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A comparative study on quantitation of measles vaccine virus in Vero cells maintained in foetal bovine serum and calf serum
Journal of Biological Stanabdization
( 1986) 14, 15 3- 15 5
A comparative study on quantitation of measles vaccine virus in Vero cells maintained in foetal bovine serum and calf serum*
S. M. Saba,? D. K. Sood, “f R. Aggarwal, t S. Chakravartyt and S. N. Saxena”f
INTRODUCTION In developing countries, measles vaccine is generally imported for mass immunization under the expanded programme of immunization (EPI). It is strongly recommended by WHO that such vaccines are subjected to quality control tests by the National Quality Control Authorities responsible for immunobiologicals to ensure the successful implementation of the immunization programme. The recommended testing procedures for measles vaccine have been laid down. ’ In the test proper, the requirement for foetal bovine serum (FBS) increases when Vero cells are used as this substrate for the potency test of vaccine is propagated in medium supplemented with FBS. Although the superiority of FBS over calf serum (CS) is well documented,2,3 Seefried & MacMorine* showed that calf or adult bovine sera are suitable supplements for the preparation of the cell substrate to be used in viral vaccine production and quality control tests. The high cost and limited availability of virus- and mycoplasma-free FBS has led us to study the suitability of inactivated CS as a substitute for FBS in the quality control tests of measles vaccine. * Received for publication 12 September 1985. t The Central Research Institute, Kasaule (H.P.), 0092-1157/86/020153+03
$03.00/O
PIN-173205,
@ 1986 The Inrernational
India. Assoclarion
of Biological
Srandardmxmn
153
S. M. SAHA E7’A.L Ten lots of measles vaccine from four different manufacturers were titrated for potency using Vero cells originally propagated in FBS and Vero cells propagated in CS. Prior to assay, cells preserved in a cryostat were reconstituted in Eagle’s minimal essential medium supplemented with 10% FBS or 10% CS and serially subcultured to obtain a sufficient number of cultures in Roux bottles. The titrations were performed according to the macro method described5 and results were computed by the Reed & Muench method. In each titration series a freeze-dried local standard of measles vaccine was included as a reference. Table 1 shows the results of virus titration of measles vaccines performed in Vero cells propagated in CS or FBS and the results claimed by manufacturers. The geometric mean titre, standard deviation and coefficient of variation of ten measles vaccine samples were 3.76 and 3.77, kO.37 and kO.47 and 9.8% to 12.5% in the two The correlation coefficient was +0*99 with a regression systems, respectively. equation of y = 0.37x + 2.38. The F-test (Table 2) revealed that there was no statistically significant difference (P > O-05) between the methods of vaccine testing and between samples. TABLE 1. Virus titres of samples of measles virus titrated in Vero cells propagated in foetal bovine serum (FBS) and calf serum (CS)
Sample No.
Manufacturer
1 2 3 4 5 6 7 8 9 10
Method used by manufacturer
A
Micro
B
Micro
Mean virus titre assigned* (Log IO TCID50 per dose)
Not Not C
Macro
D
Pfu
Not Not
3.96 3.65 3.81 submitted 4.10 submitted 4.10 submitted submitted 3.98
Virus titre (Log,, TCIDSo per dose obtained at the Central Research Institute FBS
cs
4.10 4.10 4.10 3.59 3.86 3.30 4.10 3.50 3.70 3.40
4.10 3.75 4.10 3.68 4-10 3.50 3.32 3.80 3.92 3.44
* The results have been computed from the protocols submitted by the manufacturers. TABLE 2. Analysis of variance of virus titres of measles vaccine samples claimed by the manufacturer and in the Central Research Institute Source of variation
Sum of squares
Degrees of freedom
Mean squares
Between methods Between samples Residual
0.0750 O-4270 O-7596
2 5 10
0.0375 O-0854 0.0759
Total
l-2616
17
F5.b; = 4-10; F%.‘d: = 3.33.
154
F o-49 1.12
QUANTITATION
OF MEASLES
It can thus be concluded that inactivated CS with good growth-promoting potential may be substituted for the expensive FBS for the maintenance of the substrate without affecting the sensitivity of the test system in quality control tests of measles vaccine. This helps to implement the recommendations of WHO for the quality control tests of measles vaccines used under EPI by those countries with readily available indigenous material
REFERENCES 1. WHO Manual of detail tests required on final vaccines used in the WHO Expanded programmes of immunisation. Unpublished document BLGIUNDPI82.1, 1982: I17123. 2. Mitchell JT, Anderson WF, Evans VJ. Comparative effect of horse, calfand foetal serum on chromosomal characteristics and neoplastic conversion of mouse embryo cells in vitro. J Nat Cancer Inst 1969; 42: 709-72 1. 3. Price FM, Gault R, Evans VJ. Effect of fraction of horse and foetal bovine serum on neoplastic conversion of C3H mouse cells in tissue culture. In Vitro 197 1; 6: 437440. 4. Seefried AV, MacMorine HG. The use offoetal, calfand adult bovine sera for the growth of serially subcultured diploid cells. Devel Biol Stand 1976; 37: 83-89. 5. Lennette EH, Schmidt NJ. Diagnostic procedures for viral, rikettsial and chlamydial infection. Cell culture technique. 5th Edition American Public Health Association, 1979: 100.
155
( 1986) 14, 15 3- 15 5
A comparative study on quantitation of measles vaccine virus in Vero cells maintained in foetal bovine serum and calf serum*
S. M. Saba,? D. K. Sood, “f R. Aggarwal, t S. Chakravartyt and S. N. Saxena”f
INTRODUCTION In developing countries, measles vaccine is generally imported for mass immunization under the expanded programme of immunization (EPI). It is strongly recommended by WHO that such vaccines are subjected to quality control tests by the National Quality Control Authorities responsible for immunobiologicals to ensure the successful implementation of the immunization programme. The recommended testing procedures for measles vaccine have been laid down. ’ In the test proper, the requirement for foetal bovine serum (FBS) increases when Vero cells are used as this substrate for the potency test of vaccine is propagated in medium supplemented with FBS. Although the superiority of FBS over calf serum (CS) is well documented,2,3 Seefried & MacMorine* showed that calf or adult bovine sera are suitable supplements for the preparation of the cell substrate to be used in viral vaccine production and quality control tests. The high cost and limited availability of virus- and mycoplasma-free FBS has led us to study the suitability of inactivated CS as a substitute for FBS in the quality control tests of measles vaccine. * Received for publication 12 September 1985. t The Central Research Institute, Kasaule (H.P.), 0092-1157/86/020153+03
$03.00/O
PIN-173205,
@ 1986 The Inrernational
India. Assoclarion
of Biological
Srandardmxmn
153
S. M. SAHA E7’A.L Ten lots of measles vaccine from four different manufacturers were titrated for potency using Vero cells originally propagated in FBS and Vero cells propagated in CS. Prior to assay, cells preserved in a cryostat were reconstituted in Eagle’s minimal essential medium supplemented with 10% FBS or 10% CS and serially subcultured to obtain a sufficient number of cultures in Roux bottles. The titrations were performed according to the macro method described5 and results were computed by the Reed & Muench method. In each titration series a freeze-dried local standard of measles vaccine was included as a reference. Table 1 shows the results of virus titration of measles vaccines performed in Vero cells propagated in CS or FBS and the results claimed by manufacturers. The geometric mean titre, standard deviation and coefficient of variation of ten measles vaccine samples were 3.76 and 3.77, kO.37 and kO.47 and 9.8% to 12.5% in the two The correlation coefficient was +0*99 with a regression systems, respectively. equation of y = 0.37x + 2.38. The F-test (Table 2) revealed that there was no statistically significant difference (P > O-05) between the methods of vaccine testing and between samples. TABLE 1. Virus titres of samples of measles virus titrated in Vero cells propagated in foetal bovine serum (FBS) and calf serum (CS)
Sample No.
Manufacturer
1 2 3 4 5 6 7 8 9 10
Method used by manufacturer
A
Micro
B
Micro
Mean virus titre assigned* (Log IO TCID50 per dose)
Not Not C
Macro
D
Pfu
Not Not
3.96 3.65 3.81 submitted 4.10 submitted 4.10 submitted submitted 3.98
Virus titre (Log,, TCIDSo per dose obtained at the Central Research Institute FBS
cs
4.10 4.10 4.10 3.59 3.86 3.30 4.10 3.50 3.70 3.40
4.10 3.75 4.10 3.68 4-10 3.50 3.32 3.80 3.92 3.44
* The results have been computed from the protocols submitted by the manufacturers. TABLE 2. Analysis of variance of virus titres of measles vaccine samples claimed by the manufacturer and in the Central Research Institute Source of variation
Sum of squares
Degrees of freedom
Mean squares
Between methods Between samples Residual
0.0750 O-4270 O-7596
2 5 10
0.0375 O-0854 0.0759
Total
l-2616
17
F5.b; = 4-10; F%.‘d: = 3.33.
154
F o-49 1.12
QUANTITATION
OF MEASLES
It can thus be concluded that inactivated CS with good growth-promoting potential may be substituted for the expensive FBS for the maintenance of the substrate without affecting the sensitivity of the test system in quality control tests of measles vaccine. This helps to implement the recommendations of WHO for the quality control tests of measles vaccines used under EPI by those countries with readily available indigenous material
REFERENCES 1. WHO Manual of detail tests required on final vaccines used in the WHO Expanded programmes of immunisation. Unpublished document BLGIUNDPI82.1, 1982: I17123. 2. Mitchell JT, Anderson WF, Evans VJ. Comparative effect of horse, calfand foetal serum on chromosomal characteristics and neoplastic conversion of mouse embryo cells in vitro. J Nat Cancer Inst 1969; 42: 709-72 1. 3. Price FM, Gault R, Evans VJ. Effect of fraction of horse and foetal bovine serum on neoplastic conversion of C3H mouse cells in tissue culture. In Vitro 197 1; 6: 437440. 4. Seefried AV, MacMorine HG. The use offoetal, calfand adult bovine sera for the growth of serially subcultured diploid cells. Devel Biol Stand 1976; 37: 83-89. 5. Lennette EH, Schmidt NJ. Diagnostic procedures for viral, rikettsial and chlamydial infection. Cell culture technique. 5th Edition American Public Health Association, 1979: 100.
155