Flow cytometry evaluation for unassociated virus-size particles having an adenovirus epitope

Abstract

A method for evaluating a biological material for unassociated virus-size particles having an adenovirus epitope uses a fluorescent antibody stain specific for binding with the epitope and a fluid sample with the virus-size particles and fluorescent antibody stain is subjected to flow cytometry with identification of fluorescent emission detection events indicative of passage through a flow cell of a flow cytometer of unassociated labeled particles of virus size including such a virus-size particle and fluorescent antibody stain.

Claims

1. A flow cytometry method for evaluating a biological material sample for unassociated virus-size particles having an adenovirus epitope, the method comprising: subjecting to flow cytometry a fluid sample comprising at least a portion of the biological material sample, wherein the fluid sample comprises a fluorescent antibody stain capable of binding, directly or indirectly, with an adenovirus epitope, the flow cytometry comprising: flowing the fluid sample through a flow cell of a flow cytometer; subjecting the fluid sample flowing through the flow cell to excitation radiation capable of causing a fluorescent emission response from the fluorescent antibody stain; and detecting radiation from the flow cell within a wavelength range of the fluorescent emission and evaluating the detected radiation to identify detection events indicative of passage through the flow cell of unassociated labeled particles of virus size with the adenovirus epitope bound with a portion of the fluorescent antibody stain; wherein the unassociated labeled particles of virus size are of a particle size in a range of from 10 nanometers to 200 nanometers; and wherein the fluid sample as fed to the flow cytometer comprises a concentration of the fluorescent antibody stain free in solution and not bound in the unassociated labeled particles in a range of from 0.5 microgram per milliliter to 10 micrograms per milliliter.

2. A flow cytometry method according to claim 1, wherein the fluid sample as fed to the flow cytometer comprises a concentration of the fluorescent antibody stain not bound in the unassociated labeled particles in a range of from 0.5 microgram per milliliter to 4 micrograms per milliliter.

3. A flow cytometry method according to claim 1, wherein the fluid sample as fed to the flow cytometer includes a total concentration of the fluorescent antibody stain in a range of from 0.5 microgram per milliliter to 10 micrograms per milliliter.

4. A flow cytometry method according to claim 1, comprising manufacturing a product comprising the virus-size particles having an adenovirus epitope, the manufacturing comprising: production processing to prepare a purified product including the virus-size particles having an adenovirus epitope, the production processing including: generating the virus-size particles in a biological production operation; harvesting from the biological production operation crude product comprising the virus-size particles generated during the generating; and purifying at least a portion of material of the crude product to prepare a purified product including the virus-size particles; collecting the biological material sample from a stage of the production processing during which the virus-size particles are expected to be present in the biological material sample; evaluating the biological material sample for presence of the virus-size particles having an adenovirus epitope, the evaluating comprising preparing the fluid sample for the flow cytometry.

5. A flow cytometry method according to claim 4, comprising performing the collecting multiple times to collect different said biological material samples to be evaluated from multiple different said stages and performing a said evaluating on a different said fluid sample prepared from each said collected biological material sample to be evaluated.

6. A flow cytometry method according to claim 5, wherein the multiple different said stages include at least a first stage during the generating or the harvesting and a second stage during or after the purifying.

7. A flow cytometry method according to claim 3, wherein the flow cytometry is in the absence of detecting for light scatter.

8. A flow cytometry method according to claim 3, wherein the fluid sample as fed to the flow cytometer has a concentration of the unassociated labeled particles up to 110.sup.9 particles per milliliter.

9. A flow cytometry method according to claim 3, wherein the fluorescent antibody stain comprises a fluorophore attached to antibody molecules and the fluorescent antibody stain in the fluid sample comprises an average of from 3 to 8 of the fluorophore attached per said antibody molecule in the fluorescent antibody stain.

10. A flow cytometry method according to claim 1, wherein; the fluorescent antibody stain is a first fluorescent stain and the fluid sample comprises a second fluorescent stain having a second fluorescent emission response, different than the first fluorescent emission response of the first fluorescent stain, caused by the excitation radiation; and the second fluorescent stain comprises a fluorescent nucleic acid stain not specific to particle type.

11. A flow cytometry method according to claim 1, wherein: the fluorescent antibody stain is a first fluorescent stain and the fluid sample comprises a second fluorescent stain having a second fluorescent emission response, different than the first fluorescent emission response of the first fluorescent stain, caused by the excitation radiation; the unassociated labeled particles are first unassociated labeled particles, the epitope is a first epitope, the virus-size particles are first virus-size particles and the unassociated labeled particles are first unassociated labeled particles; the second fluorescent stain is a second fluorescent antibody stain that is different than the first fluorescent antibody stain and is capable of binding, directly or indirectly, with a second epitope of second virus-size particles that are different than the first particles, wherein the second epitope that is different than the first epitope to form second unassociated labeled particles of virus size with the second fluorescent antibody stain; the fluid sample as fed to the flow cytometer comprises a total concentration of the second fluorescent antibody stain and a concentration of the second fluorescent antibody stain free in solution and not bound in the unassociated labeled particles each in a range of from 0.5 microgram per milliliter to 4 micrograms per milliliter; and the method comprises detecting radiation from the flow cell within a wavelength range of the second fluorescent emission and evaluating the detected radiation to identify detection events indicative of passage of the second unassociated labeled particles through the flow cell.

12. A flow cytometry method according to claim 11, wherein the second fluorescent emission has a peak wavelength at least 20 nanometers different than a peak wavelength of the first fluorescent emission and the flow cytometry comprises separately detecting for at least the first and second fluorescent emissions and not detecting for light scatter.

13. A flow cytometry method according to claim 1, wherein the virus-size particles comprise virus-like particles with the adenovirus epitope.

14. A flow cytometry method according to claim 1, wherein the adenovirus epitope is adenovirus hexon protein and the fluorescent antibody stain comprises a monoclonal antibody.

15. A flow cytometry method according to claim 1, wherein the virus-size particles comprise adenovirus modified to express recombinant protein.

16. A flow cytometry method according to claim 7, wherein the flow cytometry comprises detecting only for fluorescent emission response.

17. A flow cytometry method according to claim 3, comprising preparing the fluid sample, the preparing the fluid sample comprising: mixing biological material to be evaluated for presence of the unassociated virus-size particles with the fluorescent antibody stain; and after the mixing, not removing the fluorescent antibody stain free in solution and not bound in the unassociated labeled particles from the fluid sample prior to the flow cytometry.

18. A flow cytometry system to evaluate properties of virus-size particles, the system comprising a flow cytometer and a fluid sample with unassociated labeled particles of virus size in an aqueous liquid medium for flow cytometry evaluation by the flow cytometer, wherein the flow cytometer comprises a flow cell to receive flow of the fluid sample to investigate properties of the unassociated labeled particles, an excitation radiation source to direct excitation radiation to the flow cell and a detector system to detect radiation coming from the flow cell within a wavelength range, and wherein the fluid sample is in fluid communication with the flow cell and the fluid sample comprises: the unassociated labeled particles of virus size of particle size in a range of from 10 nanometers to 200 nanometers in the aqueous liquid medium, wherein the unassociated labeled particles each includes; a virus-size particle having an adenovirus epitope; and a fluorescent antibody stain specific for binding, directly or indirectly with the adenovirus epitope, the fluorescent antibody stain having a fluorescent emission response in the wavelength range in response to excitation by the excitation radiation; and an unbound portion of the fluorescent antibody stain in the aqueous liquid medium, free in solution and not bound in the unassociated labeled particles, at a concentration in a range of from 0.5 microgram per milliliter to 10 micrograms per milliliter.

19. A flow cytometry system according to claim 18, wherein the fluid sample includes a total concentration of the fluorescent antibody stain in a range of from 0.5 microgram per milliliter to 10 micrograms per milliliter.

20. A flow cytometry system according to claim 19, wherein the fluorescent antibody stain comprises fluorophore attached to antibody molecules and the fluorescent antibody stain in the fluid sample comprises an average of 3 to 8 of the fluorophores attached per antibody molecule of the fluorescent antibody stain.

21. A flow cytometry system according to claim 19, wherein: the fluorescent antibody stain is a first fluorescent stain having a first fluorescent emission; and the fluid sample includes a second fluorescent stain for nucleic acid, the second fluorescent stain having a second fluorescent emission, different than the first fluorescent emission.

22. A flow cytometry system according to claim 19, wherein: the fluorescent antibody stain is a first fluorescent antibody stain, the adenovirus epitope is a first epitope, the fluorescent emission is a first fluorescent emission, and the unassociated labeled particles are first unassociated labeled particles of virus size; the fluid sample includes a second fluorescent antibody stain different than the first fluorescent antibody stain and capable of binding, directly or indirectly, with a second epitope different than the first epitope, the second epitope being indicative of virus other than adenovirus and the second fluorescent antibody stain having a second fluorescent emission response that is different than the first fluorescent emission response.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a general process block diagram for some implementations of a flow cytometry method of the disclosure.

(2) FIG. 2 shows a process block diagram illustrating some more specific processing implementations within the general processing of FIG. 1.

(3) FIG. 3 illustrates an example of processing that may be performed during flow cytometry with the processing shown in FIG. 2.

(4) FIG. 4 shows a process block diagram illustrating some other more specific processing implementations within the general processing of FIG. 1.

(5) FIG. 5 illustrates an example of processing that may be performed during flow cytometry with the processing shown in FIG. 4.

(6) FIG. 6 shows a process block diagram illustrating some other more specific processing implementations within the general processing of FIG. 1.

(7) FIG. 7 illustrates an example of processing that may be performed during flow cytometry with the processing shown in FIG. 6.

(8) FIG. 8 shows a process block diagram illustrating some other more specific processing implementations within the general processing of FIG. 1.

(9) FIG. 9 shows a process block diagram illustrating some more specific processing implementations within a purifying crude sample step of the processing of FIG. 8.

(10) FIG. 10 is a plot summarizing results for Example 1 presented below.

(11) FIG. 11 is a plot summarizing some results for Example 2 presented below.

(12) FIG. 12 is a plot summarizing some other results for Example 2 presented below

(13) FIG. 13 is a plot summarizing results for Example 3 presented below.

(14) FIG. 14 is a plot summarizing results for Example 4 presented below.

DETAILED DESCRIPTION

(15) FIG. 1 shows a general process block diagram of processing for a flow cytometry method for evaluating biological material for the presence of unassociated viral particles of targeted adenovirus or adeno-associated virus viral type. The processing shown in FIG. 1 includes a step of preparing a fluid sample 102 to prepare a fluid sample 104 that is then subjected to flow cytometry 106. The fluid sample 104 resulting from the preparing fluids sample 102 step includes a fluorescent antibody stain capable of binding, directly or indirectly, with the unassociated viral particles of the targeted viral type to form unassociated labeled particles that may be detected during the flow cytometry 106. The preparing fluid sample 102 may include mixing the biological material to be tested with at least one fluorescent antibody stain. FIG. 2 shows a process block diagram of a more specific example implementation of the general processing shown in FIG. 1, in which the preparing fluid sample 102 includes a mixing 108 step during which biological material 110 to be evaluated is mixed with a fluorescent antibody stain 112 that is capable of binding, directly or indirectly, with epitopes indicative of the targeted adenovirus or adeno-associated virus viral type for which the biological material 110 is being evaluated. As introduced to the mixing 108, the biological material 110 may be in a buffered solution at appropriate concentration for processing, and may be the result of prior processing from an initial crude sample of biological material. Such a solution in which the biological material 110 may be fed to the mixing 108 may also include other additives or reagents useful during the processing. Likewise, the fluorescent antibody stain may be provided to the mixing 108 step in an appropriately buffered solution with one or more additives or other reagents. For example, the fluorescent antibody stain 112 may be provided in buffer solution also including reagents to stabilize, help preserve or disperse the fluorescent antibody stain 112 prior to the mixing 108. For example, the fluorescent antibody stain 112 may be provided in a buffered solution containing an anti-agglomeration agent (e.g., bovine albumin) and/or an anti-microbial agent (e.g., sodium azide). The buffered-solution may be a phosphate buffered solution or other buffered solution at an appropriate PH.

(16) FIG. 3 illustrates an example of processing that may be performed during the flow cytometry 106 of the example implementation of FIG. 2. As shown in FIG. 3, a flow of the fluid sample 104 through a flow cell 114 of a flow cytometer is subjected in the flow cell 114 to excitation radiation 116, for example from a laser, LED or other light source. In the example shown in FIG. 3, the flow of fluid sample 104 through the flow cell 114 is in the direction of the flow arrow 118. A detector system 120 includes optical componentry for detecting radiation coming from the flow cell within a wavelength range of the fluorescent emission of the fluorescent antibody stain 112. Illustrated in FIG. 3 is passage of an unassociated labeled particle 122 of virus size including a viral particle of adenovirus or adeno-associated virus viral type and having fluorescent antibody stain bound thereto. FIG. 3 also illustrates an example time plot 124 of output (C1) of a photodetector detecting for the fluorescent emission and showing a peak voltage at time t.sub.1 corresponding with passage of such an unassociated labeled particle 122 through the flow cell 114. Also shown in FIG. 3 for illustration purposes are residual, unbound fluorescent antibody stain 112 in the fluid sample 104 that may contribute to some of the larger background peaks in the time plot 124.

(17) The fluid sample 104 of FIG. 1 subjected to the flow cytometry 106 may be made using two or more different fluorescent stains, at least one of which is a fluorescent antibody stain for an adenovirus or adeno-associated virus viral type. FIG. 4 shows example processing that is the same as that shown in FIG. 2, except that during the mixing 108 a second fluorescent stain 126 is provided to the mixing 108 step for inclusion in the fluid sample 104 that is subjected to the flow cytometry 106. In the example shown in FIG. 4, the second fluorescent stain 126 is a stain for nucleic acid that is not selective as to viral type. The second fluorescent stain 126 may be a fluorogenic stain and may be provided in a single formulation with the fluorescent antibody stain 112 or may be provided in a separate formulation. In a preferred processing situation, the fluorescent antibody stain 112 and the second fluorescent stain 126 may be provided in a single formulation, such as a single buffered solution in desired proportions for inclusion in the fluid sample 104. FIG. 5 illustrates an example of processing for the flow cytometry 106 of FIG. 4. As shown in FIG. 5, a flow of the fluid sample 104 through the flow cell 114 is subjected in the flow cell 114 to the excitation radiation 116. In the illustration shown in FIG. 5, the detection system 120 includes a first photodetector for detecting fluorescent emission from the fluorescent antibody stain 112 and a second photodetector for detecting a different fluorescent emission from the second fluorescent stain. FIG. 5 shows an example unassociated labeled particle 128 including a viral particle of an adenovirus or adeno-associated virus viral type having attached thereto both the fluorescent antibody stain 112 and the second fluorescent stain 126. FIG. 5 shows a first time plot 130 with an example output (C1) of the first photodetector detecting for fluorescent emission from the fluorescent antibody stain 112 and a second time plot 132 showing example output (C2) from the second photodetector detecting for fluorescent emission from the second fluorescent stain 126. The time coincidence of voltage peaks at time t.sub.2 in the first time plot 130 and the second time plot 132 are indicative of passage of the unassociated labeled particle 128 through the flow cell 114. The voltage peaks at times t.sub.1 and t.sub.3 shown in the second time plot 132 are indicative of passage of particles through the flow cell 114 that contain the second fluorescent stain but that do not contain the fluorescent antibody stain 112. Such particles that may include the second fluorescent stain 126 and not the fluorescent antibody stain 112 may be, for example, viral particles of a different viral type than the viral type of the viral particle of the unassociated labeled particle 128. Greater magnitude of background and larger background peaks shown in the time plot 130 may be indicative of the passage of unbound fluorescent antibody stain 112 through the flow cell 114. For illustration purposes, FIG. 5 shows the presence of such unbound fluorescent antibody stain 112 in the fluid sample 104 passing through the flow cell 114. The second time plot 132 includes a background pattern with smaller background peaks indicative of the second fluorescent stain being a fluorogenic material that provides very little background fluorescent emission when not attached to nucleic acid with a particle.

(18) FIG. 6 shows example processing that is the same as that shown in FIG. 4, except that the second fluorescent stain provided to the mixing 108 is a second fluorescent antibody stain 134 that is specific for binding to epitopes of viral particles of a different viral type (e.g., different than the adenovirus or adeno-associated virus or of a different serotype of adenovirus or adeno-associated virus) than the fluorescent antibody stain 112. Processing such as that shown in FIG. 6 may be used to discriminate between the presences of viral particles of different viral types present in the biological material 110 and to quantify the presence of viral particles of each of the two different types in the fluid sample. FIG. 7 illustrates an example of processing that may occur during the flow cytometry 106 of the processing example shown in FIG. 6. FIG. 7 is the same as FIG. 5, except showing the fluid sample 104 including an example first type of unassociated labeled particle 136 including a viral particle of a first viral type of adenovirus or adeno-associated virus with the fluorescent antibody stain 112 attached thereto and a second type of unassociated labeled particle 138 including a viral particle of a second viral type having the second antibody stain 134 attached thereto. FIG. 7 shows a first time plot 140 illustrating example output (C1) of a first photodetector detecting fluorescent emissions of the fluorescent antibody stain 112 and a second time plot 142 illustrating example output (C2) from a second photodetector detecting fluorescent emissions from the second antibody stain 134. For illustration purposes, the fluid sample 104 in FIG. 7 is shown including unassociated first fluorescent antibody stain 112 and unassociated second fluorescent antibody stain 134. As shown in FIG. 7, a voltage peak on the first time plot 140 at time t.sub.1 is indicative of passage of the first type of unassociated labeled particle 136 through the flow cell 114 and the voltage peak at t.sub.2 shown in the second time plot 142 is indicative of passage of the second type of unassociated labeled particle 138 through the flow cell 114.

(19) FIG. 8 shows an example of processing according to the general processing shown in FIG. 1 in which the preparing a fluid sample 102 includes a purifying crude sample step 144 prior to the mixing 108. During the purifying crude sample 144, a crude sample of biological material 146 is subjected to purification processing to remove at least a portion of impurities that may interfere with the flow cytometry 106, to prepare the biological material 110 in a more pure form to be mixed with the fluorescent antibody stain 112 during the mixing 108 to prepare the fluid sample 104. For illustration purposes, the processing in FIG. 8 includes providing a second fluorescent stain 148 to the mixing 108 for inclusion in the fluid sample. The second fluorescent stain 148 may be a second fluorescent antibody stain that is different than the fluorescent antibody stain 112 or may be a non-specific fluorescent stain, for example a nucleic acid stain. The crude sample of biological material 146 may or may not be provided in a diluted form with buffer solution or other reagents and may or may not have been subjected to processing prior to introduction to the purifying crude sample 144 step.

(20) FIG. 9 shows an example of processing that may be performed during the purifying crude sample 144 step of the general processing shown in FIG. 8. In the example processing shown in FIG. 9, the purifying crude sample 144 includes a chromatographic removal 150 step followed by a filtration 152 step. During the chromatographic removal 150, smaller-size impurities 154 are removed from the crude sample of biological material 146 by chromatography, with the smaller-size impurities 154 being retained within chromatographic media. The smaller-size impurities may be impurities smaller than a virus size. Processed liquid 156 from the chromatographic removal 150 is then subjected to the filtration 152 to filter out larger-size impurities 158. The larger-size impurities 158 may be removed as filter retentate and the biological material 110 provided to the mixing 108 may be or include filtrate from the filtration 152 or portions thereof, with or without further processing.

EXAMPLES

(21) In the examples below, flow cytometry tests are performed on a Virus Counter 3100 flow cytometer (ViroCyt, Inc.). Plots in the Figures referred to in these examples that plot virus particle concentration versus dilution factor are log-log plots. Flow cytometry tests using fluorescent antibody stain are performed without washing stained virus samples following staining and without detection of light scatter to assist with particle detection. Except as otherwise stated below, fluorescent antibody stains in these examples generally include an average number of attached dye molecules (fluorophores) per antibody molecule in the fluorescent antibody stain (F/P ratio) of 3 to 7. Concentrations of fluorescent antibody stains provided in these examples are total concentrations in the samples. As will be appreciated, some of this fluorescent antibody stain attaches to and stains virus particles in the samples, but most of fluorescent antibody stain in the samples will typically be in an unbound state when the samples are subjected to flow cytometry. All sample fluids in these examples are prepared to a pH within a range of pH 7 to pH8 with buffered solution.

Examples 1-2

(22) In Examples 1 and 2 below flow cytometry tests are performed for evaluation of adeno-associated virus serotype 2 (AAV-2) using a fluorescent antibody stain including anti-AAV-2 monoclonal antibody A20 (PROGEN Biotechnik) conjugated with Alexa Fluor 532 dye (identified below as CF-A20 antibody stain). A20 is an antibody for a conformational epitope of assembled capsid of AAV-2 and AAV-3.

Example 1

(23) Comparative flow cytometry tests are performed on AAV-2 test formulations using the fluorescent antibody stain CF-A20 relative to controls of AAV-5 and AAV-9 which are not antigenic for the A20 antibody. Two different AAV-2 test formulations are prepared using AAV-2 obtained from different commercial sources. For all tests, the total concentration of the CF-A20 antibody stain (both bound and unbound to AAV-2) in the test solutions is about 2.5 micrograms per milliliter. Five test solutions are prepared at different dilutions of stock virus solution for each for each dilution series, to provide five different virus concentrations for testing in buffered solution. For a specificity control, the fluorescent antibody stain was tested against the AAV-5 and AAV-9 serotypes, as well as buffered solution including CF-A20 but absent virus of any type.

(24) Results are summarized in FIG. 10, which shows a plot of AAV-2 concentration for each AAV-2 sample determined by flow cytometry as a function of dilution factor for the CF-A20 stain tests and for the controls. Both the vertical and horizontal axes are log-scale. As seen in FIG. 10, AAV-2 concentrations indicated for the CF-A20 tests are close for each of the two AAV-2 test formulations. FIG. 10 also summarizes data for the control formulations relative to an approximate lower detection limit for the flow cytometer (about 10.sup.5.5 particles per milliliter). The results demonstrate that data from the antibody stain-treated AAV-2 samples were reasonably consistent between different sources of AAV-2. In addition, the antibody failed to bind to AAV-5 or AAV-9, confirming the specificity of the fluorescent antibody stain for AAV-2 relative to those other serotypes. Furthermore, the fluorescent antibody stain did not increase background to unacceptable levels when added to the blank medium.

Example 2

(25) Flow cytometry dilution series using a variety of antibody stain concentrations are performed against a titration of AAV-2 solution containing about 110.sup.5 to 110.sup.8 AAV-2 particles per milliliter. Each dilution series includes flow cytometry of 200 microliter samples prepared at different dilutions of the stock AAV-2 solution, with all samples in each dilution series containing the same concentration of the fluorescent antibody stain. Three dilution series are run at antibody stain concentrations of about 0.91, 1.82 and 2.73 micrograms of CF-A20 antibody stain per milliliter of sample. Flow cytometry tests are performed to detect and evaluate for fluorescent emissions from stained AAV-2. Results of the flow cytometry tests for each dilution series are summarized in FIGS. 11 and 12. FIG. 11 shows a plot of uncorrected AAV-2 concentration (virus particles per milliliter) as determined for the samples by the flow cytometry tests, whereas FIG. 12 shows a plot of the same data but corrected the dilution factor. Both figures are log-log plots. FIGS. 11 and 12 both show generally good results (close to linear fit) at intermediate dilution factors, with the flow cytometry results corrected for dilution shown in FIG. 12 being close to the actual AAV-2 concentrations of the AAV-2 stock solution for the intermediate dilution factors. At lower dilution factors (higher AAV-2 concentrations in the sample) and higher dilution factors (lower AAV-2 concentrations in samples) results tend to degrade, which may indicate lower counts due to possible agglomeration of some AAV-2 particles at the lower dilution factors and higher counts due to possible interference from background signals from unbound antibody stain at the higher dilution factors.

Examples 3-4

(26) In Examples 3 and 4 below flow cytometry tests are performed to evaluate for multiple adenovirus serotypes using as the fluorescent antibody stain an 8C4 monoclonal antibody-CF 532 conjugate (identified below CF-8C4). The 8C4 antibody is specific for the hexon protein of adenovirus, and is effective across different adenovirus serotypes (tested against at least serotypes 2 through 6).

Example 3

(27) Comparative flow cytometry tests are performed on adenovirus test formulations using the fluorescent antibody stain CF-8C4 on two different test formulations containing different adenovirus serotypes. One test formulation included adenovirus serotype 4 and the other test formulation included adenovirus serotype 5. For all tests, the total concentration of the CF-8C4 antibody stain (both bound and unbound to adenovirus) in the test solutions is about 2.5 micrograms per milliliter. For each test formulation four test solutions are prepared at different dilutions of stock adenovirus solution for each for the dilution series, to provide four different virus concentrations for testing in buffered solution for each test formulation.

(28) Results are summarized in FIG. 13, which shows a plot of adenovirus concentration for each dilution of the dilution series for each of the different serotypes determined by flow cytometry as a function of dilution factor. Both the vertical and horizontal axes are log-scale. As seen in FIG. 13, the concentrations for each dilution series fall almost on a line indicating that the CF-8C4 binds with both serotypes, although data tend to diverge at high dilution factors where concentrations are at or below the detection limit of the flow cytometer (about 10.sup.5.5 particles per milliliter).

Example 4

(29) Comparative flow cytometry tests are performed on adenovirus test formulations using the same fluorescent antibody stain as used in Example 1 (CF-8C4) relative to controls of an influenza virus and lentivirus, which are not antigenic for the 8C4 antibody. The adenovirus used in the test formulations is serotype 4. For all tests, the total concentration of the CF-8C4 antibody stain (both bound and unbound to adenovirus) in the test solutions is about 2.5 micrograms per milliliter. Six test solutions are prepared at different dilutions of stock adenovirus solution for each for the dilution series, to provide six different virus concentrations for testing in buffered solution. For a specificity control, the fluorescent antibody stain was tested against unrelated viruses of influenza and lentivirus types.

(30) Results are summarized in FIG. 14, which shows a plot of adenovirus concentration for each dilution determined by flow cytometry as a function of dilution factor for the different test formulations. Both the vertical and horizontal axes are log-scale. As seen in FIG. 14, for adenovirus the plot of concentrations for the dilution series show good linearity. FIG. 14 also summarizes data for the control formulations relative to an approximate lower detection limit for the flow cytometer (about 10.sup.5.5 particles per milliliter), indicating that the fluorescent antibody stain failed to bind to the influenza virus and lentivirus controls, confirming the specificity of the fluorescent antibody stain.

Example Implementation Combinations

(31) Some example implementation combinations, and for various types of implementation applications, which may be the subject of claims with or without additional features as disclosed above, are summarized as follows:

(32) 1. A method for evaluating a biological material sample for unassociated virus-size particles having a particular epitope of a viral type selected from the group consisting of an adenovirus viral type or an adeno-associated virus viral type, the method comprising:

(33) subjecting to flow cytometry a fluid sample comprising at least a portion of the biological material sample, wherein the fluid sample comprises a fluorescent antibody stain capable of binding, directly or indirectly, with the particular epitope, the flow cytometry comprising: flowing the fluid sample through a flow cell of a flow cytometer; subjecting the fluid sample flowing through the flow cell to excitation radiation capable of causing a fluorescent emission response from the fluorescent antibody stain; and detecting radiation from the flow cell within a wavelength range of the fluorescent emission and evaluating the detected radiation to identify detection events indicative of passage through the flow cell of unassociated labeled particles of virus size including a said virus-size particle having the epitope and the fluorescent antibody stain.

(34) 2. A method according to example implementation combination 1, wherein the biological material sample is suspected of including virus-like particles.

(35) 3. A method according to example implementation combination 2, wherein the virus-size particles are the virus-like particles.

(36) 4. A method according to example implementation combination 3, wherein the biological material comprises virus particles of a different viral type than the virus-like particles.

(37) 5. A method according to example implementation combination 4, wherein:

(38) the fluorescent antibody stain is a first fluorescent antibody stain, the epitope is a first epitope, the unassociated labeled particles are first unassociated labeled particles, the fluorescent emission is a first fluorescent emission, the detecting is first detecting, the detection event is a first detection event and the virus particles have a second epitope that is different than the first epitope;

(39) the fluid sample includes a second fluorescent antibody stain different than the first fluorescent antibody stain and capable of binding, directly or indirectly, with the second epitope, the second fluorescent antibody stain having a second fluorescent emission response to the excitation radiation that is different than the first fluorescent emission response; and

(40) the flow cytometry comprises second detecting for second radiation from the flow cell within a wavelength range of the second fluorescent emission and evaluating the detected second radiation for detection events indicative of passage through the flow cell of second unassociated labeled particles of virus size including a said virus particle and the second fluorescent antibody stain.

(41) 6. A method according to either one of example implementation combination 4 or implementation combination 5, wherein the virus particles are of a baculovirus viral type.

(42) 7. A method according to any one of example implementation combinations 4-6, wherein the epitope is a first epitope, the flow cytometry is first flow cytometry, the fluid sample is a first fluid sample including a first portion of the of the biological material sample, the unassociated labeled particles are first unassociated labeled particles and the excitation radiation is first excitation radiation, and the method comprises:

(43) subjecting to flow cytometry a second fluid sample comprising a second portion of the biological material sample, wherein the fluid sample comprises a second fluorescent antibody stain that is different than the first antibody stain and is capable of binding, directly or indirectly, with a second epitope that is different than the first epitope, the second flow cytometry comprising: flowing the second fluid sample through a flow cell of a flow cytometer; subjecting the second fluid sample flowing through the flow cell to second excitation radiation, which is the same as or different than the first excitation radiation, capable of causing a second fluorescent emission response that is different than the first fluorescent emission response from the second fluorescent antibody stain; and detecting radiation from the flow cell within a wavelength range of the fluorescent emission and evaluating the detected radiation to identify detection events indicative of passage through the flow cell of second unassociated labeled particles of virus size including a particle of virus size having the second epitope and the second fluorescent antibody stain.

(44) 8. A method according to example implementation combination 7, wherein:

(45) the first and second fluid samples each includes at least two different fluorescent antibody stains for binding, directly or indirectly, with two different epitopes; and

(46) the first and second fluid samples each includes at least one fluorescent antibody stain that is not in the other said fluid sample.

(47) 9. A method according to example implementation combination 8, wherein the at least two fluorescent antibody stains of each said fluid sample are targeted to detecting at least two different types of particles of virus size in the said fluid sample and at least one said type of particle is different between the first sample fluid and the second sample fluid.

(48) 10. A method according to any one of example implementation combinations 4-9, wherein the biological material sample is a sample collected from a stage of a production operation for producing the virus-like particles during which the virus-like particles are expected to be present in the biological material.

(49) 11. A method for manufacturing a product comprising virus-like particles, the method comprising:

(50) production processing to prepare a purified product including the virus-like particles, the production processing including: generating the virus-like particles in a biological production operation; harvesting from the biological production operation crude product comprising the virus-like particles generated during the generating; purifying at least a portion of material of the crude product to prepare a purified product including the virus-like particles;

(51) collecting a biological material sample from a stage of the production processing during which virus-like particles would be expected to be present in the biological material;

(52) evaluating the sample for presence of particles of virus size having a particular epitope, the evaluating comprising performing the method according to any one of example implementation combinations 4-10.

(53) 12. A method according to example implementation combination 11, comprising performing the collecting multiple times to collect different said biological material samples to be evaluated from multiple different said stages and performing a said evaluating on each said collected biological material sample to be evaluated.

(54) 13. A method according to example implementation combination 12, wherein the multiple different said stages include at least a first stage during the generating or the harvesting and a second stage during or after the purifying.

(55) 14. A method according to example implementation combination 1, wherein the virus-size particles are virus particles of adenovirus or adeno-associated virus.

(56) 15. A method according to example implementation combination 14, wherein:

(57) the fluorescent antibody stain is a first fluorescent stain, the fluorescent emission is a first fluorescent emission, the detecting is first detecting and the detection event is a first detection event;

(58) the fluid sample includes a second fluorescent stain for nucleic acid, the second fluorescent stain having a second fluorescent emission response, different than the first fluorescent emission response, to the excitation radiation when attached to nucleic acid; and

(59) the flow cytometry comprises second detecting for second radiation from the flow cell within a wavelength range of the second fluorescent emission and evaluating the detected second radiation for detection events indicative of passage of particles of virus size containing nucleic acid stained with the second fluorescent stain; and

(60) comparing results for the first detecting and the second detecting for identification of occurrences of a said first detection event that temporally coincides with a said second detection event that is further indicative of passage through the flow cell of a said unassociated labeled particle including a said virus particle.

(61) 16. A method according to example implementation combination 15, wherein:

(62) the fluorescent antibody stain is a first fluorescent antibody stain, the viral type is a first viral type, the epitope is a first epitope, the fluorescent emission is a first fluorescent emission, the detecting is first detecting and the detection event is a first detection event;

(63) the fluid sample includes a second fluorescent antibody stain different than the first fluorescent antibody stain and capable of binding, directly or indirectly, with a second epitope different than the first epitope, the second fluorescent antibody stain having a second fluorescent emission response to the excitation radiation that is different than the first fluorescent emission response; and

(64) the flow cytometry comprises second detecting for second radiation from the flow cell within a wavelength range of the second fluorescent emission and evaluating the detected second radiation for detection events indicative of passage through the flow cell of unassociated labeled particles of virus size including a particle of virus size with the second epitope and the second fluorescent antibody stain.

(65) 17. A method according to example implementation combination 16, wherein the first and the second epitope is indicative of a second viral type other than the first viral type.

(66) 18. A method according to example implementation combination 17, wherein the first viral type is an adeno-associated virus viral type and the second viral type is an adenovirus viral type.

(67) 19. A method according to example implementation combination 17, wherein the first viral type is the adenovirus viral type and the second viral type is an adeno-associated virus viral type.

(68) 20. A method according to example implementation combination 17, wherein the first epitope is indicative of a first adeno-associated virus serotype and the second epitope is indicative of a second, different adeno-associated virus serotype.

(69) 21. A method according to example implementation combination 17, wherein the first epitope is indicative of a first adenovirus serotype and the second epitope is indicative of a second, different adenovirus serotype.

(70) 22. A method according to example implementation combination 14, wherein the epitope is a first epitope, the flow cytometry is first flow cytometry, the fluid sample is a first fluid sample including a first portion of the of the biological material sample, the unassociated labeled particles are first unassociated labeled particles and the excitation radiation is first excitation radiation, and the method comprises:

(71) subjecting to flow cytometry a second fluid sample comprising a second portion of the biological material sample, wherein the fluid sample comprises a second fluorescent antibody stain that is different than the first antibody stain and is capable of binding, directly or indirectly, with a second epitope that is different than the first epitope, the second flow cytometry comprising: flowing the second fluid sample through a flow cell of a flow cytometer; subjecting the second fluid sample flowing through the flow cell to second excitation radiation, which is the same as or different than the first excitation radiation, capable of causing a second fluorescent emission response that is different than the first fluorescent emission response from the second fluorescent antibody stain; and detecting radiation from the flow cell within a wavelength range of the fluorescent emission and evaluating the detected radiation to identify detection events indicative of passage through the flow cell of second unassociated labeled particles of virus size including a particle of virus size with the second epitope and the second fluorescent antibody stain.

(72) 23. A method according to example implementation combination 22, wherein:

(73) the first and second fluid samples each includes at least two different fluorescent antibody stains for binding, directly or indirectly, with two different epitopes; and

(74) the first and second fluid samples each includes at least one fluorescent antibody stain that is not in the other said fluid sample.

(75) 24. A method according to example implementation combination 23, wherein the at least two fluorescent antibody stains of each said fluid sample are targeted to detecting at least two different types of particles of virus size in the said fluid sample and at least one said type of particle is different between the first sample fluid and the second sample fluid.

(76) 25. A method according to example implementation combination 24, wherein at least one of the fluorescent antibody stains in the first fluid sample is targeted to detecting an epitope of a first viral type of the adenovirus viral type or the adeno-associated virus viral type and at least one of the fluorescent antibody stains in the second fluid sample is targeted to detecting an epitope of a second viral type that is different than the first viral type.

(77) 26. A method according to example implementation combination 24, wherein either:

(78) at least one of the fluorescent antibody stains in the first fluid sample is targeted to detecting an epitope of an adeno-associated virus serotype and at least one of the fluorescent antibody stains in the second fluid sample is targeted to detecting a different adeno-associated virus serotype; or

(79) at least one of the fluorescent antibody stains in the first fluid sample is targeted to detecting an epitope of an adenovirus serotype and at least one of the fluorescent antibody stains in the second fluid sample is targeted to detecting a different adenovirus serotype.

(80) 27. A method according to any one of example implementation combinations 14-26, wherein the biological material sample is a sample collected from a stage of a production operation for producing the virus particles during which the virus particles are expected to be present in the biological material.

(81) 28. A method for manufacturing a product comprising virus particles of adenovirus adeno-associated virus, the method comprising:

(82) production processing to prepare a purified product including the virus particles, the production processing including: generating the virus particles in a biological production operation; harvesting from the biological production operation crude product comprising the virus particles generated during the generating; purifying at least a portion of material of the crude product to prepare a purified product including the adeno-associated virus particles;

(83) collecting a biological material sample from a stage of the production processing during which the virus particles would be expected to be present in the biological material;

(84) evaluating the sample for presence of particles of virus size having a particular epitope, the evaluating comprising performing the method according to any one of example implementation combinations 14-28.

(85) 29. A method according to example implementation combination 28, comprising performing the collecting multiple times to collect different said biological material samples to be evaluated from multiple different said stages and performing a said evaluating on each said collected biological material sample to be evaluated.

(86) 30. A method according to example implementation combination 29, wherein the multiple different said stages include at least a first stage during the generating or the harvesting and a second stage during or after the purifying.

(87) 31. A method according to any one of example implementation combinations 28-29, wherein the adenovirus or adeno-associated virus of the virus particles has been modified to express recombinant protein.

(88) 32. A method according to any one of example implementation combinations 1-31, wherein the biological material in a said fluid sample is purified biological material with larger-size components removed to a filtration size of not larger than 2 microns, and the fluid sample comprises a concentration of fluorescent antibody stain not bound in the unassociated labeled particles in a range of from 0.25 microgram per milliliter to 10 micrograms per milliliter of the fluid sample.

(89) 33. A method according to any one of example implementation combinations 1-32, wherein a said fluid sample as fed to the flow cytometer includes a total concentration of the fluorescent antibody stain in a range of from 0.25 microgram per milliliter to 10 micrograms per milliliter.

(90) 34. A method according to any one of example implementation combinations 1-33, wherein the virus-size particles are of the adeno-associated virus viral type and the unassociated labeled particles have a maximum cross dimension in a range of from 10 nanometers to 100 nanometers.

(91) 35. A method according to any one of example implementation combinations 1-33, wherein the virus-size particles are of the adenovirus viral type and the unassociated labeled particles have a maximum cross dimension in a range of from 80 nanometers to 200 nanometers.

(92) 36. A method according to any one of example implementation combinations 1-35, wherein the excitation radiation includes radiation within a wavelength range of from 520 nanometers to 550 nanometers.

(93) 37. A method according to any one of example implementation combinations 1-36, wherein the fluorescent emission has a Stokes shift in wavelength of at least 10 nanometers.

(94) 38. A method according to any one of example implementation combinations 1-37, wherein a said fluid sample as fed to the flow cytometer comprises a concentration of the fluorescent antibody stain not bound in the unassociated labeled particles in a range of from 0.25 microgram per milliliter to 10 micrograms per milliliter.

(95) 39. A method according to any one of example implementation combinations 1-38, wherein a said fluid sample as fed to the flow cytometer has a concentration of the unassociated labeled particles in a range of from 110.sup.5 to 110.sup.9 particles per milliliter.

(96) 40. A method according to any one of example implementation combinations 1-39, wherein a said fluorescent antibody stain comprises a fluorophore attached to an antibody molecule.

(97) 41. A method according to example implementation combination 40, wherein a said fluorescent antibody stain comprises an average of from 3 to 8 of the fluorophores attached per antibody molecule of the fluorescent antibody stain.

(98) 42. A flow method according to either one of example implementation combination 40 or example implementation combination 41, wherein the antibody molecules are monoclonal.

(99) 43. A method according to either one of example implementation combination 40 or example implementation combination 41, wherein the antibody molecules are polyclonal.

(100) 44. A method according to any one of example implementation combinations 1-43, wherein the flow cytometry comprises flowing a said fluid sample through the flow cell at a fluid sample flow rate in a range of from 250 to 3000 nanoliters per minute.

(101) 45. A method according to any one of example implementation combinations 1-44, wherein the viral type is an adenovirus viral type.

(102) 46. A method according to example implementation combination 45, wherein a said fluorescent antibody stain comprises 8C4 monoclonal antibody.

(103) 47. A method according to any one of example implementation combinations 1-46, wherein the viral type is the adeno-associated virus viral type.

(104) 48. A method according to example combination 47, wherein a said fluorescent antibody stain includes one or more than one monoclonal antibody selected from the group consisting of anti-AAV-1 antibody (optionally ADK1a), antiAAV-2 antibody (optionally A20), anti-AAV-3 antibody (optionally A20), anti-AAV-4 antibody (optionally ADK4), anti-AAV-5 antibody (optionally ADK5a and/or ADK5b), anti-AAV-6 antibody (optionally ADK6), anti-AAV-8 antibody (optionally ADK8 and/or ADK8/9), anti-AAV-9 antibody (optionally ADK9 and/or ADK 8/9), and combinations thereof.

(105) 1A. A flow cytometry method for evaluating biological material for the presence of unassociated particles of a virus size having a particular epitope indicative of a virus type selected from the group consisting of an adenovirus viral type or an adeno-associated virus viral type, the method comprising:

(106) preparing a fluid sample, comprising mixing biological material to be evaluated for presence of the unassociated particles with a fluorescent antibody stain, the fluorescent antibody stain being capable of binding, directly or indirectly, with the unassociated particles through the epitope to form unassociated labeled particles of virus size;

(107) subjecting the fluid sample to flow cytometry, comprising: flowing the fluid sample through a flow cell of a flow cytometer under flow conditions for passage of virus-size particles individually through the flow cell; subjecting the fluid sample flowing through the flow cell to excitation radiation capable of causing a fluorescent emission response from the fluorescent antibody stain; and detecting radiation from the flow cell within a wavelength range of the fluorescent emission and evaluating the detected radiation to identify detection events indicative of passage of said unassociated labeled particles through the flow cell.

(108) 2A. A flow cytometry method according to example implementation combination 1A, wherein the preparing a fluid sample comprises, prior to the mixing, purifying a crude sample of biological material to prepare the biological material for the fluid sample as subjected to the flow cytometry, the purifying comprising filtering out particles of the crude sample at a filtration size of not larger than 2 microns and chromatographic removal of at least a portion of impurities smaller than virus size.

(109) 3A. A flow cytometry method according to example implementation combination 2A, wherein the chromatographic removal comprises spin chromatography in a centrifuge.

(110) 4A. A flow cytometry method according to any one of example implementation combinations 1A-3A, wherein the preparing a fluid sample comprises, after the mixing, not removing fluorescent antibody not bound in the unassociated labeled particles from the fluid sample prior to flow cytometry.

(111) 5A. A flow cytometry method according to any one of example implementation combination 1A-4A, wherein the fluorescent antibody stain comprises a biotinylated antibody and a fluorophore conjugated streptavidin.

(112) 6A. A flow cytometry method according to example implementation combination 5A, wherein the fluorophore conjugated streptavidin is attached to the biotinylated antibody when provided to the mixing.

(113) 7A. A flow cytometry method according to example implementation combination 6A, wherein the fluorophore conjugated streptavidin and the biotinylated antibody are provided in separate formulations to the mixing and the fluorophore conjugated streptavidin attaches to the biotinylated antibody in solution during the mixing.

(114) 8A. A flow cytometry method for evaluating biological material for the presence of unassociated particles of a virus size having a particular epitope indicative of a viral type selected from the group consisting of an adenovirus viral type and an adeno-associated virus viral type, the method comprising:

(115) subjecting a fluid sample comprising biological material to be evaluated and a fluorescent antibody stain to flow cytometry, fluorescent antibody stain being capable of binding, directly or indirectly, with the unassociated particles through the epitope to form unassociated labeled particles of virus size, the flow cytometry comprising: flowing the fluid sample through a flow cell of a flow cytometer under flow conditions for passage of virus-size particles individually through the flow cell; subjecting the fluid sample flowing through the flow cell to excitation radiation capable of causing a fluorescent emission response from the fluorescent antibody stain; and

(116) detecting radiation from the flow cell within a wavelength range of a fluorescent emission of the fluorescent antibody stain and evaluating the detected radiation to identify detection events indicative of passage of said unassociated labeled particles through the flow cell;

(117) wherein the biological material in the fluid sample is purified biological material with larger-size components removed to a filtration size of not larger than 2 microns, and the fluid sample comprises a concentration of fluorescent antibody stain not bound in the unassociated labeled particles in a range of from 0.25 microgram per milliliter to 10 micrograms per milliliter of the fluid sample.

(118) 9A. A method according to any one of example implementation combinations 1A-8A, wherein the flow cytometry is in the absence of detecting for light scatter.

(119) 10A. A flow cytometry method according to any one of example implementation combinations 1A-9A, wherein the fluid sample as fed to the flow cytometer includes a total concentration of the fluorescent antibody stain in a range of from 0.25 microgram per milliliter to 10 micrograms per milliliter.

(120) 11A. A flow cytometry method according to any one of example implementation combinations 1A-10A, wherein the fluid sample as fed to the flow cytometer comprises a concentration of the fluorescent antibody stain not bound in the unassociated labeled particles in a range of from 0.25 microgram per milliliter to 10 micrograms per milliliter.

(121) 12A. A flow cytometry method according to any one of example implementation combinations 1A-11A, wherein the fluid sample as fed to the flow cytometer has a concentration of the unassociated labeled particles in a range of from 110.sup.5 to 110.sup.9 particles per milliliter.

(122) 13A. A flow cytometry method according to any one of example implementation combinations 1A-12A, wherein the excitation radiation includes radiation within a wavelength range of from 520 nanometers to 550 nanometers.

(123) 14A. A flow cytometry method according to any one of example implementation combinations 1A-13A, wherein the fluorescent emission has a Stokes shift in wavelength of at least 10 nanometers.

(124) 15A. A flow cytometry method according to any one of example implementation combinations 1A-14A, wherein the fluorescent antibody stain comprises a fluorophore attached to an antibody molecule.

(125) 16A. A flow cytometry method according to example implementation combination 15A, wherein the fluorescent antibody stain comprises an average of from 3 to 8 of the fluorophores attached per antibody molecule of the fluorescent antibody stain.

(126) 17A. A flow cytometry method according to either one of example implementation combination 15A or example implementation combination 16A, wherein the antibody molecules are monoclonal.

(127) 18A. A flow cytometry method according to either one of example implementation combination 15A or example implementation combination 16A, wherein the antibody molecules are polyclonal.

(128) 19A. A flow cytometry method according any one of example implementation combinations 1A-18A, wherein the fluorescent antibody stain is a first fluorescent stain and the fluid sample comprises a second fluorescent stain having a second fluorescent emission response, different than the first fluorescent emission response of the first fluorescent stain, caused by the excitation radiation.

(129) 20A. A flow cytometry method according to example implementation combination 19A, wherein the second fluorescent stain comprises a fluorescent nucleic acid stain not specific to particle type.

(130) 21A. A flow cytometry method according to example implementation combination 19A, wherein:

(131) the unassociated particles are first unassociated particles, the epitope is a first epitope, the unassociated particles are of a first particle type and the unassociated labeled particles are first unassociated labeled particles;

(132) the second fluorescent stain is a second fluorescent antibody stain that is different than the first fluorescent antibody stain and is capable of binding with second unassociated particles of virus size having a second epitope indicative of a second particle type that is different than the first particle type to form second unassociated labeled particles of virus size; and

(133) the method comprises detecting radiation from the flow cell within a wavelength range of the second fluorescent emission and evaluating the detected radiation to identify detection events indicative of passage of the second unassociated labeled particles through the flow cell.

(134) 22A. A flow cytometry method according to any one of example implementation combinations 19A-21A, wherein the second fluorescent emission has a peak wavelength at least 20 nanometers different than a peak wavelength of the first fluorescent emission nanometers.

(135) 23A. A flow cytometry method according to any one of example implementation combinations 1A-22A, wherein each said fluorescent antibody stain is capable of binding directly with a corresponding said unassociated particle to form the corresponding said unassociated labeled particle.

(136) 24A. A flow cytometry method according to any one of example implementation combinations 1A-23A, wherein the unassociated labeled particles have a maximum cross dimension in a range of from 10 nanometers to 200 nanometers.

(137) 25A. A flow cytometry method according to any one of example implementation combinations 1A-24A, wherein the flow cytometry comprises flowing the fluid sample through the flow cell at a fluid sample flow rate in a range of from 250 to 3000 nanoliters per minute.

(138) 26A. A flow cytometry method according to any one of example implementation combinations 1A-25A, wherein the flow cytometry comprises hydrodynamically focusing flow of the fluid sample with a sheath fluid and flowing the fluid sample and the sheath fluid through the flow cell.

(139) 27A. A flow cytometry method according to any one of example implementation combinations 1A-26A, wherein the flow cytometry comprises separately detecting for at least two different fluorescent emissions and not detecting for light scatter.

(140) 28A. A flow cytometry method according to any one of example implementation combinations 1A-27A, wherein the viral type is the adeno-associated virus viral type.

(141) 29A. A flow cytometry method according to any one of example implementation combinations 1A-27A, wherein the viral type is the adenovirus viral type.

(142) 30A. A flow cytometry method according to either one of example implementation combination 28A or example implementation combination 29A, wherein the viral particles comprise a member selected from the group consisting of virus particles of the viral type, virus-like particles of the viral type and combinations thereof.

(143) 31A. A flow cytometry method according to any one of example implementation combinations 1A-30A, wherein the viral type is the adeno-associated virus viral type and the unassociated labeled particles have a maximum cross-dimension in a range of from 10 nanometers to 100 nanometers.

(144) 32A. A flow cytometry method according to any one of example implementation combinations 1A-30A, wherein the viral type is the adenovirus viral type and the unassociated labeled particles have a maximum cross-dimension in a range of from 80 nanometers to 200 nanometers.

(145) 33A. A fluid formulation comprising;

(146) an aqueous liquid medium;

(147) unassociated labeled particles of virus size in the aqueous liquid medium at a concentration of from 110.sup.5 to 110.sup.9 of the unassociated labeled particles per milliliter, wherein the unassociated labeled particles each includes a virus-size particle having a particular epitope indicative of a viral type selected from the group consisting of an adenovirus viral type and an adeno-associated virus viral type and a fluorescent antibody stain specific for binding, directly or indirectly with the epitope; and

(148) a concentration of the unbound fluorescent antibody stain in the aqueous liquid medium, not bound in the unassociated labeled particles, is in a range of from 0.25 microgram per milliliter to 10 micrograms per milliliter.

(149) 34A. A fluid formulation according to example implementation combination 33A, wherein the unassociated labeled particles are unassociated labeled particles of any one of example implementation combinations 1-48 and 1A-33A.

(150) 1B. A flow cytometry method for evaluating a biological material sample for unassociated virus-size particles of a viral type selected from the group consisting of an adenovirus viral type and an adeno-associated virus viral type and having a particular epitope indicative of the viral type, the method comprising:

(151) subjecting to flow cytometry a fluid sample comprising at least a portion of the biological material sample, wherein the fluid sample comprises a fluorescent antibody stain capable of binding, directly or indirectly, with the particular epitope, the flow cytometry comprising: flowing the fluid sample through a flow cell of a flow cytometer; subjecting the fluid sample flowing through the flow cell to excitation radiation capable of causing a fluorescent emission response from the fluorescent antibody stain; and detecting radiation from the flow cell within a wavelength range of the fluorescent emission and evaluating the detected radiation to identify detection events indicative of passage through the flow cell of the unassociated labeled particles of virus size including a said virus-size particle of the viral type having the epitope and the fluorescent antibody stain.

(152) 2B. A method according to example implementation combination 1B, wherein the fluid sample as fed to the flow cytometer comprises a concentration of the fluorescent antibody stain not bound in the unassociated labeled particles in a range of from 0.25 micrograms per milliliter to 10 micrograms per milliliter.

(153) 3B. A flow cytometry method according to either one of example implementation combination 1B or 2B, wherein the fluid sample as fed to the flow cytometer includes a total concentration of the fluorescent antibody stain in a range of from 0.25 microgram per milliliter to 10 micrograms per milliliter.

(154) 4B. A flow cytometry method according to any one of example implementation combinations 1B-3B, wherein the epitope is a conformational epitope of assembled capsid of an adeno-associated virus serotype and the fluorescent antibody stain comprises a monoclonal antibody for the conformational epitope.

(155) 5B. A flow cytometry method according to any one of example implementation combinations 1B-4B, wherein the virus-size particles comprise adeno-associated virus particles.

(156) 6B. A flow cytometry method according to example implementation combination 5B, wherein the adeno-associated virus particles express recombinant protein.

(157) 7B. A flow cytometry method according to any one of example implementation combinations 1B-3B, wherein the epitope is an adenovirus hexon protein.

(158) 8B A flow cytometry method according to example implementation combination 7B, wherein the fluorescent antibody stain comprises 8C4 monoclonal antibody.

(159) 9B. A flow cytometry method according to any one of example implementation combinations 1B-4B, 7B and 8B, wherein the virus-size particles comprise adenovirus particles.

(160) 10B. A flow cytometry method according to example implementation combination 9B, wherein the adenovirus particles express recombinant protein.

(161) 11B. A flow cytometry method according to any one of example implementation combinations 1B-10B, wherein:

(162) the fluorescent antibody stain is a first fluorescent stain, the fluorescent emission is a first fluorescent emission, the detecting is first detecting and the detection event is a first detection event;

(163) the fluid sample includes a second fluorescent stain for nucleic acid, the second fluorescent stain having a second fluorescent emission response, different than the first fluorescent emission response, to the excitation radiation when attached to nucleic acid; and

(164) the flow cytometry comprises second detecting for second radiation from the flow cell within a wavelength range of the second fluorescent emission and evaluating the detected second radiation for detection events indicative of passage of particles of virus size containing nucleic acid stained with the second fluorescent stain; and

(165) comparing results for the first detecting and the second detecting for identification of occurrences of a said first detection event that temporally coincides with a said second detection event that is further indicative of passage through the flow cell of a said unassociated labeled particle including a said virus-size particle of the viral type.

(166) 12B. A flow cytometry method according to any one of example implementation combinations 1B-10B, wherein:

(167) the fluorescent antibody stain is a first fluorescent antibody stain, the epitope is a first epitope, the fluorescent emission is a first fluorescent emission, the detecting is first detecting, the viral type is a first viral type, the unassociated labeled particles are first unassociated labeled particles of virus size and the detection event is a first detection event;

(168) the fluid sample includes a second fluorescent antibody stain different than the first fluorescent antibody stain and capable of binding, directly or indirectly, with a second epitope different than the first epitope, the second epitope being indicative of a second viral type that is other than the first viral type and the second fluorescent antibody stain having a second fluorescent emission response to the excitation radiation that is different than the first fluorescent emission response; and

(169) the flow cytometry comprises second detecting for second radiation from the flow cell within a wavelength range of the second fluorescent emission and evaluating the detected second radiation for detection events indicative of passage through the flow cell of the second unassociated labeled particles of virus size including a particle of virus size with the second epitope and the second fluorescent antibody stain.

(170) 13B. A flow cytometry method according to example implementation combination 12B, wherein the particles of virus size with the first epitope comprise virus-like particles.

(171) 14B. A flow cytometry method according to example implementation combination 13B, wherein the second viral type is a baculovirus viral type.

(172) 15B. A flow cytometry method according to example implementation combination 12B, wherein the first viral type is an adeno-associated virus serotype and the second viral type is a different adeno-associated virus serotype.

(173) 16B. A flow cytometry method according to example implementation combination 12B, wherein the first viral type is the adeno-associated virus viral type and the second viral type is an adenovirus viral type.

(174) 17B. A flow cytometry method according to example implementation combination 12B, wherein the first viral type is the adenovirus viral type and the second viral type is an adeno-associated virus viral type.

(175) 18B. A flow cytometry method according to example implementation combination 12B, wherein the first viral type is an adenovirus serotype and the second viral type is a different adenovirus serotype.

(176) 19B. A flow cytometry method according to any one of example implementation combinations 1B-10B, wherein the epitope is a first epitope, the viral type is a first viral type, the flow cytometry is first flow cytometry, the fluid sample is a first fluid sample including a first portion of the of the biological material sample, the unassociated labeled particles are first unassociated labeled particles and the excitation radiation is first excitation radiation, and the method comprises:

(177) subjecting to flow cytometry a second fluid sample comprising a second portion of the biological material sample, wherein the fluid sample comprises a second fluorescent antibody stain that is different than the first fluorescent antibody stain and is capable of binding, directly or indirectly, with a second epitope that is different than the first epitope, the second epitope being indicative of a second viral type other than the first viral type and the second flow cytometry comprising: flowing the second fluid sample through a flow cell of a flow cytometer; subjecting the second fluid sample flowing through the flow cell to second excitation radiation, which is the same as or different than the first excitation radiation, capable of causing a second fluorescent emission response that is different than the first fluorescent emission response from the second fluorescent antibody stain; and detecting radiation from the flow cell within a wavelength range of the fluorescent emission and evaluating the detected radiation to identify detection events indicative of passage through the flow cell of second unassociated labeled particles of virus size including a particle of virus size with the second epitope and the second fluorescent antibody stain.

(178) 20B. A flow cytometry method according to example implementation combination 19B, wherein:

(179) the first and second fluid samples each includes at least two different fluorescent antibody stains for binding, directly or indirectly, with two different epitopes; and

(180) the first and second fluid samples each includes at least one fluorescent antibody stain that is not in the other said fluid sample.

(181) 21B. A flow cytometry method according to example implementation combination 20B, wherein the at least two fluorescent antibody stains of each said fluid sample are targeted to detecting at least two different types of particles of virus size in the said fluid sample and at least one said type of particle is different between the first sample fluid and the second sample fluid.

(182) 22B. A flow cytometry method according to any one of example implementation combinations 1B-21B, wherein the biological material sample is a sample collected from a stage of a production operation for producing virus particles during which the virus-size particles of viral type are expected to be present in the biological material.

(183) 23B. A flow cytometry method for manufacturing a product comprising virus-size particles of a viral type selected from the group consisting of an adenovirus viral type and adeno-associated virus viral type, the method comprising:

(184) production processing to prepare a purified product including the virus-size particles of the viral type, the production processing including: generating the virus-size particles in a biological production operation; harvesting from the biological production operation crude product comprising the virus-size particles generated during the generating; purifying at least a portion of material of the crude product to prepare a purified product including the virus-size particles;

(185) collecting a biological material sample from a stage of the production processing during which the virus-size particles would be expected to be present in the biological material;

(186) evaluating the sample for presence of particles of virus size having a particular epitope indicative of the viral type, the evaluating comprising performing the method according to any one of example implementation combinations 1B-21B.

(187) 24B. A flow cytometry method according to example implementation combination 23B, comprising performing the collecting multiple times to collect different said biological material samples to be evaluated from multiple different said stages and performing a said evaluating on each said collected biological material sample to be evaluated.

(188) 25B. A flow cytometry method according to example implementation combination 24B, wherein the multiple different said stages include at least a first stage during the generating or the harvesting and a second stage during or after the purifying.

(189) 26B. A flow cytometry method according to any one of example implementation combinations 1B-25B, wherein the biological material in the fluid sample is purified biological material with larger-size components removed to a filtration size of not larger than 2 microns.

(190) 27B. A flow cytometry method according to any one of example implementation combinations 1B-26B, wherein a said fluid sample as fed to the flow cytometer has a concentration of the unassociated labeled particles in a range of from 110.sup.5 to 110.sup.9 particles per milliliter.

(191) 28B. A flow cytometry method according to any one of example implementation combinations 1B-27B, wherein a said fluorescent antibody stain comprises fluorophore attached to antibody molecules and the fluorescent antibody stain in the fluid sample comprises an average of from 3 to 8 of the fluorophores attached per said antibody molecule of the fluorescent antibody stain.

(192) 29B. A flow cytometry method according to any one of example implementation combinations 1B-28B, wherein antibody of the fluorescent antibody stain is monoclonal.

(193) 30B. A flow cytometry method according to any one of example implementation combinations 1B-29B, wherein the flow cytometry comprises flowing a said fluid sample through the flow cell at a fluid sample flow rate in a range of from 250 to 3000 nanoliters per minute.

(194) 31B. A flow cytometry method according to any one of example implementation combinations 1B-30B comprising:

(195) preparing the fluid sample, comprising mixing biological material to be evaluated for presence of the unassociated virus-size particles with the fluorescent antibody stain.

(196) 32B. A flow method according to example implementation combination 31B, wherein the preparing the fluid sample comprises, prior to the mixing, purifying a crude sample of biological material to prepare the biological material for the fluid sample as subjected to the flow cytometry, the purifying comprising filtering out particles of the crude sample at a filtration size of not larger than 2 microns and comprising spin chromatography in a centrifuge to remove at least a portion of impurities smaller than virus size.

(197) 33B. A flow cytometry method according to either one of example implementation combination 31B or example implementation combination 32B, wherein the preparing a fluid sample comprises, after the mixing, not removing fluorescent antibody stain not bound in the unassociated labeled particles from the fluid sample prior to the flow cytometry.

(198) 34B. A flow cytometry method according to any one of example implementation combinations 31B-33B, wherein the fluorescent antibody stain comprises a biotinylated antibody and a fluorophore conjugated streptavidin.

(199) 35B. A flow cytometry method according to example implementation combination 34B, wherein the fluorophore conjugated streptavidin is attached to the biotinylated antibody when provided to the mixing.

(200) 36B. A flow cytometry method according to example implementation combination 35B, wherein the fluorophore conjugated streptavidin and the biotinylated antibody are provided in separate formulations to the mixing and the fluorophore conjugated streptavidin attaches to the biotinylated antibody in solution during the mixing.

(201) 37B. A flow cytometry method according to any one of example implementation combinations 1B-36B, wherein the flow cytometry is in the absence of detecting for light scatter.

(202) 38B. A flow cytometry method according to any one of example implementation combinations 1B-37B, wherein the fluorescent antibody stain is a first fluorescent stain and the fluid sample comprises a second fluorescent stain having a second fluorescent emission response, different than the first fluorescent emission response of the first fluorescent stain, caused by the excitation radiation.

(203) 39B. A flow cytometry method according to any one of example implementation combinations 1B-38B wherein the viral type is an adeno-associated virus viral type and the epitope is a conformational epitope of assembled capsid of an adeno-associated virus serotype and the fluorescent antibody stain comprises a monoclonal antibody for the conformational epitope, and the method comprises any one or more of the following:

(204) (i) a said serotype is AAV-1 and a said monoclonal antibody is ADK1a;

(205) (ii) a said serotype is AAV-2 and a said monoclonal antibody is A20;

(206) (iii) a said serotype is AAV-3 and a said monoclonal antibody is A20;

(207) (iv) a said serotype is AAV-4 and a said monoclonal antibody is ADK4;

(208) (v) a said serotype is AAV-5 and a said monoclonal antibody is selected from the group consisting of ADK5a, ADK5b and combination thereof;

(209) (vi) a said serotype is AAV-6 and a said monoclonal antibody is ADK6;

(210) (vii) a said serotype is AAV-8 and a said monoclonal antibody is selected from the group consisting of ADK8, ADK8/9 and combinations thereof; and

(211) (viii) a said serotype is AAV-9 and a said monoclonal antibody is selected from the group consisting of ADK9, ADK8/9 and combinations thereof.

(212) 40B. A flow cytometry method according to any one of example implementation combinations 1B-38B wherein the viral type is an adenovirus viral type and the epitope is an adenovirus hexon protein and the fluorescent antibody stain comprises 8C4 monoclonal antibody.

(213) 41B. A fluid sample for feed to a flow cytometer for flow cytometry evaluation, comprising;

(214) an aqueous liquid medium;

(215) unassociated labeled particles of virus size in the aqueous liquid medium at a concentration of from 110.sup.5 to 110.sup.9 of the unassociated labeled particles per milliliter, wherein the unassociated labeled particles each includes; a virus-size particle of viral type selected from the group consisting of an adenovirus viral type and an adeno-associated virus viral type having a particular epitope indicative of the viral type; and a fluorescent antibody stain specific for binding, directly or indirectly with the epitope; and

(216) a concentration of the unbound fluorescent antibody stain in the aqueous liquid medium, not bound in the unassociated labeled particles, in a range of from 0.25 microgram per milliliter to 10 micrograms per milliliter.

(217) 42B. A fluid sample according to example implementation combination 41B, wherein the fluid sample includes a total concentration of the fluorescent antibody stain in a range of from 0.25 microgram per milliliter to 10 micrograms per milliliter.

(218) 43B. A fluid sample according to either one of example implementation combination 41B or example implementation combination 42B, wherein the biological material in the fluid sample is purified biological material with larger-size components removed to a filtration size of not larger than 2 microns.

(219) 44B. A fluid sample according to any one of example implementation combinations 41B-43B, wherein the unassociated labeled particles have a maximum cross dimension of not larger than 100 nanometers.

(220) 45B. A fluid sample according to any one of example implementation combinations 41B-44B, wherein the fluorescent antibody stain comprises fluorophore attached to antibody molecules and the fluorescent antibody stain in the fluid sample comprises an average of 3 to 8 of the fluorophores attached per antibody molecule of the fluorescent antibody stain.

(221) 45B. A fluid sample according to any one of example implementation combinations 41B-45B, wherein antibody of the antibody stain is monoclonal.

(222) 46B. A fluid sample according to any one of example implementation combinations 41B-45B, wherein the virus-size particles comprise adeno-associated virus modified to express recombinant protein.

(223) 47B. A fluid sample according to any one of example implementation combinations 41B-46B, wherein the virus-size particles comprise adenovirus modified to express recombinant protein.

(224) 48B. A fluid sample according to any one of example implementation combinations 41B-45B, wherein the viral type is an adeno-associated virus viral type and the epitope is a conformational epitope of assembled capsid.

(225) 49B. A fluid sample according to any one of example implementation combinations 41B-45B, wherein the viral type is an adenovirus viral type and the epitope is a hexon protein.

(226) 50B. A fluid sample according to any one of example implementation combinations 41B-49B, wherein the fluorescent antibody stain comprises a biotinylated antibody and a fluorophore conjugated streptavidin.

(227) 51B. A fluid sample according to any one of example implementation combinations 41B-50B, wherein:

(228) the fluorescent antibody stain is a first fluorescent stain having a first fluorescent emission; and

(229) the fluid sample includes a second fluorescent stain for nucleic acid, the second fluorescent stain having a second fluorescent emission, different than the first fluorescent emission.

(230) 52B. A fluid sample according to any one of example implementation combinations 41B-50B, wherein:

(231) the fluorescent antibody stain is a first fluorescent antibody stain, the epitope is a first epitope, the fluorescent emission is a first fluorescent emission, the viral type is a first viral type and the unassociated labeled particles are first unassociated labeled particles of virus size;

(232) the fluid sample includes a second fluorescent antibody stain different than the first fluorescent antibody stain and capable of binding, directly or indirectly, with a second epitope different than the first epitope, the second epitope being indicative of a second viral type that is other than the first viral type and the second fluorescent antibody stain having a second fluorescent emission response that is different than the first fluorescent emission response.

(233) 53B. A fluid sample according to example implementation combination 52B, wherein the particle of virus size with the first epitope is a virus-like particle.

(234) 54B. A fluid sample according to example implementation combination 52B, wherein the particle of virus size with the second epitope is a virus-like particle.

(235) 55B. A fluid sample according to any one of example implementation combinations 52B-54B, wherein the first viral type is an adenovirus viral type and the second viral type is an adeno-associated virus viral type.

(236) 56B. A fluid sample according to any one of example implementation combinations 52B-54B, wherein the first viral type is an adeno-associated viral type and the second viral type is an adenovirus viral type.

(237) 57B. A fluid sample according to any one of example implementation combinations 52B-54B, wherein the first epitope is indicative of a first adeno-associated virus serotype and the second epitope is indicative of a second, different adeno-associated virus serotype.

(238) 58B. A fluid sample according to any one of example implementation combinations 52B-54B, wherein the first epitope is indicative of a first adenovirus serotype and the second epitope is indicative of a second, different adenovirus serotype.

(239) 59B. A fluid sample according to any one of example implementation combinations 41B-58B, wherein the unassociated labeled particles are unassociated labeled particles of any one of example implementation combinations 1-48, 1A-32A and 1B-40B.

(240) 60B. A method or fluid sample according to any one of example implementation combinations 1-48, 1A-34A and 1B-59B, wherein the viral type is the adenovirus viral type.

(241) 61B. A method or fluid sample according to any one of example implementation combinations 1-48, 1A-34A and 1B-59B, wherein the viral type is an adeno-associated virus viral type.

(242) 1C. A method according to any one of example implementation combinations 1-48, 1A-32A, 1B-40B and 60B-61B, wherein the flow cytometry comprises:

(243) counting the detection events as occurrences of individual ones of the unassociated labeled particles passing through the flow cell to determine a count for unassociated labeled particles in a volume of the fluid sample passing through the flow cell;

(244) determining the volume of fluid sample passing through the flow cell that corresponds with the count; and

(245) determining a concentration of the unassociated labeled particles in the volume of the fluid sample passing through the flow cell using the count of unassociated labeled particles.

(246) 2C. A method according to example implementation combination 1C, wherein the flow cytometry comprises:

(247) performing the counting of a said unassociated labeled particle in real time relative to the detecting of the radiation corresponding with the detection event; and

(248) determining the concentration in real time relative to passage of the volume of the fluid sample through the flow cell.

(249) 3C. A method according to either one of example implementation combination 1C or example implementation combination 2C, wherein the determining the volume of fluid sample passing through the flow cell comprises measuring in real time with a flow sensor the flow rate of the fluid sample to the flow cell and integrating resulting measured flow rate data over time.

(250) 4C. A method according to any one of example implementation combinations 1C-3C, wherein the identification of detection events indicative of passage through the flow cell of the unassociated labeled particles is determined in the absence of correlating with light scatter detection information.

(251) The foregoing discussion of the invention and different aspects thereof has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to only the form or forms specifically disclosed herein. Consequently, variations and modifications commensurate with the above teachings, and the skill or knowledge of the relevant art, are within the scope of the present invention. The embodiments described hereinabove are further intended to explain best modes known for practicing the invention and to enable others skilled in the art to utilize the invention in such, or other, embodiments and with various modifications required by the particular applications or uses of the present invention. It is intended that the appended claims be construed to include alternative embodiments to the extent permitted by the prior art. Although the description of the invention has included description of one or more possible embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter. Furthermore, any feature described or claimed with respect to any disclosed variation may be combined in any combination with one or more of any other features of any other variation or variations, to the extent that the features are not necessarily technically compatible, and all such combinations are within the scope of the present invention. The description of a feature or features in a particular combination do not exclude the inclusion of an additional feature or features. Processing steps and sequencing are for illustration only, and such illustrations do not exclude inclusion of other steps or other sequencing of steps. Additional steps may be included between illustrated processing steps or before or after any illustrated processing step.

(252) The terms comprising, containing, including and having, and grammatical variations of those terms, are intended to be inclusive and nonlimiting in that the use of such terms indicates the presence of some condition or feature, but not to the exclusion of the presence also of any other condition or feature. The use of the terms comprising, containing, including and having, and grammatical variations of those terms in referring to the presence of one or more components, subcomponents or materials, also include and is intended to disclose the more specific embodiments in which the term comprising, containing, including or having (or the variation of such term) as the case may be, is replaced by any of the narrower terms consisting essentially of or consisting of or consisting of only (or the appropriate grammatical variation of such narrower terms). For example, a statement that something comprises a stated element or elements is also intended to include and disclose the more specific narrower embodiments of the thing consisting essentially of the stated element or elements, and the thing consisting of the stated element or elements. Examples of various features have been provided for purposes of illustration, and the terms example, for example and the like indicate illustrative examples that are not limiting and are not to be construed or interpreted as limiting a feature or features to any particular example. The term at least followed by a number (e.g., at least one) means that number or more than that number. The term at at least a portion means all or a portion that is less than all. The term at least a part means all or a part that is less than all.

(253) A portion or a material, e.g., of a biological material sample, refers to some component or components of such material and includes both equal composition aliquots of a material and processed portions of the material that no longer have the same composition but that have one or more components from the material and which may be mixed with other components (e.g., buffer solution, reagents) not from the material. For example, a fluid sample including biological material that is to be subjected to flow cytometry includes a portion of a collected crude biological material sample even if the fluid sample includes only some purified biological components separated from the crude sample. As another example, if a batch of fluid sample is prepared including some biological material for evaluation is divided into separate aliquots for separate flow cytometry runs of the individual aliquots, each such aliquot includes both a portion of the biological material of the batch and includes a portion of the biological material that originated from a crude sample of biological material that may have been subjected to purification.