Viral clearance by low pH hold

Patent metadata

Field	Value
Publication number	US-12077782-B2
Application number	US-202117317602-A
Country	US
Kind code	B2
Filing date	May 11, 2021
Priority date	May 11, 2020
Publication date	Sep 3, 2024
Grant date	Sep 3, 2024

Abstract

Methods for viral clearance using low pH hold based on a statistical design of experiment are provided. Several factors are evaluated to characterize the impacts of a low pH hold step for virus inactivation, including the factors of pH conditions, conductivity conditions, protein type, temperature, acid titrant, spike timing, and post-spike filtration. In addition to the effect of pH on virus inactivation, an increase in ionic strength through manipulating the conductivity can be a key component that influences virus inactivation kinetics.

First claim

What is claimed is:

1. A method for purifying a peptide or protein from a sample, the method comprising:
subjecting the sample to increasing ionic strength by addition of sodium chloride, wherein the concentration of the sodium chloride is from about 1 mM to about 100 mM;
subjecting the sample to an acidic pH, and
subsequently maintaining the sample at the ionic strength condition and the pH condition for at least about 15 minutes to inactivate a quantity of viral particles;
wherein the sample comprises one or more impurities including the viral particles.

2. The method of claim 1 , wherein the quantity of the viral particle inactivation is at least about 3 LRF (logarithmic reduction factor).

3. The method of claim 1 , wherein the quantity of the viral particle inactivation is at least about 4 LRF.

4. The method of claim 1 , wherein the pH condition of the sample is less than or equal to about pH 3.90.

5. The method of claim 1 , wherein the pH condition of the sample is in a range of from about pH 3.60 to about pH 3.90.

6. The method of claim 1 , wherein the pH condition of the sample is in a range of from about pH 3.65 to about pH 3.80.

7. The method of claim 1 , wherein the peptide or protein is an antibody produced in a host cell.

8. The method of claim 1 , wherein the sample is maintained at the ionic strength condition and the pH condition for at least about 30 minutes to inactivate the quantity of the viral particles.

9. The method of claim 1 , wherein the sample is maintained at the ionic strength condition and the pH condition for from about 15 minutes to about 30 minutes to inactivate the quantity of the viral particles.

10. The method of claim 1 further comprising optimizing the ionic strength and the pH condition of the sample for inactivation of the quantity of the viral particles by running a D-Optimal design of experiment.

11. The method of claim 10 , wherein the D-Optimal design of experiment evaluates the pH condition of the sample and the ionic strength of the sample and adjusts the pH condition of the sample and the ionic strength of the sample to inactivate a quantity of viral particles.

12. The method of claim 11 , wherein the D-Optimal design of experiment further evaluates and adjusts one or more of:
a conductivity of the sample;
a type of the peptide or protein;
a temperature of the sample;
an acid titrant to adjust the pH condition of the sample;
a method for spiking the viral particles to the sample; or
a presence of a post-spike filtration.

13. The method of claim 1 , wherein the sample is an eluent from protein A chromatography.

14. The method of claim 1 , wherein the ionic strength of the sample is adjusted using an addition of sodium chloride, wherein a concentration of the sodium chloride is in a range of from about 1 mM to about 100 mM, or is about 25 mM, about 50 mM or about 100 mM.

15. The method of claim 1 , wherein the concentration of the sodium chloride is in a range of from about 1 mM to about 50 mM.

16. The method of claim 1 , wherein the concentration of the sodium chloride is about 25 mM, about 50 mM, or about 100 mM.

17. The method of claim 1 , wherein the pH condition of the sample is adjusted using phosphoric acid or glycine HCl.

18. The method of claim 1 , wherein the peptide or protein is an antibody having an IgG1 isotype or having an IgG4 isotype.

19. The method of claim 1 , wherein the peptide or protein is a monoclonal antibody or a bispecific antibody.

20. The method of claim 1 , wherein the peptide or protein is an antibody, an antibody fragment, a Fab region of an antibody, an antibody-drug conjugate, a fusion protein, a protein pharmaceutical product or a drug.

21. A method of producing a preparation comprising a protein of interest and a reduced amount of viral particles from a sample having the protein of interest and an viral particle, comprising:
subjecting the sample to a pH of greater than about 3.6;
subjecting the sample to an increase in ionic strength condition by addition of sodium chloride to the starting solution, wherein the concentration of sodium chloride is from about 1 mM to about 100 mM; and
maintaining the sample at the pH and ionic strength condition for an appropriate amount of time to produce the preparation comprising the protein of interest and the reduced amount of viral particles.

22. The method of claim 21 , wherein the concentration of the protein of interest in the sample is greater than about 25 g/L.

23. The method of claim 21 , wherein the appropriate amount of time is about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes.

24. The method of claim 21 , wherein the method reduces the amount of infectious viral particles from a sample by about 3 LRF (logarithmic reduction factor).

25. The method of claim 21 , wherein the method reduces the amount of infectious viral particles from a sample by about 4 LRF (logarithmic reduction factor).

26. The method of claim 21 , wherein the pH condition of the sample is greater than about pH 3.70, about 3.80, about pH 3.90 or about pH 4.0.

27. The method of claim 21 , wherein the pH condition of the sample is in a range of from about pH 3.60 to about pH 4.0.

28. The method of claim 21 , wherein the sample is an eluent from protein A chromatography.

29. The method of claim 21 , wherein the ionic strength of the sample is adjusted using an addition of sodium chloride, wherein a concentration of the sodium chloride is in a range of from about 1 mM to about 200 mM.

30. The method of claim 21 , wherein the concentration of the salt is greater than about 50 mM, or about 100 mM.

31. The method of claim 21 , wherein the pH condition of the sample is adjusted using phosphoric acid or glycine HCl.

32. A method for purifying a peptide or protein from a sample containing IgG1 or IgG4, the method comprising:
subjecting the sample to increasing ionic strength by addition of a salt, wherein the concentration of the salt is from about 1 mM to about 100 mM;
subjecting the sample to an acidic pH, and
subsequently maintaining the sample at the ionic strength condition and the pH condition for at least about 15 minutes to inactivate a quantity of viral particles;
wherein the sample comprises one or more impurities including the viral particles.

33. The method of claim 32 further comprising optimizing the ionic strength and the pH condition of the sample for inactivation of the quantity of the viral particles by running a D-Optimal design of experiment.

34. The method of claim 33 , wherein the D-Optimal design of experiment evaluates the pH condition of the sample and the ionic strength of the sample and adjusts the pH condition of the sample and the ionic strength of the sample to inactivate a quantity of viral particles.

35. The method of claim 34 , wherein the D-Optimal design of experiment further evaluates and adjusts one or more of:
a conductivity of the sample;
a type of the peptide or protein;
a temperature of the sample;
an acid titrant to adjust the pH condition of the sample;
a method for spiking the viral particles to the sample; or
a presence of a post-spike filtration.

36. The method of claim 32 , wherein the pH condition of the sample

Assignees

• Regeneron Pharma

Inventors

• Daya Jena
• Cusick Valerie Ann
• Mattila John

Classifications

• C07K16/112

  Retroviridae (F), e.g. leukemia viruses · CPC title

• C12N2740/13063

  by chemical treatment · CPC title

• C07K1/14

  Extraction; Separation; Purification · CPC title

• C07K1/22

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• C07K16/065 Primary

  Purification, fragmentation · CPC title