonly used alkaline titrants include sodium hydroxide (NaOH), which is commercially available in impure solid form and as a solution of approximately 50% w / v. NaOH solutions can be standardized against weak acid standards to determine their concentration. Other commonly used alkaline titrants include tromethamine (also known as tris(hydroxymethyl)aminomethane, C4H11NO3). Exemplary alkaline titrants include tromethamine at concentrations between 0.5 M and 3.0 M, 1.0 M and 2.5 M, 1.0 M and 2.0 M, or 1.5 M and 2.0 M. For example, sulphuric acid ammonium in concentrations of 0.50 M, 0.60 M, 0.70 M, 0.80 M, 0.90 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, 2.1 M, 2.2 M, 2.3 M, 2.4 M, 2.5 M, 2.6 M, 2.7 M, 2.8 M, 2.9 M, or 3.0 M is used as an alkaline titrant.

[0051] pH measurement is based on the activity of hydrogen ions in a solution of water, indicating its acidity or alkalinity, expressed as pH. pH measurement measures the potential difference between a pH electrode and a reference electrode. A pH "probe" refers to the measuring part containing both the pH electrode and the reference electrode. Typically, the pH electrode is a glass electrode, a type of ion-selective electrode made of a doped glass membrane sensitive to specific ions. An exemplary pH electrode is a hydrogen ion-sensitive glass electrode. The voltage of the glass electrode relative to some reference value (i.e., the reference electrode) is sensitive to changes in hydrogen ion activity. In other words, the hydrogen ion activity in the measured solution affects the electrochemical potential between the reference electrode and the hydrogen ion-sensitive electrode. The pH meter is calibrated to correlate the electrochemical potential to the pH value.

[0052] "pH meter calibration" refers to a method of calibrating a pH meter against one or more standardized buffer solutions of known pH values, as the known pH electrode deviates from its calibration setting. Typical calibration methods use calibration curves generated from at least three standard buffer solutions, but two-point calibration can also be used. An exemplary calibration procedure involves cleaning the electrode, immersing the rinsed electrode in a first standard of pH 4.0, followed by immersion in a second standard of pH 7.0 and a final standard of pH 10.0, and cleaning the electrode between measurements.

[0053] As used herein, "slipstream" or "slip stream" refers to a sampling method in which, for example, a tube inserted into a master sample is used to extract or separate subsamples from the master sample and to measure the subsamples. Slipstream can be continuous, i.e., continuously extracting from the sample, or discontinuous, extracting from the sample only at discrete time points during the process.

[0054] As used herein, "online probe" or "online pH probe" refers to a probe (online pH) that measures the pH of a sample during pH changes, and uses the information from the model described herein to determine the amount of titrant added to the sample during the titrant addition step. The online probe may be, for example, a slipstream probe mounted in a flow cell coupled to a slipstream. Alternatively, the online probe may be inserted directly into the reactor.

[0055] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to molecules containing two or more amino acid residues linked together by peptide bonds. Peptides, polypeptides, and proteins may also include modifications such as glycosylation, lipid attachment, sulfation, γ-carboxylation, alkylation, hydroxylation, and ADP ribosylation of glutamic acid residues. Peptides, polypeptides, and proteins may have scientific or commercial interest, including protein-based pharmaceuticals (biologics). Peptides, polypeptides, and proteins particularly include antibodies and chimeric or fusion proteins. Peptides, polypeptides, and proteins may be produced from recombinant animal cell lines (such as mammalian cell lines) using cell culture methods.

[0056] As used herein, the phrase "viral reduction / inactivation" is intended to refer to a reduction in the number of viral particles in a particular sample ("reduction") and a reduction in the activity of viral particles in a particular sample, such as, but not limited to, a reduction in infectivity or replication capacity ("inactivation"). Such reduction in the number of viral particles and / or activity can be approximately 50% to approximately 99%, preferably approximately 60% to approximately 99%, preferably approximately 70% to approximately 99%, preferably approximately 80% to 99%, preferably approximately 90% to approximately 99%, preferably approximately 95% to 99%, preferably approximately 95% to 99.9%, preferably approximately 95% to 99.99%, and preferably approximately 98% to 99.99%. In some non-limiting embodiments, the amount of virus (if present) in the purified antibody product is less than the virus ID50 (the amount of virus that will infect 50 percent of the target population), preferably at least 10 times smaller than the virus ID50, more preferably at least 100 times smaller than the virus ID50, and more preferably at least 1000 times smaller than the virus ID50.

[0057] All publications and patents mentioned herein are incorporated herein by reference in their entirety, just as individual publications or patents are specifically and independently incorporated herein by reference. In case of conflict, the application (including any definitions herein) shall prevail. However, any references, articles, publications, patents, patent publications, and patent applications cited herein are not and should not be construed as an admission or representation in any form that they constitute valid prior art or form part of the ordinary knowledge in any country worldwide. [, pH , ] [, Calibration , ] [, , ]

[0058] The method described herein can be used to determine whether there are errors in the pH meter calibration or function when changing the pH of a sample. This method can be used to verify the accuracy of the pH meter, thereby improving the reliability of methods for changing the pH of a sample.

[0059] Alternatively, if an error in pH meter calibration or function is detected, the process of adding titrant to the sample to change its pH can be stopped until the pH meter error is resolved, thereby improving process accuracy and preventing sample damage. When the sample is a protein, pH changes exceeding the protein's tolerance can cause protein denaturation and potentially damage the sample. Therefore, the ability to quickly and reliably identify pH meter calibration errors is an advantage of the method disclosed herein compared to other methods known in this technique. For example, if an error in pH meter calibration is detected, the pH meter can be recalibrated, replaced with a new pH meter, or the measurement obtained from a second pH meter can be mathematically corrected for the measurement obtained from an inaccurate pH meter.

[0060] In some embodiments, calibrating the pH meter includes sending a signal to the operator indicating a malfunction in the pH probe. In some embodiments, calibrating the pH meter includes stopping the addition of titrant to the sample when an error is detected. For example, when adding a corresponding amount of titrant, if the measured sample pH differs from the pH predicted by the model, a signal is sent to the operator, and / or the process of adding titrant to the sample is stopped until the operator corrects the error. In some embodiments, when the difference between the measured sample pH and the pH predicted by the model is > 0.01 pH units, > 0.02 pH units, > 0.03 pH units, > 0.04 pH units, > 0.05 pH units, > 0.06 pH units, > 0.07 pH units, > 0.08 pH units, > 0.09 pH units, or > 0.10 pH units, a signal is sent to the operator, and / or the process of adding titrant to the sample is stopped. In some embodiments, when the difference between the measured sample pH and the pH predicted by the model is > 0.01 pH units, a signal is sent to the operator, and / or the process of adding titrant to the sample is stopped. In some embodiments, when the difference between the measured sample pH and the pH predicted by the model is > 0.05 pH units, a signal is sent to the operator, and / or the process of adding titrant to the sample is stopped. In some embodiments, when the difference between the measured sample pH and the pH predicted by the model is > 0.10 pH units, a signal is sent to the operator, and / or the process of adding titrant to the sample is stopped.

[0061] In some embodiments, calibrating a pH meter after detecting an error includes recalibrating the pH meter. In some embodiments, calibrating a pH meter includes replacing the pH meter or pH probe. In some embodiments, calibrating a pH meter includes measuring the sample pH using a second independently calibrated pH meter, comparing the measurements between the pH meter and the second pH meter to determine the amount of difference between the measurements, and applying that difference to the pH meter measurement to determine the accurate pH.

[0062] In some embodiments, after pH meter calibration, the method further includes adding an additional amount of titrant to the sample and measuring the additional pH; applying a model and comparing the standardized titrant and pH with the model; and when the measured sample pH corresponds to the pH predicted by the model, continuing to add titrant to the sample to change the sample pH.

[0063] In some embodiments, after pH meter calibration, the method further includes adding an additional amount of titrant to the sample and measuring the additional pH; applying a model and comparing the standardized titrant and pH with the model; and when the measured pH corresponds to the model, adding the remaining amount of titrant to the sample to achieve the final pH.

[0064] In some embodiments, the difference between the measured sample pH and the model pH identifies an error in the calibration of the pH meter used to measure the sample pH. In some embodiments, the difference between the measured pH and the pH predicted by the model of >0.01 pH units, >0.02 pH units, >0.03 pH units, >0.04 pH units, >0.05 pH units, >0.06 pH units, >0.07 pH units, >0.08 pH units, >0.09 pH units, or >0.10 pH units indicates an error related to the pH meter, such as calibration error. In some embodiments, a difference of >0.05 pH units indicates pH meter error.

[0065] In some embodiments, the correspondence between the measured pH value and the value predicted by the model indicates that the pH meter is functioning correctly. This correspondence can be determined with the pH meter initially calibrated, i.e., without calibration, or after the pH meter has been calibrated, i.e., indicating that the calibrated pH meter is functioning correctly. In some embodiments, a difference between the measured pH and the pH predicted by the model of ≤ 0.01 pH units, ≤ 0.02 pH units, ≤ 0.03 pH units, ≤ 0.04 pH units, ≤ 0.05 pH units, ≤ 0.06 pH units, ≤ 0.07 pH units, ≤ 0.08 pH units, ≤ 0.09 pH units, or ≤ 0.10 pH units indicates a correspondence between the measured pH value and the predicted pH value, and that the pH meter is functioning correctly. In some embodiments, a difference between the measured pH and the pH predicted by the model of ≤ 0.01 pH units indicates a correspondence between the measured pH value and the predicted pH value, and that the pH meter is functioning correctly. In some embodiments, the difference between the measured pH and the pH predicted by the model is ≤ 0.05 pH units, indicating the correspondence between the measured pH value and the predicted pH value, and that the pH meter is functioning properly.

[0066] In some embodiments, the method further includes, in accordance with the method described herein, correcting for known quantifiable errors in pH meter calibration when measuring the pH of a sample or the pH of at least one reference sample used to generate a model. When the calibration errors are consistent and quantifiable across a range of pH values ​​analyzed while varying the sample pH, the pH meter can be mathematically calibrated using independent offline pH measurements. For example, measurements can be performed using a subsample aspirated from the sample using an independent pH meter.

[0067] In some embodiments, correcting a pH meter calibration includes: (a) removing a first portion of the sample or reference sample before adding titrant and measuring the pH of that first portion using an independently calibrated pH meter to generate an offline initial pH value (pH initial_offline); (b) removing a second portion of the sample or reference sample after adding the full amount of titrant and measuring the pH of that second portion using an independently calibrated pH meter to generate an offline final pH value (pH final_offline); and (c) applying the relationship between the offline pH value and the measured pH value to determine a corrected pH for the reference sample. The independently calibrated pH meter may be the same pH meter used for the initial measurement after another round of calibration. Alternatively, the independently calibrated pH meter may be a different pH meter.

[0068] Offline measurements can be used to calculate the corrected pH according to the following formula, where the corrected pH of the sample (or reference sample) is determined by the following formula: (Equation 16) Here, pH_initial_offline refers to the initial pH of the sample measured by an offline pH meter, pH_final_offline refers to the final pH of the sample measured by an offline pH meter, and pH_initial and pH_final refer to the initial and final pH values ​​measured by an online pH meter (a meter with calibration error), and pH_n is an uncalibrated pH measurement from an uncalibrated pH meter.

[0069] If a calibrated pH meter is used to measure a reference sample, the calibrated pH and the uncalibrated pH maintain the same relationship described above for the sample in some embodiments of the methods described herein. In these embodiments, when calibrating a pH meter for a reference sample, the method includes: (a) removing a first portion of the reference sample before adding titrant and measuring the pH of the first portion with an independently calibrated pH meter to generate an offline initial pH value (pH initial_offline_reference); (b) removing a second portion of the reference sample after adding the full amount of titrant and measuring the pH of the second portion with an independently calibrated pH meter to generate an offline final pH value (pH final_offline_reference); and (c) determining the calibrated pH of the reference sample by applying the relationship between the offline pH value and the measured pH value. In some embodiments, the calibrated pH n_reference of the reference sample is determined by the following formula: (Equation 22) Here, pH Initial_Offline_Reference refers to the initial pH of the reference sample measured by an offline pH meter, pH Final_Offline_Reference refers to the final pH of the reference sample measured by an offline pH meter, pH Initial_Reference and pH Final_Reference refer to the initial and final pH values ​​of the reference sample measured by an online pH meter (a meter with calibration error), and pH n_ref is an uncalibrated pH measurement value from an uncalibrated pH meter.

[0070] Errors in pH meter calibration can be detected at the start of a process used to change the pH of a sample, for example, by comparing the initial pH value of the sample with the initial pH value of a reference sample that is expected to behave the same as or substantially the same as the sample. If the sample and the reference sample are substantially the same and subjected to the same conditions, then if the difference in their initial pH values ​​is, for example, > 0.05 pH units, > 0.06 pH units, > 0.07 pH units, > 0.08 pH units, > 0.09 pH units, > 0.10 pH units, > 0.20 pH units, > 0.30 pH units, > 0.40 pH units, > 0.50 pH units, > 0.60 pH units, or > 0.70 pH units, then an error in pH meter calibration can be suspected. In some embodiments, the method further includes comparing an initial pH value with an initial pH value previously measured for at least one reference sample before adding the titrant, and calibrating the pH meter when the difference between the initial pH value and the previously measured initial pH value indicates a calibration error in the pH meter. In some cases, a statistical titration model, i.e., a model based on historical titration data of similar or substantially similar samples, can be used. A statistical titration model predicts the pH (or pH change) produced by adding a predetermined amount of titrant to the sample. Therefore, a statistical model can be used to verify that the pH change of the sample is within the expected limits after the first titrant addition. A statistical model can also be used to assess whether the initial pH is within normal limits. In some embodiments, the method includes comparing an initial pH value from the sample with an initial pH value predicted by a statistical titration model, and calibrating the pH meter when the difference between the initial pH value and the initial pH value predicted by the statistical titration model indicates a calibration error in the pH meter. [, Change sample , ] [, pH , ]

[0071] This disclosure provides a method for detecting errors in pH meter calibration when the pH of a sample is changed. In some embodiments, the method includes calibrating the pH meter when a measurement obtained by a pH meter used to measure the pH of a sample differs from a measurement predicted by a model. In some embodiments, the method includes performing an initial pH measurement on the sample, adding a first amount of titrant to the sample, measuring a first additional pH value, and applying a model to correlate the sample pH to a standardized amount of titrant added to the sample, adding a second amount of titrant to the sample and measuring a second additional pH value, applying the model, and calibrating the pH meter when the difference between the second pH measurement and the model-predicted second pH measurement indicates a calibration error in the pH meter used to measure the pH of the sample. In some embodiments, the method further includes adding a third or another amount of titrant to the sample and measuring a third or another pH value, and applying the model after each addition of titrant. In some embodiments, the method further includes adding a fourth or another amount of titrant, measuring the pH value after each addition, and applying the model. Additional titration steps can confirm errors in pH meter calibration, or, if performed after pH meter calibration, verify the accuracy of subsequent pH measurements. The additional steps of adding a conservative amount of titrant and checking the pH can also be used to verify that the sample behavior is as predicted by the model and that there are no additional errors in the process. After adding titrant to the sample two or more times and using the corresponding measurements to validate the pH meter, these measurements and the model can be used to determine the amount of titrant to be added to the sample to bring the sample pH to the final or target pH.

[0072] By adding discrete, conservative amounts of titrant, measuring the resulting pH change, and applying the model described herein to determine whether the actual pH change is related to the pH change predicted by the model, the method disclosed herein verifies the accuracy of a pH meter without removing the pH meter from the sample or relying on a second pH meter measurement to confirm accuracy. As an example of applying the method described herein, the initial pH of the sample is 4.30, and the target pH is 3.60. After the first addition of acid titrant, the sample pH may be approximately 4.0, i.e., between 4.3 and 3.6. Using the model and the measured pH and standardized titrant values, a second amount of titrant to be added to the sample to achieve a second intermediate pH, such as 3.80, is calculated. After adding the second predetermined amount of titrant, the second intermediate pH is measured, and the correspondence between the measured pH and the intermediate target of 3.80 is determined. If the two values ​​correspond, the pH sequence continues. If the two values ​​do not correspond, appropriate corrective actions are taken, such as recalibrating the pH meter. Therefore, the method described in this paper improves the accuracy and reliability of pH measurement when the pH of a sample is changed, reduces sample waste, and increases the efficiency of the process for changing the pH of a sample.

[0073] Furthermore, the method disclosed herein can achieve this by using a relatively small number of discrete measurements and a model to determine the amount of titrant added to the sample to change the pH. Compared to methods that measure pH by inserting a pH probe into the sample, these methods can improve the accuracy of reaching the final pH of the sample by using the model. The difference between the measured pH value and the model can also be used to identify errors in the process, such as pH meter calibration or functional errors.

[0074] In some embodiments, the sample pH is changed by adding multiple amounts of titrant. In some embodiments, the sample pH is changed to a final pH by adding titrant 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the sample pH is changed to a final pH by adding titrant 2 times. In some embodiments, the sample pH is changed to a final pH by adding titrant 3 times. In some embodiments, the sample pH is changed to a final pH by adding titrant 4 times. In some embodiments, the sample pH is changed to a final pH by adding titrant 5 times. In some embodiments, the sample pH is changed to a pH value within 0.01 to 0.20, 0.01 to 0.15, 0.01 to 0.10, 0.05 to 0.20, 0.05 to 0.15, 0.05 to 0.10, 0.01 to 0.07, or 0.05 to 0.07 pH units of the final pH, and then a final titrant is added to reach the final pH. In some embodiments, the sample pH is changed to a pH value within 0.05 to 0.10 pH units of the final pH, followed by a final addition of titrant to achieve the final pH. For example, the sample pH can be changed to a target pH within 0.05 to 0.10 pH units of the final pH by adding titrant 1, 2, 3, 4, or 5 times, followed by a final addition of titrant to achieve the final pH. In some embodiments, such as those where the sample pH is decreased, the titrant is an acid. In alternative embodiments, such as those where the sample pH is increased, the titrant is a base. A mismatch between the target pH predicted by the model described herein and the measured pH at any of the addition steps described herein may indicate a calibration error in the pH meter used to perform the measurement. For example, if the difference between the predicted and measured pH value for a given addition step is greater than 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, or a larger pH unit, it indicates that the pH meter used to measure the sample is giving an incorrect reading. As another example, if the difference between the predicted and measured pH value for a given addition step is greater than 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.10 pH units, it indicates that the pH meter used to measure the sample is giving an incorrect reading. In some embodiments, the method includes stopping the process of changing the sample pH when a difference occurs between the predicted pH value and the measured pH value, until the pH meter is recalibrated or the pH probe is replaced.

[0075] The method disclosed herein can be used whenever a change in sample pH is required. For example, if a process such as protein purification produces a liquid sample containing a protein of interest (sometimes referred to as a protein pool) with a pH unsuitable for downstream purification steps or applications, the method described herein can be used to change the sample pH to the desired pH. As another example, the method disclosed herein can be used to lower the pH of a protein sample to a pH low enough to inactivate viruses that may contaminate the protein sample, and then raise the pH to neutral for further protein purification and analytical processes.

[0076] In some embodiments, the sample contains a protein of interest, such as a therapeutic protein, and the methods are used to verify the accuracy of the pH meter used or to calibrate the pH meter when the sample pH is changed to inactivate the virus in the sample.

[0077] Methods for inactivating pH-dependent viruses include (but are not limited to) incubating a mixture at a low pH for a period of time, followed by pH neutralization and removal of particulates by filtration. In some embodiments, the sample pH is lowered to a pH between about 2 and 5, preferably between about 3 and 4, and even more preferably at about 3.6, and the sample is incubated at this pH to inactivate any present viruses. The pH of the sample mixture can be lowered by any suitable acid, including (but not limited to) phosphate, glycine salt, perchloric acid, hydrochloric acid, citric acid, acetic acid, caprylic acid, or other suitable acids. The choice of pH level depends primarily on the stability profile of the proteins in the sample and the buffer composition.

[0078] In an exemplary method for inactivating the virus in a sample, a conservative initial amount of acid titrant is added, the pH is assessed, and then an additional conservative amount of acid titrant is added, followed by another pH assessment. This can be repeated using small amounts of acid over a process that can take between 30 minutes and 2 hours until the target pH is reached. The sample is held at the target pH for a period sufficient to inactivate the virus, and the sample pH is increased by the same process described above.

[0079] In some embodiments, the duration of the low-pH incubation period for virus inactivation will be 0.5 hours to 2 hours, or 0.5 hours to 1.5 hours, or 0.5 hours to 1 hour. In some embodiments, the low-pH incubation period is approximately 20 minutes, approximately 30 minutes, approximately 40 minutes, approximately 50 minutes, approximately 60 minutes, approximately 70 minutes, approximately 80 minutes, or approximately 90 minutes. Therefore, depending on the protein of interest, those skilled in this art will be able to select the appropriate protein concentration, pH, and duration to achieve virus inactivation.

[0080] In some embodiments, altering the pH of a sample containing the protein of interest involves lowering the sample pH. The methods described herein are used to verify the accuracy of a pH meter used to measure or calibrate a pH meter when lowering the sample pH by adding an acid titrant. For example, the final pH of the sample (pH final) is less than the initial pH of the sample (pH initial), and the titrant is acid. Any suitable acidic solution can be used, as long as the pH of the titrant is less than the initial pH of the sample.

[0081] In some embodiments, such as those involving lowering pH, the initial pH of the sample (initial pH) is between about 4.0 and 4.7, between about 4.0 and 4.5, between about 4.0 and 4.3, between about 4.1 and 4.6, between about 4.1 and 4.5, between about 4.1 and 4.4, between about 4.1 and 4.3, between about 4.1 and 4.2, between about 4.2 and 4.5, between about 4.3 and 4.5, between about 4.1 and 4.4, or between about 4.2 and 4.4. In some embodiments, the initial pH is between about 4.0 and 4.5, between about 4.1 and 4.5, between about 4.2 and 4.5, between about 4.3 and 4.5, between about 4.1 and 4.4, or between about 4.2 and 4.4. In some embodiments, the initial pH is about 4.1. In some embodiments, the final pH of the sample is between about 3.0 and 3.8, between about 2.0 and 3.7, between about 3.0 and 3.6, between about 3.0 and 3.5, between about 3.0 and 3.4, between about 3.0 and 3.3, between about 3.1 and 3.8, between about 3.3 and 3.8, between about 3.5 and 3.8, between about 3.2 and 3.8, between about 3.3 and 3.7, between about 3.4 and 4.0, between about 3.5 and 4.0, between 3.4 and 3.9, between about 3.4 and 3.8, between about 3.4 and 3.7, between about 3.4 and 3.6, between about 3.5 and 3.9, between about 3.5 and 3.8, between about 3.5 and 3.7, or between about 3.5 and 3.6. In some embodiments, the final pH is between about 3.5 and 3.7. In some embodiments, the final pH is between about 3.0 and 3.8, between about 3.1 and 3.8, between about 3.2 and 3.8, between about 3.3 and 3.7, between about 3.4 and 3.7, or between about 3.5 and 3.7. In some embodiments, the final pH is about 3.6.

[0082] In some embodiments, altering the pH of a sample containing the protein of interest involves raising the sample pH. The methods described herein are used to verify the accuracy of a pH meter used to measure or calibrate a pH meter when raising the pH by adding an alkaline titrant. For example, the final pH of the sample (pH final) is greater than the initial pH of the sample (pH initial), and the titrant is alkaline. Any suitable alkaline solution can be used, as long as the pH of the titrant is greater than the initial pH of the sample.

[0083] In some embodiments, such as those involving raising the pH, the initial pH of the sample (initial pH) is between about 3.0 and 3.8, between about 2.0 and 3.7, between about 3.0 and 3.6, between about 3.0 and 3.5, between about 3.0 and 3.4, between about 3.0 and 3.3, between about 3.1 and 3.8, between about 3.3 and 3.8, between about 3.5 and 3.8, between about 3.2 and 3.8, between about 3.3 and 3.7, between about 3.4 and 4.0, and between about 3.5 and 4.0. The pH is between 3.4 and 3.9, between about 3.4 and 3.8, between about 3.4 and 3.7, between about 3.4 and 3.6, between about 3.5 and 3.9, between about 3.5 and 3.8, between about 3.5 and 3.7, or between about 3.5 and 3.6. In some embodiments, the pH ultimately falls between about 3.0 and 3.8, between about 3.1 and 3.8, between about 3.2 and 3.8, between about 3.3 and 3.7, between about 3.4 and 3.7, or between about 3.5 and 3.7. In some embodiments, the pH initially falls between about 3.0 and 3.8, between about 3.1 and 3.8, between about 3.2 and 3.8, between about 3.3 and 3.7, between about 3.4 and 3.7, or between about 3.5 and 3.7. In some embodiments, the initial pH is between about 3.1 and 3.8. In some embodiments, the initial pH is between about 3.3 and 3.8. In some embodiments, the initial pH is between about 3.5 and 3.7. In some embodiments, the initial pH is about 3.6.In some embodiments, the final pH (final pH) is between 5.1 and 8.5, between about 5.1 and 8.3, between about 5.1 and 8.1, between about 5.1 and 8.0, between about 5.1 and 7.7, between about 5.1 and 7.5, between about 5.1 and 7.3, between about 5.1 and 7.0, between about 5.3 and 8.5, between about 5.3 and 8.3, between about 5.3 and 8.1, etc. Between approximately 5.3 and 8.0, between approximately 5.3 and 7.7, between approximately 5.3 and 7.5, between approximately 5.3 and 7.3, between approximately 5.3 and 7.0, between approximately 5.5 and 8.5, between approximately 5.5 and 8.3, between approximately 5.5 and 8.1, between approximately 5.5 and 8.0, between approximately 5.5 and 7.7, between approximately 5.5 and 7.0, between approximately 6. Between 0 and 8.5, between approximately 6.0 and 8.3, between approximately 6.0 and 8.0, between approximately 6.0 and 7.7, between approximately 6.0 and 7.0, between approximately 6.5 and 8.5, between approximately 6.5 and 8.3, between approximately 6.5 and 8.0, between approximately 6.5 and 7.7, between approximately 6.5 and 7.0, between approximately 7.0 and 8.5, between approximately 7.0 and 8. The pH values ​​are between 3, between about 7.5 and 8.0, between about 7.7 and 8.0, between about 7.7 and 8.5, between about 7.7 and 8.3, between about 7.9 and 8.2, between about 7.0 and 8.0, between about 7.0 and 7.9, between about 7.0 and 7.5, between about 6.8 and 7.8, between about 6.8 and 7.6, or between about 6.8 and 7.4. In some embodiments, the final pH value is between about 5.3 and 8.5, between about 5.1 and 8.1, between about 5.5 and 8.0, or between about 7.5 and 8.0. In some embodiments, the final pH value is between about 5.5 and 8.0. In some embodiments, the final pH value is between about 7.0 and 8.0.

[0084] This disclosure provides a method for determining the accuracy of a pH meter and, if necessary, calibrating the pH meter when inactivating a virus in a sample. In some embodiments, the method includes providing a sample containing a protein of interest, such as a sample purified from cultured cells by column chromatography, and lowering the pH. An exemplary sample may have an initial pH of about 4.1 to 4.5 and a final pH of about 3.5 to 3.7, and, if necessary, about 3.6. The initial pH will depend on the protein of interest, the purification method used, and the composition of the sample after the protein purification step (e.g., dissolution buffer and the like). After lowering the pH and holding it for a period of time to inactivate the virus, the pH is then raised to a final alkaline pH of about 7.5 to 8.5, or about 7.5 to 8.0, or about 7.6. The final alkaline pH will depend on the protein of interest and the choice of buffer and the like, which will depend on the desired downstream application. The method described herein for determining the accuracy of a pH meter can be used to lower the sample pH, raise the sample pH, or both.

[0085] Therefore, this disclosure provides a method for inactivating viruses in a sample. In some embodiments, the sample contains the protein of interest. In some embodiments, the method includes providing a sample at an initial pH (pH initial) of 4.0 or greater, such as 4.1, 4.2, 4.3, 4.4, or 4.5. In some embodiments, the method includes measuring the initial pH before adding an acid titrant. In some embodiments, the method includes adding a first amount of acid titrant (titer n_acid) to the sample and measuring a first additional acid pH (pH n_acid), the amount of acid titrant added to the sample to reach pH n_acid, wherein pH n_acid differs from pH initial. The first amount of titrant is typically a conservative amount of titrant. For example, the first amount of titrant is a titration dose predicted based on a previous reference sample, sufficient to cause the sample pH change to not exceed half the way to reach the target or final pH, or not exceed two-thirds or three-quarters of the way to reach the target pH. Those skilled in this art will understand that the amount of acid titrant added each time can be adjusted depending on the sample, the initial pH of the sample, the final target pH, and the number of times acid titrant is added to the sample to change the sample pH. In some embodiments, the method includes applying a first model to determine a standardized titrant, i.e., a standardized titrant dosage corresponding to the initial pH and the pH after adding the first amount of acid titrant, wherein the first model links the standardized titrant to the sample pH. After adding the first amount of titrant, the first model can be used to determine a second amount of titrant required to achieve the pH between pH n_acid and the final acid pH (pH_acid_final, referred to herein as pH_n+1_acid). In some embodiments, the method includes adding a second amount of acid titrant (titer n+1_acid) to the sample and measuring a second additional acid pH (pH_n+1_acid), where titrant n+1_acid is the amount of acid titrant added to the sample to achieve pH_n+1_acid, wherein pH_n+1_acid is different from pH_n_acid. In some embodiments, the method includes measuring the sample pH after adding a second amount of titrant to determine the measured pH n+1 acid, and comparing the measured pH n+1 acid with a pH n+1 acid predicted by a first model. In some embodiments, the method includes correcting the pH meter when the difference between the measured pH n+1 acid and the pH n+1 from the first model indicates a calibration error in the pH meter used to measure the sample pH.In some embodiments, the difference between the pH n+1 acid and the first model is > 0.01 pH units, > 0.02 pH units, > 0.03 pH units, > 0.04 pH units, > 0.05 pH units, > 0.06 pH units, > 0.07 pH units, > 0.08 pH units, > 0.09 pH units, or > 0.10 pH units. In some embodiments, the difference between the measured pH n+1 acid and the pH n+1 from the first model is > 0.05 pH units. Depending on the circumstances, a certain amount of titrant may be added at least once, twice, three times, four times, five times, or more to confirm that the behavior of the sample corresponds to the model and that, if corrected, the pH meter now behaves as predicted by the first model. If the sample does not conform to the model after adding a second amount of titrant, or if pH meter calibration error is suspected, a skilled practitioner can reduce the amount of titrant added and increase the number of titrations to more accurately measure pH during the process and avoid exceeding the target pH.

[0086] In some embodiments, the method includes determining, based on a standardized titrant, pH, and a first model, the remaining amount of titrant to be added to the sample to achieve a final acid pH (pH acid - final) between 3.1 and 3.8. In some embodiments, the method includes adding the remaining amount of titrant to the sample to achieve pH acid - final. In some embodiments, the method includes maintaining the sample at pH final - acid for a period of time sufficient to inactivate the virus, such as the incubation time described above.

[0087] Therefore, this disclosure provides, for example, a method for raising the pH of a sample after inactivating a virus as described above. In some embodiments, the sample contains the protein of interest. In some embodiments, the method includes providing a sample at an initial pH (pH initial - base) of 3.1 to 3.8. For example, pH initial - base may be the same as pH acid - final when the virus is inactivated as described above. In some embodiments, the method includes measuring the initial pH before adding an alkaline titrant. In some embodiments, the method includes adding a first amount of alkaline titrant (titrant n - base) to the sample and measuring a first additional base pH value (pH n - base), wherein titrant n - base is the amount of titrant added to the sample to achieve pH n - base, and pH n - base is different from pH initial - base. The first amount of alkali titrant added to the sample is typically a conservative amount, i.e., a titration dose predicted based on a previously referenced sample, sufficient to cause the sample pH change to be no more than half, two-thirds, or three-quarters of the way to the target alkaline pH. Those skilled in the art will understand that the amount of alkali titrant added to the sample each time can be adjusted depending on the sample, the initial pH of the sample, the final target pH, and the number of times alkali titrant has been added to the sample to change the sample pH. In some embodiments, the method includes applying a second model to determine a standardized titrant, i.e., a standardized titration dose corresponding to the initial pH and the pH after the addition of the first amount of alkali titrant, wherein the second model links the standardized titrant to the sample pH. After the addition of the first amount of alkali titrant, the second model can be used to determine a second amount of alkali titrant required to achieve the pH between pH n_alkali and the final alkaline pH (pH final_alkali, referred to herein as pH n+1_alkali). In some embodiments, the method includes adding a second amount of alkali titrant (titrant n+1_alkali) to the sample and measuring a second additional alkali pH value (pH n+1_alkali), wherein titrant n+1_alkali is the amount of alkali titrant added to the sample to achieve pH n+1_alkali, and pH n+1_alkali is different from pH n_alkali. In some embodiments, the method includes measuring the sample pH after adding the second amount of titrant to determine the measured pH n+1_alkali, and comparing the measured pH n+1_alkali with the pH n+1_alkali predicted by a second model. In some embodiments, the method includes correcting the pH meter when the difference between the measured pH n+1_alkali and the pH n+1_alkali from the second model indicates a calibration error in the pH meter used to measure the sample pH.In some embodiments, the difference between the measured pH n+1 base and the pH n+1 base from the second model is > 0.01 pH unit, > 0.02 pH unit, > 0.03 pH unit, > 0.04 pH unit, > 0.05 pH unit, > 0.06 pH unit, > 0.07 pH unit, > 0.08 pH unit, > 0.09 pH unit, or > 0.10 pH unit. In some embodiments, the difference between the pH n+1 base and the second model is > 0.05 pH unit. In some embodiments, the method includes repeating the addition and measurement steps at least once, twice, three times, four times, five times, or more to confirm that the sample behavior corresponds to the second model. If the sample does not conform to the second model, or if pH meter calibration error is suspected, a person skilled in this technique can reduce the amount of titrant added and increase the number of titrant additions to more accurately measure pH during the process and avoid exceeding the target pH. In some embodiments, the method includes determining, based on standardized titrant, pH, and a model, the remaining amount of alkaline titrant to be added to the sample to change the sample pH to a final pH between 5.0 and 8.5 (pH final - alkaline). In some embodiments, the method includes adding the remaining amount of titrant to the sample to achieve pH final - alkaline.

[0088] In some embodiments, the method includes adding one or more conservative amounts of titrant, i.e., the amount of titrant intended to change the sample pH to no more than half of the target pH, measuring the pH, and applying a model to determine the remaining amount of titrant to be added to the sample to achieve the target or final pH. In some embodiments, the final amount of titrant to be added to the sample is determined by the following formula: (Equation 15). In this formula, the standardized titrant is always the total amount added to the sample to achieve the final pH after standardization; the initial standardized titrant is the amount added to the sample to achieve the initial pH (this value can be 0 before standardization); and the standardized titrant n is the amount of titrant added to the sample to achieve an intermediate pH n using this model, where pH n falls between the initial and final pH values. Those familiar with this technique will understand that when multiple intermediate amounts of titrant are added to the sample and the corresponding pH values ​​are measured, the remaining amount of titrant to be added to the sample to achieve the final pH will be recalculated according to the formula described above.

[0089] In some embodiments, the method includes adding a first amount of titrant (titer n) to a sample and measuring at least a first additional pH value (pH n), where titrant n is the amount of titrant added to the sample to achieve pH n, wherein pH n is different from an initial pH (pH initial); applying a model to determine an initial amount of standardized titrant (titer initial) and standardized titrant n, wherein the model relates the standardized titrant added to the sample to the sample pH; and determining another additional amount (titer n+1) of titrant to be added to the sample to achieve a target pH (pH n+1), where pH n+1 is the pH achieved by adding the other additional amount of titrant (titer n+1) to the sample. In some embodiments, the method includes applying a model and calculating an additional amount (titer n+2) of titrant to be added to the sample to achieve a second target pH (pH n+2). In some embodiments, the method further includes adding an additional amount of titrant (titer n+2) to the sample, thereby changing the sample pH to the second target pH (pH n+2). In some embodiments, the method further includes applying a model and calculating an additional amount (titer n+3) of titrant added to the sample to achieve a third target pH (pH n+3). In some embodiments, the method includes adding titrant n+3, thereby changing the sample pH to pH n+3. In some embodiments, the method further includes applying a model and calculating an additional amount (titer n+4) of titrant added to the sample to achieve a fourth target pH (pH n+4). In some embodiments, adding titrant n+1, titrant n+2, titrant n+3, or titrant n+4 produces a target pH within 0.05 to 0.10 pH units of the final target pH (pH final). In some embodiments, an additional amount of titrant determined by applying a model is added to the sample to achieve the final target pH. For example, pH n+2 is within 0.05 to 0.10 pH units of the final target pH, which is achieved by adding titrant n+3, wherein the amount of titrant added for titrant n+3 is determined by applying a model. As another example, pH n+3, within 0.05 to 0.10 pH units of the final target pH, is achieved by adding titrant n+4, wherein the amount of titrant added for titrant n+4 is determined by applying a model. Those skilled in this art will understand that, depending on the desired degree of pH change and the properties of the sample and titrant, more or less titrant addition than described above can be used to achieve the final target pH. In some embodiments, the final pH is achieved by adding 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 titrant additions, wherein the pH is measured after each addition, and a model is applied to determine the amount of titrant to be added to achieve the additional target pH and, where appropriate, the final pH required.In some embodiments, the method includes comparing the target pH (e.g., pH n+1, pH n+2, pH n+3, pH n+4, etc.) of any of the addition steps described herein with the target pH predicted by the model for that corresponding step.

[0090] When performing the pH alteration methods described herein, a pH probe inserted into a subsample removed from the sample can be used to measure the pH value of the sample. The subsample can be removed from the sample via a slipstream, for example, by connecting a reaction vessel containing the sample to a flow channel in which the pH probe is inserted. In some embodiments, the slipstream is continuous. In some embodiments, the slipstream is discontinuous or intermittent. In some embodiments, the sample pH is not measured using a pH probe directly inserted into the sample. [, Model , ] [, , ]

[0091] This disclosure provides models for the methods of this disclosure and methods for generating such models.

[0092] In some embodiments, the generative model includes dimensionless transformation, such as dimensionless transformation of titrant values ​​from a reference titration curve. Dimensionless transformation is the process of removing physical dimensions from or completely removing from the equations involving physical quantities by replacing appropriate variables. For example, the volume of titrant added to the sample can be determined by pump rotations per kilogram or the number of milliliters of titrant added per kilogram of total sample, and these dimensions can be removed by dimensionless transformation techniques. Dimensionless transformation simplifies and parameterizes issues involving units of measurement. In some cases, scaling can be interchanged with dimensionless transformation when dimensionless transformation is used to convert multiple datasets to a common scale.

[0093] In some embodiments, the generative model includes regression analysis. Regression analysis is a set of statistical procedures used to estimate the relationship between a dependent variable (often called the 'response' variable) and a more independent variable (in this case, pH and the standardized titrant). A common form of regression analysis is linear regression, in which those skilled in the technique find the line that best fits the data according to specific mathematical criteria. For example, the ordinary least squares method calculates a unique line that minimizes the sum of squared differences between the true data and this line.

[0094] In some embodiments, the fitted model includes linear regression. Linear regression is a linear method used to model the relationship between a scalar response variable and one or more explanatory variables. The case of one explanatory variable is called simple linear regression. In linear regression, the relationship is modeled using linear prediction functions whose unknown model parameters are estimated from the data. Such models are called linear models.

[0095] Linear regression is the first type of regression analysis, which has been rigorously studied and widely used in practical applications. This is because models that are linearly dependent on their unknown parameters are easier to fit than models that are nonlinearly related to their parameters, and because the statistical properties of the obtained estimates are easier to determine.

[0096] In some embodiments, regression analysis includes multinomial regression. Multinomial regression is a form of regression analysis in which the relationship between independent variables (e.g., a standardized titrant) and dependent variables (e.g., pH) is modeled as an nth-degree polynomial. Multinomial regression fits a nonlinear relationship between the values ​​of the independent variables and the corresponding conditional means of the dependent variables. Although multinomial regression fits a nonlinear model to the data, as a statistical estimation problem, it is linear, meaning that the regression function is linear with respect to the unknown parameters estimated from the data. For this reason, multinomial regression is considered a type of multiple linear regression.

[0097] The least squares method can be used to fit a polynomial regression model. Under the conditions of the Gauss-Markov theorem, the least squares method minimizes the variance of the unbiased estimates of the coefficients.

[0098] In some embodiments, the fitting model includes curve fitting. Curve fitting is the process of constructing a curve or mathematical function that best fits a set of data points. Curve fitting may involve interpolation, which requires an accurate fit to the data, or smoothing, in which a "smooth" function is constructed that roughly fits the data. Curves may be extrapolated, that is, extended beyond the range of the observed data, but extrapolated curves have a certain degree of uncertainty.

[0099] The fitted model can be performed using any suitable program known in this technique, such as Microsoft Excel, MATLAB, or R.

[0100] In some embodiments, one or more reference samples are used to generate one or more titration profiles to determine the model. The reference sample may be consistent with the sample, such as a subsample of a larger sample that has undergone a consistent pH process. Alternatively, the reference sample may be similar to the sample. Examples of such reference samples include a previously purified batch of protein consistent with the protein of interest, purified using similar or identical methods, and subjected to substantially the same pH process. Alternatively, the reference protein may be a protein similar to but not identical to the protein of interest, such as two antibodies or two Fc receptor fusion proteins, provided that the two proteins behave similarly when subjected to similar pH change protocols. Due to the dimensionless and modeling methods described herein, the initial and final pH values ​​of all reference samples and samples need not be exactly the same. For example, the initial and / or final pH values ​​of one or more reference samples and samples may differ by approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 pH units. Alternatively, one or more reference samples and the initial and / or final pH values ​​of the samples can be consistent.

[0101] Therefore, this disclosure provides one or more reference samples for generating the model used herein. This disclosure provides titration curves generated by varying the pH of the reference sample and relating the pH of the reference sample to the amount of titrant added to the reference sample. Methods for generating and plotting titration curves are generally known to those skilled in the art.

[0102] In some embodiments, the method includes: (i) generating at least one reference titration curve from at least one reference sample, relating the amount of titrant added to the reference sample to the pH of the reference sample; (ii) normalizing the at least one reference titration curve; and (iii) generating a model to fit the at least one reference titration curve. In some embodiments, generating at least one reference titration curve includes measuring the initial pH of the reference sample (pH initial_reference). A titrant sufficient to change the pH of the reference sample is then added to the reference sample (titer n_reference), and an additional reference pH value is measured after the addition of this titrant (pH n_reference). These steps may be repeated until a final pH is reached, and a graph of the amount of titrant versus the pH of the reference sample is plotted using any suitable procedure known in this art. When generating the reference titration curve, any suitable method for adding the titrant may be used. The titrant may be added in a discrete step, for example by adding a discrete amount of titrant, stirring for a certain period of time to mix it into the reference sample (e.g., until the pH of the reference sample stabilizes), and performing pH measurements. Alternatively, titrant can be added continuously, and pH can be measured continuously. When generating a reference sample or a titration profile of the sample, any suitable method for measuring pH can be used. For example, the pH of the reference sample can be measured by inserting a pH probe directly into the reference sample, or by inserting a pH probe into a slipstream of continuous or discrete samples drawn from the reference sample.

[0103] In some embodiments, the amount of titrant added to the reference sample is standardized by the following formula: (Equation 17) In this formula, titrant 1_reference is the amount of titrant added to the reference sample to achieve a first pH 1_reference, and titrant 2_reference is the amount of titrant added to the reference sample to achieve a pH 2_reference.

[0104] In some embodiments, such as when a single reference sample and its corresponding titration curve are used to generate a model, pH 1_reference may be the same as the initial pH of the reference sample, and pH 2_reference may be the same as the final pH of the reference sample.

[0105] In an alternative embodiment, a plurality of reference samples and corresponding titration curves are used to generate the model. When the plurality of titration curves do not have the same initial and / or final pH values, pH 1_reference is not the same as some or all of the initial pH values ​​of the reference samples, and pH 2_reference is not the same as some or all of the final pH values ​​of the reference samples. Each reference titration curve includes an initial pH_reference and a final pH_reference, and pH 1_reference is the initial pH_reference from one of the plurality of reference titration curves, pH 2_reference is the final pH_reference from one of the plurality of reference titration curves, and pH 1_reference and pH 2_reference are selected to cover the largest difference in values, while still covering the pH values ​​covered by all the curves in the plurality of reference titration curves. Therefore, pH 1_reference and pH 2 are as far apart as possible, but offset from the initial pH and final pH to a certain extent. For example, when the reference titration curve includes an increase in pH, pH 1_reference can be the pH initial_reference of the reference titration curve with the highest initial pH, and pH 2_reference can be the pH final_reference of the reference titration curve with the lowest final pH. Those skilled in this technique will understand that the opposite relationship holds when the reference titration curve includes a decrease in pH.

[0106] In some embodiments, the initial pH of the sample (pH initial) and pH 1_ reference are approximately the same.

[0107] For example, pH values ​​that are within approximately 0.05 units of each other can be considered approximately the same. Alternatively, pH values ​​that are within 10%, 5%, or 3% of each other can be considered approximately the same.

[0108] In some embodiments, the initial pH of the sample (pH initial) is not the same as the pH 1 reference, that is, the difference between the initial pH and the pH 1 reference is about 0.05 to 1.5, about 0.05 to 1, about 0.1 to 1, about 0.1 to 0.5, or about 0.1 to 0.3 pH units. In some embodiments, the difference between the initial pH and the pH 1 reference is about 0.1 to 0.5 pH units.

[0109] In some embodiments, the final pH of the sample (pH final) and pH 2_ reference are approximately the same.

[0110] In some embodiments, the final pH value is not the same as the pH 2 reference, i.e., the difference between the final pH value and the pH 2 reference is about 0.5 to 1.5, which is about 0.05 to 1, about 0.1 to 1, about 0.1 to 0.5, or about 0.1 to 0.3 pH units. In some embodiments, the difference between the final pH value and the pH 2 reference is about 0.5 to 1.5 pH units. In some embodiments, the difference between the final pH value and the pH 2 reference is about 0.5 to 1.0 pH units. In some embodiments, the difference between the final pH value and the pH 2 reference is about 0.1 to 0.5 pH units.

[0111] In some embodiments, pH initial, pH initial_reference, and pH 1_reference are the same, and pH final, pH final_reference, and pH 2_reference are the same.

[0112] Those familiar with this technique should understand that the selection of pH 1 reference and pH 2 reference depends on the specific reference sample, the corresponding reference titration curve, and the amount of change in pH relative to the initial and final values ​​contained therein.

[0113] In some embodiments, the final pH of the sample (pH final) is less than the initial pH of the sample (pH initial), and the titrant is acid. In some embodiments, the sample and a plurality of reference samples contain the protein of interest. In some embodiments, pH 1 reference is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, or 4.9, and pH 2 reference is about 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.8, or 3.9. In some embodiments, pH 1 reference is about 4.1, and pH 2 reference is about 3.6. In some embodiments, the initial pH of the sample and a plurality of reference samples is between about 4.1 and 4.5. In some embodiments, the final pH of the sample and a plurality of reference samples is between about 3.5 and 3.7, and, where appropriate, about 3.6. In some embodiments, the amount of titrant added to the plurality of reference samples is normalized to a scale of about -0.76 to about 1.49. In some embodiments, the model generated from multiple reference samples includes a fitting polynomial. In some embodiments, the polynomial includes a fourth-order polynomial: (Equation 18) In some embodiments, the polynomial includes: (Equation 19) The polynomials generated by the model can be used to calculate the standardized titrant to be added to the sample based on the measured pH value.

[0114] In some embodiments, the final pH of the sample (final pH) is greater than the initial pH (initial pH), and the titrant is base. In some embodiments, the sample and a plurality of reference samples contain the protein of interest. In some embodiments, the pH 1 reference is between about 3.1 and 3.8. In some embodiments, the pH 1 reference is between about 3.4 and 4.1. In some embodiments, the pH 1 reference is about 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.8, or 3.9. In some embodiments, the pH 1 reference is about 3.6. In some embodiments, the pH 1 reference is about 3.7. In some embodiments, the pH 2 reference is between about 7.5 and 8.5. In some embodiments, the pH 2 reference is about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, or 8.4. In some embodiments, the pH 2 reference is about 7.6. In some embodiments, the initial pH is between about 3.5 and 3.7. In some embodiments, the final pH is between about 5.1 and 8.5. In some embodiments, the final pH is between about 7.5 and 8.0. In some embodiments, the final pH is between about 7.5 and 8.0. In some embodiments, the final pH is between about 7.0 and 8.0, between about 7.1 and 7.9, between about 7.2 and 7.8, between about 7.3 and 7.7, or between about 7.4 and 7.6. In some embodiments, the amount of titrant added to the reference sample is normalized to a scale of approximately -0.06 to approximately 1.53. In some embodiments, the model generated from multiple reference samples includes a fitting polynomial. In some embodiments, the polynomial includes a fifth-order polynomial of the following: (Equation 20) In some embodiments, the polynomial includes: Standardized titrant n = 12.256725 - 10.723277 * pH n + 3.3662386 * pH n 2 - 0.4588175 * pH n 3 + 0.0255417 * pH n 4 - 0.0003153 * pH n 5. (Equation 21) The polynomials generated by the model can be used to calculate the standardized titrant to be added to the sample based on the measured pH value.

[0115] The models described above are intended to be exemplary and not limiting. Those skilled in the art will understand that, depending on one or more reference samples or the initial and final pH values ​​of the samples, other models (including other polynomials) generated from reference samples by the methods described herein will be suitable for use in the methods described herein. The aforementioned model links a standardized titrant to a pH value. This disclosure provides a method comprising comparing one of a sample or a measured pH value to a model. In some embodiments, the method comprises measuring an initial pH, adding a first conservative amount of titrant, measuring a first additional pH value, and then applying the model to determine a first standardized amount of titrant, for example, a standardized amount of titrant corresponding to the initial pH and / or the pH after adding the first amount of titrant. In some embodiments, the method comprises determining a second conservative amount of titrant, the second conservative amount corresponding to the pH between the pH after adding the first amount of titrant and a final target pH, adding the second conservative amount of titrant, measuring a second additional pH value, and comparing the measured second pH value to a second pH value predicted by the model. If, after fitting the data on the initial pH, the first additional pH, and the standardized titrant to the model, the difference between the second measured pH value and the pH value predicted by the model is, for example, > 0.01 pH units, > 0.02 pH units, > 0.03 pH units, > 0.04 pH units, > 0.05 pH units, > 0.06 pH units, > 0.07 pH units, > 0.08 pH units, > 0.09 pH units, or > 0.10 pH units, then the model pH does not correspond to the sample pH, indicating an error in the pH meter. Conversely, if the difference between the predicted pH after fitting the sample data and the sample pH is, for example, ≤ 0.01 pH units, ≤ 0.02 pH units, ≤ 0.03 pH units, ≤ 0.04 pH units, ≤ 0.05 pH units, ≤ 0.06 pH units, ≤ 0.07 pH units, ≤ 0.08 pH units, ≤ 0.09 pH units, or ≤ 0.10 pH units, then the model pH corresponds to the sample pH, indicating that the pH meter is functioning correctly. In some embodiments, the method includes determining a third conservative amount of titrant, which corresponds to the pH between the pH after adding a second amount of titrant and the final target pH, adding a third amount of titrant, measuring a third pH value, and comparing the measured third pH value with the third pH value predicted by the model. In some embodiments, the method includes adding 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amounts of titrant, measuring the pH value after each addition of titrant, comparing the measured pH value with the pH value predicted by the model, and determining whether the measured pH value corresponds to the pH value predicted by the model.If the difference between the pH values ​​from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional pH measurements and the pH predicted by the model for the addition of the additional titrant in these 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional measurements is > 0.01 pH units, > 0.02 pH units, > 0.03 pH units, > 0.04 pH units, > 0.05 pH units, > 0.06 pH units, > 0.07 pH units, > 0.08 pH units, > 0.09 pH units, or > 0.10 pH units, then the model pH does not correspond to the sample pH, indicating an error in the pH meter. [, Proteins of interest , ] [, , ]

[0116] This disclosure provides samples containing the protein of interest for use in the methods described herein. The protein of interest may be a therapeutic protein, i.e., a protein administered to an individual for the treatment of a disease or condition. Exemplary proteins of interest include (but are not limited to) antibodies, receptor Fc fusion proteins (such as trap proteins), cytokines, chemokines, growth factors, and analogues thereof.

[0117] In some embodiments, the protein of interest is an antigen-binding protein, such as an antibody.

[0118] The term "antigen-binding protein" includes proteins that have at least one complementarity-determining region (CDR) and are capable of selectively recognizing antigens, i.e., binding antigens at a KD of at least in the micromolar range. Therapeutic antigen-binding proteins (e.g., therapeutic antibodies) often require a KD in the nanomolar or picomolar range. Typically, antigen-binding proteins include two or more CDRs, such as 2, 3, 4, 5, or 6 CDRs. Examples of antigen-binding proteins include antibodies, antigen-binding fragments of antibodies, such as polypeptides containing variable regions of the heavy and light chains of antibodies (e.g., Fab fragments, F(ab')2 fragments), and proteins containing variable regions of the heavy and light chains of antibodies and additional amino acids (such as one or more constant domains, i.e., one or more of the CL, CH1, hinge, CH2, and CH3 domains) from constant regions of the heavy and / or light chains.

[0119] "Antibody" refers to an immunoglobulin molecule composed of four polypeptide chains, two heavy (H) chains linked by disulfide bonds, and two light (L) chains. Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain has a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The term "antibody" includes any isotype or subtype of glycosylated and non-glycosylated immunoglobulin. The term "antibody" includes antibody molecules prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from host cells transfected with nucleotide sequences to express antibodies. The term "antibody" also includes bispecific antibodies, which include heterotetrameric immunoglobulins capable of binding to more than one antigenic determinant. As used herein, the term "antibody" also includes the antigen-binding fragment of a complete antibody molecule and fusion proteins containing either an antibody or an antigen-binding fragment.

[0120] The term "antigen-binding portion" (or "antibody fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Non-limiting examples of protein-binding fragments covered under the term "antigen-binding portion" of antibody include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments comprising two Fab fragments linked by disulfide bridging in the hinge region; (iii) Fd fragments, which consist of VH and CH1 domains; (iv) Fv fragments, which consist of the VL and VH domains of an antibody arm; (v) dAb fragments (Ward et al., Nature (1989) 241:544-546), which consist of the VH domain; (vi) isolated CDRs; and (vii) scFv, which consists of the two domains VL and VH of an Fv fragment, which are joined by a synthetic linker to form a single protein chain, wherein the VL and VH regions pair to form a monovalent molecule. Other forms of single-chain antibodies, such as bifunctional antibodies, are also covered under the term "antibody". See, for example, Holliger et al., PNAS USA (1993) 90:6444-6448; Poljak et al., Structure (1994) 2:1121-1123.

[0121] Furthermore, antibodies or their antigen-binding portions can be part of larger immunoadhesion molecules formed by covalent or non-covalent association of antibodies or antibody portions with one or more other proteins or peptides. Non-limiting examples of such immunoadhesion molecules include tetrameric scFv molecules prepared using the streptavidin core region (Kipriyanov et al., Human Antibodies and Hybridomas (1995) 6:93-101) and divalent, biotin-labeled scFv molecules prepared using cysteine ​​residues, labeled peptides, and C-terminal polyhistamine tags (Kipriyanov et al., Mol. Immunol. (1994) 31:1047-1058). Antibody portions such as Fab and F(ab')2 fragments can be prepared from whole antibodies using known techniques, such as papain or pepsin digestion of the whole antibody. In addition, antibodies, antibody fractions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques commonly known in this field (see Sambrook et al., 1989).

[0122] The term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of this disclosure may include, for example, amino acid residues in CDR, and especially CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations induced by random or site-specific mutations in vitro or introduced by somatic mutations in vivo).

[0123] As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into host cells; antibodies isolated from a library of recombinant human antibodies; antibodies isolated from animals transfected with human immunoglobulin genes (e.g., mice) (see, for example, Taylor et al., Nucl. Acids Res. (1992) 20:6287-6295); or antibodies prepared, expressed, produced, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies possess variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies can be induced by in vitro mutations (or by in vivo somatic mutations when using animals transgenic human Ig sequences), and therefore, although the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and associated with human germline VH and VL sequences, these amino acid sequences are sequences that are not naturally present in the human antibody germline library in vivo.

[0124] Consideration is given to additional therapeutic proteins within the scope of the cell culture methods and therapeutic protein production methods disclosed in this invention. In some embodiments, the therapeutic protein is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen-binding antibody fragment, a single-chain antibody, a bifunctional antibody, a trifunctional or tetrafunctional antibody, a Fab fragment or an F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody, an IgG2 antibody, an IgG4 antibody, a chimeric IgG2 / IgG4 antibody, a chimeric IgG2 / IgG1 antibody, or a chimeric IgG2 / IgG1 / IgG4 antibody.

[0125] In some embodiments, the antibody system is selected from the group consisting of: anti-planned cell death 1 antibody (e.g., an anti-PD1 antibody as described in U.S. Patent Application Publication No. US2015 / 0203579A1), anti-planned cell death ligand-1 (e.g., an anti-PD-L1 antibody as described in U.S. Patent Application Publication No. US2015 / 0203580A1), anti-Dll4 antibody, anti-angiogenic-2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Patent No. 9,402,898), and anti-angiogenic-3 antibody (e.g., an anti-AngPtl antibody as described in U.S. Patent No. 9,018,356). 3) Antibodies, antiplatelet-derived growth factor receptor antibodies (e.g., antiPDGFR antibodies as described in U.S. Patent No. 9,265,827), antiErb3 antibodies, antiprolactin receptor antibodies (e.g., antiPRLR antibodies as described in U.S. Patent No. 9,302,015), anticomplement 5 antibodies (e.g., antiC5 antibodies as described in U.S. Patent Application Publication No. US2015 / 0313194A1), antiTNF antibodies, antiepidermal growth factor receptor antibodies (e.g., antiEGFR antibodies as described in U.S. Patent No. 9,132,192 or as described in U.S. Patent Application Publication No. US2015 / 0259423A1). The described antibodies include anti-EGFRvIII antibodies, anti-proprotein convertase subtilisin Kexin-9 antibodies (e.g., anti-PCSK9 antibodies as described in U.S. Patent No. 8,062,640 or U.S. Patent Application Publication No. US2014 / 0044730A1), anti-glucagon-8 antibodies (e.g., anti-GDF8 antibodies, also known as anti-myosin antibodies, as described in U.S. Patent Nos. 8,871,209 or 9,260,515), and anti-glucagon receptors (e.g., as described in U.S. Patent Application Publication No. US2015 / 0337045A1 or US2016 / 0075778A1). Anti-GCGR antibody), anti-VEGF antibody, anti-IL1R antibody, interleukin 4 receptor antibody (e.g., the anti-IL4R antibody described in U.S. Patent Application Publication No. US2014 / 0271681A1 or U.S. Patent No. 8,735,095 or 8,945,559), anti-interleukin 6 receptor antibody (e.g., the anti-IL6R antibody described in U.S. Patent Nos. 7,582,298, 8,043,617 or 9,173,880), anti-IL1 antibody, anti-IL2 antibody, anti-IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-interleukin 33 (e.g., anti-GCGR antibody), anti-VEGF antibody, anti-IL1R antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-interleukin 33 (e.g., anti-IL1R antibody), anti-IL2 antibody, anti-IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-interleukin 33 (e.g., anti-IL1R antibody), anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-interleukin 33 (e.g., anti-IL1R antibody), anti-IL2 antibody, anti-IL3 antibody, anti-IL4 ...Anti-IL33 antibodies, such as those described in U.S. Patent Application Publication No. US2014 / 0271658A1 or US2014 / 0271642A1, anti-respiratory fusion virus antibodies (e.g., anti-RSV antibodies described in U.S. Patent Application Publication No. US2014 / 0271653A1), and anti-differentiation cluster 3 antibodies (e.g., anti-CD3 antibodies, such as those described in U.S. Patent Application Publication No. US2014 / 0088295A1 and US20150266966). Anti-differentiation cluster 20 (e.g., anti-CD20 antibodies as described in U.S. Patent Application Publication No. 62 / 222,605), anti-CD19 antibodies, anti-CD28 antibodies, anti-differentiation cluster-48 (e.g., anti-CD48 antibodies as described in U.S. Patent Application Publication No. 9,228,014), anti-Fel d1 antibody (e.g., as described in U.S. Patent No. 9,079,948), anti-Middle East Respiratory Syndrome Virus (e.g., anti-MERS antibody as described in U.S. Patent Application Publication No. US2015 / 0337029A1), anti-Ebola virus antibody (e.g., as described in U.S. Patent Application Publication No. US2016 / 0215040), anti-Zika virus antibody, anti-lymphocyte activation gene 3 antibody (e.g., anti-LAG3 antibody or anti-CD223 antibody), anti-nerve growth factor antibody (e.g., anti-NGF antibody as described in U.S. Patent Application Publication No. US2016 / 0017029 and U.S. Patent Nos. 8,309,088 and 9,353,176), and anti-activator A antibody. In some embodiments, the bispecific antibody system is selected from the group consisting of: anti-CD3×anti-CD20 bispecific antibodies (as described in U.S. Patent Application Publication Nos. US2014 / 0088295A1 and US20150266966A1), anti-CD3×antimucin 16 bispecific antibodies (e.g., anti-CD3×anti-Muc16 bispecific antibodies), and anti-CD3×antiprostate-specific membrane antigen bispecific antibodies (e.g., anti-CD3×anti-PSMA bispecific antibodies). In some embodiments,The proteins of interest are selected from the following group: alirocumab, sarilumab, fasinumab, nesvacumab, dupilumab, trevogrumab, evinacumab, and rinucumab. All disclosures mentioned in this disclosure are incorporated herein by reference in their entirety.

[0126] In other embodiments, the therapeutic protein is a recombinant protein containing an Fc moiety and another domain (e.g., an Fc fusion protein). In some embodiments, the Fc fusion protein is a receptor Fc fusion protein containing one or more extracellular domains of a receptor coupled to the Fc moiety. In some embodiments, the Fc moiety includes a hinge region followed by the CH2 and CH3 domains of IgG. In some embodiments, the receptor Fc fusion protein contains two or more unique receptor chains that bind to a single ligand or multiple ligands. For example, Fc fusion proteins are trap proteins, such as IL-1 traps (e.g., rilonacept, which contains an IL-1RAcP ligand-binding domain fused to the extracellular domain of Il-1R1, which is fused to the Fc of hIgG1; see U.S. Patent No. 6,927,004, which is incorporated herein by reference in its entirety), VEGF traps (e.g., aflibercept or ziv-aflibercept, which contains an Ig domain 2 of VEGF receptor Flt1 fused to the Ig domain 3 of VEGF receptor Flk1, which is fused to the Fc of hIgG1). Fusion; see U.S. Patent Nos. 7,087,411 and 7,279,159; or conbercept, which contains an Ig domain 2 of VEGF receptor Flk1 fused to Ig domain 3 of VEGF receptor Flk1, the Ig domain 3 of VEGF receptor Flk1 fused to Ig domain 4 of VEGF receptor Flk1, the Ig domain 4 of VEGF receptor Flk1 fused to the Fc of hIgG1; see U.S. Patent No. 8,216,575) or TNF trap (e.g., etanercept, which contains a TNF receptor fused to the Fc of hIgG1; see U.S. Patent No. 5,610,279). In other embodiments, the Fc fusion protein is a ScFv-Fc fusion protein containing one or more of one or more antigen-binding domains, such as variable heavy chain fragments and variable light chain fragments of antibodies partially coupled to the Fc.

[0127] In some embodiments, the protein of interest is a glycoprotein. Glycoproteins with aspartic acid-linked (N-linked) glycans are common in eukaryotic cells. The biosynthesis of these glycans and their transfer to polypeptides occur in the endoplasmic reticulum (ER). The N-glycan structure is further modified by various glycosidases and glycosyltransferases in the ER, as well as the Golgi complex. Glycosylation of therapeutic proteins can be critical to the quality and efficacy of therapeutic proteins. For example, antibody glycosylation is a common post-translational modification and plays a role in antibody effector function and antibody stability. Those skilled in this art will know methods for analyzing glycosylation patterns and the percentage of glycosylated proteins in protein samples. [, , ] [, Protein purification , ]

[0128] Those skilled in this art will know methods for purifying the protein of interest produced by the cell and cell culture methods described herein. Methods for purifying the protein of interest from cell culture media or from cells include chromatographic and non-chromatographic methods. Chromatographic methods involve passing a solution containing an antibody through a solid phase (e.g., silica resin or beads, monolithic column, or cellulose membrane) and, depending on whether "binding and dissociation" or "flow-through" chromatography is used, allowing the protein of interest to bind or pass through. Chromatographic methods include (but are not limited to) affinity-tag binding, protein A binding, ion exchange chromatography (such as anion exchange chromatography), size exclusion chromatography, or immunoaffinity chromatography. Purification can also be achieved using purification tags based on gene fusion (such as polyhistamine tags or FLAG tags).

[0129] Exemplary protein purification protocols include obtaining a clear solution containing the protein of interest and performing a combination of different purification techniques, including ion exchange separation steps and hydrophobic interaction separation steps. Separation steps separate protein mixtures based on the protein's charge, degree of hydrophobicity, or size. In one embodiment of the invention, separation is performed using chromatography involving cations, anions, and hydrophobic interactions. Different chromatographic resins can be used in each of these steps, allowing the purification protocol to be precisely modified for the specific protein in question. The essence of each separation method lies in causing proteins to migrate down the column at different rates, achieving physical separation that increases as the protein is further delivered down the column, or selectively adhering to the separation medium and subsequently dissolving differentially with different solvents. In some cases, when impurities specifically adhere to the column and proteins do not, i.e., the protein of interest is present in the flow-through, the protein of interest is separated from the impurities.

[0130] In some embodiments, the purification of the protein of interest involves a primary recovery step. In some embodiments, the primary recovery step involves a chromatography column, such as an affinity column. The primary recovery step may also be followed by a time when the virus is inactivated using methods described herein, such as by subjecting the pool of proteins of interest in the elution solution to the pH changes described herein.

[0131] In some embodiments, the protein sample recovered from the main recovery step undergoes an additional purification step to further purify the protein of interest. For example, affinity chromatography may be used. Non-limiting examples of chromatographic materials that may be used include: protein A, protein G, chromatographic materials containing an antigen bound by an antibody of interest, or chromatographic materials containing an antibody bound to the protein of interest and an Fc-binding protein. As another example, hydrophobic interaction columns may be used to remove impurities such as aggregates.

[0132] Any purification step involving changes to sample pH can be subjected to the pH meter calibration and error correction methods described herein. Potential viruses can be inactivated using pH control procedures including the methods described herein after any suitable purification step. Additionally, the pH of solutions containing proteins of interest can be changed using pH control procedures including the methods described herein, for example, by changing the pH to the pH required for the next purification step or other downstream applications using the methods described herein. [, Cells and cell culture , ]

[0133] This disclosure provides a population of cells for producing the proteins of interest described herein. Suitable cells include bacterial cells, yeast cells, and mammalian cells.

[0134] In some embodiments, the cell population is isolated from or obtained from a cell line capable of producing the protein of interest. Non-limiting examples of cell lines used to produce therapeutic proteins include, in particular, primary cells, BSC cells, HeLa cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, young hamster kidney (BHK) cells, BHK-21 cells, CHO cells, CHO-K1 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, and chicken embryonic cells. In some embodiments, the cell population comprises CHO cells. In some embodiments, CHO cells comprise CHO cells derived from one or more of several specific CHO cell variants optimized for large-scale protein production, such as CHO-K1, CHO-K1-derived EESYR® (enhanced performance and stability region) cells (U.S. Patent No. 7,771,997), or the FASTR technology described in U.S. Patent No. 6,919,183, which provides isolation of cells that produce secretory proteins.

[0135] In some embodiments, the cell population cultured and expressing the protein of interest is a cell population obtained by pure-line amplification of cells (i.e., pioneer cells) that have and express polynucleotides encoding the therapeutic protein of interest. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or about 100% of the constituent cells of the cell population obtained by pure-line amplification from pioneer cells or derived from pure-line amplification contain polynucleotides encoding the protein and express the protein of interest.

[0136] In some embodiments, a population of cells cultured and exhibiting therapeutic proteins is generated by culturing frozen and stored cells. Mammalian cells can be frozen and cryopreserved in a cryopreservation medium, for example, containing dimethyl sulfoxide (DMSO) and cell culture medium. In an exemplary cryopreservation protocol, mammalian cells are transferred to a cryopreservation medium and slowly frozen, subsequently stored in liquid nitrogen. For example, cells can be expanded and cryopreserved to create a cell bank, which is a group of cells generated from a single cell pool having the desired characteristics. [, , ]

[0137] This disclosure provides a method for culturing cells that express a protein of interest prior to purification.

[0138] "Cell culture" or "culture" means the growth and proliferation of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in this art. See, for example, *Animal cell culture: A Practical Approach*, edited by D. Rickwood, Oxford University Press, New York (1992). Mammalian cells can be cultured in suspension or simultaneously attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, roller flasks, shake flasks, or stirred tank bioreactors, with or without microcarriers and operating in batch, batch-feed, continuous, semi-continuous, or perfusion modes, can be used for mammalian cell culture.

[0139] In some embodiments, culturing a cell population that expresses the protein of interest includes an expansion or growth phase, wherein the cell population expands to a size sufficient to produce the required quantity of the protein of interest in a production phase.

[0140] In some embodiments, the cultivation of a cell population exhibiting the protein of interest includes a production phase, wherein the cell population is cultured in a production cell culture medium under conditions sufficient to produce the protein of interest. The production phase can be carried out at any culture scale, from individual flasks and shake flasks or wave bags to one-liter bioreactors and large-scale industrial bioreactors. Large-scale processes can be carried out in volumes of about 100 liters to 20,000 liters or greater. One or more of several means can be used to control protein production, such as temperature variations or chemical induction. The growth phase can be carried out at higher temperatures than the production phase. For example, the growth phase can be carried out at a first temperature of about 35°C to 38°C, and the production phase can be carried out at a second temperature of about 29°C to 37°C, and, where applicable, about 30°C to 36°C or about 30°C to 34°C. In addition, chemical inducers of protein production, such as caffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline, and hexamethylene diacetamide (HMBA), can be added at the same time as, before, or after the temperature change. If the inducer is added after the temperature change, it can be added one hour to five days after the temperature change, such as one to two days after the temperature change. Cell cultures can be produced as a continuous feed culture system, such as in a chemostat (see C. Altamirano et al., Biotechnol Prog. 2001 Nov-December; 17(6):1032-41), or according to a batch feed process (Huang, 2010).

[0141] As used herein, the terms "cell culture media," "media," "cell media," "cell culture medium," or "culture medium" refer to any nutrient solution used for the growth of cells, such as animal or mammalian cells, and generally provide at least one or more of the following components: an energy source (usually in the form of carbohydrates, such as glucose); one or more of all essential amino acids, and generally twenty basic amino acids; vitamins and / or other organic compounds, usually required in low concentrations; lipids or free fatty acids; and trace elements, such as inorganic compounds or naturally occurring elements, usually required in very low concentrations, typically in the micromolar range. In some embodiments, cell culture media are formed by combining hydrolysates of soybean or other plant proteins with one or more additional ingredients.

[0142] As used herein, "additional components" include any or more of the components of a cell culture medium, including but not limited to water; an energy source; one or more of all essential amino acids, typically the twenty basic amino acids; vitamins and / or other organic compounds, usually required in low concentrations; lipids or free fatty acids; trace elements; and polyamines, such as ornithine and putrescine. For example, a cell culture medium can be formed by combining soybean hydrolysate with a basic cell culture medium and supplementing the medium with additional polyamines.

[0143] In some embodiments, the cell culture medium contains a basal culture medium with a defined chemical composition, such as a custom formulation or a commercially available basal culture medium.

[0144] Commercially available culture media will be known to those skilled in this technique, and in particular include Eagle's MEME (Eagle, Science, 1955, 112(3168):501-504), Ham's F12 (Ham, Proc. Nat'l. Acad. Sci. USA, 1965, 53:288-293), F-12 K medium, Dulbecco's medium, Dulbecco's Modified Eagle Medium (Proc. Natl. Acad. Sci. USA., August 1952; 38(8): 747-752), DMEM / Ham's F12 1:1, Trowell's T8, A2 medium (Holmes and Wolf, Biophys. Biochem. Cytol., 1961, 10:389-401), Waymouth medium (Davidson and Waymouth, Biochem. J., 1945, 39(2):188-199), Williams E medium (Williams et al., Exp. Cell Res., 1971, 69:105 and below), RPMI 1640 (Moore et al., J. Amer. Med. Assoc., 1967, 199:519-524), MCDB 104 / 110 medium (Bettger et al., Proc. Nat'l. Acad. Sci. USA, 1981, 78(9):5588-5592), Ventrex HL-1 medium, albumin-globulin medium (Orr et al., Appl. Microbiol., 1973, 25(1):49-54), RPM I-1640 medium, RPMI-1641 medium, Iscove's Modified Dulbecco's Medium, McCoy's 5 A medium, Leibovitz's L-15 medium, and serum-free media, such as the EX-CELL™ 300 series (JRH Biosciences, Lenexa, Kans).Protamine-zinc-insulin medium (Weiss et al., 1974, US Patent No. 4,072,565), biotin-folate medium (Cartaya, 1978, US Re30,985), transferrin-fatty acid medium (Baker, 1982, US Patent No. 4,560,655), transferrin-EGF medium (Hasegawa, 1982, US Patent No. 4,615,977; Chesbeuf, 1984, US Patent No. 4,786,599) and other medium replacements (see Inlow, US Patent No. 6,048,728; Drapeau, US Patent No. 7,294,484; Mather, US Patent No. 5,122,469; Furukawa, US Patent No. 5,976,833; Chen, US Patent No. 6,180,401; Chen, U.S. Patent No. 5,856,179; Etcheverry, U.S. Patent No. 5,705,364; Etcheverry, U.S. Patent No. 7,666,416; Ryll, U.S. Patent No. 6,528,286; Singh, U.S. Patent No. 6,924,124; Luan, U.S. Patent No. 7,429,491; and the like.

[0145] In some embodiments, the cell culture medium is serum-free. In some embodiments, the cell culture medium is serum-free and free of hydrolysis products.

[0146] In some embodiments, the culture medium contains at its applicable concentration (i.e., 1×) a mixture of at least 40 ± 6 mM or at least 55 ± 10.5 mM of amino acids or amino acid salts. In one embodiment, the culture medium contains at least 40 mM of the amino acid mixture. In this or another embodiment, the culture medium contains at least 55 mM of the amino acid mixture. In one embodiment, the amino acid mixture (which, except for glutamic acid, may be added back to the culture medium as a point-of-use additive) contains alanine, arginine, aspartic acid, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0147] In some embodiments, the culture medium contains one or more fatty acids. In a particular embodiment, the culture medium contains a mixture of fatty acids (or fatty acid derivatives) and α-tocopherol. The fatty acids or fatty acid derivatives are selected from the group consisting of: linoleic acid, alpha-linolenic acid, lipoic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, lauric acid, dodecanoic acid, decanoic acid, dodecanoic acid, hexanoic acid, tetracosanoic acid, myristic acid, and caprylic acid.

[0148] In some embodiments, the culture medium contains a mixture of nucleosides. In one embodiment, the culture medium contains adenosine, guanosine, cytidine, uridine, thymidine, and hypoxanthine.

[0149] In some embodiments, the culture medium contains a mixture of salts. The salts include divalent cations, such as calcium and magnesium. In one embodiment, the culture medium contains calcium chloride and magnesium sulfate. Other salts may include phosphate salts.

[0150] Depending on the cell culture process, different cell culture media may be used at different times during cell culture. For example, an expansion cell culture medium may be used when expanding an initial cell population from a frozen aliquot to produce a cell population for generating the protein of interest. A second production cell culture medium may be used to culture the expanded cell population to produce the protein of interest, and a third "feed" cell culture medium may be used to feed the cell culture during production. Alternatively, the same cell culture medium may be used throughout the cell culture process. As another alternative, the expansion medium may differ from the production and feed media, which may have the same or similar composition.

[0151] In some embodiments, one or more point-of-use additives may be added to any of the cell culture media as described herein during cell culture.

[0152] In some embodiments, culturing a cell population includes adding a feed medium to a production cell culture. As used herein, "feed medium" refers to a culture medium added to cultured cells to replenish depleted nutrients. The feed medium may be concentrated. For example, one or all components of the feed medium may be concentrated compared to the production cell culture medium. Alternatively, the concentration of the feed medium may be similar to that of the production cell culture medium. The feed medium may be added to the culture continuously or at intervals during culture, such as daily or every other day, or the cell culture may be fed when the concentration of a particular culture medium component monitored exceeds the desired range.

[0153] In some embodiments, the culture medium is replenished at intervals during cell culture according to a batch feeding process. Batch feeding culture is generally known in this art and is used to optimize protein production. See, for example, YM Huang et al., Biotechnol Prog. (2010) 26(5) pp. 1400-1410.

[0154] The percentage of viable cells can be measured at any time point during the cell culture methods described herein. Methods for determining viable cell counts and cell density include (but are not limited to) imaging cells and quantifying cell number, density, diameter, and biomarker performance.

[0155] Mammalian cells (such as CHO cells) can be cultured in small-scale cell culture vessels, such as 125 ml vessels with approximately 25 ml of culture medium, 250 ml vessels with approximately 50 to 100 ml of culture medium, or 500 ml vessels with approximately 100 to 200 ml of culture medium. For example, such small-scale vessels can be shake flasks. Cell culture flasks are known in this art and are available from suppliers such as Corning, Fisher Scientific, and others.

[0156] Alternatively, cell cultures can be grown on a laboratory scale. These include, for example, 1000 ml containers with approximately 300 to 1000 ml of culture medium, 3000 ml containers with approximately 500 to 3000 ml of culture medium, 8000 ml containers with approximately 2000 to 8000 ml of culture medium, and 15000 ml containers with approximately 4000 to 15000 ml of culture medium. Suitable cell culture systems are commercially available.

[0157] Cultures used for manufacturing (i.e., production cell cultures) may contain 10,000 L or more of culture medium. Large-scale cell cultures, or "production cell cultures," such as those used for the manufacture of protein therapeutics, are typically maintained for several days or even weeks while the cells produce the required proteins. During this time, the culture may be replenished with concentrated feed medium containing components consumed during the culture process, such as nutrients and amino acids.

[0158] In some embodiments, during cell growth or protein production, the cell culture medium is supplemented with one or more "point-of-use additives," also referred to as additives, point-of-use components, or point-of-use chemicals. Point-of-use additives include any and more of growth factors or other proteins, buffers, energy sources, salts, amino acids, metals, osmotic agents, and chelating agents. Other proteins include transferrin and albumin. Growth factors, including cytokines and chemokines, are generally known in the art and are known to stimulate cell growth or, in some cases, cell differentiation. Growth factors are typically proteins (e.g., insulin), small peptides, or steroid hormones such as estrogen, DHEA, testosterone, and analogues.

[0159] In some embodiments, the cell culture medium is supplemented with one or more or all of the following point-of-use additives: sodium bicarbonate, dextrose, L-glutamic acid, L-tyrosine, an amino acid mixture, and sodium phosphate.

[0160] The buffer is generally known in this art. This invention is not limited to any particular buffer, and anyone generally skilled in the art can choose an appropriate buffer or buffering system for use with a particular cell line that produces a particular protein.

[0161] Energy sources for additives used in cell cultures are also well known in this art. Without limitation, in some embodiments, the energy source for the additive is glucose. In other embodiments, the energy source for the additive is dextrose.

[0162] Chelating agents are also well-known in cell culture and protein production techniques. EDTA tetrasodium dehydrate and citrate are two commonly used chelating agents in this technique, but other chelating agents may be used in the practice of this invention.

[0163] Other additives include one or more of various metal salts, such as iron, nickel, zinc, and copper salts. In one embodiment, the cell culture medium is supplemented with one or more of copper sulfate, zinc sulfate, ferric chloride, and nickel sulfate. [, equipment , ] [, , ]

[0164] This disclosure provides an apparatus for use in the methods described herein.

[0165] In some embodiments, the device can be used to control pH and implement pH sequencing (changing the order of sample pH), while also detecting and correcting errors in the pH meter used in the process. The device can be used to effectively inactivate viruses in protein samples in a reactor. Extracting samples from the reactor and measuring sample pH can resolve several problems arising from the use of probes inserted into the reactor. For example, probes are typically not calibrated after sterilization. Sterilization can affect probe calibration profiles. Autoclaving often involves coordination with a third party and is therefore time-consuming. Probes can be stored in a dry environment after sterilization, which can reduce probe efficacy and shelf life. Probe use typically involves using additional sterile connection orifices. Additionally, probe use includes the risk of probe breakage and leakage of reference solution into the protein products measured by the probe.

[0166] Figure 19 is a block diagram of an apparatus 100 for pH control according to one embodiment. As shown, apparatus 100 includes a reactor 110, a pH flow channel 120, an acid titrant supply 130, an alkali titrant supply 140, and a waste receiver 150, optionally. The pH flow channel 120 contains a pH probe 121 disposed therein to measure the pH of a fluid sample in the flow channel. Piping between components indicates fluid coupling. Apparatus 100 may include one or more controllers to control any of its process units. The controllers may control the sequence of steps (e.g., the use of acid / alkali titrants). In some embodiments, one or more controllers may control the sequence based on pH measurements in the pH flow channel. The controllers may be accessible via a user interface. In some embodiments, the user interface may include a computer, laptop, mobile device, tablet, mobile phone, or any other suitable device.

[0167] In reactor 110, pH is controlled based on a user-defined sequence, such as a user-defined sequence of pH changes designed to inactivate viruses in a sample containing purified or partially purified proteins. In some embodiments, reactor 110 may be a batch reactor or have the characteristics of a batch reactor. In some embodiments, reactor 110 may be a continuously stirred tank reactor (CSTR) or have the characteristics of a CSTR. In some embodiments, reactor 110 may be a plug flow reactor (PFR) or have the characteristics of a PFR. In some embodiments, reactor 110 may include a mixer disposed therein. In some embodiments, the mixer may include an impeller. The mixer may homogenize the contents of reactor 110 at the time of, after, or during the addition of acid and / or base titrants. In some embodiments, the mixer is controlled by a controller, i.e., the controller sends signals to the mixer to start the impeller, stop the impeller, or control the impeller speed. In some embodiments, device 100 may include a mixer disposed outside reactor 110. In other words, the mixer may be a separate unit from reactor 110. In some embodiments, an acid mixer (not shown) may be fluidly coupled to an acid titrant supply 130 and mix the acid titrant before it is added to reactor 110. In some embodiments, an alkali mixer (not shown) may be fluidly coupled to an alkali titrant supply 140 and mix the alkali titrant before it is added to reactor 110. In some embodiments, reactor 110 may be without a pH measuring probe disposed therein. Reactor 110 may be maintained at a desired pH value by delivering acid titrant and / or alkali titrant from acid titrant supply 130 and / or alkali titrant supply 140.

[0168] In some embodiments, the controller sends a signal to the mixer to turn on the mixer before starting the acid titrant pump (for acid titrant addition) or before starting the alkali titrant pump (for alkali titrant addition). In some embodiments, the controller sends a signal to the mixer to stop for a fixed period of time after the acid titrant pump or alkali titrant pump stops. For example, the controller may send a signal to the mixer to stop for 15 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, or 1 hour after the acid titrant pump or alkali titrant pump stops.

[0169] In some embodiments, reactor 110 may have a volume of at least about 1 L, at least about 2 L, at least about 3 L, at least about 4 L, at least about 5 L, at least about 6 L, at least about 7 L, at least about 8 L, at least about 9 L, at least about 10 L, at least about 20 L, at least about 30 L, at least about 40 L, at least about 50 L, at least about 60 L, at least about 70 L, at least about 80 L, at least about 90 L, at least about 100 L, at least about 200 L, at least about 300 L, at least about 400 L, at least about 500 L, at least about 600 L, at least about 700 L, at least about 800 L, at least about 900 L, at least about 1 m³, at least about 2 m³, at least about 3 m³, at least about 4 m³, at least about 5 m³, at least about 6 m³, at least about 7 m³, at least about 8 m³, at least about 9 m³, at least about 10 m³, at least about 20 m³. 3. A volume of at least about 30 m3, at least about 40 m3, at least about 50 m3, at least about 60 m3, at least about 70 m3, at least about 80 m3, or at least about 90 m3. In some embodiments, reactor 110 may have a capacity of up to about 100 m³, up to about 90 m³, up to about 80 m³, up to about 70 m³, up to about 60 m³, up to about 50 m³, up to about 40 m³, up to about 30 m³, up to about 20 m³, up to about 10 m³, up to about 9 m³, up to about 8 m³, up to about 7 m³, up to about 6 m³, up to about 5 m³, up to about 4 m³, up to about 3 m³, up to about 2 m³, up to about 1 m³, up to about 900 L, up to about 800 L, up to about 700 L, up to about 600 L, up to about 500 L, up to about 400 L, up to about 300 L, up to about 200 L, up to about 100 L. Volumes of L, up to approximately 90 L, up to approximately 80 L, up to approximately 70 L, up to approximately 60 L, up to approximately 50 L, up to approximately 40 L, up to approximately 30 L, up to approximately 20 L, up to approximately 10 L, up to approximately 9 L, up to approximately 8 L, up to approximately 7 L, up to approximately 6 L, up to approximately 5 L, up to approximately 4 L, up to approximately 3 L, or up to approximately 2 L.

[0170] Combinations of the aforementioned volumes of reactor 110 are also possible (e.g., at least about 1 L and at most about 100 m3 or at least about 10 L and at most about 500 L), including all values ​​and ranges therein. In some embodiments, reactor 110 may have a volume of approximately 1 L, approximately 2 L, approximately 3 L, approximately 4 L, approximately 5 L, approximately 6 L, approximately 7 L, approximately 8 L, approximately 9 L, approximately 10 L, approximately 20 L, approximately 30 L, approximately 40 L, approximately 50 L, approximately 60 L, approximately 70 L, approximately 80 L, approximately 90 L, approximately 100 L, approximately 200 L, approximately 300 L, approximately 400 L, approximately 500 L, approximately 600 L, approximately 700 L, approximately 800 L, approximately 900 L, approximately 1 m³, approximately 2 m³, approximately 3 m³, approximately 4 m³, approximately 5 m³, approximately 6 m³, approximately 7 m³, approximately 8 m³, approximately 9 m³, approximately 10 m³, approximately 20 m³, approximately 30 m³, approximately 40 m³, approximately 50 m³, approximately 60 m³, approximately 70 m³, etc. 3. A volume of approximately 80 m³, approximately 90 m³, or approximately 100 m³. In some embodiments, reactor 110 may include a level indicator.

[0171] In some embodiments, a pH probe 121 may be disposed in a pH flow channel 120. In some embodiments, the pH in reactor 110 may be determined or confirmed based on readings from the pH probe 121 in the pH flow channel 120. The pH probe 121 in the pH flow channel 120 measures the potential difference between a reference electrode and a hydrogen ion selective electrode when inserted into the effluent from reactor 110. In other words, the activity of hydrogen ions in the effluent affects the electrochemical potential between the reference electrode and the hydrogen ion selective electrode, and a pH transmitter (not shown) is calibrated to correlate the potential difference to pH. In some embodiments, device 100 may include a pH transmitter (not shown). In some embodiments, the pH transmitter is coupled to the pH probe 121 and configured to receive a signal generated by the pH probe 121. In some embodiments, the pH transmitter converts the signal received from the pH probe 121 into a pH measurement. In some embodiments, the pH transmitter includes a user interface configured to display the pH measurement. In some embodiments, the pH transmitter may be a component separate from the pH flow channel 120. In some embodiments, the pH transmitter can transmit pH readings from pH probe 121 to a controller. In some embodiments, the controller sends pH readings from the pH transmitter to a user interface (not shown) configured to display pH readings. Based on the pH transmitted by the pH transmitter, the device 100 can maintain its current operation or initiate changes to its operation (e.g., adding acid titrant from acid titrant supply 130). In some embodiments, changes to operation can be implemented automatically. In some embodiments, changes to operation can be implemented via user input.

[0172] In some embodiments, a pH probe 121 disposed in a pH flow channel 120 can measure pH between a lower limit pH value and an upper limit pH value. In some embodiments, the lower limit pH value may be about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.0, including all values ​​and ranges therebetween. In some embodiments, the upper limit pH value may be about 8.5, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10.0, about 10.2, about 10.5, about 10.7, about 11.0, about 11.5, about 12.0, or about 12.5, including all values ​​and ranges therebetween.

[0173] A pH probe measures the pH of a sample volume from reactor 110 in pH flow cell 120. In some embodiments, the sample volume may be at least about 0.1 mL, at least about 0.2 mL, at least about 0.3 mL, at least about 0.4 mL, at least about 0.5 mL, at least about 0.6 mL, at least about 0.7 mL, at least about 0.8 mL, at least about 0.9 mL, at least about 1 mL, at least about 2 mL, at least about 3 mL, at least about 4 mL, at least about 5 mL, at least about 6 mL, at least about 7 mL, at least about 8 mL, at least about 9 mL, at least about 10 mL, at least about 20 mL, at least about 30 mL, at least about 40 mL, at least about 50 mL, at least about 60 mL, at least about 70 mL, at least about 80 mL, at least about 90 mL, at least about 100 mL, at least about 110 mL, at least about 120 mL, at least about 130 mL, at least about 140 mL, or at least about 150 mL. In some embodiments, the sample volume may be up to about 150 mL, up to about 140 mL, up to about 130 mL, up to about 120 mL, up to about 110 mL, up to about 100 mL, up to about 90 mL, up to about 80 mL, up to about 70 mL, up to about 60 mL, up to about 50 mL, up to about 40 mL, up to about 30 mL, up to about 20 mL, up to about 10 mL, up to about 9 mL, up to about 8 mL, up to about 7 mL, up to about 6 mL, up to about 5 mL, up to about 4 mL, up to about 3 mL, up to about 2 mL, up to about 1 mL, up to about 0.9 mL, up to about 0.8 mL, up to about 0.7 mL, up to about 0.6 mL, up to about 0.5 mL, up to about 0.4 mL, up to about 0.3 mL, or up to about 0.2 mL. Combinations of the above sample volumes are also possible (e.g., at least about 0.1 mL and at most about 100 mL or at least about 10 mL and at most about 20 mL), including all values ​​and ranges therein. In some embodiments, the sample volume may be about 0.1 mL, about 0.2 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 70 mL, about 80 mL, about 90 mL, or about 100 mL.

[0174] In some embodiments, the sample volume may be a fixed volume. In some embodiments, the sample volume may be a variable volume. In some embodiments, the sample volume may vary based on a variety of factors, including but not limited to the amount of fluid in reactor 110, the type of protein in reactor 110, and / or the size of reactor 110. In some embodiments, samples may be extracted from reactor 110 and measured at predetermined time intervals. In some embodiments, samples may be extracted from reactor 110 and measured at user-specified spontaneous time intervals.

[0175] In some embodiments, one or more controllers may trigger actions based on the pH value measured by the pH flow channel 120. For example, if the pH measured by the pH flow channel 120 is greater than the desired pH value, the controller may trigger the delivery of acid titrant from the acid titrant supply 130 to the reactor 110. In some embodiments, if the pH measured by the pH flow channel 120 is less than the desired pH value, the controller may trigger the delivery of alkali titrant from the alkali titrant supply 140 to the reactor 110. In some embodiments, the controller may maintain the pH at the desired value for a desired period of time. In some embodiments, the action may be manually triggered (i.e., by user intervention). In some embodiments, the action may be automatically triggered (i.e., based on a predetermined sequence). In some embodiments, the predetermined sequence may include lowering the pH in the reactor 110 to a first pH value and then raising the pH in the reactor to a second pH value. In some embodiments, the first pH value may be between about 3.0 and about 4.5, between about 3.5 and about 4.3, between about 3.5 and about 4.0, between about 3.1 and about 3.9, between about 3.2 and about 3.8, between about 3.3 and about 3.7, between about 3.4 and about 3.7, between about 3.3 and about 3.6, between about 3.4 and about 3.6, between about 3.4 and about 3.5, or between about 3.5 and about 3.6. In some embodiments, the second pH value may be between about 7 and about 8.5, between about 7.1 and about 8.4, between about 7.2 and about 8.3, between about 7.3 and about 8.2, between about 7.4 and about 8.2, between about 7.4 and about 8.1, between about 7.4 and about 8.0, between about 7.5 and about 8.2, between about 7.5 and about 8.1, between about 7.5 and about 8.0, between about 7.6 and about 8.1, between about 7.6 and about 8.0, between about 7.6 and about 7.9, between about 7.7 and about 8.0, between about 7.7 and about 7.9, or between about 7.7 and about 7.8.

[0176] In some embodiments, the acid titrant may be delivered in multiple phases to achieve a first pH value. In other words, a particulate acid titrant may be added, allowing for precise monitoring of the pH of the contents within reactor 110. In some embodiments, the acid titrant may be delivered in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, about 15, about 20, about 35, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 phases, including all values ​​and ranges therebetween, to achieve the first pH value. In some embodiments, the acid titrant may be delivered in 1, 2, 3, or 4 phases to achieve the first pH value. This solves the difficulty of checking pH meters because the addition of small amounts of titrant allows for more predictable changes in the pH of the contents within reactor 110.

[0177] In some embodiments, the alkali titrant may be delivered in multiple phases to achieve a second pH value. In other words, a particulate alkali titrant may be added, allowing for precise monitoring of the pH of the contents within reactor 110. In some embodiments, the alkali titrant may be delivered in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, about 15, about 20, about 35, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 phases, including all values ​​and ranges therebetween, to achieve a second pH value. In some embodiments, the alkali titrant may be delivered in 1, 2, 3, or 4 phases to achieve a second pH value.

[0178] If necessary, acid titrant can be delivered from acid titrant supply 130. In some embodiments, acid titrant can be delivered via a pump (not shown). In some embodiments, acid titrant supply 130 may include a container. In some embodiments, acid titrant supply 130 may include a tank. In some embodiments, the acid titrant supply 130 may have at least about 10 mL, at least about 20 mL, at least about 30 mL, at least about 40 mL, at least about 50 mL, at least about 60 mL, at least about 70 mL, at least about 80 mL, at least about 90 mL, at least about 100 mL, at least about 200 mL, at least about 300 mL, at least about 400 mL, at least about 500 mL, at least about 600 mL, at least about 700 mL, at least about 800 mL, at least about 900 mL, at least about 1 L, at least about 2 L, at least about 3 L, at least about 4 L, at least about 5 L, at least about 6 L, at least about 7 L, at least about 8 L, at least about 9 L, at least about 10 L, at least about 20 L, at least about 30 L, at least about 40 L, at least about 50 L, at least about 60 L, at least about 70 L, at least about 80 L, at least about 90 L, at least about 100 L, at least about 200 L. Volume of at least L, at least about 300 L, at least about 400 L, at least about 500 L, at least about 600 L, at least about 700 L, at least about 800 L, at least about 900 L, at least about 1 m3, at least about 2 m3, at least about 3 m3, at least about 4 m3, at least about 5 m3, at least about 6 m3, at least about 7 m3, at least about 8 m3 or at least about 9 m3.In some embodiments, the acid titrant supply 130 may have a capacity of up to about 10 m³, up to about 9 m³, up to about 8 m³, up to about 7 m³, up to about 6 m³, up to about 5 m³, up to about 4 m³, up to about 3 m³, up to about 2 m³, up to about 1 m³, up to about 900 L, up to about 800 L, up to about 700 L, up to about 600 L, up to about 500 L, up to about 400 L, up to about 300 L, up to about 200 L, up to about 100 L, up to about 90 L, up to about 80 L, up to about 70 L, up to about 60 L, up to about 50 L, up to about 40 L, up to about 30 L, up to about 20 L, up to about 10 L, up to about 9 L, up to about 8 L, up to about 7 m³, up to about 7 m³, up to about 10 m³, up to about 9 L, up to about 8 L, up to about 7 m³, up to about 10 m³, up to about 9 m³, up to about 8 L, up to about 7 m³, up to about 10 m³ ...10 Volumes of approximately 6 L, 5 L, 4 L, 3 L, 2 L, 1 L, 900 mL, 800 mL, 700 mL, 600 mL, 500 mL, 400 mL, 300 mL, 200 mL, 100 mL, 90 mL, 80 mL, 70 mL, 60 mL, 50 mL, 60 mL, 50 mL, 40 mL, 30 mL, or 20 mL.

[0179] Combinations of the above volumes of acid titrant 130 are also possible (e.g., at least about 10 mL and at most about 10 m3 or at least about 1 L and at most about 5 L), including all values ​​and ranges therein. In some embodiments, the acid titrant supply 130 may have approximately 10 mL, approximately 20 mL, approximately 30 mL, approximately 40 mL, approximately 50 mL, approximately 60 mL, approximately 70 mL, approximately 80 mL, approximately 90 mL, approximately 100 mL, approximately 200 mL, approximately 300 mL, approximately 400 mL, approximately 500 mL, approximately 600 mL, approximately 700 mL, approximately 800 mL, approximately 900 mL, approximately 1 L, approximately 2 L, approximately 3 L, approximately 4 L, approximately 5 L, approximately 6 L, approximately 7 L, approximately 8 L, approximately 9 L, approximately 10 L, approximately 20 L, approximately 30 L, approximately 40 L, approximately 50 L, approximately 60 L, approximately 70 L, approximately 80 L, approximately 90 L, approximately 100 L, approximately 200 L, approximately 300 L, approximately 400 L, approximately 500 L, approximately 600 L, approximately 700 L, approximately 800 L, approximately 900 L, etc. Volumes of L, approximately 1 m³, approximately 2 m³, approximately 3 m³, approximately 4 m³, approximately 5 m³, approximately 6 m³, approximately 7 m³, approximately 8 m³, or approximately 9 m³, or approximately 10 m³.

[0180] In some embodiments, the acid titrant supply 130 may be maintained at a pH of at least about 0.5, at least about 1, at least about 1.5, at least about 2, at least about 2.5, at least about 3, at least about 3.5, at least about 4, at least about 4.5, at least about 5, at least about 5.5, at least about 6, or at least about 6.5. In some embodiments, the acid titrant supply 130 may be maintained at a pH of at most about 7, at most about 6.5, at most about 6, at most about 5.5, at most about 5, at most about 4.5, at most about 4, at most about 3.5, at most about 3, at most about 2.5, at most about 2, at most about 1.5, at most about 1, or at most about 0.5. Combinations of the above pH values ​​in the acid titrant supply 130 are also possible (e.g., at least about 0.5 and at most about 7 or at least about 2 and at most about 6), including all values ​​and ranges therebetween. In some embodiments, the acid titrant supply 130 may be maintained at a pH of about 0, about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5 or about 7.

[0181] If necessary, alkali titrant can be delivered from alkali titrant supply 140. In some embodiments, the alkali titrant can be delivered via a pump (not shown). In some embodiments, alkali titrant supply 140 may include a container. In some embodiments, alkali titrant supply 140 may include a tank. In some embodiments, the alkaline titrant supply 140 may have at least about 10 mL, at least about 20 mL, at least about 30 mL, at least about 40 mL, at least about 50 mL, at least about 60 mL, at least about 70 mL, at least about 80 mL, at least about 90 mL, at least about 100 mL, at least about 200 mL, at least about 300 mL, at least about 400 mL, at least about 500 mL, at least about 600 mL, at least about 700 mL, at least about 800 mL, at least about 900 mL, at least about 1 L, at least about 2 L, at least about 3 L, at least about 4 L, at least about 5 L, at least about 6 L, at least about 7 L, at least about 8 L, at least about 9 L, at least about 10 L, at least about 20 L, at least about 30 L, at least about 40 L, at least about 50 L, at least about 60 L, at least about 70 L, at least about 80 L, at least about 90 L, at least about 100 L, at least about 200 L. Volume of at least L, at least about 300 L, at least about 400 L, at least about 500 L, at least about 600 L, at least about 700 L, at least about 800 L, at least about 900 L, at least about 1 m3, at least about 2 m3, at least about 3 m3, at least about 4 m3, at least about 5 m3, at least about 6 m3, at least about 7 m3, at least about 8 m3 or at least about 9 m3.In some embodiments, the alkaline titrant supply 140 may have a capacity of up to about 10 m³, up to about 9 m³, up to about 8 m³, up to about 7 m³, up to about 6 m³, up to about 5 m³, up to about 4 m³, up to about 3 m³, up to about 2 m³, up to about 1 m³, up to about 900 L, up to about 800 L, up to about 700 L, up to about 600 L, up to about 500 L, up to about 400 L, up to about 300 L, up to about 200 L, up to about 100 L, up to about 90 L, up to about 80 L, up to about 70 L, up to about 60 L, up to about 50 L, up to about 40 L, up to about 30 L, up to about 20 L, up to about 10 L, up to about 9 L, up to about 8 L, up to about 7 m³, up to about 7 m³, up to about 10 m³, up to about 9 L, up to about 8 L, up to about 7 m³, up to about 10 m³, up to about 9 m³ ... Volumes of approximately 6 L, 5 L, 4 L, 3 L, 2 L, 1 L, 900 mL, 800 mL, 700 mL, 600 mL, 500 mL, 400 mL, 300 mL, 200 mL, 100 mL, 90 mL, 80 mL, 70 mL, 60 mL, 50 mL, 60 mL, 50 mL, 40 mL, 30 mL, or 20 mL.

[0182] Combinations of the above-mentioned volumes of alkali titrant supply 140 are also possible (e.g., at least about 10 mL and at most about 10 m3 or at least about 1 L and at most about 5 L), including all values ​​and ranges therein. In some embodiments, the alkaline titrant supply 140 may have approximately 10 mL, approximately 20 mL, approximately 30 mL, approximately 40 mL, approximately 50 mL, approximately 60 mL, approximately 70 mL, approximately 80 mL, approximately 90 mL, approximately 100 mL, approximately 200 mL, approximately 300 mL, approximately 400 mL, approximately 500 mL, approximately 600 mL, approximately 700 mL, approximately 800 mL, approximately 900 mL, approximately 1 L, approximately 2 L, approximately 3 L, approximately 4 L, approximately 5 L, approximately 6 L, approximately 7 L, approximately 8 L, approximately 9 L, approximately 10 L, approximately 20 L, approximately 30 L, approximately 40 L, approximately 50 L, approximately 60 L, approximately 70 L, approximately 80 L, approximately 90 L, approximately 100 L, approximately 200 L, approximately 300 L, approximately 400 L, approximately 500 L, approximately 600 L, approximately 700 L, approximately 800 L, approximately 900 L, etc. Volumes of L, approximately 1 m³, approximately 2 m³, approximately 3 m³, approximately 4 m³, approximately 5 m³, approximately 6 m³, approximately 7 m³, approximately 8 m³, or approximately 9 m³, or approximately 10 m³.

[0183] In some embodiments, the alkaline titrant supply 140 may be maintained at a pH of at least about 7, at least about 7.5, at least about 8, at least about 8.5, at least about 9, at least about 9.5, at least about 10, at least about 10.5, at least about 11, at least about 11.5, at least about 12, at least about 12.5, at least about 13, or at least about 13.5. In some embodiments, the alkaline titrant supply 140 may be maintained at a pH of at most about 14, at most about 13.5, at most about 13, at most about 12.5, at most about 12, at most about 11.5, at most about 11, at most about 10.5, at most about 10, at most about 9.5, at most about 9, at most about 8.5, at most about 8, or at most about 7.5. Combinations of the pH values ​​described above in the alkaline titrant supply 140 are also possible (e.g., at least about 7.5 and at most about 14 or at least about 8 and at most about 10), including all values ​​and ranges therebetween. In some embodiments, the alkaline titrant supply 140 may be maintained at a pH of about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, or about 14.

[0184] Waste receiver 150 is present as appropriate and can receive effluent from a sample measured in pH flow channel 120. In some embodiments, waste receiver 150 may include a container, tank, treatment facility, or any other suitable device for receiving effluent from pH flow channel 120.

[0185] Figure 20 is a schematic diagram of a pH control device 200 according to one embodiment. As shown, the device 200 includes a reactor 210, a pH flow channel 220 with a pH probe 221, a sampling pump 222, a check valve 224 (also called a non-return valve), an acid titrant supply 230, an acid titrant pump 232, an acid titrant flow meter 234, an alkali titrant supply 240, an alkali titrant pump 242, an alkali titrant flow meter 244, a waste receiver 250, a controller 260, and a user interface 262. In some embodiments, the check valve 224 allows flow in only one direction from the reactor 210, thereby preventing the reactor 210 from being contaminated by that volume of sample in the pH flow channel 220. In some embodiments, reactor 210, pH flow channel 220, acid titrant supply 230, alkali titrant supply 240, and waste receiver 250 may be the same as or substantially similar to reactor 110, pH flow channel 120, acid titrant supply 130, alkali titrant supply 140, and waste receiver 150, as described above with reference to FIG. 19. Therefore, certain configurations of reactor 210, pH flow channel 220, acid titrant supply 230, alkali titrant supply 240, and waste receiver 250 are not described in more detail herein. As shown, arrows indicate fluid flow.

[0186] As shown, the material stream can flow from reactor 210 to waste receiver 250. Reactor 210 receives the material stream from acid titrant supply 230 and alkali titrant supply 240. In some embodiments, reactor 210 may include a mixer disposed therein. In some embodiments, apparatus 200 may include one or more mixers located outside reactor 210.

[0187] In some embodiments, pH flow channel 220 may be a Mettler Toledo pH flow channel. In some embodiments, pH probe 221 may be disposed in pH flow channel 220. Exemplary pH probes suitable for the devices described herein include inline pH probes from Mettler Toledo, Thermo Fisher Scientific, or Cole-Parmer. In some embodiments, pH probe 221 is coupled to a pH transmitter. The pH transmitter may be, for example, a Mettler Toledo M400 pH transmitter. In some embodiments, the pH transmitter may visually display a pH reading on it. In some embodiments, the pH transmitter transmits the pH reading to a controller 260. In some embodiments, the controller 260 transmits the pH reading to a user interface 262, which displays the pH reading. In some embodiments, the pH transmitter may transmit the pH reading to the user interface 262. The flow rate through flow channel 220 may be controlled by sampling pump 222.

[0188] Sampling pump 222 pumps a certain volume of sample fluid (i.e., slipflow) from reactor 210, allowing that volume of sample fluid to enter pH flow channel 220. In some embodiments, sampling pump 222 may include a peristaltic pump, diaphragm pump, gear pump, multi-lobe pump, piston pump, centrifugal pump, or any other suitable pump or combination thereof. In some embodiments, sampling pump 222 may include a Watson-Marlow 120 pump.

[0189] Check valve 224 allows flow in only one direction, exiting reactor 210, thereby preventing reactor 210 from being contaminated by the sample volume in flow channel 220. As shown, valve 224 is upstream of sampling pump 222. In some embodiments, device 200 may include a shut-off valve (not shown) downstream of reactor 210 and fluidly coupled to reactor 210. In some embodiments, the shut-off valve may be physically coupled to the reactor. Acid titrant supply 230 contains acid titrant. Acid titrant can be drawn from acid titrant supply 230 via acid titrant pump 232. Acid titrant pump 232 draws fluid from acid titrant supply 230 and facilitates the flow of acid titrant to reactor 210. In some embodiments, the acid titration pump 232 can facilitate a flow rate of at least about 1 mL / min, at least about 2 mL / min, at least about 3 mL / min, at least about 4 mL / min, at least about 5 mL / min, at least about 6 mL / min, at least about 7 mL / min, at least about 8 mL / min, at least about 9 mL / min, at least about 10 mL / min, at least about 20 mL / min, at least about 30 mL / min, at least about 40 mL / min, at least about 50 mL / min, at least about 60 mL / min, at least about 70 mL / min, at least about 80 mL / min, at least about 90 mL / min, at least about 100 mL / min, at least about 200 mL / min, at least about 300 mL / min, at least about 400 mL / min, at least about 500 mL / min, at least about 600 mL / min, at least about 700 mL / min, at least about 800 mL / min, at least about 900 mL / min, at least about 1 L / min, at least about 2 L / min, at least about 5 Acid titrant flow rate of at least L / min, at least about 10 L / min, at least about 20 L / min, at least about 30 L / min, at least about 40 L / min or at least about 50 L / min.In some embodiments, the acid titration pump 232 can facilitate a flow rate of up to about 50 L / min, up to about 40 L / min, up to about 30 L / min, up to about 20 L / min, up to about 10 L / min, up to about 5 L / min, up to about 2 L / min, up to about 1 L / min, up to about 900 mL / min, up to about 800 mL / min, up to about 700 mL / min, up to about 600 mL / min, up to about 500 mL / min, up to about 400 mL / min, up to about 300 mL / min, up to about 200 mL / min, up to about 100 mL / min, up to about 90 mL / min, up to about 80 mL / min, up to about 70 mL / min, up to about 60 mL / min, up to about 50 mL / min, up to about 40 mL / min, up to about 30 mL / min, up to about 20 L / min. Acid titrant flow rates of up to approximately 10 mL / min, up to approximately 9 mL / min, up to approximately 8 mL / min, up to approximately 7 mL / min, up to approximately 6 mL / min, up to approximately 5 mL / min, up to approximately 4 mL / min, up to approximately 3 mL / min, or up to approximately 2 mL / min.

[0190] Combinations of the above-mentioned acid titrant flow rates are also possible (e.g., at least about 1 mL / min and at most about 1 L / min or at least about 10 mL / min and at most about 50 mL / min), including all values ​​and ranges therein. In some embodiments, the acid titrant pump 232 can facilitate an acid titrant flow rate of about 1 mL / min, about 2 mL / min, about 3 mL / min, about 4 mL / min, about 5 mL / min, about 6 mL / min, about 7 mL / min, about 8 mL / min, about 9 mL / min, about 10 mL / min, about 20 mL / min, about 30 mL / min, about 40 mL / min, about 50 mL / min, about 60 mL / min, about 70 mL / min, about 80 mL / min, about 90 mL / min, about 100 mL / min, about 200 mL / min, about 300 mL / min, about 400 mL / min, about 500 mL / min, about 600 mL / min, about 700 mL / min, about 800 mL / min, about 900 mL / min, or about 1 L / min.

[0191] The flow rate of the acid titrant can be measured by an acid titrant flow meter 234. In some embodiments, the acid titrant flow meter 234 may include an ultrasonic meter, a vortex mixer, a magnetometer, a Coriolis meter, or any other suitable flow meter or combination thereof. Suitable acid titrant flow meters are commercially available; for example, the acid titrant flow meter 234 may include a Sonotec ultrasonic flow meter.

[0192] In some embodiments, the acid titrant pump 232 may include a peristaltic pump, a diaphragm pump, a gear pump, a multi-lobe pump, a piston pump, a centrifugal pump, or any other suitable pump or combination thereof. A suitable acid titrant pump (232) is commercially available, such as the Watson-Marlow 530 pump.

[0193] Alkali titrant supply 240 contains alkali titrant. Alkali titrant can be drawn from alkali titrant supply 240 via alkali titrant pump 242. Alkali titrant pump 242 draws fluid from alkali titrant supply 240 and promotes the flow of alkali titrant to reactor 210. In some embodiments, the alkaline titration pump 242 can facilitate a flow rate of at least about 1 mL / min, at least about 2 mL / min, at least about 3 mL / min, at least about 4 mL / min, at least about 5 mL / min, at least about 6 mL / min, at least about 7 mL / min, at least about 8 mL / min, at least about 9 mL / min, at least about 10 mL / min, at least about 20 mL / min, at least about 30 mL / min, at least about 40 mL / min, at least about 50 mL / min, at least about 60 mL / min, at least about 70 mL / min, at least about 80 mL / min, at least about 90 mL / min, at least about 100 mL / min, at least about 200 mL / min, at least about 300 mL / min, at least about 400 mL / min, at least about 500 mL / min, at least about 600 mL / min, at least about 700 mL / min, at least about 800 mL / min, at least about 900 mL / min, at least about 1 L / min, at least about 2 L / min, at least about 5 Alkali titrant flow rate of at least L / min, at least about 10 L / min, at least about 20 L / min, at least about 30 L / min, at least about 40 L / min or at least about 50 L / min.In some embodiments, the alkaline titration pump 242 can be accelerated to a maximum of about 50 L / min, a maximum of about 40 L / min, a maximum of about 30 L / min, a maximum of about 20 L / min, a maximum of about 10 L / min, a maximum of about 5 L / min, a maximum of about 2 L / min, a maximum of about 1 L / min, a maximum of about 900 mL / min, a maximum of about 800 mL / min, a maximum of about 700 mL / min, a maximum of about 600 mL / min, a maximum of about 500 mL / min, a maximum of about 400 mL / min, a maximum of about 300 mL / min, a maximum of about 200 mL / min, a maximum of about 100 mL / min, a maximum of about 90 mL / min, a maximum of about 80 mL / min, a maximum of about 70 mL / min, a maximum of about 60 mL / min, a maximum of about 50 mL / min, a maximum of about 40 mL / min, a maximum of about 30 mL / min, a maximum of about 20 L / min. Acid titrant flow rates of up to approximately 10 mL / min, up to approximately 9 mL / min, up to approximately 8 mL / min, up to approximately 7 mL / min, up to approximately 6 mL / min, up to approximately 5 mL / min, up to approximately 4 mL / min, up to approximately 3 mL / min, or up to approximately 2 mL / min.

[0194] The above-mentioned combinations of alkaline titrant flow rates are also possible (e.g., at least about 1 mL / min and at most about 1 L / min or at least about 10 mL / min and at most about 50 mL / min), including all values ​​and ranges therein. In some embodiments, the alkali titrant pump 242 can achieve an alkali titrant flow rate of about 1 mL / min, about 2 mL / min, about 3 mL / min, about 4 mL / min, about 5 mL / min, about 6 mL / min, about 7 mL / min, about 8 mL / min, about 9 mL / min, about 10 mL / min, about 20 mL / min, about 30 mL / min, about 40 mL / min, about 50 mL / min, about 60 mL / min, about 70 mL / min, about 80 mL / min, about 90 mL / min, about 100 mL / min, about 200 mL / min, about 300 mL / min, about 400 mL / min, about 500 mL / min, about 600 mL / min, about 700 mL / min, about 800 mL / min, about 900 mL / min, or about 1 L / min.

[0195] The flow rate of the alkali titrant can be measured by an alkali titrant flow meter 244. In some embodiments, the alkali titrant flow meter 244 may include an ultrasonic meter, a vortex mixer, a magnetometer, a Coriolis meter, or any other suitable flow meter or combination thereof. Suitable alkali titrant flow meters are commercially available; for example, the alkali titrant flow meter 244 may include a Sonotec ultrasonic flow meter.

[0196] In some embodiments, the alkali titrant pump 242 may include a peristaltic pump, a diaphragm pump, a gear pump, a multi-lobe pump, a piston pump, a centrifugal pump, or any other suitable pump or combination thereof. A suitable alkali titrant pump is commercially available; for example, the alkali titrant pump 242 may include a Watson-Marlow 530 pump.

[0197] As shown, the components are enclosed in dotted lines, and the user interface 262 provides a certain level of control over these components. In other words, the user interface 262 can operate via controller 260 or multiple controllers to control any of the components of device 200. In some embodiments, the user interface 262 can communicate with and control reactor 210, pH flow tank 220, sampling pump 222, valve 224, acid titrant supply 230, acid titrant pump 232, acid titrant flow meter 234, alkali titrant supply 240, alkali titrant pump 242, alkali titrant flow meter 244, and / or waste receiver 250. In some embodiments, control of any of the above components can be initiated by the user. In other words, the user can manually control any of the components of device 200 to initiate the action of at least one of the components. In some embodiments, control of any component of device 200 may be performed automatically in response to conditions within device 200 (e.g., pH measured in pH flow cell 220). In some embodiments, control of any component of device 200 may be performed without any user intervention. In some embodiments, a user may send instructions to controller 260 via user interface 262 to control or advance a pre-programmed pH sequence or add a predetermined amount of acid or alkali titrant.

[0198] In some embodiments, the controller 260 may communicate with the acid titrant flow meter 234, the alkali titrant flow meter 244, the pH probe 221, the acid titrant pump 232, and / or the alkali titrant pump 242. In some embodiments, the controller 260 may receive a signal from the acid titrant flow meter 244, thereby determining the amount of acid titrant added to the sample. In some embodiments, the controller 260 may receive a signal from the alkali titrant flow meter 244, thereby determining the amount of alkali titrant added to the sample. In some embodiments, the controller 260 may receive a signal from the pH probe 221, thereby transmitting a pH measurement value to the controller 260, and the controller 260 associates the pH measurement value with the corresponding amount of acid or alkali titrant added to the sample. In some embodiments, the controller 260 may send a signal to the acid titrant pump 232 to start the pump, stop the pump, or change the pump speed. In some embodiments, the controller 260 may send a signal to the alkali titrant pump 242 to start the pump, stop the pump, or change the pump speed. In some embodiments, the controller 260 may apply a model to the pH measurement value and the corresponding amount of acid or alkali titrant added to the sample.

[0199] In some embodiments, the user interface 262 may communicate with the reactor 210 and / or a mixer disposed therein via the controller 260. In some embodiments, the controller 260 may communicate with the mixer to control the mixing timing and mixing rate. For example, the controller 260 may send signals to the mixer to initiate mixing at the moment of addition of acid titrant and / or base titrant and during the addition of acid titrant and / or base titrant. Mixing during titrant addition prevents the formation of large amounts of high titrant concentrations in the sample, which could damage the sample. In some embodiments, the mixing timing may be modified based on the amount of acid titrant and / or base titrant added. In some embodiments, the controller 260 may communicate with the valve 224 to stop or allow sampling fluid to flow through it. In some embodiments, the controller 260 may communicate with the sampling pump 222 to initiate the pumping of sampling fluid through it or to change the flow rate of the sampling fluid through it. In some embodiments, the sequence of access to the sampling pump 222 and the valve 224 may be based on data transmitted to the user interface 262 via a pH transmitter and the controller 260. The controller 260 is configured to receive signals from components of the device described herein and to send instructions to those components. In some embodiments, the controller 260 may communicate with the acid titrant pump 232 to pump acid titrant through it. For example, the controller 260 sends a signal to start the acid titrant pump 232, stop the acid titrant pump, or change the speed of the acid titrant pump. In some embodiments, communication with the acid titrant pump 232 may be based on data transmitted to the controller 260 via a pH transmitter and / or an acid titrant flow meter 234. In some embodiments, the controller 260 may communicate with the alkali titrant pump 242 to pump alkali titrant through it. For example, the controller 260 sends a signal to start the alkali titrant pump 242, stop the alkali titrant pump, or change the speed of the alkali titrant pump. In some embodiments, communication with the alkali titrant pump 242 may be based on data transmitted to the controller 260 via a pH transmitter and / or an alkali titrant flow meter 244. In some embodiments, controller 260 is configured to receive pH values ​​from pH probe 221 (e.g., via a pH transmitter), and controller 260 applies a mathematical model to the pH value when measuring the pH, applying it to the corresponding amount of acid or base titrant added to the sample. In some embodiments, controller 260 is configured to apply the model described herein to one or more pH values ​​and the corresponding amounts of titrant added to the sample. In some embodiments, controller 260 is configured to determine the remaining amount of titrant to be added to the sample to achieve a target pH based on the measured pH value, the amount of titrant added to the sample, and the model. These steps may be repeated one or more times, depending on the situation, until the final target pH is reached. For example, controller 260 is configured to receive an initial pH reading, send a signal to acid or base titrant pump 242 to add a predetermined amount of acid or base titrant to the sample, and then pH probe 221 acquires a reading and sends the measured pH value to controller 260 via pH transmitter. The controller 260 then applies the model to the initial pH value, the pH value measured after adding the titrant, and the amount of titrant added to the sample, and determines the additional amount of titrant added to the sample. In some embodiments, such as when a predetermined pH sequence exists, the controller 260 may send instructions to automatically advance the pH sequence. In other embodiments, the user sends instructions to the controller 260 via a user interface to advance the pH sequence, and the controller 260 relays these instructions to the device, for example, by activating the acid titrant pump 232 or the alkali titrant pump 242, acquiring pH readings, and similar methods.

[0200] In some embodiments, controller 260 may include a server, computer, laptop, mobile device, tablet computer, mobile phone, or any other suitable device. Controller 260 may include one or more central processing units ("processors"), memory, and input / output devices. In some embodiments, controller 260 includes one or more memory and / or storage devices. The memory and storage devices may be one or more computer-readable storage media that can store computer-executable instructions implementing at least a portion of the various embodiments described herein. In some embodiments, controller 260 includes computer-readable storage media that stores computer-executable instructions, including (but not limited to) instructions to start, stop, or change the speed of acid titrant pump 232; instructions to start, stop, or change the speed of alkali titrant pump 242; instructions to receive and store pH values ​​from pH probe 221 and / or pH transmitter; and instructions to receive and store data from acid titrant flow meter 234 and / or alkali titrant flow meter 244. In some embodiments, the computer-executable instructions include instructions to receive and store user-inputted pH values, such as offline pH values ​​measured and input by the user when an error is detected in the pH probe 221 placed in the pH flow tank 220. In some embodiments, the computer-executable instructions include instructions to calculate the amount of acid or alkali titrant to be added to the sample based on data from the acid titrant flow meter 234 or alkali titrant flow meter 244 and, as appropriate, the acid titrant pump 232 or alkali titrant pump 242. In some embodiments, the computer-executable instructions include instructions to apply the model described herein to the pH value and the amount of titrant to be added to the sample. In some embodiments, the computer-executable instructions include instructions to perform one or more steps of a pH sequence in response to a user command via a user interface, as appropriate. In some embodiments, the controller 260 includes a processor configured to execute the instructions described above.

[0201] This disclosure provides a user interface 262 configured to receive signals from a controller 260 and send instructions to the controller 260. In some embodiments, the user interface 262 is an industrial human-machine interface. Suitable user interfaces include visual interfaces (computer monitors, flat-panel screens, touch screens, and the like) as well as keyboards, pointing devices (such as mice), and equivalent devices. In some embodiments, the user interface 262 is configured to display pH measurements from the controller 260. In some embodiments, the user interface 262 is configured to receive one or more offline pH measurements from a user. In some embodiments, the user interface 262 is configured to receive instructions from a user, thereby sending the instructions to the controller 260 to advance a pH sequence.

[0202] In some embodiments, the user interface is configured to display a warning signal and / or pause the pH sequence or titrant addition when the pH measurement from the pH probe differs from the pH measurement predicted by the model. For example, when the user interface includes a graphical interface, the user interface may display a warning signal on that graphical interface. In some embodiments, a warning signal is displayed when the difference between the pH measurement and the model is > 0.01 pH units, > 0.02 pH units, > 0.03 pH units, > 0.04 pH units, > 0.05 pH units, > 0.06 pH units, > 0.07 pH units, > 0.08 pH units, > 0.09 pH units, or > 0.10 pH units. In some embodiments, a warning signal is displayed when the difference between the pH measurement and the model is > 0.05 pH units.

[0203] This specification describes numerous exemplary configurations, methods, parameters, and the like. However, it should be understood that such descriptions are not intended to limit the scope of this disclosure, but are provided as descriptions of exemplary embodiments. Embodiments of the subject matter of the invention described above may be advantageous alone or in combination with one or more other forms or embodiments. Without limiting the foregoing description, certain non-limiting embodiments of the invention are provided below. As will be apparent to those skilled in the art upon reading this disclosure, each of the individually numbered embodiments may be used, or in combination with any of the previously or subsequently individually numbered embodiments. This is intended to provide support for all such combinations of embodiments, and not only the combinations of embodiments explicitly provided below. [Examples provided]

[0204] The invention can be understood by referring to the following examples:

[0205] 1. A method comprising: [(a)] Measure the initial pH of the sample (initial pH); [(b)] A first amount of titrant (titer n) is added to the sample and a first additional pH value (pH n) is measured, where titrant n is the amount of titrant added to the sample to achieve pH n, and pH n is different from the initial pH; [(c)] A model is applied to determine the initial amount of standardized titrant (titer initial) and the standardized titrant n, wherein the model relates the amount of standardized titrant added to the sample to the pH of the sample; [(d)] A second amount of titrant is added to the sample to achieve a second pH value (pH n+1), wherein the second amount of titrant is determined by the initial pH, pH n, initial standardized titrant, and standardized titrant n; [(e)] After the addition of the second amount of titrant, the pH of the sample is measured to determine the measured pH n+1; and [(f)] When the difference between the measured pH n+1 and the pH n+1 from the model indicates a calibration error in the pH meter used to measure the pH of the sample, the pH meter is calibrated.

[0206] 2. The method of Example 1, wherein the difference between the measured pH n+1 and the pH n+1 from the model is > 0.01 pH unit, > 0.02 pH unit, > 0.03 pH unit, > 0.04 pH unit, > 0.05 pH unit, > 0.06 pH unit, > 0.07 pH unit, > 0.08 pH unit, > 0.09 pH unit, or > 0.10 pH unit.

[0207] 3. The method of Example 1, wherein the difference between the measured pH n+1 and the pH n+1 from the model is >0.05 pH units.

[0208] 4. The method of any one of Examples 1 to 3, comprising adding a third amount of titrant (titrant n+2) to the sample and measuring a third additional pH (pH n+2), and repeating steps (c) to (e), wherein the difference between the measured pH n+2 and the pH n+2 from the model indicates a calibration error in the pH meter.

[0209] 5. The method of Example 4, wherein the difference between pH n+2 and the model is > 0.01 pH unit, > 0.02 pH unit, > 0.03 pH unit, > 0.04 pH unit, > 0.05 pH unit, > 0.06 pH unit, > 0.07 pH unit, > 0.08 pH unit, > 0.09 pH unit, or > 0.10 pH unit.

[0210] 6. The method of Example 4, wherein the difference between pH n+2 and the model is > 0.05 pH units.

[0211] 7. The method of Example 1, which includes repeating steps (d) to (f) until the final pH is reached.

[0212] 8. The method of Example 7, which includes repeating steps (d) to (f) at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.

[0213] 9. The method of Example 7 or 8, wherein a difference of >0.01 pH unit, >0.02 pH unit, >0.03 pH unit, >0.04 pH unit, >0.05 pH unit, >0.06 pH unit, >0.07 pH unit, >0.08 pH unit, >0.09 pH unit, or >0.10 pH unit between the pH measured at any repetition and the pH from the model indicates a calibration error in the pH meter.

[0214] 10. The method of any one of Examples 1 to 9, comprising: (i) Generate at least one reference titration curve from at least one reference sample, relating the amount of titrant added to the reference sample to the pH of the reference sample; (ii) standardize the at least one reference titration curve as appropriate; and (ii) Generate the model to fit the at least one reference titration curve.

[0215] 11. The method of Example 10, wherein generating the at least one reference titration curve comprises: [(i)] Measure the initial pH of the reference sample (pH initial_reference); [(ii)] A certain amount of titrant is added to the reference sample (titer n_reference) and an additional reference pH value (pH n_reference) is measured, where titrant n_reference is the amount of titrant added to the sample to achieve pH n_reference, and pH n_reference is different from pH initial_reference; [(iii)] Repeat step (ii) until the at least one reference sample reaches the final pH (pH final - reference) by adding the full amount of titrant to the reference sample (total titrant - reference); and [(iv)] Plot the amount of titrant added relative to the pH of the reference sample.

[0216] 12. The method of Example 10 or 11, wherein the titrant is added to the reference sample in a discrete step during a plurality of time periods.

[0217] 13. The method of Example 10 or 11, wherein the titrant is continuously added to the reference sample.

[0218] 14. The method of any one of Examples 10 to 12, wherein the pH of the reference sample is measured by means of a pH probe directly inserted into the reference sample.

[0219] 15. The method of any one of Examples 10 to 12, wherein the pH of the reference sample is measured by means of a pH probe inserted into a slipstream of continuous or discrete sampling from the reference sample.

[0220] 16. The method of any one of Examples 10 to 15, wherein the pH meter calibration is performed when the model is generated.

[0221] 17. The method of Example 16, wherein the calibration for pH meter calibration includes: [(a)] Before the addition of the titrant, a first portion of the reference sample is removed, and the pH of the first portion is measured using an independently calibrated pH meter to generate an offline initial pH value (pH initial_offline_reference); [(b)] After the addition of the full amount of titrant, the second portion of the reference sample is removed and the pH of the second portion is measured using an independently calibrated pH meter, thereby generating an offline final pH value (pH final_offline_reference); and [(c)] The relationship between the offline pH value and the measured pH value is used to determine the corrected pH of the reference sample.

[0222] 18. The method of Example 17, wherein the calibrated pH n_reference of the reference sample is determined by: (Equation 22).

[0223] 19. The method of any one of Examples 10 to 18, wherein the amount of titrant added to the reference sample is standardized by means of: (Equation 17), where titrant 1_reference is the amount of titrant added to the reference sample to achieve pH 1_reference, and titrant 2_reference is the amount of titrant added to the reference sample to achieve pH 2_reference.

[0224] 20. The method of any one of Examples 10 to 19, wherein the at least one reference titration curve comprises a single titration curve, and wherein pH 1_reference = pH initial_reference, and pH 2_reference = pH final_reference.

[0225] 21. The method of any one of Examples 10 to 19, wherein the at least one reference titration curve comprises a plurality of reference titration curves.

[0226] 22. The method of Example 21, wherein each reference titration curve includes an initial pH reference and a final pH reference, and wherein: [(a)]pH 1_Reference is the initial pH of one of the plurality of reference titration curves. [(b)]The pH 2 reference is the final pH reference from one of the plurality of reference titration curves, and [(c)] Where pH 1_reference and pH 2_reference are selected to cover the greatest difference in values, while still covering the pH values ​​covered by all of the plurality of reference titration curves.

[0227] 23. The method of any one of Examples 19 to 22, wherein the initial pH (pH initial) of the sample is approximately the same as that of pH 1 reference.

[0228] 24. The method of any one of Examples 19 to 22, wherein the initial pH (pH initial) of the sample is different from that of pH 1 reference.

[0229] 25. The method of Example 24, wherein the difference between the initial pH and the pH 1 reference is about 0.05 to 1, about 0.1 to 1, about 0.1 to 0.5, or about 0.1 to 0.3 pH units.

[0230] 26. The method of any one of Examples 19 to 25, wherein the final pH (pH final) of the sample is approximately the same as that of the pH 2 reference.

[0231] 27. The method of any one of Examples 19 to 25, wherein the final pH is different from the pH 2 reference.

[0232] 28. The method of Example 27, wherein the final pH value is about 0.05 to 1, about 0.1 to 1, about 0.1 to 0.5, or about 0.1 to 0.3 pH units from the pH value to the pH 2 reference.

[0233] 29. The method of any one of Examples 19 to 28, wherein the initial pH, initial pH_reference, and pH 1_reference are approximately the same, and wherein the final pH, final pH_reference, and pH 2_reference are approximately the same.

[0234] 30. The method of any one of Examples 1 to 29, wherein the final pH (pH final) of the sample is less than the initial pH (pH initial) of the sample, and the titrant is an acid.

[0235] 31. The method of Example 30, wherein the amount of titrant added to the reference sample is standardized to a scale of about -0.76 to about 1.49.

[0236] 32. The method of Example 30 or 31, wherein the pH 1 reference is between about 4.0 and 4.3, and the pH 2 reference is between about 3.4 and 3.9.

[0237] 33. The method of Example 30 or 31, wherein pH 1 is referenced at about 4.1 and pH 2 is referenced at about 3.7.

[0238] 34. The method of any one of Examples 30 to 32, wherein the initial pH is between about 4.0 and 4.5, between about 4.1 and 4.5, between about 4.2 and 4.5, between about 4.3 and 4.5, between about 4.1 and 4.4, or between about 4.2 and 4.4.

[0239] 35. The method of any one of Examples 30 to 34, wherein the pH is ultimately between about 3.0 and 3.8, between about 3.1 and 3.8, between about 3.2 and 3.8, between about 3.3 and 3.7, between about 3.4 and 3.7, or between about 3.5 and 3.7.

[0240] 36. The method of any one of Examples 30 to 34, wherein the final pH is about 3.6.

[0241] 37. The method of any one of Examples 30 to 36, wherein the model comprises a polynomial.

[0242] 38. The method of Example 37, wherein the model comprises a fourth-order polynomial of the following: (Equation 18)

[0243] 39. The method of embodiment 37 or 38, wherein the polynomial comprises: (Equation 19).

[0244] 40. The method of any one of Examples 1 to 29, wherein the final pH (pH final) of the sample is greater than the initial pH (pH initial) of the sample, and the titrant is a base.

[0245] 41. The method of Example 40, wherein the amount of titrant added to the reference sample is standardized to a scale of about -0.06 to about 1.53.

[0246] 42. The method of Example 40 or 41, wherein pH 1_ref is between about 3.0 and 3.8 or between about 3.1 and 3.8, between about 3.2 and 3.8, between about 3.3 and 3.7, between about 3.4 and 3.7 or between about 3.5 and 3.7, and pH 2_ref is between about 5.3 and 8.5, between about 5.1 and 8.1, between about 5.5 and 8.0 or between about 7.5 and 8.0.

[0247] 43. The method of Example 40 or 41, wherein pH 1 is a reference of about 3.7 and pH 2 is a reference of about 7.6.

[0248] 44. The method of any one of Examples 40 to 43, wherein the initial pH is between about 3.0 and 3.8, between about 3.1 and 3.8, between about 3.2 and 3.8, between about 3.3 and 3.7, between about 3.4 and 3.7, or between about 3.5 and 3.7.

[0249] 45. The method of any one of Examples 40 to 44, wherein the pH is ultimately between about 5.3 and 8.5, between about 5.1 and 8.1, between about 5.5 and 8.0, or between about 7.5 and 8.0.

[0250] 46. ​​The method of any one of Examples 40 to 45, wherein the model comprises a polynomial.

[0251] 47. The method of Example 46, wherein the model comprises a 5th-order polynomial of the following: (Equation 20).

[0252] 48. The method of embodiment 46 or 47, wherein the polynomial comprises: The standardized titrant n = 12.256725 - 10.723277*pH n + 3.3662386*pH n^2 - 0.4588175*pH n^3 + 0.0255417*pH n^4 - 0.0003153*pH n^5 (Equation 21).

[0253] 49. The method of any one of Examples 1 to 48, wherein calibrating the pH meter includes sending a signal to an operator indicating that the pH probe is faulty.

[0254] 50. The method of any one of Examples 1 to 49, wherein calibrating the pH meter includes recalibrating or replacing the pH probe.

[0255] 51. The method of any one of Examples 1 to 50, wherein the pH of the sample is measured using a pH probe inserted into a subsample removed from the sample, a separate sampling slipstream, or a continuous slipstream.

[0256] 52. The method of any one of Examples 1 to 50, wherein the pH of the reference sample is not measured using a pH probe directly inserted into the sample.

[0257] 53. The method of any one of Examples 1 to 52, further comprising, before measuring the initial pH in step (a), comparing the initial pH with a previously measured initial pH for at least one reference sample, and correcting the pH meter when the difference between the initial pH and the previously measured initial pH indicates a calibration error in the pH meter.

[0258] 54. The method of any one of Examples 1 to 53, further comprising comparing the initial pH value from the sample with an initial pH value predicted by a statistical titration model, and correcting the pH meter when the difference between the initial pH value and the initial pH value predicted by the statistical titration model indicates a calibration error in the pH meter.

[0259] 55. The method of any one of Examples 1 to 54, further comprising comparing the pH change from pH initial to pH n with the pH change predicted by a statistical titration model, and correcting the pH meter when the difference between the measured pH change and the pH change predicted by the statistical titration model indicates a calibration error in the pH meter.

[0260] 56. The method of any one of Examples 1 to 55, wherein the sample contains a first protein of interest and the at least one reference sample contains a second protein of interest.

[0261] 57. The method of Example 56, wherein the first protein of interest is the same as the second protein of interest.

[0262] 58. The method of Example 56, wherein the first protein of interest is different from the second protein of interest, but both react similarly with respect to the addition of the titrant to the sample and the reference sample.

[0263] 59. The method of any one of Examples 56 to 58, wherein the method prevents damage to the protein of interest caused by the addition of excessive titrant to the sample.

[0264] 60. An apparatus configured to perform the method of any one of Embodiments 1 to 59.

[0265] 61. The apparatus of embodiment 60, comprising: One reactor; A pH flow channel includes a pH probe disposed therein, the pH flow channel being fluidly coupled to the reactor, the pH flow channel being configured to receive a sample flow from the reactor and measure the pH of the flow; An acid titrant supply, fluidly coupled to the reactor, is configured to provide acid titrant to the reactor to lower the pH in the reactor; and / or An alkaline titrant supply, fluidly coupled to the reactor, is configured to provide alkaline titrant to the reactor to increase the pH in the reactor.

[0266] 62. An apparatus comprising: One reactor; A pH flow channel includes a pH probe disposed therein, the pH flow channel being fluidly coupled to the reactor, the pH flow channel being configured to receive a sample flow from the reactor and measure the pH of the flow; An acid titrant supply, fluidly coupled to the reactor, is configured to provide acid titrant to the reactor to lower the pH in the reactor; and / or An alkaline titrant supply, fluidly coupled to the reactor, is configured to provide alkaline titrant to the reactor to increase the pH in the reactor.

[0267] 63. The device of embodiment 61 or 62, further comprising: A sampling pump is configured to deliver the slipstream from the reactor to the pH flow channel.

[0268] 64. The device of embodiment 61 or 62, further comprising: A waste receiver configured to receive effluent from the pH flow channel.

[0269] 65. The device of embodiment 61 or 62, further comprising: An acid titrant pump, configured to supply and deliver the acid titrant to the reactor; and An acid titrant flow meter is configured to measure the flow rate of the acid titrant supplied to the reactor.

[0270] 66. The device of embodiment 61 or 62, further comprising: A base titrant pump, configured to supply and deliver the base titrant to the reactor; and An alkaline titrant flow meter is configured to measure the flow rate of the alkaline titrant supplied to the reactor.

[0271] 67. The apparatus of any one of Examples 61 to 66 further includes a controller that communicates with the acid titrant flow meter, the alkali titrant flow meter, the pH probe, the acid titrant pump and the alkali titrant pump.

[0272] 68. The device of Example 67, wherein the controller is configured to: (a) The controller receives a signal from one of the acid titrant flow meters, thereby determining the amount of acid titrant added to the sample; (b) Receives a signal from one of the alkaline titrant flow meters, thereby the controller determines the amount of alkaline titrant added to the sample; (c) Receive a signal from one of the pH probes, thereby transmitting a pH measurement value to the controller, and the controller associates the pH measurement value with the corresponding amount of acid or alkali titrant added to the sample; (d) Send a signal to the acid titrant pump to start the pump, stop the pump, or change the pump speed; and (e) Send a signal to the alkali titrant pump to start the pump, stop the pump, or change the pump speed.

[0273] 69. The device of embodiment 67 or 68, wherein the controller communicates with the sampling pump, wherein the controller is configured to send a signal to the sampling pump to start the pump, stop the pump, or change the pump speed.

[0274] 70. The apparatus of any one of Examples 67 to 69, wherein the controller is configured to apply a model to the pH measurement and the corresponding amount of acid or base titrant added to the sample.

[0275] 71. The apparatus of any one of embodiments 67 to 70, wherein the controller is configured to start the acid titrant pump and add a predetermined amount of acid titrant when the pH measured in the pH flow cell is greater than the desired value, and the controller is further configured to start the alkali titrant pump and add a predetermined amount of alkali titrant when the pH measured in the pH flow cell is less than the desired value.

[0276] 72. The apparatus of any one of Examples 67 to 71, wherein the controller is configured to send a signal to the acid titrant pump to stop the pump when a predetermined amount of acid titrant has been added to the sample, and to send a signal to the alkali titrant pump to stop the pump when a predetermined amount of alkali titrant has been added to the sample.

[0277] 73. The device of any one of Examples 67 to 72, wherein the controller is configured to maintain the pH value for a desired period of time.

[0278] 74. The device of Example 73, wherein the required value changes over time in a manner consistent with pH sequence.

[0279] 75. The apparatus of Example 74, wherein the pH sequence is adapted to inactivate viruses that may be present in the reactor.

[0280] 76. The apparatus of Example 75, wherein the pH sequence comprises (a) To lower the pH to a first target pH between approximately 3.0 and 3.8, between approximately 3.1 and 3.8, between approximately 3.2 and 3.8, between approximately 3.3 and 3.7, between approximately 3.4 and 3.7, or between approximately 3.5 and 3.7. (b) Maintain the pH value at the first target pH for a period of time. (c) Raise the pH to a second target pH between about 5.3 and 8.5, between about 5.1 and 8.1, between about 5.5 and 8.0, or between about 7.5 and 8.0, and (d) Maintain the pH at the second target pH.

[0281] 77. The apparatus of Example 76, wherein lowering the pH in step (a) or raising the pH in step (c) comprises adding one or more amounts of titrant sufficient to change the pH of the sample and measuring the pH of the sample.

[0282] 78. The apparatus of any one of embodiments 61 to 77, further comprising: A check valve is fluidly coupled between the reactor and the pH flow channel, and the check valve is configured to prevent the reactor from being contaminated by backflow from the pH flow channel.

[0283] 79. The apparatus of any one of Examples 61 to 78, wherein the reactor does not include a pH measuring probe disposed therein.

[0284] 80. The apparatus of any one of Examples 61 to 79 further comprises a mixer disposed in the reactor, configured to mix the contents in the reactor at the time of, during and after the addition of the acid titrant and / or the alkali titrant.

[0285] 81. The apparatus of Example 80, wherein the controller communicates with the mixer, and wherein the controller is configured to send a signal to the mixer before turning on the acid pump or the alkali pump to start the mixer.

[0286] 82. The apparatus of Example 81, wherein the controller is configured to send a signal to the mixer, thereby causing the mixer to stop for a fixed period of time after the acid pump or alkali pump is stopped.

[0287] 83. The device of any one of embodiments 61 to 82, further comprising a user interface configured to receive and display pH measurements from the controller.

[0288] 84. The apparatus of Example 83, wherein the user interface is configured to send a signal to the controller, thereby the controller sending a signal to the acid titrant pump or the alkali titrant pump to add a predetermined volume of acid or alkali titrant to the sample.

[0289] 85. The device of embodiment 83 or 84, wherein the user interface is configured to send a signal to the controller, thereby allowing the user to issue a command to the controller to advance a step in the pH sequence.

[0290] 86. The device of any one of Examples 83 to 85, wherein the user interface is configured to receive one or more offline pH measurements from the user, wherein the one or more offline pH measurements include pH measurements of the sample independent of the pH probe disposed in the pH flow cell.

[0291] 87. The device of any one of Examples 83 to 86, wherein the user interface is configured to display a warning signal when the pH measurement from the pH probe differs from the corresponding pH predicted by a model.

[0292] 88. The device of Example 87, wherein the warning signal is displayed when the difference between the pH measurement value and the model is > 0.01 pH unit, > 0.02 pH unit, > 0.03 pH unit, > 0.04 pH unit, > 0.05 pH unit, > 0.06 pH unit, > 0.07 pH unit, > 0.08 pH unit, > 0.09 pH unit, or > 0.10 pH unit.

[0293] 89. The device of Example 87, wherein the warning signal is displayed when the difference between the pH measurement value and the model is >0.05 pH units.

[0294] 90. The device of any one of Examples 67 to 89, wherein the controller is configured to automatically terminate the pH sequence when the pH measurement from the pH probe differs from the corresponding pH predicted by a model.

[0295] 91. The apparatus of any one of Examples 67 to 90, wherein the volume of the acid titrant or alkali titrant delivered to the reactor has an error percentage of 10% or less. [Example] [, Example , ] [, 1 , ] [, : Reduce protein solution , ] [, pH , ] [, Modeling of value titration , ] [, , ]

[0296] Cells exhibiting five different proteins were grown in a bioreactor, and the proteins were secreted into the cell culture medium. After an initial harvesting step involving the removal of cells and cell debris, the proteins were captured using a protein A chromatography system, washed, and dissociated. The dissociated solution containing partially purified proteins was then transferred to an automated system to inactivate the virus by lowering the pH to 3.6.

[0297] To measure pH, a Mettler Toledo InPro 3253 pH probe was calibrated and then sterilized in a sealed bellows with a Kleenpak connector via autoclave or gamma irradiation to allow aseptic connection to a pool container containing a partially purified protein solution. The pH probe was inserted into the pool container, and acid was added until a target pH of 3.6 was achieved. Direct insertion of the pH probe into the pool container allows for feedback control of the titration to regulate the final pH. In this example, titrant was administered continuously, and pH was measured continuously. For 11 viral operations spanning 5 different proteins, pH was measured against the amount of acid added, and the results are plotted in Figure 1. In Figure 1, the amount of alkali added is indicated by the number of pump rotations per kilogram of product in the container before any titrant was added, and pump speed data has been time-shifted to account for the delay between alkali addition and pH reaction. As can be seen in Figure 1, all titration curves are visually similar in shape. The titration curves are essentially linear, with a slightly downward curve observed. The titration curves also differ in terms of their X and Y intercepts.

[0298] A linear transformation of the axis is applied to the titration curve shown in Figure 1.

[0299] Transform the Y-axis (pH) to a scale of 1 → 0 (initial pH → final pH) using the following equation: (Equation 1). Transform the X-axis (the added acid) to a scale of 0→1 (initial→final) using the following equation: (Equation 2). A linear transformation of the two axes that causes convergence at two points on each curve should result in the contraction of all curves, as shown in Figure 2. The transformed data shown in Figure 2 is modeled to produce a second-order polynomial: Standardized pH v1 = 1.0584433-1.0049047*Standardized titrant addition v1 - 0.214062*(Standardized titrant addition v1-0.56403)^2 (Equation 3). An overview of polynomial fitting is shown in Table 1 below: Table 1. Overview of Fitting R-squared value 0.996943 R-squared value adjustment 0.996936 Root mean square error 0.017471 average reaction value 0.470191 Observations (or weighted sum) 830 The RMSE (root mean square error) of the quadratic fit is 0.0175 in standardized pH units, which is approximately: 0.011 pH (Equation 4). The initial and final pH values ​​varied slightly between operations. [, Example , ] [, 2 , ] [, : Increase the protein solution , ] [, pH , ] [, Modeling of value titration , ] [, , ]

[0300] After inactivating the virus, the pH of the solution was raised to neutral or near-neutral by adding an alkaline solution, using the same method described in Example 1.

[0301] For 12 viral inactivation operations spanning 7 different proteins, the amount of alkali added was measured to compare pH levels, and the results are plotted in Figure 4. In Figure 4, the amount of alkali added is indicated by the number of pump rotations per kilogram of product in the container before any titrant is added, and the pump speed data has been time-shifted to account for the delay between alkali addition and pH reaction. As can be seen in Figure 4, the curves have similar shapes, with a consistent inflection point around pH 6. The position of this inflection point on the X-axis is variable.

[0302] A linear transformation of the axes is applied to the titration curves shown in Figure 4. Theoretically, a linear transformation that causes convergence at two select points on each curve should result in all curves shrinking into a single curve. The initial method is based on the acid titration model described in Example 1 above.

[0303] In this first method, the Y-axis (pH) is transformed to a 0 → 1 scale (initial pH → final pH) using the following equation: (Equation 5).

[0304] Transform the X-axis (the added alkali) to a scale of 0→1 (initial→final) using the following equation: (Equation 6)

[0305] The result of this transformation is shown in Figure 5. As can be seen in Figure 5, this initial standardization method did not cause the alkali titration curve to shrink to the extent that the acid titration curve in Example 1 was (compare Figure 5 with Figure 2).

[0306] One explanation for this variation is that while the target pH for acid titration is the same across the protein system (pH 3.6), the target pH for base titration varies between proteins, ranging from 7.7 to 8.0, depending on the protein. Therefore, the final data point of the titration curve should not converge under standardization.

[0307] Therefore, the second standardization method takes into account that the titration endpoint should not converge. In this second method, the Y-axis is set to 0 at time = 0, and to 1 for pH 7.60, which is close to but less than the target pH. Similarly, the X-axis is fixed at 0 for time = 0, and to 1 for the amount of titrant required to reach pH 7.60.

[0308] In this second method, the Y-axis (pH) is transformed to a scale of 0 → 1+ (initial → pH 7.60) using the following equation: (Equation 7).

[0309] Transform the X-axis (added base) to a 0 → 1+ scale (initial → pH 7.60) using the following equation: (Equation 8)

[0310] The results of this second transformation are shown in Figure 6. As shown in Figure 6, the convergence at time = 0 and pH = 7.60 substantially improves the fit. However, the two titration curves still deviate from the rest of the curves.

[0311] This deviation could be caused by a problem with the pH probe calibration. For example, the pH probe used to generate the titration curves is sterilized in a sealed bag using an autoclave or gamma irradiation. Sterilization is performed after calibration but before use so that the probe dries for a period of time between calibration and its first measurement. Problems with the pH probe could cause these two titration curves to deviate from the model. Offline pH measurements were taken at the beginning and end of all pH titration operations. That is, at the beginning and end of each operation that decreased or increased the pH, a small amount of protein solution was removed from the tank, and the pH was measured separately using an unsterilized probe. As shown in Figure 7, the two operations with the largest deviation from the model also showed the largest differences in the initial online and offline measurements, indicating that the problem is most likely caused by the probe used to generate these two titration curves.

[0312] Problems with online pH measurements can be corrected using two offline measurements (initial and final) via a linear transformation. This is equivalent to post-hoc two-point pH normalization. A third normalization method considers (1) the titration endpoints not converging and (2) the difference between online and offline measurements. The corrected online pH is calculated using the following equation: (Equation 9) The results are shown in Figure 8. As shown in Figure 8, the correction for the online pH value improves the fit.

[0313] A fourth method was used to further tighten the fit. Because the different operations had slightly different initial pH values, the different titration curves were forced to converge at two midpoints, rather than a single midpoint (pH 7.60) as in previous attempts. Convergence was forced at two midpoints: pH 3.70 and 7.60. No pH transformation (Y-axis) was needed because convergence was forced at two fixed pH values. The amount of alkali added (X-axis) was transformed to approximately a 0 → 1+ scale (pH 3.70 → pH 7.60) using the following calculations: (Equation 10).

[0314] The results are shown in Figure 10. This corrected and transformed dataset was used to generate a model for adding alkali (line, Figure 10). Because the shape of the alkali addition titration curve is more complex than that of the acid addition titration curve, a 6+ order polynomial is required for fitting. pH (online, corrected) = 2.5460075 + 5.5157799 * titrant - 0.1694419 * (titer - 0.63949)^2 - 11.009696 * (titer - 0.63949)^3 + 2.1806629 * (titer - 0.63949)^4 + 13.771715 * (titer - 0.63949)^5 - 7.7443576 * (titer - 0.63949)^6 (Equation 11). (The titrant value is a standardized value derived from Equation 10).

[0315] The alkali-adjusted model has an RMSE of 0.080 pH units. An overview of the fit is shown in Table 2 below.

[0316] Table 2. Overview of Fitting R-squared value 0.997091 R-squared value adjustment 0.997069 Root mean square error 0.079609 average reaction value 6.035137 Observations (or weighted sum) 773

[0317] In summary, the methods described in Examples 1 and 2 enable accurate automatic pH adjustment using a discontinuous flow measurement method. Small samples can be taken when adjusting the pH, and the pH model is used to determine with high accuracy how much additional acid or base should be added to achieve the target pH, without the need for continuous sampling or measurement. Titration curves for modeling can be developed using as few as one experimental dataset. This method also allows titration to any pH value within the training dataset. Finally, pH control accuracy can be improved by adding additional intermediate sampling points. [, Example , ] [, 3 , ] [, Used for , ] [, pH , ] [, Development of adjustment and control systems , ] [, , ]

[0318] A system was developed to measure pH during virus inactivation, which uses a pH probe inserted into a discontinuous slipstream and a dimensionless model linking the pH to a titrant added to the sample. An exemplary list of components used in this system is shown in Figure 11. A diagram of the system is shown in Figures 19-20.

[0319] In this example of the system, the addition of acid and base titrants to the reaction vessel containing the sample is controlled by separate Watson Marlow 530 pumps connected to a Sonotec CO. 55 ultrasonic flow meter. The pumps receive the titrant from a tubing connected to the titrant bag and then deliver it to the reaction vessel via the flow meter. A sampling line from the reaction vessel is connected to the sampling pump via a check valve to prevent contamination of the reaction vessel in case of incorrect installation of the sampling assembly. The sampling pump delivers the sample from the reaction vessel to a pH flow channel containing a pH probe. The pH probe is connected to a pH transmitter. The sample is then delivered from the flow channel to a waste receiver. All pumps are connected to a controller, allowing the controller's programmed control logic to control the volume and flow rate of the acid and base titrants, as well as whether and how much sample is drawn from the reaction vessel for analysis.

[0320] The advantages of this system include the following: When the probe is inserted into the reaction vessel, it cannot be calibrated after sterilization (and before insertion). Sterilization can affect the probe calibration profile. In this system, the probe is inserted into a separate flow channel and sterilization is not required. There is no need for an autoclave or Kleenpak aseptic connection port, eliminating the risk of probe breakage or leakage of probe solution into the protein product within the reaction vessel. The sampling pump and check valve generate discrete samples for pH measurement, thereby reducing product waste. The system can also be connected to any reaction vessel via sampling lines, making it highly flexible.

[0321] An example of the acid conditioning workflow is shown in Figure 12. After equipment setup, including probe calibration and automatic flow meter zeroing, an initial pH measurement is automatically performed before any addition of acid titrant, and a first volume of acid titrant is added to the sample. The volume added is controlled using feedback from the flow meter. This initial amount of acid is typically conservative to ensure that the target pH is not exceeded. Acid is added at a constant rate (mL of acid per kg of protein pool in the reaction vessel), allowing the calculation of the total initial addition volume. Then, after the full volume of titrant from this initial addition has been added, the pH is automatically measured, and the initial and measured pH values, along with the amount of acid added to obtain the second pH value, are recorded.

[0322] The model is fed in to determine the corresponding dimensionless titrant addition value. An intermediate target pH is selected between the final target pH and the pH after the first titrant addition, and the corresponding dimensionless titrant addition value is calculated using the model. The amount of dimensionless titrant to be added to the sample to reach this intermediate pH is calculated according to the following formula: (Equation 15).

[0323] In this formula, the standardized titrant is always the total amount added to the sample after standardization to achieve the intermediate target pH. The initial standardized titrant is the amount of titrant added to the sample after standardization to achieve the initial pH (this value can be 0 before standardization), and the standardized titrant n is the amount of titrant added to the sample to achieve the first intermediate pH, standardized using a model. It should be noted that the model is also used to calculate the initial standardized titrant. Using the two standardized titrants and the actual (dimensionary) amount of titrant added, conversion between standardized and unstandardized titrants is possible. After maintaining a low pH to inactivate the virus, a series of similar steps are used to adjust the pH back to neutral. Add a second volume of acid, repeat these steps to verify the accuracy of the model and pH meter, and then add the final volume of acid to achieve the target pH.

[0324] A similar workflow for raising pH is shown in Figure 13. After the flow meter is automatically zeroed, a first volume of alkali titrant is added to the sample, the pH is measured, and the first volume is calculated using a linear function relating the added alkali (mL / kg) to the target neutral pH. The initial pH, the measured pH, and the volume of alkali titrant are fitted to the model. A second volume of alkali is added, and these steps are repeated to verify the accuracy of the model and the pH meter, followed by the addition of the final volume of alkali to achieve the target pH.

[0325] In these processes, after each measurement, the slipstream pH value is compared to the expected value. If the measured pH exceeds the expected range, the user is prompted to obtain a sample, measure the offline pH on an independently calibrated pH meter, and input the offline value into the model. If the two measurements differ by >0.10 pH units, the user should complete the process using the offline pH measurement, employing conservative volume and additional measurement steps. During the acid adjustment step, conservative additions are made to compensate for potential inaccuracies in the initial automated pH measurement from the online pH probe.

[0326] Although the process remains automated, instead of automatically sampling pH after each titrant addition, the user interface prompts the user to sample, for example, using an independent pH probe to measure pH offline, and inputting the offline value into the user interface. In other words, if it is determined midway through the adjustment steps (e.g., during acid adjustment after the first acid addition), a miscalibrated online probe in the pH flow cell is used, and the remainder of the adjustment steps (e.g., acid adjustment) is performed using conservative addition and offline pH measurement. In this scenario, alkali adjustment is also performed using a dimensionless titration model, but the user should continue to use an independent pH probe and input the offline pH value in the user interface, instead of measuring the pH value in the flow cell. The model is then applied to the offline pH value to conclude the pH sequence.

[0327] If the difference is ≤0.10 pH units, the process can continue using the slipstream pH measurement method. An overview of the process steps, volume calculation methods, and acceptable online pH is shown in Table 3 below: Table 3. Acceptance Criteria for Online pH Process steps Add volume calculation method Acceptable online pH Protein pool pH measurement N / A 4.00-4.50 Add #1 Conservative addition Acid - constant mL acid / kg capture tank Alkali - mL / kg target neutral pH linear function ∆pH within the possible range predicted by the statistical VI model Add #2 Dimensionless titration model Target: Half of the final pH The difference from the target is ≤ 0.10 pH units. Add #3 Dimensionless titration model Target: Final pH The difference from the target is ≤ 0.10 pH units. Table 3 applies to pH sequences with three addition steps. For example, if four (or more) additions are used, the target pH for each addition should be adjusted accordingly relative to the final pH. [, Example , ] [, 4 , ] [, Used in online manufacturing processes , ] [, pH , ] [, Calibration of the meter , ] [, , ]

[0328] One issue that needs to be addressed with the current system is pH probe calibration and how to perform calibration externally to the pH transmitter. Industrial pH transmitters (such as the Mettler Toledo M400) are typically limited to a 2-point calibration process. However, a 4-point calibration across pH 2 to 10 to ensure accuracy is desirable, and the standard procedure uses an offline probe.

[0329] Therefore, a 4-point calibration process for online probes was developed, which accepts the same calibration procedures as those accepted by offline probes.

[0330] Develop a calibration procedure that operates within the system's control logic (e.g., via MATLAB) instead of using a transmitter calibration function. In this procedure, instead of a calculated pH signal, the transmitter sends raw probe mV and probe temperature signals to the controller. The control logic guides the user through the calibration steps and records the mV and temperature of each buffer standard. The following calibration acceptance criteria are used: Slope: 95 - 105% Offset: ± (0 - 15 mV) Temperature: All standards were set at 20-25℃ Linearity test: After calibration, all standards are within 0.05 pH units.

[0331] During calibration, temperature compensation is also calculated within the control logic to account for the temperature dependence of the pH of the buffer standard. Temperature compensation is also calculated during in-process measurements during the titration process. An exemplary online probe calibration curve is shown in Figure 14. [, Example , ] [, 5 , ] [, In the absence , ] [, pH , ] [, Standardized for , ] [, pH , ] [, Additional models for adjustment , ] [, , ]

[0332] Another model was developed that correlates titrants with pH, ​​but it does not use pH normalization for the addition of acids or bases.

[0333] In this method, for acid addition, two pH values ​​are selected from the historical titration curves described in Example 1, offset from the endpoint of the titration curve. These pH reference values ​​(pH 1 and pH 2) are selected so that they are as far apart as possible, while still being included in each reference titration curve. The standardized titrant is then calculated using the following equation: Standardized titrant = (titrant added - titrant added at reference pH 1) / (titrant added at reference pH 2 - titrant added at reference pH 1) (Equation 12). For the acid titrator model, reference pH 1 is 4.1 and reference pH 2 is 3.7. For the alkali titrator model, reference pH 1 is 3.7 and reference pH 2 is 7.6.

[0334] This standardized titrant is in the range of -0 to +1. The dataset used to generate the acid-adjustment model ranges from -0.76 to 1.49, which produces a scale for standardizing the acid titrant. The dataset used to generate the base-adjustment model ranges from -0.06 to 1.53, which produces a scale for standardizing the base titrant. The ranges used depend on the variability of the initial and final pH values ​​in the dataset. The two reference pH values ​​used for standardization are chosen to be as far apart as possible, while remaining within the limits of the reference titration curve set. One advantage of this method is that the final pH of the sample can be anywhere along the titration curve generated from the reference sample, as long as it is contained within the reference titration curve. For example, the final pH used for acid adjustment can be greater than or equal to the final pH of the reference. Similarly, the final pH used for base adjustment can be less than or equal to the final pH of the reference.

[0335] This standardization strategy yields the following fourth-order polynomial, used to standardize the acid titrant during acid addition: Standardized acid titrant addition = 283.35764 - 279.43987*pH + 104.25395*pH^2 - 17.257125*pH^3 + 1.0589067*pH^4 (Equation 13).

[0336] A similar model with a fifth-order polynomial was used to standardize the alkali titrant during alkali addition: Standardized addition of alkali titrant = 12.256725 - 10.723277*pH + 3.3662386*pH^2 - 0.4588175*pH^3 + 0.0255417*pH^4 - 0.0003153*pH^5 (Equation 14).

[0337] Performance was tested using the equipment described in Example 3 and the model described herein in a small-scale protein pool. The accuracy of the slipstream pH measurement and ultrasonic flowmeter was tested. Results from five test operations are shown in Figure 15. For each of the five test operations, ΔpH and given error (%) by process step are plotted in Figure 16.

[0338] As shown in Figure 15, for all addition steps, the pH difference observed between the online and offline probes was 0.05 or less. Except for the final alkali addition in the first test operation, the yield error was generally less than 5%. Figure 16 shows the data from Figure 15, with the difference between online and offline pH at the top and the yield error at the bottom, plotted sequentially across viral pH levels for inactivation. The yield error is the error in the volume of titrant added at each step, reported as the difference between the target volume and the actual volume.

[0339] The impact of the flow-through cell on the pH probe performance was also analyzed during the testing process, comparing the online probe, offline probe, and online probe used in this method that measures outside the flow-through cell. In this case, there are four acid addition steps because the offline pH is not within the range after the third acid addition step.

[0340] For each of the three probe conditions, the pH relative to the titrant addition steps is plotted in Figure 17. After each flow-through pH measurement, during the test operation, the probe was removed from the flow channel and directly inserted into the sample to evaluate the effect of the flow channel on the measurement. As shown in Figure 17, pH measurements performed by an online probe in the flow channel, an online probe outside the flow channel, and an offline probe show good correspondence. A real-time visual representation of one of the test procedures using a 55.1 kg protein pool is shown in Figure 18. [, Example , ] [, 6 , ] [, Add using an automated titration system and model , ] [, 3 , ] [, or , ] [, 4 , ] [, Second , ] [, pH , ] [, Adjustment , ] [, , ]

[0341] The automated titration system described in Example 3 was used to inactivate 18 batches of proteins at low pH, representing seven different protein products. Using the model described in Example 5 (without pH normalization), the amount of titrant during the pH adjustment process was correlated to the pH. The goal in inactivating the 18 batches of proteins was to achieve a pH less than 0.10 pH units different from the final target pH when the pH was lowered to inactivate the latent virus and then raised back to neutral after inactivation.

[0342] When acid or base was added 3 or 4 times in each adjustment step, all 18 batches of proteins met the requirement of a pH difference of <0.10 pH units from the target pH, based on the offline reference probe used for the acid and base adjustment steps. The results are shown in Figure 21, plotted as the difference between the pH measured by the offline reference probe and the target pH after acid (left) or base (right) adjustment. Acid or base was added in 3-times (circles) or 4-times (crosses), respectively. The target acid pH for virus inactivation was between 3.50 and 3.60. The target neutralization pH after virus inactivation was between 5.50 and 8.00, depending on the protein.

[0343] When three additions were used in each adjustment step, all protein batches were within <0.10 pH units of the target pH after the addition of acid or base (Figure 21). However, when the pH was adjusted to neutral after a low-pH virus inactivation step, the more stringent target of a final pH within <0.05 pH units of the target pH was consistently not met, according to online control probes, when three additions were used in each adjustment step (Figure 22, right).

[0344] A four-addition strategy was implemented to improve the accuracy of the method. Using this revised method, the third addition of acid or base adjusts the pH to within 0.05 to 0.10 pH units of the target pH. A small fourth addition is then performed to accurately achieve the target pH. As shown in Figures 21 and 22, the four-addition strategy improves the accuracy of the method, ensuring that both acid and base adjustments consistently achieve a final pH within 0.05 pH units of the target pH. As shown in Figure 22, all 13 batches adjusted using the four-addition strategy met the target of less than 0.05 pH units from the online probe.

[0345] The automated system is also capable of accurately measuring the pH of protein samples throughout the virus inactivation process. When determining the difference between the pH measured by the offline reference probe and the pH measured by the online control probe at each addition step, 147 out of 151 discrete pH measurements were found to be within a 0.05 pH unit difference between the offline reference probe and the online control probe inserted in the flow cell (Figure 23). Therefore, the model can accurately determine the amount of acid or base added at each step, and for any given addition step, the system can add the required amount of acid or base to consistently produce a pH change within 0.05 pH units of the target pH during the acid or base titration process.

[0346] Furthermore, the automated system can accurately add the volume of acid or base titrant determined by the model. As shown in Figure 24, the titrant volume added in 133 out of 133 additions has an error of less than 10%.

[0347] 100: Equipment 110: Reactor 120: pH flow tank 121: pH probe 130: Supply of acid titrant 140: Supply of alkaline titrant 150: Waste Receiver 200: Equipment 210: Reactor 220: pH flow tank 221: pH probe 222: Sampling pump 224: Valve 230: Supply of acid titrant 232: Acid titrant pump 234: Acid Titrator Flow Meter 240: Supply of alkaline titrant 242: Alkali Titration Pump 244: Alkali Titrant Flow Meter 250: Waste Receiver 260: Controller 262: User Interface

[0348] none

Claims

1. A method for calibrating and correcting a pH meter, comprising: generating at least one reference titration curve from at least one reference sample to correlate the amount of titrant added to the at least one reference sample to the pH of the at least one reference sample; normalizing the at least one reference titration curve; generating a model to fit the at least one reference titration curve; measuring the initial pH of the sample (pH initial); adding a first amount of titrant (titer n) to the sample and measuring a first additional pH value (pH n), wherein titrant n is the amount of titrant added to the sample to achieve pH n, and pH n is different from pH initial; applying the model to determine a second amount of titrant (titer n+1) to achieve a target pH value based on pH initial, titrant n, and pH n; and adding the second amount of titrant (titer n+1) to the sample. After adding the second amount of titrant (titrant n+1), measure the second pH of the sample to determine the measured pH n+1; and when the difference between the measured pH n+1 and the target pH value indicates a calibration error in the pH meter used to measure pH n+1, calibrate the pH meter.

2. As in claim 1, wherein the difference between the measured pHn+1 and the target pH value is > 0.01 pH unit, > 0.02 pH unit, > 0.03 pH unit, > 0.04 pH unit, > 0.05 pH unit, > 0.06 pH unit, > 0.07 pH unit, > 0.08 pH unit, > 0.09 pH unit, or > 0.10 pH unit.

3. The method of request item 1, wherein the difference between the measured pHn+1 and the target pH value is > 0.05 pH units.

4. The method of any one of claims 1 to 3, wherein the target pH value is an intermediate target pH value, and wherein the method further comprises: adding a third amount of titrant (titer n+2) to the sample; measuring the third pH (pH n+2) of the sample; and applying the model to determine a fourth amount of titrant to achieve the target final pH based on the initial pH, titrant n+2, and pH n+2.

5. The method of claim 4, further comprising adding the fourth amount of titrant to the sample and measuring the fourth pH of the sample to determine the measured pHn+3, wherein the difference between the measured pHn+3 and the target final pH is > 0.01 pH unit, > 0.02 pH unit, > 0.03 pH unit, > 0.04 pH unit, > 0.05 pH unit, > 0.06 pH unit, > 0.07 pH unit, > 0.08 pH unit, > 0.09 pH unit, or > 0.10 pH unit.

6. The method of request 4, wherein the difference between the measured pHn+3 and the target final pH is > 0.05 pH units.

7. The method of claim 5, wherein the difference between the measured pHn+3 and the target final pH indicates the calibration error in the pH meter.

8. The method of claim 1, wherein generating the at least one reference titration curve comprises: measuring the initial pH of the at least one reference sample (pH initial_reference); adding a quantitative amount of titrant to the at least one reference sample (titer n_reference) and measuring an additional reference pH value (pH n_reference), titrant n_reference being the amount of titrant added to the at least one reference sample to reach pH n_reference, wherein pH n_reference is different from pH initial_reference; repeating step (ii) until the at least one reference sample reaches a final pH (pH final_reference) by adding the full amount of titrant to the at least one reference sample (titer total_reference); and plotting the amount of titrant added relative to the pH of the at least one reference sample.

9. The method of request item 1, which includes calibration for pH meter calibration when generating the model.

10. The method of claim 9, wherein correcting the pH meter calibration comprises: removing a first portion of the at least one reference sample before the addition of the quantitative titrant, and measuring the pH of the first portion with an independently calibrated pH meter to generate an offline initial pH value (pH initial_offline_reference); removing a second portion of the at least one reference sample after the addition of the full amount of titrant, and measuring the pH of the second portion with the independently calibrated pH meter to generate an offline final pH value (pH final_offline_reference); and applying the relationship between (pH final_offline_reference) and the measured pH value of the at least one reference sample to determine a corrected pH for the at least one reference sample.

11. The method of claim 10, wherein the calibrated pHn_reference of the at least one reference sample is determined by: .

12. The method of claim 10 or 11, wherein the amount of titrant added to the at least one reference sample is standardized by means of: , wherein titrant 1_reference is the amount of titrant added to the at least one reference sample to achieve a first reference pH (pH1_reference), and titrant 2_reference is the amount of titrant added to the at least one reference sample to achieve a second reference pH (pH2_reference).

13. The method of claim 10 or 11, wherein the at least one reference titration curve comprises a single titration curve, and wherein pH1_reference = pH initial_reference, and pH2_reference = pH final_reference.

14. The method of claim 10 or 11, wherein the at least one reference titration curve comprises a plurality of reference titration curves.

15. The method of claim 14, wherein each reference titration curve includes an initial pH reference and a final pH reference, and wherein: pH1_reference is the initial pH reference from one of the plurality of reference titration curves, and pH2_reference is the final pH reference from one of the plurality of reference titration curves. The pH1_reference and pH2_reference are selected to cover the largest difference in values, while still covering the pH values ​​covered by all of the plurality of reference titration curves.

16. The method of claim 12, wherein the initial pH (pH_initial) of the sample is approximately the same as that of pH1_reference, or wherein the initial pH (pH_initial) of the sample is not the same as that of pH1_reference.

17. The method of claim 12, wherein the target final pH of the sample is approximately the same as the pH2_reference, or wherein the target final pH of the sample is not the same as the pH2_reference.

18. The method of claim 12, wherein the initial pH, initial pH_reference, and pH1_reference are approximately the same, and wherein the target final pH, final pH_reference, and pH2_reference of the sample are approximately the same.

19. The method of any one of claims 8 to 11, wherein the target final pH of the sample is less than the initial pH (pH initial) of the sample, and wherein the titrant is an acid.

20. The method of claim 19, wherein the pH1 reference is between about 4.0 and 4.3, and the pH2 reference is between about 3.4 and 3.

9.

21. The method of claim 19, wherein the pH is initially between about 4.0 and 4.5, between about 4.1 and 4.5, between about 4.2 and 4.5, between about 4.3 and 4.5, between about 4.1 and 4.4, or between about 4.2 and 4.

4.

22. The method of claim 19, wherein the target final pH of the sample is between about 3.0 and 3.8, between about 3.1 and 3.8, between about 3.2 and 3.8, between about 3.3 and 3.7, between about 3.4 and 3.7, or between about 3.5 and 3.

7.

23. The method of request item 19, wherein the model comprises a fourth-order polynomial.

24. The method of any one of claims 8 to 11, wherein the target final pH of the sample is less than the initial pH of the sample (pH initial), and the titrant is a base.

25. The method of claim 24, wherein the pH1 reference is between about 3.0 and 3.8 or between about 3.1 and 3.8, between about 3.2 and 3.8, between about 3.3 and 3.7, between about 3.4 and 3.7 or between about 3.5 and 3.7, and the pH2 reference is between about 5.3 and 8.5, between about 5.1 and 8.1, between about 5.5 and 8.0 or between about 7.5 and 8.

0.

26. The method of claim 24, wherein the pH is initially between about 3.0 and 3.8, between about 3.1 and 3.8, between about 3.2 and 3.8, between about 3.3 and 3.7, between about 3.4 and 3.7, or between about 3.5 and 3.

7.

27. The method of claim 24, wherein the target final pH of the sample is between about 5.3 and 8.5, between about 5.1 and 8.1, between about 5.5 and 8.0, or between about 7.5 and 8.

0.

28. The method of request item 24, wherein the model contains a fifth-order polynomial.

29. The method of any of claims 1 to 3 and 8 to 11, wherein calibrating the pH meter includes sending a signal to the operator indicating that the pH probe is faulty.

30. The method of claim 29, wherein calibrating the pH meter includes recalibrating or replacing the pH probe.

31. The method of claim 29, wherein the pH value of the sample is measured by means of a slipstream or a continuous slipstream in which the pH probe is directly inserted into the sample, or in a subsample removed from the sample, or by means of ...

32. The method of any one of claims 9 to 11, further comprising, prior to measuring the initial pH, comparing the initial pH with a previously measured pH for the at least one reference sample, and correcting the pH meter when the difference between the initial pH and the previously measured pH for the at least one reference sample indicates the calibration error in the pH meter.

33. The method of any one of claims 1 to 3 and 8 to 11, further comprising comparing the initial pH from the sample with an initial pH value predicted by a statistical titration model, and correcting the pH meter when the difference between the initial pH and the initial pH predicted by the statistical titration model indicates the calibration error in the pH meter.

34. The method of any one of claims 1 to 3 and 8 to 11, further comprising comparing the pH change from pH initial to pHn with the pH change predicted by a statistical titration model, and correcting the pH meter when the difference between the pH change from pH initial to pHn and the pH change predicted by the statistical titration model indicates the calibration error in the pH meter.

35. The method of any one of claims 9 to 11, wherein the sample contains a first protein of interest and the at least one reference sample contains a second protein of interest.

36. The method of claim 35, wherein the first protein of interest is the same as the second protein of interest, or wherein the first protein of interest is not the same as the second protein of interest, but the reaction is similar for the addition of the titrant to the sample and the at least one reference sample.

37. The method of claim 35, wherein the method prevents damage to the first protein of interest caused by the addition of excessive titrant to the sample.

38. The method of claim 1, wherein the sample contains dupilumab.

39. The method of claim 9, wherein the at least one reference sample contains dupilumab.

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