Also, Greiff and. Rightsel (14) have shown thatthere was a rela- tionship between the maintenanceof viability of influenza virus and the type of atmosphere in.
JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1980, p. 483-489
0095-1137/80/10-0483/07$02.00/0
Vol. 12, No. 4
Influence of Residual Moisture and Sealing Atmosphere on Viabiity of Two Freeze-Dried Viral Vaccines PIERRE M. PRECAUSTA,l* DENISE SIMATOS,2 MARTINE LE PEMP,2 BERNARD DEVAUX,' AND FERENC KATO' IFFA-Mérieux, Département Vétérinaire de l'Institut Mérieux, 69007 Lyon,' and Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation, Centre Universitaire Montmuzard,
21000 Dijon,' France
This study demonstrated the complexity of the factors leading to changes in the infectivity titers of freeze-dried canine distemper and poultry infectious bronchitis viral vaccines. The change in moisture content during the storage period was an additional parameter which may influence the infectivity titer. The results emphasized the difficulty of predetermining variations in infectivity titers from the initial residual moisture. The analysis of the variations in infectivity titers during the storage of the two vaccines led to the formulation of a hypothesis of the presence of two components of different thermostability. Moreover, the temporary increase in the infectivity titer of infectious bronchitis vaccine stored at 6°C seemed to indicate the existence of aggregates of infectious particles progressively dissociating during storage concurrent with a progressive inactivation of infectious particles. There are two considerations involved in the freeze drying of microorganisms which are constituents of live virus vaccines (23): the degree of survival after the freeze drying (freeze-drying efficiency) and the maintenance of viability during storage. Many factors have an effect on the stability of the product (25): the intrinsic properties of the active component, the composition of the freezedrying medium, the deep-freezing and drying methods (the latter resulting in higher or lower residual moisture [RM]), the sealing atmosphere, the tightness of the stoppers, and the storage temperature. The influence of RM on viability during storage has been studied on bacterial or viral products (5, 8, 11, 19, 26-29), and its importance was found to vary according to the study and the products. Also, Greiff and Rightsel (14) have shown that there was a relationship between the maintenance of viability of influenza virus and the type of atmosphere in contact with the product. The present work dealt with the influence of the level of RM and the sealing atmosphere on the viability of two freeze-dried products (canine distemper virus vaccine and poultry infectious bronchitis virus vaccine). These two viral agents were selected because they were attenuated strains, easily titrated by simple laboratory techniques. MATERIALS AND METHODS Virus. Canine distemper vaccine (DV), B10 strain, was propagated on primary chicken embryo fibroblast
raw viral material. Infectious bronchitis vaccine (IBV) H120, Huybden's strain (Centraal Diergeneeskundig Instituut, Rotterdam), specific to gallinaceae, was prepared in embryonated chicken eggs (17). The infected allantoic fluid was the raw viral material. Freeze-drying media. A lactose solution was added to the preparation of the DV to a final concentration of 75 mg/ml. The IBV contained 40 mg of mannitol per ml. Vials and stoppers. The characteristics of the vials and the type of stopper are summarized in Table 1. They were sterilized by heat. Freeze drying. Freeze drying was carried out in an SMJ apparatus (Usifroid). The vials containing the products were put into special boxes, enabling them to be sealed at various stages of dehydration or in different atmospheres (16). Cooling was carried out so that crystallization was obtained after supercooling (about -10°C). The crystalline structure obtained in this way facilitated sublimation and made it possible to obtain a freeze-dried product having a homogeneous and finely porous appearance. The temperature below which the products had to be maintained during sublimation was determined by preliminary differential thermic analysis. During the drying process the temperature was controlled with thermometric probes with platinum resistors placed into control vials. To obtain different levels of RM, the vials were placed in four boxes, which were sealed separately in the sublimation chamber. After removing some of the vials, the remaining boxes were opened again under vacuum so that a longer dehydration period was possible. The two heating elements could be regulated separately; thus, the four boxes made it possible to cell cultures (21); the cell culture fluid constituted the achieve four types of drying, differing only in the 483
484
J. CLIN. MICROBIOL.
PRECAUSTA ET AL.
TABLE 1. Characteristics of the vials and type of stopper Characteristics of vials Vaccine
Distemper
Infectious bronchitis
Vol dis- Type of stopper Class" Capacity per vial (mid) tributedT (mi) I 3 1 Butyl rubber Il
" See reference 3.
5
2
Halogen-treated isobutylene rubber
Fischer's method and the constant-weight drying method, under vacuum and under different temperatures. The second method had some disadvantages (20): in particular, it was difficult to obtain full dehydration while avoiding heat alteration of the product. Karl Fischer's method seemed to be quicker, more reproducible, and more accurate, provided that no constituent of the product, other than water, reacted with Karl Fischer's reagent. No such reaction was observed in the two vaccines under study. Three to four samples were titrated at each time; the results were given as the mean. The confidence interval was calculated at P = 0.66 (± a standard error).
RESULTS DV. The standard error for one titration was 0.22 logo. The determinations of RM carried out during the storage period revealed a variation as illustrated by Fig. 1. The freeze-drying efficiency was studied on one hand in 90 batches of vaccine. Figure 2 shows the variations in the efficiency and indicates an optimum for a level of RM of about 2.2%. On the other hand, the analysis of results obtained with the four samples having different levels of RM (Table 2) showed a significant difference in the freeze-drying efficiency with the best results corresponding to levels of 3.7 and 6.4% (Snedecor test, F3 = 3.66, significant
duration and temperature. Preliminary trials made it possible to determine, for each product, the conditions of temperature and time required to attain four levels of RM ranging from 6% to about 1%. The vials were sealed in the boxes under a nitrogen atmosphere (U quality, L'Air Liquide) at a pressure of 600 torr. In the case of the DV, the study also included results obtained with a number of industrial batches of vaccine. To study the influence of the sealing atmosphere, we subjected each product to the above-mentioned treatment, except that the secondary drying was carried out so as to obtain an RM of about 1%. The vials in each of the four boxes were sealed directly at the end of the drying stage in four different atmospheres: (i) residual air at a very low pressure of about 0.025 torr; (ii) nitrogen (U quality, L'Air Liquide) at a pressure of about 600 torr; (iii) nitrogen (quality N48, L'Air Liquide) at a pressure of about 600 torr (a molecular sieve filtration device [Dow Chemical Co.] was placed between the nitrogen cylinder and the sealing box to allow a further reduction in the water and oxygen content of the gas [less than 1 ,g/g] and to compensate for the gas contamination between the cylinder and the vials); and (iv) argon (L'Air Liquide) at a pressure of 600 torr. In each case, a class-A pressure reducer was fitted onto the cylinder to limit the risk of contamination. Tests. Biological activity tests were carried out by means of infectivity titrations appropriate for the product. The DV was titrated on primary chicken embryo cell cultures (22). The titer was calculated by Karber's method and expressed in log1o cell culture 50% infective doses (CCID50) per vial of vaccine. The IBV was titrated in embryonated chicken eggs (17). The titer was calculated by Karber's method and expressed in logio 50% egg infective doses per vial of vaccine. The loss of titer during the freeze-drying process was a measure of the freeze-drying efficiency. The loss of titer during storage was an indication of the stabiity of the product and was related to its shelf life. The schedule of titrations and storage times is summarized in Tables 2 and 3 for DV and in Table 5 and Fig. 3-5 for IBV. storage RM was determined immediately after freeze drying and according to the schedule given in Table 2 for DV FIG. 1. Effect of the storage of DV on RM. Karl and Table 5 for IBV. Karl Fischer's method was used Fischer's method has been used for determining RM. in spite of some disadvantages reported by several The values were expressed as a percentage of the authors (1, 2, 6, 24). In fact, RM in the same vaccines weight of dry product with one standard error. Verwas compared in a preliminary study using Karl tical markers indicate standard error.
VIABILITY OF FREEZE-DRIED VACCINES
VOL. 12, 1980 o
o
,.
0.!
51-
._
>0
._
e ._
0
u1
c
c
Wb
o
1.5
%O
2.5
2
initial residual moisture
FIG. 2. Effect of RM on freeze-drying efficiency in DV. Vertical markers indicate standard error.
TABLE 2. Effect of initial RM on the infectivity titer of DV under different storage conditions Storage conditions
Infectivity titer' at initial RM of:
Temp
Time
1.8%
3.7%
4.3%
6.4%
6
Months 0 2 4 8 12
4.47 4.40 4.20 3.85 3.10
4.72 4.40 4.30 4.05 3.10
4.52 4.60 4.50 3.65 3.50
4.88 4.70 4.55 3.90 3.50
22
1 2 4 8 16
37
4.0 4.0 3.50 3.05 2.75
4.05 4.10 3.75 3.20 2.20
4.20 4.0 4.0 2.95 2.85
4.55 4.20 3.55 3.10 2.70
Days 2 4 8 16
riod) was 0.5 logio. The differences in the variations of titer between the four series were not significant for the samples stored at 6 and 22°C (Snedecor test, F93 = 3.11 and F32 = 3.38, not significant), but they were significant for the samples stored at 37°C (Snedecor test, F39 = 12.23, significant at 1%). It should be noted that the samples at RM of 3.7 and 6.4% were the least stable. The influence of sealing atmosphere is shown in Table 3. The infectivity titers obtained immediately after freeze drying were not significantly different; neither were the differences in the changes of the infectivity titer during the storage period. The sealing atmosphere did not seem to have any influence on the stability during storage. IBV. The standard deviation for one titration was 0.26 logo. As for DV, RM increased during the storage period (Table 4). As for the freeze-drying efficiency, the mean loss in titer between 1.4 and 6.4% moisture was 0.7 to 0.95 logo. The difference between the losses was low (0.25 logoo. It can be assumed that the freeze-drying efficiency was similar, regardless of the initial level of RM. The results concerning the stability during storTABLE 3. Effect of the type of sealing atmosphere on the infectivity titer of DV under different storage conditions . condition Storage conditions
Weeks
3.70 3.40 3.25 3.25
4.10 3.50 3.55 3.05
4.0 3.55 3.45 3.20
ResidTîmesid
N48 U niUn-nitro-
Time
(1.1)
~~gen ~ (1.3)
6
Months 0 2 4 8 12
3.95 3.70 3.50
4.60 4.20 4.15 4.25 3.50
4.70 4.45 4.0 4.35 3.30
4.63 4.20 4.10 4.20 3.40
1 2 4 8
4.0 3.65 2.40 2.95
4.10 4.15 3.30 3.0
3.95 3.95 3.20 3.0
4.20 3.70 3.35 3.0
Days 2 4 8 16
3.90 3.65 3.25 2.85
4.10 3.50 3.40 2.65
3.95 3.35 3.35 2.55
3.95 3.50 3.55 2.80
3.90 3.35 3.20 2.55 22
at 5%). These four batches were a small proportion compared with the preceding 90 batches; however, the heterogeneity of these two series of results implied that the RM parameter was not the only one concerned in maintaining infec-
37
As for viability during the storage, an analysis of variance was carried out on the titer differences with the initial titer (Table 2). The lowest significant difference between two of these values (for the given temperature and storage pe-
with sealing atmoInfectivity titer0 sphere of:
(OC)
Temp
aInfectivity titers were expressed in logo CCID50 per vial. The infectivity titer before freeze drying was 5.1 CCID50 per vial.
tivity.
485
Argon (0-9) (0.9)((1.4)
4.67 4.40
Weeks
a Infectivity titers were expressed in logo CCIDso per vial. The infectivity titer before freeze drying was 5.1 CCID50 per vial. Numbers in parentheses indicate the percent RM.
486
J. CLIN. MICROBIOL.
PRECAUSTA ET AL. TABLE 4. Effect of storage on RM of IBV
Expt no. 1 2 3 4
RM (%)' after storage at a temp of 60C for: 4 mo 8 mo 0 mo 12 mo 1.4 2.8 4.2 6.1
± ± ± ±
2.1 ± 3.8 ± 5.0 ± 6.4 ±
0.10 0.20 0.52 0.06
0.12 0.25 0.15 0.37
3.4 4.3 6.3 7.3
± ± ± ±
0.37 0.21 0.21 0.15
4.2 5.4 6.4 8.3
± ± ± ±
0.57 0.27 0.80 0.51
" Karl Fischer's method has been used for determining RM. The values were expressed as a percentage of the weight of dry product with one standard error.
0
o1
;
A
o
2 0 3
i à
_
._
.'
4)
.E
4
laf t-
5
.E
ol
o\
o
o
_
p
iI
2
4
î
6
I
8
10
12 months
r
42
sto rage
FIG. 3. Effect of initial RM on infectivity titer of IB V stored at 6° C. Initial percent moisture contents were 1.4 (A), 2.9 (-), 4.2 (O), and 6.1 (O). The symbol + means that it was below or at most equal to that shown on the curve.
age were not as homogeneous. In fact, for a storage temperature of 6°C (Fig. 3), the variations of the infectivity titer seemed to be illogical since they were in complete opposition between 2 and 4 months of storage. After 12 months, the losses in infectivity titer varied with the RM content. But it should be noted that some titrations carried out at intermediate stages indicated a certain stability or even an increase in titer. Such an increase might be attributed to the errors inherent in the technique, to the heterogeneity of the vials, or to a more specific phenomenon such as a dissociation of virion aggregates (4). In fact, these parameters might well have a cumulative effect. For a storage temperature of 22 or 37°C, the losses in infectivity titer increased with the initial level of RM (Fig. 4 and
3
2
4
8
16 days
sto roge
FIG. 5. Effect of initial RM on infectivity titer of IB V stored at 37°C. Initial percent moisture contents were 1.4 (A), 2.9 (-), 4.2 (O), and 6.1 (U). The symbol J. means that it was below or at most equal to that shown on the curve.
5). An analysis of variance was carried out on the losses of infectivity titer for each of the temperatures considered; it permitted the influence of the initial RM on the loss in titer to be defined. At 60C, the differences between the losses in titer were almost significant (F3 = 2.1, threshhold at 3.6); at 22°C (F: = 9.4) and at 37°C (F3
= 10.4), the lowest significant difference was 0.6 logo. Differences were significant at 1% with the best results for the lowest level of RM. The analysis of variance showed that the sealing atmosphere had no significant influence on the freeze-drying efficiency (Table 5). Regarding the losses in titer during storage, the analysis of variance revealed a significant difference in favor of N48 nitrogen (F3 > 3.86 at 37°C), whereas the differences between the other three types of atmospheres were negligible.
DISCUSSION The kinetics of infectivity titer decrease during the storage period of DV (see Table 3) were calculated by grouping the results obtained with these four levels of RM. None of the kinetics could be ascribed to the various models of decrease expressed by the following equations: (i) C = at + Co (order 0); (ii) log C = at + log Co (order 1); and (iii) 1/C = at + 1/Co (order 2); where Co = initial infectivity titer, C = infectivity titer at time t, and a = constant of the rate of decrease. An acceptable model is that representing the simultaneous variation of two exponential kinetics, attributable to the existence of two compoTABLE 5. Effect of the type of sealing atmosphere on the infectivity tier of IB V during storage conditions
Temp (OC) 6
Time
Months 0 2 4 8 12
22
37
sphere of:
Such a model is represented by the equation C! C0 = Ae-at + Be-bt, where e = exponential; Co and C = infectivity titers at times 0 and t, respectively; A and B = respective proportions of the two hypothetical components, A, the least thermostable, and B, the most thermostable; and a and b = rate constants of these two components.
Arrhenius' law applied to each of the two constants of decrease in titer for each temperature enables Fig. 6 to be plotted; the half-life of the infectivity titers for the two hypothetical components could be calculated for the three storage temperatures, and their respective proportions could be determined (Table 6). Arrhenius' law makes it possible to obtain the time/ temperature ratio for a product stored at various temperatures. That is why an equivalent time scale could be plotted for the same product stored at the three temperatures under consideration. Results could be plotted on the same curve (Fig. 7) to illustrate the decrease in titer for DV. This curve made it possible to calculate -~o o
Residual air (0.9)
(1.2)
(1*2)
0
N48 U nitrogen nto gen (1.1)
3__
-b
c
6.95 7.10 7.0 7.10 6.90
7.0 7.40 7.0 7.10 6.90
7.25 7.30 7.20 6.85 6.80
6.75 7.50 7.20 6.90 6.60
6.60 6.40 6.60 6.40 6.0
7.0 6.60 6.20 6.60 6.0
7.0 7.0 7.60 7.0 6.20
6.80 7.0 7.0 6.60 6.0
Days 7.50 6.70 6.70 6.0
7.0 6.80 6.90 6.50 Infectivity titers were expressed in logo 50% egg infective doses per vial. The infectivity titer of the product before freeze drying was 8.3. Numbers in parentheses indicate the percent RM. 2 4 8 16
nents with different thermostabiity. The existence of these two components is hypothesized, in an attempt to explain the observed results.
Infectivity titer' with sealing atmo-
Weeks 1 2 4 8 12
487
VIABILITY OF FREEZE-DRIED VACCINES
VOL. 12,1980
7.0 6.90 6.70 6.0
6.70 6.60 6.90 6.20
c
37C
22'C
6-c
o
temperature
FIG. 6. Relation between the temperature and the slope of the decrease for DV. (a) Decrease constant values, calculated for the least thermostable component; (b) decrease constant values, calculated for the most thermostable component. The figure was established by plotting on the y axis the logo of the decrease constant in terms of the inverse of the absolute temperature (1/T).
TABLE 6. Calculated half-life of the infectivity titer of DV for the two components of different thermostability Half-life (days) at following storage temp (0C: Component A (96%) B (4%)
6
22
37
71 414
10.9 63
1.33 7.7
488
J. CLIN. MICROBIOL.
PRECAUSTA ET AL.
100
e
c
à.
._
c
0
._
10
o
c
4.
1
2
1
1
4
8
1
31
2
2
1
dys months yea rs
16
storage conditions
FIG. 7. Effect of storage temperature of DV on kinetics of the decrease of infectivity titer. Experimental results obtained with the stored products, for various types of sealing atmosphere, at 6'C (-), 22°C (M), and 37°C (O), were plotted on the same curve. a, 4-week month. TABLE 7. Calculated half-life of the infectivity titer of IBV for the two components of different thermostability Half-life (days) at following storage temp (°C): Component A (88%) B (12%)
6
22
37
136 1,207
8 71
0.47 4.17
the assumed titer after a given period of storage at one of these three temperatures and, for either of the three temperatures under consideration, the storage period required to obtain a given infectivity titer. For IBV, as for DV, a model of kinetics compatible with the variation in the infectivity titer during storage was that representing the simultaneous variation of two exponential kinetics, which could correspond to the existence of two components of different thermostability. The calculated figures for these two possible components and the half-lives of their respective infectivity titers are shown in Table 7, for each of the storage temperatures. In contrast with DV, the variations in the infectivity could not be integrated on the same curve because of the temporary increase in titer observed for the latter temperature. The freeze-drying efficiency for DV seemed to be optimal at an RM content of about 2.2%. This optimum was in agreement with the observa-
tions made by several authors showing that over-drying of freeze-dried products is not always desirable (9, 10, 12, 13, 18, 29). It has not been possible to demonstrate such an optimum for IBV under these conditions. The stability of IBV seemed to be better at the lowest initial level of RM. The heterogeneity of the results obtained with DV emphasized the difficulty in relating RM and stability. No constant relationship could be established between the stability of the product and the RM under our observation conditions. These results underlined the different behavior of one product when compared with another (different biological agent, different medium) or of the same product when stored at different temperatures. Thus, in accord with Dayan et al. (6), we think that the determination of RM cannot replace the biological test for accelerated aging of vaccines; at the most, it could be a warning of a change in the manufacturing process. The tests of RM revealed variations during storage. In all cases these changes in excess of those introduced by the test method could be interpreted as the result of exchanges taking place between the product, the atmosphere in the vial, and the stopper (7, 15). With IBV stored at 6°C, the hypothesis of a progressive dissociation of particle aggregates, already considered by Cowdery et al. (4) would explain the phenomenon of infectivity titer increase during storage. If we accept this hypothesis, it could be assumed that more or less advanced drying would faciitate the aggregation of particles by the suppression of the water pellicule surrounding them and, possibly, the reduction of the water content in the surface layers of the virion. In the first stage, the particles would be aggregated and inactivated, resulting in a loss in infectivity without relation to the RM content. In the second stage, the relative rehydration of the product would cause the dissociation of the aggregates, resulting in a temporary increase in the titer. But the simultaneous phenomenon of inactivation would become prevalent again, and the infectivity titer would drop anew. Thus, the variation in the infectivity titer of the product would be the result of two phenomena acting simultaneously in two opposite directions: inactivation of the virions (the loss in infectivity titer could be detected as early as after 2 months of storage) and dissociation of virion aggregates, resulting from the increase in the water content. This phenomenon was attributed to the exchanges taking place between the product and its environment. The dissociation phenomenon would prevail over the inactivation phenomenon for relatively low moisture levels; the temporary
VOL. 12, 1980
VIABILITY OF FREEZE-DRIED VACCINES
increase in titer during the storage period would thus vary inversely with the initial moisture. Conversely, inactivation would predominate in case of high levels of RM. Such a variation was particularly noticeable during storage at 6°C, because storage at 22 and 37°C probably sped up the inactivation process, which predominated. The sealing of the vials in four types of atmosphere had no influence on the freeze-drying efficiency. This would seem logical, as the period of storage was extremely reduced. The variation during the storage period revealed no significant difference between the sealing atmospheres for DV, whereas a difference did exist for IBV with a slight advantage in favor of N48 nitrogen for storage at 37°C. ACKNOWLEDGMENT We are grateful to C. Stellmann and G. Tixier (IFFAMérieux), who made the statistical analysis of the results.
LITERATURE CITED 1. Bervelt, E., and E. Van Kerchove. 1975. Dosage de l'humidité résiduelle dans les vaccins lyophilisés. J. Biol. Stand. 3:321-330. 2. Caisey, P., and J. Balis. 1975. Dosage de l'humidité résiduelle des vaccins lyophilisés par le réactif de Karl Fischer. Technique et causes d'erreurs. Rev. Elev. Med. Vet. Pays Trop. 28:459-462. 3. Conseil de l'Europe. 1971. Récipients en verre pour préparations injectables, p. 65-70. In Pharmacopée européenne, vol. 2. Maison neuve S.A., Sainte Ruffine, France. 4. Cowdery, S., M. Frey, S. Orlowski, and A. Gray. 1976. Stability characteristics of freeze dried human live virus vaccines. International symposium on freeze-drying of biological products, Washington, D.C. Dev. Biol. Stand. 36:297-303. 5. Damjanovic, V. 1974. Kinetics of thermal death and prediction of the stabilities of freeze dried streptomycin dependent live Shigella vaccines. J. Biol. Stand. 2:297311. 6. Dayan, J., S. Chaniot, and R. Netter. 1975. Dosage de l'humidité résiduelle dans les vaccins lyophilisés par chromatographie gazeuse. J. Biol. Stand. 3:171-179. 7. Farley, J. J., and J. N. Drummond. 1976. Moisture vapor transmission measurement by gas chromatography. Bull. Parenter. Drug Assoc. 30:187-195. 8. Faucon, P. 1969. Etude préliminaire sur l'application de la lyophilisation à la conservation de Rhizobium meliloti. Mémoire présenté à ENSBANA. Centre Universitaire Montmuzard, Dijon, France. 9. Fry, R. M., and R. I. N. Greaves. 1961. The survival of bacteria during and after drying. J. Hyg. 49:240-246. 10. Greiff, D. 1969. The effects of residual moisture on the predicted stabilities of suspensions of viruses dried by sublimation of ice in vacuo, p. 92-109. In T. Nei (ed.), Freezing and drying of microorganisms. University of Tokyo Press, Tokyo. 11. Greiff, D. 1971. Protein structure and freeze drying. Ef-
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fects of residual moisture and gases. Cryobiology 8:145152. Greiff, D. 1976. Freeze-drying cycles. International symposium on freeze-drying of biological products, Washington, D.C. Dev. Biol. Stand. 36:105-115. Greiff, D., and W. A. Rightsel. 1968. Stability of suspensions of influenza virus dried to different contents of residual moisture by sublimation in vacuo. Appl. Microbiol. 16:835-840. Greiff, D., and W. A. Rightsel. 1969. Stabilities of dried suspensions of influenza virus sealed in a vacuum or under different gases. Appl. Microbiol. 17:830-835. Held, H. R., and S. Landi. 1977. Water permeability of elastomers. J. Biol. Stand. 5:111-119. Le Floc'h, L. 1976. Freeze-drying equipment. International symposium on freeze-drying of biological products, Washington, D.C. Dev. Biol. Stand. 36:105-115. National Academy of Sciences. 1971. Methods for examining poultry biologist and for identifying and quantifying avian pathogens. Committee on Animal Health Agricultural Board, Subcommittee on Avian Diseases. A. S. N. R. C. Publ. Nei, T. 1964. Freezing and freeze-drying of microorganisms. Cryobiology 1:87-93. Nomura, M., C. Nishimura, and M. Kitaoka. 1965. Stability and preservabiity of freeze-dried small pox vaccine with different moisture contents. Jpn. J. Med. Sci. Biol. 18:249-256. Pemberton, J. R. 1976. Critical factors of the vacuum oven technique which influence the estimation of moisture in veterinary biologist. International symposium on freeze-drying of biological products, Washington, D.C. Dev. Biol. Stand. 36:191-199. Précausta, P., J. Terré, and E. Lefthériotis. 1968. Multiplication avec effet cytopathogène du virus de Carré sur culture cellulaire. Application à la vaccination. Table ronde sur les maladies des chiens, Lyon, 1967, p. 34-60. In L'Animal de compagnie. Précausta, P., C. Stellmann, P. Bornarel, J. Terré, and E. Leftheriotis. 1969. International symposium on biological assay methods of vaccines and sera, London. Symp. Ser. Immunobiol. Stand. 10:141-156. Redway, K. F., and S. P. Lapage. 1974. Effect of carbohydrates and related compounds on the long term preservation of freeze dried bacteria. Cryobiology 11: 73-79. Rey, L 1964. L'humidité résiduelle des produits lyophilisés. Nature, origine et méthodes d'étude, p. 199-234. In L. Rey (ed.), Aspects théoriques et industriels de la lyophilisation. Hermann, Paris. Simatos, D., G. Blond, Ph. Dauvois, and F. Sauvageot. 1974. La lyophilisation. Principes et applications. Association Nationale de la Recherche Technique, Bre-
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26. Sparkes, J. D., and P. Fenje. 1972. The effect of residual moisture in lyophilized smallpox vaccine on its stability at different temperatures. Bull. W. H. O. 46:729-734. 27. Suzuki, M. 1973. Stability and residual moisture content of dried vaccinia virus. Cryobiology 10:432-434. 28. Topa, P. K. 1974. War against smallpox, development of stable vaccine. J. Commun. Dis. 6:169-176. 29. Valette, L, D. Simatos, P. Précausta, C. Stellmann, and Ph. Desmettre. 1976. Freeze-drying of Brucella vaccine. International symposium on freeze-drying of biological products, Washington, D.C. Dev. Biol. Stand. 36:313-322.
