Process Development and Spiking Studies for Virus Filtration of r-hFSH - This study on a recombinant human follicle stimulating hormone demonstrates the use of virus filters to reduce the risk of cont

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Process Development and Spiking Studies for Virus Filtration of r-hFSH
This study on a recombinant human follicle stimulating hormone demonstrates the use of virus filters to reduce the risk of contamination.


BioPharm International
pp. 24-30

Determination of virus titer by endpoint titration

To determine the virus titer of a sample, serial three-fold dilutions were made with cell-culture medium. 100 μl aliquots of each dilution were added to 8 wells of a 96-well-MTP with cells (in 100 μl cell culture medium per well). The cells were cultivated for a specified incubation period. Then, they were inspected microscopically for virus-induced changes in cell morphology.

Large-Volume Plating (LVP)

The detection limit of a sample depends on its volume incubated with the indicator cells. To improve the detection limit, a large volume of the sample was analyzed (LVP). Briefly, 200 μl of the minimal diluted sample was added to a defined number of wells containing the indicator cells in 100 μl cell culture medium. The cells were cultivated for a specified incubation period. Then, they were inspected microscopically for virus-induced changes in cell morphology.

For LVP determination. If virus-induced changes are observed in some wells of the large volume plating (15-50% of all wells) for a sample, the virus titer is calculated according to the Spearman and Kärber formula. It is assumed that a 1:3 higher concentrated dose compared to the highest dose analyzed leads to an infection of all parallel cultures.

Only two reaction rates (i.e., number of virus positive wells divided through the number of wells tested per dilution) are reported: the reaction rate determined by the LVP and the reaction rate of the virtual 1:3 higher concentrated dose (mode C of calculation).




If virus-induced changes are observed in only a few wells of the LVP (< 15 % of all wells) for a sample, the virus titer is calculated according to the following formula (mode D of calculation):

D: pre-dilution factor of the sample
np: number of virus-positive wells
n: number of all wells tested
Vw: sample volume per well (0.2 mL)




If no virus-induced changes are observed for a sample, the virus titer is determined by the Poisson distribution at the 95% confidence limits derived from "Note for guidance on quality of biotechnological products" (5, 6) (mode E of calculation):

p: 0.05
v: tested sample volume in mL
V: process fraction volume in mL




For endpoint titration. The virus titer (TCID50/mL), which causes a positive result in 50% of the tested cultures (TCID50) of an endpoint titration, is calculated according to the method of Spearman and Kärber (mode A of calculation):

Y0: decadic logarithm of highest dilution factor of the sample, which causes the infection of all parallel cultures (= 8 parallel cultures for mode A and B of calculation)
d: decadic logarithm of dilution step (=log10 3)
Pi: observed reaction rate starting at Y0. Observed reaction rate (number of virus positive wells divided through the number of wells tested) per dilution i starting at Y0
v: decadic logarithm of volume conversion factor (= log10(5) for mode A and B, log10 (3 1/3) for mode C of calculation)




Calculation of the standard error:

se: standard error
Pi: observed reaction rate (number of virus positive wells divided through the number of wells tested, per dilution)
ni: number of determinations
d: decadic logarithm of dilution step (=log10 3)




Calculation of the confidence limits:

c: confidence limits
se: standard error

If the highest dose tested in the endpoint titration does not result in the infection of parallel cultures, the virus titer for this sample is calculated according to the Spearman and Kärber formula. It is assumed that a 1:3 higher concentrated dose, compared to the highest dose analyzed, leads to an infection of all parallel cultures (mode B of calculation).




Determination of the reduction

For virus filtration:

Reduction factor of hold:
R: reduction factor
A0: total virus load of load, pre-filtered
An: total virus load of hold, pre-filtered

MuLV, PRV, Reo-3

Reduction factor of virus filtration:
R: reduction factor
A0: total virus load of load, pre-filtered
An: total virus load of filtrate fraction A

MVM

Reduction factor of virus filtration (filtrate fraction A):
R: reduction factor
A0: total virus load of load of fraction A, pre-filtered
An: total virus load of filtrate fraction A

Reduction factor of the virus filtration (sum of filtrate fraction A, B, and C):
R: reduction factor
A0: total virus load of load pre-filtered
An: Sum of 'log10 total virus load' of fraction A, B and C

After each filtration the Vpro filters were tested for integrity by gross leak test.


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