Table Of ContentPhysical Methods to
Characterize
Pharmaceutical
Proteins
Pharmaceutical Biotechnology
Series Editor: Ronald T. Borchardt
The University ofK ansas
Lawrence, Kansas
Volume 1 PROTEIN PHARMACOKINETICS AND
METABOLISM
Edited by Bobbe L. Ferraiolo, Marjorie A. Mohler, and
Carol A. Gloff
Volume 2 STABILITY OF PROTEIN PHARMACEUTICALS,
Part A: Chemical and Physical Pathways of Protein
Degradation
Edited by Tim J. Ahem and Mark C. Manning
Volume 3 STABILITY OF PROTEIN PHARMACEUTICALS,
Part B: In Vivo Pathways of Degradation and Strategies
for Protein Stabilization
Edited by Tim J. Ahem and Mark C. Manning
Volume 4 BIOLOGICAL BARRIERS TO PROTEIN DELIVERY
Edited by Kenneth L. Audus and Thomas J. Raub
Volume 5 STABILITY AND CHARACTERIZATION OF
PROTEIN AND PEPTIDE DRUGS: Case Histories
Edited by Y. John Wang and Rodney Pearlman
Volume 6 VACCINE DESIGN: The Subunit and Adjuvant
Approach
Edited by Michael F. Powell and Mark J. Newman
Volume 7 PHYSICAL METHODS TO CHARACTERIZE
PHARMACEUTICAL PROTEINS
Edited by James N. Herron, Wim Jiskoot,
and Daan J. A. Crommelin
Physical Methods to
Characterize
Pharmaceutical
Proteins
Edited by
James N. Herron
University of Utah
Salt Lake City. Utah
WimJiskoot
National Institute ofP ublic Health and Environmental Protection
Bilthoven. The Netherlands
and
Daan J. A. Crommelin
Utrecht University. and
Utrecht Institutefor Pharmaceutical Sciences
Groningen-Utrecht Institutefor Drug Exploration
Utrecht. The Netherlands
Springer Science+Business Media, LLC
Library of Congress Cataloging-in-Publication Data
On file
ISBN 978-1-4899-1081-3 ISBN 978-1-4899-1079-0 (eBook)
DOI 10.1007/978-1-4899-1079-0
© Springer Science+Business Media New York 1995
Originally published by Plenum Press, New York in 1995
Softcover reprint of the hardcover 1st edition 1995
109 8 765 43 2 1
All rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form
or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise,
without written permission from the Publisher
Contributors
Michael Bloemendal • Department of Protein and Molecular Biology, Royal
Free Hospital School of Medicine, London NW3 2PF, England
E. A. Cooper • Department of Bioengineering, University of Utah, Salt Lake
City, Utah 84112
Reinout J. Driebergen • Ares Serono, Chemin des Mines, Geneva, Switzer
land
Ernesto Freire • Department of Biology and Biocalorimetry Center, The
Johns Hopkins University, Baltimore, Maryland 21218
Paul T. Hamilton • Becton Dickinson Research Center, Research Triangle
Park, North Carolina 27709
James N. Herron • Department of Pharmaceutics and Pharmaceutical Chemis
try, University of Utah, Salt Lake City, Utah 84112
Vladimir Hlady • Department of Bioengineering, University of Utah, Salt
Lake City, Utah 84112
Joost J. M. Holthuis • OctoPlus b.v., 2300 AS Leiden, The Netherlands
Wim Jiskoot • Department of Pharmaceutics and Pharmaceutical Chemistry,
University of Utah, Salt Lake City, Utah 84112; present address: Laboratory
for Product and Process Development, National Institute of Public Health
and Environmental Protection, BA Bilthoven, The Netherlands
W. Curtis Johnson, Jr. • Department of Biochemistry and Biophysics, Oregon
State University, Corvallis, Oregon 97331
K. Knutson • Department of Pharmaceutics and Pharmaceutical Chemistry,
University of Utah, Salt Lake City, Utah 84112
Mark C. Manning • School of Pharmacy, University of Colorado Health Sci
ences Center, Denver, Colorado 80262
v
vi Contributors
James Matsuura • School of Pharmacy, University of Colorado Health Sci
ences Center, Denver, Colorado 80262
Kenneth P. Murphy • Department of Biochemistry, University of Iowa, Iowa
City, Iowa 52242
John J. Naleway • Marker Gene Technologies, University of Oregon, Eugene,
Oregon 97403
Peter Roepstorjf • Department of Molecular Biology, Odense University,
DK-5230 Odense M, Denmark
Tom A. A. M. van de Goor • Eindhoven University of Technology, Laboratory
of Instrumental Analysis, 5600 MB Eindhoven, The Netherlands; present
address: Hewlett Packard Laboratories, Palo Alto, California 94303-0867
David G. Vander Velde • NMR Laboratory, University of Kansas, Lawrence,
Kansas 66045
Preface
Proteins are still gaining importance in the pharmaceutical world, where they are
used to improve our arsenal of therapeutic drugs and vaccines and as diagnostic
tools. Proteins are different from "traditional" low-molecular-weight drugs. As a
group, they exhibit a number of biopharmaceutical and formulation problems.
These problems have drawn considerable interest from both industrial and aca
demic environments, forcing pharmaceutical scientists to explore a domain previ
ously examined only by peptide and protein chemists.
Biopharmaceutical aspects of proteins, e.g., low oral bioavailability, have
been extensively investigated. Although all possible conventional routes of ad
ministration have been examined for proteins, no real, generally applicable alter
native to parenteral administration in order to achieve systemic effects has yet
been discovered. Several of these biopharmaceutical options have been discussed
in Volume 4 of this series, Biological Barriers to Protein Delivery.
Proteins are composed of many amino acids, several of which are notorious
for their chemical instability. Rational design of formulations that optimize the
native structure and/or bioactivity of a protein is therefore of great importance
when long shelf life is required, as it is for pharmaceutical products. This issue has
also been examined in two prior volumes of this series: Volume 2: Stability of
Protein Pharmaceuticals (Part A) and Volume 5: Stability and Characterization of
Protein and Peptide Drugs.
Of equal importance for the therapeutic or diagnostic success of proteins is
their physical stability. The integrity of their secondary, tertiary, and quaternary
structure should be guaranteed during their shelf life (e.g., liquid formulations) or
upon administration to the patient (e.g., freeze-dried products). This means that
techniques that provide information about various structural aspects of proteins
have to be used. These aspects include, but are not limited to, three-dimensional
structure, hydrodynamic properties, physicochemical behavior, kinetics, thermo
dynamic properties, and dynamic behavior.
Unfortunately, no one technique in the present arsenal of structural methods
is able to provide 100% of this information. Therefore, a rational strategy is to
vii
viii Preface
employ a concerted approach in which the protein is examined using several
different structural techniques. The resulting information is cross-correlated to
provide a more complete picture of the chemical and physical state and/or bio
activity of the protein under different conditions. The most frequently used
techniques for structural analysis of proteins are described in this volume. The
impressive recent progress in all these techniques and expected future develop
ments are also discussed.
Several spectroscopic techniques can be employed for studying the structure
and function of proteins. These include fluorescence spectroscopy (Chapter I),
circular dichroism (Chapter 2), and infrared spectroscopy (Chapter 3). The first of
these, fluorescence spectroscopy, exhibits a level of sensitivity (subnanomolar)
unachievable by any other technique described in this volume. For that reason, it is
especially well suited for protein function studies where information about
ligand-receptor interactions (including antigen-antibody binding) and enzyme
kinetics is required. Furthermore, recent advances in time-resolved fluorescence
have made it possible to study dynamic processes that occur in proteins in the
nanosecond to microsecond time scale.
Circular dichroism (CD) is a spectroscopic technique that has long been part
of the concerted approach. Different parts of the spectrum (far UV, near UVNIS,
infrared) provide information on the secondary and tertiary structure of proteins.
The degree of structural information is less detailed than with new nuclear
magnetic resonance (NMR) or X-ray diffraction techniques, but CD has the
advantage that relatively simple equipment is used, scans are rapidly obtained, and
the (semi) empirical interpretation is not complicated.
It has long been known that certain transitions in the infrared spectrum of a
protein were due to the vibrations of the carbonyl and amide functions of the
peptide bond, but only in the last decade have instrumentation and analytical
techniques improved to the point where these transitions could be effectively used
to predict the secondary structure of a protein. Thus, infrared spectroscopy, or
more correctly, Fourier transform infrared (FTIR) spectroscopy, has become a
viable technique for examining secondary structure. Although the information
obtained is similar to that provided by CD spectroscopy, FTIR offers a greater
flexibility in detection that facilitates measurements with specimens such as cells,
crystals, tissue slices, and thin films, in addition to aqueous solutions of proteins.
In the last decade, the role of mass spectrometry for protein characterization
changed from only a marginal one to a core position. This dramatic change is
mainly the result of the development of a number of new ionization methods.
Potentials and limitations of these new mass spectrometry approaches and future
developments are discussed in Chapter 4.
As with mass spectrometry, the role of NMR spectroscopy in protein charac
terization has changed dramatically in the last decade and now rivals that of X-ray
diffraction, at least in the case of small-to medium-sized proteins (10-50 kDa).
This has largely been due to three developments: the availability of high-
Preface ix
resolution spectrometers (both high magnetic field and high frequency), the
effective usage of spin-coupling relaxation times to provide two-dimensional data,
and the labeling of proteins with isotopes such as 13C and 15N to provide data of
even higher dimensionality (three- and four-dimensional) than is available with
standard proton (IH) NMR experiments. These developments and their applica
tion to proteins of pharmaceutical interest, are discussed in Chapter 5.
X-ray diffraction has been and continues to be the definitive method for
determining the three-dimensional structure of a protein. However, the technique
is usually performed by specialists, and four excellent works reviewing the state
of the art in X-ray diffraction analysis of proteins appeared recently. Besides,
X-ray crystallography is not a technique that can be easily used in quality control
protocols of pharmaceutical proteins because of the problems encountered with
crystallization. Therefore, we have not included a chapter on X-ray diffraction in
the present volume and refer interested readers to recent works by Ducruix and
Giege (1992), Rhodes (1993), McRee (1994), and Drenth (1994).
Thermodynamic parameters provide valuable information about stability,
and differential scanning calorimetry has been used for many years in the charac
terization of small molecules. However, it has only been in the last 5 to 10 years
that microcalorimetry instrumentation has progressed to the point where similar
measurements could be obtained for macromolecules. Recent developments in
this field and their application to both protein folding and protein-ligand inter
actions are discussed in Chapter 6.
Chromatographic approaches have been the standard-bearers of the con
certed approach mentioned above. As discussed in Chapter 7, several fundamen
tally different chromatographic separation approaches, each with its own pros and
cons, have been developed over the years. Reversed-phase chromatography,
hydrophobic interaction chromatography, ion-exchange chromatography, size
exclusion chromatography, affinity and immunoaffinity chromatography, and
perfusion chromatography all provide characteristic information about the peptide
or protein involved. This tendency to diverge has made chromatography a highly
flexible tool for monitoring different aspects of proteins. Equally important for
optimization of the separation process is the further improvement of detectors.
The reader is therefore informed about, for example, "the state of the art" in
hyphenated high-performance liquid chromatography-mass spectrometry config
urations (Chapters 4 and 7).
A new branch on the tree of protein characterization methodologies is
capillary electrophoresis (Chapter 8). The potential of capillary electrophoresis
for the characterization of peptides and proteins has not yet been fully explored,
but it is clear that upon "maturation" of the technique in the future, capillary
electrophoresis will be recognized as extremely important because it provides data
complementary to other approaches.
We would be remiss not to include a chapter about the impact of molecular
biology on the characterization of pharmaceutical proteins. First, a number of such
