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Thermal stability of freeze-dried mammalian interferons
CRYOBIOLOGY
16,
301-314
Thermal Analysis
PATRICIA Departments
(1979)
Stability
of Freeze-Dried
of Freeze-Drying
JAMESON,
Conditions and Accelerated for Murine Interferon
DONALD
GREIFF,*
of Microbiology
and * Pathology, P.O. Box 26509, Milwaukee,
INTRODUCTION
Interferon is an antiviral protein released from appropriately stimulated animal cells; recent clinical use suggests fulfillment of its great promise as an antiviral and antitumor agent. The necessity for standard reference interferon preparations has been clearly established (6, 7, 21). For example, they provide a basis for comparison of results obtained in different laboratories throughout the world studying interferon with a multitude of assay methods. Although interferon is a relatively stable protein as compared with many enzymes, some preparations were rather labile under the conditions of storage employed to preserve interferon samples intended for use as reference reagents or in clinical trials. Previous freeze-dried reference preparations have lost potency at variable and unknown rates during storage or shipping (7). Our specific objectives, therefore, were to develop procedures for stabilizing interferon by freeze-drying, and to predict the rate at which inactivation would occur under various storage conditions. This report describes an evaluation of factors that may contribute to loss of interferon Received 10, 1979.
November
20,
1978;
Mammalian
accepted
May
AND
SIDNEY
lnterferons Storage
Tests
E. GROSSBERG
The Medical College Wisconsin 53226
of
Wisconsin,
activity following freeze-drying. Murine interferon was selected because interferon of high potency could be prepared readily. Methods of interferon preparation and partial purification as well as methods of freezedrying were evaluated to obtain the most stable preparation of murine interferon possible. To do this it was necessary to adapt accelerated storage tests to predict the rate at which activity would be lost by freeze-dried preparations of interferon. MATERIALS
Preparation
AND
of Murine
METHODS
Interferon
Confluent monolayers of L cells in 32-0~ (1.05 1) prescription bottles or 0.5 gallon (2.1 1) roller bottles were washed with GLB (Hanks’ balanced salt solution, HBSS, containing 1% gelatin and 0.5% lactalbumin hydrolysate), then inoculated with high multiplicities ( 10 to 100 plaqueforming units/cell) of a velogenic Newcastle disease virus (NDV) strain CC diluted in GLB, which was allowed to adsorb for 1 hr at 36°C. Eagle’s minimal essential medium (MEM) supplemented with 2% fetal bovine serum (FBS), MEMFB&, was added at one-half the volume used for cell growth, and cultures were incubated 16 to 24 hr at 36°C. Harvests were pooled and frozen at -20°C until. use. 301 OOll-2240/79/040301-14$02.00/0 Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form resewd.
302 Interferon
JAMESON,
GREIFF,
Assay
Two methods of interferon assay were used. The first was a plaque-reduction assay in monolayers of L cells inoculated with vesicular stomatitis virus (VSV) after overnight exposure to interferon dilutions made in MEM-FBS,; titers were expressed in units representing the median plaquedepressing dose (PDDBo) obtained from dose-response curves. The second method was the CDVII virus yield-reduction assay of Oie et al. (19) which was used for the majority of the studies involving comparison of stabilities of various types of interferon preparations; titers were expressed in units of the 0.5 log hemagglutinin yield-reduction endpoint. The laboratory reference interferon included in all titrations was a mouse plasma interferon preparation obtained by injection of adult mice with NDV according to the method of Baron and Buckler (1). This laboratory standard was used to monitor the relative consistency of the assay. No adjustment was made in the titers of individual test samples. This assay measures essentially the same unitage (12,251; standard deviation l.Cfold) as that assigned (12,000) to the National Institutes of Health (NIH) interferon standard (GOO2-904-511). Treatment
of Interferon
Processing of crude interferon in cell culture supernatant fluids or plasma consisted of the inactivation and precipitation of NDV by the addition of 2 N perchloric acid at a ratio of 7.5 ml per 100 ml of fluid at 4°C (16). The mixture was stirred at 4°C for 39 min, then centrifuged in plastic tubes at SOOg for 30 min at 4°C. The supernatant fluids were decanted into a plastic bottle, and the pH adjusted to neutrality with 5 N potassium hydroxide. The resulting precipitate, optimized by reduction of temperature to near freezing, was removed by centrifugation as above, and
AND
GROSSBERG
the supernatant fluid again decanted into a small plastic bottle for storage at -70°C. A small portion was placed in a plastic tube for use in titration, and frozen at -20’ C until assayed. Alternatively, inducing virus was inactivated by dialysis of the interferon preparation against 0.15 N HCI-KC1 buffer at pH 2 for 2 days at 4°C. Partial purification of the murine interferon was obtained by several different methods. Alcohol precipitation of interferon in the presence of 0.02 M zinc acetate was accomplished by slow addition of acetate-buffered 24% alcohol to the -5°C sample with continuous stirring (2). The precipitate was dissolved in unbuffered HBSS (without bicarbonate) with 0.1% gelatin and stored at 4°C. Trichloroacetic acid precipitation of interferon in the presence of 0.1% bovine serum albumin (BSA) was done according to the method of Davies (4). Precipitate containing interferon was dissolved in unbuffered HBSS and stored at 4°C. L cell interferon purified by SE-Sephadex column chromatography (20) was provided by Dr. Kurt Paucker. Two different lots containing 0.5% bovine serum albumin ( BSA) were supplied: one was freeze-dried (FD) and the other frozen (L) in pH 7 sodium phosphate buffer (0.1 M). We also tested a batch method adsorption with SE-Sephadex under conditions based on those described for the column method by Paucker and Stancek (20) to increase the rate of processing and avoid the addition of protein stabilizers. Results were similar to those reported for the SE-Sephadex column method (20) : approximately lo-fold purification was obtained with recovery of about 10yO of the initial activity. Another method of interferon processing was the nonspecific adsorption to zeolite as developed by Sonnabend and Katsoyannis (personal communication, 1971) as a modification of the Doucil process for
STABILIZATION
OF MURINE
chick interferon described by Fantes (5). Interferon was adsorbed to zeolite at low pH in low ionic strength buffer and eluted in high molar@ neutral buffer. The eluant was concentrated by removal of water using carbowax and extensively dialyzed to yield a partially purified interferon in a selected buffer supplemented with a desired stabilizing agent. The resulting interferon solution was filter sterilized using a 0.2.2 ,uM membrane with a prefilter; filtration was expedited by quickly warming the solution to 37°C prior to filtration. The final buffer composition was either 0.1 M potassium phosphate or 0.1 M sodium phosphate. Cell Cultures The mouse L cell line was obtained from Microbiological Associates (Walkersville, Md.) and was propagated in MEM with Hanks’ salt solution supplemented with 10% FBS. After treatment of cells with viruses to induce interferon, the cultures were maintained in medium containing 2% FBS. Viruses The GDVII virus, VSV and NDV were obtained and propagated as previously described ( 14, 18, 19). Freeze-Drying
of Interferon
Samples of interferon were dispensed in I-ml portions in glass vials which were then held at 4°C until placed in the freeze-drying apparatus. Each type of interferon sample was freeze-dried separately by a process determined by the nature of the suspending medium and the size of the lot, using methods previously described by Greiff and Rightsel ( 12 ) . The freeze-drying cycle used in the present studies was based on that found most effective for influenza virus (8). Lyophilization vials containing 1 ml of interferon
INTERFERON
303
were placed in a specially designed chamber freeze-dryer (Edwards High Vacuum, Ltd., Crawley, Sussex, England) as described by Greiff (8) and cooled to a terminal temperature of -30°C. No attempt was made to modify or suppress supercooling. After the samples had been frozen at -30°C the chamber was evacuated. Although the shelf temperature was maintained at -30°C the product temperature declined to -40°C within approximately 1 hr because of evaporative cooling. At this time, the pressure within the chamber was approximately lo-? Torr (10 p Hg). Evaporative cooling maintained product temperature at -40°C for approximately 12 hr, after which time the product temperature began to rise. At this point in the cycle, approximately 85% of the water had been removed from the frozen sample. When the product temperature reached -30°C shelf temperature was set at 0°C and maintained at that temperature throughout the remainder of the drying cycle. Between the 14th and 17th hr of the drying cycle, product temperature rose slowly and stabilized at 0°C. During this time, the vacuum within the chamber dropped from 10m2 Torr to approximately 10m3 Torr. Drying by sublimation of ice in VacUo at 0°C was continued until the vacuum within the chamber was 10m5Torr. The desired residual moisture may be obtained by adjusting the length of time for the freeze-drying cycle (8). In general, with the freeze-dryer used, approximate terminal pressures within the chamber and corresponding residual moistures were as follows: 5 X 1O-5 Torr, 3$%0; lO-‘j Torr, 2%; and 5 X lo-’ Torr, 1%. The vacuum obtained in preliminary experiments corresponded to a residual moisture of about 1 to 3%; total elapsed time for freeze-drying 50 to 100 ampoules of 1 ml interferon in solutions containing gelatin was approximately 22 hr, with BSA about 30 hr, and with serum about 26 hr. Larger loads required longer times.
304 Accelerated Stability
JAMESON,
GREIFF,
Storage Tests of Interferon
Two accelerated storage tests were used to obtain data for predicting the stabilities of freeze-dried preparations of interferon. Tests developed for the purpose of predicting stability of pharmaceutical preparations (3, 24) and certain freeze-dried viruses (9) were adapted for the evaluation of stability of interferon samples. The linear nonisothermal stability (LNS) test was used primarily to compare stabilities of two or more samples of interferon prepared in different ways; a starting temperature of 50°C was obtained by a 30min equilibration period after which the temperature was allowed to rise to 90°C at a rate of 1.5”C per hr (~27 hr in duration). Samples were taken at the end of the equilibration period and during the anticipated inactivation phase of the heating. The multiple isothermal stability (MIS) test was used primarily to predict the storage life of a given interferon sample after freeze-drying (11). Five or more samples of the interferon preparation were placed at each of three temperatures selected to provide appropriate intervals for subsequent analysis; the temperatures used were 52, 60, and 68°C. Samples were taken at intervals from 10 days through 6 months. Samples taken during either of these two types of accelerated storage tests were placed at -70°C until the time at which all samples within a group were assayed simultaneously. Samples were reconstituted for titration by the addition of 1 ml of sterile water and dilutions were made in maintenance medium ( MEM-FBSZ). Rubber-stoppled vials that had lost vacuum, as observed during reconstitution, were replaced by intact replicates exposed to the same storage conditions.
AND
GROSSBERG
Confkmatory Stability
Storage Tests of Interferon
In order to determine how accurate the predictive MIS test would be, several heatsealed glass ampoules of freeze-dried interferon were placed at each of the following temperatures: -70, -20, 4, 22, 37, and 56°C. In this test, still in progress, samples are taken at intervals of several months for many years in order to determine the long-range stability of interferon stored at these various temperatures and compare the observed stability with the predicted activities obtained from the MIS accelerated storage test. Characterization
of Interferon
In addition to measurements of interferon potency and stability, the interferon freeze-dried to provide the reference reagent to the NIH was tested for physicochemica1 properties, purity, and microbial sterility. The physico-chemical characterization included molecular exclusion chromatography using G-100 Sephadex, ultracentrifugation at 100,OOOg for 2 hr, and trypsinization. Purity was assessed by the amount of antiviral activity which could be detected using heterologous cell cultures, such as primary chicken embryo fibroblast and human cell strain (BUD-S) cultures, as well as by specific tests for residual inducing virus including plaque assays in chicken embryo cell cultures and blind passage in embryonated eggs. The interferon was tested for the presence of bacteria by cultivation on brain-heart infusion agar for 2 weeks at 36°C for fungi by cultivation on Sabouraud’s agar for 3 weeks at 22°C and for mycoplasma by cultivation on appropriate medium for 2 weeks at 36°C in an atmosphere of nitrogen. Vials and Ampoules for Freeze-Dying During the preliminary experiments testing various methods of processing and
STABILIZATION
OF MURINE TABLE
305
INTERFERON
1
Recovery of interferon activity during several experiments testing various methods of virus inactivation, partial purification, and freeze-drying Part
Interferon
Treatment I
A
Part
None6 Perchlorate pH 2 dialysis Perchlorate, then : zinc-alcohol precipitation TCA precipitation Treatment
activity
(units/mk)
II
III
None Perchlorate pH 2 dialysis Perchlorate, then : zinc-alcohol precipitation TCA precipitation
IV
V
< 1,000 3,300 nt
nt 2,000 nt
8,300 6,800 8,600
4,700d 3,500 nt
440’ 440’
< 1,000 < 1,000
13,000 3,400
<500 1,700
2,700 1,500
activity in samples after freeze-drying
III
B
shown
ntc 610 nt
Interferon
Liquid
in experiments
from the indicated or storage at +4”C.
experiments
IV F-D/
Liquid
V F-Dr
Liquid
F-D,
28,000 3,500 nt
18,000 4,600 nt
3,500 2,300 3,500
3,300 2,100 720
nt 3,900 nt
nt 1,500 nt
180 340
<40 230
nt 1,000
nt 380
3,100 1,400
3,100 1,000
QInterferon was titrated by the GDVII hemagglutinin yield-reduction method (19). b A portion of the lot was stored at +4’C during the processing of the rest of the lot. c Not tested. d This titer was obtained by adjusting for the dilution made of the original sample to provide the raw material to be tested by the various procedures. e This titer ~a.8 obtained by correction for the concentration factor relative to the original sample, which was introduced during processing. f The original samples were preserved as a liquid at +4”C; and the freeze-dried (F-D) samples were stored at -20°C until titrated.
additives for freeze-drying, interferon samples were placed in neutral glass vials obtained from Johnsen and Jorgensen (Trident) Ltd., London, England. These vials were sealed under vacuum at the conclusion of the freeze-drying process and the butyl rubber stopples held in place by metal closures. Interferon for use as a reference reagent was dispensed in l-ml volumes in borosilicate glass ampoules obtained from Wheaton Scientific (Millville, N.J. ) and freezedried to a residual moisture of approximately 3%. These ampoules were heat-sealed containing an atmosphere of argon provided by back-flushing at the conclusion of the freeze-drying process (13). In order to insure complete closure,
the heat-sealed tips of the vials were then dipped in neoprene solution (10) to close the small channels which often remain after heat-sealing of glass vials under atmospheric pressure. RESULTS
Inactivation
of Residual Inducer
Virus
NDV remaining in the interferon induced in L cell cultures was inactivated either by perchloric acid treatment or by dialysis against pH 2 buffer. There was usually no remarkable change in interferon activity of the preparation by either of these methods (Table 1) although the NDV was completely inactivated as determined by infectivity tests using embryo-
JAMESON,
306
GREIFF,
AND
GROSSBERG
nated eggs. The perchloric acid method is shorter, does not involve placing infectious material into dialysis tubing, and provides an interferon that is more stable after freeze-drying than a preparation exposed to the pH 2 treatment (Fig. 1). One sample exposed to pH 2 lost about 80% of its activity during freeze-drying alone (Table 1). The percblorate method was therefore used in our subsequent studies of murine interferon. Purification
IO280 50
after storage at -20°C as long as 14 months, and during storage for 7 months at 4°C (Table 2). Samples were therefore held at 4°C for the few hours or days between the completion of processing and the initiation of freeze-drying to avoid an additional freeze-thaw cycle. As much as 50 to 90% of the activity was lost in sam-
TABLE
Treatment
2
of Various Interferon Samples Stored as a Liquid -2O”C, or in Freeze-Dried Form at -20°C. Initial activity (units/ml) ----Liquid
Remaining months
Freezedried
Liquid, 1-3
Liquid.
13-15
at +4”C
or
activity after storage for the number shown under the conditions described.
at 4°C 6-8
(‘C)
FIG. 1. Linear nonisothermal storage test of crude interferon samples after freeze-drying. The original 1, cell inteferon ( 0) was treated to inactivate the residual inducing virus (NDV) by dialysis against pH 2 buffer (A) or by the perchlorate precipitation method ( 0 ).
Four methods for partial preparation of interferon were evaluated: zeolite adsorption, co-precipitation by trichloroacetic acid treatment in the presence of albumin, zinc-alcohol precipitation at low temperature, and ion exchange adsorption. Untreated or perchlorate-treated interferon samples did not lose activity during freeze-drying or the corresponding short term storage of control samples at 4°C (Table 1B). Liquid crude and perchloratetreated interferon samples were stable
Sample
70
TEMPERATLRE
Methods
Stability
60
l-3
at 6-8
-20°C
of
Freeze-dried, at -20°C 13-15 1-3
A
NOIE Zn-alcohol precipitated TCA precipitated
B
NOlIE
C
NOID?
Perchlorate
Perchlorate D
NOW2
3,500
1,500
3,900
420
2.700
3,100
3,100
1,800
1,500
1,000
1,400
NOW
8,600
10,000 3,300 2,800
1,700
1,600
680
8,500 3.800
11,000 8,200
860
3,900
740
2,400 3,900 (-7OOC)
150 2,700 7,600
13-15
150
17,000 7,400 14,000
Perchlorate E
6-8
5,300 1,200 6.800
1,100 3.000 620 800
580 740
540 1,200
STABILIZATIOPi
L 1 -7’0 +50 Cant~l
c
” 60
k 70
TEMPERATURE
8
’ 60
t
OF MURIiYE
a 90
(“C)
FIG. 2. Linear nonisothermal storage test of two different partially purified interferon samples supplemented with 0.5% BSA. One was prepared by SE-Sephadex column chromatography and supplied by Dr. K. Paucker ( -0); and the other was processed by the zeolite adsorption method (- -A- -.). Lines were calculated by linear regression of y on x, using eight points.
pies stored at 4°C for 14 months. Recovery of interferon activity by precipitation was relatively poor and inconsistent using
307
INTERFERON
zinc-alcohol, or co-precipitation either with albumin in the presence of trichloroacetic acid (TCA) (Table 1A). Interferons prepared by both of these methods were inactivated to various extents during freeze-drying or storage at 4°C (Table 1B). Two methods of charge-dependent purification of interferon yielded products after with similar stability properties freeze-drying, as determined by the LNS test. Both were consistently more stable than interferon which had been precipitated by alcohol or TCA (Fig. 2). The SE-Sephadex column eluant, stabilized by the addition of BSA during the collection of fractions, provided about a 10% recovery of the original activity (personal communication, K. Paucker ) ; recovery of activity by our batch method of SE-Sephadex adsorption was similarly low. Inter-
B
0
Titw below dksn L F-D
L
3.1 Recovery
n
3.0
q
0 ,caro,
2.9
2.8
L
2.7
F-D
t
3.1
0
3.0
2.9
00,
2.8
2.7
R-m I/T
x IO3
I/T
x IO3
FIG. 3. Linear nonisothermal storage test of zeolite treated interferon freeze-dried in the presence of various stabilizing agents. The relative recovery of activity after the freezedrying process (F-D recovery) is shown as log of the percentage of the activity in the sample with the highest titer, set equal to 2 log ( 100%). Stability is expressed by the plot of log of the percent of activity in the sample relative to the freeze-dried control stored at -70°C (set equal to 2 log, or 100%) at each temperature where samples were taken. The temperature is represented as 1000 times the reciprocal of the absolute temperature (l/T X 109). Two different lots of interferon were tested and are shown separately in parts A and B. Stabilizers were tested as follows: none (O), 0.1% gelatin (A), 0.5% BSA ( l ), 1.0% and 1.0% calcium lactobionate (0). BSA (A), 1.0% sorbitol (Cl), 0.25 M sucrose (l), Lines were calculated by linear regression of y on x using the points directly above or below the line as drawn.
30s
JAMESON,
GREIFF,
AND
feron recovery from the zeolite adsorption process was usually 50 to 106% of original activity, with a purification of about lofold (Table 3). The effects of many other variables (pH, residual moisture, stabilizers, etc.) on freeze-drying and stability of the dried interferon were assessed using interferon partially purified ‘by the zeolite adsorption method. Conditions
activity) (Fig. 4B). With some lots of interferon a loss of 25 to 75% of the activity was observed during freeze-drying at pH 7; and in one experiment, even greater inactivation occurred at lower pH ( Fig. 4A). Therefore, during preliminary experiments the buffer used for suspension of interferon samples was 0.1 M potassium phosphate at neutral pH. The potassium phosphate buffer was selected because it undergoes less change in pH during freezing in contrast to sodium phosphate buffers which tend to have an acidic shift during freezing (22). However, two different lots of interferon purified by SEsubSephadex column chromatography jected to the LNS test, exhibited markedly different stabilities, which appeared to be related to the buffer solution present during the freeze-drying process. One lot (FD) had been freeze-dried in 0.1 M SOdium phosphate buffer by Dr. Paucker, then reconstituted in 0.1 M potassium phosphate buffer in this laboratory for use in comparison with the zeolite-purified interferon contained in that buffer. The other lot (L) was received as a frozen sample in 0.1 M sodium phosphate buffer and used without further dilution or treat-
of Freeze-D Tying
Several cryoprotective agents were tested as additives for the purpose of stabilizing interferon during and after freezedrying: calcium lactobionate, lactose, sucrose, sorbitol, gelatin, and BSA. Since the best stabilization was obtained with BSA, it was used in the majority of freeze-drying studies testing the influence of such other conditions as buffer solution composition, and gaseous phase (Fig. 3). Inactivation of murine interferon in citrate-phosphate buffer was retarded during the early phases (50 to 80°C) of the LNS test at pH 4 compared with pH 7, 6, or 5 (in order of decreasing stabilization); but the extent of degradation at all four pH values in samples taken at 90°C was similar (a loss of 90% of the original TABLE Partial Treatment
Purification
of &Iurine
3
Interferon
by Zeolite Adsorption Fold purification
Interferon~
Proteina mg/ml
GROSSBERG
% of original
units/ml
units/mg
Perchlorate inactivation of virus
0.140
100
16,000
114,000
Supernatant fluid after zeolite adsorptionc
0.160
116
5,200
32,500
Eluant pH 7.4 1 M sodium phosphate
0.016
11
18,000
1,130,000
% of original
100
32.5
113
1.0
0.29
10.9
a Protein was estimated optically using the correction factors determined by Warburg and Christian b Interferon was titrated by the GDVII hemagglutinin yield-reduction method (19). c Results were corrected for 1: 4 dilution with pH 3.5 buffer.
(23).
STABILIZATION
+4 -70 ccdds
OF MURINE
-!-
+50
60
70
TEMPERATURE
80
,‘,,-..*.I
_ ------aless Man 50
I;\
PC)
309
INTERFERON
90
-70 CClhl
+50
60
1
70
TEMPERATURE
80
-&90
(“3
FIG. 4. Recovery of interferon activity and linear nonisothermal storage test of different lots of zeolite treated interferon supplemented with 0.5% BSA and freeze-dried in 0.1 M buffers at several different hydrogen ion concentrations. B. The activity in the original sample stored at -t4”C is plotted next to the activity detected in freeze-dried samples stored at -70°C. One lot of interferon was freeze-dried after dialysis to equilibrium with potassium phosphate buffer alone or in combination with citric acid to obtain the lower hydrogen ion concentrations and provide adequate buffering capacity: pH 7 (0), pH 6 ( l ), pH 5 (A), and pH 4 ( A). Results with pH 3 were similar to those obtained with pH 5 and are not shown. A. Three different lots of interferon were freeze-dried at pH 7 in 0.1 M potassium phosphate buffer; lot 050571 (0), lot 121870 ( l ), and lot 040472 ( 0). The last lot was also tested at pH 6 (a), and pH 5 (0) in 0.1 M potassium phosphate buffer. The activity in the original sample stored as a liquid at +4”C is plotted next to the activity recovered after freeze-drying and storage at -70°C.
ment. The lot which contained only sodium salts appeared to be more stable than the sample containing both sodium and potassium salts (Fig. 5). The effects of sodium and potassium containing phosphate buffer solutions were therefore tested in two separate experiments by using a sing1.e lot of zeolite-purified interferon supplemented with 0.5% BSA which was divided into two portions for the final dialysis steps and equilibrated with either sodium or potassium phosphate buffer, 0.1 M, pH 7. In each experiment, the sodium phosphate sample was much more stable than the one containing potassium salts (Fig. 6). All of the inactivation rates in this representation were linear (T 2 0.82) at the 95% probability level, except for one sodium phosphate sample (r = 0.66)
for which all titers obtained fell within a two-fold range. The final interferon preparation for use as a reference reagent therefore contained 0.1 M sodium phosphate at pH 7. The conditions of freeze-drying, which had previously been developed for preservation of viruses such as influenza and measles (8), provided adequate conditions for the preservation of interferon; no further adjustments were made in the procedure. Samples dried to residual moistures of 0.1, 1, 3, or 6% exhibited similar inactivation profiles in the LNS test. In order to provide optimal protection of the interferon during long-term storage, it was preferable to seal the ampoules at atmospheric pressure rather than under a vacuum. Therefore, we tested the stability
JAMESON,
310
GREIFF,
AND
GROSSBERG
of freeze-dried interferon in the presence of highly purified argon at one atmosphere as compared with interferon sealed under the vacuum achieved by the freeze-drying process. There was no apparent difference in the stability of interferon preserved under these two different atmospheres, as determined by the LNS test. IL
I 3.1
Murine
Interferon
Reference Reagent
Interferon was produced in the presence of MEM-FBS2 after induction of L cells with NDV. The interferon was treated with perchloric acid to inactivate the inducing virus and partially purified by the zeolite adsorption technique. The resulting solution of this partially purified interferon preparation contained 0.1 M sodium phosphate buffer at pH 7 with 0.5%
I.1
F-D RWJVery
3.1
3.0 I/T
2.9
2.0
2.7
x IO3
FIG. 5. Linear nonisothermal storage test of SE-Sephadex purified interferon supplemented with 0.5% BSA and freeze-dried in the presence of different buffers at pH 7. Two different lots of L cell interferon partially purified by SESephadex column chromatography were supplemented with 0.5% BSA and freeze-dried in 0.1 M sodium phosphate buffer, and reconstituted in 0.1 M potassium phosphate buffer for freezedrying immediately ( -c) or after storage for 1 month at 4°C (- -A- -); lot L was stored at -20°C in 0.1 M sodium phosphate buffer until thawed and dispensed for freeze-drying (- 0 -). The interferon activity and temperature of each sample are represented as described in Fig. 3. Lines were calculated by linear regression of y on x using those points directly above or below the line as drawn.
3.0
Id
29 I/T
I
28
t
2.7
x IO3
FIG. 6. Linear nonisothermal storage test of stability of murine interferon freeze-dried in medium containing either sodium phosphate or potassium phosphate buffer. Solid symbols designate interferon in a buffer made with sodium salts ( l , A ) and open symbols refer to samples in a buffer with potassium salts (0, A). Results obtained in two separate experiments with pH 7 buffers prepared with 0.1 M phosphate salts are distinguished as circles (0, o ) and triangles (A, A). The interferon activity and temperature of each sample are represented as described in Fig. 3. Linear correlation coefficients (r) were determined for the lines calculated by linear regression of y on x: A, r = 0.66; 0, r = 0.92; 0, r = 0.99; and A, r = 0.93.
BSA. The interferon activity in the final preparation possessed those properties attributed to murine interferon (17) as follows: a molecular weight of 25,006 daltons (by G-100 Sephadex chromatography); nonsedimentable (100,000 g for 2 hr); stable at pH 2 for 48 hr at 4°C; stable at 56°C for 1 hr; and destroyed by trypsin. There was no antiviral activity detectable in primary chicken embryo fibroblast cultures or in the BUD-8 diploid human cell strain. There was no residual NDV or mycoplasma contamination in the preparation; and the many freeze-dried samples tested contained no bacteria or fungi, as determined by accepted techniques described in materials and methods. Several types of stability tests were performed using the freeze-dried interferon. At a temperature of 90°C it was stable for more than 18 hr. During the LNS test there was no loss in activity. The MIS test (Fig. 7) provided results that made it possible to calculate conservative esti-
STABILIZATION
Cl
20
OF MURINE
40
60
a0
311
INTERFERON
100
120
I40
160 180
DAYS FIG. 7. Multiple (G002-904-5117. 52 (O), 60 (O),
isothermal storage Samples were hid or 68°C (A).
test of freeze-dried for the indicated
mates of stability (11) and predict that the freeze-dried interferon would lose 1000 units of activity during storage at 37°C for IO0 days, at 20°C for 5.5 years, or at 4°C for 110 years. To determine the usefulness of these predictions, a confirmatory stability test, with samples placed at 37”C, was initiated and is still in progress (Fig. 8). During the first 6 years of this test, the murine interferon reference reagent was, as expected, more stable than the intentionally conservative prediction. The reference reagent interferon, available from the National Institutes of Health (NIH), Catalog No. G002-904-511, was assigned an activity of 12,000 units/ml; the assignment was made from results pooled from several laboratories that titrated simultaneously this reagent with the previous interim reference reagent provided by the NIH as a frozen mouse serum. DISCUSSION
From the data presented, it is clear that the methods used for treatment and purification of the interferon can influence the ultimate stability of the interferon even
murine periods
interferon reference reagent of time at temperatures of
after it has been freeze-dried. In particular, the manner of inactivation of inducing virus, the method of purification of the interferon, the composition of the suspending medium, and the presence of stabilizing additives may all contribute independently to the ultimate stability of the freeze-dried product. In view of the rela-
YEARS
FIG. 8. Confirmatory stability test of freeze-dried murine interferon reference reagent (G002-904-511) by storage at 37°C. The predicted inac-tivation rate (0) was obtained from the multipleisothermal storage test shown in Fig. 7. The ob-served loss of activity ( l ) was expressed as ti percentage of the activity measured for the control sample which had been stored at -70°C for the same length of time as the test sample, and was titrated in the same assay.
312
JAMESON,
GREIFF,
tive impurity of the interferon used for the reference reagent, it is not possible to determine whether these effects are directly on the interferon molecule or on other molecules remaining in the preparation at the time of freeze-drying. For example, the greater stability in the presence of sodium ions may not necessarily indicate that potassium ions are directly detrimental to the stability or activity of the interferon molecule but may reflect the consequence of an interaction of potassium with yet another substance in the preparation, or may simply be a result of the acidic change in pH which probably occurs during the freezing of the mixture of sodium phosphate salts present in that buffer solution (22). Low pH does enhance stability of liquid (15) and freezedried interferon ( reported here). One difficulty in testing and predicting the stability of freeze-dried materials is, paradoxically, their exceptional stability as compared with the liquid counterpart. For example, if the test interferons had stability properties similar to the NIH reference reagent, predicted to be stable for 5.5 years at 20°C it would not have been possible to conduct direct stability tests requiring such exceptional lengths of time to detect even very small changes in activity. Therefore, accelerated storage tests were adapted for these studies and made it possible not only to compare rapidly the relative stabilities of different types of freeze-dried interferon preparations but also to predict stabilities at low temperatures of storage. The methods described herein have provided a basis for the development of freeze-drying processes for the preservation of other types of interferons to be used as reference reagents as well as medication in clinical trials. The results and conclusions of these studies which led to the selection of procedures and conditions for the preparation of the NIH freeze-dried murine interferon reference reagent should not be interpreted as
AND
GROSSBERG
excluding the existence of other methods which may also provide satisfactory stabilization of interferon, It should be stressed that the prospect of stabilizing interferons for clinical use is one of great concern in view of the many problems investigators have experienced in unpredictable loss of activity of interferon samples prior to their use in the treatment of patients. To the present time, freeze-drying, a method acceptable in the preparation of biological or pharmaceutical substances for clinica application, remains the best available method of stabilizing interferon for longterm storage. SUMMARY
Conditions of interferon processing were analyzed to select those that promote stability after freeze-drying. The effects of various preparative methods and treatment conditions were assessed by measuring the retention of biological activity by lyophilized interferon samples in two kinds of accelerated storage tests: the linear nonisothermal stability (LNS) test, a rapid method used for direct comparison of two or more preparations of interferon, and the multiple isothermal storage (MIS) test, a slow method requiring weeks to months to obtain data for the prediction of stability of a given preparation stored under various conditions. The most stable preparations of Newcastle-disease-virus-induced mouse L cell interferon were obtained using the following conditions: 1) perchlorate treatment to inactivate residual inducing virus, 2) nonspecific adsorption using zeolite for partial purification, 3) suspending medium of 0.5% bovine serum albumin in 0.1 M sodium phosphate buffer at pH 7, and 4) sublimation of ice in vacua with a starting temperature of -30°C to a final residual moisture of about 3%. The final product, reference reagent GOO2-QO4511, was stable throughout the course of the LNS test. From an extensive MIS test,
STABILIZATION
OF MURINE
this reference interferon was predicted to lose 1000 units of activity in 110 years at 4°C and 1000 units in 100 days at 37°C. After 6 years of storage at 37°C when the predicted residual activity would be about 2070 of the original potency, 35% of initial interferon activity remained, confirming the usefulness of the short-term predictive test. ACKNOWLEDGMENTS We thank Mrs. Patricia Z. Johnson and Mrs. Christine K. Schoenherr for their excellent technical assistance. We are grateful to Dr. J. J. Sedmak for his help in chromatographic estimation of the molecular weight of the interferon. This work was supported by award NOlAI42514 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health. REFERENCES 1. Baron, S., and Buckler, C. E. Circulating interferon in mice after intravenous injection of virus. Science 141, 1061-1063 (1963). 2. Cohn, E. J., Gurd, F. R. N., Surgenor, D. M., Barnes, B. A., Brown, R. K., Derouaux, G., Gillespie, J. M., Kahnt, F. W., Lever, W. F., Liu, C. H., Mittelman, D., Moutin, R. F., Schmid, K., and Uroma, E. A system for the separation of the components of human blood: Quantitative procedures for the separation of the protein components of human plasma. J. Amer. Chem. Sot. 72,
465-474 ( 1950). 3. Cole, B. R., and Leadbeater,
4. 5.
6.
7.
L. A critical assessment of an accelerated storage test. I. Plzarm. Pharmacol. 18, 101-111 (1966). purification of calf Davies, A. The partial interferon. Biochem. J. 90, 29P-30P (1964). Fantes, K. H. Purification of interferon from chick embryonic allantoic fluids and fibroblast tissue infected with influenza virus. J. Gen. Viral. 1, 257-267 (1967). Finter, N. B. The assay and standardization of interferon and interferon inducers. In “Interferons and Interferon Inducers” (N. B. Fin&x, Ed.), Chapter 7, pp. 135-169. North-Holland, Amsterdam; American Elsevier, New York, 1973. reagents in interGalasso, G. J. Standard feron research, In “International Symposium on Interferon and Interferon Inducers, London, 1969” (F. T. Perkins and R. H.
INTERFERON
Regamy, Eds.),
313
S. Karger, Basel. Symp. Ser. Immunobiol. Stand. 14, 272-276 (1970). 8. Greiff, D. Freeze-drying cycles. In “International Symposium on Freeze-Drying of Biological Products, 1976” (V. J. Cabasso and R. H. Regamey, Eds.), S. Karger, Basel. Den. Biok Stand. 37, 105-115 (1977). 9. Greiff, D., and Greiff, C. Linear nonisothermal, single-step, stability studies of dried preparations of influenza virus. Cryobiology 9, 34-37 ( 1972). 10. Greiff, D., Melton, H., and Rowe, T. W. G. On the sealing of gas-filled ampoules. Cryobiology 12, 1-14 ( 1975). 11. Creiff, D., and Rightsel, W. A. An accelerated storage test for predicting the stability of suspensions of measles virus dried by sublimation in vucuo. j. lmmunol. 94, 395-400 ( 1965 ). 12. Greiff, D., and Rightsel, W. A. Stability of suspensions of influenza virus dried to different contents of residual moisture by sub16, limation in vacua. Appl. Microbial. 835-840 ( 1968). 13. Greiff, D., and Rightsel, W. A. Stabilities of dried suspensions of influenza virus sealed in a vacuum or under different gases. Appl. Microbial. 17, 830-835 ( 1969). 14. Jameson, P., Dixon, M. A., and Grossberg, S. E. A sensitive interferon assay for many species of cells: encephalomyocarditis virus hemagglutinin yield reduction. PTOC. Sot. Exp. Biol. Med. X5.5, 173-178 ( 1977). 15. Jariwalla, R., Grossberg, S. E., and Sedmak, J. J. The influence of physicochemical factors on the thermal inactivation of murine interferon. Arch. Viral. 49, 261-272 (1975). 16. Lampson, G. P., Tytell, A. A., Nemes, M. M., and Hilleman, M. R. Purification and characterization of chick embryo interferon. Proc. Sot. Exp. Biol. Med. 112, 468-478 (1963). 17. Lockhart, R. Z., Jr. Criteria for acceptance of a viral inhibitor as an interferon and a general description of the biological properties of known interferons. In “Lnterferons and Interferon Inducers” (N. B. Finter, Ed.), Chapter 2, pp. 11-27. North-Holland, Amsterdam; American Elsevier, New York, 1973. 18. Morahan, P. S., and Grossberg, S. E. Agerelated cellular resistance of the chicken embryo to viral infections. I. Interferon and natural resistance to myxoviruses and vesicular stomatitis virus. J. Infect. Dis. 121, 615-623 ( 1970). 19. Oie, H. K., Buckler, C. E., Uhlendorf, C. P.,
314
JAMESON,
GREIFF,
Hill, D. A., and Baron, S. B. Improved assays for a variety of interferons. Proc. Sot. Exp. Biol. Med. 140, 1178-1181 (1972). 20. Paucker, K., and Stancek, D. Characterization of interferon-associated proteins. J. Gen. Viral. 15, 129-138 ( 1972). 21. Perkins, F. T. Discussion on recommendations. In “International Symposium on Interferon and Interferon Inducers, London, 1969” (F: T. Perkins and R. H. Regamy, Eds.), S. Karger, Basel. Symp. Ser. Immunobioz. Stand 14, 297-325 ( 1970). 22. Smith, A. U. Effects of subzero temperatures on water and on physiological media. In
AND
GROSSBERG
“Biological Effects of Freezing and Supercooling” (A. U. Smith, Ed.), Chapter 11, pp. 369-405. Williams and Wilkins, Baltimore, 1961. 23. Williams, C. A., and Chase, M. W. Chemical analyses. In “Methods in Immunology and Immunochemistry” (C. A. Williams and M. W. Chase, Eds.), Vol. 2, Chapter 12, pp. 286-282. Academic Press, New York, 1968. 24. Zoglio, M. A., Windheuser, J. J., Vatti, R., Maulding, H. V., Komblum, S. S., Jacobs, A., and Hamot, H. Linear nonisothermal stability studies. J. Pharm. Sci. 57, .20802085 ( 1968).
16,
301-314
Thermal Analysis
PATRICIA Departments
(1979)
Stability
of Freeze-Dried
of Freeze-Drying
JAMESON,
Conditions and Accelerated for Murine Interferon
DONALD
GREIFF,*
of Microbiology
and * Pathology, P.O. Box 26509, Milwaukee,
INTRODUCTION
Interferon is an antiviral protein released from appropriately stimulated animal cells; recent clinical use suggests fulfillment of its great promise as an antiviral and antitumor agent. The necessity for standard reference interferon preparations has been clearly established (6, 7, 21). For example, they provide a basis for comparison of results obtained in different laboratories throughout the world studying interferon with a multitude of assay methods. Although interferon is a relatively stable protein as compared with many enzymes, some preparations were rather labile under the conditions of storage employed to preserve interferon samples intended for use as reference reagents or in clinical trials. Previous freeze-dried reference preparations have lost potency at variable and unknown rates during storage or shipping (7). Our specific objectives, therefore, were to develop procedures for stabilizing interferon by freeze-drying, and to predict the rate at which inactivation would occur under various storage conditions. This report describes an evaluation of factors that may contribute to loss of interferon Received 10, 1979.
November
20,
1978;
Mammalian
accepted
May
AND
SIDNEY
lnterferons Storage
Tests
E. GROSSBERG
The Medical College Wisconsin 53226
of
Wisconsin,
activity following freeze-drying. Murine interferon was selected because interferon of high potency could be prepared readily. Methods of interferon preparation and partial purification as well as methods of freezedrying were evaluated to obtain the most stable preparation of murine interferon possible. To do this it was necessary to adapt accelerated storage tests to predict the rate at which activity would be lost by freeze-dried preparations of interferon. MATERIALS
Preparation
AND
of Murine
METHODS
Interferon
Confluent monolayers of L cells in 32-0~ (1.05 1) prescription bottles or 0.5 gallon (2.1 1) roller bottles were washed with GLB (Hanks’ balanced salt solution, HBSS, containing 1% gelatin and 0.5% lactalbumin hydrolysate), then inoculated with high multiplicities ( 10 to 100 plaqueforming units/cell) of a velogenic Newcastle disease virus (NDV) strain CC diluted in GLB, which was allowed to adsorb for 1 hr at 36°C. Eagle’s minimal essential medium (MEM) supplemented with 2% fetal bovine serum (FBS), MEMFB&, was added at one-half the volume used for cell growth, and cultures were incubated 16 to 24 hr at 36°C. Harvests were pooled and frozen at -20°C until. use. 301 OOll-2240/79/040301-14$02.00/0 Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form resewd.
302 Interferon
JAMESON,
GREIFF,
Assay
Two methods of interferon assay were used. The first was a plaque-reduction assay in monolayers of L cells inoculated with vesicular stomatitis virus (VSV) after overnight exposure to interferon dilutions made in MEM-FBS,; titers were expressed in units representing the median plaquedepressing dose (PDDBo) obtained from dose-response curves. The second method was the CDVII virus yield-reduction assay of Oie et al. (19) which was used for the majority of the studies involving comparison of stabilities of various types of interferon preparations; titers were expressed in units of the 0.5 log hemagglutinin yield-reduction endpoint. The laboratory reference interferon included in all titrations was a mouse plasma interferon preparation obtained by injection of adult mice with NDV according to the method of Baron and Buckler (1). This laboratory standard was used to monitor the relative consistency of the assay. No adjustment was made in the titers of individual test samples. This assay measures essentially the same unitage (12,251; standard deviation l.Cfold) as that assigned (12,000) to the National Institutes of Health (NIH) interferon standard (GOO2-904-511). Treatment
of Interferon
Processing of crude interferon in cell culture supernatant fluids or plasma consisted of the inactivation and precipitation of NDV by the addition of 2 N perchloric acid at a ratio of 7.5 ml per 100 ml of fluid at 4°C (16). The mixture was stirred at 4°C for 39 min, then centrifuged in plastic tubes at SOOg for 30 min at 4°C. The supernatant fluids were decanted into a plastic bottle, and the pH adjusted to neutrality with 5 N potassium hydroxide. The resulting precipitate, optimized by reduction of temperature to near freezing, was removed by centrifugation as above, and
AND
GROSSBERG
the supernatant fluid again decanted into a small plastic bottle for storage at -70°C. A small portion was placed in a plastic tube for use in titration, and frozen at -20’ C until assayed. Alternatively, inducing virus was inactivated by dialysis of the interferon preparation against 0.15 N HCI-KC1 buffer at pH 2 for 2 days at 4°C. Partial purification of the murine interferon was obtained by several different methods. Alcohol precipitation of interferon in the presence of 0.02 M zinc acetate was accomplished by slow addition of acetate-buffered 24% alcohol to the -5°C sample with continuous stirring (2). The precipitate was dissolved in unbuffered HBSS (without bicarbonate) with 0.1% gelatin and stored at 4°C. Trichloroacetic acid precipitation of interferon in the presence of 0.1% bovine serum albumin (BSA) was done according to the method of Davies (4). Precipitate containing interferon was dissolved in unbuffered HBSS and stored at 4°C. L cell interferon purified by SE-Sephadex column chromatography (20) was provided by Dr. Kurt Paucker. Two different lots containing 0.5% bovine serum albumin ( BSA) were supplied: one was freeze-dried (FD) and the other frozen (L) in pH 7 sodium phosphate buffer (0.1 M). We also tested a batch method adsorption with SE-Sephadex under conditions based on those described for the column method by Paucker and Stancek (20) to increase the rate of processing and avoid the addition of protein stabilizers. Results were similar to those reported for the SE-Sephadex column method (20) : approximately lo-fold purification was obtained with recovery of about 10yO of the initial activity. Another method of interferon processing was the nonspecific adsorption to zeolite as developed by Sonnabend and Katsoyannis (personal communication, 1971) as a modification of the Doucil process for
STABILIZATION
OF MURINE
chick interferon described by Fantes (5). Interferon was adsorbed to zeolite at low pH in low ionic strength buffer and eluted in high molar@ neutral buffer. The eluant was concentrated by removal of water using carbowax and extensively dialyzed to yield a partially purified interferon in a selected buffer supplemented with a desired stabilizing agent. The resulting interferon solution was filter sterilized using a 0.2.2 ,uM membrane with a prefilter; filtration was expedited by quickly warming the solution to 37°C prior to filtration. The final buffer composition was either 0.1 M potassium phosphate or 0.1 M sodium phosphate. Cell Cultures The mouse L cell line was obtained from Microbiological Associates (Walkersville, Md.) and was propagated in MEM with Hanks’ salt solution supplemented with 10% FBS. After treatment of cells with viruses to induce interferon, the cultures were maintained in medium containing 2% FBS. Viruses The GDVII virus, VSV and NDV were obtained and propagated as previously described ( 14, 18, 19). Freeze-Drying
of Interferon
Samples of interferon were dispensed in I-ml portions in glass vials which were then held at 4°C until placed in the freeze-drying apparatus. Each type of interferon sample was freeze-dried separately by a process determined by the nature of the suspending medium and the size of the lot, using methods previously described by Greiff and Rightsel ( 12 ) . The freeze-drying cycle used in the present studies was based on that found most effective for influenza virus (8). Lyophilization vials containing 1 ml of interferon
INTERFERON
303
were placed in a specially designed chamber freeze-dryer (Edwards High Vacuum, Ltd., Crawley, Sussex, England) as described by Greiff (8) and cooled to a terminal temperature of -30°C. No attempt was made to modify or suppress supercooling. After the samples had been frozen at -30°C the chamber was evacuated. Although the shelf temperature was maintained at -30°C the product temperature declined to -40°C within approximately 1 hr because of evaporative cooling. At this time, the pressure within the chamber was approximately lo-? Torr (10 p Hg). Evaporative cooling maintained product temperature at -40°C for approximately 12 hr, after which time the product temperature began to rise. At this point in the cycle, approximately 85% of the water had been removed from the frozen sample. When the product temperature reached -30°C shelf temperature was set at 0°C and maintained at that temperature throughout the remainder of the drying cycle. Between the 14th and 17th hr of the drying cycle, product temperature rose slowly and stabilized at 0°C. During this time, the vacuum within the chamber dropped from 10m2 Torr to approximately 10m3 Torr. Drying by sublimation of ice in VacUo at 0°C was continued until the vacuum within the chamber was 10m5Torr. The desired residual moisture may be obtained by adjusting the length of time for the freeze-drying cycle (8). In general, with the freeze-dryer used, approximate terminal pressures within the chamber and corresponding residual moistures were as follows: 5 X 1O-5 Torr, 3$%0; lO-‘j Torr, 2%; and 5 X lo-’ Torr, 1%. The vacuum obtained in preliminary experiments corresponded to a residual moisture of about 1 to 3%; total elapsed time for freeze-drying 50 to 100 ampoules of 1 ml interferon in solutions containing gelatin was approximately 22 hr, with BSA about 30 hr, and with serum about 26 hr. Larger loads required longer times.
304 Accelerated Stability
JAMESON,
GREIFF,
Storage Tests of Interferon
Two accelerated storage tests were used to obtain data for predicting the stabilities of freeze-dried preparations of interferon. Tests developed for the purpose of predicting stability of pharmaceutical preparations (3, 24) and certain freeze-dried viruses (9) were adapted for the evaluation of stability of interferon samples. The linear nonisothermal stability (LNS) test was used primarily to compare stabilities of two or more samples of interferon prepared in different ways; a starting temperature of 50°C was obtained by a 30min equilibration period after which the temperature was allowed to rise to 90°C at a rate of 1.5”C per hr (~27 hr in duration). Samples were taken at the end of the equilibration period and during the anticipated inactivation phase of the heating. The multiple isothermal stability (MIS) test was used primarily to predict the storage life of a given interferon sample after freeze-drying (11). Five or more samples of the interferon preparation were placed at each of three temperatures selected to provide appropriate intervals for subsequent analysis; the temperatures used were 52, 60, and 68°C. Samples were taken at intervals from 10 days through 6 months. Samples taken during either of these two types of accelerated storage tests were placed at -70°C until the time at which all samples within a group were assayed simultaneously. Samples were reconstituted for titration by the addition of 1 ml of sterile water and dilutions were made in maintenance medium ( MEM-FBSZ). Rubber-stoppled vials that had lost vacuum, as observed during reconstitution, were replaced by intact replicates exposed to the same storage conditions.
AND
GROSSBERG
Confkmatory Stability
Storage Tests of Interferon
In order to determine how accurate the predictive MIS test would be, several heatsealed glass ampoules of freeze-dried interferon were placed at each of the following temperatures: -70, -20, 4, 22, 37, and 56°C. In this test, still in progress, samples are taken at intervals of several months for many years in order to determine the long-range stability of interferon stored at these various temperatures and compare the observed stability with the predicted activities obtained from the MIS accelerated storage test. Characterization
of Interferon
In addition to measurements of interferon potency and stability, the interferon freeze-dried to provide the reference reagent to the NIH was tested for physicochemica1 properties, purity, and microbial sterility. The physico-chemical characterization included molecular exclusion chromatography using G-100 Sephadex, ultracentrifugation at 100,OOOg for 2 hr, and trypsinization. Purity was assessed by the amount of antiviral activity which could be detected using heterologous cell cultures, such as primary chicken embryo fibroblast and human cell strain (BUD-S) cultures, as well as by specific tests for residual inducing virus including plaque assays in chicken embryo cell cultures and blind passage in embryonated eggs. The interferon was tested for the presence of bacteria by cultivation on brain-heart infusion agar for 2 weeks at 36°C for fungi by cultivation on Sabouraud’s agar for 3 weeks at 22°C and for mycoplasma by cultivation on appropriate medium for 2 weeks at 36°C in an atmosphere of nitrogen. Vials and Ampoules for Freeze-Dying During the preliminary experiments testing various methods of processing and
STABILIZATION
OF MURINE TABLE
305
INTERFERON
1
Recovery of interferon activity during several experiments testing various methods of virus inactivation, partial purification, and freeze-drying Part
Interferon
Treatment I
A
Part
None6 Perchlorate pH 2 dialysis Perchlorate, then : zinc-alcohol precipitation TCA precipitation Treatment
activity
(units/mk)
II
III
None Perchlorate pH 2 dialysis Perchlorate, then : zinc-alcohol precipitation TCA precipitation
IV
V
< 1,000 3,300 nt
nt 2,000 nt
8,300 6,800 8,600
4,700d 3,500 nt
440’ 440’
< 1,000 < 1,000
13,000 3,400
<500 1,700
2,700 1,500
activity in samples after freeze-drying
III
B
shown
ntc 610 nt
Interferon
Liquid
in experiments
from the indicated or storage at +4”C.
experiments
IV F-D/
Liquid
V F-Dr
Liquid
F-D,
28,000 3,500 nt
18,000 4,600 nt
3,500 2,300 3,500
3,300 2,100 720
nt 3,900 nt
nt 1,500 nt
180 340
<40 230
nt 1,000
nt 380
3,100 1,400
3,100 1,000
QInterferon was titrated by the GDVII hemagglutinin yield-reduction method (19). b A portion of the lot was stored at +4’C during the processing of the rest of the lot. c Not tested. d This titer was obtained by adjusting for the dilution made of the original sample to provide the raw material to be tested by the various procedures. e This titer ~a.8 obtained by correction for the concentration factor relative to the original sample, which was introduced during processing. f The original samples were preserved as a liquid at +4”C; and the freeze-dried (F-D) samples were stored at -20°C until titrated.
additives for freeze-drying, interferon samples were placed in neutral glass vials obtained from Johnsen and Jorgensen (Trident) Ltd., London, England. These vials were sealed under vacuum at the conclusion of the freeze-drying process and the butyl rubber stopples held in place by metal closures. Interferon for use as a reference reagent was dispensed in l-ml volumes in borosilicate glass ampoules obtained from Wheaton Scientific (Millville, N.J. ) and freezedried to a residual moisture of approximately 3%. These ampoules were heat-sealed containing an atmosphere of argon provided by back-flushing at the conclusion of the freeze-drying process (13). In order to insure complete closure,
the heat-sealed tips of the vials were then dipped in neoprene solution (10) to close the small channels which often remain after heat-sealing of glass vials under atmospheric pressure. RESULTS
Inactivation
of Residual Inducer
Virus
NDV remaining in the interferon induced in L cell cultures was inactivated either by perchloric acid treatment or by dialysis against pH 2 buffer. There was usually no remarkable change in interferon activity of the preparation by either of these methods (Table 1) although the NDV was completely inactivated as determined by infectivity tests using embryo-
JAMESON,
306
GREIFF,
AND
GROSSBERG
nated eggs. The perchloric acid method is shorter, does not involve placing infectious material into dialysis tubing, and provides an interferon that is more stable after freeze-drying than a preparation exposed to the pH 2 treatment (Fig. 1). One sample exposed to pH 2 lost about 80% of its activity during freeze-drying alone (Table 1). The percblorate method was therefore used in our subsequent studies of murine interferon. Purification
IO280 50
after storage at -20°C as long as 14 months, and during storage for 7 months at 4°C (Table 2). Samples were therefore held at 4°C for the few hours or days between the completion of processing and the initiation of freeze-drying to avoid an additional freeze-thaw cycle. As much as 50 to 90% of the activity was lost in sam-
TABLE
Treatment
2
of Various Interferon Samples Stored as a Liquid -2O”C, or in Freeze-Dried Form at -20°C. Initial activity (units/ml) ----Liquid
Remaining months
Freezedried
Liquid, 1-3
Liquid.
13-15
at +4”C
or
activity after storage for the number shown under the conditions described.
at 4°C 6-8
(‘C)
FIG. 1. Linear nonisothermal storage test of crude interferon samples after freeze-drying. The original 1, cell inteferon ( 0) was treated to inactivate the residual inducing virus (NDV) by dialysis against pH 2 buffer (A) or by the perchlorate precipitation method ( 0 ).
Four methods for partial preparation of interferon were evaluated: zeolite adsorption, co-precipitation by trichloroacetic acid treatment in the presence of albumin, zinc-alcohol precipitation at low temperature, and ion exchange adsorption. Untreated or perchlorate-treated interferon samples did not lose activity during freeze-drying or the corresponding short term storage of control samples at 4°C (Table 1B). Liquid crude and perchloratetreated interferon samples were stable
Sample
70
TEMPERATLRE
Methods
Stability
60
l-3
at 6-8
-20°C
of
Freeze-dried, at -20°C 13-15 1-3
A
NOIE Zn-alcohol precipitated TCA precipitated
B
NOlIE
C
NOID?
Perchlorate
Perchlorate D
NOW2
3,500
1,500
3,900
420
2.700
3,100
3,100
1,800
1,500
1,000
1,400
NOW
8,600
10,000 3,300 2,800
1,700
1,600
680
8,500 3.800
11,000 8,200
860
3,900
740
2,400 3,900 (-7OOC)
150 2,700 7,600
13-15
150
17,000 7,400 14,000
Perchlorate E
6-8
5,300 1,200 6.800
1,100 3.000 620 800
580 740
540 1,200
STABILIZATIOPi
L 1 -7’0 +50 Cant~l
c
” 60
k 70
TEMPERATURE
8
’ 60
t
OF MURIiYE
a 90
(“C)
FIG. 2. Linear nonisothermal storage test of two different partially purified interferon samples supplemented with 0.5% BSA. One was prepared by SE-Sephadex column chromatography and supplied by Dr. K. Paucker ( -0); and the other was processed by the zeolite adsorption method (- -A- -.). Lines were calculated by linear regression of y on x, using eight points.
pies stored at 4°C for 14 months. Recovery of interferon activity by precipitation was relatively poor and inconsistent using
307
INTERFERON
zinc-alcohol, or co-precipitation either with albumin in the presence of trichloroacetic acid (TCA) (Table 1A). Interferons prepared by both of these methods were inactivated to various extents during freeze-drying or storage at 4°C (Table 1B). Two methods of charge-dependent purification of interferon yielded products after with similar stability properties freeze-drying, as determined by the LNS test. Both were consistently more stable than interferon which had been precipitated by alcohol or TCA (Fig. 2). The SE-Sephadex column eluant, stabilized by the addition of BSA during the collection of fractions, provided about a 10% recovery of the original activity (personal communication, K. Paucker ) ; recovery of activity by our batch method of SE-Sephadex adsorption was similarly low. Inter-
B
0
Titw below dksn L F-D
L
3.1 Recovery
n
3.0
q
0 ,caro,
2.9
2.8
L
2.7
F-D
t
3.1
0
3.0
2.9
00,
2.8
2.7
R-m I/T
x IO3
I/T
x IO3
FIG. 3. Linear nonisothermal storage test of zeolite treated interferon freeze-dried in the presence of various stabilizing agents. The relative recovery of activity after the freezedrying process (F-D recovery) is shown as log of the percentage of the activity in the sample with the highest titer, set equal to 2 log ( 100%). Stability is expressed by the plot of log of the percent of activity in the sample relative to the freeze-dried control stored at -70°C (set equal to 2 log, or 100%) at each temperature where samples were taken. The temperature is represented as 1000 times the reciprocal of the absolute temperature (l/T X 109). Two different lots of interferon were tested and are shown separately in parts A and B. Stabilizers were tested as follows: none (O), 0.1% gelatin (A), 0.5% BSA ( l ), 1.0% and 1.0% calcium lactobionate (0). BSA (A), 1.0% sorbitol (Cl), 0.25 M sucrose (l), Lines were calculated by linear regression of y on x using the points directly above or below the line as drawn.
30s
JAMESON,
GREIFF,
AND
feron recovery from the zeolite adsorption process was usually 50 to 106% of original activity, with a purification of about lofold (Table 3). The effects of many other variables (pH, residual moisture, stabilizers, etc.) on freeze-drying and stability of the dried interferon were assessed using interferon partially purified ‘by the zeolite adsorption method. Conditions
activity) (Fig. 4B). With some lots of interferon a loss of 25 to 75% of the activity was observed during freeze-drying at pH 7; and in one experiment, even greater inactivation occurred at lower pH ( Fig. 4A). Therefore, during preliminary experiments the buffer used for suspension of interferon samples was 0.1 M potassium phosphate at neutral pH. The potassium phosphate buffer was selected because it undergoes less change in pH during freezing in contrast to sodium phosphate buffers which tend to have an acidic shift during freezing (22). However, two different lots of interferon purified by SEsubSephadex column chromatography jected to the LNS test, exhibited markedly different stabilities, which appeared to be related to the buffer solution present during the freeze-drying process. One lot (FD) had been freeze-dried in 0.1 M SOdium phosphate buffer by Dr. Paucker, then reconstituted in 0.1 M potassium phosphate buffer in this laboratory for use in comparison with the zeolite-purified interferon contained in that buffer. The other lot (L) was received as a frozen sample in 0.1 M sodium phosphate buffer and used without further dilution or treat-
of Freeze-D Tying
Several cryoprotective agents were tested as additives for the purpose of stabilizing interferon during and after freezedrying: calcium lactobionate, lactose, sucrose, sorbitol, gelatin, and BSA. Since the best stabilization was obtained with BSA, it was used in the majority of freeze-drying studies testing the influence of such other conditions as buffer solution composition, and gaseous phase (Fig. 3). Inactivation of murine interferon in citrate-phosphate buffer was retarded during the early phases (50 to 80°C) of the LNS test at pH 4 compared with pH 7, 6, or 5 (in order of decreasing stabilization); but the extent of degradation at all four pH values in samples taken at 90°C was similar (a loss of 90% of the original TABLE Partial Treatment
Purification
of &Iurine
3
Interferon
by Zeolite Adsorption Fold purification
Interferon~
Proteina mg/ml
GROSSBERG
% of original
units/ml
units/mg
Perchlorate inactivation of virus
0.140
100
16,000
114,000
Supernatant fluid after zeolite adsorptionc
0.160
116
5,200
32,500
Eluant pH 7.4 1 M sodium phosphate
0.016
11
18,000
1,130,000
% of original
100
32.5
113
1.0
0.29
10.9
a Protein was estimated optically using the correction factors determined by Warburg and Christian b Interferon was titrated by the GDVII hemagglutinin yield-reduction method (19). c Results were corrected for 1: 4 dilution with pH 3.5 buffer.
(23).
STABILIZATION
+4 -70 ccdds
OF MURINE
-!-
+50
60
70
TEMPERATURE
80
,‘,,-..*.I
_ ------aless Man 50
I;\
PC)
309
INTERFERON
90
-70 CClhl
+50
60
1
70
TEMPERATURE
80
-&90
(“3
FIG. 4. Recovery of interferon activity and linear nonisothermal storage test of different lots of zeolite treated interferon supplemented with 0.5% BSA and freeze-dried in 0.1 M buffers at several different hydrogen ion concentrations. B. The activity in the original sample stored at -t4”C is plotted next to the activity detected in freeze-dried samples stored at -70°C. One lot of interferon was freeze-dried after dialysis to equilibrium with potassium phosphate buffer alone or in combination with citric acid to obtain the lower hydrogen ion concentrations and provide adequate buffering capacity: pH 7 (0), pH 6 ( l ), pH 5 (A), and pH 4 ( A). Results with pH 3 were similar to those obtained with pH 5 and are not shown. A. Three different lots of interferon were freeze-dried at pH 7 in 0.1 M potassium phosphate buffer; lot 050571 (0), lot 121870 ( l ), and lot 040472 ( 0). The last lot was also tested at pH 6 (a), and pH 5 (0) in 0.1 M potassium phosphate buffer. The activity in the original sample stored as a liquid at +4”C is plotted next to the activity recovered after freeze-drying and storage at -70°C.
ment. The lot which contained only sodium salts appeared to be more stable than the sample containing both sodium and potassium salts (Fig. 5). The effects of sodium and potassium containing phosphate buffer solutions were therefore tested in two separate experiments by using a sing1.e lot of zeolite-purified interferon supplemented with 0.5% BSA which was divided into two portions for the final dialysis steps and equilibrated with either sodium or potassium phosphate buffer, 0.1 M, pH 7. In each experiment, the sodium phosphate sample was much more stable than the one containing potassium salts (Fig. 6). All of the inactivation rates in this representation were linear (T 2 0.82) at the 95% probability level, except for one sodium phosphate sample (r = 0.66)
for which all titers obtained fell within a two-fold range. The final interferon preparation for use as a reference reagent therefore contained 0.1 M sodium phosphate at pH 7. The conditions of freeze-drying, which had previously been developed for preservation of viruses such as influenza and measles (8), provided adequate conditions for the preservation of interferon; no further adjustments were made in the procedure. Samples dried to residual moistures of 0.1, 1, 3, or 6% exhibited similar inactivation profiles in the LNS test. In order to provide optimal protection of the interferon during long-term storage, it was preferable to seal the ampoules at atmospheric pressure rather than under a vacuum. Therefore, we tested the stability
JAMESON,
310
GREIFF,
AND
GROSSBERG
of freeze-dried interferon in the presence of highly purified argon at one atmosphere as compared with interferon sealed under the vacuum achieved by the freeze-drying process. There was no apparent difference in the stability of interferon preserved under these two different atmospheres, as determined by the LNS test. IL
I 3.1
Murine
Interferon
Reference Reagent
Interferon was produced in the presence of MEM-FBS2 after induction of L cells with NDV. The interferon was treated with perchloric acid to inactivate the inducing virus and partially purified by the zeolite adsorption technique. The resulting solution of this partially purified interferon preparation contained 0.1 M sodium phosphate buffer at pH 7 with 0.5%
I.1
F-D RWJVery
3.1
3.0 I/T
2.9
2.0
2.7
x IO3
FIG. 5. Linear nonisothermal storage test of SE-Sephadex purified interferon supplemented with 0.5% BSA and freeze-dried in the presence of different buffers at pH 7. Two different lots of L cell interferon partially purified by SESephadex column chromatography were supplemented with 0.5% BSA and freeze-dried in 0.1 M sodium phosphate buffer, and reconstituted in 0.1 M potassium phosphate buffer for freezedrying immediately ( -c) or after storage for 1 month at 4°C (- -A- -); lot L was stored at -20°C in 0.1 M sodium phosphate buffer until thawed and dispensed for freeze-drying (- 0 -). The interferon activity and temperature of each sample are represented as described in Fig. 3. Lines were calculated by linear regression of y on x using those points directly above or below the line as drawn.
3.0
Id
29 I/T
I
28
t
2.7
x IO3
FIG. 6. Linear nonisothermal storage test of stability of murine interferon freeze-dried in medium containing either sodium phosphate or potassium phosphate buffer. Solid symbols designate interferon in a buffer made with sodium salts ( l , A ) and open symbols refer to samples in a buffer with potassium salts (0, A). Results obtained in two separate experiments with pH 7 buffers prepared with 0.1 M phosphate salts are distinguished as circles (0, o ) and triangles (A, A). The interferon activity and temperature of each sample are represented as described in Fig. 3. Linear correlation coefficients (r) were determined for the lines calculated by linear regression of y on x: A, r = 0.66; 0, r = 0.92; 0, r = 0.99; and A, r = 0.93.
BSA. The interferon activity in the final preparation possessed those properties attributed to murine interferon (17) as follows: a molecular weight of 25,006 daltons (by G-100 Sephadex chromatography); nonsedimentable (100,000 g for 2 hr); stable at pH 2 for 48 hr at 4°C; stable at 56°C for 1 hr; and destroyed by trypsin. There was no antiviral activity detectable in primary chicken embryo fibroblast cultures or in the BUD-8 diploid human cell strain. There was no residual NDV or mycoplasma contamination in the preparation; and the many freeze-dried samples tested contained no bacteria or fungi, as determined by accepted techniques described in materials and methods. Several types of stability tests were performed using the freeze-dried interferon. At a temperature of 90°C it was stable for more than 18 hr. During the LNS test there was no loss in activity. The MIS test (Fig. 7) provided results that made it possible to calculate conservative esti-
STABILIZATION
Cl
20
OF MURINE
40
60
a0
311
INTERFERON
100
120
I40
160 180
DAYS FIG. 7. Multiple (G002-904-5117. 52 (O), 60 (O),
isothermal storage Samples were hid or 68°C (A).
test of freeze-dried for the indicated
mates of stability (11) and predict that the freeze-dried interferon would lose 1000 units of activity during storage at 37°C for IO0 days, at 20°C for 5.5 years, or at 4°C for 110 years. To determine the usefulness of these predictions, a confirmatory stability test, with samples placed at 37”C, was initiated and is still in progress (Fig. 8). During the first 6 years of this test, the murine interferon reference reagent was, as expected, more stable than the intentionally conservative prediction. The reference reagent interferon, available from the National Institutes of Health (NIH), Catalog No. G002-904-511, was assigned an activity of 12,000 units/ml; the assignment was made from results pooled from several laboratories that titrated simultaneously this reagent with the previous interim reference reagent provided by the NIH as a frozen mouse serum. DISCUSSION
From the data presented, it is clear that the methods used for treatment and purification of the interferon can influence the ultimate stability of the interferon even
murine periods
interferon reference reagent of time at temperatures of
after it has been freeze-dried. In particular, the manner of inactivation of inducing virus, the method of purification of the interferon, the composition of the suspending medium, and the presence of stabilizing additives may all contribute independently to the ultimate stability of the freeze-dried product. In view of the rela-
YEARS
FIG. 8. Confirmatory stability test of freeze-dried murine interferon reference reagent (G002-904-511) by storage at 37°C. The predicted inac-tivation rate (0) was obtained from the multipleisothermal storage test shown in Fig. 7. The ob-served loss of activity ( l ) was expressed as ti percentage of the activity measured for the control sample which had been stored at -70°C for the same length of time as the test sample, and was titrated in the same assay.
312
JAMESON,
GREIFF,
tive impurity of the interferon used for the reference reagent, it is not possible to determine whether these effects are directly on the interferon molecule or on other molecules remaining in the preparation at the time of freeze-drying. For example, the greater stability in the presence of sodium ions may not necessarily indicate that potassium ions are directly detrimental to the stability or activity of the interferon molecule but may reflect the consequence of an interaction of potassium with yet another substance in the preparation, or may simply be a result of the acidic change in pH which probably occurs during the freezing of the mixture of sodium phosphate salts present in that buffer solution (22). Low pH does enhance stability of liquid (15) and freezedried interferon ( reported here). One difficulty in testing and predicting the stability of freeze-dried materials is, paradoxically, their exceptional stability as compared with the liquid counterpart. For example, if the test interferons had stability properties similar to the NIH reference reagent, predicted to be stable for 5.5 years at 20°C it would not have been possible to conduct direct stability tests requiring such exceptional lengths of time to detect even very small changes in activity. Therefore, accelerated storage tests were adapted for these studies and made it possible not only to compare rapidly the relative stabilities of different types of freeze-dried interferon preparations but also to predict stabilities at low temperatures of storage. The methods described herein have provided a basis for the development of freeze-drying processes for the preservation of other types of interferons to be used as reference reagents as well as medication in clinical trials. The results and conclusions of these studies which led to the selection of procedures and conditions for the preparation of the NIH freeze-dried murine interferon reference reagent should not be interpreted as
AND
GROSSBERG
excluding the existence of other methods which may also provide satisfactory stabilization of interferon, It should be stressed that the prospect of stabilizing interferons for clinical use is one of great concern in view of the many problems investigators have experienced in unpredictable loss of activity of interferon samples prior to their use in the treatment of patients. To the present time, freeze-drying, a method acceptable in the preparation of biological or pharmaceutical substances for clinica application, remains the best available method of stabilizing interferon for longterm storage. SUMMARY
Conditions of interferon processing were analyzed to select those that promote stability after freeze-drying. The effects of various preparative methods and treatment conditions were assessed by measuring the retention of biological activity by lyophilized interferon samples in two kinds of accelerated storage tests: the linear nonisothermal stability (LNS) test, a rapid method used for direct comparison of two or more preparations of interferon, and the multiple isothermal storage (MIS) test, a slow method requiring weeks to months to obtain data for the prediction of stability of a given preparation stored under various conditions. The most stable preparations of Newcastle-disease-virus-induced mouse L cell interferon were obtained using the following conditions: 1) perchlorate treatment to inactivate residual inducing virus, 2) nonspecific adsorption using zeolite for partial purification, 3) suspending medium of 0.5% bovine serum albumin in 0.1 M sodium phosphate buffer at pH 7, and 4) sublimation of ice in vacua with a starting temperature of -30°C to a final residual moisture of about 3%. The final product, reference reagent GOO2-QO4511, was stable throughout the course of the LNS test. From an extensive MIS test,
STABILIZATION
OF MURINE
this reference interferon was predicted to lose 1000 units of activity in 110 years at 4°C and 1000 units in 100 days at 37°C. After 6 years of storage at 37°C when the predicted residual activity would be about 2070 of the original potency, 35% of initial interferon activity remained, confirming the usefulness of the short-term predictive test. ACKNOWLEDGMENTS We thank Mrs. Patricia Z. Johnson and Mrs. Christine K. Schoenherr for their excellent technical assistance. We are grateful to Dr. J. J. Sedmak for his help in chromatographic estimation of the molecular weight of the interferon. This work was supported by award NOlAI42514 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health. REFERENCES 1. Baron, S., and Buckler, C. E. Circulating interferon in mice after intravenous injection of virus. Science 141, 1061-1063 (1963). 2. Cohn, E. J., Gurd, F. R. N., Surgenor, D. M., Barnes, B. A., Brown, R. K., Derouaux, G., Gillespie, J. M., Kahnt, F. W., Lever, W. F., Liu, C. H., Mittelman, D., Moutin, R. F., Schmid, K., and Uroma, E. A system for the separation of the components of human blood: Quantitative procedures for the separation of the protein components of human plasma. J. Amer. Chem. Sot. 72,
465-474 ( 1950). 3. Cole, B. R., and Leadbeater,
4. 5.
6.
7.
L. A critical assessment of an accelerated storage test. I. Plzarm. Pharmacol. 18, 101-111 (1966). purification of calf Davies, A. The partial interferon. Biochem. J. 90, 29P-30P (1964). Fantes, K. H. Purification of interferon from chick embryonic allantoic fluids and fibroblast tissue infected with influenza virus. J. Gen. Viral. 1, 257-267 (1967). Finter, N. B. The assay and standardization of interferon and interferon inducers. In “Interferons and Interferon Inducers” (N. B. Fin&x, Ed.), Chapter 7, pp. 135-169. North-Holland, Amsterdam; American Elsevier, New York, 1973. reagents in interGalasso, G. J. Standard feron research, In “International Symposium on Interferon and Interferon Inducers, London, 1969” (F. T. Perkins and R. H.
INTERFERON
Regamy, Eds.),
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S. Karger, Basel. Symp. Ser. Immunobiol. Stand. 14, 272-276 (1970). 8. Greiff, D. Freeze-drying cycles. In “International Symposium on Freeze-Drying of Biological Products, 1976” (V. J. Cabasso and R. H. Regamey, Eds.), S. Karger, Basel. Den. Biok Stand. 37, 105-115 (1977). 9. Greiff, D., and Greiff, C. Linear nonisothermal, single-step, stability studies of dried preparations of influenza virus. Cryobiology 9, 34-37 ( 1972). 10. Greiff, D., Melton, H., and Rowe, T. W. G. On the sealing of gas-filled ampoules. Cryobiology 12, 1-14 ( 1975). 11. Creiff, D., and Rightsel, W. A. An accelerated storage test for predicting the stability of suspensions of measles virus dried by sublimation in vucuo. j. lmmunol. 94, 395-400 ( 1965 ). 12. Greiff, D., and Rightsel, W. A. Stability of suspensions of influenza virus dried to different contents of residual moisture by sub16, limation in vacua. Appl. Microbial. 835-840 ( 1968). 13. Greiff, D., and Rightsel, W. A. Stabilities of dried suspensions of influenza virus sealed in a vacuum or under different gases. Appl. Microbial. 17, 830-835 ( 1969). 14. Jameson, P., Dixon, M. A., and Grossberg, S. E. A sensitive interferon assay for many species of cells: encephalomyocarditis virus hemagglutinin yield reduction. PTOC. Sot. Exp. Biol. Med. X5.5, 173-178 ( 1977). 15. Jariwalla, R., Grossberg, S. E., and Sedmak, J. J. The influence of physicochemical factors on the thermal inactivation of murine interferon. Arch. Viral. 49, 261-272 (1975). 16. Lampson, G. P., Tytell, A. A., Nemes, M. M., and Hilleman, M. R. Purification and characterization of chick embryo interferon. Proc. Sot. Exp. Biol. Med. 112, 468-478 (1963). 17. Lockhart, R. Z., Jr. Criteria for acceptance of a viral inhibitor as an interferon and a general description of the biological properties of known interferons. In “Lnterferons and Interferon Inducers” (N. B. Finter, Ed.), Chapter 2, pp. 11-27. North-Holland, Amsterdam; American Elsevier, New York, 1973. 18. Morahan, P. S., and Grossberg, S. E. Agerelated cellular resistance of the chicken embryo to viral infections. I. Interferon and natural resistance to myxoviruses and vesicular stomatitis virus. J. Infect. Dis. 121, 615-623 ( 1970). 19. Oie, H. K., Buckler, C. E., Uhlendorf, C. P.,
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Hill, D. A., and Baron, S. B. Improved assays for a variety of interferons. Proc. Sot. Exp. Biol. Med. 140, 1178-1181 (1972). 20. Paucker, K., and Stancek, D. Characterization of interferon-associated proteins. J. Gen. Viral. 15, 129-138 ( 1972). 21. Perkins, F. T. Discussion on recommendations. In “International Symposium on Interferon and Interferon Inducers, London, 1969” (F: T. Perkins and R. H. Regamy, Eds.), S. Karger, Basel. Symp. Ser. Immunobioz. Stand 14, 297-325 ( 1970). 22. Smith, A. U. Effects of subzero temperatures on water and on physiological media. In
AND
GROSSBERG
“Biological Effects of Freezing and Supercooling” (A. U. Smith, Ed.), Chapter 11, pp. 369-405. Williams and Wilkins, Baltimore, 1961. 23. Williams, C. A., and Chase, M. W. Chemical analyses. In “Methods in Immunology and Immunochemistry” (C. A. Williams and M. W. Chase, Eds.), Vol. 2, Chapter 12, pp. 286-282. Academic Press, New York, 1968. 24. Zoglio, M. A., Windheuser, J. J., Vatti, R., Maulding, H. V., Komblum, S. S., Jacobs, A., and Hamot, H. Linear nonisothermal stability studies. J. Pharm. Sci. 57, .20802085 ( 1968).