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- Herausgeber: Wiley-VCH Verlag GmbH & Co. KGaA
- Kategorie: Fachliteratur
- Sprache: Englisch
This completely updated and enlarged third edition of the classic text adopts a practical approach to describe the fundamentals of freeze-drying, backed by many explanatory examples.
Following an introduction to the fundamentals, the book goes on to discuss process and plant automation as well as methods to transfer pilot plant qualifications and process data to production. An entire section is devoted to a large range of different pharmaceutical, biological, and medical products. New to this edition are chapters on antibodies, freeze-dry microscopy, TEMPRIS, microwave freeze-drying, spray freeze-drying, and PAT.
Their many years of experience in freeze-drying enable the authors to supply valuable criteria for the selection of laboratory, pilot and production plants, discussing the advantages, drawbacks and limitations of different plant designs. Alongside guidelines for the evaluation and qualification of plants and processes, the author also includes a troubleshooting section.
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CONTENTS
Cover
Title Page
Copyright
Preface to Third Edition and Acknowledgment
Preface to the Second Edition
Chapter 1: Foundations and Process Engineering
1.1 Freezing
1.2 Drying
1.3 Storage
References
Chapter 2: Installation and Equipment Technique
2.1 Freezing Installation
2.2 Components of a Freeze-Drying Plant
2.3 Installations Up to 10 kg Ice Capacity
2.4 Production Plants
2.5 Production Plants for Food
2.6 Process Automation
References
Further Reading
Chapter 3: Pharmaceutical, Biological, and Medical Products
3.1 Proteins and Hormones
3.2 Viruses, Vaccines, Bacteria, and Yeasts
3.3 Antibiotics, Cytostatics, Ibuprofen
3.4 Liposomes and Nanoparticles
3.5 Antibody
3.6 Transplants, Collagen
3.7 Freeze-Drying Subject Terms – Overview and Summary
References
Chapter 4: Metal Oxides, Ceramic Powders
References
Chapter 5: Trouble Shooting
5.1 Prolonged Evacuation Time
5.2 Sublimation Front Temperature Too High
5.3 Sublimation Front Temperature Irregular
5.4 Slow Pressure Increase in the Chamber during Main Drying
5.5 Stoppers ›Pop Out‹ or Slide into the Vials
5.6 Traces of Highly Volatile Solvents (Acetone, Ethanol)
5.7 Different Structures of the Dried Product in the Center and Border of a Shelf
Chapter 6: Regulatory Issues
6.1 PAT (Practical Analytical Technology)
6.2 Quality of the Product
6.3 Description of the Process Developed for Manufacturing of the Product
6.4 Description of Production Installations and Their Handling
6.5 Equipment Performance Tests
6.6 Quality of Installation to Document the Ability of Equipment to Operate Processes (Described in Section 6.3)
6.7 Documentation of the Quality of the Products Manufactured (in Comparison with Section 6.2)
References
Appendix: Abbreviations, Symbols, and Unit of Measure
Index
End User License Agreement
List of Tables
Table 1.1
Table 1.2
Table 1.3
Table 1.4
Table 1.5
Table 1.6
Table 1.7
Table 1.8
Table 1.9
Table 1.10
Table 1.11
Table 1.12
Table 1.13
Table 1.14
Table 1.15
Table 1.16
Table 1.17
Table 1.18
Table 1.19
Table 1.20
Table 1.21
Table 1.22
Table 1.23
Table 1.24
Table 1.25
Table 1.26
Table 1.27
Table 1.28
Table 1.29
Table 1.30
Table 1.31
Table 1.32
Table 1.33
Table 2.1
Table 2.2
Table 2.3
Table 2.4
Table 2.5
Table 2.6
Table 2.7
Table 2.8
Table 2.9
Table 2.10
Table 2.11
Table 3.1
Table 3.2
Table 3.3
Table 3.4
Table 3.5
Table 3.6
List of Illustrations
Figure 1.1
Figure 1.2
Figure 1.3
Figure 1.4
Figure 1.5
Figure 1.6
Figure 1.7
Figure 1.8
Figure 1.9
Figure 1.10
Figure 1.11
Figure 1.12
Figure 1.13
Figure 1.14
Figure 1.15
Figure 1.16
Figure 1.17
Figure 1.18
Figure 1.19
Figure 1.20
Figure 1.21
Figure 1.22
Figure 1.23
Figure 1.24
Figure 1.25
Figure 1.26
Figure 1.27
Figure 1.28
Figure 1.29
Figure 1.30
Figure 1.31
Figure 1.32
Figure 1.33
Figure 1.34
Figure 1.35
Figure 1.36
Figure 1.37
Figure 1.38
Figure 1.39
Figure 1.40
Figure 1.41
Figure 1.42
Figure 1.43
Figure 1.44
Figure 1.45
Figure 1.46
Figure 1.47
Figure 1.48
Figure 1.49
Figure 1.50
Figure 1.51
Figure 1.52
Figure 1.53
Figure 1.54
Figure 1.55
Figure 1.56
Figure 1.57
Figure 1.58
Figure 1.59
Figure 1.60
Figure 1.61
Figure 1.62
Figure 1.63
Figure 1.64
Figure 1.65
Figure 1.66
Figure 1.67
Figure 1.68
Figure 1.69
Figure 1.70
Figure 1.71
Figure 1.72
Figure 1.73
Figure 1.74
Figure 1.75
Figure 1.76
Figure 1.77
Figure 1.78
Figure 1.79
Figure 1.80
Figure 1.81
Figure 1.82
Figure 1.83
Figure 1.84
Figure 1.85
Figure 1.86
Figure 1.87
Figure 1.88
Figure 1.89
Figure 1.90
Figure 1.91
Figure 1.92
Figure 1.93
Figure 1.94
Figure 1.95
Figure 1.96
Figure 1.97
Figure 1.98
Figure 1.99
Figure 1.100
Figure 1.101
Figure 1.102
Figure 1.103
Figure 1.104
Figure 1.105
Figure 1.106
Figure 1.107
Figure 1.108
Figure 1.109
Figure 1.110
Figure 1.111
Figure 1.112
Figure 1.113
Figure 1.114
Figure 1.115
Figure 1.116
Figure 1.117
Figure 1.118
Figure 1.119
Figure 1.120
Figure 1.121
Figure 1.122
Figure 1.123
Figure 1.124
Figure 1.125
Figure 1.126
Figure 1.127
Figure 1.128
Figure 1.129
Figure 1.130
Figure 1.131
Figure 1.132
Figure 1.133
Figure 1.134
Figure 1.135
Figure 1.136
Figure 1.137
Figure 1.138
Figure 1.139
Figure 1.140
Figure 1.141
Figure 1.142
Figure 1.143
Figure 1.144
Figure 1.145
Figure 1.146
Figure 1.147
Figure 1.148
Figure 1.149
Figure 1.150
Figure 1.151
Figure 1.152
Figure 1.153
Figure 1.154
Figure 1.155
Figure 1.156
Figure 1.157
Figure 1.158
Figure 1.159
Figure 2.1
Figure 2.2
Figure 2.3
Figure 2.4
Figure 2.5
Figure 2.6
Figure 2.7
Figure 2.8
Figure 2.9
Figure 2.10
Figure 2.11
Figure 2.12
Figure 2.13
Figure 2.14
Figure 2.15
Figure 2.16
Figure 2.17
Figure 2.18
Figure 2.19
Figure 2.20
Figure 2.21
Figure 2.22
Figure 2.23
Figure 2.24
Figure 2.25
Figure 2.26
Figure 2.27
Figure 2.28
Figure 2.29
Figure 2.30
Figure 2.31
Figure 2.32
Figure 2.33
Figure 2.34
Figure 2.35
Figure 2.36
Figure 2.37
Figure 2.38
Figure 2.39
Figure 2.40
Figure 2.41
Figure 2.42
Figure 2.43
Figure 2.44
Figure 2.45
Figure 2.46
Figure 2.47
Figure 2.48
Figure 2.49
Figure 2.50
Figure 2.51
Figure 2.52
Figure 2.53
Figure 2.54
Figure 2.55
Figure 2.56
Figure 2.57
Figure 2.58
Figure 2.59
Figure 2.60
Figure 2.61
Figure 2.62
Figure 2.63
Figure 2.64
Figure 2.65
Figure 2.66
Figure 2.67
Figure 2.68
Figure 2.69
Figure 2.70
Figure 2.71
Figure 2.72
Figure 2.73
Figure 2.74
Figure 2.75
Figure 2.76
Figure 2.77
Figure 2.78
Figure 2.79
Figure 2.80
Figure 2.81
Figure 2.82
Figure 2.83
Figure 2.84
Figure 2.85
Figure 2.86
Figure 2.87
Figure 2.88
Figure 2.89
Figure 2.90
Figure 2.91
Figure 2.92
Figure 2.93
Figure 2.94
Figure 2.95
Figure 2.96
Figure 2.97
Figure 2.98
Figure 2.99
Figure 2.100
Figure 2.101
Figure 2.102
Figure 2.103
Figure 2.104
Figure 2.105
Figure 2.106
Figure 2.107
Figure 2.108
Figure 2.109
Figure 2.110
Figure 2.111
Figure 2.112
Figure 2.113
Figure 2.114
Figure 2.115
Figure 2.116
Figure 2.117
Figure 2.118
Figure 2.119
Figure 2.120
Figure 2.121
Figure 2.122
Figure 2.123
Figure 3.1
Figure 3.2
Figure 3.3
Figure 3.4
Figure 3.5
Figure 3.6
Figure 3.7
Figure 3.8
Figure 3.9
Figure 3.10
Figure 3.11
Figure 3.12
Figure 3.13
Figure 3.14
Figure 3.15
Figure 3.16
Figure 3.17
Figure 3.18
Figure 3.19
Figure 3.20
Figure 3.21
Figure 3.22
Figure 3.23
Figure 3.24
Figure 3.25
Figure 3.26
Figure 3.27
Figure 3.28
Figure 3.29
Figure 3.30
Figure 3.31
Figure 3.32
Figure 3.33
Figure 3.34
Figure 3.35
Figure 3.36
Figure 3.37
Figure 3.38
Figure 3.39
Figure 3.40
Figure 3.41
Figure 3.42
Figure 4.1
Figure 4.2
Figure 4.3
Figure 4.4
Figure 4.5
Figure 5.1
Figure 6.1
Figure 6.2
Guide
Cover
Table of Contents
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Chapter 1
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Freeze-Drying
Third, Completely Revised and Enlarged Edition
Peter Haseley and Georg-Wilhelm Oetjen
All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.
Library of Congress Card No.: applied for
British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.
Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at <http://dnb.d-nb.de>.
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany
All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.
Print ISBN: 978-3-527-34306-5
ePDF ISBN: 978-3-527-80891-5
ePub ISBN: 978-3-527-80893-9
Mobi ISBN: 978-3-527-80892-2
oBook ISBN: 978-3-527-80894-6
Preface to Third Edition and Acknowledgment
This third, revised edition I would like to dedicate to Dr. Georg-Wilhelm Oetjen, who passed away in 2015. He introduced me to Freeze-Drying and essentially influenced my professional career.
I would like to acknowledge my supporters who helped me to complete this book. Without their continued support and effort I probably would have not been able to bring my work to a successful completion. I would like to thank Mr. Mars Ho – CEO Austar Group, Hong Kong; Mr. Austin McDonald – CEO Sterile Technology LLC, USA; Mr. Manfred Steiner – GEA Lyophil GmbH, Germany; and Mr. Anton Mangold – CEO iQ-mobil solutions, Germany.
Without the professional support of Mrs. Regine Fisher, MD Semicom Media & Communications, Germany, I would have had difficulties to finish the book. She was responsible for the text finalization and proofreading as well as for the editing and handling of images, tables, and graphics.
Peter HaseleyDipl.-Ing.(FH)
Preface to the Second Edition
Drying of food and herbs is one of the oldest preservation methods of humanity.
Freeze-drying was first carried out, as K.H. Neumann wrote in his book Grundriß der Gefriertrocknung, 1954, by Altmann, who freeze-dried parts of organs in 1890. In 1932, Gersh designed an effective vacuum plant for freeze-drying of histological preparations with the help of the diffusion pump just invented by Gaede at that time. Sawyer, Lloyd, and Kitchen successfully freeze-dried yellow fiber viruses in 1929. Industrial freeze-drying began, as E.W. Flosdorf shows in his book Freeze-Drying, 1949, with the production of preserved blood plasma and penicillin.
Vacuum technology and penicillin were also my own first encounter with freeze-drying. After my studies of physics at the university in Göttingen, I worked in the development department of E. Leybold's Nachf. where I had to build a freeze-drying plant for penicillin. Since that time I was engaged in vacuum process technology for almost 25 years, from 1952 on as managing director of Leybold Hochvakuum Anlagen GmbH. From this time I know Peter Haseley, whom I employed for the freeze-drying department. Later, as “Geschäftsführer” of Steris GmbH, he was actively involved with engineering modern freeze-drying plants with all their complex requirements of documentation and qualification. Together we have developed an old idea of mine to control the freeze-drying process not by predetermined time, pressure, and temperature data, but by the data measured during the process. Therefore, I was very happy when Peter Haseley agreed to rewrite the chapters “Installation and Equipment Technique” and “Trouble-Shooting and Regulatory Issues” for this second edition.
Freeze-drying has always fascinated me as the most complex vacuum process. Mechanical and chemical engineering, chemistry and biology, and sterility and regulatory issues are all part of the freeze-drying process.
After my retirement from the managing board of Drägerwerke AG, I had the time to write the German edition of this book, Gefriertrocknen, published in 1997, and the first English edition in 1999. In 2004, a translation into Japanese has also been published.
When I started to write the German edition, Mr. Wolfgang Suwelack, managing partner of Dr. Otto Suwelack Nachf. GmbH & Co., asked me to work for him as consultant in freeze-drying, and I have to thank him for the permission to use some of the results achieved in the last years. This activity was the new start of my work in freeze-drying, and I would therefore like to dedicate this book to Mr. Wolfgang Suwelack out of gratitude for a harmonic cooperation lasting for over a decade.
Several companies and publishing houses have granted permission to use drawings and photographs to which they own the copyright. Mr. Haseley and I are grateful to all of them because they have thus made it possible to present freeze-drying under many aspects.
We have tried to show the interconnection between the properties of the product, the goal to make it stable, and the necessary processes to achieve this. The problems of the different process steps are discussed with examples and the parameters influencing each step are described. We have avoided to follow the many theoretical attempts to describe one or more of the freeze-drying steps, but have restricted ourselves to a few equations that permit calculating process and product data with sufficient accuracy, or to at least allow an estimate based on measuring some of the data.
The freezing of a product is a very important step. The structure in the frozen product decides whether the product can be freeze-dried at all, and under which conditions it can be done. Therefore, the consequences of freezing rate, layer thickness of the product, and excipients are discussed in some detail. The second main point is the measurement and control of the two drying phases, the main and secondary drying, and the third point concentrates on the residual moisture content, its measurement, and the consequences during storage of the dry product. There will be critical opinions that some of the processes are unilaterally represented. I have tried to show the limits and advantages of certain procedures to enable the reader to decide for himself whether the ideas of the quoted authors or my own can be applied best to his particular task.
The approximately 270 literature references in the 1999 edition have been in part replaced and furthermore supplemented to a new total of 370.
Dr. Georg-Wilhelm Oetjen
1Foundations and Process Engineering
Freeze-drying or lyophilization is a drying process in which the solvent and/or the suspension medium is crystallized at low temperatures and thereafter sublimed from the solid state directly into the vapor phase.
Freeze-drying is mostly done with water as solvent. Figure 1.1 shows the phase diagram of water and the area in which this transfer from solid to vapor is possible.
Figure 1.1 Phase diagram of water.
Table 1.1 shows the relation of temperature (°C), mTorr, and mbar.
Table 1.1 Vapor pressure of water.
Temperature (°C)
mTorr
mbar
Temperature (°C)
mTorr
mbar
0
4579
6.108
−40
96.6
0.1238
−4
3280
4.372
−44
60.9
0.0809
−8
2326
3.097
−48
37.8
0.0502
−12
1832
2.172
−52
23.0
0.0300
−16
1132
1.506
−56
13.8
0.0183
−20
930
1.032
−60
8.0
0.0107
−24
526
0.6985
−64
4.6
0.0061
−28
351
0.4669
−68
2.8
0.0034
−32
231
0.3079
−72
1.4
0.0018
−36
150
0.2020
This step is relatively straightforward for pure water. If the product contains two or more components in true solutions or suspensions, the situation can become so complicated that simplified model substances have to be used to make the process more understandable. Such complex systems occur ubiquitously in biological substances.
The drying transforms the ice or water in an amorphous phase into vapor. Owing to the low vapor pressure of the ice, the vapor volumes become large, as can be seen in Figure 1.2. During the second step of the drying, the water adsorbed on the solids is desorbed.
Figure 1.2 Specific volume of water vapor as a function of the water vapor pressure. The temperature of the vapor in this diagram is that of ice.
The goal of freeze-drying is to produce a substance with good shelf stability and which is unchanged after reconstitution with water, although this depends also very much on the last step of the process: the packing and conditions of storage.
The advantages of freeze-drying can be summarized as follows:
The drying at low temperatures reduces degradation of heat-sensitive products.
The liquid product can be accurately dosed.
The moisture content of the final product can be controlled during the process.
The dry product can have an appealing physical form.
The dry product with a high specific surface area is rapidly reconstituted.
The disadvantages are as follows:
The high investment, operating and maintenance costs.
The complexity of the process and the equipment requires a team of skilled and permanently trained collaborators.
1.1 Freezing
To freeze a substance, it must be cooled to a temperature at which the water and the solids are fully crystallized or at which areas of crystallized ice and solids are enclosed in zones in which amorphous concentrated solids and water remain in a mechanically solid state (see Section 1.1.2). In the zone of freezing, the ice crystals first grow, thus concentrating the remaining solution, which can vary the pH value. In many substances an eutectic temperature can be determined, but in many others this value does not exist. The crystallization depends on several factors that influence each other: cooling rate, initial concentration, end temperature of cooling, and the time at this temperature. In several products, no crystallization takes place and the product remains in an amorphous, glass-like phase or a mixture of both occurs.
1.1.1 Amount of Heat, Heat Conductivity, Heat Transfer, and Cooling Rate
For pure water, the melting heat to be withdrawn for freezing (Qtot) can be calculated by Eq. (1.1), if the starting and the desired final temperatures are known:
where
c
w
=
specific heat capacity of water;
Q
e
=
melting heat of ice;
c
e
=
specific heat capacity of ice;
T
0
=
freezing temperature of ice;
T
1
=
initial temperature of water;
T
2
=
final temperature of ice.
The temperature dependences of cw between +20 and 0 °C and ce between 0 and −50 °C have to be adopted as average values.
For solutions and suspensions, the solid content has to be recognized. This is reflected in Eq. (1.2):
where
x
w
=
part of water above 0 °C;
c
f
=
specific heat of solids, for example:
for animal products
≈ 1.47 kJ/kg °C
for plant products
≈ 1.34 kJ/kg °C
for some solids:
carbohydrates
≈ 1.42 kJ/kg °C
proteins
≈ 1.55 kJ/kg °C
fats
≈ 1.7 kJ/kg °C
salts
≈ 0.8 kJ/kg °C;
x
f
=
part of solids;
x
w′
=
part of ice, which freezes until temperature
T
2
is reached. If not all water is frozen at
T
2
, an additional term has to be introduced, which reflects the cooling of the unfrozen water.
Table 1.2 shows the unfreezable water (UFW) in various foods. The reasons and the consequences are described in Sections 1.1.3 and 1.1.4. In comparing these data with other publications, for example, Ref. [1], smaller values may be found. This can depend not only on the different raw materials and the history of the probe until measurement but also on the methods of measurement.
Table 1.2 Percentage of water frozen out at various temperatures for some foods.
Product
Frozen out water at °C (% of the total water)
UFW (% of total water)
−10
−15
−20
−30
Lean beef
82
85
87
88
12
Haddock
84
87
89
91
9
Whole eggs, liquid
89
91
92
93
7
Yolk
85
86
87
87
13
Egg white
91
93
94
6
Yeast
80
85
88
89
11
Fruit juice
85
90
93
96
(3)
Peas
80
86
89
92
(7)
Part of
Table 1.1
in Refs [2,3].
For meat with less than 4% fat content, Riedel [2] has published an enthalpy diagram (shown in Figure 1.3). For some other foods, Table 1.3 shows enthalpy data at various temperatures. At −40 °C the enthalpy is set at 0 kJ/kg.
Figure 1.3 Enthalpy of lean beef meat as a function of its water content (0 kJ/kg at −40 °C). The temperatures at the beginning of cooling and the desired end temperatures for freezing are plotted as parameters. The dotted lines indicate the percentage of water frozen at the end temperatures (see also Figure 1 from Refs [2,3]). Example: Beef meat has 74% water. At +10 °C, the enthalpy is ∼325 kJ/kg; at −20 °C, the enthalpy is ∼40 kJ/kg; therefore, 285 kJ/kg have to be removed and 83% of the water frozen. The maximum possible (88%) (see Table 1.1) is reached at ≃30 °C.
Table 1.3 Enthalpy of meat, fish, and egg products.
Product
Water content (weight %)
Enthalpy (kJ/kg) at a temperature of °C
−30
−20
−10
0
+5
+20
Beef, 8% fat
74.0
19.2
41.5
72.4
298.5
314.8
368.4
Cod
80.3
20.1
41.9
74.1
322.8
341.2
381.0
Egg white
86.5
18.4
38.5
64.5
351.3
370.5
427.1
Whole egg
74.0
18.4
38.9
66.2
308.1
328.2
386.9
Part of
Table 1.3
in Refs [2,3].
In Table 1.4 the UFW data for products used in pharmaceuticals are listed in Ref. [6] of Chapter 3.
Table 1.4 Percentage of unfrozen water (UFW) , which cannot be frozen by lower temperature (see Figure 1.20).
Excipient
UFW (%)
Trehalose
16.7
Sorbitol
18.7
Maltose
20
Glycerin
27
Glucose
29.1
Sucrose
35.9
Lactose
40.8
Glycerol
45.9
Fructose
49.0
The transport of the calculated energy from the freezing zone of the product to the cooling medium can be described in a simplified way by the following steps: the product is an infinite plate, which is cooled from one site only, and the energy flows only perpendicular to its infinite expansion. The crystallization energy flows from the crystallization zone, through the already frozen ice, through the container bottom to a shelf, and finally into the cooling brine.
The freezing time (te) is approximately given by Eq. (1.3) [4]:
where
t
e
=
freezing time;
Δ
J
=
enthalpy difference between the initial freezing point and the final temperature;
Δ
T
=
difference of temperature between the freezing point and the cooling medium;
D
=
thickness of the product parallel to direction of prevailing heat transfer;
ρ
g
=
density of the frozen product;
λ
g
=
thermal conductivity of the frozen product;
K
su
=
surface heat transfer coefficient between cooling medium and the freezing zone.
The thermal conductivity of ice and of dried products are relatively well known, but the surface heat transfer coefficient Ksu during freezing and the total heat transfer coefficient Ktot during freeze-drying vary largely, as described in the various chapters. Table 1.5 gives a survey of some data of interest in freeze-drying.
Table 1.5 Surface heat transfer coefficient, total heat transfer coefficient, and thermal conductivity.
K
su
From gases to a solid surface (kJ/m
2
h °C): free convection
17–21
Laminar flow 2 m/s
50
Laminar flow 5 m/s
100
K
su
Between the shelf of a freeze-drying plant and a product in vials or trays during freezing (kJ/m
2
h °C)
200–400
K
su
Between a liquid and a solid surface (kJ/m
2
h °C): oil in tubes, laminar
160–250
LN
2
by drops on the product
a)
900
From liquids similar to water
b)
1600
From water at 1 bar, temperature difference <7 °C
c)
3600
K
tot
Between the shelf of a freeze-drying plant and the sublimation front in the product contained in vials or trays under vacuum
d)
(kJ/m
2
h °C)
60–130
λ
Thermal conductivity (kJ/m
2
h °C)
λ
g
Frozen product (ice)
e)
5.9–6.3
λ
tr
Dry product
f)
0.059–0.29
a)
Reinsert, A.P.: Factors affecting the erythrocyte during rapid freezing and thawing.
Ann. N. Y. Acad. Sci.
85
, 576–594, 1960.
b)
From Ref. [3].
c)
From VDI- = Wärmeatlas 5. Auflage, Bild 38, P. A 26, VDI-Verlag, Düsseldorf, 1988.
d)
Figures 1.116
and
1.117
.
e)
From Ref. [3].
f)
From Refs [5–8].
The influence of the variables in Eq. (1.3) can be studied by an example. A slice of lean beef with a thickness that is small compared with its horizontal dimensions is to be frozen to −20 °C. The influences of the border of the slice are neglected. The thickness of the slice is d = 2 cm. As can be seen in Figure 1.3, the enthalpy difference for beef with 74% water is approximately 240 kJ/kg. If the freezing process starts between 0 and −3 °C and is mostly finished at −20 °C, the cooling medium has a temperature of −43 °C and an average λ = 1.38 × 10−2 J/°C cm s is used when the slice is in contact with a liquid, having a similar behavior to water at 20 °C, Ksu = 4.61 × 10−2 J/°C cm2 s can be used for the calculation. The freezing time is
As shown in Eqs. (1.3) and (1.3a), the thickness d has a major influence if the conductivity term w, which includes d2, is large compared with the transfer term u, which includes only d.
In Eq. (1.4), w:u = 1.7:1, showing that the influence of the conductivity is almost double that of the transfer. Assuming that d is only 0.2 cm, the freezing time falls to
In this case, w:u = 1:6 and the transfer term is overwhelming. The freezing time is neither reduced by d2 nor by d, since the importance of w and u has changed. An increase in d by a factor of 3, to 6 cm, prolongs the freezing time:
Here w:u = 5:1, and the freezing time depends mostly on the heat conductivity of the material.
The freezing of a slice of beef in direct contact with a model liquid has been used to demonstrate the influence of the two terms w and u. To freeze a product for freeze-drying, two methods are mainly used: (i) freezing of the product in trays or in vials on cooled surfaces; or (ii) in a flow of cold air. If these methods do not result in a sufficient freezing rate, liquid nitrogen (LN2) in direct contact with the vials is used (see Figures 2.2 and 2.3) or droplets of the product are sprayed into LN2 (see Section 2.1.4).
The heat transfer coefficient Ksu in air varies strongly with the gas velocity, surface conditions of the product, and the geometry of the installation. In practical operations, it will be difficult to achieve Ksu values of 1.7–2.5 × 10−3 J/cm2 s °C or ∼75 kJ/m2 h °C and in many applications only half of this value (or less) may be possible. However, even with this high Ksu, the above-discussed slice of beef (2 cm thick) has a freezing time
compared with 12 min when cooled by a liquid, since the Ksu of a gas is ≤10% that of a liquid.
The time to reach a desired temperature level can be expressed as freezing rate vf, the change in temperature per unit time, for example, °C/min. Thus, the results of Eqs. (1.4)–(1.7) are approximately as follows:
(4)
v
f
= 1.7 °C/min
(5)
v
f
= 43 °C/min
(6)
v
f
= 0.3 °C/min
(7)
v
f
= 0.2 °C/min
These data are calculated by using 0 °C as the start and −20 °C as the end temperature to show the relative data. The exact calculation requires more information, as given below.
Figure 1.4 is the cooling curve of vials filled with a solution of 4% solid content and 27 mm filling height. From the curve, vf can be estimated:
0 to −10 °C
∼0.15 °C/min
0 to −14° C
∼0.18 °C/min
−14 to −30 °C
∼0.73 °C/min
0 to −30 °C
∼0.3 °C/min
Figure 1.4 Temperatures during freezing as a function of time. 1, shelf temperature; 2, product temperatures in a product with d = 2.7 cm, solid content ∼4%. (From Steris GmbH, 50354 Hürth, Germany.)
During the freezing of the main part of the water, vT is only 25% compared with the value after most of the water is crystallized. Taking the average value between 0 and −30 °C can therefore be misleading: The intention to freeze at a rate of 0.3 °C/min has not occurred during an important part of the operation. The difference between 0.15 and 0.7 °C/min influences the structure of the product. How important the change is has to be checked from case to case, but the difference between 0.15 and 0.7 °C/min is most likely important.
With Eq. (1.3), it is also possible to estimate Ksu. The uncertainties are the differences between the freezing of the product around the temperature sensor and in the undisturbed product, the position of the sensors, the correlation between time and temperature, and occasionally also the actual amount of frozen water. From Figure 1.4, the estimated Ksu is approximately 480 kJ/m2 h °C with a possible error of ±10% and maximum error of ±20%. Such high values can only be expected if the vials are carefully selected for their uniformity, especially with respect to a very even and flat bottom. Otherwise, the Ksu can be much smaller, for example, 230 kJ/m2 h °C as calculated from data shown in Table 1.6.
Table 1.6 Cooling time and freezing rate as a function of layer thickness for well manufactured vials, not selected for the flatness of the bottom.
Layer (mm)
Time from 0 °C to −10 °C (min)
Cooling rate (°C/min)
Time from −10 °C to −30 °C (min)
a)
Cooling rate (°C/min)
Cooling rate from 0 °C to −30 °C (°C/min)
6
14
0.71
9.3
2.1
1.29
12
32
0.31
12.9
1.6
0.67
20
60
0.17
19.0
1.1
0.38
30
105
0.095
28.3
0.7
0.23
a)
In this time the cooling of the glass of the vials from 0 °C to −30 °C is included.
If the vials are placed in trays and these are loaded on the shelves, Ksu will be reduced, very likely to <100 kJ/m2 h °C, with the consequence that the freezing time is twice or three times longer and freezing rates of 1 °C/min cannot be achieved.
Equation (1.3) can be used to estimate the influence of the variation of the layer thickness and the shelf temperature, if the Ksu values are measured for the type of vials used.
As shown, for example, in Figures 1.5–1.7, the temperature as a function of time can vary. Therefore, the calculation of freezing rates and the resulting Ksu contain a certain error. Table 1.7 shows a comparison of cooling rates [9]. Run 1 is from Figure 1.5, run 3 from Figure 1.6, and run 5 from Figure 1.7. The percentage indicates the maximum differences between the measurements with three temperature sensors in three vials.
Figure 1.5 Temperatures during freezing as a function of time for two different runs in the same plant, with the same product, Tsh cooled as quickly as possible. 1, shelf temperature; 2, product temperature.
Figure 1.6 See Figure 1.5.
Figure 1.7 As Figure 1.5, but Tsh cooled controlled. (From Ref. [9].)
Table 1.7 Comparison of cooling rates, measured in the same installation, with comparable vials and comparable d.
Run
Time from 0 °C to −10 °C (min)
Cooling rate (°C/min)
Time from −10 °C to −30 °C (min)
Cooling rate (°C/min)
1
34 ± 5
0.29 ± 15%
13 ± 5
1.5 ± 38%
2
25
0.4
17
1.2
3
23 ± 1.5
0.4 ± 6%
15 ± 2
1.3 ± 13%
4
a)
19 ± 2.5
0.5 ± 13%
21 ± 3.5
0.95 ± 17%
5
b)
79 ± 7
0.13 ± 9%
38 ± 5
0.5 ± 13%
a)
During the cooling phase −10 to −30 °C Δ
T
≈ 13 °C instead of ≈ 20 °C in run 1–3; taking this into account, the value of 0.95 corresponds to 1.4 in run 1–3.
b)
The shelf temperature was constantly lowered at ≈10 °C/30 min. Therefore, Δ
T
is only ≈ 8 °C during the freezing phase, compared with ≈ 30 °C in run 1–3. 0.13 °C/min therefore corresponds to ≈ 0.48 °C/min. The same applies to the 0.5 °C/min during the cooling phase, making it comparable to 1.3 °C/min.
To increase vt, the following possibilities can be used: (i) reducing d; (ii) reducing the shelf temperature; (iii) precooling of the vials, for example, to −80 °C, and filling the precooled product, for example, +4 °C, into the cold vials; (iv) cooling of the vials directly with LN2; and (v) dropping the product into LN2. With precooled vials, vt can be on the order of 10–20 °C/min, and with direct cooling by LN2 40–60 °C/min and more is possible. With droplet freezing, up to 1000 °C/min can be achieved.
For laboratory work, different cooling liquids can be used as shown in Table 1.8. However, these substances are not easy to use, they boil and are partially explosive. The cooling method shown in Figure 1.8 can be helpful. LN2 is evaporated under vacuum, freezing part of the N2 as a solid. In this mixture the solid melts, if energy is produced from cooling and crystallization. Thus, the formation of gaseous N2 is greatly reduced, which otherwise limits the heat transfer. Figure 1.9 shows the relative cooling rates for different forms of N2.
Table 1.8 Physical data of cooling liquids.
Medium
Boiling point,
T
s
(°C)
c
p
of liquid at
T
s
(kJ/kg °C)
λ
of liquid at
T
s
(kJ/mh °C)
Heat of vaporization at
T
s
(kJ/kg)
Helium (He
4
)
−268.9
4.41
0.098
20.5
Nitrogen
−195.8
2.05
0.506
197.6
Propane
−42.3
2.19
426.2
n
-Pentane
+36.1
2.2
234.1
(Figure 2 from Umrath, W. Kurzbeitrag für die Tagung Raster-Elektronenmikroskopie in Medizin and Biologie, unpublished, Brühl.)
Figure 1.8 Apparatus to produce a mixture of liquid and solid nitrogen. 1, inner container with LN2; 2, external container with LN2 connected to a vacuum pump; 3, the container 2 is evacuated to ∼124 mbar and kept at that pressure. The evaporating nitrogen reduces the temperature in 2 and thereby also in 1, since the two containers are in close thermal contact. A temperature of −210 °C is reached in container 1 after ∼5 min. (From Umrath, 1974 [10]. Reproduced with permission of John Wiley & Sons.)
Figure 1.9 Relative cooling rate of a small sample in different forms of N2. (The plot for LN2 depends mostly on the successful removal of the nitrogen gas.) Melting solid nitrogen reduces the formation of gaseous N2, since the crystallization energy melts the solid nitrogen and does not evaporate the liquid. (Note: Theoretically, cooling in solid N2 would be the fastest method, but liquid N2 will be formed and the heat transfer is not stable.) 1, LN2; 2, LN2 + solid N2; 3, melting of solid N2. (See also Umrath, W., unpublished results, Brühl.)
Riehle [11] has calculated the theoretically possible cooling velocities for small objects between 1 and 10−3 mm as shown in Figure 1.10. These calculations are made for a substance consisting of water only and Ksu is assumed to be infinitely large for the geometric dimensions shown in (a) a sphere, (b) a square cylinder of infinite length, and (c) a plate of infinite length and the thickness X, cooled only from one side. For the plate (c), vf is also calculated for three limited Ksu: 103, 104, and 105 W/m2 s (Chain lines). The purpose of this calculation is to show that freezing rates of 103–104 °C/s (6 × 104–6 × 105 °C/min) cannot be achieved. However, these rates are necessary to reduce the velocity of crystal growth in pure water sufficiently to obtain water in a glass-like phase with irregular particle size <10−8 m.
Figure 1.10 Maximum theoretical cooling rate for different geometric configurations (a–c) of water by cooling with LN2, if α is assumed to be ∞. The dotted lines are calculated for three values: α = 103, 104, 105 W/m2 s. (From Riehle, 1986 [11]. Reproduced with permission of John Wiley & Sons.)
Riehle showed that such freezing rates can only be reached for layers of <0.1 mm under a pressure of 1.5–2.5 kbar.
A different way of obtaining short cooling and freezing times is to evaporate part of the water in the product under vacuum. The evaporation energy of water at 0 °C is approximately 2.5 × 103 kJ/kg. To cool 1 kg of beef from 0 to −20 °C, 240 kJ have to be removed, which corresponds to ∼0.1 kg of water to be evaporated or 15% of the water in the beef. This quick evaporation will produce foam or bubbles in the product. This is unacceptable in most cases, since the original structure is changed and that part of the product that is vacuum dried will have different qualities to the freeze-dried part. Often the product frozen in this way cannot be freeze-dried at all.
1.1.2 Structure of Ice, Solutions, and Dispersions
The water molecule has a configuration as shown in Figure 1.11 [12], having a pronounced dipole moment, which produces the liquid phase at relatively high temperatures and ensures a structure in the envelope of molecules that surrounds ions [13]. However, clusters are also in water without ions; these consist of approximately 10 water molecules in a tetrahedral geometry surrounded by O—H—O groups. The clusters are not stable units with always the same molecules and they are constantly exchanging molecules with their surroundings, having an average lifetime of between 10−10 and 10−1 s. The number of clusters decreases as the temperature is lowered until freezing occurs.
Figure 1.11 Configuration of the electrical charges in a water molecule. (From de Quervain, 1975 [12]. Reproduced with permission of Elsevier.)
In water that is very well cleared of all foreign particles, the clusters begin to crystallize in the subcooled water at −39 °C; this is called homogeneous nucleation. Foreign, undissolved particles in water act as nuclei for the crystallization of ice and this is called heterogeneous nucleation. In normal water there exist approximately 106 particles per cm3 and these act as nuclei for crystallization. They become increasingly effective if their structure is similar to that of water. If a nucleus has formed, it grows faster at the outside than at the inside, producing (depending on subcooling and cooling velocity) structures of ice stars (Figure 1.12). During further freezing, branches grow at an angle of 60°, well known as frost flowers. For a crystal of 1 × 10−9 mm3, 2.7 × 1010 molecules have to be brought into position. It is difficult to visualize how such a crystal can be formed in a small fraction of a second, but it is obvious that the growth of such a crystal will be influenced or disturbed by many factors.
Figure 1.12 Growth of ice crystals in water. The subcooling is increased from left to right. (From de Quervain, 1975 [12]. Reproduced with permission of Elsevier.)
Figure 1.13 shows logJ* (J* = nuclei per unit time and volume) as a function of the temperature of the water–ice phase transition at different pressures of 1 and 2100 bar according to Riehle. At 2100 bar, J* is comparable to J* at an approximately 35 °C higher temperature. Under pressure, water can be subcooled further, with a delayed formation of nuclei.
Figure 1.13 Nucleation rate J* (nuclei/volume time) as a function of the temperature of the water–ice phase transformation. (From Riehle, 1986 [11]. Reproduced with permission of John Wiley & Sons.)
The growth of crystals is determined by the diffusion of molecules to the surface of the nucleus, the finding of a proper place, and the distribution of the freed energy to the surroundings. Under normal conditions (cooling speed vf < 102 °C/s and subcooling Tsc < 10 °C), Eq. (1.8) can be used:
where n = 1 if the energy transport and n = 1.7 if the surface reactions are decisive (Hillig and Turnbull, J. Chem. Physiol., 1956, 24, 914). If Tsc > 10 °C, the diffusion process has to be taken into account. Since vk is furthermore dependent on the concentration, the calculation of vk is insecure.
To summarize, the following can be stated:
To produce large crystals,
the rate of nucleation should be small, therefore the subcooling should be small;
the freezing should take place in a quasi-equilibrium situation between solution and crystals;
the temperature should be as high as possible, since the crystals grow with the function
e
−1/
T
;
the time given for crystallization has to be increased, since
v
K
is inversely proportional to the size of the crystal.
To produce only very few or no crystals,
freezing should take place under high pressure (
Figure 1.13
);
the freezing rate should be as high as possible, to produce a large degree of subcooling.
As can be seen from Figure 1.14, in pure water Ic and the other phases can only be reached under high pressures.
Figure 1.14 Phase diagram of water. L = liquid water; Ih = hexagonal ice; Ic = cubic ice; III–IX crystal configurations of ice. (From de Quervain, 1978 [12]. Reproduced with permission of Elsevier.)
Dowell and Rinfret [14] demonstrated that the phase at temperatures above −160 °C consists of small crystals ∼400 Å in size and having cubic and pseudohexagonal structures.
Figure 1.15 shows the three phases of ice that exist under normal pressure as a function of temperature, indicating also the time it takes to change from one type to another. If water vapor is condensed on a cold surface in a very thin film, amorphous ice is formed and remains stable at −160 °C for a long time. As shown in Figure 1.15, the change from amorphous to cubic ice will take ∼5 × 105 min or more than a year. The rate of change depends very much on the temperature: at −135 °C the same change takes only 1 min. This change is called devitrification. At −125 °C the change from cubic to hexagonal ice takes ∼1000 h, while at −65 °C only hexagonal ice is stable.
Figure 1.15 Water–glycerin phase diagram. On the left-hand side, the dependence of the phase transformation time on the ice temperature is shown: At −140 °C, amorphous ice transforms into cubic ice in ∼10 min. (See also Figure 8 from Ref. (Umrath, W., Kurzbeitrag für die Tagung Raster-Elektronenmikroskopie in Medizin and Biologie, unpublished, Brühl.))
To summarize, amorphous ice is stable below −160 °C, until −125 °C when cubic ice is formed irreversibly from the amorphous phase; above this temperature, hexagonal ice develops. Between −160 and −130 °C, cubic ice can be embedded in an amorphous surrounding. During warming, it is likely that some amorphous ice changes directly into the hexagonal form. Between −130 and −65 °C all three phases could be present, depending on the time–temperature function. This behavior of pure water changes if water solutions, suspensions in water, and mixtures with water are studied, as will be the case for virtually all products to be freeze-dried.
The freezing process will be discussed with model substances, which will be used as cryoprotective agents (CPAs). If a solution of water and glycerol is cooled quickly, a 10% solution in a layer of 3 × 10−3 mm and vf = 106 °C/s can be vitrified ([11], p. 218), but in a 5% solution crystals of 1000 Å are formed. At high pressures (1.5–2.1 kbar), 4 × 103 °C/s is sufficient for a 10% solution and 2 × 104 C/s for a 5% solution to achieve vitrification. For these measurements, the absence of foreign particles must be presumed in order to use the subcooling effect fully. Foreign particles could also come from containers, holding devices, and so on.
Riehle has proved the existence of such vitrification by electron microscopy. With higher concentrations of glycerol, vitrification becomes simpler. Luyet [15] showed diagrammatically (Figure 1.16) how various phase changes take place at different glycerol concentrations. At 60% glycerol devitrification takes place at ≃115 °C and increases with increasing glycerol concentration to ≃85 °C. However, such high concentrations of glycerol can normally not be used to freeze-dry organic substances.
Figure 1.16 Temperature as a function of the concentration of water–glycerin mixture at which phase transformations occur. (Figure 14 from Ref. [15].) Definitions by Luyet: AE, formation of small crystals or molecular groups; E, eutectic point; EB, formation of clusters; R, eruptive recrystallization; G, glass transition.
As shown for pure water, the phase transitions depend on the cooling rate, the end temperature of cooling, and the temperature and time of the treatment after cooling. The rate of rewarming is especially critical. One has to differentiate between quasi-static situations, which are independent of time and all other dynamic states, in which the history of the present situation and the rate of the further changes play an important role.
Freezing processes can be divided into two categories: one type is so slow that they run under almost equilibrium conditions; others are too fast to approach the equilibrium situation. Figures 1.17–1.19 show the effect of the freezing rate on the structure of the dried product. In Figure 1.17, milk has been frozen slowly (0.2–0.4 °C/min) in trays. In Figure 1.18, mannitol solution has been frozen in vials at a rate of ∼1 °C/min; the arch at the bottom represents the vial bottom. In Figure 1.19, γ-globulin has been frozen in LN2 (∼10–15 °C/min). This shows only the upper part of the dry product. The cake has been frozen so quickly from the bottom and the walls that the concentrated liquid has been pushed to the center, where it has been pressed to form a cone. The cake is cut and in the center of the cone a channel can be seen, in which highly concentrated solution has been included, leaving a channel. Since the solids of this part are agglomerated to the surrounding areas, the structure of the channel is partially collapsed during drying.
Figure 1.17 Milk frozen slowly (0.2–0.4 °C/min) in a tray. (Courtesy of Steris GmbH, Hürth, Germany.)
Figure 1.18 Mannitol solution frozen at ∼1 °C/min in a vial on precooled shelf. (Courtesy of Steris GmbH, Hürth, Germany.)
Figure 1.19 γ-Globulin solution frozen in a vial by LN2 at ∼10 °C/min (only the upper part of the product is shown). (Courtesy of Steris GmbH, Hürth, Germany.)
The nonequilibrium status can be seen during a slow cooling of a water–glycerol solution. Starting with a 20% glycerol solution, pure ice crystals will first be formed until at −46.5 °C when the glycerol concentration has reached 66.7%. At this temperature, the eutectic should solidify. However, it is possible to reduce the temperature to −58 °C with a glycerol concentration of 73%. A further decrease in temperature does not crystallize any more water. The solution is so highly concentrated and viscous and the mobility of the water molecules is so much reduced that the remaining water is unfreezable (UFW) in an amorphous state between the glycerol and ice molecules.
Figure 1.20 ([16], p. 286) shows diagrammatically at a given starting concentration which parts will be ice, unfrozen water, and glycerol at a freezing temperature actually used under equilibrium conditions. A solution of 20% initial glycerol contains, when cooled to −50 °C, 70% ice, 10% UFW, and 20% glycerol. At −58 °C, the line marked UFW is effective; 72% is glycerol and 27% UFW.
Figure 1.20 Rate of ice, water, and dissolved substance in the state of equilibrium of a glycerin–water solution as a function of the initial glycerin concentration, plotted at different freezing temperatures between −5 and −50 °C. A 40% glycerin solution frozen at −30 °C contains in the state of equilibrium ∼32% ice, 30% water, and 38% glycerin. The line marked UFW represents the temperature at which the glycerin concentration becomes so high that no more water can be frozen (the water molecules become highly unmovable). The glycerin concentration is ∼73% and the UFW concentration 27%. The diagram shows the equilibrium conditions, which may not exist during quick freezing. (See also Figure 1 from Ref. [16].)
The fact that a certain amount of water cannot crystallize in a highly concentrated solution, and that the molecules cannot move any more to the existing crystals, is important during the freezing of biological substances. Tables 1.2 and 1.4 show this for some food products and CPAs.
The combination of this knowledge and the results of quick-freezing processes provide a theoretical opportunity to freeze products into a solid, amorphous state. If the freezing velocity is smaller than required for vitrification, but large enough to avoid an equilibrium state, an amorphous mixture will result of hexagonal ice, concentrated solids, and UFW.
1.1.3 Influence of Excipients
The freezing of complex organic solutions and suspensions is often difficult to predict theoretically. The methods to analyze the freezing process and the structure formed are described in Section 1.1.5. The freezing is influenced by several factors, which often act in opposing directions:
Freezing rate
– slow: quasi-equilibrium
– very fast: dynamically governed
Number and geometry of foreign particles, which influence the heterogeneous nucleation: the closer their structure is similar to the ice structure, the better is their effectiveness as nuclei.
The degree of subcooling, which depends on the substance, but is strongly influenced by the two points above.
The rate of growth of the ice crystals, which depends on temperature and the viscosity of the solution; the latter increases strongly with increasing concentration of the solution.
That part of the water that is not frozen due to high freezing rate forms highly viscous occlusions in between the ice crystals.
The crystallization of the solved substance(s) (or part of it) or the subcooling and the delay of this crystallization, which depends again not only on the temperature but also very much on the viscosity of mixture.
By adding excipients not only is it possible to influence the cooling and solidification processes, but also they may be necessary to obtain one or more of the following objectives:
To grow stable structures if the amount of solids is small, for example, <3% in the solution, to prevent solid particles from being carried out of the vials by the water vapor stream (bulking agents).
To adjust pH data (buffers).
To avoid or induce crystallization.
To protect the active constituent during freezing (cryoprotectants).
To protect the active constituent during freeze-drying (lyoprotectants).
To reduce changes of the active constituent during storage (e.g., unfolding or aggregation of proteins.
An example of avoiding crystallization of sucrose by adding polyvinylpyrrolidone (PVP) was given by Shamblin and Zografi [17] even if a significant level of absorbed water is present. Zeng et al. [18] described the effect of the molecular weight (MW) and the added amount of PVP on the glass transition temperature Tg and the crystallization of sucrose; 5% of PVP of MW 300 K increased Tg from 48.3 °C for freeze-dried sucrose alone to 58.8 °C; 2.5% of PVP of MW 24 K or 40 K showed smaller or no effects on Tg. Shalaev et al. [19] freeze-dried sucrose in the presence of citric acid (citric acid:sucrose 1: 10) to RM <0.1% w/w. At 50 °C, the sucrose undergoes significant inversion in spite of the low RM. The rate of inversion is directly related to the citric acid concentration in the solution before freeze-drying. The authors concluded that the freeze-drying of sucrose with acidic substances may lead to substances that could react with other ingredients. Kouassi and Roos [20] freeze-dried maltodextrin–sucrose (2: 1) and maltodextrin–lactose–sucrose solutions (1: 1: 1) with invertase (10 mg/17.2 g). Sorption isotherms and Tg values of the amorphous dried products were measured. Sucrose hydrolysis was observed significantly at 24 °C and 0.662 aw. Saleki-Gerhardt and Zografi [21] studied the crystallization of sucrose from the amorphous state, influenced by absorbed water and additives (lactose, trehalose, and raffinose). Table 1.9 shows the data for Tg and Tc with absorbed water, and Table 1.10 the respective data with additives.
Table 1.9 Glass transition temperature and crystallization temperature for amorphous sucrose, trehalose, lactose, raffinose, and amorphous sucrose in the presence of absorbed water.
Product
T
g
(°C)
T
c
(°C)
Sucrose
74
130
Sucrose, 0.99% H
2
O
60
125
Sucrose, 1.47% H
2
O
58
115
Sucrose, 1.98% H
2
O
50
100
Sucrose, 3.13% H
2
O
32
92
Trehalose
115
–
a)
Lactose
108
185
Raffinose
102
–
a)
a)
Did not crystallize.Table I and II from Ref. [23].
Table 1.10 Crystallization temperature of sucrose with various proportions of additives.
Additives (% w/w)
Crystallization temperature (°C)
Lactose
Trehalose
Raffinose
0.0
130
130
130
1.0
131
128
128
5.0
137
145
148
10.0
156
161
160
Table IV in Ref. [23].
Mannitol, a frequently used excipient, shows complexity in its application. Yu et al. [22] reported the formation of a metastable mannitol hydrate during freeze-drying. The amount of mannitol hydrate varies from vial to vial in one batch. It reduces the drying rate, it can be converted to anhydrous polymorphs, redistributing the residual hydrate water, and it shows varying moisture levels from vial to vial. Cannon and Trappler [23] found at least three different polymorphs of mannitol. Under all studied process conditions, all three polymorphs were present, but in different ratios, strongly dependent on the freezing technique.
Pyne and Suryanarayanan [24] followed the phase transitions of glycine during freeze-drying among other methods in the sample chamber of an X-ray diffractometer. Freezing rates of 20 and 2 °C/min of a 15% wt/wt glycin solution resulted in the crystallization of 2-glycin with an increasing amount after annealing to −10 °C. Glycin immersed in LN2 formed an amorphous product. Upon heating to −65 °C, an unidentified crystalline phase of glycin was observed, which transformed at ≃55 °C to 2-glycin. After annealing, 3-glycin appeared to an extent that depended on the annealing temperature. Cooling rate, annealing, and the temperature during MD influence the solid state of glycin.
Hinrichs et al. [25] compared inulin of various degrees of polymerization with trehalose as glass-forming agents. Inulin above a certain degree of polymerization, DPn/DPw > 5.5/6.0 and trehalose stabilize alkaline phosphatase equally well. The Tg and values for inulin of <5.5/6.0 were higher than those for trehalose.
Glucose-6-phosphate dehydrogenase (G6PDH) freeze-dried with sucrose/raffinose at different mass ratios showed a higher Tg at higher mass ratios of raffinose than sucrose [26]. Different mass ratios did not influence the recovery of G6PDH after freeze-drying, but during storage low sucrose offered the best enzyme stability.
Fakes et al. [27] evaluated the moisture sorption behavior of mannitol, anhydrous lactose, sucrose, D-(+)-trehalose, dextran 40, and povidine (PVP K24) as bulking agents. Mannitol was found to be crystalline and nonhygroscopic
