Viral clearance evaluation for biological medical product preparation processes
View Patent ↗Fluorescent viral particles, including viral particles and a fluorescent dye conjugated to the viral particles, are used for viral contaminant removal testing as part of viral clearance evaluation for biological medical product preparation processes. The conjugated fluorescent dye adds only marginally to viral particle size, but provides versatility both for rapid screening of alternative viral removal approaches during process development and for final process validation, as the fluorescent viral particles have fluorescent activity throughout the viral clearance evaluation process, permitting rapid and consistent quantification of initial test solutions, resulting purified solutions, and intermediate solutions of interest at any point during processing under evaluation.
1. A method for viral clearance evaluation, the method comprising:
subjecting an evaluation solution to a purification processing to prepare create a treated solution, wherein the evaluation solution comprises fluorescent viral particles, and wherein the purification process comprises at least one viral removal technique for separation of a viral contaminant; and
determining a degree of removal of the fluorescent viral particles between the evaluation solution and the treated solution;
wherein the fluorescent viral particles comprise viral particles and a fluorescent dye conjugated to the viral particles;
wherein the determining a degree of removal of the fluorescent viral particles comprises subjecting a sample of the treated solution to flow cytometry to analytically quantify a residual concentration of the fluorescent viral particles in the treated solution; and
wherein the flow cytometry comprises detecting for a fluorescent emission signature of the fluorescent dye of the fluorescent viral particles; and
after the purification process no additional fluorescent stain is added to the treated solution prior to the flow cytometry.
2. The method of claim 1 , wherein the at least one removal technique comprises chromatography, filtration, centrifugation, membrane separation, dialysis, or combinations thereof.
3. The method of claim 2 , further comprising:
subjecting each of a plurality of different said evaluation solutions, each comprising the fluorescent viral particles, to a different said purification process to create different said treated solutions, wherein each different said purification process includes a different said viral removal technique; and
determining a said degree of removal of the fluorescent viral particles between each said corresponding pair of a said evaluation solution and a said treated solution for each different said purification process.
4. The method of claim 3 , wherein at least two of the different said evaluation solutions have the same composition for evaluation of viral clearance alternatives for the different said evaluation solutions having the same composition.
5. The method of claim 3 , wherein at least two of the different said evaluation solutions have different compositions for evaluation of viral clearance.
6. The method of claim 1 , wherein:
the fluorescent viral particles are first said fluorescent viral particles comprising first said viral particles and first said fluorescent dye;
the evaluation solution comprises second said fluorescent viral particles comprising second said viral particles and second said fluorescent dye;
the first said viral particles have a first viral epitope of a first virus and the second said viral particles have a second viral epitope of a second virus different than the first virus;
the first fluorescent dye has a first fluorescent emission signature and the second fluorescent dye has a second fluorescent emission signature, different than the first fluorescent emission signature; and
the method comprises second determining a degree of removal of the second said fluorescent viral particles between the evaluation solution and the treated solution.
7. The method of claim 1 , wherein:
the evaluation solution is a first said evaluation solution, and the fluorescent viral particles are first said fluorescent viral particles comprising first said viral particles and first said fluorescent dye, the treated solution is a first said treated solution and the determining a degree of removal is a first said determining a degree of removal; and
the method further comprises:
subjecting a second said evaluation solution to the purification process to create a second said treated solution, wherein the second said evaluation solution comprises second said fluorescent particles comprising second said viral particles, different than the first said viral particles, and second said fluorescent dye, which second said fluorescent dye may be the same or different than the first said fluorescent dye, and wherein the first fluorescent dye has a first fluorescent emission signature and the second fluorescent dye has a second fluorescent emission signature, which said second fluorescent emission signature may be the same or different than the first fluorescent emission signature; and
second said determining a degree of removal of the second said fluorescent viral particles between the second said evaluation solution and the second said treated solution.
8. The method claim 1 , wherein the evaluation solution comprises biological contaminants, other than the fluorescent viral particles.
9. The method of claim 1 , wherein the determining a degree of removal of the fluorescent viral particles comprises:
determining a feed concentration of the fluorescent viral particles in the evaluation solution.
10. A method for viral clearance evaluation of a biological medical product preparation process, the method comprising:
subjecting an evaluation solution to a purification process to create a treated solution, wherein the evaluation solution comprises a biological medical product and fluorescent viral particles, and wherein the purification processing comprises a viral removal technique for separation of a viral contaminant from the biological medical product; and
determining a degree of removal of the fluorescent viral particles between the evaluation solution and the treated solution;
wherein the fluorescent viral particles comprise viral particles and a fluorescent dye conjugated to the viral particles;
wherein the determining a degree of removal of the fluorescent viral particles comprises determining a feed concentration of the fluorescent viral particles in the evaluation solution and a residual concentration of the fluorescent viral particles in the treated solution;
wherein the determining the residual concentration comprises subjecting a sample of the treated solution to flow cytometry to analytically quantify the residual concentration of the fluorescent viral particles in the evaluation solution; and
wherein the flow cytometry comprises detecting for a fluorescent emission signature of the fluorescent dye of the fluorescent viral particles; and
after the purification process no additional fluorescent stain is added to the treated solution prior to the flow cytometry.
11. The method of claim 9 , wherein the determining a degree of removal of the fluorescent viral particles comprises subjecting a sample volume of the evaluation solution to flow cytometry to analytically quantify a feed concentration of the fluorescent viral particles in the evaluation solution from the fluorescent properties of the fluorescent viral particles.
12. The method of claim 1 , comprising preparing the evaluation solution, wherein the preparing the evaluation solution comprises:
combining a feed volume of a preliminary feed solution with a reagent volume of a viral standard solution comprising the fluorescent viral particles dispersed in a standard liquid medium at an analytically quantified number concentration.
13. The method of claim 12 , wherein the determining a degree of removal of fluorescent viral particles comprises calculating a feed concentration (C F ) of the fluorescent viral particles in the evaluation solution by the following calculation:
C F =C AQ ×V R /( V F +V R )
where V F is the feed volume of the preliminary feed solution, V R is the reagent volume of the viral standard solution combined to make the evaluation solution, and C AQ is the analytically quantified number concentration of the viral standard solution.
14. The method of claim 1 , wherein the viral particles are non-infectious and non-replicating.
15. The method of claim 1 , wherein the viral particles comprise a viral epitope of a virus family selected from the group consisting of Parvoviridae, Adenoviridae, Retroviridae, Reoviridae and Herpesviridae.
16. The method of claim 1 , wherein the fluorescent viral particles are of a size with a maximum cross-dimension of no more than 4 nanometers larger than an original maximum cross-dimension of the viral particles.
17. The method of claim 16 , wherein:
the fluorescent dye is conjugated to a viral protein of the viral particles and the fluorescent dye is in a moiety including a residual nitrogen of a reacted primary amine of the viral protein, and wherein the moiety has a molecular mass of no larger than 2,500 Daltons.
18. The method of claim 1 , wherein the evaluation solution further comprises a biological medical product.
19. The method of claim 18 , wherein the biological medical product is derived from a cell culture of a characterized cell line.
20. The method of claim 18 , wherein the evaluation solution comprises biological contaminants, other than the fluorescent viral particles, representative of a biological medical product preparation process to make the biological medical product.
21. A method for viral clearance evaluation for a biological medical product preparation process, the method comprising:
subjecting an evaluation solution to a purification process to create a treated solution, wherein the evaluation solution comprises a biological medical product and fluorescent viral particles, and wherein the purification process comprises a viral removal technique for separation of a viral contaminant from the biological medical product; and
determining a degree of removal of the fluorescent viral particles between the evaluation solution and the treated solution;
wherein the fluorescent viral particles comprise viral particles and a fluorescent dye conjugated to the viral particles;
wherein the determining a degree of removal of the fluorescent viral particles comprises subjecting a sample of the treated solution to flow cytometry to analytically quantify a residual concentration of the fluorescent viral particles in the treated solution; and
wherein the flow cytometry comprising detecting for a fluorescent emission signature of the fluorescent dye of the fluorescent viral particles; and
after the purification process no additional fluorescent stain is added to the treated solution prior to the flow cytometry.
22. The method of claim 21 , wherein the determining a degree of removal of the fluorescent viral particles comprises subjecting a sample volume of the evaluation solution to an analytical technique to analytically quantify a feed concentration of the fluorescent viral particles in the evaluation solution from the fluorescent properties of the fluorescent viral particles.
23. The method of claim 1 , wherein after the purification process there is no chemical modification of the treated solution prior to the flow cytometry.