JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1980, p. 483-4890095-1137/80/10-0483/07$02.00/0

Vol. 12, No. 4

Influence of Residual Moisture and Sealing Atmosphere on

Viabiity of Two Freeze-Dried Viral VaccinesPIERRE 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 NationaleSupérieure 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 inthe infectivity titers of freeze-dried canine distemper and poultry infectiousbronchitis viral vaccines. The change in moisture content during the storageperiod was an additional parameter which may influence the infectivity titer. Theresults emphasized the difficulty of predetermining variations in infectivity titersfrom the initial residual moisture. The analysis of the variations in infectivitytiters during the storage of the two vaccines led to the formulation of a hypothesisof the presence of two components of different thermostability. Moreover, thetemporary increase in the infectivity titer of infectious bronchitis vaccine storedat 6°C seemed to indicate the existence of aggregates of infectious particlesprogressively dissociating during storage concurrent with a progressive inactiva-tion of infectious particles.

There are two considerations involved in thefreeze drying of microorganisms which are con-stituents of live virus vaccines (23): the degreeof survival after the freeze drying (freeze-dryingefficiency) and the maintenance of viability dur-ing storage.Many factors have an effect on the stability of

the product (25): the intrinsic properties of theactive component, the composition of the freeze-drying medium, the deep-freezing and dryingmethods (the latter resulting in higher or lowerresidual moisture [RM]), the sealing atmo-sphere, the tightness of the stoppers, and thestorage temperature. The influence of RM onviability during storage has been studied onbacterial or viral products (5, 8, 11, 19, 26-29),and its importance was found to vary accordingto the study and the products. Also, Greiff andRightsel (14) have shown that there was a rela-tionship between the maintenance of viability ofinfluenza virus and the type of atmosphere incontact with the product.The present work dealt with the influence of

the level of RM and the sealing atmosphere onthe viability of two freeze-dried products (caninedistemper virus vaccine and poultry infectiousbronchitis virus vaccine).These two viral agents were selected because

they were attenuated strains, easily titrated bysimple laboratory techniques.

MATERIALS AND METHODSVirus. Canine distemper vaccine (DV), B10 strain,

was propagated on primary chicken embryo fibroblastcell cultures (21); the cell culture fluid constituted the

raw viral material.Infectious bronchitis vaccine (IBV) H120, Hu-

ybden's strain (Centraal Diergeneeskundig Instituut,Rotterdam), specific to gallinaceae, was prepared inembryonated chicken eggs (17). The infected allantoicfluid was the raw viral material.

Freeze-drying media. A lactose solution wasadded to the preparation of the DV to a final concen-tration of 75 mg/ml. The IBV contained 40 mg ofmannitol per ml.

Vials and stoppers. The characteristics of thevials and the type of stopper are summarized in Table1. They were sterilized by heat.Freeze drying. Freeze drying was carried out in an

SMJ apparatus (Usifroid). The vials containing theproducts were put into special boxes, enabling themto be sealed at various stages of dehydration or indifferent atmospheres (16).

Cooling was carried out so that crystallization wasobtained after supercooling (about -10°C). The crys-talline structure obtained in this way facilitated sub-limation and made it possible to obtain a freeze-driedproduct having a homogeneous and finely porous ap-pearance. The temperature below which the productshad to be maintained during sublimation was deter-mined by preliminary differential thermic analysis.During the drying process the temperature was con-trolled with thermometric probes with platinum resis-tors placed into control vials.To obtain different levels of RM, the vials were

placed in four boxes, which were sealed separately inthe sublimation chamber. After removing some of thevials, the remaining boxes were opened again undervacuum so that a longer dehydration period was pos-sible. The two heating elements could be regulatedseparately; thus, the four boxes made it possible toachieve four types of drying, differing only in the

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484 PRECAUSTA ET AL.

TABLE 1. Characteristics of the vials and type ofstopper

Characteristics of vials

Vaccine Vol dis- Type of stopperClass" Capacity tributedT(mid) per vial

(mi)

Distemper I 3 1 Butyl rubber

Infectious Il 5 2 Halogen-treatedbronchitis isobutylene

rubber

" See reference 3.

duration and temperature. Preliminary trials made itpossible to determine, for each product, the conditionsof temperature and time required to attain four levelsof RM ranging from 6% to about 1%. The vials weresealed in the boxes under a nitrogen atmosphere (Uquality, L'Air Liquide) at a pressure of 600 torr. In thecase of the DV, the study also included results ob-tained with a number of industrial batches of vaccine.To study the influence of the sealing atmosphere,

we subjected each product to the above-mentionedtreatment, except that the secondary drying was car-ried out so as to obtain an RM of about 1%. The vialsin each of the four boxes were sealed directly at theend of the drying stage in four different atmospheres:(i) residual air at a very low pressure of about 0.025torr; (ii) nitrogen (U quality, L'Air Liquide) at a pres-sure of about 600 torr; (iii) nitrogen (quality N48, L'AirLiquide) at a pressure of about 600 torr (a molecularsieve filtration device [Dow Chemical Co.] was placedbetween the nitrogen cylinder and the sealing box toallow a further reduction in the water and oxygencontent of the gas [less than 1 ,g/g] and to compensatefor the gas contamination between the cylinder andthe vials); and (iv) argon (L'Air Liquide) at a pressureof 600 torr. In each case, a class-A pressure reducerwas fitted onto the cylinder to limit the risk of contam-ination.

Tests. Biological activity tests were carried out bymeans of infectivity titrations appropriate for theproduct. The DV was titrated on primary chickenembryo cell cultures (22). The titer was calculated byKarber's method and expressed in log1o cell culture50% infective doses (CCID50) per vial of vaccine. TheIBV was titrated in embryonated chicken eggs (17).The titer was calculated by Karber's method andexpressed in logio 50% egg infective doses per vial ofvaccine. The loss of titer during the freeze-dryingprocess was a measure of the freeze-drying efficiency.The loss of titer during storage was an indication ofthe stabiity of the product and was related to its shelflife. The schedule of titrations and storage times issummarized in Tables 2 and 3 for DV and in Table 5and Fig. 3-5 for IBV.RM was determined immediately after freeze drying

and according to the schedule given in Table 2 for DVand Table 5 for IBV. Karl Fischer's method was usedin spite of some disadvantages reported by severalauthors (1, 2, 6, 24). In fact, RM in the same vaccineswas compared in a preliminary study using Karl

J. CLIN. MICROBIOL.

Fischer's method and the constant-weight dryingmethod, under vacuum and under different tempera-tures. The second method had some disadvantages(20): in particular, it was difficult to obtain full dehy-dration while avoiding heat alteration of the product.Karl Fischer's method seemed to be quicker, morereproducible, and more accurate, provided that noconstituent of the product, other than water, reactedwith Karl Fischer's reagent. No such reaction wasobserved in the two vaccines under study. Three tofour samples were titrated at each time; the resultswere given as the mean. The confidence interval wascalculated at P = 0.66 (± a standard error).

RESULTSDV. The standard error for one titration was

0.22 logo.The determinations of RM carried out during

the storage period revealed a variation as illus-trated by Fig. 1.The freeze-drying efficiency was studied on

one hand in 90 batches of vaccine. Figure 2shows the variations in the efficiency and indi-cates an optimum for a level of RM of about2.2%.On the other hand, the analysis of results

obtained with the four samples having differentlevels of RM (Table 2) showed a significantdifference in the freeze-drying efficiency withthe best results corresponding to levels of 3.7and 6.4% (Snedecor test, F3 = 3.66, significant

storage

FIG. 1. Effect of the storage of DV on RM. KarlFischer's method has been used for determining RM.The values were expressed as a percentage of theweight of dry product with one standard error. Ver-tical markers indicate standard error.

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VIABILITY OF FREEZE-DRIED VACCINES 485

o

o

,. 0.!._

>0

._

e._

u10c

c

o

Wb

51-

1.5 2 2.5 %O

initial residual moisture

FIG. 2. Effect ofRM on freeze-drying efficiency inDV. Vertical markers indicate standard error.

TABLE 2. Effect of initial RM on the infectivity titerofDV under different storage conditions

Storage conditions Infectivity titer' at initial RM of:

Temp Time 1.8% 3.7% 4.3% 6.4%

6 Months0 4.47 4.72 4.52 4.882 4.40 4.40 4.60 4.704 4.20 4.30 4.50 4.558 3.85 4.05 3.65 3.9012 3.10 3.10 3.50 3.50

22 Weeks1 4.0 4.05 4.20 4.552 4.0 4.10 4.0 4.204 3.50 3.75 4.0 3.558 3.05 3.20 2.95 3.1016 2.75 2.20 2.85 2.70

37 Days2 3.70 4.10 4.0 3.904 3.40 3.50 3.55 3.358 3.25 3.55 3.45 3.2016 3.25 3.05 3.20 2.55

aInfectivity titers were expressed in logo CCID50per vial. The infectivity titer before freeze drying was5.1 CCID50 per vial.

at 5%). These four batches were a small propor-tion compared with the preceding 90 batches;however, the heterogeneity of these two seriesof results implied that the RM parameter wasnot the only one concerned in maintaining infec-tivity.As for viability during the storage, an analysis

of variance was carried out on the titer differ-ences with the initial titer (Table 2). The lowestsignificant difference between two of these val-ues (for the given temperature and storage pe-

riod) was 0.5 logio.The differences in the variations of titer be-

tween the four series were not significant for thesamples stored at 6 and 22°C (Snedecor test, F93= 3.11 and F32 = 3.38, not significant), but theywere significant for the samples stored at 37°C(Snedecor test, F39 = 12.23, significant at 1%). Itshould be noted that the samples at RM of 3.7and 6.4% were the least stable.The influence of sealing atmosphere is shown

in Table 3. The infectivity titers obtained im-mediately after freeze drying were not signifi-cantly different; neither were the differences inthe changes of the infectivity titer during thestorage period. The sealing atmosphere did notseem to have any influence on the stability dur-ing 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 effi-ciency, the mean loss in titer between 1.4 and6.4% moisture was 0.7 to 0.95 logo. The differ-ence between the losses was low (0.25 logoo. Itcan be assumed that the freeze-drying efficiencywas similar, regardless of the initial level of RM.The results concerning the stability during stor-

TABLE 3. Effect of the type of sealing atmosphereon the infectivity titer ofDV under different storage

conditions.condition Infectivity titer0 with sealing atmo-Storage conditions sphere of:

Resid- U ni- N48Temp Tîmesid Argon Un-nitro-(OC) Time (0-9) ((1.4) (1.1) gen(0.9) ~~~~~(1.3)6 Months

0 4.67 4.60 4.70 4.632 4.40 4.20 4.45 4.204 3.95 4.15 4.0 4.108 3.70 4.25 4.35 4.2012 3.50 3.50 3.30 3.40

22 Weeks1 4.0 4.10 3.95 4.202 3.65 4.15 3.95 3.704 2.40 3.30 3.20 3.358 2.95 3.0 3.0 3.0

37 Days2 3.90 4.10 3.95 3.954 3.65 3.50 3.35 3.508 3.25 3.40 3.35 3.5516 2.85 2.65 2.55 2.80

a Infectivity titers were expressed in logo CCIDsoper vial. The infectivity titer before freeze drying was5.1 CCID50 per vial. Numbers in parentheses indicatethe percent RM.

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TABLE 4. Effect of storage on RM ofIBV

Expt RM (%)' after storage at a temp of 60C for:no. 0 mo 4 mo 8 mo 12 mo

1 1.4 ± 0.10 2.1 ± 0.12 3.4 ± 0.37 4.2 ± 0.572 2.8 ± 0.20 3.8 ± 0.25 4.3 ± 0.21 5.4 ± 0.273 4.2 ± 0.52 5.0 ± 0.15 6.3 ± 0.21 6.4 ± 0.804 6.1 ± 0.06 6.4 ± 0.37 7.3 ± 0.15 8.3 ± 0.51

" Karl Fischer's method has been used for determining RM.The values were expressed as a percentage of the weight ofdry product with one standard error.

ol<._ \

o\a>

_

p iI î I2 4 6 8 10 12 months

sto rage

FIG. 3. Effect of initial RM on infectivity titer ofIBV stored at 6° C. Initial percent moisture contentswere 1.4 (A), 2.9 (-), 4.2 (O), and 6.1 (O). The symbol+ means that it was below or at most equal to thatshown on the curve.

age were not as homogeneous. In fact, for astorage temperature of 6°C (Fig. 3), the varia-tions of the infectivity titer seemed to be illogicalsince they were in complete opposition between2 and 4 months of storage. After 12 months, thelosses in infectivity titer varied with the RMcontent. But it should be noted that some titra-tions carried out at intermediate stages indicateda certain stability or even an increase in titer.Such an increase might be attributed to theerrors inherent in the technique, to the hetero-geneity of the vials, or to a more specific phe-nomenon such as a dissociation of virion aggre-gates (4). In fact, these parameters might wellhave a cumulative effect. For a storage temper-ature of 22 or 37°C, the losses in infectivity titerincreased with the initial level of RM (Fig. 4 and

0

o 1o

2

0

3

._

.' 44)

.E 5

.EtA u

1 4

A ;

ià_

laf t-

16 weeksstora ge

FIG. 4. Effect of initial RM on infectivity titer ofIBVstored at 22°C. Initial percent moisture contentswere 1.4 (A), 2.9 (-), 4.2 (O), and 6.1 (M). The symbol

, means that it was below or at most equal to thatshown on the curve.

o

o r

42

3

2 4 8 16 dayssto roge

FIG. 5. Effect of initial RM on infectivity titer ofIBV stored at 37°C. Initialpercent moisture contentswere 1.4 (A), 2.9 (-), 4.2 (O), and 6.1 (U). The symbolJ. means that it was below or at most equal to thatshown on the curve.

5).An analysis of variance was carried out on the

losses of infectivity titer for each of the temper-atures considered; it permitted the influence ofthe initial RM on the loss in titer to be defined.At 60C, the differences between the losses intiter were almost significant (F3 = 2.1, thresh-hold at 3.6); at 22°C (F: = 9.4) and at 37°C (F3

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VIABILITY OF FREEZE-DRIED VACCINES 487

= 10.4), the lowest significant difference was 0.6logo. Differences were significant at 1% with thebest results for the lowest level of RM.The analysis of variance showed that the seal-

ing atmosphere had no significant influence onthe freeze-drying efficiency (Table 5). Regardingthe losses in titer during storage, the analysis ofvariance revealed a significant difference in favorof N48 nitrogen (F3 > 3.86 at 37°C), whereas thedifferences between the other three types ofatmospheres were negligible.

DISCUSSIONThe kinetics of infectivity titer decrease dur-

ing the storage period of DV (see Table 3) werecalculated by grouping the results obtained withthese four levels of RM. None of the kineticscould be ascribed to the various models of de-crease 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 = infectivitytiter at time t, and a = constant of the rate ofdecrease.An acceptable model is that representing the

simultaneous variation of two exponential kinet-ics, attributable to the existence of two compo-

TABLE 5. Effect of the type of sealing atmosphereon the infectivity tier ofIBV during storage

conditions Infectivity titer' with sealing atmo-sphere of:

Resid- U ni- N48Temp Time ual air (1.2) trogen nto(OC) (0.9) (1*2) (1.1) gen

6 Months0 6.95 7.0 7.25 6.752 7.10 7.40 7.30 7.504 7.0 7.0 7.20 7.208 7.10 7.10 6.85 6.9012 6.90 6.90 6.80 6.60

22 Weeks1 6.60 7.0 7.0 6.802 6.40 6.60 7.0 7.04 6.60 6.20 7.60 7.08 6.40 6.60 7.0 6.6012 6.0 6.0 6.20 6.0

37 Days2 7.0 6.70 7.50 7.04 6.90 6.60 6.70 6.808 6.70 6.90 6.70 6.9016 6.0 6.20 6.0 6.50

Infectivity titers were expressed in logo 50% egginfective doses per vial. The infectivity titer of theproduct before freeze drying was 8.3. Numbers inparentheses indicate the percent RM.

nents with different thermostabiity. The exis-tence of these two components is hypothesized,in an attempt to explain the observed results.Such a model is represented by the equation C!C0 = Ae-at + Be-bt, where e = exponential; Coand C = infectivity titers at times 0 and t,respectively; A and B = respective proportionsof the two hypothetical components, A, the leastthermostable, and B, the most thermostable;and a and b = rate constants of these twocomponents.

Arrhenius' law applied to each of the twoconstants of decrease in titer for each tempera-ture enables Fig. 6 to be plotted; the half-life ofthe infectivity titers for the two hypotheticalcomponents could be calculated for the threestorage temperatures, and their respective pro-portions could be determined (Table 6). Arrhen-ius' law makes it possible to obtain the time/temperature ratio for a product stored at varioustemperatures. That is why an equivalent timescale could be plotted for the same productstored at the three temperatures under consid-eration. Results could be plotted on the samecurve (Fig. 7) to illustrate the decrease in titerfor DV. This curve made it possible to calculate

-~oo

0

3__c -b

c 37C 22'C 6-co

temperature

FIG. 6. Relation between the temperature and theslope of the decrease for DV. (a) Decrease constantvalues, calculated for the least thermostable compo-nent; (b) decrease constant values, calculated for themost thermostable component. The figure was estab-lished by plotting on the y axis the logo of the de-crease constant in terms ofthe inverse ofthe absolutetemperature (1/T).

TABLE 6. Calculated half-life of the infectivity titerofDV for the two components of different

thermostabilityHalf-life (days) at following storage

Component temp (0C:6 22 37

A (96%) 71 10.9 1.33B (4%) 414 63 7.7

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100

c

._

._

à.

c

4.

10

e

c 0

o

2 1 4 8 dys 161 1 2 31 months

1 2 yea rs

storage conditions

FIG. 7. Effect of storage temperature of DV onkinetics ofthe decrease ofinfectivity titer. Experimen-tal results obtained with the stored products, forvarious types ofsealing 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 titerofIBVfor the two components of different

thermostabilityHalf-life (days) at following storage

Component temp (°C):6 22 37

A (88%) 136 8 0.47B (12%) 1,207 71 4.17

the assumed titer after a given period of storageat one of these three temperatures and, for eitherof the three temperatures under consideration,the storage period required to obtain a giveninfectivity titer.For IBV, as for DV, a model of kinetics com-

patible with the variation in the infectivity titerduring storage was that representing the simul-taneous variation of two exponential kinetics,which could correspond to the existence of twocomponents of different thermostability. Thecalculated figures for these two possible com-ponents and the half-lives of their respectiveinfectivity titers are shown in Table 7, for eachof the storage temperatures. In contrast withDV, the variations in the infectivity could notbe integrated on the same curve because of thetemporary increase in titer observed for the lat-ter temperature.The freeze-drying efficiency for DV seemed to

be optimal at an RM content of about 2.2%. Thisoptimum was in agreement with the observa-

tions made by several authors showing thatover-drying of freeze-dried products is not al-ways desirable (9, 10, 12, 13, 18, 29). It has notbeen possible to demonstrate such an optimumfor IBV under these conditions.The stability of IBV seemed to be better at

the lowest initial level of RM. The heterogeneityof the results obtained with DV emphasized thedifficulty in relating RM and stability. No con-stant relationship could be established betweenthe stability of the product and the RM underour observation conditions. These results under-lined the different behavior of one product whencompared with another (different biologicalagent, different medium) or of the same productwhen stored at different temperatures. Thus, inaccord with Dayan et al. (6), we think that thedetermination of RM cannot replace the biolog-ical test for accelerated aging of vaccines; at themost, it could be a warning of a change in themanufacturing process.The tests of RM revealed variations during

storage. In all cases these changes in excess ofthose introduced by the test method could beinterpreted as the result of exchanges takingplace between the product, the atmosphere inthe vial, and the stopper (7, 15). With IBV storedat 6°C, the hypothesis of a progressive dissocia-tion of particle aggregates, already consideredby Cowdery et al. (4) would explain the phenom-enon of infectivity titer increase during storage.If we accept this hypothesis, it could be assumedthat more or less advanced drying would facii-tate the aggregation of particles by the suppres-sion of the water pellicule surrounding themand, possibly, the reduction of the water contentin the surface layers of the virion. In the firststage, the particles would be aggregated andinactivated, resulting in a loss in infectivity with-out relation to the RM content. In the secondstage, the relative rehydration of the productwould cause the dissociation of the aggregates,resulting in a temporary increase in the titer.But the simultaneous phenomenon of inactiva-tion would become prevalent again, and theinfectivity titer would drop anew. Thus, the var-iation in the infectivity titer ofthe product wouldbe the result of two phenomena acting simulta-neously in two opposite directions: inactivationof the virions (the loss in infectivity titer couldbe detected as early as after 2 months of storage)and dissociation of virion aggregates, resultingfrom the increase in the water content. Thisphenomenon was attributed to the exchangestaking place between the product and its envi-ronment. The dissociation phenomenon wouldprevail over the inactivation phenomenon forrelatively low moisture levels; the temporary

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VIABILITY OF FREEZE-DRIED VACCINES 489

increase in titer during the storage period wouldthus vary inversely with the initial moisture.Conversely, inactivation would predominate incase of high levels of RM. Such a variation wasparticularly noticeable during storage at 6°C,because storage at 22 and 37°C probably spedup the inactivation process, which predomi-nated.The sealing of the vials in four types of at-

mosphere had no influence on the freeze-dryingefficiency. This would seem logical, as the periodof storage was extremely reduced. The variationduring the storage period revealed no significantdifference between the sealing atmospheres forDV, whereas a difference did exist for IBV witha slight advantage in favor of N48 nitrogen forstorage at 37°C.

ACKNOWLEDGMENTWe are grateful to C. Stellmann and G. Tixier (IFFA-

Mérieux), who made the statistical analysis of the results.

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3. Conseil de l'Europe. 1971. Récipients en verre pourpréparations injectables, p. 65-70. In Pharmacopée eu-ropéenne, 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 virusvaccines. International symposium on freeze-drying ofbiological products, Washington, D.C. Dev. Biol. Stand.36:297-303.

5. Damjanovic, V. 1974. Kinetics of thermal death andprediction of the stabilities of freeze dried streptomycindependent live Shigella vaccines. J. Biol. Stand. 2:297-311.

6. Dayan, J., S. Chaniot, and R. Netter. 1975. Dosage del'humidité résiduelle dans les vaccins lyophilisés parchromatographie gazeuse. J. Biol. Stand. 3:171-179.

7. Farley, J. J., and J. N. Drummond. 1976. Moisturevapor transmission measurement by gas chromatogra-phy. Bull. Parenter. Drug Assoc. 30:187-195.

8. Faucon, P. 1969. Etude préliminaire sur l'application dela lyophilisation à la conservation de Rhizobium meli-loti. Mémoire présenté à ENSBANA. Centre Universi-taire Montmuzard, Dijon, France.

9. Fry, R. M., and R. I. N. Greaves. 1961. The survival ofbacteria during and after drying. J. Hyg. 49:240-246.

10. Greiff, D. 1969. The effects of residual moisture on thepredicted stabilities of suspensions of viruses dried bysublimation of ice in vacuo, p. 92-109. In T. Nei (ed.),Freezing and drying of microorganisms. University ofTokyo Press, Tokyo.

11. Greiff, D. 1971. Protein structure and freeze drying. Ef-

fects of residual moisture and gases. Cryobiology 8:145-152.

12. Greiff, D. 1976. Freeze-drying cycles. International sym-posium on freeze-drying of biological products, Wash-ington, D.C. Dev. Biol. Stand. 36:105-115.

13. Greiff, D., and W. A. Rightsel. 1968. Stability of sus-pensions of influenza virus dried to different contents ofresidual moisture by sublimation in vacuo. Appl. Micro-biol. 16:835-840.

14. Greiff, D., and W. A. Rightsel. 1969. Stabilities of driedsuspensions of influenza virus sealed in a vacuum orunder different gases. Appl. Microbiol. 17:830-835.

15. Held, H. R., and S. Landi. 1977. Water permeability ofelastomers. J. Biol. Stand. 5:111-119.

16. Le Floc'h, L. 1976. Freeze-drying equipment. Interna-tional symposium on freeze-drying of biological prod-ucts, Washington, D.C. Dev. Biol. Stand. 36:105-115.

17. National Academy of Sciences. 1971. Methods for ex-amining poultry biologist and for identifying and quan-tifying avian pathogens. Committee on Animal HealthAgricultural Board, Subcommittee on Avian Diseases.A. S. N. R. C. Publ.

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23. Redway, K. F., and S. P. Lapage. 1974. Effect of car-bohydrates and related compounds on the long termpreservation of freeze dried bacteria. Cryobiology 11:73-79.

24. Rey, L 1964. L'humidité résiduelle des produits lyophi-lisés. Nature, origine et méthodes d'étude, p. 199-234.In L. Rey (ed.), Aspects théoriques et industriels de lalyophilisation. Hermann, Paris.

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27. Suzuki, M. 1973. Stability and residual moisture contentof dried vaccinia virus. Cryobiology 10:432-434.

28. Topa, P. K. 1974. War against smallpox, development ofstable vaccine. J. Commun. Dis. 6:169-176.

29. Valette, L, D. Simatos, P. Précausta, C. Stellmann,and Ph. Desmettre. 1976. Freeze-drying of Brucellavaccine. International symposium on freeze-drying ofbiological products, Washington, D.C. Dev. Biol. Stand.36:313-322.

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