Processing biological material for flow cytometry evaluation for virus particles

1. A method for processing biological material for flow cytometry evaluation for virus particles, the method comprising:

centrifuging a mixture comprising biological material for evaluation and purification particles to prepare a centrifuged composition including a more-dense phase concentrated in the purification particles and a less-dense supernatant, wherein the biological material comprises larger-size components and smaller-size components and the purification particles comprise:

a porous core functionalized to capture at least some non-virus impurities of the smaller-size components; and

a porous size-exclusion shell surrounding the core and having a pore structure to exclude the larger-size components from entering into the core through the pore structure of the shell while permitting the smaller-size components to enter into the core through the pore structure of the shell; and

filtering a liquid-containing composition comprising at least a portion of the supernatant comprising at least a portion of the larger-size components, wherein retentate of the filtering includes at least a portion of the larger-size components from the liquid-containing composition and the filtering comprises filtration at a separation size to pass at least a portion of particles of virus size in the filtrate of the filtering.

2. A method according to claim 1 , wherein the centrifuging a mixture comprises simultaneously centrifuging a plurality of said mixtures with each said mixture disposed in a different fluid container of a multi-container sample purification unit.

3. A method according to claim 1 , wherein the filtering comprises simultaneous centrifugal filtration at a said separation size of no larger than 2 microns of a plurality of said liquid-containing compositions, each comprising at least a portion of a different said supernatant from the centrifuging a plurality of said mixtures, to prepare a plurality of said retentates with each said retentate retained in a different filter well of a sample filtration unit and to prepare a plurality of said filtrates with each said filtrate collected in a different filtrate collection container of the sample filtration unit.

4. A method according to claim 1 , comprising flow cytometry evaluation for presence of virus particles of a sample comprising at least a portion of the filtrate; and

contacting the at least a portion of the filtrate with at least one fluorescent marker prior to the flow cytometry evaluation.

5. A method according to claim 4 , wherein the flow cytometry evaluation comprises sequentially subjecting to flow cytometry each of a plurality of said samples with each said sample comprising at least a portion of a different one of a plurality of said filtrates prepared by simultaneously filtering a plurality of said liquid-containing compositions.

6. A method according to claim 5 , wherein the sequentially subjecting to flow cytometry comprises sequentially removing said samples from a multi-container plate with an autosampler and delivering said samples from the autosampler to a flow cytometer.

7. A method according to claim 1 , comprising preparing the mixture, the preparing the mixture comprising:

mixing together the biological material and the purification particles; and

prior to the mixing, unsealing a sealed container containing the purification particles mixed with a storage liquid, wherein the sealed container contains a unit quantity of the purification particles for the mixture.

8. A method according to claim 7 , wherein:

the preparing the mixture comprises preparing a plurality of said mixtures with each said mixture disposed in a separate container of a multi-container sample purification unit; and

the unsealing comprises unsealing each of the said separate containers of the sample purification unit, wherein each of the said separate containers of the sample purification unit comprises a said unit quantity of said purification particles mixed with said storage liquid.

9. A method according to claim 7 , comprising prior to the mixing:

centrifuging the unit quantity of the purification particles and storage liquid to prepare a centrifuged composition; and

separating at least a portion of the storage liquid from the centrifuged composition.

10. A method according to claim 7 , wherein the storage liquid comprises a Tris-HCl buffer solution;

the preparing the mixture comprises mixing the biological material, the unit quantity of the purification particles and a buffer solution reagent, the buffer solution reagent comprising a Tris-HCl buffer solution reagent; and

the mixture has a pH in a range of from pH 7 to pH 9.

11. A method according to claim 10 , wherein the preparing the mixture comprises processing the biological material, wherein the processing comprises:

centrifuging a composition comprising crude biological material sample comprising the biological material and diluted with buffer solution reagent comprising a Tris-HCl buffer solution; and

recovering at least a portion of resulting supernatant comprising the biological material for inclusion in the mixture.

12. A method according to claim 7 , wherein the pore structure of the shell is configured with a size exclusion cutoff of not larger than 1,000,000 Daltons and the core is functionalized with a hydrophobic ligand having a positive charge.

13. A method according to claim 7 , wherein the mixture comprises from 35 microliters to 70 milliliters bulk volume of the purification particles and from 45 microliters to 90 milliliters of the biological material.

14. A method according to claim 13 , wherein the mixture comprises a ratio of bulk volume of the purification particles to volume of the biological material in a range of from 0.5:1 to 1:1.

15. A method according to claim 1 , wherein:

the biological material comprises material from a chicken egg;

the non-virus impurities comprise protein and nucleic acid impurities; and

the larger-size components comprise a member selected from the group consisting of cell debris, chicken embryo debris, bacteria, protein aggregates, lipids, lipid assemblies, lipid-protein assemblies, lecithins, lipid-protein aggregates, liposomes, ribosomes, vesicles, protein-nucleic acid complexes and combinations thereof.

16. A method according to claim 1 , wherein:

the method comprises preparing the mixture, comprising mixing the biological material, the purification particles and a buffer solution reagent having a pH in a range of from pH 7 to pH 9;

the supernatant is concentrated in the larger size components of the mixture; and

the preparing the mixture comprises, prior to the mixing:

unsealing a sealed container containing the purification particles mixed with a buffer storage liquid, wherein the sealed container contains a unit quantity of the purification particles for the mixing; and

centrifuging the unit quantity of the purification particles and storage liquid to prepare a centrifuged composition and removing at least a portion of the storage liquid from the centrifuged composition.

17. A method according to claim 16 , wherein:

the centrifuging a mixture comprises simultaneously centrifuging a plurality of said mixtures with each said mixture disposed in a different fluid container of a multi-container sample purification unit;

the filtering comprises simultaneously filtering a plurality of said liquid-containing compositions, each comprising at least a portion of a different said supernatant from the centrifuging a plurality of said mixtures, to prepare a plurality of said retentates with each said retentate retained in a different filter well of a sample filtration unit and to prepare a plurality of said filtrates with each said filtrate collected in a different filtrate collection container of the sample filtration unit;

the preparing, a mixture comprises preparing the plurality of said mixtures with each said mixture disposed in a separate container of the multi-container sample purification unit; and

the unsealing comprises unsealing each of the said separate containers of the sample purification unit, wherein each of the said separate containers of the sample purification unit comprises a said unit quantity of said purification particles mixed with said storage liquid.

18. A method according to claim 17 , comprising:

flow cytometry evaluation for virus particles of samples each comprising at least a portion of a different said filtrate, wherein the flow cytometry evaluation comprises hydrodynamically focusing a flow of each said sample and flowing each hydrodynamically focused sample through a flow cytometry investigation cell at a flow rate maintained in a range of from 500 to 3000 nanoliters per minute; and

prior to the flow cytometry evaluation, marking the at least a portion of each said filtrate with at least one fluorescent marker;

wherein:

the flow cytometry evaluation comprises sequentially subjecting to flow different ones of the plurality of said samples; and

the sequentially subjecting to flow cytometry comprises sequentially removing said samples from different filtrate collection containers of a filtrate collection plate of the sample filtration unit with an autosampler and sequentially delivering said samples from the autosampler to a flow cytometer.

19. A method according to claim 17 , wherein the buffer solution reagent is a Tris-HCl buffer solution reagent and the buffer storage liquid is a Tris-HCl buffer storage liquid.