Methods of reducing or eliminating protein modification and degradation arising from exposure to UV light

Patent metadata

Field	Value
Publication number	US-10634589-B2
Application number	US-201214353704-A
Country	US
Kind code	B2
Filing date	Oct 25, 2012
Priority date	Oct 26, 2011
Publication date	Apr 28, 2020
Grant date	Apr 28, 2020

Abstract

Methods of inactivation of a virus in a sample comprising a protein component are provided. Also provided are methods of reducing protein degradation or modification in to the presence of a reactive species, such as a reactive species generated as a result of UV exposure, are also provided. In another aspect, a method of reducing oxidation of methionine residues, tryptophan residues or both methionine and tryptophan residues in a protein subjected to UV light is provided. The disclosed methods can be performed at any scale and can be automated as desired.

First claim

What is claimed is:

1. A method of inactivating a virus in a sample comprising an antibody comprising:
(a) providing a sample comprising the antibody, wherein the sample is known or suspected to contain a virus;
(b) adding a protectant to the sample to form a stabilized mixture, wherein the protectant comprises one or more of tyrosine, tryptophan, and methionine;
(c) measuring the absorbance of the stabilized mixture at 254 nm by in-line absorption spectroscopy;
(d) passing the stabilized mixture through a device configured to provide continuous exposure to UVC light such that a target dose of UVC light under which the virus is inactivated is delivered to the mixture;
(e) adjusting the UVC light exposure based on the absorbance measurement in (c) such that the target dose of UVC light is continually delivered to the stabilized mixture passing through the device, wherein steps (c), (d) and (e) are conducted as part of a real time automated feedback loop; and
(f) removing the protectant from the mixture following exposure to UVC light.

2. The method of claim 1 , wherein the sample comprises a chromatography column pool.

3. The method of claim 2 , wherein the pool comprises one or more of a Protein A column eluant pool comprising the antibody, a Protein G column eluant pool comprising the antibody, a HIC column pool comprising the antibody, a SEC column pool comprising the antibody, an IEC column pool comprising the antibody, and a hydroxyapatite column pool comprising the antibody.

4. The method of claim 1 , wherein the sample comprises a chromatography column effluent stream.

5. The method of claim 4 , wherein the effluent stream comprises one or more of a Protein A column effluent stream comprising the antibody, a Protein G column effluent stream comprising the antibody, a HIC column effluent stream comprising the antibody, a SEC column effluent stream comprising the antibody, an IEC column effluent stream comprising the antibody, and a hydroxyapatite column effluent stream comprising the antibody.

6. The method of claim 1 , wherein the antibody is a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a single chain antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

7. The method of claim 1 , wherein the virus comprises one or more of a dsDNA virus, a ssDNA virus, a dsRNA virus and a ssRNA virus.

8. The method of claim 7 , wherein the virus comprises a virus of one or more of the virus families adenoviridae, asfarviridae, herpesviridae, iridoviridae, papillomaviridae, polyomaviridae, poxviridae, circoviridae, hepadnaviridae, parvoviridae, birnaviridae, reoviridae, arenaviridae, vornaviridae, bunyaviridae, deltaviridae, filoviridae, orthomyxoviridae, paramyxoviridae, rhabdoviridae, arterioviridae, astroviridae, caliciviridae, cornonavirdae, flaviviridae, REV-like viruses, nodaviridae, picornaviridae, togaviridae, and retroviridae.

9. The method of claim 8 , wherein the virus is the parvovirus MVM, the retrovirus MuLV or the bunya virus CVV.

10. The method of claim 1 , wherein the protectant is added to the sample in a concentration ratio of greater than 1 part protectant to 200 parts antibody.

11. The method of claim 1 , wherein the protectant comprises (i) tyrosine; (ii) tryptophan; or (iii) tyrosine and tryptophan.

12. The method of claim 1 , wherein the UVC light has a wavelength in the range of about 200 nm to about 280 nm.

13. The method of claim 12 , wherein the UVC light has a wavelength of about 254 nm.

14. The method of claim 1 , wherein the target dose is about 1 mJ/cm 2 , about 10 mJ/cm 2 , about 25 mJ/cm 2 , about 50 mJ/cm 2 , about 75 mJ/cm 2 , about 100 mJ/cm 2 , about 125 mJ/cm 2 , about 200 mJ/cm 2 , about 250 mJ/cm 2 , about 300 mJ/cm 2 , about 350 mJ/cm 2 , about 400 mJ/cm 2 , about 450 mJ/cm 2 , about 500 mJ/cm 2 , about 600 mJ/cm 2 , about 700 mJ/cm 2 , about 800 mJ/cm 2 , about 900 mJ/cm 2 , about 1000 mJ/cm 2 or greater than about 1000 mJ/cm 2 .

15. The method of claim 1 , wherein the method provides a viral log reduction value (LRV) of greater than or equal to about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5 or greater than about 6.5.

16. The method of claim 1 , wherein the method is performed as a step in an antibody purification operation.

17. A method for inactivating a virus in a therapeutic antibody preparation comprising:
(a) providing a sample comprising the therapeutic antibody, wherein the sample is an effluent stream from an antibody purification step and the sample is known or suspected to contain a virus;
(b) introducing a protectant into the effluent stream to form a stabilized mixture, wherein the protectant comprises tyrosine, tryptophan, methionine, or combinations thereof,
(c) measuring the absorbance of the stabilized mixture at 254 nm by in-line absorption spectroscopy;
(d) passing the stabilized mixture through a device configured to provide continuous exposure to UVC light such that a target dose of UVC light under which the virus is inactivated is delivered to the mixture;
(e) adjusting the flow rate of the stabilized mixture through the device or the lamp power of the device based on the absorbance measurement in (c) such that the target dose of UVC light is continually delivered to the stabilized mixture passing through the device, wherein steps (c), (d) and (e) are conducted as part of a real time automated feedback loop; and
(f) removing the protectant from the mixture following UVC light exposure.

18. The method of claim 17 , wherein the absorbance of the stabilized mixture is measured at 254 nm by in-line absorption spectroscopy.

19. The method of claim 17 , wherein the sample is an effluent stream from a Protein A column.

20. The method of claim 17 , wherein the sample is an effluent stream from a depth filtration step.

21. The method of claim 17 , wherein the therapeutic antibody is a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a single chain antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

22. The method of claim 17 , wherein the target dose is about 1 mJ/cm 2 , about 10 mJ/cm 2 , about 25 mJ/cm 2 , about 50 mJ/cm 2 , about 75 mJ/cm 2 , about 100 mJ/cm 2 , about 125 mJ/cm 2 , about 200 mJ/cm 2 , about 250 mJ/cm 2 , about 300 mJ/cm 2 , about 350 mJ/cm 2 , about 400 mJ/cm 2 , about 450 mJ/cm 2 , about 500 mJ/cm 2 , about 600 mJ/cm 2 , about 700 mJ/cm 2 , about 800 mJ/cm 2 , about 900 mJ/cm 2 , about 1000 mJ/cm 2 or greater than about 1000 mJ/cm 2 .

23. The method of claim 22 , wherein the target dose is about 100 mJ/cm 2 or greater.

24. The method of claim 22 , wherein the target dose is about 1000 mJ/cm 2 or greater.

25. The method of claim 17 , wherein the method provides a viral LRV of greater than or equal to about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5 or greater than about 6.5.

26. The method of claim 17 , wherein the UVC light has a wavelength in the range of about 200 nm to about 280 nm.

27. The method of claim 26 , wherein the UVC light has a wavelength of about 254 nm.

28. The

Assignees

• Amgen Inc

Inventors

• Shultz Joseph Edward
• Hart Roger
• Welborn Brent

Classifications

• B01D15/24

  relating to the treatment of the fractions to be distributed · CPC title

• G01N1/30 Primary

  Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis} · CPC title

• C07K1/22

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

• C07K1/16

  by chromatography · CPC title

• A61L2/10 Primary

  Ultraviolet [UV] radiation · CPC title