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1. WO2020197644 - FLOW CYTOMETRY EVALUATION FOR UNASSOCIATED NON-ENVELOPED VIRAL PARTICLES

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[ EN ]

CLAIMS

What Is Claimed Is:

1. A method for flow cytometry evaluation of a biological material for unassociated non-enveloped viral particles having a non-enveloped viral capsid, the method comprising: preparing a fluorescently-stained fluid sample for flow cytometry evaluation, the fluorescently-stained fluid sample comprising:

an aqueous liquid medium;

a sample of biological material to be evaluated by flow cytometry for the unassociated non-enveloped viral particles having the non-enveloped viral capsid;

at least one fluorescent stain to fluorescently slain the unassociated non- enveloped viral particles to prepare unassociated labeled particles of virus size dispersed in the aqueous liquid medium, wherein each said unassociated labeled particle comprise a said unassociated non-enveloped viral particle stained with the at least one fluorescent stain, and wherein each said fluorescent stain has a fluorescent emission response when the unassociated labeled particle is subjected to a stimulation radiation;

after the preparing, subjecting the fluorescently-stained fluid sample to flow cytometry evaluation in a flow cytometer, the flow cytometry evaluation comprising flowing the fluorescently-stained fluid sample through an investigation zone of the flow cytometer and in the investigation zone subjecting the fluorescently-stained fluid sample to the stimulation radiation and detecting for the fluorescent emission response from the investigation zone and counting identified occurrences of the unassociated labeled particles; and

the preparing the fluorescently-stained fluid sample comprising fluorescent staining the biological material in a fluid sample composition with a said fluorescent stain to prepare a stained fluid sample composition at an acidic pH in an acidic pH range of from pH 3.0 to pH

6.5.

2. The method of claim I , wherein the acidic pH range is from pH 4.0 to pH 6.0.

3. The method of either one of claim 1 or claim 2, wherein the at least one fluorescent stain comprises a fluorogenic dye for staining of nucleic acid content inside the viral capsid of the non-enveloped viral particles.

4. The method of any one of claims 1-3, wherein the fluorogenic dye is a first fluorogenic dye and the at least one fluorescent stain comprises a second fluorogenic dye for staining of protein content of the viral capsid.

5. The method of any one of claims 1-4, wherein the fluorescently-stained fluid sam ple as prepared during the preparing and as fged to the flow ev >T tometer com 1prises an or 4eanic liquid component as a minor component of the aqueous liquid medium.

6. The method of claim 5, wherein the organic liquid component is dimethyl sulfoxide (DMSO).

7. The method of any one of claims 1 -6 if, wherein the aqueous liquid medium comprises a weight ratio of water to the organic liquid component in a range of from 30: 1 to 3000: 1.

8. The method of any one of claims 1 -7, wherein the preparing the fluorescently-stained fluid sample comprises:

first preparatory processing to prepare a concentrated stain formulation with the fluorogenic dye containing at least one aromatic group susceptible to pi stacking interactions in aqueous liq Iuids, the first preparatpry porcessin g comprising dissolving the fluorogenic dye into a first liquid medium from a dry powder dye composition with the fluorogenic dye, wherein the first liquid medium comprises the organic liquid component;

after the first preparatory processing, second preparatory processing while the fluorogenic dye remains in solution to prepare an aqueous diluted stain formulation comprising the fluorogenic dye dissolved in a diluted aqueous liquid comprising the organic liquid component, the second preparatory processing comprising diluting the first liquid medium with aqueous stain dilution liquid at a volume ratio of the aqueous stain dilution liquid to the first liquid medium of at least 10: 1 to prepare the diluted aqueous liquid with a weight ratio of water to the organic liquid component of at least 10: 1 ; and

during the fluorescent staining, mixing at least a portion of the aqueous diluted stain formulation with the fluid sample composition.

9. The method of claim 8, wherein;

the fluorogenic dye is a first fluorogenic dye with a first fluorescent emission signature and the at least one stain comprises a second fluorogenic dye for staining of protein content of the viral capsid, the second fluorogenic dye containing at least one aromatic group susceptible to pi slacking interactions in aqueous liquids and having a second fluorescent emission signature that is different than the first fluorescent emission signature;

the dry powder dye composition comprises a dry powder mixture including the first fluorogenic dye and the second fluorogenic dye;

the first preparatory processing comprises dissolving the second fluorogenic dye into the first liquid medium from the dry powder dye composition to prepare the concentrated stain

formulation including the second fluorogenic dye, wherein the stained fluid sample composition includes both the first fluorogenic dve and the second fluorogenic dve:

the second preparatory processing comprises, while the second fluorogenic dye remains in solution, preparing the aqueous diluted stain formulation comprising the first fluorogenic dye and the second fluorogenic dye dissolved in the diluted aqueous liquid: and

the flow cytometry evaluation comprises detecting and counting occurrences of the unassociated labeled particles stained with both the first fluorogenic dye and the second fluorogenic dye.

10. The method of any one of claims 1 -7, wherein the preparing the fluorescently-stained fluid sample comprises:

providing a liquid dye concentrate comprising the first fluorogenic dye and the second fluorogenic dye in a stain liquid medium: and

durin vg the fluorescent stainin vg, " mixin vg the liq ·uid dv w' e concentrate with the fluid samp a le composition;

and wherein the method comprises at least one member selected from the group consisting of:

(i) the stain liquid medium comprising a mixture including water and liquid phase organic material and with the, with the slain liquid medium comprising more than 50 percent by moles of water;

(ii) the liquid dye concentrate comprising disaccharide dissolved in the slain liquid medium;

(iii) the providing the liquid dye concentrate being in the absence of reconstituting the fluorogenic dyes from a dry form into the stain liquid medium; and

(iv) combinations including two or more of any of (i)-(iii).

1 1. The method of claim 10, comprising the providing the liquid dye concentrate being in the absence of reconstituting the first and second fluorogenic dyes from a dry form into the stain liquid medium.

12. The method of claim 10, wherein the liquid dye concentrate comprises both (i) and (ii).

13. The method of any one of claims 1-12, wherein the preparing the fluorescently-stained fluid sample comprises diluting a preliminary fluid sample with aqueous sample dilution liquid to prepare a diluted preliminary fluid sample, wherein the aqueous sample dilution liquid has a pH in the acidic pH range.

14. The method of claim 13, wherein the diluted preliminary fluid sample has a pH in the acidic a pH ran e agnd the fluorescent stainin ¼e comp i rises addine the said fluorescent stain to the diluted preliminary fluid sample as the fluid sample composition.

15. The method of any one of claims 1 - 14, wherein the at least one fluorescent stain comprises a fluorescent antibody stain for binding with an epitope of the viral capsid of the unassociated non-enveloped viral particle.

16. The method of claim 15, wherein the fluorescent antibody stain comprises a said fluorescent slain in the fluorescent staining and the stained fluid sample composition at the acidic pH includes the fluorescent antibody stain.

17. The method of any one of claims 1 - 16, wherein as fed to the flow cytometer the fluorescently-stained fluid sample has a pH in the acidic pH range.

18. The method of any one of claims 1-17, wherein:

the at least one fluorescent stain comprises a fluorogenic dye for staining of nucleic acid content inside the viral capsid of the non-enveloped viral particles

the fluorescent staining is a first fluorescent staining the biological material and the stained fluid sample composition is a first stained fluid sample composition, and the preparing the fluorescently-stained fluid sample comprises second fluorescent staining the biological material afier the first fluorescent staining; and

the second fluorescent staining comprises staining the biological material with the fluorescent antibody stain to prepare a second stained fluid sample composition including both the fluorescent antibody stain and the fluorogenic dye for staining of nucleic acid content, wherein the second stained fluid sample composition is at a second pH that is larger than the acidic pH by at least 0.5 pH unit, and the second pH is pH 5.5 or larger.

19. The method of any one of claims 1 - 18, wherein the flow cytometry evaluation comprises:

flowing the fluorescenlly-siained fluid sample through the investigation zone at a flow rate in a range of from 300 nanolilers per minute to 6000 nanoliters per minute while subjecting the fluorescently-stained fluid sample to the stimulation radiation, and preferably the flow rate is in a range of from 600 nanolilers per minute to 3000 nanolilers per minute; and

evaluating only fluorescent emission response from the investigation zone to identify occurrences of the unassociated labeled particles, and not evaluating light scatter from the investigation zone.

20. The method of any one of claims 1 - 19, wherein;

the at least one fluorescent stain includes a plurality of fluorescent stains and the flow

cytometry evaluation comprises detecting for a separate fluorescent emission response from each of the said pluralit ev of fluorescent stains:

the flow cytometry evaluation comprises identifying coincidences of at least two different fluorescent emission responses from the plurality of fluorescent stains indicative of passage through the investigation zone of a said unassociated labeled particle including the at least two different fluorescent stains; and

the flow cytometry evaluation comprises counting as occurrences of said unassociated non-enveloped viral particles identified coincidences of the at least two different fluorescent emission responses.

21. The method of any one of claims 1 -20, wherein the unassociated non-enveloped viral particles are virions.

22. The method of any one of claims 1 -20, wherein the unassociated non-enveloped viral particles are virus-like particles.

23. The method of any one of claims 1 -20, wherein the unassociated non-enveloped viral particles are genetically modified.

24. The method of any one of claims 1 -23, wherein the viral capsid is of a bacteriophage.

25. The method of any one of claims 1 -24. wherein the unassociated non-enveloped viral particles have a particle size in a range of from 30 nanometers to 1 micron.

26. The method of any one of combinations 1-25, wherein the stained fluid sample comprises dissolved disaccharide.

27. A kit for preparing a fluorescenlly-stained fluid sample for flow cytometry evaluation of biological material for quantification of unassociated non-enveloped viral particles of virus size, having a non-enveloped viral capsid, the kit comprising:

a plurality of sealed containers;

a fluorescent stain composition for fluorescent staining the unassocialed non-enveloped viral particles to prepare unassociated labeled particles of virus size, each said unassociated labeled particle of virus size comprising a said unassociated non-enveloped viral particle stained with a fluorescent slain from the fluorescent slain composition, the fluorescent stain composition comprising at least one fluorescent stain and being contained in a first said sealed container; an aqueous dilution liquid for preparing a pH-adjusled fluid sample for fluorescent staining biological material at in an acidic pH of up to pH 6.5 during preparation of the unassociated labeled particles of virus size dispersed in an aqueous medium for flow cytometry evaluation of a fluorescently-stained fluid sample with the biological material, the aqueous dilution liquid having an acidic pH of no larger than pH 6.5 and being contained in a second said sealed container.

28. The kit of claim 27, wherein the first said sealed container and the second said sealed container are packaged in a common packaging enclosure.

29. The kit of either one of claim 27 or claim 28, wherein the at least one fluorescent stain of the fluorescent stain composition comprises a fluorogenic dye for staining nucleic acid content inside the viral capsid of the non-enveloped viral particles.

30. The kit of claim 29, wherein;

the fluorogenic dye is a first fluorogenic dye with a first fluorescent emission signature and the at least one fluorescent slain of the fluorescent stain composition comprises a second fluorogenic dye for staining of protein content of the viral capsid, the second fluorogenic dye having a second fluorescent emission signature different than the first fluorescent emission signature; and

each said fluorogenic dye in the fluorescent stain composition contains at least one aromatic group susceptible to pi stacking interactions in aqueous liquids;

31 . The kit of either one of claim 29 or claim 30 comprising a reconstitution liquid medium, the reconstitution liquid medium being a solvent for each said fluorogenic dye to provide each said fluorogenic dye in solution in the reconstitution liquid medium prior to mixing with the aqueous dilution liquid to prepare the pH-adjusted fluid sample, the reconstitution liquid medium comprising an organic liquid component; and

the organic liquid component is dimethyl sulfoxide (DMSO), and the reconstitution liquid medium comprises at least 34 percent by moles of DMSO.

32. The kit of any one of claims 29-31 , wherein the fluorescent stain composition comprises a dry powder dye composition including each said fluorogenic dye.

33. The kit of any one of claims 29-32, wherein the fluorescent stain composition comprises the reconstitution liquid medium and each said fluorogenic dye dissolved in the reconstitution liquid medium.

34. The kit of any one of claims 27-33, wherein;

the fluorescent stain composition is a first fluorescent stain composition and the kit comprises a second fluorescent stain composition comprising a fluorescent antibody stain; and the fluorescent antibody stain is either:

specific for binding with an epitope of the viral capsid for direct staining of the unassociated non -enveloped viral particles; or

specific for binding with a primary antibody that is specific for binding with an epitope of the viral ca sid fopr indirect stainin Vg of the unassociated non- enveloped viral particles.

35. The kit of claim 34, wherein the kit comprises another said sealed container, other than the first sealed container or the second sealed container, containing the second fluorescent stain composition.

36. The kit of claim 30, wherein the fluorescent stain composition comprises a liquid dye concentrate comprising the first fluorogenic dye and the second fluorogenic dye in a slain liquid medium, and wherein the liquid dye concentrate comprises at least one member selected from the group consisting of:

(i) the slain liquid medium comprising a liquid mixture including water and liquid phase organic material, with the stain liquid medium comprising more than 50 percent by moles of water;

(ii) disaccharide dissolved in the stain liquid medium; and

(iii) a combination including both (i) and (ii)

37. The kit of claim 36, wherein the slain liquid medium comprises the liquid phase organic material and the liquid phase organic material comprises dimethyl sulfoxide (DMSO).

38. The kit of claim 36 or claim 37, wherein the liquid dye concentrate comprises the disaccharide dissolved in the slain liquid medium.

39. The kit of claim 38, wherein a concentration of the disaccharide in the slain liquid medium is in a range of from 1 weight percent to 45 weight percent.

40. The kit of any one of claims 27-33, wherein the at least one fluorescent stain comprises a fluorescent antibody stain, and the fluorescent antibody slain is either:

specific for binding with an epitope of the viral capsid for direct staining of the unassociated non -enveloped viral particles; or

specific for binding with a primary antibody that is specific for binding with an epitope of the viral capsid for indirect staining of the unassociated non- enveloped viral particles.

41 . The kit of any one of claims 27-40, wherein the acidic pH of the aqueous dilution liquid is in a range of from pH 4.0 to pH 5.6.

42. The kit of any one of claims 27-41 , wherein the aqueous dilution liquid is an aqueous sample dilution liquid to dilute a preliminary fluid sample including the biological material prior to a fluorescent staining step.

43. The kit of claim 42, comprising another said sealed container, other than the first sealed container and the second sealed container, containing an aqueous slain dilution liquid to prepare an aa *ueous diluted stain formulation to fiuorescentlv * stain the biolo Vgical material: the aqueous sample dilution liquid and the aqueous stain dilution liquid have different compositions;

the aqueous stain dilution liquid has a pH of at least pH 7.0; and

the aqueous sample dilution liquid has a pH in an acidic pH range of from pH 3.0 to pH

6.5.

44. The kit of claim 42, wherein;

the aqueous sample dilution liquid is a first aqueous sample dilution liquid and the kit comprises a second aqueous sample dilution liquid to dilute a preliminary stained fluid sample including the biological material following a fluorescent staining step;

the second aqueous sample dilution liquid has a pH that is higher than the acidic pH of the first aqueous sample dilution liquid; and

the second aqueous sample dilution liquid is contained in another sealed container, other than the first sealed container and the second sealed container; and

the second aqueous sample dilution liquid has a pH that is at least 0.5 pH units larger than the acidic pH of the first aqueous sample dilution liquid.

45. The kit of any one of claims 27-44, comprising a disaccharide dissolved in the aqueous dilution liquid.