Freeze-Drying/lyophilization

of Pharmaceutical
and Biological Products
Second Edition, Revised and Expanded
edited by

Louis Rey
Cabinet d 'Etudes
Lausanne, Switzerland

Joan C. May
Center for Biologics Evaluation and Research
Food and Drug Administration
Rockville, Maryland, U.S.A.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Although great care has been taken to provide accurate and current information,
neither the author(s) nor the publisher, nor anyone else associated with this publication, shall be liable for any loss, damage, or liability directly or indirectly caused or
alleged to be caused by this book. The material contained herein is not intended to
provide specic advice or recommendations for any specic situation.
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trademarks and are used only for identication and explanation without intent to
infringe.
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ISBN: 0-8247-4868-9
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DRUGS AND THE PHARMACEUTICAL SCIENCES

Executive Editor

James Swarbrick
PharmaceuTech, Inc.
Pinehurst, North Carolina

Advisory Board
Larry L. Augsburger
University of Maryland
Baltimore, Maryland

Harry G. Brittain
Center for Pharmaceutical Physics
Milford, New Jersey

Jennifer B. Dressman Anthony J. Hickey

Johann Wolfgang Goethe-University University of North Carolina School of Pharmacy
Frankfurt, Germany Chapel Hill, North Carolina
Jeffrey A. Hughes Ajaz Hussain
University of Florida College of Pharmacy U.S. Food and Drug Administration
Gainesville, Florida Frederick, Maryland
Trevor M. Jones Hans E. Junginger
The Association of the LeidedAmsterdam Center
British Pharmaceutical Industry for Drug Research
London, United Kingdom Leiden, The Netherlands
Vincent H. L. Lee
University of Southern California
Los Angeles, California
Jerome P. Skelly
Alexandria, Virginia
Geoffrey T. Tucker
University of Sheffield
Royal Hallamshire Hospital
Sheffield, United Kingdom

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Stephen G. Schulman
University of Florida
Gainesville, Florida
Elizabeth M. Topp
University of Kansas School of Pharmacy
Lawrence, Kansas
Peter York
University of Bradford School of Pharmacy
Bradford, United Kingdom

DRUGS AND THE PHARMACEUTICALSCIENCES

A Series of Textbooks and Monographs

1. Pharmacokinetics, Milo Gibaldi and Donald Perrier

2. Good Manufacturing Practices for Pharmaceuticals: A Plan for
Total Quality Control, Sidney H. Willig, Murray M. Tuckerman, and
William S. Hitchings IV
3. Microencapsulation, edited by J. R. Nixon
4. Drug Metabolism: Chemical and Biochemical Aspects, Bernard
Testa and Peter Jenner
5. New Drugs: Discovery and Development, edited by Alan A. Rubin
6. Sustained and Controlled Release Drug Delivery Systems, edited
by Joseph R. Robinson
7. Modern Pharmaceutics, edited by Gilbert S. Banker and
Christopher T. Rhodes
8. Prescription Drugs in Short Supply: Case Histories, Michael A.
Schwartz
9. Activated Charcoal: Antidotal and Other Medical Uses, David 0.
Cooney
10. Concepts in Drug Metabolism (in t w o parts), edited by Peter
Jenner and Bernard Testa
11. Pharmaceutical Analysis: Modern Methods (in t w o parts), edited by
James W. Munson
12. Techniques of Solubilization of Drugs, edited by Samuel H.
Yalkowsky
13. Orphan Drugs, edited by Fred . Karch
14. Novel Drug Delivery Systems: Fundamentals, Developmental Concepts, Biomedical Assessments, Yie W. Chien
15. Phar maco kinet ic s : Second Editio n, Revised and Expanded, Milo
Gibaldi and Donald Perrier
16. Good Manufacturing Practices for Pharmaceuticals: A Plan for Total
Quality Control, Second Edition, Revised and Expanded, Sidney H.
Willig, Murray M. Tuckerman, and William S. Hitchings IV
17. Formulation of Veterinary Dosage Forms, edited by Jack Blodinger
18. Der mat oIogic aI FormuIat io ns : Percut aneous Absorption, Brian W.
Barry
19. The Clinical Research Process in the Pharmaceutical Industry,
edited by Gary M. Matoren
20. Microencapsulation and Related Drug Processes, Patrick B. Deasy
21. Drugs and Nutrients: The Interactive Effects, edited by Daphne A.
Roe and T. Colin Campbell
22. Biotechnology of Industrial Antibiotics, rick J. Vandamme

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23. Pharmaceutical Process Validation, edited by Bernard T. Loftus and

Robert A. Nash
24. Anticancer and Interferon Agents: Synthesis and Properties, edited
by Raphael M. Ottenbrite and George B. Butler
25. Pharmaceutical Statistics: Practical and Clinical Applications, Sanford Bolton
26. Drug Dynamics for Analytical, Clinical, and Biological Chemists,
Benjamin J. Gudzinowicz, Burrows T. Younkin, Jr., and Michael J.
Gudzinowicz
27. Modern Analysis of Antibiotics, edited by Adjoran Aszalos
28. Solubility and Related Properties, Kenneth C. James
29. Controlled Drug Delivery: Fundamentals and Applications, Second
Edition, Revised and Expanded, edited by Joseph R. Robinson and
Vincent H. Lee
30. New Drug Approval Process: Clinical and Regulatory Management,
edited by Richard A. Guarino
3 1 . Transdermal Controlled Systemic Medications, edited by Yie W. Chien
3 2 . Drug Delivery Devices: Fundamentals and Applications, edited by
Praveen Tyle
33. Pharmacokinetics: Regulatory 0 Industrial 0 Academic Perspectives,
edited by Peter G. Welling and Francis L. S. Tse
34. Clinical Drug Trials and Tribulations, edited by Allen E. Cat0
3 5 . Transdermal Drug Delivery: Developmental Issues and Research
Initiatives, edited by Jonathan Hadgraft and Richard H. Guy
36. Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms,
edited by James W. McGinity
3 7 . Pharmaceutical Pelletization Technology, edited by Isaac GhebreSellassie
38. Good Laboratory Practice Regulations, edited by Allen F. Hirsch
39. Nasal Systemic Drug Delivery, Yie W. Chien, Kenneth S. E. Su,
and Sh yi-Feu Chang
40. Modern Pharmaceutics: Second Edition, Revised and Expanded,
edited by Gilbert S. Banker and Christopher T. Rhodes
4 1 . Specialized Drug Delivery Systems: Manufacturing and Production
Technology, edited by Praveen Tyle
42. Topical Drug Delivery Formulations, edited by David W. Osborne
and Anton H. Amann
43. Drug Stability: Principles and Practices, Jens T. Carstensen
44. Pharmaceutical Statistics: Practical and Clinical Applications, Second Edition, Revised and Expanded, Sanford Bolton
4 5 . Biodegradable Polymers as Drug Delivery Systems, edited by Mark
Chasin and Robert Langer
46. Preclinical Drug Disposition: A Laboratory Handbook, Francis L. S.
Tse and James J. Jaffe
47. HPLC in the Pharmaceutical Industry, edited by Godwin W. Fong
and Stanley K. Lam

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48. Pharmaceutical Bioequivalence, edited by Peter G. Welling, Francis

L. S. Tse, and Shrikant V. Dinghe
49. Pharmaceutical Dissolution Testing, Umesh V. Banakar
50. Novel Drug Delivery Systems: Second Edition, Revised and
Expanded, Yie W. Chien
51. Managing the Clinical Drug Development Process, David M. Cocchetto and Ronald V. Nardi
52. Good Manufacturing Practices for Pharmaceuticals: A Plan for
Total Quality Control, Third Edition, edited by Sidney H. Willig and
James R. Stoker
53. Prodrugs: Topical and Ocular Drug Delivery, edited by Kenneth B. Sloan
54. Pharmaceutical Inhalation Aerosol Technology, edited by Anthony
J. Hickey
55. Radiopharmaceuticals: Chemistry and Pharmacology, edited by
Adrian D. Nunn
56. New Drug Approval Process: Second Edition, Revised and
Expanded, edited by Richard A. Guarino
57. Pharmaceutical Process Validation: Second Edition, Revised and
Expanded, edited by Ira R. Berry and Robert A. Nash
58. Ophthalmic Drug Delivery Systems, edited by Ashim K. Mitra
59. Pharmaceutical Skin Penetration Enhancement, edited by Kenneth
A. Walters and Jonathan Hadgraft
60. Colonic Drug Absorption and Metabolism, edited by Peter R. Bieck
6 1 . Pharmaceutical Particulate Carriers: Therapeutic Applications,
edited by Alain Rolland
62. Drug Permeation Enhancement: Theory and Applications, edited by
Dean S. Hsieh
63. Glycopeptide Antibiotics, edited by Ramakrishnan Nagarajan
64. Achieving Sterility in Medical and Pharmaceutical Products, Nigel
A. Halls
65. Multiparticulate Oral Drug Delivery, edited by Isaac GhebreSellassie
66. Colloidal Drug Delivery Systems, edited by Jlirg Kreuter
67. Pharmacokinetics: Regulatory Industrial Academic Perspectives,
Second Edition, edited by Peter G. Welling and Francis L. S. Tse
68. Drug Stability: Principles and Practices, Second Edition, Revised
and Expanded, Jens T. Carstensen
69. Good Laboratory Practice Regulations: Second Edition, Revised and
Expanded, edited by Sandy Weinberg
70. Physical Characterization of Pharmaceutical Solids, edited by Harry
G. Brittain
7 1 . Pharmaceutical Powder Compaction Technology, edited by Goran
Alderborn and Christer Nystrom
72. Modern Pharmaceutics: Third Edition, Revised and Expanded,
edited by Gilbert S. Banker and Christopher T. Rhodes

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

73. Microencapsulation: Methods and Industrial Applications, edited by

Simon Benita
74. Oral Mucosal Drug Delivery, edited by Michael J, Rathbone
75. Clinical Research in Pharmaceutical Development, edited by Barry
Bleidt and Michael Montagne
7 6 The Drug Development Process: Increasing Efficiency and Cost Effectiveness, edited by Peter G. Welling, Louis Lasagna, and Umesh
V. Banakar
77 Microparticulate Systems for the Delivery of Proteins and
Vaccines, edited by Smadar Cohen and Howard Bernstein
78 Good Manufacturing Practices for Pharmaceuticals: A Plan for
Total Quality Control, Fourth Edition, Revised and Expanded, Sidney H. Willg and James R. Stoker
79 Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms:
Second Edition, Revised and Expanded, edited by James W. McGinity
8 0 Pharmaceutical Statistics: Practical and Clinical Applications, Third
Edition, Sanford Bolton
81 Handbook of Pharmaceutical Granulation Technology, edited by
Dilip M. Parikh
82 Biotechnology o f Antibiotics: Second Edition, Revised and Expanded, edited by William R. Strohl
83 Mechanisms of Transdermal Drug Delivery, edited by Russell 0.
Potts and Richard H. Guy
84. Pharmaceutical Enzymes, edited by Albert Lauwers and Simon
Scharpe
85. Development of Biopharmaceutical Parenteral Dosage Forms,
edited by John A. Bontempo
86. Pharmaceutical Project Management, edited by Tony Kennedy
87. Drug Products for Clinical Trials: A n International Guide t o Formulation 0 Production 0 Quality Control, edited by Donald C. Monkhouse and Christopher T. Rhodes
88. Development and Formulation of Veterinary Dosage Forms: Second
Edition, Revised and Expanded, edited by Gregory E. Hardee and J.
Desmond Baggot
89. Receptor-Based Drug Design, edited by Paul Leff
90. Automation and Validation of Information in Pharmaceutical Processing, edited by Joseph F. deSpautz
91. Dermal Absorption and Toxicity Assessment, edited by Michael S.
Roberts and Kenneth A. Walters
92. Pharmaceutical Experimental Design, Gareth A. Lewis, Didier
Mathieu, and Roger Phan-Tan-Luu
93. Preparing for FDA Pre-Approval Inspections, edited by Martin D.
Hynes Ill
94. Pharmaceutical Excipients: Characterization b y IR, Raman, and
NMR Spectroscopy, David E. Bugay and W. Paul Findlay
95. Polymorphism in Pharmaceutical Solids, edited by Harry G. Brittain

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

96. Freeze-Drying/Lyophilizationof Pharmaceutical and Biological Products, edited by Louis Rey and Joan C. May
97. Percutaneous Absorption: Drugs-Cosmetics-Mechanisms-Methodology, Third Edition, Revised and Expanded, edited by Robert L.
Bronaugh and Howard I. Maibach
98. Bioadhesive Drug Delivery Systems: Fundamentals, Novel Approaches, and Development, edited by Edith Mathiowitz, Donald E.
Chickering Ill, and Claus-Michael Lehr
99. Protein Formulation and Delivery, edited by Eugene J. McNally
100. New Drug Approval Process: Third Edition, The Global Challenge,
edited by Richard A. Guarino
101. Peptide and Protein Drug Analysis, edited by Ronald E. Reid
102. Transport Processes in Pharmaceutical Systems, edited by Gordon
1. Amidon, Ping I. lee, and Elizabeth M. Topp
103. Excipient Toxicity and Safety, edited by Myra L. Weiner and Lois
A. Kotkoskie
104. The Clinical Audit in Pharmaceutical Development, edited by Michael R. Hamrell
105. Pharmaceutical Emulsions and Suspensions, edited by Francoise
Nielloud and Gilberte Marti-Mestres
106. Oral Drug Absorption: Prediction and Assessment, edited by Jennifer
B. Dressman and Hans Lennernas
107. Drug Stability: Principles and Practices, Third Edition, Revised and
Expanded, edited by Jens T. Carstensen and C. T. Rhodes
108. Containment in the Pharmaceutical Industry, edited by James P. Wood
109. Good Manufacturing Practices for Pharmaceuticals: A Plan for
Total Quality Control from Manufacturer t o Consumer, Fifth
Edition, Revised and Expanded, Sidney H. Willig
110. Advanced Pharmaceutical Solids, Jens T. Carstensen
111. Endotoxins: Pyrogens, LAL Testing, and Depyrogenation, Second
Edition, Revised and Expanded, Kevin L. Williams
112. Pharmaceutical Process Engineering, Anthony J. Hickey and David
Ganderton
113. Pharmacogenomics, edited by Werner Kalow, Urs A. Meyer, and
Rachel F. Tyndale
114. Handbook of Drug Screening, edited by Ramakrishna Seethala and
Prabhavathi B. Fernandes
115. Drug Targeting Technology: Physical Chemical Biological Methods, edited by Hans Schreier
116. Drug-Drug Interactions, edited by A. David Rodrigues
117. Handbook of Pharmaceutical Analysis, edited by Lena Ohannesian
and Anthony J. Streeter
118. Pharmaceutical Process Scale-Up, edited by Michael Levin
119. Dermatological and Transdermal Formulations, edited by Kenneth
A. Walters

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

120. Clinical Drug Trials and Tribulations: Second Edition, Revised and
Expanded, edited by Allen Cato, Lynda Sutton, and Allen Cat0 Ill
121. Modern Pharmaceutics: Fourth Edition, Revised and Expanded, edited by Gilbert S. Banker and Christopher T. Rhodes
122. Surfactants and Polymers in Drug Delivery, Martin Malmsten
123. Transdermal Drug Delivery: Second Edition, Revised and Expanded,
edited by Richard H. Guy and Jonathan Hadgraft
1 24. Good Laboratory Practice Regulations: Second Edition, Revised and
Expanded, edited by Sandy Weinberg
1 25. Parenteral Quality Control: Sterility, Pyrogen, Particulate, and
Package Integrity Testing: Third Edition, Revised and Expanded,
Michael J. Akers, Daniel S. Larrimore, and Dana Morton Guazzo
1 26. Modified-Release Drug Delivery Technology, edited by Michael J.
Rathbone, Jonathan Hadgraft, and Michael S. Roberts
1 27. Simulation for Designing Clinical Trials: A Pharmacokinetic-Pharmacodynamic Modeling Perspective, edited by Hui C. Kimko and Stephen B. Duffull
1 28. Affinity Capillary Electrophoresis in Pharmaceutics and Biopharmaceutics, edited by Reinhard H. H. Neubert and Hans-Hermann Rii ttinger
1 29. Pharmaceutical Process Validation: An International Third Edition, Revised and Expanded, edited by Robert A , Nash and Alfred H. Wachter
130. Ophthalmic Drug Delivery Systems: Second Edition, Revised and
Expanded, edited by Ashim K. Mitra
1 3 1 . Pharmaceutical Gene Delivery Systems, edited by Alain Rolland
and Sean M. Sullivan
132. Biomarkers in Clinical Drug Development, edited by John C. Bloom
and Robert A. Dean
133. Pharmaceutical Extrusion Technology, edited by Isaac GhebreSellassie and Charles Martin
134. Pharmaceutical Inhalation Aerosol Technology: Second Edition, Revised and Expanded, edited by Anthony J. Hickey
135. Pharmaceutical Statistics: Practical and Clinical Applications,
Fourth Edition, Sanford Bolton and Charles Bon
136. Compliance Handbook for Pharmaceuticals, Medical Devices, and
Biologics, edited by Carmen Medina
1 37. Freeze-Drying/Lyophilization of Pharmaceutical and Biological
Products: Second Edition, Revised and Expanded, edited by Louis
Rey and Joan C. May

ADDITIONAL VOLUMES IN PREPARATION

New Drug Approval Process: Fourth Edition, Accelerating Global

Registrations, edited by Richard A. Guarino
Microbial Contamination Control in Parenteral
edited by Kevin L. Williams

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Manufacturing,

Foreword
Like most of todays technological success stories, the history of freezedrying has been pretty much limited to the 20th century. Whereas Altmann
(1), in 1890, reported drying frozen tissues to make histological sections, it
was not until 1909 that an application to biologicals was rst reported (2).
Although the rst patent was led in 1927 (3), there appears to have been
little interest in commercial uses until 1935 with a publication by Flosdorf
and Mudd (4) that introduced the concept of the cold trap. In the early
1940s Flosdorf et al. (5) in the United States and Greaves (6), working quite
independently in England, constructed plants for the large-scale production
of dried plasma for wartime use, establishing both the principles and the
commercial potential of a new industry.
During the subsequent half-century, the potential of freeze-drying
captured the imagination of both scientists and industrial engineers, often
without full appreciation of the economic limitations imposed by the
immutable thermodynamic costs of freezing and the subsequent sublimation
of the frozen water. The food industry in particular was attracted by the
potential of prolonged room-temperature storage at a time when home
freezers were not yet a staple in every kitchen. It is surprising how long
it took to recognize the inappropriateness of this demanding technology for
use with a low-cost, high-volume product.
It is the biological and pharmaceutical industries that have been
best able to capitalize on the unique virtues of lyophilization and that
have stimulated continuing research into the biophysics of both freezing
and freeze-drying, some of which is displayed in the initial chapters of
this volume. These are the studies that are progressively converting the
pioneering and somewhat brute force demonstrations by Flosdorf
and Greaves into modern, more nely tuned procedures, the importance
of which cannot be underestimated. At present it is the pharmaceutical
industry with its high-cost product that drives the development of this
demanding technology that is increasingly delivering great benets to our
society.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

This volume provides clear evidence of the mature state of

freeze-drying technology, from the mundane to the sophisticated, all
of which are essential to a quality product. And, paradoxically, it is
the potential for a high-quality product that will stimulate eorts for still
higher quality. The more faithfully the lyophilized product resembles the
starting material, the more the focus will fall on the quality of the starting
material.
The ability to maintain the complex and delicate structural relationships of biologically active compounds during storage at ambient
temperatures has been a boon to manufacturers but with an impact well
beyond the manufacturing process itself. Analyses of the freeze-drying
process are beginning to shift from the physical aspects of freezing and
sublimation to a more sophisticated examination of the eects of the process
on the chemical structures and the biological properties of the products
themselves.
It is here that the technology of freeze-drying may ultimately make
its greatest contribution. Protein function and protein conformation
are inexorably linked, and the forces that maintain functional conformations can be substantially and often irreversibly altered by cold and by
dehydration (7,8). As those in the pharmaceutical industry look more
closely at how these forces may be altering the structure and jeopardizing
the function of biologicals during lyophilization, they will inevitably nd
that many of these alterations, particularly those induced by cold, are not
limited only to the freeze-drying process but are inherent in many of the
isolation and purication procedures conducted well before nal processing
begins.
An active site on a protein may be a very small proportion of the
total molecule. Is maintaining the function of the active site good enough?
Why is the rest of the molecule there? Does it make an unrecognized
contribution and should we worry about its integrity? The human body
is exquisitely designed to reject malformed and altered proteins. Will
denaturation of inactive portions of a protein alter its physiological
function? Can such denaturation be responsible for unrelated side eects?
The resolution of technical concerns such as freezing rates, drying temperatures, and product solubility will permit a more critical reassessment of
product quality.
As the technology of lyophilization is perfected, it is creating an
environment in which attention will be increasingly focused on the stability
of product at the molecular level, not just after or even during lyophilization
but throughout the entire manufacturing process. The benets to society of
safe and more eective pharmaceuticals are indisputable and for those of us

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

who have participated in the development of freeze-drying technology, it is

a privilege to have been part of that history.
Harold T. Meryman, M.D.
Founding Member and Past-President
The Society of Cryobiology
Rockville, Maryland, U.S.A.

REFERENCES
1.
2.
3.
4.
5.
6.
7.

8.

R Altmann. Die Elementarorganismen und ihre Beziehungen zu den Lellen.

Veit and Co., Leipzig, 1890.
L Shackell. Amer. J. Physiol. 24:325, 1909.
HL Tival. U.S. Patent No. 1,630,985, 1927 (and 1932, No. RE 18,364).
EW Flosdorf and S Mudd. J. Immunol. 29:389, 1935.
EW Flosdorf, F Stokes, and S Mudd. J. Amer. Med. Ass. 115:1095, 1940.
RIN Greaves. The Preservation of Proteins by Drying, H.M.S.O., London,
1946.
JF Carpenter, BS Chang. Lyophilization of protein pharmaceuticals. Biotechnology and Biopharmaceutical Manufacturing, Processing and Preservation.
Edited by KE Avis and VL Wu, Volume 2, 199264, 1996.
LI Tsonev and AG Hirsh. Fluorescence ratio intrinsic basic states analysis:
a novel approach to monitor and analyze protein unfolding by uorescence.
J. of Biochemical and Biophysical Methods 45, Issue 1, 121.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Foreword
When I rst became aware of the freezing process, some 30 years ago, the
process appeared to me to be rather simple and straightforward. At
that time I was employed by a major manufacturer of freeze-dryers but
working in the eld of microelectronics. Given my background in physical
chemistry, I found myself becoming increasingly involved in the eld of
freeze-drying. I must admit I was surprised and somewhat puzzled to learn
that some of those using this process were experiencing diculties. Many
formulations of lyophilized products were made isotonic and contained
a host of other recipients such as bulking compounds, cryoprotectants,
surfactants, and pH modiers. It was then that I started to read publications
concerning the freeze-drying process, particularly those that oered any
explanation regarding why some freeze-drying processes were not successful. It was at that time that I realized that the freeze-drying process was
more complex than my rst impression. That viewpoint has not been altered
and perhaps has only been reinforced as the years have passed. However,
a great deal of research has been and continues to be done in this eld to
enhance our understanding.
The actual number of factors we should take into account with a
freeze-drying or lyophilization process I simply never stopped to count. But
I am certain that they will exceed the 10 ngers on our hands. While not
wishing to examine every possible aspect of this process, let me just share
with the reader just three areas that I feel to be of major concern.
The rst and foremost is the thermal properties of the formulation,
without which one is reduced to process development by trial and error.
With knowledge of the thermal properties, one is able to quickly develop
and validate a lyophilization process. Without such knowledge one has no
reference point on which to rely should a change occur in the properties of
the formulation. Knowledge of the thermal properties is paramount to the
development of a lyophilization process.
The stability of a lyophilized product will be dependent on the residual
moisture content. The industry is certainly very much in need of a means

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

of determining the residual moisture in a product that is both nondestructive and noninvasive. The method should determine the moisture without
destroying the dried product, nor should the measurement cause any
changes in the product properties such as a loss in activity.
Finally, although the equipment should only provide a safe environment for the product and the necessary operating parameters for the
lyophilization process, dierences in freeze-drying equipment can aect
the process. One should be aware of such dierences, especially when
transferring a process from one dryer to another. So in any discussion
of the lyophilization process, the freeze-drying equipment should not be
overlooked.
It is the intent of this Foreword to provide the reader with an
appreciation that considerable eorts are being made to enhance our
knowledge of the lyophilization process and its associated instrumentation
and equipment. While admittedly we will need more information to
complete our knowledge of this process, the advances described in this book
will take us closer to achieving this nal objective.
Thomas A. Jennings, Ph.D.
President
The International Society for LyophilizationFreeze-Drying, Inc.
Bala Cynwyd, Pennsylvania, U.S.A.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Preface
In the middle of the 1950s, when I was actively engaged in low-temperature
preservation of living tissues and organs, and tissue banking, I discovered
with surprise that in 1902, in St. Petersburg, the participants of the
International Congress on Paleontology were given mammoth meat at the
banquet. That curiosity actually came from Iakoutia, where a whole frozen
mammoth suddenly appeared in the collapsed bank of a Siberian river.
Apparently this body had been stored there in the permafrost for 15,000
years and the wolves still found it palatable, as did the conference
participants.
I learned then that this discovery was not uncommon and that, from
time to time, well-preserved mammoth were found in Northern Siberia.
Quite excited by this news I started to investigate whether I could get a
sample of that unique material. Numerous requests made at all levels of the
administration were fruitless and I had almost forgotten the issue when, in
the late 1960s, I received in Switzerland a big box from the USSR Academy
of Sciences andwhat a surpriseinside there was a big mammoth steak
with its fur still attached to the skin. Instead of being frozen it was perfectly
freeze-dried and, of course, naturally so. Apparently the big animal had
broken its backbone falling into a crevasse and was buried under snow,
almost immediately and relatively close to the surface, where it sublimed
for millennia, which kept its anatomical features almost intact. We made
many scientic investigations of this sample and obtained excellent electron
micrographs of the dried muscles. Less successful, by far, was the stew
that the Nestle cooks managed to prepare with it : it denitely was no
delicacy!
Much more recently, when opening a microbiological conference in
Morocco, I came across in my preparation work, on several interesting
papers dealing with lithopanspermia, the fantastic ride of living cells in
suspended animation, dashing throughout outer space on a rocks back.
Quite certainly freeze-dried, they traveled there for maybe millions of years
before being captured by the earths gravity eld and falling into the depths

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

of the ocean, whereit is claimed by some scientiststhey seeded life on

our planet. Freeze-drying, as a natural phenomenon, was again in the
limelight.
A scientic curiosity for almost 40 years after the publication of
Bordas and dArsonval in 1906, freeze-drying, later called lyophilization by
Earl Flosdorf at the time of World War II, has bloomed again within the
past 20 years to become an almost unavoidable technology to preserve rare
and sensitive biochemicals and drugs. Concomitantly, new challenges
appeared in basic and applied engineering elds and required more and
more sophisticated approaches.
This is the reason that Joan May and I found it useful, if not
compulsory, to prepare a second edition of our 1999 book Freeze-Drying/
Lyophilization of Pharmaceutical and Biological Products. Indeed, our
understanding of the fundamentals of freeze-drying has been continuously
improving in such areas as conned water, annealing, NMR assessment of
mobility in dried products, formulation, protein stabilization, and the role
of additives. We witness rising interest in some elds which have been long
considered as collateral but which are of prime importance today:
properties and behavior of glass and elastomeres in the always present
container-closer system. In parallel, industrial operations are becoming
more diversied and oer numerous dierent problems: scaling up towards
production from the laboratory bench throughout the pilot plant, cleaning
and environmental concerns, and sterile handling of bulk material with the
associated qualication and validation strategies. Moreover, new technologies are starting to develop: the use of co-solvents and irradiation.
However, in all cases, an absolute duty of care still remains for the operators
to provide both security and quality and keep their outgoing products in line
with the international standards, a eld which is quickly expanding.
Thus, year after year, lyophilization is becoming a vast, diversied
eld for research and development, engineering, and production, still under
the close eye of the administration and of the compliance ocers.
Therefore, it comes as no surprise that the publisher and the editors
decided to prepare this revised and expanded second edition. In so doing, it
is a privilege and pleasure for Joan May and I to extend our warm
appreciation to all our devoted, competent contributors and acknowledge
once more the generous support and professional skill of the sta at
Marcel Dekker, Inc.
Louis Rey, Ph.D.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Contents
Foreword
Harold T. Meryman
Foreword
Thomas A. Jennings
Preface
Louis Rey
Contributors
1

Glimpses into the Realm of Freeze-Drying: Fundamental Issues

Louis Rey

Structural and Dynamic Properties of Bulk and Conned Water

M.-C. Bellissent-Funel and J. Teixeira

Mechanisms of Protein Stabilization During Freeze-Drying

and Storage: The Relative Importance of Thermodynamic
Stabilization and Glassy State Relaxation Dynamics
Michael J. Pikal

Freezing and Annealing Phenomena in Lyophilization

James A. Searles

Freezing- and Drying-Induced Perturbations of

Protein Structure and Mechanisms of Protein
Protection by Stabilizing Additives
John F. Carpenter, Ken-ichi Izutsu, and Theodore W. Randolph

Molecular Mobility of Freeze-Dried Formulations as Determined

by NMR Relaxation, and its Eect on Storage Stability
Sumie Yoshioka

Formulation Characterization
D. Q. Wang

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Practical Aspects of Freeze-Drying of Pharmaceutical and

Biological Products Using Non-Aqueous Co-Solvent Systems
Dirk L. Teagarden and David S. Baker

10

Closure and Container Considerations in Lyophilization

Frances L. DeGrazio
Advanced Glassware for Freeze-Drying
Jurgen Thurk and Peter Knaus

11

Critical Steps in the Preparation of Elastomeric Closures for

Biopharmaceutical Freeze-Dried Products
Maninder S. Hora and Sidney N. Wolfe

12

Development of a New Concept for Bulk Freeze-Drying:

LYOGUARD Freeze-Dry Packaging
Meagan Gassler and Louis Rey

13

Regulatory Control of Freeze-Dried Products: Importance and

Evaluation of Residual Moisture
Joan C. May

14

Freeze-Drying of Biological Standards

Paul Matejtschuk, Michelle Andersen, and Peter Phillips

15

Industrial Freeze-Drying for Pharmaceutical Applications

Georg-Wilhelm Oetjen

16

Development of Process Data in a Pilot Plant Transferable

to Production
Hanna Willemer

17

Technical Procedures for Operation of

Cleaning-in-Place/Sterilization-in-Place Process for Production
Freeze-Drying Equipment
Gilles A. Beurel

18

Global Validation of Freeze-Drying Cycle Parameters

by Using Integral HFT Systems
Gilles A. Beurel

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

19

Lyophilizer Qualication: Some Practical Advice

Thorsten Fischer

20

Lyophilization Process Validation

Christian Bindschaedler

21

A New Development: Irradiation of Freeze-Dried

Vaccine and Other Select Biological Products
Louis Rey and Joan C. May

22

Some Leading Edge Prospects in Lyophilization

Louis Rey

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Contributors
Michelle Andersen Standards Division, National Institute for Biological
Standards & Control (NIBSC), Hertfordshire, England
David S. Baker

Pfizer Corporation, Kalamazoo, Michigan, U.S.A.

M.-C. Bellissent-Funel Laboratoire Leon-Brillouin (CEA-CNRS), CEA

Saclay, Gif-sur-Yvette, France
Gilles A. Beurel

S.G.D. Serail, Argenteuil, France

Christian Bindschaedler

Serono Laboratories S.A., Aubonne, Switzerland

John F. Carpenter University of Colorado Health Sciences Center,

Denver, Colorado, U.S.A.
Frances L. DeGrazio
Pennsylvania, U.S.A.

West Pharmaceutical Services, Inc., Lionville,

Thorsten Fischer

Aventis Behring GmbH, Marburg, Germany

Meagan Gassler
U.S.A.

W. L. Gore & Associates, Inc., Elkton, Maryland,

Maninder S. Hora

Chiron Corporation, Emeryville, California, U.S.A.

Ken-ichi Izutsu University of Colorado Health Sciences Center, Denver,

Colorado, U.S.A.
Peter Knaus
Switzerland

Forma Vitrum, Schott Pharmaceutical Packaging, St. Gallen,

Paul Matejtschuk Standards Division, National Institute for Biological

Standards & Control (NIBSC), Hertfordshire, England
Joan C. May Center for Biologics Evaluation and Research, Food and
Drug Administration, Rockville, Maryland, U.S.A.
Georg-Wilhelm Oetjen

Lubeck, Germany

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Peter Phillips Standards Division, National Institute for Biological

Standards & Control (NIBSC), Hertfordshire, England
Michael J. Pikal School of Pharmacy, University of Connecticut, Storrs,
Connecticut, U.S.A.
Theodore W. Randolph
Louis Rey

University of Colorado, Boulder, Colorado, U.S.A.

Cabinet dEtudes, Lausanne, Switzerland

James A. Searles Global Parenteral Products, Manufacturing Science

and Technology, Eli Lilly and Company, Indianapolis, Indiana, U.S.A.
Dirk L. Teagarden

Pfizer Corporation, Kalamazoo, Michigan, U.S.A.

J. Teixeira Laboratoire Leon-Brillouin (CEA-CNRS), CEA Saclay,

Gif-sur-Yvette, France
Jurgen Thurk Forma Vitrum, Schott Pharmaceutical Packaging, St.
Gallen, Switzerland
D. Q. Wang

Bayer Corporation, Berkeley, California, U.S.A.

Hanna Willemer

Koln, Germany

Sidney N. Wolfe

Chiron Corporation, Emeryville, California, U.S.A.

Sumie Yoshioka

National Institute of Health Sciences, Tokyo, Japan

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.