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JP-2026076177-A - Methods for inactivating viruses

JP2026076177AJP 2026076177 AJP2026076177 AJP 2026076177AJP-2026076177-A

Abstract

[Problem] To provide a method for developing an acid-based inactivation protocol for viruses in a mixture. [Solution] A method comprising: preparing a pool of eluate samples of a mixture containing a target molecule purified by a chromatography process; generating a pool of acidified samples having a pH below a first inactivation pH; generating a first data point for the acidified samples; generating a second data point for the inactivated samples; and using the first and second data points, regressing the relationship between the protein concentration of the mixture, the pH of the mixture, the inactivation pH range, and the amount of acid required to bring the mixture into the inactivation pH range. [Selection Diagram] Figure 1

Inventors

  • マッティラ、 ジョン
  • バルマッセ、 アンドリュー
  • チボロスキ、 マーク

Assignees

  • リジェネロン・ファーマシューティカルズ・インコーポレイテッド

Dates

Publication Date
20260511
Application Date
20251225
Priority Date
20190801

Claims (20)

  1. A method for inactivating viruses in a mixture, Elute the mixture from the chromatography column at a pH higher than 3.9 and less than 8.5; To measure the protein concentration of the aforementioned mixture; Calculating the amount of acid required to lower the pH of the mixture to an inactivating pH, based on the protein concentration of the mixture; A method comprising: adding a first portion of an acid to a mixture, wherein the first portion of the acid is 68% to 99% of the amount of acid necessary to lower the pH of the mixture to an inactivating pH; and adding an additional portion of the acid to the mixture such that the pH of the mixture and the acid combination is below the inactivating pH.
  2. Maintaining the combination at the inactivation pH for the inactivation time; and, after the inactivation time, adding the first portion of the acid to the mixture less than one hour later, The method according to claim 1, further comprising titrating the combination until the pH is 4.5 or higher and 8.5 or lower.
  3. The method according to claim 1, further comprising measuring the pH of the mixture before adding the first portion of the acid to the mixture.
  4. The method according to claim 3, wherein the amount of acid required to lower the pH of the mixture to the inactivating pH is calculated according to the following formula. w = Ax + By + C (In the formula, w is the amount of acid expressed as the number of moles of acid per kilogram of the mixture, and x is 1 (The protein concentration of the mixture is expressed in grams per liter, where y is the inactivation pH, and A, B, and C are constants.)
  5. A is approximately 0.0003 L/mol/g/kg or more, or approximately 0.0006 L/mol/g/kg The following: B is between approximately -0.1 mol/kg and approximately 0.0 mol/kg; and C is between approximately 0.02 and approximately 0.1. The method according to claim 4.
  6. The method according to claim 3, wherein the amount of acid required to lower the pH of the mixture to the inactivation pH is calculated based on the protein concentration of the mixture and the pH of the mixture.
  7. The method according to claim 1, wherein the amount of acid required to lower the pH of the mixture to the inactivation pH is about 0.002 moles to about 0.025 moles per kilogram of the mixture.
  8. The method according to claim 1, wherein the inactivation pH is approximately 3.8 or less and approximately 3.0 or more.
  9. The method according to claim 1, further comprising measuring the conductivity of the mixture; and adding a sufficient amount of one or more salts to the mixture to adjust the conductivity of the mixture.
  10. The method according to claim 1, wherein the chromatography column is configured to perform a protein affinity capture process.
  11. The method according to claim 1, wherein the pH of the mixture is about 3.5 to about 3.75 after the first portion of the acid is added and before the additional portion of the acid is added.
  12. A method for inactivating viruses in a mixture, A mixture containing the target molecule, with a pH of approximately 5.0 to approximately 8.5, is loaded into a chromatography column; From the chromatography column, an elution mixture containing the target molecule is obtained, approximately 3. To elute a elution mixture having a pH higher than 9 and approximately 5.0 or lower; Adding a certain amount of acid to the elution mixture to form a combination of the elution mixture and the acid that is configured to exhibit effective virus inactivation and has a pH of approximately 3.8 or less and approximately 3.0 or more; The predicted pH of the above combination is determined in advance using a pH verification model; Record the pH of the aforementioned combination; A method comprising: calculating the difference between the predicted pH and the recorded pH; and taking corrective action based on the calculated difference between the predicted pH and the recorded pH.
  13. The method according to claim 12, wherein the correction measure is taken when the difference between the predicted pH and the recorded pH is approximately 0.15 or more.
  14. The method according to claim 12, wherein the corrective measures include adjusting a pH meter, adjusting the composition of the mixture, adjusting one or more environmental conditions, or a combination thereof.
  15. The method according to claim 12, wherein the combination is maintained at a pH of approximately 3.8 or less and approximately 3.0 or more for approximately 30 minutes.
  16. After adding the aforementioned amount of acid to the aforementioned mixture, the combination is reduced to approximately 4.5 to approximately 8 in less than one hour. The method according to claim 12, further comprising titrating to a pH of 5 or less.
  17. The method according to claim 12, further comprising measuring the conductivity of the mixture; and adding a sufficient amount of one or more salts to the mixture to adjust the conductivity of the mixture.
  18. The method according to claim 12, further comprising measuring the pH of the mixture before adding the aforementioned amount of acid to the mixture; and measuring the protein concentration of the mixture.
  19. The pH confirmation model according to claim 18, comprising the following formula. y=Kx+Lw+Mz+N (In the formula, y is the pH of the combination, x is the protein concentration of the mixture expressed in grams per liter, z is the pH of the mixture before adding the aforementioned amount of acid, w is the amount of acid added to the mixture expressed in moles of acid per kilogram of eluate, and K, L, M, and N are constants.)
  20. A method for developing an acidic deactivation protocol for a mixture, A pool of eluate samples of the mixture, wherein each eluate sample in the pool contains a target molecule purified by a protein affinity capture process; Measure the pH and protein concentration of each eluate sample; To determine the amount of acid required to bring each eluate sample to an inactivating pH by titrating each eluate sample in the pool of eluate samples; Determining data points using each eluate sample in the pool of eluate samples; and determining the amount of acid required to bring the mixture to an inactivating pH using the data points. A method comprising regression of the relationship between the protein concentration of the mixture, the pH of the mixture, and the inactivation pH.

Description

Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62/881,692, filed on 1 August 2019, and its entire disclosure is incorporated herein by reference. Technical field This disclosure generally relates to a method for achieving a target pH in a polypeptide-containing mixture. More specifically, this disclosure relates to an enveloped virus (envelope virus) The present invention relates to a method for achieving a target pH in a polypeptide-containing mixture to help ensure that viruses (or virus-like particles) are inactivated. In the production of polypeptides, target molecules (e.g., pharmaceutical products (drug product) In some cases, the target polypeptide component of t) may be separated from the culture medium. For example, a separation process such as affinity chromatography may be carried out as part of the target molecule preparation process. After such a separation process, the resulting mixture containing polypeptides may contain potentially undesirable viruses or other contaminants that are undesirable to be included in the pharmaceutical product. Therefore, a method for removing or inactivating such contaminants is desirable. In some commercial-scale target molecule synthesis processes, process analysis techniques (Process ss Analytical Technology (PAT) may be implemented. T includes systems and methods involved in the design, analysis, and control of the manufacturing process of the target molecule. PAT includes identifying process parameters that affect product quality and regularly monitoring those parameters to ensure product quality is maintained. PAT is encouraged by regulatory bodies to reduce the overall risks associated with the target molecule and pharmaceutical products. PAT can provide statistical verification or confirmation that one or more process conditions are met that can improve or maintain the quality of the target molecule and/or product. The methods and systems disclosed herein can improve the efficiency and/or productivity of polypeptide preparation methods, including virus inactivation. The methods and systems disclosed herein can also improve the efficiency and/or productivity of pharmaceutical product preparation methods and can address one or more of the problems identified above. Overview Embodiments of this disclosure may relate to a method for inactivating a virus in a mixture, for example, an eluate. This method involves 3.9 from a chromatography column. This method may include eluting the mixture at a pH higher than 8.5 and below 8.5. This method may further include measuring the protein concentration of the mixture and measuring the pH of the mixture. Next, based on the protein concentration of the mixture, the pH of the mixture is set to the inactivation pH (inact). The amount of acid needed to lower the pH to IV (pH) can be calculated. After calculating the amount of acid added, the first part of the acid, which is 68% to 99% of the total amount of acid added, can be calculated. The portion may be added to the mixture. This method may further include adding an additional portion of acid to the mixture so that the pH of the mixture is below the inactivation pH. In the method of this disclosure, The mixture is inactivated at an inactivation pH and for an inactivation time (inactivation interval). It may be maintained for l) and configured to inactivate the virus in the mixture. In some embodiments of this disclosure, a method for inactivating a virus in a mixture may include loading a mixture containing a target molecule onto a chromatography column, the loading of which may be performed at a pH of about 5.0 to about 8.5. This method further involves eluting the mixture containing the target molecule from the chromatography column at a pH of about 3.9 to about 5.0. This method may include eluting the eluted mixture. This method may also include adding an acid to the mixture to form a mixture-acid combination, which is configured to exhibit effective virus inactivation, and which has a pH of about 3.8 or less and about 3.0 or more. Using a pH confirmation model, the expected pH of the combination is determined. The pH can be predetermined. Furthermore, the pH of the combination may be measured and/or recorded. The difference between the predicted pH and the recorded pH is calculated, and the predicted pH and the recorded pH Corrective measures may be taken based on the calculated difference. Further embodiments of this disclosure include an acid inactivation protocol. The method may include a method for developing a vacation protocol. This method may include preparing a pool of elutes, where each elute in the pool contains a target molecule purified in a protein affinity capture process. This method may include the pH of each elute in the pool. This may include measuring the protein concentration and/or the eluate concentration. Furthermore, each eluate in the eluate pool can be titrated to determine