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 shell.

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 , comprising 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 filtrate of the filtering.

4 - 8 . (canceled)

9 . A method according to claim 3 , 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.

10 - 13 . (canceled)

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

mixing together the biological material and the purification particles;

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;

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.

15 - 16 . (canceled)

17 . A method according to claim 14 , wherein the storage liquid comprises a Tris-HCl buffer solution;

the method comprises, prior to the centrifuging a mixture, preparing the mixture, comprising mixing the biological material, 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.

18 - 20 . (canceled)

21 . A method according to claim 30 , 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.

22 . (canceled)

23 . A method according to claim 1 , 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.

24 - 29 . (canceled)

30 . A method according to claim 1 , 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.

31 - 32 . (canceled)

33 . 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.

34 - 35 . (canceled)

36 . 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;

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

37 - 38 . (canceled)

39 . A method according to claim 36 , 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.

40 . A method according to claim 39 , comprising:

flow cytometry evaluation for virus particles of a-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.

41 . (canceled)

42 . A method according to claim 39 , wherein the buffer solution reagent is a Tris-HCl buffer solution reagent and the buffer storage liquid is a Tris-HCl buffer storage liquid.

43 . A product for use to prepare samples of biological material for flow cytometry evaluation for virus particles, wherein the biological material may comprise larger-size components and smaller-size components, the product comprising:

a sealed container containing a mixture sealed within the container, the mixture comprising:

a unit quantity of purification particles for processing a single sample of the biological material for flow cytometry evaluation for virus particles, the purification particles comprising:

a porous core functionalized to capture at least some non-virus impurities that may be in the smaller-size components of the biological material; 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;

a Tris-HCl buffer solution storage liquid.

44 - 46 . (canceled)

47 . A product according to claim 43 , wherein;

the unit quantity of purification particles has a bulk volume in a range of from 35 microliters to 70 milliliters;

the mixture has a total volume of from 70 microliters to 170 milliliters; and

the pore structure of the shell is configured to exclude at least a majority of molecules having a molecular weight greater than 1,000,000 Daltons and the core is functionalized with sites having hydrophobicity and a positive charge.

48 - 53 . (canceled)

54 . A product according to claim 47 , wherein the storage liquid comprises at least 5 millimoles per liter of Tris, at least 20 millimoles per liter of sodium chloride and a pH in a range of from pH 7 to pH 9.

55 - 56 . (canceled)

57 . A kit for use to prepare samples of biological material for flow cytometry evaluation for virus particles, wherein the biological material may comprise larger-size components and smaller-size components, the kit comprising:

at least one sealed container containing a unit quantity of purification particles for processing a single sample of the biological material for flow cytometry evaluation for virus particles, the purification particles comprising:

a porous core functionalized to capture at least some biological impurities that may be in the smaller-size components of the biological material; 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;

at least one centrifugal filter for centrifugal filtration of at least a portion of the biological material after treatment with the purification particles, to prepare filtrate for flow cytometry evaluation for virus particles.

58 - 62 . (canceled)

63 . A kit for use to prepare samples of biological material for flow cytometry evaluation for virus particles and which biological material may contain larger-size components including virus particles and smaller-size components with non-virus impurities, the kit comprising a plurality of multi-sample processing units, wherein the plurality of multi-sample processing units comprise:

a sample clarification unit comprising a plurality of first fluid containers each having a fluid containment volume with a first portion of the fluid containment volume occupied by a volume of buffer solution reagent and a second portion of the fluid containment volume available to receive a sample of the biological material to mix with a said volume of buffer solution reagent in a said first fluid container;

a sample purification unit comprising a plurality of second fluid containers each having disposed therein a volume of a mixture, wherein each said volume of the mixture comprises:

a unit quantity of purification particles for processing a clarified biological material sample following processing in a said first fluid container, the purification particles comprising a porous core functionalized to capture at least some of the non-virus impurities 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 and to permit the smaller-size components to enter into the core through the pore structure of the shell; and

buffer solution storage liquid.

64 . A kit according to claim 63 , wherein:

each said second fluid container of the sample purification unit is sealed with a said volume of the mixture disposed therein;

each said first fluid container of the sample clarification unit is sealed with a said volume of the buffer solution reagent disposed therein;

the sample purification unit and the sample clarification unit are each individually packaged in separate sterile packaging enclosures.

65 - 67 . (canceled)

68 . A kit according to claim 63 , wherein:

the sample clarification unit comprises a first tube rack and the first fluid containers comprise first tubes received in the first tube rack;

the first tubes are comprised in a plurality of first tube strips, each said first tube strip comprising multiple ones of the first tubes connected together;

the sample purification unit comprises a second tube rack and the second fluid containers comprise second tubes received in the second tube rack; and

the second tubes are comprised in a plurality of second tube strips, each said second tube strip comprising multiple ones of the second tubes connected together.

69 . (canceled)

70 . A kit according to claim 79 , wherein:

each said volume of buffer solution reagent is in a range of from 45 microliters to 90 milliliters; and

each said first fluid container has a total containment volume of at least 2 times as large as the volume of the buffer solution reagent.

71 - 78 . (canceled)

79 . A kit according to claim 63 , wherein:

each said second fluid container comprises a bulk volume of the purification particles in a range of from 35 microliters to 70 milliliters;

each said second fluid container has a total containment volume of at least two times as large as the bulk volume of the purification particles in the second fluid container;

the mixture in each said second fluid container comprises a ratio of volume of buffer solution storage liquid to the bulk volume of the purification particles of at least 0.75; and

each said volume of the mixture is in a range of from 70 microliters to 170 milliliters.

80 - 85 . (canceled)

86 . A kit according to claim 63 , wherein the buffer solution reagent in the first fluid containers and the buffer solution storage liquid in the second fluid containers are each a Tris-HCl buffer solution having a pH in a range of from pH 7 to pH 9; and

the buffer solution reagent in the first fluid containers comprises a greater concentration of Tris and a greater concentration of sodium chloride than a concentration of Tris and a concentration of sodium chloride in the buffer solution storage liquid.

87 . A kit according to claim 86 , wherein the buffer solution storage liquid comprises a concentration of Tris of at least 5 millimoles per liter and a sodium chloride concentration of at least 20 millimoles per liter.

88 - 89 . (canceled)

90 . A kit according to claim 63 , comprising a sample filtration unit, the sample filtration unit comprising:

a plurality of filter wells with filter media to filter a mixture comprising purified sample and a said unit volume of purification particles following processing in a said second fluid container; and

a plurality of filtrate collection containers each to receive filtrate passing through a said filter media of a different one of said filter wells.

91 - 94 . (canceled)

95 . A kit according to claim 90 , wherein the filter media of each said filter well has a filtration separation size in a range of from 0.5 micrometers to 2 micrometers.

96 . A kit according to claim 95 , wherein:

each said filter well has a volume in a range of from 10 microliters to 15 milliliters; and

each said filtrate collection container has a fluid containment volume in a range of from 25 microliters to 50 milliliters.

97 . (canceled)

98 . A kit according to claim 90 , wherein:

the sample clarification unit comprises at least six of the first fluid containers;

the sample purification unit comprises at least six of the second fluid containers;

the sample filtration unit comprises at least six of the filter wells; and

the sample clarification unit, the sample purification unit and the sample filtration unit are each individually packaged in separate sterile packaging enclosures.

99 . A kit according to claim 98 , wherein the sample clarification unit, the sample purification unit and the sample filtration unit are all disposed within a single common packaging container.

100 . (canceled)