Systems and methods for pH modeling and control - Patents.com

Abstract

The present disclosure relates to systems and methods for controlling the pH of a sample, including measuring an initial pH, adding an amount of titrant, measuring a second pH, and using non-dimensionalized modeling to normalize the titrant and determine the amount of titrant needed to reach a final pH. The systems and methods can be used to control pH during viral inactivation or titration of protein samples.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 253,281, filed October 7, 2021, the contents of which are incorporated by reference herein in their entirety. [Background technology]

[0002] Conventional methods of measuring and controlling pH during processes that involve altering the pH of a sample can be problematic. Methods of sterilizing pH probes and inserting the probes into samples are frequently incompatible with pH probe calibration. Thus, there is a need in the art for systems and methods for controlling the pH of a sample. Summary of the Invention

[0003] The present disclosure provides systems and methods for measuring and controlling pH in processes that involve altering the pH of a sample, such as a protein sample.

[0004] In some embodiments of the methods of the present disclosure, the method further comprises: (a) determining an initial pH (pH initial (b) measuring at least a first amount of a titrant; n ) is added to the sample and at least a first additional pH value (pH n ), measuring Titrant n But pH n is the amount of titrant added to the sample to reach a pH of n But pH initial (c) Applying the model to obtain the normalized initial amount of Titrant (Titrant initial ) and normalized Titrant n (d) determining a target pH (pH n+1determining an additional amount of titrant to be added to the sample to reach a pH of n+1 is the pH achieved by addition of the entire amount of titrant to the sample.

[0005] In some embodiments of the methods of the present disclosure, the method further comprises administering a second amount of a titrant n+2 ) was added to the sample and a second additional pH (Titrant n+2 ), and repeating steps (c) and (d). In some embodiments, the method includes adding a third amount of titrant to the sample, measuring a third additional pH, and repeating steps (c) and (d). In some embodiments, the addition of a third amount of titrant to the sample determines a final target pH (pH final In some embodiments, the method includes adding a fourth amount of titrant to the sample and measuring a fourth additional pH. In some embodiments, the method includes measuring the pH of the sample by pH final This involves adding no more than three or four titrants to change the concentration to

[0006] In some embodiments of the methods of the present disclosure, the method includes generating a model. In some embodiments, the method includes (i) generating at least one reference titration curve from at least one reference sample that relates 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 that fits the at least one reference titration curve. In some embodiments, the model includes a polynomial that relates the normalized titrant to pH.

[0007] In some embodiments of the disclosed methods, the difference between the measured sample pH and the model identifies an error in the calibration of the pH meter used to measure the sample pH. In some embodiments, the method includes the steps of recalibrating the pH meter; (a) adding an additional amount of titrant to the sample and measuring the additional pH; (b) applying the model and comparing the normalized titrant and pH to the model; and (c) if the pH matches the model, determining whether the pH is correct. final adding a remaining amount of titrant to the sample to reach titrant = 0; thereby preventing damage to the sample from adding too much titrant to the sample. In some embodiments, the sample comprises a protein and the method prevents damage to the protein.

[0008] The present disclosure provides a method for inactivating viruses in a sample, comprising: (a) detecting an initial pH of 4.0 or greater (pH initial (b) providing a sample of a first amount of an acid titrant; n_acid ) was added to the sample and the first additional acid pH value (pH n_acid ), measuring Titrant n_acid But pH n_acid is the amount of titrant added to the sample to reach a pH of n_acid But pH initial (c) applying the model to determine a normalized titrant, the model relating the normalized titrant added to the sample to the pH of the sample; (d) determining a target acid pH (pH acid_target (e) determining the amount of titrant to be added to the sample to achieve a pH acid_target (f) adding an amount of titrant to the sample to reach a final acid pH (pH acid_final (g) subjecting the sample to pH 7.0 or below for a period of time sufficient to inactivate the virus; final_acid (h) holding a first amount of a base titrant n_base ) is added to the sample and a first additional base pH value (pHn_base ), measuring Titrant n_base But pH n_base is the amount of titrant added to the sample to reach a pH of n_base But pH acid_final (i) applying the second model to Titrant n_base (j) normalizing the pH of the sample to the target basic pH (pH target_base (k) determining the amount of base titrant to add to the sample to change the pH target_base (l) adding an amount of basic titrant to the sample to reach a final basic pH (pH final_base In some embodiments, the method includes repeating steps (j) and (k) until a pH value of 0.1 is reached. In some embodiments, the method includes repeating steps (b) and (c) at least once to confirm that the behavior of the sample is consistent with the model. In some embodiments, the method includes repeating steps (d) and (e) one, two or three times. In some embodiments, the method includes repeating steps (d) and (e) two or three times, wherein the two or three times of repeating steps (d) and (e) is a pH value of 0.1. acid_final In some embodiments, the method results in a target acid pH that is within 0.05 to 0.10 pH units of pH. acid_final In some embodiments, the method includes repeating steps (d) and (e) an additional number of times to reach pH 6. In some embodiments, the method includes no more than three or four additions of acid titrant in total. In some embodiments, the method includes repeating steps (h) and (i) at least once to confirm that the behavior of the sample is consistent with the model. In some embodiments, the method includes repeating steps (j) and (k) one, two or three times. In some embodiments, the method includes repeating steps (j) and (k) two or three times, where the repeating steps (j) and (k) two or three times determines whether the pH 6. final_base In some embodiments, the method results in a pH that is within 0.05 to 0.10 pH units of pH 6. final_baseIn some embodiments, the method includes repeating steps (j) and (k) an additional number of times to reach pH 6. In some embodiments, the method includes up to 3 or 4 additions of the base titrant. acid_final In some embodiments, the pH is about 3.0 to 4.0, about 3.1 to 3.9, about 3.2 to 3.8, about 3.3 to 3.7, about 3.4 to 3.7, or about 3.5 to 3.7. final_base is about 5.3 to 8.5, about 5.1 to 8.1, about 5.5 to 8.0, or about 7.0 to 8.5.

[0009] The present disclosure provides an apparatus configured for the methods of the present disclosure.

[0010] The present disclosure provides an apparatus for pH control in protein purification. In some embodiments, the apparatus may include a reactor and a pH flow cell including a pH probe disposed therein, the pH flow cell being in fluid communication with the reactor. The pH flow cell can receive a slipstream of sampling from the reactor and contains a pH probe disposed therein that measures the pH of the slipstream. The apparatus includes an acid titrant supply in fluid communication with the reactor. The acid titrant supply provides an acid titrant to the reactor to reduce the pH in the reactor. The apparatus further includes a base titrant supply in fluid communication with the reactor. The base titrant supply provides a base titrant to the reactor to increase the pH in the reactor. In some embodiments, the apparatus may further include a sampling pump that delivers the slipstream from the reactor to the pH flow cell. In some embodiments, the apparatus may include a waste receiver that receives the effluent from the pH flow cell. [Brief description of the drawings]

[0011] [Figure 1]FIG. 1 is a plot showing 11 titration curves generated using five different proteins. The pH was lowered through the addition of acid solution to reach a target pH of 3.6. The pH is shown on the Y-axis while the X-axis shows the amount of Acid Titrant Added as pump revolutions per kilogram of eluate (rot / kg). A time shift was applied to the pump speed data to account for the lag between acid addition and the pH response.

[0012] [Diagram 2] 2 is a plot showing the titration curves from FIG. 1 after linear transformation of the X and Y axes using Equation 1 (Y axis) and Equation 2 (X axis).

[0013] [Diagram 3] FIG. 1 illustrates the application of pH modeling in one embodiment of the present disclosure.

[0014] [Figure 4] Figure 1 is a plot showing 12 titration curves generated using seven different proteins. Depending on the protein, the pH was increased through the addition of base solution to reach a target pH of 7.5-8.0. pH is shown on the Y-axis, while the X-axis shows the amount of acid titrant added as pump revolutions per kilogram of eluate (rot / kg). A time shift was applied to the pump speed data to account for the lag between acid addition and the pH response.

[0015] [Diagram 5] 5 is a plot showing the titration curves from FIG. 4 after linear transformation of the X and Y axes using Equation 5 (Y axis) and Equation 6 (X axis).

[0016] [Figure 6] 5 is a plot showing the titration curves from FIG. 4 after linear transformation of the X and Y axes using Equation 7 (Y axis) and Equation 8 (X axis).

[0017] [Figure 7]7 is a plot showing the normalized titration curves from FIG. 6, color-coded by the difference between the initial online and offline measurements for the pH probe used to collect the data for each titration curve. The curve with the greatest deviation from the model (black line) also had the greatest difference in the initial online and offline measurements.

[0018] [Figure 8] 7 is a plot showing the normalized titration curve from FIG. 6 after online pH correction using Equation 9.

[0019] [Figure 9] 5 is a plot showing the titration curves from FIG. 4 after forced convergence for two fixed pH values ​​(pH 3.70 and pH 7.60) and linear transformation of the X-axis using Equation 10. The online pH values ​​(Y-axis) were corrected using Equation 9.

[0020] [Figure 10] 5 is a plot showing the titration curves from FIG. 4 after forced convergence for two fixed pH values ​​(pH 3.70 and pH 7.60) and linear transformation of the X-axis using Equation 10. The online pH values ​​(Y-axis) were corrected using Equation 9. A sixth order polynomial fit to the data is shown as the solid line.

[0021] [Figure 11] 1 is a list of parts for an exemplary system for controlling pH while varying the pH of a sample.

[0022] [Figure 12] VI: Viral inactivation. Flow chart showing an exemplary control strategy for lowering the pH of a protein sample for viral inactivation.

[0023] [Figure 13]FIG. 1 is a flow chart showing an exemplary control strategy for increasing the pH of a protein sample after viral inactivation. VIP: viral inactivated pool (sample after addition of base).

[0024] [Figure 14] 1 shows an exemplary calibration curve for a pH meter used in an exemplary method of the present disclosure.

[0025] [Figure 15] 1 is a table showing results from five test runs lowering and raising pH using the apparatus and method of one embodiment of the present disclosure.

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

[0027] [Figure 17] 1 is a plot comparing pH probe conditions in a viral inactivation test run using an apparatus and method of one embodiment of the present disclosure.

[0028] [Figure 18] 1 shows real-time process monitoring of a sample viral inactivation process.

[0029] [Figure 19] FIG. 1 is a block diagram of an apparatus for pH control, according to one embodiment.

[0030] [Figure 20] FIG. 1 is a schematic diagram of an apparatus for pH control, according to one embodiment.

[0031] [Figure 21]FIG. 1 is a plot showing the difference between actual pH (measured by an offline reference probe) and target pH for 18 protein batches after pH adjustment using 3 (circles) or 4 (crosses) acid or base additions. On the left x-axis, the difference between the measured pH and the target pH is shown after the pH was lowered to a pH of 3.50-3.60 for viral inactivation depending on the protein. On the right x-axis, the difference between actual pH and target pH is shown after the pH was raised to a pH of 5.50-8.00 depending on the protein. The dashed line indicates the final pH target after additions that is within 0.10 pH units of the target pH.

[0032] [Figure 22] FIG. 1 is a plot showing the difference between the pH measured by the online control probe and the target pH for 18 protein batches after pH adjustment using 3 (circles) or 4 (crosses) acid or base additions. On the left x-axis, the difference between the measured pH and the target pH is shown after the pH was lowered to a pH of 3.6 for viral inactivation. On the right x-axis, the difference between the actual pH and the target pH is shown after the pH was raised to pH 7.7-8.0 depending on the protein. The dashed lines indicate the final pH target after additions that are within 0.05 pH units of the target pH.

[0033] [Diagram 23] 1 is a plot showing the difference (ΔpH) between the pH measured by an offline reference probe and the pH measured by an online control probe mounted in the flow cell at each addition step for 18 protein batches.

[0034] [Figure 24] 15 is a plot showing the percent error in addition volume (sometimes called drug injection error) at each addition step (total of 133 additions) for 18 protein batches. The formula for the percent error in addition volume is shown in FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present disclosure relates to a method of controlling pH during a process that involves changing the pH of a sample. One example of a process that involves a pH change is the large-scale production of biopharmaceuticals, such as antibodies or other therapeutic proteins. The production 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 therapeutic protein production. Most mammalian cells have a specific pH range that supports optimal cell growth, metabolism, and protein production. Additionally, cells used in the production of therapeutic proteins may harbor viruses, which may be harmful if they contaminate the drug substance or drug product. One method of inactivating potentially harmful viruses is by transiently lowering the pH during purification of the therapeutic protein. Many viruses are irreversibly denatured and effectively destroyed at a pH of about 5.0-5.5. Some enveloped viruses are effectively inactivated at a pH range of about 3.5-4.0. However, lowering the pH of a protein sample carries too high a risk of denaturing the therapeutic protein, which may cause the destruction of the protein batch and increase the production cost. Therefore, there is a need to measure and control the pH during the production of therapeutic proteins, both during cell culture and during protein purification.

[0036] Traditional methods of measuring pH during protein purification can be unreliable and result in wastage of protein product. In one method, pH is measured during protein purification by inserting a sterile pH probe directly into a reaction vessel containing the protein solution. However, maintaining sterility and probe accuracy using this approach can be difficult. Typically, the pH probe is calibrated and sealed in a bag with a bellows-type connector used to insert the probe into the reaction vessel and sterilized by autoclaving or gamma irradiation. However, this results in a period between calibration and use during which the pH probe is dry, which can affect the accuracy of the probe. In addition, pH probes are made of glass and can break when inserted into a vessel. Inserting the probe while maintaining sterility can be difficult. In another method of indirectly measuring the pH of a protein solution, a "slip stream" is removed from the main protein solution and the pH of the slip stream is measured using a pH probe. However, without a direct measurement of the main protein solution pool, direct feedback control of the titration to adjust the pH is not possible. Furthermore, any protein that is withdrawn from the main pool into the slipstream to measure pH is not returned to the main pool and ends up being wasted. When making pH adjustments to protein solutions during the manufacturing process, statistical titration models can be used to predict the amount of acid or base to add, but these models require manual protein concentration input by the user, and each titration type (acid or base) requires multiple historical data sets to generate the model. Additionally, these models are not universally accurate for all processes and protein types.

[0037] Thus, there is a need for additional methods of pH measurement and control in protein production that do not require direct insertion of a pH probe into the protein solution pool or continuous withdrawal of a slipstream of material from the protein pool. The present disclosure provides methods and systems for modeling and controlling pH during protein production. The disclosed methods are accurate across a wide range of proteins, do not require operator input or off-line concentration measurements, and do not require large amounts of historical data. The disclosed methods can be used to extrapolate the amount of acid or base needed to adjust pH during the production process. Furthermore, the methods and systems disclosed herein can reproducibly and accurately achieve pH values ​​that are within 0.05-0.10 pH units of the desired target pH during the process of changing the pH of a sample. The final target pH, e.g., a target acid pH for viral inactivation of a protein sample, or a target basic pH after inactivation, can be accurately and reliably achieved with only 3-4 titrant additions. Additionally, the methods and systems disclosed herein can also accurately determine and add the amount of acid or base titrant to be added to a sample, and can add the desired volume of titrant with an accuracy of 10% or less volumetric error per titrant addition.

[0038] The present disclosure provides a method that includes measuring an initial pH of a protein pool, adding a conservative amount of a titrant, such as an acidic or basic solution, measuring the intermediate pH, optionally adding a second amount of titrant and repeating the pH measurement, and determining the additional amount of titrant needed to reach a target pH based on the initial measurement and a model relating pH to a normalized amount of titrant based on a reference sample. The present disclosure further provides an apparatus for performing the method of the present disclosure.

[0039] Thus, the present disclosure provides: (a) determining the initial pH of a sample (pH initial (b) measuring at least a first amount of a titrant; n ) is added to the sample and at least a first additional pH value (pHn ), measuring Titrant n But pH n is the amount of titrant added to the sample to reach a pH of n But pH initial (c) Applying the model to the normalized Titrant n (d) determining a final pH (pH final determining the remaining amount of titrant to be added to the sample to reach a pH of final However, the total amount of titrant added to the sample total The method includes the steps of:

[0040] [Definition] As used herein, the term "initial pH" refers to the pH of a sample prior to the addition of a titrant used to change the pH, i.e., a solution that is acidic or basic compared to the initial pH of the sample.

[0041] As used herein, "final pH" refers to the desired pH of a sample. For example, a sample may have a pH of 3.6, but needs to be at a pH of 7.5 to be suitable for a particular purpose, and the process used herein is used to change the pH from 3.6 to 7.5 through the controlled addition of a basic titrant. In this case, 3.6 is the initial pH, and 7.5 is the final or target pH. Those skilled in the art will understand that the initial and final pH values ​​of any particular sample may differ depending on the sample, sample conditions, and application. Those skilled in the art will understand that when a process for changing pH is performed, the process may include multiple steps, each of which has a target pH associated with it before the final pH (or final target pH) of the sample is reached.

[0042] As used herein, "Titrant total)" refers to the amount of titrant added to a sample to change the pH from the initial pH to the final pH.

[0043] As used herein, "pH n " refers to the change in the sample from its previous pH (pH n-1 ) to pH n It refers to the pH of a sample after the addition of a portion of the titrant required to change the pH from, for example, an initial pH to pH n The amount of titrant needed to convert n Those skilled in the art will appreciate that the measured pH values ​​and the corresponding amounts of titrant added to the sample to change the pH of the sample to these measured pH values ​​can be repeatable. n+1 A further amount of titrant is added to change the pH n may be added to the sample at pH n+1 The amount of titrant added to the sample to change it to Titrant n+1 Similarly, the pH of a sample is called pH n+2 To change the pH to n+1 may be added to the sample at pH 6.0, and so on until the target pH is achieved.

[0044] The term "sample" refers to a sample that has been subjected to the methods described herein to change its pH. In some cases, the sample contains proteins, such as purified or partially purified proteins, in a liquid solution. However, other types of samples are contemplated within the scope of the present disclosure, including DNA, RNA, and drugs. Those skilled in the art will understand that as used herein, a sample refers to a liquid solution, such as a liquid solution that contains multiple biological molecules (DNA, RNA, or proteins) or analytes (compounds, drugs, etc.). The sample may be at any suitable concentration or initial pH and include any suitable buffer or carrier.

[0045] The term "reference sample" refers to a reference sample with similar or identical properties as the sample, which has been subjected to a pH change similar to that of the sample, from which data on pH and titrant addition, and the relationship between the two, were collected. The reference sample may be identical to the sample, e.g., the reference sample is taken from a larger sample (i.e., a sub-sample as the reference sample). However, the reference sample need not be identical to the sample, provided that it behaves similarly to the sample when titrant is added. For example, the sample and reference sample may be different batches of the same protein produced and purified by the same or similar process. As a further example, the sample and reference sample may be similar but not identical proteins, e.g., two antibodies, or two proteins with similar glycosylation patterns, that behave similarly when subjected to a similar titration process.

[0046] As used herein, the term "titration curve" refers to a graph (or series of measurements) that relates the volume of titrant added to a sample, as the independent variable, to the pH of the solution, as the independent variable. A titration curve can be generated by continuous measurements, for example by inserting a pH probe directly into the sample and taking continuous measurements. Alternatively, a titration curve can be generated from non-continuous measurements followed by fitting an appropriate curve to the measured data points.

[0047] As used herein, "normalize" refers to adjusting values ​​measured on different scales to a common scale.

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

[0049] "Acid titrant" refers to a titrant that has a pH that is more acidic than the sample. Typically, an acid titrant has a pH less than 7.0. Common acid titrants include phosphoric acid (H3PO4), glycine hydrochloride (C2H6ClNO2 or glycine HCl), acetic acid (CH3COOH), hydrochloric acid (HCl), perchloric acid (HClO4), and sulfuric acid (H2SO4). Acid titrant solutions may be prepared by diluting a commercially available concentrated stock solution and determining the concentration by standardizing against a standard weak base. Exemplary acid titrants include phosphoric acid in concentrations of 0.20M-2.0M, 0.25M-1.5M, or 0.5M-1.0M. For example, phosphoric acid at a concentration of 0.10M, 0.20M, 0.25M, 0.30M, 0.35M, 0.40M, 0.45M, 0.50M, 0.60M, 0.70M, 0.80M, 0.90M, 1.0M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M or 2.0M can be used as the acid titrant. Further exemplary acid titrants include glycine HCl at concentrations of 0.1M to 1.0M, 0.2M to 0.75M, 0.25M to 0.75M or 0.25M to 0.5M. For example, glycine HCl at a concentration 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 an acid titrant. Further exemplary acid titrants include acetic acid at concentrations of 0.5 M to 3.0 M, 1.0 M to 2.5 M, 1.0 M to 2.0 M or 1.5 M to 2.0 M. For example, acetic acid at a concentration of 0.50M, 0.60M, 0.70M, 0.80M, 0.90M, 1.0M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2.0M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, or 3.0M is an acid titrant. "Base titrant" or "base titrant" refers to a titrant that has a pH that is more basic than the sample. Common base titrants include sodium hydroxide (NaOH), which is commercially available as an impure solid and as a solution of approximately 50% w / v. Solutions of NaOH can be standardized against weak acid standards to determine concentration.An additional common base titrant is tromethamine (also called tris(hydroxymethyl)aminomethane, or Tris base, CH). 11 NO3). Exemplary base titrants include tromethamine at concentrations of 0.5M to 3.0M, 1.0M to 2.5M, 1.0M to 2.0M, or 1.5M to 2.0M. For example, tromethamine at concentrations of 0.50M, 0.60M, 0.70M, 0.80M, 0.90M, 1.0M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2.0M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, or 3.0M is a base titrant.

[0050] A pH meter measures hydrogen ion activity in a water-based solution, which indicates its acidity or alkalinity, expressed as pH. A pH meter measures the difference in potential between a pH electrode and a reference electrode. A pH "probe" refers to the portion of the meter that contains the pH electrode and the reference electrode. Typically, a pH electrode is a glass electrode, which is a type of ion-selective electrode consisting of a doped glass membrane that is sensitive to specific ions. An exemplary pH electrode is a glass electrode that is sensitive to hydrogen ions. The voltage of the glass electrode relative to some reference value (i.e., the reference value from the reference electrode) is sensitive to changes in the activity of hydrogen ions. In other words, the hydrogen ion activity in the solution being measured affects the electrochemical potential between the reference electrode and the hydrogen ion-sensitive electrode. A pH meter is calibrated to correlate the electrochemical potential with a pH value.

[0051] "pH meter calibration" refers to the process of calibrating a pH meter against one or more standardized buffer solutions of known pH, since pH electrodes are known to drift from their calibrated settings. A typical calibration process uses a calibration curve generated by at least three standard buffer solutions, although a two-point calibration may also be used. An exemplary calibration protocol includes cleaning the electrode, immersing the rinsed electrode in a first standard at pH 4.0, then a second standard at pH 7.0, and a final standard at pH 10.0, cleaning the electrode between measurements.

[0052] "Slipstream" or "slip stream," as used herein, refers to a sampling method in which a sub-sample is withdrawn or isolated from a main sample, for example, using a tube inserted into the main sample, and measurements are made on the sub-sample. The slipstream may be continuous, i.e., constantly withdrawn from the sample, or it may be discontinuous, withdrawn from the sample only at discrete times during the process.

[0053] As used herein, "online probe" or "online pH probe" refers to a probe that measures the pH of a sample during a pH change (online pH) and the information therefrom is used with the models described herein to determine the amount of titrant to add to the sample during the titrant addition step. An online probe can be a slipstream probe, for example, attached to a flow cell coupled to a slipstream. Alternatively, an online probe can be inserted directly into the reactor.

[0054] As used herein, "peptide", "polypeptide" and "protein" are used interchangeably throughout and refer to molecules comprising two or more amino acid residues joined together by peptide bonds. Peptides, polypeptides and proteins may also include modifications such as glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, alkylation, hydroxylation and ADP-ribosylation. Peptides, polypeptides and proteins may be of scientific or commercial interest and include protein-based drugs (biologics). Peptides, polypeptides and proteins include, among others, antibodies and chimeric or fusion proteins. Peptides, polypeptides and proteins may be produced by recombinant animal cell systems, such as mammalian cell systems, using cell culture methods.

[0055] The phrase "virus reduction / inactivation" as used herein is intended to refer to a reduction in the number of virus particles in a particular sample ("reduction"), and a reduction in the activity of the virus particles in a particular sample, such as, but not limited to, the ability to infect or replicate ("inactivation"). Such a reduction in the number and / or activity of virus particles can be a reduction of about 50% to about 99%, even more preferably about 60% to about 99%, also more preferably about 70% to about 99%, also more preferably about 80% to 99%, also more preferably about 90% to about 99%, also more preferably about 95% to 99%, also more preferably about 95% to 99.9%, also more preferably about 95% to 99.99%, and also more preferably about 98% to 99.99%. In certain non-limiting embodiments, the amount of virus in the purified antibody product, if present, is less than the ID50 for that virus (the amount of virus that infects 50 percent of a target population), preferably at least 10-fold less than the ID50 for that virus, more preferably at least 100-fold less than the ID50 for that virus, and even more preferably at least 1000-fold less than the ID50 for that virus.

[0056] All publications and patents mentioned in this specification are hereby incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, this application, including any definitions herein, will control. However, the mention of any references, articles, publications, patents, patent publications and patent applications cited in this specification is not, and should not be construed as, an admission or any form of suggestion that they constitute legitimate prior art or form part of the common general knowledge in any country in the world.

[0057] [Changing the sample pH] The present disclosure provides a method of changing the pH of a sample, comprising the steps of taking an initial pH measurement, adding at least a first amount of titrant to the sample, measuring at least a first additional pH value, and applying a model that relates the sample pH to a normalized amount of titrant added to the sample. In some embodiments, the method further comprises adding a second amount of titrant to the sample, measuring the second pH, and applying the model. In some embodiments, the method further comprises adding a third, fourth, or further amount of titrant, measuring the pH after each addition, and applying the model. The additional steps of adding a conservative amount of titrant and checking the pH can be used to verify that the sample is behaving as predicted by the model and that there were no errors in the process, such as errors due to pH meter calibration. After one or more additions and measurements of titrant, these measurements and the model can be used to determine the amount of titrant to add to the sample to change the pH of the sample to a final or target pH.

[0058] The disclosed method can be accomplished using a relatively small number of separate measurements and a model to determine the amount of titrant to add to a sample to change the pH. By using a model, the method can improve the accuracy with which the final pH of the sample is achieved when compared to methods that measure pH by inserting a pH probe into the sample. The difference between the measured pH value and the model can also be used to identify errors in the process, such as errors in pH meter calibration or function.

[0059] In some embodiments, the pH of the sample is changed by the addition of a large amount of titrant. In some embodiments, the pH of the sample is changed by the addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 titrants to reach the final pH. In some embodiments, the pH of the sample is changed by the addition of 2 titrants to reach the final pH. In some embodiments, the pH of the sample is changed by the addition of 3 titrants to reach the final pH. In some embodiments, the pH of the sample is changed by the addition of 4 titrants to reach the final pH. In some embodiments, the pH of the sample is changed by the addition of 5 titrants to reach the final pH. In some embodiments, the pH of the sample is changed to a pH value within 0.01-0.20, 0.01-0.15, 0.01-0.10, 0.05-0.20, 0.05-0.15, 0.05-0.10, 0.01-0.07, or 0.05-0.07 pH units of the final pH, followed by a final addition of titrant to reach the final pH. In some embodiments, the pH of the sample is changed to a pH value within 0.05-0.10 pH units of the final pH, followed by a final addition of titrant to reach the final pH. As an example, the pH of the sample may be changed to a target pH within 0.05-0.10 pH units of the final pH by the addition of 1, 2, 3, 4, or 5 additions of titrant, followed by a final addition of titrant to reach the final pH. In some embodiments, such as those in which the pH of the sample is lowered, the titrant is an acid. In alternative embodiments, such as those in which the pH of the sample is increased, the titrant is a base. A mismatch between the target pH predicted by the model described herein and the measured pH in any of the addition steps described herein may indicate that the pH meter used to measure has a calibration error. For example, if the predicted and measured pH values ​​for a given addition step differ by more 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 pH units, it indicates that the pH meter used to measure the sample is making an incorrect reading.As a further example, if the predicted and measured pH values ​​for a given addition step differ by more than 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.10 pH units, it is an indication that the pH meter used to measure the sample is making an erroneous reading. In some embodiments, the method includes stopping the process of altering the pH of the sample if a difference between the predicted and measured pH values ​​occurs until the pH meter is recalibrated or the pH probe is replaced.

[0060] The disclosed method can be used whenever it is necessary to change the pH of a sample. For example, when a process such as protein purification produces a liquid sample (sometimes called a protein pool) containing a protein of interest that has an unsuitable pH for downstream purification steps or applications, the method described herein can be used to change the pH of the sample to the desired pH. In a further example, the disclosed method can be used to lower the pH of a protein sample to a pH low enough to inactivate viruses that potentially contaminate the protein sample, and then raise the pH to a neutral pH for further protein purification and analysis processes.

[0061] In some embodiments, the sample contains a protein of interest, e.g., a therapeutic protein, and the method is used to inactivate viruses in the sample containing the therapeutic protein.

[0062] Methods of pH virus inactivation include, but are not limited to, incubating the mixture at a low pH for a period of time, followed by neutralizing the pH and removing particulates by filtration. In some embodiments, the pH of the sample is lowered to a pH of about 2-5, preferably about 3-4, more preferably about 3.6, and the sample is incubated at this pH to inactivate any viruses present. The pH of the sample mixture can be lowered by any suitable acid, including, but not limited to, phosphoric acid, glycine hydrochloride, perchloric acid, hydrochloric acid, citric acid, acetic acid, caprylic acid, or other suitable acids. The choice of pH level depends largely on the stability profile of the proteins in the sample, and the buffer components.

[0063] In an exemplary method of inactivating viruses in a sample, a conservative initial amount of acid titrant is added, the pH is assessed, then an additional conservative amount of acid titrant is added, followed by a further pH assessment. This may be repeated using smaller amounts of acid until the target pH is achieved, in a process that may take from 30 minutes to 2 hours. The sample is held at the target pH for a period of time sufficient to inactivate the viruses, and the sample pH is raised by the same process described above.

[0064] In some embodiments, the duration of the incubation period at low pH to inactivate viruses is 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 incubation at low pH is about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, or about 90 minutes. Thus, depending on the protein of interest, one skilled in the art can select the appropriate protein concentration, pH, and duration to achieve virus inactivation.

[0065] In some embodiments, changing the pH of a sample containing a protein of interest comprises lowering the pH of the sample. For example, the final pH of the sample (pH final ) is the initial pH of the sample (pH initial) and the titrant is an acid. Any suitable acidic solution may be used, so long as the pH of the titrant is less than the initial pH of the sample.

[0066] In some embodiments, e.g., embodiments in which the pH is lowered, the initial pH of the sample (pH initial ) is about 4.0 to 4.7, about 4.0 to 4.5, about 4.0 to 4.3, about 4.1 to 4.6, about 4.1 to 4.5, about 4.1 to 4.4, about 4.1 to 4.3, about 4.1 to 4.2, about 4.2 to 4.5, about 4.3 to 4.5, about 4.1 to 4.4, or about 4.2 to 4.4. In some embodiments, the pH initial In some embodiments, the initial pH is about 4.1. In some embodiments, the final pH of the sample (pH final ) is about 3.0 to 3.8, about 2.0 to 3.7, about 3.0 to 3.6, about 3.0 to 3.5, about 3.0 to 3.4, about 3.0 to 3.3, about 3.1 to 3.8, about 3.3 to 3.8, about 3.5 to 3.8, about 3.2 to 3.8, about 3.3 to 3.7, about 3.4 to 4.0, about 3.5 to 4.0, 3.4 to 3.9, about 3.4 to 3.8, about 3.4 to 3.7, about 3.4 to 3.6, about 3.5 to 3.9, about 3.5 to 3.8, about 3.5 to 3.7, or about 3.5 to 3.6. final is about 3.0 to 3.8, about 3.1 to 3.8, about 3.2 to 3.8, about 3.3 to 3.7, about 3.4 to 3.7, or about 3.5 to 3.7. In some embodiments, the final pH is about 3.5 to 3.7. In some embodiments, the final pH is about 3.6.

[0067] In some embodiments, changing the pH of a sample containing a protein of interest comprises increasing the pH of the sample. For example, the final pH of the sample (pH final ) is the initial pH of the sample (pH initial ) and the titrant is a base. Any suitable basic solution may be used, so long as the pH of the titrant is greater than the initial pH of the sample.

[0068] In some embodiments, e.g., embodiments in which the pH is increased, the initial pH of the sample (pH initial ) is about 3.0 to 3.8, about 2.0 to 3.7, about 3.0 to 3.6, about 3.0 to 3.5, about 3.0 to 3.4, about 3.0 to 3.3, about 3.1 to 3.8, about 3.3 to 3.8, about 3.5 to 3.8, about 3.2 to 3.8, about 3.3 to 3.7, about 3.4 to 4.0, about 3.5 to 4.0, 3.4 to 3.9, about 3.4 to 3.8, about 3.4 to 3.7, about 3.4 to 3.6, about 3.5 to 3.9, about 3.5 to 3.8, about 3.5 to 3.7, or about 3.5 to 3.6. final is about 3.0 to 3.8, about 3.1 to 3.8, about 3.2 to 3.8, about 3.3 to 3.7, about 3.4 to 3.7, or about 3.5 to 3.7. initial is about 3.0 to 3.8, about 3.1 to 3.8, about 3.2 to 3.8, about 3.3 to 3.7, about 3.4 to 3.7, or about 3.5 to 3.7. In some embodiments, the initial pH is about 3.1 to 3.8. In some embodiments, the initial pH is about 3.3 to 3.8. In some embodiments, the initial pH is about 3.5 to 3.7. In some embodiments, the initial pH is about 3.6. In some embodiments, the final pH (pH final ) is about 5.1 to 8.5, about 5.1 to 8.3, about 5.1 to 8.1, about 5.1 to 8.0, about 5.1 to 7.7, about 5.1 to 7.5, about 5.1 to 7.3, about 5.1 to 7.0, about 5.3 to 8.5, about 5.3 to 8.3, about 5.3 to 8.1, about 5.3 to 8.0, about 5.3 to 7.7, about 5.3 to 7.5, about 5.3 to 7.3, about 5.3 to 7.0, about 5.5 to 8.5, about 5.5 to 8.3, about 5.5 to 8.1, about 5.5 to 8.0, about 5.5 to 7.7, about 5.5 to 7.0, about 6.0 to 8.5, about 6.0 to 8.3, about 6.0 to 8.0, about 6.0 to 7.7, about 6.0 to 7.0, about 6.5 to 8.5, about 6.5 to 8.3, about 6.5 to 8.0, about 6.5 to 7.7, about 6.5 to 7.0, about 7.0 to 8.5, about 7.0 to 8.3, about 7.5 to 8.0, about 7.7 to 8.0, about 7.7 to 8.5, about 7.7 to 8.3, about 7.9 to 8.2, about 7.0 to 8.0, about 7.0 to 7.9, about 7.0 to 7.5, about 6.8 to 7.8, about 6.8 to 7.6, or about 6.8 to 7.4.final is about 5.3 to 8.5, about 5.1 to 8.1, about 5.5 to 8.0, or about 7.5 to 8.0. In some embodiments, the final pH is about 5.5 to 8.0. In some embodiments, the final pH is about 7.0 to 8.0.

[0069] The present disclosure provides a method for inactivating viruses in a sample. In some embodiments, the method includes providing a sample containing a protein of interest, e.g., a sample purified from cultured cells by column chromatography, and lowering the pH. Exemplary samples may have an initial pH of about 4.1-4.5, with a final pH of about 3.5-3.7, and optionally about 3.6. The initial pH depends on the protein of interest, the purification method used, and the composition of the sample after the protein purification step (e.g., elution buffer, etc.). After a reduction in pH and a period of hold to inactivate viruses, the pH is then increased to a final basic pH of about 7.5-8.5, or about 7.5-8.0, or about 7.6. The final basic pH depends on the protein of interest, and the choice of buffer depending on the desired downstream application, etc.

[0070] Thus, the present disclosure provides a method for inactivating viruses in a sample. In some embodiments, the sample comprises a protein of interest. In some embodiments, the method comprises inactivating a virus in a sample at an initial pH of 4.0 or higher (pH initial In some embodiments, the method includes providing a sample of a first amount of an acid titrant. In some embodiments, the method includes measuring an initial pH prior to addition of the acid titrant. In some embodiments, the method includes providing a sample of a first amount of an acid titrant. n_acid ) was added to the sample and the first additional acid pH value (pH n_acid ), measuring Titrant n_acid But pH n_acid is the amount of titrant added to the sample to reach a pH of n_acid But pH initialThe first amount of titrant is generally a conservative amount of titrant. For example, the first amount of titrant is an amount of titrant predicted based on a previous reference sample to be sufficient to change the pH of the sample half or less to the target pH, or two-thirds or less to the target pH, or three-quarters or less to the target pH. Those skilled in the art will appreciate that the amount of acid titrant to be added to the sample in each addition may be adjusted depending on the sample, the initial pH of the sample, the final target pH, and the number of additions of acid titrant to be added to the sample to change the sample pH. In some embodiments, the method includes normalizing the pH and applying a model to determine a normalized titrant, i.e., a normalized amount of titrant corresponding to the initial pH and the pH after the addition of the first amount of acid titrant, where the model relates the normalized titrant added to the sample to the pH of the sample. Optionally, the addition of an amount of titrant may be repeated at least one, two, three, four, five or more times to confirm that the sample's behavior is consistent with the model. If the sample does not match the model or pH meter calibration error is suspected, one skilled in the art may decrease the amount of titrant added and increase the number of times the titrant is added to more accurately measure the pH and to avoid overshooting the target pH in the process. In some embodiments, the method determines a final acid pH of 3.4 to 3.7 (pH 3.0) based on the normalized titrant, pH and model. acid_final In some embodiments, the method includes determining the remaining amount of titrant to be added to the sample to reach a pH acid_final adding the remaining amount of titrant to the sample to reach

[0071] In some embodiments, the method comprises inactivating the virus at a pH of 0.1 or 0.2 for a period of time sufficient to inactivate the virus, such as an incubation period described above. final_acid In some embodiments, the method includes retaining the sample in a first amount of a base titrant. n_base ) is added to the sample and a first additional base pH value (pH n_base ), measuring Titrantn_base But pH n_base is the amount of titrant added to the sample to reach a pH of n_base But pH acid_final The amount of base added to the sample is generally a conservative amount of titrant, i.e., the amount of titrant predicted based on a previous reference sample to be sufficient to change the pH of the sample by half or less to the target basic pH, or two-thirds or less to the target basic pH, or three-quarters or less. Those skilled in the art will appreciate that the amount of base titrant added to the sample in each addition may be adjusted depending on the sample, the initial pH of the sample, the final target pH, and the number of additions of base titrant to be added to the sample to change the sample pH. In some embodiments, the method includes applying a second model to calculate the titrant pH by averaging the titrant pH. n_base In some embodiments, the method includes repeating the adding and measuring steps at least one, two, three, four, five or more times to confirm that the sample's behavior is consistent with the model. If the sample does not match the model or pH meter calibration error is suspected, one skilled in the art may decrease the amount of titrant added and increase the number of times the titrant is added to more accurately measure the pH and to avoid overshooting the target pH in the process. In some embodiments, the method includes adjusting the pH of the sample based on the normalized titrant, pH and model to a final pH of 7.0 to 8.5 (pH final_base In some embodiments, the method includes determining the remaining amount of base titrant to add to the sample to change the pH of the sample to 0.5. final_base adding the remaining amount of base titrant to the sample to reach

[0072] In some embodiments, the method includes adding one or more conservative amounts of titrant, i.e., an amount of titrant that is expected to change the pH of the sample no more than halfway to the target pH, measuring the pH, and applying a model to determine the remaining amount of pH to add to the sample to reach the target or final pH. In some embodiments, determining the final amount of titrant to add to the sample is based on the formula:

number

[0073] In some embodiments, the method further comprises administering a first amount of a titrant n ) is added to the sample and at least a first additional pH value (pH n ), measuring Titrant n But pH n is the amount of titrant added to the sample to reach a pH of n is the initial pH (pH initial ) and step; Apply the model to the normalized initial amount of Titrant (Titrant initial ) and normalized Titrant n determining a target pH (pH n+1 The further additional amount of titrant to be added to the sample to reach n+1 ) determining pH n+1 However, additional titrants are n+1) to the sample. In some embodiments, the method includes applying the model and determining a second target pH (pH n+2 The additional amount of titrant added to the sample to reach n+2 In some embodiments, the method includes calculating an additional amount of titrant (Titrant) to the sample. n+2 ), thereby changing the pH of the sample to a second target pH (pH n+2 In some embodiments, the method further comprises applying the model and changing the pH to a third target pH (pH n+3 The additional amount of titrant added to the sample to reach n+3 In some embodiments, the method further comprises calculating Titrant n+3 This changes the pH of the sample to pH n+3 In some embodiments, the method includes applying the model and changing the fourth target pH (pH n+4 The additional amount of titrant added to the sample to reach n+4 In some embodiments, the method further comprises calculating Titrant n+1 , Titrant n+2 , Titrant n+3 or Titrant n+4 Adding the 100% 15 ... final ) to produce a target pH that is within 0.05 to 0.10 pH units of the titrant. In some embodiments, an additional amount of titrant, determined by applying the model, is added to the sample to reach the final target pH. For example, pH n+2 is Titrant n+3 is within 0.05 to 0.10 pH units of the final target pH achieved by the addition of Titrant n+3 The amount of titrant to add for pH is determined by applying the model. n+3 is Titrant n+4is within 0.05 to 0.10 pH units of the final target pH achieved by the addition of Titrant n+4 The amount of titrant to add for is determined by applying the model. Those skilled in the art will appreciate that depending on the magnitude of pH change desired and the nature of the sample and titrant, more or less titrant additions than those described above may be used to reach 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 additions of titrant, with the pH measured after each addition, and the model applied to determine the additional target pH, and optionally the amount of titrant to add to reach the desired final pH. In some embodiments, the method includes determining the target pH (e.g., pH ) at any of the addition steps described herein. n+1 , pH n+2 , pH n+3 , pH n+4 etc.) to the target pH predicted for the corresponding step by the model.

[0074] When performing the methods of altering pH described herein, the pH measurement of the sample may be measured using a pH probe inserted into a subsample removed from the sample. The subsample may be removed from the sample via a slipstream, such as a slipstream connecting a reaction vessel containing the sample to a flow cell 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 pH of the sample is not measured using a pH probe inserted directly into the sample.

[0075] [pH meter calibration] The methods described herein can be used to determine if there has been an error in pH meter calibration or function. If the sample is a protein, pH changes outside of those tolerated by the protein can cause protein denaturation and potentially destroy the sample. Thus, the ability to quickly and reliably identify pH meter calibration errors is an advantage of the methods disclosed herein compared to other methods known in the art. For example, if an error in pH meter calibration is detected, the pH meter may be recalibrated or replaced with a new one, or measurements made with an inaccurate pH meter may be mathematically corrected using measurements made with a second pH meter. In some embodiments, the method includes recalibrating the pH meter. In some embodiments, the method includes replacing the pH meter or pH probe. In some embodiments, the method includes adding an additional amount of titrant to the sample and measuring the additional pH; applying a model and comparing the normalized titrant and pH or normalized pH to the model; and if the pH or normalized pH matches the model, measuring the pH. final adding a remaining amount of titrant to the sample to reach a titer of 0.01; thereby preventing damage to the protein of interest caused by adding too much titrant to the sample.

[0076] In some embodiments, the difference between the measured sample pH and the model identifies an error in the calibration of the pH meter used to measure the sample pH. In some embodiments, a difference between the measured pH and the pH predicted by the model that is more than 0.01 pH units, more than 0.02 pH units, more than 0.03 pH units, more than 0.04 pH units, more than 0.05 pH units, more than 0.06 pH units, more than 0.07 pH units, more than 0.08 pH units, more than 0.09 pH units, or more than 0.10 pH units indicates an error associated with the pH meter, such as a calibration error. In some embodiments, a difference of more than 0.01 pH units indicates a pH meter error. In some embodiments, a difference of more than 0.05 pH units indicates a pH meter error. In some embodiments, a difference of more than 0.10 pH units indicates a pH meter error.

[0077] In some embodiments, the method further comprises the step of correcting for pH meter calibration when determining the pH value of the sample or the pH value of at least one reference sample used to generate the model. In some embodiments, the step of correcting for pH meter calibration comprises: (a) removing a first portion of the sample or reference sample prior to addition of titrant and measuring the pH of said first portion with an independently calibrated pH meter, thereby determining an offline initial pH value (pH initial_off (b) removing a second portion of the sample or reference sample after the addition of the entire amount of titrant and measuring the pH of said second portion with an independently calibrated pH meter, thereby generating an off-line final pH value (pH final_off ) and (c) applying a 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 to make the initial measurement after a further round of calibration. Alternatively, the independently calibrated pH meter may be a different pH meter.

[0078] The offline measurements may be used to calculate a corrected pH according to the following formula, where the corrected pH for the sample (or reference sample) is:

number

[0079] [Model] The present disclosure provides models for use in the methods of the present disclosure, and methods for generating these models.

[0080] In some embodiments, generating the model includes non-dimensionalization, such as non-dimensionalization of the titrant values ​​of the reference titration curve. Non-dimensionalization is the partial or complete removal of physical dimensions from an equation involving physical quantities by substitution of suitable variables. For example, the volume of titrant added to a sample can be determined by pump revolutions per kg, or mL of titrant added per kg of total sample, and these dimensions can be removed by non-dimensionalization techniques. Non-dimensionalization can simplify and parameterize problems involving measured units. In some cases, scaling is compatible with non-dimensionalization when non-dimensionalization is used to convert multiple data sets to a common scale.

[0081] In some embodiments, generating the model comprises regression analysis. Regression analysis is a set of statistical processes for estimating the relationship between a dependent variable (often called a "response" variable) and a more independent variable, in this case, pH and normalized titrant, respectively. One common form of regression analysis is linear regression, in which a person skilled in the art finds the line that most closely fits the data according to a certain mathematical standard. For example, the method of least squares estimation calculates the unique line that minimizes the sum of the squared difference between the true data and the line.

[0082] In some embodiments, fitting the model comprises linear regression. Linear regression is a linear approach to modeling the relationship between a scalar response variable and one or more explanatory variables. In the case of one explanatory variable, it is called simple linear regression. In linear regression, the relationship is modeled using a linear predictor function, where the unknown model parameters are estimated from the data. Such a model is called a linear model.

[0083] Linear regression was the first type of regression analysis to be rigorously studied and to be used extensively in practical applications because models that depend linearly on the unknown parameters are easier to fit than models that relate nonlinearly to the parameters, and because the statistical properties of the resulting estimators are easier to determine.

[0084] In some embodiments, the regression analysis includes polynomial regression. Polynomial regression is a form of regression analysis in which the relationship between an independent variable (e.g., a normalized titrant) and a dependent variable (e.g., pH) is modeled as an nth order polynomial. Polynomial regression fits a non-linear relationship between the values ​​of the independent variables and the corresponding conditional mean values ​​of the dependent variables. Although polynomial regression fits a non-linear model to the data, as a matter of statistical estimation it is linear in the sense that the regression function is linear in the unknown parameters that are estimated from the data. For this reason, polynomial regression is considered a type of linear multiple regression.

[0085] Polynomial regression models can be fitted using the least squares method, which minimizes the variance of the unbiased estimators of the coefficients under the conditions of the Gauss-Markov theorem.

[0086] In some embodiments, fitting the model includes curve fitting. Curve fitting is the process of constructing a curve or mathematical function that has the best fit to a set of data points. Curve fitting may include either interpolation, where an exact fit to the data is required, or smoothing, where a "smooth" function is constructed that approximately fits the data. Curves may be extrapolated, i.e., extended beyond the range of the observed data, but the extrapolated curve is subject to uncertainty.

[0087] Fitting the model can be performed using any suitable program known in the art, for example Microsoft excel, MATLAB, or R.

[0088] In some embodiments, the model is determined from one or more titration curves generated from one or more reference samples. The reference sample may be identical to the sample, e.g., a subsample of a larger sample subjected to the same pH process. Alternatively, the reference sample may be similar to the sample. Examples of such reference samples include previously purified batches of the same protein as the protein of interest, purified using similar or identical methods and subjected to substantially the same pH process. As yet a further alternative, the reference protein may be a protein similar to but not identical to the protein of interest, e.g., two antibodies or two Fc receptor fusion proteins, so long as the reference protein and the protein of interest behave similarly when subjected to similar pH change regimes. For the purposes of the non-dimensionalization and modeling methods described herein, the initial and final pH values ​​of all reference samples and samples do not need to be completely identical. For example, the initial and / or final pH values ​​of the one or more reference samples and the samples may differ by about 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, the initial and / or final pH values ​​of the one or more reference samples and the samples may be the same.

[0089] Thus, the present disclosure provides one or more reference samples that are used to generate the models used herein. The present disclosure provides titration curves that are 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 known to those skilled in the art.

[0090] In some embodiments, the method includes the steps of: (i) generating at least one reference titration curve from the at least one reference sample that relates an 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 the at least one reference titration curve includes normalizing the initial pH (pH initial_ref This is followed by adding a sufficient amount of titrant to the reference sample to change the pH of the reference sample (Titrant n_ref ), the additional reference pH value after the addition of this titrant (pH n_ref ) is measured. These steps may be repeated until a final pH is achieved, and the amount of titrant is plotted against the pH of the reference sample using any suitable program known in the art. When generating a reference titration curve, any suitable method of adding titrant may be used. The titrant may be added in separate steps - for example, by adding a separate amount of titrant, stirring for a period of time to mix it into the reference sample (e.g., until the pH of the reference sample stabilizes), and taking a pH measurement. Alternatively, the titrant may be added continuously and the pH may be measured continuously. When generating a reference sample or a titration curve for a sample, any suitable method of measuring pH may be used. For example, the pH of the reference sample may be measured by a pH probe inserted directly into the reference sample, or by a pH probe inserted into a continuous or individually sampled slipstream drawn from the reference sample.

[0091] In some embodiments, the amount of titrant added to the reference sample is determined according to the following formula:

number

[0092] In some embodiments, for example, when a single reference sample and corresponding titration curve are used to generate the model, the pH 1_ref may be the same as the initial pH of the reference sample, pH 2_ref may be the same as the final pH of the reference sample.

[0093] In an alternative embodiment, multiple reference samples and corresponding titration curves are used to generate the model. If the multiple titration curves do not have the same initial and / or final pH values, the pH 1_ref is not the same as some or all of the initial pH value of the reference sample, 2_ref is not the same as some or all of the final pH values ​​of the reference samples. Each reference titration curve is initial_ref and pH final_ref Contains pH 1_ref is the pH value from one of several standard titration curves initial_ref and pH 2_Ref is the pH value from one of several standard titration curves final_ref and pH 1_ref and pH 2_ref is selected to encompass the maximum difference in values ​​while still encompassing the pH values ​​encompassed by all of the multiple reference titration curves. 1_ref and pH2 are as far apart as possible, but pH initial and pH final As an example, if the reference titration curve involves increasing pH, 1_ref is the pH of the reference titration curve with the highest initial pH initial_ref may be, pH 2_ref is the pH of the reference titration curve with the lowest final pH final_ref Those skilled in the art will appreciate that if the reference titration curve involves decreasing pH, the opposite relationship may apply.

[0094] In some embodiments, the initial pH of the sample (pH initial ) and pH 1_ref are almost the same.

[0095] As an example, pH values ​​can be considered to be about the same if they are within about 0.05 units of each other. Alternatively, pH values ​​within 10%, 5%, or 3% of each other may be considered to be about the same.

[0096] In some embodiments, the initial pH of the sample (pH initial ) and pH 1_ref are not the same, i.e., pH initial and pH 1_ref The difference between 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. initial and pH 1_ref The difference between is approximately 0.1 to 0.5 pH units.

[0097] In some embodiments, the final pH of the sample (pH final ) and pH 2_ref are almost the same.

[0098] In some embodiments, the pH final and pH 2_ref are not the same, i.e., pH final and pH 2_ref In some embodiments, the difference between the pH and the pH is about 0.5 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. final and pH 2_ref In some embodiments, the difference between the pH and the pH is about 0.5 to 1.5 pH units. final and pH 2_ref In some embodiments, the difference between the pH final and pH 2_ref The difference between is approximately 0.1 to 0.5 pH units.

[0099] In some embodiments, the pH initial , pH initial_ref and pH 1_ref is the same, and pH final , pH final_ref and pH 2_ref is the same.

[0100] Those skilled in the art will appreciate that pH 1_ref and pH 2_ref It will be understood that the selection of will depend on the particular reference sample, the corresponding reference titration curve, and the variables contained therein with respect to the initial and final pH values.

[0101] In some embodiments, the final pH of the sample (pH final ) is the initial pH of the sample (pH initial ) and the titrant is an acid. In some embodiments, the sample and the plurality of reference samples contain a protein of interest. In some embodiments, the pH 1_ref is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8 or 4.9, 2_ref is about 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.8 or 3.9. 1_ref is about 4.1, and the pH 2_ref is about 3.6. In some embodiments, the initial pH of the sample and the plurality of reference samples is about 4.1-4.5. In some embodiments, the final pH of the sample and the plurality of reference samples is about 3.5-3.7, optionally 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, generating a model from the plurality of reference samples includes fitting a polynomial. In some embodiments, the polynomial is of the formula:

number

number

[0102] In some embodiments, the final pH of the sample (pH final ) is the initial pH (pH initial ) and the titrant is a base. In some embodiments, the sample and the plurality of reference samples contain a protein of interest. In some embodiments, the pH 1_ref In some embodiments, the pH is about 3.1 to 3.8. 1_ref In some embodiments, the pH is about 3.4 to 4.1. 1_ref is about 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.8 or 3.9. 1_ref is about 3.6. 1_ref In some embodiments, the pH is about 3.7. 2_ref In some embodiments, the pH is about 7.5 to 8.5. 2_ref 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. 2_ref In some embodiments, the pH is about 7.6. initial In some embodiments, the pH is about 3.5 to 3.7. final In some embodiments, the pH is about 5.1 to 8.5. final In some embodiments, the pH is about 7.5 to 8.0. final In some embodiments, the pH is about 7.5 to 8.0. finalis about 7.0 to 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, or about 7.4 to 7.6. In some embodiments, the amount of titrant added to the reference sample is normalized to a scale of about -0.06 to about 1.53. In some embodiments, generating a model from the plurality of reference samples includes fitting a polynomial. In some embodiments, the polynomial is of the formula:

number

number

[0103] [Protein of interest] The present disclosure provides a sample containing a 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 a subject for the treatment of a disease or disorder.Exemplary proteins of interest include, but are not limited to, antibodies, receptor Fc fusion proteins, such as trap proteins, cytokines, chemokines, growth factors, and the like.

[0104] In some embodiments, the protein of interest is an antigen binding protein, such as an antibody.

[0105] The phrase "antibody binding protein" includes proteins that have at least one complementarity determining region (CDR) and are capable of selectively recognizing an antigen, i.e., capable of binding to an antigen with a KD that is at least in the micromolar range. Therapeutic antigen binding proteins (e.g., therapeutic antibodies) frequently require a KD that is in the nanomolar or picomolar range. Typically, an antigen binding protein comprises two or more CDRs, e.g., 2, 3, 4, 5, or 6 CDRs. Examples of antigen binding proteins include antibodies, antigen-binding fragments of antibodies, e.g., polypeptides containing the variable regions of the antibody heavy and light chains (e.g., Fab fragments, F(ab')2 fragments), and proteins that contain the variable regions of the antibody heavy and light chains and additional amino acids from the constant regions of the heavy and / or light chains (e.g., one or more constant domains, i.e., one or more of the CL, CH1, hinge, CH2, and CH3 domains).

[0106] "Antibody" refers to an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. 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 regions of hypervariability called complementarity determining regions (CDRs) that disperse these into more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, which are arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The term "antibody" includes both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term "antibody" includes antibody molecules prepared, expressed, produced or isolated by recombinant means, e.g., antibodies isolated from a host cell transfected with a nucleotide sequence to express the antibody. The term "antibody" also includes bispecific antibodies, including heterotetrameric immunoglobulins capable of binding to two or more epitopes. The term "antibody", as used herein, also includes antigen-binding fragments of full-length antibody molecules, and fusion proteins comprising an antibody or antigen-binding fragment.

[0107] The term "antigen-binding portion" of an antibody (or antibody fragment) 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 encompassed by the term "antigen-binding portion" of an antibody include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., Nature (1989) 241:544-546); (vi) an isolated CDR; and (vii) an scFv consisting of the two domains VL and VH of an Fv fragment joined by a synthetic linker to form a single protein chain in which the VL and VH domains pair to form a monovalent molecule. Other forms of single chain antibodies, such as diabodies, are also encompassed by the term "antibody". See, e.g., Holliger et al., PNAS USA (1993) 90:6444-6448; Poljak et al., Structure (1994) 2:1121-1123.

[0108] Furthermore, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule formed by covalent or non-covalent binding of the antibody or antibody portion to one or more other proteins or peptides. Non-limiting examples of such immunoadhesion molecules include the use of streptavidin core regions to produce tetrameric scFv molecules (Kipriyanov et al., Human Antibodies and Hybridomas (1995) 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to produce bivalent biotinylated scFv molecules (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 conventional techniques, for example, by papain or pepsin digestion of whole antibodies. Furthermore, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques generally known in the art (see Sambrook et al., 1989).

[0109] The term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) that are not encoded by human germline immunoglobulin sequences, for example in the CDRs, particularly CDR3.

[0110] The term "recombinant human antibody", as used herein, is intended to include all human antibodies prepared, expressed, created or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. Nucl. Acids Res. (1992) 20:6287-6295), or antibodies prepared, expressed, created or isolated by any other means, including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally exist within the human antibody germline repertoire in vivo.

[0111] Additional therapeutic proteins are contemplated within the scope of the disclosed methods of cell culture and therapeutic protein production. In certain 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 diabody, a triabody or a tetrabody, a Fab fragment or a 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 certain 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.

[0112] In some embodiments, the antibody is an anti-Programmed Cell Death 1 antibody (e.g., the anti-PD1 antibody described in U.S. Patent Application Publication No. US2015 / 0203579A1), an anti-Programmed Cell Death Ligand-1 (e.g., the anti-PD-L1 antibody described in U.S. Patent Application Publication No. US2015 / 0203580A1), an anti-Dll4 antibody, an anti-angiopoietin-2 antibody (e.g., the anti-ANG2 antibody described in U.S. Patent No. 9,402,898), an anti-angiopoietin-like 3 antibody (e.g., the anti-AngPtl3 antibody described in U.S. Patent No. 9,018,356), an anti-platelet derived growth factor receptor antibody (e.g., the anti-PDGFR antibody described in U.S. Patent No. 9,265,827), an anti-Erb3 antibody, an anti-prolactin receptor antibody (e.g., the anti-PDGFR antibody described in U.S. Patent No. No. 9,302,015), anti-complement 5 antibodies (e.g., the anti-C5 antibodies described in U.S. Patent Application Publication No. US2015 / 0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., the anti-EGFR antibodies described in U.S. Patent Application Publication No. US2015 / 0259423A1 or the anti-EGFRvIII antibodies described in U.S. Patent Application Publication No. US2015 / 0259423A1), anti-proprotein convertase subtilisin kexin-9 antibodies (e.g., the anti-PCSK9 antibodies described in U.S. Patent No. 8,062,640 or U.S. Patent Application Publication No. US2014 / 0044730A1), anti-growth differentiation factor-8 antibodies (e.g., the anti-PRLR antibodies described in U.S. Patent Application Publication No. Anti-GDF8 antibodies, also known as anti-myostatin antibodies, as described in U.S. Patent Application Publication Nos. US2015 / 0337045A1 or US2016 / 0075778A1, anti-glucagon receptor (e.g., anti-GCGR antibodies, as described in U.S. Patent Application Publication Nos. US2015 / 0337045A1 or US2016 / 0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, interleukin 4 receptor antibodies (e.g., anti-IL4R antibodies, as described in U.S. Patent Application Publication Nos. US2014 / 0271681A1 or U.S. Patent Nos. 8,735,095 or 8,945,559), anti-interleukin 6 receptor antibodies (e.g., anti-IL4R antibodies, as described in U.S. Patent Application Publication Nos.Nos. 7,582,298, 8,043,617 or 9,173,880), anti-IL1 antibodies, anti-IL2 antibodies, anti-IL3 antibodies, anti-IL4 antibodies, anti-IL5 antibodies, anti-IL6 antibodies, anti-IL7 antibodies, anti-interleukin 33 (e.g., the anti-IL33 antibodies described in U.S. Patent Application Publication Nos. US2014 / 0271658A1 or US2014 / 0271642A1), anti-respiratory syncytial virus antibodies (e.g., the anti-RSV antibodies described in U.S. Patent Application Publication No. US2014 / 0271653A1), anti-surface cluster 3 antibodies (e.g., the anti-IL6R antibodies described in U.S. Patent Application Publication Nos. US2014 / 0088295A1 and US20150266966A1, and U.S. Application No. 62 / 222,605), anti-cluster of chromosome 20 (e.g., anti-CD20 antibodies described in U.S. Patent Application Publication Nos. US2014 / 0088295A1 and US20150266966A1, and U.S. Patent No. 7,879,984), anti-CD19 antibodies, anti-CD28 antibodies, anti-cluster of chromosome 48 (e.g., anti-CD48 antibodies described in U.S. Patent No. 9,228,014), anti-Fel d1 antibodies (e.g., as described in U.S. Patent No. 9,079,948), anti-Middle East Respiratory Syndrome virus (e.g., anti-MERS antibodies described in U.S. Patent Application Publication No. US2015 / 0337029A1), anti-Ebola virus antibodies (e.g., as described in U.S. Patent Application Publication No. US2016 / 0215040), anti-Zika virus antibodies, anti-lymphocyte activation gene 3 antibodies (e.g., anti-LAG3 antibodies, or anti-CD223 antibodies), anti-nerve growth factor antibodies (e.g., anti-NGF antibodies described in U.S. Patent Application Publication No. US2016 / 0017029 and U.S. Patent Nos. 8,309,088 and 9,353,176), and anti-activin A antibodies. In some embodiments, the bispecific antibody is selected from the group consisting of an anti-CD3 x anti-CD20 bispecific antibody (e.g., anti-CD3 x anti-CD20 antibody described in U.S. Patent Application Publication No.US2014 / 0088295A1 and US20150266966A1), anti-CD3 x anti-mucin 16 bispecific antibodies (e.g., anti-CD3 x anti-Muc16 bispecific antibodies), and anti-CD3 x anti-prostate specific membrane antigen bispecific antibodies (e.g., anti-CD3 x anti-PSMA bispecific antibodies). In some embodiments, the protein of interest is selected from the group consisting of alirocumab, sarilumab, fasinumab, nesvacumab, dupilumab, trevogrumab, evinacumab, and rinucumab. All publications mentioned throughout this disclosure are incorporated herein by reference in their entirety.

[0113] In other embodiments, the therapeutic protein is a recombinant protein containing an Fc portion and another domain (e.g., an Fc fusion protein). In some embodiments, the Fc fusion protein is a receptor-Fc fusion protein that contains one or more extracellular domains of a receptor linked to an Fc portion. In some embodiments, the Fc portion contains a hinge region followed by the CH2 and CH3 domains of IgG. In some embodiments, the receptor-Fc fusion protein contains two or more separate receptor chains that bind either a single ligand or multiple ligands. For example, the Fc fusion protein can be a trap protein, such as an IL-1 trap (e.g., rilonacept, which contains the IL-1RAcP ligand binding region fused to the Il-1R1 extracellular domain fused to the Fc of hIgG1; see U.S. Patent No. 6,927,004, which is incorporated herein by reference in its entirety), a VEGF trap (e.g., aflibercept or ziv-aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to the Fc of hIgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159; or conbercept, which contains the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to the Ig domain 4 of the VEGF receptor Flk1 fused to the Fc of hIgG1; U.S. Patent No. 8,216,575) or a 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 that contains one or more antigen binding domains of an antibody, e.g., one or more of a variable heavy chain fragment and a variable light chain fragment, linked to the Fc portion.

[0114] In some embodiments, the protein of interest is a glycoprotein. Glycoproteins with asparagine-linked (N-linked) glycans are ubiquitous in eukaryotic cells. The biosynthesis of these glycans and their transfer to polypeptides occurs in the endoplasmic reticulum (ER). N-glycan structures are further modified by several glycosidases and glycosyltransferases in the ER and Golgi complex. Glycosylation of therapeutic proteins can be important for the quality and efficacy of therapeutic proteins. For example, antibody glycosylation is a common post-translational modification and can play a role in antibody effector function and antibody stability. Methods for analyzing glycosylation patterns and the proportion of glycosylated proteins in protein samples are known to those skilled in the art.

[0115] [Protein purification] Methods for purifying a protein of interest produced by the cells and cell culture methods described herein that produce the protein of interest are known to those skilled in the art. Methods for purifying a protein of interest from cell culture medium or from cells include chromatographic and non-chromatographic methods. Chromatographic methods include passing a solution containing an antibody through a solid phase (e.g., silica resin or beads, monolithic column, or cellulose membrane) and allowing the protein of interest to bind or pass through, depending on whether a "bind and elute" or "flow-through" chromatographic method is used. Chromatographic methods include, but are not limited to, affinity tag binding, protein A binding, ion exchange chromatography (e.g., anion exchange chromatography), size exclusion chromatography, or immunoaffinity chromatography. Purification can also be achieved through the use of genetically fused purification tags, such as polyhistidine tags or FLAG tags.

[0116] An exemplary protein purification protocol involves obtaining a clarified solution containing the protein of interest and performing a combination of different purification techniques, including an ion exchange separation step and a hydrophobic interaction separation step. The separation step separates the protein mixture based on the charge, hydrophobicity or size of the proteins. In one aspect of the invention, the separation is performed using chromatography, including cationic, anionic and hydrophobic interaction. Different chromatographic resins are available for each of these steps, allowing precise adjustment of the purification scheme for the specific protein involved. The crux of each of the separation methods is that the proteins can pass through the column at different rates, achieving a physical separation that increases as they pass further through the column, or selectively adhere to the separation medium and are then differentially eluted by different solvents. In some cases, the protein of interest is separated from the impurities when the impurities specifically adhere to the column and the protein does not, i.e., the protein of interest is present in the flow-through.

[0117] In some embodiments, purification of the protein of interest includes a first recovery step. In some embodiments, the first recovery step includes a chromatography column, such as an affinity column. The first recovery step may also be the point at which viruses are inactivated using the methods described herein, for example by subjecting the pool of protein of interest in the eluate to a pH change as described herein.

[0118] In some embodiments, the protein sample recovered from the primary recovery step is subjected to 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: chromatographic materials comprising protein A, protein G, an antigen bound by the antibody of interest or an antibody that binds to the protein of interest, and chromatographic materials comprising Fc binding proteins. As a further example, a hydrophobic interaction column may be used to remove impurities such as aggregates.

[0119] Any purification step may produce a sample that can be subjected to the methods described herein. Potential viruses may be inactivated after any suitable purification step using the pH control processes described herein after any suitable purification step. In addition, or the pH of the solution containing the protein of interest may be changed to a desired pH for the next purification step or other downstream application, for example, using the methods described herein.

[0120] [Cells and cell cultures] The present disclosure provides populations of cells for use in producing the proteins of interest described herein. Suitable cells include bacterial cells, yeast cells and mammalian cells.

[0121] In some embodiments, the population of cells is isolated or derived from a cell line capable of producing the protein of interest. Non-limiting examples of cell lines used to produce therapeutic proteins include, among others, 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, baby 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 embryo cells. In some embodiments, the population of cells comprises CHO cells. In some embodiments, the CHO cells include one or more of several specific CHO cell variants optimized for large-scale protein production, such as CHO-K1, CHO-K1 derived EESYR (enhanced expression and stability regions)® cells (U.S. Patent No. 7,771,997), or CHO cells from the FASTR technology described in U.S. Patent No. 6,919,183, which provides for the isolation of cells that produce secreted proteins.

[0122] In some embodiments, the population of cultured cells expressing a protein of interest is a population of cells obtained by clonal expansion of a cell (i.e., a progenitor cell) that contains and expresses a polynucleotide encoding a 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 population of cells obtained by clonal expansion from or descended from a progenitor cell contain the polynucleotide encoding the protein and express the protein of interest.

[0123] In some embodiments, a population of cultured therapeutic protein-expressing cells is produced by culturing frozen and stored cells. Mammalian cells can be frozen and cryopreserved, for example, in a cryopreservation medium containing dimethyl sulfoxide (DMSO) and cell culture medium. In an exemplary cryopreservation protocol, mammalian cells are transferred to a cryopreservation medium, slowly frozen, and then stored under liquid nitrogen. For example, cells can be expanded and cryopreserved to create a cell bank, which is a bank of cells generated from a single pool of cells with desired characteristics.

[0124] The present disclosure provides methods for culturing cells expressing a protein of interest prior to purification.

[0125] "Cell culture" or "culturing" refers to the growth and propagation of cells outside a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. See, for example, Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells may be cultured in suspension or alternatively attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks or stirred tank bioreactors, with or without microcarriers and operating in batch, fed-batch, continuous, semi-continuous or perfusion modes, can be used for mammalian cell culture.

[0126] In some embodiments, culturing the population of cells expressing a protein of interest includes an expansion or growth step in which the population of cells is grown to a size sufficient to produce a desired amount of the protein of interest in a production step.

[0127] In some embodiments, culturing a population of cells expressing a protein of interest includes a production stage in which the population of cells is cultured in a production cell culture medium under conditions sufficient to produce the protein of interest. The production stage can be performed at any culture scale, from individual flasks and shake flasks or wave bags to 1 liter bioreactors to large scale industrial bioreactors. Large scale processes can be performed in volumes of about 100 liters to 20,000 liters or more. One or more of several means may be used to control protein production, such as temperature shift or chemical induction. The growth stage can occur at a higher temperature than the production stage. For example, the growth stage may be performed at a first temperature of about 35°C to 38°C, and the production stage may be performed at a second temperature of about 29°C to 37°C, optionally 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 bisacetamide (HMBA), may be added simultaneously with, before, or after the temperature shift. If inducers are added after the temperature shift, they may be added 1 hour to 5 days after the temperature shift, such as 1 to 2 days after the temperature shift. The production cell cultures may be run as continuous fed culture systems, in chemostats (see C. Altamirano et al., Biotechnol Prog. 2001 November-December; 17(6):1032-41), or according to a fed-batch process (Huang, 2010).

[0128] As used herein, the terms "cell culture media", "culture medium", "cell culture medium", "cell culture medium" or "culture medium" refer to any nutrient solution used to grow cells, e.g., animal or mammalian cells, and generally providing at least one or more of the following components: an energy source (usually in the form of a carbohydrate such as glucose); one or more of all essential amino acids, and generally the 20 basic amino acids; vitamins and / or other organic compounds, typically required at low concentrations; lipids or free fatty acids; and trace elements, e.g., inorganic compounds or naturally occurring elements, typically required at very low concentrations, usually in the micromolar range. In some embodiments, the cell culture medium is formed by combining soy or other plant protein hydrolysates with one or more additional ingredients.

[0129] As used herein, "additional components" include, but are not limited to, any one or more of the cell culture medium components including water, an energy source, one or more of all essential amino acids, and generally the 20 basic amino acids; vitamins and / or other organic compounds typically 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 soy hydrolysate with a basic cell culture medium and supplementing the medium with additional polyamines.

[0130] In some embodiments, the cell culture medium contains a chemically defined basal medium, such as a conventionally formulated or commercially available basal medium.

[0131] Commercially available culture media are known to those skilled in the art and include, among others, Eagle's MEME (Minimum Essential Medium) (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., 1952 August; 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's medium (Davidson and Waymouth, Biochem. J., 1945, 39(2):188-199), Williams E medium (William's et al., Exp. Cell Res., 1971, 69:105 et seq.), 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 EX-CELL® 300 Series (JRH Biosciences, Lenexa, Kans.), protamine-zinc-insulin medium (Weiss et al., 1974, U.S. Pat. No.4,072,565), biotin-folic acid 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; Chessebeuf, 1984, US Patent No. 4,786,599), as well as other medium replacements (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, US Patent No. (see, e.g., 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).

[0132] In some embodiments, the cell culture medium is serum-free. In some embodiments, the cell culture medium is serum-free and hydrolysate-free.

[0133] In some embodiments, a medium at a useful concentration (i.e., 1x) contains at least 40±6 mM or at least 55±10.5 mM of the mixture of amino acids or amino acid salts. In one embodiment, the medium contains at least 40 mM of the mixture of amino acids. In this or another embodiment, the medium contains at least 55 mM of the mixture of amino acids. In one embodiment, the mixture of amino acids (excluding glutamine, which may be added back to the medium as a point-of-use addition) contains alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0134] In some embodiments, the medium contains one or more fatty acids. In a particular embodiment, the medium contains a mixture of fatty acids (or fatty acid derivatives) and alpha tocopherol. The fatty acid or fatty acid derivative is selected from the group consisting of linoleic acid, linolenic acid, thioctic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, lauric acid, behenic acid, decanoic acid, dodecanoic acid, hexanoic acid, lignoceric acid, myristic acid, and octanoic acid.

[0135] In some embodiments, the medium contains a mixture of nucleosides, hi one embodiment, the medium contains adenosine, guanosine, cytidine, uridine, thymidine and hypoxanthine.

[0136] In some embodiments, the medium contains a mixture of salts. The salts include divalent cations, such as calcium and magnesium. In one embodiment, the medium contains calcium chloride and magnesium sulfate. Other salts may include those of phosphates.

[0137] Depending on the cell culture process, different cell culture media may be used at different times during cell culture. For example, when growing an initial population of cells from a frozen aliquot to produce a population of cells for the production of a protein of interest, a growth cell culture medium may be used. Second, a production cell culture medium may be used to culture the grown population of cells for the production of 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 a further alternative, the growth medium may be different from the production and feed media, with the production and feed media having the same or similar composition.

[0138] In some embodiments, one or more point-of-use additions may be added to any of the cell culture media during cell culture as described herein.

[0139] In some embodiments, culturing the population of cells includes adding a feed medium to the production cell culture. As used herein, "feed medium" refers to a medium that is added to the cells being cultured to replenish depleted nutrients. The feed medium may be concentrated. For example, one or all components of the feed medium may be concentrated when compared to the production cell culture medium. Alternatively, the feed medium may be at a similar concentration to the production cell culture medium. The feed medium may be added to the culture continuously or occasionally during the culture, for example, every day, every other day, or may be fed to the cell culture when the concentration of a particular medium component being monitored falls outside the desired range.

[0140] In some embodiments, the medium is replenished from time to time during cell culture according to a fed-batch process. Fed-batch culture is commonly known in the art and is used for optimized protein production. See, for example, YM Huang et al., Biotechnol Prog. (2010) 26(5) pp.1400-1410.

[0141] The percent viable cells may be measured at any point during the cell culture methods described herein. Methods for determining viable cell count and cell density include, but are not limited to, imaging the cells and quantifying cell number, density, diameter and biomarker expression.

[0142] Mammalian cells, e.g., CHO cells, may be cultured in small-scale cell culture vessels, e.g., 125 ml vessels with about 25 ml of medium, 250 ml vessels with about 50-100 ml of medium, or 500 ml vessels with about 100-200 ml of medium. For example, these small-scale vessels can be shake flasks. Cell culture flasks are known in the art and are available, e.g., from Corning, Fisher Scientific, and other suppliers.

[0143] Alternatively, cell cultures may be grown on a bench scale. These include, for example, 1000 ml vessels with about 300-1000 ml of medium, 3000 ml vessels with about 500-3000 ml of medium, 8000 ml vessels with about 2000-8000 ml of medium, and 15,000 ml vessels with about 4000-15,000 ml of medium. Suitable cell culture systems are commercially available.

[0144] Cultures for manufacturing (i.e., production cell cultures) can contain 10,000 L or more of medium. Large-scale cell cultures, or "production cell cultures," for example for the manufacture of protein therapeutics, are typically maintained for days or even weeks while the cells produce the desired protein. During this time, the cultures may be supplemented with a concentrated feed medium containing components such as nutrients and amino acids that are consumed during the culture process.

[0145] In some embodiments, cell culture media is supplemented with one or more "point-of-use additives", also known as additives, point-of-use components or point-of-use chemicals, during the course of cell growth or protein production. Point-of-use additives include any one or more of growth factors or other proteins, buffers, energy sources, salts, amino acids, metals, osmolytes and chelators. 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 usually proteins (e.g., insulin), small peptides or steroid hormones, such as estrogen, DHEA, testosterone, etc.

[0146] In some embodiments, the cell culture medium is supplemented with any one or more or all of the following point-of-use additives: sodium bicarbonate, dextrose, L-glutamine, L-tyrosine, a mixture of amino acids, and sodium phosphate.

[0147] Buffers are generally known in the art. The present invention is not limited to any particular buffer or buffers, and one of skill in the art can select an appropriate buffer or buffer system for use with a particular cell line producing a particular protein.

[0148] Energy sources for use as point-of-use additions in cell cultures are also well known in the art. Without limitation, in some embodiments, the point-of-use added energy source is glucose. In other embodiments, the point-of-use added energy source is dextrose.

[0149] Chelators are likewise well known in the art of cell culture and protein production. Tetrasodium EDTA dihydrate and citrate are two common chelators used in the art, although other chelators may be used in the practice of the present invention.

[0150] Other point-of-use additives include various metal salts, such as one or more of the following salts: iron, nickel, zinc, and copper. In one embodiment, the cell culture medium is supplemented with any one or more of copper sulfate, zinc sulfate, ferric chloride; and nickel sulfate.

[0151] [Device] The present disclosure provides devices for use in the methods described herein.

[0152] In some embodiments, the device may be used to effectively inactivate viruses in protein samples in a reactor by controlling the pH and performing a pH sequence (a sequence that changes the pH of the sample). Drawing a sample from the reactor and measuring the pH of the sample can address some of the issues that arise from using a probe inserted into the reactor. For example, the probe often cannot be calibrated after sterilization. Sterilization can affect the probe calibration curve. Sterilization by autoclaving often involves coordination with a third party and is therefore time consuming. The probe may be stored in a dry environment after sterilization, which can reduce the probe performance and shelf life. The use of a probe often involves the use of an additional sterile connection port. In addition, the use of a probe involves the risk of the probe breaking and the leakage of the reference solution into the protein product measured by the probe.

[0153] FIG. 19 is a block diagram of an Apparatus 100 for pH control, according to one embodiment. As shown, the Apparatus 100 includes a Reactor 110, a pH Flow Cell 120, an Acid Titrant Supply 130, a Base Titrant Supply 140, and optionally a Waste Receiver 150. The pH Flow Cell 120 contains a pH Probe 121 disposed therein for measuring the pH of a liquid sample in the flow cell. Lines between components represent fluid connections. The Apparatus 100 may include one or more controllers for controlling any of its process units. The controller may control the sequence of steps (e.g., development of acid / base titrants). In some embodiments, the one or more controllers control the sequence, which may be based on pH measurements in the pH Flow Cell. The controller may be accessible via a user interface. In some embodiments, the user interface may include a computer, a laptop, a mobile device, a tablet, a mobile phone, or any other suitable device.

[0154] In the reactor 110, the 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, the reactor 110 may be a batch reactor or may have the characteristics of a batch reactor. In some embodiments, the reactor 110 may be a constantly stirred tank reactor (CSTR) or may have the characteristics of a CSTR. In some embodiments, the reactor 110 may be a plug flow reactor (PFR) or may have the characteristics of a PFR. In some embodiments, the reactor 110 may include a mixer disposed therein. In some embodiments, the mixer may include an impeller. The mixer may homogenize the contents of the reactor 110 immediately before, after, or during the addition of the acid titrant and / or base titrant. In some embodiments, the mixer is controlled by a controller, i.e., the controller sends a signal to the mixer to start the impeller, stop the impeller, or control the speed of the impeller. In some embodiments, the apparatus 100 may include a mixer disposed outside the reactor 110. In other words, the mixer may be a separate unit from the reactor 110. In some embodiments, an acid mixer (not shown) may be fluidly coupled to the acid titrant supply 130 to mix the acid titrant prior to addition to the reactor 110. In some embodiments, a base mixer (not shown) may be fluidly coupled to the base titrant supply 140 to mix the base titrant prior to addition to the reactor 110. In some embodiments, the reactor 110 may not have a pH measurement probe disposed therein. The reactor 110 may be maintained at a desired pH via delivery of acid titrant and / or base titrant from the acid titrant supply 130 and / or base titrant supply 140.

[0155] In some embodiments, the controller sends a signal to the mixer to start before starting the acid titrant pump (for acid titrant addition) or before starting the base titrant pump (for base titrant addition). In some embodiments, the controller sends a signal to the mixer to stop after a fixed period of time after the acid titrant pump or base titrant pump is stopped. For example, the controller may send a signal to the mixer to stop 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 base titrant pump is stopped.

[0156] In some embodiments, the reactor 110 is 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 m 3 , at least about 2m 3 , at least about 3m 3 , at least about 4m 3 , at least about 5m 3 , at least about 6m 3 , at least about 7m 3 , at least about 8m 3 , at least about 9m 3 , at least about 10m 3 , at least about 20m 3 , at least about 30m 3 , at least about 40m 3 , at least about 50m 3 , at least about 60m 3 , at least about 70m 3 , at least about 80m3 Or at least about 90m 3 In some embodiments, the reactor 110 may have a volume of about 100 m 3 Below, approximately 90m 3 Below, about 80m 3 Below, about 70m 3 Below, approximately 60m 3 Below, about 50m 3 Below, about 40m 3 Below, about 30m 3 Below, approximately 20m 3 Below, approximately 10m 3 Below, about 9m 3 Below, approximately 8m 3 Below, approximately 7m 3 Below, approximately 6m 3 Below, about 5m 3 Below, approximately 4m 3 Below, approximately 3m 3 Below, approximately 2m 3 Below, approximately 1m 3 Below, about 900L or less, about 800L or less, about 700L or less, about 600L or less, about 500L or less, about 400L or less, about 300L or less, about 200L or less, about 100L or less, about 90L or less, about 80L or less, about 70L or less, about It can have a volume of 60 L or less, about 50 L or less, about 40 L or less, about 30 L or less, about 20 L or less, about 10 L or less, about 9 L or less, about 8 L or less, about 7 L or less, about 6 L or less, about 5 L or less, about 4 L or less, about 3 L or less, or about 2 L or less.

[0157] Combinations of the above-mentioned volumes of the reactor 110 are also possible, including all values ​​and ranges therebetween (e.g., at least about 1 L and at least about 100 m 3 In some embodiments, the reactor 110 can be about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 20 L, about 30 L, about 40 L, about 50 L, about 60 L, about 70 L, about 80 L, about 90 L, about 100 L, about 200 L, about 300 L, about 400 L, about 500 L, about 600 L, about 700 L, about 800 L, about 900 L, about 1 m 3 , about 2m 3 , about 3m 3 , about 4m3 , about 5m 3 , about 6m 3 , about 7m 3 , about 8m 3 , about 9m 3 , about 10m 3 , about 20m 3 , about 30m 3 , about 40m 3 , about 50m 3 , about 60m 3 , about 70m 3 , about 80m 3 , about 90m 3 Or about 100m 3 In some embodiments, the reactor 110 may include a level indicator.

[0158] In some embodiments, the pH probe 121 may be disposed in the pH flow cell 120. In some embodiments, the pH in the reactor 110 may be determined or ascertained based on a reading from the pH probe 121 in the pH flow cell 120. The pH probe 121 in the pH flow cell 120, when inserted into the effluent from the reactor 110, measures the difference in potential between a reference electrode and a hydrogen ion selective electrode. In other words, hydrogen ion activity 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 with a pH value. In some embodiments, the 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 produced 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 separate component from the pH flow cell 120. In some embodiments, the pH transmitter may transmit a pH reading from the pH probe 121 to a controller. In some embodiments, the controller sends the pH reading from the pH transmitter to a user interface (not shown) configured to display the pH reading. Based on the pH transmitted by the pH transmitter, the device 100 may maintain its current operation or may initiate a change in operation (e.g., addition of an acid titrant from the acid titrant supply 130). In some embodiments, the change in operation may be performed automatically. In some embodiments, the change in operation may be performed by user input.

[0159] In some embodiments, the pH probe 121 disposed in the pH flow cell 120 may measure a 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.

[0160] The pH probe measures the pH of a sample volume in the pH flow cell 120 from the reactor 110. In some embodiments, the sample volume can 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 is about 150 mL or less, about 140 mL or less, about 130 mL or less, about 120 mL or less, about 110 mL or less, about 100 mL or less, about 90 mL or less, about 80 mL or less, about 70 mL or less, about 60 mL or less, about 50 mL or less, about 40 mL or less, about 30 mL or less, about 20 mL or less, about 10 mL. less than or equal to about 9 mL, less than or equal to about 8 mL, less than or equal to about 7 mL, less than or equal to about 6 mL, less than or equal to about 5 mL, less than or equal to about 4 mL, less than or equal to about 3 mL, less than or equal to about 2 mL, less than or equal to about 1 mL, less than or equal to about 0.9 mL, less than or equal to about 0.8 mL, less than or equal to about 0.6 mL, less than or equal to about 0.5 mL, less than or equal to about 0.4 mL, less than or equal to about 0.3 mL, or less than or equal to about 0.2 mL. Combinations of the above-referenced sample volumes are also possible, including all values ​​and ranges therebetween (eg, at least about 0.1 mL and not more than about 100 mL, or at least about 10 mL and not more than about 20 mL).In some embodiments, the sample volume can 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.

[0161] 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 number of factors, including, but not limited to, the amount of fluid in the reactor 110, the type of protein in the reactor 110, and / or the size of the reactor 110. In some embodiments, samples may be withdrawn from the reactor 110 and measured at prescribed intervals. In some embodiments, samples may be withdrawn from the reactor 110 and measured at spontaneous, user-specified intervals.

[0162] In some embodiments, the one or more controllers may trigger an action based on the pH value measured by the pH flow cell 120. For example, if the pH measured by the pH flow cell 120 is greater than a desired pH value, the controller may trigger delivery of an acid titrant from the acid titrant supply 130 to the reactor 110. In some embodiments, if the pH measured by the pH flow cell 120 is less than a desired pH value, the controller may trigger delivery of a base titrant from the base titrant supply 140 to the reactor 110. In some embodiments, the controller may hold the pH at a desired value for a desired period of time. In some embodiments, the action may be triggered manually (i.e., via a user interface). In some embodiments, the action may be triggered automatically (i.e., based on a prescribed sequence). In some embodiments, the prescribed sequence may include decreasing the pH in the reactor 110 to a first pH value and then increasing the pH in the reactor to a second pH value. In some embodiments, the first pH value can be about 3.0 to about 4.5, about 3.5 to about 4.3, about 3.5 to about 4.0, about 3.1 to about 3.9, about 3.2 to about 3.8, about 3.3 to about 3.7, about 3.4 to about 3.7, about 3.3 to about 3.6, about 3.4 to about 3.6, about 3.4 to about 3.5, or about 3.5 to about 3.6. In some embodiments, the second pH value can be about 7 to about 8.5, about 7.1 to about 8.4, about 7.2 to about 8.3, about 7.3 to about 8.2, about 7.4 to about 8.2, about 7.4 to about 8.1, about 7.4 to about 8.0, about 7.5 to about 8.2, about 7.5 to about 8.1, about 7.5 to about 8.0, about 7.6 to about 8.1, about 7.6 to about 8.0, about 7.6 to about 7.9, about 7.7 to about 8.0, about 7.7 to about 7.9, or about 7.7 to about 7.8.

[0163] In some embodiments, the delivery of the acid titrant to reach the first pH value can be multi-step. In other words, the acid titrant can be added in granular form so that the pH of the contents of the reactor 110 can be precisely monitored. In some embodiments, the delivery of the acid titrant to reach the first pH value can be in a number of steps including 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 steps, and all values ​​and ranges therebetween. In some embodiments, the delivery of the acid titrant to reach the first pH value can be in 1, 2, 3, or 4 steps. This can address the difficulty of checking the pH meter, since the addition of a small amount of titrant can change the pH of the contents of the reactor 110 more predictably.

[0164] In some embodiments, the delivery of the base titrant to reach the second pH value can be multi-step. In other words, the base titrant can be added in a granular manner so that the pH of the contents of the reactor 110 can be precisely monitored. In some embodiments, the delivery of the acid titrant to reach the second pH value can be in a number of steps including 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 steps, and all values ​​and ranges therebetween. In some embodiments, the delivery of the base titrant to reach the second pH value can be in 1, 2, 3 or 4 steps.

[0165] The acid titrant may be delivered from acid titrant supply 130, if desired. In some embodiments, the acid titrant may 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, acid titrant supply 130 may include a reservoir. In some embodiments, acid titrant supply 130 may include a reservoir. In some embodiments, acid titrant supply 130 may include a reservoir. In some embodiments, acid titrant supply 130 may include a reservoir. In some embodiments, acid titrant supply 130 may include a reservoir. about 3L, at least about 4L, at least about 5L, at least about 6L, at least about 7L, at least about 8L, at least about 9L, at least about 10L, at least about 20L, at least about 30L, at least about 40L, at least about 50L, at least about 60L, at least about 70L, at least about 80L, at least about 90L, at least about 100L, at least about 200L, at least about 300L, at least about 400L, at least about 500L, at least about 600L, at least about 700L, at least about 800L, at least about 900L, at least about 1m 3 , at least about 2m 3 , at least about 3m 3 , at least about 4m 3 , at least about 5m 3 , at least about 6m 3 , at least about 7m 3 , at least about 8m 3 Or at least about 9m 3 In some embodiments, the acid titrant supply 130 may have a volume of about 10 ml. 3 Below, about 9m 3 Below, approximately 8m 3 Below, approximately 7m 3 Below, approximately 6m 3 Below, about 5m 3 Below, approximately 4m 3Below, approximately 3m 3 Below, approximately 2m 3 Below, approximately 1m 3 Below, about 900L or less, about 800L or less, about 700L or less, about 600L or less, about 500L or less, about 400L or less, about 300L or less, about 200L or less, about 100L or less, about 90L or less, about 80L Below, about 70L or less, about 60L or less, about 50L or less, about 40L or less, about 30L or less, about 20L or less, about 10L or less, about 9L or less, about 8L or less, about 7L or less, about 6L or less, about 5L or less, about 4L or less , about 3 L or less, about 2 L or less, about 1 L or less, about 900 mL or less, about 800 mL or less, about 700 mL or less, about 600 mL or less, about 500 mL or less, about 400 mL or less, about 300 mL or less, about 200 mL or less, about 100 mL or less, about 90 mL or less, about 80 mL or less, about 70 mL or less, about 60 mL or less, about 50 mL or less, about 40 mL or less, about 30 mL or less, or about 20 mL or less.

[0166] Combinations of the above-referenced volumes of acid titrant supply 130 are also possible, including all values ​​and ranges therebetween (e.g., at least about 10 mL and at least about 10 mL). 3 or less, or at least about 1 L and less than about 5 L). In some embodiments, acid titrant supply 130 may be 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, about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about L, about 1L, about 2L, about 3L, about 4L, about 5L, about 6L, about 7L, about 8L, about 9L, about 10L, about 20L, about 30L, about 40L, about 50L, about 60L, about 7 0L, approximately 80L, approximately 90L, approximately 100L, approximately 200L, approximately 300L, approximately 400L, approximately 500L, approximately 600L, approximately 700L, approximately 800L, approximately 900L, approximately 1m 3 , about 2m 3 , about 3m 3 , about 4m 3 , about 5m 3 , about 6m 3 , about 7m 3 , about 8m 3 Or about 9m 3 Or about 10m3 The volume of the slit may be 0.01 to 0.05.

[0167] 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 about 7 or less, about 6.5 or less, about 6 or less, about 5.5 or less, about 5 or less, about 4.5 or less, about 4 or less, about 3.5 or less, about 3 or less, about 2.5 or less, about 2 or less, about 1.5 or less, about 1 or less, or about 0.5 or less. Combinations of the above-referenced pH values ​​in the acid titrant supply 130 are also possible, including all values ​​and ranges therebetween (e.g., at least about 0.5 and about 7 or less, or at least about 2 and about 6 or less). 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.

[0168] The base titrant may be delivered from the base titrant supply 140, if desired. In some embodiments, the base titrant may be delivered via a pump (not shown). In some embodiments, the base titrant supply 140 may include a container. In some embodiments, the base titrant supply 140 may include a tank. In some embodiments, the base titrant supply 140 may include a reservoir. In some embodiments, the base titrant supply 140 may include a reservoir. In some embodiments, the base titrant supply 140 may include a reservoir. In some embodiments, the base titrant supply 140 may include a reservoir. In some embodiments, the base titrant supply 140 may include a reservoir. about 3L, at least about 4L, at least about 5L, at least about 6L, at least about 7L, at least about 8L, at least about 9L, at least about 10L, at least about 20L, at least about 30L, at least about 40L, at least about 50L, at least about 60L, at least about 70L, at least about 80L, at least about 90L, at least about 100L, at least about 200L, at least about 300L, at least about 400L, at least about 500L, at least about 600L, at least about 700L, at least about 800L, at least about 900L, at least about 1m 3 , at least about 2m 3 , at least about 3m 3 , at least about 4m 3 , at least about 5m 3 , at least about 6m 3 , at least about 7m 3 , at least about 8m 3 Or at least about 9m 3 In some embodiments, the base titrant supply 140 may have a volume of about 10 m 3 Below, about 9m 3 Below, approximately 8m 3 Below, approximately 7m 3 Below, approximately 6m 3 Below, about 5m 3 Below, approximately 4m3 Below, approximately 3m 3 Below, approximately 2m 3 Below, approximately 1m 3 Below, about 900L or less, about 800L or less, about 700L or less, about 600L or less, about 500L or less, about 400L or less, about 300L or less, about 200L or less, about 100L or less, about 90L or less, about 80L Below, about 70L or less, about 60L or less, about 50L or less, about 40L or less, about 30L or less, about 20L or less, about 10L or less, about 9L or less, about 8L or less, about 7L or less, about 6L or less, about 5L or less, about 4L or less , about 3 L or less, about 2 L or less, about 1 L or less, about 900 mL or less, about 800 mL or less, about 700 mL or less, about 600 mL or less, about 500 mL or less, about 400 mL or less, about 300 mL or less, about 200 mL or less, about 100 mL or less, about 90 mL or less, about 80 mL or less, about 70 mL or less, about 60 mL or less, about 50 mL or less, about 40 mL or less, about 30 mL or less, or about 20 mL or less.

[0169] Combinations of the above-referenced volumes of base titrant supply 140 are also possible, including all values ​​and ranges therebetween (e.g., at least about 10 mL and at least about 10 mL). 3 In some embodiments, the base titrant supply 140 may be 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, about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1000 mL, about 2000 mL, about 30 ...0 mL, about 5000 mL, about 6000 mL, about 700 mL, about 800 mL, about 900 mL, about 1000 mL, about 2000 mL, about 3000 mL, about 4000 mL, about 5000 mL, about 5000 mL, about 5000 mL, about 6000 mL, about 7000 mL, about 8000 mL, about 9000 mL, about 10000 mL, about 20000 mL, about 30000 mL, about 40000 mL, about 50000 mL, about 50000 mL, about 50000 mL, about 50000 mL, about 50000 mL, about 50000 mL, about 5 L, about 1L, about 2L, about 3L, about 4L, about 5L, about 6L, about 7L, about 8L, about 9L, about 10L, about 20L, about 30L, about 40L, about 50L, about 60L, about 7 0L, approximately 80L, approximately 90L, approximately 100L, approximately 200L, approximately 300L, approximately 400L, approximately 500L, approximately 600L, approximately 700L, approximately 800L, approximately 900L, approximately 1m 3 , about 2m 3 , about 3m 3 , about 4m 3 , about 5m 3 , about 6m 3 , about 7m 3 , about 8m 3 Or about 9m 3Or about 10m 3 The volume of the slit may be 0.01 to 0.05.

[0170] In some embodiments, the base titrant supply 140 can 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 base titrant supply 140 can be maintained at a pH of about 14 or less, about 13.5 or less, about 13 or less, about 12.5 or less, about 12 or less, about 11.5 or less, about 11 or less, about 10.5 or less, about 10 or less, about 9.5 or less, about 9 or less, about 8.5 or less, about 8 or less, or about 7.5 or less. Also, combinations of the above-referenced pH values ​​in the base titrant supply 140 are possible, including all values ​​and ranges therebetween (e.g., at least about 7.5 and not more than about 14, or at least about 8 and not more than about 10). In some embodiments, the base titrant supply 140 can 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.

[0171] The waste receiver 150 is optional and may receive effluent from a sample being measured in the pH flow cell 120. In some embodiments, the waste receiver 150 may include a container, tank, treatment facility, or any other suitable device capable of receiving effluent from the pH flow cell 120.

[0172] 20 is a schematic diagram of an apparatus 200 for pH control, according to one embodiment. As shown, the apparatus 200 includes a reactor 210, a pH flow cell 220 with a pH probe 221, a sampling pump 222, a check valve 224 (also known as a check valve), an acid titrant supply 230, an acid titrant pump 232, an acid titrant flow meter 234, a base titrant supply 240, a base titrant pump 242, a base 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 out of the reactor 210 in only one direction, thereby preventing contamination of the reactor 210 with the volume of sample in the pH flow cell 220. In some embodiments, reactor 210, pH flow cell 220, acid titrant supply 230, base titrant supply 240, and waste receiver 250 may be the same as or substantially similar to reactor 110, pH flow cell 120, acid titrant supply 130, base titrant supply 140, and waste receiver 150, described above with reference to Figure 19. Accordingly, certain aspects of reactor 210, pH flow cell 220, acid titrant supply 230, base titrant supply 240, and waste receiver 250 will not be described in greater detail herein. As shown, arrows represent fluid flow.

[0173] As shown, the streams may flow from reactor 210 to waste receiver 250. Reactor 210 receives streams from acid titrant supply 230 and base 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 external to reactor 210.

[0174] In some embodiments, the pH flow cell 220 can be a Mettler Toledo pH flow cell. In some embodiments, the pH probe 221 can be disposed within the pH flow cell 220. Exemplary pH probes suitable for the devices described herein include in-line pH probes manufactured by Mettler Toledo, Thermo Fisher Scientific, or Cole-Parmer. In some embodiments, the pH probe 221 is coupled to a pH transmitter. The pH transmitter can be, for example, a Mettler Toledo M400 pH transmitter. In some embodiments, the pH transmitter can visually display a pH reading thereon. In some embodiments, the pH transmitter communicates the pH reading to the controller 260. In some embodiments, the controller 260 communicates the pH reading to the user interface 262, which displays the pH reading. In some embodiments, the pH transmitter can communicate the pH reading to the user interface 262. The flow through the flow cell 220 can be controlled by a sampling pump 222.

[0175] The sampling pump 222 pumps a volume of sample fluid (i.e., a slipstream) from the reactor 210 so that the volume of sample fluid can enter the pH flow cell 220. In some embodiments, the sampling pump 222 may include a peristaltic pump, a diaphragm pump, a gear pump, a lobe pump, a piston pump, a centrifugal pump, or any other suitable pump or combination thereof. In some embodiments, the sampling pump 222 may include a Watson-Marlow 120 pump.

[0176] Check valve 224 allows flow in only one direction out of reactor 210, thereby preventing contamination of reactor 210 with the volume of sample in flow cell 220. As shown, valve 224 is upstream of sampling pump 222. In some embodiments, valve 224 may be downstream of sampling pump 222. In some embodiments, apparatus 200 may include a shutoff valve (not shown) downstream of and fluidly connected to reactor 210. In some embodiments, the shutoff valve may be physically connected to the reactor.

[0177] Acid titrant supply 230 contains an acid titrant. The acid titrant may be drawn from acid titrant supply 230 by acid titrant pump 232. Acid titrant pump 232 draws fluid from acid titrant supply 230 to facilitate the flow of acid titrant to reactor 210. In some embodiments, acid titrant pump 232 draws fluid from acid titrant supply 230 to facilitate the flow of acid titrant to reactor 210. In some embodiments, acid titrant pump 232 draws fluid from acid titrant supply 230 to facilitate the flow of acid titrant to reactor 210 at a 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 ...10 mL / min, at least about 15 mL / min, at least about 10 mL / min, at least about 15 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 8 The acid titrant flow rate may be facilitated to be 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 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 titrant pump 232 is about 50 L / min or less, about 40 L / min or less, about 30 L / min or less, about 20 L / min or less, about 10 L / min or less, about 5 L / min or less, about 2 L / min or less, about 1 L / min or less, about 900 mL / min or less, about 800 mL / min or less, about 700 mL / min or less, about 600 mL / min or less, about 500 mL / min or less, about 400 mL / min or less, about 300 mL / min or less, about 200 mL / min or less, about 10 In one embodiment, the acid titrant flow rate may be promoted as follows: 0 mL / min or less, about 90 mL / min or less, about 80 mL / min or less, about 70 mL / min or less, about 60 mL / min or less, about 50 mL / min or less, about 40 mL / min or less, about 30 mL / min or less, about 20 mL / min or less, about 10 mL / min or less, about 9 mL / min or less, about 8 mL / min or less, about 7 mL / min or less, about 6 mL / min or less, about 5 mL / min or less, about 4 mL / min or less, about 3 mL / min or less, or about 2 mL / min or less.

[0178] Combinations of the above-referenced acid titrant flow rates are also possible, including all values ​​and ranges therebetween (e.g., at least about 1 mL / min and up to about 1 L / min, or at least about 10 mL / min and up to about 50 mL / min). In some embodiments, the acid titrant pump 232 may 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.

[0179] 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 can include an ultrasonic meter, a vortex mixer, a magnetic meter, 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 can include a Sonotec ultrasonic flow meter.

[0180] In some embodiments, the acid titrant pump 232 may include a peristaltic pump, a diaphragm pump, a gear pump, a lobe pump, a piston pump, a centrifugal pump, or any other suitable pump or combination thereof. Suitable acid titrant pumps (232) are commercially available, for example, the Watson-Marlow 530 pump.

[0181] The base titrant supply 240 contains a base titrant. The base titrant may be drawn from the base titrant supply 240 by a base titrant pump 242. The base titrant pump 242 draws fluid from the base titrant supply 240 to facilitate the flow of the base titrant to the reactor 210. In some embodiments, the base titrant pump 242 draws fluid from the base titrant supply 240 to facilitate the flow of the base titrant to the reactor 210. In some embodiments, the base titrant pump 242 draws fluid from the base titrant supply 240 to facilitate the flow of the base titrant to the reactor 210 at a 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 30 ... The base titrant flow rate may be facilitated to be 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 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 base titrant pump 242 is about 50 L / min or less, about 40 L / min or less, about 30 L / min or less, about 20 L / min or less, about 10 L / min or less, about 5 L / min or less, about 2 L / min or less, about 1 L / min or less, about 900 mL / min or less, about 800 mL / min or less, about 700 mL / min or less, about 600 mL / min or less, about 500 mL / min or less, about 400 mL / min or less, about 300 mL / min or less, about 200 mL / min or less, about 1 L / min or less, about 200 mL / min or less, about 300 mL ... 00mL / min or less, approx. 90mL / min or less, approx. 80mL / min or less, approx. 70mL / min or less, approx. 60mL / min or less, approx. 50mL / min or less, approx. 40mL / min or less, approx. 30mL / min or less, approx. 20mL / min or less, approx. The acid titrant flow rate may be facilitated to be less than or equal to mL / min, less than or equal to about 9 mL / min, less than or equal to about 8 mL / min, less than or equal to about 7 mL / min, less than or equal to about 6 mL / min, less than or equal to about 5 mL / min, less than or equal to about 3 mL / min, or less than or equal to about 2 mL / min.

[0182] Combinations of the above-referenced base titrant flow rates are also possible, including all values ​​and ranges therebetween (e.g., at least about 1 mL / min and up to about 1 L / min, or at least about 10 mL / min and up to about 50 mL / min). In some embodiments, the base titrant pump 242 can facilitate a base 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.

[0183] The flow rate of the base titrant can be measured by a base titrant flow meter 244. In some embodiments, the base titrant flow meter 244 can include an ultrasonic meter, a vortex mixer, a magnetic meter, a Coriolis meter, or any other suitable flow meter or combination thereof. Suitable base titrant flow meters are commercially available, for example, the base titrant flow meter 244 can include a Sonotec ultrasonic flow meter.

[0184] In some embodiments, the base titrant pump 242 may include a peristaltic pump, a diaphragm pump, a gear pump, a lobe pump, a piston pump, a centrifugal pump, or any other suitable pump or combination thereof. Suitable base titrant pumps are commercially available, for example, the base titrant pump 242 may include a Watson-Marlow 530 pump.

[0185] As shown, dotted boxes enclose components over which user interface 262 may have some level of control. In other words, user interface 262 may act to control any of the components of device 200 via controller 260 or multiple controllers. In some embodiments, user interface 262 may be in communication with and may exercise control over reactor 210, pH flow cell 220, sampling pump 222, valve 224, acid titrant supply 230, acid titrant pump 232, acid titrant flow meter 234, base titrant supply 240, base titrant pump 242, base titrant flow meter 244, and / or waste receiver 250. In some embodiments, control of any of the above listed components may be initiated by a user. In other words, a user may manually control any of the components to initiate an action on at least one of the components of device 200. In some embodiments, control of any of the components of the device 200 may be automatic in response to conditions in the device 200 (e.g., pH measured in pH flow cell 220). In some embodiments, control of any of the components of the device 200 may be without any user involvement. In some embodiments, a user can send commands to the controller 260 via the user interface 262 to control or advance a preprogrammed pH sequence or to add a predetermined amount of an acid or base titrant.

[0186] In some embodiments, the controller 260 may be in communication with the acid titrant flow meter 234, the base titrant flow meter 244, the pH probe 221, the acid titrant pump 232, and / or the base titrant pump 242. In some embodiments, the controller 260 may receive a signal from the acid titrant flow meter 244, which causes the controller 260 to determine the amount of acid titrant to add to the sample. In some embodiments, the controller 260 may receive a signal from the base titrant flow meter 244, which causes the controller 260 to determine the amount of base titrant to add to the sample. In some embodiments, the controller 260 may receive a signal from the pH probe 221, which causes the signal to communicate the pH measurement to the controller 260, which relates the pH measurement to a corresponding amount of acid titrant or base titrant to add 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 base 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 measurements and the corresponding amount of acid or base titrant added to the sample.

[0187] In some embodiments, the user interface 262 may be in communication with the reactor 210 and / or a mixer disposed therein via the controller 260. In some embodiments, the controller 260 may be in communication with the mixer to control the timing and speed of mixing. For example, the controller 260 may send a signal to the mixer to begin just before and during the addition of the acid and / or base titrants. Mixing during titrant addition may prevent high titrant concentration volumes from occurring in the sample that could potentially damage the sample. In some embodiments, the timing of mixing may be modified based on how much acid and / or base titrant has been added. In some embodiments, the controller 260 may be in communication with the valve 224 to stop or enable the flow of sampling fluid through the valve. In some embodiments, the controller 260 may be in communication with the sampling pump 222 to activate the pumping of the sampling fluid through the pump or to vary the flow rate of the sampling fluid through the pump. In some embodiments, commands to the sampling pump 222 and valve 224 may be based on data communicated via the pH transmitter and controller 260 to the user interface 262 .

[0188] The controller 260 is configured to receive signals from and send commands to components of the devices described herein. In some embodiments, the controller 260 may be in communication with the acid titrant pump 232 to pump the acid titrant through the pump. 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, the communication to the acid titrant pump 232 may be based on data communicated to the controller 260 via the pH transmitter and / or the acid titrant flow meter 234. In some embodiments, the controller 260 may be in communication with the base titrant pump 242 to pump the base titrant through the pump. For example, the controller 260 sends a signal to start the base titrant pump 242, stop the base titrant pump, or change the speed of the base titrant pump. In some embodiments, the transmission to the base titrant pump 242 may be based on data transmitted to the controller 260 via a pH transmitter and / or a base titrant flow meter 244. In some embodiments, the controller 260 is configured to receive a pH value from the pH probe 221 (e.g., via a pH transmitter), and the controller 260 applies a mathematical model to the pH value and the corresponding amount of acid or base titrant added to the sample when the pH was measured. In some embodiments, the controller 260 is configured to apply a model described herein to one or more pH values ​​and the corresponding amount of titrant added to the sample. In some embodiments, the controller 260 is configured to determine from the measured pH value, the amount of titrant added to the sample, and the model, the remaining amount of titrant to be added to the sample to reach the target pH. Optionally, these steps may be repeated one or more times until the final target pH is achieved.For example, the controller 260 may be configured to receive an initial pH reading and send a signal to the acid or base titrant pump 242, which will cause a predetermined amount of acid or base titrant to be added to the sample, after which the pH probe 221 takes a reading and sends the measured pH value via the pH transmitter to the controller 260. The controller 260 then applies a model to the initial pH value, the pH value measured after titrant addition, and the amount of titrant added to the sample to determine an additional amount of titrant to add to the sample. In some embodiments, for example, if there is a predetermined pH sequence, the controller 260 may send a command to automatically advance the pH sequence. In other embodiments, the user commands the controller 260 to advance the pH sequence through a user interface, and the controller 260 relays the command to the device, for example, by activating the acid titrant pump 232 or the base titrant pump 242, taking a pH reading, etc.

[0189] In some embodiments, the controller 260 may include a server, computer, laptop, mobile device, tablet, mobile phone, or any other suitable device. The controller 260 may include one or more central processing units ("processors"), memory, and input / output devices. In certain embodiments, the controller 260 includes one or more memories and / or storage devices. The memory and storage devices may be one or more computer readable storage media that may store computer executable instructions to perform at least a portion of the various embodiments described herein. In some embodiments, the controller 260 includes a computer readable storage medium that stores computer executable instructions, including, but not limited to, instructions to activate, stop, or vary the speed of the acid titrant pump 232, instructions to activate, stop, or vary the speed of the base titrant pump 242, instructions to receive and store pH values ​​from the pH probe 221 and / or pH transmitter, and instructions to receive and store data from the acid titrant flow meter 234 and / or the base titrant flow meter 244. In some embodiments, the computer executable instructions include instructions to accept and store a user-entered pH value, e.g., an error in a pH probe 221 disposed in the pH flow cell 220, and an off-line pH value measured and entered by a user when an error is detected. In some embodiments, the computer executable instructions include instructions to calculate an amount of acid or base titrant added to a sample from data from the acid titrant flow meter 234 or the base titrant flow meter 244, and optionally the acid titrant pump 232 or the base titrant pump 242. In some embodiments, the computer executable instructions include instructions to apply a model described herein to the pH value and the amount of titrant added to the sample. In some embodiments, the computer executable instructions include instructions to perform one or more steps of a pH sequence, optionally in response to a command from a user through a user interface.In some embodiments, the controller 260 includes a processor configured to execute the instructions described above.

[0190] The present disclosure provides a user interface 262 configured to receive signals from and send commands 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 screens, touch screens, etc.), as well as pointing devices such as keyboards, mice, and the like. 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 off-line pH measurements from a user. In some embodiments, the user interface 262 is configured to receive commands from a user, whereby the commands are sent to the controller 260 to advance the pH sequence.

[0191] The present description sets forth many exemplary configurations, methods, parameters, and the like. However, it should be recognized that such description is not intended as a limitation on the scope of the present disclosure, but is instead provided as a description of exemplary embodiments. The above-described embodiments of the present subject matter may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the above description, certain non-limiting embodiments of the present disclosure are provided below. As would be apparent to one of ordinary skill in the art upon reading the present disclosure, each of the individually numbered embodiments may be used or combined with any of the preceding or subsequent individually numbered embodiments. This is intended to support all such combinations of embodiments, and is not limited to the combinations of embodiments expressly provided below.

[0192] Enumeration of embodiments The present disclosure can be understood with respect to the following enumerated embodiments:

[0193] 1. (a) Initial pH of the sample (pH initial ) measuring the (b) at least a first amount of a titrant n ) is added to the sample and at least a first additional pH value (pH n ), measuring Titrant n But pH n is the amount of titrant added to the sample to reach a pH of n But pH initial and different,steps; (c) The model was applied to measure the normalized initial amount of Titrant (Titrant initial ) and normalized Titrant n determining a pH of the sample, the model relating the normalized titrant added to the sample to the pH of the sample; and (d) Target pH (pH n+1 The further additional amount of titrant to be added to the sample to reach n+1 ) determining pH n+1 is the pH achieved by the addition of an additional amount of titrant to the sample. The method includes:

[0194] 2. The second amount of titrant n+2 ) is added to the sample and a second additional pH (pH n+2 2. The method of embodiment 1, comprising measuring the concentration of β-aminobutyric acid in the culture medium (C) and repeating steps (c) and (d).

[0195] 3. The third amount of titrant n+3 ) was added to the sample and a third additional pH (pH n+3 3. The method of embodiment 2, comprising measuring the concentration of β-aminobutyric acid in the blood and repeating steps (c) and (d).

[0196] 4. The addition of a third amount of titrant to the sample results in a final target pH (pH final 4. The method of claim 3, wherein the pH is within 0.05 to 0.10 pH units of the pH of the aqueous solution.

[0197] 5. The method of any one of embodiments 1 to 4, comprising adding a fourth amount of titrant to the sample and measuring a fourth additional pH.

[0198] 6. Determine the pH of the sample by pH final 6. The method of any one of the preceding claims, comprising no more than three or four titrant additions to change the titer to

[0199] 7. (i) generating at least one reference titration curve from the at least one reference sample relating the amount of titrant added to the reference sample to the pH of the reference sample; (ii) optionally normalizing at least one reference titration curve; and (iii) generating a model that fits the at least one reference titration curve; The method according to any one of embodiments 1 to 6, comprising:

[0200] 8. Generating at least one reference titration curve comprises: (i) The initial pH of the reference sample (pH initial_ref ) measuring the (ii) Add an amount of titrant to the reference sample (Titrant n_ref ), additional reference pH value (pH n_ref ), measuring Titrant n_ref But pH n_ref is the amount of titrant added to the sample to reach a pH of n_ref But pH initial_ref and different,steps; (iii) Add the entire amount of titrant to the reference sample. tot_ref ) ensures that at least one reference sample meets the final pH (pH final_ref repeating steps (i)-(ii) until (iv) plotting the amount of titrant added versus the pH of the reference sample. 8. The method of embodiment 7, comprising:

[0201] 9. The method of embodiment 7 or 8, wherein the titrant is added to the reference sample in separate steps over multiple time periods.

[0202] 10. The method of embodiment 7 or 8, wherein the titrants are added sequentially to the reference sample.

[0203] 11. A method according to any one of embodiments 7 to 10, wherein the pH of the reference sample is measured by a pH probe inserted directly into the reference sample.

[0204] 12. The method of any one of embodiments 7 to 10, wherein the pH of the reference sample is measured by a pH probe inserted into a continuous or individually sampled slipstream from the reference sample.

[0205] 13. The amount of titrant added to the reference sample is:

number

[0206] 14. At least one reference titration curve includes a single titration curve and is 1_ref =pH initial_ref , and pH 2_ref =pH final_ref The method according to any one of embodiments 7 to 13, wherein

[0207] 15. The method of any one of embodiments 7 to 13, wherein the at least one reference titration curve comprises a plurality of reference titration curves.

[0208] 16. Each standard titration curve is initial_ref and pH final_ref Contains, where: (a) pH 1_ref is the pH value from one of several standard titration curves. initial_ref and (b) pH 2_Ref is the pH value from one of several standard titration curves. final_ref and pH 1_ref and pH 2_ref is selected to encompass the maximum difference in values ​​while still encompassing the pH values ​​encompassed by all of the multiple reference titration curves; 16. The method of embodiment 15.

[0209] 17. Initial pH of the sample (pH initial ) and pH 1_ref The method according to any one of embodiments 13 to 16, wherein the are approximately the same.

[0210] 18. Initial pH of the sample (pH initial ) and pH 1_ref 17. The method according to any one of embodiments 13 to 16, wherein:

[0211] 19. pH initial and pH 1_ref 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.

[0212] 20. Final pH of the sample (pH final ) and pH 2_ref The method according to any one of embodiments 13 to 19, wherein the are approximately the same.

[0213] 21. pH final and pH 2_ref20. The method according to any one of embodiments 13 to 19, wherein:

[0214] 22. pH final and pH 2_ref 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.

[0215] 23. pH initial , pH initial_ref and pH 1_ref However, the pH is almost the same. final , pH final_ref and pH 2_ref The method according to any one of embodiments 13 to 22, wherein the are approximately the same.

[0216] 24. Final pH of the sample (pH final ) is the initial pH of the sample (pH initial 24. The method of any one of the preceding claims, wherein the titrant is an acid.

[0217] 25. The method of embodiment 24, wherein the amount of titrant added to the reference sample is normalized to a scale of about -0.76 to about 1.49.

[0218] 26. pH 1_ref is about 4.0 to 4.3, and in some cases pH 1_ref is about 4.1, and the pH 2_ref is about 3.4 to 3.9, and in some cases pH 2_ref 26. The method of embodiment 24 or 25, wherein the R is about 3.7.

[0219] 27. pH initial is about 4.0 to 4.5, about 4.1 to 4.5, about 4.2 to 4.5, about 4.3 to 4.5, about 4.1 to 4.4, or about 4.2 to 4.4.

[0220] 28. pH finalis about 3.0 to 3.8, about 3.1 to 3.8, about 3.2 to 3.8, about 3.3 to 3.7, about 3.4 to 3.7, or about 3.5 to 3.7.

[0221] 29. pH final The method of any one of embodiments 24 to 27, wherein the β-amino acid is about 3.6.

[0222] 30. The method of any one of embodiments 24 to 29, wherein the model includes a polynomial.

[0223] 31. The model is:

number

[0224] 32. If a polynomial is:

number

[0225] 33. Final pH of the sample final ) is the initial pH (pH initial 24. The method of any one of the preceding claims, wherein the titrant is a base.

[0226] 34. The method of embodiment 33, wherein the amount of titrant added to the reference sample is normalized to a scale of about -0.06 to about 1.53.

[0227] 35. pH 1_ref is about 3.0 to 3.8, or about 3.1 to 3.8, about 3.2 to 3.8, about 3.3 to 3.7, about 3.4 to 3.7, or about 3.5 to 3.7, and pH 2_ref is about 5.3 to 8.5, about 5.1 to 8.1, about 5.5 to 8.0, or about 7.5 to 8.0.

[0228] 36. pH 1_ref is about 3.7, and the pH 2_ref The method of embodiment 33 or 34, wherein the β-amino acid is about 7.6.

[0229] 37. pH initial is about 3.0 to 3.8, about 3.1 to 3.8, about 3.2 to 3.8, about 3.3 to 3.7, about 3.4 to 3.7, or about 3.5 to 3.7.

[0230] 38. pH final is about 5.3 to 8.5, about 5.1 to 8.1, about 5.5 to 8.0, or about 7.5 to 8.0.

[0231] 39. The method of any one of embodiments 33 to 38, wherein the model includes a polynomial.

[0232] 40. The model is:

number

[0233] 41. If a polynomial is:

number

[0234] 42. The method of any one of embodiments 1 to 41, further comprising a step of correcting for pH meter calibration when determining the pH value of the sample or at least one reference sample.

[0235] 43. The steps to correct for pH meter calibration include: (a) removing a first portion of the sample or reference sample prior to the addition of the titrant and measuring the pH of said first portion with an independently calibrated pH meter, thereby determining an off-line initial pH value (pH initial_off ) generation step; (b) removing a second portion of the sample or reference sample after the addition of the entire amount of titrant and measuring the pH of said second portion with an independently calibrated pH meter, thereby determining an off-line final pH value (pH final_off ) ; and (c) applying the relationship between the offline pH value and the measured pH value to determine a corrected pH for the reference sample. 43. The method of embodiment 42, comprising:

[0236] 44. The corrected pH for the reference sample is:

number

[0237] 45. The corrected pH for the sample is:

number

[0238] 46. ​​The step of determining the remaining amount of titrant to be added to the sample comprises, in step (d):

number

[0239] 47. The method of any one of embodiments 1 to 46, wherein the pH of the sample is measured using a pH probe inserted into a subsample removed from the sample, or into a separately sampled slipstream.

[0240] 48. A method according to any one of embodiments 1 to 46, wherein the pH of the sample is measured using a pH probe inserted directly into the sample or into a continuous slipstream.

[0241] 49. A method according to any one of embodiments 1 to 48, wherein the sample contains a first protein of interest and at least one reference sample contains a second protein of interest.

[0242] 50. The method of embodiment 49, wherein the first protein of interest and the second protein of interest are the same.

[0243] 51. The method of embodiment 49, wherein the first protein of interest and the second protein of interest respond similarly, although not identically, to the addition of a titrant to the sample and the reference sample.

[0244] 52. The method of any one of embodiments 49 to 51, wherein the first protein of interest and the second protein of interest are glycosylated proteins.

[0245] 53. The method of any one of embodiments 49 to 52, wherein the first and second proteins of interest are each antibodies.

[0246] 54. Antibodies include anti-PD1 antibody, anti-PDL-1 antibody, anti-Dll4 antibody, anti-ANG2 antibody, anti-AngPtl3 antibody, anti-PDGFR antibody, anti-Erb3 antibody, anti-PRLR antibody, anti-TNF antibody, anti-EGFR antibody, anti-PCSK9 antibody, anti-GDF8 antibody, anti-GCGR antibody, anti-VEGF antibody, anti-IL1R antibody, anti-IL4R antibody, anti-IL6R antibody, anti-IL1 antibody, anti-IL2 antibody, anti-IL3 antibody , anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-RSV antibody, anti-NGF antibody, anti-CD3 antibody, anti-CD20 antibody, anti-CD19 antibody, anti-CD28 antibody, anti-CD48 antibody, anti-CD3 / anti-CD20 bispecific antibody, anti-CD3 / anti-MUC16 bispecific antibody, and anti-CD3 / anti-PSMA bispecific antibody.

[0247] 55. The method of any one of embodiments 49 to 54, wherein the first and second proteins of interest are each receptor Fc fusion (TRAP) proteins.

[0248] 56. The method of embodiment 55, wherein the TRAP protein comprises VEGF TRAP or IL-1 TRAP.

[0249] 57. A method according to any one of embodiments 1 to 56, which improves the accuracy of arriving at the final pH of the sample compared to methods in which the pH is measured by inserting a pH probe directly into the sample or into a continuous slipstream drawn from the sample.

[0250] 58. A method according to any one of embodiments 1 to 57, which reduces sample waste compared to a method in which the pH is measured by a pH meter inserted in a continuous slipstream drawn from the sample.

[0251] 59. A method according to any one of embodiments 1 to 58, wherein the difference between the measured sample pH and the model identifies an error in the calibration of the pH meter used to measure the sample pH.

[0252] 60. (a) Recalibrate the pH meter; (b) adding an additional amount of titrant to the sample and measuring the additional pH; (c) applying the model and comparing the normalized titrant and pH or the normalized pH to the model; and (d) If the pH or normalized pH matches the model, pH final adding the remaining amount of titrant to the sample to reach This prevents damage to the protein of interest caused by adding too much titrant to the sample. 60. The method of embodiment 59, comprising:

[0253] 61. A method for inactivating viruses in a sample, comprising: (a) an initial pH of 4.0 or greater (pH initial providing a sample of (b) a first amount of an acid titrant n_acid ) was added to the sample and the first additional acid pH value (pH n_acid ), measuring Titrant n_acid But pH n_acid is the amount of titrant added to the sample to reach a pH of n_acid But pH initial and different,steps; (c) The model was applied to measure the normalized initial amount of Titrant (Titrant initial ) and determining a normalized titrant, the model relating the normalized titrant added to the sample to the pH of the sample; (d) Based on the normalized titrant, pH, and model, the target acid pH (pH acid_target determining the amount of titrant to be added to the sample to achieve a titration of 0.1; (e) pH acid_target adding an amount of titrant to the sample so as to reach a titration of (f) Final acid pH (pH acid_final repeating steps (d) and (e) until (g) subjecting the sample to pH 7.0 for a period of time sufficient to inactivate the virus. final_acid holding at (h) a first amount of a base titrant n_base ) is added to the sample and a first additional base pH value (pH n_base ), measuring Titrant n_base But pH n_base is the amount of titrant added to the sample to reach a pH of n_base But pH acid_final and different,steps; (i) By applying the second model, Titrant n_base A step to normalize (j) Based on the normalized titrant, pH, and model, adjust the pH of the sample to the target basic pH (pH target_base determining the amount of base titrant to add to the sample to change the pH of the sample to pH 5; (k) pH target_base adding an amount of a basic titrant to the sample to reach a titration of (l) Final basic pH (pH final_base repeating steps (j) and (k) until

[0254] 62. The method of embodiment 61, comprising repeating steps (b) and (c) at least once to verify that the behavior of the sample is consistent with the model.

[0255] 63. The method of embodiment 61 or 62, comprising repeating steps (d) and (e) one, two or three times.

[0256] 64. The method includes repeating steps (d) and (e) two or three times, wherein the step of repeating steps (d) and (e) two or three times is performed at a pH acid_final 64. The method of any one of embodiments 61-63, wherein the method results in a target acid pH that is within 0.05 to 0.10 pH units of

[0257] 65. pH acid_final 65. The method of embodiment 64, comprising repeating steps (d) and (e) a further number of times to reach

[0258] 66. The method of any one of embodiments 61 to 65, comprising repeating steps (h) and (i) at least once to verify that the behavior of the sample is consistent with the model.

[0259] 67. The method of any one of embodiments 61 to 66, comprising repeating steps (j) and (k) 1, 2 or 3 times.

[0260] 68. The method includes repeating steps (j) and (k) two or three times, wherein the step of repeating steps (j) and (k) two or three times is performed at a pH final_base 67. The method of any one of embodiments 61 to 66, wherein the pH is within 0.05 to 0.10 pH units of

[0261] 69. pH final_base 69. The method of embodiment 68, comprising repeating steps (j) and (k) a further number of times to reach

[0262] 70. pH acid_final is about 3.0 to 3.8, about 3.1 to 3.8, about 3.2 to 3.8, about 3.3 to 3.7, about 3.4 to 3.7, or about 3.5 to 3.7.

[0263] 71. pH final_base is about 5.3 to 8.5, about 5.1 to 8.1, about 5.5 to 8.0, or about 7.0 to 8.5.

[0264] 72. The method of any one of embodiments 61-71, wherein the first model comprises a polynomial.

[0265] 73. If a polynomial is:

number

[0266] 74. The method of any one of embodiments 61 to 73, wherein the second model includes a polynomial.

[0267] 75. If a polynomial is:

number

[0268] 76. The method of any one of embodiments 61 to 75, further comprising correcting for pH meter calibration.

[0269] 77. The method of any one of embodiments 61 to 76, wherein the pH of the sample is measured using a pH probe inserted into a subsample removed from the sample or into a separately sampled slipstream.

[0270] 78. The method of any one of embodiments 61 to 77, wherein measuring the pH of the sample does not include a pH probe inserted directly into the sample.

[0271] 79. A method according to any one of embodiments 61 to 78, wherein the sample contains a protein of interest.

[0272] 80. The method of embodiment 79, wherein the protein of interest is a therapeutic protein.

[0273] 81. The method of embodiment 79 or 80, wherein the protein of interest is an antibody.

[0274] 82. The method of embodiment 79 or 80, wherein the protein of interest is a receptor Fc fusion (TRAP) protein.

[0275] 83. A method according to any one of embodiments 61 to 82, which improves the accuracy of arriving at the final pH of the sample compared to methods in which the pH is measured by inserting a pH meter into the sample or into a continuous slipstream drawn from the sample.

[0276] 84. A method according to any one of embodiments 61 to 83, which reduces waste of sample compared to a method in which the pH is measured by a pH meter inserted in a continuous slipstream drawn from the sample.

[0277] 85. The method of any one of embodiments 61 to 84, wherein the difference between the measured sample pH and the model identifies an error in the calibration of the pH meter used to measure the sample pH.

[0278] 86. (a) Steps for recalibrating the pH meter; (b) adding an additional amount of titrant to the sample and measuring the additional pH; (c) applying the model and comparing the normalized titrant and pH to the model; and (d) If the pH matches the model, pH final adding the remaining amount of titrant to the sample to reach This prevents damage to the protein of interest from being caused by adding too much titrant to the sample. 86. The method of embodiment 85, comprising:

[0279] 87. An apparatus configured to perform a method according to any one of embodiments 1 to 86.

[0280] 88. Reactor; a pH flow cell including a pH probe disposed therein, the pH flow cell fluidly connected to the reactor and configured to receive a sampling slipstream from the reactor and measure a pH of the slipstream; an acid titrant supply fluidly connected to the reactor, the acid titrant supply configured to provide an acid titrant to the reactor to reduce the pH in the reactor; and / or a base titrant supply fluidly connected to the reactor, the base titrant supply configured to provide a base titrant to the reactor to increase the pH in the reactor. 88. The apparatus of embodiment 87, comprising:

[0281] 89. Reactor; a pH flow cell including a pH probe disposed therein, the pH flow cell fluidly coupled to the reactor and configured to receive a sampling slipstream from the reactor and measure a pH of the slipstream; an acid titrant supply fluidly connected to the reactor, the acid titrant supply configured to provide an acid titrant to the reactor to reduce the pH in the reactor; and / or An apparatus comprising: a base titrant supply fluidly connected to the reactor, the base titrant supply configured to provide a base titrant to the reactor to increase a pH in the reactor.

[0282] 90. A sampling pump configured to deliver a slipstream from the reactor to a pH flow cell. 90. The apparatus of embodiment 88 or 89, further comprising:

[0283] 91. A waste receptacle configured to receive effluent from the pH flow cell. 90. The apparatus of embodiment 88 or 89, further comprising:

[0284] 92. an acid titrant pump configured to deliver an acid titrant from an acid titrant supply to the reactor; and an acid titrant flow meter configured to measure the flow rate of the acid titrant from the acid titrant supply to the reactor; 90. The apparatus of embodiment 88 or 89, further comprising:

[0285] 93. A base titrant pump configured to deliver a base titrant from a base titrant supply to the reactor; and a base titrant flow meter configured to measure a flow rate of the base titrant from the base titrant supply to the reactor; 93. The apparatus of embodiment 92, further comprising:

[0286] 94. The apparatus of embodiment 93, further comprising a controller in communication with the acid titrant flow meter, the base titrant flow meter, the pH probe, the acid titrant pump, and the base titrant pump.

[0287] 95. If the controller: (a) receiving a signal from the acid titrant flow meter, whereby the controller determines the amount of acid titrant added to the sample; (b) receiving a signal from the base titrant flow meter, whereby the controller determines the amount of base titrant added to the sample; (c) receiving a signal from the pH probe, whereby the signal communicates a pH measurement to a controller, which relates the pH measurement to a corresponding amount of acid or base titrant added to the sample; (d) sending a signal to the acid titrant pump to start the acid titrant pump, stop the pump, or change the pump speed; and (e) The device of embodiment 94, configured to send a signal to the base titrant pump to start the base titrant pump, stop the pump, or change the pump speed.

[0288] 96. An apparatus as described in embodiment 94 or 95, wherein the controller is in communication with the sampling pump and is configured to send a signal to the sampling pump to start the sampling pump, stop the pump, or change the pump speed.

[0289] 97. An apparatus described in any one of embodiments 94 to 96, wherein the controller is configured to apply the model to the pH measurements and the corresponding amount of acid or base titrant added to the sample.

[0290] 98. The apparatus of any one of embodiments 94 to 97, wherein the controller is configured to activate an acid titrant pump to add a predetermined amount of acid titrant if the pH measured in the pH flow cell is greater than the desired value, and wherein the controller is further configured to activate a base titrant pump to add a predetermined amount of base titrant if the pH measured in the pH flow cell is less than the desired value.

[0291] 99. An apparatus described in any one of embodiments 94 to 98, 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 base titrant pump to stop the pump when a predetermined amount of base titrant has been added to the sample.

[0292] 100. An apparatus described in any one of embodiments 94 to 99, wherein the controller is configured to maintain the pH at a desired value for a period of time.

[0293] 101. The device of embodiment 100, wherein the desired value varies over time in accordance with a pH sequence.

[0294] 102. The apparatus of embodiment 101, wherein the pH sequence is suitable for inactivating viruses that may be present in the reactor.

[0295] 103. pH sequence is (a) lowering the pH to a first target pH of about 3.0-3.8, about 3.1-3.8, about 3.2-3.8, about 3.3-3.7, about 3.4-3.7, or about 3.5-3.7; (b) holding the pH at a first target pH for a period of time; (c) increasing the pH to a second target pH of about 5.3-8.5, about 5.1-8.1, about 5.5-8.0, or about 7.5-8.0; and (d) maintaining the pH at a second target pH. 103. The apparatus of embodiment 102, comprising:

[0296] 104. The apparatus of embodiment 103, wherein lowering the pH in step (a) or increasing the pH in step (c) comprises adding one or more amounts of a titrant sufficient to change the pH of the sample and measuring the pH of the sample.

[0297] 105. An apparatus described in any one of embodiments 88 to 104, further comprising a check valve for the fluid connection between the reactor and the pH flow cell, the check valve being configured to prevent contamination of the reactor by backflow from the pH flow cell.

[0298] 106. An apparatus described in any one of embodiments 88 to 105, wherein the reactor does not include a pH measurement probe disposed therein.

[0299] 107. The apparatus of any one of embodiments 88-106, further comprising a mixer disposed in the reactor configured to mix the contents in the reactor immediately prior to, during, and / or after addition of the acid titrant and / or base titrant.

[0300] 108. The apparatus of embodiment 107, wherein the controller is in communication with the mixer and is configured to send a signal to the mixer to activate the mixer prior to starting the acid pump or the base pump.

[0301] 109. The apparatus of embodiment 108, wherein the controller is configured to send a signal to the mixer, causing the mixer to be stopped a defined period of time after stopping the acid pump or the base pump.

[0302] 110. A device described in any one of embodiments 88 to 109, further comprising a user interface configured to receive and display pH measurements from the controller.

[0303] 111. The apparatus of embodiment 110, wherein the user interface is configured to send a signal to the controller, whereby the controller sends a signal to an acid titrant pump or a base titrant pump to add a predetermined volume of an acid or base titrant to the sample.

[0304] 112. An apparatus as described in embodiment 110 or 111, wherein the user interface is configured to send a signal to the controller, thereby enabling a user to instruct the controller to advance a step in the pH sequence.

[0305] 113. An apparatus described in any one of embodiments 110 to 112, wherein the user interface is configured to accept one or more offline pH measurements from a user, and the one or more offline pH measurements include a pH measurement of the sample independent of a pH probe disposed in the pH flow cell.

[0306] 114. An apparatus described in any one of embodiments 88 to 113, wherein the volume of acid titrant or base titrant delivered to the reactor has a percent error of 10% or less. EXAMPLES

[0307] Example 1: Modeling the titration of a protein solution to decrease its pH Cells expressing five different proteins were grown in a bioreactor and the proteins were secreted into the cell culture medium. After an initial harvesting step to remove cells and cell debris, the proteins were captured, washed, and eluted using a Protein A chromatography system. The eluate containing the partially purified proteins was then transferred to an automated system for viral inactivation by lowering the pH to 3.6.

[0308] To measure pH, a Mettler Toledo InPro 3253 pH probe was calibrated and then sterilized by autoclaving or gamma irradiation in a closed bellows with a Kleenpak connection that allows for a sterile connection to the pool bath containing the partially purified protein solution. The pH probe was inserted into the pool bath and the acid solution was added until a target pH of 3.6 was achieved. Insertion of the pH probe directly into the pool bath allowed for feedback control of the titration to control the final pH. In this example, the titrant was continuously dosed and the pH was continuously measured.

[0309] For 11 virus inactivation runs across five different proteins, pH versus amount of acid added was measured and plotted in Figure 1. In Figure 1, the amount of base added is shown in units of pump revolutions per kg of product in the bath before any titrant was added, and the pump speed data is time shifted to account for the lag between base addition and the pH response. As can be seen in Figure 1, all titration curves were visually similar in shape. The titration curves were generally linear with a slight downward curvature observed. The titration curves also differed in terms of X and Y intercepts.

[0310] A linear transformation of the axes was applied to the titration curves shown in FIG.

[0311] The following formula:

number

number

number

number

[0312] Example 2: Modeling the titration of increasing pH of a protein solution After viral inactivation, the pH of the solution was raised to neutral or near neutral pH by addition of a basic solution using the same procedure as described in Example 1.

[0313] For 12 viral inactivation runs across 7 different proteins, pH versus amount of base added was measured and plotted in Figure 4. In Figure 4, the amount of base added is shown in units of pump revolutions per kg of product in the bath before any titrant addition, and the pump speed data is time shifted to account for the lag between base addition and pH response. As can be seen in Figure 4, the curves were similar in shape and had a consistent inflection point at approximately pH 6. The location of this inflection point on the x-axis was variable.

[0314] A linear transformation of the axes was applied to the titration curves shown in Figure 4. In theory, a linear transformation that converges two well-chosen points on each curve should result in all curves collapsing into a single curve. An initial approach was based on modeling of acid titrants, as described in Example 1 above.

[0315] In this first approach, the following formula:

number

[0316] The following formula:

number

[0317] The results of this transformation are shown in Figure 5. As can be seen in Figure 5, this initial normalization approach did not result in as much collapse of the base titration curves as the acid titration curves in Example 1 (compare Figure 5 with Figure 2).

[0318] One explanation for this variation is that the target pH for the acid titration was the same across proteins (pH 3.6), while the target pH for the base titration varied between proteins, ranging from 7.7 to 8.0 depending on the protein, and therefore the final data points of the titration curves should not converge upon normalization.

[0319] Therefore, a second approach to normalization took into account that the titration endpoints should not converge. In this second approach, the Y-axis was set to 0 at time=0, and a pH close to but lower than the target pH, 7.60, was set to 1. Similarly, the X-axis was fixed at 0 for time=0, and fixed at 1 for the amount of titrant needed to reach pH 7.60.

[0320] In this second approach, the formula:

number

[0321] formula:

number

[0322] The results of this second transformation are shown in Figure 6. As shown in Figure 6, forcing convergence at time=0 and pH=7.60 improves the fit substantially. However, two titration curves still deviate from the rest of the curves.

[0323] This deviation may be caused by a problem with the pH probe calibration. For example, the pH probe used to generate the titration curves was sterilized in a sealed bag using an autoclave or gamma irradiation. Since the sterilization was performed after calibration but before use, the probe was dry during the period from calibration to when it made its first measurement. A problem with the pH probe may cause these two titration curves to deviate from the model. Offline pH was measured at the beginning and end of every pH titration run. That is, at the beginning and end of each run that lowered or raised the pH, a small amount of protein solution was removed from the pool and the pH was measured separately with a probe that did not experience sterilization. Also, as can be seen in Figure 7, the two runs with the greatest deviation from the model showed the greatest difference in the initial online and offline measurements, indicating that the problem was most likely due to the probe used to generate these two titration curves.

[0324] Problems with the online pH measurements can be corrected by a linear transformation using two offline measurements (initial and final). This is equivalent to after-the-fact 2-point pH standardization. A third approach to normalization took into account both (1) that the titration endpoint should not converge, and (2) the difference between the online and offline measurements. The corrected online pH is calculated using the following formula:

number

[0325] A fourth technique was used to further tighten the fit. Because the different runs had slightly different initial pHs, the different titration curves were forced to converge at two midpoints, rather than a single midpoint (pH 7.60) as in the previous attempt. Convergence was forced at two midpoints: pH 3.70 and 7.60. No pH conversion (Y-axis) was required since convergence was forced at two fixed pH values. The amount of base added (X-axis) was calculated using the following:

number

[0326] The results are shown in Figure 10. This corrected and transformed data set was used to generate a model for the addition of base (line, Figure 10). Due to the more complex curve shape for the base addition titration curve compared to the acid addition titration curve, a 6+ degree polynomial is required for the fit:

number

[0327] Base adjustment model RMSE=0.080 pH units. A summary of the fits is shown in Table 2 below.

[0328] [Table 2]

[0329] In summary, the method described in Examples 1 and 2 allows for accurate automated pH adjustment with a non-continuous slipstream measurement approach. When adjusting pH, a few samples may be taken and a pH model is used to determine how much additional acid or base should be added to reach the target pH with high accuracy, without requiring continuous sampling or measurement. The titration curves used in the modeling can be generated with data sets as few as one experimental data set. This approach also allows for titration to any pH value within the training data set. Finally, pH control accuracy can be improved by adding additional intermediate sampling points.

[0330] Example 3: Development of a system for pH regulation and control A system was developed to measure pH during viral inactivation using a pH probe inserted in the discontinuous slipstream and a non-dimensionalized model relating pH to a titrant added to the sample. An exemplary list of parts used in this system is shown in Figure 11. A diagram of the system is shown in Figures 19-20.

[0331] In this example of the system, the addition of acid and base additives to the reaction vessels containing the sample is controlled by separate Watson Marlow 530 pumps, each connected to a Sonotec CO.55 ultrasonic flow meter. The pumps receive titrant from tubing connected to the titrant bag and then deliver the titrant to the reaction vessel through the flow meter. The sample collection lines from the reaction vessels are connected to the sample collection pumps through check valves that can be used to prevent contamination of the reaction vessel in the event the sample collection assembly is installed incorrectly. The sample collection pump delivers the sample from the reaction vessel to a pH flow cell, into which a pH probe is inserted. The pH probe is connected to a pH transmitter. The sample is delivered from the flow cell to a waste container. All pumps are connected to a controller so that the programmed control logic of the controller can control the volume and flow rate of the acid and base titrants, as well as whether and how much sample is withdrawn from the reaction vessel for analysis.

[0332] Advantages of this system include: When the probe is inserted into the reaction vessel, it cannot be calibrated after sterilization (and before insertion). Sterilization can affect the probe calibration curve. In this system, the probe is inserted into a separate flow cell and sterilization is not required. No autoclave or Kleenpak sterile connection ports are required, eliminating the risk of the probe breaking in the reaction vessel or the risk of the probe solution leaking into the protein product. The sampling pump and check valve produce a separate sample for pH measurement, reducing product waste. Also, the system can be connected to any reaction vessel via a sampling line, making it very flexible.

[0333] An example of an acid adjustment workflow is shown in FIG. 12. After equipment setup, including probe calibration and automatic zeroing of the flow meter, an initial pH measurement is automatically taken before any addition of acid titrant, and a first amount of acid titrant is added to the sample. The volume of addition is controlled using feedback from the flow meter. The initial amount of acid is typically conservative to ensure that the target pH is not overshot. Acid is added at a constant rate (mL of acid per kg of protein pool in the reaction vessel), allowing the total initial addition volume to be calculated. The pH is then automatically measured after the entire volume of titrant in this initial addition has been added, and the initial and measured pH values, as well as the amount of acid added to obtain the second pH value, are fed into a model to determine the corresponding dimensionless titrant addition value. An intermediate target pH between the final target pH and the pH after the first addition of titrant is selected, and the model is used to calculate the corresponding dimensionless titrant addition value. The amount of dimensionless titrant that needs to be added to the sample to reach this intermediate pH is given by the following formula:

number

[0334] In this formula, the normalized Titrant total is the total amount added to the sample to achieve the intermediate target pH after normalization, and Normalized Titrant initial is the amount of titrant added to the sample for the initial pH after normalization (this value can be 0 before normalization), and the normalized Titrant n is the amount of titrant added to the sample to reach the first intermediate pH, normalized using the model. The model also uses the normalized Titrant initialNote that the normalized titrant is used to calculate the pH of the solution. With the two normalized titrants and the actual (dimensional) amount of titrant added, it is possible to convert between normalized and non-normalized titrants. A second volume of acid is added and these steps are repeated to validate the accuracy of the model and pH meter, then a final volume of acid is added to reach the target pH. After the low pH hold to inactivate the virus, the pH is adjusted back to neutral using a similar series of steps.

[0335] A similar workflow for increasing the pH is shown in Figure 13. After auto-zeroing the flow meter, a first amount of base titrant is added to the sample, the pH is measured, and a first volume is calculated using a linear function relating mL / kg of base added to the target neutral pH. The initial pH, measured pH, and volume of base titrant are fit to a model. A second volume of base is added and these steps are repeated to validate the accuracy of the model and pH meter, after which a final volume of base is added to reach the target pH.

[0336] In these processes, the slipstream pH value is compared to an expected value after each measurement. If the measured pH is outside the expected range, the user is prompted to take a sample and measure the offline pH on an independently calibrated pH meter and enter the offline value into the model. If the two measurements differ by more than 0.10 pH units, the user should complete the process with the offline pH measurement and use conservative volume and additional measurement steps to complete the process. The conservative addition during the acid adjustment step compensates for potential inaccuracies in the earlier autosampled pH measurements from the online pH probe.

[0337] While the process is still automated, after each addition of titrant, instead of automatically sampling pH, the user interface prompts the user to take a sample and measure the pH offline, for example with a separate pH probe, and enter the offline value into the user interface. In other words, if it is determined that the online probe in the pH flow cell was improperly calibrated midway through the adjustment step (e.g., during the acid adjustment after the first acid addition has been made), the remainder of the adjustment step (e.g., the acid adjustment) is made using conservative additions and offline pH measurements. In this scenario, the base adjustment is also made using the dimensionless titration model, but the user should continue to use a separate pH probe and offline pH values ​​entered into the user interface instead of measuring pH in the flow cell. The model is applied to the offline pH values ​​to complete the pH sequence.

[0338] If the difference is less than or equal to 0.10 pH units, the process may continue using the slipstream pH measurement technique. A summary of the process steps, volume calculation techniques and acceptable on-line pH is shown in Table 3 below: [Table 3] Table 3 applies to a pH sequence with three addition steps. If, for example, four (or more) additions are used, the target pH for each addition relative to the final pH is adjusted accordingly.

[0339] Example 4: Calibration of a pH meter used in an online process One issue that needed to be addressed with current systems was pH probe calibration and how to perform the calibration external to the pH transmitter. Industrial pH transmitters (e.g., Mettler Toledo M400) are typically limited to a two-point calibration process. However, a four-point calibration spanning pH 2-10 is desirable to ensure accuracy, and standard procedure uses an offline probe.

[0340] Therefore, we developed a four-point calibration process for the online probes that undergoes a calibration procedure equivalent to that undergone by the offline probes.

[0341] Instead of using the transmitter's calibration capabilities, a calibration procedure was developed that is performed within the system's control logic (e.g., through MATLAB). In this calibration procedure, the transmitter sends raw probe mV and probe temperature signals to the controller rather than a calculated pH signal. The control logic prompts the user to perform the calibration steps and records the mV and temperature for each buffer standard. The following calibration acceptance criteria were used: Tilt: 95~105% Offset: ±(0~15mV) Temperature: 20~25℃ for all standards Linearity Test: All standards within 0.05 pH units after calibration

[0342] Temperature compensation is also calculated within the control logic to account for the temperature dependence of the pH of the buffer standards during calibration, and during in-process measurements during the titration process.

[0343] An exemplary Online Probe Calibration curve is shown in FIG.

[0344] Example 5: Additional models for pH regulation without pH normalization An additional model was developed for relating titrants to pH that did not use pH normalization for either acid or base addition.

[0345] In this approach, two pH values ​​from the historical titration curve described in Example 1 were selected for acid addition, which were outliers from the end points of the titration curve. These pH reference pH values ​​(pH1 and pH2) were selected such that they were as far apart as possible, while still being pH values ​​that would be included on any reference titration curve. The following formula was then used:

number

[0346] This normalization titrant ranges from less than 0 to greater than 1. The data set used to generate the acid adjustment model ranged from -0.76 to 1.49, which resulted in the scale to which the acid titrants were normalized. The data set used to generate the base adjustment model ranged from -0.06 to 1.53, which resulted in the scale to which the base titrants were normalized. The range used depends on the variability of the initial and final pH values ​​in the data set. The two reference pH values ​​selected for normalization were as far apart as possible while remaining within the range of the set of reference titration curves. One advantage of this approach is that the final pH of the sample can be anywhere along the titration curve generated by the reference sample, as long as it is contained in the reference titration curve. For example, the final pH for the acid adjustment can be greater than or equal to the final pH of the reference. Similarly, the final pH for the base adjustment can be less than or equal to the final pH of the reference.

[0347] Using this normalization strategy, the following fourth-order polynomial for normalizing the acid titrant during acid addition was obtained:

number

[0348] A similar model, a fifth order polynomial, was used for base titrant normalization during base addition:

number

[0349] The apparatus described in Example 3 and the model described herein were used on a small scale Protein Pool to test performance. Slipstream pH measurements and ultrasonic flow meter accuracy were tested. Results from five Test Runs are shown in Figure 15. The ΔpH and % Dosage Error by Process Step for each of the five Test Runs are plotted in Figure 16.

[0350] As shown in Figure 15, the difference in pH observed with the online probe and the offline probe was 0.05 or less for all dosing steps. Dosing Error was generally less than 5% except for the final Base Add of the first test run. Figure 16 shows the data from Figure 15 plotted across the viral inactivation pH sequence, showing the difference between online and offline pH (top) and dosing error (bottom). Dosing Error is the error in the volume of titrant added at each step and is reported as the % difference between Target Volume and Volume Dosed.

[0351] The effect of the flow cell on pH probe performance was also assayed in a test run that compared the online probe used in the method, an offline probe, and an online probe that makes measurements outside of the flow cell. In this case, there were four Acid Add steps, since the offline pH was not within range after the third Acid Add step.

[0352] The pH versus titrant addition step for each of the three probe conditions is plotted in Figure 17. After each slipstream pH measurement during the test run, the probe was removed from the flow cell and inserted directly into the sample to evaluate the effect of the flow cell on the measurement. As shown in Figure 17, the pH measurements made by the Online Probe in flow Cell, the Online Probe out of Flow Cell, and the Offline Probe showed excellent agreement.

[0353] A real-time visualization of one of the test runs using a 55.1 kg protein pool is shown in FIG.

[0354] Example 6: pH Control with 3 or 4 Additions Using an Automated Titration System and Model Low pH viral inactivation was performed on 18 batches of proteins representing 7 different protein products using the automated titration system described in Example 3. The amount of titrant during the pH adjustment process was related to pH using the model described in Example 5, which does not use pH normalization. In viral inactivation of the 18 batches of proteins, the goal was to achieve a pH that was less than 0.10 pH units from the final target pH, both when lowering the pH to inactivate potential viruses and when raising the pH back to neutral after viral inactivation.

[0355] When using 3 or 4 additions of acid or base per Adjustment Step, all 18 batches of protein met the target within 0.10 pH units from the target pH by the offline reference probe for both the acid and base adjustment steps. The results are shown in Figure 21, which plots 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 additions (circles) or 4 additions (crosses), respectively. The target acid pH for viral inactivation was 3.50-3.60. The target neutralization pH after viral inactivation was 5.50-8.00, depending on the protein.

[0356] When 3 additions per adjustment step were used, all protein batches were within less than 0.10 pH units of the target pH after the addition of acid or base (Figure 21). However, when 3 additions per adjustment step were used, the more stringent goal of a final pH within less than 0.05 pH units of the target pH was not consistently met when adjusting the pH to neutral after the low pH viral inactivation step, according to the online control probe (Figure 22, right side).

[0357] A four-addition strategy was implemented to improve the precision of the method. With this modified approach, the third addition of acid or base adjusted the pH to within 0.05-0.10 pH units of the target pH. A small fourth addition was made to precisely achieve the target pH. As can be seen in Figures 21 and 22, the four-addition strategy increased the precision of the method, such that a final pH within 0.05 pH units of the target pH was consistently achieved for both the acid and base adjustment steps. As can be seen in Figure 22, all 13 batches in which the pH was adjusted using the four-addition step strategy met the target within 0.05 pH units from the target according to the online probe.

[0358] The automated system was also able to accurately measure the pH of the protein samples throughout the viral inactivation process. When the difference between the pH measured by the offline reference probe and the pH measured by the online control probe was determined at each addition step, 147 of the 151 separate pH measurements were found to be within 0.05 pH units of the difference between the offline reference probe and the online control probe inserted into the flow cell (FIG. 23). Thus, the model can accurately determine the amount of acid or base to add at each step, and the system can add the required amount of acid or base to consistently produce a pH change that is within 0.05 pH units of the target pH for any given addition step during both the acid or base titration process.

[0359] In addition, the automated system can accurately add the volume of acid or base titrant determined by the model. As shown in Figure 24, 133 of the 133 additions had less than 10% error in the volume of titrant added.

Claims

1. A step of generating a reference titration curve from at least one reference sample; generating a model based on the regression of the reference titration curve; The initial pH of the sample (pH initial ) measuring the At least the first amount of titrant n ) to the sample, and adjusting the sample to at least a first pH value (pH n ) measuring titrant n is pH n is the amount of titrant added to the sample to reach pH n But pH initial different from; and Applying the model to determine a second amount of titrant (Titrant n+1) to reach the target pH based on pH initial , Titrant n , and pH n . A method comprising:

2. The method according to claim 1, wherein the target pH is a first intermediate target pH, and the method further comprises: adding a second amount of titrant (Titrant n+1) to the sample; measuring a second pH (pH n+1); and applying the model to determine a third amount of titrant (Titrant n+2) to reach a second intermediate target pH based on pH initial , Titrant n+1 , and pH n+1 . The method of claim 1 , comprising:

3. The method of claim 2, further comprising the step of adding the third amount of titrant (Titrant n+2) to the sample and measuring a third pH (pH n+2).

4. The addition of the third amount of titrant (Titrant n+2) to the sample achieves a final target pH (pH final 4. The method of claim 3, wherein the pH is within 0.05 to 0.10 pH units of the pH of the aqueous solution.

5. The method of claim 4, further comprising the step of adding a fourth amount of titrant (Titrant n+3) to the sample to reach pH final.

6. The pH of the sample was pH initial pH final 5. The method of claim 4, comprising adding no more than four titrants to change the titrant concentration to

7. The method of claim 1, further comprising normalizing the reference titration curve to generate a normalized reference titration curve, and generating a model based on regression of the reference titration curve comprises generating a model based on regression of the normalized reference titration curve.

8. The method of claim 7, wherein the step of generating a reference titration curve comprises: (i) the initial pH (pH initial_ref ) measuring the (ii) adding an amount of titrant to said at least one reference sample (Titrant n_ref ), additional reference pH value (pH n_ref ) measuring titrant n_ref is pH n_ref is the amount of titrant added to the sample to reach pH n_ref But pH initial_ref different from,step; (iii) adding the entire amount of titrant to the at least one reference sample to determine whether the at least one reference sample has a final pH (pH final_ref repeating step (ii) until (iv) plotting the amount of titrant added versus the pH of said at least one reference sample. The method of claim 4, comprising:

9. The amount of titrant added to the at least one reference sample is: [Equation 1] (In the formula, Titrant 1_ref is pH 1_ref the amount of titrant added to the at least one reference sample to achieve 2_ref is pH 2_ref 9. The method of claim 8, wherein the titration is normalized by a titration amount (wherein the titration amount is the amount of titrant added to the at least one reference sample to achieve a titration amount of 0.01).

10. The reference titration curve comprises a single titration curve, and 1_ref = pH initial_ref , and pH 2_ref = pH final_ref The method of claim 8, wherein

11. The reference titration curve comprises a plurality of reference titration curves, Each standard titration curve is initial_ref and pH final_ref Contains, where: pH 1_ref is the pH value from one of several standard titration curves initial_ref and pH 2_Ref is the pH value from one of several standard titration curves final_ref and pH 1_ref and pH 2_ref is selected to encompass the largest difference in values ​​while still encompassing the pH values ​​encompassed by all of the multiple reference titration curves, The method of claim 8.

12. The initial pH of the sample (pH initial ) and pH 1_ref are approximately the same or the initial pH of the sample (pH initial ) and pH 1_ref The method of claim 9 , wherein:

13. The final target pH of the sample (pH final ) and pH 2_ref are approximately the same or pH final and pH 2_ref The method of claim 9 , wherein:

14. pH initial , pH initial_ref and pH 1_ref are almost the same, and pH final , pH final_ref and pH 2_ref 10. The method of claim 9, wherein are approximately the same.

15. The final target pH of the sample (pH final ) is the initial pH of the sample (pH initial 5. The method of claim 4, wherein the titrant is an acid.

16. pH 1_ref is about 4.0 to 4.3, and optionally pH 1_ref is about 4.1, and the pH 2_ref is about 3.4 to 3.9, and in some cases pH 2_ref 16. The method of claim 15, wherein is about 3.

7.

17. pH initial is about 4.0 to 4.5, about 4.1 to 4.5, about 4.2 to 4.5, about 4.3 to 4.5, about 4.1 to 4.4, or about 4.2 to 4.

4.

18. pH final is about 3.0 to 3.8, about 3.1 to 3.8, about 3.2 to 3.8, about 3.3 to 3.7, about 3.4 to 3.7, or about 3.5 to 3.

7.

19. The method of claim 15 , wherein the model comprises a fourth-order polynomial, a fifth-order polynomial, or a sixth-order polynomial.

20. The final target pH of the sample (pH final ) is the initial pH (pH initial 5. The method of claim 4, wherein the titrant is a base.

21. pH 1_ref is about 3.0 to 3.8, or about 3.1 to 3.8, about 3.2 to 3.8, about 3.3 to 3.7, about 3.4 to 3.7, or about 3.5 to 3.7, and pH 2_ref is about 5.3 to 8.5, about 5.1 to 8.1, about 5.5 to 8.0, or about 7.5 to 8.

0.

22. pH initial is about 3.0 to 3.8, about 3.1 to 3.8, about 3.2 to 3.8, about 3.3 to 3.7, about 3.4 to 3.7, or about 3.5 to 3.

7.

23. pH final is about 5.3 to 8.5, about 5.1 to 8.1, about 5.5 to 8.0, or about 7.5 to 8.

0.

24. 21. The method of claim 20, wherein the model comprises a fifth-order polynomial.

25. 10. The method of claim 1, further comprising correcting for pH meter calibration when determining the pH value of the sample or at least one reference sample.

26. 2. The method of claim 1, wherein the sample contains a first protein of interest and at least one reference sample contains a second protein of interest.

27. 27. The method of claim 26, wherein the first protein of interest and the second protein of interest are glycosylated proteins.

28. 28. The method of claim 27, wherein the first protein of interest and the second protein of interest are each an antibody or a receptor Fc fusion (TRAP) protein.

29. 5. The method of claim 4, which improves the accuracy of reaching the final target pH (pH final) of the sample compared to methods in which the pH is measured by inserting a pH probe directly into the sample or into a continuous slipstream drawn from the sample.

30. 10. The method of claim 1, which reduces sample waste compared to a method in which pH is measured by a pH meter inserted in a continuous slipstream drawn from the sample.

31. 10. The method of claim 1, wherein the difference between the measured sample pH and the model identifies an error in the calibration of the pH meter used to measure the sample pH.

32. 1. A method for inactivating viruses in a sample, comprising: Initial pH of 4.0 or higher (pH initial providing a sample of First dose of acid titrant (Titrant n_acid ) is added to the sample and the first acid pH value (pH n_acid ) measuring titrant n_acid is pH n_acid is the amount of titrant added to the sample to reach pH n_acid But pH initial different from,step; pH initial , Titrant n_acid , and pH n_acid Based on the target acid pH (pH acid_target determining a second amount of acid titrant to be added to the sample to achieve an acid titration of 0.01; pH acid_target adding said second amount of titrant to the sample to achieve The sample was incubated at pH 7.0 for a period sufficient to inactivate the virus. acid_target holding at First amount of base titrant (Titrant n_base ) is added to the sample to obtain a first base pH value (pH n_base ) measuring titrant n_base is pH n_base is the amount of titrant added to the sample to reach pH n_base is different from pH acid_target, step; pH acid_target , pH n_base , and Titrant n_base Based on this, pH n_base is the target basic pH (pH target_base determining a second amount of base titrant to add to the sample to change the pH of the sample to pH 4; pH target_base adding said second amount of basic titrant to the sample to achieve A method comprising:

33. The method of claim 32, further comprising the step of comparing the measured pH n_acid with a predicted target pH value to confirm that the sample behavior is consistent with the model, and the model is used to determine the second amount of acid titrant.

34. The method of claim 32, wherein adding the second amount of acid titrant to the sample comprises adding the acid titrant to the sample one, two or three separate times.

35. The method of claim 35, wherein adding the second amount of acid titrant to the sample includes a first addition of acid titrant, a second addition of acid titrant, and a third addition of acid titrant, wherein uptake of the second addition of acid titrant into the sample results in a pH acid_target 33. The method of claim 32, wherein the pH is within 0.05 to 0.10 pH units of 36. The method of claim 35, wherein the third addition of an acid titrant adjusts the pH of the sample to pH acid_target.

37. The method of claim 33, further comprising the step of comparing the measured pH n_base with a predicted target pH value to confirm that the sample behavior is consistent with the model, and the model is used to determine the second amount of base titrant.

38. The method described in claim 32, wherein adding the second amount of basic titrant to the sample to reach pH target_base includes adding the basic titrant to the sample one, two or three separate times.

39. The method of claim 32, wherein adding the second amount of basic titrant to the sample to reach pH target_base comprises a first addition of basic titrant, a second addition of basic titrant, and a third addition of basic titrant, and wherein adding the second amount of basic titrant to the sample results in a pH of the sample that is within 0.05 to 0.10 pH units of pH target_base.

40. The method of claim 32, wherein pH acid_target is about 3.0 to 3.8, about 3.1 to 3.8, about 3.2 to 3.8, about 3.3 to 3.7, about 3.4 to 3.7, or about 3.5 to 3.

7.

41. The method of claim 32, wherein the pH target_base is about 5.3 to 8.5, about 5.1 to 8.1, about 5.5 to 8.0, or about 7.0 to 8.

5.

42. The method of claim 32, wherein the model includes a polynomial.

43. 33. The method of claim 32, further comprising correcting for pH meter calibration.

44. 33. The method of claim 32, wherein the sample comprises a protein of interest.

45. 45. The method of claim 44, wherein the protein of interest is a therapeutic protein.

46. 33. The method of claim 32, which improves the accuracy of reaching the final target pH (pH final) of the sample compared to methods in which the pH is measured by inserting a pH sensor into the sample or into a continuous slipstream drawn from the sample.

47. 33. The method of claim 32, which reduces sample waste compared to a method in which pH is measured by a pH sensor inserted in a continuous slipstream withdrawn from the sample.

48. 34. The method of claim 33, wherein the difference between the measured sample pH and the model identifies an error in the calibration of the pH meter used to measure the sample pH.

49. 49. Apparatus configured to perform the method of any one of claims 1 to 48.

50. The method described in claim 44 or 45, wherein the target protein is an antibody or receptor Fc fusion (TRAP) protein.

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

52. The method of claim 32, wherein the sample contains dupilumab.

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