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Stability and residual moisture content of dried vaccinia virus
CRYOBIOLOGY
10,
432434
(1973)
Stability
and Residual Moisture Content Dried Vaccinia Virus ’ MASATOSHI
Department
of Veterinary
MATERIALS
AND
METHODS
Dried products of unpurified vaccinia virus were prepared by means of our routine method (8). The protectants used were 5% sodium glutamate or 5% peptone in the final concentration. TWO groups of the products, the one containing the protectant and the other without any protectant, were employed in the study. Products with different moisture content were obtained by modification of the drying time and the drying temperature. The residual moisture content of dried product was determined gravimetrically 1 From Proceedings of Japan-U.S.A. Conference on Freezing and Freeze-Drying, held October 15 20, 1972, at Cacapon State Park, Berkeley Springs, West Virginia, sponsored by the Japan Society for the Promotion of Science, The National Science Foundation, and the American Type Culture Collection.
Copyright All rights
0 1973 by Academic of reproduction in any
Press, Inc. form reserved.
SUZUKI
Medicine, Faculty of Agriculture, Kagamigahara-shi, Gifu, Japan
The residual moisture content of dried biological products is one of the important factors affecting stability on storage (1). In regard to the relation between the moisture and stabilities of dried viral materials, Greiff (2, 3), and Greiff and Rightsel (4, 5) indicated that the stability of dried influenza virus was maximal at the residual moisture content of approximately 1.7%, by an accelerated storage test. This study is concerned with the stability and the residual moisture content of unpurified vaccinia virus dried in order to elucidate the relation between them.
of
Gifu
University,
by the method of Abderhalden, after desiccation at 60” C for 3 hr under 0.1 mm Hg of vacuum (9). Virus titer was estimated by the method of Westwood et al. (lo), and expressed by the log pock-forming units/ml of the reconstituted virus suspension. Stability of the products was evaluated by the reduction of titer in the boiling test, which involved heating in a boiling water bath for 60 min, and in the preservation test, which involved storing at 37” C and 45” C, respectively, for 12 months. And the reduction of titer was calculated from the titers obtained before and after each test. RESULTS
Boiling test. The relation between the reduction of the titer in the test and the residual moisture content of the product was observed. In the case of the product with 5% SOdium glutamate, the reduction in titer in 18 batches could not be correlated with the residual moisture content in the range of 1.4%3.71% ( r = 0.3156, P > 0.10). The titer of four batches of the product, which contained 5% peptone, with 0.11, 2.26, 3.31, and 4.24% of residual moisture reduced to Iog 0.74, 0.49, 0.62, and 0.41. In these cases, no significant differences were observed. So a residual moisture content below 4.24% did not affect the stability of the dried product. Preservation test. Dried products with-
STABILITY
OF DRIED
VACCINIA
TABLE
-----.--
~.~..__~_ Residwl moisture content
-~_.
.-
~~-
1
~~.~~_-~-.~-.-
~.~
temperature 1
3
---
4.61(;i _____
._
fi __-__-___--
12
0.8“ J .60
1.60
2.08
5.20
>6 .OS” t
-
37" c 45°C
0.66 l.;i4
1.81 3.57
2.G7 >6.11*
3.02 -
37°C 4G°C I_--.-
a > = larger reduction
.~---
45°C
37” c
3.7-ra /o'
-
Reduction of titer (log I’l~‘U/ml) in preservation period (months)0
l’rewxvatlon
0.97” ,O
433
VIRUS
1.23 2.88 -__-..
of titer than the given point;
1.x4 >5.41" -
2.96
2.81 >6.41a -- -~-
3.53 >6.41a
not tested.
out any protectant, having several residual stability of the unpurified product was not moisture values were preserved at 37” C remarkable. and 45” C, respectively, for 12 months, and As to the residual moisture of dried viral stabilities were compared with the residual products, Greiff (2, 3), and GreifE and moisture content. As shown in Table 1, the Rightsel (4, 5) observed a certain degree reduction in titer after preservation for 6 of correlation between the residual moismonths reached a similar level among sam- ture content and stability of dried influenza ples with different residual moisture con- virus. The results obtained in the present tents both at 37” C and 45” C. study did not correspond with their results In the product with added sodium gluta- using dried in%uenza virus, because the mate or peptone, the reduction in titer in presence of extraneous nonviral substances the preservation test at 45” C for 12 months in the unpurified product might act as a again did not correlate with different re- factor obscuring the relationships under sidual moisture contents. Table 2 indicates test. that the residual moisture content in a cerAccording to the Minimum Requiretain range did not bring about any marked ments (9), a residual moisture content difference in reduction of titer during below 3% is acceptable for unpurified vacpreservation. DISCUSSION
The effect of protectant on purified and unpurified vaccinia virus products on freeze-drying and subsequent preservation was investigated in a previous paper (8). The results showed the protective effect of sodium glutamate in a single and combined state with several substances. The study investigated the relation between stability and the residual moisture content in order to make a more stable dried product of unpurified vaccinia virus. However, the effect of the residual moisture content, as shown in Table 3, on the
_~ I’rutech,t
37;, Sodimn glulubanmle
.il’r Peptonc
I
~~-
Reduction of titer (I”B I’l~l‘/ml) in preservation period (months) at 45” c I
6
12
0.65’1 /Cl 3.02:;, 4.07':;
0.66 1.12 1.07
0.6" 0.84 1.22
1.96 1.78 1.37
1.23:;> 2.277; 3.039;
1.16 1.04 0.64
1.38 1.75 1.25
2.00 2.45 1.66
SUZUKI
434
Not t.est,ed 0.97y;,4.61”’ C’ __-..-
Boiling test Preservation test
cinia virus product. However, present work suggests that the satisfactory range of residual moisture in the product will be beIow 4% in view of stability on preservation. SUMMARY
Dried products of unpurified vaccinia virus containing 5% sodium glutamate or 5% peptone as a protectant, and the product without any protectant were prepared from calf dermal pulp. In these products the relation between the stabilities and the residual moisture content were examined. The stability of all dried products remained unaffected when the residual moisture content was kept below about 4%. ACKNOWLEDGMENTS The study was carried out in the Japan BCG Laboratory, Kiyose-shi, Tokyo, Japan. The author wishes to express his gratitude to the director, Dr. Tetsuji Sawada of the Japan BCG Laboratory. The paper was read as an additional paper to Dr. Greiff’s paper i’n the International Symposium on “Mechanisms of Cellular Injury by Freezing and Drying in Microorganisms,” organized by the Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan in 1968 (6). The original paper appeared in the Kitasato Adz. E?;p. Med. 43, 23-28 ( 1970). REFERENCES 1. Beckett, L. G. The effect of residual moisture on freeze-dried materials, and its measurement. In “Biological Applications of Freezing and Drying” (R. J. C. Harris, Ed.), pp. 285-301. Academic Press, New York, 1954.
2. Greiff, D. The effect of residual moisture on predicted stabilities of suspension of virus dried by sublimation of ice in vacua. in “Freezing and Drying of Microorganisms” (T. Nei, Ed.), pp. 93-109. Univ. of Tokyo Press, Tokyo, 1969. 3. Greiff, D. An accelerated test for predicting the stabilities of freeze-dried or liquid suspension of virus stored at low temperature. In “Proceedings of the First International Conference on Culture Collections” (H. Iizuka and T. Hasegawa, Eds.), pp. 287307. Univ. of Tokyo Press, Tokyo, 1970. 4. Greiff, D., and Rightsel, W. A. Stability of suspension of virus after freezing or drying by vacuum sublimation and storage. cryobiology 3, 432444 ( 1967). 5. Greiff, D., and Rightsel, W. A, Stability of suspension of Influenza virus dried to different contents of residual moi#sture by sublimation in vacua. Appl. Microbial. 16, 835840 ( 1968). 6. Suzuki, M. Relation between reduction of vaccinia virus titer and residual moisture content. In “Freezing and Drying of Microorganisms” (T. Nei, Ed.), pp. 111-115. Univ. of Tokyo Press, Tokyo, 1969. 7. Suzuki, M. Effect of residual moisture content on stability of dried smallpox vaccine. Kitasato
Arch. Exp. Med. 43, 23-28
( 1970).
8. Suzuki, M. Effect ,of suspending media on freeze-drying and preservation of vaccinia virus. J. Hyg. 68, 29-41 (1970). 9. The Ministry of Health and Welfare. Smallpox vaccine and dried smallpox vaccine. In “Minimum Requirements of Biological Products in Japan” (The Ministry of Health and Welfare, Ed.), pp. 289-298, The Ministry of Health and Welfare, Japanese Government, Tokyo, 1968. 10. Westwood, J. C. N., Phipps, P. H., and Boulter, E. A. The titration of vaccinia virus on the chorioallantoic membrane of developing chick embryo. J. Hyg. 55, 123-139 ( 1957).
10,
432434
(1973)
Stability
and Residual Moisture Content Dried Vaccinia Virus ’ MASATOSHI
Department
of Veterinary
MATERIALS
AND
METHODS
Dried products of unpurified vaccinia virus were prepared by means of our routine method (8). The protectants used were 5% sodium glutamate or 5% peptone in the final concentration. TWO groups of the products, the one containing the protectant and the other without any protectant, were employed in the study. Products with different moisture content were obtained by modification of the drying time and the drying temperature. The residual moisture content of dried product was determined gravimetrically 1 From Proceedings of Japan-U.S.A. Conference on Freezing and Freeze-Drying, held October 15 20, 1972, at Cacapon State Park, Berkeley Springs, West Virginia, sponsored by the Japan Society for the Promotion of Science, The National Science Foundation, and the American Type Culture Collection.
Copyright All rights
0 1973 by Academic of reproduction in any
Press, Inc. form reserved.
SUZUKI
Medicine, Faculty of Agriculture, Kagamigahara-shi, Gifu, Japan
The residual moisture content of dried biological products is one of the important factors affecting stability on storage (1). In regard to the relation between the moisture and stabilities of dried viral materials, Greiff (2, 3), and Greiff and Rightsel (4, 5) indicated that the stability of dried influenza virus was maximal at the residual moisture content of approximately 1.7%, by an accelerated storage test. This study is concerned with the stability and the residual moisture content of unpurified vaccinia virus dried in order to elucidate the relation between them.
of
Gifu
University,
by the method of Abderhalden, after desiccation at 60” C for 3 hr under 0.1 mm Hg of vacuum (9). Virus titer was estimated by the method of Westwood et al. (lo), and expressed by the log pock-forming units/ml of the reconstituted virus suspension. Stability of the products was evaluated by the reduction of titer in the boiling test, which involved heating in a boiling water bath for 60 min, and in the preservation test, which involved storing at 37” C and 45” C, respectively, for 12 months. And the reduction of titer was calculated from the titers obtained before and after each test. RESULTS
Boiling test. The relation between the reduction of the titer in the test and the residual moisture content of the product was observed. In the case of the product with 5% SOdium glutamate, the reduction in titer in 18 batches could not be correlated with the residual moisture content in the range of 1.4%3.71% ( r = 0.3156, P > 0.10). The titer of four batches of the product, which contained 5% peptone, with 0.11, 2.26, 3.31, and 4.24% of residual moisture reduced to Iog 0.74, 0.49, 0.62, and 0.41. In these cases, no significant differences were observed. So a residual moisture content below 4.24% did not affect the stability of the dried product. Preservation test. Dried products with-
STABILITY
OF DRIED
VACCINIA
TABLE
-----.--
~.~..__~_ Residwl moisture content
-~_.
.-
~~-
1
~~.~~_-~-.~-.-
~.~
temperature 1
3
---
4.61(;i _____
._
fi __-__-___--
12
0.8“ J .60
1.60
2.08
5.20
>6 .OS” t
-
37" c 45°C
0.66 l.;i4
1.81 3.57
2.G7 >6.11*
3.02 -
37°C 4G°C I_--.-
a > = larger reduction
.~---
45°C
37” c
3.7-ra /o'
-
Reduction of titer (log I’l~‘U/ml) in preservation period (months)0
l’rewxvatlon
0.97” ,O
433
VIRUS
1.23 2.88 -__-..
of titer than the given point;
1.x4 >5.41" -
2.96
2.81 >6.41a -- -~-
3.53 >6.41a
not tested.
out any protectant, having several residual stability of the unpurified product was not moisture values were preserved at 37” C remarkable. and 45” C, respectively, for 12 months, and As to the residual moisture of dried viral stabilities were compared with the residual products, Greiff (2, 3), and GreifE and moisture content. As shown in Table 1, the Rightsel (4, 5) observed a certain degree reduction in titer after preservation for 6 of correlation between the residual moismonths reached a similar level among sam- ture content and stability of dried influenza ples with different residual moisture con- virus. The results obtained in the present tents both at 37” C and 45” C. study did not correspond with their results In the product with added sodium gluta- using dried in%uenza virus, because the mate or peptone, the reduction in titer in presence of extraneous nonviral substances the preservation test at 45” C for 12 months in the unpurified product might act as a again did not correlate with different re- factor obscuring the relationships under sidual moisture contents. Table 2 indicates test. that the residual moisture content in a cerAccording to the Minimum Requiretain range did not bring about any marked ments (9), a residual moisture content difference in reduction of titer during below 3% is acceptable for unpurified vacpreservation. DISCUSSION
The effect of protectant on purified and unpurified vaccinia virus products on freeze-drying and subsequent preservation was investigated in a previous paper (8). The results showed the protective effect of sodium glutamate in a single and combined state with several substances. The study investigated the relation between stability and the residual moisture content in order to make a more stable dried product of unpurified vaccinia virus. However, the effect of the residual moisture content, as shown in Table 3, on the
_~ I’rutech,t
37;, Sodimn glulubanmle
.il’r Peptonc
I
~~-
Reduction of titer (I”B I’l~l‘/ml) in preservation period (months) at 45” c I
6
12
0.65’1 /Cl 3.02:;, 4.07':;
0.66 1.12 1.07
0.6" 0.84 1.22
1.96 1.78 1.37
1.23:;> 2.277; 3.039;
1.16 1.04 0.64
1.38 1.75 1.25
2.00 2.45 1.66
SUZUKI
434
Not t.est,ed 0.97y;,4.61”’ C’ __-..-
Boiling test Preservation test
cinia virus product. However, present work suggests that the satisfactory range of residual moisture in the product will be beIow 4% in view of stability on preservation. SUMMARY
Dried products of unpurified vaccinia virus containing 5% sodium glutamate or 5% peptone as a protectant, and the product without any protectant were prepared from calf dermal pulp. In these products the relation between the stabilities and the residual moisture content were examined. The stability of all dried products remained unaffected when the residual moisture content was kept below about 4%. ACKNOWLEDGMENTS The study was carried out in the Japan BCG Laboratory, Kiyose-shi, Tokyo, Japan. The author wishes to express his gratitude to the director, Dr. Tetsuji Sawada of the Japan BCG Laboratory. The paper was read as an additional paper to Dr. Greiff’s paper i’n the International Symposium on “Mechanisms of Cellular Injury by Freezing and Drying in Microorganisms,” organized by the Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan in 1968 (6). The original paper appeared in the Kitasato Adz. E?;p. Med. 43, 23-28 ( 1970). REFERENCES 1. Beckett, L. G. The effect of residual moisture on freeze-dried materials, and its measurement. In “Biological Applications of Freezing and Drying” (R. J. C. Harris, Ed.), pp. 285-301. Academic Press, New York, 1954.
2. Greiff, D. The effect of residual moisture on predicted stabilities of suspension of virus dried by sublimation of ice in vacua. in “Freezing and Drying of Microorganisms” (T. Nei, Ed.), pp. 93-109. Univ. of Tokyo Press, Tokyo, 1969. 3. Greiff, D. An accelerated test for predicting the stabilities of freeze-dried or liquid suspension of virus stored at low temperature. In “Proceedings of the First International Conference on Culture Collections” (H. Iizuka and T. Hasegawa, Eds.), pp. 287307. Univ. of Tokyo Press, Tokyo, 1970. 4. Greiff, D., and Rightsel, W. A. Stability of suspension of virus after freezing or drying by vacuum sublimation and storage. cryobiology 3, 432444 ( 1967). 5. Greiff, D., and Rightsel, W. A, Stability of suspension of Influenza virus dried to different contents of residual moi#sture by sublimation in vacua. Appl. Microbial. 16, 835840 ( 1968). 6. Suzuki, M. Relation between reduction of vaccinia virus titer and residual moisture content. In “Freezing and Drying of Microorganisms” (T. Nei, Ed.), pp. 111-115. Univ. of Tokyo Press, Tokyo, 1969. 7. Suzuki, M. Effect of residual moisture content on stability of dried smallpox vaccine. Kitasato
Arch. Exp. Med. 43, 23-28
( 1970).
8. Suzuki, M. Effect ,of suspending media on freeze-drying and preservation of vaccinia virus. J. Hyg. 68, 29-41 (1970). 9. The Ministry of Health and Welfare. Smallpox vaccine and dried smallpox vaccine. In “Minimum Requirements of Biological Products in Japan” (The Ministry of Health and Welfare, Ed.), pp. 289-298, The Ministry of Health and Welfare, Japanese Government, Tokyo, 1968. 10. Westwood, J. C. N., Phipps, P. H., and Boulter, E. A. The titration of vaccinia virus on the chorioallantoic membrane of developing chick embryo. J. Hyg. 55, 123-139 ( 1957).