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Freeze drying and residual moisture
CRYOBIOLOGY,
8, 138-144 (1971)
FREEZE
DRYING
EDWARD
B. SELIGMANN,
Laboratory
of Control
National Institutes
AND RESIDUAL
MOISTURE1
JR. AND JANE F. FARBER
Activities, Division of Biologics Xtasdards, of Health, Bethesda, Maryland SOOl/,
Drying of materials in order to preserve desired properties has been in use from the early ages of mankind. Undoubtedly, the first attempts involved the sun drying of food. Development of methods for freeze drying on a commercial scale began with the work of Flosdorf and Mudd reported in 1935 (2). Since that time, many types of equipment have been developed to meet specific needs of individuals and types of materials being dried. This is not intended to be a review of the fields of freeze drying and residual moisture detection nor is it solely a presentation of methods. It is a presentation of some of the problems which have been encountered and solved to varying degrees in recent years in the day-to-day control of freeze-dried biological products such as plasma, serum, toxins, vaccines, and cultures of microorganisms. For these types of materials generally the objective is to remove practically all of the water as rapidly as possible. Thus all water in the material must remain frozen until removed by sublimation. In the usual freeze-drying cycle the material is frozen rapidly and held on shelves cooled to approximately -50°C. After the material has reached the shelf temperature a high vacuum of 10 p or less is drawn. It is important that all of the material to be dried has reached the shelf temperature before vacuum is applied. If some of it has not, when heat is added to the shelves the portions at a higher temperature may thaw and sublimation will not occur. After achieving vacuum the condenser is brought to approximately -75°C and then the temperature of the shelves can be raised gradually. The rate of increase of the shelf temperature depends upon many factors associated with the material to be dried. While sublimation may be relatively rapid at first even with only a slight difference between the temperature of the material and that of the condenser, sublimation will cause further cooling of the material which in turn slows the rate of sublimation. Therefore, the shelf temperature is raised at a rate which will maintain a steady rate of sublimation. As the material dries, the solids will take on a physical form influenced by the chemical makeup of the material. When whole serum is freeze dried a porous plug remains. Water vapor escapesfrom this relatively open matrix with ease, even from the deepest part of the plug. When materials with relatively high salt and low protein content such as crude bacterial toxins are freeze dried, a glassy layer may form on the surface hampering the release of water vapor and resulting in trapped water remaining below the surface. In attempts to preserve microorganisms it was discovered that some cells survived freezing in water but most often cells were killed by such t’reatment or had a low rate of survival. The damage was due mainly to events occurring during freezing. Mazur (3) in summary stated that permeability of the cell membrane to water determines whether equilibration within the cell occurs by dehydration or formation of intracellular ice. Additives to protect living cells from damage due to freezing and thawing have been used for about 35 years. Varying concentrations of sugar solutions have been used with the idea that they act as dehydrating agents and thus reduce the amount of intracellular ice crystal formation. The addition of glycerol has been found to be superior to sugar solutions in its protective qualities in the freezing process but it is not suitable for use ‘Presented at the Sixth Cryopreservation Conference during the Annual Meeting of the Society for Cryobiology, Los Angeles, California, August 12-14, 1970, sponsored in part by the Office of Naval Research, Contract ONR3700. 138
FREEZE
DRYING
AND RESIDUAL
MOISTURE
in freeze drying. It is the suspending medium currently used in one method of preserving frozen red blood cells. Otten (4) working with Vibrio cholera, which is relatively sensitive to the rigors of freeze drying, discovered meat extract to be more protective than saline and also that survival was enhanced when bacteria were dried in a suspension of dead organisms (5). From this he concluded that the addit’ional protein provided by the meat extract and dead cells enhanced the survival rate. Investigators experimenting with different microorganisms have tried many types of additives such as broth, serum, sugars, gums, peptone, blood, gelatine, starch, and milk. Ahhough we have not performed quantitative survival studies in our laboratory, we have been successful in recovering viable cells from a variety of cultures suspended in skim milk. Some dried cultures have been recovered after 30 years of storage under vacuum at +4”C. These cultures include various species of Bacteroides, Bord&llo, Pasteurella, Vibrio, and many less difficult forms. Clostridium, Corynebacterium, Suspensions of aerobic cultures are prepared by scraping the growth from 1%24hr agar slants into an overlay of skim milk. Anaerobic microorganisms are grown in fluid thioglycollate medium. This broth culture is then diluted 1: 2 with double strength skim milk. The heavy concentration of cells in the suspension adds greatly to the protection afforded by the milk. Bacterial suspensions are freeze dried routinely on a manifold type dryer with 0.1-0.2 ml suspension/l ml glass ampule. Troughs containing Dry Ice in alcohol are raised to immerse the lower portion of the tubes and freeze the suspensions before vacuum is drawn. For convenience, the drying process continues overnight after which time the ampules are flame sealed under the original vacuum. This procedure has proven to be a simple and effective means for preserving seed stocks of bncterilt. The apparatus is shown in Figs. 1 and 2. The freeze drying of vaccines has proven to be a more complex problem. In actual pm&cc each product presents new problems. Ingredients of t,he vaccine, size of the contn,iner, volume of fill, and the freeze-drying equipment all interact to influence the final results. As a result some amount of development work is necessary to arrive at the best procedure for drying each new product. Research and development expended on determining the best additives may be wasted if, during production runs, errors are made in operating the freeze-drying apparatus. Heat transfer between the product and the shelf is most important. Irregular contact of the vials with the shelf surface due to buckling of the bottom of the tray on which the vials rest may cause nonuniformity of drying throughout the lot. This problem can be eliminated by use of a tray assembly developed by Seligmann and Berkeley (6) ha,ving a moveable frame provided to hold vials securely in place regardless of the number in the tray. It is constructed so that the bot,tom may be withdrawn from under the vials and thus allow them to rest directly on the shelf. The tray is shown in Fig. 3. Another type of frame resembling a large t’est-tube rack fits on the same type tray. Specially designed Z-ml ampules with slit stoppers developed by Center Laboratories, Inc. (Supported by PSH Contract No. PH 43-64-40, National Institute of Allergy and Infectious Diseases) are securely held in the holes when the bottom of the tray is removed. This ampule must be sealed under vacuum. The stoppers are seated in the usual manner for vials. After removing from the chamber, the ampule is flame sealed below the rubber stopper. This permits the preservation of freeze-dried materials under the original vacuum and with a glass seal. This frame is shown in Fig. 4. Some chamber type freeze dryers are still in use which do not permit internal stoppcring. Difficulty in maintaining the product at the original low residual moisture content, has been experienced when the stoppers were not dried in the chamber with t,he prodnrt. When the product was dried in vials without stoppers, removed from the chamber, and then stoppers inserted, residual moisture determinations made shortly after indicated less than the maximum level permitted. However, when tested some
139
140
SELIGMANN
AND FARBER
FIQ. 1. Manifold freeze dryer with cultures being frozen in constricted ampules.
FIG. 2. Manifold freeze dryer with dried cultures being sealed under vacuum.
FREEZE
FIG.
DRYING
AND RESIDUAL
MOISTURE
3. Tray with removable bottom loaded with vials.
FIG. 4. Frame holding special ampules with butyl slit stoppers.
142
SELIGMANN
SND FARBER
weeks later, it was found that the maximum level had been exceeded. It was shown subsequently that moisture in or on the surface of the stoppers gradually transferred t’o the product resulting in an eventual excess of moisture. This occurred in spite of precautions taken to assure the unsealed vials were only exposed to very low humidity. More recently we have learned that some silicone-treated stoppers must be dried at high temperature even though they are in the vials during the freeze-drying cycle. If this is not) done, moisture may transfer rapidly from the stopper to the product if the vial is exposed to a relatively high temperature at any time or the transfer may be more gradual over a long period of storage in a refrigerator. It is preferable to seat the stoppers under the original vacuum in the freeze-drying chamber or replace the vacuum with dry, oil-free inert gas and then seat the stoppers in the chamber. After removing the vials from the chamber an aluminum seal should be applied to hold the stopper in place. Enough pressure should be used in applying the seal so that the top of the stopper is compressed against the lip of the vial and the seal cannot be rotated by hand. Both loss of vacuum and contamination have been detected in vials on which the aluminum seal was loose enough to be capable of being rotat,ed by hand. While there is more risk associated with relying on a rubber sealed vial for long-term storage, we have not experienced difficulties with preparations dried and sealed in butyl rubber stoppered vials in our laboratory. One such preparation is the U.S. Standard Pertussis Vaccine. Dried in 1961, it is still in use as the Standard. It was stored at 4°C and was stoppered under the original vacuum with butyl rubber, slit st,oppersand sealed with aluminum seals. Difficulty has been experienced in freeze drying bacterial toxins unless additional protein is added. When nothing has been added to the toxin very little solid residue remains. It is generally glassy in appearance and may show evidence of having melted partially and foamed during drying. Frequently after a storage period of several days to weeks most of the containers will show signs of rehydrating to the point of containing a syrupy fluid. Additives such as albumin which increase the bulk of the dried mat’erial will correct this problem. One goal of freeze drying is to reduce the moisture in the product to 1% or less. In some biological products residual moisture contents up to 3% are permitted. Once a technique has been developed which will achieve the desired residual moisture, it remains for the control laboratory to test samples to assure that each lot is acceptable. Because most products are hygroscopic to some degree, samples must be handled with extreme care in order to assure valid results. The method for testing biological products used in man is specified in the U.S. Public Health Service Regulations (7). This is simply drying to constant weight at room temperature under vacuum over P,O,. It has been found that the absolute value determined by the met’hod of Fischer (1) and the many more recent modifications of that method may not agree with the value obtained by the gravimetric method although for a given product a constant relationship may be established. For this reason only problems associated with the gravimetric method will be included here. In our laboratory samples are transferred from the original containers to tared weighing dishes in an airtight, low-humidity glove box. The box is shown in Fig. 5. It has been found that a sample of at least 200 mg is needed to obtain accuracy when the residual moisture is expected to be less than 1%. It is extremely important to transfer the sample in an atmosphere free of moisture. When first constructed the low-humidity box may continue to release enough moisture to invalidate determinations. Some glues used in plywood, some paints, and some types of rubber gloves will releasemoisture for considerable periods of time. If such a problem is encountered, it is better to use other construction materials rather than trying to correct the situation. In any event the box should not be used as long as there is evidence that hygroscopic materials exposed in the sealed box will pick up any amount of moisture. P,O, has been found to be satisfactory as a
FREEZE
DRYI?JG
FIG. 5. Low-humidity
AND RESIDITAI,
MOISTIiRE
transfer box with dishes of desiccant.
desiccant providing it is fresh. Moisture can be released from P,O, and taken up b\ hygroscopic samples. The desiccant should be present in relatively large volume wit,11the greatest possible surface a.rea exposed. Only one lot of a product should be opened at a time in the box. The samples, tools necessary to transfer the contents, and tared weighing dishes are placed in the box at least 3 hr before the first sample is opened. If the amount of material which can be removed from a single container is less than 200 mg, several containers must, be opened and the contents pooled in a tared weighing dish in order to obt,ain a sample of 200 mg. A preliminary weighing will indicate how many containers are needed. With the sample in it, the covered weighing dish is removed from the box to the balance and the initial weighing is made. The weighing dish is then placed in a drsiccator, the cover of the dish removed, the desiccator sealed and vacuum drawn. Again because of t)he possibility of error due to transfer of moisture from one sample to another only one sample is placed in each desiccator. Periodically, as indicated by ~xperience, the sample is reweighed until a constant weight has been reached. Besides the usual precautions taken in handling weighing dishes, it is extremely important to follon all of t’he steps just, outlined in order to prevent exposing t,he sample to moisture. When all precautions have been taken, we have found it is possible t,o have a very good agrcement among laboratories testing the same lot of a product. Invariably when differences have occurred one or more of the basic principles outlined here have bren violated. Sot all products are equally hygroscopic and slightly less than optimum test conditions ma> suffice for one product but may be entirely unsatisfactory for a more hygroscopic product. Therefore a technique must be tested with a material known to be extremely hygroscopio before it can be considered satisfactory for determining residual moisture. SUMMARY Several factors associated with operation of freeze-drying equipment and residiial moisture determinations have been discussed in relation to their influence on t,he residual moist’ure content of the product or on the determination of the residual moistrite.
144
SELIGMANS
.4ND FSRBER
Factors which should be achieved in t,he freeze-drying process are a relatively open physical structure of the mass as it is being dried, the presence of additives which improve t#he physical structure of the mass or provide protection to otherwise labile components, good heat transfer from the shelves to the product, and removal of moisture from rubber stoppers. Important factors which can influence the results from residual moisture determinations are the sample size which preferably should be at least 200 mg, transfer of samples which should be in a low-humidity atmosphere, isolation of samples by handling only one at a time in a low-humidity box and by use of separate desiccators, and monit,oring the procedure with highly hygroscopic material as a test of its ability to yield valid results. REFERENCES 1. Fischer, K. A new method for the analytical determination of the water content of liquids and solids. Angew. Chem., 48: 394-396, 1935. 2. Flosdorf, E. W., and Mudd, S. Procedure and apparatus for preservation in “Lyophile” form of serum and other biological substances. J. Immunol., 29: 389425, 1935. 3. Mezur. P. Cryobiology: The freezing of biological systems. Science, 168: 939-949, 1970. 4. Otten. L. The preservation of viability and virulence in dried pathogenic bacteria. Trans. 8th Congr. Far East. Ass. Trop. Med. 1930, p. 89, 1932. 5. Ot,ten, L. Die Trockenkonservierung von pathogenen Bakterien. Zentr. Bakteriol. Parasitenk., I. Orig., 116: 199, 1930. 6. Seligmann, E. B., Jr., and Berkeley, W. H. Direct contact tray assembly for freezedrying. Appl. Microbial., 5: 830, 1965. 7. U.S. Department of Health, Education, and Welfare, Public Health Service Reg., Biol. Products, Title 42, Part 73, Section 73.74 (a), p. 21, rev. ed., 1969.
8, 138-144 (1971)
FREEZE
DRYING
EDWARD
B. SELIGMANN,
Laboratory
of Control
National Institutes
AND RESIDUAL
MOISTURE1
JR. AND JANE F. FARBER
Activities, Division of Biologics Xtasdards, of Health, Bethesda, Maryland SOOl/,
Drying of materials in order to preserve desired properties has been in use from the early ages of mankind. Undoubtedly, the first attempts involved the sun drying of food. Development of methods for freeze drying on a commercial scale began with the work of Flosdorf and Mudd reported in 1935 (2). Since that time, many types of equipment have been developed to meet specific needs of individuals and types of materials being dried. This is not intended to be a review of the fields of freeze drying and residual moisture detection nor is it solely a presentation of methods. It is a presentation of some of the problems which have been encountered and solved to varying degrees in recent years in the day-to-day control of freeze-dried biological products such as plasma, serum, toxins, vaccines, and cultures of microorganisms. For these types of materials generally the objective is to remove practically all of the water as rapidly as possible. Thus all water in the material must remain frozen until removed by sublimation. In the usual freeze-drying cycle the material is frozen rapidly and held on shelves cooled to approximately -50°C. After the material has reached the shelf temperature a high vacuum of 10 p or less is drawn. It is important that all of the material to be dried has reached the shelf temperature before vacuum is applied. If some of it has not, when heat is added to the shelves the portions at a higher temperature may thaw and sublimation will not occur. After achieving vacuum the condenser is brought to approximately -75°C and then the temperature of the shelves can be raised gradually. The rate of increase of the shelf temperature depends upon many factors associated with the material to be dried. While sublimation may be relatively rapid at first even with only a slight difference between the temperature of the material and that of the condenser, sublimation will cause further cooling of the material which in turn slows the rate of sublimation. Therefore, the shelf temperature is raised at a rate which will maintain a steady rate of sublimation. As the material dries, the solids will take on a physical form influenced by the chemical makeup of the material. When whole serum is freeze dried a porous plug remains. Water vapor escapesfrom this relatively open matrix with ease, even from the deepest part of the plug. When materials with relatively high salt and low protein content such as crude bacterial toxins are freeze dried, a glassy layer may form on the surface hampering the release of water vapor and resulting in trapped water remaining below the surface. In attempts to preserve microorganisms it was discovered that some cells survived freezing in water but most often cells were killed by such t’reatment or had a low rate of survival. The damage was due mainly to events occurring during freezing. Mazur (3) in summary stated that permeability of the cell membrane to water determines whether equilibration within the cell occurs by dehydration or formation of intracellular ice. Additives to protect living cells from damage due to freezing and thawing have been used for about 35 years. Varying concentrations of sugar solutions have been used with the idea that they act as dehydrating agents and thus reduce the amount of intracellular ice crystal formation. The addition of glycerol has been found to be superior to sugar solutions in its protective qualities in the freezing process but it is not suitable for use ‘Presented at the Sixth Cryopreservation Conference during the Annual Meeting of the Society for Cryobiology, Los Angeles, California, August 12-14, 1970, sponsored in part by the Office of Naval Research, Contract ONR3700. 138
FREEZE
DRYING
AND RESIDUAL
MOISTURE
in freeze drying. It is the suspending medium currently used in one method of preserving frozen red blood cells. Otten (4) working with Vibrio cholera, which is relatively sensitive to the rigors of freeze drying, discovered meat extract to be more protective than saline and also that survival was enhanced when bacteria were dried in a suspension of dead organisms (5). From this he concluded that the addit’ional protein provided by the meat extract and dead cells enhanced the survival rate. Investigators experimenting with different microorganisms have tried many types of additives such as broth, serum, sugars, gums, peptone, blood, gelatine, starch, and milk. Ahhough we have not performed quantitative survival studies in our laboratory, we have been successful in recovering viable cells from a variety of cultures suspended in skim milk. Some dried cultures have been recovered after 30 years of storage under vacuum at +4”C. These cultures include various species of Bacteroides, Bord&llo, Pasteurella, Vibrio, and many less difficult forms. Clostridium, Corynebacterium, Suspensions of aerobic cultures are prepared by scraping the growth from 1%24hr agar slants into an overlay of skim milk. Anaerobic microorganisms are grown in fluid thioglycollate medium. This broth culture is then diluted 1: 2 with double strength skim milk. The heavy concentration of cells in the suspension adds greatly to the protection afforded by the milk. Bacterial suspensions are freeze dried routinely on a manifold type dryer with 0.1-0.2 ml suspension/l ml glass ampule. Troughs containing Dry Ice in alcohol are raised to immerse the lower portion of the tubes and freeze the suspensions before vacuum is drawn. For convenience, the drying process continues overnight after which time the ampules are flame sealed under the original vacuum. This procedure has proven to be a simple and effective means for preserving seed stocks of bncterilt. The apparatus is shown in Figs. 1 and 2. The freeze drying of vaccines has proven to be a more complex problem. In actual pm&cc each product presents new problems. Ingredients of t,he vaccine, size of the contn,iner, volume of fill, and the freeze-drying equipment all interact to influence the final results. As a result some amount of development work is necessary to arrive at the best procedure for drying each new product. Research and development expended on determining the best additives may be wasted if, during production runs, errors are made in operating the freeze-drying apparatus. Heat transfer between the product and the shelf is most important. Irregular contact of the vials with the shelf surface due to buckling of the bottom of the tray on which the vials rest may cause nonuniformity of drying throughout the lot. This problem can be eliminated by use of a tray assembly developed by Seligmann and Berkeley (6) ha,ving a moveable frame provided to hold vials securely in place regardless of the number in the tray. It is constructed so that the bot,tom may be withdrawn from under the vials and thus allow them to rest directly on the shelf. The tray is shown in Fig. 3. Another type of frame resembling a large t’est-tube rack fits on the same type tray. Specially designed Z-ml ampules with slit stoppers developed by Center Laboratories, Inc. (Supported by PSH Contract No. PH 43-64-40, National Institute of Allergy and Infectious Diseases) are securely held in the holes when the bottom of the tray is removed. This ampule must be sealed under vacuum. The stoppers are seated in the usual manner for vials. After removing from the chamber, the ampule is flame sealed below the rubber stopper. This permits the preservation of freeze-dried materials under the original vacuum and with a glass seal. This frame is shown in Fig. 4. Some chamber type freeze dryers are still in use which do not permit internal stoppcring. Difficulty in maintaining the product at the original low residual moisture content, has been experienced when the stoppers were not dried in the chamber with t,he prodnrt. When the product was dried in vials without stoppers, removed from the chamber, and then stoppers inserted, residual moisture determinations made shortly after indicated less than the maximum level permitted. However, when tested some
139
140
SELIGMANN
AND FARBER
FIQ. 1. Manifold freeze dryer with cultures being frozen in constricted ampules.
FIG. 2. Manifold freeze dryer with dried cultures being sealed under vacuum.
FREEZE
FIG.
DRYING
AND RESIDUAL
MOISTURE
3. Tray with removable bottom loaded with vials.
FIG. 4. Frame holding special ampules with butyl slit stoppers.
142
SELIGMANN
SND FARBER
weeks later, it was found that the maximum level had been exceeded. It was shown subsequently that moisture in or on the surface of the stoppers gradually transferred t’o the product resulting in an eventual excess of moisture. This occurred in spite of precautions taken to assure the unsealed vials were only exposed to very low humidity. More recently we have learned that some silicone-treated stoppers must be dried at high temperature even though they are in the vials during the freeze-drying cycle. If this is not) done, moisture may transfer rapidly from the stopper to the product if the vial is exposed to a relatively high temperature at any time or the transfer may be more gradual over a long period of storage in a refrigerator. It is preferable to seat the stoppers under the original vacuum in the freeze-drying chamber or replace the vacuum with dry, oil-free inert gas and then seat the stoppers in the chamber. After removing the vials from the chamber an aluminum seal should be applied to hold the stopper in place. Enough pressure should be used in applying the seal so that the top of the stopper is compressed against the lip of the vial and the seal cannot be rotated by hand. Both loss of vacuum and contamination have been detected in vials on which the aluminum seal was loose enough to be capable of being rotat,ed by hand. While there is more risk associated with relying on a rubber sealed vial for long-term storage, we have not experienced difficulties with preparations dried and sealed in butyl rubber stoppered vials in our laboratory. One such preparation is the U.S. Standard Pertussis Vaccine. Dried in 1961, it is still in use as the Standard. It was stored at 4°C and was stoppered under the original vacuum with butyl rubber, slit st,oppersand sealed with aluminum seals. Difficulty has been experienced in freeze drying bacterial toxins unless additional protein is added. When nothing has been added to the toxin very little solid residue remains. It is generally glassy in appearance and may show evidence of having melted partially and foamed during drying. Frequently after a storage period of several days to weeks most of the containers will show signs of rehydrating to the point of containing a syrupy fluid. Additives such as albumin which increase the bulk of the dried mat’erial will correct this problem. One goal of freeze drying is to reduce the moisture in the product to 1% or less. In some biological products residual moisture contents up to 3% are permitted. Once a technique has been developed which will achieve the desired residual moisture, it remains for the control laboratory to test samples to assure that each lot is acceptable. Because most products are hygroscopic to some degree, samples must be handled with extreme care in order to assure valid results. The method for testing biological products used in man is specified in the U.S. Public Health Service Regulations (7). This is simply drying to constant weight at room temperature under vacuum over P,O,. It has been found that the absolute value determined by the met’hod of Fischer (1) and the many more recent modifications of that method may not agree with the value obtained by the gravimetric method although for a given product a constant relationship may be established. For this reason only problems associated with the gravimetric method will be included here. In our laboratory samples are transferred from the original containers to tared weighing dishes in an airtight, low-humidity glove box. The box is shown in Fig. 5. It has been found that a sample of at least 200 mg is needed to obtain accuracy when the residual moisture is expected to be less than 1%. It is extremely important to transfer the sample in an atmosphere free of moisture. When first constructed the low-humidity box may continue to release enough moisture to invalidate determinations. Some glues used in plywood, some paints, and some types of rubber gloves will releasemoisture for considerable periods of time. If such a problem is encountered, it is better to use other construction materials rather than trying to correct the situation. In any event the box should not be used as long as there is evidence that hygroscopic materials exposed in the sealed box will pick up any amount of moisture. P,O, has been found to be satisfactory as a
FREEZE
DRYI?JG
FIG. 5. Low-humidity
AND RESIDITAI,
MOISTIiRE
transfer box with dishes of desiccant.
desiccant providing it is fresh. Moisture can be released from P,O, and taken up b\ hygroscopic samples. The desiccant should be present in relatively large volume wit,11the greatest possible surface a.rea exposed. Only one lot of a product should be opened at a time in the box. The samples, tools necessary to transfer the contents, and tared weighing dishes are placed in the box at least 3 hr before the first sample is opened. If the amount of material which can be removed from a single container is less than 200 mg, several containers must, be opened and the contents pooled in a tared weighing dish in order to obt,ain a sample of 200 mg. A preliminary weighing will indicate how many containers are needed. With the sample in it, the covered weighing dish is removed from the box to the balance and the initial weighing is made. The weighing dish is then placed in a drsiccator, the cover of the dish removed, the desiccator sealed and vacuum drawn. Again because of t)he possibility of error due to transfer of moisture from one sample to another only one sample is placed in each desiccator. Periodically, as indicated by ~xperience, the sample is reweighed until a constant weight has been reached. Besides the usual precautions taken in handling weighing dishes, it is extremely important to follon all of t’he steps just, outlined in order to prevent exposing t,he sample to moisture. When all precautions have been taken, we have found it is possible t,o have a very good agrcement among laboratories testing the same lot of a product. Invariably when differences have occurred one or more of the basic principles outlined here have bren violated. Sot all products are equally hygroscopic and slightly less than optimum test conditions ma> suffice for one product but may be entirely unsatisfactory for a more hygroscopic product. Therefore a technique must be tested with a material known to be extremely hygroscopio before it can be considered satisfactory for determining residual moisture. SUMMARY Several factors associated with operation of freeze-drying equipment and residiial moisture determinations have been discussed in relation to their influence on t,he residual moist’ure content of the product or on the determination of the residual moistrite.
144
SELIGMANS
.4ND FSRBER
Factors which should be achieved in t,he freeze-drying process are a relatively open physical structure of the mass as it is being dried, the presence of additives which improve t#he physical structure of the mass or provide protection to otherwise labile components, good heat transfer from the shelves to the product, and removal of moisture from rubber stoppers. Important factors which can influence the results from residual moisture determinations are the sample size which preferably should be at least 200 mg, transfer of samples which should be in a low-humidity atmosphere, isolation of samples by handling only one at a time in a low-humidity box and by use of separate desiccators, and monit,oring the procedure with highly hygroscopic material as a test of its ability to yield valid results. REFERENCES 1. Fischer, K. A new method for the analytical determination of the water content of liquids and solids. Angew. Chem., 48: 394-396, 1935. 2. Flosdorf, E. W., and Mudd, S. Procedure and apparatus for preservation in “Lyophile” form of serum and other biological substances. J. Immunol., 29: 389425, 1935. 3. Mezur. P. Cryobiology: The freezing of biological systems. Science, 168: 939-949, 1970. 4. Otten. L. The preservation of viability and virulence in dried pathogenic bacteria. Trans. 8th Congr. Far East. Ass. Trop. Med. 1930, p. 89, 1932. 5. Ot,ten, L. Die Trockenkonservierung von pathogenen Bakterien. Zentr. Bakteriol. Parasitenk., I. Orig., 116: 199, 1930. 6. Seligmann, E. B., Jr., and Berkeley, W. H. Direct contact tray assembly for freezedrying. Appl. Microbial., 5: 830, 1965. 7. U.S. Department of Health, Education, and Welfare, Public Health Service Reg., Biol. Products, Title 42, Part 73, Section 73.74 (a), p. 21, rev. ed., 1969.