44. The volume of PBS for the cell resuspension can be adapted on
the applied FACS equipment, to provide a sufficient cell
concentration.
45. If the sample virus concentration is beyond the linear range of
the calibration curve, a further dilution of the virus sample
must be analyzed with the assay.
46. For the size determination using light scattering detection, a
sample concentration above 1E+07 particles/mL is recommended. Whether lower virus concentrations can be analyzed
by this method, depends on the sample composition, its purity
and most important the actual size of the nanoplex.
47. There are multiple ways to determine the size and shape of the
virus particles. Light scattering is only one possibility. Alternatives include transmission electron microscopy, tunable resistive
pulse sensing, disc centrifugation and others. Each method has
advantages and drawbacks, and should be applied according to
the product’s requirements.
Quantification of process contaminants
48. As for most types of assays, the protein standards must be
dissolved in a buffer resembling your sample buffer the most,
or the buffer differences must be considered during assay
validation.
49. Incubation can be done at room temperature, if a heating
device is not available. However, in this case, the incubation
time needs to be adjusted.
50. Directly after mixing in the sample with the protein assay
reagent, a color change is observed. However, the reaction
does not have a true endpoint. After 30 min of incubation at
37
C, the reaction is nearly completed, and the further color
development will be too slow to affect the measurement significantly. However, corresponding samples must be measured at
the same time point.
51. A new calibration curve should be prepared for each plate
measured. Although the assay is robust, deviations over time,
or differences in individual measurements, can be reduced by a
corresponding calibration curve on every plate.
52. Nonused standards can be stored at 4
C for one week.
53. If sample concentrations are beyond of the validated calibration
range (absorbance higher than the highest calibration), a dilution must be prepared and remeasured. An extrapolation of
values is not valid.
54. If samples contain varying amounts of salts or assay-interfering
components, the samples must be dialyzed to ensure a valid
analysis. However, the molecular weight cut off (MWCO) of
Viral Nanoplex Vaccine Production
243
the applied FACS equipment, to provide a sufficient cell
concentration.
45. If the sample virus concentration is beyond the linear range of
the calibration curve, a further dilution of the virus sample
must be analyzed with the assay.
46. For the size determination using light scattering detection, a
sample concentration above 1E+07 particles/mL is recommended. Whether lower virus concentrations can be analyzed
by this method, depends on the sample composition, its purity
and most important the actual size of the nanoplex.
47. There are multiple ways to determine the size and shape of the
virus particles. Light scattering is only one possibility. Alternatives include transmission electron microscopy, tunable resistive
pulse sensing, disc centrifugation and others. Each method has
advantages and drawbacks, and should be applied according to
the product’s requirements.
Quantification of process contaminants
48. As for most types of assays, the protein standards must be
dissolved in a buffer resembling your sample buffer the most,
or the buffer differences must be considered during assay
validation.
49. Incubation can be done at room temperature, if a heating
device is not available. However, in this case, the incubation
time needs to be adjusted.
50. Directly after mixing in the sample with the protein assay
reagent, a color change is observed. However, the reaction
does not have a true endpoint. After 30 min of incubation at
37
C, the reaction is nearly completed, and the further color
development will be too slow to affect the measurement significantly. However, corresponding samples must be measured at
the same time point.
51. A new calibration curve should be prepared for each plate
measured. Although the assay is robust, deviations over time,
or differences in individual measurements, can be reduced by a
corresponding calibration curve on every plate.
52. Nonused standards can be stored at 4
C for one week.
53. If sample concentrations are beyond of the validated calibration
range (absorbance higher than the highest calibration), a dilution must be prepared and remeasured. An extrapolation of
values is not valid.
54. If samples contain varying amounts of salts or assay-interfering
components, the samples must be dialyzed to ensure a valid
analysis. However, the molecular weight cut off (MWCO) of
Viral Nanoplex Vaccine Production
243